<%BANNER%>

Magnitude of the Oxidative Stress Response Influences Species Distributions

xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID E20101207_AAAACN INGEST_TIME 2010-12-07T16:38:18Z PACKAGE UFE0020084_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES
FILE SIZE 2064 DFID F20101207_AABSNH ORIGIN DEPOSITOR PATH matos_j_Page_034.txt GLOBAL false PRESERVATION BIT MESSAGE_DIGEST ALGORITHM MD5
412f629140f2fd95c23c958afa825318
SHA-1
96595e2358748d0902149bd1c2bff5f41c1063c8
2146 F20101207_AABSMS matos_j_Page_018.txt
fe941df96f4bdcea352d224bb9315977
2ddcda1602b458a6b1bf4806674ce372143994b5
139816 F20101207_AABRKF matos_j_Page_154.jp2
69d14cd5ab9f069ebd2905ab8f964e86
10811b8e1f4b96b0b0a7b9af27f89aa4ab148a15
86889 F20101207_AABRJQ matos_j_Page_083.jpg
27b380afb87d63dc77391309f08bd945
6576b12235b675d9fa2d71611a6875fc0ccbfc24
2200 F20101207_AABSMT matos_j_Page_019.txt
988254f1e53857f6a9ce713929d10e0e
5b9dcfcf4a794e442363287d4443c796316f062a
7183 F20101207_AABRJR matos_j_Page_159thm.jpg
37b4d5fe033afa4f2cd1a78b341348cd
512ca62eee659220882ae413aeb6f106f79e21f5
1983 F20101207_AABSNI matos_j_Page_035.txt
5ddeddd95e51e1ecd0f54be7b4459841
1112c333bb9eec64622e4456af7a188aa919b0b5
2215 F20101207_AABSMU matos_j_Page_020.txt
7292641e5a47ecfd5aafe99df387f0b0
770ad90b99c3fc66f2b564b21584b5a0b15be395
8217 F20101207_AABRKG matos_j_Page_055.QC.jpg
fd78b0c9a649e080e77459656145ae4a
ab2c36e12429d3865fac15b7ae31fe5a1c25fcae
1053954 F20101207_AABRJS matos_j_Page_029.tif
1c6caf8a319090b262a735e60d335a63
d32de0f8ad76738024ad620dad66a2fc82e6a78e
2102 F20101207_AABSNJ matos_j_Page_036.txt
e1649c9334c24e1b5d24570f3b495d7a
6220282e8ad05d506b0b1edada589c760b117569
2195 F20101207_AABSMV matos_j_Page_021.txt
889e06cc19c37e40717a198d8e36594a
13c72078ba4b100870c2b1edb59f64728b1c6c96
F20101207_AABRKH matos_j_Page_126.tif
7a8dab5d9cd2d411d8193f60788f8c5b
2bb36c99e60e46aab3d8f6e8f0cbd7ee5a4a6642
28354 F20101207_AABRJT matos_j_Page_160.QC.jpg
7c77db1aaeea743c76627af1e4479c16
91ed384cf6b1e6a0069cdf4fe754761b450efe7c
2111 F20101207_AABSNK matos_j_Page_037.txt
2e23f4e745ddafcc83065bc8586dabd7
f7b0f0e2a96228cd4d4b433cd57db5c37bc66d3f
2192 F20101207_AABSMW matos_j_Page_022.txt
1735b4aea3b68f5a756715a88eed5572
83767d71b954fe5ec8d8e77ba4caef9d258029bb
92162 F20101207_AABRKI matos_j_Page_045.jpg
37a640c7874aa9cdeba0a90edc44a570
744acddfb0f4b57cda4ba140d644cdeed6717b6b
76798 F20101207_AABRJU matos_j_Page_067.jpg
4415d054f9f7c9197eefdc7c2555a9d7
3c30854dd6427175c77a1618f5285a7a6292d547
1198 F20101207_AABSOA matos_j_Page_056.txt
731df17a1cc332ef418e5ca22205b1a4
32acdc0fe0dc3b0726e4d5376d0dc31e20d8d787
2088 F20101207_AABSNL matos_j_Page_038.txt
e3399f4e542e874ede63bd8e7814a60f
45907fd4fbcadc7cd69002f0096e21f8a2b43296
2207 F20101207_AABSMX matos_j_Page_023.txt
d3f8b0d6f009cc20242b61c5b6d6805b
3f0159d1dbfff0cbce34ff06d828856e8eb2307c
1393 F20101207_AABRKJ matos_j_Page_173.txt
2a9e3daabfcf157b7f5e5f2c39b114ab
e629e97a1b65ae48220104aa994e6f4e05bc8f2c
605413 F20101207_AABRJV matos_j_Page_053.jp2
2058e609e513bcc94fb11a0e8dd1676c
db2a6c6d54f876e8754f11088ef5a10d55b383c8
820 F20101207_AABSOB matos_j_Page_057.txt
4ebcb9dc10c0d70cee0b476bb414f920
e0951ae2cdf1f70824e2eb7b6e6bb6f0b1dab2b7
2001 F20101207_AABSNM matos_j_Page_040.txt
2b7c110ded04d1144a41599e2629a41a
44f435817c11bf4aeff4810d9658e1c95985b3cd
2252 F20101207_AABSMY matos_j_Page_024.txt
e67febccb39b4db75b78d94a1818571a
f4dec299a92b4cfe95dd838c593e506dd5686e3b
3867 F20101207_AABRKK matos_j_Page_002.jpg
298120393a03095d2462f16ccfd1ed44
6225d0814993c5e0bc7367786c8c4c70d667475d
7029 F20101207_AABRJW matos_j_Page_149thm.jpg
ab63a59e01c510f2512b57cae20f3d67
edb3a56ecf472402451975abaca71c41614a0956
1115 F20101207_AABSOC matos_j_Page_058.txt
1294fde6bc7995df7efd7558f5bac35c
5080a9634b9ec9ed9517f176a5cd239bf233f9d9
2045 F20101207_AABSNN matos_j_Page_041.txt
965e90a5e39a5f8a120a42b1932c899b
49ceb8350b390674c593f6309189a150d78fb38e
2087 F20101207_AABSMZ matos_j_Page_025.txt
0c8762bd84b556d8582f67119dd4e668
18f7042f526f53e0cbfc70c06941fdc3448f2970
24687 F20101207_AABRLA matos_j_Page_086.QC.jpg
8fd0fc0ec479f35f059f867d714bafb2
84bf600f283ad1ac490d4a2560785f65e612dae7
27235 F20101207_AABRKL matos_j_Page_065.QC.jpg
f511d62b47d5cd599cc25f29a33003bf
855473a9f945e28102d52482ce14c928eed1d67f
5566 F20101207_AABRJX matos_j_Page_141thm.jpg
a0bd4059ccd43ec9bce543bf2328efaf
b1531fb95b1ed8029f84e87fedd655be9816f477
742 F20101207_AABSOD matos_j_Page_059.txt
32c04e2d449c45554a1c7c7b532f72a4
0e1a98003f8c2522b3450db6a808fdfe5df3c3d4
2269 F20101207_AABSNO matos_j_Page_042.txt
c23163ef200265e15b648ba78f64ca27
4bc49adada0c5d4291003a69e6bca4572e5ab0d8
12421 F20101207_AABRLB matos_j_Page_003.jpg
618c18b7ef3245927a0482e45491eb62
cf4109d7124a3bc0e21c6b74d7f137b68dabec3c
2157 F20101207_AABRKM matos_j_Page_080.txt
f49e5832def4de63d4169e1fd7daf7a6
f8a4da2e42bc0333319ddc4bbff78b00516800a7
105188 F20101207_AABRJY matos_j_Page_163.jpg
43879aa0e4f1a4d71ba82ba70332f704
bfb076c552da0fc9d969ecfa325bb0746ffb65f1
1038 F20101207_AABSOE matos_j_Page_060.txt
d34b2f9c4027f370390afe4539799e8a
f0c209cbd05a1fb345f3b3a0bda98ab6759878f7
2095 F20101207_AABSNP matos_j_Page_043.txt
231c3b0e700c822222e6084011969bb1
5c51586b29013432e6927021f39701624a04e377
28981 F20101207_AABRLC matos_j_Page_034.QC.jpg
e61dce29cd5b47063bdf420e790e0479
2a87183c6ffea52588d2ade0348ca4509f9c309b
6709 F20101207_AABRKN matos_j_Page_131thm.jpg
82f33ab0ba5b44175f33aee6c1ad88ea
c630b5868017cb60b233a40536b05f1945d1cd0b
91106 F20101207_AABRJZ matos_j_Page_015.jpg
48755af186657dd1b1a301166b450a09
d78e607bde48c2f0739593473cfe73416f97a0ff
2179 F20101207_AABSOF matos_j_Page_061.txt
872258bb6732ced4397fa923b057c3dd
7d730d41870f995c7489985c8efd66cd2e794b2c
2169 F20101207_AABSNQ matos_j_Page_044.txt
78e75898819fa1d41ce1ddeb14e5f9f7
ba59d40bc903aabed6c5e5c488cf24e5de60423d
1372 F20101207_AABRLD matos_j_Page_111.txt
9f9e27844e5ed5ef1d9aacb8b59c6cb5
f637bff9daa05c7e3dc4e9a840cbcf54b771df75
1244 F20101207_AABRKO matos_j_Page_115.txt
e686a3e5c944ea142e9ba875f9798d10
b559a754ec8b8fab4889681160ea8a5753c22432
2218 F20101207_AABSOG matos_j_Page_062.txt
54047077fd6deb1318c618f0e5d2b895
a4d023a72e18b0ae20c2cf5ae22f2687088f0aad
2193 F20101207_AABSNR matos_j_Page_045.txt
3e85cf4be9ec77828ed9513f11d2329c
a33048f623ba7fe37acbca6cc6551cee5999d663
51130 F20101207_AABRLE matos_j_Page_052.pro
2e7e6222054f1171c3325ecad5d8c47e
fd66e40ef7c3e7544c82ade1d36ff53c480401e0
4211 F20101207_AABRKP matos_j_Page_147.QC.jpg
006a98cccacdc2f55de7df9de1e9165f
23575cc74690c4f6178c960283f09766dc027ba1
1897 F20101207_AABSOH matos_j_Page_063.txt
95848f2a2be6b4219403348283e76bb4
18f7004a887e385dc35d4087be50aa793ceb8192
F20101207_AABSNS matos_j_Page_047.txt
311596afb1755cae254ecc481cca55c5
73acc80949eb51dbbe3eb3624d21b9e708ed1b72
122004 F20101207_AABRLF matos_j_Page_126.jp2
f8eb4b33470bf46b6558fb1591f707ae
7384e0a0761b3636fc7add69cfa7720eb3bc22e8
27532 F20101207_AABRKQ matos_j_Page_073.QC.jpg
8db030f24e3af49df12f0ab094d60301
d2c25c808914d20b6c19d8a7840536158fa8b8b6
2093 F20101207_AABSOI matos_j_Page_065.txt
6c5245dcad4f9a9163885a3bfd346e6b
14e6a1012fc82b2f2f778497aa34d528c169a615
2180 F20101207_AABSNT matos_j_Page_049.txt
e7e57e4fdd7ab76c0c34987128879097
61339aca14b75920717d2354e838a16f847e8072
7149 F20101207_AABRLG matos_j_Page_162thm.jpg
9eba3878b9850becb8b560fa0d78649e
8144cb212fb3ad3fef33c4d00101713d6f98b8e1
F20101207_AABRKR matos_j_Page_098.tif
9be479d0b11a09641d3089b74610aad8
73d6f13698cf8384130c2415bd5ff79bf472286e
1031 F20101207_AABSNU matos_j_Page_050.txt
1e7cd7260ef5c5a2391338ce9c49d622
7a2127bf4712abfb98686a0572ae2f912790cdca
F20101207_AABRKS matos_j_Page_132.tif
1863a17a8f746210b20941f4956b8588
03c36973c2267a2d2777a1f96d6dbcd7550eccb0
2070 F20101207_AABSOJ matos_j_Page_066.txt
1be462955c0c985dc19dc2bfdea791db
3e147e524ab96303cd553b0123ef22c33a2352ce
1773 F20101207_AABSNV matos_j_Page_051.txt
a7d1193755620cd04320d6c7ebac2414
e962d77dafe5e60faf1a0f91d7d802d3f470e670
36277 F20101207_AABRLH matos_j_Page_102.jpg
427805087286e375aa6d28a0a9908566
1630f927aa4f700bb0a2c8cd57c39ab25e77d93c
31287 F20101207_AABRKT matos_j_Page_166.QC.jpg
a98825c9d419cf0bb7e21b9f9b97ea84
df1f838c7bb554a8c95e669fdb2140c7f1370f66
1870 F20101207_AABSOK matos_j_Page_067.txt
b4363bcd4c5d5d2932d07bc225946abd
6b421ea25fd32887d5440347add533e05a2778ef
2250 F20101207_AABSNW matos_j_Page_052.txt
fbd9f86f33ece15572b0fabbd4362cd1
51b723763e788d558018b584958f93c527e8ad7b
2793 F20101207_AABRLI matos_j_Page_162.txt
0539b943674e9d1c1466626ab8df5a11
d7db1a48c1c229d2c0bf121bc7cd9173a2714e32
108818 F20101207_AABRKU matos_j_Page_071.jp2
3563209b88c20421b9d2409b37cdbaf2
71baad02c5a6e52fd93d3e553697dec53079a507
1891 F20101207_AABSPA matos_j_Page_086.txt
e6fd9ec4c55953d80bb4c3acfa7fc13b
ed24320ccad03647ae57df10c48c7345ea04055e
2245 F20101207_AABSOL matos_j_Page_068.txt
3e14a1507449ef237766c735e298cfe0
cf6d21714f912987594dfcb5acf76ef670bafbe4
1070 F20101207_AABSNX matos_j_Page_053.txt
98d6631eb03b8bde29233b4ad1f29445
d5a9fb7684a81b1b49fa4826dd67685f409c8d5b
2162 F20101207_AABRLJ matos_j_Page_039.txt
70bb190a341fdf331efa06250cc871a3
5fec01431f02b966efc637761125777ee06a516f
56611 F20101207_AABRKV matos_j_Page_020.pro
185f1b3c419a4beffafdaa837153c436
7d9a49b20957203cc77f6f3c6c95afa494e1e10b
2163 F20101207_AABSPB matos_j_Page_087.txt
429575fdc8bdcdd0bde159b7e8fc74ce
aceb410539cd5573b583cfef11559a93daf45ec2
1894 F20101207_AABSOM matos_j_Page_070.txt
3b5e3d698dacda2c706afca3dbc6c58b
da9f7cb3883ea903a3ea10dac79623a4a9a7d0b5
1002 F20101207_AABSNY matos_j_Page_054.txt
2f6f0d37b84e8c07faca8335fef3e677
bb2bf1b46fdad291e22c7888b99ecc54231e8aac
6620 F20101207_AABRLK matos_j_Page_018thm.jpg
8e57bd4c607ac15bb0bb920b352d25c8
8b2ffad28b4275efdde8609c20173b895e30f335
57803 F20101207_AABRKW matos_j_Page_130.pro
adcf1cbd8834f9e102ac0b2ce1c10248
1d126a70073ea0842751c46a0abbbd48f5d37ffe
2145 F20101207_AABSPC matos_j_Page_089.txt
d9fb563ba938c84b542f7890e1bbb3c4
c32999a852f34e98f65caa3cdccdf89ed26e646a
2021 F20101207_AABSON matos_j_Page_071.txt
047f54fb7e43392f3f385d5ded79dc4f
dafb6f4f27d0340750ce9b3ae1b30687f9f7a277
959 F20101207_AABSNZ matos_j_Page_055.txt
66acf3d9407cadaa602ff90ac669e4d8
5af021023c767a6e4c4a33bd3131b9f583eef04d
F20101207_AABRLL matos_j_Page_033.tif
e9c6d1c68f467cdaab295c5085761027
6ee278c4cab414d514eac9a8b41037565273fe24
F20101207_AABRKX matos_j_Page_128.tif
097c79cd8eef16d68cc3eda4787fdb56
b78d8fb302e9f74edcc1509286b2816f7fe6278d
6714 F20101207_AABRMA matos_j_Page_085thm.jpg
b8b8c84785330c1de604c79755aec46a
d23f7b36884c73cc0746eee04e657c4c67599575
F20101207_AABSPD matos_j_Page_090.txt
be4bc484f834a14626250dfd97ffe0fb
01e6f0f875ce9c0f24e64044a14c871e42f40694
2140 F20101207_AABSOO matos_j_Page_072.txt
88c536a932842b8d115a66c19360426a
c6a5b1274e09ab7fe7507a8b02e6a8fd32cd0e38
6757 F20101207_AABRLM matos_j_Page_096thm.jpg
90586ceabe29681fa83de840159ab54b
68a1405586b7b649a513a0e7d748d05b45d11ae6
26530 F20101207_AABRKY matos_j_Page_071.QC.jpg
96ba1a0b112cd8e06b7744829b30ae3a
d16bf5a597e9b45bf1f47c4954c752a116ca0446
2081 F20101207_AABRMB matos_j_Page_082.txt
0720df50269650667a20409652ef8568
2efea74796e459f389ac3ab083f6fe5d4a6f2bcd
2136 F20101207_AABSPE matos_j_Page_091.txt
c637c507dcae189746297da5f25449c6
5feec35a79f301c6b7d16d652fef8746836d500a
2090 F20101207_AABSOP matos_j_Page_073.txt
8c27e24f74fc2f0d52ca0565eb47b193
fae74907d87a6a5f423432f37d81855f89dcefa3
6495 F20101207_AABRLN matos_j_Page_075thm.jpg
84945ee408846f0725a6179d1e006d57
ea445e4eb7fc3366404bd59be31e626997d1cb18
F20101207_AABRKZ matos_j_Page_137.tif
16336884d8216f8f6470645cd5803534
aeb3e289b83a173f52e2812bed55cc5e20a174aa
98601 F20101207_AABRMC matos_j_Page_160.jpg
3c551de89a63a4606be77a7311322a65
e183b44b9a10475f24478a2895a55101b6a8c852
2105 F20101207_AABSPF matos_j_Page_092.txt
315e0b46cbe8a86802518472ded81415
b006a2b3f40b581910bf334cec40dcd608876468
2030 F20101207_AABSOQ matos_j_Page_074.txt
5dfdd078a24604e2f3d921d139692fbd
d8aa20857c1baeaff22d58077faddbbfa3ec5dde
141351 F20101207_AABRLO matos_j_Page_159.jp2
e52380f0db9c0bf58c739379d505f650
34449eaef5eab7a77911bc329dbbe1ee026ae44b
2209 F20101207_AABRMD matos_j_Page_046.txt
206db773cc36f19e82a1c19ca3464b89
622ff7a201953dfc27dfb4b1a161576b72983195
2204 F20101207_AABSPG matos_j_Page_094.txt
3ab2a9a566eefaacf740d1153d26e9db
daede57c883246052c3bacc3552735d51f0bfd5a
F20101207_AABSOR matos_j_Page_075.txt
9a8916961927d06b753227b474be471e
70b6a707664b28384c34542da2252db35b77d3fd
1284 F20101207_AABRLP matos_j_Page_012thm.jpg
a80e3550c260101067e468b843d610d4
828e7bb52fa0fd343c1ebc6b6d304438c712ce8b
2533 F20101207_AABRME matos_j_Page_165.txt
b4eb806fdf95aff2749d2432e6dd6515
de01636591f41df7a2c675bd85e4c683e3983e6d
F20101207_AABSPH matos_j_Page_095.txt
3f2cebe5496ccb9de95a313375b50445
1299cc3597f3e114a85d2c8d321df1107f3ba6d2
F20101207_AABSOS matos_j_Page_076.txt
b83e2724f65d5f89376f7280cfa2cc9d
fd7e6405449a7cc4f26f247b1a59353cee33ffc2
84537 F20101207_AABRLQ matos_j_Page_074.jpg
8bb522c15e31d7ab02610629427d6905
d7ca4e68795c8da78e51b32838481d9ce2074115
88313 F20101207_AABRMF matos_j_Page_098.jpg
82d24fa546d1f0a8911ab02962ded32f
fd00a2ad9bc70d0c84b788a105d4a4a816868b59
F20101207_AABSPI matos_j_Page_096.txt
940db7bc655d3810bb88f3f40b8e0f86
34317321439e69a05977fff89b8fb4f20cbd2038
2003 F20101207_AABSOT matos_j_Page_077.txt
6045b8c4843f8d5bdfa75af8c418229b
881d752a32827790ffab70d2c5db8096c4d302c3
6499 F20101207_AABRLR matos_j_Page_098thm.jpg
3162fabe321f5ede350416bbf5e598f4
87dea11adf0ccb907265082b787ba19513fe6a71
772 F20101207_AABRMG matos_j_Page_101.txt
cbb40d5ea8a17f9474dd28d231ed24aa
fbc579bdb7173d05fc072afb4507c36ab33fa17c
2188 F20101207_AABSPJ matos_j_Page_097.txt
74b623a9105147c5f65de99f46147c9e
7efec5be4d4b4995ed3c79057eb5ba72e6b6b131
1981 F20101207_AABSOU matos_j_Page_078.txt
f5c11c9958f142df4ab53a955c48d982
b3f35e39e49c89976dc96194e3f7e3eaccc0d535
29283 F20101207_AABRMH matos_j_Page_047.QC.jpg
8cc4e4091433d63d436e818f619ddd4a
a5f0468268ba16f3595071d362c478e5fe95ffa2
27661 F20101207_AABRLS matos_j_Page_033.QC.jpg
5243973f3fc88c79df2afbbc767af3b4
1f55264b06d7181f6d056cfb5c9decd262c6169a
2164 F20101207_AABSOV matos_j_Page_079.txt
9b4958b757f4edffbe15a093e1156dd1
369edf8e40f3977d31d99214b4d97fce418ba4a2
27197 F20101207_AABRLT matos_j_Page_009.QC.jpg
9b4990631152831a8af2fe18a487d5a7
c073d68045b4553376e028c0b00a73401feb5aaa
2120 F20101207_AABSPK matos_j_Page_098.txt
239b9d2821d5cf56f2e6f2597da43b66
34f4d91c2b182cee00c372becb9294046e463aed
2246 F20101207_AABSOW matos_j_Page_081.txt
892a3315437df8bee25bb549a28baa69
adbca5c2859be26da0693a726153190b7d4a1d11
5622 F20101207_AABRMI matos_j_Page_052thm.jpg
23a7d710de1e35543079c737b9e04b7c
0411e04705351395e92f2c36da44e42e1d46315a
25804 F20101207_AABRLU matos_j_Page_058.pro
38de986f0a1729bcae2186f5a3fec8f4
220b99976ea8eac3a6fa4ebbd288811096c635e7
2176 F20101207_AABSQA matos_j_Page_117.txt
ae97ee5976eb62dca4be8cd01dc09743
5f5a2677195abf7f9a4b740a519a60547b5d8d5a
1926 F20101207_AABSPL matos_j_Page_099.txt
e6eb76bc9f40c4ce613fb2ae5c9b761c
ea138c7d73930c11b7623f53b16268715e2f87e4
F20101207_AABSOX matos_j_Page_083.txt
5c3007b4f77456e8916a58571234860a
8ecb3396d5a428edc70622798eac2ba6e9d25cb2
120493 F20101207_AABRMJ matos_j_Page_027.jp2
84be0cd8f240bdf345723e1ef14955c3
b4a3d822dcf1c3c80fe2647c64c95c3433b63d1c
26009 F20101207_AABRLV matos_j_Page_078.QC.jpg
dd7090af42a1ac2218082d430cbea2a2
df8d7a4e604dfb84a7982363c13679543e5398db
1980 F20101207_AABSQB matos_j_Page_118.txt
b1363d789dac5fcb0c7848c0ae90c510
9216154b471861006b35adc5797274393bb0b47b
516 F20101207_AABSPM matos_j_Page_100.txt
c5ca5a9d2e3633dcfff2f71129ba06b1
037d5d8b8d73b5669c08551e9721db97618c511e
2109 F20101207_AABSOY matos_j_Page_084.txt
9737446985326ecdc06861e1ce10ed12
4397de750a76f592217b06e9cd6c29a27b95d551
F20101207_AABRMK matos_j_Page_034.tif
34cbcde76cfb147131a01bc33fc6956f
3893689f6319210f0facc966af529293e10d0b24
F20101207_AABRLW matos_j_Page_151.tif
48ad3c44069a81f016a05dc365bf72ce
65fd401f709d018fb9c583419f8362056788d535
2012 F20101207_AABSQC matos_j_Page_119.txt
8e1f94ff05647c659c812f5c8ed2e26f
6952f137cfb768d4b551a9c2f7e95f0eb2936561
575 F20101207_AABSPN matos_j_Page_102.txt
c2685ee74d70b38664925799cb5fcb1e
b3ed8d320b7ebdc02be386bda282a1fcbfccdce4
2196 F20101207_AABSOZ matos_j_Page_085.txt
a05c416e78c6c0f7a488b6fce5659553
860af423e6b73539e6518b6d2ece193f1f6f3806
6468 F20101207_AABRNA matos_j_Page_065thm.jpg
2313888e051c2b3622414ced384b99cf
9ccb3957475cf951097379008311b56822031202
104534 F20101207_AABRML matos_j_Page_099.jp2
dbadc63485c6e7bfcfe79595b50a5e48
66d06be06452b2aba94a62a3ad02ef056183355f
39141 F20101207_AABRLX matos_j_Page_108.jpg
569318666f4c1663213499ec02fd3629
e4ea26b2751c4a660337f943e600034833597785
2127 F20101207_AABSQD matos_j_Page_120.txt
42665c753ca4a5ee350545a70878e861
ab296b6ee32e47097f8309a72a36b5b446e61f3b
1767 F20101207_AABSPO matos_j_Page_103.txt
2c2ad747f33fa1053c8823a40b4d23cc
a4c83cdb2e9da8df61eb6aaa83e4d7f597e64789
F20101207_AABRNB matos_j_Page_050.tif
83a3962fb779a00a506cbbab39c97c93
2ac19541857f414e488dbaae07c06e270d4a16c8
6724 F20101207_AABRMM matos_j_Page_156thm.jpg
1fba3ac667d75d0fc1249ac433eb10cb
3a92c91a2f611dfb4837bff56a2ae2a803c4ca8d
118018 F20101207_AABRLY matos_j_Page_039.jp2
0d22edc6a5d5679fc19f74b7d3ccb441
c4068425b3ea9f15206a27f2ad20ca62b38ab42c
2076 F20101207_AABSQE matos_j_Page_121.txt
24928a73408827a2ebb1faf41c7974f4
87a25c66353015e4dc82a042b2f937db207f54d2
1521 F20101207_AABSPP matos_j_Page_104.txt
924abc4e549442c8774b1186e8f9367c
4217ba445445cc1949d32f5a14e625d21d1e2691
13513 F20101207_AABRNC matos_j_Page_053.QC.jpg
66592be4600035597f807ce06e9f221e
01eacd018dbcebe3e7d363e06e94807133ed1f2d
45675 F20101207_AABRMN matos_j_Page_050.jpg
ef5e0c7af100614002fdf89d8be819a8
95f3102de09ee50616dae79b59a2acc4b355fe5f
F20101207_AABRLZ matos_j_Page_074.tif
31bf74d688c3f59a95b337c24ee78eef
6193bc656e9223112591a1f801bcc9912fe7002c
2074 F20101207_AABSQF matos_j_Page_122.txt
cd186eb838c8793e761eb5d00c4b0f4b
7c54046cb6af3228f6cefb9db473be62f57be96a
1552 F20101207_AABSPQ matos_j_Page_105.txt
7cdcb5e92a5d469d6f323704474d169f
98e964bd35b30991e6da7d93176fa4f080c9009f
F20101207_AABRND matos_j_Page_123.tif
73ca02dfc8fe83a7aad819e30d05d5d7
1d253feb98b7f5f5e78da4589116e631a7fb7881
24608 F20101207_AABRMO matos_j_Page_011.QC.jpg
3cf7aae444bfda65be708544c40bf0d5
d0241d79f3f97e3eed7894122f7913b001213ea4
2138 F20101207_AABSQG matos_j_Page_123.txt
5bbdff9c3740946c6f64af44e99db5a0
09cdd5598f666eadcea3a5e9c16bbb5b6c62f6d9
849 F20101207_AABSPR matos_j_Page_106.txt
0c92dcb443d1797295f4d14f58df5d95
91dbebbbf6ecc3e76ccd9b41688eea7a099aac7c
120894 F20101207_AABRNE matos_j_Page_044.jp2
662943e29749385ae4396f560c8be698
de0429f246e4104ab3f760424c624e2bcf433059
53998 F20101207_AABRMP matos_j_Page_014.pro
20ae43fc30331e4151209a357df4417d
583acc69ac828d58a2d36e2ac48d5f3d711faabe
2073 F20101207_AABSQH matos_j_Page_124.txt
d070b5682cc2d9cee9f9c9ede32eb2d9
cbd3ad3cb56dab295b6fd9f869ce59d011c45f2c
764 F20101207_AABSPS matos_j_Page_107.txt
f507622dbc8b8bed2ef36eed9a45830f
21b15821ed0e8ebce639f399a9231d37802d4a60
931653 F20101207_AABRNF matos_j.pdf
e4ebee50be96d6e1bd9ff20bfe9f9e0d
37461de2ff7ae65df95104ba0a476617c1f42bef
F20101207_AABRMQ matos_j_Page_135.txt
7418a23174494b3a828414b00fd92ddb
c480bd252bfca8897ccca63aa1e83ba4ebcf5f4a
1937 F20101207_AABSQI matos_j_Page_125.txt
eeee818625160ff08f0b1f50472bd54a
72a22d4ac040fe57facbc759ef9874734acdae73
664 F20101207_AABSPT matos_j_Page_108.txt
bab8a1f13007fa7955ae462536589cf0
865c1a67b393d08aa3bff3f20f47641e45d79275
F20101207_AABRNG matos_j_Page_162.tif
d52a40a1782762867be2a43871feb0f2
37872320b08e8c9cf621e71827c4e681ded995e9
6652 F20101207_AABRMR matos_j_Page_036thm.jpg
6d72adf40810e5c2e9c1a3305c1f6f08
d1f87f6626969434f0fa89c4a1c72b7c18340b8d
F20101207_AABSQJ matos_j_Page_126.txt
bbdf8b724f0aeb1f2d46e85af6b523bd
53e9ca3cf38380fb8fbe0c08da6c9ebf17cf19d9
F20101207_AABSPU matos_j_Page_109.txt
57ddaa073ba2a9609afe147db1f95065
b31e1eaef4df835c5bc34523578fe4d8dd4e4214
30619 F20101207_AABRNH matos_j_Page_042.QC.jpg
45cdb5dd3101d9d4f12b8e7f2825c0a5
9befedba1b4f1e7050e01a87b9652c1011e00a8d
114830 F20101207_AABRMS matos_j_Page_076.jp2
98bdd4b2d7fe70d18d13ef26a440169b
ee826c861b348fbc5a705aa2a422e1a8fcb2b21a
2124 F20101207_AABSQK matos_j_Page_127.txt
260830ee6b12b9e947cc348c3da4fd70
cc25332f91e89a1633bc7178f77cfb647eeb1328
1714 F20101207_AABSPV matos_j_Page_110.txt
d03beecdebae5748c56a8132668211de
dbf6fda46bb41dd0eddf5ec9f80985cc35e45288
14934 F20101207_AABRNI matos_j_Page_147.jp2
f09c8c846866874ac750cafb2851e5c2
d48706d7b6294f35d3ceb4a139941fecb915fed8
53478 F20101207_AABRMT matos_j_Page_036.pro
1786e0cf8a1701fab50ba89a6b7001c5
cb7e6dde6dec2e376eb926ec56e38110038adec1
930 F20101207_AABSPW matos_j_Page_112.txt
13fba74764501e62887cfecfe61ad14f
26c06e727cc7e33fd9af7d8b089f66a4b7c08aa5
1987 F20101207_AABRMU matos_j_Page_029.txt
17fe1c29f3235369e8868c8492c2bedc
d981ceac532e8e0f80bdfc4a565e56d874b210b9
2323 F20101207_AABSRA matos_j_Page_144.txt
ae83324c64be08022977e6ccda866b81
a28fb65c2105e7d86f353d0f9e6e1ee40f9d0abf
2114 F20101207_AABSQL matos_j_Page_128.txt
d4eb18bae6008c364563a0bbaddd864e
11ee9d69d7980acf893e79289f9ec628f7481447
1217 F20101207_AABSPX matos_j_Page_113.txt
2f0341c75b979895f58e9dc9f5523fab
16367c7488d3a393d88de5fb12793d2ce8eb3c72
55596 F20101207_AABRNJ matos_j_Page_117.pro
ab85a7869f472677cb796d017073ace5
c1dd07d44a739f2e18d84b9b0d99389558bd6872
102848 F20101207_AABRMV matos_j_Page_067.jp2
dab8bd0b2b7eeb2a68261b27b46fd398
bfa0d30f762307573dd2b9e8f18f0c05812400d0
F20101207_AABSRB matos_j_Page_145.txt
441715bd7befdc230dbb7a450a7066ec
ece1e78e27e903fac7d87dc8a00982d2fd2a9b95
2139 F20101207_AABSQM matos_j_Page_129.txt
afcbf3f79d6c350c025b8054ab100191
45056b289239280708890118e04685571dd4b824
897 F20101207_AABSPY matos_j_Page_114.txt
1e639d5291a18eded73666ce1c029d6c
450ff8f4b14dcd3b53b4a2b7887ea6ccda749928
94358 F20101207_AABRNK matos_j_Page_048.jpg
9da73fc4ef8e7a2621d5dd0b8ed68c6b
9ab2dab9107579082b856d1d8ae36cec8e978d93
97020 F20101207_AABRMW matos_j_Page_052.jpg
a5e1b478ec60ef0d1b3c26efd568ff0a
e0cc65787f830c3ea0a09f1e8d8e99d0948c5cb9
2174 F20101207_AABSRC matos_j_Page_146.txt
66e76690890b13fe86fe5bb168162446
7f960b743ab16684527860802b4668cc7056750b
2262 F20101207_AABSQN matos_j_Page_130.txt
5303a37e62de9ff471dbdd714b736055
a3fe56cd0ab3ce880b099b0cd9eca97a18d1eaf9
2205 F20101207_AABSPZ matos_j_Page_116.txt
f8ff448d9339ed75e69cd171c95f984a
8f10dc62dc846f21b9f222a1fe53dc4511febac1
25928 F20101207_AABRNL matos_j_Page_077.QC.jpg
5cd5679327aa94015a35c2b8194ad05a
f0f75e490d067856e80027133d583c22cf5d1798
88354 F20101207_AABRMX matos_j_Page_123.jpg
ba2d88e11aa8e31a0c5b7b8438a6881d
82e786d5de7e7edee2224fb8fcbc60b72914709f
7006 F20101207_AABROA matos_j_Page_068thm.jpg
0e0baf1fe32f0de897ee8bcb1631702b
024ec687041c85bc933803999e7fb9ad6ca4df61
215 F20101207_AABSRD matos_j_Page_147.txt
68efa3a5c6deb9d528bac487e0860755
6cd1d58530c66954eed727faea06f828652e2ad8
2101 F20101207_AABSQO matos_j_Page_131.txt
28d09c47bf1dec9ef799d52dec4bb009
d991e9cd79c0d0f09405262d5558e0727b5546c9
2123 F20101207_AABRNM matos_j_Page_093.txt
249b379aac3eb2d9f416086b3dab93af
d5bc11b732a9d6121d83c3bbcf0bef5a5efab832
6783 F20101207_AABRMY matos_j_Page_122thm.jpg
53fbe9bec24b3a5b11f77dd5d6da45e7
91b384792e309982a6b26ead82002e9435501b46
90947 F20101207_AABROB matos_j_Page_116.jpg
3301cfaf136c5ae894f01aa685fbc896
8fae513b94d39943a2fb48e2c208da955a571ee0
2107 F20101207_AABSRE matos_j_Page_148.txt
5d48eb840ad1a069d848a5f082e26e4e
09d2434257e7c0e3b72395af3165e5515fbf787b
F20101207_AABSQP matos_j_Page_132.txt
c576db478a51718df50e37720bc281f9
0a21fb42e8497044c6715520008a286a22aa0a29
89370 F20101207_AABRNN matos_j_Page_079.jpg
c9b8d55008db77a5389354677261bd23
f6e60afc7a02292390eee7c7332ba3193c222c34
1051934 F20101207_AABRMZ matos_j_Page_016.jp2
0085d6ad8a61bd63bd3b58692cd889bd
d002c3e03e824b5c080310183d58d7e75d5454d1
27831 F20101207_AABROC matos_j_Page_084.QC.jpg
aff9bf64cf61dd35eeae28438c750d70
88798d43ec18a3aa5417f1ee9cb67e1517a0efde
2150 F20101207_AABSRF matos_j_Page_149.txt
ca06b357b7d79805cfa9ea6884e2d630
e16159bdffc4851b94523669eb13ad8af0e584c5
2155 F20101207_AABSQQ matos_j_Page_133.txt
1b7e34170b8ae9acf6fc589c462bb198
f09cbdbbde8f32c3a86cc97fcbd3e28a7df3fb2d
F20101207_AABRNO matos_j_Page_060.tif
ffab0fdedd4fcc3a88650a4a19e50bd1
d61585ccaf14d6dcd16ea2eee295e31b040d3c90
112061 F20101207_AABROD matos_j_Page_119.jp2
b395fcd9a06574ecdc0c771f9b7cc1a6
2f55515db4c7709e7ec5bc568eb8cc45ae43ada2
2182 F20101207_AABSRG matos_j_Page_150.txt
a95857dcd5c251c803381c3487e73c7f
de002a32f80281cafab43cbc86386dd208231fd2
579 F20101207_AABSQR matos_j_Page_134.txt
032c5a2e70fb5711e6bcf72b43f9c492
7d63cc5326a731aa213d7b42245f091aeb2d719d
71841 F20101207_AABRNP matos_j_Page_009.pro
8b85175374d922b2d7dc34f42ed7b058
f8f6bf14ef9479df71be1ea23d71100697847494
8423998 F20101207_AABROE matos_j_Page_138.tif
3cb3697397cc957074295ec6382f15a9
28c2e49b422efbac9c246876c9498c4f0c4f86b3
1607 F20101207_AABSRH matos_j_Page_151.txt
caac08009c14a02d415a0c2e9faefccc
6c550cfd35e2fedc55dd49e31c780a7c4e1ce4c9
615 F20101207_AABSQS matos_j_Page_136.txt
b8243a14964afaf7674698dd41e1e53f
3629a9efa550a8cdbcf838c8862737dee49c44f0
38525 F20101207_AABRNQ matos_j_Page_153.jpg
397bb508c8ecbf2462406320aaeb837b
23dd549358a3b87e72d6152f6306510658a32375
90697 F20101207_AABROF matos_j_Page_039.jpg
1ee6c2eace3884e2e5ec399caab16de3
b17eacf12f9e14909ba7a98fbce502d17b05067b
824 F20101207_AABSRI matos_j_Page_152.txt
7f1cfd8f8967407ef9677cc6f7fa2e30
fee93017231873766c2be794db08ee417fa99e88
804 F20101207_AABSQT matos_j_Page_137.txt
c831f9df8d3caecf74fe3ce7117c4d85
24a5ffb61c62d3f7d8638b7d2583d03edce8dfcd
88870 F20101207_AABRNR matos_j_Page_146.jpg
e3e0dff679c48d33030a4c6e844e9e0e
b8dc05326f0cdf228e08d7be796c160bdb41d5e6
38698 F20101207_AABROG matos_j_Page_054.jpg
218e3ba302d8e44f4bb1fbf32fd32fb7
6de1be40594a1a32ab5778b815b2bc761a773150
836 F20101207_AABSRJ matos_j_Page_153.txt
2bb6eff1de84c221ad99533946a6b920
58a8e45d5e6ca842fc3c6ccae93ea26d4490f4de
913 F20101207_AABSQU matos_j_Page_138.txt
d5fae92b379aedb896eb4279e60beaee
42efa4eafaefbf0d28a56a17090b15bc1422a6e6
3612 F20101207_AABRNS matos_j_Page_138thm.jpg
acf77fc2aede17b8c5c1d710ae0d5fb6
9f36018ce367220838374f407a520f745de7de41
92428 F20101207_AABROH matos_j_Page_145.jpg
86178a810a40065859b95ab16fc212d0
61377745c0802dc36948cb1ff81084207a2bad00
2724 F20101207_AABSRK matos_j_Page_154.txt
1a0f9b14eaa10f226708b84f7628f6b6
6e7e85b517b69944e1270addc9b9ae88be0e4811
911 F20101207_AABSQV matos_j_Page_139.txt
ad6f9e581ae0d5290c12b91a42e9641c
038bb37d2216da12af852b9e900be785705e69b1
2201 F20101207_AABRNT matos_j_Page_088.txt
f2a21a2e96490dcf9a67c09a17c8ab62
9b40e5d831b8a81e5f1905732f774a56492e2f0b
6660 F20101207_AABROI matos_j_Page_148thm.jpg
49ced00af568d9e6ccb68963d645ed6f
66c67f87e3d4b171d3f41d366f7cc0e23beb70f4
2774 F20101207_AABSRL matos_j_Page_155.txt
c856c662c648a8c6068e0f9f560dfecb
21589bfe33803a1d52053b2db35f6b9ca51f03d8
1492 F20101207_AABSQW matos_j_Page_140.txt
6f431abc4679e0c70ec681928daa8cb4
20937f1fa5f63fd251c2a06962b59b340951dea4
12175 F20101207_AABRNU matos_j_Page_106.QC.jpg
b9a99f25bc523bcbeb7146123aa003c2
72cea5da6668375ceac40ff5f3539c68d29df333
89551 F20101207_AABROJ matos_j_Page_072.jpg
ebe70605a393401b0b4c24852b75739c
f8082b3ab41ef94e0d648d073bc63f2b50a0c2c2
27788 F20101207_AABSSA matos_j_Page_038.QC.jpg
7ea2241b8c56b3edf7a5a208659bbb67
445d17643f090b2d99373ba5279505b30e567718
1447 F20101207_AABSQX matos_j_Page_141.txt
85835bbeb1846923ee7b8cd81f46cc86
d7b759ff77c53d49eeb591ab6083d3fd0790c6bd
17243 F20101207_AABRNV matos_j_Page_141.QC.jpg
5aa6f30fbd409acf99e3332adc974308
83b52bf9056b32b94a593c32c3922e6a7bfc6b8e
28614 F20101207_AABSSB matos_j_Page_039.QC.jpg
6dd5c201b0feaab5486b02ecca4782e4
3c3f2db6c480a23b659e32aa9d2ed528ff2883a3
2483 F20101207_AABSRM matos_j_Page_156.txt
bf373583b8e4264eea10b8640abf63ea
5d903ca24df2655d7177e2f2b7257f1aff94e364
2211 F20101207_AABSQY matos_j_Page_142.txt
30ccae88a12ec686b350ebc3ad144df1
f1d620fcd593bddfb36a32cbc8560b4c64c0d822
117643 F20101207_AABRNW matos_j_Page_004.jp2
d6519894a796c82322f96b3b4109f558
fd9be02dec5c21e483d4d090b14f1e2db0c3bcaf
16474 F20101207_AABROK matos_j_Page_055.pro
be81ee54f82be31a64a3f8e162ad4011
a9be9335c04bf723c343ad488a95729853b32bd6
28769 F20101207_AABSSC matos_j_Page_081.QC.jpg
bfc3d36328ef18937c14b0cb759b72de
7f487eb3e2c36ebf79757a03c99883e2f965167f
2574 F20101207_AABSRN matos_j_Page_157.txt
c7b9d37fe188618d72067a29e1eb53b2
c6870cbdb91a3fb5bafa4d5899190a32d938d066
2298 F20101207_AABSQZ matos_j_Page_143.txt
a6c00747895aed9d1927095b1678ce3e
11c52e0f57eae3ea3151b73ca1af07c02356aafe
62434 F20101207_AABRNX matos_j_Page_165.pro
879b597c908166b97cc4349910cc6055
466a23d7c926a5a1d5f0ae9f252f98f14ba8d6b8
289 F20101207_AABRPA matos_j_Page_012.txt
c0f6fb8646629880c8cd7946154644c5
f9e1ac8275e0b3c2722a56bc41846e8f04aaf1bd
84135 F20101207_AABROL matos_j_Page_078.jpg
888625828b67009186b46745cdb9da1b
ffadfe5654274aa129e0835992cdd9ac86d0e5ec
6653 F20101207_AABSSD matos_j_Page_117thm.jpg
f0cba0e582b0dc0b58e616489f4ff0ca
eedb60c544d92bab06410e213f8e937986925bc1
2530 F20101207_AABSRO matos_j_Page_158.txt
f4c374c29eda6264ee5c22b3803a2928
2b16bb8bfee346cf18a23e7aad6ae7863a8dc9a2
6718 F20101207_AABRNY matos_j_Page_020thm.jpg
5ee92d5c4d8c2b5b903d522f0f0f54e5
a552683cb99dae8d6eeb3b352faa3803abf44841
117286 F20101207_AABRPB matos_j_Page_131.jp2
c3677fbbd802eb5cac8e1c666c7e4475
d5a9e1330230e16b3c0107f8d0604568e4a8e2cf
222 F20101207_AABROM matos_j_Page_175.txt
32c86cc62b1a39afea5b83c206229b11
7f3d7a8b4577ad6dabbf711577349ca24752d00b
6774 F20101207_AABSSE matos_j_Page_023thm.jpg
6cf5c627887440b0513563c6bdcf1acb
574ab0bf3caea812b091e5707909e1226abf90bc
2764 F20101207_AABSRP matos_j_Page_159.txt
e982339548cbb5e472ade8623d26c0e9
5867042deadd78e272e34ba2530cbe3153361531
25271604 F20101207_AABRNZ matos_j_Page_133.tif
1cbd0b97b9fac9abf20f39b201e792eb
8903a80c446f12736633a9588f5e045c2ae46bed
54455 F20101207_AABRPC matos_j_Page_129.pro
f6533c10c2c818a6ef52a2bfa5ccbe29
55ffd80d841caca6c5938ff228cc7c8ae699b4e2
2063 F20101207_AABRON matos_j_Page_064.txt
6d4d202e7ae5794a99c12ffccef32f1b
78fc3f6a3782e0ab46e22f4b3ba3090de9bedf6a
3975 F20101207_AABSSF matos_j_Page_173thm.jpg
92819de02bb0cca7f438cff821e98d46
d2b1d546c26fb841aabe7f0a9664c2466931ed6d
2592 F20101207_AABSRQ matos_j_Page_160.txt
d90b3f2f920f2e836d6afa5ce2c6f121
5839ead5dbf65846a6ddb7660fa828802bab7d30
100690 F20101207_AABRPD matos_j_Page_157.jpg
ae128978b2a91522823e72cd16106b93
7f5ec2d0163b2d2efc8764cb4fde428c3eef14dc
F20101207_AABROO matos_j_Page_005.tif
2aa2a40d77156b95987719b9bb916ee5
edd658a55f3c96e2cabd2b5d249334c22b9ee7d6
3794 F20101207_AABSSG matos_j_Page_053thm.jpg
b771e5a568682bf474f25bab8a53d34d
06862ef3a42ecd5e5c49b7355405ebe8b5e2b45e
2698 F20101207_AABSRR matos_j_Page_164.txt
3fee892dc724e5a8800addb693a18c25
77b0c992ae20a75e6a15d7d8be0cf64b70986605
6806 F20101207_AABRPE matos_j_Page_145thm.jpg
d3ee28733aa050a771c714111bc05068
e12baef8dcc282594f5125bdb92beb1989cc682c
121843 F20101207_AABROP matos_j_Page_045.jp2
fd3a030581ee189405871dad4a16fd60
62d69a029fe4bc838c0d039185596db590d25871
6624 F20101207_AABSSH matos_j_Page_123thm.jpg
dcb66790b47c9856b83aecec3ddf1237
28719f869f0d1aa3c348fbc9d57b0fee7d74c224
2773 F20101207_AABSRS matos_j_Page_166.txt
144d3e3bf87dd83f83f4f083f334b53d
dae521358f6830294c90796b172f7dfef961a8bf
6707 F20101207_AABRPF matos_j_Page_127thm.jpg
ffd69a395a6fdfa902933f2aadacae1e
5473f384dbf4398a04990a739bbe76860575f876
28122 F20101207_AABROQ matos_j_Page_079.QC.jpg
294b0798155537178228c23238f68411
614379082d92eb840af5c26af2045887d86f8eec
3020 F20101207_AABSSI matos_j_Page_003.QC.jpg
ad08664014df6cd09e5fc4e34c7f360a
e8a47ec6fee79083b5f7204e4eb054355851dde6
2877 F20101207_AABSRT matos_j_Page_167.txt
4e075cdbc4a7abf59fee6aae0c431884
9e77e66bb415c52774e95689b48460dc2c782516
11983 F20101207_AABRPG matos_j_Page_102.QC.jpg
f74b27481bf55fba7ce4bb72cfb9697b
d494b3f68733be35bb17e5956e762e0aa403e901
6343 F20101207_AABROR matos_j_Page_040thm.jpg
a5adf0c671d8086be6f9165bf59f8a99
b7ed226f2b1578fd75bc73a03ea1915d6393aa17
29295 F20101207_AABSSJ matos_j_Page_154.QC.jpg
340254352a497941850dd0de7223ccff
a0085be0cab975eabdeb9b880a20d92158ba541d
2754 F20101207_AABSRU matos_j_Page_168.txt
a9d0a34e8076d21b553bd59bb08b003f
8699458643ae8ac08106781fd5386de0d8277aaa
F20101207_AABRPH matos_j_Page_003.tif
d662feb963c6cfe4343d2af96a3d133f
c872ea7bf55e62b2d995cb5c36d16351d7a1dadb
4531 F20101207_AABROS matos_j_Page_112thm.jpg
e4eb5662c8b795a84c19536edeea74f9
6b14a9b5e04ca146ace5567ad0968c5c221be896
8835 F20101207_AABSSK matos_j_Page_134.QC.jpg
0b1d1c9c83b7cf3f1939703b2d156b8b
9527df9faae6125bc7300918bdec70b5d74dd0e9
2669 F20101207_AABSRV matos_j_Page_169.txt
bc5ee58f21c755ac35cb8819c27997ba
0fcc04ed5584b17cd7f83244ef80830bc1e65762
F20101207_AABRPI matos_j_Page_099.tif
f7cf00f6c22152352e45fec556d791b6
cf61723df3ab8049be5f9f704e8e05a325931a9f
23010 F20101207_AABROT matos_j_Page_136.jpg
85a1e31179c52ee08f561555d70555fc
f0c947d8ce40b6c2b9b52609986182a9305642f8
28953 F20101207_AABSSL matos_j_Page_085.QC.jpg
30e8fce3b52231f532c9902f5ff3c763
9cc6cb23f0516022490d0a93822dd338310bd7d1
2381 F20101207_AABSRW matos_j_Page_170.txt
2caab082015fdf9202e9701d6d427862
a140b3d6f1e9f7882278aa50928ffa9ecf4a80c5
95648 F20101207_AABRPJ matos_j_Page_006.pro
09aedde54480354dc77cb112956702d2
2c7d50317e1d71989277dbaf19dbc17b61da4f60
6781 F20101207_AABROU matos_j_Page_094thm.jpg
93956b71a250ccdecb0d2c9e6242aa24
9ef346fdb45d58d5e51363df081af6c73e120219
28124 F20101207_AABSTA matos_j_Page_120.QC.jpg
f12e0a8fd9b99e2335b2d923ec8d2fc9
e07e74158d371d5abc31f4a13cd48f1af5605667
3380 F20101207_AABSSM matos_j_Page_050thm.jpg
066356aba97aafdbdbbd4741308515f3
463d6da4ac9fa9f8f4f38c3498468ac9d758d004
2770 F20101207_AABSRX matos_j_Page_171.txt
1e1b5441137ad5d541734fd88b8bbbab
a24fae3e1d0209c111c10803065ec89948d6b597
7102 F20101207_AABRPK matos_j_Page_168thm.jpg
19e4698acbeb5a21a7049a2307e5c656
8948f5789433e24ff79d6e64e028ee72c1a46dac
139827 F20101207_AABROV matos_j_Page_161.jp2
33a6459cd04afdfd903c46ffbc352ffe
cad3a0a2a106d0932cd58b6acb8d809a64e537e7
4086 F20101207_AABSTB matos_j_Page_114thm.jpg
397c11eb3ff100276d7304b5522a5359
057a46470ea7b4a4c7228f81aa61d45eb429e15e
F20101207_AABSRY matos_j_Page_174.txt
f4e6ffa643ee89da2e742d7d20d9182f
01d1cf5659b95274f103efdc07bba7961cdbb693
24456 F20101207_AABROW matos_j_Page_052.QC.jpg
fe10bec89bd34ecce0c879323c08a38c
fd793929edabcc0f8ee285f82fc598d0b67260bf
29099 F20101207_AABSTC matos_j_Page_163.QC.jpg
b39f61c7b72a64bab460eed771cd0d4a
0cd72b0f24dacc352e85bfc5b1c0e2c70e4b7369
6448 F20101207_AABSSN matos_j_Page_066thm.jpg
aa4b49489c1e67bc61fbf82c32ba6238
752fd37b41bff63fe8a9fd96557542d390208698
1582 F20101207_AABSRZ matos_j_Page_001thm.jpg
32ba5644303c90a6490ae55c22807418
2237acfb9a7e048713d5a60fdf7d19ffa19f095e
88494 F20101207_AABRQA matos_j_Page_014.jpg
ea5105e8e5041efacfb65959d3283270
39388974f7321b6bfe69b3b7517d2489a21974cc
112543 F20101207_AABRPL matos_j_Page_148.jp2
b02511c44261c1c2d443d872749399fc
afd519f3eeb408a90900f643110fefb7053aeec5
113223 F20101207_AABROX matos_j_Page_083.jp2
34a982adb0e7804a3f82beb60e76012a
7406e0742ec2c1af67e379cf2b1ec818e8dee5c9
6825 F20101207_AABSTD matos_j_Page_044thm.jpg
d8292bd13ff21f4b6184a2ea2dd0af96
d0c0597e36f00f1fa8cf47931089b50fb469da9c
6466 F20101207_AABSSO matos_j_Page_022thm.jpg
7510ef17ddb3e2fca9e4780dd029edf9
9402f471ff0bb609229db25a1c81b83566ec439c
89759 F20101207_AABRQB matos_j_Page_016.jpg
7865f4fb142ba3be5a2ec2c8ee965421
21551f72267f9b5b8453f76b1ade33a909b35cfe
200547 F20101207_AABRPM UFE0020084_00001.mets FULL
1f5d23677418173b2c198cd3d690d31f
ba83923b2817c9d8c7c85259d71b337b2e1d959c
88435 F20101207_AABROY matos_j_Page_036.jpg
4eee4ed8854522587e676ded6d1adc8d
6acb1746efc9476bac77cf96d3f0243937ff7c09
12167 F20101207_AABSTE matos_j_Page_139.QC.jpg
b1169b74fbbff587f12928032cc6928b
5bd8fcb7c933c716d8665996c691f137ee946597
30643 F20101207_AABSSP matos_j_Page_167.QC.jpg
bb80a1e58beb650b633f45bed9deadf5
a2cc680b5d9198abfb38dbe39db787680485791d
93017 F20101207_AABRQC matos_j_Page_017.jpg
5c730e03c6b91dc7464891d16bc23c94
a2108f449488312af4fe7218860b02a0614f69b2
28506 F20101207_AABROZ matos_j_Page_046.QC.jpg
8500fde8236e1b2f269d2e1452f69a01
6093d969a6e09fc873820a3e7c85329301bbb182
6412 F20101207_AABSTF matos_j_Page_014thm.jpg
080ea2cff59b27199165d79dab79114d
211824bda28efefdc5125d422f8102dd2a218ded
6882 F20101207_AABSSQ matos_j_Page_001.QC.jpg
8071fc61f9bdeb1e4241c5ca2d85b63e
15fc5352e22d953fbaf27b4db988bc961da5c0ea
90751 F20101207_AABRQD matos_j_Page_018.jpg
c76ba90d1929e1f0d462d055be2580d8
56c9ccd11526c37fe7b908005b30ccdcbb1ad540
5034 F20101207_AABSTG matos_j_Page_151thm.jpg
c229793712b036211037b619a2295af5
df573098ff74b3490399519bcd974f11b9ff5c8c
29145 F20101207_AABSSR matos_j_Page_020.QC.jpg
fd0e2d7f0eff4d965f9a4fdf500fd31f
465d5c2f921ae120d30b14eb59d77c7e68a83164
92167 F20101207_AABRQE matos_j_Page_019.jpg
4b8f12b2822d79a16e836d995bea7bb7
8cf2bef1eb4454fb5f82a9e192297d8cecdf1e56
22968 F20101207_AABRPP matos_j_Page_001.jpg
b46bfb9d09b5111dc089cca5d05177af
9a544093c432d6017f675aae980dae609123722f
5873 F20101207_AABSTH matos_j_Page_086thm.jpg
d932b27afa1e17a811565778fbb76abd
87695d11053095cda9a2f47a9041119511fb5638
12766 F20101207_AABSSS matos_j_Page_101.QC.jpg
ff47110d4421776476cc04dfedfd0ac1
88795a14ea8fed9a965782da0b3bb64925edc696
93853 F20101207_AABRQF matos_j_Page_020.jpg
17875f65fde3b8876d8893ee39feac4d
b1d27f3111caf8b9c623279c53381c8f7804940c
91512 F20101207_AABRPQ matos_j_Page_004.jpg
2f390fddd6372d4e2b12d4e649764364
1010388217816726549babc23d351db068e4b3f4
4934 F20101207_AABSTI matos_j_Page_007thm.jpg
f7e06fdc762ce4a8607d799c1c8b9b0f
1a0b0a0fd04544e8200617e02894e2858304c299
6721 F20101207_AABSST matos_j_Page_069thm.jpg
cc7f69a803a7dc3b336346ce81632272
f207d5d83a0b4372d9a9c5ed85aea68771e3f4f5
92868 F20101207_AABRQG matos_j_Page_021.jpg
bece762a652c4b195b92ca6af218b562
810d598e7b0095ec302671bd4a614be5f1a91732
75301 F20101207_AABRPR matos_j_Page_005.jpg
7e15d4ca2a6da1129200d37400f126f9
3c60fe119c99d6e92251b944ce26bf8a027bca44
16285 F20101207_AABSTJ matos_j_Page_109.QC.jpg
4eacf847df44df70b7268a2d43106104
598da10e7ae6be115cf372a4e35a9ed58f34d5c1
6924 F20101207_AABSSU matos_j_Page_154thm.jpg
7e7eea9a6fe92c999bc16e2ec39d9d13
8f338af210194ce7aaff04856cd104f9f8b334af
92211 F20101207_AABRQH matos_j_Page_023.jpg
750a1e7f9a93a9923045af31ea54bd84
cc107aaa981eca93504f35d093fab88baa564c14
90360 F20101207_AABRPS matos_j_Page_006.jpg
89b3bcac42e0a8568496a6b369458c46
85c9e6487276e894eae99123222a46a7eba86971
4299 F20101207_AABSTK matos_j_Page_175.QC.jpg
785bfd213a6607c99417ad8840781158
d9c0879347f843db7cccc0f5ad1aefe27ce13c66
6575 F20101207_AABSSV matos_j_Page_084thm.jpg
c50ff632809cbf9bba08aa8ddbad4651
1192f397b4f7097402f995675efcfc3d2daec763
93101 F20101207_AABRQI matos_j_Page_024.jpg
890430cb83d77c4496fa4592757244f9
8ee8299411549f923532333c5d0b560c6229a1f3
94032 F20101207_AABRPT matos_j_Page_007.jpg
8daeb3ac3e725a4c586e9612adbe5534
329d7b2976fdde87f6675ced9f7fdf7cb5aa1220
6570 F20101207_AABSTL matos_j_Page_116thm.jpg
ac2af1b5412361a01d3336a1d8b0508c
1d9ce2f749b0df9309d0c5f2055642d546aa08da
27183 F20101207_AABSSW matos_j_Page_127.QC.jpg
e103d88329258a8e017c6c399e9377c1
594b39b839395267b97538b71e03b93b737f6701
88082 F20101207_AABRQJ matos_j_Page_025.jpg
baa71363d152399cab1f8fe3c2d96d9f
2f778a49c186155488d094821c58319c9ecb00eb
21353 F20101207_AABRPU matos_j_Page_008.jpg
cb91bf319d2cf58e66410a35f3b942d3
ae90cfd3a38efdf61b422f0abcc267603634b39c
27497 F20101207_AABSUA matos_j_Page_091.QC.jpg
00e795936c24e4e2cabd1fa974e8e2d5
461031427d5490a4364f0d3d56aca1ea7c4dacae
F20101207_AABSTM matos_j_Page_120thm.jpg
2a13005550524af07484f0a2a386f9dd
11966e55def38a00c2d5a7a56840d281659e03b5
4233 F20101207_AABSSX matos_j_Page_057thm.jpg
f2dba25bf8401574c42ee16bc7237318
20a54ded67f53ae6599bb7af398388c86d688d5c
88557 F20101207_AABRQK matos_j_Page_026.jpg
3d3285643d394226d48f27a5b774b6ba
92f48450707de59b18c402b30a4eb48b59216ce8
102877 F20101207_AABRPV matos_j_Page_009.jpg
4bcbe6b051a65d854f2945b87be7e823
6998c1f05602ff8a79171ec304dd74800a44fc45
28067 F20101207_AABSUB matos_j_Page_128.QC.jpg
6ddb58060fc10ad3a417bc53f0c3682e
df8d9cea9d02753c419d147df0074894f09e1397
23878 F20101207_AABSTN matos_j_Page_005.QC.jpg
2cdc4efa6ee2038b5e2eedff9aab3c60
80570a42d1ec52daf36cbd9f3825aaadbe46150e
6732 F20101207_AABSSY matos_j_Page_174thm.jpg
efb96d237f85c9193ddf25634c016571
41f1d50e52f567c2810bf189ac23773d945711bf
92677 F20101207_AABRQL matos_j_Page_027.jpg
e36cf2a70e9299f9c1404289e2777fcc
8281dc2a2d5f020f714f5c28e342e4ddda343f00
18086 F20101207_AABRPW matos_j_Page_010.jpg
c7d31c94788c81813cf8eb8f5a9d694c
57eea4b9f42698102ab1372ba062c0824b3eef45
12739 F20101207_AABSUC matos_j_Page_137.QC.jpg
1f31e6f5395e71aff9fe3ecbc516482a
5a0b6e4bb76e4fe3bcce105367a5ca1b37a38435
F20101207_AABSSZ matos_j_Page_064thm.jpg
914b9028494bee2565daa1e3533021be
58659203dbc804ecdf403c52ccafebc4ed5f8a7e
83779 F20101207_AABRPX matos_j_Page_011.jpg
96344cd6eb698f52330eecefa9e94b70
3a850b6a21a354d630045e93b499bc6b1a7c1f89
91200 F20101207_AABRRA matos_j_Page_044.jpg
088bc12a2971e7f69fad6ff8005d7891
d4df3894b7c83f184bbe3c516801066c56283080
26870 F20101207_AABSUD matos_j_Page_119.QC.jpg
5df120972691dc9d7471024d60d48d68
6048e1a07e6fa7e1dde18d6df7c19181ef651146
6302 F20101207_AABSTO matos_j_Page_041thm.jpg
09038bb540b13b1c3370720596b0f9c6
f7935ee8835d5cd7f85bdce526d9c7da2664493f
92028 F20101207_AABRQM matos_j_Page_028.jpg
cfdfdeeb410bd5cd80cc06a9ae56cdd3
54ac3fa4891b4f0333ba818becab0851ac804dd7
14431 F20101207_AABRPY matos_j_Page_012.jpg
e03beab435b51cd5f57f2c84a5c8e92f
edf8da85be50270102b9b5306d7437989f87fc02
91889 F20101207_AABRRB matos_j_Page_046.jpg
04d50c3d34ac92325aa60b976215b052
1775763ed324a4f4d54d7e5733fe384f716564e4
27639 F20101207_AABSUE matos_j_Page_072.QC.jpg
05d146bbeae08baaa4875ec685fd6542
35a98eef17068733eed3503d90a4e6631427b058
24926 F20101207_AABSTP matos_j_Page_067.QC.jpg
1dfe53b63b8707f6a45578d0eb5db571
31faf8b2ebaf6112a6de79cabb25ffaf63b676b1
82729 F20101207_AABRQN matos_j_Page_029.jpg
d83153e5234b5c45f054c529fcb110a2
44fb537dfa6ae54412e726d95eb9c204c77f3f2c
100487 F20101207_AABRPZ matos_j_Page_013.jpg
1302242dccc2a179ebf3f2f257c4ab18
cc6ae056fd226d83d3f4446dd90d9c288f10ca7e
92883 F20101207_AABRRC matos_j_Page_047.jpg
bdea12109ffd1b4317d3322500c5fdf3
fcf2659fedf6b0d0f9adda2450d238ee0494f4ce
6628 F20101207_AABSUF matos_j_Page_150thm.jpg
db9537533e0138795697aa60b6f7e58e
9389748e336e7cd13c68b5305cb64cb6f4469ea7
3911 F20101207_AABSTQ matos_j_Page_113thm.jpg
65b0aad06cd95aa414f8e1e8ddb85668
e831242fbc4711cf477c2316640143927b4bb9d7
92111 F20101207_AABRQO matos_j_Page_030.jpg
e62cb9ed3306cff76a6c545e64597031
b91ceca4a66ecb7ec4435b09039ade801bc6a6cb
91364 F20101207_AABRRD matos_j_Page_049.jpg
1a525fc3ae75a6a67d8e4446ba5d8c6a
8991c07cb1a52b34f38c94f79ec2b5fb064b61af
14892 F20101207_AABTAA matos_j_Page_103.QC.jpg
fe510cfa9c2a9402333d4aa2572939f5
69ef339d69bc767c1f06c596e4644cf48febf426
6014 F20101207_AABSUG matos_j_Page_011thm.jpg
566c3badb9456af6deb63e541dd3b099
a31e0eb43f9be75cb6c8194cf9fe83f828edb22f
F20101207_AABSTR matos_j_Page_105thm.jpg
0bbcb447ed386becaa0ca3bd4f491127
d1970bd7c7be31a900a77850186d843c3d859d59
43439 F20101207_AABRRE matos_j_Page_051.jpg
a5987697f07a2117d111887dcbca10f8
705b9a275df1935e79459c3654760dcbdb533ac5
94269 F20101207_AABRQP matos_j_Page_031.jpg
ff791d156cce4b3bb1e5bb3882ecfb45
e96de41dd8cb80df1875297aecfaa559301849d4
F20101207_AABTAB matos_j_Page_104thm.jpg
8c98640638321033086a52b8e9db5223
7f2e0016924cb792e935f0c25d22fb3e0e8f8fbb
6859 F20101207_AABSUH matos_j_Page_028thm.jpg
6370d56b54cef143723e7da74f4adfee
af31668fd7bc4365ade32c6419a9ed9006b26b31
26671 F20101207_AABSTS matos_j_Page_043.QC.jpg
afa88f337a79a43f347c04f85bd424cc
c903a086bfcc4f6247d20396f034e9f9416435a5
46068 F20101207_AABRRF matos_j_Page_053.jpg
d364c25c0c011912fb2526c280bf601e
eae0ddc51cd912480c54f1d5921bb6228da711bd
91612 F20101207_AABRQQ matos_j_Page_032.jpg
8322e739c70b20a317de4109b3e8bfda
a39ab850e5737380acb92d43c58c56040645fc40
15457 F20101207_AABTAC matos_j_Page_104.QC.jpg
7c8da17f98dcf78e161a38297c722668
3e44bd24357c70430192f129c87f1ff790e7f39d
28976 F20101207_AABSUI matos_j_Page_174.QC.jpg
be8bf375adad824d9d02acf7c964fe82
5cf9bc9fcb3f0e37bfb028c71b3558b12d7c2940
2545 F20101207_AABSTT matos_j_Page_056thm.jpg
bb1aa8d7e04593b132276fb6d179f41e
9518efbf45af73f082a9d131e76a2906f66f6c3c
30944 F20101207_AABRRG matos_j_Page_055.jpg
93cc8800f35dd0616c90cdc792ac9abb
ce19ffd3eb327a3e7919d200788fc5d2c5b37a64
88760 F20101207_AABRQR matos_j_Page_033.jpg
17941998c5b4028a981330a0922e82a9
3e7a3caac347afb461755b0b90567d054ba91a33
11614 F20101207_AABTAD matos_j_Page_105.QC.jpg
f3b240f01314a52da99432c8cd774734
f74c91883acc485062e2257adf69bd0ad9fbcf9a
2816 F20101207_AABSUJ matos_j_Page_060thm.jpg
da4abe24ba26268bee9a5fb4c5fab8d2
f5a1e501852f0150210450aa0425ceef8de17991
28247 F20101207_AABSTU matos_j_Page_095.QC.jpg
5aaa85f706e5f98f088dc9448e8b11a1
34d943c732a03b9fac92ec06a8f5c62973b5b91e
48676 F20101207_AABRRH matos_j_Page_056.jpg
bccd4dd938b102427857c1aa61680dcd
d2c48922442361a26acf2e20ae6f036fcf8a7056
87989 F20101207_AABRQS matos_j_Page_034.jpg
38d23942ab008953a828c6fa9863091c
a2ef5b2be90088ae8ff912a14b22cab2a166b5c6
4229 F20101207_AABTAE matos_j_Page_107thm.jpg
5b9258a634a2b7ff900908ee50b93896
2993a17a963231899baf49a6c66edbf93a328f34
28860 F20101207_AABSUK matos_j_Page_121.QC.jpg
7ac259b30745765b8d5d2d41872a534f
768a782dbf4b73edd65e07446289cafc7410d53e
3971 F20101207_AABSTV matos_j_Page_103thm.jpg
8305c5070c0e49c0d37094b854850f1a
c1d0e743baf12054a65de62a490888c0e10db2d3
39519 F20101207_AABRRI matos_j_Page_057.jpg
49ec6cb49d395c0e63b50b5b4356be14
3f4ff0f10c2b13c44f897298d5742784db0b018f
82657 F20101207_AABRQT matos_j_Page_035.jpg
1a13bd5d0926d72e5fdfc7d9581ba0eb
fdec21fffe91af900ede960072ad43d10b9d27ef
4033 F20101207_AABTAF matos_j_Page_108thm.jpg
709d67a5a1d7333eae386e9967f87ab4
d35b95bc9af38072c775735fa68dce0998176df5
6541 F20101207_AABSUL matos_j_Page_132thm.jpg
1261489f383955cd07aea3e508181bcf
9dfa4a71e4b8a2d9ed0ef5fc7ec9cbb672459160
6989 F20101207_AABSTW matos_j_Page_143thm.jpg
030912e121aac44b6ef12f2154f90f0e
3812aef818ed4a1b7fa9462e74a2542278627c99
44801 F20101207_AABRRJ matos_j_Page_058.jpg
516971be2e3b8e179418aa3e7ac15f74
1e3ce26c1dcb06e68d09e36582bd5401c58a99f8
91397 F20101207_AABRQU matos_j_Page_037.jpg
5d583fd6c330c9b376ea785056e72c96
822d5439d50c37eb70737131463d263bc6e7be00
12502 F20101207_AABTAG matos_j_Page_108.QC.jpg
ccf5d0733b5b8c2ec2853d0357d28b27
85f1de599802231a4a4079a18318dc772be4eb40
5537 F20101207_AABSVA matos_j_Page_008.QC.jpg
bcc16f5bbcec7ca91e2424a510d13182
e07e0047fd94031e966b26155888aed1789f92ac
29152 F20101207_AABSUM matos_j_Page_158.QC.jpg
7eebbb22f75b6e2edb10e3e0c7cf64ed
8c9dcd7079f1dc148953be350c6cf0037cb15412
6313 F20101207_AABSTX matos_j_Page_074thm.jpg
39fd26458aebee3778ee0d7deb072a9e
fb1793f9b4a4311ccbb84b5d0c5ba61f3859199e
39266 F20101207_AABRRK matos_j_Page_059.jpg
65e74728ff6edb8fa25186718cebab10
17eec4b60d5dee81cd97529f9372df4857d46c9c
90152 F20101207_AABRQV matos_j_Page_038.jpg
2f12a79aa1c6b26e04f65a611e3fe6e9
b301c2133c16e80eb02ed1e7d646f7b020365067
15411 F20101207_AABTAH matos_j_Page_110.QC.jpg
114e5394309442bb2ad2bad9dc8db0e8
421fe5fbcd8c0ce09eef6c3b4476d18fa78c8854
6837 F20101207_AABSVB matos_j_Page_081thm.jpg
da5dfd1d07d2ac7e98766ad80cb8198e
1d807d51036db803dda99d13eff00e089fca5e89
27855 F20101207_AABSUN matos_j_Page_124.QC.jpg
e79f2ad74cae91b82f470e70e8900cb8
a94b6334c26766421930d896fad826739b9f68fa
28148 F20101207_AABSTY matos_j_Page_037.QC.jpg
d2efdd8649ad8fbac15edefa22cdbb59
4c8332e6e1efc1b2a2108162654566660a088f36
41241 F20101207_AABRRL matos_j_Page_060.jpg
3c795359c6afd21dcf9701b07d75d934
df994c0f3ce63736240bb32c000ef43708225194
85767 F20101207_AABRQW matos_j_Page_040.jpg
9a2cfbaf7d32e2789850b33f45ea60cf
97ab43a6d64dae72275d881c81aeee3ac2895e81
3275 F20101207_AABTAI matos_j_Page_111thm.jpg
d1c55679b0a6eedd22f2534dc474cfb4
2befde362030d5267eb1e525faa958e18500ebd4
19440 F20101207_AABSVC matos_j_Page_152.QC.jpg
52b8299c56664cebbe12185fb443753f
89f5b36cfba263f8a07c1561efdcd68812048770
6294 F20101207_AABSUO matos_j_Page_125thm.jpg
b125c2758dde84b325e354dda6f39289
c9127360452310aa3ce07937a5666866601de3f3
6525 F20101207_AABSTZ matos_j_Page_093thm.jpg
26d6a38890d3ff05ff604362bd2b069c
932b26b823d48d9973174584e1f4c88ec5d62c48
88573 F20101207_AABRSA matos_j_Page_080.jpg
76dcce9be19a792075c29d0fbb7ca937
7ed517e09e05c290ab454f3fcb43051577ba43c0
88614 F20101207_AABRRM matos_j_Page_061.jpg
4406be79cf1b93f7b9689674acce2360
528257958e421c9fb0ec1b7b9285a14f616380b6
84119 F20101207_AABRQX matos_j_Page_041.jpg
e32032c3f79af5822b52c6deff28f6bd
5bf5ad93598e45401c4757045d293abf0be10aff
12278 F20101207_AABTAJ matos_j_Page_111.QC.jpg
caf089be9f91aa64707fe5618819a02a
ad1119b89282fd6f8b7fd212a0dd74706bdb8edb
1124 F20101207_AABSVD matos_j_Page_175thm.jpg
9a03699159b7ebe5925718b28b111d06
61265b399cbf31898e49895ddd59d0c9fbf5e59e
92084 F20101207_AABRSB matos_j_Page_081.jpg
ee953e14b9be8f714e88972155f9bc4e
f8bbaa901783e0b975461ccf170620fd60920fcf
96430 F20101207_AABRQY matos_j_Page_042.jpg
e56bafdb8da56b37938227d26f31a4af
55809f6715b72e74e36ab6b706ca893466825691
15644 F20101207_AABTAK matos_j_Page_112.QC.jpg
3d097ce7ad5207bb950d1b8e3e132c3a
a3fa8ce2716fa3994f2db0516330cd29a4a36d2b
29952 F20101207_AABSVE matos_j_Page_130.QC.jpg
c13fc44d9e33377763899331d7d0aef8
d537578da34469d9d9279b7579ae4b46ee8a3d0e
17218 F20101207_AABSUP matos_j_Page_135.QC.jpg
077c8a9f22245885754e65a490b2f044
eabe9ef201ffb64dd01494759f5ad0de0eeb2bb9
86271 F20101207_AABRSC matos_j_Page_082.jpg
1312438b99545e8b00d97db8ba77cf83
96fc8a5218a67ce6f3ece77a97b2bf6cd76d797a
91498 F20101207_AABRRN matos_j_Page_062.jpg
4e01a9428f4dee6d1914e3269916e715
6eaa79961770b070f6f99f7b2b5348def542c8a7
87297 F20101207_AABRQZ matos_j_Page_043.jpg
c8c3f0221a247f00e24fb787e84d3c9b
f7f3b43bd26d160f1ad22d336a78c405dfe2a4a2
13797 F20101207_AABTAL matos_j_Page_113.QC.jpg
f4bd331f2cb3b0c24079c2c51891628c
38604bd1e41ef3fb1e57521bc1c2f021d9d3dc2b
27445 F20101207_AABSVF matos_j_Page_025.QC.jpg
49a3155bb18fe7e1d933d42498a3c746
f6676e03b3c5c91acded4de45ca777f5a141e97c
3545 F20101207_AABSUQ matos_j_Page_106thm.jpg
c13ee7d3725591470b28ab53c96ef523
71772336d1f267fd2e46ad9674b76f2f949b9b8e
88910 F20101207_AABRSD matos_j_Page_084.jpg
5369285756bacdb0ffbc78854aa53409
615d0aa6aafbb5b49bf3f1134750fbf29ff742e0
80243 F20101207_AABRRO matos_j_Page_063.jpg
966513ca11d8481369f7d018afe0eabf
089365cf97a1f74ae3b652ada10a365ae5440ab2
28355 F20101207_AABTBA matos_j_Page_131.QC.jpg
3a0c0725a38a694cf1f4b35a37a8ec4a
d2c97fe54674cc4fc012f92b4b3ed27277aaebfb
15147 F20101207_AABTAM matos_j_Page_114.QC.jpg
59981637ee501b3d03f23374815fb1ea
1f2f88e90977630e37d5713478a62c1b9182062c
27162 F20101207_AABSVG matos_j_Page_040.QC.jpg
6efdc44f398d512a33f926fdd3501e36
33b7aef4f7e28b4b3b2c10d4b13a082cbad077e1
26324 F20101207_AABSUR matos_j_Page_125.QC.jpg
9b7233f611a930e94a48e1f20b7c4ef2
8e9c935c5c98444457deebbd918190d3f75f45b8
92335 F20101207_AABRSE matos_j_Page_085.jpg
60c7c4d883a231af3025fb16df3eebec
fb3a4c4d096389039a9bca0688994acd21a4a8ea
87426 F20101207_AABRRP matos_j_Page_064.jpg
fd84c788a7b7886fdfede2876238e635
d7c1d9080e827f9b63589f9e4e6b5715ed7dd920
27513 F20101207_AABTBB matos_j_Page_132.QC.jpg
959a40c0e77d69e9511c29515b995a19
32416ac159f6e2148ce96f79cdf711df95fa1f73
3767 F20101207_AABTAN matos_j_Page_115thm.jpg
0047bd08cd914688217077b77eb620c2
97d9a15ec411012a98924c7673fdd951b2f81909
6540 F20101207_AABSVH matos_j_Page_142thm.jpg
a4f9c7c3285dda2a19bc336a771443f5
2d7a2590355997c96a85118c683fbc6091186b75
26453 F20101207_AABSUS matos_j_Page_066.QC.jpg
2b1ec001bd6c94ce54fd881ac9215c84
a270aeb267187a947fe5b80cb15cb5a2dc062659
79336 F20101207_AABRSF matos_j_Page_086.jpg
f813d768063bd4c81154e4bc05b35aa8
53e48635f80960b8f9e68b8e8b20d14f653fecb8
87676 F20101207_AABRRQ matos_j_Page_065.jpg
e9b323bf0b415f2553e301c00963731d
786e80b45c2093c1d0827759a1d7e7bcac8faae7
6634 F20101207_AABTBC matos_j_Page_133thm.jpg
31bd74fadbd469598968e5f70c907061
3fccc427bffd9d8d49c18288c6d0270464cd1d8c
28906 F20101207_AABTAO matos_j_Page_116.QC.jpg
7abbe6c33e711159174dd8c370baa3ec
786913a62e17c193fefd42fba3d4216b35ef7ef5
1093 F20101207_AABSVI matos_j_Page_147thm.jpg
b2e510c19f9fe993d8d47261138aff3c
9a6f587a63cb9d3ed153142500cf9be29120c535
27249 F20101207_AABSUT matos_j_Page_148.QC.jpg
b70d4da48341621f5f54ecb83ee735de
d7efdf40dc22c58122d983125a45fcdf14c7d7ea
86685 F20101207_AABRSG matos_j_Page_087.jpg
3e770dd24851670636a88af11458796c
c55a31cdf40eed5e25c065e1f016e1dd886d9720
84900 F20101207_AABRRR matos_j_Page_066.jpg
df0991ab70f838ee092400668495f96f
bc1a7710d312f1e0265b694d05a1302f2ed0c06f
28551 F20101207_AABTBD matos_j_Page_133.QC.jpg
1c5c71e9abb9477cdd4e841b0eee8f7e
86038bc55a2c6644d02c6ab60389d8de13a8562c
6221 F20101207_AABTAP matos_j_Page_118thm.jpg
996df831d9e942c113c73d5c01d8145b
c38406007c13322d0e74dfa50de3bc2c3f363f38
27793 F20101207_AABSVJ matos_j_Page_061.QC.jpg
d975a20bdbd6e22f4e55b9e692699e81
f25a3a31dacee4e2682eecb6e95a8d24bd1cb06a
6480 F20101207_AABSUU matos_j_Page_021thm.jpg
3203c389c36276a0454680ef554c2a55
0f610a60ca66d2714c554bd4127992337484eb8d
92367 F20101207_AABRSH matos_j_Page_088.jpg
2f2809afbbbdcd3e4fb98e2da4594af4
841b580d2175c3f40051aa75ca381be0673a27c6
94600 F20101207_AABRRS matos_j_Page_068.jpg
366fb580dfec91a3ae7f4d8808280925
f689debc20ed09940329e1b934aaa4a149121540
2002 F20101207_AABTBE matos_j_Page_134thm.jpg
8bdd0f94e1e41826b7633011ad7cb7f4
c565bc6c725b81e5c0d419028fa4cf4c61db9941
26314 F20101207_AABTAQ matos_j_Page_118.QC.jpg
e3ba24fc90e274dade64354565528573
b5351feb5233246eab1ff3f4d290b967fae65018
28062 F20101207_AABSVK matos_j_Page_117.QC.jpg
a3bddc0c4f11dd956f7641a4cdf1ffc2
3e01841f6aa033a4eeccc3234302e3be28273bb2
6642 F20101207_AABSUV matos_j_Page_013thm.jpg
6c3d72006103ed23efd629c2bc0b170d
94b27b06d44c8159759c1e1e9b29d232e716e22f
89364 F20101207_AABRSI matos_j_Page_089.jpg
022ca87164dfbf20ae2473ead92e604a
0edd644effee4a9cd72fdb465c32e0a461dcaf76
88256 F20101207_AABRRT matos_j_Page_069.jpg
b078002b8f051d7483f009bd346b6082
df88c6f42a4cf98f5a42c7e6e3e46ca77ee1bbc0
4048 F20101207_AABTBF matos_j_Page_135thm.jpg
c0a5545d28e5419767a5d565d216092f
82e1d0b2cfa1f1351e2f3498ec0104bd75df49cf
6585 F20101207_AABTAR matos_j_Page_119thm.jpg
8593d9a365ebdfd91213437158b512d7
5083995ad9333954627ab8caa3e111ee193bdb0a
5742 F20101207_AABSVL matos_j_Page_152thm.jpg
0e1dc424022d26a2ff947a0721d92eca
b96de4baf041d65f49fa59a6a21397b013523163
F20101207_AABSUW matos_j_Page_145.QC.jpg
b2ed5ee7228f61ca643837fb7b2703a3
27fb23c32d062a0694527f41eb843ca8fcc52e1b
92379 F20101207_AABRSJ matos_j_Page_090.jpg
f244daf6aef849ba4e283f4113b08141
fd6599e54b6b9c15fb3c859f0f03bf4337391c1a
82721 F20101207_AABRRU matos_j_Page_070.jpg
e7d8584977a5375df77f5f85402e52b3
f1f80f0e00445d77d77c5a784eda53d6856d9da4
1828 F20101207_AABTBG matos_j_Page_136thm.jpg
ccaa6602af89004fde2029e2f51277dc
eb49db6c4133bf3fea384492279e966365e6bc71
6821 F20101207_AABTAS matos_j_Page_121thm.jpg
5e43398dd2f0fb8b22e69281eae7c4e7
0f2e2172f3e029759873898959a0984ce1305acd
6394 F20101207_AABSWA matos_j_Page_009thm.jpg
74dd5eda42bcc7655c17a7ea84b1b76e
03dcbc45fc74298424e68468418934c418b52977
10703 F20101207_AABSVM matos_j_Page_060.QC.jpg
a22ce6811c20a926781798ca12b3414b
d1a8314b2dbca2cac73e4be2ae54c4b6bcec4604
19679 F20101207_AABSUX matos_j_Page_006.QC.jpg
8664d478fd006141706f218b9edce645
8e51c1796d5715939d91461fe9b4e85bec84e3e1
87532 F20101207_AABRSK matos_j_Page_091.jpg
7f9388fa2fd7c37dc6496583fbaa8a45
ef14cd9a35650d63547c023faf595c5a8be2698d
84521 F20101207_AABRRV matos_j_Page_071.jpg
769180e91b721252ea430afc9d99d23f
69d03b56fedbad3f95a24532377a8ec170532b3e
6700 F20101207_AABTBH matos_j_Page_136.QC.jpg
5ddad45c85acb5a1d31dc57a36dde488
2bbed9244592df98e50c042fbc4e0ff7424ed260
28475 F20101207_AABTAT matos_j_Page_122.QC.jpg
a81a7beeeb1323331b5d45b96d1bf75e
8643ec7ac05e97850141e5a4f746adaac29c88bd
1543 F20101207_AABSWB matos_j_Page_010thm.jpg
5d9199d2ec52dc0e9212fb82c382ef37
51be851e35a6fe3e939d99d3e430c7fc14fc5eeb
28482 F20101207_AABSVN matos_j_Page_172.QC.jpg
dca90bdb61378e3a244da1f129676f36
0ec9df6feedc8b5a9702f8de578b23898e4b2317
4823 F20101207_AABSUY matos_j_Page_012.QC.jpg
73174fb1e8f168ef1934c8b4d7ccd815
7018df8591befadf24b7448ae3672b08197766a3
87274 F20101207_AABRSL matos_j_Page_092.jpg
c83fdd48d4876e514dc251340a695835
cb92a77e59288effc4bae213e47eb92498d5f1fe
87445 F20101207_AABRRW matos_j_Page_073.jpg
07ca31c0d2a6eb753a8982ec9ae854d9
a40efc117cdeb96784d9703420cd3d3c264aea8e
3588 F20101207_AABTBI matos_j_Page_137thm.jpg
7081e63c7cc5d3a8debc627504ba9855
0fd28d4441a3bf085332010f83895ab7518f2810
27295 F20101207_AABTAU matos_j_Page_123.QC.jpg
0a342e5057ad2a8d7f486fe70b613e5b
dc069674f44d1dc46dabef74419bff6e9ed300ec
5103 F20101207_AABSWC matos_j_Page_010.QC.jpg
fa2c0e01e613ca32e4eb302ba6ac93ad
2fd3f0ba01ef57598d28abd8b7e7a24ba8d6c5c4
4081 F20101207_AABSVO matos_j_Page_110thm.jpg
e6fa04e02ae8f186ae557754f9cb9396
050e8ac9896903d46271058b625a1c8952e29474
27691 F20101207_AABSUZ matos_j_Page_064.QC.jpg
d1fdd9ef3b294e43055d3d5114dacf28
9b41d7b3e5b0046e429d96b8c0d8cebc9ec421fc
88520 F20101207_AABRSM matos_j_Page_093.jpg
54ee40b7945ebf189100610433d517c9
0ff667d54221ae331e0401844fc094219bebc0ee
86846 F20101207_AABRRX matos_j_Page_075.jpg
093ea08deceaec98a36d8c3dd40d9323
0939d438511ced6974cde7b051e2a4e3ee7c6d1e
42498 F20101207_AABRTA matos_j_Page_111.jpg
113e379f59320f7e5603ab38ee9ca81d
d3f49f86ec76edc54baf08fec70eaf415ea8854b
13558 F20101207_AABTBJ matos_j_Page_138.QC.jpg
b90d491d65154b22bd18073937fa0712
f2d9582e2d342c4b572b95b5a6971192e809c7bc
6657 F20101207_AABTAV matos_j_Page_124thm.jpg
c84d4449e0993e6439160745da05dffd
f6c40c14ba9122183f4f3d0910a74b578ad8040b
29206 F20101207_AABSWD matos_j_Page_013.QC.jpg
f27fb47371de06f0ed8e35a074ef728b
f94da4daa82788e2652addd963ae3eea490e3541
27600 F20101207_AABSVP matos_j_Page_089.QC.jpg
d5acd8eb9f9c27e5c18c5ebcf42e3d2a
8e38d1304430cac1a18da04ce35626803bdad446
91013 F20101207_AABRSN matos_j_Page_094.jpg
777c5941d1bd6ce6a68ea1ecb0e3458a
c0fb5fcace3763707f8b3a4b17adc5bd9225639c
88449 F20101207_AABRRY matos_j_Page_076.jpg
0d0affa9aaec9367bd94bcad725218b7
88c2c47cc6ade91cd72f967d8e20c8291c25f138
48937 F20101207_AABRTB matos_j_Page_112.jpg
d58eff424202a8840dd237a9d7139b5f
2e68767ef79c6efe57c450b4a3da0d5bfd32f1fa
3500 F20101207_AABTBK matos_j_Page_139thm.jpg
e8d61b0f0a208598854752603984e5a0
31ee616ffc8bc47c87bd7dcb63099d06acbbbba8
6750 F20101207_AABTAW matos_j_Page_126thm.jpg
acfc6f93aaec0ec2de2acb324359730f
752575bc0f22650750c8a45e547bfe71579156df
27385 F20101207_AABSWE matos_j_Page_014.QC.jpg
c1ba0fc878798c451c2c1a38d2076d57
ce839cefdff0eccf54e5077eca567f4fbf33a0e2
83577 F20101207_AABRRZ matos_j_Page_077.jpg
c1530aabc5ac1e709f4bd6db367268b8
28f1cd14f597d202edf9c85e4231431de31653f3
47121 F20101207_AABRTC matos_j_Page_113.jpg
67fd0c6348f68d9b9262cbb801e88a33
8709e617b1c0149cbdf1c67eb4098f5864de7568
2950 F20101207_AABTBL matos_j_Page_140thm.jpg
0ce5dc560868268b10ea645598adf16a
fd6b4d5b275b2ee201b232b9893d826ba6d8d3ef
6737 F20101207_AABTAX matos_j_Page_128thm.jpg
daa1dc2d19c16e328f3212d8c7e07275
679ef3eca4ff934a3957bc0db6b6ff7fcee9625d
28454 F20101207_AABSWF matos_j_Page_015.QC.jpg
24433ef14212863ef61fc9fc0ccc8377
4366baff1a2c3cc0216662acd90cd00ec43c1de7
259693 F20101207_AABSVQ UFE0020084_00001.xml
646e33ff86d9f517d89a9c962824508b
98c772121a9ddb502aa5e1ff922ab7f4ee385848
91206 F20101207_AABRSO matos_j_Page_095.jpg
10075c965313dea2bd98b4ae3456e249
a9254dfcefaaa8795937ff9d4c2256cce08d1354
53280 F20101207_AABRTD matos_j_Page_114.jpg
9d84b60566217255206740b037c6e532
4b50204e14a73b2edabc253b17d93424f9e18a30
28770 F20101207_AABTCA matos_j_Page_157.QC.jpg
3a5ba8b07781f71b272f0bedfdb4d2a0
48567e57678d32543f606b5e1a80c36bb47c4134
13418 F20101207_AABTBM matos_j_Page_140.QC.jpg
2d9fab43fe9c86aac1f81129369cd175
6bb10db4a1316e32fd52ec2135789cea6e462def
6753 F20101207_AABTAY matos_j_Page_129thm.jpg
21908a93dca967613b4f2f1308f96ec4
e2c9348e3cc260b24dd8eb377fbf56d780bba075
6582 F20101207_AABSWG matos_j_Page_016thm.jpg
ab2fe2a5cb71bdee56b269d5720d8130
d3de6d6c83fc20919ce67c8eeab8d9a53e31c717
474 F20101207_AABSVR matos_j_Page_002thm.jpg
8d6c94581eb1a8941005a5e3ddfccb11
4cc630b56d90273fffe2fb07596df2fe0768b5f6
89609 F20101207_AABRSP matos_j_Page_096.jpg
61aa80ce104331a4dc9019e15d448b51
64e5a2a8926e050d0b7c018dbd8df7f61f1c4210
36620 F20101207_AABRTE matos_j_Page_115.jpg
f8c63a5c921eb330a2a7fb999f280292
1a9715d1f6aa2f292eaa269e7db6591888ed6c4d
F20101207_AABTCB matos_j_Page_158thm.jpg
8fa343d85dbd7a33e3cb2da6cbc2cc24
875254880c82e206c15592fdd8aa53dcd763e7da
26878 F20101207_AABTBN matos_j_Page_142.QC.jpg
b78ae84f095f55b4334f5b432cd3a527
6432c730a9d8b5ce651c695babe69f4c5adb9844
28104 F20101207_AABTAZ matos_j_Page_129.QC.jpg
3559d16b5d47be013ac9bc78c1f84463
b67ea75c83c09500c41c6a3e163cd894e46a86e6
27674 F20101207_AABSWH matos_j_Page_016.QC.jpg
6761d63ec3b4f8d87e682f39aaa0133d
a0b75a70df8e2200b8eac0c2666126d54df6df8b
1097 F20101207_AABSVS matos_j_Page_002.QC.jpg
ac977e2be39cf15a96b120dd4ef904e8
b30a884899a82f20de4bbe57ef5cf3342a1d5604
78970 F20101207_AABRSQ matos_j_Page_099.jpg
42dc19a513398b495063e2edd6610fcb
f42e5f12cd615c918b85f87433f575a24ec60e44
90629 F20101207_AABRTF matos_j_Page_117.jpg
08a35082a249655b84312edea2f56bf1
2401f8cd33cea6e237db8164f0a0a7e15af020dc
31307 F20101207_AABTCC matos_j_Page_159.QC.jpg
08d0e6a2711f765cc094dd4dcad85ec7
297b81d44a29198c691ca2ca0fab9e473f6879b8
29536 F20101207_AABTBO matos_j_Page_143.QC.jpg
74c8747aafa1a6d9ead7a8674794e007
342d57b3127859835a4680da914723962c6194da
6874 F20101207_AABSWI matos_j_Page_017thm.jpg
041c7ffd5b4ff4b3f24596435e07c2ef
b84cb6742dabf0a94bfbd79f6c220b1c3e0985b7
1082 F20101207_AABSVT matos_j_Page_003thm.jpg
b4fe30b7776ae706936c072d28ac8d30
0b538a712716ed70e66a017be39108a552375fca
27660 F20101207_AABRSR matos_j_Page_100.jpg
c5c64786f500f9ebc2e5f07af909830c
15fd7d1ba34394910eb6646723d43c6bc88d9621
84477 F20101207_AABRTG matos_j_Page_118.jpg
c259c960d802a40249389f9d6fc2e96f
cad54f76b3e1093a0c31971f7117ea968f4ebaca
7129 F20101207_AABTCD matos_j_Page_160thm.jpg
4c82b0440a2e270493735a7176282a37
4999d96651fb168209fa1ef5c267b865829a44ff
29451 F20101207_AABTBP matos_j_Page_144.QC.jpg
b3bbb3c97bf2cc3f57344781bc23a0b3
d155cd8889a1d954ca7d98abfaf9b3ff824a613e
29656 F20101207_AABSWJ matos_j_Page_017.QC.jpg
7bf3bb05850ed546c4bd31c194eff058
519994d0f80d11f77f026f4fc8aad92287955580
6586 F20101207_AABSVU matos_j_Page_004thm.jpg
851b623e41da43a0672e0a8f9486b7a1
84ff4b5fe5f39e9c4c26b1047b871b642b2799cf
36558 F20101207_AABRSS matos_j_Page_101.jpg
ac607c0eb42c842f5df1a87714ef3c96
9ab9705fc8fe65eb2a8f29acadd61ad06aeeb907
85281 F20101207_AABRTH matos_j_Page_119.jpg
a8e4a93a8b92acdec87c5ba327e21eda
6ac2080805b0aa4d3c01a50f35769a74e40fefa6
6979 F20101207_AABTCE matos_j_Page_161thm.jpg
0dc9df5f779b9d51c20ba4db9ec2b9f9
be10c94747b01b248f89168647fb98a1f230c45e
6584 F20101207_AABTBQ matos_j_Page_146thm.jpg
2e957bb6af3f642050e6e6fbf6ef92b2
7995bb492d57be61fcfd656f14df2898d4cdaf21
29083 F20101207_AABSWK matos_j_Page_018.QC.jpg
4c93025b9e02a4bdba01e5264182c8bb
992c04ab3d1ac4d23dd9bc3131198a9375eb3511
28314 F20101207_AABSVV matos_j_Page_004.QC.jpg
b7375161b43682969244b6acc6924604
a2c8fcab8363dce6d8555683665ee3b9281c5e9a
49332 F20101207_AABRST matos_j_Page_103.jpg
0193c7ffad90c99edb111505f4f9e568
10817e75c2a80bc5daa838832f449b5bb85b0bd0
89064 F20101207_AABRTI matos_j_Page_120.jpg
f5310594b2e2aae6e3dbc7e776bd90d9
7b9b9f6741c23b2576cb70d297dd5714bf690f0c
29624 F20101207_AABTCF matos_j_Page_161.QC.jpg
533f0ece5dd3923c7c51c32e7b1732aa
fd06a309bb13f3519474048bb0032faab3dc72c1
27342 F20101207_AABTBR matos_j_Page_146.QC.jpg
b5b27a2c57737ce06a619eaed756c629
6c4bcae533f4409b590ff279fb7562492664bc55
6457 F20101207_AABSWL matos_j_Page_019thm.jpg
cdf8a04c6930f32dc3a3fb6ad2f18530
04fdc15e016ea0193e92a3c36b1001c6cb2a1b47
5820 F20101207_AABSVW matos_j_Page_005thm.jpg
3c1fd0a3960b4af522fb76c85dfdebe0
010673938c7d09125617703124fa976da4d3742d
50065 F20101207_AABRSU matos_j_Page_104.jpg
28825106b9fb783811e94ab081c64836
fd5839573f7bc9ae66761ce092dbb2ac797dba7d
92772 F20101207_AABRTJ matos_j_Page_121.jpg
1c00b2d8e7b0d192af4e47c77ef2c781
569b7575093fc7e1d43ff940290d03dcb85f714c
30630 F20101207_AABTCG matos_j_Page_162.QC.jpg
767cf057710d64d5b6b66a2380e95295
dd9c625b6fdf9c92988fa64df9796d60a4afeace
29412 F20101207_AABTBS matos_j_Page_149.QC.jpg
69bf476c9e27684af84cd2ef5f19ec2b
202093541d51162911d3563f2de503f5d0390f8c
28630 F20101207_AABSXA matos_j_Page_030.QC.jpg
b4e8fb51204aff2b3b0f7bef3c6830f7
65c0f0dbd4ecfc760fd8340ef694ceb8f2ba205a
29127 F20101207_AABSWM matos_j_Page_019.QC.jpg
3fde1a63c6edc7494ab7e5ce024ab2bf
f81b89cf2cdea8f79e86668878696e4e4871057f
4968 F20101207_AABSVX matos_j_Page_006thm.jpg
3711eda87737aa0fb352638280f20239
3a6eeff2b980b8118441487f71b9be14a749edd5
39177 F20101207_AABRSV matos_j_Page_105.jpg
a0196aa86e68f63a678ae04bbaf13f2f
ca1b56ebc3f42f99c9d132a76c76c37ccc276a5a
90850 F20101207_AABRTK matos_j_Page_122.jpg
50c568ec4dd43a5eaf3a0c3dd5ce514f
c4bfd5fbc77fc29aa2b67c1b22d14f1f77623c50
7037 F20101207_AABTCH matos_j_Page_164thm.jpg
7617c2af068fff164a74c3b2697b007f
721db5dd8ddc81df89c45729d466fa96527944ef
28449 F20101207_AABTBT matos_j_Page_150.QC.jpg
c31d76da1e5076f2dcfa69fa138ecd87
58284e40afd8eb11b03f422044eed0c48b49953c
F20101207_AABSXB matos_j_Page_031thm.jpg
1d74178994d0c0db0418b9c5c5afc46f
aa2252e66dda5843d934d17f08927d599213de55
29460 F20101207_AABSWN matos_j_Page_021.QC.jpg
cbc88e3e8ab1992bc603ef1d6559ef83
15092db5f4eb4f614e31db7e8cd205e3e483dfc2
20941 F20101207_AABSVY matos_j_Page_007.QC.jpg
e1b3616f43c45e6edc862ff4a8f773a8
cea8f5e0676d2092590b82d834a1c8482731858f
41309 F20101207_AABRSW matos_j_Page_106.jpg
2576d30e6c92be77615ad677ec5dea3c
e43a9a1eee498dd1a107bc0a11c0094973b9f550
88392 F20101207_AABRTL matos_j_Page_124.jpg
0d45840aee4538379a2302416f57de10
246039ce5d32f2a16bdcfef7c4bad87e621d814b
6778 F20101207_AABTCI matos_j_Page_165thm.jpg
656236f28af51f95f4b6f013b8277dc3
7f95c4dbe86f12207e9a80c384a3317407a1f573
21557 F20101207_AABTBU matos_j_Page_151.QC.jpg
bd47064ffcaaa1fd805ffbb5b75becb2
6672c9f1a4cc1f3ec09219c10929bcee2d4bf7f3
29441 F20101207_AABSXC matos_j_Page_031.QC.jpg
e49f2d14b50626cec03310e00a856fae
6488d736bd66af4353e1602e0e3331cc37ea0c6e
28650 F20101207_AABSWO matos_j_Page_022.QC.jpg
9c75bf4b3d65d1683a656e2419a45ee2
4e079e2eead965b52e949b3bcac65e29b843bdc9
F20101207_AABSVZ matos_j_Page_008thm.jpg
0ec2a9440be5cbe34c8c6e6d5c803395
748fd8137d79e06d15678bd7f90bb19d18e9c508
40948 F20101207_AABRSX matos_j_Page_107.jpg
5516f22e4d2d3df30d99386593b073be
22287b5116fc7cb30155f5a89765f319011ad87f
94464 F20101207_AABRUA matos_j_Page_143.jpg
b6cf9148fa0c2ae67fe81a093def6ecf
9a0df819e50cec0cc12d8f0db3890b9bbe3af805
93564 F20101207_AABRTM matos_j_Page_126.jpg
b3992f7b482218b8aad4677c55d122a2
15b82d9118950239eaeeba97ff5caa49f4205d49
27531 F20101207_AABTCJ matos_j_Page_165.QC.jpg
b3b223322b3bb665f0bdd1cc342dfe09
f0a3e216cb9f622c609001943dd88be5ca1ffb28
3382 F20101207_AABTBV matos_j_Page_153thm.jpg
e5816067259b748fd0d742f5ee8888b5
9d85a34355c88b4c442e0dcd59017b122dd71241
6798 F20101207_AABSXD matos_j_Page_032thm.jpg
8a37ffcd7da563fad9fcc1603946de7d
f2e925212d536a431f25b46ede91db8329950727
29155 F20101207_AABSWP matos_j_Page_023.QC.jpg
d957ba39168fa70d872760aff5d0bd6d
849dc978c7c4a4760bb043bd7d7ec36a4eaff175
53775 F20101207_AABRSY matos_j_Page_109.jpg
19b8d39442e9f3c181ed48ea134b8f0a
b6dbddee6195046088c65633af069e399ee95c23
93964 F20101207_AABRUB matos_j_Page_144.jpg
69bc02612dc0bd1b1bb511df8756a0e8
d46fe72cc67a0134f14a69303e3e3c1ed7942c16
88280 F20101207_AABRTN matos_j_Page_127.jpg
2f171df697506c2010877cae8452e2ab
1495759e3ba8f7255cef130ec0d5e314b0390fae
7080 F20101207_AABTCK matos_j_Page_166thm.jpg
30b12b040df726fa7a2dff03b11d8839
a6dc06636bf4bdb2a23074be6f2c58612f3e6559
11622 F20101207_AABTBW matos_j_Page_153.QC.jpg
c4ca0f45b8654570f471642d41503595
e3bacfebbcfca0ab93ce81f79193c586cc6b36b7
28428 F20101207_AABSXE matos_j_Page_032.QC.jpg
c29dbcae3b79bc03b4bc827e10658485
68abf7612fb9445cbc2c49c63eef7f4e969e8b90
29332 F20101207_AABSWQ matos_j_Page_024.QC.jpg
499668206c12dfb6d98cb8c4d330e719
abe32bc09f329b983aa9d7d424f0b1d1b40bbbd6
49413 F20101207_AABRSZ matos_j_Page_110.jpg
87bc62924587295f775cf8def2189158
9194ad6287377b613ddf3aced457dd63113d58b2
86511 F20101207_AABRUC matos_j_Page_148.jpg
a2c2290eab56c8cae94a416aefabda77
7e736b09f71dc069e1cddc6ca457390cfd06f74f
90840 F20101207_AABRTO matos_j_Page_128.jpg
61ad695ea34d86ed4f892b3513d2239d
75376a8678007d27637691cfac5b1fea1e6f84a3
7068 F20101207_AABTCL matos_j_Page_167thm.jpg
32729c4c4d4dea01ffeedce23e80a968
19c946bf6056a31ffb8590b3575055a8ca2fb935
7069 F20101207_AABTBX matos_j_Page_155thm.jpg
45fe82f66a7eec277be345463c43c140
c71e06cb726f360fd57d307a36532ea8397c859c
6608 F20101207_AABSXF matos_j_Page_033thm.jpg
fc67b2f1cb1881a874f24d96398ab4bc
4ba1e5931cf3aa5f9cbc7119e76ac2b21eadb7e6
92931 F20101207_AABRUD matos_j_Page_149.jpg
aaa3c569e96bcd63ddb6fa66a93bcf71
7ac65eec8abd8287b748c0187c9ca141a7def86a
30177 F20101207_AABTCM matos_j_Page_168.QC.jpg
e31c730e8ec91b1ef82d4b52d92543e6
0d225b7361b569d8c5505493f6c50ea79e49a280
31279 F20101207_AABTBY matos_j_Page_155.QC.jpg
3cbf9c131c15947cc20c84d2f7a2f705
f0d366ff16b8c032aabebfce99969e366f2a6ff4
6535 F20101207_AABSXG matos_j_Page_034thm.jpg
4533098b43fb65d04cefa65d81540187
b1bc3698b9c6f063a8dd00f0f164cf4371887bc6
6703 F20101207_AABSWR matos_j_Page_025thm.jpg
42e5b360cc634e2b06280eb421284559
5c1b7275be06d11ee63dd1c7975bd24758346d50
89233 F20101207_AABRUE matos_j_Page_150.jpg
bdd49ea11e818e77a20b1d4ea20a8da1
8ec929de23506c210cf7166dedd50057e806ffa2
89114 F20101207_AABRTP matos_j_Page_131.jpg
a339194694bbe156a2e9a9af3d831fab
8e281271e340b6fecf15aab2e904a0b3dd4a4937
6901 F20101207_AABTCN matos_j_Page_169thm.jpg
1672e82b90e5b926849ae515f58359db
6b7aa8d0df8c71280eccf1b594706f353b580dd9
28442 F20101207_AABTBZ matos_j_Page_156.QC.jpg
d225ad7c303bdb91983770e9a71440e4
e75b5496a4401c1e2ca30df027ee7d63d14c9028
6329 F20101207_AABSXH matos_j_Page_035thm.jpg
a80cbdb074adc778713d8067fd8e3d24
eb2e0096822b42b1c9d81e35b90105fd944af3b7
F20101207_AABSWS matos_j_Page_026thm.jpg
23274448672e06d350aae5123d4c2238
ae0da3d25d308d78ddbc595938de716381b5e98b
66751 F20101207_AABRUF matos_j_Page_151.jpg
255eea965cf81b06934cfe2a71d66f66
4fd5d6e5c20c76470acc423b7d93a5f4c30d5b5d
88465 F20101207_AABRTQ matos_j_Page_132.jpg
8f3712f82310a93856a52455abf719f6
4525f0cac08f8f04bbd084802b5c2a897cd6dcfc
29338 F20101207_AABTCO matos_j_Page_169.QC.jpg
b8601cea542a493e55baeb1a52eabe63
8ce024720a257754bd7e220caa5118df837155f7
26634 F20101207_AABSXI matos_j_Page_035.QC.jpg
4a90d17709fff1cbfd867305901584ef
60e06d88567f0cba113e03bd94cf366e8ebb8f9c
27369 F20101207_AABSWT matos_j_Page_026.QC.jpg
742edba7bc75c45f0da30fd6bc804a55
9818b040b2a72f2f43d231a452a730696e1965e3
59935 F20101207_AABRUG matos_j_Page_152.jpg
0e0f0d235b357deabdff194b245bdad7
835d17ce0098692ba0b96731caa4fc8e3c16280a
90164 F20101207_AABRTR matos_j_Page_133.jpg
b5050562f2310198c545b58f7f721ea8
77843628d5afcb53b68c3ecf1e03f03b1e20bddc
128112 F20101207_AABSAA matos_j_Page_156.jp2
f141627ecb07d5eb99a15885ed2e1742
4dae7ccbd2e67d61c29d347aff0e9b79f99c1e7c
27754 F20101207_AABTCP matos_j_Page_170.QC.jpg
d1ca72d53015bb8be45665470699fde3
7ed86adbd340d430b21ec5779988af31a8339421
27974 F20101207_AABSXJ matos_j_Page_036.QC.jpg
6036fb4f1600d768bb165fe29f12fdfe
4b5571273e8023e34b6d06342562dfeafa3c2a94
F20101207_AABSWU matos_j_Page_027thm.jpg
7b9b614ad3a8b79bcaa9189302f46c36
9158d9429d307b96e399535cc454e32e5a803dec
107789 F20101207_AABRUH matos_j_Page_154.jpg
872ffdb59b043de3765e567c4eab0519
c2f9cb84f0ff92e4f7da2da191d66773e5790b9a
27077 F20101207_AABRTS matos_j_Page_134.jpg
3546e66890d9988d33e40f49dd286537
d463c582883c979804cc10f6e24098724bf9a233
131307 F20101207_AABSAB matos_j_Page_157.jp2
8be0e53b467e9a2b6f9249839135efe8
c48700b38d4512cd63de45893804c413144e67d9
31093 F20101207_AABTCQ matos_j_Page_171.QC.jpg
4fb1366780b16c9eb078bde198bc9147
19b91e66511f5debb392e66c0cc6d9fecefeb84f
6799 F20101207_AABSXK matos_j_Page_037thm.jpg
80afd89c217789664b73e38c779eba5e
7849297829f1676102f85bef3548d4f71996cb94
29326 F20101207_AABSWV matos_j_Page_027.QC.jpg
01c2070eb131009b78ef8134d9bbbc5b
19fd205dbc0e2e75983ef8cf4478b5ff9c744b3f
117541 F20101207_AABRUI matos_j_Page_155.jpg
dff0723b19c42cb4d5ffaa7915ebb78f
a120d1cb199841f9ed0c2445fc4123fed36bd94e
63445 F20101207_AABRTT matos_j_Page_135.jpg
09b0c645e8c1ce66d2084486ba7ae8f9
11f02a4bb24d4e26e8deb051422afae40790bb07
131266 F20101207_AABSAC matos_j_Page_158.jp2
b63f189ef934c5e5e0058a2f4d151d91
570697cdd7a11adaca00ba13fdf1e7c77b4997b9
6976 F20101207_AABTCR matos_j_Page_172thm.jpg
927447e6d8359d14b1af298159ccbca6
fac47b75e55ccef8268a46251b9f1a4198ee4676
6408 F20101207_AABSXL matos_j_Page_038thm.jpg
683925aa44df8a9a18e98302f17374d2
2cbe8aa356b638c63448712ac9e66494662b6d46
28725 F20101207_AABSWW matos_j_Page_028.QC.jpg
16da24eb5107591eae930ccafe2b38da
d151b4d2876498da16384efff51a57af93adba7d
101726 F20101207_AABRUJ matos_j_Page_156.jpg
d27d80534e01744c48105ecef7c282c0
833365c29c90216fdba46769836bda26f111ad95
41805 F20101207_AABRTU matos_j_Page_137.jpg
b67d6b52481d9bcac31a08c1768660f9
1054ed9eda0e9d34bf1a032c03699021a3ea37ed
133247 F20101207_AABSAD matos_j_Page_160.jp2
7af938331219748d667192e78b62bc4b
3e7fb88c8decb579ce894a2bd6c2533eca2396ae
16553 F20101207_AABTCS matos_j_Page_173.QC.jpg
98aba5677391b0beab33a5a569d1f876
b7658df4318f50ccdc8aa14a9afbada0c614ec37
11025 F20101207_AABSYA matos_j_Page_051.QC.jpg
97ddf433ee4f174ca633ea8052d4c4df
0347755f7d95c9c2fcad4ab634bfc43a1f7d2a46
6561 F20101207_AABSXM matos_j_Page_039thm.jpg
04efec9619839e96209f8a638a7d32ce
c20e9ff25cd7e0e086d09ef9214f769b48ce363c
6492 F20101207_AABSWX matos_j_Page_029thm.jpg
7cb742f6efff1a1ddfb755275678a25a
b67b37c468717ed05e076a5b8edd3fae9106633b
105942 F20101207_AABRUK matos_j_Page_158.jpg
dda1f201761f2612ce69056016a1b620
dacf0f8226ecd2863b9062c5c9c5d308ec69bd27
45762 F20101207_AABRTV matos_j_Page_138.jpg
55652046e25cf9d02da787cf4795e0ca
0d9f976fa29774005f0a80e6afb0324f6112db39
130693 F20101207_AABSAE matos_j_Page_163.jp2
f770553b2d5fd8aa073422752ef3d08d
4a8f2cab451174e56b43bdab08545234ce1d1891
2699 F20101207_AABSYB matos_j_Page_054thm.jpg
14563115bb5258d49c1c11044a44a1bf
96d86b14d60ec78758d70bf098fbdb5d13fcff5c
27073 F20101207_AABSXN matos_j_Page_041.QC.jpg
348a9066a1bd647fa4d69fa2fd25719e
0a38097e639a6e7b9d6717fd6975b744d54f4199
25569 F20101207_AABSWY matos_j_Page_029.QC.jpg
48056d5cecf14ba96100885ec1cf5c2c
2550d5eed8aacfe0f754430c5fe43e1be7ca260d
115631 F20101207_AABRUL matos_j_Page_159.jpg
c7fafc6d9e443378c25691043393b70d
7e1ad091aeef0167d42749a41309cfc325368ed4
41820 F20101207_AABRTW matos_j_Page_139.jpg
cd4708421abb1936363ab7d290512692
20f75ace56bd9c044830660cd210301880e72a35
137456 F20101207_AABSAF matos_j_Page_164.jp2
05bf3a00018706e6cad27325103fff61
481ef3bfd059645b650ac20607d7c2db2d75e59a
2271 F20101207_AABSYC matos_j_Page_055thm.jpg
94ed2699af2ca4331dd3a8e9249befa9
9cabad15252982b0c573ed3b1eb895484232e5ac
6954 F20101207_AABSXO matos_j_Page_042thm.jpg
304f39a1036a79dc850b1318d27dcfa2
386bce2f392c8ccb84285918eeb5a32e30fc21a0
6830 F20101207_AABSWZ matos_j_Page_030thm.jpg
60de7f65b9708362e25b2943f25d785c
f13ca47fc2c149269b9599a1991b17320f15ea0b
24999 F20101207_AABRVA matos_j_Page_001.jp2
61dea3713f374742c6a1bd66118f1962
c56a17d355b4059df22b6cf8246f0dfb7a3a01cb
102425 F20101207_AABRUM matos_j_Page_161.jpg
3e9267e1cff6b0bfb614f710a75942a6
e99b82b76afac4068fcc14c7db812c2bc70ae7e1
56967 F20101207_AABRTX matos_j_Page_140.jpg
131041a467dbf1007166b0ced44c6bc5
b268c084f2857edd9a2c1c2516c63bf7286c9da9
129566 F20101207_AABSAG matos_j_Page_165.jp2
98db4a298df6d1d18fd5c9641567f63b
1eff850fbc58579a4bc166e5fec6c9f35cff5165
11776 F20101207_AABSYD matos_j_Page_056.QC.jpg
c5c6d9e204d5bdbcf1158a79098602b8
c82b0407096f694561d7e073c626b2d4a11efcf2
6317 F20101207_AABSXP matos_j_Page_043thm.jpg
a5291b89b0b58411644e01932e0de4d0
7589c666ac46ad84dcaa8ad6c0a938603b1b983d
5664 F20101207_AABRVB matos_j_Page_002.jp2
fa4985cda926a491577cf8143271b90b
5a9664cd871ae9fc51d663c708c54f0889e6678a
108928 F20101207_AABRUN matos_j_Page_162.jpg
5def02d85076e20feb4cb66ba0c6cad4
fd26c9a5288402ef2a249f924aded1338831f3e3
48058 F20101207_AABRTY matos_j_Page_141.jpg
90c863c39b17d4c82aaafbfca22af4ae
26d304bbcafc5bf0a90f76e8c934e3b7d4c4a8c6
142658 F20101207_AABSAH matos_j_Page_166.jp2
63ed7e02f4dbea0dc712d63783238998
50fce69fab885fac3e995a57388bd311fb56dcd7
10910 F20101207_AABSYE matos_j_Page_058.QC.jpg
f46a08fe595bfc929953e5eacd3f88b9
42840da25b362a81adb67f7eaf1fc31386856365
28696 F20101207_AABSXQ matos_j_Page_044.QC.jpg
5d40d771d1740de78f127cab0260b7a4
83269aa3071fc78f90f8f4751b8ad407aefa6360
14356 F20101207_AABRVC matos_j_Page_003.jp2
301792ad8b9f5824c2ee23071f456501
a7f175264493fb2fdfaf72fe67b802fd723d1a65
109430 F20101207_AABRUO matos_j_Page_164.jpg
c1a915a824fe1787811bea95f0fd2f0d
0be017b1d1ed10d1f7d2a8a4d59164f54f010cbc
86247 F20101207_AABRTZ matos_j_Page_142.jpg
a8892b4e281e3a3515ae435a1d097a23
a87f4feddb8b4dd3a2d256d6880bd071dfc8a17d
145072 F20101207_AABSAI matos_j_Page_167.jp2
ead690ee9679897db5c8007dacabea62
878e9505b93d90a4b88d5be345f9fa0ad8092229
4311 F20101207_AABSYF matos_j_Page_059thm.jpg
aa1c12fb4000fc3fcf59d4f43459b15e
836247dd0eaff7bb29bc0866a7d0a0a20b49990c
6720 F20101207_AABSXR matos_j_Page_045thm.jpg
2abcff1239a4645ddc841a2090b55934
344e73c0978ae84f3d087ae98fe7cfb353fd0048
1051939 F20101207_AABRVD matos_j_Page_007.jp2
1094a549809380f5590817f35203cdef
7e99527aeaae246877c2f49e3a20b9d3b8348531
99239 F20101207_AABRUP matos_j_Page_165.jpg
fdd5a6a95a3568be9abdda50b538f6c1
b4b66fbad6624d81ae74602c4706785ac2c9d25f
139541 F20101207_AABSAJ matos_j_Page_168.jp2
f3d2af9768e47710942099a87b311aae
10ae8de582260063b5886b5e5902037a3f7d633a
13725 F20101207_AABSYG matos_j_Page_059.QC.jpg
09f4faa014be34afd1f0641651d4f243
40b4a464825279021edf6e88266bfa840c1681e8
402184 F20101207_AABRVE matos_j_Page_008.jp2
c080161f9d7d4ff396181c389ba8e7f7
c4380b7253c02b72ef1dc9dabc59b70243ce4190
134638 F20101207_AABSAK matos_j_Page_169.jp2
d8dcd006789827208b2d2f08a5315c1a
3fbc6dede1a22b82a1565487ed80730efe0a7703
6322 F20101207_AABSYH matos_j_Page_061thm.jpg
66a8769fcaf5472394191707d08cf1b9
a2fce159f08a4a6aacfa8a08a15887d4fe047b29
28836 F20101207_AABSXS matos_j_Page_045.QC.jpg
74d7532433e5cc3de8773f5d09715ab2
7116747d71b43c9c624b0ed947f4191a1cb74eea
1051981 F20101207_AABRVF matos_j_Page_009.jp2
1c2c1498878fffc1a6c122a9734c455f
0eb5a44cecd900a913ea9e8539197f3463b8d78d
114807 F20101207_AABRUQ matos_j_Page_166.jpg
e873f90df54c135c87218148998be0a6
7befa3d474d6090973a6341c271fc94dc144c103
123256 F20101207_AABSAL matos_j_Page_170.jp2
5a407447e731ea3522038b202a33ba83
bda34128832573d5650c8b8d48dd1d9139e08cb9
6754 F20101207_AABSYI matos_j_Page_062thm.jpg
6a9afcabcb3d0559d5f91953d930a06a
4745fcb6ad35202b742d2150eb8ad2377bc2e898
6834 F20101207_AABSXT matos_j_Page_046thm.jpg
f77c60bf82bb6cee793677e23ded0959
dcfad1f2d06a34f491058a748ec1c69ea4e2c3bf
385939 F20101207_AABRVG matos_j_Page_010.jp2
2b7917db8fca672acea5e67f960f9bc3
f0ad692737788a531e4822c7ece23aa2f0e542e0
112728 F20101207_AABRUR matos_j_Page_167.jpg
7cd16d9567373e1cfcf85cc632c613f6
c57a851a73ffb6d102b9e5091d1ba3fe30e8f669
F20101207_AABSBA matos_j_Page_014.tif
3859a734256d94440aa23604e61f62b5
120f83824c5aed04c5b4116923f9f238271f87a1
141100 F20101207_AABSAM matos_j_Page_171.jp2
7841b6218044e83e73212e12c7908186
2c88b69b97714d0f94ebbe49c52e3cde19254e6c
28481 F20101207_AABSYJ matos_j_Page_062.QC.jpg
fa1eb6ff9c8d375e5c95031afa3193c6
3f94a3364c2ed5bb8d1d0dc8c07d48e6f62684eb
6780 F20101207_AABSXU matos_j_Page_047thm.jpg
d8191cd05d6a3146a1414d7e67b6d06b
d74d306af55c93779cf8cec71562461270ec81b6
107433 F20101207_AABRVH matos_j_Page_011.jp2
c69f1b3da49d8c5563723d8b75db3c0f
61f3b6841d5f6491077135f887015d9ea2331627
110324 F20101207_AABRUS matos_j_Page_168.jpg
0193818249a6e92dba59906ef2201144
0e61faa32b7118ce0b6361fd6f89391e9e887cd6
F20101207_AABSBB matos_j_Page_015.tif
8701f344cd0d2f022fbd56a6c3568863
cc2601f07d8cba5d2d963823da84494766b028bb
131942 F20101207_AABSAN matos_j_Page_172.jp2
f616e5e91e820e7d8d8973a8ee972f28
921589992697680a248ae20f017ad2a941930fe3
5766 F20101207_AABSYK matos_j_Page_063thm.jpg
9a9bf9f11f93f6ffefc9fc4abbda186f
e5c2b5b32e35c111f1a54a56e2597ddef63d919a
6712 F20101207_AABSXV matos_j_Page_048thm.jpg
3ed56f1cd84978001b0c2d7bf153b2c4
0e0a8103ebc436a28a16f4505bdc6ce2294fd1ed
18726 F20101207_AABRVI matos_j_Page_012.jp2
06443e9824fb3ec2632dff935de3c83e
ac28aedabbb1a33002cd8fbb461133dff35a332b
105706 F20101207_AABRUT matos_j_Page_169.jpg
8a7c4c0b76283c226b025fa9b3508cd3
2d24df2ad5722a475d0de6d912cfd309f559b71f
F20101207_AABSBC matos_j_Page_016.tif
521abb28ca2f7b38f7d4f55691c29791
ed2f5e3313ad97a35fa0d61c32a2d268be3154b1
73074 F20101207_AABSAO matos_j_Page_173.jp2
50bd8498ff010c90ea4a96bfd57aa407
109e4ec1764548fc5f2e5f49b8c90f58401174c2
25292 F20101207_AABSYL matos_j_Page_063.QC.jpg
e702890ec77ab4ef285252bc6bf30d91
5eb2222d399beb8506734db5447d908a03f30eec
6613 F20101207_AABSXW matos_j_Page_049thm.jpg
c8fee84cae8d603a49b9c8feffda3636
fdf6532041d3d905e4fe37dc371948b1e044ecac
130259 F20101207_AABRVJ matos_j_Page_013.jp2
72cb18f61b32156c0202787c7e99f44a
46a3b2a6ff354f706002788ef6f07e4d2569e61f
99569 F20101207_AABRUU matos_j_Page_170.jpg
3a08aa83ec0f4e5cf031b7d5204a5004
a7c45535e37d21cf8978669b1c5eea523a44db18
F20101207_AABSBD matos_j_Page_018.tif
02fc39ceb84b61effb5d9342ae73951c
6db50ca4322b98da16a217e1b3327468546778bd
117591 F20101207_AABSAP matos_j_Page_174.jp2
f4837139c977a645340f56438db942df
697646c82de2afbb21198334b44d8a9c15e1eaa8
6410 F20101207_AABSZA matos_j_Page_080thm.jpg
7fc7699ee2497c4b5d17a687dcb94894
74fa65090dc7c2abfcc9f7eaeff9cc8da02e5203
5954 F20101207_AABSYM matos_j_Page_067thm.jpg
4fcd78308ce77b60e35828759a9c820c
caa507f89920de0d081a0087a44446aa93142950
29058 F20101207_AABSXX matos_j_Page_049.QC.jpg
0fbcb21edae0ff0b44af43c70b2b239c
29cc7b38f5fe46cce8b7d1bb7d598da892362c92
117053 F20101207_AABRVK matos_j_Page_014.jp2
04ff4f53a1dc753f470bfc85a5793062
079c0c612b9d05c34ca7f3eb70627629626629e4
117488 F20101207_AABRUV matos_j_Page_171.jpg
24dc19b07cd022aa0353af111c53a6b2
a8f02d7fff75413cc128389a4c72c722d3610bb7
F20101207_AABSBE matos_j_Page_019.tif
e97d1763da1753593c0a57a7fe982a8a
ec42969f3782f7801bf9b7efdc5f375ee0f1a157
F20101207_AABSAQ matos_j_Page_001.tif
a2c940ae9806663344d2fb2dacc7cf97
da4872e777324d85dab29212f3b5df6c4910f5f8
27442 F20101207_AABSZB matos_j_Page_080.QC.jpg
283cef176b909a9abc5cdae5a51cdbc8
28a1a035627f711967e0896dc250e35748aa8c70
29602 F20101207_AABSYN matos_j_Page_068.QC.jpg
a364b272e42c0e0852066a2128b99eaa
07e8891363040ee2ad6df7522dae614b5fb1dded
14401 F20101207_AABSXY matos_j_Page_050.QC.jpg
d77e643d8c2dde483d422c1ea5d4b584
2d7fa01be99bb46f3bde711b1a17d1ec108086c5
119328 F20101207_AABRVL matos_j_Page_015.jp2
5dc1e63f208da23147045d26481ae035
06b8e7750e653b7f9da3bd43b9dfa133bcad308e
101870 F20101207_AABRUW matos_j_Page_172.jpg
8c0d545bee6aa4b2fc444a0466c4488d
a9eea04fdcb6b554df1bb1a5ecd9a92b99562345
F20101207_AABSBF matos_j_Page_020.tif
230af8ea14611276f6c102645580d597
639441afebac390d7ce702fb1ec29fc04519c583
F20101207_AABSAR matos_j_Page_002.tif
4153682491c271ca972d78d32d8921ed
d6e0b01edcc7bb87f099d6871652db64db93b1aa
26793 F20101207_AABSZC matos_j_Page_082.QC.jpg
bb9d21fad5b313f8294e04b3687cba06
bd3d6c06f32ca0f2146db98819afb84def9e702e
27731 F20101207_AABSYO matos_j_Page_069.QC.jpg
4b635d1b23c9d4935d5d27213df87dfc
f1ae03f09002e1c39a1410c118a489cd5c96b1d5
2694 F20101207_AABSXZ matos_j_Page_051thm.jpg
8448f2d7a814e5638d3f1c7f2dab14d8
0cdb61c78d13c52502702825d43e1bf1ba4a5da6
123367 F20101207_AABRVM matos_j_Page_017.jp2
4bd75770ad53adfe3a45a083dd67c7f3
44cc84be9857d166db84152b807add5598d8e0df
56865 F20101207_AABRUX matos_j_Page_173.jpg
5734fb961e887a792ff22ca72a09d2ca
91733a54e8d8d9644dcd3dcffea5dfe1bb5e1877
F20101207_AABSBG matos_j_Page_021.tif
b4e497bbb2d2262c0136eaafb5a57a3a
661afc4de380d4937f26484a339317d45cd6123e
1051986 F20101207_AABRWA matos_j_Page_032.jp2
22eabdeaa9375a7a75300e1c282a2be4
b8f26d7e0efb29d1e479401a470fa3cd351f8076
F20101207_AABSAS matos_j_Page_004.tif
a6d926fc36154c57ff59479ece489ccb
0f0a0d4636f25594aceac162c0a001744e9fce0b
6483 F20101207_AABSZD matos_j_Page_083thm.jpg
cabc69bdf6ccc43b030125360752b7ca
1062925ba5873fde64fb693a751610adda55166a
6025 F20101207_AABSYP matos_j_Page_070thm.jpg
65fb2b42305343bf87d8e4225427e9cb
a09d53a1e00ee1fe296abd325f81010f7dbedb44
90806 F20101207_AABRUY matos_j_Page_174.jpg
2dd2f8856d40cf5b0f04c201a4e722fc
ee7f57fe2725af449b882c6070a7f77508bfb747
F20101207_AABSBH matos_j_Page_022.tif
ec6bae4dfaa5b710810b04951bc178a5
c20d01cd507486e91a05593476652fe4c2ac7a5f
114674 F20101207_AABRWB matos_j_Page_033.jp2
e04f97f6cb0e68d18168f708864b24b0
23d81476e01a2f7f7cad0a96e6f2ed2a9067c33f
F20101207_AABSAT matos_j_Page_006.tif
ee1ba77d16bc7486f98ad8c0cf69fbb2
24cab1c70d8fcdf5770817ee42281e67438fbe13
119422 F20101207_AABRVN matos_j_Page_018.jp2
3256062fd708c4833c6a04298343d92d
1e7732c04d396d1e23584ecdd1c8fd820ac14ded
26933 F20101207_AABSZE matos_j_Page_083.QC.jpg
06465e6aaae978e07d58fc01521fe6e4
3dba6d7a1f100882b93b0e1ee86c7e57f6267871
26058 F20101207_AABSYQ matos_j_Page_070.QC.jpg
50fcceebf7752f344d7c3db92abd0ed6
900046c863980e5f06dcb6a39bd7d5f774ebd68b
12362 F20101207_AABRUZ matos_j_Page_175.jpg
06300eb58cd0043b05120a8a7538af4d
32390f7d258c02ca4169ba0af7ccbc73d7e31fa8
F20101207_AABSBI matos_j_Page_023.tif
3d0ba85a816dc63a07fa8338826e09fe
a025722217014924a2cb742fc7cc51bf2c1dc617
116340 F20101207_AABRWC matos_j_Page_034.jp2
bb14901ddc2ae0efae277beb56dd480b
de20eaadc882c2bd545dd77690c9016c1703fcbf
F20101207_AABSAU matos_j_Page_007.tif
b5c6eb04b2bf78935ef7b01a5d8d2d5d
b137b973fb079fa953433dba5b24010d099c714d
121438 F20101207_AABRVO matos_j_Page_019.jp2
4e91390e54a3569ff8cbca9adcf17ec6
6f36318774a32c1599a5af426516623bf714e067
6403 F20101207_AABSZF matos_j_Page_087thm.jpg
2a2c2409cc9e4fc7950504106d3b392f
a28f691fd113105d66342a605e487b848f32cbcf
6219 F20101207_AABSYR matos_j_Page_071thm.jpg
bbf523066866761c3204bf5a1a462614
0a3f6dce861b83f50a74fc48d3bc49137a20f027
F20101207_AABSBJ matos_j_Page_024.tif
18ac11c775f275018d46fe92c2db182e
e9248dd48f4789dc16d6184542660e1e4279a1d3
106569 F20101207_AABRWD matos_j_Page_035.jp2
be3b94c18dbafc2d3f9f267afb33f1af
d5b68ac90c9072bb9527dc51cf4fad805c1549e2
F20101207_AABSAV matos_j_Page_008.tif
87e4d38c5b6db6b84906e1ad7b7998e2
c7eb556bd591cfb4bb78aeda97e64a9db8feeb53
123101 F20101207_AABRVP matos_j_Page_020.jp2
64d6cec98b541ab008c646901b58d1ef
bb30005f85704578ae76dbe60837501ed4820b73
26294 F20101207_AABSZG matos_j_Page_087.QC.jpg
73c09f2c8538ade633f8f87272d5d4e6
e1e0f3b3f50edf14b545695a486fdd6b32219693
6764 F20101207_AABSYS matos_j_Page_072thm.jpg
d32e1a97c9c3835f64200173c09a423f
744dabdadef7de629951ec5e93a81f1d8bf31163
F20101207_AABSBK matos_j_Page_025.tif
1547486bf5330c15bcecdc9de43f8ef4
adbb945780d595b72e76b21bdb02335ea1c236e7
114510 F20101207_AABRWE matos_j_Page_036.jp2
36e6ade56c04200201e88d129360591c
b89be78857617304583e97e1e7e6708048cfb87a
F20101207_AABSAW matos_j_Page_009.tif
e0ed26b4ff98cb4afb2c1ad3b179f296
fb7f2b7deb4bc82ae04f3270604d0d248b216022
1051960 F20101207_AABRVQ matos_j_Page_021.jp2
965635f1eb8bf3088fdda022baf8d00c
1a581e3a7b44a8dcbc523fde6bf6a464d2d32337
6883 F20101207_AABSZH matos_j_Page_088thm.jpg
cac79b4b9926e70b785f11e264192923
1005aaa5b5e837584efab75e33314cd3bd3d15c3
F20101207_AABSBL matos_j_Page_026.tif
4fb6e8c218bcd1c473eb3387ececcc87
d25bde9f8db5430b6a070d15bfd3268c17e46eb5
118015 F20101207_AABRWF matos_j_Page_037.jp2
101a58f12c7c828d3c2f1ab7edad8770
747ee27d19fcd3e09c7c1f56df148ea840fd1f1d
F20101207_AABSAX matos_j_Page_010.tif
0beb51c222c23fecfcc302b7e360cf14
39a56115da175f0d16bc201a846f4d382017eab0
28711 F20101207_AABSZI matos_j_Page_088.QC.jpg
7b45d28614be22ad4f60e41578a06c93
95f380e71fcfc5f60ee0f28c90f3ea88c6461b14
6618 F20101207_AABSYT matos_j_Page_073thm.jpg
eba343925bd9c5f7270c132cbbc6e9dd
72cf9cb237960e0e1434c101b9a12ae720d16426
F20101207_AABSCA matos_j_Page_044.tif
6a771de7b2c3c8bda0f7ff33653ed8f3
799e10cc168493f2e76904ade1eb828c96c3e7cc
F20101207_AABSBM matos_j_Page_027.tif
94c6567ae621d287df2bb361d57bdfe0
0fe69b5f20b36793064f6296b830aa80911f290d
114771 F20101207_AABRWG matos_j_Page_038.jp2
3b50834a590d810abfa372f4dfed5c88
e8b12bff099842d70b2665de1a2185cc8a1607d9
F20101207_AABSAY matos_j_Page_011.tif
b2af42917d56fbe526fe122f48d22d87
ee8ee5da2fc40758645dd4086718a7cdf83dcbf2
120315 F20101207_AABRVR matos_j_Page_022.jp2
9801a63183f107286f050b79211911b2
9fc97ea06b900a45aed8d9c5e32cd8093214d740
6502 F20101207_AABSZJ matos_j_Page_089thm.jpg
c73539957e6d32dc750de43b83f3c923
dfa1b67b11e7aba8e9fa6eaebeb6096bc16d2663
25744 F20101207_AABSYU matos_j_Page_074.QC.jpg
240aaeed2779397281d674fb2ada3d33
742844aeb2ca95bbef5b9be00fe1d91da2127c76
F20101207_AABSCB matos_j_Page_045.tif
270c0c578916adc5e5a01d6e3ca045ec
fc46642dc38b5923360dc97672c966f31e46c482
F20101207_AABSBN matos_j_Page_028.tif
edc0606a29b5421a98bef5dd81a89b40
ef42a55e213356bec07325693d5f13864019a758
111352 F20101207_AABRWH matos_j_Page_040.jp2
3d8c188f31402a42c32af8a54e04fe3f
a3f537b3e60d8b4dd8853fda446f2038f878ecc1
F20101207_AABSAZ matos_j_Page_012.tif
7791796673b7d3326ba6b54c2a2ba8ea
93ccc10de7b20db9312040eb993a54294ca8714b
122052 F20101207_AABRVS matos_j_Page_023.jp2
f1a7c7c5bb093036180fa5c8ba29929c
ec50f19eda70248ca47da1c3d84682b31f0f7618
6889 F20101207_AABSZK matos_j_Page_090thm.jpg
3f4ee8f1528d5e4151f0c3f213574c7a
3937d504167a32d457c8fce8f32723d774b7fade
27207 F20101207_AABSYV matos_j_Page_075.QC.jpg
5e8ef8ac9c76d3e4e16a75c941ead8de
25a88989ca0abb9898c7493106e63ccb3f850654
F20101207_AABSCC matos_j_Page_046.tif
4be159c85ebe91b76bd0fdd61cc348e3
205db35059faf7e643886d09db82035356fe7cb6
F20101207_AABSBO matos_j_Page_030.tif
a11ac62e0539f7f00e6a648003526e15
2da72a4c2c27dde82526169c091eea1d42eed2e0
115409 F20101207_AABRWI matos_j_Page_043.jp2
dba050aac69f7e52e39cf9b3aac221c2
bc312efcd05bfa647790cd7061d75886501563a9
121427 F20101207_AABRVT matos_j_Page_024.jp2
5b9599b6802d6719ed10f90d583b441b
61bfbd5793d82806eaf14f7eeb0f3a9f3fdf3a0e
28606 F20101207_AABSZL matos_j_Page_090.QC.jpg
1d0a213f12483de26c63432a83b6e18a
23979a9b3ea468250bec16afe2f04b0e734455bd
27090 F20101207_AABSYW matos_j_Page_076.QC.jpg
88d180e75c4329a7f260d26ff6dafb2e
adb3d474f045fe6eb96b755cd11611014a59c99c
F20101207_AABSCD matos_j_Page_047.tif
f80ad5433656a945938abcbc9cbfe4d1
fac65468aafa97e56920000c3f19fa3f66754e7f
F20101207_AABSBP matos_j_Page_031.tif
e48618ad2017d8437961e46f63eb5dab
5447d3c386bcff9620a35da0a43e23c38aa76c4f
120984 F20101207_AABRWJ matos_j_Page_046.jp2
a9f9ab567e22212035281c17ac9ebf61
89b319659c0ae91fe7157bcb8f5fd2c95df22b7b
115765 F20101207_AABRVU matos_j_Page_025.jp2
93a10b687f96bdd439cd15eb779be59f
4d42b15dc32b05fecba076890ec941e29313f07c
F20101207_AABSZM matos_j_Page_091thm.jpg
1d3ad78da436b4ab6e38099bfda41885
7860715968407db78db8b00df5f6c24704c10dcc
6021 F20101207_AABSYX matos_j_Page_077thm.jpg
6439b937605ecddc9efbb80c69242165
5e0d9225f1e50f1ee9405a81dfcef951b239518f
F20101207_AABSCE matos_j_Page_048.tif
0dc0c3e6972992add272f5f07b16d46d
42701af2b3616669001f4cd3bf4090f3f8bebd7c
F20101207_AABSBQ matos_j_Page_032.tif
8eac30a8a8ab18167af46b61691d9127
21626c242c7adfeaa98403a8347b63141072beb2
120569 F20101207_AABRWK matos_j_Page_047.jp2
0d6f7c20fe93e9951d1b1237b5d81252
26bddf6e905df0d48e67f2f36564f35ccb590787
115951 F20101207_AABRVV matos_j_Page_026.jp2
4a5c7a2d504e078544068adb82b7c97c
35eaedd2694a03611b3524905ee8a8d2a4d6f979
6496 F20101207_AABSZN matos_j_Page_092thm.jpg
18377da9d335c6a37184b5aad11c96d2
fe4c05c9be2be341211064aca9909fd1e4835455
6118 F20101207_AABSYY matos_j_Page_078thm.jpg
e9973933da01c533d1219c1ef9532317
d7a1149fc69600242335ebf05df6090f1025d8a9
F20101207_AABSCF matos_j_Page_049.tif
ba57eee097bbf896d180be6db8076ed2
4dd2e2f4eb6a9e27130cfdaef5a44f36a33e3292
F20101207_AABSBR matos_j_Page_035.tif
b27c618b0cedf2eb3e40c660b1b7578d
fc8c7789ce53614f8e86cbfe0815a698917bccb1
123199 F20101207_AABRWL matos_j_Page_048.jp2
049f099ab0bf872ad8a37ee6f1541ee3
2892c70f6388c7a53a31c652fced60738ef02d1d
121939 F20101207_AABRVW matos_j_Page_028.jp2
1c7e2e8437f5e76a10d389090aff6e3f
734193ba36be6efa2f4030087292bdb70f1a1df2
27291 F20101207_AABSZO matos_j_Page_093.QC.jpg
7b651daaede909f7ef30fca42a49d7b3
068589e35b814067126c8a567ec92a4ca2596b81
6353 F20101207_AABSYZ matos_j_Page_079thm.jpg
865cf31bbf710ebff5daf16267cd4b30
706644ffb01e5537632299963218206338407de3
1054428 F20101207_AABSCG matos_j_Page_051.tif
304833a004763221964dff461222b26d
d8b15cfe104219357b3adc542d4926ae6cd8e6e2
114575 F20101207_AABRXA matos_j_Page_064.jp2
238db5d11b83b8b7252928fb3ab2f6c9
14b5172c40272115a7e06cfcbc1309d042d89dc8
F20101207_AABSBS matos_j_Page_036.tif
f622657d4c47a356bb63dd990346e6ac
9e577506ccd904a5910b251aff0c52076807ace6
120723 F20101207_AABRWM matos_j_Page_049.jp2
b09e99406c47091a7aea1099434bbce6
64a7336953bdda46f1632c63e99fd30621a3beab
108229 F20101207_AABRVX matos_j_Page_029.jp2
ee1a567a4afe8236b7c8b968c01a885d
6c8936bc62df6a00abc4c31ef6682e3260198726
28583 F20101207_AABSZP matos_j_Page_094.QC.jpg
4746c23a4b7c3ebd85b69c0458596eac
a6fc6c91a9e68641a922e36d3a0a399c594eb0e0
F20101207_AABSCH matos_j_Page_052.tif
1a165daf8b72dbc42bc29641c83fcf95
fc850ad9d2b7fb37060f6c8c10c34e2a86138801
117694 F20101207_AABRXB matos_j_Page_065.jp2
7911ddb183b25123d92f1bb4839f41b5
3070c4a05302ca757d841e160a07641f9e9b5ab5
F20101207_AABSBT matos_j_Page_037.tif
47342e614afaa49ab558a03dae66eef4
5f16caadf6466cf3e595f5f703ef2dcc74b2bf0b
59256 F20101207_AABRWN matos_j_Page_050.jp2
25cf8bc68d08d4eefd7b9d2f616e63bf
8a722d232c2975f0e92dd55bc20755c3983d483e
1051983 F20101207_AABRVY matos_j_Page_030.jp2
94a5306a731ca0367a81241e3ed6639a
87161b6ab50d34a1d369d81172aa3eedb74c5f5d
27832 F20101207_AABSZQ matos_j_Page_096.QC.jpg
dcc21a94d08294f0ca591ea65ece8000
b9e997e517b1196879a37c0c7e36ca5f6f92a634
F20101207_AABSCI matos_j_Page_053.tif
1185a186f43c6c1951ccaa84ac4aee13
49100474c9f437a92d19e166e6f6b2bea39d7f8c
110729 F20101207_AABRXC matos_j_Page_066.jp2
9b75e63e3366a2de9d7c284474c84cff
664210b1f160b0c29a905684c113c6a97d8be1d3
F20101207_AABSBU matos_j_Page_038.tif
16fd68398632eb4624b216cc00508460
38af80a6ca22f366e5f8106b40e02a0e15eafa7a
81773 F20101207_AABRWO matos_j_Page_051.jp2
142bd7d8321c836b108583af52f6f4cd
a5f6d56c855b704aed48b9fcb72bdef051def790
121534 F20101207_AABRVZ matos_j_Page_031.jp2
5a64e450469d11d1d04a21ecc6c7761f
aa1fd2f4d147fdfdb29e325f92f6bcca8c7976b9
F20101207_AABSZR matos_j_Page_097thm.jpg
5bf2ce28ee9159d36f14072bda8a2538
75c87dfeb455f426316161bfd72df5ac257c1bd0
F20101207_AABSCJ matos_j_Page_054.tif
703ea610a9f492855a7edd0df240e5b9
c0fbcffdfd0eaae824664873cf349722b7edff26
124243 F20101207_AABRXD matos_j_Page_068.jp2
5743d3534af8f20b539dcc398b6b8954
ad7a6077bca76bb1406251de4110e67cc39343d1
F20101207_AABSBV matos_j_Page_039.tif
e723a5d88e24a81d500e4b9707555f5f
5aafad12c7c1e138e8a8d2a2767a5103adcdd66d
1051887 F20101207_AABRWP matos_j_Page_052.jp2
54358cc8fb5ed208c719d7dfd8b6c636
f6b5bc3a010e5ed5ee7819d0e6fd315a1b5d0ca1
28461 F20101207_AABSZS matos_j_Page_097.QC.jpg
cab49928f9affc2f590f1d3522fb9f45
f8dd352c9ad7d413848938b730eab08a022adb35
115164 F20101207_AABRXE matos_j_Page_069.jp2
6d67a8ee4bbcde5933e56e270d4cd14c
1f246eb9d4e3f6cae3503b985859cd0739477926
F20101207_AABSBW matos_j_Page_040.tif
873defd3a37384b189c856891e50a2fe
0fe9c9af086baedaaa0f2e064d839b3c2a9f2345
43963 F20101207_AABRWQ matos_j_Page_054.jp2
5caa9717d0c03a2e4e4ec834a567275e
22769ec32a9bcf953be8b9defd9c89b3b1178f30
F20101207_AABSCK matos_j_Page_055.tif
a350a4c5f3b8460805c266d08ad6c6a1
fca21e26bdbdb5ece495a2f977126615647e8f12
27499 F20101207_AABSZT matos_j_Page_098.QC.jpg
8939167a90b11ff157598271cbdc1429
28758720251df91416e93e13d7bb799d8e7446e2
107488 F20101207_AABRXF matos_j_Page_070.jp2
6c3348d3cc1b4f812f80b90f039f1e36
d36b00d099a3e2b26b93d840af34767320baf7a9
F20101207_AABSBX matos_j_Page_041.tif
b7f24ea28320dbad2d6e42abd8bbc2b3
623a54f6225e4ebbe9d2eb71e445626ca12bb4fd
37187 F20101207_AABRWR matos_j_Page_055.jp2
5f186daf4d3e71d0218801a2462fb029
83d134cdcb33bfa1508feb214dfadb965f0c671e
F20101207_AABSCL matos_j_Page_056.tif
a056dfa525d182f298425f54f3944502
e6f43d4abd3911630768a0114647fc6fdcc20c66
116251 F20101207_AABRXG matos_j_Page_072.jp2
db518f0f4dd7bb0f623a86475c8d6d83
2141582758c062f40c677d9f322cfef407dc6532
F20101207_AABSBY matos_j_Page_042.tif
4b2bad25840b28e9cbe4d83be0c4aff3
2c6363e1d51364703944ce249c6ffe32413431fa
F20101207_AABSDA matos_j_Page_072.tif
18c32523977248754297a407c102c6cd
a23be5a5f445ac2e8df914fd9469991ef1e792d2
F20101207_AABSCM matos_j_Page_057.tif
7ef9690f32e6033807e7895a45ffea0e
904e92c8016c2550592dcfd4a55cb3b24d55efa8
5750 F20101207_AABSZU matos_j_Page_099thm.jpg
960c09521ed835102b8dbabd6cdcd611
1b679c29376cc6b8190bcddaf2b249bb0c86f622
115325 F20101207_AABRXH matos_j_Page_073.jp2
b2d473edcb69ceeff5137f09a68da223
06b83f3a5e8c6e96e22b54355e0c43fd682a1849
F20101207_AABSBZ matos_j_Page_043.tif
1007ad06d49d86651a71687c398ffe19
b44e9e1e0538ff4204c11778f3138532816bd5dd
60693 F20101207_AABRWS matos_j_Page_056.jp2
e8acc9ce12a54b3214b1d5dd4a28c760
fe0b8e96fa37378131b66f2dddf7a0a28b6217e3
F20101207_AABSDB matos_j_Page_073.tif
9176f6a35a7449fdb544c3cd49b71e68
d902448ff8684ee6eae90169d85a532241dc20d0
F20101207_AABSCN matos_j_Page_058.tif
e51704554c83e5aa26e60fb7121cd414
91c669421dde8c277b7802220130cb7e9a3d959b
24390 F20101207_AABSZV matos_j_Page_099.QC.jpg
9d5ab6ec61d0a91e8a113c17ceeaaac1
5d8455cbee49ebef2e3c20f6214d168b09ff93ef
110655 F20101207_AABRXI matos_j_Page_074.jp2
0bb6b6ecb3e7de05a5d6d34ba8f05027
8a3536ab1cb583c18194d10fb566ff1b9d6f0f5e
40684 F20101207_AABRWT matos_j_Page_057.jp2
8de2449a25ec16a4a367e1f3c9fe9c3c
cdef543c4ee8eda321ca17ac39a940978bc1baa3
F20101207_AABSDC matos_j_Page_075.tif
bf37cbf33ab305ab99f63c20f76c51fc
fd4fd06fce1b8cb1727caf866f128a9385cfb8a8
F20101207_AABSCO matos_j_Page_059.tif
9640e91925ea21e9547c771baa61c2ab
6c1c3577570a91f69aded789b29e9c84f5f5a1d8
2581 F20101207_AABSZW matos_j_Page_100thm.jpg
3213eeafaa3db06823d83c3ca3a425b0
caec49ce1c2b1da224ae440cc9b75a416b9c3a8a
113196 F20101207_AABRXJ matos_j_Page_075.jp2
2d32d13fdd5220316b36382fc121cb87
8d3decfea52cf9e24979a168aaad1d0d095b035c
54950 F20101207_AABRWU matos_j_Page_058.jp2
22753867ba6e8cc02536c6952ab5f3d2
d7f06ff455f68056782f60c50734924cc6505e57
F20101207_AABSDD matos_j_Page_077.tif
3b4985050095c57e3cc3be57e2555038
64492bb5b7f621cd6ee228cc708e9ac3b2a04076
F20101207_AABSCP matos_j_Page_061.tif
e9bab4a0ceed9767318e37ab12650490
3e1c0b4d0906f8ce4861d6fc42f30dd7e85ed722
8865 F20101207_AABSZX matos_j_Page_100.QC.jpg
7b014155e382bef527c015235212ce4b
17b44d81629083d6eb0dd8599319916502455c09
1051966 F20101207_AABRXK matos_j_Page_077.jp2
c44b2c1a514e22a4877356a2f1c86893
24175e4d827cebdb7a7b86e04032799eefa8ffae
40372 F20101207_AABRWV matos_j_Page_059.jp2
f3c72e205b62e3e24daf72ce91a692ca
a3238df15d75f96349d03a3c9d0a8222ef241d8c
F20101207_AABSDE matos_j_Page_078.tif
d3a0053a7cc63c611a8ad3c90011236b
5ce0728c1ce1c884b360dc75332ddd66e62ce6e3
F20101207_AABSCQ matos_j_Page_062.tif
441f72049a548d000a26f9fb5de3a6de
2cebc0047a984d6bf64575eca5fde0ad2e767e6a
4008 F20101207_AABSZY matos_j_Page_101thm.jpg
e93674fb01a2a69da55696ad780bdff8
5716cc3eaa6eced45d1de4327cbb95d5c57f1c6e
109323 F20101207_AABRXL matos_j_Page_078.jp2
793ead35b9201386eaca0efa69e9c98a
492679ba530ca376512601ea91fea0ab9730aca9
50350 F20101207_AABRWW matos_j_Page_060.jp2
9022baf88e2780d9982200ac59bff437
076f809b65af19c3141d48d06517229e32065ad7
F20101207_AABSDF matos_j_Page_079.tif
0bb665133f1f343920b5dc56aa6e28b1
3107af53e433818531f0326cfec70ac8bb8f5687
F20101207_AABSCR matos_j_Page_063.tif
4bb96306f75dd8db51c2a0402d8e3b8d
a3597029f30d238d9d1c2b38b24c0c0834441db5
3998 F20101207_AABSZZ matos_j_Page_102thm.jpg
55f79926ddf2fede6e86c4ced6ac3d5f
ce9f0dd131fdb0a4343ea65a2c159b4e7ee0eb77
115444 F20101207_AABRXM matos_j_Page_079.jp2
8401d921a805951c21ac9cc664545478
10f9d9564348603fa51b1193c41236c14a4403eb
114130 F20101207_AABRWX matos_j_Page_061.jp2
259ad1cddbbc1f88bf27d8cd2a354763
89940d8705930f7499fcee900695dd49d5737ddb
F20101207_AABSDG matos_j_Page_080.tif
c1f3f595ffc5c1cb4fdb2a9c03b3c4fa
c46b56823afd0f321bd4f60bffaf6cc8e196cdf6
118067 F20101207_AABRYA matos_j_Page_096.jp2
f7e26f527fee0fab07daab64ffb5f48d
b76ccd13118a12804f1e567c715f502025c49362
F20101207_AABSCS matos_j_Page_064.tif
e5e29b17c0c1669aeedad1dcf2f3a664
b9ec5d61f18322a854f9b57c77b727f107749c37
113426 F20101207_AABRXN matos_j_Page_080.jp2
71629ad256f499835a186ff434c4ad4e
927ddec8688d7942f483417346c7c9ae896b482d
120543 F20101207_AABRWY matos_j_Page_062.jp2
8029c17b137c0275a1cb3d5332c49418
a3379c05d0c5f451dc5670863ed908d8774f2985
F20101207_AABSDH matos_j_Page_081.tif
502b955af509bfd3efef981435c44c52
f2c42e0126696cf46b74d2f746c2cefd08ea9c40
120134 F20101207_AABRYB matos_j_Page_097.jp2
1f2ff98711c162507d48bd2bbb30047a
9c73b18406351ab66b0297009cb03021f1ace591
F20101207_AABSCT matos_j_Page_065.tif
868182e367d1661f48add07349a6752e
77f09f1a244f9c3cec2602a2218734e3428a8414
119315 F20101207_AABRXO matos_j_Page_081.jp2
04995b72bbc80080b23cdb8a5b3e25a3
eb7378cebd14d8d4d665e1f4a8549937cd51a345
105836 F20101207_AABRWZ matos_j_Page_063.jp2
2916181d60381ea379b90ec52dab0294
1a186d5a4f2ea1e07dd2f893c91efc50384b90d6
F20101207_AABSDI matos_j_Page_082.tif
fc73f60ffbc1d3bf0e661e4d6f687590
1f595505f083a4ba6ae7404abac5c1f53ed953b4
116164 F20101207_AABRYC matos_j_Page_098.jp2
9575be2d4491f514e67c1ebb31364fa8
a506b4d5fed7a489d394e27cc7b26f06b70895d4
F20101207_AABSCU matos_j_Page_066.tif
029c1b0f6ea5b5bdd8d213d4ffcf2d33
f8198f75c3ec65b0cebce398a8429bfecf23ba22
112287 F20101207_AABRXP matos_j_Page_082.jp2
7bebe0ec682a9bcb0efc8645d36ad420
797fcd86dfb7fbfec15d89b67272c6e4e0ad062e
F20101207_AABSDJ matos_j_Page_083.tif
b7689cbf6aeeedbd4b56a73b80c58755
091c96d69f208844436d4283779b0ac371d81ab2
364896 F20101207_AABRYD matos_j_Page_100.jp2
9c4882498faa36bb8a9599a31f7a6fb3
9b49f885a1612ea2899147c4d2dc855f9604faaa
F20101207_AABSCV matos_j_Page_067.tif
e816f97932ec31002512572e1823b23e
45c15d46a8c31d4dd2f56b0cc88afa71899a1a78
116163 F20101207_AABRXQ matos_j_Page_084.jp2
5e2748b71a18c547849b08d3fe2e1cb0
ff93c95332fd66bb711c1009272937c1560178df
F20101207_AABSDK matos_j_Page_084.tif
a6178150c567183aa4685862b6724987
d8ab5c18fa51c979865ee900bc9439ec12acec07
355475 F20101207_AABRYE matos_j_Page_101.jp2
02ab96cc2df9b8fea0c9f945515ae5cc
462b931be01c9906038be5a36662f15dce62c4ec
F20101207_AABSCW matos_j_Page_068.tif
f491b245b00364777b2beb1fb378ab24
3f251e7ba2c25841bf92ab4bc5c3989f4dde5618
104261 F20101207_AABRXR matos_j_Page_086.jp2
79f22b9e6c9a7dc2238b2f17ae0be9f7
379a7092ad067d84313da23b892ba64edeea0919
F20101207_AABSDL matos_j_Page_085.tif
d931f10e334fa37ba14faf01a4858993
f4f1e4f877effc95b61d285411913898983aaf3c
365366 F20101207_AABRYF matos_j_Page_102.jp2
c5539715be0de8cfec641ac31a80512f
32178f3f13535116617091c850d84bccd64b5ebf
F20101207_AABSCX matos_j_Page_069.tif
65eafbd12a60532e7cd9d31572097b3b
611336421bcb7d8b2b9e40e9e53043aead2f04da
115098 F20101207_AABRXS matos_j_Page_087.jp2
fa440c120a8889fd9a960b55fc7d865a
b863fa0706c7560b1bb6174f1efb6200f5225762
F20101207_AABSEA matos_j_Page_103.tif
c731dec34148e9cc856cdb1a892d0c3e
d90019c6d531d7f7809c01cf68871fb4d7438917
F20101207_AABSDM matos_j_Page_086.tif
09256129c8acf4fe00c9171211bc3bef
2d73fbd9c13741ceadcdd1b8a2353e8771c88147
79417 F20101207_AABRYG matos_j_Page_103.jp2
859c68d4e8c5cb3f022d6e8d589ca19a
b904ba88a7999beca9cd267c58c0dce04c0deac4
F20101207_AABSCY matos_j_Page_070.tif
7a08facab716b11c1b58cb257d531998
6c82ec3bedafc9b0065cae72b5ec4ded670810e2
F20101207_AABSEB matos_j_Page_104.tif
eb151a105dd3afcd7d9b8bd2bef5d59b
4344b5110af6c8ab319c060e497091c0f7802061
F20101207_AABSDN matos_j_Page_087.tif
4e6f56f3ee92fcfb4c00e642050ea56c
49a9a9479c0747bb32b252b992df23834bdbf6db
79797 F20101207_AABRYH matos_j_Page_104.jp2
edd8dc7bc9ecce4d512804f96181f652
7dc719a95ae2fb9c9434af261663409103c5ed01
F20101207_AABSCZ matos_j_Page_071.tif
1fba30686d71f0d279135582747e2fd4
f9463cff42698d7cc5ea705288a58f4b84fba46c
117867 F20101207_AABRXT matos_j_Page_089.jp2
acb85cee7a57938220538f9a8e45eaaa
7d1f11fc73d671a30598f470e458fad2c262d875
F20101207_AABSEC matos_j_Page_105.tif
5ee301a6ebe85d1f028f835df8cf9510
bddd35dc433f1ed41f39bd564766028f01eff726
F20101207_AABSDO matos_j_Page_088.tif
3268ef744792a02e928698cd02cadccf
5bd00bbb6bbe5e4dbe321545fa265067e2455679
64591 F20101207_AABRYI matos_j_Page_105.jp2
d335701ecf3cd8d6e8f64620009a2962
9ecbf5dca87f41c1044715f8926720dee3bf7461
122515 F20101207_AABRXU matos_j_Page_090.jp2
9116ae94d4c3e90c8b63de4d46aa062d
7ba7f13a56c11c1897d28568573666d0db9c994e
F20101207_AABSED matos_j_Page_106.tif
28a67e9fae3e942757f88fcb2221d4e9
d53922b47f9ea8983a6488b1c597511bcb682b0c
F20101207_AABSDP matos_j_Page_089.tif
9b71d37d3b1c749f2cad372e4ccf1643
f779bcbdec5677ffdc75576fcb34a4b8a14064ee
468317 F20101207_AABRYJ matos_j_Page_106.jp2
85be58c22b21da461c5be83bd7875517
747e338cc6c71d8c32d7e328f5f65b7c221f49e0
116480 F20101207_AABRXV matos_j_Page_091.jp2
c5079cb2e91419f020e49e9a0658fb04
875ff71e82a2872546458f6741806ad591112e67
F20101207_AABSEE matos_j_Page_108.tif
9ae1d4e56ffcbf34cec0745b97331786
73d23e1c6a57ae0bc4c5c339387d585917c3ba21
F20101207_AABSDQ matos_j_Page_090.tif
52c0b989a1ef5f4759d57727fb1fdc3c
9015fe38e2f3f55d8aa32e0afbb6c312f559d283
417018 F20101207_AABRYK matos_j_Page_107.jp2
6bbebb696e9af7eadb3b675bf0a4648c
dcf5d6fefbcdde2a3c0ea26b516fe22ab6cf7495
1051982 F20101207_AABRXW matos_j_Page_092.jp2
baafd2c84cb4c824b1558e32be3aefa4
d88f81968302e792563eda41bf55e719fcb2a8f2
F20101207_AABSEF matos_j_Page_109.tif
6684a0de1f5da3e372fac613ef82a748
ec687b8d397cd4121681f0ddb688c7b9eebd80e9
F20101207_AABSDR matos_j_Page_092.tif
e01b3690940b5f38263efbadc86cdfbd
0ba7dc6f77e7cea86f497d1719bfc2c51a6b5d26
398769 F20101207_AABRYL matos_j_Page_108.jp2
2552f188df3be14114f93da9eb399a02
7cbb8ffd6164d926774abb781b905b4c093f3203
115748 F20101207_AABRXX matos_j_Page_093.jp2
9ad45c0c9f3848c31e8e62dc4e9f15b1
30f28b31895df42d187be9a9b7a2bf882f6e7a84
F20101207_AABSEG matos_j_Page_110.tif
6e0863551fe940bb9459f155cbb45003
01d20e2466bc590452c101870c3ff7ef674d05af
114451 F20101207_AABRZA matos_j_Page_124.jp2
939e566c09835b0b34cbf6af58e53ea6
d9ccf9add6df698ca26bdb7c73d5382b59a13ec8
F20101207_AABSDS matos_j_Page_093.tif
154b51a84bc9422a393df2e91372ab64
7be3b649505f80fd0e07dd4157df7f10a2276b31
86855 F20101207_AABRYM matos_j_Page_109.jp2
b885e4a1924d7c8809bc97ba5ed090c0
f841cbb860ffb59aee1b539f19bcec801b8111f5
120112 F20101207_AABRXY matos_j_Page_094.jp2
b801bdc1988c992569f66e844d5e1c86
10592b5ac4f78161453bf086e5e910dbb8072de2
F20101207_AABSEH matos_j_Page_112.tif
88b5fbddd2a853a28f591d707d1c05e8
edec10ec0c5f5ced3c4f5230fa963555f878002b
107323 F20101207_AABRZB matos_j_Page_125.jp2
86307e6746773c169093dfe4810d4185
0d596640127cc8ce8e585c8f30bda220a65d0b22
F20101207_AABSDT matos_j_Page_094.tif
a907558842a532e0411a0d23f0cec5ed
b331935032c2ee4f2fdb5a71aa5f097d1df1e5af
83499 F20101207_AABRYN matos_j_Page_110.jp2
0807e5c4d9b798c19b42536fc4ecb892
64f6d138b119fe8b0e895d7c8a6f23885d5b2990
119175 F20101207_AABRXZ matos_j_Page_095.jp2
9f56e76cac9e668d143575b1c7e58eee
fc05912c52ab1d496ff66c4eee24cbe6355b3d04
F20101207_AABSEI matos_j_Page_113.tif
eb3ffba240542c4a5903bf3acb7b004c
f6d9da4bbaeb8c9d3212c21e2d5e5ea5acaf82f4
115937 F20101207_AABRZC matos_j_Page_127.jp2
5ba29c285bddc13a78d2916541b444e2
4de9e71f695ed2aceb89397bcfe71019746e34c1
F20101207_AABSDU matos_j_Page_095.tif
234f2182c6d8731d8aa4fdbbb3ac0601
449e3950846d872076d053d70dc7712afb65bb8d
69899 F20101207_AABRYO matos_j_Page_111.jp2
1ac5cae58096473fb4bfe0baf67c64ab
411686dc7d103c3785b074fa287d049a9616d21e
F20101207_AABSEJ matos_j_Page_114.tif
85786418f5d1b9a76a67f6bff97220d1
9981e7942d184a22aa7354afd757e53d115609cf
117931 F20101207_AABRZD matos_j_Page_128.jp2
259f3ed82ca28d186b3608a92fc2e561
14de3547b28c488ced7bb3e623ab9ca26af82053
F20101207_AABSDV matos_j_Page_096.tif
ecdb564b1ad0b3f2eecfb2796faadab7
ed784b5ea2e702f7e8a1a7875ac8f49a79acadd8
599263 F20101207_AABRYP matos_j_Page_112.jp2
12b0494fe06378d7192cc6d2fd698658
fcc6d9b6caeecfe9e3d314ada26427f21cd6bd3f
F20101207_AABSEK matos_j_Page_115.tif
f022e03788ddfc5bc3e42b49667454a2
c94ee39aa10469f0e2001b3cf42685a41beee6c7
F20101207_AABRZE matos_j_Page_130.jp2
c7cbabf63d5d60196106eadaf40f59b2
cf1c34a2105d74d5ea4b8b96305c24e1fbab8c31
F20101207_AABSDW matos_j_Page_097.tif
251f07145eae76b47dc8cc734b1d2226
db96ea6ce25f09c80d0c9b5785055feda945ac3a
599693 F20101207_AABRYQ matos_j_Page_113.jp2
a2e15b0b9227e2c7c9c3f7f88f40b3ae
b6ad7811ed2b845b7ff6f5989be991fe212f66ef
F20101207_AABSEL matos_j_Page_116.tif
8f6e4a792c25197bc80bb04d0282fa40
d9906a1219c8c04f309eb5dbd49f6b345b48abcf
116588 F20101207_AABRZF matos_j_Page_132.jp2
43e1ec4796768f63142a14cecb86f531
9b1a1c69045096380d9f4a97657f61bda9872435
F20101207_AABSDX matos_j_Page_100.tif
6c9de5b7aa0967d1cad2fbe62e48f2e1
12cbce052b9567caed6d798557e96afbc092b6a6
703553 F20101207_AABRYR matos_j_Page_114.jp2
ba3ef4a0e08b4278cee60d3318411244
e0ba92643e239b32261d16ec84ca99771ae6c91b
F20101207_AABSEM matos_j_Page_117.tif
c104fd98f4b3b18fada1d33ad9e88da4
c98eeb1a0629fb9e2f410ab715892bdfa31ebe6e
F20101207_AABRZG matos_j_Page_133.jp2
d40af49ec008557c5fc754cfc9c5b55b
8639fa0406c517b48c1fea9b2b38d542603e0a1a
F20101207_AABSDY matos_j_Page_101.tif
9ce63670d35cca7e432b5ab76feaff89
70749a0910786e53a5dd66fef578d1bdb896c725
39510 F20101207_AABRYS matos_j_Page_115.jp2
622bb4dc257cd78259f7b7c1ee09aec9
bd48657e1841888596a46a0de3181e34952f646e
F20101207_AABSFA matos_j_Page_141.tif
9903a96c25069614211abfd717a43610
35fe3518ebdc324d8b14887860a8a9e8b1759f7c
F20101207_AABSEN matos_j_Page_118.tif
148938f5eb5011b2f1f07f7951f08aab
a4bb46e6ce5b0f83ddd2d99c1104e6514c7ac4fd
35276 F20101207_AABRZH matos_j_Page_134.jp2
7afccdb2e109b59ee0c31349b0b14d99
73b6a9dd91e2a785f3dd38335ac9bee542b72974
F20101207_AABSDZ matos_j_Page_102.tif
c209ee6a72b6227717847231b5095489
9d73909d54176434483f92c83cdb1526f1b31742
117187 F20101207_AABRYT matos_j_Page_116.jp2
65d09a540b42ebf8c5af074a274db530
298ea7f9505fe25dc4ef9009f8ff77e5be06cf02
F20101207_AABSFB matos_j_Page_142.tif
15be27be2dfbec81e1877b38841cd66f
f1fdd5f59b7afb3e0850fc063db186334b4fe1e5
F20101207_AABSEO matos_j_Page_119.tif
31880eec67f52272f701c37795808d64
e927d206036ebfa98f31ca85c4224856617671d5
77690 F20101207_AABRZI matos_j_Page_135.jp2
b1640d3b9657fd5f38cf6492cf41f722
5defbf222c014cb4797fe76954ae8235e6a96acb
F20101207_AABSFC matos_j_Page_143.tif
afce2f06b95486ad292aef45159be478
b6e43d2f6259187a1dd05f8aee5ddbbe26effe32
F20101207_AABSEP matos_j_Page_120.tif
5da3eda9cb7704a3a388e332ddf2b5c2
498059c12ac655eb324326da50dbce032d8a1149
271625 F20101207_AABRZJ matos_j_Page_136.jp2
3d6599a78a8f2ff266bcf9413486b4dc
f1e856e58de1a536d7048f75a2cecd377e9a761b
F20101207_AABRYU matos_j_Page_117.jp2
55fa7dafad0ed8e27ed9e6a5966280c2
02ae03a06a0ad1356b104dc46d645eada4a5c5fb
F20101207_AABSFD matos_j_Page_144.tif
eec7f50006b7c27e020f2656370ae0f3
f51a3ef9c4c7f4abe2ab2b8df3fd025ec7e7e894
F20101207_AABSEQ matos_j_Page_121.tif
c7599c20ef37522ee8aaf18ed6e6af9a
e91a90ab72590bd9b6d943ade11dab1360805f3b
43019 F20101207_AABRZK matos_j_Page_137.jp2
4f10eef92ab4f77abd7a595b3d1ff761
6fee423963661428b4c85c31467bb079a158f102
109608 F20101207_AABRYV matos_j_Page_118.jp2
793fb2cdec9ae0fd5642bd1a082e8008
ee5bff37543d402c4601dd4b60c47b49fbfcd633
F20101207_AABSFE matos_j_Page_145.tif
121f42d5cc8dfa71c79f6d967b120c53
ef1328cfe59a4a396cd30f8f63cb06db5178e6f2
F20101207_AABSER matos_j_Page_122.tif
336e6bf5bfa7784b20ffdd467462cc0e
27c730f493758eb44a168d6feb737681d1cec6ea
508827 F20101207_AABRZL matos_j_Page_138.jp2
d02db48a99bf6cea4a54de500fb507c9
3d5b51c4413f43d64edb481c6765777665f50df3
115469 F20101207_AABRYW matos_j_Page_120.jp2
8718d938bd6c5429d3173b716931ddf6
4e1d3f0b8f4ff95e8bf59ab2d4ab007782810a35
F20101207_AABSFF matos_j_Page_146.tif
896cd05a0ef56a11092727f5cfbc1e70
ec5096ecb731bff1419b1cc8f56c23c230400d8b
F20101207_AABSES matos_j_Page_124.tif
32173f63ff9c92f53147beff291d1104
b7ef242f27bcb30563d26a887b3d31c69c1e5d27
461382 F20101207_AABRZM matos_j_Page_139.jp2
047ba39ba7184f63f91af6caa40bd064
80c74dbdb39068f4b2d724fe79f7b7790db8cd17
119736 F20101207_AABRYX matos_j_Page_121.jp2
f0be25078d90a7d7c523100e57e95a3b
dcea1596e34d33e47c2690cb1630062311ae5c7b
F20101207_AABSFG matos_j_Page_147.tif
915bd86b0b0cc8eb2efaa4a0d3e9b267
6b0f35e1d7fcdf2ad2715c512e7e21c22e03be97
F20101207_AABSET matos_j_Page_125.tif
dbbf6d2a3cccbcc3159215ffee939ff5
3b1a3aba192f5861459f10496f77bc7e1b581470
71884 F20101207_AABRZN matos_j_Page_140.jp2
2a85de536b623fc95be0564c6f772405
6e935ea8d143a6bb02682705ab45a900cb17e7cd
1051975 F20101207_AABRYY matos_j_Page_122.jp2
c7aca0c6429d609db7a04b7b616feae3
d0539eed454eceeb7f3941a88e5d4713fd4c5539
F20101207_AABSFH matos_j_Page_148.tif
cb3407f919553d7ba34b8b382a4eed30
b8116eaaaa197b99136cdbc23c26cc66b2579a80
F20101207_AABSEU matos_j_Page_127.tif
07f2f8b66bff2371fa96028f680e7d5a
8c63aef95861cfbad58d0c2dd306db85bd5c72e0
504030 F20101207_AABRZO matos_j_Page_141.jp2
cd70ae6fcdc763acf10d8b551e0f39c8
cfc59d574ce3cac9ae57974418d0e677fbbe45df
114308 F20101207_AABRYZ matos_j_Page_123.jp2
28778bf118a592a0a21b0c955ea572ed
05c425c755f63e1098505aba4115e0ddd91521e8
F20101207_AABSFI matos_j_Page_149.tif
3ea8069b0d61c90e2da243bcd70dc092
e23650a98835fe4c5a17c0bf81c4feef60aad4df
F20101207_AABSEV matos_j_Page_130.tif
b54f8e80feeae0cb028b8fee0658dfa8
679b1868b41cd57884530f0849260894963d6549
114500 F20101207_AABRZP matos_j_Page_142.jp2
760af470d465ad0b57ae4b7dbb7b9c24
124302770747ce8e780ec9e75c92852abbe46c13
F20101207_AABSFJ matos_j_Page_150.tif
9a5e0e653e50288ea0c3f55f76274d6b
cb78a5ff889053336aa557afc55fe477a3b0f446
F20101207_AABSEW matos_j_Page_134.tif
08a3a497188eb98c776dc899101d6d7e
9d78bff4d3f9e21141aede0f0287e8cc9518e432
124290 F20101207_AABRZQ matos_j_Page_143.jp2
dba35309ebfef5143d31e2c1062427a9
7b6c194028c1b1dfa4e3a04e319c52e9dee6939d
F20101207_AABSFK matos_j_Page_152.tif
e9e0dba0f780374aff0bedb82342f43b
2b358d1668eb3efb8d79f0df10f6b61bc17b4449
F20101207_AABSEX matos_j_Page_136.tif
80f9c318216819dacd613e13102c124a
73dc48548df0a5b1c0ae82a1b3ac135ef49d0f47
123943 F20101207_AABRZR matos_j_Page_144.jp2
79ca66a2d38aa3af9f8b8f98b7b31ba6
80cbee169a01864097aa39231c7640e94cc52d07
F20101207_AABSGA matos_j_Page_170.tif
e8b5d11d2a58ed82c7fab5bb3ed2fa5f
215734c94da916098ebcf115519f445bc3e47705
F20101207_AABSFL matos_j_Page_153.tif
51348dde060327e7d91b45a2f4172aa7
622a5db14e801cda83115a6ee70e122d51e78b1d
F20101207_AABSEY matos_j_Page_139.tif
a69b5d2513d9e61e05fe41507cce8d51
5e7b9d8b23a4d67cd2f693bf5f13c00820abe378
122505 F20101207_AABRZS matos_j_Page_145.jp2
08eb77d72e137cf3775dcd03ae728d7e
43697ad70fe96db89aa9dd2df8191c2a1510fb0e
F20101207_AABSFM matos_j_Page_154.tif
bd3d4cfdce63179ec7097607391adfe9
4d02a27815ca0fb9217f6a264a84a69565c576b8
F20101207_AABSEZ matos_j_Page_140.tif
1fffdddd20c60ab4ebb6808f4906e9b9
be7331663f1f808473d7d75454a22eac1579219c
117472 F20101207_AABRZT matos_j_Page_146.jp2
9e208170f109c4bdf4425e6a84c029b7
d9701a80b6df76ba0306ac8fe800be7b77d2f606
F20101207_AABSGB matos_j_Page_171.tif
e259185ae713f49ba3e55d6a642c833d
3cf9bde6eca0f8a58f21d1417166d907f0b26d45
F20101207_AABSFN matos_j_Page_155.tif
45dc1a7557802da5cefdb7e139216ef2
843d3126a2f741742c9ee2ec864ca2565635242d
1051967 F20101207_AABRZU matos_j_Page_149.jp2
2547b53503c71a4531a5cf597c3577ef
927f5569d3e535c31fdc76d77f1efd242b9749ea
F20101207_AABSGC matos_j_Page_172.tif
50b79dfdd2c30b8cda4beae79b28f54b
e4a8725090182c6ef166dc89dd8a322f92e76bfc
F20101207_AABSFO matos_j_Page_156.tif
a41c147bf22713b6002da13ff696458c
a8543ea69f00692aa6deb70f7c1541557e191c8b
F20101207_AABSGD matos_j_Page_173.tif
2f78170890c7ac9b4d76c1b1a5ba1ba3
893c7073b49ccaf61adea81c89938a91cb17eee4
F20101207_AABSFP matos_j_Page_157.tif
b3091a630e023c74077c9ba514daba00
ece0a4bfed62ac0f4e56cb0d0e590bc76044faf2
F20101207_AABRZV matos_j_Page_150.jp2
b764d4a4b4fe1001ec4507722c01aabc
e1ee945e13e720944c79a0f6c75aa45079d3342f
F20101207_AABSGE matos_j_Page_174.tif
1fd39a3eab604c02def67df449a4e1b1
da0415db9bf5fd2db623550b11d82736579fc1fc
F20101207_AABSFQ matos_j_Page_158.tif
59c2bdb99c3547655f7e17062b30bd20
cdf0487247c74b9837bdf8ad17825f357f3e7a5d
89394 F20101207_AABRZW matos_j_Page_151.jp2
20f2a1fd45c63384a44991665a65b52d
8117253c1e0ffd4b071f29ad8cde076d2c7c0ab6
F20101207_AABSGF matos_j_Page_175.tif
e26d33e4478ea599809f46584462f028
c2408d9388a7c9df080a601e6810b117a418ad1f
F20101207_AABSFR matos_j_Page_159.tif
d881c6130f68f7fea0a660d031b3ed12
a56feb0754e532bc841794c1c293fb651928476c
652468 F20101207_AABRZX matos_j_Page_152.jp2
306e84ed79857b012a425829046a17c3
48ad165a5760d86df97a6405a7a16db727e6ce7f
7953 F20101207_AABSGG matos_j_Page_001.pro
515f6daee72959eaa4709e52975d8ac0
cd3b0c4e271940f607c56193d910dcf60ba0c293
F20101207_AABSFS matos_j_Page_160.tif
383430c9ec7bdd72039193b07c23bd05
5bd27475b5fef570d3efa4febe53cf2c7533f937
433008 F20101207_AABRZY matos_j_Page_153.jp2
850d2a1731142f6254e4c84a23c9b000
07c2d0b9d2717f26ccc83d757ab5553929999c00
964 F20101207_AABSGH matos_j_Page_002.pro
6f6ab9f3c2ff65bdb51c61a40efc4111
e85c4359dd9a1929ec1e975f3440a63db93600d1
F20101207_AABSFT matos_j_Page_161.tif
de90469c35affaa1ce6b74d88ae3a601
91ff41e6079fe7620887e35ac10a4f5299d720a0
140042 F20101207_AABRZZ matos_j_Page_155.jp2
f1ac7daa322048429db588855d717191
b0e4b228467c618574398dfded290132ceb6915c
5386 F20101207_AABSGI matos_j_Page_003.pro
1e64d902b7a586b8567f876c2271d7c6
0ecae7efd9affd6d4e0f370b887e5516e5d2df97
F20101207_AABSFU matos_j_Page_164.tif
9578810c66a664492b742b8237a63bb1
9022e28f60751091dde367142ee0cd4ea79233a5
54994 F20101207_AABSGJ matos_j_Page_004.pro
c78044a2b08c76737afe5d8bb69ab501
771ddcb5edd56a874625d655879ba403174b809b
F20101207_AABSFV matos_j_Page_165.tif
f35159e79bc930eeb86470a73b88a0d2
7f7c4f7edb7b58c111ed9f8485fe84a8d674ce16
42531 F20101207_AABSGK matos_j_Page_005.pro
e8415227a61b676ffc19f06aca6e96a0
192b3e7a0216ea093679fe59f437d8c227b0196e
F20101207_AABSFW matos_j_Page_166.tif
8f75242f0190eea7f487aa2769eb8cf4
28fa27a586cfe9bfe28af21481d3d7d286e6fd36
49484 F20101207_AABSHA matos_j_Page_029.pro
8946ae3b587d1e5cb44a8fa6ab30dc2b
51878c225fe565df10e23972badae6db557f0a3f
88805 F20101207_AABSGL matos_j_Page_007.pro
c19a90bd711ef2e234e9fb8e8f4fc117
9e5ae3df4979990d7e3397a219f4c1289ece5a72
F20101207_AABSFX matos_j_Page_167.tif
4eb0b629c3124a1201c970d28cd82f0f
dac376dee082006071481587aaa3c59f1914edb4
53756 F20101207_AABSHB matos_j_Page_030.pro
1338911f98566e62927f496d8e85fa94
8b17ee7f3e334f409cf083f89668bf8d076f24b7
11262 F20101207_AABSGM matos_j_Page_008.pro
47fd8b552c722842f85dd0019e195ccd
57250350e0dabe4479a506c34ab68648fb72b991
F20101207_AABSFY matos_j_Page_168.tif
dcad431583dc03b5df7c38731b2bb1ba
ddff7631244295ac3d33c625ac33837912217796
10599 F20101207_AABSGN matos_j_Page_010.pro
38a5f3a646c1ccd02d68a26c9e6479f7
c0c03a13daad80ab12a2d6beee2bd9b2b24edc09
F20101207_AABSFZ matos_j_Page_169.tif
f172314c4bdfcd2c7fdec57e7272cd5d
1dcb670cdda7f1425a2a0cd09043f0060532178e
56238 F20101207_AABSHC matos_j_Page_031.pro
66c28c75b864165c0f99065f3adb54b2
a781d8626a8b1fb29bba3dfc7ea51b6d6875dda8
49713 F20101207_AABSGO matos_j_Page_011.pro
df961b5d0f37c28bfeeb3304550426b8
309afd0664642c36b241b13f3ac857b977f55ded
50482 F20101207_AABSHD matos_j_Page_032.pro
a66a78d1532f3540636308b57b576774
97e0e43a6975ce1371f69168898698832524581d
7232 F20101207_AABSGP matos_j_Page_012.pro
6a94fe69ae2c2d82f5bf02f3ce52f456
4e12173aa7baecd625a4e89fad5452ead16af94c
52832 F20101207_AABSHE matos_j_Page_033.pro
9f54c054795db6bf2035bf23fd80444d
d36936abb0479b78ed605cd5bd806cea43f95b0b
61655 F20101207_AABSGQ matos_j_Page_013.pro
48a3b2fc3db605e2ed02705faa46a3be
42b8274d752e38304368e5b4060ece51cf7c4b2d
52685 F20101207_AABSHF matos_j_Page_034.pro
1baae44a7e43792732a84c7baea463db
bb32d2d37a00e36084bca0a1e487c1b5c4bc9355
54969 F20101207_AABSGR matos_j_Page_018.pro
89cffd12e35795f42479d48b085d35a4
2d3310da51cc0fdca7f31035844711895c7d9a89
49134 F20101207_AABSHG matos_j_Page_035.pro
b7da38ad6b4ff5baec872f9e4f0635de
3f9092cf2a6cffec3905903fe404240ad552bb21
56172 F20101207_AABSGS matos_j_Page_019.pro
1ae5539f35ad5a86b3a04ac2d1971fe1
c2c8d08e7ef144bb85325b592e1d22db37aba939
55969 F20101207_AABSGT matos_j_Page_022.pro
378a8523c59abd7172f74bd84ed88006
8120f1f7c33f104adaf4d1671ef54f9575be1142
53557 F20101207_AABSHH matos_j_Page_037.pro
1a3fb99b2b8e15318a19f0f922f392db
7b9a1a4215aa642b50fbe3b4fbc1d89af688c2b5
56351 F20101207_AABSGU matos_j_Page_023.pro
1750ec98e8646b8bad0cb838d28c39dd
38a8d696e05a8941a353adb4fac88429f7a018f0
53058 F20101207_AABSHI matos_j_Page_038.pro
4f9bd75e386e0ed91b31138ab9edca02
ed9e1bfb995cc9a8fe0bbd80bff0ae644975a3ca
56779 F20101207_AABSGV matos_j_Page_024.pro
2dfb97a23f0d5fec18828bfea0201b06
a23c1c88dd202f6674175f3dcb7cb9232850eae3
55068 F20101207_AABSHJ matos_j_Page_039.pro
085a222ee233f3c630cd557db0619942
4bf0ce6a8782ae332cac7a466bc208750bdf1476
53127 F20101207_AABSGW matos_j_Page_025.pro
fbc227e8f1ffa216658a249609fb5015
27e659af0ec9ebab22c72270f7c2c702022bb5fa
50795 F20101207_AABSHK matos_j_Page_040.pro
a6482000e4a9097a228a7e4c2c395e03
3f836d6b8a259dccbfc5eaedcd043291d7575aa2
54220 F20101207_AABSGX matos_j_Page_026.pro
d8305a4f21d595b48ec6041a6c798918
8c8f3ef9f61510dd66b0fc1351d80375d1fffdf5
12597 F20101207_AABSIA matos_j_Page_059.pro
f720203100fd40355308e0fe40226035
8345e170eaedf81a80001c25f6be68d45a00818c
50930 F20101207_AABSHL matos_j_Page_041.pro
684758e3d31874adcd6b1b011a8958d8
a2c581b4759183fb0bd7aa2f36e9e746032f5e24
54485 F20101207_AABSGY matos_j_Page_027.pro
d2de68ece944350c985b1a384b17b8f5
ed0571b2d33e64b89dc70cfa5e0c95aea3a4cc5e
22996 F20101207_AABSIB matos_j_Page_060.pro
c60f468ac12a8df126e055ddc350883b
c15853436af6f7dbe46f1cad186e44379201c142
58126 F20101207_AABSHM matos_j_Page_042.pro
14ef53deb4797da71bc78a93a16b054e
9c8a47d8dc361c06f69f309f801d291d8ffe1606
56490 F20101207_AABSGZ matos_j_Page_028.pro
ce112bd040ed9ec34d7d690efa162710
8002f8945e6ba4f30c09c827d850e0ae24fb8ed9
52577 F20101207_AABSIC matos_j_Page_061.pro
91b0bfea3cf4f5a03bb161f78e40a43e
efca631058e348930be381e5e27edb3bf7d056d6
53231 F20101207_AABSHN matos_j_Page_043.pro
8696507b14d2da52c96bd696c8c005fe
cfd8eb629743377c42c7da96d346441ee075f544
55274 F20101207_AABSHO matos_j_Page_044.pro
e66ae78a2673161993007dbfb99d8d26
717cd01fef84d86f660d25fcea1f1d06fa57253c
47934 F20101207_AABSID matos_j_Page_063.pro
814446965980e1f0e6309676c52cfc0e
44f446455a93136abb43a79e75eafe3051176957
56044 F20101207_AABSHP matos_j_Page_045.pro
d9f736091eb2544bb610d237ecebc35c
e287b4dd1c4ee7b44dfa7606202130e9c6c32ebf
52375 F20101207_AABSIE matos_j_Page_064.pro
27bdd3c1381a02578297ac16aec0ced9
f5ca2e44e13187fc902f127bd1dcdf027b5e9954
56305 F20101207_AABSHQ matos_j_Page_046.pro
1129ad44d61353ac19cead06a81c3cf0
a5a3e144a4e886ea168684e9833a4a8618dbc4ac
53278 F20101207_AABSIF matos_j_Page_065.pro
8d527427703815b517820550b56b809e
18cef1e4dc4fc906bc76a43f7e6a4ca24175e89c
56847 F20101207_AABSHR matos_j_Page_047.pro
f174999f075d0b51919ece71763486c5
28342fc5a4459d4785712aa58fafc4ba28118fe3
51721 F20101207_AABSIG matos_j_Page_066.pro
d667ffc10a39db21579a16a2c9d8fd4d
91fff6a40e0e2d10358b303b734f865763e5bc7e
57048 F20101207_AABSHS matos_j_Page_048.pro
1bfdcdbbaf1d94b0b7b7531c12fc0096
65536671ed4ac53b2b48c7673317eccedb4c8fb4
46709 F20101207_AABSIH matos_j_Page_067.pro
49496fb62d88c3c28766561e1dc48d50
258dc05b3d0c73a3525ff0dcdc1e62bb18efe062
55809 F20101207_AABSHT matos_j_Page_049.pro
df799069a0255a5749ce253a8b52e7de
4b35e9c0dafce612e77203df60030f8d6daa2246
57545 F20101207_AABSII matos_j_Page_068.pro
8db1934ce98981c1afc7cb07b2a3274f
30a33156021fab22d01b22bb3d0a1c3f5f665b89
26021 F20101207_AABSHU matos_j_Page_050.pro
82d502eceda535d93b7b4c580e5ad01f
398bb5fb1e230c9c729ab85870cd0a65480c8e44
52272 F20101207_AABSIJ matos_j_Page_069.pro
cfc0e938835e060174557332210f2179
3e2a60d4cafe5e7e9da7bfe1a2d8b7513dbda9b0
36663 F20101207_AABSHV matos_j_Page_051.pro
9eade0d5922b439ff7d03b807274eb81
e70db467313514bf83461aac333822ad100527b9
50707 F20101207_AABSIK matos_j_Page_071.pro
63730185b493edda2b38e08f8a2e50a0
25eabb46bc1262621b4f9974897aa3dc9942e917
23181 F20101207_AABSHW matos_j_Page_053.pro
8a16baee28dac4b1907ad6df2bc2fdc7
188128fb3f770fe4f82155128d5ff4b3be4745d0
54628 F20101207_AABSJA matos_j_Page_089.pro
74bec7ba4b616fa2923c84a1fe6d14a2
c145eb4fa7881a3acd8def6449d19acdccefe63d
54364 F20101207_AABSIL matos_j_Page_072.pro
bb871b0a8fd41a1dd7289186ce5e022a
ed2215111bc3610b1b5b964770aade40791c86b6
19035 F20101207_AABSHX matos_j_Page_054.pro
3fe660191bc657457013fef4dfe305d5
e07ccb69362d01ec592c991be4ac03a3323824f9
54284 F20101207_AABSJB matos_j_Page_091.pro
45b758263e28b0532476660407c5768b
2cb808c256c22758bc7a13e81dae64ecd4633397
53057 F20101207_AABSIM matos_j_Page_073.pro
1f94c3c8ae65ad7457afd7db79aa2321
5a0ef191940e04fab020f819ad29d6d3ad4506ab
27741 F20101207_AABSHY matos_j_Page_056.pro
95462014f033c0966637136226f6f3e5
8427e512b0798f6b1ffe6cb66b6e94fcfd95cde5
53329 F20101207_AABSJC matos_j_Page_092.pro
849f53c2dfe61c73cf82e21bd1862fb2
85c93fd03d58c0a75f3f5279613ec15ac2eb97f7
51385 F20101207_AABSIN matos_j_Page_074.pro
3e18ae1926e163fac7ec7748bc30860e
675e2e4d16d3124a9b55834532fa426d81a6ba59
13662 F20101207_AABSHZ matos_j_Page_057.pro
37418d917b794ce0f908300565bd6da8
2436f5dfd33fe614489e85f4760cf931e0b517c7
53993 F20101207_AABSJD matos_j_Page_093.pro
137896ff72a7768c98ea7b939a7f5331
71dee21c134db57188598d4fc7a8413ebbc5754f
53036 F20101207_AABSIO matos_j_Page_075.pro
7a7637447fee7e399ae44af2a0f95a79
7c46a25ebdbb9771218586be8c7ec72d7f5bdaf0
54140 F20101207_AABSIP matos_j_Page_076.pro
effc090a6f5d960b300065a12ac59309
5fbca25ac6c9d44e5c05be7f776039a6cfd6ae13
56178 F20101207_AABSJE matos_j_Page_094.pro
d194f0fe0c37515090e53c1fd059231f
a8d18b539f847c2068df1b73f2ff1d3ef1a06420
50742 F20101207_AABSIQ matos_j_Page_077.pro
3b1963aae0a7d8ab763ff72323389fb3
572c3101f238361650ff1a0b538942ba48a4db72
56181 F20101207_AABSJF matos_j_Page_095.pro
98baf7911083f062f2974b6df6fa7778
fad96a63e32dc57e56d75e8dd645a832fc731eb3
50042 F20101207_AABSIR matos_j_Page_078.pro
2bedfa481af32fdea6547e33f3c1ecd4
587de6041d04a22c49123b9fa81a09f5b8d9cd92
55502 F20101207_AABSJG matos_j_Page_096.pro
9a757df820b5b154f0866dc9335a519e
1d5efcf6b970a6760af4a2f7b94b502c6e165567
53710 F20101207_AABSIS matos_j_Page_080.pro
610201076bb67c0355432794fa2a592c
8f4b2f7d7df4fe34a82702ceade8c3f09903e84a
55913 F20101207_AABSJH matos_j_Page_097.pro
f47c3840729bf5c0d7386a78fca8cd05
861c5e808e0d129ccb20d2809970f388c395cf93
57300 F20101207_AABSIT matos_j_Page_081.pro
b974be1e2ef4587a7fceed9c97e9a9f8
1b5aa5d175b4420823365065e672d65527a64f9a
54028 F20101207_AABSJI matos_j_Page_098.pro
df5283cc2357d1fc6247dccdf23583a7
23a1552bdcdd29c0025b6d354797a09ad2b1954c
52881 F20101207_AABSIU matos_j_Page_082.pro
f8ce06d09be0d0c62fac29beb87317fe
08189612aa13c77768e013fa94e95b408ed41d3e
48553 F20101207_AABSJJ matos_j_Page_099.pro
26576652a531ca246491600f9acb6cbe
83ee3ff7b34bce553b0d90b3503aa07cd2b9cad4
53130 F20101207_AABSIV matos_j_Page_083.pro
f2594217331cd881e55a6a824b58a0d6
6e2f6f3080d4480d9ceb21937f0a1ce6db89785d
10933 F20101207_AABSJK matos_j_Page_100.pro
152e3c696dca250d66869a7e7a4dfe9d
6dc508af14a2f13793b04e3c086feb7ca9656b2e
53723 F20101207_AABSIW matos_j_Page_084.pro
38a99037b94b418d9c2f68f65ca92ac7
5ab988eac1317d7b313d22f80d7e8e2fa971cb26
12683 F20101207_AABSJL matos_j_Page_101.pro
b3fec8b6df7a65de75503a015bdd64b7
abbbc16a85aee979b470b8a1b4aa75c8a314fbc6
55759 F20101207_AABSIX matos_j_Page_085.pro
b96ce9deac7b2d519a889eaab271292c
e3368d229162bbd41ed8b73320d203806011b56d
49287 F20101207_AABSKA matos_j_Page_118.pro
321b5dc2ad2db7f9d6db2fc5d3adfcde
d68acf7cc9c5e56bcbad333c9f63cec8c079eb37
11214 F20101207_AABSJM matos_j_Page_102.pro
2f225bf319eeedb0ea7a8939347a2d2c
071804a0284982f62234d24e43da769ceb62bdbb
47741 F20101207_AABSIY matos_j_Page_086.pro
c322c94437388e6e0051c3ff94dfa22f
a843c6f5a3dddb48bdb87dcb6ae308324ee23707
50981 F20101207_AABSKB matos_j_Page_119.pro
302b2ff84342e6df1f3da8ba79c5de24
ded7b375b814144fffdbefbdf4d04975ad19b7f2
38104 F20101207_AABSJN matos_j_Page_103.pro
76835b5009febf9ab860980b724d275b
42936b3c90137ffb27d7b78aff86d5dc904ce958
56094 F20101207_AABSIZ matos_j_Page_088.pro
47eaed790b3bef6717829f1a3801682c
7f1e4e620c5987603aca1da2d7aee948ca0276f7
54162 F20101207_AABSKC matos_j_Page_120.pro
eed3ea08180c7375e0d8ef4b25304a14
1044743e962bd9fd081ea9e3e2ab402fb7d1fae5
33926 F20101207_AABSJO matos_j_Page_104.pro
eeec0994911b9e113dfb009ae165f8c7
05efc7ed9079abcdbf6c4072b4afc6b679224973
52879 F20101207_AABSKD matos_j_Page_121.pro
2f656b817ca048106174df37c4e42662
caa56ab81178585d03f8ba0061c70e0236b73609
33612 F20101207_AABSJP matos_j_Page_105.pro
2ac1cbc1d5d3efc29f99695c4c019e9e
c0da1be815561e9d6872109727746509f1675f93
52896 F20101207_AABSKE matos_j_Page_122.pro
e3abf9364868044d0010a8a20d7ce9c1
82cb5081da44e2b4aa4e86f1e3d69bc77ff292aa
19042 F20101207_AABSJQ matos_j_Page_106.pro
c647c717cabb0c363252a0ba2d010150
c2ddf24007ce4d55b241d01ee85057f76577cf75
14030 F20101207_AABSJR matos_j_Page_107.pro
b1fb5a0247eec80ffa334b091ca2770b
50f80063f377c9693a45346459be1f5d47ef637e
53153 F20101207_AABSKF matos_j_Page_123.pro
7bf7ee13b2fff307951d56092cc4dfbb
f989decc3b51bfd91cb0f34f4c093028c8820ff4
39188 F20101207_AABSJS matos_j_Page_109.pro
f739df1adef1196d6a28c37e2d35230d
8bf684fc6e68cb1a54a86cb511871d7e1ccc06a4
52546 F20101207_AABSKG matos_j_Page_124.pro
cefec667879ece05ccd6a9305de1c59d
c3d578628e93d9e914430f9375a687e460675d06
38622 F20101207_AABSJT matos_j_Page_110.pro
79444f0d89a5054a946b650b3997130c
2204226cc18f789efa1ddfc7e81f134be334630a
49092 F20101207_AABSKH matos_j_Page_125.pro
e0951f8bd758d66af961ab07a91a2918
a8d5ae7aaa4f9f3b6f31e9f115bc924ae4fe2eb5
30879 F20101207_AABSJU matos_j_Page_111.pro
e5b5a6cbaefde2b87e81361daf9ae9df
ccc90043742c863a78634192f8dd214eec9520ec
56221 F20101207_AABSKI matos_j_Page_126.pro
7a4bcc5718925c6b143519621dfaf969
2e05941a5b66a90a931af150c61809c3345be7d9
20508 F20101207_AABSJV matos_j_Page_112.pro
b3b3c6a6dfe66778d3324b15632a0bfd
77e8e0eb0a7a8562de0598da22af5e7929a123ce
54091 F20101207_AABSKJ matos_j_Page_127.pro
f2dd25a138be310c862a5c9b1baf3aec
8fe8d09dd105029bb401e7e8bc58bafe57a20691
21315 F20101207_AABSJW matos_j_Page_113.pro
678a8378f6aeebcf5fedaa883b51facc
b5a17db90b9d00f0cf2abe068ee84fa1e7e6ebbb
53782 F20101207_AABSKK matos_j_Page_128.pro
b4e244bd146bd9cf8981579f7ee42f51
b96e64527c1fd1e226486b5fa0dd1d8035551c5d
19178 F20101207_AABSJX matos_j_Page_114.pro
905f0d90b136aa13490a7e238e50eea1
c0866d63488c6cc722aebd62b9538c71f395c1ad
55394 F20101207_AABSLA matos_j_Page_146.pro
cf51f1c204afe67a54801733d881baa6
5be96333f588e041b799a4e145a0aa3298569bef
53468 F20101207_AABSKL matos_j_Page_131.pro
7a56a7aa847c51434b258cd560494a93
a78d4030899da6d6fed24d56d1cbe162c8afcc05
14601 F20101207_AABSJY matos_j_Page_115.pro
ae8c759a7da4361928a898ec89b3f49d
daf6422dc3d2ecb4e84fdd3be71b277f5a347829
51454 F20101207_AABSLB matos_j_Page_148.pro
868caa217d599ba9912f3de8378e247d
706565c9f2d6e0fbc8f20e9889813dfcce11d7e3
53500 F20101207_AABSKM matos_j_Page_132.pro
f8609673e70f4af8ba3bb5774404046d
47c4a44c8c561ed872a8e03ac48952aba2be5dae
53624 F20101207_AABSJZ matos_j_Page_116.pro
b32c49e38b0a8da9d0d202fe8332f088
b4ca5f7da87aefac43cbb31666cdc4104de226eb
13903 F20101207_AABRIA matos_j_Page_108.pro
33b13fb0dbffced718fb6b6d289b5d49
9aa203f128098fc8177224170256023f797bc2ca
54432 F20101207_AABSLC matos_j_Page_149.pro
4f7d2d04cd7018d3720c63255c079b38
89633a2fa149c0bf7a24575f34d29639550e7fc5
F20101207_AABRHL matos_j_Page_135.tif
0f63c6b5109c45475be325e8306f6cbb
4ebe709908eab4f911bb3af00ef2de0323e2e73b
55025 F20101207_AABSKN matos_j_Page_133.pro
729827d95cc7a7bae98529d15587f0ff
45785cdf3c94ff3794662c096f1da917a0e347e2
29730 F20101207_AABRIB matos_j_Page_048.QC.jpg
829f4f9be4d834e3a5fbf60555864919
5a529677b44042961227ff7fcbb041035d284f3b
54652 F20101207_AABSLD matos_j_Page_150.pro
91adfaad53d78d9d1e2c2b2b9831bb30
af72be2b01921fc14d155761a74dad719dfd22e6
6532 F20101207_AABRHM matos_j_Page_024thm.jpg
944e3e60343fcdafedb25f71836d4b9e
dad83e2e290934a7d81f0d89375d7697fb0976b9
14583 F20101207_AABSKO matos_j_Page_134.pro
1e455910f40e8a47dfca11b94bc26bba
1a7e37065c371c9af9f733604214f800ad1f5682
91636 F20101207_AABRIC matos_j_Page_022.jpg
c34dfd94a84784f456795258bbbc01bc
8f4f9e494edbc77582f864f54bef321dd4348481
40244 F20101207_AABSLE matos_j_Page_151.pro
b31459bafa82ab4e721eea26953799e6
fac14694651ad1553ccf788ec38f9615d45b37da
110513 F20101207_AABRHN matos_j_Page_041.jp2
513800df2bcaa54d57f17cc6eba7e229
6a037d454feb1ff4a4f379e160ef3046f8165dd0
37801 F20101207_AABSKP matos_j_Page_135.pro
ab42de622fa325699c04ec5a90ba7829
cd8c417719c47ec88e3039f7300a085cc98bb6bf
F20101207_AABRID matos_j_Page_076thm.jpg
37899ecef4df833db57a4961f19e20b2
adc38683b7986a312dca84cc2d3420e496ba68c3
20150 F20101207_AABSLF matos_j_Page_152.pro
25b09cb8e1fb11e01bf5e6c2cf15870c
a332b1e5af3dfecd606bfa0c5848d9c034a2c3e2
54530 F20101207_AABRHO matos_j_Page_079.pro
ff7964cc9eeb5b05837c554325e4900a
a2fd07d8a1747d36112efb41def22f0d509b1712
12192 F20101207_AABSKQ matos_j_Page_136.pro
55b565da7bfe2ba88dc0537e92fda00a
644994b2415714b201c28019a1b1c1876262e862
142123 F20101207_AABRHP matos_j_Page_162.jp2
e179dc44e8767f74248664157441a6c7
71fb64a82ee605c53b12242274b2e76796278a58
14335 F20101207_AABSKR matos_j_Page_137.pro
93a1d39251a2aa3e21db1dab1c329c16
b833c45c0dc016b6f877c09701b4868a4b623f07
121415 F20101207_AABRIE matos_j_Page_085.jp2
eff8e957f29678fba83c32c9c5c790c0
acf4db6a488a42114af21396464d5f970b7a06d9
18288 F20101207_AABSLG matos_j_Page_153.pro
9f7f202671f75b5b4db09dd47c9c56fa
4ec63f5f914f124d7ef30c47dc773dd4965f6adc
6969 F20101207_AABRHQ matos_j_Page_171thm.jpg
5a336bf5344daa2547eb5c50b33546a0
c2f5cc2f8d5f8281872d5dd86834cc25cd6d9a1b
19332 F20101207_AABSKS matos_j_Page_138.pro
b804f68541294cf7608849c617f21c10
71ff125bf6b605a57a4c3d6939e144fb114569f9
27083 F20101207_AABRIF matos_j_Page_092.QC.jpg
d802d1b3b9b979807f7be0fcfdf9421e
8ddfc90398f56df8dabd2e0531791baabb98e8c2
67576 F20101207_AABSLH matos_j_Page_154.pro
9afee708b30f34b395bcb99da4b4d582
7d99c01e1f11a1c818a933d69140cac1414377ee
121053 F20101207_AABRHR matos_j_Page_088.jp2
9dd74e677878bc3c4a3e8188456fc2dd
98964722d5b0d9133fc1095e7d2ff451ec843654
17961 F20101207_AABSKT matos_j_Page_139.pro
6babf8358509bfece43a5ee2531d5884
a13f01c64410d18690a1930f1f578cbd564e18db
95466 F20101207_AABRIG matos_j_Page_005.jp2
8e86d8b79ae6b670575b5a52fc004ed9
be0690b284f097af9f0aa0fe96401f941fd72536
68728 F20101207_AABSLI matos_j_Page_155.pro
c5b119383449c4f7e5c66d550644ec1b
ec436f868b6f85a0b6bf1008181749285c3da10b
53635 F20101207_AABRHS matos_j_Page_016.pro
0919caa8d789821d007f7502046748c0
81b52abcd9dee4bb1a1b4949c122cba1bfd4071b
34176 F20101207_AABSKU matos_j_Page_140.pro
d50161617d5c8051e634a179afd12bc1
0c0e40b8ae2919cbf00b1c35fe4fb8e28f90cd2b
6567 F20101207_AABRIH matos_j_Page_170thm.jpg
dea06f21f08483f9e750b36e1903cdfb
47cc41a492cd5169a0cee2cd632c1d3f73fcff25
61557 F20101207_AABSLJ matos_j_Page_156.pro
17f4ca51e4cbac15420bfd25dde281a4
35ed083c4970425d4d0f5d3b833a8300718d45d3
F20101207_AABRHT matos_j_Page_076.tif
27acf9c4d9f9a3e5a96a188e25069ad8
5353f8aa1d88da24e8553d9cd6e26802f70ee83f
24911 F20101207_AABSKV matos_j_Page_141.pro
116cc0d17b50ddacf8ec2400332f1364
1a82cc2c73de2cb6b94a313b7ebde72700656612
54043 F20101207_AABRII matos_j_Page_087.pro
47f1a4473f7a610fde79c8d9963cc0d8
9229ff8a33c4baf3edea259cf1dcc3c1e3f38e81
63757 F20101207_AABSLK matos_j_Page_157.pro
3131691bdb17329913431fb84245be40
f561e488d6b3abc7599e90d2513c7067294e1fff
2464 F20101207_AABRHU matos_j_Page_058thm.jpg
53d8160db1cab7509e338f7f1ef489eb
fef354ff193ddf59c70b5efe59be2450f20cc2ca
53225 F20101207_AABSKW matos_j_Page_142.pro
7cf54e024488b06f8679293c7560edcc
57749ded8d2d6c3ff68547dbcba93b89e3050ca9
54378 F20101207_AABSMA matos_j_Page_174.pro
23e286d99e028857f73cc8f86fbb0928
5447210e8155d456b62169a1ac05212348e1f897
6735 F20101207_AABRIJ matos_j_Page_144thm.jpg
ef659e332bd54c1e89f3923053b65d6b
083cb708fe1636d7e55c470ca9f4fa9e51f2cba5
62777 F20101207_AABSLL matos_j_Page_158.pro
55a223a0198f121b9553084dc2b2375b
3f34873a888f0443e73db33f4734d1ae0522b757
91017 F20101207_AABRHV matos_j_Page_097.jpg
b1ce5ce6ffdf74d4bb9fa609707582ef
63cd8373e28a0af9405d796a2c2f4d2cb4b27790
58781 F20101207_AABSKX matos_j_Page_143.pro
209eb67bd8d04cba42c374df5bdfdd5c
a7edd4ddbcdce8d0dabbd55a201dfb546627cfe2
5490 F20101207_AABSMB matos_j_Page_175.pro
a71afc95cdf21c167bce8895057d6571
e5934a81dfc5bb3ec27b8d66089ef9c870896704
2239 F20101207_AABRIK matos_j_Page_048.txt
7a4d4b0ceb4d64af1e612ce4637f9206
2fcf2d8845c5d7012f5cdc473c8b1e44874d630b
68752 F20101207_AABSLM matos_j_Page_159.pro
e31628c30b6d2c669b5378409bec8a6a
07b3d90e2fa0229f111224a90292859038668677
56430 F20101207_AABRHW matos_j_Page_090.pro
3032333ff102ab860f626eec08a1a8a3
727d20ceaaebb7dd08bdd6b69d447064c68b9db8
58464 F20101207_AABSKY matos_j_Page_144.pro
c53c8d915098e1c6e2a115b766332f1a
f66e19a0280613c61c376dd74fed1100b91ac05d
481 F20101207_AABSMC matos_j_Page_001.txt
e45eded49ca1e0c016559bfddfadaf69
3f99f041eb36b5a5a5fa7a1a46ea55c4687bce66
4054 F20101207_AABRIL matos_j_Page_109thm.jpg
a1e925757b40c77de0b561f4114bb707
d683d7b55fac7d91df6a4649e16ef8496b5b69bf
64410 F20101207_AABSLN matos_j_Page_160.pro
e5d3c83ae82e4ed51de3dd8946f14f0f
4e3ccb3d752fe9df3cb66042dc8cc4ffeee04c9e
2054 F20101207_AABRHX matos_j_Page_069.txt
a849368a92fc7ff1c290165e141743ca
a29eb01e6ac25ff3c5f8f3ef2c57a48013dfd30d
57401 F20101207_AABSKZ matos_j_Page_145.pro
255c6003f6b8e47bde52ae5315aa3992
6199398cbc976b4daac0ef39a2d39f0cdf437149
12455 F20101207_AABRJA matos_j_Page_115.QC.jpg
c67bf7fd649a146f05d8eb046e483729
45ed5f7fa2df20490b85cddb8675aa795fd79288
97 F20101207_AABSMD matos_j_Page_002.txt
d6996beb1bde9973ae449b712e19df38
c350640d1c5f093d4fceae96a770eae3667e8501
14060 F20101207_AABRIM matos_j_Page_107.QC.jpg
630dc8fcf34069f8a9265f13a6a69370
b870e9681e7cc88b0044a96d5fe4137d1dc26b90
67882 F20101207_AABSLO matos_j_Page_161.pro
27e6e6ace4769474747a253020912564
c15b5e40b4209adeb6ae6f5f736c81e887c4f976
2552 F20101207_AABRHY matos_j_Page_172.txt
fb34ed71da614b6ca89f62cba23b46a3
4d4b7cee62169062f89af6596989853eee81d4df
F20101207_AABRJB matos_j_Page_017.tif
c4f5a05eccb933c14feee312982da0f8
edd1180778d582df851c8ffe76bc6a869fb84c44
305 F20101207_AABSME matos_j_Page_003.txt
365ef80c100a3123a16cb9a5e920b55a
e6bb97badce9ed9ba0cdbbca1e91373da08f93d1
6726 F20101207_AABRIN matos_j_Page_015thm.jpg
d01f1ae7cd5ef209dd60dfaa8819a90d
b27ad727bec5834c01c95d8721947a2c5716c84c
69717 F20101207_AABSLP matos_j_Page_162.pro
1c1f69593172d7536f3756ed86c9295c
8e9a2205037e10db431cd80c4c99c648f088dd6e
6835 F20101207_AABRHZ matos_j_Page_157thm.jpg
efb6701143b8c87b9d85ecf5edd0f03c
babbea22cdeac99cb30035312b971093feec94f1
56199 F20101207_AABRJC matos_j_Page_021.pro
316ccfda137ad44825d7ded183c68670
d5cc356405f098fb9860e25b48ff818f939f57f2
2194 F20101207_AABSMF matos_j_Page_004.txt
eec0d3e18bf688fa760c02b5c726ee8d
f77affa7fc3feb4d1373763dbbaa737183bd5131
11859 F20101207_AABRIO matos_j_Page_147.jpg
6663720a04a19b32c99594e71d8793da
6da8d77ecc2a7243c0ec13b596c54184328cc384
62866 F20101207_AABSLQ matos_j_Page_163.pro
2a5f24750763977dcbf153d30dd4af6d
94acc3b6fb0dcbf4a7b0d3e9332c8ad060eeb85f
29727 F20101207_AABRJD matos_j_Page_126.QC.jpg
73c9a994d18fe8bac9da3cf538e7920a
383cad52bd4065c4f3f281219a1bdbf07edec31e
1699 F20101207_AABSMG matos_j_Page_005.txt
1714621cbc3c05529e1da88f967c900f
ccd11ab032aff6708e13bc59fd46a427983f5ab2
66774 F20101207_AABSLR matos_j_Page_164.pro
8f4becb04387b8f0e27726071cbc27ce
520899ab33c349f3046334ac5c49972e2f9144c2
15498 F20101207_AABRIP matos_j_Page_175.jp2
d87391eb32ae8df683e9e5b1ffeb5139
48c1416fffc0a185ccca9aad36aeefa77a1f7342
30447 F20101207_AABRJE matos_j_Page_164.QC.jpg
a224168d5f830f5557fe43c36cd4084d
1709f138533b9ba2e29e8be49d1e426668747d11
69010 F20101207_AABSLS matos_j_Page_166.pro
105006a348f212ef39cedb4560e237b1
e69d6718273a08c49d2f40186926a5d7bb44df06
F20101207_AABRIQ matos_j_Page_006.jp2
3711dc7f74b6fdfeca1129bf6b72641c
2afb97e35097c2bbfd26c57222c57c15337d4f94
4009 F20101207_AABSMH matos_j_Page_006.txt
62e65c10a8048590b791a04b5807b56f
0c13608e796dd118e88867b6b0564f990d00f150
71311 F20101207_AABSLT matos_j_Page_167.pro
cb562b057bc9cabaf06c7bb8cde8b5c2
237ec414e5042d001047f3078dca6b9625e40b33
55942 F20101207_AABRIR matos_j_Page_015.pro
6cc15d88f331502084b66a9a7d9c62b0
7e29ad43b6609948e3806e06a888510ba822c786
90406 F20101207_AABRJF matos_j_Page_129.jpg
040c78b2703e3f52285f8ba85c52daea
df0cff8891e290f95af18e0eb8518e7ff663b20d
3622 F20101207_AABSMI matos_j_Page_007.txt
b3b7757c46c95767d72dfcced1a2bb08
952add318021ce729818fa3cf4b844616936ba50
68378 F20101207_AABSLU matos_j_Page_168.pro
3c6ea7aaf269b9e8b70c3d06c1a5ffb0
17b6fc0161d92e0d4748161fb98ddfdc3ee94873
2535 F20101207_AABRIS matos_j_Page_163.txt
6ddef3a4430da0bb2da5ae285339adbb
c4f6b467fddf42a0d98089c342bd4cf384784a8b
F20101207_AABRJG matos_j_Page_013.tif
9b3c96feeddd27e75ebc3c45beb77623
f7fcb82ae2b3f5561a7085eaf2ddacbe170f64c7
498 F20101207_AABSMJ matos_j_Page_008.txt
67d03a2bb4e5c1590a379ebf2d7238fb
cf26b74920256ee5afa730599a335a2c1541b3bd
66059 F20101207_AABSLV matos_j_Page_169.pro
cac5ebc8b313651e99c131e3c6610a70
3e1edf109133f160f09721514b6e77358920f44e
47878 F20101207_AABRIT matos_j_Page_070.pro
aea588960b2a72a139f5d8edf6fdb9e9
b7c2a887272f0b0ac319e707c7b0d914c40933a7
10626 F20101207_AABRJH matos_j_Page_054.QC.jpg
ba837bc0a9e1f0bdec40e6a410a6aaff
a0cb2825d69d5244e1c5c689a9b7c30771bdc6a4
2895 F20101207_AABSMK matos_j_Page_009.txt
afd490e48f2f0a912046ed74affbb8fb
f0615df265a752444686b6029e6051248341c0cb
58852 F20101207_AABSLW matos_j_Page_170.pro
41c6f87a31074d26e6962b9db9b400ab
61656feaae44b6896f62492652f9c74c82a1846e
94583 F20101207_AABRIU matos_j_Page_130.jpg
bbeb501e30ca5317953b2781a7c128d8
4930ff49a2d3a83156aa662b90bbeca29c6c8b6a
F20101207_AABRJI matos_j_Page_129.tif
bbd8d1315679578d577ac847c506b10c
b8925d6566549087e709e2d4e8e2460ee9a4e157
2129 F20101207_AABSNA matos_j_Page_026.txt
0b405955d7c1c31d131c18a6f787b694
9e00e266f45ca6caf191a39932a6448e5491d96d
434 F20101207_AABSML matos_j_Page_010.txt
1e9658a140f06d9a52043d790ab8e9b3
e4eb621d99501c7ee342c5e052e88ccf2ca841dc
68927 F20101207_AABSLX matos_j_Page_171.pro
89fb1e0ce336b2ecd5bb0512b5c98fd0
eef9161f364ab80e359b84a3e8a9d9d4b378aed6
6565 F20101207_AABRIV matos_j_Page_082thm.jpg
f1ef69c548ea92263d68f417bca84c0d
b27f2de0941f8a81892ceefc2f53f27faa47f1fe
5308 F20101207_AABRJJ matos_j_Page_147.pro
724e44a40f78117a2bb501f97c6ae8dc
5f33ba67034f87f64651f774b073ff0048cfa162
2229 F20101207_AABSNB matos_j_Page_027.txt
dd760d8c255684b017d01a4e79aebb24
80041c4c6a6aad580d27630a903789aee5bc2f08
2167 F20101207_AABSMM matos_j_Page_011.txt
b8f9059b601fde83fdd6447c6831980f
62ea5dee9bc9e4c7bbf66752cca32ef35ff80be6
63350 F20101207_AABSLY matos_j_Page_172.pro
a6b75706cdce7edbb52dbfccfff397b4
72a68e1a9bdc107953490ea9e8e74fea814956d0
F20101207_AABRIW matos_j_Page_111.tif
65cc79b5fba8b91de7342fb1fbd55b4c
ac4044a55462174910b88541256902c4f267bf1a
13532 F20101207_AABRJK matos_j_Page_057.QC.jpg
bbaf7365dd4da84e8d6ada976a64ec04
d66d55fba4c4cf23ef76f0ec8499fb0db061d928
2210 F20101207_AABSNC matos_j_Page_028.txt
bc68a00c74240ebaa92d9d4bfa79978d
bd8ae1b5707c7bccab4030719402cd6054c68c69
2529 F20101207_AABSMN matos_j_Page_013.txt
fdc0befa0052eb328baab866d10325fd
c348fcbdb7514995de6d2642a174aa91e2983ddd
34223 F20101207_AABSLZ matos_j_Page_173.pro
3500f918e5aae3b229e6b386a671311f
3b7cacb8b8d20ee838133aafed842be67c916304
F20101207_AABRIX matos_j_Page_131.tif
853fb203f24fd7cad709ce019c8dad7f
0b85e01bdac5d3feb5c6b3dc954845a88b26c6dd
118557 F20101207_AABRKA matos_j_Page_129.jp2
211d1cc767ea37224cf5ea2c200269d8
fbcbbae7eaabc935787579a1629fb53d9b9a0c6a
6797 F20101207_AABRJL matos_j_Page_095thm.jpg
57430ece2ab57964c70f700b202c5b29
a20fcf94ae932d8c8670594152b6df5e3647886d
2110 F20101207_AABSND matos_j_Page_030.txt
b1a44f00b956ad5fb4021d0e7f55818b
17306f4854855dbfb4a01926e529bd4929ec8822
2125 F20101207_AABSMO matos_j_Page_014.txt
b1b6529f7f875110de9afa8e8ecb2540
f455c64af98a083ffa378aebb1d92b2a32893b3e
F20101207_AABRIY matos_j_Page_163.tif
c02de84f3e9a341165b35cc6fbe57096
5ae944c5b77152f28b6e9427b5f198cac488e559
125578 F20101207_AABRKB matos_j_Page_042.jp2
8550bcbc43339fee5fb1d2462a002ef2
b3256a607e71913c0683dd927ee89ef1eb82b1fc
6693 F20101207_AABRJM matos_j_Page_130thm.jpg
cf6396cd5d2045953a27e72025bea0d4
d3a2bf59d0279dee99431e1f870febe15c50e4a1
F20101207_AABSNE matos_j_Page_031.txt
b9428c3bfcb6435eb65364a3d4a087d5
63210f30f213d4b51bc960f72b47b30f7b1b29e2
F20101207_AABSMP matos_j_Page_015.txt
74c18233473939fb4518e1de5c010d04
0982435dbe79bf5f12e8b7a6b8163da0f91aa43c
2726 F20101207_AABRIZ matos_j_Page_161.txt
263702509f5b45a86ac24c7100e6c737
4b982e609641794ed5cf0a9f3fc79817419e99bc
56634 F20101207_AABRKC matos_j_Page_017.pro
aa70d500c277e48b3245816b1b6b4a3f
7aeaf63793a82daf34bd1441ab7e1340ec29b337
F20101207_AABRJN matos_j_Page_107.tif
b882b500dca12f73f364a27071e107b1
25d9acd3227edf08f4cc892de4d62eb4c831ad4d
1985 F20101207_AABSNF matos_j_Page_032.txt
1cbd63f298e8f112ecad2a24d38cad45
40d0bb15b51968706e55194d9cd702bed0967f72
F20101207_AABSMQ matos_j_Page_016.txt
90a6e4bb2b6c3c5bfb7f9ecd3a43e4af
d7346e2b1aad62e4509ec7600ae75c74a8284174
83227 F20101207_AABRKD matos_j_Page_125.jpg
acfbeca467d8d3de8962d35ca8bfec18
58708a8aed95a79ca8094e9de42dbf8279c2d021
F20101207_AABRJO matos_j_Page_091.tif
baee5edefc0176319b4a0b6faef46c3b
7b81c6b4f5c993d06880f0f03c4b3e561e60656b
2075 F20101207_AABSNG matos_j_Page_033.txt
f024429d357393b63f32fc03f940595c
4e5bbc036f594bb51249eeaaa3a3680f50abea7b
2219 F20101207_AABSMR matos_j_Page_017.txt
e15d944e055e94548d2be9bcfdfbb2cc
71c5a775899aa934ec7586e766a406dd16c3f28b
56517 F20101207_AABRKE matos_j_Page_062.pro
1e8266d954840b3933173897d42d0843
054ee48f0bcdcdb253b5555f91137db052ddff4e
6787 F20101207_AABRJP matos_j_Page_163thm.jpg
5aacbe6795cc5f96c308119b3962f6c8
738cc3df4bba3b1f2f66007480219ab0d2e90b23



PAGE 1

1 MAGNITUDE OF THE OXIDATIVE STRESS RESPONSE INFLUENCES SPECIES DISTRIBUTIONS By JOANNA JOYNER MATOS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

PAGE 2

2 2007 Joanna Joyner Matos

PAGE 3

3 To my parents, David and Pamela Joyner, who so strongly encouraged the pursuit of higher education in their daughters that they will have th e joy of seeing both of us complete Ph.D.s this spring.

PAGE 4

4 ACKNOWLEDGMENTS I first thank my dissertation committee: Shirle y M. Baker, Lauren J. Chapman, Robert D. Holt, David Julian, and Christiaan Leeuwenbur gh. They are an amazingly diverse group of scientists and each one played a key role in the development and execution of the projects described herein. They all were good-naturedly tolerant of my “quick questions” and I am looking forward to continuing conversations with th em in the future. I wish to thank Lauren for her patience with me during my first foray into fiel d work. Lastly, I particularly wish to thank the chair of my dissertation committee, David Juli an. While some advisors seemingly limit their involvement to providing financ ial and logistical support, Dave engaged me in a five-year conversation that touched upon nu merous topics beyond those of the dissertation research, including leadership and managerial skills, depa rtmental politics, work/life balance issues, and many other aspects of an academic career. He has not just mentored me in how to conduct experiments and interpret results, but taught me how to be a scientist and a faculty member. I owe a great deal to a wonde rful group of undergraduate st udents: Jenessa Andrzejewski, Laura Briggs, Michaela Hogan, Jennifer Rivas, and Nicole Scheys. The bulk of the data presented in Chapter 3 was collected by these ta lented and industrious ladies. Working with these students (“The Mercenaria Group”) was an excellent l earning experience and I am indebted to them for their patience, diligence, and enthusiasm. I also must thank Craig A. Downs, former ly of EnVirtue Biotechnologies, Inc., and currently of Haereticus Envir onmental Laboratories, for his ge nerosity and expertise. Craig taught me how to measure stress pr otein expression levels, a technique that figures largely in my dissertation research. For almost a year I peppered him with ques tions and he answered all of them. Most importantly, Craig has been unbelievably generous with the an tibodies I used in all of my dissertation research as well as several si de projects.

PAGE 5

5 I would also like to thank th e following people for many forms of assistance: Drs. Ben Bolker and Craig Osenberg (m y unofficial committee members), Dr. Stephanie Wohlgemuth, Dr. Derk Bergquist, Dr. David Evan s, Andrea Martinez and the rest of my cohort, Benjamin Predmore, Michael McCoy, Nat S eavy, Pete Ryschkewitsch, Cathy Moore, Karen Pallone, and Vitrell Sherif. My research was funded by a Florida Sea Gran t Pilot Proposal grant, a national Sea Grant Industry Fellowship, a Sigma XI Grant-in-Aid of Research, and the Department of Zoology. This research also was supported by NSF IB N-0422139 (to David Julian), NSF IBN-0094393 (to Lauren Chapman), the Wildlife Conservation Soci ety (to Lauren Chapman), and start-up funds (to Christiaan Leeuwenburgh) from the Genomics and Biomarkers Core of The Institute on Aging, University of Florida. Permission to cond uct research in Uganda was acquired from the National Council for Science and Technology, the Office of the President, and Makerere University (Uganda). I thank Luis F. Matos and th e field assistants of the Kibale Fish Project, particularly Jovan, who provided in valuable assistance during the field work. I thank Cheryl M. Woodley (NOAA National Ocean Service, Center for Coastal Environmental Health and Biomolecular Research, Charleston, SC) for the loan of a liquid N2 dry shipper, and Robert H. Richmond, Kewalo Marine Laborat ory, University of Hawaii, for use of the Biomek robotic workstation. Finally, I thank my family, particularly my husband, Luis, for their never-ending support and love. I would not have made it this far without them!

PAGE 6

6 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES................................................................................................................ .........9 ABSTRACT....................................................................................................................... ............11 CHAPTER 1 INTRODUCTION................................................................................................................. .13 Abiotic Factors in Aquatic Habitats and Oxidative Damage..................................................15 Hypoxia........................................................................................................................ ...17 Hyperoxia...................................................................................................................... ..18 Hydrogen Sulfide.............................................................................................................19 Elevated Temperature......................................................................................................21 Salinity....................................................................................................................... ......22 Stress Proteins as Indicators of Organism Health...........................................................23 Overview of Dissertation Research........................................................................................24 2 INCREASED EXPRESSION OF STRESS PROTEINS IN THE SURF CLAM Donax variabilis FOLLOWING HYDROGEN SULFIDE EXPOSURE..........................................27 Introduction................................................................................................................... ..........27 Materials and Methods.......................................................................................................... .29 Exposure of Clams to Abiotic Stressors..........................................................................29 Analyses of Stress Protein Expression............................................................................31 Data Analysis and Statistics............................................................................................34 Results........................................................................................................................ .............35 Experimental Conditions.................................................................................................35 Survival Analysis.............................................................................................................3 5 Exposure to Normoxia.....................................................................................................36 Antioxidant Protein, Lipid Pe roxidation, and Oxidative Repair Enzyme Expression....36 Protein Rescue and/or Degradation.................................................................................39 Cytoskeletal Protein Content...........................................................................................41 Discussion..................................................................................................................... ..........41 Abiotic Factors are Linked to Free Radical Production..................................................42 Sources of Variance.........................................................................................................47 Physiological Responses to Stress Vary by Season........................................................48 Conclusions.................................................................................................................... .49

PAGE 7

7 3 PHYSIOLOGICAL RESPONSES OF Mercenaria mercenaria TO SINGLE AND MULTIPLE ABIOTIC FACTORS........................................................................................61 Introduction................................................................................................................... ..........61 Materials and Methods.......................................................................................................... .64 Laboratory Exposures......................................................................................................64 Schedule....................................................................................................................... ...66 Tissue Processing............................................................................................................67 Survival Analyses............................................................................................................67 Analyses....................................................................................................................... ...67 Results........................................................................................................................ .............71 Hypoxia Experiments......................................................................................................71 Temperature Experiments...............................................................................................75 Dual stressor Experiments...............................................................................................79 Discussion..................................................................................................................... ..........87 4 STRESS RESPONSE OF A FRESHWATER CLAM ALONG AN ABIOTIC GRADIENT: TOO MUCH OXYGEN MAY LIMIT DISTRIBUTION.............................116 Introduction................................................................................................................... ........116 Materials and Methods.........................................................................................................1 18 Study Site..................................................................................................................... ..118 Sampling Methods.........................................................................................................119 Sampling for RNA/DNA, Nucleic Acid Oxidation and Stress Proteins.......................120 Statistical analyses.........................................................................................................12 2 Results........................................................................................................................ ...........123 Discussion..................................................................................................................... ........126 Limnological Characters and Rela tionship with Clam Density....................................126 Cellular-level Indicators................................................................................................128 The Extreme Edge of the Distribution...........................................................................130 Relationships Between Limnological Ch aracters and Cellular-Level Stress Indicators....................................................................................................................1 31 Conclusions...................................................................................................................1 33 5 SYNTHESIS.................................................................................................................... .....142 APPENDIX....................................................................................................................... ...........148 LIST OF REFERENCES............................................................................................................. 154 BIOGRAPHICAL SKETCH.......................................................................................................174

PAGE 8

8 LIST OF TABLES Table page 2-1 Summary of statistical results from comparisons between samples from Donax variabilis exposed to norm oxia treatment and samples from animals exposed to hypoxia, hyperoxia, and sulfide.........................................................................................51 4-1 Overview of stress protein functions...............................................................................135

PAGE 9

9 LIST OF FIGURES Figure page 2-1 Diagram of flow-through system.......................................................................................52 2-2 Antibody specificity tests................................................................................................. ..53 2-3 Fraction of surviving Donax variabilis clams in survival experiments in fall and spring......................................................................................................................... .........54 2-4 Expression levels of Hsp70 in Donax vari abilis exposed to normoxia for 0, 1, 3, and 5 days......................................................................................................................... ........55 2-5 Expression levels of three antioxidant pr oteins, a lipid peroxidation marker, and an oxidative repair enzyme in Donax variabilis exposed to normoxia (normox), hypoxia (hypox), hyperoxia (hyperox), and sulfide........................................................................56 2-6 Expression levels of five proteins involved in protein rescue and/or degradation in Donax variabilis exposed to normoxia (normox), hypoxia (hypox), hyperoxia (hyperox), and sulfide........................................................................................................5 8 2-7 Expression levels of total actin in Donax variabilis exposed to normoxia (normox), hypoxia (hypox), hyperoxia ( hyperox), and sulfide...........................................................60 3-1 Antibody specificity tests in M. mercenaria whole clam homogenates..........................100 3-2 Fraction of clams that buried in the three hypoxia experiments......................................101 3-3 Glycogen content of whole clams in the three hypoxia experiments..............................102 3-4 Stress protein expression levels in clams from hypoxia experiments..............................103 3-5 Survivorship of clams in the winter (A) and spring (B) temperature experiments..........106 3-6 Fraction of clams that buried in the three temperature experiments................................107 3-7 Glycogen content of whole clams in the three temperature experiments........................108 3-8 Stress protein expression levels in clams from temperature experiments.......................109 3-9 Survivorship of clams in dual stressor experiments........................................................112 3-10 Fraction of clams that buried in the dual stressor experiments........................................113 3-11 Glycogen content of whole clams in the dual stressor experiments................................114 3-12 RNA oxidation after 24 hour exposur e in dual stressor experiments..............................115

PAGE 10

10 A-1 Relative stress protei n expression levels in Sphaerium sp. clams from the 14-day transplant experiment.......................................................................................................152 A-2 Levels of oxidatively damaged DNA and RNA in Sphaerium sp. clams from the 14day transplant experiment................................................................................................153

PAGE 11

11 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy MAGNITUDE OF THE OXIDATIVE STRESS RESPONSE INFLUENCES SPECIES DISTRIBUTIONS By Joanna Joyner Matos May 2007 Chair: David Julian Major: Zoology Animals must employ diverse physiological strate gies to survive in aquatic habitats with extreme or fluctuating abiotic factors. Whether these physiol ogical strategies include the oxidative stress response, and whether ability to ma intain an oxidative stress response influences species distribution, is not fully understood. In the resear ch projects described in this dissertation, I take three approaches to addres sing this topic. I first test wh ether abiotic factors typical of aquatic habitats cause a cellula r stress response consistent wi th free radical production in the marine bivalve Donax variabilis These results demonstrate th at exposure to hydrogen sulfide causes an oxidative stress response in a non-sulf ide-adapted bivalve and highlight the importance of examining seasonal variation in stress physiol ogy. I next test whether organisms can mitigate the cellular-level damage associated with exposure to single or multiple stressful abiotic factors. This study was conducted in the estuarine bivalve, Mercenaria mercenaria. These results demonstrate that physiological stra tegies at higher levels of or ganization buffer the need for a cellular-level oxidative stress response in a stresstolerant organism. Fina lly, I test whether the capacity to produce an oxidative stress response affects species distribution. This combined field and laboratory study of the freshwater bivalve Sphaerium sp. demonstrates that individuals in a population that overlies an environmental gradient s how a variation in their ability to maintain a

PAGE 12

12 cellular stress response that reflects position alo ng the gradient. These three interrelated studies demonstrate that abiotic factors found in aquati c habitats do cause oxidative stress and that ability to respond to the abiotic f actors correlates with distribution.

PAGE 13

13 CHAPTER 1 INTRODUCTION Much of the work that is done under the name of ecology is not ecology at all, but either pure physiology – i.e. finding out how animals work internally – or pure geology or meteorology…In solving ecological pr oblems we are concerned with what animals do in their capacity as whole, living animals…We ha ve next to study the circumstances under which they do these things, and, most important of all, the limiting factors which prevent them from doing certain other things. By solv ing these questions it is possible to discover the reasons for the distribution and numbers of different animals in nature. (Elton, 1927, p. 33-34) The examination of how physical attributes of the environment interact with physiology to influence the distributio n of organisms was an early goal of ecology, as described above in the introduction to one of the first animal ecology textbooks. During the devel opment of the field of ecology in the first half of the twentieth century, many inve stigators used physiological techniques to address fundamental ecological to pics including “the dist ribution and abundance of organisms” (Spicer and Gaston, 1999; McNab, 2002). Decades later, the two fields split and ‘ecologists’ began focusing on bi otic interactions and demogra phic processes as the causative agents of distribution and abundance patte rns (Andrewartha and Birch, 1984), while ‘physiologists’ focused on mechanisms occurring at lower levels of biological organization (Hochachka and Somero, 2002). The relatively recen t re-integration of these two approaches in the field of ecological physiology utilizes newl y developed molecular and biochemical tools to examine how environmental variables limit repr oduction and survival and therefore impact distribution and abundance (Spicer a nd Gaston, 1999; Chown and Storey, 2006). The set of environmental variables that limits reproduction and survival of a species is the species’ niche (Brown et al., 1996). Traditionally, studies that have attempted to define a species’ niche involved either in situ manipulations combining biotic interactions with environmental variables (exemplified by Connell, 1961) or laboratory studies of physiol ogical responses to abiotic factors. Unlike in situ manipulations, which allow the experimental subjects to be

PAGE 14

14 impacted by all aspects of a complex environm ent, laboratory studi es typically involve acclimation to one select environmental variab le. However, the limitations of extrapolating laboratory studies of single variables to populat ion-level processes have long been identified (e.g., Hall, 1964), including the tendency of la boratory studies to over-estimate physiological tolerances (Nordlie, 2006). What is most likely to be successful, therefore, is a combined field and laboratory approach that u tilizes biochemical and molecula r tools to investigate biotic interactions and speci es distributions. A combined field/laboratory appr oach has been successfully a pplied to the study of species distributions and physiological adap tations of invertebrates in roc ky intertidal ha bitats along the Pacific coast of the United States. This habitat has well-characterized communities and steep gradients of multiple abiotic factors that exhib it diel and seasonal patterns. It has become a model system for ecological physiology, and has been the setting for much of the recent progress in linking population distribution with cellularlevel physiology. For exam ple, latitudinal and vertical zonation pattern s of snails, mussels, and crabs reflect the thermal tolerances of the individual species, which themselves rely upon a complex web of responses including gene expression, protein repair and degradation processes, mitoc hondrial energetics, and heart function (for review, Hofmann et al., 2002; Somero, 2002; Stillman, 2002; Tomanek, 2002). These physiological parameters and the resultant patterns in mortality, in turn, influence such biotic processes as predation (Dahlhoff et al., 2001) and competition (Menge and Sutherland, 1987; Menge and Olson, 1990). Most studies that could be termed ‘ecologi cal physiology’ or ‘comparative physiology,’ as exemplified by the work noted above in rocky in tertidal habitats, take a common approach to understanding how organisms respond to a given ab iotic factor. Many of these studies are built

PAGE 15

15 upon the assumption that the abiotic factor imposes a stress on the organism, resulting in a perturbation to homeostasis at either the cellu lar or organismic level (Hoffmann and Parsons, 1991). Suites of physiological respon ses to this abiotic factor ar e assumed to be energetically costly, and if the cost of maintenance of th ese responses grows too high, the animal cannot survive (Parsons, 1991). To understand the effects of an abiotic factor therefore, the optimal study organism is typically considered to be one that tolerates extremes of th e abiotic factor (Hoffmann and Parsons, 1991; Spicer and Gaston, 1999), because these organisms are most likely to employ protective physiological st rategies that can be detected in laboratory studies. However, the usefulness of such organisms may be limited when the goal is to understand the mechanisms by which the stressor exerts its effects, since th ese same protective physiological strategies will minimize the stressor’s impact. In such cases, an alternate and under-utilized approach is to study the effects of an abiotic factor on an organism that is not adapted to the factor. In this ‘vulnerable’ or ‘naive’ organism one could be more successful at detecting the consequences of the abiotic factor in the absen ce of protective responses. Both of these approaches, the study of stress-tolerant and of ‘vulnera ble’ organisms, are employed in the work described in this dissertation, which addresses how abiotic factors in aquatic hab itats affect th e physiology of bivalves from coastal and inland aquatic habitats. Abiotic Factors in Aquatic Habitats and Oxidative Damage Coastal and inland aquatic habitats vary widely in their characteriza tion as stressful or benign. Potentially stressful ab iotic factors in aquatic hab itats include low dissolved O2 availability (terme d hypoxia), elevated O2 availability (termed hype roxia), hydrogen sulfide, thermal extremes, and salinity fluctuations. Thes e factors can occur singly, but more often are found in combination, including the widesp read combination of low dissolved O2 and hydrogen

PAGE 16

16 sulfide in marine sediments (Fenchel and Riedl, 1970) and the combination of high temperature, hypoxia, and salinity extremes in estuaries in su mmertime (Millie et al., 2004; Caccia and Boyer, 2005). Free radicals have been suggested as a mechanism by which many abiotic factors, including hypoxia, hyperoxia, hydr ogen sulfide, high temperatur e, and salinity extremes, cause cellular damage and thus influence the distribu tion of marine inverteb rates in extreme or otherwise stressful habitats. Fr ee radicals are atoms or molecule s that contain one or more unpaired electrons and are ther efore highly reactive (Halliwe ll and Gutteridge, 1999). During aerobic metabolism, the mitochondria of nearly all eukaryotic cells convert 0.1% (Fridovich, 2004) to 3% (Boveris and Chance, 1973) of O2 into free radicals such as superoxide. This endogenous free radical producti on can be increased by many environmental stressors, particularly fluctuations in O2 availability (Boveris and Chan ce, 1973; Li and Jackson, 2002). Free radicals spontaneously react in a number of ways, one of which is to strip electrons from cellular macromolecules, particularly proteins, lipids, and nucleic acids, causing oxidative damage. For example, oxidative damage from free radical attacks on nucleic acids include strand breaks and oxidation of nitrogenous bases (Shi genaga et al., 1989; Evans and Cooke, 2004). If free radical production overwhelms a cell’s ability to detoxify the free radicals, oxidative damage occurs (Halliwell and Gutteridge, 1999). Cellular oxi dative damage, if not repaired, leads to cell death, disease, and aging (Hal liwell and Gutteridge, 1999). To minimize oxidative damage, eukaryotic ce lls use a variety of protective mechanisms, including the expression of stre ss proteins. These proteins fall in to three general categories: (1) antioxidants such as manganese superoxide dism utase (MnSOD), copper/zinc superoxide dismutase (Cu/Zn SOD), glutathi one, glutathione peroxidase (G Px) and catalase, that convert

PAGE 17

17 free radicals into less toxic or nontoxic forms; (2) chaperone proteins such as ubiquitin, the heat shock proteins (e.g., Hsp60, Hsp70 and Grp75) and sm all heat shock protein (sHsp), that aid the folding or removal of damaged proteins; and (3) oxidative repair enzymes, such as OGG1, that repair oxidatively damaged DNA (Halliwell and Gutteridge, 1999). Hypoxia Conditions of low dissolved O2 are widespread and occur na turally in aquatic habitats, particularly in coastal areas a ffected by upwelling, rock pools in th e intertidal zone, all marine sediments, and in standing freshwater bodies (Grieshaber et al., 1994; Diaz and Rosenberg, 1995). Hypoxia results from a variety of processes, including elevated water temperature, low mixing, dense animal populations, and high organi c decomposition (Grieshaber et al., 1994). The importance of maintaini ng adequate access to O2 centers on its function as the final electron acceptor in the mitochondrial electron tr ansport chain. In the absence of O2, mitochondrial production of ATP via oxidative phosphorylation ceases, and cells must rely upon the reduced ATP production available from glycolysis and other pathways of anaerobic metabolism (for review of pathways in inverteb rates, see Grieshaber et al., 19 94). Hypoxia affects a wide variety of ecological processes in aquatic organisms, including recruitment (Marinelli and Woodin, 2002), distribution (Rosenberger and Chapman, 2000), seasonal migration along a vertical gradient (Pihl et al., 1991), predator-prey interact ions (Breitburg et al., 19 97), the use of refuges (Chapman et al., 2002), and larv al settlement and growth rates (Baker and Mann, 1992). Most of the well-studied instances of hypoxia tolerance are found in the i nvertebrates, particularly the molluscs, annelids, nematodes, and platyhelminths (Hochachka and Somero, 2002). Whether hypoxia can induce free radical pr oduction directly, or whether reoxygenation following a period of hypoxia is necessary for free radical production, is not yet understood (Kukreja and Janin, 1997; Hermes-Lima et al., 1998; Halliwell and Gutteridge, 1999; Semenza,

PAGE 18

18 2000; Hermes-Lima and Zenteno-Savin, 2002; Li and Jackson, 2002). During hypoxia, the absence of O2 as the final electron acceptor causes accu mulation of electrons in mitochondrial electron transport chains (i.e., the chai ns are reduced). A sudden return of O2 can cause the production of superoxide due to nearly instantaneous reactions between O2 and the free electrons that accumulated in proteins of the electron tr ansport chain (Du et al., 1998; Li and Jackson, 2002). Such a scenario might occur during tidal e bb and flow for intertidal animals. However, several recent studies have also shown free radical pr oduction during hypoxia without subsequent reoxygenation. The evidence for he ightened free radical production during hypoxia comes from direct measurement of free radicals (Vanden Hoek et al., 199 7; Becker et al., 1999) and measurement of oxidative DNA damage in mammalian cells (Englander et al., 1999), and changes in antioxidant expressi on and/or activity in goldfish (Lushchak et al., 2001) and an estuarine crab (de Oliveira et al., 2005). Howe ver, several studies of both vertebrates and invertebrates have noted decrease s or a lack of changes in an tioxidant expression or activity during hypoxia that are consistent with an ove rall metabolic depression during hypoxia (Hass and Massaro, 1988; Willmore and Storey, 1997; Joan isse and Storey, 1998; Larade and Storey, 2002). Hyperoxia While less common in marine habitats th an hypoxic conditions, hyperoxic conditions include rocky intertidal pools with photosynthe tically active algae (Truchot and Duhamel-Jouve, 1980), boundary layers of intertidal seaweed (Irwin and Davenport, 2002) and brown algae (Pohn et al., 2001), the cold seawater of polar regions (Viarengo et al., 1995; Abele and Puntarulo, 2004), and within the tissues of some algal-cnidaria n symbioses (Dykens et al., 1992; Richier et al., 2003, 2005). The physiological effect s of hyperoxia are not well-characterized, but it is evident that hyperoxia cause s mitochondrial-induced cellular death (Chandel and Budinger,

PAGE 19

19 2007) and decreased cellular metabolism, which is lik ely due to the inactivation of the citric acid cycle enzyme aconitase (Gardner et al., 1994). The effects of hyper oxia on biotic interactions have been characterized in only a few systems. Fo r example, cnidarian hosts of intracellular algal symbionts utilize protective strategies to mi nimize damage from hyperoxic conditions induced by excessive algal photosynthesis during periods of elevated temperature (Dykens et al., 1992; Nii and Muscatine, 1997; Richier et al., 2003). Elevated cellular O2 levels induce mitochondrial fr ee radical production (Boveris and Chance, 1973; Akbar et al., 2004) and oxidative damage (Dennog et al., 1999). Exposure to hyperoxia has been linked to elevated antioxida nt responses in tissues of both vertebrates (O'Donovan et al., 2002; Cho et al., 2005) and inve rtebrates (Dykens et al., 1992; Viarengo et al., 1995; Abele and Puntarulo, 2004). However, this rela tionship is not consiste nt, as demonstrated by a variety of in vitro and in vivo studies in invertebrates and vertebrates (Abele et al., 1998b; Dennog et al., 1999; Allen and Balin, 2003; Freiberger et al., 2004). Hydrogen Sulfide Hydrogen sulfide (referred to here simply as “sulfide”) occurs in a variety of aquatic habitats, including mudflats, mangrove swamps deep-sea hydrothermal vents, hydrocarbon seeps, and anoxic basins, where animals may be pe riodically or continuous ly exposed to sulfide at levels up to 12 mM (for review, Fenchel a nd Riedl, 1970; Somero et al., 1989; Bagarinao, 1992). Sulfide is a highly reactive toxin, and the H2S form diffuses freely across respiratory surfaces and therefore cannot be excluded from tissues (Denis and Reed, 1927; Julian and Arp, 1992). Sulfide has several mechanisms of toxici ty, including the reversible inhibition of cytochrome c oxidase, the final enzyme of the mitochondrial elec tron transport chain (Lovatt Evans, 1967; Nicholls, 1975), reduction in hemogl obin oxygen affinity (Carrico et al., 1978) and inhibition of approximately 20 enzymes (Bagar inao, 1992). Invertebrates inhabiting sulfidic

PAGE 20

20 environments employ a variety of strategies to detoxify sulfide, of which the most widely demonstrated is the oxidation of sulfide to th iosulfate or other com pounds (for review, Lovatt Evans, 1967; Vismann, 1991; Grieshaber and Vlkel 1998). Marine invert ebrates that are not found in sulfidic habitats and do not employ sulfide detoxificati on strategies typically show increased mortality upon exposure to hydrogen sulf ide (Grieshaber and Vlkel, 1998). A number of interspecific interactions are as sociated with sulfidic habitats, the best-studied of which is the widespread relationship between invertebrate hosts and chemoaut otrophic bacterial symbionts that use the chemical energy from sulfide oxi dation to fix carbon dioxide into carbohydrates (Felbeck et al., 1981; Ruby et al., 1981; Ca vanaugh, 1983). Additionally, sulfide structures coastal communities (Gamenick et al., 1996) and methane seep communities (Levin et al., 2006) by influencing recruitment and spatial patter ns, and sulfide gradients influence food web dynamics in deep sea hydrothermal vent communities (Levesque et al., 2006). Hydrogen sulfide oxidizes spontaneously in the pr esence of divalent metals (both dissolved and in metalloenzymes), genera ting oxygen-centered ra dicals (likely supe roxide) and sulfurcentered radicals in aqueous solutions (Chen a nd Morris, 1972; Tapley et al., 1999) and in animal tissues (Tapley, 1993; Abele-Oeschger and Oeschger, 1995; Eghbal et al., 2004; Julian et al., 2005). Several studies have specifically addressed the lin k between oxidative damage and sulfide exposure at the organismal level. Fo r example, antioxidant enzyme activities were proportional to the sulfide tolera nces of thiobiotic meiofauna (Morrill et al., 1988). Similarly, MnSOD activity increased in res ponse to sulfide exposure in th e chemoautotrophic symbiotic bivalve Solemya velum but not in the related nonsymbiotic Yoldia limatula (Tapley, 1993). In contrast, a relationship between sulfide tolera nce and antioxidant activity was not found in a survey of sulfide-tolerant polychaetes and bi valves (Abele-Oeschger, 1996). A relationship

PAGE 21

21 between sulfide and free radical production also has been investigated at the cellular level. In isolated erythrocytes fr om the marine polychaete Glycera dibranchiata sulfide exposure causes mitochondrial depolarization, increased cellular oxidative stress, and increased mitochondrial superoxide production (Julian et al., 2005). These studies suggest th at increased oxidative stress is an additional mechanism by which sulfide exposure could cause toxicity (Morrill et al., 1988; Abele-Oeschger et al., 1994; Abele-Oeschger, 1996). Elevated Temperature The importance of temperature in regulating nearly all physiologica l processes and the resulting impacts on species distribution and abu ndance is highlighted by extensive discussions of this topic in recent books describing bioc hemical adaptations (160 pages, Hochachka and Somero, 2002) and physiological ecology ( 100 pages, McNab, 2002). Although aquatic organisms encounter extremes of high and lo w temperature, only consequences of, and adaptations to, high temperature wi ll be addressed in this diss ertation. Distribution is tightly linked with tolerance to heat stress across a wide variety of aquatic organisms (Gilchrist, 1995; Prtner, 2002; Chown and St orey, 2006; Nordlie, 2006). This relationship is dependent upon an array of cellular and biochemical processes, including changes in protein structure, enzyme activity and cell membrane composition (Hocha chka and Somero, 2002). For example, in ectothermic organisms, the expression levels and activities of enzymes such as lactate dehydrogenase are temperature adaptive, as exhi bited by correlations between environmental temperature and activation energy, and conservati on of catalytic rate c onstants and substrate binding ability, over a broad range of temperatures (but not at upper leth al temperatures; for review, Somero, 2004). Temperatures much high er than the environmental or acclimation temperature of an organism cause damage at the cellular level, incl uding mitochondrial swelling and distortion (Cole and Armour 1988) and structural damage to proteins and cell membranes

PAGE 22

22 (Hochachka and Somero, 2002). Heat stress and the ability to respond to heat stress affect functional responses including buria l ability in bivalves (Savag e, 1976), development (Mahroof et al., 2005; McMillan et al., 2005) heart function (Stillman, 2002), and the ability to respond to other abiotic factors such as hypoxia (Chang et al., 2000) and low salinity (Cain, 1973). Ability to respond to thermal stresses may affect a variety of biotic processes in addition to distribution, including recruitment success (Menge, 2000), faculta tive interspecific interactions (Burnaford, 2004), and resistance to predation (Mes a et al., 2002; Pauw els et al., 2005). A mechanistic link between exposure to hi gh temperature and fr ee radical production likely results from alterations in mitochondrial respira tion, as demonstrated in both vertebrates (Zuo et al., 2000; Zhang et al., 2003 ; Mujahid et al., 2006) and inve rtebrates (Abele et al., 2002; Heise et al., 2003; Keller et al., 2004 ). Heat stress affects the ability of organisms to minimize or repair oxidative damage, typically by enhanci ng this ability by preconditioning the cells, as demonstrated in studies of cardi oprotection in vertebrates (Arna ud et al., 2002; Joyeux-Faure et al., 2003). Salinity The influence of extremes of salinity on phys iological processes, particularly when external salinity is not equivale nt to intracellular osmotic concentr ation, is nearly as pervasive as that of temperature (Hochachka and Somero, 2002). Internal osmo tic concentrations of aquatic organisms are typically regulated with extensive a rrays of ‘compatible osmolytes’ such as free amino acids, sugars, polyhydric alcohols, a nd urea (Yancey et al., 1982; Yancey, 2001). Insufficient cellular-level responses to fluctuating or extreme salin ity result in changes in cell volume (Neufeld and Wright, 1996), structural and functional damage to mitochondria (Suresh and Jayaraman, 1983) and lysosomes (Pipe and Moore, 1985; Hauton et al., 1998), decreased ability to regulate O2 consumption during hypoxia (Hawkins et al., 1987), and decreased ability

PAGE 23

23 to respond to elevated temperatures (Cain, 1973; Hauton et al., 1998; Werner, 2004). Ability to tolerate salinity extremes or fluctuations and th e energetic costs associat ed with osmoregulatory strategies affect a wide variet y of ecological processes, includ ing foraging behavior (Webster and Dill, 2007), predation and recruitment (Wit man and Grange, 1998), and species distributions (Nordlie, 2006; Lowe et al., 2007). Several studies of stress protei n expression (particularly heat shock protein expression) in estuarine invertebrates exposed to hyposalinity or hyposalinity/high temperature treatments have produced conflicting results (Kul tz, 1996; Clark et al., 2000; Wern er and Hinton, 2000; Spees et al., 2002; Werner, 2004; Blank et al., 2006). A causative relations hip between hypersalinity and free radical production is well-esta blished in plant physiology and biomedical fields such as nephrology and immunology (e.g., He rnndez et al., 1993; Qin et al., 1999; Hizoh and Haller, 2002). However, whether hyposalinity is linked to alterations in free radical metabolism and oxidative damage is not understood. A recent stu dy of hyposalinity responses of a marine alga detected an elevation in glut athione but not in an tioxidant enzymes such as catalase and superoxide dismutase (J ahnke and White, 2003). Stress Proteins as Indicators of Organism Health Several characteristics of stress proteins make them useful for asse ssing the effects of abiotic factors. Many stress proteins are consti tutively expressed at low levels, but this expression is upregulated in response to conditions that result in elevat ed free radical production (Feder and Hofmann, 1999; Downs et al., 2001a; Kultz, 2005) and ev en to biotic interactions such as predation (Pauwels et al., 2005). Patterns of expression of multiple stress proteins can indicate the stressor to which the organi sm is exposed (Downs et al., 2000, 2001a, 2001b, 2002b). However, the interpretation and applicability of stress protein expr ession is hampered by our poor understanding of the temporal profiles of stress protein induction and how this

PAGE 24

24 induction affects the long-term health of th e organism. Very few studies have coupled physiological condition assays with stress protein expression levels (Brown et al., 1995; HamzaChaffai et al., 2003; Romo et al., 2003b), and even these studies treated the condition assays only as indicators of overall health rather th an investigating whethe r the protein expression patterns were accurate predictors of the functi onal assays and vice versa. Additionally, some stress proteins like Hsp70 have a large but s hort-term response to a stressor (Tomanek and Sanford, 2003), which limits the practical applicati on of monitoring expression of this protein in the field because timing of sampling is crucial to detecting a stress pr otein response. Finally, there are conditions under which down regulation (rather than upregul ation) of stress protein expression indicates physiological stress (Werner and Hinton, 1999), pa rticularly if the organism is stressed to such an extreme that all metabolic processes are decreasing and death is imminent (Bierkens, 2000). Overview of Dissertation Research Animals that must employ diverse physiological strategies to survive in habitats with extreme or fluctuating abiotic factors tend to be generalists with broad tolerance ranges (Lynch and Gabriel, 1987). Whether these ph ysiological strategies include the oxidative stress response, and whether animals from benign versus extreme ha bitats exhibit differences in the magnitude of their oxidative stress re sponses is not fully understood. As noted above, accumulating evidence suggests that free radical metabolism and oxida tive stress are linked to many of the abiotic factors typically faced by invertebrates in coas tal and inland aquatic habitats. However, whether the ability of these organisms to respond to oxida tive stress influences th eir tolerance to these abiotic factors, and ultimately infl uences species distribution, is unknown. In the research projects described in this di ssertation, I address three interrelated questions, each of which is examined in a different bivalv e species. In the selection of species for these

PAGE 25

25 projects I followed what is typically termed the August Krogh Principle, which Krogh articulated in his opening address on “The Progress of P hysiology” to the Thir teenth International Physiological Congress with the comment, “For a large number of problems there will be some animal of choice or a few such animals on whic h it can be most conveniently studied” (Krogh, 1929, p. 202). In Chapter 2 I test whether a subset of the abiotic factors discussed above triggers an oxidative stress response in the marine bivalve Donax variabilis I examine the effects of 24hour exposures to hypoxia, hyperoxia, and hydrogen su lfide on the seasonal patterns of survival and stress protein expression in D. variabilis Since these clams do not encounter these abiotic factors in the high-energy sandy beaches they inhabit, these clams are unlikely to possess protective mechanisms that would minimize the impact of the stressor and reduce my ability to detect a cellular-level oxidative stress res ponse. This study therefore follows the alternate approach discussed above: examining a ‘vulnera ble’ species for evidence of oxidative stress resulting from an abiotic factor. Results from Ch apter 2 were published in the following paper: J. Joyner-Matos, C.A. Downs, and D. Julian, Increased expression of stress pr oteins in the surf clam Donax variabilis following hydrogen sulfide exposure, Comparative Biochemistry and Physiology 145:245-257, 2006. In Chapter 3 I test whether the ability to in itiate an oxidative st ress response following exposure to single or multiple abiotic factors correlates with changes in whole-organism, functional, metrics of condition. The effects on the cellularand or ganismal-level responses of hypoxia, high temperature, and the combinati on of high temperature and hyposalinity were examined in the estuarine bivalve Mercenaria mercenaria These clams exemplify the generalist strategy described by Lynch (1987), with demonstr ated broad tolerances for extremes of and

PAGE 26

26 fluctuations in temperature, dissolved O2 level, salinity, and pH that reflect the species’ widespread distribution in intertid al and subtidal coastal habitats. With the selection of such a stress-tolerant species, I am able to examine wh ether physiological strategi es at higher levels of organization buffer the need for a cellular-level oxidative stress response or correlate with the cellular-level responses. The resu lts from Chapter 3 are in prep aration for submission, likely to the Journal of Shellfish Research In Chapter 4 I take the tools developed from the two previous chap ters, which were in essence purely physiological studi es, and in a combined field a nd laboratory study I address how the capacity to produce an oxidative stress res ponse affects species distribution. This study specifically examines whether the distribution of a freshwater clam over a complex and stable gradient of several abiotic fact ors is related to the physiologi cal condition and oxidative stress response of the clams. The clams used in this project, Sphaerium sp., live in a swamp-river system in Uganda and cannot be classified into e ither the ‘vulnerable’ or ‘tolerant’ categories. They clearly are not vulnerable to abiotic factors like D. variabilis since they tolerate a range of pH and dissolved O2 concentrations; however, it is not know n whether these clams can tolerate the extremely broad ranges of multiple abiotic factors in the manner demonstrated by M. mercenaria Therefore, I could not form a priori predictions about whether clams experiencing different conditions along the envi ronmental gradient would exhibit differences in their oxidative stress responses or levels of oxidative damage. The results from Chapter 4 are in press as the following manuscript: J. Joyner-Matos, L.J. Chap man, C.A. Downs, T. Hofer, C. Leeuwenburgh, and D. Julian, Stress response of an African fres hwater clam along a natu ral abiotic gradient: Too much oxygen can be a limiting factor in aquatic environments, Functional Ecology 21:344355, 2007.

PAGE 27

27 CHAPTER 2 INCREASED EXPRESSION OF STRESS PROTEINS IN THE SURF CLAM Donax variabilis FOLLOWING HYDROGEN SULFIDE EXPOSURE Introduction Abiotic factors in marine habitats include th ermal extremes, salinity fluctuations, hypoxia, hyperoxia, and sulf ide (sum of H2S, HSand S2-). Such factors may influence species distribution by stressing organisms to their physiological li mits (Parsons, 1991). However, the mechanisms by which many abiotic factors cause stress are not completely understood. Free radicals have been suggested as a mechanism by which some abio tic factors, including temperature (Abele et al., 1998a, 2002; Downs et al., 2002a; Heise et al., 2003), hypoxia (Greenway and Storey, 1999), hyperoxia (Dykens et al., 1992; Vi arengo et al., 1995), and sulfide (Morrill et al., 1988; AbeleOeschger et al., 1994, 1996; Tapley et al., 1999), cause cellular damage and thus influence the distribution of marine in vertebrates in extreme or otherwis e stressful habitats. Free radicals, which are atoms or molecules that contain one or more unpaired el ectrons (Halliwell and Gutteridge, 1999), cause cellular damage, termed oxi dative damage, by stri pping electrons from cellular macromolecules. In the mitochondria of nearly all eukaryotic cells, a fraction of O2 consumption is converted into the free radical superoxide during aerobic metabolis m; estimates range from 0.1% (Fridovich, 2004) to 3% (Bove ris and Chance, 1973) of O2 consumption. To minimize oxidative damage, eukaryotic cells utili ze a variety of protective mechan isms, including the expression of an assortment of proteins which, for the purposes of this paper, are coll ectively referred to as “stress proteins.” These fall into three general categories: (1) antioxidants such as manganese superoxide dismutase (MnSOD), copper/zinc superoxide dismut ase (Cu/Zn SOD), glutathione, glutathione peroxida se (GPx) and catalase, which convert fr ee radicals into less toxic or nontoxic forms; (2) proteins involved in prot ein rescue and/or degradation, such as the heat shock

PAGE 28

28 proteins (e.g., Hsp60, Hsp70 and Grp75) and small heat shock protein (s Hsp), and the protein ubiquitin, which aid in the folding or removal of damaged proteins (Downs et al., 2005); and (3) oxidative repair enzymes, such as 8-oxoguanine DNA glycos ylase (OGG1), which repair oxidatively-damaged DNA (Halliwell and Gutteridge 1999). Expression of some stress proteins is upregulated in response to conditions that result in elevated free radical produc tion (Downs et al., 2001a, 2001b) and to other en vironmental stressors such as thermal stresses (Hofmann and Somero, 1995). The measurement of stress protein expression levels has served as a cellularlevel indicator of elevated free radical produc tion in marine invertebrates (e.g., Abele and Puntarulo, 2004), and in comparative physiolo gical (e.g., Willmore and Storey, 1997) and biomedical studies (e.g., Maga lhes et al., 2005). The aim of this study was to investigate whet her sulfide, as well as hypoxia and hyperoxia, have the potential to stimulate a cellular response consis tent with increased oxidative stress in a marine invertebrate. To test th is, we exposed the marine clam Donax variabilis (the coquina clam) to these abiotic stressors in controlled laboratory conditions and assessed the animals’ overall tolerance (i.e., survival), stress prot ein expression, and lipid peroxidation. Additionally, we examined whether the abiotic stressors affect expression of the cyto skeletal protein actin, which has traditionally been measured as a contro l for sample protein content, but recently has been shown to decrease in vertebrate cells following exposure to hypoxia, hyperoxia, and freeradical generating toxins (Alla ni et al., 2004; Brown and Davi s, 2005; Cho et al., 2005). We conducted identical experiments in fall and spring to investigate seasona l differences (Hofmann and Somero, 1995; Chapple et al ., 1998; Sheehan and Power, 1999) Unlike previous studies of oxidative stress in marine invertebrates, we selected our study species based on the high probability that it does not encounter hypoxia, hyperoxia or sulf ide in its habitat and therefore

PAGE 29

29 likely does not employ additional protective mechanisms, such as those in invertebrates adapted to hypoxia (Grieshaber et al., 1994) and sulfide (Grieshaber and V lkel, 1998). Such adaptations would be expected to minimize the impact of th e stressor, reducing our ability to detect a cellular-level oxidative stress response. D. variabilis inhabits sandy beaches with moderate to high waves along the southeastern coast of North America (Mi kkelsen, 1981; Ellers, 1995). The clams migrate up and down the beach, following th e tidal cycle, generally remaining buried in the upper 4 cm of sand. The wave activity and hi gh porosity of sandy beaches likely maintains the seawater surrounding the clams sulfide-free a nd at or approaching air-saturation. Therefore, we considered it probable that D. variabilis is more vulnerable to these stressors than would be expected of invertebrates from habitats such as tide pools, mudflats or marshes (Grieshaber and Vlkel, 1998). Materials and Methods Clam collection and maintenance. Donax variabilis clams were collected at Crescent Beach, FL (approx. 29.7N, 81.2W), within 30 minutes of high tide during September 2003 (“fall”) and March 2004 (“spring”). Water temperat ures at all collections were 28 3C. All clams were between 1.0-1.5 cm in length. Im mediately after collec tion, the clams were transported to the University of Florida in aerated seawater in an insulated container ( ca 90 minutes transport time). Exposure of Clams to Abiotic Stressors Flow-through system. Exposure to abiotic stressors was achieved with a constanttemperature flow-through system that used electr onic gas flow controllers to regulate water PO 2 and sulfide concentration (Fig. 21). Seawater in the system was obtained from the University of Florida Whitney Marine Labor atory (Marineland, FL, USA), and was pretreated with

PAGE 30

30 chloramphenicol (2 mg L-1) to prevent the growth of sulfate reducing bacteria. This pretreatment has been shown to markedly increase survival of marine bivalves in respirometry experiments (De Zwaan et al., 2002). The flow -through system consisted of four channels, with each having an animal chamber designed to contain eight clam s. Seawater for each of the four channels was continuously equilibrated with ai r (normoxia) for channel one, N2 (hypoxia) for channel two, a mixture of O2 and air (hyperoxia) for channel three, and a mixture of air and hydrogen sulfide gas (from a compressed tank of 2% H2S, balance N2) for channel four. Th e equilibrated water was pulled through the animal chambers at 2 mL min-1. Further details of the apparatus construction and gas equilibration are provide d in the legend to Fig. 2-1. Dissolved O2 was measured twice daily in each channe l using a fluorometric dissolved O2 probe (FOXY probe; Ocean Optics, Inc., Dunedin, FL, USA). The sulfide concentration in the H2S-channel was measured twice daily using the methylene blue method (Cline, 1969) Survival during exposure to stressors. D. variabilis were exposed to normoxia, hypoxia, hyperoxia and sulfide in each season (fall a nd spring), during which survival was assessed. Exposures lasted 4-7 days and were conducted with 8 clams. The clams were checked twice daily for mortality, and were presumed to have di ed when they did not close their valves when disturbed or when obvious tissu e degradation had begun (dead clams were immediately removed from the chambers). Surviving clams and tissues from these experiments were not used for any other experiments. Exposure to normoxia. In a preliminary experiment in the fall, we tested whether maintenance in the flow-through exposure system under normoxic conditions resulted in changes in expression of Hsp70. This protein has shown sens itivity to a variety of stressors, and therefore was used as a general indicator of stress (Fed er and Hofmann, 1999). For this experiment, one

PAGE 31

31 additional channel was added to the flow-through system and all channels contained seawater equilibrated with air. Fifty clams were placed in the system initially, and the experiment continued for 9 days. The clams were not fe d. Every day, 1 clam was removed from each channel, and tissues from clams removed at days 1, 3 and 5, as well as tissues from control clams collected and frozen in liquid N2 at the beach collection site, we re prepared, stored and assessed for Hsp70 expression, as described below. Exposure to stressors. For tissues to be used for dete rmination of the remaining stress proteins, clams were exposed to normoxia, hypoxi a, hyperoxia and sulfide for 24 hours in each season (fall and spring). A total of 8 clams were exposed to each stressor in each season. At the end of the exposure, the clams were removed a nd immediately processed, as described below. Analyses of Stress Protein Expression Tissue sample processing. Immediately upon removal fr om the flow-through system, clams were opened by severing their adductor musc les, and their whole tissues were quickly blotted dry and frozen in liquid N2, followed by storage at –80 C fo r further processing. Of the eight clams per treatment, four we re processed for stress protein e xpression analysis, as modified from Downs et al. (2002b), and th e remaining four clams per treatm ent were archived at –80 C. For homogenization, whole tissues, frozen and stor ed as described above, were individually ground in liquid nitrogen to a fine powder in a mortar and pestle pre-cooled by liquid N2, and then immediately returned to –80 C storage. For resuspension of these homogenates, a small volume of each individual powdered sample wa s dissolved in denaturi ng SDS buffer (50 mmol L-1 Tris, 15 mmol L-1 EDTA, 2% SDS, 15 mmol L-1 DTT, 0.5% DMSO, and 0.01% Halt protease inhibitor cockta il from Pierce Biotechnology, Inc. Ro ckford, IL, USA; pH 7.8). Each suspension was then vortexed for 30 seconds, inc ubated at 85 C for 3 minutes, vortexed for 15 seconds, incubated again at 85 C for 3 minutes vortexed for 15 seconds, and centrifuged at

PAGE 32

32 12,000 g for 10 minutes. Total soluble protein concentration of each sample was assayed by the method of Ghosh et al. (1988). Ti ssues from fall and spring expe riments were homogenized and resuspended at the same time. The resuspended sa mples were then aliquotted, frozen in liquid N2 and stored at –80 C. Unless noted otherwise, a ll chemicals were obtained from Sigma Chemical Company and were the highest quality available. Antibodies. Samples were assayed for stress pr otein expression using mono-specific, ELISA-grade polyclonal antibodies generated by and donated by EnVirtue Biotechnologies, Inc. (Winchester, VA, USA). The antibodies were raised in rabbits against 8-15 amino-acid polypeptides (conjugated to bovine serum albumin) derived from each target protein sequence of the bivalve Mya arenaria (Downs et al., 2002b). The following antibodies were used: Cu/Zn SOD (Cat. # AB-SOD-1516), GPx (Cat. # AB-GPX-1433 ) MnSOD (Cat. #AB-1976), ubiquitin (Cat. #AB-U100), invertebrate small heat s hock protein homologues (s Hsp; Cat. #AB-H105), heat shock protein 70 (Hsp70; Cat. #ABHsp70-1519), mitochondrial Hsp70 (Grp75; log 3219), Hsp60 (Cat. # AB-H100-IN), OGG1-mito (lot 2916), and 4-hydroxy-2 E -nonenol-adducted protein (HNE; lot 156). Total actin pool wa s determined with a polyclonal antibody from Stressgen Bioreagents (Victoria, BC, Canada). Specificity of each antibody was verified by SDSPAGE and western blotting with goat anti-rabbit, alkaline phosphatase-conjugated secondary antibody (Sigma) and a chemilumine scent reporter system (DuoLux Chemiluminescent/Fluorescent Substrate for Al kaline Phosphatase, Vector Laboratories, Burlingame, CA, USA) on samples from both fall and spring exposures (Downs et al., 2002b). Representative antibody verification resu lts for Cu/Zn SOD, GPx, MnSOD, Hsp70, mitochondrial Hsp70, Hsp60, OGG1-mito and actin are presented in Fig. 2-2 Note that

PAGE 33

33 ubiquitin, sHsp and HNE interact with other prot eins and therefore typically form “smears” rather than single bands. After verification of specific ity, stress protein expression for each tissue sample was determined by dry-dotting (Cu/Zn SOD, GPx, MnSOD, ubiquitin, Hsp70, Hsp60, Grp75, HNE and actin) or ELISA (sHsp and OGG1-mito), bot h as described below. All assays were performed on 16 clams from each season (four per treatment), and tissues from both seasons were assayed at the same time to minimize experimental variation. Dry-dotting. The following dry-dotting technique was developed for this study. Samples were diluted to 30 ng or 100 ng of total sol uble protein (TSP) in 50 l TBS (50 mmol L-1 Tris, 10 mmol L-1 NaCl, pH 7.5) and triplicate 1 L volumes were dotted onto dry nitrocellulose membrane (Fisher Scientific, Fairlawn, NJ, US A) with a multichannel micropipette (0.5-10 L, Eppendorf, Westbury, NY, USA). On each membra ne, an eight-fold serial dilution from one sample was dotted in triplicate to allow subsequent confirmation that sample concentrations were within the linear detection range and semi-quantitative comparisons of samples across membranes (see below). Approximately 48 sample s could typically be dotted onto a 7 cm by 11 cm membrane. Once dry, the membranes were subse quently treated identical ly to a western blot. Specifically, each membrane was blocked in TBS-T (TBS with 0.05% Tween-50) with 5% milk (Carnation instant dry milk) or 5% acid-hydrol yzed casein for 30 minutes and then incubated with primary antibody at 1:10,000 in TBS-T for 1 hour at room temperature. Each membrane was then washed with TBS-T, incubated with secondary antibody at 1:10 ,000 in TBS-T with 5% milk or 5% casein for 1 hour at room temperatur e, and finally washed three times with TBS-T followed by TBS and then Tris solution (100 mmol L-1, pH 9.5). Chemiluminescence substrate (as above) was then added as per the manufact urer’s instructions and each membrane was

PAGE 34

34 visualized on a GeneGnome Chemiluminescent Detection System (Syngene, Frederick, MD, USA). Images were analyzed using GeneTools application software (Syngene). The serial dilution of one sample on each membrane was used to determine the correlation between concentration and luminescence intensity within a membrane using one site, saturation ligandbinding regression curve fits (performed by SigmaPlot 8.02, Systat Software, Inc., Point Richmond, CA, USA). Drydotting of tissues from fall and sp ring experiments were performed at the same time and on the same piece of me mbrane; allowing direct comparisons among samples. ELISA. OGG1-m and sHsp expression levels we re determined by ELISA at EnVirtue Biotechnologies, Inc. A Biomek 2000 robotic wo rkstation (Beckman Cou lter, Inc., Fullerton, CA, USA) was used to conduct the ELISA assays using 384-we ll microplates. Samples were assayed in triplicate with intra-specific variati on of less than 7.5% for all samples combined for each assay. An eight-point calibrant curve using a protein standard relevant to each antibody was added in sextuplicate for each plate (Downs et al., 2002b). ELISA assays of tissues from fall and spring experiments were performed at the same time and on the same plates, allowing direct comparisons among samples. Data Analysis and Statistics All stress protein expression leve ls were standardized by the st andard curves run with each dry-dotting or ELISA assay. Data generated by dry-dotting are expr essed as relative units per nanogram of total soluble protein (RU ng TSP-1). Data generated by ELISA are expressed as fmol mg TSP-1 or fmol ng TSP-1. The data were not normally distributed, as confirmed by the Shapiro-Wilk W test for non-normality (p value < 0.05 for at least one season for each protein), and therefore they were analyzed by the nonparametric Kruskal-Wallis one-way ANOVA. T values for each ANOVA are listed in Table 21. Significantly different groups were then

PAGE 35

35 analyzed using the Conover-Inman post hoc comparison test, which is a form of the Fisher’s least significant diffe rence (LSD) method performed on ranks (Conover, 1999). This analysis was conducted pairwise for each stress protein and individually for each season. We did not analyze the data with two-way ANOVAs with season and treatment as factors because we expected to find extensive seasonal differences in expression patterns within and among proteins and we considered the samples collected in fall and spring to be truly independent. P values less than 0.05 were considered significant and are listed in Table 2-1. All sta tistical analyses were performed with StatsDirect version 2.4.4 (Cheshir e, UK). Stress protein data are presented as scatterplots, with the data sym bol signifying the median, and vert ical asymmetrical error bars denoting minimum and maximum values. Results Experimental Conditions. The conditions of the four chambers in the flow-through exposure system for experiments with the three environmental stresso rs were: 1) normoxia, 21.6 1.9 kPa PO 2 (mean s.d.; n = 2 readings per 24 hour period) ; 2) hypoxia, 12.3 1.4 kPa PO 2; 3) hyperoxia, 36.6 3.0 kPa PO 2; and 4) sulfide, 98 2.9 mol l-1 total sulfide and 24.0 1.8 kPa PO 2. Average pH in both the gas equilibration chambers and the outflow from the animal chambers for th e four treatments was 8.11 0.14 with no significant differences between treatments. Survival Analysis. To determine the survival tolerance of D. variabilis to each exposure condition in each season, we exposed 8 clams per treatment to nor moxia, hypoxia, hyperoxia and sulfide for up to 7 d. Clams collected in the fall showed 100% mo rtality during hypoxia exposure, with 6 of 8 clams dying on the third day and the remaining 2 cl ams dying on the fourth day (Fig. 2-3). In the

PAGE 36

36 spring, mortality was also 100%, although the clams su rvived slightly longer, with 4 of 8 clams dying on the fifth day, 1 clam dying on the sixt h day, and the remaining clam dying on the seventh day (Fig. 2-3). Exposure to hyperoxia caused no mortality in the fall (4 d exposure). In the spring, 1 of 8 clams died at day 3 during hyp eroxia exposure, with no additional mortality (7 d exposure). Hydrogen sulfide exposure showed sim ilar mortality between seasons; in the fall, 6 of 8 clams died on the second day and the remain ing 2 clams died on the third day, whereas in the spring, 1 clam died on the second day, 3 more died on the third day and the remaining 4 clams died on the fourth day. Ther e was no mortality in the normoxia treatments in either season (4 d exposure in fall and 7 d exposure in spring). Exposure to Normoxia To confirm that changes in stress protein e xpression were due to the stressors and not simply a result of the clams being maintained in the flow-through system, we conducted a 9 d normoxia exposure study in the fall. We measured expression levels of Hsp70 in clams from days 1, 3, 5, and compared those to expression in control clams that were collected and frozen at the beach (time 0; see Fig. 2-4). We found no signi ficant changes in Hsp70 expression levels ( p = 0.064). However, Hsp70 levels were slightly increased in clams sampled at day 5. Based on this, we assume that any changes in stress pr otein expression in clams from the remaining exposure experiments (which lasted 24 h) are du e to the stressor(s) themselves rather than representing an artifact of the clams bei ng maintained in the flow-through system. Antioxidant Protein, Lipid Peroxidation, and Oxidative Repair Enzyme Expression To test for evidence that 24 hour exposure to hypoxia, hyperoxia or sulfide induced a cellular-level response consiste nt with oxidative stress in D. variabilis with respect to normoxia exposure, we measured changes in the expression of the antioxidant stress proteins MnSOD,

PAGE 37

37 Cu/Zn SOD and GPx, and the DNA repair enzyme OGG1, as well as the concentration of 4-hydroxy-2 E -nonenol adducted to protein (HNE), with the results as follows. MnSOD. MnSOD is typically located in the mito chondrial matrix of eukaryotic cells. It catalyzes the dismutation of superoxide to the less reactive pro-oxidant H2O2 (Halliwell and Gutteridge, 1999). The appropriate banding pattern for this antibody is a single dominant band at approximately 25 kD (EnVirtue Biotechnologies, In c. product information; Fig. 2-2). In clams collected and exposed to stress ors in the fall, exposure to hypoxia had no effect on MnSOD expression, whereas exposure to hyperoxia or su lfide resulted in twice the MnSOD expression levels compared to clams exposed to normoxi a (Fig. 2-5A, Table 2-1). Clams collected and exposed to these stressors in the spring show ed no significant change in MnSOD expression (Fig. 2-5B, Table 2-1). Cu/Zn SOD. Cu/Zn SOD is primarily located in the cytoplasm but may also be detected in lysosomes, mitochondria, peroxisomes and the nucle us, but the isoform detected in this study is expressed in the cytoplasm. As with MnS OD, Cu/Zn SOD catalyzes the dismutation of superoxide into H2O2 (Halliwell and Gutteridge, 1999). The a ppropriate banding pattern for this antibody is a single dominant band at approximate ly 19 kD (EnVirtue Biotechnologies, Inc. product information; Fig. 2-2).T here were no signifi cant changes in Cu/Zn SOD expression in clams collected and exposed to any stressors in the fall (Fig. 2-5C, Table 2-1), whereas in clams collected and exposed to stressor s in the spring, exposure to sulfid e caused significantly elevated Cu/Zn SOD (Fig. 2-5D, Tabl e 2-1), Exposure to hypoxia or hyperoxia had no effect. GPx. GPx is located primarily in the cytoplas m (60-75%) and to a lesser extent in the mitochondria. This selenoprotei n catalyzes the reduction of H2O2 to water with the concomitant oxidation of reduced glutathione (Halliwell and Gutteridge, 19 99). The appropriate banding

PAGE 38

38 pattern for this antibody is several bands at ap proximately 20 35 kD with additional bands from tetramer formation possible in the 70 – 90 kD range (EnVirtue Biotechnologies, Inc. product information; Fig. 2-2). The response of GPx was similar to that of Cu/Zn SOD. There were no significant changes in expression of GPx in clams collected and e xposed to any stressors in the fall (Fig. 2-5E, Table 2-1), whereas in clams co llected and exposed to stressors in the spring, exposure to sulfide caused significantly elevated GPx (Fig. 2-5F, Table 2-1) in comparison to clams exposed to normoxia. Exposure to hypoxi a or hyperoxia had no significant effect. HNE. HNE-adducted protein is a peroxidation pr oduct of polyunsaturated fatty acids and indicates increased oxidative damage to lip ids (Halliwell and Gutte ridge, 1999). The antibody detects all HNE-adducted proteins and therefor e does not produce a di stinct banding pattern. There were no significant change s in HNE-adducted protein in cl ams collected and exposed to stressors in the fall, regardless of the stress or (Fig. 2-5G, Table 2-1). In the spring, clams exposed to sulfide, but not hypoxia or hyperoxia, had significantly lower HNE levels than did clams in normoxia (Fig. 2-5H, Table 2-1). OGG1-m. OGG1-m is a DNA repair enzyme located in the mitochondria. It catalyzes the removal of the highly mutagenic 8-hydroxyguani ne (8-OH-G) lesion (Boiteux and Radicella, 2000), which can be generated by oxidative stress and ionizing radiati on and, if not removed, causes GC to TA transversions upon replication (Boiteux and Ra dicella, 2000). The appropriate banding pattern for this antibody is one or two bands at approximately 38 45 kD (EnVirtue Biotechnologies, Inc. product information; Fig. 22). Expression levels of OGG1-m were near the lower detection limit in clams collected and ex posed to stressors in the fall, regardless of the stressor (Fig. 2-5I, Table 2-1). In contrast, clam s collected and exposed to hyperoxia or sulfide,

PAGE 39

39 but not hypoxia, in the spring ha d significant increases in OGG1 -m expression (Fig. 2-5J, Table 2-1). Protein Rescue and/or Degradation To determine whether D. variabilis exposed to the stressors for 24 hour showed responses characteristic of increased protein denaturation c onditions, we measured the expression levels of ubiquitin, small heat shock prot ein (sHsp), Hsp60, Hsp70 and Grp75. Ubiquitin. Ubiquitin is a small (76 amino acids ), highly conserved protein that is expressed in the nucleus, cytoplasm, and cell memb rane of eukaryotic ce lls. It facilitates the degradation of proteins damaged by oxidation (or by other processes) by at taching to the target proteins and aiding in their transport to the 26S proteasome (Wilkinson, 2000; Pickart, 2001; Schnell and Hicke, 2003; Herrmann et al., 2004). Th e antibody detects all ubiquitinated proteins and therefore does not produce a distinct bandin g pattern. In clams collected and exposed to stressors in the fall, hypoxia and hyperoxia had no effect on ubiquitin expression compared to clams exposed to normoxia, whereas clams expos ed to sulfide had si gnificantly increased ubiquitin expression (Fig. 2-6A, Ta ble 2-1). In clams collected a nd exposed to stressors in the spring, ubiquitin expression was not significan tly affected (Fig. 2-6B, Table 2-1). sHsp. sHsp are a group of proteins found in the cytosol, nucleus, and mitochondria (Downs et al., 1999). They bind denatured proteins preventing irreversible protein aggregation, and participate in the ubiquiti n/proteasome system (Parcellie r et al., 2005)..The sHsp are involved in protective responses to a wide range of stressors, in cluding oxidative stress, heat shock and environmental toxins (Downs et al ., 2001a, 2001b; Basha et al., 2004; Arrigo et al., 2005). Since the antibody detects all sHsp-adducted pr oteins, as well as tes ting for the proteins themselves, which form up to five bands ra nging from 10 kD to 45 kD, testing for antibody specificity in the manner shown for the other an tibodies is not appropria te. Clams collected and

PAGE 40

40 exposed to stressors in the fall did not show any significant differe nces in sHsp expression (Fig. 2-6C, Table 2-1). However, clams collected and exposed to sulfide in the spring had elevated sHsp expression, whereas exposure to hypoxia and hyperoxia had no effect (Fig. 2-6D, Table 21). Hsp70. Hsp70 family proteins are present in prokaryotes and in most cellular compartments in eukaryotes. They have numerous roles involving chaper one functions, protein degradation (Chapple et al., 2004) and protein folding (Fr ydman, 2001; Kregel, 2002). The appropriate banding pattern for this antibody is two bands at approximately 70 kD (EnVirtue Biotechnologies, Inc. product information; Fig. 2-2) In clams collected and exposed to stressors in the fall, exposure to hyperoxia caused si gnificantly higher Hsp70 expression compared to clams exposed to normoxia, whereas exposure to hypoxia and sulfide had no effect (Fig. 2-6E, Table 2-1). There were no signi ficant differences in Hsp70 e xpression among clams collected and exposed to stressors in the spring (Fig. 2-6F, Table 2-1). Hsp60 and Grp75. Hsp60 is expressed in the mitochondria. It aids in the folding of newlyformed proteins under normal physiological co ndition and refolds damaged proteins during stress (Hartl, 1996; Kregel, 2002). The appropria te banding pattern for Hsp60 is one band at approximately 60 kD (EnVirtue Biotechnologies Inc. product information; Fig. 2-2). Grp75, which is also known as mitochondrial hsp70, is primarily expressed in the mitochondria. The appropriate banding pattern for Grp75 is one band at approximately 75 kD (EnVirtue Biotechnologies, Inc. product information; Fig. 2-2) .It is involved in severa l processes, including responses to oxidative stress (M itsumoto et al., 2002), chaperone functions, and intracellular trafficking (Wadhwa et al., 2002).We did not detect any significant differences in Hsp60 (Fig. 2-

PAGE 41

41 6G and H, Table 2-1) or Grp75 (F ig. 2-6I and J, Table 2-1) ex pression in clams collected and exposed to stressors in either fall or spring. Cytoskeletal Protein Content We also measured the total actin pool to assess whether D. variabilis exposed to the stressors experienced cellular damage in the form of disruption of the cyto skeleton or changes in actin production, which could indicate a change in metabolic activity. The appropriate banding pattern for this antibody is one band at appr oximately 43 kD (Stressg en Bioreagents product information; Fig. 2-2).Clams collected and exposed to stressors in the fa ll showed no significant differences in total actin pool (Fig. 2-7A, Table 2-1). However, clams collected in the spring and exposed to hypoxia and sulfide, but not hyperoxi a, had significantly decreased total actin pool (Fig. 2-7B, Table 2-1). Discussion Donax variabilis upregulated expression of some antioxi dants, proteins involved in protein rescue and/or degradation, and re pair enzymes in response to 24 hour exposure to sulfide and, to a much lesser extent, to hyperoxia. We also found elevated levels of the lipid peroxidation endproduct, HNE, in clams exposed to hyperoxia but not to sulfide. However, there was a marked seasonality in the response to stressors, with clams collected a nd tested in the spring showing greater expression of many stress proteins and signi ficant decreases in HNE-adducted proteins and actin. Finally, we f ound that hypoxia and sulfide were le thal stressors for the clams, although clams in the spring experiment tolerated th e stressors for a longer duration. In a marine invertebrate that likely does not experience sulfide, hypoxia or hyperoxia in its habitat, these results indicate that 1) exposure to sulfide, and probably hyperoxia, i nduces increased stress protein expression and lipid pero xidation in a pattern consistent with oxidative stress, and 2)

PAGE 42

42 clams in the spring had an increased stress pr otein response and decrea sed evidence of injury (decreased HNE and increased survival ) compared to clams in the fall. Abiotic Factors are Linked to Free Radical Production Hypoxia. Hypoxia is widespread and occurs naturall y in marine habitats particularly in coastal areas affected by upwelli ng, rock pools in the intertidal zone, and all marine sediments (Grieshaber et al., 1994; Diaz a nd Rosenberg, 1995). Hypoxia has b een linked to increased free radical production, but whether hypoxia can induce free radical pr oduction directly, or whether reoxygenation following a period of hypoxia is nece ssary for free radical production is not yet understood (Kukreja and Janin, 1997; Hermes-Lima et al., 1998; Halliwell and Gutteridge, 1999; Semenza, 2000; Hermes-Lima and Zenteno-Savin, 2002; Li and Jackson, 2002). During hypoxia, the absence of O2 as the final electron acceptor causes accumulation of electrons in mitochondrial electron transport chai ns (i.e., the chains are reduced), with the result that a sudden return of O2 can cause the production of superoxide due to nearly instantaneous reactions between O2 and the accumulated free el ectrons (Du et al., 1998; Li and Jackson, 2002). Such a scenario might occur during tidal flow for inter tidal animals. However, several recent studies have also shown free radical production dur ing hypoxia without subsequent reoxygenation. These are based on direct measurement of free radicals (Vanden Hoek et al., 1997; Chandel et al., 1998; Becker et al., 1999), measurement of oxidative DNA damage in mammalian cells (Englander et al., 1999) and yeast cells (Dirmeier et al., 2002), a nd the indirect measures of changes in antioxidant expressi on and/or activity in goldfish (Lushchak et al., 2001) and an estuarine crab (de Oliveira et al., 2005). Severa l studies of both vertebrates and invertebrates have noted decreased or unchange d antioxidant expression or ac tivity during hypoxia, consistent with an overall metabolic depression during hypoxia (Hass and Massaro, 1988; Willmore and Storey, 1997; Joanisse and Storey, 1998; Larade and Storey, 2002). In the current study, clams

PAGE 43

43 exposed to hypoxia did not show significant changes in antioxidant protein expression, regardless of the season in which the experiment s were performed. Similarly, we did not detect significant changes in proteins involved in expression levels of proteins involved in protein rescue and/or degradation in hypoxia-exposed clams, consistent with some vertebrate studies (Gupta and Knowlton, 2002) but not others (Currie and Boutilie r, 2001; Magalhes et al., 2004, 2005). We detected a significant decreas e in total actin expression in D. variabilis exposed to hypoxia in the spring but not the fa ll. A decrease in total actin pr otein expression is consistent with previous studies of bovine brain endothelial cel ls exposed to hypoxia (Brown and Davis, 2005) and human cortical neurons e xposed to a free radical generati ng neurotoxin (Allani et al., 2004). Interestingly, although D. variabilis exposed to hypoxia did not show evidence of oxidative damage, which could ha ve included alterations in an tioxidant or OGG1-m expression or increases in HNE, hypoxia none theless constituted a lethal stress in both fall and spring survival experiments. This is consistent with a previous study of hypoxia exposure in the congener D. serra (Laudien et al., 2002). Therefore, D. variabilis are vulnerable to moderately hypoxic conditions, which they typically do not enc ounter in their habitat. The absence of a stress protein response consistent with elevat ed free radical production suggests that oxidative stress does not play a large role in the mechan ism of hypoxic death, or that these clams were so severely stressed that they were unable to ap propriately respond to hypoxia-induced oxidative stress (Werner and Hinton, 1999). Hyperoxia. Hyperoxic conditions are pr esent in a variety of ma rine habitats, including rocky intertidal pools with photosynthetically active algae (Truchot and Duhamel-Jouve, 1980), boundary layers of intertidal seaweed (Irwin and Davenport, 2002) and brown algae (Pohn et al.,

PAGE 44

44 2001), in the cold seawater of polar regions (Viarengo et al., 1995; Ab ele and Puntarulo, 2004), and within some algal-cnidarian symbioses (D ykens et al., 1992; Richier et al., 2003, 2005). Elevated cellular O2 levels increase mitochondrial free ra dical production (Boveris and Chance, 1973; Akbar et al., 2004), oxidative damage (Den nog et al., 1999), and antioxidant responses in tissues of both vertebrates (O'Donovan et al., 2002; Cho et al., 2005) and invertebrates (Viarengo et al., 1995; Abele and Puntarulo, 2004). Noneth eless, hyperoxic exposure is not linked to changes in SOD activity in the polychaete Heteromastus filiformis (Abele et al., 1998b) or in humans exposed to hyperbaric oxygen treatment (Dennog et al., 1999). In the current study, we found a significant increase in MnSOD expression in D. variabilis that were exposed to hyperoxia in the fall experi ment. We did not detect significant changes in the expression levels of the other two antioxidant s, Cu/Zn SOD (Freiberger et al., 2004) and GPx (Allen and Balin, 2003), or the marker of lipid peroxidation (HNE). Howe ver, we did detect significant increases in expression of the mitochondrial DNA repair enzyme OGG1-m in hyperoxia-exposed clams from the spring expe riment. One possible explanation for this discrepancy is that MnSOD did not blunt the in creased mitochondrial free radical production in clams exposed to hyperoxia in the spring, thereby resulting in DNA damage and a consequent stimulation of increased OGG1-m expression. Among the proteins involved in protein resc ue and/or degradati on, the only significant change in expression was an increase in Hsp70 expression in D. variabilis exposed to hyperoxia in the fall. A link between Hsp70 expression and hyperoxia is well supporte d by studies utilizing a number of different organisms and cell types (Wong et al., 1998; Dennog et al., 1999; Akbar et al., 2004; Shyu et al., 2004; Cho et al., 2005) and may have contribu ted to the clams’ increased survival in response to hyperoxia. We did not detect an effect of hyperoxia on total actin

PAGE 45

45 expression. This contrasts with studies of cultu red mammalian cells, which show that hyperoxia causes decreased actin gene expression (Cho et al., 2005) and that toxi n-induced free radical production (although not necessarily hyperoxia) causes decreased actin protein expression but not decreased gene expression (Allani et al., 2004). While the stress protein results in this study present some evidence that exposure to hype roxia caused a stress re sponse indicative of increased free radical production, the survival experiments showed th at exposure to the hyperoxic condition was a sublethal stressor. Hydrogen sulfide. Animals in a variety of marine ha bitats, including mudflats, mangrove swamps, deep-sea hydrothermal vents, hydrocarbon seeps and anoxic basins, are periodically or continuously exposed to sulfide at levels up to 12 mmol L-1 (Fenchel and Riedl, 1970; Somero et al., 1989; Bagarinao, 1992). Hydrogen sulfide is a highly reactive toxin that diffuses freely across respiratory surfaces and therefore cannot be excluded from tissues (Denis and Reed, 1927; Julian and Arp, 1992). Hydrogen sulfide has severa l mechanisms of toxi city, including the reversible inhibition of cytochrome c oxidase, the final enzyme of the mitochondrial electron transport chain (Lovatt Evans, 1967; Nicholls, 1975), reduction in hemoglobin oxygen affinity (Carrico et al., 1978) and inhi bition of approximately 20 enzy mes (Bagarinao, 1992). Hydrogen sulfide oxidizes spontaneously in the presence of divalent metals (b oth dissolved and in metalloenzymes), generating oxygen-cen tered (likely superoxide) and sulfur-centered radicals in aqueous solutions (Chen and Morris, 1972; Tapley et al., 1999) and in animal tissues (Tapley, 1993; Abele-Oeschger and Oeschger, 1995; Eghbal et al., 2004; Julian et al., 2005). Organisms inhabiting sulfide-rich environments employ a vari ety of strategies to detoxify sulfide (Lovatt Evans, 1967; Vismann, 1991; Grieshaber and Vlkel 1998). Marine invert ebrates that are not found in sulfide-rich habitats a nd that do not employ sulfide det oxification strategies typically

PAGE 46

46 show increased mortality upon exposure to sulfid e, such as can occur in upwelling events (Grieshaber and Vlkel, 1998). We found that 0.1 mmol L-1 sulfide was a lethal stressor for D. variabilis in both fall and spring surviv al experiments. These results are consistent with a previous study of sulfid e tolerance in juvenile Donax serra which documented a LT50 of 80 hour with exposure to 0.1 mmol L-1 under hypoxic conditions (Laudien et al., 2002). Of the three stressors we tested, we found the greatest evidence for a stress protein response, consistent with a cellula r response to oxidative stress in D. variabilis exposed to sulfide, and the response was st rongest in the spring experiment. Specifically, clams exposed to sulfide had elevated expression of MnSOD (f all), Cu/Zn SOD (spring), GPx (spring), and OGG1-m (spring). Two of the prot eins involved in protein rescue and/or degrad ation, ubiquitin (fall) and sHsp (spring), also increased in clams exposed to su lfide. In contrast, the lipid peroxidation marker HNE and total actin expres sion levels were significantly decreased in sulfide-exposed clams in the spring experiment. These results suggest th at cellular response of D. variabilis to sulfide is consistent w ith a response to oxidative st ress and that the mortality detected in the sulfide exposure treatment in bot h fall and spring survival experiments could be linked to oxidative stress in D. variabilis which do not normally encounter sulfide. Several studies have specifically addressed the link between oxidative damage and sulfide exposure at the organismal level. For example, antioxidant enzyme activities were proportional to the sulfide toleranc es of thiobiotic meiofauna (Mo rrill et al., 1988). Similarly, MnSOD activity increased in response to sulfide exposure in the chemoautotrophic symbiotic bivalve Solemya velum but not in the related nonsymbiotic Yoldia limatula (Tapley, 1993). In contrast, a relationship between sulfide tole rance and antioxidant activity was not found in a survey of sulfide-tolerant polychaetes and bivalves (A bele-Oeschger, 1996). A relationship between

PAGE 47

47 sulfide and free radical production has also been i nvestigated at the cellu lar level; in isolated erythrocytes from the marine polychaete Glycera dibranchiata 1 hour sulfide exposure causes mitochondrial depolarization, increased superoxide production and increased cellular oxidative stress (Julian et al. 2005). Recently, Eghbal et al (2004) showed that free radical production in rat hepatocytes was two-to-three times faster wh en the cells were exposed to 0.5 mmol L-1 sulfide than when they were exposed to cyanide or control conditions, and that the addition of ROS scavengers decreased cell death by up to 40% in hepatocytes exposed to 0.5 mmol L-1 sulfide for 3 hours. These studies, in conjunction with the results of the current study, support the theory that increased oxidative stress is a mechanism by which sulfide exposure causes toxicity in marine animals (Morrill et al ., 1988; Tapley, 1993; Abele-Oesc hger et al., 1994, 1996; Julian et al., 2005). Sources of Variance A number of factors, both in the collection and treatment of the clams as well as in the tissue processing procedure, could have contributed to the varian ce detected within treatment groups. We employed several methods to minimi ze the influence of th ese factors. These included: 1) To control for effects of daily cycles in stress protei n production (Podrabsky and Somero, 2004), we collected the clams within 30 minutes of high tide. 2) We did not determine the ages of the individuals, which in mammals is closely linked to both endogenous free radical production (Barja, 2002) and ability to synthesize functional stress prot eins (Szczesny et al., 2003), but we did control for sh ell length. Populations of D. variabilis from sites on the eastern Florida coast reach maturity in spring and fall an d most individuals live fo r one year (Mikkelsen, 1985). Given these patterns in abundance and size-f requency, it is likely that the clams sampled in the current study were adult and of similar age. 3) We selected D. variabilis as a study species because it inhabits a habitat that lacks extremes of dissolved O2 or sulfide, which allowed us

PAGE 48

48 minimize the potential complications of pr econditioning from exposure to environmental stressors (Kultz, 2005). 4) An additional met hod to limit preconditioning effects would have been to acclimate the clams in the laboratory prior to the experiment. However, because we found slightly elevated Hsp70 expression levels in clams maintained in normoxic conditions for several days, laboratory acclimation would likely ha ve introduced additional variables. Given the short survival times demonstrated in the su rvival experiments and the potential confounding effect of starvation (Morales et al., 2004), we exposed the clams to the stressors for only 24 hours. 5) To minimize the effects of daily variation in tissue processing methodology, we homogenized and suspended all samples on the sa me day and with the same batch of buffer solution. 6) Stress protein expression levels were determined fo r all samples at the same time and on the same piece of membrane to minimi ze inter-membrane staining differences. We found that variance in the stress treatment groups was elevated in comparison to variance in the normoxia samples in all stress pr oteins that had signifi cant changes (excluding actin). This relationship between stressful treatm ents and elevated variance has been documented in ecotoxicological studies (Orl ando and Guillette, 2001) and may even be useful as a biomarker (Callaghan and Holloway, 1999). For example, Callaghan and Holloway (1999) found that when weevils ( Sitophilus oryzae ) were transferred to a toxic food s ource, the mean activity levels of glutathioneS -transferase and two naphthyl acetate estera ses did not change si gnificantly, but the variances about the means increased up to 5 fold. The elevated variance that we detected in clams exposed to stressors is cons istent with the concep t that, at the population level, variance in a physiological metric is indicative of stress. Physiological Responses to Stress Vary by Season The importance of assessing seasonal cha nges in the physiological stress response, particularly stress protein expres sion and activity levels, is well es tablished. A number of factors,

PAGE 49

49 including availability of nutrien ts, temperature variation, reproducti ve status and growth cycle, and seasonal patterns in envir onmental stressors, shape season al changes in bivalve stress physiology (Sheehan and Power, 1999). For exampl e, warm summer conditions were linked to higher catalase and glutathioneS -transferase activities and higher condition index in Mytilus galloprovincialis (Romo et al., 2003a), higher hsp70 levels in M. edulis (Chapple et al., 1998), and higher ubiquitin conjugate and hsp70 levels in M. trossulus (Hofmann and Somero, 1995). Seasonal differences in catalase, metallothionein, and gluathioneS -transferase levels in M. edulis and Macoma balthica corresponded with patterns of temperat ure and food availability as well as reproductive phase (Leini and Lehtonen, 2005). Helix aspersa snails estivating during the summer have a greater stress protein response, lower lipid peroxida tion and lower protein carbonyl levels than those esti vating during the winter (Ramos -Vasconcelos et al., 2005). Similarly, we detected greater changes in expressi on levels of the antioxida nt proteins and some of the proteins involved in protei n rescue and/or degradation in cl ams from the spring experiment in comparison to clams from the fall experiment. Th is increased expression of stress proteins in clams collected in the spring may have contributed to their enhanced survival (Chapple et al., 1998). Conclusions Marine invertebrates from su lfidic environments are like ly to have physiological or biochemical adaptations to limit their susceptibilit y to these abiotic stressors (Grieshaber et al., 1994; Grieshaber and Vlkel, 1998). Such adapta tions would reduce the need for upregulated expression of stress proteins dur ing experimental exposure to sulfide, thereby reducing our ability to detect whether this stressor has the ca pacity to cause oxidative damage. The surf clam D. variabilis does not experience hypoxia, hyperoxia or su lfide in its habitat and therefore is likely to be more sensitive to these stressors. Over the course of the survival experiments,

PAGE 50

50 particularly in the fall experiment, both sulf ide exposure and hypoxia e xposure were lethal, whereas hyperoxia and normoxia were not. Therefor e, increased expression of key antioxidants and repair enzymes following 24 hour exposure to sulfide, particularly during the spring experiment, but not hypoxia, sugge sts that the expression changes were a specific response. It remains to be determined how this protein expre ssion pattern differs at sh orter and longer time points, although substantially longer time point s in hypoxia and sulfide treatments resulted in mortality. Consequently, these data are consistent w ith sulfide, and to a lesser extent hyperoxia, causing oxidative stress. It remain s to be determined whether anim als evolutionarily adapted to sulfide exposure have increased capacity for stre ss protein expression to limit oxidative damage, as has been shown for Hsp70 expression in inte rtidal mussels exposed to thermal stress (Hofmann and Somero, 1995; Hofmann, 1999).

PAGE 51

51Table 2-1. Summary of statistical resu lts from comparisons between samples from Donax variabilis exposed to normoxia treatment and samples from animals exposed to hypoxia, hyperoxia, and sulfid e. Data were analyzed by Kruskal-Wallis ANOVA followed by pairwise Conover-Inman post hoc comparison tests. Significant values ( p < 0.05) are highlighted with bold text T values are for data pooled across treatment within each season. p value from post-hoc pairwise comparison T value (df=3) Hypoxia Hyperoxia Sulfide Protein type Protein Fall Spring Fall Spring Fall Spring Fall Spring MnSOD 8.112 0.948 0.181 0.389 0.033 0.593 0.003 0.639 Cu/Zn SOD 1.743 4.346 0.413 0.26 0.629 0.326 0.253 0.047 Antioxidant GPx 3.331 4.787 0.178 0.316 0.713 0.173 0.158 0.038 Lipid peroxidation HNE 2.978 6.794 0.391 0.132 0.145 0.736 0.828 0.038 Oxidative repair OGG1-m 1.261 11.206 0.44 0.089 0.421 0.0007 0.865 0.0002 Ubiquitin 6.728 2.713 0.0621 0.359 0.436 0.359 0.014 0.134 sHsp 2.027 5.581 0.337 0.297 0.321 0.119 0.235 0.023 Hsp70 7.434 2.051 0.468 0.834 0.027 0.626 0.648 0.222 Hsp60 0.618 0.132 0.691 0.794 0.51 >0.999 0.597 >0.999 Protein rescue and/or degradation Grp75 0.684 0.088 0.509 0.896 0.739 >0.999 0.552 0.896 Cytoskeletal protein Actin 0.485 9.529 0.44 0.048 0.153 0.668 0.401 0.004

PAGE 52

52 Air O2 N2 H2Swater pump MFC3 hypoxia hyperoxia normoxia H2S animal chambers peristaltic pump water bath 55.3 4.75 1.22 58.0 42.0 MFC2 MFC1 reservoir Air Air O2 O2 N2 N2 H2S H2Swater pump MFC3 hypoxia hyperoxia normoxia H2S animal chambers peristaltic pump water bath 55.3 4.75 1.22 55.3 4.75 1.22 58.0 58.0 42.0 42.0 MFC2 MFC1 reservoir reservoir Figure 2-1. Diagram of flow-through system. F iltered, chloramphenicol-treated seawater was pumped from a 20 L reservoir to a 200 ml upper reservoir at 10 ml min-1. Water from this reservoir then drained into each of 4 vertical ga s equilibration chambers. These chambers were constructed of 18 mm inner diameter (i.d.) clear, cast acrylic tube, each 25 cm in length. A sintered glass aerat or in each chamber was used to bubble either air (normoxia), N2 (hypoxia), a mixture of O2 and air (hyperoxia) or a mixture of air and hydrogen sulfide gas (from a compressed tank of 2% H2S, balance N2). Gas mixtures were controlled with three digita l mass flow controllers, indicated in the figure as MFC1, MFC2 (both being FMA5400 single-channel controllers, Omega Engineering, Inc., Stamford, CT, USA) and MFC3 (three-channel controller from Cameron Instrument Co., Port Aransas, TX USA). Controllers that handled hydrogen sulfide gas were customized with corro sion-resistant fitti ngs and O-rings. Gasequilibrated water from each chamber wa s continuously pulled into an animal chamber by a 4-channel peristaltic pump (M asterflex cartridge system, Cole Parmer Instrument Co., Vernon Hills, IL, USA) at 2 ml min-1 per chamber. These chambers were constructed of 18 mm i.d., 15 cm long clear, cast acrylic tube with one-hole rubber stoppers at end, through which the s eawater flowed through 1/8” i.d., 1/16” wall Tygon tubing. All tubing connections were via nylon Luer fittings (Cole Parmer Instrument Co.). Effluent water from the peristaltic pump was monitored periodically for PO 2 and pH. The gas equilibration chambers and animal chambers were housed in a polycarbonate water bath maintained at 24 C. The entire system (except for compressed air, N2 and O2 supplies) was housed in a fume hood to minimize the hazards associated with handling H2S gas.

PAGE 53

53 Figure 2-2. Antibody specificity te sts. Two random samples of Donax variabilis were pooled, subjected to SDS-PAGE, western blotted, and assayed with the antibodies to the following proteins: manganese superoxi de dismutase (MnSOD), copper/zinc superoxide dismutase (Cu/Zn SOD), glut athione peroxidase (GPx), OGG1-mito (OGG1), heat shock protein 70 (Hs p70), heat shock protein 60 (Hsp60), mitochondrial heat shock protein 70 (G rp75), and actin. The positions of known molecular weights standards are indicated by bars to the left of each individual band image and the molecular weight masses in kiloda ltons (kD) are listed at the far left of the figure.

PAGE 54

54 exposure duration (d) 01234567 0 20 40 60 80 100 exposure duration (d) 01234 % surviving 0 20 40 60 80 100 normox hyperox hypox sulfideA. Fall B. Springnormox hyperox hypox sulfide Figure 2-3. Fraction of surviving Donax variabilis clams in survival experiments in fall and spring. The fall experiment was conducted fo r 4 d and the spring experiment for 7 d. The treatments were: normoxia (solid line; 21.6 1.9 kPa PO 2), hypoxia (dotted line; 12.3 1.4 kPa PO 2), hyperoxia (dashed line; 36.6 3.0 kPa PO 2), and normoxic sulfide (alternating dashes and dots; 98 2.9 mol L-1 total sulfide, 24.0 1.8 kPa PO 2). Eight clams were exposed to each condition in each season.

PAGE 55

55 Figure 2-4. Expression levels of Hsp70 in Donax variabilis exposed to normoxia for 0, 1, 3, and 5 days. Clams used in the 0 time point were frozen in liquid N2 immediately after collection at the beach. Data are presented as a scatterplot with asymmetrical error bars denoting minimum and maximum values and central dot signifying the mean of five clams per day. Data are given as re lative units per nanogram of total soluble protein (RU ng TSP-1). Abbreviations: day, d. Data were analyzed by KruskalWallis ANOVA but were not st atistically significant. exposure duration (d) 0135 RU ng TSP-1 0 50 100 150 200 250 300

PAGE 56

56 Figure 2-5. Expression levels of three antioxidant proteins, a lip id peroxidation marker, and an oxidative repair enzyme in Donax variabilis exposed to normoxia (normox), hypoxia (hypox), hyperoxia (hyperox), and sulfide. Da ta are presented as scatterplots with asymmetrical error bars denoting minimu m and maximum values and central dot signifying the mean of four clams per tr eatment in fall (left) and spring (right) experiments. Data for manganese supe roxide dismutase (MnSOD), copper-zinc superoxide dismutase (Cu/Zn SOD), glutat hione peroxidase (GPx), and (4-hydroxy2 E -nonenol-adducted protein) HNE are given as relative un its per nanogram of total soluble protein (RU ng TSP-1), and data for OGG1-mitochondria (OGG1-m) are given as fmoles mg TSP-1. Data were analyzed by Kruskal-Wallis ANOVA and Conover-Inman post hoc pairwise comparisons. Similar letters denote statistically indistinguishable samples in data sets with significant ANOVAs. Data sets with no significant differences by ANOVA contain no letters adjacent to the symbols.

PAGE 57

57 Fall Spring C Cu/Zn SOD normoxhypoxhyperoxsulfide RU ng TSP-1 0 4 8 12 E GPx normoxhypoxhyperoxsulfide RU ng TSP-1 0 4 8 12 G HNE normoxhypoxhyperoxsulfide RU ng TSP-1 0 1 2 3 4 5 6 I OGG1-m normoxhypoxhyperoxsulfide fmol mg TSP-1 0 20 40 60 80 normoxhypoxhyperoxsulfide J OGG1-ma a b b A MnSOD normoxhypoxhyperoxsulfide RU ng TSP-1 0 2 4 6 8 normoxhypoxhyperoxsulfide B MnSOD normoxhypoxhyperoxsulfide D Cu/Zn SODa a a b normoxhypoxhyperoxsulfide F GPxa a a b normoxhypoxhyperoxsulfide H HNEa ab a bc a ab bc c

PAGE 58

58 Figure 2-6. Expression levels of fi ve proteins involved in protei n rescue and/or degradation in Donax variabilis exposed to normoxia (normox), hypoxia (hypox), hyperoxia (hyperox), and sulfide. Data ar e presented as scatterplots wi th asymmetrical error bars denoting minimum and maximum values and ce ntral dot signifying the mean of four clams per treatment in fall (left) and spri ng (right) experiments. Data for ubiquitin, heat shock protein 70 (Hsp70), heat shock protein 60 (Hsp60), and mitochondrial heat shock protein 70 (GRP75) are given as relati ve units per nanogram of total soluble protein (RU ng TSP-1). Data for small heat shock protein (sHsp) are given as fmol mg TSP-1. Data were analyzed by Kruskal-Wallis ANOVA and Conover-Inman post hoc pairwise comparisons. Similar letters denote statistically indistinguishable samples in data sets with significant ANOVAs. Data sets with no significant differences by ANOVA cont ain no letters adjacent to the symbols.

PAGE 59

59 Fall Spring C sHSP normoxhypoxhyperoxsulfide fmol ng TSP -1 0.00 0.05 0.10 0.15 E Hsp70 normoxhypoxhyperoxsulfide RU ng TSP -1 0 1 2 3 4 G Hsp60 normoxhypoxhyperoxsulfide RU ng TSP -1 0 10 20 30 I Grp75 normoxhypoxhyperoxsulfide RU ng TSP -1 0 2 4 6 8 10 normoxhypoxhyperoxsulfide A Ubiquitin normoxhypoxhyperoxsulfide RU ng TSP -1 0 1 2 3 4 normoxhypoxhyperoxsulfide normoxhypoxhyperoxsulfide normoxhypoxhyperoxsulfide normoxhypoxhyperoxsulfide D sHSP F Hsp70 H Hsp60 J Grp75 B .Ubiquitina a b a a a a b a a a b

PAGE 60

60 FallSpring A Actin normoxhypoxhyperoxsulfide RU ng TSP -1 0 1 2 3 4 normoxhypoxhyperoxsulfide B Actina b a b Figure 2-7. Expression leve ls of total actin in Donax variabilis exposed to normoxia (normox), hypoxia (hypox), hyperoxia (hyperox) and sulfide. Data are pr esented as scatterplots with asymmetrical error ba rs denoting minimum and maxi mum values and central dot signifying the mean of four clams per tr eatment in fall (left) and spring (right) experiments. Data are given as relative un its per nanogram of total soluble protein (RU ng TSP-1). Data were analyzed by Kruskal-Wallis ANOVA and Conover-Inman post hoc pairwise comparisons. Similar letters denote statistically indistinguishable samples in data sets with significant ANOVAs. Data sets with no significant differences by ANOVA cont ain no letters adjacent to the symbols.

PAGE 61

61 CHAPTER 3 PHYSIOLOGICAL RESPONSES OF Mercenaria mercenaria TO SINGLE AND MULTIPLE ABIOTIC FACTORS Introduction The examination of how physical conditions in the environment interact with physiology to influence the distribution of organisms is a l ong-standing goal of ecologi sts, particularly of physiological ecologists (Brown et al., 1996; Sp icer and Gaston, 1999). Numerous studies have attempted to define a species’ niche, the set of environmental variables that limits reproduction and survival and therefore impacts dist ribution and abundance (Brown et al., 1996). Traditionally, these studies involved either in situ manipulations combining biotic interactions with environmental variables (exemplified by Connell, 1961) or la boratory studies of physiological responses to abiotic factors. Unlike in situ manipulations, which allow the experimental subjects to be imp acted by all aspects of a complex environment, laboratory studies involve acclimation to one, or at most two, select environmental variables. Although the limitations of extrapolating laboratory studies of single variables to popu lation-level processes have long been identified (e.g., Hall, 1964), recent reviews conti nue to emphasize the importance of investigating multiple abiotic factors in the laboratory (e.g., Brown et al., 1996). The interplay between tolerances of abiotic f actors with biotic inte ractions and species distributions have been rigorously studied in the rocky intertidal habitat (for review, Menge and Olson, 1990; Benson, 2002; Tomanek and Helmuth, 2002). This habitat, with its steep gradients of multiple abiotic factors, diel and seasonal patterns, accessibility, and well-characterized communities, serves as a mode l system for experimental population and community ecology. Additionally, since most of the st udy organisms are sessile as adu lts, distribution patterns are not confounded by behavioral responses to stressors, such as use of refuges. This habitat has

PAGE 62

62 facilitated many recent advances in physiol ogical ecology, such as the linking of population distribution with cellular-leve l responses including heat shoc k protein production (Hofmann et al., 2002). The approaches taken and conclusions reached in studies of rocky intertidal invertebrates can be applied to organisms inhabiting other variab le aquatic habitats. Estuaries share many similarities with the ro cky intertidal. Estuaries have seasonal and daily fluctuations in a variety of abiotic factors, including te mperature, salinity, dissolved O2 levels, and pH (Hubertz and Cahoon, 1999; Beck and Bruland, 2000). The organisms inhabiting estuaries, like those of the rocky intertidal, must be broadly tolerant of environmental stressors (Fisher, 1977; Parsons, 1994). Although the phys iological strategies employed by estuarine organisms are not as well understood as those of rocky intertidal i nvertebrates, several similarities are apparent. For example, alterati ons in heat shock protei n expression and function in estuarine organisms have been linked to e nvironmentally-relevant vari ations in temperature (Zippay et al., 2004) and salinity (Blank et al., 2006). Therefor e, estuarine organisms, like intertidal invert ebrates, could be developed as models for studies of how physiological responses to multiple environmental variables interact to affect distribution and abundance. One such invertebrate species that is tolera nt of multiple environmental stressors, wellstudied, broadly distributed in coastal habita ts, and accessible year-round is the northern quahog or hard clam, Mercenaria mercenaria (Kraeuter and Castagna, 2001). These clams live at intertidal and subtidal depths in bays and estuaries along the A tlantic coast of North America, from the Gulf of St. Lawrence to the s outhern Florida coas tline (Harte, 2001). Mercenaria mercenaria is a generalist in its tolerance of temperature extremes, low dissolved O2 levels (hypoxia), and salinity extremes (Grizzle et al., 2001), as w ould be expected given its distribution (Lynch and Gabriel, 1987; Gilchrist, 1995). It is th erefore an appropriate model

PAGE 63

63 organism for an investigation of how abiotic f actors, singly and in combination, affect the physiology of aquatic inverteb rates (Grizzle et al., 2001). The current study examined how hypoxia, high temperature, and hyposalinity (reduced salinity), singly and in combination, a ffect the physiological responses of M. mercenaria The physiological responses were divided into two categories, traditional functional markers and cellular-level indicators. Tradit ional functional markers measure long-term growth and fecundity responses to sublethal environmental stressors (Widdows, 1985). These assays measure general, whole-organismal responses, integrating the eff ects of the environmenta l stressor over several hierarchical levels of biol ogical organization (Stebbing, 1985). Traditional assays measure behavioral responses such as valve-closure in bivalves (Heinonen et al., 1997), burial ability (Savage, 1976), and metabolic responses such as glycogen content (Ham za-Chaffai et al., 2003), and condition index (Romo et al., 2003a). In recent years, cellular-level markers, such as stress protein expression (Sanders, 1993; Feder and Ho fmann, 1999), oxidative damage markers, and RNA:DNA ratio (Elser et al., 2000), have become increasingl y powerful and popular tools in part because they test for evidence of stress at the level of organizati on primarily affected; the molecular level (Stebbing, 1985; Bierkens, 2000). However, few studies have attempted to investigate both cellular-level indicators and tr aditional functional tec hniques (Brown et al., 1995; Hamza-Chaffai et al., 2003; Romo et al ., 2003b). This study attemp ted to delineate the links between cellular-level and whole-organismal responses to individual and multiple abiotic stressors and detail how an integrat ed stress profile changes over time.

PAGE 64

64 Materials and Methods Laboratory Exposures Hard clams (average 12 mm shell length) were obtained from Southern Cross Sea Farms, Inc. (12170 SR 24 Cedar Key, FL) and maintained in 2.5 gallon aquaria with half water changes daily. The seawater was obtained from the Whitney Marine Lab (Marineland, FL) by the Department of Zoology and diluted to 26 ppt salinity (standard concentration for hypoxia and temperature experiments) or the appropriate salin ity (dual stressor experiments) with ultrapure water. The water was pretreated with chloramphenicol (2 mg L-1) to prevent the growth of bacteria. Clams were fed a mixed shellfish diet (1800 formula, Reed Mariculture) at 2-5% of estimated dry weight per day. The single stressor experiments were designe d to examine responses to hypoxia and to elevated temperature. In the hypoxia experiments the clams were exposed to three levels of dissolved oxygen: 1) air saturated (normoxia, approximately 0.25 mmol L-1), 2) mild hypoxia (approx. 0.16 mmol L-1), and 3) moderate hypoxia (approx. 0.07 mmol L-1). Each aquarium was bubbled with air or air/N2 mixes (one aquarium per treatment ). Experimental conditions were monitored daily with a fiber optic O2 probe (Ocean Optics, Inc., Dunedin, FL). All exposures were conducted at room temperature in all seas ons (20-22C). Experime nts were conducted in three seasons: fall (August 2004), winter (Janua ry 2005), and spring (April 2005). Given the lack of a measurable effect of the mild and moderate hypoxia treatments in the fall hypoxia experiment (see results), dissolv ed oxygen levels were decrease d by 50% in both treatments in the winter and spring experiments in co mparison to the fall hypoxia experiment. In the second set of single -stressor experiments, the cl ams were exposed to three temperature treatments: 1) room temperature (approximately 24C), 2) mild temperature elevation (approx. 28C), and 3) moderate temp erature elevation (appr ox. 33C). The water

PAGE 65

65 temperature was maintained by 15 W or 25W a quarium heaters and was monitored daily. All aquaria were bubbled with air and the di ssolved oxygen content was monitored daily. Experiments were conducted during three seas ons: fall (September 2004) winter (February 2005), and spring (May 2005). Experimental conditi ons used in the single-stressor experiments were consistent with environmental values recorded in Florida estuaries (see Discussion for details). The dual stressor experiments were designed to examine responses to elevated temperature at each of three salinities. The clams were e xposed to the following treatments: 1) high temperature/ambient salinity (measurements take n at the hatchery; approximately 33C/24 ppt), 2) high temperature/mild hyposalinity (approx. 33 C/15 ppt), 3) high temperature/moderate hyposalinity (approx. 33C/5ppt), 4) room temperature/ambient sa linity (approx. 24C/24ppt), 5) room temperature/mild hyposalin ity (approx. 24C/15 ppt), and 6) room temperature/moderate hyposalinity (approx. 24C/5 ppt). The water temperature in the high temperature treatment was maintained by 25W aquarium heaters and was monito red in all treatments daily. All aquaria were bubbled with air and the dissolved oxygen conten t was monitored daily. Salinity was measured daily using a portable refractometer. Experime nts were conducted during three seasons: fall (September 2005), winter (January 2006), and spring (May 2006). In all experiments, two aquaria (one holding, one depuration) and one water jug (to provide fresh water of treatment conditions) were mainta ined at the experimental conditions for each treatment. Dissolved oxygen cont ent, temperature, and salinity did not differ among the three water containers for any treatment in any of th e nine experiments (data not shown). Daily half water changes were conducted using water from the jugs, which minimized abrupt changes in temperature or salinity. In all experiments, le vels of ammonia, nitrite, and nitrate were

PAGE 66

66 monitored. The pH of the water was monitored dur ing the first several experiments and averaged 8.17 0.11, which is within the pH range associ ated with optimal growth for this clam (Calabrese and Davis, 1966). The clams were not provided with burrowing substrate except during timed burial analysis tests, which is cons istent with the conditions in which they were acclimated at the hatchery for 1-2 weeks prior to each experiment. Due to space limitations in a shared aquarium room, only one aquarium set was used per treatment. However, the clams could be considered independent experi mental units for the purposes of statistical analyses since we maintained a large ratio of wate r volume (6 L) to clam tissue (m aximum 15.7 g), daily half water changes, sufficient feeding, and constant aeration. Clams were maintained with feeding in experimental aquaria until 24 hours before they were scheduled to be sacrificed (see schedule below), at which time they were transferred to the matching depuration aquaria. The experiments la sted 14 days, with clams sampled on days 1, 5, 9, and 14. The schedule of the experiments is outlined below and was identical for all experiments (actual sample sizes varied with clam availability and effects of the treatments, see details below in the results section): Schedule. Day 0: 20 clams were preserved for Time 0 measurements, < 20 clams were placed into depuration aquaria (for 1 day sampling), the re mainder of the clams were placed into the large aquaria and fed daily. Day 1: The clams in the three depuration aqua ria (< 20 per treatment) were processed. Day 4: 20 clams from each large aquarium were transferred to the matching depuration aquarium. Day 5: The clams in the three de puration aquaria were processed. Day 8: < 20 clams from each large aquarium were transferred to the matching depuration aquarium. Day 9: The clams in the three de puration aquaria were processed.

PAGE 67

67 Day 13: < 20 clams from each large aquarium were transferred to the matching depuration aquarium. Day 14: The clams in the three depuration aquaria were processed. Tissue Processing Once the burial tests were completed (see below), all clams were removed from the depuration tanks. The clams were opened by se vering their adductor muscles were severed. Whole clam tissues were blotted a nd weighed, then flash-frozen in liquid nitrogen and stored at – 80C. All of the biochemical assays were conducted on whol e clams and/or whole clam homogenates from individual clams. When the samp le size was sufficiently large, clams used in the burial tests were not used for glycogen content or stress protein analyses. In several experiments, all available clams were used for bur ial analyses so the clams were gently rinsed before opened and frozen for the biochemical analyses. Survival Analyses In some of the experiments, clams in high te mperature treatments died (see appropriate results sections). To maintain consistency, the experiments were continued through fourteen days with the surviving clams. In experiments with treatment-associated mort ality, survival in all treatments was analyzed using an extension of the nonparametric Gehan’s generalized Wilcoxon test, which assigns a score to each individual’s su rvival time and then calculates and analyzes a Chi-square value for each treatment group (S tatistica version 7.1, Tulsa, OK). Pairwise comparisons were made, when appropriate, using the nonparametric Gehan’s generalized Wilcoxon test (Statistica). Analyses: Ability to bury. Burial rate, which is the proportion of clams that bury themselves in a given period of time when placed on a substrate, can serve as an index of total clam health

PAGE 68

68 (Byrne and O'Halloran, 2000). Sand was utilized as th e substrate, since it is a favorable substrate type for burial of M. mercenaria (Grizzle et al., 2001). On each sampling day, burial analysis was conducted on up to thirteen of the clams in each depuration chamber. Small containers with sand (approximately 2.5 cm deep) were placed into the depuration aquaria and randomly selected clams were placed flat on top of the sand. In the fall hypoxia experiment (first experiment we conducted) only, the containers of sand were reused throughout the experiment. In all subsequent experiments, the containers were filled with new sand on each sampling day. On days 1, 5, 9, and 14, we analyzed burial rate at 5, 10, 20, 30, and 60 min. Burial rate analyses were not conducted on Time 0 clams. Data collected at the 60 minut e time point only were modeled as total number of clams that buried as a function of day and treatment by a generalized linear model with Poisson distribution and log link, Type III likelihood test, with the offset set as the total number of live clams tested on any given day (Statistica) In cases where clam death was high, the model was run with zeros listed for these treatments in both the number of clams buried and the total number of clams, since the analysis is weight ed by the total number tested and requires a balanced design across treatment and day. Glycogen content. Glycogen content, which is a direct measure of a major energy storage reserve, was measured using standard techniques (Byrne and O'Halloran, 2000). Glycogen analyses were conduc ted on homogenates of whole clams, with a sample size of 3-5 clams per treatment per day. Briefly, glycogen was extracted from whole clam homogenates and hydrolyzed overnight by am yloglucosidase. Glucose monomers were quantified by a colorimetric assay that utilizes glucose oxidase/peroxid ase and the substrate o dianosidine. Sample absorbances were read at 450 nm on a Biotek S ynergy-HT plate reader (Biotek, VT). Data from single stressor experi ments were analyzed by a general linear model with treatment nested within experiment dura tion with Fisher’s LSD post hoc comparisons for

PAGE 69

69 those experiments for which there was a balanced number of samples (Statistica). Data from dual stressor experiments could not be analyzed using a nested design given the mortality in multiple treatments and therefore were analyzed within each day by one-way ANOVA with Fisher’s LSD post hoc comparisons. RNA oxidative damage. Amounts of total tissue oxidized RNA bases were determined in 5 clams per treatment (same clams as those used for stress protein assa ys) from dual stressor experiments only using a protoc ol optimized for bivalves (J oyner-Matos et al., 2007). RNA oxidative damage results from increased producti on or decreased detoxification of free radicals (Halliwell and Gutteridge, 1999). RNA gua nine base oxidation produces 8-oxo-7,8dihydroguanosine (8-oxoGuo). Data from dual stre ssor experiments could not be analyzed using a nested design given the mortality in multiple treatments. Data from the 1-day samples are presented and analyzed by 2-factor ANOVA with Fisher’s LSD post hoc comparisons. The 1-day time point was the only sampling time in all thre e experiments that had samples available from every treatment. Stress protein biomarkers. Five clams from each treatm ent group in the single stressor experiments were processed according to EnVirtue Biotechnologies Inc. standard bivalve sample preparation protocols (Downs et al., 2002b; Joyner-Matos et al., 2006). The whole clams were individually homogenized in liquid nitrogen. Small amounts ( 80-100 mg) of homogenized tissue were suspended in suspension buffer [50 mmol L-1 Tris, 15 mmol L-1 EDTA, 2% sodium dodecyl sulfate (SDS), 15 mmol L-1 dithiothreitol (DTT), 0.5% dimethyl sulfoxide (DMSO), and 0.01% Halt protease inhibitor co cktail]. After small amounts of tissue were suspended in suspension buffer, the solutions were vortexed, heat ed (3 minutes at 85C), vortexed, heated (3 minutes at 85C), kept at room temperature for 10 minutes, and then cen trifuged for 10 minutes

PAGE 70

70 at 12,000 rpm (room temperature). Supernatants were aliquotted, frozen in liquid nitrogen, and stored at –80C. Protei n concentrations were determined using a modified Ghosh method (Ghosh et al., 1988) prior to freezing. The samples from the single stressor experi ments were assayed in cooperation with C. Downs using high-throughput ELISA analysis. Af ter thawing, samples were diluted so that application of all samples was 25 nanogram s of total soluble protein per well (30 L volume) on 384-well microtiter plates. Sample dilutions, sa mple application bloc king buffer application, primary and secondary antibody ap plication, plate washes, and de velopment solution were all applied to the microtiter plates by a Beckma n-Coulter Biomek 2000 liquid handling system. Carousels, plate washers, and plate readers were all integrated with the Biomek 2000 to produce a functional high-throughput system Standard curves of one sample diluted over an eight-point curve were dispersed across the plates to accomm odate minor artifact formation associated with microtiter plate ELISA systems (e.g., edge-effects) Eight stress proteins were analyzed in samples from the single stressor experiments: copper/zinc superoxide dismutase (Cu/ZnSOD), glutathione peroxidase (GPx), manganese superoxide dismutase (MnSOD), heat shock protein 60 (Hsp60), heat shock protein 70 (Hsp70), small heat shock proteins (sHsp), ubiquitin, and 8oxoguanine DNA glycosylase (OGG1m, mitochondrial isoform). Cro ss-reactivity of the stress protein antibodies was validated (Figure 3-1) by polyacrylamide gel elec trophoresis and western blotting. Given the large variances in all stress protein data sets, data were analyzed by nonparametric Kruskal-Wallis ANOVA with post hoc multiple comparisons of mean ranks when appropriate (Statistica).

PAGE 71

71 Results Hypoxia Experiments Experimental conditions. In the fall experiment, the clams were exposed to three levels of dissolved oxygen: 1) air saturated (normoxia; average 0.247 0.024 mmol L-1), 2) mild hypoxia (0.191 0.016 mmol L-1), and 3) moderate hypoxia (0.162 0.021 mmol L-1). Following the hurricanes in the fall of 2004, the availability of grow -out size (average 12 mm) M. mercenaria was severely decreased. For the wint er hypoxia experiment, sample size was decreased to nine clams per tr eatment per sampling day. Since th ere were no significant effects of the hypoxia treatment in the fall hypoxia expe riment (see appropriate sections below), the mean dissolved oxygen levels of the two treatment groups for the remaining experiments were decreased. The clams in the winter experiment we re exposed to three levels of dissolved oxygen: normoxia (0.247 0.007 mmol L-1), mild hypoxia (0.161 0.011 mmol L-1), and moderate hypoxia (0.078 0.015 mmol L-1). In the spring experiment the clams were expos ed to three levels of dissolved oxygen: 1) normoxia (0.247 0.011 mmol L-1), 2) mild hypoxia (0.158 0.016 mmol L-1), and 3) moderate hypoxia (0.074 0.009 mmol L-1). In all three experiments levels of amm onia, nitrite, and nitrate were within acceptable/sublethal levels (data not shown). These experiments were conducted at room temperature and the temperature was monitored daily (data not shown). Ability to bury. Burial analysis was conducted on a randomly selected subset ( n = 6-10) of clams in the fall hypoxia experi ment (Figure 3-2A). Burial abi lities at the final sampling time (60 minutes) were affected by experiment duration ( p < 0.0001), but not by treatment ( p = 0.085). At all time points, more control (normoxia; black bars) clams buried than clams from the mild hypoxia (gray bars) or moderate hypoxia (white bars) treatments, but this difference was

PAGE 72

72 not statistically significant. Surprisingly, burial ability decreased markedly between days 5 and 9, even in the control clams. This might have i ndicated that even these clams were experiencing some stress. However, additional sand was added to the burial rate testing dishes prior to day 9, and this may have caused or at least contributed to the overall decreased burial on days 9 and 14 because the sand became quite densely packed. Burial analysis was conducted on all clams ( n = 8-9) from each depuration aquarium during the winter hypoxia experiment (Figure 3-2B). Burial abilities at the final sampling time (60 minutes) were affected by experiment duration ( p = 0.023), but not by treatment ( p = 0.123). As seen in the fall hypoxia experiment, overall bur ial in the late r two sampling days were lower than in the first two sampling dates. Burial analysis was conducted on all clams (n = 10-14) from each depuration aquarium during the spring hypoxia experime nt (Figure 3-2C). Burial ab ilities were affected by both experiment duration ( p < 0.0001) and treatment ( p < 0.0001). Across all four sampling days, clams in the mild hypoxia treatment buried more ( p < 0.0001) than clams in the moderate hypoxia treatment. Since none of the clams in th e control treatment burie d at the 14-day time point, the overall burial of control cl ams was not signifi cantly different ( p = 0.0755) from the burial ability of the clams in the moderate hypoxia treatment or the mild hypoxia treatment. Glycogen content. The glycogen content of whole clams ( n = 3 per treatment, per sampling day) from the fall experiment were anal yzed (Figure 3-3A). There was no overall effect of treatment on glycogen content ( p = 0.211) when data were analy zed by a general linear model with treatment nested within experiment duration. In this complete model, experiment duration had a large and significant effect ( p < 0.0001), with glycogen cont ent decreasing by fourteen days, regardless of treatment. There was also a significant effect of the interaction between

PAGE 73

73 experiment duration and treatment on glycogen content ( p = 0.0324). Since there were equal numbers of replicates in all samples, post hoc comparisons were conduc ted. The only significant effect of treatment was detect ed in the 9-day samples, wher e clams in the moderate hypoxia treatment had significantly less glyc ogen than did control clams ( p = 0.042). When the glycogen content of whole clams ( n = 3 per treatment, per sampling day) from the winter hypoxia experiment were analyzed (F igure 3-3B) by a general linear model with treatment nested within experime nt duration, there was a signifi cant effect of both experiment duration ( p < 0.0001) and treatment ( p = 0.047), as well as a significant interaction term ( p = 0.0024). Overall, glycogen contents decreased over the duration of the experiment. There were no significant differences among treatments on sampling days 1, 9, and 14. At five days, however, clams in the moderate hypoxia treatment had significantly less gl ycogen than clams in the other two treatments ( p < 0.0055). When the glycogen content of whole clams ( n = 3 per treatment, per sampling day) from the spring hypoxia experiment were analyzed (F igure 3-3C) by a general linear model with treatment nested within experi ment duration, there was no significant effect of experiment duration ( p = 0.082) or treatment ( p = 0.847). Unlike previous experi ments, glycogen content in all three treatments tended to increase over time, with no significant differences among treatments at any of the sampling days. Stress protein expression. Since stress protein expression levels were determined via ELISA, only those proteins with little to no nonspecific cross-reactivity were analyzed. The banding patterns presented in Fi gure 3-1 are consistent with antibody specificity information provided by the manufacturer of the antibodies, EnVirtue Bi otechnologies, Inc. and are consistent with other studies utilizing these an tibodies with bivalve tissues (Joyner-Matos et al.,

PAGE 74

74 2006, 2007). It is not necessary to test antibody sp ecificity for ubiquitin or small heat shock protein (Joyner-Matos et al., 2007). Stress protein expression levels were analyzed in clams ( n = 5) from all three treatments on all four sampling days, as well as in clams collect ed and frozen on the first day of the experiment (0d; Figure 3-4, left column). Since there was high variance in all samples for all eight stress proteins, there were no consistent patterns in the effects of di ssolved oxygen levels on stress protein expression in clams from the fall hypoxia experiment. Although overall significant differences were detected by Kruska l-Wallis ANOVA in some data sets (e.g., p = 0.0007 for GPx, Figure 3-4D and p = 0.0165 for Hsp70, Figure 3-4M), there were no biologically relevant significant differences among treatments in any stress protein data set. A similar lack of treatment effect was found in the eight stress protei ns analyzed in clams ( n = 5) from the winter and spring hypoxia experi ments (Figure 3-4, center and right columns, respectively). In both experiments, there were isolated cases of overall significant ANOVAs for some stress proteins, but either no statistically significant or no biologi cally relevant post hoc comparisons were detected. Summary. Hypoxia treatments were sublethal in a ll seasons. In general, clams exposed to the moderate hypoxia treatment had a lower burial ability than clams in the normoxia or mild hypoxia treatments, but this difference was only si gnificant in the spring experiment. In both the fall and winter experiments, glycogen levels in clams in the moderate hypoxia treatment declined one sampling day before glycogen levels decreas ed in clams from the other two treatments. There were no treatment-associated changes in glycogen content in the spring experiment. Both functional markers, burial ability and glycogen co ntent, decreased significantly over the duration

PAGE 75

75 of the fall and winter experiments. There were no significant, biological ly-relevant changes in stress protein expression in clams from any of the three hypoxia experiments. Temperature Experiments Experimental conditions. In the fall temperature experime nt, the clams were exposed to three treatments: room temperature (24.2 1.2 C), mild temperature elevation (28.3 0.8C), and moderate temperature elevation (32.7 1.2C) In the winter temperature experiment the clams were exposed to three treatments: room temperature (23.21.4 C ), mild temperature elevation (29.61.2C), and moderate temperat ure elevation (33.90.7C). In the spring temperature experiment the clams were exposed to three treatments: room temperature (24.4 1.1 C), mild temperature elev ation (27.9 1.8C), and moderate temperature elevation (34.0 1.1C). In all three experiments levels of am monia, nitrite, and nitrate were within acceptable/sublethal levels (data not shown). Dissolved O2 levels in the three treatments were monitored daily and averaged 0.235 0.013 mmol L-1 (data not shown). Survival analysis. In the winter temperature experiment, the clams in the moderate temperature elevation treatment (Figure 3-5A, wh ite squares) experienced mortality starting at day 2 and were all dead by day 7 The mean surv ival time of clams in the moderate temperature elevation treatment was 3.33 1.6 days. There was no mortality in the room temperature (black squares) or mild temperature elevation treatments (gray squares). Clams in the room temperature and mild temperature elevation treatments surviv ed for an average of 7.25 4.9 days. Survival rates among the three treatments we re significantly different (Chi2 = 59.588, df = 2, p < 0.0001). In pairwise comparisons, clams in the moderate temperature elevation treatment had significantly lower survival than clams in both the ambient temperature ( z = 5.808, p < 0.0001) and the mild temperature elevation treatments ( z = 5.808, p < 0.0001).

PAGE 76

76 In the spring temperature experiment, the clams in the moderate temperature elevation treatment experienced mortality starting at day 8. Mean survival time of clams in the moderate temperature elevation treatment was 7.31 4.9 days. There was no mortality in the room temperature or mild temperature elevation treatm ents. Clams in these two treatments survived approximately 7.25 4.9 days. The mean survival time for the moderate temperature elevation treatment is slightly higher than for the ot her two treatments because a smaller-than-normal number of clams from this treatment were sacrif iced on day 9, resulting in a greater-than-normal proportion of clams maintained until 14 days. Su rvival rates among the three treatments were significantly different (Chi2 = 9.693, df = 2, p = 0.0079). In pairwise comparisons, clams in the moderate temperature elevation treatmen t had significantly lower survival time ( z = 2.261, p = 0.0238) than clams in the other two treatments. Ability to bury. Burial analysis was conducted on a randomly selected subset (n = 8-10) of clams in the fall temperature experiment (Figur e 3-6A). Burial abilitie s at the final sampling time (60 minutes) were affect ed by experiment duration ( p < 0.00025), but not by treatment ( p = 0.993). As seen in the fall hypoxia experiment, overall burial abilities were lower in later days regardless of treatment. In the winter temperature experiment bur ial analysis was conducted on all clams ( n = 1114) from each depuration aquarium on sampling days 1 and 5, and on clams from the room temperature (black bars) and mild temperature el evation (gray bars) treatments on days 9 and 14 (Figure 3-6B). Overall burial abilities at the fi nal sampling time (60 minu tes) were affected by experiment duration ( p = 0.0142) and by treatment ( p = 0.022). Since treatment comparisons can only be made at time points with clams presen t in all three treatments (1 day and 5 day), significant differences between treatments reflect only the first two sampling days. On these

PAGE 77

77 early sampling days, clams in the mild temp erature elevation treatment buried more ( p = 0.0058) than clams in the moderate temperature elevation tr eatment (white bars). In contrast, clams in the room temperature treatment did not have elevated burial ( p = 0.287) in comparison to clams in the moderate temperature elevation treatment. On Days 9 and 14, overall burial abilities of clams in both the room temperature and mild temper ature elevation treatment increased, with no differences detected between treatments. In the spring temperature experiment, burial analysis was conducted on randomly selected clams ( n = 4-10) from each treatment on all sampling days (Figure 3-6C). Burial ability was significantly affected by treatment ( p = 0.034) but not by experiment duration ( p = 0.3386). In contrast to previous experiment s, overall burial ability did not decrease in later time points. Clams in the room temperature treatment buried more than clams in the moderate temperature elevation treatment on days 5, 9, and 14 ( p = 0.013). Burial ability of clams in the mild temperature elevation treatment were similar ( p = 0.069). Glycogen content. The glycogen content of whole clams ( n = 1-3 per treatment, per sampling day) from the fall temperature experime nt were analyzed (Figure 3-7A) by a general linear model with treatment nested within e xperiment duration. We were unable to assess glycogen content in clams from the room temper ature (black bars) and moderate temperature elevation (white bars) treatments on the first sampling date due to loss of the samples during sample processing. The statistical model, theref ore, includes the data only from days 5, 9, and 14. There was a significant eff ect of experiment duration ( p = 0.00029), with glycogen content decreasing with time in clams fr om all three treatments. There were no significant differences among treatments at any sampling day ( p = 0.968).

PAGE 78

78 The glycogen content of whole clams ( n = 3 per treatment, per sampling day) from all treatments with surviving clams from the winter temperature experiment were analyzed (Figure 3-7B). Since mortality occurred in the moderate temperature el evation treatment (indicated by the number symbol), this data set is unbalanced and could not be analyzed by a nested ANOVA. Individual one-way ANOVAs conducted on the 1day and 5-day data sets did not show a significant effect of the elev ated temperature treatment ( p > 0.05). When the glycogen content of clams ( n = 3) from each treatment and sampling day of the spring temperature experiment (Figure 3-7C) were analyzed by a general linear model with treatment nested within experi ment duration, there was a signif icant effect of experiment duration ( p = 0.0039), but not treatment ( p = 0.409). The significant effect of experiment duration likely was influenced by increases in glycogen content of clams from the room temperature and mild temperature elevation (g ray bars) treatments at sampling day 9 in comparison to other days. There were no signi ficant differences among treatments at any sampling day. Stress protein expression. Stress protein expression levels were analyzed in clams ( n = 5) from all three treatments on all sampling days of the fall temperature experiment, as well as in clams collected and frozen on the first day of the experiment (0d; Figure 38, left column). Since there was either unequal or high va riance in all samples, there were no consistent patterns in the effects of elevated temperature on stress pr otein expression. Although overall significant differences were detected by Kruska l-Wallis ANOVA in some data sets (e.g., p = 0.0032 for MnSOD, Figure 3-8G and p = 0.0041 for sHsp, Figure 3-8P), ther e were no biologically relevant significant differences among treatments in any stress protein data set.

PAGE 79

79 A similar lack of treatment effect was found in the eight stress protei ns analyzed in clams ( n = 5) from the winter and spring temperat ure experiments (Figur e 3-8, center and right columns, respectively). In both experiments, ther e were isolated cases of overall significant ANOVAs for some stress proteins but either no statistically si gnificant or no biologically relevant post hoc comparisons were detected. Summary. The mild temperature elevation tr eatment was sublethal in all three experiments, but the moderate temperature eleva tion treatment caused signi ficant mortality in the winter and spring experiments. In general, clams in the moderate temperature elevation treatment had lower burial ability than clams in the othe r two treatments, although this difference was not always statistically significant. In contrast, there were no tr eatment-associated changes in glycogen content. Both burial ability and glycog en content tended to de crease with experiment duration. There were no significant, biologically-relevant changes in stress protein expression in clams from any of the three temperature experiments. Dual Stressor Experiments Experimental conditions. In the fall dual stressor experime nt, the clams were exposed to three levels of hyposalinity at each of two temperatures: 1) high temperature/ambient salinity (37.1 2.6C, 24.9 0.9 ppt), 2) high temperat ure/mild hyposalinity (35.6 0.6C, 15.9 0.9 ppt), 3) high temperature/moderate hyposa linity (35.0 1.0C, 5.3 0.6 ppt), 4) room temperature/ambient salinity (26.1 1.2C, 22.5 1.5 ppt), 5) room temperature/mild hyposalinity (25.9 0.7C, 15.3 1.2 ppt), and 6) room temperature/moderate hyposalinity (23.7 0.6C, 4.5 0.7 ppt). Ambient salinity was define d as the salinity at th e hatchery where the clams had been maintained. Daily dissolved O2 levels in the six treatments averaged 0.242 0.016 mmol L-1 (data not shown). Levels of ammonia, nitrite, and nitrate were within acceptable/sublethal levels [data not shown; Epifanio and Srna, 1975].

PAGE 80

80 In the winter dual stressor experiment, the clams were exposed to the following six treatments: 1) high temperature/ambient sa linity (34.5 0.6C, 24.1 2.1 ppt), 2) high temperature/mild hyposalinity (33.8 0.8C, 15.4 2.5 ppt), 3) high temperature/moderate hyposalinity (33.0 1.6C, 5.8 2.2 ppt ), 4) room temperature/ambient salinity (22.5 1.3C, 22.6 1.5 ppt), 5) room temperature/mild hypos alinity (22.3 1.1C, 14.2 1.5 ppt), and 6) room temperature/moderate hyposalinity (21.7 1.2C, 5.2 0.5 ppt). In the spring dual st ressor experiment, the clams were exposed to the following six treatments: 1) high temperature/ambient sa linity (36.6 1.5C, 25.9 1.1 ppt), 2) high temperature/mild hyposalinity (34.1 0.3C, 15.1 0.6 ppt), 3) high temperature/moderate hyposalinity (33.8 0.8C, 5.0 0.1 ppt ), 4) room temperature/ambient salinity (25.0 0.5C, 25.6 0.9 ppt), 5) room temperature/mild hypos alinity (24.8 0.6C, 15.0 0.4 ppt), and 6) room temperature/moderate hyposalinity (22.3 0.5C, 5.2 0.6 ppt). The high temperature/ambient salinity treatment was terminated after 24 hours due to a heater malfunction. Survival analysis. In the fall dual stressor experiment (Figure 3-9A), clams in the high temperature/moderate hyposalinity treatment (white triangles) e xperienced mortality starting at day 4 and were all dead by day 5, resulting in a mean survival time of 2.69 0.9 days. Clams in the high temperature/ambient salinit y treatment (black triangles) experienced mortality starting at day 7 and were all dead by day 13 (mean survival time of 7.06 3.9 days). Similarly, clams in the high temperature/mild hyposalinity treatment (gray triangles) experien ced mortality starting on day 7, but clams in this treatment did not die before the experiment end (mean survival time of 7.50 4.6 days). Among the clams maintained at room temperature, those in the moderate hyposalinity treatment (white circles) experienced mortality starting at day 7 and were all dead

PAGE 81

81 by day 14 (mean survival times of 7.18 4.0 days), a pattern that was si gnificantly different from that of the other two room temperature treatments ( z = 5.98, p < 0.0001). In contrast, none of the clams in the room temperature/ambient sali nity (black circles) or room temperature/mild hyposalinity (gray circles) treatments died (mean survival time 7.25 4.9 days). Overall, the mean survival times of the six treatm ents were significantly different (Chi2 = 291.8, df = 5, p < 0.0001). In pairwise comparisons, clams in the hi gh temperature/moderate hyposalinity treatment had a significantly shorter survival time than clams in any other treatment ( z 10.1, p < 0.0001). Clams in the high temperature/mild hyposalinity treatment had a significantly longer survival time than clams in the high temper ature/ambient salinity treatment ( z = 1.97, p = 0.0484). In the winter dual stressor experiment (Figure 3-9B), clams in the high temperature/moderate hyposa linity treatment and the high temperature/mild hyposalinity treatments experienced mortality starting at day 2 and were all d ead by day 5, resulting in mean survival times of 2.81 1.2 days and 2.73 1.2 days, respectively. Clams in the high temperature/ambient salinity treatment experience d mortality starting at day 3 and were all dead by day 7 (mean survival time 4.06 1.9 days). There was some death in the room temperature/moderate hyposalinity treatment after day 11 (mean survival time 7.12 4.7 days). In contrast, none of the clams in the room temper ature/ambient salinity or room temperature/mild hyposalinity treatments died (mean survival time 7.25 4.9 days). Overall, mean survival times of the six treatments were significantly different (Chi2 = 174.12, df = 5, p < 0.0001). Clams in the high temperature/ambient salinity treatment su rvived significantly longer than clams in the other two high temperature treatments ( z 6.11, p < 0.0001) and significantly shorter than clams in the room temperature/am bient salinity treatment ( z = 5.69, p < 0.0001). Despite the mortality at day 12, survival times of clams in the room temperature/moderate hyposalinity treatment were

PAGE 82

82 not significantly different than clams in the other room temperature treatments ( z = 1.19, p = 0.230). In the spring dual stressor experiment (Figure 3-9C), clams in the high temperature/moderate hyposalinity treatment began to die at day 3 and were completely dead by day 5 (mean survival time 3.06 1.3 days). Clam s in the high temperature/mild hyposalinity treatment began to die at day 4 and were comp letely dead by day 10 (mean survival time 4.19 2.4 days). Clams in the room temperature/modera te hyposalinity treatment began to die at day 9 and were all dead by day 12 (mean survival time 6.16 3.5 days). In contrast, none of the clams in the room temperature/ambient salinity or room temperature/m ild hyposalinity treatments died (mean survival time 7.25 4.9 days). Overall, su rvival times were significantly different among treatments (Chi2 = 82.663, df = 4, p < 0.0001). Clams in the high temperature/moderate hyposalinity treatment had significantly shorter survival times than clams in all other treatments ( z 3.71, p 0.00021). Clams in the high temperatur e/mild hyposalinity treatment had significantly shorter survival time than clams in the mild hyposalinity/room temperature treatment ( z = 4.25, p = 0.00002). Clams in the room temperature/moderate hyposalinity experiment had a significantly shorter mean su rvival time than did clams in the other room temperature treatments ( z = 2.98, p = 0.0029). Ability to bury. Burial analysis was conducted on clams ( n = 6-10) from each treatment with surviving clams on each sampling day of th e fall dual stressor experiment (Figure 3-10A). There was a significant effect of treatment ( p = 0.0035) but not of experiment duration ( p = 0.739) on the ability of the clams to bury. Although the burial ability of th e clams in the room temperature/ambient salinity treatment was highe r than those of any ot her treatment at all sampling days, pairwise differences am ong treatments were not significant ( p > 0.05). Although

PAGE 83

83 clams in the high temperature/ambient salinity (black bar) showed the same ability to bury as those in the room temperature/ambient salinity treatment (thinly striped bar) on day 1, clams experiencing dual stressors were una ble to bury at later time points. In the winter dual stressor experiment, burial analysis was conducted on all ( n = 9-10) surviving clams at all treatment times (figure 3-10B). There was a significant effect of treatment ( p = 0.0029) but not of experiment duration ( p = 0.741) on burial ability. On all sampling days, clams in the room temperature/ambient salini ty treatment exhibited burial ability, which decreased after day 5. On the first sampling day, the only day with clams present in all treatments, clams exposed to high temperature bu ried less than those at room temperature, regardless of salinity level. In the spring dual stressor expe riment, burial analysis was co nducted on a subset (n = 4-8 clams) of surviving clams at all treatment time s (Figure 3-10C). There was no significant effect of either experiment duration ( p = 0.507) or treatment ( p = 0.266), likely because burial was only detected in two of the six treatments. Glycogen content. The glycogen content of whole clams ( n = 5) from all treatments on day 1 and from treatments with a sufficient surv iving clams on subsequent days of the fall dual stressor experiment were analyzed (Figure 3-11 A). Since this data set is unbalanced (due to mortality in some treatments) each day was analyzed individually by one-way ANOVA with Fisher’s LSD where appropriate. There were no significant differences among the six treatment groups on sampling day 1 ( p = 0.873). On sampling day 5, there was a significant effect of treatment ( p = 0.021). The clams in the high temperatur e/ambient salinity (black bars) and high temperature/mild hyposalinity (gray bars) treatm ents had significantly less glycogen than did clams in the room temperature/ambient sa linity (thinly striped bars) treatment ( p < 0.026;

PAGE 84

84 significances not indicated in gr aph). Additionally, the clams expos ed to room temperature/mild hyposalinity (thickly striped bar) had significantly less glycogen than clams in the other two salinity (and room temperature) treatments ( p < 0.035). At the day 9 sampling date, treatment again had a significant eff ect on glycogen content ( p = 0.033). Clams exposed to high temperature/mild hyposalinity treatment had signi ficantly less glycogen than clams exposed to room temperature/mild hyposalinity ( p = 0.0045). There were no significant differences in glycogen content of clams surviving to 14 days ( p = 0.061). Unlike most of the single stressor experiments, overall glycogen levels did not de crease over the duration of the fall dual stressor experiment. The glycogen content of whole clams ( n = 5 for all but 14 day room temperature/moderate hyposalinity treatment, for which n = 2) from all six treatments on sampling day 1, and from the room temperature treatments on subsequent sampli ng days of the winter dual stressor experiment were analyzed (Figure 3-11B). We did not find any significant differences among treatments on day 1 ( p = 0.301), the only sampling da y with all six treatments pres ent. Similarly, there were no significant differences in glycogen content among the three room temperature treatments on day 5 ( p = 0.774). In contrast, we found significant eff ects of the hyposalinity tr eatments at both day 9 ( p = 0.0055; significant differences are not indicated on the graph) and day 14 ( p = 0.0012). On both days, the clams in the ambient salinity had si gnificantly more glycogen than clams in either hyposalinity treatment (9 day, p < 0.045; 14 day, p < 0.039). Additionally, the clams in the moderate hyposalinity treatment on sampling day 14 had significantly less glycogen than clams in the mild hyposalinity treatment ( p = 0.0046). Glycogen content in clams from the room temperature/ambient salinity and room temperat ure/mild hyposalinity treatments did not decline with experiment duration.

PAGE 85

85 The glycogen content of whole clams ( n = 2-3 clams) from all six treatments on sampling day 1 and of the room temperature treatments on subsequent sampling days of the spring dual stressor experiment were analyzed (Figure 311C). There was no significant treatment effect on glycogen content of clams on sampling day 1 ( p = 0.779) or day 5 ( p = 0.72). Glycogen content of clams sampled on days 9 or 14 were not sta tistically analyzed due to low sample sizes. RNA oxidation. The levels of oxidatively damaged RNA bases, 8-oxoGuo, were measured in clams ( n = 5) from each treatment on day 1 of the fall dual stressors experiment (Figure 3-12A). RNA oxidat ion was significantly affected by salinity treatment ( p = 0.0157) and a marginally affected by temperature ( p = 0.0734), but not by the inte raction between the two factors ( p = 0.809). Several post hoc comparisons were statistically significan t; of those only one was biologically significant. Clams exposed to th e room temperature/mild hyposalinity treatment (triangle) had significantly more oxidized RNA th an did clams in the room temperature/ambient salinity treatment (diamond; p = 0.0211, significances not noted on graph). Levels of oxidatively damaged RNA were measured in clams ( n = 5 for all except room temperature/mild hyposalinity, which had n = 2) from each treatment on day 1 of the winter dual stressors experiment (Figure 3-12B). RNA oxidati on was significantly affected by temperature ( p < 0.0001) but not by salinity ( p = 0.142) or the interaction be tween temperature and salinity ( p = 0.210). In general, RNA oxidation levels were higher in clams in the high temperature treatments in comparison to clams in the room temperatur e experiments. Specifically, clams exposed to high temperature/ambient salinity (circle) or high temperature/mild hyposalinity (inverted triangle) had significantly more oxidized guanine bases than did clams from any room temperature treatment ( p < 0.00091). The clams exposed to high temperature/moderate hyposalinity had more oxidized RNA than did cl ams in the two room temperature/hyposalinity

PAGE 86

86 treatments ( p < 0.012), but this difference was only marginally significant ( p = 0.0587) in comparison to clams from the room temperatur e/ambient salinity treatment. There were no significant differences among the three room temperature treatments ( p > 0.229). Levels of oxidatively damaged RNA were measured in clams ( n = 5) from each treatment on day 1 of the spring dual stresso r experiment (Figure 3-12C). Unlike in previous seasons, there were no significant effects of e ither treatment factor or the in teraction between the factors on RNA oxidation ( p > 0.104). However, the post hoc multiple comparison test did detect two significant comparisons to the RNA oxidation levels of the clams in the room temperature/ambient salinity (diamond) treatmen t. Clams exposed to high temperature/ambient salinity had significantly more oxidized RNA bases than did clams exposed to room temperature/ambient salinity ( p = 0.0151). Similarly, the cl ams in the two hyposalinity treatments (room temperature) had more oxidized RNA bases than did the room temperature/ambient salinity clams, a increase that was significant in the mild hyposalinity treatment ( p = 0.0429) and marginally si gnificant in the moderate hyposalinity treatment ( p = 0.0541). Summary. The dual stressor treatments resulted in significant mortality in all three seasons. Additionally, the high temperature/am bient salinity treament caused significant mortality, particularly in the wi nter experiment. Clams exposed to dual stressors had decreased burial in all three experiments, but no significant changes in gl ycogen content. Although there was no consistent pattern in RNA oxidation acro ss season, in several cases the levels of RNA oxidation were higher in clams exposed to hypos alinity in comparison to clams in ambient salinity.

PAGE 87

87 Discussion These results support several overall conclu sions concerning how one should investigate the ability of an organism to respond to indivi dual and multiple abiotic factors. First, a strong influence of season was detected in all experiments, with an overall decrease in the ability of the clams to tolerate the abiotic st ressors evident during the winter experiments. Second, there was no clear relationship between cellu lar-level and functional markers, with most of the treatment effects detected at the whole-or ganism level rather than the cellular level. Third, many of the functional responses were negatively impacted by experiment duration, cal ling into question the utility of long-term laboratory exposures. Four th, high temperature and hyposalinity were found to have an additive effect on most metrics, pa rticularly whole-organism metrics. Since this combination of abiotic factors occurs in estuar ine environments, particularly in the summer, these results highlight the importance of investigating the effects of multiple stressors in controlled laboratory conditions to more accurately determine how tolerance of abiotic factors may influence species distribution. Hypoxia. Conditions of low dissolved O2, or hypoxia, are prevalent in estuaries, generally resulting from nutrient enrichments, tidal cy cles, and excessive rainfall (Hubertz and Cahoon, 1999; Beck and Bruland, 2000). Since temperature affects O2 solubility, elevated water temperatures cause both seasonal and diel cycles of hypoxia and normoxia. Mercenaria mercenaria thrive in estuarine habitats which, along the Florida coast, have typically have O2 levels of 0.19 – 0.31 mmol L-1 (Millie et al., 2004; Harris et al., 2005) but can be as low as 0.09 mmol L-1 (Caccia and Boyer, 2005). Hard clams are hypoxia tolerant and ar e oxyregulators until dissolved O2 levels reach the critical PO 2 of 0.15 mmol L-1, at which point the clams close their valves and maintain anaerobic respiration for ex tended periods of time (re viewed in Grizzle et

PAGE 88

88 al., 2001). During these periods of anaerobic metabol ism, hard clams, like many bivalves, rely upon glycogen fermentation as a major energy sour ce (Grieshaber et al., 1994; Grizzle et al., 2001). While survival of M. mercenaria is not strongly affected by hypoxia (Winn and Knott, 1992; Grizzle et al., 2001; Carmic hael et al., 2004), functional responses of hard clams are impacted by hypoxia, but only at very low dissolved O2 levels. Pumping rate, which directly affects feeding ability, is linearl y correlated with dissolved O2 levels from 0.03 – 0.15 mmol L-1 (reviewed in Grizzle et al., 2001). Clam grow th is negatively impacted by dissolved O2 levels below 0.15 mmol L-1 (Appleyard and Dealteris, 2002), an effect that may be confounded by decreased feeding under these c onditions. Burial time in M. mercenaria also is inversely related to dissolved O2 levels, with burial time doubli ng over a range of dissolved O2 levels from 0.21 mmol L-1 to 0.03 mmol L-1 (Savage, 1976). In the fall hypoxia experiment, both hypoxia tr eatments were well above the threshold dissolved O2 level determined to affect functional respon ses of hard clams, a nd it is therefore not surprising that there were no treatment-associated declines in burial abi lity or glycogen content in clams from this experiment. However, both burial ability and glycogen content decreased over the duration of the experiment, a trend that has been documented for glycogen levels in bivalves in field (Byrne and O'Halloran, 2000) and labora tory (de Zwann and Zandee, 1972) studies. In the winter and spring experiments, hypoxia tr eatments were modified to encompass the threshold dissolved O2 level (mild hypoxia treatment, approximately 0.16 mmol L-1) as well as a level significantly below threshold (moderate hypoxia treatment, approximately 0.08 mmol L-1), both of which are representative of conditions in estuaries with hard clams (Winn and Knott, 1992; Ringwood and Keppler, 2002). Although there wa s no treatment-associated mortality in the hypoxia experiments, there was some evidence fo r a decline in functional responses. In most

PAGE 89

89 sampling days of the winter and spring experiments, the burial abilities of clams in the moderate hypoxia treatment were lower than those of the normoxia and mild hypoxia treatments, which is consistent with previous characterizations of hard clam burial abilities (Savage, 1976). In both the fall and winter experiments, glycog en levels in clams in the moderate hypoxia treatment declined one sampling day before glyc ogen levels decreased in clams from the other two treatments, a trend that occurred earlier in the winter experiment In contrast, glycogen levels in the spring experiment increased or showed no change with experiment duration, a pattern repeated in the spring temperature and du al stressor experiments, regardless of treatment. Since the feeding regimen did not differ among e xperiments, it appears that maintaining the clams in the aquarium system was most detrim ental to clam health (assessed by glycogen content) in the winter, somewh at detrimental in the fall, a nd not detrimental in the spring. Seasonal differences in glycogen storage in bivalves are well-established (e.g., Byrne and O'Halloran, 2000; Hamburger et al., 2000), and are typically li nked to reproductive status. Mercenaria mercenaria are consecutive hermaphrodites and at the size used for these experiments (12 – 15 mm) are likely in the male juvenile stage. Clams living in estuaries along the Florida coast typically have a semiannual sp awning cycle, with spawns in March/April and October/November (Eversole, 2001). The spring experiment was conducted in April, and therefore these clams may have ut ilized their glycogen stores diffe rently than the clams in the fall and winter experiments. Short-term exposure to hypoxia has been linked to increased free radical production (Hermes-Lima et al., 1998; Halliwell and Gu tteridge, 1999; Hermes-Lima and Zenteno-Savin, 2002; Li and Jackson, 2002). Du ring hypoxia, the absence of O2 as the final electron acceptor causes accumulation of electrons in mitochondrial electron transpor t chains (i.e., the chains are

PAGE 90

90 reduced), with the result that a sudden return of O2 can cause the production of free radicals due to nearly instantaneous reactions between O2 and the accumulated free electrons (Du et al., 1998; Li and Jackson, 2002). Although most studies of free radical production during hypoxia include a period of reoxygenation, recent studies have documented free radical production occurring during hypoxia without subsequent reoxygenation (Vanden Hoek et al., 1997; Chandel et al., 1998; Becker et al., 1999). Hypoxia-associated fr ee radical production, if it occurs, can be accompanied by oxidative damage (Englander et al., 1999; Dirmeier et al., 2002) and changes in antioxidant expression and/or activ ity (Lushchak et al., 2001; de O liveira et al., 2005). However, changes in antioxidant expression or activity are not always detect ed in animals or cells exposed to hypoxia (Hass and Massaro, 1988; Willmore a nd Storey, 1997; Joanisse and Storey, 1998; Larade and Storey, 2002). In the current study, M. mercenaria exposed to hypoxia did not show signifi cant changes in stress protein expression, rega rdless of the season in which th e experiments were performed. There are two possible interpretations for these re sults. First, it is possible, in light of the conflicting literature summarized above, that prolonged hypoxi a in the absence of reoxygenation does not induce free radical production or cellu lar damage. Upregulation of stress protein expression, therefore, would be counterproductiv e and metabolically expensive (Somero, 2002). Alternatively, if hypoxia exposure can cause oxidative damage, then it is possible that the degree of hypoxia utilized in these studies was not sufficien tly severe to incite a stress protein response. These results are not consistent with those suggesting that ce llular-level responses are more likely to be detected than organismal responses, which integrate over severa l levels of biological organization (Stebbing, 1985; Bierkens 2000). Most of the studies ci ted above, particularly those that showed cellular damage following hypoxia, were performed on organisms that are not

PAGE 91

91 hypoxia-tolerant. It is possible th at even though the severity of the hypoxia stress utilized in the winter and spring experiments was sufficient to ca use declines in functiona l responses, the stress was not severe enough to cause damage or in itiate responses at the cellular level. The relationship between the timing and degree of ce llular-level responses such as stress protein expression and/or activity and whol e-animal tolerance of hypoxia ha s not been expl icitly studied for any organisms. These results suggest that for animals as tolerant of a stressor as M. mercenaria is tolerant of hypoxia, ce llular-level markers alone ar e not sufficient to detect detrimental effects of exposure to individual abiotic stressors and should not be used to investigate species distribution. Temperature. Diel and seasonal temperature fluctuat ions are typical of estuaries. The estuaries along the Florida coast line experience ‘wet’ and ‘dry’ s easons that can differ by 8C in average temperature (Millie et al., 2004). Mercenaria mercenaria are tolerant of a wide range of temperatures, from 0 – 30C, and typically more tolerant of warm temperatures than cold. Maximum growth and pumping rates of hard cl ams occur at temperatures ranging from 20 – 26C, with both functional res ponses declining dramatically above 32C and below 7C (for review, Grizzle et al., 2001). This pattern of thermal tolerance closely matches both their distribution (Wells, 1957; Harte, 2001) and their reproductive activity, which in warmer southern latitudes has a semiannual pattern (Eversole, 2001). Burrowing time in M. mercenaria mirrors that of growth and pumping rates, with shorte st burrowing times detected at 21 – 29C and longer burrowing times at 37C (Savage, 1976). In the current study, the functiona l responses of hard clams to temperature elevation varied strongly by season, with the greatest susceptibility to high temperat ure detected during the winter temperature experiment. Whereas none of the cl ams died during the fall temperature experiment,

PAGE 92

92 all clams exposed to moderate temperature el evation (34C) died by day 7 in the winter experiment and some clams in this treatment died on day 9 of the spring experiment. Seasonal differences in susceptibility to thermal extremes has been documented previously and linked to ability to express heat shock proteins (Chapple et al., 1998). Burial abilities did not follow clear seasonal pa tterns, which is consistent with a previous study (Savage, 1976). Burial abilities decreased over time in the fall experiment, but this pattern was not repeated in the winter and spring experiments. In the wi nter experiment, clams in the moderate temperature elevation treatment had ve ry low burial ability on day 1 and did not bury on day 5. However, burial abilities in the room temperature and mild temperature elevation treatments were low on these days, so the lack of burial in the moderate temperature elevation treatment cannot be considered predictive of subsequent mortality. Patterns in glycogen content did not vary by s eason and were not significantly affected by elevated temperature. However, in contra st to the hypoxia experiments, there were no a priori expectations for changes in glycogen content w ith exposure to elevated temperature. In the winter experiment, glycogen leve ls were not significantly different among clams in the three treatments in the first two sampling days, and th erefore cannot serve as a predictor of imminent clam death. In the winter and spring experiments, glycogen levels in the room temperature clams increased over time, which for the spring experime nt is consistent with the hypoxia experiments. According to a large body of literature, mild a nd moderate elevations in temperature result in increased heat shock protei n expression and, if cellular prot eins are damaged beyond repair, elevations in ubiquitin expre ssion (for review, Feder and Ho fmann, 1999; Hofmann et al., 2002; Kregel, 2002; Dahlhoff, 2004). The degree of upre gulation and temperatur e at which expression

PAGE 93

93 is stimulated varies seasonally (e.g., Hofmann and Somero, 1995; Lesser a nd Kruse, 2004) and is influenced by previous exposure to stressors (Somero, 2002). Statistically significant treatmentor season-de pendent changes in expression levels were not detected for any of the stress proteins meas ured in this study, in cluding the heat shock proteins. Although these results may seem surprising, they are consistent w ith recent studies of thermotolerant organisms, such as those dem onstrating a temperature-insensitivity of Hsc70 function over the environmentally-relevant range of temperatures for an estuarine fish (Hofmann et al., 2002; Zippay et al., 2004). The extreme thermotolerance exhibited by M. mercenaria may result from physiological strategi es that buffer the need for a ce llular-level response to elevated temperature. The temperatures ut ilized in this experiment ma y not have been high enough to trigger a stress protein response, which typically requires thermal denaturation of proteins (Feder and Hofmann, 1999). Even though the moderate temperature eleva tion treatment resulted in mortality in the winter experiment, it did not indu ce a significant stress pr otein response. Clams in this treatment experienced a substantial (but not significant) increase in sHsp expression between 1 and 5 days and decreases in Hsp60, Hsp70, and ubiquitin ex pression over the same period. While this pattern could be consistent w ith extreme cellular stress (Werner and Hinton, 1999; Joyner-Matos et al., 2007), given the la ck of statistical significance and la ck of detectable response in other experiments, it is unlikely that these changes reflect anything more than random variation. The physiological parameters examined in this study ca nnot rule out the possibili ty that the cause of death in the winter experiment clams was a loss of function at a higher level of organization than the cellular level, similar to th e “heart failure” documented in in tertidal crabs exposed to high temperature (reviewed in Somero, 2002).

PAGE 94

94 Dual stressors. Given the lack of detectable cel lular-level response or consistent functional response to hypoxia or hi gh temperature, the ability of M. mercenaria to respond to a combination of high temperature and hyposalinity wa s examined in a second set of experiments. The combined effects of high temperature and hy posalinity are well-studied, likely because this combination is common in coastal habitats, particularly during summer periods of high temperature and substantial rainfall (e.g., Mil lie et al., 2004; Caccia and Boyer, 2005). While these two factors have been show n to interact in their effects on hard clam pumping rate (for review, Grizzle et al., 2001), it is not clear whether these stressor s have an additive effect on bivalve growth, development, or surviv al (Cain, 1973; Lough and Gonor, 1973). While M. mercenaria are tolerant of a wi de range of temperatures, their distribution patterns suggest the clams are not as tolera nt of hyposalinity (We lls, 1957; Harte, 2001). Mercenaria mercenaria function as osmoconformers, a llowing their pallial fluids and hemolymph to be isosmotic with the environmen t when their valves are open (reviewed in Grizzle et al., 2001). The clams usually close th eir valves, however, when the environmental salinity drops by 50%, a response that impacts the ability of the clams to feed and grow (Carmichael et al., 2004). Pumpi ng rates, ability to bury, and gr owth are maximized between 20 – 30 ppt, and decrease dramatically above 32 ppt and below 15 ppt (Grizzle et al., 2001). Survival is significantly decrea sed at salinities ranging from 5 – 10 ppt (Winn and Knott, 1992). Hard clams are particularly susceptible to rapi d decreases in salinity, experiencing substantial mortality following a decrease of approximately 20 ppt over a 24-hour period (Baker et al., 2005). Such rapid fluctuations ofte n occur where hard clams live, due either to rainfall events or anthropogenic activities such as water discharg es (Caccia and Boyer, 2005; Wilson et al., 2005). A multiyear survey of salinity at sites close to where the clams utilized for this study are

PAGE 95

95 maintained revealed daily and seasonal variati on in salinity (Baker et al., 2005). Across three years, salinities along the Florida Gulf coast ra nged from approximately 5 ppt to 35 ppt, with daily fluctuations ranging from 5 to 24 ppt in magnitude (Baker et al., 2005). In the dual stressor experiments, the effects of three levels of salinity (app roximately 25, 15, and 5 ppt) at each of two temperatures, room temperature and high temperature (average 34 C) were examined. A strong additive effect of th e two stressors was detect ed in the pattern of survivorship of clams in all three seasons. Regardless of s eason, the clams exposed to the most extreme set of conditions (high temperature/mode rate hyposalinity) had the lowest survivorship, with all clams in this treatment dead by day 4 or 5. In the winter experiment, a strong effect of temperature on survival was again documented, with clams in all of the high temperature treatments dead by day 7. A similar additive eff ect of high temperature and hyposalinity has been documented for growth of M. mercenaria larvae (discussed in Lough and Gonor, 1973) and survival of larvae of the estuarine clam Rangia cuneata (Cain, 1973). Among the room temperature treatments, only the moderate hyposalin ity treatment resulted in clam death, with the greatest effect of this treatment detected during the spring experiment, which is consistent with reports of hard clam susceptibility to low salinity. It is less clear whether salinity alone or salinity in combination with high temperature affected the burial abilities of clams in the dual stressor expe riments. In the fall and spring experiments, none of the clams exposed to high temperature and hyposalinity buried after 24 hours of exposure, the only time point with surviv ing clams in all six treatments. In the winter, one clam in the high temperature/mild hyposalin ity treatment buried, and none of the clams in the high temperature/moderate hyposalinity treatmen t buried. In contrast, some clams exposed to high temperature/ambient salinity in the fall a nd winter experiments bur ied on the first day of

PAGE 96

96 sampling, which suggests that high temperature alone was not sufficient to inhibit burial. Interestingly, burial ability was strongly in fluenced by hyposalinity. On ly two of the clams exposed to room temperature/moderate hyposalinity buried in the fall experiment and none of the clams in this treatment buried in the winter or spring experiments, even though they survived to nearly the end of the experiment In nearly all cases, burial ab ilities of clams in the room temperature/mild hyposalinity treatments were lower than those of clams in the room temperature/ambient salinity treatments. The fe w studies that have examined the effects of hyposalinity on burial ab ility or burial rate of bivalves ha ve produced conflicting results but have the general trend of decreased burial in hyposaline conditions, rega rdless of temperature (Grizzle et al., 2001; Lardies et al., 2001; Matthews and Fairweather, 2004) In contrast to the significant effects of the single and dual st ressors on survival and burial ability, there were no clear effect s of the dual stressor treatments on glycogen content. At day 1, the only sampling day with all treatments presen t, there were no signifi cant differences in glycogen content in any of the th ree seasons. Unlike in previous experiments, glycogen content did not decrease over time. A cau sal relationship between glycoge n content and salinity or high temperature has not been established for bivalv es, but several studies have reported seasonal correlations between reproductive cycle, glycogen content, a nd environmental temperatures, salinities and chlorophyll leve ls (e.g., Li et al., 2006). A causative relationship betw een hypersalinity and free radical production is wellestablished in plant physiology and biomedical fields such as nephr ology and immunology (e.g., Hernndez et al., 1993; Qin et al., 1999; Hizoh and Haller, 2002). However, whether hyposalinity is liked to alterations in free radical metabo lism and oxidative damage is not understood. A recent study of hyposalinity responses of a marine alga detected an elevation in

PAGE 97

97 glutathione but not in antioxidant enzymes such as catalas e and superoxide dismutase (Jahnke and White, 2003). Similarly inconsistent results in other studies, all of which have been conducted on plants, have not yet determined wh ether hyposalinity causes elevated free radical production and oxidative damage. In the curr ent study, some evidence of a link between hyposalinity and oxidative damage to RNA was dete cted in the fall and spring experiments, but not in the winter experiment. In the fall expe riment, clams exposed to hyposalinity had more 8oxoGuo than clams exposed to ambient salinity, re gardless of temperature treatment. In the spring experiment, clams in the room temperat ure/hyposalinity treatments again had higher 8oxoGuo levels than clams in the ambient salinity tr eatment. In neither fall nor spring was there a significant effect of temperature or an interaction between temper ature and salinit y. These results suggest that clams exposed to hyposalinity experi enced greater oxidative damage to their RNA, either due to elevated free ra dical production or to a diminish ed ability to repair oxidative damage. In the winter experime nt, in contrast, RNA oxidation was closely linked to temperature (higher oxidative damage in clams in the high te mperature treatment) but not related to salinity level. These results suggest that the high temperat ure treatment severely limited the ability of the clams to minimize or repair oxid ative damage, but do not give any indication of what may have caused the elevated oxidative damage. An examination of stress protein expressi on levels in clams from the dual stressors experiment may provide further evidence for a link between hyposalinity and oxidative damage and also may provide further evidence for an additive effect of high temperature and hyposalinity. The tissue samp les for this analysis have been processed but the stress protein analyses are not yet complete. Several studies of stress protein expressi on (particularly heat shock protein expression) in es tuarine invertebrates exposed to hyposalinity or hyposalinity/high

PAGE 98

98 temperature treatments have produced conflicti ng results (Kultz, 1996; Clark et al., 2000; Werner and Hinton, 2000; Spees et al., 2002; Werner, 2004; Blank et al., 2006). The combination of high temperature and hyposalinity was lethal for M. mercenaria particularly in the winter experiment. While the sublethal hypo xia treatments did not ca use detectable stress protein responses and no significant pattern of stress protein expression was detected in the sublethal or lethal temperature treatments, th e combination of high te mperature and hyposalinity may trigger a stress protein respon se. The oxidative damage results suggest that the clams in the dual stressor experiments are experiencing cellular-l evel damage, and this damage may prove to be sufficient to induce change s in stress protein expression. Conclusions. The effects of the singleand dual -stressor treatments on functional and cellular-level responses in M. mercenaria illustrate the importance of examining a variety of physiological responses, across several levels of biological organization. For example, based on the extensive studies of heat shock protein expr ession and thermotolerance in rocky intertidal invertebrates, the seasonal susceptibility to high temperature demonstrated in this study would be predicted to be accompanied by changes in heat s hock protein expression. It is unlikely that the high variance in stress protein expression levels masked any treatment -related changes as a similar level of variance (with the same sample size and methodol ogy) existed in the data set presented in Chapter 2, and significant change s were detected in that study. Many of the potential sources of variance in stress protein ex pression that were discussed in Chapter 2 (page 48), as well as the methodological steps taken to mi nimize that variance, are applicable to this study as well. The fact that no significant patte rn in stress protein expression was detected, particularly during those seas ons in which the treatments caused mortality, highlights the

PAGE 99

99 importance of assessing multiple physiological res ponses, particularly the more sensitive wholeanimal responses such as burial ability. Not surprisingly, a strong effect of season was de tected in all experiments and in nearly all measured parameters. Several environmental pa rameters, including salinity, temperature, food availability, and dissolved O2 levels, vary seasonally in estuar ine habitats and thus influence the growth, reproductive cycle, and stress re sponsiveness of estuarine organisms. In all seasons, functional responses tended to decrease over the durati on of the experiment, a trend that has been documented previously. It cannot be determined from this study whether these declines resulted from prolonged maintenan ce in laboratory conditions or whether they would occur in the field. In their habitat th e clams likely would not experience prolonged exposure to the equivalent of the most extreme treatments, particularly the high temperature/moderate hyposalinity treatment, since abiotic conditions in estuaries fluctuate daily. However, since the functional declines occurred in the control treatments as well as the experimental treatments, it cannot be concluded that the functional dec lines represent reduced tolerance of the stressors. Finally, although an additive effect of high te mperature and hyposalinity was apparent in the survival analyses, we were unable to detect additive effects on cellular-level or functional markers. These results suggest th at we did not characterize the cause of mortality in the dual stressor experiment. Examination of a wider va riety of functional metrics and stress protein expression levels may lead to an understanding of how the dual stressors affect the hard clams in the laboratory and how toleran ce of these abiotic factors may influence species distribution.

PAGE 100

100 MWM Copper/zinc superoxide dismutase Glutathione peroxidase Manganese superoxide dismutase Heat shock protein 60 Heat shock protein 70 8-oxoguanine DNA glycosylase Figure 3-1. Antibody specificity tests in M. mercenaria whole clam homogenates. Molecular weight markers (in kilodaltons) are specif ied in the first column.

PAGE 101

101 A. Fall 15914Fraction buried 0.0 0.2 0.4 0.6 0.8 1.0 Normoxia Mild hypoxia Mod. hypoxia B. Winter 15914Fraction buried 0.0 0.2 0.4 0.6 0.8 1.0 C. SpringExperiment duration (d) 15914Fraction buried 0.0 0.2 0.4 0.6 0.8 1.0 Figure 3-2. Fraction of clams that buried in the three hypoxia experi ments. Data are expressed as a summary of fraction buried at the 60 minute time point of each sampling date. Experiment duration is in days.

PAGE 102

102 C. SpringExperiment duration (d) 15914Glycogen (mg/ml) 0 10 20 30 40 B. Winter 15914Glycogen (mg/ml) 0 10 20 30 40 A. Fall 15914Glycogen (mg/ml) 0 20 40 60 80 100 Normoxia Mild hypoxia Mod. hypoxia Figure 3-3. Glycogen content of whole clams in the three hypoxia experiments. Data are expressed as mean SD. Experiment dur ation is in days. Abbreviations: mg, milligrams; ml, milliliters.

PAGE 103

103 Fall Winter Spring A. Cu/ZnSOD 0d1d5d9d14d1d5d9d14d1d5d9d14d RU/ng TSP 0 70 140 210 280 350 B. Cu/ZnSOD 0d1d5d9d14d1d5d9d14d1d5d9d14d 0 70 140 210 280 350 C. Cu/ZnSOD 0d1d5d9d14d1d5d9d14d1d5d9d14d 0 70 140 210 280 350 D. GPx 0d1d5d9d14d1d5d9d14d1d5d9d14d RU/ng TSP 0 100 200 300 E. GPx 0d1d5d9d14d1d5d9d14d1d5d9d14d 0 100 200 300 F. GPx 0d1d5d9d14d1d5d9d14d1d5d9d14d 0 100 200 300 G. MnSOD 0d1d5d9d14d1d5d9d14d1d5d9d14d 0 100 200 300 RU/ng TSP H. MnSOD 0d1d5d9d14d1d5d9d14d1d5d9d14d 0 100 200 300 I. MnSOD 0d1d5d9d14d1d5d9d14d1d5d9d14d 0 100 200 300 Normoxia Mild hypoxia Moderate hypoxia Control Normoxia Mild hypoxia Moderate hypoxia Control Normoxia Mild hypoxia Moderate hypoxia Control Figure 3-4. Stress protein e xpression levels in clams from hyp oxia experiments. Data are expre ssed as relative units of expres sion per nanogram of total soluble protein (RU/ ng TSP) and are presented as scatterplo ts with asymmetrical error bars. The dot represents the median and the error bars define the minimum and maximum. Abbrevia tions: d, day; mod, moderate; Cu/Zn SOD, copper/zinc super oxide dismutase; GPx, glutathione peroxidase; MnSOD, ma nganese superoxide dismutase.

PAGE 104

104 J. Hsp60 0d1d5d9d14d1d5d9d14d1d5d9d14d RU/ng TSP 0 40 80 120 160 Fall Winter Spring K. Hsp60 0d1d5d9d14d1d5d9d14d1d5d9d14d 0 40 80 120 160 200 L. Hsp60 0d1d5d9d14d1d5d9d14d1d5d9d14d 0 50 100 150 200 250 M. Hsp70 0d1d5d9d14d1d5d9d14d1d5d9d14d RU/ng TSP 0 100 200 300 N. Hsp70 0d1d5d9d14d1d5d9d14d1d5d9d14d 0 100 200 300 O. Hsp70 0d1d5d9d14d1d5d9d14d1d5d9d14d 0 100 200 300 P. sHSP 0d1d5d9d14d1d5d9d14d1d5d9d14d RU/ng TSP 0 100 200 300 Q. sHSP 0d1d5d9d14d1d5d9d14d1d5d9d14d 0 100 200 300 R. sHSP 0d1d5d9d14d1d5d9d14d1d5d9d14d 0 100 200 300 Normoxia Mild hypoxia Moderate hypoxia Control Normoxia Mild hypoxia Moderate hypoxia Control Normoxia Mild hypoxia Moderate hypoxia Control Figure 3-4 continued. Stress prot ein expression levels in clams from hypoxia experiments. Data are expressed as relative units of expression per nanogram of total soluble pr otein (RU/ng TSP) and are presented as scatterplots with asymmetrical error bars. The dot represents the median a nd the error bars define the minimum a nd maximum. Abbreviations: d, day; mod, moderate; Hsp60, heat shock protein 60; Hsp70, heat s hock protein 70; sHsp, small heat shock protein.

PAGE 105

105 Normoxia Mild hypoxia Moderate hypoxia ControlFall Winter Spring S. Ubiquitin 0d1d5d9d14d1d5d9d14d1d5d9d14d RU/ng TSP 0 50 100 150 200 250 T. Ubiquitin 0d1d5d9d14d1d5d9d14d1d5d9d14d 0 50 100 150 200 250 U. Ubiquitin 0d1d5d9d14d1d5d9d14d1d5d9d14d 0 50 100 150 200 250 V. OGG1m 0d1d5d9d14d1d5d9d14d1d5d9d14d RU/ng TSP 0 70 140 210 280 350 W. OGG1m 0d1d5d9d14d1d5d9d14d1d5d9d14d 0 70 140 210 280 350 X. OGG1m 0d1d5d9d14d1d5d9d14d1d5d9d14d 0 70 140 210 280 350 Normoxia Mild hypoxia Moderate hypoxia Control Normoxia Mild hypoxia Moderate hypoxia Control Figure 3-4 continued. Stress prot ein expression levels in clams from hypoxia experiments. Data are expressed as relative units of expression per nanogram of total soluble pr otein (RU/ng TSP) and are presented as scatterplots with asymmetrical error bars. The dot represents the median a nd the error bars define the minimum a nd maximum. Abbreviations: d, day; mod, moderate; OGG1m, 8-oxoguanine DNA glycosylase.

PAGE 106

106 02468101214 0.0 0.2 0.4 0.6 0.8 1.0Cumulative Proportion SurvivingA. Winter 02468101214 Experiment duration (d) 0.0 0.2 0.4 0.6 0.8 1.0Cumulative Proportion Surviving Room temp. Mild temp. elevation Mod. temp. elevationB. Spring Figure 3-5. Survivorship of clams in the wint er (A) and spring (B) temperature experiments. Data are expressed as cumulative propor tion of surviving clams. Each symbol represents one clam on the day the clam died. Experiment dur ation is in days. Symbols for treatments with identical survival patterns (room temperature and mild temperature elevation) are overlaid, with only one treatment visible.

PAGE 107

107 C. SpringExperiment duration (d) 15914Fraction buried 0.0 0.2 0.4 0.6 0.8 1.0 B. Winter 15914Fraction buried 0.0 0.2 0.4 0.6 0.8 1.0 ## A. Fall 15914Fraction buried 0.0 0.2 0.4 0.6 0.8 1.0 Room temp. Mild temp. elevation Mod. temp. elevation Figure 3-6. Fraction of clams th at buried in the three temper ature experiments. Data are expressed as a summary of fraction buried at the 60 minute time point of each sampling date. Experiment duration is in days Number signs indicate that all clams in that treatment were dead.

PAGE 108

108 C. SpringExperiment duration (d) 15914Glycogen (mg/ml) 0 25 50 75 B. Winter 15914Glycogen (mg/ml) 0 10 20 30 40 # # A. Fall 15914Glycogen (mg/ml) 0 10 20 30 Room temp. Mild temp. elevation Mod. temp. elevation Figure 3-7. Glycogen content of whole clams in the three temperature experiments. Data are expressed as mean SD. Experiment dur ation is in days. Abbreviations: mg, milligrams; ml, milliliters. Number signs indicate that all clams in that treatment were dead.

PAGE 109

109 A. Cu/Zn SOD 0d1d5d9d14d1d5d9d14d1d5d9d14d RU/ng TSP 0 100 200 300 B. Cu/Zn SOD 0d1d5d9d14d1d5d9d14d1d5d9d14d 0 100 200 300 C. Cu/Zn SOD 0d1d5d9d14d1d5d9d14d1d5d9d14d 0 100 200 300 D. GPx 0d1d5d9d14d1d5d9d14d1d5d9d14d RU/ng TSP 0 50 100 150 200 250 E. GPx 0d1d5d9d14d1d5d9d14d1d5d9d14d 0 50 100 150 200 250 F. GPx 0d1d5d9d14d1d5d9d14d1d5d9d14d 0 50 100 150 200 250 G. MnSOD 0d1d5d9d14d1d5d9d14d1d5d9d14d RU/ng TSP 0 50 100 150 200 250 ## H. MnSOD 0d1d5d9d14d1d5d9d14d1d5d9d14d 0 50 100 150 200 250 ##Control Room temp. Mild temp. elevation Mod. temp. elevation Control Room temp. Mild temp. elevation Mod. temp. elevation I. MnSOD 0d1d5d9d14d1d5d9d14d1d5d9d14d 0 50 100 150 200 250 Control Room temp. Mild temp. elevation Mod. temp. elevation# #Fall Winter Spring Figure 3-8. Stress protein e xpression levels in clams from te mperature experiments. Data are e xpressed as relative units of ex pression per nanogram of total soluble protein (RU/ ng TSP) and are presented as scatterplots with asymmetrical error bars. The dot represents the median and the error ba rs define the minimum and maximum. A bbreviations: d, day; mod, moderate; Cu/Zn SOD, copper/zinc superoxide dismutase; GPx, glutathione peroxidase ; MnSOD, manganese super oxide dismutase. Number signs indicate clam death.

PAGE 110

110 Control Room temp. Mild temp. elevation Mod. temp. elevation Control Room temp. Mild temp. elevation Mod. temp. elevation Control Room temp. Mild temp. elevation Mod. temp. elevationFall Winter Spring K. Hsp60 0d1d5d9d14d1d5d9d14d1d5d9d14d 0 100 200 300 L. Hsp60 0d1d5d9d14d1d5d9d14d1d5d9d14d 0 100 200 300 J. Hsp60 0d1d5d9d14d1d5d9d14d1d5d9d14d RU/ng TSP 0 100 200 300 M. Hsp70 0d1d5d9d14d1d5d9d14d1d5d9d14d RU/ng TSP 0 100 200 300 N. Hsp70 0d1d5d9d14d1d5d9d14d1d5d9d14d 0 100 200 300 O. Hsp70 0d1d5d9d14d1d5d9d14d1d5d9d14d 0 100 200 300 P. sHSP 0d1d5d9d14d1d5d9d14d1d5d9d14d RU/ng TSP 0 70 140 210 280 350 Q. sHSP 0d1d5d9d14d1d5d9d14d1d5d9d14d 0 70 140 210 280 350 R. sHSP 0d1d5d9d14d1d5d9d14d1d5d9d14d 0 70 140 210 280 350 # # # # # # Figure 3-8 continued. Stress protei n expression levels in clams from temperature experiments. Data are expressed as relative u nits of expression per nanogram of total soluble pr otein (RU/ng TSP) and are presented as scatterplots with asymmetrical error bars. The dot represents the median a nd the error bars define the minimum a nd maximum. Abbreviations: d, day; mod, moderate; Hsp60, heat shock protein 60; Hsp70, heat shock protein 70; sHsp, sma ll heat shock protein. Number signs indicate clam death.

PAGE 111

111 S. Ubiquitin 0d1d5d9d14d1d5d9d14d1d5d9d14d RU/ng TSP 0 50 100 150 200 250 Fall Winter Spring Control Room temp. Mild temp. elevation Mod. temp. elevation T. Ubiquitin 0d1d5d9d14d1d5d9d14d1d5d9d14d 0 50 100 150 200 250 U. Ubiquitin 0d1d5d9d14d1d5d9d14d1d5d9d14d 0 50 100 150 200 250 V. OGG1m 0d1d5d9d14d1d5d9d14d1d5d9d14d RU/ng TSP 0 50 100 150 200 250 W. OGG1m 0d1d5d9d14d1d5d9d14d1d5d9d14d 0 50 100 150 200 250 X. OGG1m 0d1d5d9d14d1d5d9d14d1d5d9d14d 0 50 100 150 200 250 Control Room temp. Mild temp. elevation Mod. temp. elevation Control Room temp. Mild temp. elevation Mod. temp. elevation# # # # Figure 3-8 continued. Stress protei n expression levels in clams from temperature experiments. Data are expressed as relative u nits of expression per nanogram of total soluble pr otein (RU/ng TSP) and are presented as scatterplots with asymmetrical error bars. The dot represents the median a nd the error bars define the minimum a nd maximum. Abbreviations: d, day; mod, moderate; OGG1m, 8-oxoguanine DNA glycosylas e. Number signs indicate clam death.

PAGE 112

112 02468101214 0.0 0.2 0.4 0.6 0.8 1.0Cumulative Proportion SurvivingA. Fall 02468101214 0.0 0.2 0.4 0.6 0.8 1.0Cumulative Proportion Surviving High temp./Ambient salinity High temp./Mild hyposalinity High temp./Mod. hyposalinity Room temp./Ambient salinity Room temp./Mild hyposalinity Room temp./Mod. hyposalinityB. Winter 02468101214 Experiment duration (d) 0.0 0.2 0.4 0.6 0.8 1.0Cumulative Proportion SurvivingC. Spring Figure 3-9. Survivorship of clam s in dual stressor experiments. Data are expressed as cumulated proportion of surviving clams. Each symbol represents one clam on the day it died. Experiment duration is in days. Treatmen ts with identical survival patterns are overlaid. The high temperature/ambient salinit y treatment is missing from the spring.

PAGE 113

113 A. Fall 15914Fraction buried 0.0 0.2 0.4 0.6 0.8 1.0 High temp./Ambient salinity High temp./Mild hyposalinity High temp./Mod. hyposalinity Room temp./Ambient salinity Room temp./Mild hyposalinity Room temp./Mod. hyposalinity # # # # C. SpringExperiment duration (d) 15914Fraction buried 0.0 0.2 0.4 0.6 0.8 1.0 # # # # # # # # B. Winter 15914Fraction buried 0.0 0.2 0.4 0.6 0.8 1.0 # # # # # # # # #Figure 3-10. Fraction of clams that buried in the dual stressor experiment s. Data are expressed as a summary of fraction buried at the 60 minute time point of each sampling date. Experiment duration is in days Number signs indicate that all clams in that treatment were dead.

PAGE 114

114 C. SpringExperiment duration (d) 15914Glycogen (mg/ml) 0 10 20 30 # # # # # # # # B. Winter 15914Glycogen (mg/ml) 0 5 10 15 20 25 # # ## # # # # # A. Fall 15914Glycogen (mg/ml) 0 10 20 30 40 50 High temp./Ambient salinity High temp./Mild hyposalinity High temp./Mod. hyposalinity Room temp./Ambient salinity Room temp./Mild hyposalinity Room temp./Mod. hyposalinity # # # #Figure 3-11. Glycogen content of whole clams in the dual stressor experiments. Data are expressed as mean SD. Experiment dur ation is in days. Abbreviations: mg, milligrams; ml, milliliters. Number signs indicate that all clams in that treatment were dead.

PAGE 115

115 8-oxoGuo/10 6 Guo 0 4 8 12 C. Spring 8-oxoGuo/10 6 Guo 0 20 40 60 B. Winter 8-oxoGuo/10 6 Guo 0 5 10 15 20 25 A. FallRoom Temperature: High Salinity: Amb.Mild Mod.Amb.Mild Mod. Figure 3-12. RNA oxidation after 24 hour expos ure in dual stressor experiments. Data are presented as scatterplot of median with uneven error bars denoting minimum and maximum values. Note different scales on the three graphs. Abbreviations: 8oxoGuo, 8-oxo-7,8-dihydroguanosine; Guo, guanosine; amb, ambient; mod, moderate.

PAGE 116

116 CHAPTER 4 STRESS RESPONSE OF A FRESHWATER CLAM ALONG AN ABIOTIC GRADIENT: TOO MUCH OXYGEN MAY LIMIT DISTRIBUTION Introduction The size and location of a species’ range ar e dynamic and influenced by evolutionary constraints (Holt, 2003), biotic in teractions (Case et al., 2005) and abiotic factors (Brown et al., 1996), acting either singly or in combination. Abiotic factor s may influence a species’ distribution by stressing organi sms beyond their physiological lim its (Parsons, 1991). Near the edge of a species’ distribution, partic ularly if that edge is influen ced by abiotic factors, we expect to find decreased abundance as well as decr eased body condition of individuals. Condition has traditionally been assessed using whole-organism characters such as body size and fecundity (Caughley et al., 1988), but cellula r-level indicators may be at le ast equally useful for assessing condition. Such cellular-level indicators include : RNA:DNA ratio as an indicator of protein synthesis and growth (Dahlhoff, 2004); DNA a nd RNA oxidation as indicators of cellular damage from free radicals (Halliwell and Gu tteridge, 1999); and upre gulated expression of specific stress proteins, such as heat shock proteins, as indica tors of a homeostatic cellular response to a stressor (e.g., Hofmann and Somero, 1995; Downs et al., 2001a; Abele and Puntarulo, 2004). Therefore, these indicators pr ovide information about cellular metabolic and homeostatic responses to stress, and thus may be more sensitive than whole-organism characters for determining how abiotic factors affect the condition and distribution of individuals in a population. In the current study we used cel lular-level indicators to test the following hypothesis: if a population’s distribution overlies a stressful environmental gr adient, then abundance should decrease near the distribution e dge, and edge individuals should have increased stress and lower condition. In selecting the appropr iate system, we looked for a stable population of sessile

PAGE 117

117 animals in an environment with a stable gradie nt of a small number of potentially stressful abiotic factors. For this study, we examined th e distribution and physiolo gy of a population of small freshwater clam, Sphaerium sp., which inhabits a swamp/ stream system in western Uganda. This habitat contains a relatively stable dissolved O2 (DO) gradient, from freshwater tributary streams with normoxic water (equilibrated with atmospheric O2 levels) to a papyrus swamp with very hypoxic water (DO less than 10% of fu lly-aerated water), and a pH gradient from the neutral stream wate r to acidic swamp water. A 2year, broad-scale survey of macroinvertebrates in this system (Chapman et al., 2004) revealed that the clams were very abundant in the swamp but largely absent from re gions of tributary stre ams with normoxic water and neutral pH, despite the absence of any appare nt physical or biotic barriers limiting access to stream sites. Since sphaeriid clams are ovovivi parous (i.e., they in ternally brood their young; Mackie, 1978), it appears that thes e clams do not spend any part of their life cycle in normoxic, neutral pH water. This distri bution pattern could arise becaus e hypoxic and acidic conditions are correlated with other factors that make the swam p a habitat of high suitab ility or, alternatively, because the hypoxic and/or acidic conditions are favor able for these clams. This latter possibility is especially intriguing because it is typically assumed that marine and freshwater invertebrates avoid hypoxic and/or acidic conditions when po ssible, or utilize physiological mechanisms to compensate for the resulting hypometabolism a nd acidosis when the conditions cannot be avoided (Burnett, 1997). To test whether the sphaeriid clams, whic h are abundant in the hypoxic and acidic swamp, show evidence of physiological stress in the norm oxic and neutral pH conditions of the tributary streams, we measured clam distribution and cellu lar-level indicators along a gradient from the swamp into a tributary stream. In addition, we measured the limnological characters of DO,

PAGE 118

118 conductivity, pH, temperature and transparency, and we quantif ied the relationship between these characters and clam density. To examine how the abiotic gradient along this transect affects the physiological condition of the clams, we sampled clams from a reference site from the dense swamp interior and from three sites along the transect, in cluding the extreme edge of the population’s distribution. We assessed RNA:DNA ratio, oxidized DNA and RNA bases, and stress protein expression levels, including several stress proteins that indicate generalized cellular stress, as well as those that specifically respond to increased free radical production, which has been linked to fluctuations in DO and pH (Bove ris and Chance, 1973; Li et al., 2002; Vesel and Wilhelm, 2002). Our goal was to determine whet her the patterns of ce llular indicators were consistent with increased stress in clams near the edge of their dist ribution and furthermore whether the pattern was consistent with increased oxidative damage. Materials and Methods Study Site This study was conducted in a swamp/stream sy stem in Kibale National Park in western Uganda (013’ –041’N, 3019’ –3032’E). The park is an equatorial moist evergreen forest, transitional between lowland rain forest and mont ane forest, with distinct bimodal wet and dry seasons (Chapman et al., 1997). The Rwembaita Swamp is a large (approximately 6.5 km in length) papyrus ( Cyperus papyrus ) swamp, with dense papyrus stands that can reach 5 m in height and form a closed canopy. The low rates of mixing and low incident light induced by the heavy forest of papyrus sedge, and high rate s of organic decompos ition produce hypercapnic (elevated dissolved CO2) and hypoxic water (Chapman et al., 2001). Over a 3-year period, Chapman et al. (2000) reported DO in the Rwemba ita Swamp averaging 0.0466 0.0072 mmol

PAGE 119

119 L-1 across dry and rainy seasons.1 The swamp is fed by several small streams that have intermediate DO, which can vary among streams from 0.118 0.0056 mmol L-1 to 0.222 0.0059 mmol L-1 (Chapman et al., 2004). The streams fo rm ecotonal gradients of decreasing DO concentration as they enter the swamp. In May 2004, at the beginning of the summe r dry period, we measured limnological characters and clam abundance along a tran sect from the Rwembaita Swamp, through an ecotonal region and into a tributar y of the swamp, Inlet Stream We st (see Figure 4-1). Data from the broad range surveys conducted by Chapma n and colleagues (Chapman et al., 2000, 2004) were used to design our sampling regime. Sites on the transect were separated by 10 m. Five sites (sites 1-5) with papyrus canopy were designat ed as swamp sites; five sites (sites 6-10) with emergent (non-papyrus) vegetation mixed with fo rest understory vegetation were designated as ecotone sites; and six sites (sit es 11-16) were designated as stream sites. Water depth was shallow (10.2 4.3 cm, n = 48) across all 16 sites. The width of the water stream at ecotone and stream sites ( n =11), was 1.15 1.33 m. The substrate wa s mud mixed with vegetation in all sites, with some sand also present in the stream sites. Sampling Methods Triplicate readings of all limnological ch aracters were collected between 900 and 1200 hour at each site along the tr ansect, following previous prot ocols (Chapman and Liem, 1995). DO, water temperature and conductivity readings were collected using YSI meters, and pH was measured using an Oaktron meter. Water transp arency was measured using a transparent tube marked off in cm, with a miniature Secchi disc at the bottom; all measurements were taken by the same individual. At each site we reco rded maximum water depth and water velocity 1 To convert mmol O2 L-1 to mg O2 L-1, multiply by 32 (molecular weight of O2).

PAGE 120

120 (categorical variables ra nked on a scale of 0 to 3; 0 = no curr ent, 1 = low, 2 = medium, 3 = high; Chapman, 1995). At each site along the transect, clams were sa mpled using triplicate scoop nets (frame size 30.5 x 40.6 cm, mesh size of 0.32 cm), with triplicates separate d by approximately 0.5 m. The samples were taken by disturbing and scooping th e bottom substrate. Scoop net samples were sorted directly in the field. We calculated an i ndex of clam density (cat ch per unit effort; CPUE) as the average number of clams per scoop. All clams collected in the initial survey were used for the size/frequency distribution analysis. For size measurements, shell length was measured from umbo to ventral margin using digital calipers. Sampling for RNA/DNA, Nucleic Acid Oxidation and Stress Proteins Clams used for nucleic acid analyses and stre ss proteins were collect ed from four sites, three of which were on the transect: a swamp s ite (site 2), an ecotone site (site 8) with comparable clam abundance to the swamp site but higher DO, and the stream site (site 11) with the highest DO of any site with clams. The four th site (our reference site) was in the swamp interior, approximately 500 m downstream from stream water input. At this site, multi-year surveys indicated presence of clams in all seas ons (Chapman et al., 2004). DO at this site averaged 0.0337 mmol L-1 over a 2-year period (Chapman et al., 2004). Clams were collected with a scoop net, briefly rinsed in water from their habitat, pier ced with a dissecting needle to release water held in the mantle cav ity, and flash-frozen in a liquid N2 dry shipper (CX100, Taylor Wharton Cryogenics, Theodore, AL, USA). The time from scoop netting to flash-freezing was less than 1 minute. Clams were collected on two consecutive days between 900 and 1200 h. Clams were maintained in the dry shipper during transport back to the University of Florida, where they were stored at -80C. All clams used for nucleic acid and stress protein analyses were 5 – 7 mm in shell length.

PAGE 121

121 Amounts of total tissue DNA, RN A, oxidized DNA and RNA bases were determined in 45 clams per site. DNA guanine base oxidati on produces 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodGuo), and RNA guanine base oxida tion produces 8-oxo-7,8-dihydroguanosine (8oxoGuo). Samples consisting of 80 – 100 mg whol e clam homogenate were processed as described previously (Hofer et al., 2006). Briefly, we simu ltaneously extracted both DNA and RNA from whole clam homogena tes using guanidine thiocyanat e and phenol/chloroform at neutral pH, after which nucleic acids were hydrolyzed with Nuclease P1 and alkaline phosphatase. Hydrolytic enzymes were then removed by filtration, and the hydrolysate was analyzed by high performance liquid chromatogr aphy coupled to electrochemical detection (HPLC-ECD; ESA Inc., Chelmsford, MA, USA). Stress protein expression le vels were determined using mono-specific, ELISA-grade polyclonal antibodies generated by and donated by EnVirtue Biotechnologies, Inc. (Winchester, VA, USA). The following antibodies were used : manganese superoxide dismutase (MnSOD; Cat. #AB-1976), copper/zinc superoxide dism utase (Cu/Zn SOD; Cat. # AB-SOD-1516), glutathione peroxidase (GPx; Cat. # AB-GPX-1433 ) mitochondrial 8-oxoguanine DNA glycosylase (OGG1-mito; lot 2916), heat shock protein 60 (Hsp60; Cat. # AB-H100-IN), heat shock protein 70 (Hsp70; Cat. #AB-Hsp70-1519), a nd invertebrate small heat shock protein homologues (sHsp; Cat. #AB-H105) A summary of the functions of these proteins is presented in Table 4-1. The antibodies were raised in rabbits against 8-15 amino-acid polypeptides (conjugated to bovine serum albumi n) derived from each target protein sequence of the bivalve Mya arenaria (Downs et al., 2002b). Stress proteins were analyzed in whole clam homogenates (Joyner-Matos et al., 2006) from 10 clams from each of the four sites, in cluding those clams used for nucleic acid analyses. Br iefly, whole clams (with shells ) were homogenized in liquid N2.

PAGE 122

122 Homogenized tissue from each clam was suspende d in protein denaturing buffer, and total soluble protein concentration was determined (Ghosh et al., 1988). Each clam was processed individually, and all clam samples were prep ared on the same day using the same buffer. Antibody specificity of seven stress proteins wa s verified by polyacrylamide gel electrophoresis (PAGE) and western blotting, as described in the Results section. Expression levels were analyzed in triplicate by enzyme-li nked-immunosorbent assay (ELISA). Statistical Analyses The nonparametric Spearman’s rank correlation was used to test for a significant relationship between each limnological variable a nd distance along the transe ct, and the index of clam density (CPUE) and distan ce. Linear regression was used to evaluate the relationship between clam CPUE and the following variab les: DO, conductivity, pH and transparency. Multiple regression was used to quantify the rela tionship between clam CPUE and the same suite of independent variables when entered into th e regression model together. RNA oxidation data and stress protein expression levels were anal yzed by one-way ANOVA with Fisher’s LSD posthoc multiple comparison test. The DNA oxidation da ta were not normally distributed (ShapiroWilks test W = 0.77764 and p < 0.0007) and were therefore analyzed using a Kruskal-Wallis one-way ANOVA with Conover-Inman pairwise comparisons, using StatsDirect (v. 2.4.5, www.statsdirect.com ). To better understand the relations hip between overall stress protein expression pattern and site, a principal component s analysis was performe d on the stress protein data. Factor scores resu lting from an analysis with two unrot ated factors were then analyzed by one-way ANOVA as above. Except as noted all st atistical analyses were performed with Statistica (v. 7.1, Statsoft Inc., Tulsa OK, USA). Except as indicated, all values are reported as mean standard error. Statis tical significance was accepted at = 0.05.

PAGE 123

123 Results Limnological survey. DO in the swamp sites (sites 1-5) averaged 0.023 mmol L-1, which is less than one-tenth that of aerated stream water (0.25 mmol L-1). DO increased along the transect from the swamp into the stream ( rs = 0.944, p < 0.0001, Figure 4-2a), reaching 0.19 mmol L-1 at the most upstream site (site 16). Th e surface water temperat ures averaged 17.5 1.5C ( n = 48; data not shown) across all sites. C onductivity was not related to distance along the transect ( rs = 0.329, p = 0.226, Figure 4-2d), but pH in creased along the transect ( rs = 0.889, p < 0.0001, Figure 4-2c), as did water transparency ( rs = 0.743, p = 0.0021, Figure 4-2b). At all sites (with the exception of site 16), water velocity was low with ranks between 0 and 1, and with most sites ranked as 0.5, indicating sluggi sh water flow (data not shown). Clam density and size-frequency pattern. The catch per unit effort (CPUE) of clams was high but ‘patchy’ in the swamp (range fr om 17–144, corresponding to ca. 140-1200 clams m-2) and decreased slightly in the ecotone sites (Fig ure 4-3a). CPUE decreased along the transect ( rs = -0.889, p < 0.0001), with clams absent from stream sites with DO level higher than 0.17 mmol L-1 (i.e., sites 13-16). Clams were bur ied below the sediment surface. We examined the degree to which DO, conductivity, pH and transparency explained variation in CPUE of clams al ong the transect. In a multiple re gression, only DO was marginally significant (whole model, R2 = 0.4168, p = 0.0005; DO, partial r = -0.308, p = 0.0596); however, all four factors exhibited coll inearity (VIF ranging from 1.11 to 3.65). We therefore examined the relationship between clam CPUE and each in dependent variable using least-squares linear regression. DO explained 52.9% of the variance in clam CPUE ( p = 0.0014), pH explained 48.2% ( p = 0.0041), and transparen cy explained 39.8% ( p = 0.0117). In contrast, the relationship between conductivity and clam abundance was not significant ( p = 0.34).

PAGE 124

124 The umbo-to-margin length of the clams was qu antified to examine variation in size and size frequency among sites. For this analysis we grouped the data according to site type (Figure 4-3b). All size classes from 2 mm to 7 mm were present in samples from the swamp ( n = 1209) and the ecotone ( n = 545). In the swamp and ecotone, the di stribution of clams into size classes roughly matched a normal distribution. In th e stream site, the sample population ( n = 15) was heavily skewed towards the larger (> 5 mm) si ze classes, with only one (7%) of the 15 clams having a shell length of less than 5 mm. In the swamp and ecotone, clams with a shell length of less than 5 mm represented 40% and 59% of the samples, respectively. RNA:DNA ratio. To investigate whether clams near th e edge of the distribution exhibited decreased protein synthesis or growth in comp arison to clams from the swamp interior, we analyzed RNA:DNA ratio in 4-5 clams per site. The RNA:DNA ratio of the clams in the ecotone and stream was 25% lower than that of clams in the swamp margin ( F = 5.283, p = 0.012; swamp margin vs. ecotone, p = 0.049, swamp margin vs. stream, p = 0.054, Figure 4-4a). Nucleic acid oxidation. To determine whether clams experienced cellular oxidative damage, which results from increased production or decreased detoxification of free radicals, we measured the levels of two forms of oxida tively damaged nucleo sides: the DNA oxidation product 8-oxodGuo and the RNA oxidation produc t 8-oxoGuo. DNA yield was consistent across sites (0.472 0.07 g/mg tissue; F = 0.246, p = 0.863, data not shown), indicating that nucleoside extraction was equivalent in all samples. Clams in the ecotone and stream sites had significantly more DNA and RNA ox idation products than clams from the swamp interior (8oxodGuo, Kruskal-Wallis ANOVA H3 = 8.456, p = 0.037, Conover-Inman pairwise comparisons t14 = 2.145, p 0.0077, Figure 4-4b; 8-oxoGuo, F = 5.871, p = 0.008; for all comparisons, p

PAGE 125

125 0.036, Figure 4-4c). At each site, RNA oxidati on was proportionally more extensive than DNA oxidation. Stress protein analyses. We analyzed the expression levels of seven stress proteins (Table 4-1), which we categorized according to func tion as antioxidants (MnSOD, Cu/ZnSOD, and GPx), oxidative repair (OGG1m) and chaperones (Hsp60, Hsp70, sHsp). Antioxidants: MnSOD levels were higher in clams from the swamp margin and ecotone sites than in clams from either the swamp interior or stream sites (Figure 4-5a; n = 40, F = 2.83, p = 0.052; swamp margin vs. stream p = 0.014, ecotone vs. stream p = 0.020). Cu/ZnSOD levels were lower in the clams from the stream site than in clams from the other three sites (Figure 4-5b; n = 40, F = 4.21, p = 0.012; stream vs. other sites p 0.012). There were no significant cha nges in GPx expression levels (Figure 4-5c; n = 40; F = 1.39, p = 0.262). Oxidative repair: Clams from the swamp margin expressed more OGG1m than did clams from the sw amp interior or stream sites (Figure 4-5d; n = 40; F = 3.82, p = 0.018; swamp interior vs. swamp margin p = 0.013, swamp margin vs. stream p = 0.003). Chaperones: Clams from the swamp margin an d ecotone expressed more Hsp60 than clams from the dense interior, with clams from the swamp margin expressing more Hsp60 than clams from the stream (Figure 4-5e; n = 40; F = 5.333, p = 0.004; for all significant comparisons, p 0.036). Clams from the stream site had lower Hsp70 levels than clams from the swamp margin or ecotone site, but comparable expression levels to clams from the swamp interior (Figure 4-5f; n = 40; F = 3.647, p = 0.021; stream versus swamp margin and ecotone, p 0.0125). Clams from the swamp margin had higher sHsp expression levels than clams from the other three sites (Figure 4-5g; n = 40; F = 3.87, p = 0.017; swamp margin versus other sites p 0.014).

PAGE 126

126 We conducted a principal components analysis to investigate whethe r the stress protein expression showed evidence of a c oordinated response. We extracte d two factors that explained a total of 63.53% of the variance, w ith the first factor explaining 45.13% of the total variance. The first factor was composed of f our proteins with factor loadin g scores greater than 0.7: GPx, Hsp60, Hsp70, and MnSOD, all of which had pos itive loading scores ranging from 0.718 to 0.827. The second factor, consisting only of OGG1m, explained 18.4% of the variance in stress protein expression. OGG1m loaded heavily on the s econd factor with a score of -0.762. We next examined whether the mean factor scores (assigned to each indivi dual on the basis of factor 1) differed among sites. Factor 1 loading scores were significantly different among sites ( F = 4.034, p = 0.0153, Figure 4-5h), with clams from the swam p margin having a higher score than clams from the dense interior or the stream ( p = 0.035 and p = 0.039, respectively). Discussion The “abundant center” distribution pattern (Sagar in and Gaines, 2002) predicts that when a population overlies a strong environmental gradie nt, abundance will be highest where conditions are the least stressful and/or the most benefici al. Furthermore, those individuals living where abundance is highest are expected to be in better condition than those living where abundance is low (Caughley et al., 1988). Although the Sphaerium sp. distribution pattern matches the abundant center pattern, the distri bution appears to indicate that the hypoxic, acidic swamp water is less stressful (or more beneficial) th an the normoxic, neutral pH stream water. Limnological Characters and Relationship with Clam Density A previous characterization of the Rwembaita Swamp showed that the water is extremely hypoxic and mildly acidic relative to the tributar y streams feeding into it (Chapman et al., 2000), consistent with abiotic conditi ons in other papyrus swamp syst ems in Africa (Carter, 1955; Chapman et al., 2001). The ecotonal region of inte rmediate pH and DO is always present along

PAGE 127

127 the tributary streams, but the exact location of the ecotone varies with season and other local habitat characteristics (e .g., stream volume), and it can be disturbed by large animals such as elephants (Chapman et al., 2001). We conducted a fine -scale survey of a transect extending from within the Rwembaita Swamp into one of its tr ibutary streams (Figure 4-2a). We found that clams were abundant in the swamp, less abundant along the ecotone, and absent from stream sites with DO above 0.17 mmol L-1 and a pH above 6.9. In a multiple regression of our measure of abundance (CPUE) versus DO, pH, tran sparency and conductivity, DO was the only significant predictor. However, all four factors were collinear and when examined by leastsquares linear regression, DO, pH, and transpar ency each explained significant variation in CPUE. The population of clams at the distribution edge was skewed towards larger size classes (5 – 7 mm). Since this is the first assessment of a size-frequency pa ttern in this system, and it was only done at one season, these results must be in terpreted with caution. Nonetheless, these data suggest that demographic diffe rences exist along this physic ochemical gradient. Sphaeriid lifespans range from less than 1 year to 5 y ears (Burky, 1983), and all kn own species internally brood their young, which are released as shelled j uveniles (Mackie, 1978). The lack of smaller size classes in stream sites could reflect fluctua tions in the position of th e ecotone; for example, a period of low rainfall could have led to abiotic conditions in these sites that were transiently similar to the swamp, allowing successful recruitment. Other physicochemical characters, as well as biotic interactions, likely influence clam distribution and abundance in this swamp-stream system. For example, substrate type typically influences the distribution of burrowing bivalves, although a relatio nship between substrate type and habitat preference was not su pported in several st udies of sphaeriid clams (reviewed in

PAGE 128

128 Burky, 1983). In the Rwembaita Swamp-stream system, substrate type (mud mixed with vegetation) was similar across all sites with cl ams and no clams observed on the surface of the substrate, which is consistent with a previous survey in which clams were sampled from a depth of 2-3 cm (Osborne et al., 2001; Chapman et al., 2004). The extent to which the increased sandiness in the stream might negatively influen ce burial and thus influe nce dispersal to these sites is unknown. Currently, we canno t address the potential influen ce of biotic factors such as competition and predation on clam distribution. However, previous studies (Chapman, 1995; Chapman et al., 1999, 2004; Osborne et al., 2001) have additionally highl ighted the importance of considering effects of physicoc hemical gradients when assessing how biotic interactions shape this community structure (Menge and Sutherland, 1987; Case et al., 2005). Cellular-level Indicators To test whether clams at the distribution edge were under more stress and whether the condition of the clams changed along the abiotic grad ients, we investigated several ce llular-level indicators of stress: RNA:DNA ratio, nucleic aci d oxidative damage, and number of different stress proteins. Compared to clams in the swam p interior and swamp margin, we expected clams from the ecotone and stream site (distribut ion edge) to have lower RNA:DNA ratio, higher oxidative damage, and higher stre ss protein expression, all cons istent with higher stress. Decreased RNA:DNA ratio indicates reduced leve ls of protein synthesis, as could occur with stress or decreased growth rate (Elser et al., 2000; Dahlhoff, 2004) We found that clams from the ecotone and stream sites had lower RNA:DNA ratio in comparison to clams from the swamp margin. Although statistica lly significant, the difference in RNA:DNA ratio was small in comparison to studies examining changes across seasons or following experimental manipulations (Dahlhoff, 2004). Nonetheless, to our knowledge comparable studies of

PAGE 129

129 RNA:DNA ratio across a population do not exis t, so it is unknown whether even small differences are functionally signi ficant in natural populations. Oxidative damage, including damage to DNA and RNA, occurs when free radical production overwhelms a cell’s ability to detoxi fy the free radicals (Halliwell and Gutteridge, 1999). The deleterious effects of DNA oxidati on on mutation accumulation and aging are wellestablished (Hartman et al., 2004; Sanz et al., 2006), but the effects of RNA oxidation have not been well characterized (Seo et al., 2006). We found that clams from the ecotone and stream sites had significantly elevated levels of oxidatively damaged DNA and RNA in comparison to clams from the dense interior of the swamp, and furthermore that RNA oxidative damage was proportionally higher than DNA damage at all si tes. To our knowledge, this is the first quantification of DNA and RNA oxi dation in a natural population. Stress proteins are produced by nearly all eu karyotes, and patterns of stress protein expression and activity serve as cellular-level indicators of stressor type (e.g., Willmore and Storey, 1997; Abele and Puntarulo, 2004; Magalhes et al ., 2005) and indicators of how stressors influence habitat preferences (e.g., Feder a nd Hofmann, 1999; Downs et al., 2002b). We found patterns of stress protein expression in clams al ong the transect indicating that clams in the swamp margin and ecotone were ex periencing more stress than thos e in the dense interior of the swamp. The elevations in ch aperone proteins (Hsp60, Hsp70, and sHsp) in clams in the intermediate sites indicate generalized cellular stre ss, particularly that wh ich results in protein misfolding, protein aggregation, or heightened need for protei n degradation (Kregel, 2002). Furthermore, elevations in antioxidants (MnSOD) and the repair enzyme OGG1-m in clams in the swamp margin and ecotone compared to th e dense interior suggests that these clams experienced elevated free radical production and oxidative damage (Halliwell and Gutteridge,

PAGE 130

130 1999). Similar relative changes in protein expres sion were detected in bivalves exposed to abiotic stressors in controlled laboratory conditions (Joyner-Matos et al., 2006) and in bivalves at sites impacted by a crude oil sp ill (Downs et al., 2002b). However, these data on relative changes do not allow comparisons of absolute stress protein expression levels between different organisms nor of comparisons between e xpression levels and enzyme activity. Principal components analysis, which provides a composite view of a coordinated stress response, showed that clams in the swamp margin and ecotone had a larger stress response than did clams in either the dense interior or stream s ites, but that the response of clams in the swamp margin did not differ from that of clams in th e ecotone. This integrated stress response was dominated by two antioxidants, GPx and Mn SOD, and two chaperone proteins, Hsp60 and Hsp70, indicating that clams at the intermediate sites were experiencing stress at the cellular level and that at least some of that stress was due to elevated free radical production. The Extreme Edge of the Distribution Comparison of clams in the swamp margin with those in sites closer to the edge of the distribution followed the expected pattern: as abundance decreases, RNA:DNA ratio decreases, oxidative damage increases, and, for the swam p margin and ecotone sites, stress protein expression increases. Surprisingly, however, stress protein expression in clams from the stream site was equivalent to, or lower than, that of cl ams from the dense interior. One interpretation of this pattern is that clams from the stream site were less stressed than clams from the swamp margin and ecotone, but this is not consistent with the RNA:DNA ratio and nucleotide oxidative damage results, which both indicate that clam s in the stream were under more stress. The alternate interpretation is that clams from the st ream site were so stressed that they were not capable of maintaining high stre ss protein expression (Werner a nd Hinton, 1999). Therefore, the clams at the extreme edge of the distribution s how a stress response patter n that might otherwise

PAGE 131

131 be interpreted as pathologic lo ss of cellular homeostatic stress response and repair mechanisms. It is unknown whether this pattern of extreme phys iological stress in organisms at the extreme distribution edge is present in other systems with stable population distributions overlying stressful abiotic gradients. Relationships Between Limnological Characte rs and Cellular-level Stress Indicators The distribution data demonstrat ed that the swamp is less st ressful (or more beneficial) than the stream, consistent w ith the abundant center distributi on pattern. Of the limnological variables measured along the transect, DO, pH an d transparency were significant predictors of clam CPUE, and all showed a negative corr elation. When examining cellular-level stress indicators, we found a pattern cons istent with increasing oxidative stress towards the distribution edge. While any of the three limnological vari ables could contribute to oxidative stress, we believe the evidence is strongest for DO. Oxygen availability, particularly hypoxia, affect s a wide variety of ecological processes in aquatic organisms (Baker and Mann, 1992; Diaz and Rosenberg, 1995; Rosenberger and Chapman, 2000). The ability of the sphaeriid clam s in the Rwembaita Swamp to tolerate hypoxia is not surprising, given the well-documented abi lity of clams from this cosmopolitan family to tolerate DO fluctuations (Waite and Neufeld, 1977) and to aestivate duri ng seasonal dry periods in ephemeral ponds (McKee and Mackie, 1983). Howe ver, the hypoxia toleran ce of the sphaeriid clams in the Rwembaita Swamp appears to be gr eater than that demonstrated by congeners and closely related pisiid clams. For example, a European congener showed significantly decreased valve-movement behavior at DO levels comparable to that of the ecotone sites (Heinonen et al., 1997), and a North American sphaeriid a nd European pisiid had critical O2 tensions comparable to the DO level at swamp sites (Waite and Neufeld, 1977; Hamburger et al., 2000).

PAGE 132

132 While it is still unclear whether hypoxia cause s elevated free radical production (e.g., Li and Jackson, 2002; de Oliveira et al., 2005), it is well established that hyperoxia (typically defined as O2 concentrations higher than atmospheric le vels) is linked to increased free radical production and oxidative damage (Boveris a nd Chance, 1973; Abele and Puntarulo, 2004). However, the concentration of O2 necessary to cause oxidative da mage may be much less than “hyperoxia” for isolated cells and small anim als (Packer and Fuehr, 1977; Saito et al., 1995; Gray et al., 2004). Given that th e sphaeriid clams in the Rwemba ita Swamp system are adapted to hypoxia and appear to spend their entire li fe cycle in hypoxic conditions, we propose that clams living in the intermediate sites and di stribution edge experien ce the equivalent of hyperoxic stress. The pattern of cellular-level indicators in our study is consistent with biomedical studies of hyperoxia (Wong et al., 1998; Freiberger et al., 2004; Roper et al., 2004; Olsvik et al., 2005). Therefore, “hyperoxia” stress may occur in any animal exposed to oxygen levels higher than those to which it is adap ted, whether this is a normoxia-adapted animal exposed to hyperoxia, a hypoxia-adapted animal e xposed to normoxia, or even, in the case of Sphaerium sp., an animal adapted to extreme hypoxia exposed to moderate hypoxia. We cannot, however, rule out the possible in fluence of pH or transparency on clam distribution or cellular-level indi cators of stress. In aquatic ha bitats, pH is influenced by CO2 concentration, and the co-occurrence of hypoxia and acidic/hypercapnic conditions is widespread (Burnett, 1997). In the papyrus swamps of East Africa, both DO and pH are strongly influenced by organic matter decomposition, with 81% of the variance in DO explained by the concentration of free, dissolved CO2 (Chapman et al., 2001). Interestingly, CO2 may either promote or inhibit oxidative damage through a va riety of reactions (Vesel and Wilhelm, 2002).

PAGE 133

133 To our knowledge, there are no studies linking oxid ative stress to environmental alterations of pH or CO2. Water transparency, which is influenced by co ncentrations of tannins (Labieniec et al., 2003) and suspended materials (Ward and Shumwa y, 2004), also was inversely correlated with clam abundance. Since tannin levels in the Rwemba ita Swamp system are unknown, we are unable to evaluate their potenti al influence on clam physiology. Suspended materials, which may be inorganic or organic, also decrease water transparency, bu t the composition of suspended materials in this system is unknown. Since bivalv e feeding efficiency decreases in water with high concentrations of inorganic suspended materials (Ward and Shumway, 2004), we would expect clam abundance to be lowest in the swamp, where water transparency is lowest, which is not consistent with our results or multi-year surveys (Chapman et al., 2004). However, when the suspended materials are primarily organic, clam abundance would be lowest where transparency is highest, as observed in this study. Nevertheless, the observed size/frequency distribution is inconsistent with nutrient limitation influe ncing the population distribution, and nutrient restriction does not cause increased oxidative stress (Gredilla and Barja, 2005). Conclusions In the Rwembaita Swamp system we found th at the hypoxic, low-transparency, and acidic conditions of the swamp minimize oxidative dama ge, while the elevated DO, high transparency and neutral ph conditions of the st ream increase oxidative damage. We propose that in this clam, DO above the extreme hypoxia of the swamp causes a physiological condition similar to that of other animals exposed to hyperoxia. A critical test of this hyp othesis would entail long-term transplant experiments, coupled with direct manipulation of the physical environment. However, such experiments would be challenging at this s ite due to frequent disturbance by large animals and substantial fluctuations in water level during dry and rain y seasons. In addition, we found

PAGE 134

134 that clams at the extreme edge showed decrease d stress protein expres sion, despite decreased condition and elevated oxidative damage, which we propose is an indicati on of extreme stress. This demonstrates that analysis of stress prot ein expression alone may be a misleading index of stress and highlights the importance of sampling the extreme distribution edge. The integration of ecological and biochemical responses provides a useful approach to unde rstanding the role of the physicochemical environment in st ructuring population distributions.

PAGE 135

135 Table 4-1. Overview of stress protein functions. Protein type Protein Function Manganese superoxide dismutase (MnSOD) Catalyzes the dismutation of superoxide to the less reactive pro-oxidant H2O2, primarily mitochondrial (Halliwell and Gutteridge 1999). Copper/zinc superoxide dismutase (Cu/ZnSOD) Catalyzes the dismutatio n of superoxide into H2O2, primarily cytoplasmic (Halliwell and Gutteridge 1999). Antioxidant Glutathione peroxidase (GPx) Catalyzes the reduction of H2O2 to water with the concomitant oxid ation of reduced glutathione (Halliwell and Gutteridge 1999). Oxidative repair Mitochondrial 8oxoguanine DNA glycosylase (OGG1m) Catalyzes the removal of the mutagenic 8-hydroxyguanine (8-ox odGuo) base lesion (Boiteux and Radicella 2000) Heat shock protein 60 (Hsp60) Aids in the folding of newly-formed proteins under normal physiological condition and refolds damaged proteins during stress (Hartl 1996; Kregel 2002). Heat shock protein 70 (Hsp70) Has numerous roles involving chaperone functions, protein degradation and protein folding (Frydman 2001; Kregel 2002). Chaperone Small heat shock proteins (sHsp) Bind denatured proteins, preventing irreversible protein aggregation, and participate in the ubiquitin/proteasome system (Parcellier et al. 2005).

PAGE 136

136 Figure 4-1. Schematic map of the study site in the Rwembaita Swamp system of Kibale National Park, Uganda showing direction of water fl ow from the stream and into the swamp. Numbered sites are at 10 m intervals. Site s 1-5 are considered swamp sites, 6-10 as ecotone sites, and 11-16 as stream sites.

PAGE 137

137 Dissolved O2(mmol L-1) 0.00 0.05 0.10 0.15 0.20 0.25 Transparency (cm) 10 30 50 70 90 pH 5 6 7 8 Site 13579111315 Conductivity (microhms cm-1) 70 75 80 85 90 95 SwampEcotone Streama. b. c. d. Figure 4-2. Limnological features of the stream-swamp transect. Mean ( 1 SE) of triplicate measures of a dissolved O2 (mmol l-1), b transparency (cm), c pH, and d conductivity (microhms cm-1) Sites correspond to the survey s ites shown in Figure 4-1. We were unable to collect a sufficient volume of wa ter from site 4 to measure conductivity, pH or transparency.

PAGE 138

138 Site 12345678910111213141516 Catch per unit effort 1 10 100 1000 a SwampEcotone Stream 234567Number of clams 0 50 100 150 200 250 300 350 Size class 234567 0 20 40 60 80 100 120 140 160 234567 0 1 2 3 4 5 6 7 b Swamp c Ecotone d Stream Figure 4-3. Catch per unit effort and size/frequency di stribution of clams. Means ( 1 SE) of a triplicate measures of catch per unit effort of clams in each of the 16 sites on the transect and b size-frequency distribution of cl ams from pooled samples from the three characteristic site types (swamp, n = 1209 clams; ecotone, n = 545 clams; stream, n = 15 clams). Note the logarithmic scale used in the catch per unit effort graph. Shell length size classes of all co llected clams ranged from 2 – 7 mm.

PAGE 139

139 RNA/DNA ratio 0.0 0.5 1.0 1.5 2.0 2.5 a. Oxidized DNA (8-oxodGuo 10-6 dGuo) 0 5 10 15 20 25 30 35 InteriorMarginEcotoneStream Oxidized RNA (8-oxoGuo 10-6 Guo) 0 10 20 30 40 50 60 c. b.ab baa aab bb aab bb Figure 4-4. RNA:DNA rati o and oxidatively damaged nucleic acid in clams. Means ( 1 SD) of a RNA/DNA, b oxidatively damaged DNA nucleos ides (8-oxo-dGuo, oxidized bases per 106 dGuo, black bars), and c oxidatively damaged RNA nucleosides (8-oxoGuo, oxidized bases per 106 Guo, grey bars) in 4-5 clams from each site. Nucleoside analyses were conducted in a subset of th e clams used for stress protein analyses. Bars that share similar letters are not significantly different.

PAGE 140

140 Figure 4-5. Antibody specificity te sts and stress protein expression levels in clams. Levels are presented as relative unit s per nanogram of total soluble protein, (RU ng TSP-1; n = 10 from each site). a manganese superoxide dismutase (MnSOD), b copper/zinc superoxide dismutase (Cu/ZnSOD), c glutathione peroxidase (GPx), d the DNA repair enzyme mitochondrial 8-oxo guanine DNA glycosylase (OGG1-m), e heat shock protein 60 (Hsp60), f heat shock protein 70 (Hsp70), and g small heat shock proteins (sHSP). Clams were collected from the swamp interior, swamp margin (site 2), ecotone (site 8), and stream (site 11). Bars depict means 1 SE. Similar letters indicate statistically indistinguishable samples according to Fisher’s LSD post-hoc multiple comparison test. For antibody speci ficity tests, two random samples of Sphaerium sp. were pooled, subjected to SDSPAGE, western blotted, and assayed with the appropriate antibodies. The positio ns of known molecular weight standards are indicated in kilodaltons. h Factor 1 loading scores from a principal components analysis of stress protein e xpression levels presented in box-and-whiske r plots with the box defining data in the 25th to 75th percentiles, the line i ndicating the median, and the whiskers defining the 10th and 90th percentiles.

PAGE 141

141

PAGE 142

142 CHAPTER 5 SYNTHESIS The research presented in this dissertation ad dresses three aspects of the overall question, “does the magnitude of the cellular stress response influence species distribution?” This research focused on the cellular stress responses of bivalves exposed to abiotic factors present in aquatic environments. The first part of this synthesis pres ents a discussion of what overall themes can be drawn from the three studies. Th e second part of this synthesis presents a summary of findings. Sources of Variance Students of physiology are taught that a well-d esigned laboratory e xperiment examining physiological processes should succe ssfully minimize all potential s ources of variance and that any detected variability in physiological response is an error that should be addressed with the appropriate statistics. In a recent book explori ng patterns in physiologi cal diversity and their impact on ecological processes, Spicer and Gast on (1999) emphasize that physiological variation pervades all hierarchical levels, from within an individual to within a nd between populations and species. They advocate embracing an d studying this variation and its role in species distribution rather than attempting to minimize or ignore it. A number of sources of variation impact the ability of invertebrates to mainta in an oxidative stress response a nd also impact the ability of an investigator to detect the oxida tive stress response and properly interpret the results. Two of these sources are discussed below. Season As demonstrated in Chapters 2 and 3, seasonal differences in bivalve physiology affect both cellular-level and whol e-organismal responses and suscep tibilities to abiotic factors. These seasonal differences likely reflect vari ation in body composition (protein-rich or lipidrich), reproductive versus growth cycle, degree of nourishment, age, and previous exposure to abiotic factors. All of these f actors influence the oxidative stre ss response, as reviewed in the

PAGE 143

143 previous chapters. Despit e the categorization of D. variabilis (Chapter 2) as a species that is vulnerable to abiotic factors and that of M. mercenaria (Chapter 3) as a stress-tolerant species, a common seasonal pattern in susceptibility was de tected, with the highest mortality occurring in the season for which the experimental conditions were most distinct from the environmental conditions (fall and winter). However, whether this pattern in susceptibilit y is related to cellularlevel responses is unclear, since an ox idative stress response was detected in D. variabilis but not in M. mercenaria In the case of D. variabilis the abiotic factors, par ticularly hydrogen sulfide, instigated an oxidative stress res ponse, but either this stress res ponse was unrelated to survival or it was not sufficient to prevent clam death. Mercenaria mercenaria did not have a detectable cellular-level response to high temperature, the tr eatment that caused significant mortality in the winter experiments, but it is not known whethe r these clams died because they did not elevate stress protein expression or if they died from other causes. The seasonal pattern in mortality and functional responses like burial ability, particularly in the high temperature experiments, suggests that the cause of mortality was at a leve l of organization higher than the cellular level. These results highlight the importance of examining stress physiology over multiple seasons, regardless of whether the study subject is vulnera ble to or tolerant of the abiotic factors. Multiple levels of biological organization The results from all three projects underscore the importance of examining responses at multiple levels of organization, a conclusion consistent with the views expressed by Spicer and Gast on (1999). The examination of stress protein responses as indicators of whole-animal toleranc e and species distribution is a well-established technique. However, there is a large gap between the expression levels of a given stress protein and the documented distribution of a population, a gap that is not always bridged in ecological physiology studies and which therefore limits the power of inference. As demonstrated in both

PAGE 144

144 Chapters 2 and 3, the upregulation or lack of upregulation of stress pr otein expression is not reliably predictive of whole-an imal tolerances and therefore s hould not be used exclusively to draw conclusions about species distribution. As demonstrated in Chapter 4, elevated stress protein expression should not be interpreted strictly as evidence of conditions causing oxidative damage and the converse, decreased stress pr otein expression, expect ed to be found in individuals in benign conditions. Th ese results confirm that stress protein expressi on data should not be interpreted in the absence of either cellular-level markers of damage or metabolic activity (as demonstrated in Chapter 4) or whol e-organism markers (Chapters 2 and 3). Spicer and Gaston (1999) emphasi ze that variation at all levels of biological organization are interrelated and that inves tigators should, when possible, look for correlation and causation at multiple levels. In some but not all of the expe riments described in this dissertation, multiple assays were conducted on the same individual. Had this approach been made universal, even within an experiment, then it w ould have been possible to test for within-individual correlations using multivariate statistics. Such an analysis, if successful, would have facilitated an exploration into the causes underlying treatment-, season-, and species-dependent variation in stress tolerance. However, an informal analysis (not pres ented in this dissertation) of those instances in which multiple assays were conducted on the sa me individual reveals that high levels of variation, even within treatmen t and season, would make the dete ction of correlations unlikely. Summary of Findings In Chapter 2 I tested whether abiotic factors typical of aquatic hab itats cause a cellular stress response consistent with free radical production. This study was conducted in the marine bivalve, Donax variabilis The factors examined in this study, hypoxia, hyperoxia, and hydrogen sulfide, are not present in the hi gh-energy sandy beaches inhabited by D. variabilis This species was selected to test whether one could be more successful at detecting th e consequences of the

PAGE 145

145 abiotic factor in the absence of protective res ponses in an organism that presumably is not adapted to the stressors. The results show a st rong seasonal pattern in susceptibility to and cellular-level responses to the stressors. The clams survived lo nger in the experiment conducted in the spring and, for the most part, had higher stress protein expression le vels in the spring. The pattern of stress protein expression was consis tent with elevated fr ee radical production, particularly in the hydrogen su lfide treatment. These results demonstrated that exposure to hydrogen sulfide causes an oxidati ve stress response in a non -sulfide-adapted bivalve and highlighted the importance of examining s easonal variation in stress physiology. In Chapter 3 I tested whether organisms can mitigate the cellular-level damage associated with exposure to single or multiple stressful ab iotic factors. This study was conducted in the estuarine bivalve, Mercenaria mercenaria. The abiotic factors examined in this study, hypoxia, high temperature, and high temperature combined with hyposalinity, have been shown to cause elevated free radical production in invertebrates and vertebrates. Both cellularand organismallevel responses to the abiotic factors were exam ined, and a seasonal pattern in susceptibility and magnitude of response was again detected. Although M. mercenaria did not have a detectable stress protein response to hypoxia or high temper ature, the seasonal patte rn in mortality and functional responses like burial ability, particularly in the high temperature experiments, suggests that the cause of mortality was at a leve l of organization higher than the cellular level. The additive effects of the dual f actor treatments were evident in the functional responses of the clams, which underscore the importance of exam ining multiple factors when testing whether tolerance of abiotic factors a ffects distribution in complex e nvironments. These clams have broad tolerances for extremes of temperature, dissolved O2 level, salinity, and pH that reflect the species’ widespread distribution in intertidal and subtidal coastal habitats. These results

PAGE 146

146 demonstrate that physiological stra tegies at higher levels of or ganization buffer the need for a cellular-level oxidative stress response in a stress-tolerant organism. In Chapter 4 I tested whether the capacity to produce an oxidative stress response affects species distribution. This combined field and laboratory study of th e freshwater bivalve Sphaerium sp. demonstrated that individuals in a population that overlies an environmental gradient show a variation in the ability to maintain a cellular stress response that reflects position along the gradient. These results were consistent with the “abundant cente r distribution pattern,” which dictates that abundance and individua l condition decrease near the edge of the population’s distribution if the distribution overlies a potentia lly stressful environmental gradient. In contrast to most previous studies linking stress protein expression to species distribution, these results showed that decreases in stress protein expression are more indicative of severe stress than are increases Finally, since the clams were most abundant and experienced the least oxidative stress when living in conditions of extreme hypoxi a, these results are the first whole-organism and field study to challenge long -held belief that normoxia is beneficial and hypoxia is stressful for aquatic organisms. These three interrelated studies demonstrated that abiotic fact ors found in aquatic habitats do cause oxidative stress and that ability to respond to the ab iotic factors correlates with distribution. The results highlight the importan ce of examining stress physiology over multiple seasons and underscore the importance of examining responses at multiple levels of organization. Since the upregulation or lack of change in stress protein expression was not reliably predictive of whole-animal tolerances these data should not be interpreted in the absence of either cellular-level markers of da mage or metabolic activity or whole-organism markers of physiological condition. Finally, these results demonstrate that future studies of

PAGE 147

147 ecological physiology would benefit from new ap proaches, particularly the examination of multiple abiotic factors and the inclusion of ‘ vulnerable’ species in the selection of model organisms.

PAGE 148

148 APPENDIX TRANSPLANT OF SPHAERIUM SP. ALONG A SWAMP/STREAM TRANSECT Introduction and Methods The study presented in this appendix is a c ontinuation of Chapter 4. It was conducted in May – June 2004 at the Makerere University Biol ogical Field Station in Kibale National Park, Uganda. To examine whether Sphaerium sp. clams acclimatized to the hypoxic and acidic swamp conditions experience stress when expos ed to the elevated dissolved O2 (DO) and neutral pH conditions of the stream sites, we conducted a two-week transplant experiment. For a full description of the limnological characters of the transect site s, see Chapter 4 pages 119-120. Transect sites, numbered 1 – 16, were situated at 10 m intervals starting in the swamp (sites 1-5) and continuing through the ecotone (sites 6-10) a nd into Inlet Stream We st (sites 11-16). Clams (5-8 mm shell length) were collected fr om a swamp site (Site #1 in Figure 4-1, page 133) and kept in a bucket of swamp water until placed into cages. The cages were constructed of white PVC pipe (length approximately 25 cm, inte rnal diameter 6 cm) with mesh placed over each end and secured with rubber bands. Holes were drilled at each end of the cage and fishing line was looped through the holes with enough slac k to allow the cages to be suspended from overhanging vegetation. Five cages were placed at each of four si tes (see site numbers in Figure 4-1, page 133): a) a swamp contro l site (Site 2), b) an ecotone site with a higher DO than the swamp and with clam abundance comparable to that of the swamp (Site 8), c) a stream site at the extreme edge of the clam populatio n distribution that had the highe st DO of any site with clams (Site 11), and d) a stream site with the highest DO of any site on the transect (Site 16). Thirty clams were placed into each cage, and cages were oriented to allow water flow through the cage. In most sites, the cages rested on top of the substrate.

PAGE 149

149 Five clams per cage were collected at each of six time points (total 600 clams): 2 hours, 5 hours, 1 day, 2 days, 5 days, and 14 days. At each sampling, removed clams were replaced with newly collected clams (from Site 1) that were marked on both valves with pink nail polish. Collected clams were rinsed in water from the tr ansect site, pierced with a dissecting needle to release water held in the mantle cav ity, and flash-frozen in a liquid N2 dry shipper (CX100, Taylor Wharton Cryogenics, Theodore, AL, USA). Clams were maintained in the dry shipper during transport back to the University of Fl orida, where they were stored at -80C. We analyzed stress protein expression levels in clams collected from all four sites at the six time points (3 per cage per site per sampling tim e) using Western Blotting and enzyme-linkedimmunosorbent assay (ELISA). For details on th e methods of stress protein analyses, see the descriptions in Chapters 2 and 4 (pages 31-34 and 117-119, respectively). For details about stress protein functions and the anti body binding patterns for Sphaeriid tissues, see pages 36-38 in Chapter 2 and Table 4-1 (page 132). Expression levels of the stress proteins were analyzed in triplicate by ELISA. The following proteins were examined: copper/zinc superoxide dismutase (CuZnSOD), glutathione peroxidase (GPx), mitochondrial 8-oxoguanine DNA glycosylase (OGG1-m), heat shock protein 60 (Hsp60), heat shock prot ein 70 (Hsp70), and small heat shock proteins (sHsp). Statistical results for these data are not presented as all results were nonsignificant ( p > 0.05). Each stress protein was an alyzed independently. Since ther e were no clear trends across sampling times, the data were analyzed with in sampling times by a nested ANOVA (Statistica), with cages nested within site a nd clams within cages treated as independent experimental units. To examine whether the clams experienced oxi dative damage during the transplant, we quantified the levels of oxidatively damaged RNA and DNA guanine bases in clams that had

PAGE 150

150 been transplanted for 14 days. For details about the methods of sample preparation and analysis, see Chapters 4 (pages 117-118). We quantified oxidized RNA and DNA bases in five clams from each transplant site. Data were analyzed with one-way ANOVA. Results and Discussion The stress protein expression results are presen ted in Figure A-1. The data are presented as expression levels relative to the mean of the expr ession levels of clams collected at Time 0 to allow for temporal comparisons. Expression levels of all stress proteins exce pt sHsp increased in clams transplanted to the ecotone, stream, and up stream sites in the 14-d ay period. Interestingly, the relative expression levels of all proteins (except sHSP) in clams from the swamp site tended to be close to or slightly below one. There were no significant diffe rences in stress protein expression levels following the 14day transplant experiment. Expression levels of the two antioxidants (CuZnSOD and GPx) were constant across the four sites (F igure A-1A and B). Clams transplanted to ecotone and stream sites expressed slightly (but not significantly) higher levels of the DNA repair enzyme, OGG1m, than did clams maintained in the swamp site (Figure A-1C). There wa s some indication of elevated expression in heat shoc k proteins (Hsp60, Hsp70, and sHsp) in clams transplanted to the ecotone and stream, in relation to clams maintain ed at the swamp site (Figure A-1D, C, and E, respectively). This is consistent with the results of the survey of clams collected from the ecotone and stream sites (pages 121-123). In summary, the general increase in stress protein expression in transplanted clams, particularly the elevated expr ession of the chaperone proteins Hsp70 and Hsp60, suggests that the clams experienced physiologi cal stress during the transplant. This response was marginally greater in the clams transplanted to the ecotone and stream sites in comparison to the clams maintained in cages in the swamp site. However, since the highest overall expression levels and

PAGE 151

151 the largest (but nonsignificant) differences in stress protein expression were detected in the chaperone proteins rather than in the antioxida nts, these data do not s upport the hypothesis that the clams maintained an elevated oxidative stress response followi ng transplantation to sites with elevated DO. DNA and RNA oxidation levels in clams transpla nted for 14 days are presented in Figure A-2A and B, respectively. There were no significa nt differences in the levels of oxidatively damaged DNA or RNA bases across transplant sites. Across sites, there was more RNA oxidation than DNA oxidation, which is consistent with the findings of the survey (page 121). Like the stress protein data, these data do not su pport the hypothesis that the clams experienced elevated oxidative stress following transplantatio n. An alternative interpretation is that they experienced oxidative damage to their DNA and/or RNA at earlier sampling times and repaired or removed the damaged nucleotides. However, nuc leic acid oxidation was not assessed in clams from the earlier time points. In summary, the stress protein expressi on and oxidative damage levels in clams transplanted for 14 days to sites with elevat ed DO and neutral pH do not support the hypothesis that the clams experienced elevated stress at the transplantation si tes. The results do suggest that the clams experienced generalized stress (eleva ted chaperone expression) which could result from starvation or from being maintained in cages rather than buried in the substrate.

PAGE 152

152 A. CuZnSOD Relative expression 0.0 0.5 1.0 1.5 2.0 B. GPx 0.0 0.5 1.0 1.5 2.0 C. OGG1-m Relative expression 0.0 0.5 1.0 1.5 2.0 2.5 D. Hsp60 0.0 0.5 1.0 1.5 2.0 E. Hsp70 MarginEcotoneStreamUpstream Relative expression 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 F. sHSP MarginEcotoneStreamUpstream 0.0 0.5 1.0 1.5 2.0 Figure A-1. Relative stress prot ein expression levels in Sphaerium sp. clams from the 14-day transplant experiment. Clams (15 per site ) were collected at the swamp site and held in cages at each site for 14 days. Data are expressed as rela tive to the mean of the expression levels of the five clams collected at Time 0. Data are presented as mean standard deviation. Abbreviati ons: CuZnSOD, copper/zinc superoxide dismutase; GPx, glutathi one peroxidase; OGG1m, mitochondrial 8-oxoguanine DNA glycosylase; Hsp60, heat shock prot ein 60; Hsp70, heat shock protein 70; sHsp, small heat shock protein.

PAGE 153

153 A. DNA oxidation MarginEcotoneStreamUpstream 8-oxodGuo/106 dGuo 0 6 12 18 B. RNA oxidation MarginEcotoneStreamUpstream 8-oxoGuo/106 Guo 0 10 20 30 40 Figure A-2. Levels of oxidative ly damaged DNA and RNA in Sphaerium sp. clams from the 14-day transplant experiment. Clams (5 per site) were collected at the swamp site and held in cages at each site for 14 da ys. Data are expressed as the number of oxidized bases (8-oxodGuo or 8-oxoGuo) per 106 non-oxidized bases (dGuo or Guo). Data are presented as means standard deviations. Abbreviations: 8oxodGuo, 8-oxo-7,8-dihydro-2 -deoxyguanosine; 8-oxoGuo, 8-oxo-7,8dihydroguanosine.

PAGE 154

154 LIST OF REFERENCES Abele, D., Burlando, B., Viarengo, A., Prtner, H.O., 1998a. Exposure to elevated temperatures and hydrogen peroxide elicits ox idative stress and antioxidant response in the Antarctic intertidal limpet Nacella concinna Comparative Biochemistry and Physiology 120B, 425435. Abele, D., Grosspietsch, H., Prt ner, H.O., 1998b. Temporal fluctua tions and spatial gradients of environmental PO2, temperature, H2O2 and H2S in its intertidal habitat trigger enzymatic antioxidant protection in the capitellid worm Heteromastus filiformis Mar. Ecol. Prog. Ser. 163, 179-191. Abele, D., Heise, K., Prtner, H.O., P untarulo, S., 2002. Temperature-dependence of mitochondrial function and production of reac tive oxygen species in the intertidal mud clam Mya arenaria J. Exp. Biol. 205, 1831-1841. Abele, D., Puntarulo, S., 2004. Formation of re active species and induc tion of antioxidant defence systems in polar and temperate mari ne invertebrates and fish. Comp. Biochem. Physiol. 138A, 405-415. Abele-Oeschger, D., 1996. A comparative study of superoxide dismutase activity in marine benthic invertebrates with resp ect to environmental sulphide exposure. J. Exp. Mar. Biol. Ecol. 197, 39-49. Abele-Oeschger, D., Oeschger, R., 1995. Hypoxiainduced autoxidation of haemoglobin in the benthic invertebrates Arenicola marina (Polychaeta) and Astarte borealis (Bivalvia) and the possible effects of sulphide. J. Exp. Mar. Biol. Ecol. 187, 63-80. Abele-Oeschger, D., Oeschger, R., Theed e, H., 1994. Biochemical adaptations of Nereis diversicolor (Polychaeta) to temporarily increased hydrogen peroxide leve ls in intertidal sandflats. Mar. Ecol. Prog. Ser. 106, 101-110. Akbar, M.A., Chatterjee, N.S., Sen, P., Debnat h, A., Pal, A., Bera, T., Das, P., 2004. Genes induced by a high-oxygen environment in Entamoeba histolytica Molec. Biochem. Parasit. 133, 187-196. Allani, P.K., Sum, T., Bhansali, S.G., Mukherj ee, S.K., Sonee, M., 2004. A comparative study of the effect of oxidative stress on the cytoskelet on in human cortical ne urons. Toxicol. Appl. Pharmacol. 196, 29-36. Allen, R.G., Balin, A.K., 2003. Effects of oxygen on the antioxidant responses of normal and transformed cells. Exper. Cell Res. 289, 307-316. Andrewartha, H.G., Birch, L.C., 1984. The eco logical web: More on the distribution and abundance of animals. University of Chicago Press, Chicago. Appleyard, C.L., Dealteris, J.T., 2002. Growth of the northern quahog, Mercenaria mercenaria in an experimental-scale upwelle r. J. Shellfish Res. 21, 3-12.

PAGE 155

155 Arnaud, C., Joyeux, M., Garrel, C., GodinRibuot, D., Demenge, P., Ribuot, C., 2002. Freeradical production triggered by hyperthermia contributes to he at stress-induced cardioprotection in isolated rat hearts. Br. J. Pharmacol. 135, 1776-1782. Arrigo, A.-P., Virot, S., Chaufour, S., Fird aus, W., Kretz-Remy, C., Diaz-Latoud, C., 2005. Hsp27 consolidates intracellular redox homeos tasis by upholding glutathione in its reduced form and by decreasing iron intracellul ar levels. Antioxid. Redox Signal. 7, 414-424. Bagarinao, T., 1992. Sulfide as an environmental f actor and toxicant: Tole rance and adaptations in aquatic organisms. Aquatic Toxicol. 24, 21-62. Baker, S.M., Baker, P., Heuberger, D., Sturmer, L., 2005. Short-term eff ects of rapid salinity reduction on seed clams ( Mercenaria mercenaria ). J. Shellfish Res. 24, 29-33. Baker, S.M., Mann, R., 1992. Effects of hypoxia and anoxia on larval settlement, juvenile growth, and juvenile survival of the oyster Crassostrea virginica Biol. Bull. 182, 265-269. Barja, G., 2002. Endogenous oxidative stress: rela tionship to aging, longevity, and caloric restriction. Ageing Research Reviews 1, 397-411. Basha, E., Lee, G.J., Breci, L.A., Hausrath, A.C., Buan, N.R., Giese, K.C., Vierling, E., 2004. The identity of proteins associated with a small heat shock protein during heat stress in vivo indicates that these chaper ones protect a wide range of cellular functions. J. Biol. Chem. 279, 7566-7575. Beck, N.G., Bruland, K.W., 2000. Diel biogeochemical cycling in a hyperventilating shallow estuarine environment. Estuaries 23, 177-187. Becker, L.B., Vanden Hoek, T.L., Shao, Z.-H., Li, C.-Q., Schumacker, P.T., 1999. Generation of superoxide in cardiomyocytes during ischemia before reperfusion. Am. J. Physiol. 277, H2240-H2246. Benson, K.R., 2002. The study of vert ical zonation on rocky intertid al shores A historical perspective. Integ. and Comp. Biol. 42, 776-779. Bierkens, J.G.E.A., 2000. Applications and p itfalls of stress-pro teins in biomonitoring. Toxicology 153, 61-72. Blank, M., Bastrop, R., Jrss, K., 2006. Stress pr otein response in tw o sibling species of Marenzelleria (Polychaeta:Spionidae): Is there an influence of acclimation salinity? Comp. Biochem. Physiol. 144B, 451-462. Boiteux, S., Radicella, J.P., 2000. The human OG G1 gene: structure, functions, and its implication in the process of carcino genesis. Arch. Biochem. Biophys. 377, 1-8. Boveris, A., Chance, B., 1973. The mitochondri al generation of hydroge n peroxide: General properties and effect of hyperbar ic oxygen. Biochem. J. 134, 707-716.

PAGE 156

156 Breitburg, D.L., Loher, T., Pacey, C.A., Gerstein, A., 1997. Varying effects of low dissolved oxygen on trophic interactions in an es tuarine food web. Ecol. Mono. 67, 489-507. Brown, D.C., Bradley, B.P., Tedengren, M., 1995. Genetic and environmental regulation of Hsp70 expression. Mar. E nviron. Res. 39, 181-184. Brown, J.H., Stevens, G.C., Kaufman, D.M., 1996. The geographic range: size, shape, boundaries, and internal structure. Annu. Rev. Ecol. Syst. 27, 597-623. Brown, R.C., Davis, T.P., 2005. Hypoxia/aglycemia alters expression of occludin and actin in brain endothelial cells. Biochem. Biophys. Res. Comm. 327, 1114-1123. Burky, A.J., 1983. Physiological ecology of freshwat er bivalves. In: Hochachka, P.W. (Ed.) The mollusca, vol. 6. Academic Press, New York, pp. 281-327 Burnaford, J.L., 2004. Habitat modification and re fuge from sublethal stress drive a marine plant-herbivore asso ciation. Ecology 85, 2837-2849. Burnett, L.E., 1997. The challenges of living in hypoxic and hypercapnic aquatic environments. Amer. Zool. 37, 633-640. Byrne, P.A., O'Halloran, J., 2000. Acute and sublet hal toxicity of estuarine sediments to the Manila clam, Tapes semidecussatus Environ. Toxicol. 15, 456-468. Caccia, V.G., Boyer, J.N., 2005. Spatial patterning of water quality in Biscayne Bay, Florida as a function of land use and water mana gement. Mar. Poll. Bull. 50, 1416-1429. Cain, T.D., 1973. The combined effects of temperat ure and salinity on embryos and larvae of the clam Rangia cuneata Mar. Biol. 21, 1-6. Calabrese, A., Davis, H.C., 1966. The pH tolerance of embryos and larvae of Mercenaria mercenaria and Crassostrea virginica Biol. Bull. 131, 427-436. Callaghan, A., Holloway, G.J., 1999. The relatio nship between environmental stress and variance. Ecol. Appl. 9, 456-462. Carmichael, R.H., Shriver, A.C., Valiela, I., 2004. Changes in shell and soft tissue growth, tissue composition, and survival of quahogs, Mercenaria mercenaria and softshell clams, Mya arenaria in response to eutrophic-dri ven changes in food supply a nd habitat. J. Exp. Mar. Biol. Ecol. 313, 75-104. Carrico, R.J., Blumberg, W.E., Peisach, J ., 1978. The reversible binding of oxygen to sulfhemoglobin. J. Biol. Chem. 253, 7212-7215. Carter, G.S., 1955. The papyrus swamps of Ug anda. W. Heffer and Sons Ltd., Cambridge, England.

PAGE 157

157 Case, T.J., Holt, R.D., McPeek, M.A., Keitt, T.H., 2005. The community context of species' borders: ecological and evolutiona ry perspectives. Oikos 108, 28-46. Caughley, G., Grice, D., Barker, R., Brown, B., 1988. The edge of the range. J. Animal Ecol. 57, 771-785. Cavanaugh, C.M., 1983. Symbiotic chemoautotrophic bacteria in marine invertebrates from sulphide-rich habitats. Nature 302, 58-61. Chandel, N.S., Budinger, G.R.S., 2007. The cellula r basis for diverse responses to oxygen. Free Rad. Biol. Med. 42, 165-174. Chandel, N.S., Maltepe, E., Goldwasser, E., Ma thieu, C.E., Simons, M.C., Schumacker, P.T., 1998. Mitochondrial reactive oxygen species tr igger hypoxia-induced tr anscription. Proc. Nat. Acad. Sci. USA 95, 11715-11720. Chang, J., Knowlton, A.A., Wasser, J.S., 2000. Expre ssion of heat shock prot eins in turtle and mammal hearts: relationship to anoxia tolera nce. Am. J. Physiol. Regulatory Integrative Comp. Physiol. 278, R209-R214. Chapman, C.A., Chapman, L.J., Wrangham, R ., Isabirye-Basuta, G., Ben-David, K., 1997. Spatial and temporal variability in the structur e of a tropical forest Afr. J. Ecol. 35, 287302. Chapman, L.J., 1995. Seasonal dynamics of habi tat use by an air-breathing catfish ( Clarias liocephalus ) in a papyrus swamp. Ecology of Freshwater Fish 4, 113-123. Chapman, L.J., Balirwa, J., Bugenyi, F.W.B., Chapman, C.A., Crisman, T.L., 2001. Wetlands of East Africa: Biodiversity, expl oitation, and policy perspectiv es. In: Gopal, B., Junk, W.J., Davis, J.A. (Eds.), Biodiversity in wetlands: assessment, function and conservation, vol. 2. Backhuys Publishers, Leiden, The Netherlands, pp. 101-131 Chapman, L.J., Chapman, C.A., Brazeau, D.A., McLaughlin, B., Jordan, M., 1999. Papyrus swamps, hypoxia, and faunal diversificat ion: variation among populations of Barbus neumayeri J. Fish Biol. 54, 310-327. Chapman, L.J., Chapman, C.A., Crisman, T.L., Pre nger, J., 2000. Predictors of seasonal oxygen levels in a Ugandan swamp/river system: A 3-y ear profile. Verh. Internat. Verein. Limnol. 27, 3048-3053. Chapman, L.J., Chapman, C.A., Nordlie, F.G., Ro senberger, A.E., 2002. Physiological refugia: swamps, hypoxia tolerance and maintenance of fi sh diversity in the Lake Victoria region. Comp. Biochem. Physiol. 133A, 421-437. Chapman, L.J., Liem, K.F., 1995. Papyrus sw amps and the respiratory ecology of Barbus neumayeri Environ. Biol. Fish. 44, 183-197.

PAGE 158

158 Chapman, L.J., Schneider, K.R., Apodaca, C ., Chapman, C.A., 2004. Respiratory ecology of macroinvertebrates in a swamp-river system of East Africa. Biotropica 36, 572-585. Chapple, J.P., Smerdon, G.R., Berry, R.J., Hawkins, A.J.S., 1998. Seasonal changes in stress-70 protein levels reflect thermal to lerance in the marine bivalve Mytilus edulis L. J. Exp. Mar. Biol. Ecol. 229, 53-68. Chapple, J.P., van der Spuy, J., Poopalasundara m, S., Cheetham, M.E., 2004. Neuronal DnaJ proteins HSJ1a and HSJ1b: A role in linking the Hsp70 chaperone machine to the ubiquitin-proteasome system? Bi ochem. Soc. Trans. 32, 640-642. Chen, K.Y., Morris, J.C., 1972. Oxidation of sulfide by O2: catalysis and inhi bition. Journal of the Sanitation Engineering Division Proceed ings of the American Society of Civil Engineers 98, 215-227. Cho, H.-Y., Reddy, S.P., DeBiase, A., Yamamoto M., Kleeberger, S.R., 2005. Gene expression profiling of NRF2-mediated prot ection against oxidative inju ry. Free Radic. Biol. Med. 38, 325-343. Chown, S.L., Storey, K.B., 2006. Linking molecular physiology to ecological realities. Physiol. Biochem. Zool. 79, 314-323. Clark, S.L., Teh, S.J., Hinton, D.E., 2000. Tissue and cellular alterations in Asian clam ( Potamocorbula amurensis ) from San Francisco Bay: Toxico logical indicators of exposure and effect? Mar. Environ. Res. 50, 301-305. Cline, J.D., 1969. Spectrophotometric determina tion of hydrogen sulfide in natural waters. Limnol. Oceanogr. 14, 454-458. Cole, A., Armour, E.P., 1988. Ultr astructural study of mitochondrial damage in CHO cells exposed to hyperthermia. Radiat. Res. 115, 421-435. Connell, J.H., 1961. The influence of interspe cific competition and other factors on the distribution of the barnacle Chthamalus stellatus Ecology 42, 710-723. Conover, W.J., 1999. Practical Nonparam etric Statistics. Wiley, New York. Currie, S., Boutilier, R.G., 2001. Strategies of hypoxia and anoxia toleran ce in cardiomyocytes from the overwintering common frog, Rana temporaria Physiol. Biochem. Zool. 74, 420428. Dahlhoff, E.P., 2004. Biochemical i ndicators of stress and metabolis m: Applications for marine ecological studies. Annu. Rev. Physiol. 66, 183-207. Dahlhoff, E.P., Buckley, B.A., Menge, B.A., 2001. P hysiology of the rocky intertidal predator Nucella ostrina along an environmental stress gradient. Ecology 82, 2816-2829.

PAGE 159

159 de Oliveira, U.O., Araujo, A.S. D., Bello-Klein, A., da Silva, R.S.M., Kucharski, L.C., 2005. Effects of environmental a noxia and different periods of reoxygenation on oxidative balance in gills of the estuarine crab Chasmagnathus granulata Comp. Biochem. Physiol. 140B, 51-57. De Zwaan, A., Barbarro, J.M.F., Monari, M., Ca ttani, O., 2002. Anoxic survival potential of bivalves: (arte)facts. Comp. Biochem. Physiol. 131A, 615-624. de Zwann, A., Zandee, D.I., 1972. The utilization of glycogen and accumulation of some intermediates during anaerobiosis in Mytilus edulis L. Comp. Biochem. Physiol. 43B, 4754. Denis, W., Reed, L., 1927. The action of blood on sulfides. J. Biol. Chem. 72, 385-394. Dennog, C., Radermacher, P., Barnett, Y.A., Speit, G., 1999. Antioxidant stat us in humans after exposure to hyperbaric oxygen. Mut. Res. 428, 83-89. Diaz, R.J., Rosenberg, R., 1995. Marine benthic h ypoxia: A review of its ecological effects and the behavioral responses of benthic macr ofauna. Oceanography and Marine Biology: an Annual Review 33, 245-303. Dirmeier, R., O'Brien, K.M., Engle, M., Dodd, A., Spears, E., Poyton, R.O., 2002. Exposure of yeast cells to anoxia induces transient oxidative stress. J. Biol. Chem. 277, 34773-34784. Downs, C.A., Dillon, J., R. T., Fauth, J.E ., Woodley, C.M., 2001a. A molecular biomarker system for assessing the health of gastropods ( Ilyanassa obsoleta ) exposed to natural and anthropogenic stressors. J. E xp. Mar. Biol. Ecol. 259, 189-214. Downs, C.A., Fauth, J.E., Halas, J.C., Dustan, P., Bemiss, J., Woodley, C.M., 2002a. Oxidative stress and seasonal coral bleaching. Free Radic. Biol. Med. 33, 533-543. Downs, C.A., Fauth, J.E., Robinson, C.E., Curry, R., Lanzendorf, B., Halas, J.C., Halas, J., Woodley, C.M., 2005. Cellular diagnostics and cora l health: Declining co ral health in the Florida keys. Mar. Pollut. Bull. 51, 558-569. Downs, C.A., Fauth, J.E., Woodley, C.M., 2001b. Assessing the health of grass shrimp ( Palaeomonetes pugio ) exposed to natural and anthr opogenic stressors: a molecular biomarker system. Mar. Biotechnol. 3, 380-397. Downs, C.A., Jones, L.R., Heckathorn, S.A., 199 9. Evidence for a novel set of small heat-shock proteins that associates with the mitoc hondria of murine PC12 cells and protects NADH:ubiquinone oxidoreductase from heat and oxidative stress. Arch. Biochem. Biophys. 365, 344-350. Downs, C.A., Mueller, E., Phillips, S., Faut h, J.E., Woodley, C.M., 2000. A molecular biomarker system for assessing the health of coral ( Montastraea faveolata ) during heat stress. Mar. Biotechnol. 2, 533-544.

PAGE 160

160 Downs, C.A., Shigenaka, G., Fauth, J.E., Robinson, C.E., Huang, A., 2002b. Cellular physiological assessment of bivalves after chronic exposure to spilled Exxon Valdez crude oil using a novel molecular diagnostic biotechnology. Environ. Sci. Technol. 36, 29872993. Du, G., Mouithys-Mickalad, A., Sluse, F. E., 1998. Generation of superoxide anion by mitochondria and impairment of their functi ons during anoxia and reoxygenation in vitro. Free Radic. Biol. Med. 25, 1066-1074. Dykens, J.A., Shick, J.M., Benoit, C., R., B. G., Winston, G.W., 1992. Oxygen radical production in the sea anemone Anthopleura elegantissima and its endosymbiotic algae. J. Exp. Biol. 168, 219-241. Eghbal, M.A., Pennefather, P.S., O'Brien, P.J., 2004. H2S cytotoxicity mechanism involves reactive oxygen species form ation and mitochondrial de polarisation. Toxicology 203, 6976. Ellers, O., 1995. Behavioral contro l of swash-riding in the clam Donax variabilis Biol. Bull. 189, 120-127. Elser, J.J., Sterner, R.W., Gorokhova, E., Faga n, W.F., Markow, T.A., Cotner, J.B., Harrison, J.F., Hobbie, S.E., Odell, G.M., Weider, L.J ., 2000. Biological stoichiometry from genes to ecosystems. Ecol. Lett. 3, 540-550. Elton, C., 1927. Animal ecology. The Macmillan Company, New York. Englander, E.W., Greeley, J., G. H., Wang, G., Perez-Polo, J.R., Lee, H.-M., 1999. Hypoxiainduced mitochondrial and nuclear DNA damage in the rat brain. J. of Neurosci. Res. 58, 262-269. Epifanio, C.E., Srna, R.F., 1975. Toxicity of ammonia, nitrite ion, nitrate ion, and orthophosphate to Mercenaria mercenaria and Crassostrea virginica Marine Biology 33, 241-246. Evans, M.D., Cooke, M.S., 2004. Factors contributi ng to the outcome of oxidative damage to nucleic acids. BioEssays 26, 533-542. Eversole, A.G., 2001. Reproduction in Mercenaria mercenaria In: Kraeuter, J.N., Castagna, M. (Eds.), The biology of the hard clam, Elsevier, New York, pp. 221-260 Feder, M., Hofmann, G.E., 1999. Heat-shock prot eins, molecular chaperones, and the stress response: evolutionary and ecological physiology. Annu. Rev. Physiol. 61, 243-282. Felbeck, H., Childress, J.J., Somero, G.N., 1981. Calvin-Benson cycle and sulphide oxidation enzymes in animals from sulphi de-rich habitats. Nature 293, 291-293. Fenchel, T.M., Riedl, R.J., 1970. The sulfide sy stem: A new biotic co mmunity underneath the oxidized layer of marine sand bottoms. Mar. Biol. 7, 255-268.

PAGE 161

161 Fisher, N.S., 1977. On the differential sensitivity of estuarine and open-oc ean diatoms to exotic chemical stress. Am. Nat. 111, 871-895. Freiberger, J., Coulombe, K., Suliman, H., Carraway, M., Piantadosi, C., 2004. Superoxide dismutase responds to hyperoxia in rat hippocampus. Undersea Hyperb. Med. 31, 227-232. Fridovich, I., 2004. Mitochondria: Are they the seat of sene scence? Aging Cell 3, 13-16. Frydman, J., 2001. Folding of newly translated proteins in vivo: The role of molecular chaperones. Annu. Rev. Biochem. 70, 603-647. Gamenick, I., Jahn, A., Vopel, K., Giere, O., 1996. Hypoxia and sulphide as structuring factors in a macrozoobenthic community on the Balt ic Sea shore: Col onisation studies and tolerance experiments. Mar. Ecol. Prog. Ser. 144, 73-85. Gardner, P.R., Nguyen, D.-D.H., White, C.W., 1994. Aconitase is a sensitive and critical target of oxygen poisoning in cultured mammalian cells and in rat lungs. Proc. Natl. Acad. Sci. USA 91, 12248-12252. Ghosh, S., Gepstein, S., Heikkila, J.J., Dumbroff E.B., 1988. Use of a scanning densitometer or an ELISA plate reader for measurement of na nogram amounts of protein in crude extracts from biological tissues. Anal. Biochem. 169, 227-233. Gilchrist, G.W., 1995. Sp ecialists and generalis ts in changing environments. I. Fitness landscapes of thermal sensitivity. Am. Nat. 146, 252-270. Gray, J.M., Karow, D.S., Lu, H., Chang, A.J ., Chang, J.S., Ellis, R.E., Marletta, M.A., Bargmann, C.I., 2004. Oxygen sensation a nd social feeding mediated by a C. elegans guanylate cyclase homologue. Nature 430, 317-322. Gredilla, R., Barja, G., 2005. Minireview: The ro le of oxidative stress in relation to caloric restriction and longevi ty. Endocrinology 146, 3713-3717. Greenway, S.C., Storey, K.B., 1999. The effect of prolonged anoxia on enzyme activities in oysters ( Crassostrea virginica ) at different seasons. J. Exp. Mar. Biol. Ecol. 242, 259-272. Grieshaber, M.K., Hardewig, I., Kreutzer, U., Prtner, H.O., 1994. Physiological and metabolic responses to hypoxia in invertebrates. Re v. Physiol. Biochem. Pharmacol. 125, 43-147. Grieshaber, M.K., Vlkel, S., 1998. Animal adap tations for tolerance and exploitation of poisonous sulfide. Annu. Rev. Physiol. 60, 33-53. Grizzle, R.E., Bricelj, V.M., Shum way, S.E., 2001. Physiological ecology of Mercenaria mercenaria In: Kraeuter, J.N., Castagna, M. (E ds.), The biology of the hard clam, Elsevier, New York, pp. 305-382 Gupta, S., Knowlton, A.A., 2002. Cytosolic heat shock protein 60, hypoxia, apoptosis. Circulation 106, 2727-2733.

PAGE 162

162 Hall, D.J., 1964. An experimental approach to the dynamics of a natural population of Daphnia galeata mendotae Ecology 45, 94-112. Halliwell, B., Gutteridge, J.M.C., 1999. Free ra dicals in biology and medicine. Oxford University Press, New York. Hamburger, K., Dall, P.C., Lindegaard, C., N ilson, I.B., 2000. Survival and energy metabolism in an oxygen deficient environment. Field a nd laboratory studies on the bottom fauna from the profundal zone of Lake Esro m, Denmark. Hydrobiologia 432, 173-188. Hamza-Chaffai, A., Pellerin, J., Amiard, J.C ., 2003. Health assessment of a marine bivalve Ruditapes decussatus from the Gulf of Gabs (Tunisia). Environ. Internat. 28, 609-617. Harris, J.E., Parkyn, D.C., Murie, D.J., 2005. Distri bution of gulf of Mexico sturgeon in relation to benthic invertebrate prey resources and environmental parameters in the Suwannee River Estuary, Florida. T. Am. Fish. Soc. 134, 975-990. Harte, M.E., 2001. Systematics and taxonomy. In: Kr aeuter, J.N., Castagna, M. (Eds.), Biology of the hard clam, Elsevier, New York, pp. 3-52 Hartl, F.U., 1996. Molecular chaperones in cellular protein fold ing. Nature 381, 571-579. Hartman, P., Ponder, R., Lo, H.-H., Ishii, N., 2004. Mitochondrial oxidativ e stress can lead to nuclear hypermutability. Mech. Ageing Dev. 125, 417-420. Hass, M.A., Massaro, D., 1988. Regulation of the synt hesis of superoxide dismutases in rat lungs during oxidant and hyperthermic st resses. J. Biol. Chem. 263, 776-781. Hauton, C., Hawkins, L.E., Hutchinson, S., 1998. The use of the neutral red retention assay to examine the effects of temperature and salinity on haemocytes of the European flat oyster Ostrea edulis (L). Comp. Biochem. Physiol. 119B, 619-623. Hawkins, A.J.S., Menon, N.R., Damodaran, R., Bayne, B.L., 1987. Metabolic responses of the mussels Perna viridis and Perna indica to declining oxygen tension at different salinities. Comp. Biochem. Physiol. 88A, 691-694. Heinonen, J., Kukkonen, J., Penttinen, O.P., Hol opainen, I.J., 1997. Effects of hypoxia on valveclosure time and bioaccumulation of 2,4,5-tr ichlorophenol by the freshwater clam Sphaerium corneum [L.]. Ecotox. Environ. Safety 36, 49-56. Heise, K., Puntarulo, S., Prtner, H.O., Ab ele, D., 2003. Production of reactive oxygen species by isolated mitochondria of the Antarctic bivalve Laternula elliptica (King and Broderip) under heat stress. Comp. Biochem. Physiol. 134C, 79-90. Hermes-Lima, M., Storey, J.M., Storey, K.B ., 1998. Antioxidant defenses and metabolic depression. The hypothesis of preparation fo r oxidative stress in land snails. Comp. Biochem. Physiol. 120B, 437-448.

PAGE 163

163 Hermes-Lima, M., Zenteno-Savin, 2002. Anim al response to drastic changes in oxygen availability and physiological oxidative st ress. Comp. Biochem. Physiol. 133C, 537-556. Hernndez, J.A., Corpas, F.J., Gmez, M., del Ro, L.A., Sevilla, F., 1993. Salt-induced oxidative stress mediated by activated oxygen species in pea leaf mitochondria. Physiologia Plantarum 89, 103-110. Herrmann, J., Ciechanover, A., Lerman, L.O., Lerman, A., 2004. The ubiquitin-proteasome system in cardiovascular diseasesA hypothe sis extended. Cardiovasc ular Research 61, 11-21. Hizoh, I., Haller, C., 2002. Radiocontrast-induced re nal tubular cell apoptosis: Hypertonic versus oxidative stress. Invest. Rad. 37, 428-434. Hochachka, P.W., Somero, G.N ., 2002. Biochemical adaptation: Mechanism and process in physiological evolution. Oxford University Press, New York. Hofer, T., Seo, A.Y., Prudencio, M., Leeuwe nburgh, C., 2006. A method to determine RNA and DNA oxidation simultaneously by HPLC-ECD: Gr eater RNA than DNA oxidation in rat liver after doxorubicin admini stration. Biol. Chem. 387, 103-111. Hoffmann, A.A., Parsons, P.A., 1991. Evolutionary genetics and environmen tal stress. Oxford University Press, New York. Hofmann, G.E., 1999. Ecologically relevant variati on in induction and fu nction of heat shock proteins in marine orga nisms. Amer. Zool. 39, 889-900. Hofmann, G.E., Buckley, B.A., Place, S.P., Zippay, M.L., 2002. Molecular chaperones in ectothermic marine animals: Biochemical f unction and gene expression. Integ. and Comp. Biol. 42, 808-804. Hofmann, G.E., Somero, G.N., 1995. Evidence for protein damage at environmental temperatures: Seasonal changes in levels of ubi quitin conjugates and hsp70 in the intertidal mussel Mytilus trossulus J. Exp. Biol. 198, 1509-1518. Holt, R.D., 2003. On the evolutionary ecology of species' ranges. Evol. Ecol. Res. 5, 159-178. Hubertz, E.D., Cahoon, L.B., 1999. Short-term vari ability of water qualit y parameters in two shallow estuaries of North Carolina. Estuaries 22, 814-823. Irwin, S., Davenport, J., 2002. Hyperoxic boundary la yers inhabited by the epiphytic meiofauna of Fucus serratus Mar. Ecol. Prog. Ser. 244, 73-79. Jahnke, L.S., White, A.L., 2003. Long-term hyposalin e and hypersaline stre sses produce distinct antioxidant responses in the marine alga Dunaliella tertiolecta J. Plant Physiol. 160, 11931202.

PAGE 164

164 Joanisse, D.R., Storey, K.B., 1998. Oxidative stre ss and antioxidants in st ress and recovery of cold-hardy insects. Insect Bi ochem. Molec. Biol. 28, 23-30. Joyeux-Faure, M., Arnaud, C., Godin-Ribuot, D., Ribuot, C., 2003. Heat stress preconditioning and delayed myocardial pr otection: what is new? Cardiovasc. Res. 60, 469-477. Joyner-Matos, J., Chapman, L.J., Downs, C.A., Hofer, T., Leeuwenburgh, C., Julian, D., 2007. Stress response of a freshwater clam al ong an abiotic gradien t: Too much oxygen may limit distribution. Functional Ecology. 21, 344-355. Joyner-Matos, J., Downs, C.A., Julian, D., 2006. Incr eased expression of stress proteins in the surf clam Donax variabilis following hydrogen sulfide exposur e. Comp. Biochem. Physiol. 145A, 245-257. Julian, D., April, K.L., Patel, S., Stei n, J.R., Wohlgemuth, S.E., 2005. Mitochondrial depolarization following hydrogen sulfide exposure in erythrocytes from a sulfide-tolerant marine invertebrate. J. Exp. Biol. 208, 4109-4122. Julian, D., Arp, A.J., 1992. Sulfide permeab ility in the marine invertebrate Urechis caupo J. Comp. Physiol. 162B, 59-67. Keller, M., Sommer, A.M., Prtne r, H.O., Abele, D., 2004. Seasona lity of energetic functioning and production of reactive oxygen species by lugworm ( Arenicola marina ) mitochondria exposed to acute temperature changes. J. Exp. Biol. 207, 2529-2538. Kraeuter, J.N., Castagna, M., 2001. Biology of the hard clam. In: Developments in aquaculture and fisheries science, vol. 31. Elsevier, New York, p. 751 Kregel, K.C., 2002. Heat shock proteins: modifyi ng factors in physiologica l stress responses and acquired thermotolerance. J. Appl. Physiol. 92, 2177-2186. Krogh, A., 1929. The progress of physiology. Science 70, 200-204. Kukreja, R.C., Janin, Y., 1997. Reperfusion injury : Basic concepts and protection strategies. Thromb. Thrombolysis 4, 7-24. Kultz, D., 1996. Plasticity and stressor specifici ty of osmotic and heat shock responses of Gillichthys mirabilis gill cells. Amer. J. Physiol. 271, C1181-C1193. Kultz, D., 2005. Molecular and evolutionary basis of the cellular stress response. Annu. Rev. Physiol. 67, 225-257. Labieniec, M., Gabryelak, T., Falcioni, G., 2003. An tioxidant and pro-oxidant effects of tannins in digestive cells of the freshwater mussel Unio tumidus Mut. Res. 539, 19-28. Larade, K., Storey, K.B., 2002. Reversible suppre ssion of protein synthe sis in concert with polysome disaggregation during anoxia exposure in Littorina littorea Mol. Cell. Biochem. 232, 121-127.

PAGE 165

165 Lardies, M.A., Clasing, E., Navarro, J.M., Stead, R.A., 2001. Effects of environmental variables on burial depth of two infaunal bivalves inhabiti ng a tidal flat in southern Chile. J. Mar. Biol. Ass. U.K. 81, 809-815. Laudien, J., Schiedek, D., Brey, T., Arntz, W.E. Prtner, H.O., 2002. Survivorship of juvenile surf clams Donax serra (Bivalvia, Donacidae) expo sed to severe hypoxia and hydrogen sulphide. J. Exp. Mar. Biol. Ecol. 271, 9-23. Leini, S., Lehtonen, K.K., 2005. Seasonal variability in biomarkers in the bivalves Mytilus edulis and Macoma balthica from the northern Baltic Sea. Comp. Biochem. Physiol. 140C, 408-421. Lesser, M.P., Kruse, V.A., 2004. Seasonal temp erature compensation in the horse mussel, Modiolus modiolus : metabolic enzymes, oxidative stre ss and heat shock proteins. Comp. Biochem. Physiol. 137A, 469-504. Levesque, C., Juniper, S.K., Limn, H., 2006. Spa tial organization of food webs along habitat gradients at deep-sea hydrot hermal vents on Axial Volcano, Northeast Pacific. Deep-Sea Res. I 53, 726-739. Levin, L.A., Ziebis, W., Mendoza, G.F., Gr owney-Cannon, V., Walther, S., 2006. Recruitment response of methane-seep macrofauna to sulfiderich sediments: An in situ experiment. J. Exp. Mar. Biol. Ecol. 330, 132-150. Li, C., Jackson, R.M., 2002. Reactive species mechanisms of cellular hypoxia-reoxygenation injury. Am. J. Physiol. Cell. Physiol. 282, C227-C241. Li, C., Wright, M.M., Jackson, R.M., 2002. Reactiv e species mediated injury of human lung epithelial cells after hypoxia-reoxygenation. Exp. Lung Res. 28, 373-389. Li, Q., Liu, W., Shirasu, K., Chen, W., Jina g, S., 2006. Reproductive cycle and biochemical composition of the Zhe oyster Crassostrea plicatula Gmelin in an eastern coastal bay of China. Aquaculture 261, 752-759. Lough, R.G., Gonor, J.J., 1973. A response-surface approach to the combined effects of temperature and salinity on the larval development of Adula californiensis (Pelecypoda: Mytilidae). I. Survival and gr owth of three and fifteen-day old larvae. Mar. Biol. 22, 241250. Lovatt Evans, C., 1967. The toxicity of hydrogen su lphide and other sulphides. Quart. J. Exp. Physiol. 52, 231-248. Lowe, C.D., J., K.S., Diaz-Avalos, C., Monta gnes, D.J.S., 2007. How does salinity tolerance influence the di stributions of Brachionus plicatilis sibling species? Mar. Biol. 150, 377386.

PAGE 166

166 Lushchak, V.I., Lushchak, L.P., Mota, A.A ., Hermes-Lima, M., 2001. Oxidative stress and antioxidant defenses in goldfish Carassius auratus during anoxia and reoxygenation. Am. J. Physiol. Regulatory Integr ative Comp. Physiol. 280, 100-107. Lynch, M., Gabriel, W., 1987. Environmental tolerance. Am. Nat. 129, 283-303. Mackie, G.L., 1978. Are Sphaeriid clam ovoviviparous or viviparous? Nautilus 92, 145-147. Magalhes, J., Ascenso, A., Soares, J.M., Neupa rth, M.J., Ferreira, R., Oliveir, J., Amado, F., Duarte, J.A., 2004. Acute and severe hypobaric h ypoxia-induced muscle oxidative stress in mice: the role of glutathione against oxidati ve damage. Eur. J. Appl. Physiol. 91, 185-191. Magalhes, J., Ascenso, A., Soares, J.M.C., Fe rreira, R., Neuparth, M.J., Oliveira, J., Amado, F., Marques, F., Duarte, J.A., 2005. Acute a nd chronic exposition of mice to severe hypoxia: The role of acclimatizati on against skeletal muscle oxida tive stress. Int. J. Sports Med. 26, 102-109. Mahroof, R., Zhu, K.Y., Subramanyam, B., 2005. Ch anges in expression of heat shock proteins in Tribolium castaneum (Coleoptera: Tenebrio nidae) in relation to developmental stage, exposure time, and temperature. Annals of Entomological Society of America 98, 100-107. Marinelli, R.L., Woodin, S.A., 2002. Experiment al evidence for linkages between infaunal recruitment, disturbance, and sediment surface chemistry. Limnol. Oceanogr. 47, 221-229. Matthews, T.G., Fairweather, P.G., 2004. Effect of lowered salinity on the survival, condition, and reburial of Soletellina alba (Lamarck, 1818) (Bivalvia: Psammobiidae). Austral Ecology 29, 250-257. McKee, P.M., Mackie, G.L., 1983. Respirator y adaptations of the fingernail clams Sphaerium occidentale and Musculium securis to ephemeral habitats. Can. J. Fish. Aquat. Sci. 40, 783-791. McMillan, D.M., Fearnley, S.L., Rank, N.E., Dahl hoff, E.P., 2005. Natural temperature variation affects larval survival, development and Hsp70 expression in a leaf b eetle. Funct. Ecol. 19, 844-852. McNab, B.K., 2002. The physiological ecology of vert ebrates. A view from energetics. Cornell University Press, Ithaca, NY. Menge, B.A., 2000. Recruitment vs. postrecruitment processes as determinants of barnacle population abundance. Ecol. Mon. 70, 265-288. Menge, B.A., Olson, A.M., 1990. Role of scale a nd environmental factors in regulation of community structure. Trends Ecol. Evol. 5, 52-57. Menge, B.A., Sutherland, J.P., 1987. Community regulation: Variati on in disturbance, competition, and predation in relation to environmental stress and recruitment. Am. Nat. 130, 730-757.

PAGE 167

167 Mesa, M.G., Weiland, L.K., Wagner, P., 2002. Effect s of acute thermal stress on the survival, predator avoidance, and physiology of juve nile fall chinook salmon. Northwest Sci. 76, 118-128. Mikkelsen, P.S., 1981. A comparison of two Fl orida populations of the Coquina clam, Donax variabilis Say, 1822 (Bivalvia: Donacidae). I. Intertidal densit y, distribution, and migration. The Veliger 23, 230-239. Mikkelsen, P.S., 1985. A comparison of two Fl orida populations of the Coquina clam, Donax variabilis Say, 1822 (Bivalvia: Donacidae). II. Growth rates. The Veliger 27, 308-311. Millie, D.F., Carrick, H.J., Doering, P.H., Stei dinger, K.A., 2004. Intra-annual variability of water quality and phytoplankton in the North Fork of the St. Lucie River Estuary, Florida (USA): a quantitative assessment. Es tuar. Coast. Mar. Sci. 61, 137-149. Mitsumoto, A., Takeuchi, A., Okawa, K., Nakaga wa, Y., 2002. A subset of newly synthesized polypeptides in mitochondria from human e ndothelial cells exposed to hydroperoxide stress. Free Radic. Biol. Med. 32, 22-37. Morales, A.E., Perez-Jimenez, A., Hidalgo, M. C., Abellan, E., Cardenete, G., 2004. Oxidative stress and antioxidant defenses after prolonged starvation in Dentex dentex liver. Comp. Biochem. Physiol. 139C, 153-161. Morrill, A.C., Powell, E.N., Bidigare, R.R., Shic k, J.M., 1988. Adaptations to life in the sulfide system: a comparison of oxygen detoxifying enzymes in thiobiotic and oxybiotic meiofauna (and freshwater planarians). J. Comp. Physiol. B 158, 335-344. Mujahid, A., Sato, K., Akiba, Y., Toyomi zu, M., 2006. Acute heat stress stimulates mitochondrial superoxide production in broiler skeletal muscle, possibly via downregulation of uncoupling protein content. Poult. Sci. 85, 1259-1265. Neufeld, D.S., Wright, S.H., 1996. Response of cell volume in Mytilus gill to acute salinity change. J. Exp. Biol. 199, 473-484. Nicholls, P., 1975. The effect of sulphide on cytochrome aa 3: isoteric and allosteric shifts of the reduced alpha-peak. Biochi m. Biophys. Acta 396, 24-35. Nii, C.M., Muscatine, L., 1997. Oxidative stress in the symbiotic sea anemone Aiptasia pulchella (Carlgren, 1943): Contribution of the animal to superoxide ion production at elevated temperature. Biol. Bull. 192, 444-456. Nordlie, F.G., 2006. Physicochemical environments and tolerances of cyprinodontoid fishes found in estuaries and salt marshes of eastern North America. Rev. Fish Biol. Fisheries 16, 51-106. O'Donovan, D.J., Rogers, L.K., Kelley, D.K., Welty, S.E., Ramsay, P.L., Smith, C.V., 2002. CoASH and CoASSG levels in lungs of hype roxic rats as potential biomarkers of intramitochondrial oxidant stre sses. Pediatr. Res. 51, 346-353.

PAGE 168

168 Olsvik, P.A., Kristensen, T., Waagb, R., Rosse land, B.O., Tollefsen, K.-E., Baeverfjord, G., Berntssen, M.H.G., 2005. mRNA expression of antioxidant enzymes (SOD, CAT and GSH-Px) and lipid peroxidative stre ss in liver of Atlantic salmon ( Salmo salar ) exposed to hyperoxic water during smoltification. Comp. Biochem. Physiol. 141C, 314-323. Orlando, E.F., Guillette, J., L. J., 2001. A re-e xamination of variati on associated with environmentally stressed organisms. Human Reproduction Update 7, 265-272. Osborne, T.Z., Chapman, L.J., Chapman, C.A., Crisman, T.L., Prenger, J.P., Nyguen, S., Stecker, E., 2001. Invertebrate community st ructure and oxygen availability in an intermittent stream/wetland/river system of the Ugandan highlands. Verh. Internat. Verein. Limnol. 27, 3599-3603. Packer, L., Fuehr, K., 1977. Low oxygen concentra tion extends the lifespan of cultured human diploid cells. Nature 267, 426-425. Parcellier, A., Schmitt, E., Burunet, M., Hamm ann, A., Solary, E., Garrido, C., 2005. Small heat shock proteins HSP27 and alpha-B-crystalli n: Cytoprotective and oncogenic functions. Antioxid. Redox Signal. 7, 404-413. Parsons, P.A., 1991. Evolutionary rates: stress and species boundaries. Annu. Rev. Ecol. Syst. 22, 1-18. Parsons, P.A., 1994. Habitats, stress, and evol utionary rates. J. Evol. Biol. 7, 387-397. Pauwels, K., Stoks, R., de Meester, L., 2005. Coping with predator stress: interclonal differences in induction of heat-shock pr oteins in the water flea Daphnia magna J. Evol. Biol. 18, 867-872. Pickart, C.M., 2001. Mechanisms underlying ub iquitination. Annu. Rev. Biochem. 70, 503-533. Pihl, L., Baden, S.P., Diaz, R.J., 1991. Effects of periodic hypoxia on dist ribution of demersal fish and crustaceans. Mar. Biol. 108, 349-360. Pipe, P.K., Moore, M.N., 1985. Ultrastructural ch anges in the lysosomal-vacuolar system in digestive cells of Mytilus edulis as a response to increased salinity. Mar. Biol. 87, 157-164. Podrabsky, J.E., Somero, G.N., 2004. Changes in gene expression a ssociated with acclimation to constant temperatures and fluctuating da ily temperatures in an annual killifish Austrofundulus limnaeus J. Exp. Biol. 207, 2237-2254. Pohn, M., Vopel, K., Grunberger, E., Ott, J., 200 1. Microclimate of the brown alga Feldmannia caespitula interstitium unde r zero-flow conditions. Mar. Ecol. Prog. Ser. 210, 285-290. Prtner, H.O., 2002. Climate variations and the physiological basis of temperature dependent biogeography: Systemic to molecular hierarc hy of thermal tolerance in animals. Comp. Biochem. Physiol. 132A, 739-761.

PAGE 169

169 Qin, S., Ding, J., Takano, T., Yamamura, H., 1999. Involvement of receptor aggregation and reactive oxygen species in osmotic stress-indu ced Syk activation in B cells. Biochem. Biophys. Res. 262, 231-236. Ramos-Vasconcelos, G.R., Cardoso, L.A., Hermes -Lima, M., 2005. Seasonal modulation of free radical metabolism in estivating land snails Helix aspersa Comp. Biochem. Physiol. 140C, 165-174. Richier, S., Furla, P., Plantivaux, A., Merl e, P.-L., Allemand, D., 2005. Symbiosis-induced adaptation to oxidative stre ss. J. Exp. Biol. 208, 277-285. Richier, S., Merle, P.-L., Furla, P., Pigozzi, D ., Sola, F., Allemand, D., 2003. Characterization of superoxide dismutases in a noxiaand hyperoxia-tolerant sy mbiotic cnidarians. Biochim. Biophys. Acta 1621, 84-91. Ringwood, A.H., Keppler, C.J., 2002. Water quality va riation and clam growth: is pH really a non-issue in estuaries? Estuaries 25, 901-907. Romo, M., Hoarau, P., Garello, G., GnassiaBarelli, M., Girard, J.P., 2003a. Mussel transplantation and biomarkers as useful tools for assess ing water quality in the NW Mediterranean. Environmental Pollution 122, 369-378. Romo, M., Mourgaud, Y., Geffard, A., Gnassia-Barelli, M., Amia rd, J.C., Budzinski, H., 2003b. Multimarker approach in transplanted mussels for evaluating water quality in Charentes, France, coast areas exposed to different anth ropogenic conditions. Environ. Toxicol. 18, 295-305. Roper, J.M., Mazzatti, D.J., Watkins, R.H., Maniscalco, W.M., Keng, P.C., O'Reilly, M.A., 2004. In vivo exposure to hyperoxia induces DNA damage in a population of alveolar type II epithelial cells. Am. J. Physiol. Lung Cell Mol. Physiol. 286, L1045-L1054. Rosenberger, A.E., Chapman, L.J., 2000. Respir atory characters of three species of haplochromine cichlids: Implications for use of wetland refugia. J. Fish Biol. 57, 483-501. Ruby, E.G., Wirsen, C.O., Janna sch, H.W., 1981. Chemolithotrophi c sulfur-oxidizing bacteria from the Galpagos Rift hydrothermal ve nts. Appl. Environ. Microbiol. 42, 317-324. Sagarin, R.D., Gaines, S.D., 2002. The 'abundant centr e' distribution: To what extent is it a biogeographical rule? Ecol. Lett. 5, 137-147. Saito, H., Hammond, A.T., Moses, R.E., 1995. Th e effect of low oxygen tension on the in Vitro replicative life span of human diploid fibroblast cells and their transformed derivatives. Exp. Cell Res. 217, 272-279. Sanders, B.M., 1993. Stress proteins in aquatic orga nisms: an environmental perspective. Crit. Rev. Toxic. 23, 49-75.

PAGE 170

170 Sanz, A., Pamplona, R., Barja, G., 2006. Is the m itochondrial free radical theory of aging intact? Antioxid. Redox Signal. 8, 582-599. Savage, N.B., 1976. Burrowing activity in Mercenaria mercenaria (L.) and Spisula solidissima (Dillwyn) as a function of temperature and dissolved oxygen. Mar. Behav. Physiol. 3, 221234. Schnell, J.D., Hicke, L., 2003. Non-traditional functions of ubiquiti n and ubiquitin-binding proteins. J. Biol. Chem. 278, 35857-35860. Semenza, G.L., 2000. Cellular and molecular dissec tion of reperfusion in jury: ROS within and without. Circ. Res. 86, 117-118. Seo, A.Y., Hofer, T., Sung, B., Judge, S ., Chung, H.Y., Leeuwenburgh, C., 2006. Hepatic oxidative stress during aging: Effects of 8% long-term cal orie restriction and lifelong exercise. Antioxid. Redox Signal. 8, 529-538. Sheehan, D., Power, A., 1999. Effects of seasona lity on xenobiotic and antioxidant defense mechanisms of bivalve molluscs. Comp. Biochem. Physiol. 123C, 193-199. Shigenaga, M.K., Gimeno, C.J., Ames, B.N ., 1989. Urinary 8-hydroxy-2'-deoxyguanosine as a biological marker of in vivo oxidative DNA damage. Proc. Nat. Acad. Sci. USA 86, 96979701. Shyu, W.-C., Lin, S.-Z., Saeki, K., Kubosaki, A., Matsumoto, Y., Onodera, T., Chiang, M.-F., Thajeb, P., Li, H., 2004. Hyperbaric oxygen enha nces the expression of prion protein and heat shock protein 70 in a mouse neuroblasto ma cell line. Cell. Molec. Neurobiol. 24, 25768. Somero, G.N., 2002. Thermal physiol ogy and vertical zonation of in tertidal animals: optima, limits, and costs of living. Integ. and Comp. Biol. 42, 780-789. Somero, G.N., 2004. Adaptation of enzymes to temp erature: Searching fo r basic "strategies". Comp. Biochem. Physiol. 139B, 321-333. Somero, G.N., Childress, J.J., Anderson, A.E., 1989. Transport, metabolism, and detoxification of hydrogen sulfide in animals from sulfide-ri ch marine environments. Crit. Rev. Aquat. Sci. 1, 591-614. Spees, J.L., Chang, S.A., Snyder, M.J., Chang, E.S., 2002. Osmotic induction of stressresponsive gene expression in the lobster Homarus americanus Biol. Bull. 203, 331-337. Spicer, J.I., Gaston, K.J., 1999. Phys iological diversity and its eco logical implications. Blackwell Science Ltd, London.

PAGE 171

171 Stebbing, A.R.D., 1985. A possible synthesis. In : Bayne, B.L., Brown, D.A., Burns, K., Dixon, D.R., Ivanovici, A., Livingstone, D.R., Lo we, D.M., Moore, M.N., Stebbing, A.R.D., Widdows, J. (Eds.), The effects of stress and pollution on marine animals, Praeger Scientific, New York, pp. 310-314 Stillman, J.H., 2002. Causes and consequences of thermal tolerance limits in rocky intertidal porcelain crabs, genus Petrolisthes Integ. and Comp. Biol. 42, 790-796. Suresh, N., Jayaraman, J., 1983. The adap tation to salinity response of fish Tilapia mossambica gill mitochondria to salinity stre ss. J. Bioenerg. Biomemb. 15, 363-378. Szczesny, B., Hazra, T.K., Papaconstantinou, J., Mitra, S., Boldogh, I., 2003. Age-dependent deficiency in import of mitochondrial DNA glycos ylases required for repair of oxidatively damaged bases. Proc. Natl. Acad. Sci. USA 100, 10670-10675. Tapley, D.W., 1993. Sulfide-dependent oxidative st ress in marine invert ebrates, especially thiotrophic symbioses. In: Zoology, Un iversity of Maine, Orono, ME, p. 159 Tapley, D.W., Beuttner, G.R., Shick, J.M., 1999. Free radicals and chemiluminescence as products of the spontaneous oxidation of sulfide in seawater, and their biological implications. Biol. Bull. 196, 52-56. Tomanek, L., 2002. The heat-shock response: Its variation, regulation an d ecological importance in intertidal gastropods (genus Tegula ). Integ. and Comp. Biol. 42, 797-807. Tomanek, L., Helmuth, B., 2002. Phys iological ecology of rocky inte rtidal organisms: a synergy of concepts. Integ. and Comp. Biol. 42, 771-775. Tomanek, L., Sanford, E., 2003. Heat-shock prot ein 70 (Hsp70) as a biochemical stress indicator: an experimental fi eld test in two congeneric in tertidal gastropods (Genus: Tegula ). Biol. Bull. 205, 276-284. Truchot, J.P., Duhamel-Jouve, A., 1980. Oxygen a nd carbon dioxide in the marine intertidal environment: Diurnal and tidal change s in rockpools. Resp. Phys. 39, 241-254. Vanden Hoek, T.L., Li, C., Shao, Z.-H., Schumack er, P.T., Becker, L.B., 1997. Significant levels of oxidants are generated by is olated cardiomyocytes during is chemia prior to reperfusion. J. Mol. Cell Cardiol. 29, 2571-2583. Vesel, A., Wilhelm, J., 2002. The role of carbo n dioxide in free radi cal reactions of the organism. Physiol. Res. 51, 335-339. Viarengo, A., Canesi, L., Garcia Martinez, P., Peters, L.D., Livingstone, D.R., 1995. Pro-oxidant processes and antioxidant defence systems in the tissues of the Antarctic scallop ( Adamussium colbecki ) compared with the Mediterranean scallop ( Pecten jacobaeus ). Comp. Biochem. Physiol. 111B, 119-126.

PAGE 172

172 Vismann, B., 1991. Sulfide toleranc e: physiological mechanisms and ecological implications. Ophelia 34, 1-27. Wadhwa, R., Taira, K., Kaul, S. C., 2002. An Hsp70 family chaperone, mortalin/mthsp70/PBP74/Grp75: what, when, and where? Cell Stress and Chaperone 7, 309-316. Waite, J., Neufeld, G., 1977. Oxygen consumption by Sphaerium simile Comp. Biochem. Physiol. 57A, 373-375. Ward, J.E., Shumway, S.E., 2004. Separating the grai n from the chaff: pa rticle selection in suspensionand deposit-feeding bivalv es. J. Exp. Mar. Biol. Ecol. 300, 83-130. Webster, S.J., Dill, L.M., 2007. Estimating the energetic cost of abiotic conditions using foraging behaviour. Evol. Ecol. Res. 9, 123-143. Wells, H.W., 1957. Abundance of the hard clam Mercenaria mercenaria in relation to environmental factors. Ecology 38, 123-128. Werner, I., 2004. The influence of salinity on the heat-shock protein response of Potamocorbula amurensis (Bivalvia). Mar. Environ. Res. 58, 803-807. Werner, I., Hinton, D.E., 1999. Field validation of hsp70 stress proteins as biomarkers in Asian clam (Potamocorbula amurensis): Is downregulat ion an indicator of stress? Biomarkers 4, 473-484. Werner, I., Hinton, D.E., 2000. Spatial profil es of hsp70 proteins in Asian clam ( Potamocorbula amurensis ) in northern San Francisco Bay may be linked to natural rather than anthropogenic stressors. Mar. Environ. Res. 50, 379-384. Widdows, J., 1985. Physiological measurements. In: Bayne, B.L., Brown, D.A., Burns, K., Dixon, D.R., Ivanovici, A., Li vingstone, D.R., Lowe, D.M., Moore, M.N., Stebbing, A.R.D., Widdows, J. (Eds.), The effects of stress and pollution on mari ne animals, Praeger Scientific, New York, pp. 3-45 Wilkinson, K.D., 2000. Ubiquitination and deubi quitination: Targeting of proteins for degradation by the proteasome Sem. Cell Dev. Biol. 11, 141-148. Willmore, W.G., Storey, K.B., 1997. Antioxidant systems and anoxia tolerance in a freshwater turtle Trachemys script elegans Mol. Cell. Biochem. 170, 177-185. Wilson, C., Scotto, L., Scarpa, J., Volety, A., La ramore, S., Haunert, D., 2005. Survey of water quality, oyster reproduction and oyster health stat us in the St. Lucie Estuary. J. Shellfish Res. 24, 157-165. Winn, R.N., Knott, D.M., 1992. An evaluation of the survival of expe rimental populations exposed to hypoxia in the Savannah Rive r estuary. Mar. Ecol. Prog. Ser. 88, 161-179.

PAGE 173

173 Witman, J.D., Grange, K.R., 1998. Links between rai n, salinity, and predation in a rocky subtidal community. Ecology 79, 2429-2447. Wong, H.R., Menendez, I.Y., Ryan, M.A., De nenberg, A.G., Wisp, J.R., 1998. Increased expression of heat shock protei n-70 protects A549 cells against hyperoxia. Am. J. Physiol. Lung Cell Mol. Physiol. 275, L836-L841. Yancey, P.H., 2001. Nitrogen compounds as osmolytes. Fish Physiology 20, 309-341. Yancey, P.H., Clark, M.E., Hand, S.C., Bowlus, R.D., Somero, G.N., 1982. Living with water stress: evolution of osmolyte systems. Science 217, 1214-1222. Zhang, H.J., Xu, L., Drake, V.J., Xie, L., Ober ley, L.W., Kregel, K.C., 2003. Heat-induced liver injury in old rats is associated with exagge rated oxidative stress a nd altered transcription factor activation. FASEB J. 17, 2293-2295. Zippay, M.L., Place, S.P., Hofmann, G.E., 2004. The molecular chaperone Hsc70 from a eurythermal marine goby exhibits temperature insensitivity during lu ciferase refolding assays. Comp. Biochem. Physiol. 138A, 1-7. Zuo, L., Christofi, F.L., Wright, V.P., Liu, C.Y ., Merola, A.J., Berliner, L.J., Clanton, T.L., 2000. Intraand extracellular m easurement of reactive oxygen species produced during heat stress in diaphragm muscle. Am. J. Physiol. Cell. Physiol. 279, C1058-C1066.

PAGE 174

174 BIOGRAPHICAL SKETCH Joanna Lea Joyner was born in Guelph, On tario, on November 2, 1977, to David and Pamela Joyner. Joanna has one ol der sister, Danielle. Joanna sp ent her first several years in Guelph, followed by three years in Carbondale, IL, and eighteen years in Salt Lake City, UT. She attended Skyline High School and graduate d in 1995. She earned an Honors Bachelor of Science in Biology, with minors in chemistry and history, from the University of Utah (2000). Much of the minor in history was earned during a semester abroad at the University of Wales, Swansea (1997). In her Honors th esis she described the research she conducted in pediatric immunology under the supervision of Dr. Harry R. Hill at the University of Utah School of Medicine. Joanna served as a teaching assistan t during her senior year winning the “Teaching Assistant of the Year ” award for her second semester of teaching. In 2002 Joanna earned a Master of Science in Zoology from Washington State University (Pullman, WA). Dr. Raymond W. Lee was the chai r of her thesis committee. Her research was supported by two student grants and was awar ded an honorable mention for best student presentation at an international symposium. Du ring these two years she was a teaching assistant for five different courses. She also met and marri ed Luis F. Matos, a graduate student in the Department of Entomology. In the fall of 2002 Joanna began her disserta tion research in the la boratory of Dr. David Julian in the Department of Zoology, University of Florida (Gainesville, FL). During her graduate studies she earned three research grants, won the best student presentation competition at a national meeting, was a teach ing assistant for two courses, and supervised the research experiences of five undergraduate students. She wo rked as an adjunct instructor at Santa Fe Community College (Gainesville, FL) for two se mesters, team-teaching Introductory Biology for Non-majors with her husband, Luis F. Matos. Duri ng her final year, she ce lebrated the birth of

PAGE 175

175 her son, Lucas David. After earning her Ph.D., Joanna plans to study the relationships among free radical metabolism, mutation accumulati on, and evolution in the model organism Caenorhabditis elegans


Permanent Link: http://ufdc.ufl.edu/UFE0020084/00001

Material Information

Title: Magnitude of the Oxidative Stress Response Influences Species Distributions
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0020084:00001

Permanent Link: http://ufdc.ufl.edu/UFE0020084/00001

Material Information

Title: Magnitude of the Oxidative Stress Response Influences Species Distributions
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0020084:00001


This item has the following downloads:


Full Text





MAGNITUDE OF THE OXIDATIVE STRESS RESPONSE INFLUENCES SPECIES
DISTRIBUTIONS




















By

JOANNA JOYNER MATOS


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2007



































2007 Joanna Joyner Matos

































To my parents, David and Pamela Joyner, who so strongly encouraged the pursuit of higher
education in their daughters that they will have the joy of seeing both of us complete Ph.D.s this
spring.









ACKNOWLEDGMENTS

I first thank my dissertation committee: Shirley M. Baker, Lauren J. Chapman, Robert D.

Holt, David Julian, and Christiaan Leeuwenburgh. They are an amazingly diverse group of

scientists and each one played a key role in the development and execution of the projects

described herein. They all were good-naturedly tolerant of my "quick questions" and I am

looking forward to continuing conversations with them in the future. I wish to thank Lauren for

her patience with me during my first foray into field work. Lastly, I particularly wish to thank the

chair of my dissertation committee, David Julian. While some advisors seemingly limit their

involvement to providing financial and logistical support, Dave engaged me in a five-year

conversation that touched upon numerous topics beyond those of the dissertation research,

including leadership and managerial skills, departmental politics, work/life balance issues, and

many other aspects of an academic career. He has not just mentored me in how to conduct

experiments and interpret results, but taught me how to be a scientist and a faculty member.

I owe a great deal to a wonderful group of undergraduate students: Jenessa Andrzejewski,

Laura Briggs, Michaela Hogan, Jennifer Rivas, and Nicole Scheys. The bulk of the data

presented in Chapter 3 was collected by these talented and industrious ladies. Working with

these students ("The Mercenaria Group") was an excellent learning experience and I am

indebted to them for their patience, diligence, and enthusiasm.

I also must thank Craig A. Downs, formerly of EnVirtue Biotechnologies, Inc., and

currently of Haereticus Environmental Laboratories, for his generosity and expertise. Craig

taught me how to measure stress protein expression levels, a technique that figures largely in my

dissertation research. For almost a year I peppered him with questions and he answered all of

them. Most importantly, Craig has been unbelievably generous with the antibodies I used in all

of my dissertation research as well as several side projects.









I would also like to thank the following people for many forms of assistance: Drs. Ben

Bolker and Craig Osenberg (my unofficial committee members), Dr. Stephanie Wohlgemuth, Dr.

Derk Bergquist, Dr. David Evans, Andrea Martinez and the rest of my cohort, Benjamin

Predmore, Michael McCoy, Nat Seavy, Pete Ryschkewitsch, Cathy Moore, Karen Pallone, and

Vitrell Sherif.

My research was funded by a Florida Sea Grant Pilot Proposal grant, a national Sea Grant

Industry Fellowship, a Sigma XI Grant-in-Aid of Research, and the Department of Zoology. This

research also was supported by NSF IBN-0422139 (to David Julian), NSF IBN-0094393 (to

Lauren Chapman), the Wildlife Conservation Society (to Lauren Chapman), and start-up funds

(to Christiaan Leeuwenburgh) from the Genomics and Biomarkers Core of The Institute on

Aging, University of Florida. Permission to conduct research in Uganda was acquired from the

National Council for Science and Technology, the Office of the President, and Makerere

University (Uganda). I thank Luis F. Matos and the field assistants of the Kibale Fish Project,

particularly Jovan, who provided invaluable assistance during the field work. I thank Cheryl M.

Woodley (NOAA National Ocean Service, Center for Coastal Environmental Health and

Biomolecular Research, Charleston, SC) for the loan of a liquid N2 dry shipper, and Robert H.

Richmond, Kewalo Marine Laboratory, University of Hawaii, for use of the Biomek robotic

workstation.

Finally, I thank my family, particularly my husband, Luis, for their never-ending support

and love. I would not have made it this far without them!









TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ..............................................................................................................4

L IST O F T A B L E S ...................................................................................................... . 8

L IST O F FIG U R E S ............................................................................... 9

A B S T R A C T ........................................... ................................................................. 1 1

CHAPTER

1 IN T R O D U C T IO N ............................................................................................................ 13

Abiotic Factors in Aquatic Habitats and Oxidative Damage.....................................15
H y p o x ia ..................................................................................................................... 1 7
H y p e ro x ia .................................................................................................................. 1 8
H y d ro g en S u lfid e ....................................................................................................... 19
Elevated Temperature............................. ............................21
Salinity .............. .. .. .. .. ............ ...................................... ........ .. ... ......... 22
Stress Proteins as Indicators of Organism Health .................................................23
Overview of Dissertation Research ............. ..............................24

2 INCREASED EXPRESSION OF STRESS PROTEINS IN THE SURF CLAM Donax
variabilis FOLLOWING HYDROGEN SULFIDE EXPOSURE .......................................27

Introduction ................................. ................. ..... .... ................. 27
M materials and M methods ................... ............................................................ .......... .. 29
Exposure of Clam s to Abiotic Stressors ................................................................. 29
A analyses of Stress Protein Expression ....................................... ..........................31
D ata A analysis and Statistics ............................................. ............................... 34
R e su lts ................................................................................ ......... .... 3 5
Experim mental Conditions. ........................................... .........35
Survival A analysis .................................................... 35
Exposure to N orm oxia ..................... ....................................... ... ................... 36
Antioxidant Protein, Lipid Peroxidation, and Oxidative Repair Enzyme Expression ....36
Protein Rescue and/or Degradation .................................................... ................39
C ytoskeletal Protein C content ................................................................. .... .................4 1
D iscu ssio n ........................... ... ...... ....... ... ........................................................................ 4 1
Abiotic Factors are Linked to Free Radical Production ..................................... 42
Sources of V ariance ............................................ ................... .... 47
Physiological Responses to Stress Vary by Season ...................................... ......... 48
C o n c lu sio n s .........................................................................................................4 9





6










3 PHYSIOLOGICAL RESPONSES OF Mercenaria mercenaria TO SINGLE AND
M U LTIPLE A B IO TIC FA C TO R S ............................................................. .....................61

In tro d u ctio n .............. ........... ................................................................................................6 1
M materials and M methods ...................................... .. ......... ....... ...... 64
Laboratory Exposures ........ ............... ............. ... ..................... 64
Schedule ........................................................................66
T issue e P processing .................................................................67
Survival Analyses .................. .................. .................. 67
A n aly se s .................................................................6 7
Results ........... ......... ......... ....................................71
H ypoxia E xperim ents ...............................................................7 1
T em perature E xperim ents ......................................................................................... 75
Dual stressor Experiments .................. ....... ......... .........79
D iscu ssio n ............................ ....................................... ..................................8 7

4 STRESS RESPONSE OF A FRESHWATER CLAM ALONG AN ABIOTIC
GRADIENT: TOO MUCH OXYGEN MAY LIMIT DISTRIBUTION .............................116

Introduction ......... ......... ...... ............. ....................... ........ 116
Materials and Methods ............ .. .................................118
S tu d y S ite ................................................................................................................. 1 1 8
Sam pling M methods .............. ............................ .. ..........................119
Sampling for RNA/DNA, Nucleic Acid Oxidation and Stress Proteins .....................120
S tatistic al an aly se s ................................................................................................... 12 2
R e su lts ....................... .. ............. .. ...........................................................12 3
D iscu ssion ................. ..... .. .... ......... ...........................................126
Limnological Characters and Relationship with Clam Density ...................................126
Cellular-level Indicators ................. ....... .............. 128
The Extreme Edge of the Distribution............... .. ...................... ................ 130
Relationships Between Limnological Characters and Cellular-Level Stress
In d icato rs ...................................................... 13 1
C o n c lu sio n s .............................................................................13 3

5 S Y N T H E S IS ................................ .......................................................................14 2

A P P E N D IX ............................................................................................. 14 8

LIST OF REFEREN CES ............. ...................... ... ..................... 154

B IO G R A PH IC A L SK E T C H ......... ..... .............................................................. ............... 174









LIST OF TABLES


Table page

2-1 Summary of statistical results from comparisons between samples from Donax
variabilis exposed to normoxia treatment and samples from animals exposed to
hypoxia, hyperoxia, and sulfide ......................................................... ............... 51

4-1 Overview of stress protein functions. ................................. 135









LIST OF FIGURES


Figure page

2-1 D iagram of flow -through system ............................................... ............................ 52

2-2 A ntibody specificity tests......................................................................... .................... 53

2-3 Fraction of surviving Donax variabilis clams in survival experiments in fall and
sp rin g ......... ....... .. .......... .................. ................................................ 5 4

2-4 Expression levels of Hsp70 in Donax variabilis exposed to normoxia for 0, 1, 3, and
5 day s .......................................................... ...................................55

2-5 Expression levels of three antioxidant proteins, a lipid peroxidation marker, and an
oxidative repair enzyme in Donax variabilis exposed to normoxia (normox), hypoxia
(hypox), hyperoxia (hyperox), and sulfide ............................................. ............... 56

2-6 Expression levels of five proteins involved in protein rescue and/or degradation in
Donax variabilis exposed to normoxia (normox), hypoxia (hypox), hyperoxia
(hyperox), and sulfide. .......................... ........................ .... ......... ......... 58

2-7 Expression levels of total actin in Donax variabilis exposed to normoxia (normox),
hypoxia (hypox), hyperoxia (hyperox), and sulfide................................ ............... 60

3-1 Antibody specificity tests in M. mercenaria whole clam homogenates ..........................100

3-2 Fraction of clams that buried in the three hypoxia experiments.................................. 101

3-3 Glycogen content of whole clams in the three hypoxia experiments ............. ..............102

3-4 Stress protein expression levels in clams from hypoxia experiments ...........................103

3-5 Survivorship of clams in the winter (A) and spring (B) temperature experiments ..........106

3-6 Fraction of clams that buried in the three temperature experiments.............................107

3-7 Glycogen content of whole clams in the three temperature experiments.....................108

3-8 Stress protein expression levels in clams from temperature experiments. ....................109

3-9 Survivorship of clams in dual stressor experiments ..................................................... 112

3-10 Fraction of clams that buried in the dual stressor experiments .............. .................113

3-11 Glycogen content of whole clams in the dual stressor experiments............................14

3-12 RNA oxidation after 24 hour exposure in dual stressor experiments............................115









A-1 Relative stress protein expression levels in Sphaerium sp. clams from the 14-day
transplant experim ent ......... ........................... ......... .. .......... 1.. 152

A-2 Levels of oxidatively damaged DNA and RNA in Sphaerium sp. clams from the 14-
day transplant experim ent .................................................................... ..................... 153









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

MAGNITUDE OF THE OXIDATIVE STRESS RESPONSE INFLUENCES SPECIES
DISTRIBUTIONS

By

Joanna Joyner Matos

May 2007

Chair: David Julian
Major: Zoology

Animals must employ diverse physiological strategies to survive in aquatic habitats with

extreme or fluctuating abiotic factors. Whether these physiological strategies include the

oxidative stress response, and whether ability to maintain an oxidative stress response influences

species distribution, is not fully understood. In the research projects described in this dissertation,

I take three approaches to addressing this topic. I first test whether abiotic factors typical of

aquatic habitats cause a cellular stress response consistent with free radical production in the

marine bivalve Donax variabilis. These results demonstrate that exposure to hydrogen sulfide

causes an oxidative stress response in a non-sulfide-adapted bivalve and highlight the importance

of examining seasonal variation in stress physiology. I next test whether organisms can mitigate

the cellular-level damage associated with exposure to single or multiple stressful abiotic factors.

This study was conducted in the estuarine bivalve, Mercenaria mercenaria. These results

demonstrate that physiological strategies at higher levels of organization buffer the need for a

cellular-level oxidative stress response in a stress-tolerant organism. Finally, I test whether the

capacity to produce an oxidative stress response affects species distribution. This combined field

and laboratory study of the freshwater bivalve Sphaerium sp. demonstrates that individuals in a

population that overlies an environmental gradient show a variation in their ability to maintain a









cellular stress response that reflects position along the gradient. These three interrelated studies

demonstrate that abiotic factors found in aquatic habitats do cause oxidative stress and that

ability to respond to the abiotic factors correlates with distribution.









CHAPTER 1
INTRODUCTION

Much of the work that is done under the name of ecology is not ecology at all, but either
pure physiology i.e. finding out how animals work internally or pure geology or
meteorology... In solving ecological problems we are concerned with what animals do in
their capacity as whole, living animals...We have next to study the circumstances under
which they do these things, and, most important of all, the limiting factors which prevent
them from doing certain other things. By solving these questions it is possible to discover
the reasons for the distribution and numbers of different animals in nature. (Elton, 1927, p.
33-34)

The examination of how physical attributes of the environment interact with physiology to

influence the distribution of organisms was an early goal of ecology, as described above in the

introduction to one of the first animal ecology textbooks. During the development of the field of

ecology in the first half of the twentieth century, many investigators used physiological

techniques to address fundamental ecological topics including "the distribution and abundance of

organisms" (Spicer and Gaston, 1999; McNab, 2002). Decades later, the two fields split and

'ecologists' began focusing on biotic interactions and demographic processes as the causative

agents of distribution and abundance patterns (Andrewartha and Birch, 1984), while

'physiologists' focused on mechanisms occurring at lower levels of biological organization

(Hochachka and Somero, 2002). The relatively recent re-integration of these two approaches in

the field of ecological physiology utilizes newly developed molecular and biochemical tools to

examine how environmental variables limit reproduction and survival and therefore impact

distribution and abundance (Spicer and Gaston, 1999; Chown and Storey, 2006).

The set of environmental variables that limits reproduction and survival of a species is the

species' niche (Brown et al., 1996). Traditionally, studies that have attempted to define a species'

niche involved either in situ manipulations combining biotic interactions with environmental

variables (exemplified by Connell, 1961) or laboratory studies of physiological responses to

abiotic factors. Unlike in situ manipulations, which allow the experimental subjects to be









impacted by all aspects of a complex environment, laboratory studies typically involve

acclimation to one select environmental variable. However, the limitations of extrapolating

laboratory studies of single variables to population-level processes have long been identified

(e.g., Hall, 1964), including the tendency of laboratory studies to over-estimate physiological

tolerances (Nordlie, 2006). What is most likely to be successful, therefore, is a combined field

and laboratory approach that utilizes biochemical and molecular tools to investigate biotic

interactions and species distributions.

A combined field/laboratory approach has been successfully applied to the study of species

distributions and physiological adaptations of invertebrates in rocky intertidal habitats along the

Pacific coast of the United States. This habitat has well-characterized communities and steep

gradients of multiple abiotic factors that exhibit diel and seasonal patterns. It has become a

model system for ecological physiology, and has been the setting for much of the recent progress

in linking population distribution with cellular-level physiology. For example, latitudinal and

vertical zonation patterns of snails, mussels, and crabs reflect the thermal tolerances of the

individual species, which themselves rely upon a complex web of responses including gene

expression, protein repair and degradation processes, mitochondrial energetic, and heart

function (for review, Hofmann et al., 2002; Somero, 2002; Stillman, 2002; Tomanek, 2002).

These physiological parameters and the resultant patterns in mortality, in turn, influence such

biotic processes as predation (Dahlhoff et al., 2001) and competition (Menge and Sutherland,

1987; Menge and Olson, 1990).

Most studies that could be termed 'ecological physiology' or 'comparative physiology,' as

exemplified by the work noted above in rocky intertidal habitats, take a common approach to

understanding how organisms respond to a given abiotic factor. Many of these studies are built









upon the assumption that the abiotic factor imposes a stress on the organism, resulting in a

perturbation to homeostasis at either the cellular or organismic level (Hoffmann and Parsons,

1991). Suites of physiological responses to this abiotic factor are assumed to be energetically

costly, and if the cost of maintenance of these responses grows too high, the animal cannot

survive (Parsons, 1991).

To understand the effects of an abiotic factor, therefore, the optimal study organism is

typically considered to be one that tolerates extremes of the abiotic factor (Hoffmann and

Parsons, 1991; Spicer and Gaston, 1999), because these organisms are most likely to employ

protective physiological strategies that can be detected in laboratory studies. However, the

usefulness of such organisms may be limited when the goal is to understand the mechanisms by

which the stressor exerts its effects, since these same protective physiological strategies will

minimize the stressor's impact. In such cases, an alternate and under-utilized approach is to study

the effects of an abiotic factor on an organism that is not adapted to the factor. In this

'vulnerable' or 'naive' organism one could be more successful at detecting the consequences of

the abiotic factor in the absence of protective responses. Both of these approaches, the study of

stress-tolerant and of 'vulnerable' organisms, are employed in the work described in this

dissertation, which addresses how abiotic factors in aquatic habitats affect the physiology of

bivalves from coastal and inland aquatic habitats.

Abiotic Factors in Aquatic Habitats and Oxidative Damage

Coastal and inland aquatic habitats vary widely in their characterization as stressful or

benign. Potentially stressful abiotic factors in aquatic habitats include low dissolved 02

availability (termed hypoxia), elevated 02 availability (termed hyperoxia), hydrogen sulfide,

thermal extremes, and salinity fluctuations. These factors can occur singly, but more often are

found in combination, including the widespread combination of low dissolved 02 and hydrogen









sulfide in marine sediments (Fenchel and Riedl, 1970) and the combination of high temperature,

hypoxia, and salinity extremes in estuaries in summertime (Millie et al., 2004; Caccia and Boyer,

2005).

Free radicals have been suggested as a mechanism by which many abiotic factors,

including hypoxia, hyperoxia, hydrogen sulfide, high temperature, and salinity extremes, cause

cellular damage and thus influence the distribution of marine invertebrates in extreme or

otherwise stressful habitats. Free radicals are atoms or molecules that contain one or more

unpaired electrons and are therefore highly reactive (Halliwell and Gutteridge, 1999). During

aerobic metabolism, the mitochondria of nearly all eukaryotic cells convert 0.1% (Fridovich,

2004) to 3% (Boveris and Chance, 1973) of 02 into free radicals such as superoxide. This

endogenous free radical production can be increased by many environmental stressors,

particularly fluctuations in 02 availability (Boveris and Chance, 1973; Li and Jackson, 2002).

Free radicals spontaneously react in a number of ways, one of which is to strip electrons from

cellular macromolecules, particularly proteins, lipids, and nucleic acids, causing oxidative

damage. For example, oxidative damage from free radical attacks on nucleic acids include strand

breaks and oxidation of nitrogenous bases (Shigenaga et al., 1989; Evans and Cooke, 2004). If

free radical production overwhelms a cell's ability to detoxify the free radicals, oxidative damage

occurs (Halliwell and Gutteridge, 1999). Cellular oxidative damage, if not repaired, leads to cell

death, disease, and aging (Halliwell and Gutteridge, 1999).

To minimize oxidative damage, eukaryotic cells use a variety of protective mechanisms,

including the expression of stress proteins. These proteins fall into three general categories: (1)

antioxidants such as manganese superoxide dismutase (MnSOD), copper/zinc superoxide

dismutase (Cu/Zn SOD), glutathione, glutathione peroxidase (GPx) and catalase, that convert









free radicals into less toxic or nontoxic forms; (2) chaperone proteins such as ubiquitin, the heat

shock proteins (e.g., Hsp60, Hsp70 and Grp75) and small heat shock protein (sHsp), that aid the

folding or removal of damaged proteins; and (3) oxidative repair enzymes, such as OGG1, that

repair oxidatively damaged DNA (Halliwell and Gutteridge, 1999).

Hypoxia

Conditions of low dissolved 02 are widespread and occur naturally in aquatic habitats,

particularly in coastal areas affected by upwelling, rock pools in the intertidal zone, all marine

sediments, and in standing freshwater bodies (Grieshaber et al., 1994; Diaz and Rosenberg,

1995). Hypoxia results from a variety of processes, including elevated water temperature, low

mixing, dense animal populations, and high organic decomposition (Grieshaber et al., 1994). The

importance of maintaining adequate access to 02 centers on its function as the final electron

acceptor in the mitochondrial electron transport chain. In the absence of 02, mitochondrial

production of ATP via oxidative phosphorylation ceases, and cells must rely upon the reduced

ATP production available from glycolysis and other pathways of anaerobic metabolism (for

review of pathways in invertebrates, see Grieshaber et al., 1994). Hypoxia affects a wide variety

of ecological processes in aquatic organisms, including recruitment (Marinelli and Woodin,

2002), distribution (Rosenberger and Chapman, 2000), seasonal migration along a vertical

gradient (Pihl et al., 1991), predator-prey interactions (Breitburg et al., 1997), the use of refuges

(Chapman et al., 2002), and larval settlement and growth rates (Baker and Mann, 1992). Most of

the well-studied instances of hypoxia tolerance are found in the invertebrates, particularly the

molluscs, annelids, nematodes, and platyhelminths (Hochachka and Somero, 2002).

Whether hypoxia can induce free radical production directly, or whether reoxygenation

following a period of hypoxia is necessary for free radical production, is not yet understood

(Kukreja and Janin, 1997; Hermes-Lima et al., 1998; Halliwell and Gutteridge, 1999; Semenza,









2000; Hermes-Lima and Zenteno-Savin, 2002; Li and Jackson, 2002). During hypoxia, the

absence of 02 as the final electron acceptor causes accumulation of electrons in mitochondrial

electron transport chains (i.e., the chains are reduced). A sudden return of 02 can cause the

production of superoxide due to nearly instantaneous reactions between 02 and the free electrons

that accumulated in proteins of the electron transport chain (Du et al., 1998; Li and Jackson,

2002). Such a scenario might occur during tidal ebb and flow for intertidal animals. However,

several recent studies have also shown free radical production during hypoxia without

subsequent reoxygenation. The evidence for heightened free radical production during hypoxia

comes from direct measurement of free radicals (Vanden Hoek et al., 1997; Becker et al., 1999)

and measurement of oxidative DNA damage in mammalian cells (Englander et al., 1999), and

changes in antioxidant expression and/or activity in goldfish (Lushchak et al., 2001) and an

estuarine crab (de Oliveira et al., 2005). However, several studies of both vertebrates and

invertebrates have noted decreases or a lack of changes in antioxidant expression or activity

during hypoxia that are consistent with an overall metabolic depression during hypoxia (Hass

and Massaro, 1988; Willmore and Storey, 1997; Joanisse and Storey, 1998; Larade and Storey,

2002).

Hyperoxia

While less common in marine habitats than hypoxic conditions, hyperoxic conditions

include rocky intertidal pools with photosynthetically active algae (Truchot and Duhamel-Jouve,

1980), boundary layers of intertidal seaweed (Irwin and Davenport, 2002) and brown algae

(Pohn et al., 2001), the cold seawater of polar regions (Viarengo et al., 1995; Abele and

Puntarulo, 2004), and within the tissues of some algal-cnidarian symbioses (Dykens et al., 1992;

Richier et al., 2003, 2005). The physiological effects of hyperoxia are not well-characterized, but

it is evident that hyperoxia causes mitochondrial-induced cellular death (Chandel and Budinger,









2007) and decreased cellular metabolism, which is likely due to the inactivation of the citric acid

cycle enzyme aconitase (Gardner et al., 1994). The effects of hyperoxia on biotic interactions

have been characterized in only a few systems. For example, cnidarian hosts of intracellular algal

symbionts utilize protective strategies to minimize damage from hyperoxic conditions induced

by excessive algal photosynthesis during periods of elevated temperature (Dykens et al., 1992;

Nii and Muscatine, 1997; Richier et al., 2003).

Elevated cellular 02 levels induce mitochondrial free radical production (Boveris and

Chance, 1973; Akbar et al., 2004) and oxidative damage (Dennog et al., 1999). Exposure to

hyperoxia has been linked to elevated antioxidant responses in tissues of both vertebrates

(O'Donovan et al., 2002; Cho et al., 2005) and invertebrates (Dykens et al., 1992; Viarengo et al.,

1995; Abele and Puntarulo, 2004). However, this relationship is not consistent, as demonstrated

by a variety of in vitro and in vivo studies in invertebrates and vertebrates (Abele et al., 1998b;

Dennog et al., 1999; Allen and Balin, 2003; Freiberger et al., 2004).

Hydrogen Sulfide

Hydrogen sulfide (referred to here simply as "sulfide") occurs in a variety of aquatic

habitats, including mudflats, mangrove swamps, deep-sea hydrothermal vents, hydrocarbon

seeps, and anoxic basins, where animals may be periodically or continuously exposed to sulfide

at levels up to 12 mM (for review, Fenchel and Riedl, 1970; Somero et al., 1989; Bagarinao,

1992). Sulfide is a highly reactive toxin, and the H2S form diffuses freely across respiratory

surfaces and therefore cannot be excluded from tissues (Denis and Reed, 1927; Julian and Arp,

1992). Sulfide has several mechanisms of toxicity, including the reversible inhibition of

cytochrome c oxidase, the final enzyme of the mitochondrial electron transport chain (Lovatt

Evans, 1967; Nicholls, 1975), reduction in hemoglobin oxygen affinity (Carrico et al., 1978) and

inhibition of approximately 20 enzymes (Bagarinao, 1992). Invertebrates inhabiting sulfidic









environments employ a variety of strategies to detoxify sulfide, of which the most widely

demonstrated is the oxidation of sulfide to thiosulfate or other compounds (for review, Lovatt

Evans, 1967; Vismann, 1991; Grieshaber and Volkel, 1998). Marine invertebrates that are not

found in sulfidic habitats and do not employ sulfide detoxification strategies typically show

increased mortality upon exposure to hydrogen sulfide (Grieshaber and Volkel, 1998). A number

of interspecific interactions are associated with sulfidic habitats, the best-studied of which is the

widespread relationship between invertebrate hosts and chemoautotrophic bacterial symbionts

that use the chemical energy from sulfide oxidation to fix carbon dioxide into carbohydrates

(Felbeck et al., 1981; Ruby et al., 1981; Cavanaugh, 1983). Additionally, sulfide structures

coastal communities (Gamenick et al., 1996) and methane seep communities (Levin et al., 2006)

by influencing recruitment and spatial patterns, and sulfide gradients influence food web

dynamics in deep sea hydrothermal vent communities (Levesque et al., 2006).

Hydrogen sulfide oxidizes spontaneously in the presence of divalent metals (both dissolved

and in metalloenzymes), generating oxygen-centered radicals (likely superoxide) and sulfur-

centered radicals in aqueous solutions (Chen and Morris, 1972; Tapley et al., 1999) and in

animal tissues (Tapley, 1993; Abele-Oeschger and Oeschger, 1995; Eghbal et al., 2004; Julian et

al., 2005). Several studies have specifically addressed the link between oxidative damage and

sulfide exposure at the organismal level. For example, antioxidant enzyme activities were

proportional to the sulfide tolerances of thiobiotic meiofauna (Morrill et al., 1988). Similarly,

MnSOD activity increased in response to sulfide exposure in the chemoautotrophic symbiotic

bivalve Solemya velum but not in the related nonsymbiotic Yoldia limatula (Tapley, 1993). In

contrast, a relationship between sulfide tolerance and antioxidant activity was not found in a

survey of sulfide-tolerant polychaetes and bivalves (Abele-Oeschger, 1996). A relationship









between sulfide and free radical production also has been investigated at the cellular level. In

isolated erythrocytes from the marine polychaete Glycera dibranchiata, sulfide exposure causes

mitochondrial depolarization, increased cellular oxidative stress, and increased mitochondrial

superoxide production (Julian et al., 2005). These studies suggest that increased oxidative stress

is an additional mechanism by which sulfide exposure could cause toxicity (Morrill et al., 1988;

Abele-Oeschger et al., 1994; Abele-Oeschger, 1996).

Elevated Temperature

The importance of temperature in regulating nearly all physiological processes and the

resulting impacts on species distribution and abundance is highlighted by extensive discussions

of this topic in recent books describing biochemical adaptations (160 pages, Hochachka and

Somero, 2002) and physiological ecology (100 pages, McNab, 2002). Although aquatic

organisms encounter extremes of high and low temperature, only consequences of, and

adaptations to, high temperature will be addressed in this dissertation. Distribution is tightly

linked with tolerance to heat stress across a wide variety of aquatic organisms (Gilchrist, 1995;

Partner, 2002; Chown and Storey, 2006; Nordlie, 2006). This relationship is dependent upon an

array of cellular and biochemical processes, including changes in protein structure, enzyme

activity and cell membrane composition (Hochachka and Somero, 2002). For example, in

ectothermic organisms, the expression levels and activities of enzymes such as lactate

dehydrogenase are temperature adaptive, as exhibited by correlations between environmental

temperature and activation energy, and conservation of catalytic rate constants and substrate

binding ability, over a broad range of temperatures (but not at upper lethal temperatures; for

review, Somero, 2004). Temperatures much higher than the environmental or acclimation

temperature of an organism cause damage at the cellular level, including mitochondrial swelling

and distortion (Cole and Armour, 1988) and structural damage to proteins and cell membranes









(Hochachka and Somero, 2002). Heat stress and the ability to respond to heat stress affect

functional responses including burial ability in bivalves (Savage, 1976), development (Mahroof

et al., 2005; McMillan et al., 2005), heart function (Stillman, 2002), and the ability to respond to

other abiotic factors such as hypoxia (Chang et al., 2000) and low salinity (Cain, 1973). Ability

to respond to thermal stresses may affect a variety of biotic processes in addition to distribution,

including recruitment success (Menge, 2000), facultative interspecific interactions (Burnaford,

2004), and resistance to predation (Mesa et al., 2002; Pauwels et al., 2005).

A mechanistic link between exposure to high temperature and free radical production

likely results from alterations in mitochondrial respiration, as demonstrated in both vertebrates

(Zuo et al., 2000; Zhang et al., 2003; Mujahid et al., 2006) and invertebrates (Abele et al., 2002;

Heise et al., 2003; Keller et al., 2004). Heat stress affects the ability of organisms to minimize or

repair oxidative damage, typically by enhancing this ability by preconditioning the cells, as

demonstrated in studies of cardioprotection in vertebrates (Arnaud et al., 2002; Joyeux-Faure et

al., 2003).

Salinity

The influence of extremes of salinity on physiological processes, particularly when

external salinity is not equivalent to intracellular osmotic concentration, is nearly as pervasive as

that of temperature (Hochachka and Somero, 2002). Internal osmotic concentrations of aquatic

organisms are typically regulated with extensive arrays of 'compatible osmolytes' such as free

amino acids, sugars, polyhydric alcohols, and urea (Yancey et al., 1982; Yancey, 2001).

Insufficient cellular-level responses to fluctuating or extreme salinity result in changes in cell

volume (Neufeld and Wright, 1996), structural and functional damage to mitochondria (Suresh

and Jayaraman, 1983) and lysosomes (Pipe and Moore, 1985; Hauton et al., 1998), decreased

ability to regulate 02 consumption during hypoxia (Hawkins et al., 1987), and decreased ability









to respond to elevated temperatures (Cain, 1973; Hauton et al., 1998; Werner, 2004). Ability to

tolerate salinity extremes or fluctuations and the energetic costs associated with osmoregulatory

strategies affect a wide variety of ecological processes, including foraging behavior (Webster

and Dill, 2007), predation and recruitment (Witman and Grange, 1998), and species distributions

(Nordlie, 2006; Lowe et al., 2007).

Several studies of stress protein expression (particularly heat shock protein expression) in

estuarine invertebrates exposed to hyposalinity or hyposalinity/high temperature treatments have

produced conflicting results (Kultz, 1996; Clark et al., 2000; Werner and Hinton, 2000; Spees et

al., 2002; Werner, 2004; Blank et al., 2006). A causative relationship between hypersalinity and

free radical production is well-established in plant physiology and biomedical fields such as

nephrology and immunology (e.g., Hernandez et al., 1993; Qin et al., 1999; Hizoh and Haller,

2002). However, whether hyposalinity is linked to alterations in free radical metabolism and

oxidative damage is not understood. A recent study of hyposalinity responses of a marine alga

detected an elevation in glutathione but not in antioxidant enzymes such as catalase and

superoxide dismutase (Jahnke and White, 2003).

Stress Proteins as Indicators of Organism Health

Several characteristics of stress proteins make them useful for assessing the effects of

abiotic factors. Many stress proteins are constitutively expressed at low levels, but this

expression is upregulated in response to conditions that result in elevated free radical production

(Feder and Hofmann, 1999; Downs et al., 2001a; Kultz, 2005) and even to biotic interactions

such as predation (Pauwels et al., 2005). Patterns of expression of multiple stress proteins can

indicate the stressor to which the organism is exposed (Downs et al., 2000, 2001a, 2001b,

2002b). However, the interpretation and applicability of stress protein expression is hampered by

our poor understanding of the temporal profiles of stress protein induction and how this









induction affects the long-term health of the organism. Very few studies have coupled

physiological condition assays with stress protein expression levels (Brown et al., 1995; Hamza-

Chaffai et al., 2003; Romeo et al., 2003b), and even these studies treated the condition assays

only as indicators of overall health rather than investigating whether the protein expression

patterns were accurate predictors of the functional assays and vice versa. Additionally, some

stress proteins like Hsp70 have a large but short-term response to a stressor (Tomanek and

Sanford, 2003), which limits the practical application of monitoring expression of this protein in

the field because timing of sampling is crucial to detecting a stress protein response. Finally,

there are conditions under which downregulation (rather than upregulation) of stress protein

expression indicates physiological stress (Werner and Hinton, 1999), particularly if the organism

is stressed to such an extreme that all metabolic processes are decreasing and death is imminent

(Bierkens, 2000).

Overview of Dissertation Research

Animals that must employ diverse physiological strategies to survive in habitats with

extreme or fluctuating abiotic factors tend to be generalists with broad tolerance ranges (Lynch

and Gabriel, 1987). Whether these physiological strategies include the oxidative stress response,

and whether animals from benign versus extreme habitats exhibit differences in the magnitude of

their oxidative stress responses is not fully understood. As noted above, accumulating evidence

suggests that free radical metabolism and oxidative stress are linked to many of the abiotic

factors typically faced by invertebrates in coastal and inland aquatic habitats. However, whether

the ability of these organisms to respond to oxidative stress influences their tolerance to these

abiotic factors, and ultimately influences species distribution, is unknown.

In the research projects described in this dissertation, I address three interrelated questions,

each of which is examined in a different bivalve species. In the selection of species for these









projects I followed what is typically termed the August Krogh Principle, which Krogh articulated

in his opening address on "The Progress of Physiology" to the Thirteenth International

Physiological Congress with the comment, "For a large number of problems there will be some

animal of choice or a few such animals on which it can be most conveniently studied" (Krogh,

1929, p. 202).

In Chapter 2 I test whether a subset of the abiotic factors discussed above triggers an

oxidative stress response in the marine bivalve Donax variabilis. I examine the effects of 24-

hour exposures to hypoxia, hyperoxia, and hydrogen sulfide on the seasonal patterns of survival

and stress protein expression in D. variabilis. Since these clams do not encounter these abiotic

factors in the high-energy sandy beaches they inhabit, these clams are unlikely to possess

protective mechanisms that would minimize the impact of the stressor and reduce my ability to

detect a cellular-level oxidative stress response. This study therefore follows the alternate

approach discussed above: examining a 'vulnerable' species for evidence of oxidative stress

resulting from an abiotic factor. Results from Chapter 2 were published in the following paper: J.

Joyner-Matos, C.A. Downs, and D. Julian, Increased expression of stress proteins in the surf

clam Donax variabilis following hydrogen sulfide exposure, Comparative Biochemistry and

Physiology 145:245-257, 2006.

In Chapter 3 I test whether the ability to initiate an oxidative stress response following

exposure to single or multiple abiotic factors correlates with changes in whole-organism,

functional, metrics of condition. The effects on the cellular- and organismal-level responses of

hypoxia, high temperature, and the combination of high temperature and hyposalinity were

examined in the estuarine bivalve Mercenaria mercenaria. These clams exemplify the generalist

strategy described by Lynch (1987), with demonstrated broad tolerances for extremes of and









fluctuations in temperature, dissolved 02 level, salinity, and pH that reflect the species'

widespread distribution in intertidal and subtidal coastal habitats. With the selection of such a

stress-tolerant species, I am able to examine whether physiological strategies at higher levels of

organization buffer the need for a cellular-level oxidative stress response or correlate with the

cellular-level responses. The results from Chapter 3 are in preparation for submission, likely to

the Journal of \ell, \lh Research.

In Chapter 4 I take the tools developed from the two previous chapters, which were in

essence purely physiological studies, and in a combined field and laboratory study I address how

the capacity to produce an oxidative stress response affects species distribution. This study

specifically examines whether the distribution of a freshwater clam over a complex and stable

gradient of several abiotic factors is related to the physiological condition and oxidative stress

response of the clams. The clams used in this project, Sphaerium sp., live in a swamp-river

system in Uganda and cannot be classified into either the 'vulnerable' or 'tolerant' categories.

They clearly are not vulnerable to abiotic factors like D. variabilis since they tolerate a range of

pH and dissolved 02 concentrations; however, it is not known whether these clams can tolerate

the extremely broad ranges of multiple abiotic factors in the manner demonstrated by M.

mercenaria. Therefore, I could not form apriori predictions about whether clams experiencing

different conditions along the environmental gradient would exhibit differences in their oxidative

stress responses or levels of oxidative damage. The results from Chapter 4 are in press as the

following manuscript: J. Joyner-Matos, L.J. Chapman, C.A. Downs, T. Hofer, C. Leeuwenburgh,

and D. Julian, Stress response of an African freshwater clam along a natural abiotic gradient:

Too much oxygen can be a limiting factor in aquatic environments, Functional Ecology, 21:344-

355, 2007.









CHAPTER 2
INCREASED EXPRESSION OF STRESS PROTEINS IN THE SURF CLAM Donax variabilis
FOLLOWING HYDROGEN SULFIDE EXPOSURE

Introduction

Abiotic factors in marine habitats include thermal extremes, salinity fluctuations, hypoxia,

hyperoxia, and sulfide (sum ofH2S, HS- and S2-). Such factors may influence species distribution

by stressing organisms to their physiological limits (Parsons, 1991). However, the mechanisms

by which many abiotic factors cause stress are not completely understood. Free radicals have

been suggested as a mechanism by which some abiotic factors, including temperature (Abele et

al., 1998a, 2002; Downs et al., 2002a; Heise et al., 2003), hypoxia (Greenway and Storey, 1999),

hyperoxia (Dykens et al., 1992; Viarengo et al., 1995), and sulfide (Morrill et al., 1988; Abele-

Oeschger et al., 1994, 1996; Tapley et al., 1999), cause cellular damage and thus influence the

distribution of marine invertebrates in extreme or otherwise stressful habitats. Free radicals,

which are atoms or molecules that contain one or more unpaired electrons (Halliwell and

Gutteridge, 1999), cause cellular damage, termed oxidative damage, by stripping electrons from

cellular macromolecules.

In the mitochondria of nearly all eukaryotic cells, a fraction of 02 consumption is

converted into the free radical superoxide during aerobic metabolism; estimates range from 0.1%

(Fridovich, 2004) to 3% (Boveris and Chance, 1973) of 02 consumption. To minimize oxidative

damage, eukaryotic cells utilize a variety of protective mechanisms, including the expression of

an assortment of proteins which, for the purposes of this paper, are collectively referred to as

"stress proteins." These fall into three general categories: (1) antioxidants, such as manganese

superoxide dismutase (MnSOD), copper/zinc superoxide dismutase (Cu/Zn SOD), glutathione,

glutathione peroxidase (GPx) and catalase, which convert free radicals into less toxic or nontoxic

forms; (2) proteins involved in protein rescue and/or degradation, such as the heat shock









proteins (e.g., Hsp60, Hsp70 and Grp75) and small heat shock protein (sHsp), and the protein

ubiquitin, which aid in the folding or removal of damaged proteins (Downs et al., 2005); and (3)

oxidative repair enzymes, such as 8-oxoguanine DNA glycosylase (OGG1), which repair

oxidatively-damaged DNA (Halliwell and Gutteridge, 1999). Expression of some stress proteins

is upregulated in response to conditions that result in elevated free radical production (Downs et

al., 2001a, 2001b) and to other environmental stressors such as thermal stresses (Hofmann and

Somero, 1995). The measurement of stress protein expression levels has served as a cellular-

level indicator of elevated free radical production in marine invertebrates (e.g., Abele and

Puntarulo, 2004), and in comparative physiological (e.g., Willmore and Storey, 1997) and

biomedical studies (e.g., Magalhdes et al., 2005).

The aim of this study was to investigate whether sulfide, as well as hypoxia and hyperoxia,

have the potential to stimulate a cellular response consistent with increased oxidative stress in a

marine invertebrate. To test this, we exposed the marine clam Donax variabilis (the coquina

clam) to these abiotic stressors in controlled laboratory conditions and assessed the animals'

overall tolerance (i.e., survival), stress protein expression, and lipid peroxidation. Additionally,

we examined whether the abiotic stressors affect expression of the cytoskeletal protein actin,

which has traditionally been measured as a control for sample protein content, but recently has

been shown to decrease in vertebrate cells following exposure to hypoxia, hyperoxia, and free-

radical generating toxins (Allani et al., 2004; Brown and Davis, 2005; Cho et al., 2005). We

conducted identical experiments in fall and spring to investigate seasonal differences (Hofmann

and Somero, 1995; Chapple et al., 1998; Sheehan and Power, 1999). Unlike previous studies of

oxidative stress in marine invertebrates, we selected our study species based on the high

probability that it does not encounter hypoxia, hyperoxia or sulfide in its habitat and therefore









likely does not employ additional protective mechanisms, such as those in invertebrates adapted

to hypoxia (Grieshaber et al., 1994) and sulfide (Grieshaber and Volkel, 1998). Such adaptations

would be expected to minimize the impact of the stressor, reducing our ability to detect a

cellular-level oxidative stress response. D. variabilis inhabits sandy beaches with moderate to

high waves along the southeastern coast of North America (Mikkelsen, 1981; Ellers, 1995). The

clams migrate up and down the beach, following the tidal cycle, generally remaining buried in

the upper 4 cm of sand. The wave activity and high porosity of sandy beaches likely maintains

the seawater surrounding the clams sulfide-free and at or approaching air-saturation. Therefore,

we considered it probable that D. variabilis is more vulnerable to these stressors than would be

expected of invertebrates from habitats such as tide pools, mudflats or marshes (Grieshaber and

Volkel, 1998).

Materials and Methods

Clam collection and maintenance. Donax variabilis clams were collected at Crescent

Beach, FL (approx. 29.7N, 81.2W), within 30 minutes of high tide during September 2003

("fall") and March 2004 ("spring"). Water temperatures at all collections were 28 + 3C. All

clams were between 1.0-1.5 cm in length. Immediately after collection, the clams were

transported to the University of Florida in aerated seawater in an insulated container (ca. 90

minutes transport time).

Exposure of Clams to Abiotic Stressors

Flow-through system. Exposure to abiotic stressors was achieved with a constant-

temperature flow-through system that used electronic gas flow controllers to regulate water Po2

and sulfide concentration (Fig. 2-1). Seawater in the system was obtained from the University of

Florida Whitney Marine Laboratory (Marineland, FL, USA), and was pretreated with









chloramphenicol (2 mg L1) to prevent the growth of sulfate reducing bacteria. This pretreatment

has been shown to markedly increase survival of marine bivalves in respirometry experiments

(De Zwaan et al., 2002). The flow-through system consisted of four channels, with each having

an animal chamber designed to contain eight clams. Seawater for each of the four channels was

continuously equilibrated with air (normoxia) for channel one, N2 (hypoxia) for channel two, a

mixture of 02 and air (hyperoxia) for channel three, and a mixture of air and hydrogen sulfide

gas (from a compressed tank of 2% H2S, balance N2) for channel four. The equilibrated water

was pulled through the animal chambers at 2 mL min-. Further details of the apparatus

construction and gas equilibration are provided in the legend to Fig. 2-1. Dissolved 02 was

measured twice daily in each channel using a fluorometric dissolved 02 probe (FOXY probe;

Ocean Optics, Inc., Dunedin, FL, USA). The sulfide concentration in the H2S-channel was

measured twice daily using the methylene blue method (Cline, 1969).

Survival during exposure to stressors. D. variabilis were exposed to normoxia, hypoxia,

hyperoxia and sulfide in each season (fall and spring), during which survival was assessed.

Exposures lasted 4-7 days and were conducted with 8 clams. The clams were checked twice

daily for mortality, and were presumed to have died when they did not close their valves when

disturbed or when obvious tissue degradation had begun (dead clams were immediately removed

from the chambers). Surviving clams and tissues from these experiments were not used for any

other experiments.

Exposure to normoxia. In a preliminary experiment in the fall, we tested whether

maintenance in the flow-through exposure system under normoxic conditions resulted in changes

in expression of Hsp70. This protein has shown sensitivity to a variety of stressors, and therefore

was used as a general indicator of stress (Feder and Hofmann, 1999). For this experiment, one









additional channel was added to the flow-through system and all channels contained seawater

equilibrated with air. Fifty clams were placed in the system initially, and the experiment

continued for 9 days. The clams were not fed. Every day, 1 clam was removed from each

channel, and tissues from clams removed at days 1, 3 and 5, as well as tissues from control clams

collected and frozen in liquid N2 at the beach collection site, were prepared, stored and assessed

for Hsp70 expression, as described below.

Exposure to stressors. For tissues to be used for determination of the remaining stress

proteins, clams were exposed to normoxia, hypoxia, hyperoxia and sulfide for 24 hours in each

season (fall and spring). A total of 8 clams were exposed to each stressor in each season. At the

end of the exposure, the clams were removed and immediately processed, as described below.

Analyses of Stress Protein Expression

Tissue sample processing. Immediately upon removal from the flow-through system,

clams were opened by severing their adductor muscles, and their whole tissues were quickly

blotted dry and frozen in liquid N2, followed by storage at -800 C for further processing. Of the

eight clams per treatment, four were processed for stress protein expression analysis, as modified

from Downs et al. (2002b), and the remaining four clams per treatment were archived at -800 C.

For homogenization, whole tissues, frozen and stored as described above, were individually

ground in liquid nitrogen to a fine powder in a mortar and pestle pre-cooled by liquid N2, and

then immediately returned to -800 C storage. For resuspension of these homogenates, a small

volume of each individual powdered sample was dissolved in denaturing SDS buffer (50 mmol

L-1 Tris, 15 mmol L1 EDTA, 2% SDS, 15 mmol L1 DTT, 0.5% DMSO, and 0.01% Halt

protease inhibitor cocktail from Pierce Biotechnology, Inc. Rockford, IL, USA; pH 7.8). Each

suspension was then vortexed for 30 seconds, incubated at 850 C for 3 minutes, vortexed for 15

seconds, incubated again at 850 C for 3 minutes, vortexed for 15 seconds, and centrifuged at









12,000xg for 10 minutes. Total soluble protein concentration of each sample was assayed by the

method of Ghosh et al. (1988). Tissues from fall and spring experiments were homogenized and

resuspended at the same time. The resuspended samples were then aliquotted, frozen in liquid N2

and stored at -800 C. Unless noted otherwise, all chemicals were obtained from Sigma Chemical

Company and were the highest quality available.

Antibodies. Samples were assayed for stress protein expression using mono-specific,

ELISA-grade polyclonal antibodies generated by and donated by EnVirtue Biotechnologies, Inc.

(Winchester, VA, USA). The antibodies were raised in rabbits against 8-15 amino-acid

polypeptides (conjugated to bovine serum albumin) derived from each target protein sequence of

the bivalve Mya arenaria (Downs et al., 2002b). The following antibodies were used: Cu/Zn

SOD (Cat. # AB-SOD-1516), GPx (Cat. # AB-GPX-1433), MnSOD (Cat. #AB-1976), ubiquitin

(Cat. #AB-U100), invertebrate small heat shock protein homologues (sHsp; Cat. #AB-H105),

heat shock protein 70 (Hsp70; Cat. #AB-Hsp70-1519), mitochondrial Hsp70 (Grp75; log 3219),

Hsp60 (Cat. # AB-H100-IN), OGGl-mito (lot 2916), and 4-hydroxy-2E-nonenol-adducted

protein (HNE; lot 156). Total actin pool was determined with a polyclonal antibody from

Stressgen Bioreagents (Victoria, BC, Canada). Specificity of each antibody was verified by SDS-

PAGE and western blotting with goat anti-rabbit, alkaline phosphatase-conjugated secondary

antibody (Sigma) and a chemiluminescent reporter system (DuoLux

Chemiluminescent/Fluorescent Substrate for Alkaline Phosphatase, Vector Laboratories,

Burlingame, CA, USA) on samples from both fall and spring exposures (Downs et al., 2002b).

Representative antibody verification results for Cu/Zn SOD, GPx, MnSOD, Hsp70,

mitochondrial Hsp70, Hsp60, OGGl-mito and actin are presented in Fig. 2-2. Note that









ubiquitin, sHsp and HNE interact with other proteins and therefore typically form "smears"

rather than single bands.

After verification of specificity, stress protein expression for each tissue sample was

determined by dry-dotting (Cu/Zn SOD, GPx, MnSOD, ubiquitin, Hsp70, Hsp60, Grp75, HNE

and actin) or ELISA (sHsp and OGGl-mito), both as described below. All assays were

performed on 16 clams from each season (four per treatment), and tissues from both seasons

were assayed at the same time to minimize experimental variation.

Dry-dotting. The following dry-dotting technique was developed for this study. Samples

were diluted to 30 ng or 100 ng of total soluble protein (TSP) in 50 [l TBS (50 mmol L-1 Tris, 10

mmol L-1 NaC1, pH 7.5) and triplicate 1 tL volumes were dotted onto dry nitrocellulose

membrane (Fisher Scientific, Fairlawn, NJ, USA) with a multichannel micropipette (0.5-10 tL,

Eppendorf, Westbury, NY, USA). On each membrane, an eight-fold serial dilution from one

sample was dotted in triplicate to allow subsequent confirmation that sample concentrations were

within the linear detection range and semi-quantitative comparisons of samples across

membranes (see below). Approximately 48 samples could typically be dotted onto a 7 cm by 11

cm membrane. Once dry, the membranes were subsequently treated identically to a western blot.

Specifically, each membrane was blocked in TBS-T (TBS with 0.05% Tween-50) with 5% milk

(Carnation instant dry milk) or 5% acid-hydrolyzed casein for 30 minutes and then incubated

with primary antibody at 1:10,000 in TBS-T for 1 hour at room temperature. Each membrane

was then washed with TBS-T, incubated with secondary antibody at 1:10,000 in TBS-T with 5%

milk or 5% casein for 1 hour at room temperature, and finally washed three times with TBS-T

followed by TBS and then Tris solution (100 mmol L-1, pH 9.5). Chemiluminescence substrate

(as above) was then added as per the manufacturer's instructions and each membrane was









visualized on a GeneGnome Chemiluminescent Detection System (Syngene, Frederick, MD,

USA). Images were analyzed using GeneTools application software (Syngene). The serial

dilution of one sample on each membrane was used to determine the correlation between

concentration and luminescence intensity within a membrane using one site, saturation ligand-

binding regression curve fits (performed by SigmaPlot 8.02, Systat Software, Inc., Point

Richmond, CA, USA). Dry-dotting of tissues from fall and spring experiments were performed

at the same time and on the same piece of membrane; allowing direct comparisons among

samples.

ELISA. OGG1-m and sHsp expression levels were determined by ELISA at EnVirtue

Biotechnologies, Inc. A Biomek 2000 robotic workstation (Beckman Coulter, Inc., Fullerton,

CA, USA) was used to conduct the ELISA assays using 384-well microplates. Samples were

assayed in triplicate with intra-specific variation of less than 7.5% for all samples combined for

each assay. An eight-point calibrant curve using a protein standard relevant to each antibody was

added in sextuplicate for each plate (Downs et al., 2002b). ELISA assays of tissues from fall and

spring experiments were performed at the same time and on the same plates, allowing direct

comparisons among samples.

Data Analysis and Statistics

All stress protein expression levels were standardized by the standard curves run with each

dry-dotting or ELISA assay. Data generated by dry-dotting are expressed as relative units per

nanogram of total soluble protein (RU ng TSP-1). Data generated by ELISA are expressed as

fmol mg TSP-1 or fmol ng TSP-1. The data were not normally distributed, as confirmed by the

Shapiro-Wilk W test for non-normality (p value < 0.05 for at least one season for each protein),

and therefore they were analyzed by the nonparametric Kruskal-Wallis one-way ANOVA. T

values for each ANOVA are listed in Table 2-1. Significantly different groups were then









analyzed using the Conover-Inman post hoc comparison test, which is a form of the Fisher's

least significant difference (LSD) method performed on ranks (Conover, 1999). This analysis

was conducted pairwise for each stress protein and individually for each season. We did not

analyze the data with two-way ANOVAs with season and treatment as factors because we

expected to find extensive seasonal differences in expression patterns within and among proteins

and we considered the samples collected in fall and spring to be truly independent. P values less

than 0.05 were considered significant and are listed in Table 2-1. All statistical analyses were

performed with StatsDirect version 2.4.4 (Cheshire, UK). Stress protein data are presented as

scatterplots, with the data symbol signifying the median, and vertical asymmetrical error bars

denoting minimum and maximum values.

Results

Experimental Conditions.

The conditions of the four chambers in the flow-through exposure system for experiments

with the three environmental stressors were: 1) normoxia, 21.6 1.9 kPa Po2 (mean s.d.; n = 2

readings per 24 hour period); 2) hypoxia, 12.3 + 1.4 kPa Po2; 3) hyperoxia, 36.6 + 3.0 kPa Po2;

and 4) sulfide, 98 2.9 amol 1-1 total sulfide and 24.0 1.8 kPa Po2. Average pH in both the gas

equilibration chambers and the outflow from the animal chambers for the four treatments was

8.11 0.14 with no significant differences between treatments.

Survival Analysis.

To determine the survival tolerance ofD. variabilis to each exposure condition in each

season, we exposed 8 clams per treatment to normoxia, hypoxia, hyperoxia and sulfide for up to

7 d. Clams collected in the fall showed 100% mortality during hypoxia exposure, with 6 of 8

clams dying on the third day and the remaining 2 clams dying on the fourth day (Fig. 2-3). In the









spring, mortality was also 100%, although the clams survived slightly longer, with 4 of 8 clams

dying on the fifth day, 1 clam dying on the sixth day, and the remaining clam dying on the

seventh day (Fig. 2-3). Exposure to hyperoxia caused no mortality in the fall (4 d exposure). In

the spring, 1 of 8 clams died at day 3 during hyperoxia exposure, with no additional mortality (7

d exposure). Hydrogen sulfide exposure showed similar mortality between seasons; in the fall, 6

of 8 clams died on the second day and the remaining 2 clams died on the third day, whereas in

the spring, 1 clam died on the second day, 3 more died on the third day and the remaining 4

clams died on the fourth day. There was no mortality in the normoxia treatments in either season

(4 d exposure in fall and 7 d exposure in spring).

Exposure to Normoxia

To confirm that changes in stress protein expression were due to the stressors and not

simply a result of the clams being maintained in the flow-through system, we conducted a 9 d

normoxia exposure study in the fall. We measured expression levels of Hsp70 in clams from

days 1, 3, 5, and compared those to expression in control clams that were collected and frozen at

the beach (time 0; see Fig. 2-4). We found no significant changes in Hsp70 expression levels (p

= 0.064). However, Hsp70 levels were slightly increased in clams sampled at day 5. Based on

this, we assume that any changes in stress protein expression in clams from the remaining

exposure experiments (which lasted 24 h) are due to the stressor(s) themselves rather than

representing an artifact of the clams being maintained in the flow-through system.

Antioxidant Protein, Lipid Peroxidation, and Oxidative Repair Enzyme Expression

To test for evidence that 24 hour exposure to hypoxia, hyperoxia or sulfide induced a

cellular-level response consistent with oxidative stress in D. variabilis with respect to normoxia

exposure, we measured changes in the expression of the antioxidant stress proteins MnSOD,









Cu/Zn SOD and GPx, and the DNA repair enzyme OGG1, as well as the concentration of

4-hydroxy-2E-nonenol adducted to protein (HNE), with the results as follows.

MnSOD. MnSOD is typically located in the mitochondrial matrix of eukaryotic cells. It

catalyzes the dismutation of superoxide to the less reactive pro-oxidant H202 (Halliwell and

Gutteridge, 1999). The appropriate banding pattern for this antibody is a single dominant band at

approximately 25 kD (EnVirtue Biotechnologies, Inc. product information; Fig. 2-2). In clams

collected and exposed to stressors in the fall, exposure to hypoxia had no effect on MnSOD

expression, whereas exposure to hyperoxia or sulfide resulted in twice the MnSOD expression

levels compared to clams exposed to normoxia (Fig. 2-5A, Table 2-1). Clams collected and

exposed to these stressors in the spring showed no significant change in MnSOD expression

(Fig. 2-5B, Table 2-1).

Cu/Zn SOD. Cu/Zn SOD is primarily located in the cytoplasm but may also be detected in

lysosomes, mitochondria, peroxisomes and the nucleus, but the isoform detected in this study is

expressed in the cytoplasm. As with MnSOD, Cu/Zn SOD catalyzes the dismutation of

superoxide into H202 (Halliwell and Gutteridge, 1999). The appropriate banding pattern for this

antibody is a single dominant band at approximately 19 kD (EnVirtue Biotechnologies, Inc.

product information; Fig. 2-2).There were no significant changes in Cu/Zn SOD expression in

clams collected and exposed to any stressors in the fall (Fig. 2-5C, Table 2-1), whereas in clams

collected and exposed to stressors in the spring, exposure to sulfide caused significantly elevated

Cu/Zn SOD (Fig. 2-5D, Table 2-1), Exposure to hypoxia or hyperoxia had no effect.

GPx. GPx is located primarily in the cytoplasm (60-75%) and to a lesser extent in the

mitochondria. This selenoprotein catalyzes the reduction of H202 to water with the concomitant

oxidation of reduced glutathione (Halliwell and Gutteridge, 1999). The appropriate banding









pattern for this antibody is several bands at approximately 20 35 kD with additional bands from

tetramer formation possible in the 70 90 kD range (EnVirtue Biotechnologies, Inc. product

information; Fig. 2-2). The response of GPx was similar to that of Cu/Zn SOD. There were no

significant changes in expression of GPx in clams collected and exposed to any stressors in the

fall (Fig. 2-5E, Table 2-1), whereas in clams collected and exposed to stressors in the spring,

exposure to sulfide caused significantly elevated GPx (Fig. 2-5F, Table 2-1) in comparison to

clams exposed to normoxia. Exposure to hypoxia or hyperoxia had no significant effect.

HNE. HNE-adducted protein is a peroxidation product of polyunsaturated fatty acids and

indicates increased oxidative damage to lipids (Halliwell and Gutteridge, 1999). The antibody

detects all HNE-adducted proteins and therefore does not produce a distinct banding pattern.

There were no significant changes in HNE-adducted protein in clams collected and exposed to

stressors in the fall, regardless of the stressor (Fig. 2-5G, Table 2-1). In the spring, clams

exposed to sulfide, but not hypoxia or hyperoxia, had significantly lower HNE levels than did

clams in normoxia (Fig. 2-5H, Table 2-1).

OGG1-m. OGG1-m is a DNA repair enzyme located in the mitochondria. It catalyzes the

removal of the highly mutagenic 8-hydroxyguanine (8-OH-G) lesion (Boiteux and Radicella,

2000), which can be generated by oxidative stress and ionizing radiation and, if not removed,

causes GC to TA transversions upon replication (Boiteux and Radicella, 2000). The appropriate

banding pattern for this antibody is one or two bands at approximately 38 45 kD (EnVirtue

Biotechnologies, Inc. product information; Fig. 2-2). Expression levels of OGG1-m were near

the lower detection limit in clams collected and exposed to stressors in the fall, regardless of the

stressor (Fig. 2-51, Table 2-1). In contrast, clams collected and exposed to hyperoxia or sulfide,









but not hypoxia, in the spring had significant increases in OGG1-m expression (Fig. 2-5J, Table

2-1).

Protein Rescue and/or Degradation

To determine whether D. variabilis exposed to the stressors for 24 hour showed responses

characteristic of increased protein denaturation conditions, we measured the expression levels of

ubiquitin, small heat shock protein (sHsp), Hsp60, Hsp70 and Grp75.

Ubiquitin. Ubiquitin is a small (76 amino acids), highly conserved protein that is

expressed in the nucleus, cytoplasm, and cell membrane of eukaryotic cells. It facilitates the

degradation of proteins damaged by oxidation (or by other processes) by attaching to the target

proteins and aiding in their transport to the 26S proteasome (Wilkinson, 2000; Pickart, 2001;

Schnell and Hicke, 2003; Herrmann et al., 2004). The antibody detects all ubiquitinated proteins

and therefore does not produce a distinct banding pattern. In clams collected and exposed to

stressors in the fall, hypoxia and hyperoxia had no effect on ubiquitin expression compared to

clams exposed to normoxia, whereas clams exposed to sulfide had significantly increased

ubiquitin expression (Fig. 2-6A, Table 2-1). In clams collected and exposed to stressors in the

spring, ubiquitin expression was not significantly affected (Fig. 2-6B, Table 2-1).

sHsp. sHsp are a group of proteins found in the cytosol, nucleus, and mitochondria

(Downs et al., 1999). They bind denatured proteins, preventing irreversible protein aggregation,

and participate in the ubiquitin/proteasome system (Parcellier et al., 2005)..The sHsp are

involved in protective responses to a wide range of stressors, including oxidative stress, heat

shock and environmental toxins (Downs et al., 2001a, 2001b; Basha et al., 2004; Arrigo et al.,

2005). Since the antibody detects all sHsp-adducted proteins, as well as testing for the proteins

themselves, which form up to five bands ranging from 10 kD to 45 kD, testing for antibody

specificity in the manner shown for the other antibodies is not appropriate. Clams collected and









exposed to stressors in the fall did not show any significant differences in sHsp expression (Fig.

2-6C, Table 2-1). However, clams collected and exposed to sulfide in the spring had elevated

sHsp expression, whereas exposure to hypoxia and hyperoxia had no effect (Fig. 2-6D, Table 2-

1).

Hsp70. Hsp70 family proteins are present in prokaryotes and in most cellular

compartments in eukaryotes. They have numerous roles involving chaperone functions, protein

degradation (Chapple et al., 2004) and protein folding (Frydman, 2001; Kregel, 2002). The

appropriate banding pattern for this antibody is two bands at approximately 70 kD (EnVirtue

Biotechnologies, Inc. product information; Fig. 2-2). In clams collected and exposed to stressors

in the fall, exposure to hyperoxia caused significantly higher Hsp70 expression compared to

clams exposed to normoxia, whereas exposure to hypoxia and sulfide had no effect (Fig. 2-6E,

Table 2-1). There were no significant differences in Hsp70 expression among clams collected

and exposed to stressors in the spring (Fig. 2-6F, Table 2-1).

Hsp60 and Grp75. Hsp60 is expressed in the mitochondria. It aids in the folding of newly-

formed proteins under normal physiological condition and refolds damaged proteins during

stress (Hartl, 1996; Kregel, 2002). The appropriate banding pattern for Hsp60 is one band at

approximately 60 kD (EnVirtue Biotechnologies, Inc. product information; Fig. 2-2). Grp75,

which is also known as mitochondrial hsp70, is primarily expressed in the mitochondria. The

appropriate banding pattern for Grp75 is one band at approximately 75 kD (EnVirtue

Biotechnologies, Inc. product information; Fig. 2-2).It is involved in several processes, including

responses to oxidative stress (Mitsumoto et al., 2002), chaperone functions, and intracellular

trafficking (Wadhwa et al., 2002).We did not detect any significant differences in Hsp60 (Fig. 2-









6G and H, Table 2-1) or Grp75 (Fig. 2-61 and J, Table 2-1) expression in clams collected and

exposed to stressors in either fall or spring.

Cytoskeletal Protein Content

We also measured the total actin pool to assess whether D. variabilis exposed to the

stressors experienced cellular damage in the form of disruption of the cytoskeleton or changes in

actin production, which could indicate a change in metabolic activity. The appropriate banding

pattern for this antibody is one band at approximately 43 kD (Stressgen Bioreagents product

information; Fig. 2-2).Clams collected and exposed to stressors in the fall showed no significant

differences in total actin pool (Fig. 2-7A, Table 2-1). However, clams collected in the spring and

exposed to hypoxia and sulfide, but not hyperoxia, had significantly decreased total actin pool

(Fig. 2-7B, Table 2-1).

Discussion

Donax variabilis upregulated expression of some antioxidants, proteins involved in protein

rescue and/or degradation, and repair enzymes in response to 24 hour exposure to sulfide and, to

a much lesser extent, to hyperoxia. We also found elevated levels of the lipid peroxidation

endproduct, HNE, in clams exposed to hyperoxia but not to sulfide. However, there was a

marked seasonality in the response to stressors, with clams collected and tested in the spring

showing greater expression of many stress proteins and significant decreases in HNE-adducted

proteins and actin. Finally, we found that hypoxia and sulfide were lethal stressors for the clams,

although clams in the spring experiment tolerated the stressors for a longer duration. In a marine

invertebrate that likely does not experience sulfide, hypoxia or hyperoxia in its habitat, these

results indicate that 1) exposure to sulfide, and probably hyperoxia, induces increased stress

protein expression and lipid peroxidation in a pattern consistent with oxidative stress, and 2)









clams in the spring had an increased stress protein response and decreased evidence of injury

(decreased HNE and increased survival) compared to clams in the fall.

Abiotic Factors are Linked to Free Radical Production

Hypoxia. Hypoxia is widespread and occurs naturally in marine habitats, particularly in

coastal areas affected by upwelling, rock pools in the intertidal zone, and all marine sediments

(Grieshaber et al., 1994; Diaz and Rosenberg, 1995). Hypoxia has been linked to increased free

radical production, but whether hypoxia can induce free radical production directly, or whether

reoxygenation following a period of hypoxia is necessary for free radical production is not yet

understood (Kukreja and Janin, 1997; Hermes-Lima et al., 1998; Halliwell and Gutteridge, 1999;

Semenza, 2000; Hermes-Lima and Zenteno-Savin, 2002; Li and Jackson, 2002). During hypoxia,

the absence of 02 as the final electron acceptor causes accumulation of electrons in

mitochondrial electron transport chains (i.e., the chains are reduced), with the result that a sudden

return of 02 can cause the production of superoxide due to nearly instantaneous reactions

between 02 and the accumulated free electrons (Du et al., 1998; Li and Jackson, 2002). Such a

scenario might occur during tidal flow for intertidal animals. However, several recent studies

have also shown free radical production during hypoxia without subsequent reoxygenation.

These are based on direct measurement of free radicals (Vanden Hoek et al., 1997; Chandel et

al., 1998; Becker et al., 1999), measurement of oxidative DNA damage in mammalian cells

(Englander et al., 1999) and yeast cells (Dirmeier et al., 2002), and the indirect measures of

changes in antioxidant expression and/or activity in goldfish (Lushchak et al., 2001) and an

estuarine crab (de Oliveira et al., 2005). Several studies of both vertebrates and invertebrates

have noted decreased or unchanged antioxidant expression or activity during hypoxia, consistent

with an overall metabolic depression during hypoxia (Hass and Massaro, 1988; Willmore and

Storey, 1997; Joanisse and Storey, 1998; Larade and Storey, 2002). In the current study, clams









exposed to hypoxia did not show significant changes in antioxidant protein expression,

regardless of the season in which the experiments were performed. Similarly, we did not detect

significant changes in proteins involved in expression levels of proteins involved in protein

rescue and/or degradation in hypoxia-exposed clams, consistent with some vertebrate studies

(Gupta and Knowlton, 2002) but not others (Currie and Boutilier, 2001; Magalhdes et al., 2004,

2005).

We detected a significant decrease in total actin expression in D. variabilis exposed to

hypoxia in the spring but not the fall. A decrease in total actin protein expression is consistent

with previous studies of bovine brain endothelial cells exposed to hypoxia (Brown and Davis,

2005) and human cortical neurons exposed to a free radical generating neurotoxin (Allani et al.,

2004). Interestingly, although D. variabilis exposed to hypoxia did not show evidence of

oxidative damage, which could have included alterations in antioxidant or OGG1-m expression

or increases in HNE, hypoxia nonetheless constituted a lethal stress in both fall and spring

survival experiments. This is consistent with a previous study of hypoxia exposure in the

congener D. serra (Laudien et al., 2002). Therefore, D. variabilis are vulnerable to moderately

hypoxic conditions, which they typically do not encounter in their habitat. The absence of a

stress protein response consistent with elevated free radical production suggests that oxidative

stress does not play a large role in the mechanism of hypoxic death, or that these clams were so

severely stressed that they were unable to appropriately respond to hypoxia-induced oxidative

stress (Werner and Hinton, 1999).

Hyperoxia. Hyperoxic conditions are present in a variety of marine habitats, including

rocky intertidal pools with photosynthetically active algae (Truchot and Duhamel-Jouve, 1980),

boundary layers of intertidal seaweed (Irwin and Davenport, 2002) and brown algae (Pohn et al.,









2001), in the cold seawater of polar regions (Viarengo et al., 1995; Abele and Puntarulo, 2004),

and within some algal-cnidarian symbioses (Dykens et al., 1992; Richier et al., 2003, 2005).

Elevated cellular 02 levels increase mitochondrial free radical production (Boveris and Chance,

1973; Akbar et al., 2004), oxidative damage (Dennog et al., 1999), and antioxidant responses in

tissues of both vertebrates (O'Donovan et al., 2002; Cho et al., 2005) and invertebrates (Viarengo

et al., 1995; Abele and Puntarulo, 2004). Nonetheless, hyperoxic exposure is not linked to

changes in SOD activity in the polychaete Heteromastusfiliformis (Abele et al., 1998b) or in

humans exposed to hyperbaric oxygen treatment (Dennog et al., 1999).

In the current study, we found a significant increase in MnSOD expression in D. variabilis

that were exposed to hyperoxia in the fall experiment. We did not detect significant changes in

the expression levels of the other two antioxidants, Cu/Zn SOD (Freiberger et al., 2004) and GPx

(Allen and Balin, 2003), or the marker of lipid peroxidation (HNE). However, we did detect

significant increases in expression of the mitochondrial DNA repair enzyme OGG1-m in

hyperoxia-exposed clams from the spring experiment. One possible explanation for this

discrepancy is that MnSOD did not blunt the increased mitochondrial free radical production in

clams exposed to hyperoxia in the spring, thereby resulting in DNA damage and a consequent

stimulation of increased OGGI-m expression.

Among the proteins involved in protein rescue and/or degradation, the only significant

change in expression was an increase in Hsp70 expression in D. variabilis exposed to hyperoxia

in the fall. A link between Hsp70 expression and hyperoxia is well supported by studies utilizing

a number of different organisms and cell types (Wong et al., 1998; Dennog et al., 1999; Akbar et

al., 2004; Shyu et al., 2004; Cho et al., 2005) and may have contributed to the clams' increased

survival in response to hyperoxia. We did not detect an effect of hyperoxia on total actin









expression. This contrasts with studies of cultured mammalian cells, which show that hyperoxia

causes decreased actin gene expression (Cho et al., 2005) and that toxin-induced free radical

production (although not necessarily hyperoxia) causes decreased actin protein expression but

not decreased gene expression (Allani et al., 2004). While the stress protein results in this study

present some evidence that exposure to hyperoxia caused a stress response indicative of

increased free radical production, the survival experiments showed that exposure to the

hyperoxic condition was a sublethal stressor.

Hydrogen sulfide. Animals in a variety of marine habitats, including mudflats, mangrove

swamps, deep-sea hydrothermal vents, hydrocarbon seeps and anoxic basins, are periodically or

continuously exposed to sulfide at levels up to 12 mmol L-1 (Fenchel and Riedl, 1970; Somero et

al., 1989; Bagarinao, 1992). Hydrogen sulfide is a highly reactive toxin that diffuses freely

across respiratory surfaces and therefore cannot be excluded from tissues (Denis and Reed, 1927;

Julian and Arp, 1992). Hydrogen sulfide has several mechanisms of toxicity, including the

reversible inhibition of cytochrome c oxidase, the final enzyme of the mitochondrial electron

transport chain (Lovatt Evans, 1967; Nicholls, 1975), reduction in hemoglobin oxygen affinity

(Carrico et al., 1978) and inhibition of approximately 20 enzymes (Bagarinao, 1992). Hydrogen

sulfide oxidizes spontaneously in the presence of divalent metals (both dissolved and in

metalloenzymes), generating oxygen-centered (likely superoxide) and sulfur-centered radicals in

aqueous solutions (Chen and Morris, 1972; Tapley et al., 1999) and in animal tissues (Tapley,

1993; Abele-Oeschger and Oeschger, 1995; Eghbal et al., 2004; Julian et al., 2005). Organisms

inhabiting sulfide-rich environments employ a variety of strategies to detoxify sulfide (Lovatt

Evans, 1967; Vismann, 1991; Grieshaber and Volkel, 1998). Marine invertebrates that are not

found in sulfide-rich habitats and that do not employ sulfide detoxification strategies typically









show increased mortality upon exposure to sulfide, such as can occur in upwelling events

(Grieshaber and Volkel, 1998). We found that 0.1 mmol L-1 sulfide was a lethal stressor for D.

variabilis in both fall and spring survival experiments. These results are consistent with a

previous study of sulfide tolerance in juvenile Donax serra, which documented a LT50 of 80 hour

with exposure to 0.1 mmol L-1 under hypoxic conditions (Laudien et al., 2002).

Of the three stressors we tested, we found the greatest evidence for a stress protein

response, consistent with a cellular response to oxidative stress in D. variabilis exposed to

sulfide, and the response was strongest in the spring experiment. Specifically, clams exposed to

sulfide had elevated expression ofMnSOD (fall), Cu/Zn SOD (spring), GPx (spring), and

OGG1-m (spring). Two of the proteins involved in protein rescue and/or degradation, ubiquitin

(fall) and sHsp (spring), also increased in clams exposed to sulfide. In contrast, the lipid

peroxidation marker HNE and total actin expression levels were significantly decreased in

sulfide-exposed clams in the spring experiment. These results suggest that cellular response ofD.

variabilis to sulfide is consistent with a response to oxidative stress and that the mortality

detected in the sulfide exposure treatment in both fall and spring survival experiments could be

linked to oxidative stress in D. variabilis, which do not normally encounter sulfide.

Several studies have specifically addressed the link between oxidative damage and sulfide

exposure at the organismal level. For example, antioxidant enzyme activities were proportional

to the sulfide tolerances ofthiobiotic meiofauna (Morrill et al., 1988). Similarly, MnSOD

activity increased in response to sulfide exposure in the chemoautotrophic symbiotic bivalve

Solemya velum but not in the related nonsymbiotic Yoldia limatula (Tapley, 1993). In contrast, a

relationship between sulfide tolerance and antioxidant activity was not found in a survey of

sulfide-tolerant polychaetes and bivalves (Abele-Oeschger, 1996). A relationship between









sulfide and free radical production has also been investigated at the cellular level; in isolated

erythrocytes from the marine polychaete Glycera dibranchiata, 1 hour sulfide exposure causes

mitochondrial depolarization, increased superoxide production and increased cellular oxidative

stress (Julian et al. 2005). Recently, Eghbal et al (2004) showed that free radical production in rat

hepatocytes was two-to-three times faster when the cells were exposed to 0.5 mmol L-1 sulfide

than when they were exposed to cyanide or control conditions, and that the addition of ROS

scavengers decreased cell death by up to 40% in hepatocytes exposed to 0.5 mmol L-1 sulfide for

3 hours. These studies, in conjunction with the results of the current study, support the theory

that increased oxidative stress is a mechanism by which sulfide exposure causes toxicity in

marine animals (Morrill et al., 1988; Tapley, 1993; Abele-Oeschger et al., 1994, 1996; Julian et

al., 2005).

Sources of Variance

A number of factors, both in the collection and treatment of the clams as well as in the

tissue processing procedure, could have contributed to the variance detected within treatment

groups. We employed several methods to minimize the influence of these factors. These

included: 1) To control for effects of daily cycles in stress protein production (Podrabsky and

Somero, 2004), we collected the clams within 30 minutes of high tide. 2) We did not determine

the ages of the individuals, which in mammals is closely linked to both endogenous free radical

production (Barja, 2002) and ability to synthesize functional stress proteins (Szczesny et al.,

2003), but we did control for shell length. Populations ofD. variabilis from sites on the eastern

Florida coast reach maturity in spring and fall and most individuals live for one year (Mikkelsen,

1985). Given these patterns in abundance and size-frequency, it is likely that the clams sampled

in the current study were adult and of similar age. 3) We selected D. variabilis as a study species

because it inhabits a habitat that lacks extremes of dissolved 02 or sulfide, which allowed us









minimize the potential complications of preconditioning from exposure to environmental

stressors (Kultz, 2005). 4) An additional method to limit preconditioning effects would have

been to acclimate the clams in the laboratory prior to the experiment. However, because we

found slightly elevated Hsp70 expression levels in clams maintained in normoxic conditions for

several days, laboratory acclimation would likely have introduced additional variables. Given the

short survival times demonstrated in the survival experiments and the potential confounding

effect of starvation (Morales et al., 2004), we exposed the clams to the stressors for only 24

hours. 5) To minimize the effects of daily variation in tissue processing methodology, we

homogenized and suspended all samples on the same day and with the same batch of buffer

solution. 6) Stress protein expression levels were determined for all samples at the same time and

on the same piece of membrane to minimize inter-membrane staining differences.

We found that variance in the stress treatment groups was elevated in comparison to

variance in the normoxia samples in all stress proteins that had significant changes (excluding

actin). This relationship between stressful treatments and elevated variance has been documented

in ecotoxicological studies (Orlando and Guillette, 2001) and may even be useful as a biomarker

(Callaghan and Holloway, 1999). For example, Callaghan and Holloway (1999) found that when

weevils (Sitophilus oryzae) were transferred to a toxic food source, the mean activity levels of

glutathione-S-transferase and two naphthyl acetate esterases did not change significantly, but the

variances about the means increased up to 5 fold. The elevated variance that we detected in

clams exposed to stressors is consistent with the concept that, at the population level, variance in

a physiological metric is indicative of stress.

Physiological Responses to Stress Vary by Season

The importance of assessing seasonal changes in the physiological stress response,

particularly stress protein expression and activity levels, is well established. A number of factors,









including availability of nutrients, temperature variation, reproductive status and growth cycle,

and seasonal patterns in environmental stressors, shape seasonal changes in bivalve stress

physiology (Sheehan and Power, 1999). For example, warm summer conditions were linked to

higher catalase and glutathione-S-transferase activities and higher condition index in Mytilus

galloprovincialis (Romeo et al., 2003a), higher hsp70 levels in M edulis (Chapple et al., 1998),

and higher ubiquitin conjugate and hsp70 levels in M. trossulus (Hofmann and Somero, 1995).

Seasonal differences in catalase, metallothionein, and gluathione-S-transferase levels in M edulis

and Macoma balthica corresponded with patterns of temperature and food availability as well as

reproductive phase (Leinio and Lehtonen, 2005). Helix aspersa snails estivating during the

summer have a greater stress protein response, lower lipid peroxidation and lower protein

carbonyl levels than those estivating during the winter (Ramos-Vasconcelos et al., 2005).

Similarly, we detected greater changes in expression levels of the antioxidant proteins and some

of the proteins involved in protein rescue and/or degradation in clams from the spring experiment

in comparison to clams from the fall experiment. This increased expression of stress proteins in

clams collected in the spring may have contributed to their enhanced survival (Chapple et al.,

1998).

Conclusions

Marine invertebrates from sulfidic environments are likely to have physiological or

biochemical adaptations to limit their susceptibility to these abiotic stressors (Grieshaber et al.,

1994; Grieshaber and Volkel, 1998). Such adaptations would reduce the need for upregulated

expression of stress proteins during experimental exposure to sulfide, thereby reducing our

ability to detect whether this stressor has the capacity to cause oxidative damage. The surf clam

D. variabilis does not experience hypoxia, hyperoxia or sulfide in its habitat and therefore is

likely to be more sensitive to these stressors. Over the course of the survival experiments,









particularly in the fall experiment, both sulfide exposure and hypoxia exposure were lethal,

whereas hyperoxia and normoxia were not. Therefore, increased expression of key antioxidants

and repair enzymes following 24 hour exposure to sulfide, particularly during the spring

experiment, but not hypoxia, suggests that the expression changes were a specific response. It

remains to be determined how this protein expression pattern differs at shorter and longer time

points, although substantially longer time points in hypoxia and sulfide treatments resulted in

mortality. Consequently, these data are consistent with sulfide, and to a lesser extent hyperoxia,

causing oxidative stress. It remains to be determined whether animals evolutionarily adapted to

sulfide exposure have increased capacity for stress protein expression to limit oxidative damage,

as has been shown for Hsp70 expression in intertidal mussels exposed to thermal stress

(Hofmann and Somero, 1995; Hofmann, 1999).









Table 2-1. Summary of statistical results from comparisons between samples from Donax variabilis exposed to
normoxia treatment and samples from animals exposed to hypoxia, hyperoxia, and sulfide. Data were analyzed by
Kruskal-Wallis ANOVA followed by pairwise Conover-Inman post hoc comparison tests. Significant values (p <
0.05) are highlighted with bold text. T values are for data pooled across treatment within each season.

p value from post-hoc pairwise comparison


Protein type Protein Fall Spring Fall Spring Fall Spring Fall Spring
MnSOD 8.112 0.948 0.181 0.389 0.033 0.593 0.003 0.639
Antioxidant Cu/Zn SOD 1.743 4.346 0.413 0.26 0.629 0.326 0.253 0.047
GPx 3.331 4.787 0.178 0.316 0.713 0.173 0.158 0.038
Lipid peroxidation HNE 2.978 6.794 0.391 0.132 0.145 0.736 0.828 0.038
Oxidative repair OGG1-m 1.261 11.206 0.44 0.089 0.421 0.0007 0.865 0.0002
Ubiquitin 6.728 2.713 0.0621 0.359 0.436 0.359 0.014 0.134
sHsp 2.027 5.581 0.337 0.297 0.321 0.119 0.235 0.023
Pdegraation Hsp70 7.434 2.051 0.468 0.834 0.027 0.626 0.648 0.222
degradation
Hsp60 0.618 0.132 0.691 0.794 0.51 >0.999 0.597 >0.999
Grp75 0.684 0.088 0.509 0.896 0.739 >0.999 0.552 0.896
Cytoskeletal protein Actin 0.485 9.529 0.44 0.048 0.153 0.668 0.401 0.004


T value (df=3)


Hypoxia


Hyperoxia


Sulfide






















normoxia
hyperoxia
hypoxia
. . . . . . . .


Figure 2-1. Diagram of flow-through system. Filtered, chloramphenicol-treated seawater was
pumped from a 20 L reservoir to a 200 ml upper reservoir at 10 ml min-'. Water from
this reservoir then drained into each of 4 vertical gas equilibration chambers. These
chambers were constructed of 18 mm inner diameter (i.d.) clear, cast acrylic tube,
each 25 cm in length. A sintered glass aerator in each chamber was used to bubble
either air (normoxia), N2 (hypoxia), a mixture of 02 and air (hyperoxia) or a mixture
of air and hydrogen sulfide gas (from a compressed tank of 2% H2S, balance N2). Gas
mixtures were controlled with three digital mass flow controllers, indicated in the
figure as MFC1, MFC2 (both being FMA-5400 single-channel controllers, Omega
Engineering, Inc., Stamford, CT, USA) and MFC3 (three-channel controller from
Cameron Instrument Co., Port Aransas, TX, USA). Controllers that handled hydrogen
sulfide gas were customized with corrosion-resistant fittings and O-rings. Gas-
equilibrated water from each chamber was continuously pulled into an animal
chamber by a 4-channel peristaltic pump (Masterflex cartridge system, Cole Parmer
Instrument Co., Vernon Hills, IL, USA) at 2 ml min' per chamber. These chambers
were constructed of 18 mm i.d., 15 cm long clear, cast acrylic tube with one-hole
rubber stoppers at end, through which the seawater flowed through 1/8" i.d., 1/16"
wall Tygon tubing. All tubing connections were via nylon Luer fittings (Cole Parmer
Instrument Co.). Effluent water from the peristaltic pump was monitored periodically
for PO2 and pH. The gas equilibration chambers and animal chambers were housed in
a polycarbonate water bath maintained at 24 C. The entire system (except for
compressed air, N2 and 02 supplies) was housed in a fume hood to minimize the
hazards associated with handling H2S gas.










Cu/Zn


SOD SOD
_r


GPx OGG

6-M


1


Hsp Hsp Grp
70 60 75 Actin


80-

39-

31-


17-


a


- - m-


-a-


7- -


Figure 2-2. Antibody specificity tests. Two random samples ofDonax variabilis were pooled,
subjected to SDS-PAGE, western blotted, and assayed with the antibodies to the
following proteins: manganese superoxide dismutase (MnSOD), copper/zinc
superoxide dismutase (Cu/Zn SOD), glutathione peroxidase (GPx), OGGl-mito
(OGG1), heat shock protein 70 (Hsp70), heat shock protein 60 (Hsp60),
mitochondrial heat shock protein 70 (Grp75), and actin. The positions of known
molecular weights standards are indicated by bars to the left of each individual band
image and the molecular weight masses in kilodaltons (kD) are listed at the far left of
the figure.


MW Mn


(kD)
203-
126-


K-


m













B. Spring
normox
S.. '' hyperox
-L ..h..yp


:hypox

sulfide:


0 1 2 3 4 0 1 2 3 4 5 6 7


exposure duration (d)


exposure duration (d)


Figure 2-3. Fraction of surviving Donax variabilis clams in survival experiments in fall and
spring. The fall experiment was conducted for 4 d and the spring experiment for 7 d.
The treatments were: normoxia (solid line; 21.6 + 1.9 kPa Po2), hypoxia (dotted line;
12.3 + 1.4 kPa Po2), hyperoxia (dashed line; 36.6 + 3.0 kPa Po2), and normoxic
sulfide (alternating dashes and dots; 98 2.9 kmol L-1 total sulfide, 24.0 1.8 kPa
PO2). Eight clams were exposed to each condition in each season.


A. Fall


normox
hyperox




ihypox

|sulfide:













250
S200
150
100
50
0
0 1 3 5

exposure duration (d)

Figure 2-4. Expression levels of Hsp70 in Donax variabilis exposed to normoxia for 0, 1, 3, and
5 days. Clams used in the 0 time point were frozen in liquid N2 immediately after
collection at the beach. Data are presented as a scatterplot with asymmetrical error
bars denoting minimum and maximum values and central dot signifying the mean of
five clams per day. Data are given as relative units per nanogram of total soluble
protein (RU ng TSP-1). Abbreviations: day, d. Data were analyzed by Kruskal-
Wallis ANOVA but were not statistically significant.









Figure 2-5. Expression levels of three antioxidant proteins, a lipid peroxidation marker, and an
oxidative repair enzyme in Donax variabilis exposed to normoxia (normox), hypoxia
(hypox), hyperoxia (hyperox), and sulfide. Data are presented as scatterplots with
asymmetrical error bars denoting minimum and maximum values and central dot
signifying the mean of four clams per treatment in fall (left) and spring (right)
experiments. Data for manganese superoxide dismutase (MnSOD), copper-zinc
superoxide dismutase (Cu/Zn SOD), glutathione peroxidase (GPx), and (4-hydroxy-
2E-nonenol-adducted protein) HNE are given as relative units per nanogram of total
soluble protein (RU ng TSP-1), and data for OGGl-mitochondria (OGG1-m) are
given as fmoles mg TSP-1. Data were analyzed by Kruskal-Wallis ANOVA and
Conover-Inman post hoc pairwise comparisons. Similar letters denote statistically
indistinguishable samples in data sets with significant ANOVAs. Data sets with no
significant differences by ANOVA contain no letters adjacent to the symbols.











Fall

A. MnSOD






ja ab jbc +c

normox hypox hyperox sulfide

C. Cu/Zn SOD









normox hypox hyperox sulfide

E. GPx









normox hypox hyperox sulfide

G. HNE









normox hypox hyperox sulfide

I. OGG1-m









normox hypox hyperox sulfide


Spring

B. MnSOD









normox hypox hyperox sulfide

D. Cu/Zn SOD






a a a

normox hypox hyperox sulfide

F. GPx





a a a b


normox hypox hyperox sulfide

H. HNE


a a


ab bc


normox hypox hyperox sulfide

J. OGG1-m





S b hb
no ox hypox hypox sulfide

normox hypox hyperox sulfide









Figure 2-6. Expression levels of five proteins involved in protein rescue and/or degradation in
Donax variabilis exposed to normoxia (normox), hypoxia (hypox), hyperoxia
(hyperox), and sulfide. Data are presented as scatterplots with asymmetrical error bars
denoting minimum and maximum values and central dot signifying the mean of four
clams per treatment in fall (left) and spring (right) experiments. Data for ubiquitin,
heat shock protein 70 (Hsp70), heat shock protein 60 (Hsp60), and mitochondrial heat
shock protein 70 (GRP75) are given as relative units per nanogram of total soluble
protein (RU ng TSP-1). Data for small heat shock protein (sHsp) are given as fmol
mg TSP-1. Data were analyzed by Kruskal-Wallis ANOVA and Conover-Inmanpost
hoc pairwise comparisons. Similar letters denote statistically indistinguishable
samples in data sets with significant ANOVAs. Data sets with no significant
differences by ANOVA contain no letters adjacent to the symbols.











Fall
4
A. Ubiquitin





Saa a

0
normox hypox hyperox sulfide


C. sHSP








normox hypox hyperox sulfide

E. Hsp70

a b

+a aa


normox hypox hyperox sulfide

G. Hsp60








normox hypox hyperox sulfide

I. Grp75








normox hypox hyperox sulfide


Spring

B.Ubiquitin








normox hypox hyperox sulfide


U.1


0.10


S0.05


0.00

4


normox hypox hyperox sulfide

F. Hsp70








normox hypox hyperox sulfide

H. Hsp60








normox hypox hyperox sulfide

J. Grp75








normox hypox hyperox sulfide










Spring


A. Actin
3 I

o 2



0
normox hypox hyperox sulfide


B. Actin


1b Ja b


normox hypox hyperox sulfide


Figure 2-7. Expression levels of total actin in Donax variabilis exposed to normoxia (normox),
hypoxia (hypox), hyperoxia (hyperox), and sulfide. Data are presented as scatterplots
with asymmetrical error bars denoting minimum and maximum values and central dot
signifying the mean of four clams per treatment in fall (left) and spring (right)
experiments. Data are given as relative units per nanogram of total soluble protein
(RU ng TSP-1). Data were analyzed by Kruskal-Wallis ANOVA and Conover-Inman
post hoc pairwise comparisons. Similar letters denote statistically indistinguishable
samples in data sets with significant ANOVAs. Data sets with no significant
differences by ANOVA contain no letters adjacent to the symbols.


Fall









CHAPTER 3
PHYSIOLOGICAL RESPONSES OF Mercenaria mercenaria TO SINGLE AND MULTIPLE
ABIOTIC FACTORS


Introduction

The examination of how physical conditions in the environment interact with physiology

to influence the distribution of organisms is a long-standing goal of ecologists, particularly of

physiological ecologists (Brown et al., 1996; Spicer and Gaston, 1999). Numerous studies have

attempted to define a species' niche, the set of environmental variables that limits reproduction

and survival and therefore impacts distribution and abundance (Brown et al., 1996).

Traditionally, these studies involved either in situ manipulations combining biotic interactions

with environmental variables (exemplified by Connell, 1961) or laboratory studies of

physiological responses to abiotic factors. Unlike in situ manipulations, which allow the

experimental subjects to be impacted by all aspects of a complex environment, laboratory studies

involve acclimation to one, or at most two, select environmental variables. Although the

limitations of extrapolating laboratory studies of single variables to population-level processes

have long been identified (e.g., Hall, 1964), recent reviews continue to emphasize the importance

of investigating multiple abiotic factors in the laboratory (e.g., Brown et al., 1996).

The interplay between tolerances of abiotic factors with biotic interactions and species

distributions have been rigorously studied in the rocky intertidal habitat (for review, Menge and

Olson, 1990; Benson, 2002; Tomanek and Helmuth, 2002). This habitat, with its steep gradients

of multiple abiotic factors, diel and seasonal patterns, accessibility, and well-characterized

communities, serves as a model system for experimental population and community ecology.

Additionally, since most of the study organisms are sessile as adults, distribution patterns are not

confounded by behavioral responses to stressors, such as use of refuges. This habitat has









facilitated many recent advances in physiological ecology, such as the linking of population

distribution with cellular-level responses including heat shock protein production (Hofmann et

al., 2002). The approaches taken and conclusions reached in studies of rocky intertidal

invertebrates can be applied to organisms inhabiting other variable aquatic habitats.

Estuaries share many similarities with the rocky intertidal. Estuaries have seasonal and

daily fluctuations in a variety of abiotic factors, including temperature, salinity, dissolved 02

levels, and pH (Hubertz and Cahoon, 1999; Beck and Bruland, 2000). The organisms inhabiting

estuaries, like those of the rocky intertidal, must be broadly tolerant of environmental stressors

(Fisher, 1977; Parsons, 1994). Although the physiological strategies employed by estuarine

organisms are not as well understood as those of rocky intertidal invertebrates, several

similarities are apparent. For example, alterations in heat shock protein expression and function

in estuarine organisms have been linked to environmentally-relevant variations in temperature

(Zippay et al., 2004) and salinity (Blank et al., 2006). Therefore, estuarine organisms, like

intertidal invertebrates, could be developed as models for studies of how physiological responses

to multiple environmental variables interact to affect distribution and abundance.

One such invertebrate species that is tolerant of multiple environmental stressors, well-

studied, broadly distributed in coastal habitats, and accessible year-round is the northern quahog

or hard clam, Mercenaria mercenaria (Kraeuter and Castagna, 2001). These clams live at

intertidal and subtidal depths in bays and estuaries along the Atlantic coast of North America,

from the Gulf of St. Lawrence to the southern Florida coastline (Harte, 2001). Mercenaria

mercenaria is a generalist in its tolerance of temperature extremes, low dissolved 02 levels

(hypoxia), and salinity extremes (Grizzle et al., 2001), as would be expected given its

distribution (Lynch and Gabriel, 1987; Gilchrist, 1995). It is therefore an appropriate model









organism for an investigation of how abiotic factors, singly and in combination, affect the

physiology of aquatic invertebrates (Grizzle et al., 2001).

The current study examined how hypoxia, high temperature, and hyposalinity (reduced

salinity), singly and in combination, affect the physiological responses ofM mercenaria. The

physiological responses were divided into two categories, traditional functional markers and

cellular-level indicators. Traditional functional markers measure long-term growth and fecundity

responses to sublethal environmental stressors (Widdows, 1985). These assays measure general,

whole-organismal responses, integrating the effects of the environmental stressor over several

hierarchical levels of biological organization (Stebbing, 1985). Traditional assays measure

behavioral responses such as valve-closure in bivalves (Heinonen et al., 1997), burial ability

(Savage, 1976), and metabolic responses such as glycogen content (Hamza-Chaffai et al., 2003),

and condition index (Romeo et al., 2003a). In recent years, cellular-level markers, such as stress

protein expression (Sanders, 1993; Feder and Hofmann, 1999), oxidative damage markers, and

RNA:DNA ratio (Elser et al., 2000), have become increasingly powerful and popular tools in

part because they test for evidence of stress at the level of organization primarily affected; the

molecular level (Stebbing, 1985; Bierkens, 2000). However, few studies have attempted to

investigate both cellular-level indicators and traditional functional techniques (Brown et al.,

1995; Hamza-Chaffai et al., 2003; Romeo et al., 2003b). This study attempted to delineate the

links between cellular-level and whole-organismal responses to individual and multiple abiotic

stressors and detail how an integrated stress profile changes over time.









Materials and Methods


Laboratory Exposures

Hard clams (average 12 mm shell length) were obtained from Southern Cross Sea Farms,

Inc. (12170 SR 24 Cedar Key, FL) and maintained in 2.5 gallon aquaria with half water changes

daily. The seawater was obtained from the Whitney Marine Lab (Marineland, FL) by the

Department of Zoology and diluted to 26 ppt salinity (standard concentration for hypoxia and

temperature experiments) or the appropriate salinity (dual stressor experiments) with ultrapure

water. The water was pretreated with chloramphenicol (2 mg L1) to prevent the growth of

bacteria. Clams were fed a mixed shellfish diet (1800 formula, Reed Mariculture) at 2-5% of

estimated dry weight per day.

The single stressor experiments were designed to examine responses to hypoxia and to

elevated temperature. In the hypoxia experiments the clams were exposed to three levels of

dissolved oxygen: 1) air saturated (normoxia, approximately 0.25 mmol L-1), 2) mild hypoxia

(approx. 0.16 mmol L-1), and 3) moderate hypoxia (approx. 0.07 mmol L-1). Each aquarium was

bubbled with air or air/N2 mixes (one aquarium per treatment). Experimental conditions were

monitored daily with a fiber optic 02 probe (Ocean Optics, Inc., Dunedin, FL). All exposures

were conducted at room temperature in all seasons (20-22C). Experiments were conducted in

three seasons: fall (August 2004), winter (January 2005), and spring (April 2005). Given the lack

of a measurable effect of the mild and moderate hypoxia treatments in the fall hypoxia

experiment (see results), dissolved oxygen levels were decreased by 50% in both treatments in

the winter and spring experiments in comparison to the fall hypoxia experiment.

In the second set of single-stressor experiments, the clams were exposed to three

temperature treatments: 1) room temperature (approximately 240C), 2) mild temperature

elevation (approx. 280C), and 3) moderate temperature elevation (approx. 330C). The water









temperature was maintained by 15 W or 25W aquarium heaters and was monitored daily. All

aquaria were bubbled with air and the dissolved oxygen content was monitored daily.

Experiments were conducted during three seasons: fall (September 2004), winter (February

2005), and spring (May 2005). Experimental conditions used in the single-stressor experiments

were consistent with environmental values recorded in Florida estuaries (see Discussion for

details).

The dual stressor experiments were designed to examine responses to elevated temperature

at each of three salinities. The clams were exposed to the following treatments: 1) high

temperature/ambient salinity (measurements taken at the hatchery; approximately 330C/24 ppt),

2) high temperature/mild hyposalinity (approx. 330C/15 ppt), 3) high temperature/moderate

hyposalinity (approx. 330C/5ppt), 4) room temperature/ambient salinity (approx. 240C/24ppt), 5)

room temperature/mild hyposalinity (approx. 240C/15 ppt), and 6) room temperature/moderate

hyposalinity (approx. 240C/5 ppt). The water temperature in the high temperature treatment was

maintained by 25W aquarium heaters and was monitored in all treatments daily. All aquaria were

bubbled with air and the dissolved oxygen content was monitored daily. Salinity was measured

daily using a portable refractometer. Experiments were conducted during three seasons: fall

(September 2005), winter (January 2006), and spring (May 2006).

In all experiments, two aquaria (one holding, one depuration) and one water jug (to provide

fresh water of treatment conditions) were maintained at the experimental conditions for each

treatment. Dissolved oxygen content, temperature, and salinity did not differ among the three

water containers for any treatment in any of the nine experiments (data not shown). Daily half

water changes were conducted using water from the jugs, which minimized abrupt changes in

temperature or salinity. In all experiments, levels of ammonia, nitrite, and nitrate were









monitored. The pH of the water was monitored during the first several experiments and averaged

8.17 + 0.11, which is within the pH range associated with optimal growth for this clam

(Calabrese and Davis, 1966). The clams were not provided with burrowing substrate except

during timed burial analysis tests, which is consistent with the conditions in which they were

acclimated at the hatchery for 1-2 weeks prior to each experiment. Due to space limitations in a

shared aquarium room, only one aquarium set was used per treatment. However, the clams could

be considered independent experimental units for the purposes of statistical analyses since we

maintained a large ratio of water volume (6 L) to clam tissue (maximum 15.7 g), daily half water

changes, sufficient feeding, and constant aeration.

Clams were maintained with feeding in experimental aquaria until 24 hours before they

were scheduled to be sacrificed (see schedule below), at which time they were transferred to the

matching depuration aquaria. The experiments lasted 14 days, with clams sampled on days 1, 5,

9, and 14. The schedule of the experiments is outlined below and was identical for all

experiments (actual sample sizes varied with clam availability and effects of the treatments, see

details below in the results section):

Schedule.

* Day 0: 20 clams were preserved for Time 0 measurements, < 20 clams were placed into
depuration aquaria (for 1 day sampling), the remainder of the clams were placed into the
large aquaria and fed daily.

* Day 1: The clams in the three depuration aquaria (< 20 per treatment) were processed.

* Day 4: 20 clams from each large aquarium were transferred to the matching depuration
aquarium.

* Day 5: The clams in the three depuration aquaria were processed.

* Day 8: < 20 clams from each large aquarium were transferred to the matching depuration
aquarium.

* Day 9: The clams in the three depuration aquaria were processed.









* Day 13: < 20 clams from each large aquarium were transferred to the matching depuration
aquarium.

* Day 14: The clams in the three depuration aquaria were processed.

Tissue Processing

Once the burial tests were completed (see below), all clams were removed from the

depuration tanks. The clams were opened by severing their adductor muscles were severed.

Whole clam tissues were blotted and weighed, then flash-frozen in liquid nitrogen and stored at -

80C. All of the biochemical assays were conducted on whole clams and/or whole clam

homogenates from individual clams. When the sample size was sufficiently large, clams used in

the burial tests were not used for glycogen content or stress protein analyses. In several

experiments, all available clams were used for burial analyses so the clams were gently rinsed

before opened and frozen for the biochemical analyses.

Survival Analyses

In some of the experiments, clams in high temperature treatments died (see appropriate

results sections). To maintain consistency, the experiments were continued through fourteen days

with the surviving clams. In experiments with treatment-associated mortality, survival in all

treatments was analyzed using an extension of the nonparametric Gehan's generalized Wilcoxon

test, which assigns a score to each individual's survival time and then calculates and analyzes a

Chi-square value for each treatment group (Statistica version 7.1, Tulsa, OK). Pairwise

comparisons were made, when appropriate, using the nonparametric Gehan's generalized

Wilcoxon test (Statistica).

Analyses:

Ability to bury. Burial rate, which is the proportion of clams that bury themselves in a

given period of time when placed on a substrate, can serve as an index of total clam health









(Byrne and O'Halloran, 2000). Sand was utilized as the substrate, since it is a favorable substrate

type for burial ofM mercenaria (Grizzle et al., 2001). On each sampling day, burial analysis

was conducted on up to thirteen of the clams in each depuration chamber. Small containers with

sand (approximately 2.5 cm deep) were placed into the depuration aquaria and randomly selected

clams were placed flat on top of the sand. In the fall hypoxia experiment (first experiment we

conducted) only, the containers of sand were reused throughout the experiment. In all subsequent

experiments, the containers were filled with new sand on each sampling day. On days 1, 5, 9, and

14, we analyzed burial rate at 5, 10, 20, 30, and 60 min. Burial rate analyses were not conducted

on Time 0 clams. Data collected at the 60 minute time point only were modeled as total number

of clams that buried as a function of day and treatment by a generalized linear model with

Poisson distribution and log link, Type III likelihood test, with the offset set as the total number

of live clams tested on any given day (Statistica). In cases where clam death was high, the model

was run with zeros listed for these treatments in both the number of clams buried and the total

number of clams, since the analysis is weighted by the total number tested and requires a

balanced design across treatment and day.Glycogen content. Glycogen content, which is a direct

measure of a major energy storage reserve, was measured using standard techniques (Byrne and

O'Halloran, 2000). Glycogen analyses were conducted on homogenates of whole clams, with a

sample size of 3-5 clams per treatment per day. Briefly, glycogen was extracted from whole clam

homogenates and hydrolyzed overnight by amyloglucosidase. Glucose monomers were

quantified by a colorimetric assay that utilizes glucose oxidase/peroxidase and the substrate o-

dianosidine. Sample absorbances were read at 450 nm on a Biotek Synergy-HT plate reader

(Biotek, VT). Data from single stressor experiments were analyzed by a general linear model

with treatment nested within experiment duration with Fisher's LSD post hoc comparisons for









those experiments for which there was a balanced number of samples (Statistica). Data from dual

stressor experiments could not be analyzed using a nested design given the mortality in multiple

treatments and therefore were analyzed within each day by one-way ANOVA with Fisher's LSD

post hoc comparisons.

RNA oxidative damage. Amounts of total tissue oxidized RNA bases were determined in

5 clams per treatment (same clams as those used for stress protein assays) from dual stressor

experiments only using a protocol optimized for bivalves (Joyner-Matos et al., 2007). RNA

oxidative damage results from increased production or decreased detoxification of free radicals

(Halliwell and Gutteridge, 1999). RNA guanine base oxidation produces 8-oxo-7,8-

dihydroguanosine (8-oxoGuo). Data from dual stressor experiments could not be analyzed using

a nested design given the mortality in multiple treatments. Data from the 1-day samples are

presented and analyzed by 2-factor ANOVA with Fisher's LSD post hoc comparisons. The 1-day

time point was the only sampling time in all three experiments that had samples available from

every treatment.

Stress protein biomarkers. Five clams from each treatment group in the single stressor

experiments were processed according to EnVirtue Biotechnologies Inc. standard bivalve sample

preparation protocols (Downs et al., 2002b; Joyner-Matos et al., 2006). The whole clams were

individually homogenized in liquid nitrogen. Small amounts (80-100 mg) of homogenized tissue

were suspended in suspension buffer [50 mmol L-1 Tris, 15 mmol L-1 EDTA, 2% sodium

dodecyl sulfate (SDS), 15 mmol L-1 dithiothreitol (DTT), 0.5% dimethyl sulfoxide (DMSO), and

0.01% Halt protease inhibitor cocktail]. After small amounts of tissue were suspended in

suspension buffer, the solutions were vortexed, heated (3 minutes at 850C), vortexed, heated (3

minutes at 850C), kept at room temperature for 10 minutes, and then centrifuged for 10 minutes









at 12,000 rpm (room temperature). Supernatants were aliquotted, frozen in liquid nitrogen, and

stored at -800C. Protein concentrations were determined using a modified Ghosh method (Ghosh

et al., 1988) prior to freezing.

The samples from the single stressor experiments were assayed in cooperation with C.

Downs using high-throughput ELISA analysis. After thawing, samples were diluted so that

application of all samples was 25 nanograms of total soluble protein per well (30 [iL volume) on

384-well microtiter plates. Sample dilutions, sample application blocking buffer application,

primary and secondary antibody application, plate washes, and development solution were all

applied to the microtiter plates by a Beckman-Coulter Biomek 2000 liquid handling system.

Carousels, plate washers, and plate readers were all integrated with the Biomek 2000 to produce

a functional high-throughput system. Standard curves of one sample diluted over an eight-point

curve were dispersed across the plates to accommodate minor artifact formation associated with

microtiter plate ELISA systems (e.g., edge-effects). Eight stress proteins were analyzed in

samples from the single stressor experiments: copper/zinc superoxide dismutase (Cu/ZnSOD),

glutathione peroxidase (GPx), manganese superoxide dismutase (MnSOD), heat shock protein 60

(Hsp60), heat shock protein 70 (Hsp70), small heat shock proteins (sHsp), ubiquitin, and 8-

oxoguanine DNA glycosylase (OGGlm, mitochondrial isoform). Cross-reactivity of the stress

protein antibodies was validated (Figure 3-1) by polyacrylamide gel electrophoresis and western

blotting. Given the large variances in all stress protein data sets, data were analyzed by

nonparametric Kruskal-Wallis ANOVA with post hoc multiple comparisons of mean ranks when

appropriate (Statistica).









Results


Hypoxia Experiments

Experimental conditions. In the fall experiment, the clams were exposed to three levels of

dissolved oxygen: 1) air saturated (normoxia; average 0.247 0.024 mmol L-1), 2) mild hypoxia

(0.191 0.016 mmol LU1), and 3) moderate hypoxia (0.162 0.021 mmol L-1).

Following the hurricanes in the fall of 2004, the availability of grow-out size (average 12

mm) M. mercenaria was severely decreased. For the winter hypoxia experiment, sample size was

decreased to nine clams per treatment per sampling day. Since there were no significant effects

of the hypoxia treatment in the fall hypoxia experiment (see appropriate sections below), the

mean dissolved oxygen levels of the two treatment groups for the remaining experiments were

decreased. The clams in the winter experiment were exposed to three levels of dissolved oxygen:

normoxia (0.247 0.007 mmol L-1), mild hypoxia (0.161 0.011 mmol L-1), and moderate

hypoxia (0.078 0.015 mmol L-1).

In the spring experiment the clams were exposed to three levels of dissolved oxygen: 1)

normoxia (0.247 0.011 mmol L-1), 2) mild hypoxia (0.158 0.016 mmol L-1), and 3) moderate

hypoxia (0.074 0.009 mmol L-1).

In all three experiments levels of ammonia, nitrite, and nitrate were within

acceptable/sublethal levels (data not shown). These experiments were conducted at room

temperature and the temperature was monitored daily (data not shown).

Ability to bury. Burial analysis was conducted on a randomly selected subset (n = 6-10)

of clams in the fall hypoxia experiment (Figure 3-2A). Burial abilities at the final sampling time

(60 minutes) were affected by experiment duration (p < 0.0001), but not by treatment (p =

0.085). At all time points, more control (normoxia; black bars) clams buried than clams from the

mild hypoxia (gray bars) or moderate hypoxia (white bars) treatments, but this difference was









not statistically significant. Surprisingly, burial ability decreased markedly between days 5 and 9,

even in the control clams. This might have indicated that even these clams were experiencing

some stress. However, additional sand was added to the burial rate testing dishes prior to day 9,

and this may have caused or at least contributed to the overall decreased burial on days 9 and 14

because the sand became quite densely packed.

Burial analysis was conducted on all clams (n = 8-9) from each depuration aquarium

during the winter hypoxia experiment (Figure 3-2B). Burial abilities at the final sampling time

(60 minutes) were affected by experiment duration (p = 0.023), but not by treatment (p = 0.123).

As seen in the fall hypoxia experiment, overall burial in the later two sampling days were lower

than in the first two sampling dates.

Burial analysis was conducted on all clams (n = 10-14) from each depuration aquarium

during the spring hypoxia experiment (Figure 3-2C). Burial abilities were affected by both

experiment duration (p < 0.0001) and treatment (p < 0.0001). Across all four sampling days,

clams in the mild hypoxia treatment buried more (p < 0.0001) than clams in the moderate

hypoxia treatment. Since none of the clams in the control treatment buried at the 14-day time

point, the overall burial of control clams was not significantly different (p = 0.0755) from the

burial ability of the clams in the moderate hypoxia treatment or the mild hypoxia treatment.

Glycogen content. The glycogen content of whole clams (n = 3 per treatment, per

sampling day) from the fall experiment were analyzed (Figure 3-3A). There was no overall effect

of treatment on glycogen content (p = 0.211) when data were analyzed by a general linear model

with treatment nested within experiment duration. In this complete model, experiment duration

had a large and significant effect (p < 0.0001), with glycogen content decreasing by fourteen

days, regardless of treatment. There was also a significant effect of the interaction between









experiment duration and treatment on glycogen content (p = 0.0324). Since there were equal

numbers of replicates in all samples, post hoc comparisons were conducted. The only significant

effect of treatment was detected in the 9-day samples, where clams in the moderate hypoxia

treatment had significantly less glycogen than did control clams (p = 0.042).

When the glycogen content of whole clams (n = 3 per treatment, per sampling day) from

the winter hypoxia experiment were analyzed (Figure 3-3B) by a general linear model with

treatment nested within experiment duration, there was a significant effect of both experiment

duration (p < 0.0001) and treatment (p = 0.047), as well as a significant interaction term (p =

0.0024). Overall, glycogen contents decreased over the duration of the experiment. There were

no significant differences among treatments on sampling days 1, 9, and 14. At five days,

however, clams in the moderate hypoxia treatment had significantly less glycogen than clams in

the other two treatments (p < 0.0055).

When the glycogen content of whole clams (n = 3 per treatment, per sampling day) from

the spring hypoxia experiment were analyzed (Figure 3-3C) by a general linear model with

treatment nested within experiment duration, there was no significant effect of experiment

duration (p = 0.082) or treatment (p = 0.847). Unlike previous experiments, glycogen content in

all three treatments tended to increase over time, with no significant differences among

treatments at any of the sampling days.

Stress protein expression. Since stress protein expression levels were determined via

ELISA, only those proteins with little to no nonspecific cross-reactivity were analyzed. The

banding patterns presented in Figure 3-1 are consistent with antibody specificity information

provided by the manufacturer of the antibodies, EnVirtue Biotechnologies, Inc. and are

consistent with other studies utilizing these antibodies with bivalve tissues (Joyner-Matos et al.,









2006, 2007). It is not necessary to test antibody specificity for ubiquitin or small heat shock

protein (Joyner-Matos et al., 2007).

Stress protein expression levels were analyzed in clams (n = 5) from all three treatments on

all four sampling days, as well as in clams collected and frozen on the first day of the experiment

(Od; Figure 3-4, left column). Since there was high variance in all samples for all eight stress

proteins, there were no consistent patterns in the effects of dissolved oxygen levels on stress

protein expression in clams from the fall hypoxia experiment. Although overall significant

differences were detected by Kruskal-Wallis ANOVA in some data sets (e.g., p = 0.0007 for

GPx, Figure 3-4D andp = 0.0165 for Hsp70, Figure 3-4M), there were no biologically relevant

significant differences among treatments in any stress protein data set.

A similar lack of treatment effect was found in the eight stress proteins analyzed in clams

(n = 5) from the winter and spring hypoxia experiments (Figure 3-4, center and right columns,

respectively). In both experiments, there were isolated cases of overall significant ANOVAs for

some stress proteins, but either no statistically significant or no biologically relevant post hoc

comparisons were detected.

Summary. Hypoxia treatments were sublethal in all seasons. In general, clams exposed to

the moderate hypoxia treatment had a lower burial ability than clams in the normoxia or mild

hypoxia treatments, but this difference was only significant in the spring experiment. In both the

fall and winter experiments, glycogen levels in clams in the moderate hypoxia treatment declined

one sampling day before glycogen levels decreased in clams from the other two treatments.

There were no treatment-associated changes in glycogen content in the spring experiment. Both

functional markers, burial ability and glycogen content, decreased significantly over the duration









of the fall and winter experiments. There were no significant, biologically-relevant changes in

stress protein expression in clams from any of the three hypoxia experiments.

Temperature Experiments

Experimental conditions. In the fall temperature experiment, the clams were exposed to

three treatments: room temperature (24.2 + 1.2C), mild temperature elevation (28.3 0.80C),

and moderate temperature elevation (32.7 + 1.2C). In the winter temperature experiment the

clams were exposed to three treatments: room temperature (23.21.4 C), mild temperature

elevation (29.61.2C), and moderate temperature elevation (33.90.7C). In the spring

temperature experiment the clams were exposed to three treatments: room temperature (24.4 +

1.1 C), mild temperature elevation (27.9 1.80C), and moderate temperature elevation (34.0 +

1.1 C). In all three experiments levels of ammonia, nitrite, and nitrate were within

acceptable/sublethal levels (data not shown). Dissolved 02 levels in the three treatments were

monitored daily and averaged 0.235 + 0.013 mmol L-1 (data not shown).

Survival analysis. In the winter temperature experiment, the clams in the moderate

temperature elevation treatment (Figure 3-5A, white squares) experienced mortality starting at

day 2 and were all dead by day 7 The mean survival time of clams in the moderate temperature

elevation treatment was 3.33 1.6 days. There was no mortality in the room temperature (black

squares) or mild temperature elevation treatments (gray squares). Clams in the room temperature

and mild temperature elevation treatments survived for an average of 7.25 4.9 days. Survival

rates among the three treatments were significantly different (Chi2 = 59.588, df = 2,p < 0.0001).

In pairwise comparisons, clams in the moderate temperature elevation treatment had significantly

lower survival than clams in both the ambient temperature (z = 5.808, p < 0.0001) and the mild

temperature elevation treatments (z = 5.808, p < 0.0001).









In the spring temperature experiment, the clams in the moderate temperature elevation

treatment experienced mortality starting at day 8. Mean survival time of clams in the moderate

temperature elevation treatment was 7.31 4.9 days. There was no mortality in the room

temperature or mild temperature elevation treatments. Clams in these two treatments survived

approximately 7.25 4.9 days. The mean survival time for the moderate temperature elevation

treatment is slightly higher than for the other two treatments because a smaller-than-normal

number of clams from this treatment were sacrificed on day 9, resulting in a greater-than-normal

proportion of clams maintained until 14 days. Survival rates among the three treatments were

significantly different (Chi2 = 9.693, df = 2, p = 0.0079). In pairwise comparisons, clams in the

moderate temperature elevation treatment had significantly lower survival time (z = 2.261, p =

0.0238) than clams in the other two treatments.

Ability to bury. Burial analysis was conducted on a randomly selected subset (n = 8-10)

of clams in the fall temperature experiment (Figure 3-6A). Burial abilities at the final sampling

time (60 minutes) were affected by experiment duration (p < 0.00025), but not by treatment (p =

0.993). As seen in the fall hypoxia experiment, overall burial abilities were lower in later days

regardless of treatment.

In the winter temperature experiment burial analysis was conducted on all clams (n = 11-

14) from each depuration aquarium on sampling days 1 and 5, and on clams from the room

temperature (black bars) and mild temperature elevation (gray bars) treatments on days 9 and 14

(Figure 3-6B). Overall burial abilities at the final sampling time (60 minutes) were affected by

experiment duration (p = 0.0142) and by treatment (p = 0.022). Since treatment comparisons can

only be made at time points with clams present in all three treatments (1 day and 5 day),

significant differences between treatments reflect only the first two sampling days. On these









early sampling days, clams in the mild temperature elevation treatment buried more (p = 0.0058)

than clams in the moderate temperature elevation treatment (white bars). In contrast, clams in the

room temperature treatment did not have elevated burial (p = 0.287) in comparison to clams in

the moderate temperature elevation treatment. On Days 9 and 14, overall burial abilities of clams

in both the room temperature and mild temperature elevation treatment increased, with no

differences detected between treatments.

In the spring temperature experiment, burial analysis was conducted on randomly selected

clams (n = 4-10) from each treatment on all sampling days (Figure 3-6C). Burial ability was

significantly affected by treatment (p = 0.034) but not by experiment duration (p = 0.3386). In

contrast to previous experiments, overall burial ability did not decrease in later time points.

Clams in the room temperature treatment buried more than clams in the moderate temperature

elevation treatment on days 5, 9, and 14 (p = 0.013). Burial ability of clams in the mild

temperature elevation treatment were similar (p = 0.069).

Glycogen content. The glycogen content of whole clams (n = 1-3 per treatment, per

sampling day) from the fall temperature experiment were analyzed (Figure 3-7A) by a general

linear model with treatment nested within experiment duration. We were unable to assess

glycogen content in clams from the room temperature (black bars) and moderate temperature

elevation (white bars) treatments on the first sampling date due to loss of the samples during

sample processing. The statistical model, therefore, includes the data only from days 5, 9, and

14. There was a significant effect of experiment duration (p = 0.00029), with glycogen content

decreasing with time in clams from all three treatments. There were no significant differences

among treatments at any sampling day (p = 0.968).









The glycogen content of whole clams (n = 3 per treatment, per sampling day) from all

treatments with surviving clams from the winter temperature experiment were analyzed (Figure

3-7B). Since mortality occurred in the moderate temperature elevation treatment (indicated by

the number symbol), this data set is unbalanced and could not be analyzed by a nested ANOVA.

Individual one-way ANOVAs conducted on the 1-day and 5-day data sets did not show a

significant effect of the elevated temperature treatment (p > 0.05).

When the glycogen content of clams (n = 3) from each treatment and sampling day of the

spring temperature experiment (Figure 3-7C) were analyzed by a general linear model with

treatment nested within experiment duration, there was a significant effect of experiment

duration (p = 0.0039), but not treatment (p = 0.409). The significant effect of experiment

duration likely was influenced by increases in glycogen content of clams from the room

temperature and mild temperature elevation (gray bars) treatments at sampling day 9 in

comparison to other days. There were no significant differences among treatments at any

sampling day.

Stress protein expression. Stress protein expression levels were analyzed in clams (n = 5)

from all three treatments on all sampling days of the fall temperature experiment, as well as in

clams collected and frozen on the first day of the experiment (Od; Figure 3-8, left column). Since

there was either unequal or high variance in all samples, there were no consistent patterns in the

effects of elevated temperature on stress protein expression. Although overall significant

differences were detected by Kruskal-Wallis ANOVA in some data sets (e.g., p = 0.0032 for

MnSOD, Figure 3-8G andp = 0.0041 for sHsp, Figure 3-8P), there were no biologically relevant

significant differences among treatments in any stress protein data set.









A similar lack of treatment effect was found in the eight stress proteins analyzed in clams

(n = 5) from the winter and spring temperature experiments (Figure 3-8, center and right

columns, respectively). In both experiments, there were isolated cases of overall significant

ANOVAs for some stress proteins, but either no statistically significant or no biologically

relevant post hoc comparisons were detected.

Summary. The mild temperature elevation treatment was sublethal in all three

experiments, but the moderate temperature elevation treatment caused significant mortality in the

winter and spring experiments. In general, clams in the moderate temperature elevation treatment

had lower burial ability than clams in the other two treatments, although this difference was not

always statistically significant. In contrast, there were no treatment-associated changes in

glycogen content. Both burial ability and glycogen content tended to decrease with experiment

duration. There were no significant, biologically-relevant changes in stress protein expression in

clams from any of the three temperature experiments.

Dual Stressor Experiments

Experimental conditions. In the fall dual stressor experiment, the clams were exposed to

three levels of hyposalinity at each of two temperatures: 1) high temperature/ambient salinity

(37.1 2.60C, 24.9 0.9 ppt), 2) high temperature/mild hyposalinity (35.6 0.60C, 15.9 0.9

ppt), 3) high temperature/moderate hyposalinity (35.0 1.00C, 5.3 0.6 ppt), 4) room

temperature/ambient salinity (26.1 + 1.20C, 22.5 1.5 ppt), 5) room temperature/mild

hyposalinity (25.9 0.70C, 15.3 + 1.2 ppt), and 6) room temperature/moderate hyposalinity (23.7

0.60C, 4.5 0.7 ppt). Ambient salinity was defined as the salinity at the hatchery where the

clams had been maintained. Daily dissolved 02 levels in the six treatments averaged 0.242 +

0.016 mmol L-1 (data not shown). Levels of ammonia, nitrite, and nitrate were within

acceptable/sublethal levels [data not shown; Epifanio and Srna, 1975].









In the winter dual stressor experiment, the clams were exposed to the following six

treatments: 1) high temperature/ambient salinity (34.5 0.60C, 24.1 2.1 ppt), 2) high

temperature/mild hyposalinity (33.8 0.80C, 15.4 2.5 ppt), 3) high temperature/moderate

hyposalinity (33.0 1.60C, 5.8 2.2 ppt), 4) room temperature/ambient salinity (22.5 1.30C,

22.6 1.5 ppt), 5) room temperature/mild hyposalinity (22.3 1.10C, 14.2 1.5 ppt), and 6)

room temperature/moderate hyposalinity (21.7 1.20C, 5.2 0.5 ppt).

In the spring dual stressor experiment, the clams were exposed to the following six

treatments: 1) high temperature/ambient salinity (36.6 1.50C, 25.9 + 1.1 ppt), 2) high

temperature/mild hyposalinity (34.1 0.30C, 15.1 0.6 ppt), 3) high temperature/moderate

hyposalinity (33.8 0.80C, 5.0 + 0.1 ppt), 4) room temperature/ambient salinity (25.0 + 0.50C,

25.6 0.9 ppt), 5) room temperature/mild hyposalinity (24.8 0.60C, 15.0 + 0.4 ppt), and 6)

room temperature/moderate hyposalinity (22.3 0.50C, 5.2 0.6 ppt). The high

temperature/ambient salinity treatment was terminated after 24 hours due to a heater

malfunction.

Survival analysis. In the fall dual stressor experiment (Figure 3-9A), clams in the high

temperature/moderate hyposalinity treatment (white triangles) experienced mortality starting at

day 4 and were all dead by day 5, resulting in a mean survival time of 2.69 0.9 days. Clams in

the high temperature/ambient salinity treatment (black triangles) experienced mortality starting at

day 7 and were all dead by day 13 (mean survival time of 7.06 + 3.9 days). Similarly, clams in

the high temperature/mild hyposalinity treatment (gray triangles) experienced mortality starting

on day 7, but clams in this treatment did not die before the experiment end (mean survival time

of 7.50 4.6 days). Among the clams maintained at room temperature, those in the moderate

hyposalinity treatment (white circles) experienced mortality starting at day 7 and were all dead









by day 14 (mean survival times of 7.18 4.0 days), a pattern that was significantly different

from that of the other two room temperature treatments (z = 5.98, p < 0.0001). In contrast, none

of the clams in the room temperature/ambient salinity (black circles) or room temperature/mild

hyposalinity (gray circles) treatments died (mean survival time 7.25 4.9 days). Overall, the

mean survival times of the six treatments were significantly different (Chi2 = 291.8, df= 5, p <

0.0001). In pairwise comparisons, clams in the high temperature/moderate hyposalinity treatment

had a significantly shorter survival time than clams in any other treatment (z > 10.1, p < 0.0001).

Clams in the high temperature/mild hyposalinity treatment had a significantly longer survival

time than clams in the high temperature/ambient salinity treatment (z = 1.97, p = 0.0484).

In the winter dual stressor experiment (Figure 3-9B), clams in the high

temperature/moderate hyposalinity treatment and the high temperature/mild hyposalinity

treatments experienced mortality starting at day 2 and were all dead by day 5, resulting in mean

survival times of 2.81 + 1.2 days and 2.73 + 1.2 days, respectively. Clams in the high

temperature/ambient salinity treatment experienced mortality starting at day 3 and were all dead

by day 7 (mean survival time 4.06 + 1.9 days). There was some death in the room

temperature/moderate hyposalinity treatment after day 11 (mean survival time 7.12 4.7 days).

In contrast, none of the clams in the room temperature/ambient salinity or room temperature/mild

hyposalinity treatments died (mean survival time 7.25 + 4.9 days). Overall, mean survival times

of the six treatments were significantly different (Chi2 = 174.12, df = 5, p < 0.0001). Clams in

the high temperature/ambient salinity treatment survived significantly longer than clams in the

other two high temperature treatments (z > 6.11, p < 0.0001) and significantly shorter than clams

in the room temperature/ambient salinity treatment (z = 5.69, p < 0.0001). Despite the mortality

at day 12, survival times of clams in the room temperature/moderate hyposalinity treatment were









not significantly different than clams in the other room temperature treatments (z = 1.19, p =

0.230).

In the spring dual stressor experiment (Figure 3-9C), clams in the high

temperature/moderate hyposalinity treatment began to die at day 3 and were completely dead by

day 5 (mean survival time 3.06 + 1.3 days). Clams in the high temperature/mild hyposalinity

treatment began to die at day 4 and were completely dead by day 10 (mean survival time 4.19 +

2.4 days). Clams in the room temperature/moderate hyposalinity treatment began to die at day 9

and were all dead by day 12 (mean survival time 6.16 3.5 days). In contrast, none of the clams

in the room temperature/ambient salinity or room temperature/mild hyposalinity treatments died

(mean survival time 7.25 4.9 days). Overall, survival times were significantly different among

treatments (Chi2 = 82.663, df= 4, p < 0.0001). Clams in the high temperature/moderate

hyposalinity treatment had significantly shorter survival times than clams in all other treatments

(z > 3.71, p < 0.00021). Clams in the high temperature/mild hyposalinity treatment had

significantly shorter survival time than clams in the mild hyposalinity/room temperature

treatment (z = 4.25, p = 0.00002). Clams in the room temperature/moderate hyposalinity

experiment had a significantly shorter mean survival time than did clams in the other room

temperature treatments (z = 2.98, p = 0.0029).

Ability to bury. Burial analysis was conducted on clams (n = 6-10) from each treatment

with surviving clams on each sampling day of the fall dual stressor experiment (Figure 3-10A).

There was a significant effect of treatment (p = 0.0035) but not of experiment duration (p =

0.739) on the ability of the clams to bury. Although the burial ability of the clams in the room

temperature/ambient salinity treatment was higher than those of any other treatment at all

sampling days, pairwise differences among treatments were not significant (p > 0.05). Although









clams in the high temperature/ambient salinity (black bar) showed the same ability to bury as

those in the room temperature/ambient salinity treatment (thinly striped bar) on day 1, clams

experiencing dual stressors were unable to bury at later time points.

In the winter dual stressor experiment, burial analysis was conducted on all (n = 9-10)

surviving clams at all treatment times (figure 3-10B). There was a significant effect of treatment

(p = 0.0029) but not of experiment duration (p = 0.741) on burial ability. On all sampling days,

clams in the room temperature/ambient salinity treatment exhibited burial ability, which

decreased after day 5. On the first sampling day, the only day with clams present in all

treatments, clams exposed to high temperature buried less than those at room temperature,

regardless of salinity level.

In the spring dual stressor experiment, burial analysis was conducted on a subset (n = 4-8

clams) of surviving clams at all treatment times (Figure 3-10C). There was no significant effect

of either experiment duration (p = 0.507) or treatment (p = 0.266), likely because burial was only

detected in two of the six treatments.

Glycogen content. The glycogen content of whole clams (n = 5) from all treatments on

day 1 and from treatments with a sufficient surviving clams on subsequent days of the fall dual

stressor experiment were analyzed (Figure 3-11A). Since this data set is unbalanced (due to

mortality in some treatments), each day was analyzed individually by one-way ANOVA with

Fisher's LSD where appropriate. There were no significant differences among the six treatment

groups on sampling day 1 (p = 0.873). On sampling day 5, there was a significant effect of

treatment (p = 0.021). The clams in the high temperature/ambient salinity (black bars) and high

temperature/mild hyposalinity (gray bars) treatments had significantly less glycogen than did

clams in the room temperature/ambient salinity (thinly striped bars) treatment (p < 0.026;









significance not indicated in graph). Additionally, the clams exposed to room temperature/mild

hyposalinity (thickly striped bar) had significantly less glycogen than clams in the other two

salinity (and room temperature) treatments (p < 0.035). At the day 9 sampling date, treatment

again had a significant effect on glycogen content (p = 0.033). Clams exposed to high

temperature/mild hyposalinity treatment had significantly less glycogen than clams exposed to

room temperature/mild hyposalinity (p = 0.0045). There were no significant differences in

glycogen content of clams surviving to 14 days (p = 0.061). Unlike most of the single stressor

experiments, overall glycogen levels did not decrease over the duration of the fall dual stressor

experiment.

The glycogen content of whole clams (n = 5 for all but 14 day room temperature/moderate

hyposalinity treatment, for which n = 2) from all six treatments on sampling day 1, and from the

room temperature treatments on subsequent sampling days of the winter dual stressor experiment

were analyzed (Figure 3-11B). We did not find any significant differences among treatments on

day 1 (p = 0.301), the only sampling day with all six treatments present. Similarly, there were no

significant differences in glycogen content among the three room temperature treatments on day

5 (p = 0.774). In contrast, we found significant effects of the hyposalinity treatments at both day

9 (p = 0.0055; significant differences are not indicated on the graph) and day 14 (p = 0.0012). On

both days, the clams in the ambient salinity had significantly more glycogen than clams in either

hyposalinity treatment (9 day, p < 0.045; 14 day, p < 0.039). Additionally, the clams in the

moderate hyposalinity treatment on sampling day 14 had significantly less glycogen than clams

in the mild hyposalinity treatment (p = 0.0046). Glycogen content in clams from the room

temperature/ambient salinity and room temperature/mild hyposalinity treatments did not decline

with experiment duration.









The glycogen content of whole clams (n = 2-3 clams) from all six treatments on sampling

day 1 and of the room temperature treatments on subsequent sampling days of the spring dual

stressor experiment were analyzed (Figure 3-11C). There was no significant treatment effect on

glycogen content of clams on sampling day 1 (p = 0.779) or day 5 (p = 0.72). Glycogen content

of clams sampled on days 9 or 14 were not statistically analyzed due to low sample sizes.

RNA oxidation. The levels of oxidatively damaged RNA bases, 8-oxoGuo, were

measured in clams (n = 5) from each treatment on day 1 of the fall dual stressors experiment

(Figure 3-12A). RNA oxidation was significantly affected by salinity treatment (p = 0.0157) and

a marginally affected by temperature (p = 0.0734), but not by the interaction between the two

factors (p = 0.809). Several post hoc comparisons were statistically significant; of those only one

was biologically significant. Clams exposed to the room temperature/mild hyposalinity treatment

(triangle) had significantly more oxidized RNA than did clams in the room temperature/ambient

salinity treatment (diamond; p = 0.0211, significance not noted on graph).

Levels of oxidatively damaged RNA were measured in clams (n = 5 for all except room

temperature/mild hyposalinity, which had n = 2) from each treatment on day 1 of the winter dual

stressors experiment (Figure 3-12B). RNA oxidation was significantly affected by temperature (p

< 0.0001) but not by salinity (p = 0.142) or the interaction between temperature and salinity (p =

0.210). In general, RNA oxidation levels were higher in clams in the high temperature treatments

in comparison to clams in the room temperature experiments. Specifically, clams exposed to

high temperature/ambient salinity (circle) or high temperature/mild hyposalinity (inverted

triangle) had significantly more oxidized guanine bases than did clams from any room

temperature treatment (p < 0.00091). The clams exposed to high temperature/moderate

hyposalinity had more oxidized RNA than did clams in the two room temperature/hyposalinity









treatments (p < 0.012), but this difference was only marginally significant (p = 0.0587) in

comparison to clams from the room temperature/ambient salinity treatment. There were no

significant differences among the three room temperature treatments (p > 0.229).

Levels of oxidatively damaged RNA were measured in clams (n = 5) from each treatment

on day 1 of the spring dual stressor experiment (Figure 3-12C). Unlike in previous seasons, there

were no significant effects of either treatment factor or the interaction between the factors on

RNA oxidation (p > 0.104). However, the post hoc multiple comparison test did detect two

significant comparisons to the RNA oxidation levels of the clams in the room

temperature/ambient salinity (diamond) treatment. Clams exposed to high temperature/ambient

salinity had significantly more oxidized RNA bases than did clams exposed to room

temperature/ambient salinity (p = 0.0151). Similarly, the clams in the two hyposalinity

treatments (room temperature) had more oxidized RNA bases than did the room

temperature/ambient salinity clams, a increase that was significant in the mild hyposalinity

treatment (p = 0.0429) and marginally significant in the moderate hyposalinity treatment (p =

0.0541).

Summary. The dual stressor treatments resulted in significant mortality in all three

seasons. Additionally, the high temperature/ambient salinity treatment caused significant

mortality, particularly in the winter experiment. Clams exposed to dual stressors had decreased

burial in all three experiments, but no significant changes in glycogen content. Although there

was no consistent pattern in RNA oxidation across season, in several cases the levels of RNA

oxidation were higher in clams exposed to hyposalinity in comparison to clams in ambient

salinity.









Discussion

These results support several overall conclusions concerning how one should investigate

the ability of an organism to respond to individual and multiple abiotic factors. First, a strong

influence of season was detected in all experiments, with an overall decrease in the ability of the

clams to tolerate the abiotic stressors evident during the winter experiments. Second, there was

no clear relationship between cellular-level and functional markers, with most of the treatment

effects detected at the whole-organism level rather than the cellular level. Third, many of the

functional responses were negatively impacted by experiment duration, calling into question the

utility of long-term laboratory exposures. Fourth, high temperature and hyposalinity were found

to have an additive effect on most metrics, particularly whole-organism metrics. Since this

combination of abiotic factors occurs in estuarine environments, particularly in the summer,

these results highlight the importance of investigating the effects of multiple stressors in

controlled laboratory conditions to more accurately determine how tolerance of abiotic factors

may influence species distribution.

Hypoxia. Conditions of low dissolved 02, or hypoxia, are prevalent in estuaries, generally

resulting from nutrient enrichments, tidal cycles, and excessive rainfall (Hubertz and Cahoon,

1999; Beck and Bruland, 2000). Since temperature affects 02 solubility, elevated water

temperatures cause both seasonal and diel cycles of hypoxia and normoxia. Mercenaria

mercenaria thrive in estuarine habitats which, along the Florida coast, have typically have 02

levels of 0.19 0.31 mmol L-1 (Millie et al., 2004; Harris et al., 2005) but can be as low as 0.09

mmol L-1 (Caccia and Boyer, 2005). Hard clams are hypoxia tolerant and are oxyregulators until

dissolved 02 levels reach the critical Po2 of 0.15 mmol L-1, at which point the clams close their

valves and maintain anaerobic respiration for extended periods of time (reviewed in Grizzle et









al., 2001). During these periods of anaerobic metabolism, hard clams, like many bivalves, rely

upon glycogen fermentation as a major energy source (Grieshaber et al., 1994; Grizzle et al.,

2001). While survival ofM. mercenaria is not strongly affected by hypoxia (Winn and Knott,

1992; Grizzle et al., 2001; Carmichael et al., 2004), functional responses of hard clams are

impacted by hypoxia, but only at very low dissolved 02 levels. Pumping rate, which directly

affects feeding ability, is linearly correlated with dissolved 02 levels from 0.03 0.15 mmol L-1

(reviewed in Grizzle et al., 2001). Clam growth is negatively impacted by dissolved 02 levels

below 0.15 mmol L-1 (Appleyard and Dealteris, 2002), an effect that may be confounded by

decreased feeding under these conditions. Burial time in M. mercenaria also is inversely related

to dissolved 02 levels, with burial time doubling over a range of dissolved 02 levels from 0.21

mmol L-1 to 0.03 mmol L-1 (Savage, 1976).

In the fall hypoxia experiment, both hypoxia treatments were well above the threshold

dissolved 02 level determined to affect functional responses of hard clams, and it is therefore not

surprising that there were no treatment-associated declines in burial ability or glycogen content

in clams from this experiment. However, both burial ability and glycogen content decreased over

the duration of the experiment, a trend that has been documented for glycogen levels in bivalves

in field (Byrne and O'Halloran, 2000) and laboratory (de Zwann and Zandee, 1972) studies.

In the winter and spring experiments, hypoxia treatments were modified to encompass the

threshold dissolved 02 level (mild hypoxia treatment, approximately 0.16 mmol L1) as well as a

level significantly below threshold (moderate hypoxia treatment, approximately 0.08 mmol L-1),

both of which are representative of conditions in estuaries with hard clams (Winn and Knott,

1992; Ringwood and Keppler, 2002). Although there was no treatment-associated mortality in

the hypoxia experiments, there was some evidence for a decline in functional responses. In most









sampling days of the winter and spring experiments, the burial abilities of clams in the moderate

hypoxia treatment were lower than those of the normoxia and mild hypoxia treatments, which is

consistent with previous characterizations of hard clam burial abilities (Savage, 1976).

In both the fall and winter experiments, glycogen levels in clams in the moderate hypoxia

treatment declined one sampling day before glycogen levels decreased in clams from the other

two treatments, a trend that occurred earlier in the winter experiment. In contrast, glycogen

levels in the spring experiment increased or showed no change with experiment duration, a

pattern repeated in the spring temperature and dual stressor experiments, regardless of treatment.

Since the feeding regimen did not differ among experiments, it appears that maintaining the

clams in the aquarium system was most detrimental to clam health (assessed by glycogen

content) in the winter, somewhat detrimental in the fall, and not detrimental in the spring.

Seasonal differences in glycogen storage in bivalves are well-established (e.g., Byrne and

O'Halloran, 2000; Hamburger et al., 2000), and are typically linked to reproductive status.

Mercenaria mercenaria are consecutive hermaphrodites and at the size used for these

experiments (12 15 mm) are likely in the male juvenile stage. Clams living in estuaries along

the Florida coast typically have a semiannual spawning cycle, with spawns in March/April and

October/November (Eversole, 2001). The spring experiment was conducted in April, and

therefore these clams may have utilized their glycogen stores differently than the clams in the

fall and winter experiments.

Short-term exposure to hypoxia has been linked to increased free radical production

(Hermes-Lima et al., 1998; Halliwell and Gutteridge, 1999; Hermes-Lima and Zenteno-Savin,

2002; Li and Jackson, 2002). During hypoxia, the absence of 02 as the final electron acceptor

causes accumulation of electrons in mitochondrial electron transport chains (i.e., the chains are









reduced), with the result that a sudden return of 02 can cause the production of free radicals due

to nearly instantaneous reactions between 02 and the accumulated free electrons (Du et al., 1998;

Li and Jackson, 2002). Although most studies of free radical production during hypoxia include a

period of reoxygenation, recent studies have documented free radical production occurring

during hypoxia without subsequent reoxygenation (Vanden Hoek et al., 1997; Chandel et al.,

1998; Becker et al., 1999). Hypoxia-associated free radical production, if it occurs, can be

accompanied by oxidative damage (Englander et al., 1999; Dirmeier et al., 2002) and changes in

antioxidant expression and/or activity (Lushchak et al., 2001; de Oliveira et al., 2005). However,

changes in antioxidant expression or activity are not always detected in animals or cells exposed

to hypoxia (Hass and Massaro, 1988; Willmore and Storey, 1997; Joanisse and Storey, 1998;

Larade and Storey, 2002).

In the current study, M. mercenaria exposed to hypoxia did not show significant changes

in stress protein expression, regardless of the season in which the experiments were performed.

There are two possible interpretations for these results. First, it is possible, in light of the

conflicting literature summarized above, that prolonged hypoxia in the absence of reoxygenation

does not induce free radical production or cellular damage. Upregulation of stress protein

expression, therefore, would be counterproductive and metabolically expensive (Somero, 2002).

Alternatively, if hypoxia exposure can cause oxidative damage, then it is possible that the degree

of hypoxia utilized in these studies was not sufficiently severe to incite a stress protein response.

These results are not consistent with those suggesting that cellular-level responses are more

likely to be detected than organismal responses, which integrate over several levels of biological

organization (Stebbing, 1985; Bierkens, 2000). Most of the studies cited above, particularly those

that showed cellular damage following hypoxia, were performed on organisms that are not









hypoxia-tolerant. It is possible that even though the severity of the hypoxia stress utilized in the

winter and spring experiments was sufficient to cause declines in functional responses, the stress

was not severe enough to cause damage or initiate responses at the cellular level. The

relationship between the timing and degree of cellular-level responses such as stress protein

expression and/or activity and whole-animal tolerance of hypoxia has not been explicitly studied

for any organisms. These results suggest that for animals as tolerant of a stressor as M

mercenaria is tolerant of hypoxia, cellular-level markers alone are not sufficient to detect

detrimental effects of exposure to individual abiotic stressors and should not be used to

investigate species distribution.

Temperature. Diel and seasonal temperature fluctuations are typical of estuaries. The

estuaries along the Florida coastline experience 'wet' and 'dry' seasons that can differ by 8C in

average temperature (Millie et al., 2004). Mercenaria mercenaria are tolerant of a wide range of

temperatures, from 0 300C, and typically more tolerant of warm temperatures than cold.

Maximum growth and pumping rates of hard clams occur at temperatures ranging from 20 -

260C, with both functional responses declining dramatically above 320C and below 70C (for

review, Grizzle et al., 2001). This pattern of thermal tolerance closely matches both their

distribution (Wells, 1957; Harte, 2001) and their reproductive activity, which in warmer southern

latitudes has a semiannual pattern (Eversole, 2001). Burrowing time in M. mercenaria mirrors

that of growth and pumping rates, with shortest burrowing times detected at 21 290C and

longer burrowing times at 370C (Savage, 1976).

In the current study, the functional responses of hard clams to temperature elevation varied

strongly by season, with the greatest susceptibility to high temperature detected during the winter

temperature experiment. Whereas none of the clams died during the fall temperature experiment,









all clams exposed to moderate temperature elevation (340C) died by day 7 in the winter

experiment and some clams in this treatment died on day 9 of the spring experiment. Seasonal

differences in susceptibility to thermal extremes has been documented previously and linked to

ability to express heat shock proteins (Chapple et al., 1998).

Burial abilities did not follow clear seasonal patterns, which is consistent with a previous

study (Savage, 1976). Burial abilities decreased over time in the fall experiment, but this pattern

was not repeated in the winter and spring experiments. In the winter experiment, clams in the

moderate temperature elevation treatment had very low burial ability on day 1 and did not bury

on day 5. However, burial abilities in the room temperature and mild temperature elevation

treatments were low on these days, so the lack of burial in the moderate temperature elevation

treatment cannot be considered predictive of subsequent mortality.

Patterns in glycogen content did not vary by season and were not significantly affected by

elevated temperature. However, in contrast to the hypoxia experiments, there were no apriori

expectations for changes in glycogen content with exposure to elevated temperature. In the

winter experiment, glycogen levels were not significantly different among clams in the three

treatments in the first two sampling days, and therefore cannot serve as a predictor of imminent

clam death. In the winter and spring experiments, glycogen levels in the room temperature clams

increased over time, which for the spring experiment is consistent with the hypoxia experiments.

According to a large body of literature, mild and moderate elevations in temperature result

in increased heat shock protein expression and, if cellular proteins are damaged beyond repair,

elevations in ubiquitin expression (for review, Feder and Hofmann, 1999; Hofmann et al., 2002;

Kregel, 2002; Dahlhoff, 2004). The degree of upregulation and temperature at which expression









is stimulated varies seasonally (e.g., Hofmann and Somero, 1995; Lesser and Kruse, 2004) and is

influenced by previous exposure to stressors (Somero, 2002).

Statistically significant treatment- or season-dependent changes in expression levels were

not detected for any of the stress proteins measured in this study, including the heat shock

proteins. Although these results may seem surprising, they are consistent with recent studies of

thermotolerant organisms, such as those demonstrating a temperature-insensitivity of Hsc70

function over the environmentally-relevant range of temperatures for an estuarine fish (Hofmann

et al., 2002; Zippay et al., 2004). The extreme thermotolerance exhibited by M. mercenaria may

result from physiological strategies that buffer the need for a cellular-level response to elevated

temperature. The temperatures utilized in this experiment may not have been high enough to

trigger a stress protein response, which typically requires thermal denaturation of proteins (Feder

and Hofmann, 1999).

Even though the moderate temperature elevation treatment resulted in mortality in the

winter experiment, it did not induce a significant stress protein response. Clams in this treatment

experienced a substantial (but not significant) increase in sHsp expression between 1 and 5 days

and decreases in Hsp60, Hsp70, and ubiquitin expression over the same period. While this

pattern could be consistent with extreme cellular stress (Werner and Hinton, 1999; Joyner-Matos

et al., 2007), given the lack of statistical significance and lack of detectable response in other

experiments, it is unlikely that these changes reflect anything more than random variation. The

physiological parameters examined in this study cannot rule out the possibility that the cause of

death in the winter experiment clams was a loss of function at a higher level of organization than

the cellular level, similar to the "heart failure" documented in intertidal crabs exposed to high

temperature (reviewed in Somero, 2002).









Dual stressors. Given the lack of detectable cellular-level response or consistent

functional response to hypoxia or high temperature, the ability ofM mercenaria to respond to a

combination of high temperature and hyposalinity was examined in a second set of experiments.

The combined effects of high temperature and hyposalinity are well-studied, likely because this

combination is common in coastal habitats, particularly during summer periods of high

temperature and substantial rainfall (e.g., Millie et al., 2004; Caccia and Boyer, 2005). While

these two factors have been shown to interact in their effects on hard clam pumping rate (for

review, Grizzle et al., 2001), it is not clear whether these stressors have an additive effect on

bivalve growth, development, or survival (Cain, 1973; Lough and Gonor, 1973).

While M mercenaria are tolerant of a wide range of temperatures, their distribution

patterns suggest the clams are not as tolerant of hyposalinity (Wells, 1957; Harte, 2001).

Mercenaria mercenaria function as osmoconformers, allowing their pallial fluids and

hemolymph to be isosmotic with the environment when their valves are open (reviewed in

Grizzle et al., 2001). The clams usually close their valves, however, when the environmental

salinity drops by 50%, a response that impacts the ability of the clams to feed and grow

(Carmichael et al., 2004). Pumping rates, ability to bury, and growth are maximized between 20

- 30 ppt, and decrease dramatically above 32 ppt and below 15 ppt (Grizzle et al., 2001).

Survival is significantly decreased at salinities ranging from 5 10 ppt (Winn and Knott, 1992).

Hard clams are particularly susceptible to rapid decreases in salinity, experiencing substantial

mortality following a decrease of approximately 20 ppt over a 24-hour period (Baker et al.,

2005). Such rapid fluctuations often occur where hard clams live, due either to rainfall events or

anthropogenic activities such as water discharges (Caccia and Boyer, 2005; Wilson et al., 2005).

A multiyear survey of salinity at sites close to where the clams utilized for this study are









maintained revealed daily and seasonal variation in salinity (Baker et al., 2005). Across three

years, salinities along the Florida Gulf coast ranged from approximately 5 ppt to 35 ppt, with

daily fluctuations ranging from 5 to 24 ppt in magnitude (Baker et al., 2005).

In the dual stressor experiments, the effects of three levels of salinity (approximately 25,

15, and 5 ppt) at each of two temperatures, room temperature and high temperature (average

340C) were examined. A strong additive effect of the two stressors was detected in the pattern of

survivorship of clams in all three seasons. Regardless of season, the clams exposed to the most

extreme set of conditions (high temperature/moderate hyposalinity) had the lowest survivorship,

with all clams in this treatment dead by day 4 or 5. In the winter experiment, a strong effect of

temperature on survival was again documented, with clams in all of the high temperature

treatments dead by day 7. A similar additive effect of high temperature and hyposalinity has been

documented for growth ofM mercenaria larvae (discussed in Lough and Gonor, 1973) and

survival of larvae of the estuarine clam Rangia cuneata (Cain, 1973). Among the room

temperature treatments, only the moderate hyposalinity treatment resulted in clam death, with the

greatest effect of this treatment detected during the spring experiment, which is consistent with

reports of hard clam susceptibility to low salinity.

It is less clear whether salinity alone or salinity in combination with high temperature

affected the burial abilities of clams in the dual stressor experiments. In the fall and spring

experiments, none of the clams exposed to high temperature and hyposalinity buried after 24

hours of exposure, the only time point with surviving clams in all six treatments. In the winter,

one clam in the high temperature/mild hyposalinity treatment buried, and none of the clams in

the high temperature/moderate hyposalinity treatment buried. In contrast, some clams exposed to

high temperature/ambient salinity in the fall and winter experiments buried on the first day of









sampling, which suggests that high temperature alone was not sufficient to inhibit burial.

Interestingly, burial ability was strongly influenced by hyposalinity. Only two of the clams

exposed to room temperature/moderate hyposalinity buried in the fall experiment and none of the

clams in this treatment buried in the winter or spring experiments, even though they survived to

nearly the end of the experiment. In nearly all cases, burial abilities of clams in the room

temperature/mild hyposalinity treatments were lower than those of clams in the room

temperature/ambient salinity treatments. The few studies that have examined the effects of

hyposalinity on burial ability or burial rate of bivalves have produced conflicting results but have

the general trend of decreased burial in hyposaline conditions, regardless of temperature (Grizzle

et al., 2001; Lardies et al., 2001; Matthews and Fairweather, 2004)

In contrast to the significant effects of the single and dual stressors on survival and burial

ability, there were no clear effects of the dual stressor treatments on glycogen content. At day 1,

the only sampling day with all treatments present, there were no significant differences in

glycogen content in any of the three seasons. Unlike in previous experiments, glycogen content

did not decrease over time. A causal relationship between glycogen content and salinity or high

temperature has not been established for bivalves, but several studies have reported seasonal

correlations between reproductive cycle, glycogen content, and environmental temperatures,

salinities and chlorophyll levels (e.g., Li et al., 2006).

A causative relationship between hypersalinity and free radical production is well-

established in plant physiology and biomedical fields such as nephrology and immunology (e.g.,

Hernandez et al., 1993; Qin et al., 1999; Hizoh and Haller, 2002). However, whether

hyposalinity is liked to alterations in free radical metabolism and oxidative damage is not

understood. A recent study of hyposalinity responses of a marine alga detected an elevation in









glutathione but not in antioxidant enzymes such as catalase and superoxide dismutase (Jahnke

and White, 2003). Similarly inconsistent results in other studies, all of which have been

conducted on plants, have not yet determined whether hyposalinity causes elevated free radical

production and oxidative damage. In the current study, some evidence of a link between

hyposalinity and oxidative damage to RNA was detected in the fall and spring experiments, but

not in the winter experiment. In the fall experiment, clams exposed to hyposalinity had more 8-

oxoGuo than clams exposed to ambient salinity, regardless of temperature treatment. In the

spring experiment, clams in the room temperature/hyposalinity treatments again had higher 8-

oxoGuo levels than clams in the ambient salinity treatment. In neither fall nor spring was there a

significant effect of temperature or an interaction between temperature and salinity. These results

suggest that clams exposed to hyposalinity experienced greater oxidative damage to their RNA,

either due to elevated free radical production or to a diminished ability to repair oxidative

damage. In the winter experiment, in contrast, RNA oxidation was closely linked to temperature

(higher oxidative damage in clams in the high temperature treatment) but not related to salinity

level. These results suggest that the high temperature treatment severely limited the ability of the

clams to minimize or repair oxidative damage, but do not give any indication of what may have

caused the elevated oxidative damage.

An examination of stress protein expression levels in clams from the dual stressors

experiment may provide further evidence for a link between hyposalinity and oxidative damage

and also may provide further evidence for an additive effect of high temperature and

hyposalinity. The tissue samples for this analysis have been processed but the stress protein

analyses are not yet complete. Several studies of stress protein expression (particularly heat

shock protein expression) in estuarine invertebrates exposed to hyposalinity or hyposalinity/high









temperature treatments have produced conflicting results (Kultz, 1996; Clark et al., 2000;

Werner and Hinton, 2000; Spees et al., 2002; Werner, 2004; Blank et al., 2006). The

combination of high temperature and hyposalinity was lethal for M. mercenaria, particularly in

the winter experiment. While the sublethal hypoxia treatments did not cause detectable stress

protein responses and no significant pattern of stress protein expression was detected in the

sublethal or lethal temperature treatments, the combination of high temperature and hyposalinity

may trigger a stress protein response. The oxidative damage results suggest that the clams in the

dual stressor experiments are experiencing cellular-level damage, and this damage may prove to

be sufficient to induce changes in stress protein expression.

Conclusions. The effects of the single- and dual-stressor treatments on functional and

cellular-level responses in M. mercenaria illustrate the importance of examining a variety of

physiological responses, across several levels of biological organization. For example, based on

the extensive studies of heat shock protein expression and thermotolerance in rocky intertidal

invertebrates, the seasonal susceptibility to high temperature demonstrated in this study would be

predicted to be accompanied by changes in heat shock protein expression. It is unlikely that the

high variance in stress protein expression levels masked any treatment-related changes as a

similar level of variance (with the same sample size and methodology) existed in the data set

presented in Chapter 2, and significant changes were detected in that study. Many of the

potential sources of variance in stress protein expression that were discussed in Chapter 2 (page

48), as well as the methodological steps taken to minimize that variance, are applicable to this

study as well. The fact that no significant pattern in stress protein expression was detected,

particularly during those seasons in which the treatments caused mortality, highlights the









importance of assessing multiple physiological responses, particularly the more sensitive whole-

animal responses such as burial ability.

Not surprisingly, a strong effect of season was detected in all experiments and in nearly all

measured parameters. Several environmental parameters, including salinity, temperature, food

availability, and dissolved 02 levels, vary seasonally in estuarine habitats and thus influence the

growth, reproductive cycle, and stress responsiveness of estuarine organisms.

In all seasons, functional responses tended to decrease over the duration of the experiment,

a trend that has been documented previously. It cannot be determined from this study whether

these declines resulted from prolonged maintenance in laboratory conditions or whether they

would occur in the field. In their habitat the clams likely would not experience prolonged

exposure to the equivalent of the most extreme treatments, particularly the high

temperature/moderate hyposalinity treatment, since abiotic conditions in estuaries fluctuate daily.

However, since the functional declines occurred in the control treatments as well as the

experimental treatments, it cannot be concluded that the functional declines represent reduced

tolerance of the stressors.

Finally, although an additive effect of high temperature and hyposalinity was apparent in

the survival analyses, we were unable to detect additive effects on cellular-level or functional

markers. These results suggest that we did not characterize the cause of mortality in the dual

stressor experiment. Examination of a wider variety of functional metrics and stress protein

expression levels may lead to an understanding of how the dual stressors affect the hard clams in

the laboratory and how tolerance of these abiotic factors may influence species distribution.














- 203
-126
- 80


-39


-31



-17
-7


fIs


MWM Copper/zinc Glutathione Manganese Heat shock Heat shock 8-oxoguanine DNA
superoxide peroxidase superoxide protein 60 protein 70 glycosylase
dismutase dismutase
Figure 3-1. Antibody specificity tests in M. mercenaria whole clam homogenates.
Molecular weight markers (in kilodaltons) are specified in the first column.


I I I I _~ I I _