Host distribution and physiological effects of ectocommensal gill barnacle (Octolasmis muelleri) infestation on blue cra...


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Host distribution and physiological effects of ectocommensal gill barnacle (Octolasmis muelleri) infestation on blue crabs (Callinectes sapidus)
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viii, 113 leaves : ill. ; 29 cm.
Gannon, Andrew Thomas, 1958-
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Thesis (Ph. D.)--University of Florida, 1990.
Includes bibliographical references (leaves 99-112).
Statement of Responsibility:
by Andrew Thomas Gannon.
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INFESTATION ON BLUE CRABS (Callinectes sapidus).






This work is dedicated to my partner
and wife,


Many individuals contributed to the success of this
research. I would like to acknowledge the guidance of my major
professor, Dr. Michele G. Wheatly, who carried out her
responsibilities as mentor above and beyond the call of duty. She
offered unceasing help at all stages of the project including
technical advice and assistance, reading innumerable drafts, and
career counseling and guidance. I would also like to thank the
other members of my supervisory committee: Drs. John Anderson,
David Evans, Carmine Lanciani, William Lindberg and Frank
Maturo, for advice, the loan of equipment, and reasoned criticism.
Charles Jabaly is acknowledged for his photographic and technical
assistance and practical suggestions and John Pipkins for
technical assistance. Numerous graduate students in the zoology
department have assisted in data collection, including Evan
Chipouras, Vincent DeMarco, John Donald, Francesca Gross, Robert
Hueter, Charles Jabaly, Michael Lacy, Frank Lockhart, Cathleen
Norlund, John Payne, Daniel and Molly Pearson, Nikarre Redcoff,
Tes Toop and Kent Vliet. They are all gratefully thanked. Thanks
are also due to Carlos Martinez del Rio, Vincent DeMarco and
Michael Lacy for statistical and computer advice.

This research was financially supported by a Sigma Xi grant
(to ATG), teaching and research assistantships from the
Department of Zoology (to ATG) and Division of Sponsored
Research (to MGW) and NSF grants DCB 8415373 and 8916412 (to
I thank my parents and family for their continued
encouragement. Most importantly I thank my dear wife, Francesca
Gross, for her continued emotional, logistical, and editorial


ACKNOWLEDGMENTS........................................................................................ iii

ABSTRACT ...........................................................................................................vii

GENERAL INTRODUCTION................................................................................... 1
Blue Crab Respiration............................ ..............................2
ctasmis muelleri............................ .... ..................
Host:Gill-Symbiont Relationships.............. ....... ........... 8


CEDAR KEY, FLORIDA.............................................................. 11
Introduction................................................................................... 11
M ethods................................................................................... .. .....13

2 EFFECTS OF Octolasmis muelleri INFESTATION ON
BLUE CRABS....................................................................................31
Introduction............................ ..................................................... 31
M ethods...............................................................................................34
Results........................................................................................... 38

3 EFFECTS OF Octolasmis muelleri INFESTATION ON
Introduction....................................................................... .........52
Discussion ..................................................................................65

GENERAL DISCUSSION.....................................................................................89

LITERATU R E C ITED ............................................................................................ 99

BIO G RA PH ICAL SKETC H .....................................................................................113

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

INFESTATION ON BLUE CRABS (Callinectes sapidus).


Andrew Thomas Gannon

December 1990

Chair: Michele G. Wheatly
Major Department: Zoology

The relationship between an ectocommensal gill barnacle,
Octolasmis muelleri, and its host, the blue crab (Callinectes
sapidus), was studied on three levels: population, organism and
model for host/commensal systems. On the population level,
barnacle distribution was evaluated in the Cedar Key, Florida blue
crab population and within the host gill chamber. Of 529 crabs, 40%
were infested, with monthly infestation rates ranging from 17% to
67%. The mean number of barnacles per infested crab was 21.8 with
as many as 432 barnacles on one crab. The barnacles aggregated on
their expected optimal host, namely previously infested adult blue
crabs. They were also most frequent at their expected optimal site

within the host gill chamber, namely the base of the hypobranchial
side of gills 3, 4, 5 and 6. This distribution indicates that 0.
muelleri infestation is common and frequently heavy and suggests
that host and site-within-host selection occurs. At the organism
level, infested blue crabs compensate for the obstruction in their
ventilatory stream by increasing ventilation (1.8 X) and heart rates
(1.4 X) to maintain oxygen consumption at the same level (28 g.mol
kg-1 min1) as uninfested crabs. Moderately infested crabs (10-20
ectocommensals) had small differences in hemolymph pH, oxygen
tension and oxygen content. During exercise most differences
between infested and uninfested crabs disappeared. Greater
arteriovenous differences in hemolymph pH and lactate levels of
infested crabs suggest that they have a greater reliance on the Bohr
effect for oxygen delivery to the tissues, but hemocyanin-02
affinity is preserved at the gills by a counteracting lactate effect.
At the host/ectocommensal level, the barnacle has undergone
selection to choose optimal hosts and attachment sites, and to
minimize detrimental effects to the host. The blue crab has
undergone selection to compensate for environmental stresses, such
as barnacle infestation. In all but extremely heavy infestations the
barnacle has a minor effect on host crabs at rest, during sustained
swimming and during recovery.



The blue crab, Callinectes sapidus Rathbun (1896), is a widely
distributed and well-studied swimming crab (Portunidae).
Population levels are large enough to support commercial fisheries
along the Gulf coast and the Eastern seaboard of the United States.
By nature Callinectes is a very active crab, and a high rate of
respiratory gas exchange is central to its metabolism and growth.
Because this crab is relatively long-lived (up to three years as an
adult; Van Engel, 1958), moves long distances (Oesterling, 1982)
and provides a hard substratum in soft bottom estuaries (Williams,
1984), it serves as host to numerous obligate and accidental
commensals, such as the soft coral Leotoaoraia virgulata (Pearse,
1947), the bryozoan, Triticella elonaata (Maturo, 1957), the leech
Myzobdella luaubris (Overstreet, 1982), the sessile barnacle,
Chelonibia spatula, (Van Engel, 1958), the oyster, Crassostrea
virainica, (personal observation) and the tunicate Molgula
manhattensis (Pearse, 1947).
Callinectes sapidus is the favored host of Octolasmis
muelleri (Coker, 1902), an ectocommensal stalked barnacle found

on the gills or in the gill chamber of decapod crustaceans (Humes,
1941). My aim was to examine this host/ectocommensal
relationship. Initially, the distribution of 0. muelleri within the
Gulf coast blue crab population and within the blue crab gill
chamber was investigated. After characterization of the barnacle
distribution, the effects of O. muelleri infestation on
the respiratory physiology of the blue crab host were measured.
Finally, the effects of 0. muelleri infestation on the respiratory
physiology of blue crabs under the natural stress of swimming
were measured. Determination of the magnitude and ways that blue
crab respiration is affected by barnacle infestation is important in
assessing the potential threat to an important commercial
resource, understanding the mechanisms by which the crab (a model
organism for crustacean respiration studies) compensates for
barnacle infestation, and evaluating the benefits and detriments of
the host/ectocommensal relationship.

Blue Crab Respiration

The anatomy and physiology of the respiratory and circulatory
systems of the decapod crustaceans have recently been reviewed by
McLaughlin (1983) and McMahon and Wilkens (1983). In summary,
blue crabs possess eight gills on each side of the body. The gills
are phyllobranch, triangular, wide at the base and narrowing
distally as they arch dorso-posteriomedially. The gills are housed
in the branchial chamber, which is formed as a lateral outgrowth of

the carapace. The carapace curves under to fit tightly against the
lower body wall just above the insertion of the walking appendages.
This joint is tight enough to exclude water. Water flows into the
gill chamber primarily through the Milne-Edwards inhalent opening
at the base of the cheliped. Smaller inhalent holes are found at the
bases of the pereiopods.
As water enters the Milne-Edwards hole it flows posteriorly.
It first enters the branchial chamber under the gills in the
hypobranchial chamber between gills 3 and 4 (Aldridge and
Cameron, 1982). From there the water flows through the gills and,
once it enters the epibranchial chamber, it flows anteriorly exiting
the branchial chamber via the exhalent passage, which is formed by
the endo- and exopodites of the three maxillipeds. The propulsive
force for water movement is the generation of negative pressure in
the branchial chamber by the beat of the scaphognathite (modified
second maxilla) in the exhalent passage (McMahon and Wilkens,
The blue crab circulatory system, like that of other
crustaceans, is an open system. However, the hemolymph is
contained in vessels throughout much of the body. The force
necessary for hemolymph circulation is provided by the single-
chambered heart. Hemolymph in the pericardial sinus enters the
heart through openings called ostia and exits through the posterior
and anterior aortae. Numerous arteries carry the hemolymph from
the aortae to the various tissues and organs. There are no closed
venous vessels. After passing through the smaller vessels of the

arterial system, hemolymph enters the hemocoel and passes
through discrete channels before collecting in the venous
infrabranchial sinus (McLaughlin, 1982). From here hemolymph
passes through the limb bases and the afferent branchial channels
to perfuse the gill filaments. The flow of the hemolymph in the
gill lamellae is countercurrent to the flow of water in the
branchial chamber (Hughes, Knights and Scammel, 1969). After
leaving the gills via the efferent branchial channels the hemolymph
moves through the branchiopericardial "veins" to reach the
pericardial cavity (McMahon and Wilkens, 1982).
A comprehensive list of respiratory and circulatory
parameters in the blue crab was recently summarized by
Cameron (1986). On a per gram body weight basis, gill surface
area in sapidus (710 mm2 g-1) is much larger than that of
most brachyuran crabs (e. g. U., Sesarma, Ocvpode, etc. from
Gray,1957). This probably reflects the active swimming
lifestyle of the Portunidae. The oxygen consumption rate
(M02 = 85.6 ml kg-1 hr-1) is correspondingly greater than for other
crab species. The ventilation rate (V W) of 111 ml min-1 reflects a
respiratory frequency of 31.7 min-1 given a combined gill chamber
volume of 3.5 ml. The unusually low cost of ventilation (0.02% of
M02) suggests that the water pumping mechanism is highly
An unexplained aspect of decapod respiration is the
phenomenon of current reversal in the gill chamber. The
scaphognathite pump reverses the direction of its beating, causing

water to enter the normally exhalent channel, cross the gills and
exit via the Milne-Edwards openings. This may occur 0-50 times
per hour in Callinectes sapidus (Batterton and Cameron, 1978) for
brief periods of around 5 seconds (Hughes et al., 1969). Numerous
functions have been suggested for these reversals, such as
enhanced ventilation of normally poorly ventilated regions of the
gills (Arudpragasam and Naylor, 1964), removal of detritus from
the gills (Hughes et al., 1969) and cleaning of intake filters
(McMahon and Wilkens, 1983). Adequate experimental evidence has
not been presented for a general explanation for the phenomenon.
Frequency of reversals did increase when crabs were exposed to
increased salinity or C02 (Batterton and Cameron, 1978) or to air
(Taylor, Butler and Sherlock, 1973).
Other characteristic features of crustacean respiration are
intermittent cardiac and ventilatory pauses. Crustaceans at rest in
well-oxygenated, undisturbed environments often discontinue
ventilation and heart beat for periods of up to several minutes
(McMahon and Wilkens, 1977). This may be a way of conserving
energy when hemolymph oxygen levels are near maximum (Batterton
and Cameron, 1978).
Although an open circulatory system connotes sluggish
movement of hemolymph through open spaces at low velocity and
low pressure, this is not true for the decapods, particularly active
ones such as blue crabs. Resting heart rate (fH= 89 beats min-1)
and cardiac output (V B = 151 ml kg-1 min-1) for the blue crab can
increase dramatically during exercise. Cardiac stroke volume

(Svc), 1.70 ml kg-lbeat-1 at rest, also increases during exercise
(Booth, McMahon and Pinder, 1982). Although blood flow velocity
has not been measured in blue crabs, other decapods have arterial
flow velocities approaching 10 cm sec-1, and ventricular systolic
pressures of 10-36 Torr (1.3 4.8 kPa) and diastolic pressures of
4-16 Torr (0.5 2.1 kPa; McMahon and Wilkens, 1983). These
characteristics along with the large hemolymph volume
(approximately 30% wet weight; Gleeson and Zubikoff, 1977)
indicate an efficiency comparable to closed circulatory systems of
other aquatic animals of similar activity level (McMahon and
Wilkens, 1983).
Animals have been experimentally manipulated to reduce the
supply of respiratory gases or increase the demand to evaluate the
capabilities of the gas exchange system. The responses of the blue
crab to hypoxia (low 02) and hypercapnia (high C02; Batterton and
Cameron, 1978; Cameron, 1985), as well as exercise (Booth et al.,
1982; Booth, McMahon, deFur and Wilkes, 1984; Milligan et al.,
1989) has been previously examined. In each case blue crabs make
ventilatory and/or circulatory adjustments to maintain 02 delivery
and C02 elimination to and from metabolizing tissues. Hemolymph
pH and lactate levels may change, but because these allosteric
modulators have opposing effects on hemocyanin-02 affinity, 02
delivery and C02 elimination are not compromised (Milligan et al.,
1989). This is an example of enantiostatic regulation (Mangum and
Towle, 1977).

Octolasmis muelleri

Octolasmis is a genus of gooseneck (or stalked) barnacle
(Cirripedia:Thoracica). They attach to the gills of decapod
crustaceans as ectocommensals (Newman, 1967). Octolasmis
muelleri (considered synonymous with Q. lowei by Nilsson-Cantell
1927) adult stages have been found in the gill chamber of numerous
brachyuran crabs (Humes, 1941; Walker, 1974). After internal
fertilization and brooding by the parent (Jeffries and Voris, 1983),
the barnacle goes through six naupliar stages and one cyprid stage
as a free-swimming planktivore, before settling down on a crab
host (Lang, 1976). The adult barnacle, cemented to the gill
lamellae of the host crab, filter feeds on particulate matter in the
ventilatory stream. The barnacle derives no nutrients directly
from the crab and is thought to harm its host only indirectly by
occluding the ventilatory current when infestation levels are great
(Walker, 1974). Therefore the Octolasmis-Callinectes relationship
is generally considered to be a commensalism, in which the
barnacle is benefitted and the crab is unaffected. This belief will
be evaluated in the following chapters.
The barnacle should experience selection pressure to select
optimal (i. e., better ventilated) sites within the host branchial
chamber. In addition the barnacle should select optimal (non-
molting adults) hosts because, during the host molt, it would be

shed with the exoskeleton covering the gills and is not thought
capable of survival outside the host (Walker, 1974). The
possibility of Octolasmis muelleri host and site selection will be
evaluated in Chapter 1. Because the barnacle is dependent on the
host for the continued renewal of ventilatory water, selection
pressure on the barnacle should minimize any detrimental impact
on the host. This will be discussed in Chapters 2 and 3.

Host:Gill-Symbiont Relationships

The distribution of ectoparasites on gills has been studied in
several species of fish (Walkey, Lewis and Dartnall, 1970; Suydam,
1971; Hanek and Fernando, 1978; van den Broek, 1979; Davey, 1980).
Frequently, nonrandom distributions have been discovered, but the
cause of these distributions has not been satisfactorily
demonstrated. Site specificity for the best ventilated parts of the
gill chamber has been attributed to passive dispersal of parasite
larvae in the ventilatory stream (Walkey, Lewis and Dartnall, 1970;
Suydam, 1971; van den Broek, 1979). In fact, Paling (1968)
assumed that parasitic glochidia were distributed passively by the
ventilatory stream of the brown trout Salmo trutta. He used the
parasite distribution to estimate the volumes of water passing
over the parts of the gill chamber. His assumptions were validated
by more sophisticated experiments using marker dyes (Hughes and
Morgan, 1973). Davey (1980) attributed the distribution of male
copepods, Lernanthropus kroveri, on bass gills to passive

distribution, but was unable to explain satisfactorily the
preference of females for the internal hemibranchs of the gills
without invoking active site selection
The effects of endoparasites on fish respiration have been
examined (Lester, 1971; Giles, 1987), but the effects of branchial
ectosymbionts are unstudied. Even less information is available
for crustaceans and their branchial symbionts. The isopod,
Probopyrus andalicola, causes decreased oxygen consumption in
its intermediate host, Acartia tonsa (Anderson, 1975a), and in most
cases in its definitive host, Palaemonetes pugio (Anderson, 1975b).
In the former, the ectoparasitic isopod may reach a greater mass
than the host. In the latter, the isopod has an endoparasitic stage
and an ectoparasitic stage that increases in size until it takes up
the entire host branchial chamber (Anderson, 1990). In both cases
the parasite feeds on host hemolymph.
The Q. muelleri-C. sapidus system differs in several
important ways. This ectocommensal is much smaller than the
host and does not feed on host hemolymph. This allows
measurement of the effect of ectocommensal presence without the
added effects of host hemolymph loss. In this system the host's
compensatory response to a natural obstruction in the ventilatory
stream can be evaluated.
The results of this investigation will be important on three
levels: organism, population and general host/commensal
relationship. On the organismal level it will add to our
understanding of the gas exchange system in the blue crab. On the

population level it will provide preliminary information on the
impact of a potentially harmful commensal organism on a
commercially important crab species. Finally, the results of this
study should shed some light on the interaction and possible
coevolution between host/commensal physiological systems.



Adult Octolasmis muelleri live in gill chambers of decapod
crabs, generally attached to the gill lamellae (Humes, 1941). The
adults produce many broods of larvae during warmer months
(Jeffries and Voris, 1983). The larval phase consists of six
naupliar stages that feed on phytoplankton and one nonfeeding
cyprid (Lang, 1976). Under laboratory conditions (24-290 C) the
nauplius to cyprid transition takes 14-18 days (Lang, 1976). The
cyprid stage enters the branchial chamber of a crab on the inhalent
respiratory current and cements its antennules to two adjacent gill
lamellae (Walker, 1974). Metamorphosis to the adult stage then
takes place over the next 20-72 hours (Lang, 1976). Sexual
maturity is reached in the tropical species Octolasmis cor within
two weeks after metamorphosis (Jeffries, Voris, and Yang, 1985);
development may not be as rapid in 0. muelleri in temperate areas.
Because the adult barnacle cannot move once attachment is
complete, selection of the appropriate site within the host gill

chamber is critical to the barnacles survival and reproduction.
Because the barnacle is attached to the host exoskeleton, it is
shed when the host molts and ultimately dies (Walker, 1974).
Selection of a host with an intermolt period long enough to allow
barnacle maturation and reproduction is therefore essential. This
study was undertaken to determine the characteristics of natural Q.
muelleri infestations in blue crabs with a view to assessing host
and site selection by the barnacle. In addition, infestation levels in
the blue crab population of the Cedar Key area were evaluated to
examine the possibility that the barnacle has a detrimental impact
on the host population.
The settling cyprid larvae of some symbiotic cirripedes are
thought to respond to host-related chemicals and host breeding
cycles, as well as the stimuli to which free living barnacle cyprids
respond (i.e., chemicals, currents, textures, light, gravity, and
hydrostatic pressure) (Lewis, 1978). The host specificity exhibited
by some of the 10 Octolasmis species found on decapods in the seas
around Singapore (Jeffries, Voris and Yang, 1982) suggests that the
larvae exercise some discrimination in host selection. Jeffries,
Voris and Yang (1989) have found evidence that cyprid larvae of Q.
cor and Q.. angulata aggregate on premolt crabs (Scylla serrata) and
transfer to the newly molted crab for attachment, indicating fine
discrimination of host status and delayed metamorphosis. Cyprid
larvae survived up to 177 days, although their competence for
metamorphosis at that time is unknown (Jeffries et al., 1989).
Similar capabilities in the closely related Q. muelleri would allow

selection of optimal hosts and attachment sites within hosts. One
expects that the ideal host is a large adult crab already infested
with 2. muelleri. Such a host would molt infrequently, provide
greater settlement area, be least harmed by the presence of the
barnacle, and contain mating partners. Adult male blue crabs molt
two to three times after reaching maturity (Van Engel, 1958).
Females may molt after reaching maturity (Havens and McConaugha,
1990). One might expect settling barnacles to favor the
hypobranchial side of the gills where water enters the branchial
chamber, particularly in the region of gills 3, 4, 5 and 6, which are
the best ventilated in the crab Carcinus maenas (Hughes et al.,
1969) and presumably also in the blue crab. The hypotheses of
optimal site and host selection will be compared to the null
hypothesis of random settlement.
Whether the host can deter barnacle larvae from settling, or
influence subsequent survival is unknown. For example, the
grooming action of the host epipodites may affect barnacle
survival, especially during and immediately after settlement.
Otherwise molting would appear to be the only host defense against
Octolasmis (Walker, 1974).


Blue crabs were trapped monthly from June 1987 to May 1988
at Seahorse Key on the Gulf coast of Florida (290 06' N lat., 830 04'
W long.), using plastic coated wire mesh commercial crab traps.

Trapping effort was not equal for each month. Captured crabs were
sexed and their width measured. Because lateral horns were
frequently broken, short width (the distance between the notches
just anterior to the lateral horns) was used as the size
measurement. Although short width is less commonly used than
full width, it more accurately represents the blue crabs' size (Olmi
and Bishop, 1983). Crabs were visually inspected for
ectocommensal carapace barnacles (Chelonibia spatula) and gill
barnacles (. muelleri). Attachment sites were recorded with
respect to gill chamber (right or left), aspect (epibranchial or
hypobranchial), gill number (#1 8, anterior to posterior), and
distance along the gill. For the latter measurement, each gill was
arbitrarily divided into thirds (basal, medial, and distal), which,
because of the triangular nature of the phyllobranch gill, made up
56%, 33% and 11%, respectively, of the gill surface area. Because
the gills are tightly oppressed, the only area available to settling
barnacle larvae is the edges of the gill lamellae facing into the
ventilatory stream, which shall be referred to as the "leading gill
surface area." I measured the gills of four blue crabs with calipers
and the averaged the results to determine the proportion of the
total leading gill surface area made up by each gill.
The commercial traps almost exclusively captured adults. A
seagrass flat 100 m north of the trapping site was dredged in April
1988 and 105 juvenile crabs were captured. This gave the size
distribution of infested crabs from the juvenile stages up to the

adult stages. These crabs were evaluated in the same way as were
the trapped adult crabs.

Infestation Summary
The monthly crab catches are given in Table 1-1. Because
trapping effort was not equal in each month, the monthly sample
sizes are not comparable as population estimates. The trapping
appeared heavily biased towards adult male crabs, which made up
81% of the total catch. Catch sizes, sex ratios and infestation
rates fluctuated from month to month with few apparent seasonal
trends. In all months male crabs had a greater or equal infestation
rate than females. The number of amuelleri per infested crab
reached a peak in the spring with the April rate an order of
magnitude greater than other months and the mean (21.7 Q. muelleri
per infested crab). The mean number of Q. muelleri per crab was
inflated by a small number of heavily infested crabs. The overall
median infestation level was 4 barnacles per crab. No obvious age
classes of infesting barnacles existed. Heavily infested crabs
contained barnacles of many different sizes, ranging from freshly
metamorphosed adults (about 1 mm total body length) to large
mature adults (up to 2 cm total body length).

Distribution Among Hosts
The number of barnacles per infested crab host can be seen in
Figure 1-1 as the frequency distribution of 0. muelleri on crabs.
The 318 uninfested crabs are not included in the figure. The
211 infested crabs contained from 1 to 432 barnacles. This
distribution is not significantly different from the negative
binomial distribution (P < 0.01). When the left and right gill
chambers of the host crab are treated as separate entities, the
frequency distribution is similar (fig. 1-2) and again not different
from the negative binomial (P < 0.01).
For most sample months no difference existed between the
mean carapace short width of infested and uninfested crabs (t-
test, P > 0.05). However, the sampled crabs were almost
exclusively adults. Figure 1-3 contains the size distribution of the
infested and uninfested crabs from the April 1988 sample, which
contained 61% juveniles. The mean short width of infested crabs
was 11.6 cm, that of uninfested crabs was 5.3 cm. These means
were compared with a t-test and are significantly different (P <
0.001). None of the juvenile crabs were infested.
The presence of another ectocommensal barnacle, Chelonibia
patula, was observed on a significant number of our sample crabs
(21.5%). The co-occurrence of C. atula and Q.. muelleri is reported
in a contingency table (table 1-2). The Pearson chi-square test for
independence (Feinberg, 1981) of the two infestations shows that
they are not independent (P < 0.001). Crabs that contain one of

these two ectocommensal barnacles are more likely to harbor the
second than crabs not infested with the first.

Distribution Within the Host
Inside the host gill chamber Octolasmis muelleri were found
attached to either side of each of the gills, the hypobranchial gill
chamber wall, the epibranchial gill chamber membrane, the
scaphognathite, and the hypobranchial and epibranchial gill rakers.
The majority of barnacles were found on the hypobranchial side of
the gills (93%), but the epibranchial side was infested only when
the hypobranchial side was heavily infested.
Although the distribution of barnacles along the gill filament
favored the basal third (54%), the proportion of barnacles found on
each third of the gill length (medial 33%, distal 10%) was
equivalent (to the nearest 1%) to the leading surface area made up
by that region of the gill. However, the proportion of Q. muelleri
found on each of the eight gill filaments was not equivalent to the
proportional leading surface area of that gill. This comparison is
shown in Table 1-3. Using the proportion of total leading gill
surface area as the expected proportional share of barnacles for
each gill, a chi-square test indicates that the barnacle distribution
is significantly different from expected (X2 = 155, 7df; P < 0.001).
Fewer barnacles than expected were found on gills 1, 7 and 8. Gills
3, 4 and 6 were more heavily infested than expected.


The infestation rate of Octolasmis muelleri in the blue
crabs of Seahorse Key (40%) is not different from that reported
for blue crabs at Grand Isle, Louisiana (37%) by Humes (1941).
Curiously, in the present study, 45% of the males and 20% of the
females were infested, yet Humes (1941) found 19% of the males
and 43% of the females infested. This rate and the number of
barnacles per infested crab (21.8) indicate a very prevalent
symbiont. If these barnacles have a negative effect on the host,
their presence could have a major impact on the .. sapidus
population. The physiological effect of infestation on the host
is explored in Chapters 2 and 3.
The hypothesis that Q. muelleri larvae would select optimal
hosts was first tested by comparing the barnacle distribution to
the negative binomial distribution, a fundamental model
frequently used to describe host-parasite distributions (Williams,
C. B.,1964). It is used here to model the frequency distribution of
parasites on hosts, and the distribution of parasites on host gill
chambers. The negative binomial distribution is theoretically
applicable to this system because a random distribution is not
expected due to variation in host characteristics (age, size, sex,
and habitat), that makes the chance of infestation unequal
(Crofton, 1971). The close fit to the negative binomial and the
low k values (k = .14 and .13 for barnacles/crab and barnacles/gill

chamber, respectively) indicate extreme aggregation (Williams, C.
B., 1964). Barnacles are most likely to be found on previously
infested gill chambers and hosts. This may be due to barnacle
larvae being stimulated to settle by the presence of conspecifics
as has been shown in other barnacle species (Crisp, 1974) or to
certain hosts being more attractive or having greater exposure to
barnacle larvae. The low and similar k values for the host
distribution and gill chamber distribution suggest that both
effects are important. It is likely these effects enhance each
other. It is improbable that Q.. muelleri larvae are reinfesting
their parents host, since they must undergo a 2-3 week
development period as plankton (Lang, 1976).
A truncated form of the negative binomial distribution has
been successfully used to model distributions in which the
parasite incurs mortality in heavily infested hosts (Lanciani and
Boyett, 1980). Hosts with large parasite loads would die and
therefore not be sampled, causing underrepresentation in the
larger parasite load classes. The Q.. muelleri distribution on ..
sapidus exhibits overrepresentation in the larger parasite load
classes Although not conclusive, this suggests that either Q.
muelleri infestation does not affect Q. sapidus mortality or that
the effect is linear (Lanciani and Boyett, 1980).
When samples of adult crabs were compared, no difference
existed between the size (carapace short width) of infested crabs
and uninfested crabs. A large difference between the size of
infested and uninfested crabs was evident only when immature

crabs were considered. Immature crabs are unsuitable hosts
because of their short intermolt period which is less than one
month in the juveniles up to stage 14 (Millikin and Williams,
1984). Barnacles infesting immature crabs may escape detection
due to small body size (Walker, 1974). However, in this study
careful inspection of 105 immature crabs did not reveal any that
harbored Q. muelleri. Infested immature crabs have been observed
only occasionally since this study was completed (unpublished
Although the ectocommensal carapace barnacle Chelonibia
spatula does not share the microhabitat of Q. muelleri, one might
predict that certain crabs are ideal hosts for both barnacles based
on distribution criteria such as infrequency of host molting,
greater settlement area and host resistance to harmful effects.
The evidence that the two barnacle species tend to select the
same hosts supports this view: however, it is equally possible
that infestation by one barnacle species weakens the crab and
makes it more susceptible to infestation by the other. It is also
likely that the longer the duration since the last crab molt, the
more time it has had to accumulate ectocommensal barnacles of
either species. Less likely is the possibility that the planktonic
larvae of the two barnacle species complete development and
settle in the same localities, infesting crabs there.
Within crab gill chambers, adult Q. muelleri were found
attached to virtually every surface in heavily infested crabs. This
negates the hypothesis of settlement and survival only on

optimally ventilated sites. Infestations of less than 10
Octolasmis were usually confined to the actual gill tissue on the
hypobranchial side. Concentration of the barnacles on the middle
gills agrees with the distribution found for the Beaufort, North
Carolina population by Walker (1974) and Jeffries and Voris
(1983). The basal and medial portions of the hypobranchial side of
gills, 3, 4, 5 and 6, which could be considered the optimal site for
barnacle growth, contained 61.4% of the barnacles sampled. This
area constitutes only 28.6% of the available gill surface area, and
an even smaller proportion of the total surface area used for
attachment (about 5.7%). The observed aggregation of barnacles
could be explained either as site selection by gregarious settling
cyprid larvae, or simply the result of passive distribution in the
mainstream of the crabs ventilatory current.
In summary, aggregation of Octolasmis on the expected
optimal hosts and at the expected optimal sites within the gill
chamber have been demonstrated. This suggests host and site-
within-host selection by barnacle larvae. However, alternate
hypotheses have been advanced to explain the observed distribution
and warrant further investigation.



04 (D mt

T- T- T- -

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2 1-T WO ZQOuL .2<

Table 1-2. Contingency table for infestation of crabs by
Chelonibia spatula and/or Octolasmis muelleri, (values are for
observed number of crabs/expected number of crabs).
Significantly more crabs are observed with both barnacle
infestations or neither infestation than expected (p < 0.001).

Octolasmis muelleri

+ 63/31.5 19/50.4
Chelonibia oatula
84/115.4 216/184.6

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EFFECTS OF Octolasmis muelleri


The respiratory physiology of aquatic decapod crustaceans
has been well-studied (see reviews by Taylor, 1982; McMahon and
Wilkens, 1983; Mangum, 1983; Truchot, 1983; Cameron, 1986).
Experimental methodology has involved changes in the external
respiratory medium such as hyperoxia (Dejours and Beekenkamp,
1977; Sinha and Dejours, 1980; Wheatly, 1987), hypoxia (McMahon,
Burggeren and Wilkens, 1974; Butler, Taylor and McMahon, 1978;
Wheatly and Taylor, 1981) and hypercapnia (Henry, Kormanik and
Cameron, 1981; Cameron, 1985). Respiration of the blue crab,
Callinectes sapidus, has been particularly well studied with
respect to the above environmental stressors as well as molting
(Cameron and Wood, 1985), emersion (Batterton and Cameron, 1978)
and exercise (Booth et al., 1982; Booth et al., 1984). The wide
distribution of this species from fresh water to hypersaline
lagoons and temperate regions to the tropics (Williams, 1984),
confirms what physiological studies suggest, namely an ability to
tolerate a wide range of environmental conditions.

Many gill breathers are hosts to parasites and
ectocommensals that could affect respiration. Endoparasitic
cestode larvae (Schistocephalus) cause increased oxygen
consumption in stickleback fish, Gasterosteus aculeatus (Lester,
1971), and stimulate the host to breath more often at the surface
in hypoxic waters (Giles, 1987). Snails infested with larval
trematodes have higher metabolic rates than do uninfested controls
(Duerr, 1967; Vernberg and Vernberg, 1967). Crustacean
endoparasites can also have profound effects on host metabolism.
Blue crabs are hosts to rhizocephalan barnacles such as
Loxothylacus texanus. These endoparasitic barnacles stunt host
growth (Overstreet, 1982) and stimulate male hosts to develop
female secondary sex characteristics (Reinhard, 1956). Baffoni
(1953) however, was unable to show any effect of rhizocephalan
infestation on host metabolic rate.
In all of these examples the parasites feed on host tissue and
make up a significant part of the host weight. They also kill or
severely debilitate the host. The direct effects of parasite
presence on the host are not separable from the effects of
blood/hemolymph depletion, increase in biomass, or parasite
associated disease. In addition, in the first two systems, the
parasite larvae induce host behavior that make the host more
susceptible to predation by the parasites' definitive host (Lester,
1971; Giles, 1987).
The effect of gill parasites on host respiration has been
measured only for the isopod Probopyrus pandalicola. Decreased

oxygen consumption occurs in both its intermediate host, the
copepod Acartia tonsa (Anderson, 1975a), and in most cases, its
definitive host, the shrimp Palaemonetes pugio (Anderson, 1975b).
In the former host the isopod is ectoparasitic and not a gill
parasite. In the latter the female isopod has both an endoparasitic
phase of 1-2 weeks (Anderson, 1990) and a branchial phase in
which it feeds on host hemolymph (Anderson, 1975b). The male is
much smaller and associates with the female in the host branchial
chamber (Anderson, 1990). Significant host mortality occurs in the
early periods of infection, but not when the parasite is in the
branchial stage (Anderson, 1990). This is surprising, given that the
parasite grows to such a size that it distorts the shape of the host
branchial chamber (Anderson, 1975b). The decrease in metabolic
rate of parasitized shrimp is attributed to changes in host lipid
metabolism (Anderson, 1975b). This may be related to the effect
of the parasite on the host reproductive system "parasitic
castration" -inhibition of ovarian maturation and disruption of
development of male secondary sexual characteristics (Beck,
1980). Thus, the effects of the parasite on gas exchange per se are
not separable from the effects caused by feeding on host
The Octolasmis muelleri Callinectes sapidus system offers
a unique opportunity to examine the effects of a branchial
commensal on host gas exchange including determination of a
possible detrimental effect on the host as well as evaluation of the
host mechanisms of compensation.

The aim of this study was to measure the effects of O.
muelleri infestation on the following parameters of blue crab
respiration: oxygen consumption, heart rate, ventilation rate, and
the hemolymph oxygen tension, oxygen content, carbon dioxide
content, pH, and lactate concentration.


Animal Maintenance
Adult blue crabs (150 250 g) were collected in commercial
traps at Seahorse Key on the Gulf coast of Florida (29006' N. lat.,
830 04' W long.) from April 28, 88 to September 4, 1988. Less than
30 crabs were maintained at any one time in an 800 I tank of
recirculating filtered seawater (33-36 ppt salinity) at 23-260C on
a 12 hr light:12 hr dark cycle at the University of Florida in
Gainesville. The crabs were fed chopped fish 1-2 times weekly, but
food was withheld 48 hours prior to experimentation. Intermolt
crabs (stage C-4, Johnson, 1980) were given a minimum of one
week to acclimate to these holding conditions before use in

Experimental Protocol
Measurements were made on three series of experimental
blue crabs. Each series contained crabs that were naturally
infested by the barnacle, Octolasmis muelleri, and uninfested
control crabs. This was a blind experiment, infestation level could

not be determined until the crab was killed. Series A crabs (n = 30)
were used to measure whole-animal respiratory parameters
(oxygen consumption rate, 0 02; heart rate, fH; and ventilation rate,
fSC). Series B crabs (n = 40) were used to measure prebranchial
(venous,v) hemolymph characteristics (pH; oxygen tension, P02;
total carbon dioxide content, CC02; and (L+)-lactate concentration).
Series C crabs (n = 22) were used to determine both prebranchial
and postbranchial (arterial,a) hemolymph characteristics (pH, P02,
oxygen content (CO2) and (L+)-lactate concentration). After
measurements were completed, crabs were killed by freezing and
the gill chambers were opened and visually inspected for the
presence of ectocommensal barnacles (Gannon, 1990; Chapter 1).
The total number of barnacles present in the gill chamber of each
crab was used as an index of infestation level.

Analytical Techniques
Series A crabs were prepared for heart and ventilation rate
recording and placed in a continuous flow respirometer (Taylor,
Butler and AI-Wassia, 1977). Depending on body size, crabs were
placed in either a 1.7 I or 3.8 I cylindrical respirometer that was
placed in a 38 I water bath. Filtered seawater (22-250C, 33-35 ppt
salinity) was pumped through the respirometer at a constant rate
that ranged from 60 to 95 ml min-1. Sampling ports in the inflow
(incurrent) and outflow (excurrent) of the respirometer allowed
simultaneous withdrawal of 1 ml water samples for measurement

of water P02. The P02 was determined using an IL 02 electrode
(20984) thermostatted to 250C and connected to an IL 213 blood
gas analyzer. The M 02 was calculated in glmol kg-1 min-1 using
the following equation:

A PO2 oc02 f
M02 =

where A P02 is the difference between incurrent and excurrent
P02 (in kPa), cCO2 is the solubility coefficient (in l.mol 02 ml
water -1 kPa-1; Jones, 1972), f is the flow rate through the
respirometer (in ml H20 min-1), and m is body mass in kg.
The fH and fSC in both branchial chambers were measured
continuously with an impedance conversion technique (Dyer and
Uglow, 1977). In the case of heart rate, two holes were drilled
through the carapace but not penetrating the epidermis on either
side of the heart and the end of a lacquered copper wire (32 gauge)
stripped for 3 mm was inserted into each hole through the
epidermis, to a depth of 4 mm. They were held in place with
cyanoacrylate glue and latex dental dam. These wires led outside
the respirometer to an impedance converter (UFI 2991) and the
output was displayed on a Grass model 79 polygraph. The fSC was
recorded similarly, inserting the recording wire into the exhalent
canal of each branchial chamber and the reference wire onto the
carapace. Crabs were allowed at least 48 hours to recover from the

surgical procedures and to acclimate to the respirometer before
measurements were taken.
Prebranchial hemolymph samples (I ml) of series B and series
C crabs were removed from the infrabranchial sinus by penetrating
the arthrodial membrane at the base of the swimming leg with a
22 gauge needle attached to a chilled syringe. Crabs in series C
were prepared for postbranchial hemolymph sampling by drilling a
hole directly over the heart, through the carapace but not
penetrating the epidermis. The hole was sealed with a I cm2 patch
of latex dental dam and cyanoacrylate glue. The postbranchial
hemolymph sample was withdrawn by puncturing the dental dam
patch with a 22 gauge needle attached to a chilled syringe. These
crabs were give 48 hours to recover from this surgery before
Crabs in series B and C were placed individually in 12 I of
aerated filtered seawater (22-250C and 33-35 ppt salinity) in a
20 I covered container and allowed at least 2 hours to acclimate
before pre- and postbranchial hemolymph samples were taken as
outlined above. For each hemolymph sample from a series B crab,
the P02 of an 880 !l subsample was measured as outlined above for
water samples. The pH of the same subsample was also measured in
the IL 213 blood gas analyzer using an IL pH electrode (20982). The
(L+)-lactate concentration was measured on an 80 pl hemolymph
subsample using a commercial reagent kit (Sigma #826). The CC02

of a 40 pl subsample was measured with a Capnicon automatic

analyzer (Cameron Instruments Company). For each hemolymph
sample from a series C crab, the P02 and pH of a 920 .1 subsample
were measured as described above. The CO2 of an 80 p. subsample
was measured with a Cavitron Lex02con oxygen analyzer.

Statistical Analysis
Values measured for series A and C infested crabs were
compared with those recorded for uninfested crabs using Student's
t-test for independent means (Sokal and Rohlf, 1969). Values
measured for series B uninfested, moderately infested, and heavily
infested crabs were compared using a Model I single classification
ANOVA (Sokal and Rohlf, 1969). Wherever the variances were not
found to be homogeneous with the F-test or the Fmax-test, a t-test
for samples with unequal variance was used (Sokal and Rohlf,
1969). Differences were considered significant at P < 0.05.


Experimental crabs were all of similar size (mean mass =
195.7 37.2 g S. D.). This minimized effects of mass on M 02, fH
and fSC. These values were each plotted against mass on a log:log
scale but no linear relationships were found. This suggests that
the influence of mass was negligible over this narrow range.
Therefore the measured values for fH and fSC were not adjusted for
mass. To facilitate comparison with other species, M 02 was
reported per unit mass.

Oxygen consumption rates of infested series A crabs were not
significantly different from those of uninfested crabs (Table 2-1);
however the infested crabs had elevated fH (44%) and fSC (83%).
For the comparisons of prebranchial hemolymph parameters,
series B crabs were divided into three classes based upon intensity
of infestation as follows: uninfested, which includes lightly
infested crabs (from 0 to 6 barnacles), moderately infested (10-20
barnacles) and heavily infested (more than 20 barnacles). These
divisions were based on an analysis of the distribution of Q.
muelleri on 512 blue crabs (see Chapter 1; Gannon, 1990). Four
heavily infested crabs (each containing more than 50 barnacles) did
not survive handling and surgical preparation.
The mean prebranchial hemolymph pH, P02, CC02 and lactate

concentrations of the heavily infested crabs were not significantly
different from those of the uninfested crabs (Table 2-2); however
moderately infested crabs exhibited lower hemolymph pH (0.3 pH
units) and CCO2 (25% reduction) and almost double the P02 of

control crabs. Lactate concentrations were virtually identical in
all groups; however the small sample size in the moderately
infested category precludes statistical comparison.
Pre- and postbranchial hemolymph parameters were measured
for series C crabs. This group contained only uninfested and
heavily infested crabs. Moderately infested crabs did not occur in
this sample. Mean prebranchial and postbranchial hemolymph pH,
PO2 or CO2 values for heavily infested crabs were not significantly

different from those values measured in uninfested crabs (Table 2-
Arteriovenous (a-v) differences in hemolymph gas parameters
for series C crabs were calculated for crabs in which samples of
both prebranchial and postbranchial hemolymph had successfully
been obtained (Table 2-4). Because of occasional problems with
hemolymph clotting, we were not successful in measuring all
parameters for every hemolymph sample. Uninfested and heavily
infested crabs were not significantly different with respect to pH,
CO2 and P02 a-v differences.


The presence of ectocommensal barnacles on crab gills could
increase the crab's ventilatory dead space in several ways.
Firstly, by cementing several gill platelets together upon
attachment (Walker, 1974), the barnacle may prevent water
circulation and thereby gas exchange between these platelets. As
the barnacle grows, adult cement is secreted and this will fuse
several more platelets (Walker, 1974). This should have a
relatively minor effect due to the large number of platelets present
in the gill chamber (12,400 on average for a 200 g blue crab; Gray,
1957). Secondly, barnacles remove oxygen from the water in the
crab's ventilatory stream for their own metabolism. Although
oxygen consumption rates for .. muelleri have not been measured,
extrapolation from data for other barnacles such as Chthamalus

stellatus. depressus and Balanus perforatus (Hammen, 1972) at
the same experimental temperature of 25C would predict an M 02
of 0.9 1.7 nmol 02 min-1 for each muelleri barnacle, which,
even under heavy infestation, would be negligible relative to the
M 02 of the crab (28 lpmol kg" min-). Thirdly, the barnacle may
create a physical obstruction in the ventilatory stream. The
magnitude of this effect is difficult to estimate. An increase in
ventilator deadspace could lead to a simultaneous increase in
perfusion deadspace because the hemolymph perfusing the poorly
ventilated portion of the gills would not be as well oxygenated.
Control of perfusion by vasoconstriction could prevent this from
occurring, but such controls have not been described in crustaceans
(McMahon and Wilkens, 1983).
A possible long-term effect is that the presence of the
barnacle may inhibit the gill-cleaning action of the epipodites of
the second and third maxillipeds (gill rakers) allowing other
barnacles to attach and debris to build up (Walker, 1974). Many
crustaceans periodically reverse the direction of ventilatory
flow over their gills for several reasons, including removal of
detritus and particulate matter from the gill chamber (McMahon
and Wilkens, 1983). Reversal frequency increases in response to
irritants (Batterton and Cameron, 1978), particulate matter
(Arudpragasam and Naylor, 1964; Berlind, 1977), and hypoxia
(Taylor et al., 1973). During reversed ventilation, oxygen uptake
is not as effective (McDonald, 1977).

Respiratory values recorded here for uninfested crabs are
generally similar to previously reported values for resting blue
crabs (Batterton and Cameron, 1978; Aldridge and Cameron, 1979;
Booth et al., 1982; Booth et al., 1984), although slightly lower M
02 and fH values suggest that the crabs in this study may have
been more "settled". In earlier studies using restrained crabs
fitted with a respiratory mask, mean M 02 was
50 pmol kg-1 min-1 and mean fH was 89 beats min-1 (Booth et al.,

1982) compared to 28 pmol kg- min-1 and 74 beats min-1 in the
present study. However, mean fSC in this study (130 beats min1)
was above that found previously (94 beats min-1 Booth et al.,
1982; 109 beats min"1 Batterton and Cameron, 1978). The
higher fSC may be related to the P02 of the inhalent water, which
was lower in this study (17 kPa, which was further lowered by
mixing with exhalent water in the respiratory chamber) than in
the earlier study (18 kPa Booth et al., 1982). Exact
correspondence between studies is not expected because of the
effects of geographic (Mauro and Mangum, 1982) and seasonal
variation (Weiland and Mangum, 1975), as well as temperature and
salinity (Laird and Haefner, 1976) of acclimation and
experimentation on hemocyanin-oxygen affinity (Mangum, 1983).
In addition different measurement methods for
M02 (flow-through versus closed system respirometry and
masked versus unrestrained animals), may explain small
differences between studies.

It is not surprising that infested crabs maintain M 02 at the
same levels as uninfested crabs. Trout maintain resting M 02 when
up to 30% of their gill surface area has been cauterized (Duthie and
Hughes, 1987). When spiders had their respiratory surface area
halved by experimental manipulation they maintained oxygen
consumption by doubling heart rate (Anderson and Prestwich,
The elevated fH and fSC rates found in the infested crabs in this
study are greater than values reported for resting blue crabs (fH = 89,
fSC = 94 beats min-1; Booth et al., 1982), but not as great as those
found in exercising blue crabs (fH = 143, fSC = 312 beats min-1;
Booth et al., 1982)
The hemolymph parameters for uninfested controls are also
similar to reported values (Mangum and Weiland, 1975; Booth et al.,
1984). The pH and CC02 hemolymph values in the present study
exhibit similar trends to those found in fH and fSC. Values for
uninfested crabs are similar to those reported in the literature for
resting crabs. Moderately infested crabs have significantly lower
pH and CCO2, but not as low as reported for exercising blue crabs

(Booth et al., 1984). Pre- and postbranchial hemolymph pH values
are not different from each other confirming the findings of
Mangum and Weiland (1975) and Booth et al. (1984). The lactate
values in the present study, although low (1.9 mM), are higher than
reported in resting blue crabs (0.7-1.3 mM; Booth et al., 1982); as
in that study no differences between pre- and postbranchial
hemolymph lactate levels were found.

Variation exists in blue crab hemolymph P02 values in the
literature. Booth et al. (1982) found a large arteriovenous P02
difference due to a high postbranchial value (10.4 kPa) and a low
prebranchial P02 of 1.1 kPa. The present study reports
postbranchial (4.7 kPa) and prebranchial (2.0 kPa) P02 values
similar to Mangum and Weiland (1975). This is puzzling given that
similar techniques were used in all three studies. However, the
more extreme values (Booth et al., 1982) are very close to those
reported for crabs stressed by exercise (Booth et al., 1982) and
were reported as "routine" activity values rather than inactive
"settled" values.
Ventilation volume, V W, and cardiac output, V B, and
therefore gill perfusion volume, are directly related to fSC and fH,
respectively (McMahon and Wilkens, 1983). The linear relationship
between V W and fSC has been estimated (Batterton and Cameron,
1978). The 83% increase in fSC measured in infested crabs
predicts a 69% increase in VW from 128 ml min- to 216 ml min-
1 (for a 200 g crab). The Fick equation relates V W or V B (in ml

min-1 kg-1) to M 02 (in umol kg-1 min-1) and the differences in
oxygen contents between water or hemolymph entering and leaving
the branchial exchanger (in gmol ml H20-1).

VW =
C102 CE02

VB= ___=
Cao2 Cvo2

In these equations C102 and CEO2 are the oxygen contents of the
water entering and exiting the gill chamber, respectively, and Ca02
and Cvo2 the oxygen contents of the arterial and venous hemolymph.
Since M02 remains constant between uninfested and infested
crabs, and V W and V B both increase in infested crabs, then the
C02 differences must decrease in infested crabs. This is true for
Cao2 Cvo2 (Table 4); we do not have data for C102-CE02.
Decreases in C102 CEO2 and Ca02 Cvo2 values indicate that the

effectiveness of oxygen exchange has decreased. Given the
calculated increase in V W and the constancy of M 02 for infested
and uninfested crabs, the Fick equation predicts that C102 CEO2
decreases by 41%. The effectiveness of oxygen extraction from the
ventilatory current (%ExtW):

C102 CEO2
%ExtW = (100)

would also decrease by 41% on average in infested crabs.
Increasing the cardiac output would increase the gill
perfusion to compensate for increases in perfusion deadspace.

Cardiac output would be elevated by an increase in fH and stroke
volume. Using mean M 02 values from series A crabs (Table 1),
mean a-v CO2 differences for series C crabs (Table 4) and the
Fick equation, one can calculate a cardiac output (V B) of
354 ml kg1 min-1 in uninfested crabs and 482 ml kg-1 min-1
in heavily infested crabs. These values are high relative to
previously calculated values for resting blue crabs at the same
temperature (Mangum, 1977; deFur and Mangum, 1979), but
comparable (Booth et al., 1982). However, because the
calculation is based on data from two different series of crabs,
the absolute values should be viewed with caution. In relative
terms, the calculated 1.4-fold increase in V B correlates well
with the 1.4-fold increase we measured in fH.
By maintaining 0 02, infested crabs should be able to
maintain hemolymph blood gas variables at the same levels as
maintained in uninfested crabs. The prebranchial hemolymph
parameters suggest that moderately infested crabs were
disturbed (acidosis, elevated P02, and decreased CC02) while

heavily infested crabs were not. These are the same prebranchial
hemolymph characteristics exhibited by blue crabs stressed by
exercise (Booth et al., 1982; Booth et al., 1984). Moderately
infested crabs are more likely to have been recently infested and
may not have adjusted to the presence of barnacles. It is also
possible that only individual crabs that are capable of
compensating for the barnacles presence are able to survive a
heavy infestation. Those crabs that were disturbed by a heavy

infestation would die and therefore not be sampled. Moderately
infested individual crabs that are unable to compensate fully
might still survive. Compensation for the barnacle-induced
increase in ventilation and perfusion deadspace involves elevation
of ventilation and perfusion rates as discussed above. However,
increasing perfusion rate via elevated cardiac output would limit
gas exchange in the metabolizing tissues. Successful
compensation would include enhancement of tissue 02 delivery via
modification of hemocyanin oxygen binding characteristics
(Mangum, 1983; Morris, 1990).
The comparison of the pre- and postbranchial hemolymph of
infested and uninfested crabs showed that heavily infested crabs
were able to maintain hemolymph variables at similar levels to
uninfested crabs, confirming the results from the series B crabs.
Although one would expect Octolasmis muelleri to
maximally exploit its host, the barnacle is dependent on the crab's
ventilatory stream and continued survival and would therefore
undergo natural selection to minimize any detrimental effect on
its host. These results confirm that settled, infested crabs do not
show signs of significant disturbance. However, crabs with
massive infestations did not survive experimental handling and
would probably not survive long in nature if stressed. Although
the barnacle is common in Florida blue crabs, the median
infestation level is not heavy (Gannon, 1990), thus it is unlikely
that the barnacle poses a serious threat to the blue crab

Table 2-1. Mean whole-animal respiratory characteristics
of series A Callinectes sapidus, infested experimentall) and
uninfested (controls) with Octolasmis muelleri. Values are
expressed as mean S.E. (number of observations). The asterisk
denotes significant differences between infested crabs and
uninfested controls (P < 0.05).


1102 (pmol kg-1 min-1)

fH (beats min-1)

fsc (beats







106.5 *

237.6 *

--------~--- ~----~~~~~~~

Table 2-2. Mean prebranchial hemolymph parameters of
series B Callinectes sapidus at three levels of infestation by
Octolasmis muelleri. Values are expressed as mean S. E.
(number of observations). Asterisks denote values that are
significantly different from uninfested (control) crabs. (P < 0.05)

pH PO2 CC02 [La-]
Infestation Level (kPa) (mM) (mM)

Uninfested 7.50 1.96 6.0 1.9
0.05 0.27 0.4 0.1
(16) (14) (16) (21)

Moderate 7.22* 3.23* 4.5* 1.9
0.06 0.59 0.3 0.2
(7) (6) (6) (3)

Heavy 7.52 1.93 6.6 1.9
0.05 0.35 0.6 0.1
(13) (11) (13) (16)

Table 2-3. Mean prebranchial and postbranchial hemolymph
parameters of series C Callinectes sapidus, uninfested (controls)
and heavily infested with Octolasmis muelleri. Values are
expressed as means S.E. (number of observations)

Postbranchial Prebranchial
Infestation pH PO2 C02 pH P02 C02
Level (kPa) (pM) (kPa) (gIM)

Uninfested 7.50 4.71 178 7.56 2.68 71
0.13 0.96 178 0.14 0.68 99
(10) (9) (7) (8) (7) (9)

Heavily 7.42 4.08 114 7.34 2.45 51
infested 0.12 0.87 114 0.11 0.52 75
(10) (8) (10) (10) (10) (9)

Table 2-4. Mean arteriovenous differences for hemolymph
parameters of Callinectes sapidus, uninfested (controls) and
heavily infested with Octolasmis muelleri. Values are expressed
as mean S.E. (number of observations).

pH P02 C02
Infestation Level (kPa) (pM)

Uninfested -0.06 0.31 81
0.17 0.31 +120
(8) (6) (6)

Heavily Infested 0.05 1.64 62
0.16 0.68 75
(9) (7) (8)

EFFECTS OF Octolasmis muelleri


Previous reports on symbionts of commercially important
species such as the blue crab have focused on crab death and
debilitation (Overstreet, 1978) or transmission of disease to
humans (Moody, 1982). Natural selection generally acts on
symbionts to minimize damage to their host but also to maximize
the benefit they derive from the host. These opposing forces
create an evolutionary dilemma for commensal species. How this
dilemna is resolved is of theoretical importance. The effects of
gill parasites and commensals on crustacean host gas exchange
have been largely ignored. Branchial symbionts could impair host
respiration in several ways: by removing oxygen from the
ventilatory stream, through damaging branchial tissue, through
obstructing the ventilatory stream, and/or inhibiting the
ventilatory pumping mechanism.

The ectocommensal gill barnacle, Octolasmis muelleri
Coker, does not feed on the tissues of its host crab (Walker,
1974). The oxygen uptake of this barnacle is presumed to be
negligible (See Chapter 2) and it does not normally inhibit the
host's pumping appendage, the scaphognathite. However, heavily
infested crabs are more likely to die during handling and aerial
exposure, suggesting that the ectocommensal causes physiological
stress to the host (Gannon and Wheatly, 1988). In the previous
chapter I demonstrated that this barnacle stimulates
hyperventilation and tachycardia in its host Callinectes sapidus,
and in moderate infestations, causes decreased prebranchial
hemolymph pH and P02 and increased CO2. Presumably these

effects arise from obstruction of the host ventilatory stream.
These physiological effects are similar to the typical respiratory
responses to exercise.
The purpose of this phase of the study was to examine in
detail the effects of 2. muelleri infestation on the respiration of
the blue crab during exercise, a natural stress. In nature, blue
crabs are extremely active, swimming at speeds up to
1 m sec-1 (Spirito, 1972), and with well documented long range
swimming migrations (up to 500 km; Oesterling and Adams, 1982).
The effect of exercise on respiration has been well studied in
decapod crustaceans (see reviews by McMahon, 1981; Wilkens,
1981) and particularly in blue crabs (Booth et al., 1982; Booth et
al., 1984; Milligan et al., 1989).

The aerobic and anaerobic capacities of exercising decapods
are comparable to those of lower vertebrates of the same size and
activity level (Booth et al., 1982). Maximal oxygen consumption
rates (McMahon, McDonald and Wood, 1979; Rutledge, 1980; Booth
et al., 1982) are less than those reported for active fish (Brett,
1972) but greater than those reported for sluggish fish (Brett and
Blackburn, 1978; Poulson, 1963). Decapods have been studied
swimming (Booth et al., 1982), tail flipping (Rutledge, 1980),
walking on treadmills (terrestrial; Wheatly, McMahon, Burggren
and Pinder, 1985), and walking on substrate (aquatic; McDonald,
McMahon, and Wood, 1979) with similar results. Generally, during
exercise oxygen transport and delivery are enhanced through
increased oxygen uptake, facilitated by increases in ventilation,
perfusion, and the oxygen tension gradient across the gills
(McMahon, 1981). The hemolymph respiratory pigment
(hemocyanin) assumes a proportionally greater role in oxygen
delivery to the tissues (McMahon et al., 1979). Oxygen
conductance across the gills may also increase (McMahon and
Wilkens, 1983). The level of unused oxygen in the hemolymph
(venous reserve) decreases (Wood and Randall, 1981). The
hemolymph acidosis is predominantly respiratory with a
metabolic component in terrestrial crabs (Smatresk, Preslar and
Cameron, 1979). The decreased pH causes a Bohr shift, which
decreases hemocyanin-oxygen affinity thus facilitating oxygen
delivery to the tissues.
Exercising blue crabs follow the pattern observed in the
decapods but seem better adapted for aerobic exercise (Booth and

McMahon, 1985; Milligan et al., 1989). Swimming blue crabs
increase ventilation, heart rate, and oxygen uptake so drastically
and rapidly that maximum metabolic rate (227 I.mol 02 kg-1 min-1)
is larger than that reported for any other crustacean (McMahon and
Wilkens, 1983). Aerobic metabolic scope measurements for blue
crabs range from 2.6 X (Booth et al., 1982) to 3.4 X (McMahon and
Wilkens, 1983). These values are lower than actual metabolic scope
because the resting values used in these calculations were routine
metabolic rates rather than quiescent rates. If the resting oxygen
consumption rates measured in the present study (Chapter 2) had
been used in these calculations, aerobic metabolic scope would be
4.6 8.1 X.
Blue crabs will swim continuously for over an hour in
experimental conditions (Booth et al., 1982) resulting in a
hemolymph acidosis that is mainly (80%, Booth et al., 1984)
metabolic (i. e. due to metabolic acids in the blood rather than
elevated C02 in the blood) and elevated lactate levels (Booth and
McMahon, 1985). The acidosis effect is limited by the efflux of H+
at the gills (Milligan et al., 1989). Lactate counteracts the effect
of lowered pH on hemocyanin-oxygen affinity (Truchot, 1980,
Booth et al., 1982). Recovery takes several hours (Milligan et al.,
Because resting blue crabs infested with Octolasmis
muelleri compensate for the barnacles' presence by elevating
ventilation and heart rates (Gannon and Wheatly, 1988, See

Chapter 2), one would expect their ability to further increase
these parameters during exercise to be compromised.


Animal Maintenance
Adult blue crabs (150 250 g) were collected in commercial
traps at the University of Florida Marine Laboratory at Seahorse
Key on the Gulf coast of Florida (290 06' N. lat., 83004' W. long.)
from December 20, 1988 to October 5, 1989. Crabs were held at
densities of less than 30 in an 800 I tank of recirculating filtered
seawater (33-36 ppt salinity) at 22-260C on a 12 hr light:12 hr
dark cycle at the University of Florida in Gainesville. The crabs
were fed chopped fish 1-2 times weekly, but food was withheld
48 hours prior to experimentation. Intermolt crabs (stage C-4,
Johnson, 1980) were given a minimum of one week to acclimate to
these conditions before use in experiments.

Experimental Protocol
Ventilation rate (fSC), heart rate (fH) and prebranchial
(venous,v) and postbranchial (arterial,a) hemolymph characteristics
(pH; oxygen tension, P02; oxygen content, CO2; and (L+)-lactate
concentration, [La']) were measured in 39 crabs. Hemolymph
samples were withdrawn for analysis from all crabs after at least
24 hours of isolation (resting), at the end of a 15 minute exercise
period, and one hour after recovery under settled conditions. The fSC

and fH were recorded for one hour near the end of the resting period
and continuously during the exercise and recovery periods. The
animals sampled included crabs that were naturally infested by the
ectocommensal barnacle Octolasmis muelleri and controls that were
not infested. This was a blind experiment. Infestation level could
only be determined after experimentation. After measurements
were completed, crabs were killed by freezing and the gill chambers
were opened and visually inspected for the presence of Octolasmis
muelleri. The total mass of barnacles present in the gill chamber of
each crab was used as an index of infestation level.

Analytical Techniques
Experimental crabs were placed in a nalgene holding tank
containing 38 I of filtered seawater (33-36 ppt salinity) at 23-
250C. Ventilation rate, fSC, measured as frequency of
scaphognathite beating, and fH were measured using an impedance
conversion technique (Dyer and Uglow, 1977). Each crab was
prepared by drilling two holes through the carapace on either side of
the heart. The stripped end of a lacquered copper wire (32 gauge)
was inserted into each hole to a depth of 4 mm. The wires were held
in place with cyanoacrylate glue and latex dental dam. These wires
were attached to an impedance convertor (UFI 2991) and the output
was recorded on a Grass model 79 polygraph. Scaphognathite rate
was recorded similarly, with a recording wire inserted into the
exhalent canal of each branchial chamber and a reference wire
attached to the carapace directly above each scaphognathite.

Prebranchial hemolymph samples (1 ml) were taken from the
infrabranchial sinus by penetrating the arthrodial membrane at the
base of a swimming leg with a 22 guage needle on a chilled syringe.
Crabs were prepared for postbranchial hemolymph sampling by
drilling a hole through the carapace directly over the heart, through
the carapace but not penetrating the epidermis. This hole was
sealed with a 1 cm2 patch of latex dental dam and cyanoacrylate
glue. The postbranchial hemolymph sample (1 ml) was withdrawn by
puncturing the dental dam patch. The crabs were given 48 hours to
recover from this surgery before experimentation.
The CO2 of an 80 pl subsample of each hemolymph sample was
analyzed in a Lex-02-con oxygen analyzer. The [La-] of an 80 gl
subsample was measured using a commercial reagent kit (Sigma
#826). An 840 p.l subsample was injected into an IL 213 blood gas
analyzer thermostatted to 25C. The P02 of this subsample was
measured with an IL 02 electrode (20984) and the pH was measured
with an IL pH electrode (20982).
After surgery crabs were placed in a harness of plastic coated
baling wire which was looped around the lateral horns of the
carapace and attached to a 25 cm long (1 cm diameter) wooden
dowel. The dowel was attached by a clamp to a wooden pole
suspended over the nalgene holding tank. This arrangement made it
possible to hold the crab either on the floor of the tank or suspended
in the water column with minimal restraint. When crabs were
raised off the floor of the holding tank they would swim
continuously. Infrequently crabs required prodding with another

wooden dowel in order to continue swimming. Hemolymph samples
were taken from crabs after 24 hours of isolation on the floor of the
holding tank (resting), after fifteen minutes of swimming in the
water column (exercise), and after one hour of recovery on the floor
of the holding tank (recovery). A period of fifteen minutes was
selected for the exercise period because, in earlier studies on blue
crabs, (Booth et al., 1982, Booth et al., 1984) physiological variables
showed maximal change and reached or approached peak levels in the
first fifteen minutes of exercise.

Data Analysis and Statistics
Experimental crabs were divided into three classes based upon
intensity of infestation: uninfested (0.0 0.020 g of Octolasmis
muelleri in the crab gill chambers), moderately infested (0.020 -
0.100 g of muelleri), and heavily infested (0.100 1.22 g of Q,
muelleri). These divisions were based on an analysis of the
distribution of Q. muelleri on 512 blue crabs (See Chapter 1). Mass
of infesting barnacles was assumed to be a more accurate index of
potential barnacle impact than number of infesting barnacles. In
experiments described in Chapter 2, however, numerous crabs were
examined on a single day and it was impossible to carefully remove
all of the infesting barnacles and weigh them for each crab.
The continuous records of fSC and fH were analyzed by
counting the number of beats in a fifteen second interval at the
beginning of every minute. These values were expressed as beats
min-1 and combined into five minute means. For comparison of

crabs at the different infestation levels, the mean frequency of the
last fifteen minutes of the acclimation period was used for the
"resting" value. The mean of the final five minute interval of the
exercise period was used for the "exercise" value and the mean of
the last five minutes interval of the recovery period was considered
the "recovery" value. After variances were found to be homogeneous
with an F-test (Sokal and Rohlf, 1969), heart and ventilation rates
of crabs at the different infestation levels were each compared with
a one-way factorial ANOVA (Winer, 1971). Heart rate and
ventilation rate values during the full one hour recovery period were
analyzed with repeat measures ANOVA (Winer, 1971) to determine if
a significant change occurred in either of these parameters during
the recovery period.
Pre- and postbranchial blood chemistry measurements were
also compared with repeat measures ANOVA to determine
significance of any differences between the rest, exercise and
recovery periods. Prebranchial values were compared to
postbranchial values with paired t-tests to determine if a-v
differences were significant. Hemolymph chemistry data were
reported as mean + S.E. These values were also compared across
infestation levels with a factorial ANOVA. Hemolymph pH values
were converted to concentration (linear scale) before calculation of
means, S.E.s and ANOVAs.

To minimize any effect of mass on heart rate (fH) and
ventilation rate (fSC), I used crabs of similar mass (mean = 249 +
45.2 g SD; range = 128 343 g). However, fH and fSC were plotted
against mass for all crabs and for the crabs at each infestation level
separately and subjected to regression analysis. No linear
relationships were found, indicating that the effect of mass was
minor over this mass range.
At rest, fH for heavily infested crabs was significantly
greater than fH for uninfested crabs factoriall ANOVA; F2,30 = 2.4;
Fisher PLSD; P < 0.05). The heart rates of crabs at the three levels
of infestation (figure 3-1) all increased dramatically during
exercise from resting rates of around 75 100 beats min-1 up to
140 160 beats min-1 remaining at this level throughout the
exercise and recovery periods. This represented a 2.0 X increase for
uninfested crabs and a 1.4 X increase for heavily infested crabs.
Heart rates for the three groups of crabs were not significantly
different through the exercise and recovery periods. At the end of
the one hour recovery period, fH for all three groups of crabs was
not significantly different from fH at the beginning of the recovery
period (repeat measures ANOVA, P > 0.05).
Ventilation rates (fig. 3-2) followed a similar pattern for
resting and exercising crabs. At rest, heavily infested crabs had
significantly greater fSC than moderately infested and uninfested
crabs factoriall ANOVA, F2,29 = 8.3; Fisher PLSD, P < 0.05).

Variance in ventilation rates was much greater for exercising crabs
than for resting crabs and differences between infestation levels
were not significant. As exercise began, fSC increased from about
100 beats min-1 to about 300 beats min-1 in all three groups.
Unlike heart rate, ventilation rate began to decrease immediately
after exercise stopped, but as fH, did not reach resting levels by the
end of the defined recovery period. Ventilation rates at the end of
the recovery period were significantly lower than fSC at the onset
of the recovery period for all three groups of crabs (repeat measures
ANOVA; F3,44 = 83.9; P < 0.05).
The frequency of ventilatory pauses (figure 3-3) was
significantly greater for uninfested crabs than moderately or
heavily infested crabs factoriall ANOVA; F2,31 = 3.3; Fisher
PLSD; P < 0.05) at rest. During exercise, ventilatory pauses were
not observed in any crabs. In the recovery period few crabs
exhibited ventilatory pauses and no differences existed between
the groups of crabs.
The pre- and postbranchial hemolymph oxygen contents
(C02) for resting, exercising and recovering crabs at the three
infestation levels (figure 3-4) were compared with a 2-way
repeat measures ANOVA (Winer, 1971). The repeat measures
gave a significant effect (F5,10 = 11.5; P < 0.05). Means were
compared with the Fisher PLSD (Snedecor and Cochran, 1980).
The mean recovery postbranchial value was significantly
greater than the resting and exercising prebranchial means (P <
0.05). This difference was most pronounced in the uninfested

crabs. The C02 values of uninfested, moderately or heavily
infested crabs were not significantly different from each other.
For uninfested crabs the mean C02 a-v difference

increased from a resting value of 0.042 mM to a recovery value
of 0.091 mM. In heavily infested crabs the mean C02 difference

increased from a resting value of 0.079 mM to a recovery value
of 0.22 mM. In moderately infested crabs the mean C02 a-v
difference decreased from 0.092 mM to 0.054 mM. During the
recovery period heavily infested crabs had a significantly
greater a-v difference than moderately and uninfested crabs
factoriall ANOVA; F2,25 = 4.2; Fisher PLSD; P < 0.05).
Because the 2-way repeat measures ANOVA could not
include individuals with a missing measurement in the
calculations (Winer, 1971), and some samples were lost due to
blood clots, this test could not be used for P02 comparisons.
Six separatel-way repeat measures ANOVA were used to
compare pre- and postbranchial hemolymph P02 for crabs at
each infestation level over the rest, exercise and recovery
periods (figure 3-5). Only the difference between resting
prebranchial P02 and recovery prebranchial P02 for uninfested
crabs was significant (repeat measures ANOVA; F2,23 = 3.9; P <
0.05). Comparison of the P02 levels of crabs at the different
infestation levels factoriall ANOVA) were made for pre- and
postbranchial hemolymph at the three experimental periods. No
significant differences were found.

The a-v P02 differences for uninfested and moderately
infested crabs during rest, exercise and recovery were not
significant (paired t-test). Heavily infested crabs had small a-
v differences at rest, but the mean P02 a-v difference for
heavily infested exercised crabs (1.2 kPa) was significantly
greater factoriall ANOVA; F2,23 = 3.9; P < 0.05) than the mean
P02 a-v difference for uninfested exercised crabs (0.25 kPa)
and moderately infested crabs (0.09 kPa).
The trend for hemolymph pH values for all crabs was to
decrease during exercise and increase during recovery, but not
to resting levels (figure 3-6). A two-way repeat measures
ANOVA was used to compare pre- and postbranchial hemolymph
values of resting, exercising and recovering crabs at the three
infestation levels. The repeat measures gave a significant
effect (F5,10 = 6.9; P < 0.05). Mean pre- and postbranchial pH
values during exercise were significantly lower than resting
values (Fisher PLSD; P < 0.05). Samples for several crabs were
lost due to blood clotting. To include these individuals in
comparisons, I used six one-way factorial ANOVA comparisons
of pre- and postbranchial hemolymph pH values for crabs at the
different levels of infestation during rest, exercise and
recovery. No significant differences were found between
infestation levels.
The moderately and heavily infested crabs had a
significantly greater postbranchial hemolymph pH than
prebranchial pH (paired t-test; P < 0.05) during rest, exercise

and recovery. Uninfested crabs had a significant a-v difference
only during rest.
Hemolymph lactate concentrations showed the reverse of
the trend described for pH (figure 3-7). A 2-way repeat
measures ANOVA was used to compare pre- and postbranchial
lactate values of resting, exercising and recovering crabs at the
three infestation levels. Mean lactate increased significantly
during exercise and decreased significantly during recovery, but
not to resting levels (F2,19 = 25.6; P < 0.05). Differences
between pre- and postbranchial lactate were not significant,
with the exception of the recovery values for the heavily
infested crabs (paired t-test; P < 0.05). Differences between
the crabs at the three infestation levels were not significant.


The mean heart rate of uninfested crabs at rest (78.4 beats
min-1 5.6 S.E.) agrees with that found earlier for uninfested blue
crabs (73.8 beats min-1 11.2 S.E.) (See Chapter 1). In the prior
study, infested crabs (most of which would be in the heavily
infested category in this study) had an elevated mean resting fH
(106.5 beats min-1 8.7 S.E.) that also agrees with mean resting
fH in the heavily infested crabs in this study (103.6 beats min-1 +
8.7 S.E.).

These fH values for uninfested crabs are lower than those
found by Booth et al. (1982) for resting blue crabs (89 beats min-
9.0 S.E.). However, that study of Florida gulf coast blue crabs
may have included a mix of infested and uninfested blue crabs
since the underside of the gills were not inspected for infesting
barnacles (B. R. McMahon, personal communication) and infestation
levels in the Cedar Key, Florida crab population average 40% (See
Chapter 1). Another reason for an elevated heart rate among the
blue crabs in Booth's study was that they were fitted with a
respiratory mask and were restrained during measurement. Their
resting values are labelled "routine" rather than standard.
Ventilation rates differ from those found by Booth et al.
(1982) in the same way. Resting moderately infested crabs had
the same fSC as resting blue crabs in the earlier study
(93 beats min-1) with uninfested blue crabs having a significantly
lower fSC and heavily infested crabs a significantly higher fSC.
These resting heart and ventilation rates for blue crabs are higher
than those found for similar sized crab species (McMahon and
Wilkens, 1977; McMahon,McDonald and Wood, 1979); however few
of these species are as active as the blue crab (McMahon, 1981).
Crabs at all three infestation levels exhibited an immediate
elevation of heart and ventilation rates to near maximum as
exercise began and maintained elevated levels throughout the
exercise and recovery periods. Booth et al. (1982) found the same
rapid increase, with a steady state half time of about 30 seconds.
Exercise fH and fSC values found by Booth et al. (1982) were

virtually the same as those found in moderately infested crabs in
the present study, and were below values found in uninfested
crabs. Although the exercise period in the earlier study was one
hour (compared with the present exercise period of fifteen
minutes) the peak levels reached and the dynamics of recovery
were similar. Ventilation rate dropped abruptly as exercise
stopped. Heart rate did not decrease significantly but remained
high. Neither rate reached resting values after thirty minutes of
recovery in either study. Even after one hour, recovery was not
complete in the present study.
Although differences in heart rate between the three
infestation levels were not significant, uninfested crabs had
greater mean fH and fSC over all five-minute exercise and
recovery intervals but one. Variance was great during exercise
and recovery, particularly for fSC. Although heavily infested
crabs were hyperventilating at rest, they were able to elevate
fSC during exercise to about the same levels as uninfested
crabs, perhaps because this level (300 350 beats min-1) is
the maximal sustainable rate. Batterton and Cameron (1978)
measured ventilation volume in blue crabs over a large range of
fSC but could not stimulate fSC above 350 beats min-1. Values
of fSC over 300 beats min-1 are more than twice as great as
the values reported for other exercising crabs (McMahon et al.,
1979; Wood and Randall, 1981) or crayfish (Rutledge, 1980;
Booth and McMahon, 1980) indicating the great aerobic exercise
capacity of blue crabs.

Intermittent cardiac and ventilatory pauses are
characteristic of decapod crustaceans at rest in well-oxygenated
undisturbed environments (McDonald et al., 1979; McMahon and
Wilkens, 1977; Batterton and Cameron, 1978). Although
experimental evidence is lacking, these pauses may be a way of
conserving energy when hemolymph oxygen levels are near or
above saturation levels (McMahon and Wilkens, 1977). At rest,
uninfested crabs ceased ventilating more than once every two
minutes. Heavily infested crabs paused in their ventilation only
once every twenty minutes. During exercise oxygen demand
increases and ventilatory pauses were not observed.
Cardiac pauses were more difficult to evaluate. Frequently,
ventilatory and cardiac pauses would occur simultaneously. While
ventilatory pauses might last over a minute, single cardiac
contractions would typically occur intermittently throughout the
ventilatory pause, making calculation of the length or number of
cardiac pauses difficult.
Resting and exercising hemolymph oxygen contents of crabs
did not differ between infestation levels, but mean values were
only about 20% of those found by Booth et al. (1982). They found
CvO2 and CaO2 were not significantly changed after 25 minutes
of exercise. I found the same result in moderately infested crabs.
Uninfested crabs exhibited the same trend as heavily infested
crabs; CaO2 increased with exercise and recovery. Since CvO2 did
not significantly change, the CO2 a-v differences increased, and

the recovery a-v difference was greatest for heavily infested

crabs. This suggests that the metabolizing tissues of heavily
infested crabs are not reducing prebranchial CO2 below the levels
of uninfested crabs, but they are obtaining more oxygen because
the a-v difference is greater.
Resting prebranchial hemolymph oxygen tensions for all
groups of crabs were low, but agree with those reported for
uninfested resting blue crabs in Chapter 2 (Mangum and Weiland,
1975; Booth et al., 1982). Resting postbranchial P02 values
however were about 20-50% less than those reported by Mangum
and Weiland (1975) and Booth et al. (1982). Differences between
post- and prebranchial hemolymph P02 reported by Booth et al.
(1982) were large for crabs at rest (59 torr, 7.9 kPa) and during
exercise (68 torr, 9.1 kPa). In this study, uninfested and
moderately infested crabs had virtually the same P02 for post-
and prebranchial hemolymph during the rest, exercise and recovery
periods. The a-v differences found in the heavily infested crabs
during exercise and recovery are comparable to those reported by
Mangum and Weiland (1975) for resting (AP02 = 2.72 kPa) and
exercising (AP02 = 1.76 kPa) blue crabs.
Post- and prebranchial pH followed the pattern expected for
exercising animals. Both values decreased during exercise and
increased during recovery. Values for uninfested crabs were
similar to those reported for blue crabs at rest and exercise
(Mangum and Weiland, 1975; Booth et al., 1982; Booth and
McMahon, 1985). Moderately infested crabs had lower resting pH
values, but higher exercise values. Recovery values were not

available for other studies, but recovery values here were
between resting and exercise levels for all three groups of crabs,
indicating that recovery was not complete in one hour.
Respiratory acidosis accounted for less than 20% of the
total pH drop in exercising blue crabs (Booth and McMahon, 1981).
Although lactate and H+ are produced in equimolar amounts in
anaerobic glycolysis (Hochachka and Mommsen, 1983), Booth et al.
(1984) calculated a net H+ deficit in the hemolymph of exercising
crabs. However, measured H+ excretion into the external medium
exceeded that predicted from lactate accumulation (Booth et al.,
1984). Here, lactate levels in the hemolymph of resting crabs
were similar to those reported for resting blue crabs (Booth et al.,
1982; Milligan et al., 1989). During exercise, [La-] increased 2-4 X
for crabs at all infestation levels. This is significantly less than
the increases reported for blue crabs after exercise bouts of
fifteen minutes (5 X-Booth et al., 1984), 25 minutes (14 X-Booth
et al., 1982), and thirty minutes of exercise (10 X-Booth and
McMahon, 1985; 15 X-Milligan et al., 1989). In determining lactate
the samples were not buffered with EDTA to prevent interference
by Cu2+ in the hemolymph as recommended by Engel and Jones
(1978). However, this would equally affect all readings,
preserving the ratio between treatments.
In blue crabs, anaerobic metabolism is important to energy
production during the first few minutes of swimming, while
sustained exercise is fueled predominantly by aerobic metabolism
(Booth and McMahon, 1985). In some terrestrial decapods such as

Uca pugilator (Herreid, 1981) and Gecarcinus lateralis (Full and
Herreid, 1984) anaerobic metabolism is important throughout
exercise bouts. Energy expenditure during exercise is difficult to
quantify. Earlier studies of blue crabs used prodding to induce
exercise walking and swimming (Mangum and Weiland, 1975;
Booth et al., 1982; Booth et al., 1984). Intensity of exercise was
not controlled in those studies nor in the present one. The crabs
in this study all swam readily with little prodding once they were
lifted from the substrate. However there was great variation in
the rapidity of swimming leg movement. Subjective evaluation of
swimming intensity did not reveal any correlation with
infestation level. The lower lactate levels found in all three
groups of exercising crabs indicate anaerobic glycolysis was not
used to the same extent as by crabs in earlier studies (Booth et
al., 1982; Booth et al., 1984). Perhaps overall energy expenditures
here were less, resulting in lower lactate production and minimal
disturbance of blood chemistry.
During exercise the decrease in hemolymph pH affects the
hemocyanin-oxygen dissociation curve. Using the Bohr shift
values calculated for blue crab hemolymph by Booth et al. (1982):

A log P50 (torr)

-1.14 =

A pH

I found the difference between post- and prebranchial hemolymph
pH in heavily infested crabs during exercise equivalent to an
increase in P50 of 1.4 torr (0.19 kPa). For moderately infested
and uninfested crabs, the P50 increased 1.7 torr (0.23 kPa) and 0.0
torr respectively.
In blue crabs lactate concentration of the hemolymph
affects P50 independent of pH (Truchot, 1980). The effect of
increased hemolymph lactate on hemocyanin oxygen-affinity was
measured in vivo and in vitro by Booth et al. (1982) as;

P50 (torr) = -5.252 log [La-] + 11.859

Using the mean [La-] found in the post- and prebranchial
hemolymph of heavily infested crabs to calculate P50 values, I
found a change in P50 from postbranchial to prebranchial
hemolymph of -0.85 torr (-0.11 kPa), a negative Bohr shift. For
moderately infested and uninfested crabs, lactate levels would
shift the P50 -0.16 torr (-0.02 kPa) and -0.07 torr (-0.01 kPa)
respectively. Because these pH and [lactate] values and the
equations were taken from different studies and the present study
did not correct for the possible interference of Cu+ (Engel and
Jones, 1978), these absolute values are only approximations.
However, the relative effects on the hemocyanin-oxygen affinity
relationship should hold. Thus, during exercise the pH decrease in
prebranchial hemolymph would tend to shift the oxygen
dissociation curve to the right, reducing oxygen loading at the

gills and increasing oxygen unloading at the tissues. However,
this is opposed by the effect of increased lactate levels, which
would tend to shift the curve to the left by almost the same
amount for heavily infested crabs and by a lesser amount for
moderately infested and uninfested crabs.
At the end of the 1-hour recovery period, neither pH nor [La-]
have reached recovery levels but because of the opposing effects
of these two moderators, the P50 and therefore the position of the
oxygen dissociation curve is relatively unchanged. This is an
example of enantiostasis (Mangum and Towle, 1977). Lactate
produced by anaerobic metabolism counteracts the negative
effects of acidosis on hemocyanin-oxygen affinity (Milligan et al.,
1989). Exercise in the blue crab is highly aerobic (Milligan et al.,
1989) and the lactate effect preserves the "venous reserve" by
maintaining unused oxygen in the prebranchial hemolymph that can
be utilized when the intensity of exercise must be increased
(Booth et al., 1984) or some other environmental stress demands
emergency 02 transport. During exercise, the actual amount of
oxygen available to the tissues is determined by the hemocyanin-
oxygen affinity which is affected by pH and [lactate] (Booth et al.,
1982) as well as organic ion modulators (Morris, 1990). The fact
that prebranchial oxygen content remains relatively unchanged
during rest, exercise and recovery indicates that all groups of
crabs are maintaining a venous reserve. Uninfested crabs had a
significant a-v pH difference only at rest, while the prebranchial
hemolymph pH of moderately and heavily infested crabs was

significantly less than the postbranchial hemolymph pH during
rest, exercise and recovery. This suggests that moderately and
heavily infested crabs relied more on the Bohr effect to deliver
oxygen to their metabolizing tissues.
After 1 hour of recovery, fSC had dropped significantly,
while fH remained elevated. The same relationship was observed
by Booth et al. (1982) and McMahon et al. (1979), although fSC and
fH are usually coordinated in their activity (Wilkens, 1981).
During recovery M 02 dropped at about the same rate as fSC
(McMahon et al., 1979; Booth et al., 1982). This suggests that the
demand for 02 had decreased considerably. A decreased fSC could
still meet the decreased 02 need. However lactate and pH values
had not reached recovery levels at one hour of recovery. Perfusion
rates would need to be maintained at a high level to flush La- and
H+ from the muscle tissues. Excretion of H+ is presumably by
branchial exchange (Booth et al., 1984) while lactate is taken up
and metabolized by tissues that have not been identified (Milligan
et al.,1989).
Blue crabs have a well developed anaerobic capacity (Booth
and McMahon, 1985) and a large aerobic metabolic scope (Booth et
al., 1982). They are highly resistant to fatigue from sustained
swimming (Milligan et al., 1989) maintaining a level of unused
oxygen in their prebranchial hemolymph even after one hour of
continuous swimming (Booth et al., 1982). The presence of the
ectocommensal barnacle, Octolasmis muelleri in the gill chamber
does not cause a major disturbance even during the stress of

exercise. At rest, infested crabs elevate fsc and fH to maintain a
venous reserve and hemolymph variables at the levels of
uninfested crabs, thereby compensating for the physical
obstruction within the ventilatory stream. During sustained
exercise fSC and fH for all crabs approach maximum rates. Oxygen
delivery to the tissues is maintained, but oxygen uptake at the
gills is not compromised due to the effect of lactate
counteracting acidosis.
The physiology of the blue crab is extremely well designed
for sustained swimming (Booth et al., 1982). The enantiostatic
control of its oxygen delivery system (Mangum and Towle, 1977)
makes Octolasmis-infested blue crabs capable of sustained
swimming with minimal disturbance in their blood gas
parameters. Because energy expenditure while
swimming was not quantified, infested crabs may not have been
swimming as fast as were uninfested crabs, and that extremely
rapid escape swimming may be compromised by massive barnacle
infestation. Several crabs with infestation of more than 1.0 g
Octolasmis muelleri did not survive experimental stress, and
probably would not survive long in nature.





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50 -- Uninfested n = 12)

----Moderately Infested1 = 13)

40 --H Heavily Infestedn = 8)


e 20


Rest Exercise Recovery

Experimental Period

Figure 3-3. Frequency of pauses in ventilation of blue
crabs at rest, exercise and recovery
at three levels of infestation

Figure 3-4. Mean post- (a) and prebranchial (v) hemolymph
oxygen contents of crabs at rest, exercise and
recovery, at three levels of infestation. Dashed
lines indicate significant (P < 0.05) a-v
differences. Asterisk indicates an a-v difference
that is significantly greater (P < 0.05) than for
other infestation levels.





Moderately Infested
(n = 12-15)

Rest,a Rest,v




Rest,a Rest,v

Ex,a Ex,v

Rec,a Rec,v

Heavily Infested
(n = 7-10)

Ex,a Ex,v Rec,a Rec,v
Experimental Period

(n = 10-12)

Rest,aRest,v Ex,a Ex,v Rec,a Rec,v





Figure 3-5. Mean post- (a) and prebranchial (v) hemolymph
oxygen tension of crabs at rest, exercise and
recovery, at three levels of infestation. The dashed
line indicates a significant (P < 0.05) a-v
difference. The asterisk indicates an a-v
difference that is significantly greater (P < 0.05)
than for other infestation levels.

- T

T -I-

Rest,a Rest,v

Ex,a Ex,v Rec,a Rec

-r T

Rest,a Rest,v

Ex,a Ex,v

(n = 8-10)


Moderately Infested
(n = 12-15)

Rec,a Rec,v



Heavily Infested
(n = 7-10)

Rest,a Rest,v

Ex,a Ex,v Rec,a Rec,v
Experimental Period







Figure 3-6. Mean post- (a) and prebranchial (v) hemolymph pH
of crabs at rest, exercise and recovery, at three
levels of infestation. Dashed lines indicate
significant (P < 0.05) a-v differences.


I "-r-

(n = 9-12)

Rest,a Rest,v


n. 7.5


E 7.0

Rest,a Rest,v

Ex,a Ex,v

Rec,a Rec,v

Moderately Infested
(n = 12-15)

Ex,a Ex,v Rec,a Rec,v



Heavily Infested
(n = 7-9)

Rest,a Rest,v

Ex,a Ex,v

Rec,a Rec,v

Experimental Period

Figure 3-7. Mean post- (a) and prebranchial (v) hemolymph
lactate concentration of crabs at rest, exercise and
recovery, at three levels of infestation. The dashed
line indicates a significant (P < 0.05) a-v

(n = 10-11)

Rest,a Rest,v

Rest,a Rest,v

Ex,a Ex,v

Ex,a Ex,v

Rec,a Rec,v

Moderately Infested

T T (n = 11-13)

Rec,a Rec,v


Rest,a Rest,v Ex,a Ex,v

Experimental Period


Heavily Infested
(n = 7-9)

Rec,a Rec,v


This study has focused on the Octolasmis muelleri/Callinectes
sapidus relationship seeking answers to questions on three levels.
On the organism level, information was collected to evaluate the
likelihood that barnacle larvae select optimal sites and hosts and to
determine the crab's physiological response/compensation to the
barnacles presence. The optimal site and host are those that allow
the highest rate of growth, survival and reproduction. On the
population level, infestation rates were measured to determine if
the barnacle population was large enough to have a major impact on
the Cedar Key, FL crab population. On the host/ectocommensal level,
information from the other two levels was synthesized to evaluate
the selection pressures on the host and symbiont and to determine
how this system differs from other host/symbiont systems.


Octolasmis muelleri adults aggregated on the bases of the
hypobranchial aspect of gills 3,4,5 and 6, the best ventilated part of
the gill chamber (Hughes et al., 1969). This represents only a small
portion of the available attachment area on the gills (28.6%), and an

even smaller portion (5.7%) of the total suitable area. The suitable
area consists of the areas where apparently healthy adult barnacles
were found. This includes both sides of the gills, the branchial
chamber epidermal lining, and the carapace underneath the gills.
Although this suggests site selection, three pertinent assumptions
must be evaluated: 1. the best ventilated site corresponds to the
optimal site. 2. adult distribution reflects larval settlement rather
than post-settlement mortality. 3. cyprid settlement involves
active selection, not passive distribution.

Ventilation and the Optimal Site
The requirements of adult barnacles have not been researched
in detail but must include a minimal level of oxygen (Barnes and
Barnes, 1964), nutrient availability, and the presence of prospective
mates. As for the latter requirement, aggregation of the adults as
demonstrated in Chapter one should suffice. Barnacles are
uncommon among sessile animals in that they have internal
fertilization. Adult Q. muelleri are able to extend their muscular
stalk and obtain a length up to 2.0 cm (personal observation).
Coupled with a long protrusible penis, they should be able to mate
with other barnacles within a radius comprising about the area in
which they were found to aggregate, but not reaching the whole gill
With respect to oxygen availability, levels are relatively high
in all parts of the gill chamber but presumably the best ventilated
site, would be where water first enters the gill chamber (Hughes et
al., 1969). Since the blue crab takes only 50% of the oxygen out of

water passing through the gill chamber at rest (Booth et al., 1982),
oxygen availability, is probably adequate,.even in the epibranchial
In regard to nutrient requirement, little information is
available but adult blue crabs are omnivorous, cannibalistic, and
detritivorous (Laughlin, 1982) as well as being sloppy eaters. They
shred their food using the maxillipeds and mandibles before
ingesting it (Pyle and Cronin, 1950), often creating a cloud of
particulate matter (personal observation). The intake to their gill
chamber, the Milne-Edwards opening, is at the base of the cheliped
(McMahon and Wilkens, 1983) and therefore in close proximity to the
mouth. Injection of methylene blue dye into the water near the base
of the cheliped showed that particulate matter is swept into the gill
chamber (personal observation). The diet of Q. muelleri adults in
nature is unknown but they have been maintained for several months
in lab cultures on crushed Artemia (Lang, 1976) which is
nutritionally similar to the crustacean food components that make
up a large portion of blue crab gut contents (Darnall, 1959).
Therefore the best ventilated site may also be the optimal
attachment site with regards to nutrient availability.
With respect to negative impacts, Q. muelleri is remarkably
sheltered from predation in the blue crab gill chamber. Little is
known of the relative efficiency of the blue crab gill rakers
(epipodites of second and third maxillipeds (Pyle and Cronin, 1950)),
which clean the surface of the gills. However, it appears that Q.
muelleri avoids removal by attaching to the margins and bases of the

gills rather than directly on the efferent vessel (Walker, 1974).
This would also favor the proposed optimal site.

Adult Distribution and Larval Site Selection
Differential mortality at sites within the gill chamber could
cause adult distribution to be nonrepresentative of barnacle cyprid
attachment site selection. Although this question has not been
addressed directly, previous studies of Q.. muelleri distribution that
included unmetamorphosed attached cyprids, did not report a
difference between cyprid and adult distribution (Walker, 1974;
Jeffries et al., 1983).

Active Selection Rather Than Passive Distribution
The aggregated distribution of barnacles at the best ventilated
site in the gill chamber could be explained by passive distribution in
the ventilatory stream and immediate attachment of larvae to the
first surface they encounter. Barnacle cyprid larvae respond to
surface contour (Wethey, 1986), water flow patterns (Rittschof,
Branscomb and Costlow, 1984), and chemical cues (Larman, Gabbot
and East, 1982) when selecting attachment sites. Cyprids are able
to move against currents by swimming in the slow-moving boundary
layer (Crisp, 1955) or by crawling on the surface (Crisp and Barnes,
1954). Symbiotic barnacle larvae are thought to use substrate
texture cues as well as host chemical cues in settlement (Lewis,
1978). Extracts of host tissues promote settlement in several
symbiotic cirripedes (Crisp and Williams, 1960; Williams, G. B.,
1964; Hayward and Harvey, 1974). The less likely it is that a