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Morphological and Constituent Analyses of American Alligator (Alligator mississippiensis) Eggshells from Contaminated an...


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MORPHOLOGICAL AND CONSTITUENT ANALYSES OF AMERICAN ALLIGATOR (Alligator mississippiensis) EGGSHELLS FROM CONTAMINATED AND REFERENCE LAKES By TERESA A. BRYAN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFI LLMENT OF THE RE QUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by Teresa A. Bryan

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iii TABLE OF CONTENTS page LIST OF TABLES...............................................................................................................v LIST OF FIGURES...........................................................................................................vi ABSTRACT......................................................................................................................v ii CHAPTER 1 A REVIEW OF REPRODUCTIVE BIOLOGY AND SHELL FORMATION IN REPTILES....................................................................................................................1 Introduction................................................................................................................... 1 Oviducal Anatomy........................................................................................................2 The Amnio tic Egg.........................................................................................................3 Oviposition...................................................................................................................4 Environmental Perturbation to Shelling.......................................................................5 The Role of Calcium.....................................................................................................7 2 PARAMETERS OF ALLIGATOR EGGSHELLS FROM REFERENCE AND CONTAMINATED NORTH-CE NTRAL FLORIDA LAKES.................................10 Introduction.................................................................................................................10 Materials and Methods...............................................................................................13 Thickness Measurements.....................................................................................14 Pore Density........................................................................................................15 Elemental Analyses (EDS)..................................................................................15 Elemental Analyses (ICP)...................................................................................15 Scanning Electron Microscopy (SEM)................................................................16 Statistics...............................................................................................................16 Results........................................................................................................................ .16 Eggshell Thickness..............................................................................................16 Pore Density........................................................................................................17 Elemental Analyses (EDS)..................................................................................17 Elemental Analyses (ICP)...................................................................................17 Scanning Electron Microscopy (SEM)................................................................18 Discussion and Conclusions.......................................................................................19

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iv LIST OF REFERENCES...................................................................................................32 BIOGRAPHICAL SKETCH.............................................................................................37

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v LIST OF TABLES Table page 1-1 Mean avian eggshell indices in Britai n, prior to and after 1947 (modified from Ratcliffe, table 5, 1970)..............................................................................................6 2-1 Mean alligator egg viability rates and sample size of clutches collected from three study areas and incubated under 32C in an artificial incubator, 1999, 2001-2003 (Woodward, pers com.)..........................................................................11 2-2 Number of clutches from which eggs hell thickness was examined each year of the study period from 3 lakes. (Note: 3 eggs/clutch were measured)......................14

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vi LIST OF FIGURES Figure page 2-1 The mean (+/1s.e.) equatorial eggshell thickness among lakes and years (1999,2001,2002,2003). (Different superscripts indicate significant differences between lakes)..........................................................................................................23 2-2 The mean (+/1s.e.) number of eggshell pores among lakes in 2003. (similar superscripts denote no significan t difference among lakes).....................................24 2-3 The mean (+/1s.e.) number of alli gator eggshell pores per clutch, per cm2, among lakes in 2003.................................................................................................24 2-4 The mean (+/1s.e.) percent of the three most abundant elements in alligator eggshells detected by Energy Dispersive Analysis (EDS).......................................25 2-5 The mean (+/1s.e.) percent of the three most abundant elements in alligator eggshells detected by Energy Dispersive Analysis (EDS).......................................25 2-6 Scanning electron micrographs of the i nner surface of Lake Woodruff alligator eggshells (250X)......................................................................................................26 2-7 Scanning electron micrographs of the relative amount of shell fibers (F) in Apopka (a.b)And Griffin (c,d).................................................................................27 2-8 SEM images of shell membranes from a) Woodruff, b) Apop ka, c) Griffin in 2003..........................................................................................................................2 8 2-9 SEM images of eggshell pore cavities from 2003 eggshells....................................29 2-10 SEM Cross-sectional views at 5000X from lakes....................................................30 2-11 SEM image of the calcium crystalline structure on the inner eggshell surface.......31

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vii Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science MORPHOLOGICAL AND CONSTITUENT ANALYSES OF AMERICAN ALLIGATOR (Alligator mississippiensis) EGGSHELLS FROM CONTAMINATED AND REFERENCE LAKES By TERESA A. BRYAN August 2005 Chair: Louis J. Guillette, Jr. Major Department: Zoology Alligator eggs were collected from one polluted and two reference north-central Florida lakes. The eggs were artificially in cubated and their shells collected after the neonates hatched. Thickness measurements were taken from multiple eggs and clutches within each lake over a four year period (1999, 2001, 2002, 2003). In 2003, eggshells from three eggs per clutch and five clutches per lake were also an alyzed for constituent make-up, pore density, and basic morphol ogy. Constituents were determined by both inductively-coupled plasma spectroscopy (ICP) and energy-dispersive spectral analysis (EDS). Pore density was assessed using light microscopy and the morphology described through the use of electron microscopy. Egg mass and neonate morphometrics were recorded. We determined that shells from the refere nce lake were thinner than those from the contaminated lakes. Constituent analyses and morphology varied among the lakes. There

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viii was no significant difference in pore density among the lakes. Neonates from the reference lake were larger than those from the polluted lakes. We determined that eggshells from the re ference lake were co mparatively thinner, yielded less calcium, and lacked the fibrous por tion of a region of th e shell potentially to support the larger neonates. Comparativel y increased calcium mobilization from the eggshell during incubation would be critical for proper skeletogene sis and other calciumdependent functions in the larger hatchlings.

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1 CHAPTER 1 A REVIEW OF REPRODUCTIVE BI OLOGY AND SHELL FORMATION IN REPTILES Introduction Modes of reproduction vary among vertebrates. In reptiles, there were initially three reproductive modes described: vivi parity, the production of live offspring, ovoviviparity, egg retention with in the uterus and subsequent live birth, and the most common, oviparity, the laying of eggs contai ning relatively undevel oped embryos outside the body. More contemporary research sugge sts that ovoviviparity is not a discrete reproductive mode, but instead an evolutionari ly transient period between oviparity and viviparity (Guillette, 1993). Hence, species that retain eggs in-utero until late in embryonic development are considered oviparo us, whereas species with a placenta are viviparous, thus abandoning the term “ovovivipa rous” in reptiles. Oviparous reptiles lay an amniotic egg that develops within the ovi duct and it is this structure that creates an environment (the eggshell and its contents ) capable of providing for the needs of the developing embryo. In viviparous species, the embryo is retained within the uterus until development is complete (Guillette, 1993). Th erefore, the placenta must be present and provide the young with the ability to uptake nutrients, exchange gases and ions for respiration and waste removal, among other biological functions necessary for embryonic development. All vertebrate classes, excluding the Agna thans and the Aves, contain species that utilize this reproductive mode. Viviparity has evolved nearly 100 times in

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2 reptiles, though only in lizards and snakes, a nd has not been reported in any species of turtles, crocodilians, or tuatara (Shine, 1983, 1985). Oviducal Anatomy As with all amniote female vertebrates, the Crocodilian oviduct (which includes the entire reproductive tract) is derived from the embryonic Mllerian duct (Austin, 1989). The oviduct undergoes regional specialization as the female sexually matures. At maturation, the alligator oviduct consists of se ven anatomically dis tinct regions, starting anteriorly with the ampullae, followed by the infundibulum, uterin e tube, uterotubular junction, anterior and posterio r uterus, and vagina. Functiona lly, the oviduct is separated into three major regions, the uterine tube that secretes albumen (Palmer and Guillette, 1991), the anterior fiber-secreting region of the uterus, and the posterior calciumsecreting region of the uterus (Palmer and Guillette, 1992). Structurally and functionally, the anterior and posterior uterus of the alligator resemble s those of birds and not of other reptiles. The anterior fiber-s ecreting uterus is similar to the avian isthmus, whereas the posterior calcium-secreting uter us is similar to the avian sh ell gland. Endometrial glands located at the anterior and poste rior portions of the alligator uterus share similar cell types and secretory products with bird s, and not of other reptiles (Palmer and Guillette, 1992). The glands of the anterior uterus pro duce the fibrous shell membrane, which lies proximal to the embryo, whereas the posterior uterine glands are hypothesized to secrete the large amount of calcium that forms the out er, rigid calcified shell. Unlike birds, however, alligators ovulate and “shell” a comple te clutch of eggs simultaneously whereas birds ovulate and shell one egg at a time.

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3 The Amniotic Egg All amniotic eggs consist of yolk and album en derived from the maternal parent. In addition, the eggs contain membranes derived from the embryo and in alligators, this includes the chorio-allantois (a fusion betw een the chorion and the allantois) and amnion (Ferguson, 1982). During the breeding s eason, circulating estrogens increase and vitellogenin (Vtg), the major egg yolk protein, is synthesized in the liver (Guillette et al., 1997). Vitellogenin circulates through the bl oodstream and is taken up by the oocytes where it is cleaved into smaller yolk subunits (Berg et al., 2004). Th ese subunits serve as the nutritional source for the developing embr yo. In alligators, albumen is secreted by the uterine tube and serves as a water rese rvoir as well as a chemical and mechanical barrier to the embryo (Milnes et al., 2002). The amnion, a fluid-filled sac surrounding the embryo, is derived from an out-pocketing of the embryo’s hindgut and also acts as a mechanical barrier while aiding in preventi ng dessication. Finally, the chorio-allantois, also derived from the embryo, surrounds the co ntents of the egg and serves as a waste repository (Pough et al., 2001). The leathery shell membrane forms from the fiber-secreting region of the uterus and surrounds all of the egg contents. Around this fibrous membrane are four distinct layers comprising the eggshell; the innermos t mamillary layer composed of calcium and organic material (20-29 m thick), a fibrous organic layer distingui shed by blebs that serve as attachment for calcite crystals (8 -12 m thick), a porous and heavily-fibered honeycomb layer (300-400 m thick), and the outer densely calcif ied layer (100-200 m thick) of numerous calcite crystals (Fergus on, 1982; Packard et al., 1982). Ferguson (1982) also documented constituent elements in 198 alligator eggs hells through energy dispersive X-ray analysis. In all samples, the major constituents included calcium and

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4 magnesium, the minor constituent was phosphorus and trace elements included copper, silicon, sodium, aluminum, iron, zinc, and manganese. Oviposition The ancestral mode of reproduction in verteb rates is believed to involve oviparity and external fertilization. Extant forms s till displaying this mode include all agnathan fishes, most teleost fishes, and many amphibian s. Oocyte retention is short in duration (hours to days) and involves the secretion of a gelatinous coat onto the egg, the egg’s transport through the reproductive tract, and finally, it exiting the maternal parent (Pough et al., 2002). The endocrine events related to ovulation, egg transport, and oviposition, are hypothesized to be closely associated (G uillette et al., 1991). However, in some extant oviparous reptiles displaying internal fertilization (turtles and some squamates), gestation length has increased, allowing time for shell calcification (Guillette et al., 1991). The further increase in gestation time in some squamates and mammals, is presumably ancestral to viviparity (Hogarth 1976). Because of the increasing temporal separation between ovulation and oviposition, it s been suggested that hormones influence ovulation and oviposition differe ntly between oviparous and egg-retaining/viviparous vertebrates. Previous research indicates that in birds, reptiles, and mammals, hormones, particularly prostaglandins (PGs) and ar ginine vasotocin (AVT) (or oxytocin for mammals) are important in stimulating the transport of eggs or embryos through the reproductive tract (Guillette et al., 1990), regardless of parity employed. It is hypothesized that oviposition in oviparous vert ebrates is stimulated within the central nervous system (CNS) (Owman et al., 1986). Specifically, the CNS induces follicular rupture causing the release of PGF2 which stimulates oviducal contractions, induces egg-laying behavior, and stim ulates arginine vasotoci n (AVT) release from the

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5 neurohypophysis (Guillette et al., 1990). In egg-retaining / vi viparous vertebrates, it is hypothesized that a cervix has developed wh ich does not respond to PGs or AVT during ovulation but does respond during oviposition du e to a lack of AVT / PG receptors or adrenergic stimulated inhibiti on of oviducal contraction (Koob et al., 1984; Challis et al., 1988). During early-mid gesta tion, it is believed that -adrenergic neur ons (innervating the oviduct) overcome hormonal control of the oviduct, causing relaxation and inhibiting contractions. During late gestation and initial post-partum, -adrenergic nerves begin degrading. In the viviparous lizard Sceloporus jarrovi Rooney et al. (1997) observed the constant innervation of the uterus thr oughout pregnancy and subs equent denervation during vitellogenesis. This pregnancy-asso ciated innervation s upports the hypothesized mammalian role of oviductal innervation in maintaining myometrial quiescence (Rooney et al., 1997). Environmental Perturbation to Shelling Prior to the late 1940’s, it was rare to find broken eggs in the nests of peregrine falcons ( Falco peregrinus ) and sparrowhawk ( Accipiter nisus ), but from 1950 on, shells from these species were commonly found broken or destroyed (Ratcliffe, 1973). Ratcliffe (1967) was the first to correl ate eggshell breakage and thinning with the widespread use of synthetic organic chemical s used as pesticides and in industry. In particular, Ratcliffe noted that organochlori nes were accumulating in the tissues of wild raptors concurrent with the post-war use of these compounds. Since these observations, several experimental studies demonstrated e ggshell thinning in relation to exposure to the pesticide DDT (dichlorodiphenyltrichloroeth ane) and its metabolites by disturbing physiological mechanisms related to calc ium (Anderson and Hickey, 1972; Ratcliffe, 1967; Laporte; 1982; Cooke, 1973), whic h makes up approximately 90% of the

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6 eggshell (Tyler and Geake, 1953). Before 1950, British peregrines had a mean clutch size of 3.5 eggs, with an aver age of 2.5 fledglings per nest. After 1950, northern England and southern Scotland yielded 1.5 and 1.7 fle dglings per nest, respectively, yet mean clutch size remained unchanged (Ratcliffe, 1973). Another parameter implemented to document eggshell changes over time was the ‘eggshell index,’ where the weight of the shell (mg) is divided by the product of the le ngth and width of the sh ell. Not all birds studied incurred a decrease in eggshell index (table 1.1). Table 1.1. Mean avian eggshell indices in Britain, prior to and after 1947 (modified from Ratcliffe, table 5, 1970). After Ratcliffe’s discoveries in Britai n, Anderson and Hickey (1972) studied over 23,000 eggshells from 25 species in North Am erica. Like Ratcliffe, Anderson and Hickey first determined if there was a signi ficant decrease in eggshell indices before 1947. Unlike Ratcliffe, they combined data by decades and noted that eggshell changes were rare before 1940. The golden eagle was th e only species that appeared to have no significant difference in shell i ndex before 1940, which was attributed to low sample size. In addition to eggshell index, eggshell thic kness was measured with a dial micrometer. Thickness measurements were compared with eggshell indices and statistically determined to be strongly correlated (p< 0.05, Anderson and Hickey, 1972). All thickness Species # eggs before 1947 # eggs 1947Mean index before 1947 Mean index after 1947 % change in index Peregrine 509 211 1.836 1.485 19.12% Sparrowhawk 298 279 1.423 1.178 17.22% Golden Eagle 84 27 3.146 2.834 9.90% Buzzard 83 96 4.97 4.92 0.46% Raven 222 205 1.121 1.111 0.89% Razorbill 46 33 2.339 2.350 0.90%

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7 measurements were performed on shells that had thinned or broken during incubation, as opposed to fully incubated eggshells that hatc hed and produced viable offspring. It was then determined that 9 out of 25 North Am erican species underwe nt shell thinning and shell weight decreases of more than 20%. These species included the peregrine falcon, marsh hawk, brown pelican, prairie falcon, cooper’s hawk, double-crested cormorant, black-crowned night heron, ba ld eagle, and osprey. The Role of Calcium Embryonic alligators are thought to obtai n calcium required for skeletogenesis from the eggshell primarily, with additional calcium obtained from yolk stores (Packard and Packard, 1984). Initially, alligators were thought to mobilize and deposit calcium similarly to other reptiles but c onversely, the processes are more similar to those of birds. Both birds and alligators deposit calcium in the yolk prior to embryogenesis. At hatching, the neonate has a relatively subs tantial calcium yolk store for growth and development. Embryonic turtles and squamate s, however, contain less yolk calcium at hatching than at oviposition (examples include Chelydra serpentina Packard et al., 1984b; Coluber constrictor Packard et al., 1984c; Amphibolorus barbatus Packard et al., 1985). Due to this mechanistic parallel wi th birds, calcium mobilization and uptake is thought to resemble birds more closely than that of other reptiles. In either case, calcium must traverse cellular barriers including the yolk-sac and chorioallantoic membrane (CAM) to gain access to the embryo. Since al ligators lay a highly calc ified shell, and the amount of calcium in the shell decreases throughout incubation, it is believed that calcium metabolism can be regulated along the CA M interface, similarly to that in birds (Romanoff, 1960; Reider et al., 1980).

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8 Calcium transport across the chorionic ectodermal cell membranes is believed to involve both mechanistic and regulatory events. Mechanisti cally, calcium translocation across the CAM can occur pericellularly or endo cytotically. There is little evidence for the pericellular basis, leaving endocytosis as the likely tran sport model (Terepka et al., 1976; Tuan and Zrike, 1978). In the latter model, calcium binds to surface membrane calcium-binding protein (CaBP) and the liga nd is encapsulated in pinocytic vesicles, which cross the ectoderm and fuse and empty into the serosal compartment. The function of CaBP and CAM calcium transport activity are probably linked and regulated by the presence of a transport substrate (the calcium rich eggshell) and vitamin K availability (Tuan 1987). In previous studies, the CAM of shell-less in vitr o cultures of chicken embryos failed to demonstrate developmentally -specific expression of calcium transport activity or CaBP (Dunn et al., 1981; Tuan, 1980, 1984). In addition, vitamin K serves as a cofactor producing the active form of CaBP. A vitamin K deficiency results in a reduction in gamma-glutamyl carboxylation coinci dent with a decrease in CAM calcium transport activity. Interestingly, when Ca BP mRNA is experimentally increased, CaBP level in ovo increases normally as well, sugge sting disruption prior to protein translation and potentially during transcrip tion. In various in vitro and in vivo studies (see review by Tuan, 1987), Ca2+-ATPase and carbonic anhydrase are also implicated in CAM calcium transport function. CAM extracts revealed the presence of Ca2+-ATPase, which was localized on the ectoderm through histochemist ry (Tuan et al., 1984), and also aids in calcium transport. Chemically inhibiting Ca2+-ATPase decreases calcium levels in the CAM (Akins and Tuan, 1993). Carbonic anhydras e (CA) is believed to provide local

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9 acidification in the ectoderm and prom ote dissolution of e ggshell calcite (CaCO3) in vivo. In doing so, CA can produce ionized calcium ready for uptake (Tuan and Zrike, 1978)

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10 CHAPTER 2 PARAMETERS OF ALLIGATOR EGGSHELLS FROM REFERENCE AND CONTAMINATED NORTH-CENTRAL FLORIDA LAKES Introduction Although once classified as endangered, alligator populations in many wetland regions of Florida are stable or increasing (Woodward et al., 1999). Habitat degradation and changing land-use patterns, however, threaten some populations and a better understanding of the biology of this species is needed (Abercrombie, 1989). Population stability is based on reducing death and recruiting new indi viduals to the population each year. Variables affecting either of these characteristics will have a dramatic impact on the stability, growth, or d ecline of alligator populations in Florida’s wetlands. For over two decades, the Florida Fish and Wildlife Conservation Commission (FWC) has documented the hatching success of alligators from various Florida lakes. Since 1993, alligators from La ke Woodruff, part of a National Wildlife Refuge, repeatedly exhibit the highest hatch rate s (77%-86%), when compared with rates documented from other Florida lakes, partic ularly Lakes Griffin (30%-32%) and Apopka (32%-52%) (Woodward et al., 1999 ). Lake Apopka has been subject to ag ricultural and municipal run-off, extensive he rbicide and pesticide use, a nd a major pesticide spill in 1980 that contained dicofol and DDT (dic hlorodiphenyltrichloroethane), among other compounds (Schelske and Brezonik, 1992). DDT degrades into two primary metabol ites, DDE and DDD (USEPA 1996). Of the metabolites, p, p’-DDE (specifically) ha s been associated with altering the

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11 physiological process of eggshell formation th at subsequently led to eggshell thinning and population declines of numerous avian spec ies, particularly ra ptors and shorebirds (see review by Lundholm, 1997). Approximately four years after the pesticide spill in 1980, a sharp decrease in alligator egg viab ility on Lake Apopka was documented by the FWC. This decline was hypothesized to be in response to the contamination event (Woodward et al., 1993). Conversely, no ma jor point source of pollution, such as a pesticide spill, has been reported on Lake Gr iffin, but this lake has received extensive agricultural runoff, municipal storm water r unoff, and currently exchanges water with former agricultural fields that are part of a restoration program to create emergent wetlands (see Marburger et al., 2002). Clutch es of alligator eggs obtained from Lake Griffin have also exhibited decreased hatch rates along with relative ly recent increased adult alligator mortality (Ross 2000; Schoeb et al., 2002). Hatch rates for Lakes Apopka and Griffin still remain lower than those of Lake Woodruff, part icularly throughout the duration of this study (Table 2.1), though the mechanism(s) by which this occurs remains unknown. Table 2-1 Mean alligator egg viability rates and sample size of clutches collected from three study areas and incubated under 32C in an artificial incubator, 1999, 2001-2003 (Woodward, pers. com.). Study Areas Year Lake Woodruff Lake Apopka Lake Griffin Mean N Mean N Mean N 1999 0.85 18 0.56 9 0.41 21 2001 0.79 16 0.53 15 0.55 19 2002 0.74 11 0.27 7 0.56 20 2003 0.90 7 0.31 11 0.72 17 A number of factors could contribute to embryonic mortality. One of these factors could be a change in eggshell structure or composition leading to altered eggshell

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12 function during incubation. The crocodilian oviduct more cl osely resembles birds than that of other oviparous rept iles (Palmer and Guillette, 1992 ). It is a heterogeneous structure consisting of seven regions. Func tionally and ultrastruc turally, the anterior uterus of the alligator resembles the avia n isthmus where eggshell membrane formation takes place. Endometrial glands located in this region produce proteinaceous fibers that are hypothesized to compose the eggshell membra ne. The posterior uterus is similar to the avian shell gland, which secretes the ca lcareous eggshell. Unlike birds, however, alligators ‘shell’ eggs of a clutch simultaneously whereas birds ovulate and ‘shell’ each egg of a clutch individually. To date, several studies indicate that the supply of calcium to the eggshell gland in ducks is not impeded by p,p’ DDE, but rather, this organochl orine contaminant disrupts calcium transport within the eggshell gland (Lundholm, 1997, 1990a, 1990b; Kolaja, 1977). In addition, p,p’-DDE inhibits Ca2+-ATPase or Ca2+-Mg2+-ATPase activity in the eggshell gland suggesting one mechanism fo r DDE-induced eggshell thinning. Calcium transport across the eggshell gland mucosa is coupled with both sodium and bicarbonate (Eastin and Spaziani, 1978; Pearson and Go ldner, 1973) and accordingly, the impaired movement of one of these ions can influe nce the movement of others (particularly calcium). Additionally, the prostaglandins PGF2 and PGE2 have been implicated in eggshell thinning. DDE disrupts the synthesis of these two prostaglandins, which reduces bicarbonate transport in the duc k shell gland lumen, in turn reducing calcium transport. Both PGF2 and PGE2 have previously been closely associated with eggshell formation in birds (Hammond et al., 1980).

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13 Alligators from Lake Apopka lay eggs with elevated concentrations of p,p’-DDE, and juveniles and adults in the population have elevated concentrations of this contaminant in their blood and eggs (Heinz et al., 1991; Guil lette et al., 1999) presumably due to eating p,p’-DDE-contamin ated food items (Rauschenberger et al., 2004). The application of the pesticide DDT to croplands began in 1945 in the United States, and continued until th e Environmental Protection Agen cy (EPA) restricted its use in 1972. This pesticide and its metabolit es are persistent, bioaccumulating, and biomagnifying in the food chain (see review, Gu illette et al., 2005). Due to the previous affiliation between DDT and its metabolites w ith avian eggshell thinning (Anderson and Hickey, 1972; Henny and Bennett, 1990; Hick ey 1969; Ratcliffe 1967), as well as the phylogenetic relationship (Gower 2002) and simila rity in egg shell formation (Palmer and Guillette, 1991, 1992) between birds and crocod ilians, we hypothesized that alligator eggs could undergo eggshell thinning as a result of p,p’-DDE exposure. To begin to test the hypothesis that or ganochlorines, such as p,p’-DDE, could contribute to higher mortality of alligator em bryos in several Florida wetlands through altered shell function, we examined alligat or eggshell structure and composition from clutches laid on three Florid a lakes. Morphometric data were collected as well as microscopic structural data (light and scanning electron microscopy). Materials and Methods Alligator eggs were collected from three north-central Florida freshwater lakes (lakes Woodruff, Griffin, Apopka) in June of 1999, 2001, 2002, and 2003. The eggs were transported to the University of Florid a and incubated until hatching. The shells of eggs that produced viable neonates were coll ected after hatching, st ored in whirlpacks,

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14 and frozen at –20 C for processing at a la ter date. Those eggs that did not produce viable neonates were not included in any analyses. Thickness Measurements Alligator eggshells are composed of an inner tough fibrous layer composed of protein fibers covered by an outer calcium rich crystalline layer. The hard outer layer was used to determine eggshell thickness and he reafter is referred to as the ‘eggshell’. Eggshells were rinsed of organic matter with tap water, air-dried at 20 C for 72 hours, and oven dried at 42 C for 90 minutes. Thickne ss measurements were taken with a dial caliper to the nearest millimeter at fi ve different regions on each egg. Three measurements were obtained from the shell th at would have covered the equatorial region of the egg and two measurements were obtaine d from the polar ends of each egg. Three eggs were measured from each clutch so that a clutch mean could be determined (Table 2.2). The clutch mean was then us ed in all statis tical analyses. Table 2-2 Number of clutches from which e ggshell thickness was examined each year of the study period from 3 lakes. (Note: 3 eggs/clutch were measured). In 2003, five clutches (shell fragments from 3 different eggs /clutch) from each lake were further analyzed for pore density and elemental composition (including heavy metals) and were examined by scanning electron microscopy (SEM). Year Lake Woodruff Lake Apopka Lake Griffin 1999 12 12 12 2001 11 8 10 2002 10 5 15 2003 9 5 10

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15 Pore Density Eggshell fragments were dyed with 0.15 ml of fast green in 30 mls of dH2O for 2 minutes and allowed to air-dry in the laboratory overnight. A 2.4 cm2 circular region of the egg was isolated from the equatorial re gion of each egg. Pores were counted with a dissection microscope and final va lues were converted to pores/cm2. Elemental Analyses (EDS) Energy Dispersive Spectral Analysis (EDS) was performe d to detect constituent elements in each eggshell sample. Briefly, EDS operates by submitting the sample to electron bombardment producing a range of Xrays of different energy levels that correspond with a particular element. Carbon, calcium, and oxygen values were averaged for each clutch and within each lake and were compared using ANOVA. (Elements detected below 0.10% are not include d in analyses, as this represents the lowest reliable quantitative detection limit for this analysis). Elemental Analyses (ICP) Inductively coupled plasma-optical emi ssion spectrophotometry (ICP-OES, Perkin Elmer 6100) was used to detect heavy meta ls. ICP-OES uses an argon plasma to generate measurable atomic emission. The emission lines for each atom are monitored at specific wavelengths. Standard metal solutions in 5% nitric acid were obtained (Fisher Scientific Inc.) and diluted to appropriate concentrations in plastic volumetric labware to avoid the problem of metals adhering to glass. The analyses of these standard solutions were used to generate calibration curves. A lligator eggshells were dissolved in 5% nitric acid (metal analysis grade). The resu lting solution was anal yzed under identical conditions as the standards and the shell con centrations calculated using the calibration curves. All standards and samples were analyzed in triplicate. Both analysts were blind

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16 to the sample status of each eggshell. Sufficient concentrations of aluminum and magnesium were detected a nd statistically analyzed ( ANOVA) to determine possible differences among lakes. Scanning Electron Microscopy (SEM) Two fragments from the equatorial region of each shell were desiccated, mounted on SEM stubs and sputter coated with gold. One fragment was viewed in cross section (200X and 5000X), and the other fragment was oriented to view the internal surface of the shell (250X, 450X, 4000X, 8000X). The sh ells were qualitatively characterized by their crystalline structure, shell membrane st ructure, relative fibe r and node presence, pore morphology, and layer density. Statistics Analysis of Variance (ANOVA) was used to compare data among lakes (Statview for Windows, version 5.0). Statisti cal significance was determined if p 0.05. If a significant difference was determined, Fisher’s post-hoc analyses were used for pair-wise comparisons. Results Eggshell Thickness Regarding shell thickness, there was no interaction among lakes and years combined (2-way ANOVA, p=0.5117). There was a significant difference in shell thickness among lakes in 1999 and 2003, but no di fferences were detected in 2001 and 2002. This variation could be due to small sample sizes for a given year and lake. In support of this concept, if data from all ye ars were combined for each lake, a significant difference among lakes was observed (p = 0.02; Fig. 1). As hypothesized, eggshells obtained from the more contaminated Lakes Apopka and Griffin, exhibited similar shell

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17 thickness, but contrary to our predictions, th ey had thicker eggshells than those obtained from Lake Woodruff (Apopka vs. Woodruf f, p = 0.0003; Griffin vs. Woodruff, p = 0.0021; Fig. 1). It must be noted that these thickness measurements were obtained from egg shells post hatch, and represent the th ickness remaining after embryonic development was complete. Pore Density There was no significant difference in eggs hell pore density among lakes (p = 0.55; Fig 2). Further, we detected no difference in pore density among clutches from Lakes Woodruff and Griffin, whereas there was a si gnificant difference among clutches from Lake Apopka (p = 0.01; Fig. 3). Elemental Analyses (EDS) There was a significant difference in cal cium, carbon, and oxygen concentrations in the eggshells obtained from the various lakes of this study (p = 0.005, p = 0.0006, p = 0.004, respectively). That is, we observed th at shell samples from Lake Woodruff were similar in percent calcium to those from Lake Apopka but significantl y greater than those from Lake Griffin (p = 0.0047; Fig. 4). Lake Woodruff eggshells c ontained significantly lower percent carbon than both Lakes A popka (p = 0.0129) and Griffin (p = 0.0002), which were similar (Fig. 4). Eggshells from Lakes Woodruff and Apopka contained similar percentages of oxygen and both exhib ited significantly higher levels than those recorded from shells from Lake Gri ffin (p = 0.001, p = 0.0250, respectively; Fig. 4). Elemental Analyses (ICP) Lead, arsenic, iron, aluminum, zinc, nickel, manganese, chromium, calcium, magnesium, and cadmium were examined in each sample. Only calcium, aluminum, and magnesium were detectable (mgs/liter). There was a significant difference in aluminum

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18 and magnesium levels detected in the she lls from the three lakes (p = 0.025, p = 0.020, respectively). Eggshells obtained from La ke Woodruff contained significantly more aluminum than shells from Lakes Apopka (p = 0.009) and Griffin (p = 0.047), which were similar (Fig. 5). Shells obtained from Lake Griffi n contained significantly less magnesium when compared to shells from e ither of the other two lakes, Lake Woodruff (p = 0.014) or Lake Apopka (p = 0.016, Fig. 4). Scanning Electron Microscopy (SEM) A qualitative analysis of the structure of the eggshells using scanning electron microscopy revealed several interesting diffe rences when shells were compared among lakes. An irregular network of fibers is present on the inner surface of the calcareous eggshells from all three lakes, although their relative abundan ce varies (Fig 6 & 7). Eggs from Lake Woodruff can be categorized as having several intermediate few and no fibers at all (Fig. 6). They are randomly orient ated and overlap extensively. The majority of eggs from Lakes Gri ffin and Apopka contained few or no fibers at all. Several nodes are present and give each fiber a coated app earance (Fig. 8). Little is known about the make up of these fibers; howev er, it has been suggested that these nodes are made of glycosaminoglycans (GAG) (Ferguson 1982). Func tionally, they can represent a template for calcified crystalline growth similar to that in mammals where hydroxyapatite crystals give rise to bone and dentin (Brown 1975; Jenkins 1978). The fibrous arrangement and size and amount of nodes appear more simila r between samples from Lakes Apopka and Griffin (Fig. 7). Shells from La ke Woodruff, however, appear to have fewer and larger nodes (Fig. 8). There were also similarities in pore-struct ure of shells obtained from the three lakes. Pores were characteri zed by crater-like concentric ri ngs that develop on the outer-

PAGE 27

19 surface of the eggshell (Fig. 9a,d,g). The pore orifice leads to an irregular shaped cavity that extends through the calcareous shell (F ig. 9b,e,h). Within each pore cavity, the crystalline structure is apparent, though the patterns can vary among lakes (9c,f,i). SEM cross-sectional views revealed the honeycomb layer previously described for alligator eggshells (see Ferguson, 1982). Inte restingly, we observed that all eggshells from Lake Woodruff lacked the organic por tion of the honeycomb region and contained a smoother, denser region of crystallized cal cium (Fig. 10), whereas eggshells from lakes Griffin and Apopka displayed a region similar to that described by Ferguson (Fig. 10). This difference in eggshell structure was une xpected. Finally, the structural make-up of the inner eggshells appeared more dense and similar in Lakes Woodruff and Griffin when compared to Lake Apopka (Fig. 11). Discussion and Conclusions An analysis of eggshell structure and co mposition revealed that variation existed among alligator populations from central Florida lakes. Some of the variation noted appears to be correlated to contaminant e xposure but other aspects are not explained by the environmental data collect ed during this study. We observed that post hatching eggshell thickness is significantly less from eggshells from Lake Woodruff versus shells from Lakes Griffin and Apopka. Additionall y, we observed a major difference in the post hatching shell, in that shells from La ke Woodruff lacked the organic portion of the honeycomb region that lies below the dense out er cortical region composed of tightly fitted calcium crystals. Finally, we observed that shells from Lake Woodruff had higher concentrations of aluminum when compared to eggshells from other lakes. Previous studies of birds exposed to organochlorine contaminants reported thinner eggshells (for review, see Lundholm, 1997). Unlike our st udy, previous studies have examined

PAGE 28

20 eggshells that either had nonviable embryos or were obtai ned during early incubation. We have not been able, to date, to identify studies that were performed similar to ours, where eggshells were obtained after successful incubation. We hypothesize that Lake Woodruff embr yos exhibit elevated gas exchange producing larger neonates. Elevated gas ex change would presumably generate greater CO2 levels at the shell interface with the pores and honeycomb region maximizing calcium mobilization via the ge neration of carbonic acid. The lack of the organic portion in the honeycomb region can be a result of increased carbonic aci d production and its subsequent dissolution of the e ggshell. Milnes et al., (submitted) observed that mean egg mass was greater for eggs from Lake Woodruff (86.9 + 0.68g) compared to Lake Apopka (77.7 + 0.72g) in 1999. When adjusted for egg mass, hatchling body mass and SVL varied between lakes, with Woodruff hatc hlings having greater mean body mass when incubated at 32C and 33.5 oC, and greater SVL when incubated at 32oC (Milnes et al., submitted). These data suggest that even with differences in egg mass accounted for, neonates from Lake Woodruff are larger. We have previously observed that Lake Woodruff neonates are more likely to survive compared to neonates from Lake Apopka (Guillette et. al., 1994). We s uggest that vigorous, healthy neonates come from healthy embryos that utilize more calcium in the eggs hell and could have la rger chorioallantoic membranes. Future studies need to exam ine the hypothesis that embryos from Lake Woodruff generate more CO2 and greater carbonic acid leve ls that etch the inner surface of the eggshell more effectively. The lack of a honeycomb region in the out er calcium eggshell obtained from Lake Woodruff was unexpected. Again, it should be noted that thes e shells were obtained after

PAGE 29

21 successful incubation so the la ck of this region may be a post-oviposition modification, not an original structural difference. The honeycomb regi on of the eggshell lies between the outer densely calcified layer and the orga nic layer and is composed of both a fibrous organic matrix and calcite crystals (see Packard and Packard 1984). This honeycomb region would be expected to trap CO2 and water released from the chorioallantoic membrane, which lines the inner surface of the proteinaceous shell. We note above that the eggshells obtained from Lake Woodruff ne onates were thinner, possibly due to the calcified portion of the honeycomb region bei ng eroded (and mobilized to the embryo) to a greater extent than that of the same region in eggshells fr om other lakes. Additionally, Woodruff eggshells contained less carbon, as shown through en ergy dispersive analysis, which may also contribute to th eir respective thinner shells. Eggshells from Lake Woodruff also had gr eater concentrations of aluminum, a metal not required by living systems. Since al uminum is the most abundant metal in the earth’s crust (Rengal 2002), it is possible that relatively hi gher aluminum levels are natural since Lake Woodruff has undergone le ss anthropogenic influences than the other lakes. Potentially, lower aluminum levels may impede normal biological functions in the biota underexposed. Lake Griffi n eggshells contained more magnesium than shells from other lakes. This mineral is necessary for calcium metabolism and could be elevated in Lake Griffin shells due to a lack of proper calcium metabolism by the embryo. The concentrations of these minera ls could have biological impact but future studies would be required to determine if the levels measured influence alligator embryonic viability and eggshell development.

PAGE 30

22 It’s important to note that all of the sa mples of alligator eggshells were from posthatching and viable neonates, whereas bird eg gshells had to be cracked or show evidence of housing a dead embryo to be included in an alyses. It is possibl e that bird eggshells were collected at different stages of em bryonic development, and would therefore be expected to have different amounts of cal cium mobilized from the shell, thereby influencing shell thickness. This could explain why not all species were seemingly influenced by p,p’-DDE. There can also be inte rspecific and/or intraspecific variation in when, gestationally, shells begin cracking. Perhaps species that incur comparatively increased gestation times or larger clutches are more impacted by compromised calcium transport then those with shorter gestation times and smaller clutches. The same could occur within species, depending on the maturity and/or overall health of the female parent. Some species shells’ may be more prone to swelling (from ambient humidity) or dessication, which influences embryonic viabil ity and ultimately biases the sample collected. To avoid these biases, future st udies could focus on shells from post-hatching and viable avian neonates. Lastly, hormone s are known to disrupt transport channels indirectly related to calcium mobilization a nd can be another source of altered calcium transport. These potential calcium transport offenders may not be mutually exclusive of one another, thus compounding cal cium transport further. Both bird and alligator eggshells could se rve as potential biomarkers of healthy neonates since the embryos completed develo pment within them. Conversely, it would be interesting to compare alligator shells fr om early incubation to bird shells of early incubation. Shells sampled from relatively pa rallel incubation window s could be directly compared to determine relative constituents, their mobilization, and the influence of

PAGE 31

23 environmental factors on these parameters. Regardless if the shells are from early or late incubation, it could be worthy to examine the effects of other abiotic factors such as substrate, temperature, or altitude on eggs hell integrity of DDE exposed and unexposed species. 0.32 0.33 0.34 0.35 0.36 0.37 0.38 0.39 0.40 0.41 0.42 WoodruffApopkaGriffin LakeAverage equatorial thickness (mm) a b b Figure 2-1 The mean (+/1s.e.) equatori al eggshell thickness among lakes and years (1999,2001,2002,2003). (Different superscr ipts indicate significant differences between lakes).

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24 5 10 15 20 25 WoodruffApopkaGriffin LakeEggshell pores/cm2 a a a Figure 2-2 The mean (+/1s.e.) number of eggshell pores among lakes in 2003. (similar superscripts denote no signifi cant difference among lakes). 0 5 10 15 20 25 30 35 40Eggshell Pores/cm2/clutch Woodruff Apopka Griffin Figure 2-3 The mean (+/1s.e.) number of alligator eggshell pores per clutch, per cm2, among lakes in 2003. Note the variation a bout the means, particularly from Lake Apopka.

PAGE 33

25 0 10 20 30 40 50 60 70OxygenCalciumCarbonElementElemental average (% / shell ) Woodruff Apopka Griffin aa a b a a b a b Figure 2-4 The mean (+/1s.e.) percent of the three most abundant elements in alligator eggshells detected by Energy Dispersive Analysis (EDS), (n=3 shells/clutch, 5 clutches/lake; Different superscripts denote si gnificant differences among lakes). 0 500 1000 1500 2000 2500 3000 Aluminum Magnesium ElementElement Mass (g/g eggshell) Woodruff Apopka Griffiin a b b a b b Figure 2-5 The mean (+/1s.e.) percent of the three most abundant elements in alligator eggshells detected by Energy Dispersive Analysis (EDS),(n=3 shells/clutch, 5 clutches/lake; Different superscripts denote si gnificant differencesamong lakes).

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26 ab cd Figure 2-6 Scanning electron micrographs of th e inner surface of Lake Woodruff alligator eggshells (250X). Note the variation in relative fiber presence, a) no fibers b) few c) intermediate d) several.

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27 Figure 2-7 Scanning electron micrographs of the relative amount of shell fibe rs (F) in Apopka (a.b) and Griffin (c,d).Shells from both lakes displayed few to no fibers (250X, n = 3 shells/clutch, 5 clutches / lake), relativ e to some Woodruff shells. a d c

PAGE 36

28 abc Figure 2-8 SEM images of shell membranes from a) Woodruff, b) Apopka, c) Griffin in 2003. Note the numerous fibers and the relative size of the nodes (N) on Woodruff vs. Apopka and Griffin (8000x) (n = 3 shells/clutch, 5 clutches/lake).

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29 ab c def i h g Figure 2-9 SEM images of eggshell pore cavities from 2003 eggshells. W oodruff (a,b,c), a= cross section (200x), b= outer g shell surface (200x), c= internal view of pore. Apopka (d,e,f): d=cross section (200x), e= outer shell surface (200x), f=intern al view of pore (8000x). Griffin ( g,h,i,): g=cross section (200x), h=outer shell surface (200x), I=in ternal view of pore, (n= 3 shells/clutch, 5 clutches/lake).

PAGE 38

30 Figure 2-10 SEM Cross-sectional view s at 5000X from lakes a) Woodruff, b) Apopka, c) Griffin (2003). Note th e smooth surface layer (a) versus jagged and multi-edged surfaces (b & c) (n= 3 she lls/clutch, 5 clutc hes per lake). a b c

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31 Figure 2-11 SEM image of the calcium crystal line structure on the inner e ggshell surface, a) Woodruff, b) Apopka, c) Griffin 4000X, n = 3 shells/clutch, 5 clutches/lake).

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32 LIST OF REFERENCES Abercrombie, C.L., III. 1989 Population dyna mics of the American alligator. In: Crocodiles, their Ecology, Management and Conservation. IUCN Publ. New Series, No. 2-88032-987-6. Gl and, Switzerland. pp 1-16. Akins, R.E., Tuan, R.S. 1993. Transepith elial calcium transp ort in the chick chorioallantoic membrane, II. J. Cell Science. 105: 381-388. Anderson, D.W., Hickey, J.J., 1972. Oological data on egg and breeding characteristics of brown pelicans. Wilson Bull. 82: 14-28. Austin, HB. 1989. The effects of estradiol and testosterone on mullerian-duct regression in the American alligator ( Alligator mississippiensis ).Gen Comp Endocrinol, Dec., 76(3): 461-72. Berg H, Modig C, Olsson PE. 2004. 17beta-estradiol induced v itellogenesis is inhibited by cortisol at the post-transcriptional level in Arctic char ( Salvelinus alpinus ). Reprod Biol Endocrinol. Sep.2(1): 62. Brown, C.H.1975. Structural Materials in Animals. London: Pittman and Sons. Challis, J.R.G. and D.M. Olson. Parturition. 1988. In: The Physiology of Reproduction, E.Knobil and J. Neill, eds.,New York: Raven, pp 2177-2216. Cooke, A.S. 1973. Shell thinning in avian eggs by environmental pollutants. Environ. Pollut 4: 85-152. Dunn, B., Graves, J., Fitzharris, T. 1981. Active calcium transport in the chick chorioallantoic membrane requires interacti on with the shell membrane and/or shell calcium. Dev. Biol., 88: 259-268. Eastin, W.C., Spaziani, E. 1978. On the mech anism of calcium secretion in the avian shell gland (uterus). Biol. Reprod. 19: 505-518. Ferguson, M.W.J., 1982. The structure and orga nization of the eggshell and embryonic membranes of Alligator missippippiensis Trans. Zool. Soc., London. 36: 99-152. Gower, D.J., Walker, A.D., 2002. New data on th e braincase of the aetosaurian archosaur (Reptilia Diapsida) Stagonolepis robertsoni Agassiz Zoological Journal of the Linnean Society, 136(1): p. 7

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33 Guillette, L.J. Jr. 1993. The evolution of vivipa rity in lizards. BioScience, 43(11): 742751. Guillette, L.J.,Kools, S. Gunderson,M.P. in pr ess. DDT and its analogues: New insights into their endocrine disrup tive effects on wildlife. In: Endocrine disrupters: Biological basis for health effects in wildlife. D.O. Norris and J.A. Carr, Eds. Oxford University Press, NY. Guillette, L.J., Gross, T., Matter, J., Palmer, B. 1990. Arginine vasotocin-induced in vitro prostaglandin synthesis by the reproduc tive tract of the viviparous lizard Sceloporus jarrovi Prostaglandins 37: 39-51. Guillette, L.J., Dubois, D.H., Cree, A. 1991. Prostaglandins, oviducal function and parturient behavior in nonmammalian vert ebrates. American Journal of Physiology 260: R854-R861. Guillette, L .J., Jr., Gross, T.S., Masson, G.R ., Matter, J.M., Percival, H.F., Woodward, A.R. 1994. Developmental abnormalities of the gonad and abnormal sex hormone concentration in juvenile al ligators from contaminated a nd control lakes in Florida. Environ. Health Perspect. 102(8): 680-688. Guillette L.J. Jr., Woodward AR, Crain DA, Masson GR, Palmer BD, Cox MC, YouXiang Q, Orlando EF. 1997. The reproductive cycle of the female American alligator, ( Alligator mississippiensis ).Gen Comp Endocrinol. Oct.108(1): 87-101. Guillette, L. J., Jr., Brock, J. W., Roone y, A. A. & Woodward, A. R. 1999. Serum concentrations of various environmental co ntaminants and their relationship to sex steroid concentrations in juvenile American alligators. Arch. Environ. Contam. Toxicol. 36: 447-455. Hammond, R. W., D. M. Olson, R. B. Frenkel, H. V. Biellier, and F. Hertelendy, 1980. Prostaglandins and steroid hormones in plasma and ovarian follicles during the ovulation cycle of the domestic hen ( Gallus domesticus ). Gen. Comp. Endocrinol.42: 195–202. Heinz, G. H., Percival, H. F. & Jennings, M. L. 1991. Contaminants in American Alligator eggs from Lakes Apopka, Gri ffin, and Okeechobee, Florida. Environ. Monit. Assess. 16: 277-285. Henny, C.J., Bennett, J.K., 1990. Comparison of br eaking strength and shell thickness as Evaluators of white-faced ibis eggshell quality. Environ. Tox. and Chem. 9: 797-805. Hickey, J.J. 1969. The Peregrine Falcon P opulations: Their Biology and Decline. Madison, WI: University of Wisconsin Press. 596pp. Hogarth, P.J. 1976. Viviparity. Studi es in Biology, London: Arnold. 75.

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34 Jenkins, G.N. 1978. The Physiology and Biochemistry of the Mouth .4th ed. Oxford: Blackwell Scientific Publications. Kolaja, G.J., Hinton, D.E., 1977. In vitro inhibi tion of microsomal calcium ATPase from eggshell gland of mallard duck. Bull. Environ. Contam. Toxicol. 17: 591-594. Koob TJ, Laffan JJ, Callard IP. 1984. Effects of relaxin and insulin on reproductive tract size and early fetal loss in Squalus acanthias. Biol Reprod. Sep.31(2): 231-8. Laporte, P. 1982. Organochlorine residues and eggshell measurements of great blue heron eggs from Quebec. Co lon. Waterbirds 5: 95-103. Lundholm, C.E. 1990a. The eggshell thinning action of acetazolamide; relation to the binding of calcium and carbonic anhydras e activity of shell gland homogenate. Comp. Biochem. Physiol. 95C(1): 85-89. Lundholm, C.E., 1990b. The distribution of cal modulin in the mucosa of the avian oviduct and the effect of p,p’-DDE on some of its metabolic parameters. Comp. Biochem. Physiol. 96C(2): 321-326. Lundholm, C.E. 1997. DDE-induced eggshell thi nning in birds: Effects of p,p’-DDE on the calcium and prostaglandin metabolism of the eggshell gland. Review. Comp. Biol. Physiol. Vol. 118C(2): 113-128. Marburger, J. E., Johnson, W. E., Gross, T. S., Douglas, D. R. & Di, J. 2002. Residual organochlorine pesticides in soils and fish from wetland restoration areas in central Florida, USA. Wetlands 22: 705-711. Milnes MR, Woodward AR, Rooney AA, Guillette LJ. 2002. Plasma steroid concentrations in relation to size and age in juvenile alligators from two Florida lakes.Comp. Biochem. Physiol., A Mol. Integr. Physiol. Apr 131(4): 923-30. Milnes, M.R., D.S. Bermudez, T.A. Bryan, M.P. Gunderson, L.J. Guillette, Jr. submitted. Environmental contaminants alter neonata l development and endocrine function in Alligator mississippienesis Biol. Repro. Owman C, Stjernquist M, Helm G, Ka nnisto P, Sjoberg NO, Sundler F. 1986. Comparative histochemical distribution of nerve fibres storing noradrenaline and neuropeptide Y (NPY) in human ovary, fallo pian tube, and uter us. Med Biol. 64(23): 57-65. Packard, M.J., Packard, G.C., Boardman, T.J. 1982. Structure of eggshells and water relations of reptilian eggs Herpetologica 38: 136-155. Packard, M.J., Packard, G.C. 1984. Compara tive aspects of calcium metabolism in embryonic reptiles and birds. Respiration and metabolism of embryonic vertebrates. Junk Pub lishers, London, pp. 155-179.

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35 Packard, M.J., Short, T.M., Packard, G.C., Gorell, T.A. 1984a. Sources of calcium for embryonic development in eggs of the snapping turtle, Chelydra serpentina J. Experimental Zoology 230: 81-87. Packard, M.J., Packard, G.C., Gutzke, W.H.N. 1984b. Calcium metabolism in embryos of the oviparous snake, Coluber constrictor J. Exp. Biol. 110: 99-112. Packard, M.J., Packard, G.C., Miller, J.D., Jones, M.E., Gutzke, W.H.N. 1985. Calcium mobilization, water balance, and growth in embryos of the agamid lizard, Amphibolurus barbatus J. Experimental Zoology. 235: 349-357. Palmer, B. & Guillette, L. J., Jr. 1991. Ov iductal proteins and their influence on embryonic development in birds and rept iles. In: Environmental Influences on Avian and Reptilian Embryonic Development, D. Deeming & M. Ferguson, eds. Cambridge: Cambridge Univ. Press. pp. 29-46 Palmer, B. D. & Guillette, L. J., Jr. 1992. Allig ators provide evidence for the evolution of an archosaurian mode of ovipar ity. Biology of Reproduction 46: 39-47. Pearson, T.W., Goldner, A.M. 1973. Calcium tr ansport across avian uterus. 1. Effects on elecrtrolyte substitution. Am. J. Physiol. 225: 1505-1512. Pough, F.H., R.M. Andrews, J.E. Cadle, M. L. Crump, A.H. Savitzky, and K.D. Wells. 2001. Herpetology, 2nd ed. Upper Saddle River, New Jersey. Prentice-Hall, Inc 612pp. Ratcliffe, D.A., 1967. Decrease in eggshell weig ht in certain birds of prey. Nature 215: 208-210. Ratcliffe, D.A., 1973 Studies of the recent breedi ng success of the peregrine, Falco peregrinus J. Reprod. Fert. (Suppl.) 19: 337–389. Rauschenberger, R. H., Wiebe, J. J., Buckland, J. E., Smith, J. T., Sepulveda, M. S. & Gross, T. S. 2004. Achieving environmenta lly relevant organochlorine pesticide concentrations in eggs through maternal exposure in Alligator mississippiensis Mar. Environ. Res. 58: 851-856. Reider, E., Gay, C.V., Schraer, H. 1980. Autoradiographic localization of carbonic anhydrase in the developing chorioallant oic membrane. Anat. Embryol. 159: 17-31. Rengal, Z. 2004. Aluminium cycling in th e soil-plant-animal-human continuum. Biometals, Dec;17(6): 669-89. Romanoff, A.L., 1960. The Avian Embryo. New York: MacMillan. Rooney, A.A., Donald, J.A., Guillette, L.J.Jr. 1997. Adrenergic and peptidergic innervation of the oviduct of Sceloporus jarrovi during the reproductive cycle. Journal of Experimental Zoology. 278: 45-52.

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36 Ross, P. 2000. Technical Report: Effect of toxi c algae on alligators and egg development. Submitted to Florida Fish and Wildlife Consevation Commission, project no. FL 03. Schelske, C.L., Brezonik, P. 1992. Can Lake Apopka Be Restored? Restoration of Aquatic Systems, Nat’l Academic Press, Washington, D.C., pp. 393-398 Schoeb, T. R., Heaton-Jones, T. G., Clemm ons, R. M., Carbonnea u, D. A., Woodward, A. R., Shelton, D. & Poppenga, R. H. 2002. Clinical and necropsy findings associated with increased mortality am ong American alligators of Lake Griffin, Florida. J. Wildl. Dis. 38: 320-337. Shine, R. 1983. Reptilian reproductive mode s: The oviparity-viviparity continuum. Herpetologica 39: 1-8. Shine, R. 1985. The evolution of viviparity in reptiles: An ecol ogical analysis. In: Biology of the Reptilia. C. Gans and F. Billett, eds. New York. John Wiley and Sons 15: 605-694 Terepka, A.R., Coleman, J.R., Armbrecht, H.J., Gunter, T.E., 1976. Transcellular Transport of Calcium. In: Calcium in Bi ological Systems. Sy mp. Soc. Exp. Biol. 30: 117-140. Tuan, R.S., 1987. Mechanism and regulation of calcium transport by the chick embryonic membrane. J. Experimental Zoology 1: 1-13. Tuan, R.S., Knowles, K.A. 1984. Calcium-ac tivated ATPase of the chick embryonic chorioallantoic membrane. J. Bi ological Chemistry 259(5): 2754-2763. Tuan, R.S., 1980. Calcium transport and rela ted functions in the chorioallantoic membrane of cultured shell-less chick embryos. Dev Biol. 74: 196-204. Tuan, R.S., Zrike, J., 1978. Functional invo lvement of carbonic anhydrase in calcium transport of the chick chorioallant oic membrane. Biochem. J. 176: 67-74. Tyler, C. and F.H. Geake, 1953. Studies on shel ls of ratite birds. Proc. Zool. Soc. Lond., 133: 201-243. United States Environmental Protection Agency (USEPA). 1996. Method 3630, Revision C, Washington DC, USA. Woodward, A.R., Percival, H.F., Jennings, M.L. Moore, C.T., 1993. Low clutch viability of American alligators on Lake Apopka. Florida Scientist 56(1): 52-63. Woodward, A.R., Bermudez, D.S., Carbonn eau, D.A. 1999. A preliminary report on alligator clutch viability in Florida. Florida Fish and Wildlife Conservation Commission. Gainesville, FL.

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37 BIOGRAPHICAL SKETCH Teresa Bryan received her Bachelor of Science degree in wildlife ecology and conservation from the University of Flor ida, May, 2000. She worked as a laboratory technician in a wildlife reproductive biology lab for one year before being accepted into graduate school at UF under Louis J. Guillette After receiving her Master of Science degree, she plans to purs ue doctoral research in vertebrate reproduction.


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Title: Morphological and Constituent Analyses of American Alligator (Alligator mississippiensis) Eggshells from Contaminated and Reference Lakes
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Permanent Link: http://ufdc.ufl.edu/UFE0010640/00001

Material Information

Title: Morphological and Constituent Analyses of American Alligator (Alligator mississippiensis) Eggshells from Contaminated and Reference Lakes
Physical Description: Mixed Material
Copyright Date: 2008

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Source Institution: University of Florida
Holding Location: University of Florida
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MORPHOLOGICAL AND CONSTITUENT ANALYSES OF AMERICAN
ALLIGATOR (Alligator mississippiensis) EGGSHELLS FROM CONTAMINATED
AND REFERENCE LAKES














By

TERESA A. BRYAN


A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY
OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR
THE DEGREE OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2005

































Copyright 2005

by

Teresa A. Bryan















TABLE OF CONTENTS

page

L IS T O F T A B L E S ................................................. ................................. ..................... v

LIST OF FIGURES .................................................. ............................ vi

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

CHAPTER

1 A REVIEW OF REPRODUCTIVE BIOLOGY AND SHELL FORMATION IN
R E PTILE S .............. ..............................................................1......................

In tro d u ctio n .................................................................................................. ............... 1
Oviducal Anatomy ......................... ........... ...............................2
T he A m niotic E gg ............. .. .................. .................. ............... ...... ... ... ............. 3
O v ip o sitio n ........................................................................................ .................... .... 4
Environm ental Perturbation to Shelling .................................................. ...............5...
The Role of Calcium ........................ .. ........... ...............................

2 PARAMETERS OF ALLIGATOR EGGSHELLS FROM REFERENCE AND
CONTAMINATED NORTH-CENTRAL FLORIDA LAKES .............................. 10

In tro d u ctio n ............................................................................................................... .. 1 0
M materials and M ethods .. ..................................................................... ............... 13
T hickness M easurem ents....................................... ...................... ............... 14
P o re D e n sity ........................................................................................................ 1 5
E lem ental A nalyses (E D S) ..................................... ..................... ............... 15
E lem ental A nalyses (IC P ) .............................................................. ............... 15
Scanning Electron M icroscopy (SEM )........................................... ................ 16
S statistic s .......................................................................................................... . 1 6
R e su lts....................................................................................................... ....... .. 16
Eggshell Thickness ................................................... ............. 16
P o re D e n sity ........................................................................................................ 1 7
E lem ental A nalyses (E D S) ..................................... ..................... ............... 17
E lem ental A nalyses (IC P ) .............................................................. ............... 17
Scanning Electron M icroscopy (SEM )........................................... ................ 18
D discussion and C conclusions ....................................... ........................ ............... 19










L IST O F R E FE R E N C E S ... ......................................................................... ................ 32

BIOGRAPHICAL SKETCH ...................................................... 37















LIST OF TABLES


Table page

1-1 Mean avian eggshell indices in Britain, prior to and after 1947 (modified from
R atcliffe, table 5, 1970) ....................................................................................... 6

2-1 Mean alligator egg viability rates and sample size of clutches collected from
three study areas and incubated under 320C in an artificial incubator, 1999,
2001-2003 (W oodw ard, pers com .)..................................................... ................ 11

2-2 Number of clutches from which eggshell thickness was examined each year of
the study period from 3 lakes. (Note: 3 eggs/clutch were measured) ...................14















LIST OF FIGURES


Figure page

2-1 The mean (+/- Is.e.) equatorial eggshell thickness among lakes and years
(1999,2001,2002,2003). (Different superscripts indicate significant differences
b etw een lak es) .......................................................................................................... 2 3

2-2 The mean (+/- Is.e.) number of eggshell pores among lakes in 2003. (similar
superscripts denote no significant difference among lakes)................................24

2-3 The mean (+/- Is.e.) number of alligator eggshell pores per clutch, per cm2,
am ong lakes in 2003 .... ................................................................... .............. 24

2-4 The mean (+/- Is.e.) percent of the three most abundant elements in alligator
eggshells detected by Energy Dispersive Analysis (EDS)..................................25

2-5 The mean (+/- Is.e.) percent of the three most abundant elements in alligator
eggshells detected by Energy Dispersive Analysis (EDS)..................................25

2-6 Scanning electron micrographs of the inner surface of Lake Woodruff alligator
eg g sh ells (2 50X ) ...................................................................................................... 2 6

2-7 Scanning electron micrographs of the relative amount of shell fibers (F) in
A popka (a.b)A nd G riffin (c,d) .................................... ..................... ................ 27

2-8 SEM images of shell membranes from a) Woodruff, b) Apopka, c) Griffin in
2 0 0 3 ........................................................................................................ ........ .. 2 8

2-9 SEM images of eggshell pore cavities from 2003 eggshells.................29

2-10 SEM Cross-sectional views at 5000X from lakes.................................. ............... 30

2-11 SEM image of the calcium crystalline structure on the inner eggshell surface .......31















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

MORPHOLOGICAL AND CONSTITUENT ANALYSES OF AMERICAN
ALLIGATOR (Alligator mississippiensis) EGGSHELLS FROM CONTAMINATED
AND REFERENCE LAKES

By

TERESA A. BRYAN

August 2005

Chair: Louis J. Guillette, Jr.
Major Department: Zoology

Alligator eggs were collected from one polluted and two reference north-central

Florida lakes. The eggs were artificially incubated and their shells collected after the

neonates hatched. Thickness measurements were taken from multiple eggs and clutches

within each lake over a four year period (1999, 2001, 2002, 2003). In 2003, eggshells

from three eggs per clutch and five clutches per lake were also analyzed for constituent

make-up, pore density, and basic morphology. Constituents were determined by both

inductively-coupled plasma spectroscopy (ICP) and energy-dispersive spectral analysis

(EDS). Pore density was assessed using light microscopy and the morphology described

through the use of electron microscopy. Egg mass and neonate morphometrics were

recorded.

We determined that shells from the reference lake were thinner than those from the

contaminated lakes. Constituent analyses and morphology varied among the lakes. There









was no significant difference in pore density among the lakes. Neonates from the

reference lake were larger than those from the polluted lakes.

We determined that eggshells from the reference lake were comparatively thinner,

yielded less calcium, and lacked the fibrous portion of a region of the shell potentially to

support the larger neonates. Comparatively increased calcium mobilization from the

eggshell during incubation would be critical for proper skeletogenesis and other calcium-

dependent functions in the larger hatchlings.














CHAPTER 1
A REVIEW OF REPRODUCTIVE BIOLOGY AND SHELL FORMATION IN
REPTILES

Introduction

Modes of reproduction vary among vertebrates. In reptiles, there were initially

three reproductive modes described: viviparity, the production of live offspring,

ovoviviparity, egg retention within the uterus and subsequent live birth, and the most

common, oviparity, the laying of eggs containing relatively undeveloped embryos outside

the body. More contemporary research suggests that ovoviviparity is not a discrete

reproductive mode, but instead an evolutionarily transient period between oviparity and

viviparity (Guillette, 1993). Hence, species that retain eggs in-utero until late in

embryonic development are considered oviparous, whereas species with a placenta are

viviparous, thus abandoning the term "ovoviviparous" in reptiles. Oviparous reptiles lay

an amniotic egg that develops within the oviduct and it is this structure that creates an

environment (the eggshell and its contents) capable of providing for the needs of the

developing embryo. In viviparous species, the embryo is retained within the uterus until

development is complete (Guillette, 1993). Therefore, the placenta must be present and

provide the young with the ability to uptake nutrients, exchange gases and ions for

respiration and waste removal, among other biological functions necessary for embryonic

development. All vertebrate classes, excluding the Agnathans and the Aves, contain

species that utilize this reproductive mode. Viviparity has evolved nearly 100 times in









reptiles, though only in lizards and snakes, and has not been reported in any species of

turtles, crocodilians, or tuatara (Shine, 1983, 1985).

Oviducal Anatomy

As with all amniote female vertebrates, the Crocodilian oviduct (which includes the

entire reproductive tract) is derived from the embryonic Mullerian duct (Austin, 1989).

The oviduct undergoes regional specialization as the female sexually matures. At

maturation, the alligator oviduct consists of seven anatomically distinct regions, starting

anteriorly with the ampullae, followed by the infundibulum, uterine tube, uterotubular

junction, anterior and posterior uterus, and vagina. Functionally, the oviduct is separated

into three major regions, the uterine tube that secretes albumen (Palmer and Guillette,

1991), the anterior fiber-secreting region of the uterus, and the posterior calcium-

secreting region of the uterus (Palmer and Guillette, 1992). Structurally and functionally,

the anterior and posterior uterus of the alligator resembles those of birds and not of other

reptiles. The anterior fiber-secreting uterus is similar to the avian isthmus, whereas the

posterior calcium-secreting uterus is similar to the avian shell gland. Endometrial glands

located at the anterior and posterior portions of the alligator uterus share similar cell types

and secretary products with birds, and not of other reptiles (Palmer and Guillette, 1992).

The glands of the anterior uterus produce the fibrous shell membrane, which lies

proximal to the embryo, whereas the posterior uterine glands are hypothesized to secrete

the large amount of calcium that forms the outer, rigid calcified shell. Unlike birds,

however, alligators ovulate and "shell" a complete clutch of eggs simultaneously whereas

birds ovulate and shell one egg at a time.









The Amniotic Egg

All amniotic eggs consist of yolk and albumen derived from the maternal parent. In

addition, the eggs contain membranes derived from the embryo and in alligators, this

includes the chorio-allantois (a fusion between the chorion and the allantois) and amnion

(Ferguson, 1982). During the breeding season, circulating estrogens increase and

vitellogenin (Vtg), the major egg yolk protein, is synthesized in the liver (Guillette et al.,

1997). Vitellogenin circulates through the bloodstream and is taken up by the oocytes

where it is cleaved into smaller yolk subunits (Berg et al., 2004). These subunits serve as

the nutritional source for the developing embryo. In alligators, albumen is secreted by

the uterine tube and serves as a water reservoir as well as a chemical and mechanical

barrier to the embryo (Milnes et al., 2002). The amnion, a fluid-filled sac surrounding

the embryo, is derived from an out-pocketing of the embryo's hindgut and also acts as a

mechanical barrier while aiding in preventing dessication. Finally, the chorio-allantois,

also derived from the embryo, surrounds the contents of the egg and serves as a waste

repository (Pough et al., 2001).

The leathery shell membrane forms from the fiber-secreting region of the uterus

and surrounds all of the egg contents. Around this fibrous membrane are four distinct

layers comprising the eggshell; the innermost mamillary layer composed of calcium and

organic material (20-29 tm thick), a fibrous organic layer distinguished by blebs that

serve as attachment for calcite crystals (8-12 [tm thick), a porous and heavily-fibered

honeycomb layer (300-400 [tm thick), and the outer densely calcified layer (100-200 [tm

thick) of numerous calcite crystals (Ferguson, 1982; Packard et al., 1982). Ferguson

(1982) also documented constituent elements in 198 alligator eggshells through energy

dispersive X-ray analysis. In all samples, the major constituents included calcium and









magnesium, the minor constituent was phosphorus, and trace elements included copper,

silicon, sodium, aluminum, iron, zinc, and manganese.

Oviposition

The ancestral mode of reproduction in vertebrates is believed to involve oviparity

and external fertilization. Extant forms still displaying this mode include all agnathan

fishes, most teleost fishes, and many amphibians. Oocyte retention is short in duration

(hours to days) and involves the secretion of a gelatinous coat onto the egg, the egg's

transport through the reproductive tract, and finally, it exiting the maternal parent (Pough

et al., 2002). The endocrine events related to ovulation, egg transport, and oviposition,

are hypothesized to be closely associated (Guillette et al., 1991). However, in some

extant oviparous reptiles displaying internal fertilization (turtles and some squamates),

gestation length has increased, allowing time for shell calcification (Guillette et al.,

1991). The further increase in gestation time in some squamates and mammals, is

presumably ancestral to viviparity (Hogarth 1976). Because of the increasing temporal

separation between ovulation and oviposition, its been suggested that hormones influence

ovulation and oviposition differently between oviparous and egg-retaining/viviparous

vertebrates. Previous research indicates that in birds, reptiles, and mammals, hormones,

particularly prostaglandins (PGs) and arginine vasotocin (AVT) (or oxytocin for

mammals) are important in stimulating the transport of eggs or embryos through the

reproductive tract (Guillette et al., 1990), regardless of parity employed. It is

hypothesized that oviposition in oviparous vertebrates is stimulated within the central

nervous system (CNS) (Owman et al., 1986). Specifically, the CNS induces follicular

rupture causing the release of PGF2a, which stimulates oviducal contractions, induces

egg-laying behavior, and stimulates arginine vasotocin (AVT) release from the









neurohypophysis (Guillette et al., 1990). In egg-retaining / viviparous vertebrates, it is

hypothesized that a cervix has developed which does not respond to PGs or AVT during

ovulation but does respond during oviposition due to a lack of AVT / PG receptors or 3-

adrenergic stimulated inhibition of oviducal contraction (Koob et al., 1984; Challis et al.,

1988). During early-mid gestation, it is believed that 0-adrenergic neurons (innervating

the oviduct) overcome hormonal control of the oviduct, causing relaxation and inhibiting

contractions. During late gestation and initial post-partum, 3-adrenergic nerves begin

degrading. In the viviparous lizard Sceloporusjarrovi, Rooney et al. (1997) observed the

constant innervation of the uterus throughout pregnancy and subsequent denervation

during vitellogenesis. This pregnancy-associated innervation supports the hypothesized

mammalian role of oviductal innervation in maintaining myometrial quiescence (Rooney

et al., 1997).

Environmental Perturbation to Shelling

Prior to the late 1940's, it was rare to find broken eggs in the nests of peregrine

falcons (Falco peregrinus) and sparrowhawk (Accipiter nisus), but from 1950 on, shells

from these species were commonly found broken or destroyed (Ratcliffe, 1973).

Ratcliffe (1967) was the first to correlate eggshell breakage and thinning with the

widespread use of synthetic organic chemicals used as pesticides and in industry. In

particular, Ratcliffe noted that organochlorines were accumulating in the tissues of wild

raptors concurrent with the post-war use of these compounds. Since these observations,

several experimental studies demonstrated eggshell thinning in relation to exposure to the

pesticide DDT dichlorodiphenyltrichloroethanee) and its metabolites by disturbing

physiological mechanisms related to calcium (Anderson and Hickey, 1972; Ratcliffe,

1967; Laporte; 1982; Cooke, 1973), which makes up approximately 90% of the









eggshell (Tyler and Geake, 1953). Before 1950, British peregrines had a mean clutch

size of 3.5 eggs, with an average of 2.5 fledglings per nest. After 1950, northern England

and southern Scotland yielded 1.5 and 1.7 fledglings per nest, respectively, yet mean

clutch size remained unchanged (Ratcliffe, 1973). Another parameter implemented to

document eggshell changes over time was the 'eggshell index,' where the weight of the

shell (mg) is divided by the product of the length and width of the shell. Not all birds

studied incurred a decrease in eggshell index (table 1.1).

Table 1.1. Mean avian eggshell indices in Britain, prior to and after 1947 (modified from
Ratcliffe, table 5, 1970).
# eggs
before # eggs Mean index Mean index % change in
Species 1947 1947- before 1947 after 1947 index

Peregrine 509 211 1.836 1.485 19.12%
Sparrowhawk 298 279 1.423 1.178 17.22%
Golden Eagle 84 27 3.146 2.834 9.90%
Buzzard 83 96 4.97 4.92 0.46%
Raven 222 205 1.121 1.111 0.89%
Razorbill 46 33 2.339 2.350 0.90%

After Ratcliffe's discoveries in Britain, Anderson and Hickey (1972) studied over

23,000 eggshells from 25 species in North America. Like Ratcliffe, Anderson and

Hickey first determined if there was a significant decrease in eggshell indices before

1947. Unlike Ratcliffe, they combined data by decades and noted that eggshell changes

were rare before 1940. The golden eagle was the only species that appeared to have no

significant difference in shell index before 1940, which was attributed to low sample size.

In addition to eggshell index, eggshell thickness was measured with a dial micrometer.

Thickness measurements were compared with eggshell indices and statistically

determined to be strongly correlated (p<0.05, Anderson and Hickey, 1972). All thickness









measurements were performed on shells that had thinned or broken during incubation, as

opposed to fully incubated eggshells that hatched and produced viable offspring. It was

then determined that 9 out of 25 North American species underwent shell thinning and

shell weight decreases of more than 20%. These species included the peregrine falcon,

marsh hawk, brown pelican, prairie falcon, cooper's hawk, double-crested cormorant,

black-crowned night heron, bald eagle, and osprey.

The Role of Calcium

Embryonic alligators are thought to obtain calcium required for skeletogenesis

from the eggshell primarily, with additional calcium obtained from yolk stores (Packard

and Packard, 1984). Initially, alligators were thought to mobilize and deposit calcium

similarly to other reptiles but conversely, the processes are more similar to those of birds.

Both birds and alligators deposit calcium in the yolk prior to embryogenesis. At

hatching, the neonate has a relatively substantial calcium yolk store for growth and

development. Embryonic turtles and squamates, however, contain less yolk calcium at

hatching than at oviposition (examples include Chelydra serpentina, Packard et al.,

1984b; Coluber constrictor, Packard et al., 1984c; Amphibolorus barbatus, Packard et

al., 1985). Due to this mechanistic parallel with birds, calcium mobilization and uptake is

thought to resemble birds more closely than that of other reptiles. In either case, calcium

must traverse cellular barriers including the yolk-sac and chorioallantoic membrane

(CAM) to gain access to the embryo. Since alligators lay a highly calcified shell, and the

amount of calcium in the shell decreases throughout incubation, it is believed that

calcium metabolism can be regulated along the CAM interface, similarly to that in birds

(Romanoff, 1960; Reider et al., 1980).









Calcium transport across the chorionic ectodermal cell membranes is believed to

involve both mechanistic and regulatory events. Mechanistically, calcium translocation

across the CAM can occur pericellularly or endocytotically. There is little evidence for

the pericellular basis, leaving endocytosis as the likely transport model (Terepka et al.,

1976; Tuan and Zrike, 1978). In the latter model, calcium binds to surface membrane

calcium-binding protein (CaBP) and the ligand is encapsulated in pinocytic vesicles,

which cross the ectoderm and fuse and empty into the serosal compartment. The function

of CaBP and CAM calcium transport activity are probably linked and regulated by the

presence of a transport substrate (the calcium rich eggshell) and vitamin K availability

(Tuan 1987). In previous studies, the CAM of shell-less in vitro cultures of chicken

embryos failed to demonstrate developmentally-specific expression of calcium transport

activity or CaBP (Dunn et al., 1981; Tuan, 1980, 1984). In addition, vitamin K serves as

a cofactor producing the active form of CaBP. A vitamin K deficiency results in a

reduction in gamma-glutamyl carboxylation coincident with a decrease in CAM calcium

transport activity. Interestingly, when CaBP mRNA is experimentally increased, CaBP

level in ovo increases normally as well, suggesting disruption prior to protein translation

and potentially during transcription. In various in vitro and in vivo studies (see review by

Tuan, 1987), Ca2+-ATPase and carbonic anhydrase are also implicated in CAM calcium

transport function. CAM extracts revealed the presence of Ca2+-ATPase, which was

localized on the ectoderm through histochemistry (Tuan et al., 1984), and also aids in

calcium transport. Chemically inhibiting Ca2+-ATPase decreases calcium levels in the

CAM (Akins and Tuan, 1993). Carbonic anhydrase (CA) is believed to provide local






9


acidification in the ectoderm and promote dissolution of eggshell calcite (CaCO3) in vivo.

In doing so, CA can produce ionized calcium ready for uptake (Tuan and Zrike, 1978)














CHAPTER 2
PARAMETERS OF ALLIGATOR EGGSHELLS FROM REFERENCE AND
CONTAMINATED NORTH-CENTRAL FLORIDA LAKES

Introduction

Although once classified as endangered, alligator populations in many wetland

regions of Florida are stable or increasing (Woodward et al., 1999). Habitat degradation

and changing land-use patterns, however, threaten some populations and a better

understanding of the biology of this species is needed (Abercrombie, 1989). Population

stability is based on reducing death and recruiting new individuals to the population each

year. Variables affecting either of these characteristics will have a dramatic impact on

the stability, growth, or decline of alligator populations in Florida's wetlands.

For over two decades, the Florida Fish and Wildlife Conservation Commission

(FWC) has documented the hatching success of alligators from various Florida lakes.

Since 1993, alligators from Lake Woodruff, part of a National Wildlife Refuge,

repeatedly exhibit the highest hatch rates (77%-86%), when compared with rates

documented from other Florida lakes, particularly Lakes Griffin (30%-32%) and Apopka

(32%-52%) (Woodward et al., 1999). Lake Apopka has been subject to agricultural and

municipal run-off, extensive herbicide and pesticide use, and a major pesticide spill in

1980 that contained dicofol and DDT dichlorodiphenyltrichloroethanee), among other

compounds (Schelske and Brezonik, 1992).

DDT degrades into two primary metabolites, DDE and DDD (USEPA 1996). Of

the metabolites, p, p'-DDE (specifically) has been associated with altering the









physiological process of eggshell formation that subsequently led to eggshell thinning

and population declines of numerous avian species, particularly raptors and shorebirds

(see review by Lundholm, 1997). Approximately four years after the pesticide spill in

1980, a sharp decrease in alligator egg viability on Lake Apopka was documented by the

FWC. This decline was hypothesized to be in response to the contamination event

(Woodward et al., 1993). Conversely, no major point source of pollution, such as a

pesticide spill, has been reported on Lake Griffin, but this lake has received extensive

agricultural runoff, municipal storm water runoff, and currently exchanges water with

former agricultural fields that are part of a restoration program to create emergent

wetlands (see Marburger et al., 2002). Clutches of alligator eggs obtained from Lake

Griffin have also exhibited decreased hatch rates along with relatively recent increased

adult alligator mortality (Ross 2000; Schoeb et al., 2002). Hatch rates for Lakes Apopka

and Griffin still remain lower than those of Lake Woodruff, particularly throughout the

duration of this study (Table 2.1), though the mechanisms) by which this occurs remains

unknown.

Table 2-1 Mean alligator egg viability rates and sample size of clutches collected from
three study areas and incubated under 320C in an artificial incubator, 1999,
2001-2003 (Woodward, pers. com.).

Study Areas
Year Lake Woodruff Lake Apopka Lake Griffin
Mean N Mean N Mean N
1999 0.85 18 0.56 9 0.41 21
2001 0.79 16 0.53 15 0.55 19
2002 0.74 11 0.27 7 0.56 20
2003 0.90 7 0.31 11 0.72 17

A number of factors could contribute to embryonic mortality. One of these factors

could be a change in eggshell structure or composition leading to altered eggshell









function during incubation. The crocodilian oviduct more closely resembles birds than

that of other oviparous reptiles (Palmer and Guillette, 1992). It is a heterogeneous

structure consisting of seven regions. Functionally and ultrastructurally, the anterior

uterus of the alligator resembles the avian isthmus where eggshell membrane formation

takes place. Endometrial glands located in this region produce proteinaceous fibers that

are hypothesized to compose the eggshell membrane. The posterior uterus is similar to

the avian shell gland, which secretes the calcareous eggshell. Unlike birds, however,

alligators 'shell' eggs of a clutch simultaneously whereas birds ovulate and 'shell' each

egg of a clutch individually.

To date, several studies indicate that the supply of calcium to the eggshell gland in

ducks is not impeded by p,p'-DDE, but rather, this organochlorine contaminant disrupts

calcium transport within the eggshell gland (Lundholm, 1997, 1990a, 1990b; Kolaja,

1977). In addition, p,p'-DDE inhibits Ca2+-ATPase or Ca2+-Mg2+-ATPase activity in the

eggshell gland suggesting one mechanism for DDE-induced eggshell thinning. Calcium

transport across the eggshell gland mucosa is coupled with both sodium and bicarbonate

(Eastin and Spaziani, 1978; Pearson and Goldner, 1973) and accordingly, the impaired

movement of one of these ions can influence the movement of others (particularly

calcium). Additionally, the prostaglandins PGF2a and PGE2 have been implicated in

eggshell thinning. DDE disrupts the synthesis of these two prostaglandins, which reduces

bicarbonate transport in the duck shell gland lumen, in turn reducing calcium transport.

Both PGF2a and PGE2 have previously been closely associated with eggshell formation in

birds (Hammond et al., 1980).









Alligators from Lake Apopka lay eggs with elevated concentrations of p,p'-DDE,

and juveniles and adults in the population have elevated concentrations of this

contaminant in their blood and eggs (Heinz et al., 1991; Guillette et al., 1999)

presumably due to eating p,p'-DDE-contaminated food items (Rauschenberger et al.,

2004). The application of the pesticide DDT to croplands began in 1945 in the United

States, and continued until the Environmental Protection Agency (EPA) restricted its use

in 1972. This pesticide and its metabolites are persistent, bioaccumulating, and

biomagnifying in the food chain (see review, Guillette et al., 2005). Due to the previous

affiliation between DDT and its metabolites with avian eggshell thinning (Anderson and

Hickey, 1972; Henny and Bennett, 1990; Hickey 1969; Ratcliffe 1967), as well as the

phylogenetic relationship (Gower 2002) and similarity in egg shell formation (Palmer and

Guillette, 1991, 1992) between birds and crocodilians, we hypothesized that alligator

eggs could undergo eggshell thinning as a result of p,p'-DDE exposure.

To begin to test the hypothesis that organochlorines, such as p,p'-DDE, could

contribute to higher mortality of alligator embryos in several Florida wetlands through

altered shell function, we examined alligator eggshell structure and composition from

clutches laid on three Florida lakes. Morphometric data were collected as well as

microscopic structural data (light and scanning electron microscopy).

Materials and Methods

Alligator eggs were collected from three north-central Florida freshwater lakes

(lakes Woodruff, Griffin, Apopka) in June of 1999, 2001, 2002, and 2003. The eggs

were transported to the University of Florida and incubated until hatching. The shells of

eggs that produced viable neonates were collected after hatching, stored in whirlpacks,









and frozen at -20 C for processing at a later date. Those eggs that did not produce

viable neonates were not included in any analyses.

Thickness Measurements

Alligator eggshells are composed of an inner tough fibrous layer composed of

protein fibers covered by an outer calcium rich crystalline layer. The hard outer layer

was used to determine eggshell thickness and hereafter is referred to as the 'eggshell'.

Eggshells were rinsed of organic matter with tap water, air-dried at 20 C for 72 hours,

and oven dried at 42 C for 90 minutes. Thickness measurements were taken with a dial

caliper to the nearest millimeter at five different regions on each egg. Three

measurements were obtained from the shell that would have covered the equatorial region

of the egg and two measurements were obtained from the polar ends of each egg. Three

eggs were measured from each clutch so that a clutch mean could be determined (Table

2.2). The clutch mean was then used in all statistical analyses.

Table 2-2 Number of clutches from which eggshell thickness was examined each year of
the study period from 3 lakes. (Note: 3 eggs/clutch were measured).

Lake Lake Lake
Year Woodruff Apopka Griffin
1999 12 12 12
2001 11 8 10
2002 10 5 15
2003 9 5 10

In 2003, five clutches (shell fragments from 3 different eggs /clutch) from each lake

were further analyzed for pore density and elemental composition (including heavy

metals) and were examined by scanning electron microscopy (SEM).









Pore Density

Eggshell fragments were dyed with 0.15 ml of fast green in 30 mls of dH20 for 2

minutes and allowed to air-dry in the laboratory overnight. A 2.4 cm2 circular region of

the egg was isolated from the equatorial region of each egg. Pores were counted with a

dissection microscope and final values were converted to pores/cm2.

Elemental Analyses (EDS)

Energy Dispersive Spectral Analysis (EDS) was performed to detect constituent

elements in each eggshell sample. Briefly, EDS operates by submitting the sample to

electron bombardment producing a range of X-rays of different energy levels that

correspond with a particular element. Carbon, calcium, and oxygen values were

averaged for each clutch and within each lake and were compared using ANOVA.

(Elements detected below 0.10% are not included in analyses, as this represents the

lowest reliable quantitative detection limit for this analysis).

Elemental Analyses (ICP)

Inductively coupled plasma-optical emission spectrophotometry (ICP-OES, Perkin

Elmer 6100) was used to detect heavy metals. ICP-OES uses an argon plasma to

generate measurable atomic emission. The emission lines for each atom are monitored at

specific wavelengths. Standard metal solutions in 5% nitric acid were obtained (Fisher

Scientific Inc.) and diluted to appropriate concentrations in plastic volumetric labware to

avoid the problem of metals adhering to glass. The analyses of these standard solutions

were used to generate calibration curves. Alligator eggshells were dissolved in 5% nitric

acid (metal analysis grade). The resulting solution was analyzed under identical

conditions as the standards and the shell concentrations calculated using the calibration

curves. All standards and samples were analyzed in triplicate. Both analysts were blind









to the sample status of each eggshell. Sufficient concentrations of aluminum and

magnesium were detected and statistically analyzed (ANOVA) to determine possible

differences among lakes.

Scanning Electron Microscopy (SEM)

Two fragments from the equatorial region of each shell were desiccated, mounted

on SEM stubs and sputter coated with gold. One fragment was viewed in cross section

(200X and 5000X), and the other fragment was oriented to view the internal surface of

the shell (250X, 450X, 4000X, 8000X). The shells were qualitatively characterized by

their crystalline structure, shell membrane structure, relative fiber and node presence,

pore morphology, and layer density.

Statistics

Analysis of Variance (ANOVA) was used to compare data among lakes (Statview

for Windows, version 5.0). Statistical significance was determined if p < 0.05. If a

significant difference was determined, Fisher's post-hoc analyses were used for pair-wise

comparisons.

Results

Eggshell Thickness

Regarding shell thickness, there was no interaction among lakes and years

combined (2-way ANOVA, p=0.5117). There was a significant difference in shell

thickness among lakes in 1999 and 2003, but no differences were detected in 2001 and

2002. This variation could be due to small sample sizes for a given year and lake. In

support of this concept, if data from all years were combined for each lake, a significant

difference among lakes was observed (p = 0.02; Fig. 1). As hypothesized, eggshells

obtained from the more contaminated Lakes Apopka and Griffin, exhibited similar shell









thickness, but contrary to our predictions, they had thicker eggshells than those obtained

from Lake Woodruff (Apopka vs. Woodruff, p = 0.0003; Griffin vs. Woodruff, p =

0.0021; Fig. 1). It must be noted that these thickness measurements were obtained from

egg shells post hatch, and represent the thickness remaining after embryonic development

was complete.

Pore Density

There was no significant difference in eggshell pore density among lakes (p = 0.55;

Fig 2). Further, we detected no difference in pore density among clutches from Lakes

Woodruff and Griffin, whereas there was a significant difference among clutches from

Lake Apopka (p = 0.01; Fig. 3).

Elemental Analyses (EDS)

There was a significant difference in calcium, carbon, and oxygen concentrations in

the eggshells obtained from the various lakes of this study (p = 0.005, p = 0.0006, p =

0.004, respectively). That is, we observed that shell samples from Lake Woodruff were

similar in percent calcium to those from Lake Apopka but significantly greater than those

from Lake Griffin (p = 0.0047; Fig. 4). Lake Woodruff eggshells contained significantly

lower percent carbon than both Lakes Apopka (p = 0.0129) and Griffin (p = 0.0002),

which were similar (Fig. 4). Eggshells from Lakes Woodruff and Apopka contained

similar percentages of oxygen and both exhibited significantly higher levels than those

recorded from shells from Lake Griffin (p = 0.001, p = 0.0250, respectively; Fig. 4).

Elemental Analyses (ICP)

Lead, arsenic, iron, aluminum, zinc, nickel, manganese, chromium, calcium,

magnesium, and cadmium were examined in each sample. Only calcium, aluminum, and

magnesium were detectable (mgs/liter). There was a significant difference in aluminum









and magnesium levels detected in the shells from the three lakes (p = 0.025, p = 0.020,

respectively). Eggshells obtained from Lake Woodruff contained significantly more

aluminum than shells from Lakes Apopka (p = 0.009) and Griffin (p = 0.047), which

were similar (Fig. 5). Shells obtained from Lake Griffin contained significantly less

magnesium when compared to shells from either of the other two lakes, Lake Woodruff

(p = 0.014) or Lake Apopka (p = 0.016, Fig. 4).

Scanning Electron Microscopy (SEM)

A qualitative analysis of the structure of the eggshells using scanning electron

microscopy revealed several interesting differences when shells were compared among

lakes. An irregular network of fibers is present on the inner surface of the calcareous

eggshells from all three lakes, although their relative abundance varies (Fig 6 & 7). Eggs

from Lake Woodruff can be categorized as having several, intermediate, few, and no

fibers at all (Fig. 6). They are randomly orientated and overlap extensively. The majority

of eggs from Lakes Griffin and Apopka contained few or no fibers at all. Several nodes

are present and give each fiber a coated appearance (Fig. 8). Little is known about the

make up of these fibers; however, it has been suggested that these nodes are made of

glycosaminoglycans (GAG) (Ferguson 1982). Functionally, they can represent a template

for calcified crystalline growth similar to that in mammals where hydroxyapatite crystals

give rise to bone and dentin (Brown 1975; Jenkins 1978). The fibrous arrangement and

size and amount of nodes appear more similar between samples from Lakes Apopka and

Griffin (Fig. 7). Shells from Lake Woodruff, however, appear to have fewer and larger

nodes (Fig. 8).

There were also similarities in pore-structure of shells obtained from the three

lakes. Pores were characterized by crater-like concentric rings that develop on the outer-









surface of the eggshell (Fig. 9a,d,g). The pore orifice leads to an irregular shaped cavity

that extends through the calcareous shell (Fig. 9b,e,h). Within each pore cavity, the

crystalline structure is apparent, though the patterns can vary among lakes (9c,f,i).

SEM cross-sectional views revealed the honeycomb layer previously described for

alligator eggshells (see Ferguson, 1982). Interestingly, we observed that all eggshells

from Lake Woodruff lacked the organic portion of the honeycomb region and contained a

smoother, denser region of crystallized calcium (Fig. 10), whereas eggshells from lakes

Griffin and Apopka displayed a region similar to that described by Ferguson (Fig. 10).

This difference in eggshell structure was unexpected. Finally, the structural make-up of

the inner eggshells appeared more dense and similar in Lakes Woodruff and Griffin when

compared to Lake Apopka (Fig. 11).

Discussion and Conclusions

An analysis of eggshell structure and composition revealed that variation existed

among alligator populations from central Florida lakes. Some of the variation noted

appears to be correlated to contaminant exposure but other aspects are not explained by

the environmental data collected during this study. We observed that post hatching

eggshell thickness is significantly less from eggshells from Lake Woodruff versus shells

from Lakes Griffin and Apopka. Additionally, we observed a major difference in the

post hatching shell, in that shells from Lake Woodruff lacked the organic portion of the

honeycomb region that lies below the dense outer cortical region composed of tightly

fitted calcium crystals. Finally, we observed that shells from Lake Woodruff had higher

concentrations of aluminum when compared to eggshells from other lakes. Previous

studies of birds exposed to organochlorine contaminants reported thinner eggshells (for

review, see Lundholm, 1997). Unlike our study, previous studies have examined









eggshells that either had non-viable embryos or were obtained during early incubation.

We have not been able, to date, to identify studies that were performed similar to ours,

where eggshells were obtained after successful incubation.

We hypothesize that Lake Woodruff embryos exhibit elevated gas exchange

producing larger neonates. Elevated gas exchange would presumably generate greater

CO2 levels at the shell interface with the pores and honeycomb region maximizing

calcium mobilization via the generation of carbonic acid. The lack of the organic portion

in the honeycomb region can be a result of increased carbonic acid production and its

subsequent dissolution of the eggshell. Milnes et al., (submitted) observed that mean egg

mass was greater for eggs from Lake Woodruff (86.9 + 0.68g) compared to Lake Apopka

(77.7 + 0.72g) in 1999. When adjusted for egg mass, hatchling body mass and SVL

varied between lakes, with Woodruff hatchlings having greater mean body mass when

incubated at 320C and 33.5 C, and greater SVL when incubated at 32C (Milnes et al.,

submitted). These data suggest that even with differences in egg mass accounted for,

neonates from Lake Woodruff are larger. We have previously observed that Lake

Woodruff neonates are more likely to survive compared to neonates from Lake Apopka

(Guillette et. al., 1994). We suggest that vigorous, healthy neonates come from healthy

embryos that utilize more calcium in the eggshell and could have larger chorioallantoic

membranes. Future studies need to examine the hypothesis that embryos from Lake

Woodruff generate more CO2 and greater carbonic acid levels that etch the inner surface

of the eggshell more effectively.

The lack of a honeycomb region in the outer calcium eggshell obtained from Lake

Woodruff was unexpected. Again, it should be noted that these shells were obtained after









successful incubation so the lack of this region may be a post-oviposition modification,

not an original structural difference. The honeycomb region of the eggshell lies between

the outer densely calcified layer and the organic layer and is composed of both a fibrous

organic matrix and calcite crystals (see Packard and Packard 1984). This honeycomb

region would be expected to trap CO2 and water released from the chorioallantoic

membrane, which lines the inner surface of the proteinaceous shell. We note above that

the eggshells obtained from Lake Woodruff neonates were thinner, possibly due to the

calcified portion of the honeycomb region being eroded (and mobilized to the embryo) to

a greater extent than that of the same region in eggshells from other lakes. Additionally,

Woodruff eggshells contained less carbon, as shown through energy dispersive analysis,

which may also contribute to their respective thinner shells.

Eggshells from Lake Woodruff also had greater concentrations of aluminum, a

metal not required by living systems. Since aluminum is the most abundant metal in the

earth's crust (Rengal 2002), it is possible that relatively higher aluminum levels are

natural since Lake Woodruff has undergone less anthropogenic influences than the other

lakes. Potentially, lower aluminum levels may impede normal biological functions in the

biota underexposed. Lake Griffin eggshells contained more magnesium than shells from

other lakes. This mineral is necessary for calcium metabolism and could be elevated in

Lake Griffin shells due to a lack of proper calcium metabolism by the embryo. The

concentrations of these minerals could have biological impact but future studies would be

required to determine if the levels measured influence alligator embryonic viability and

eggshell development.









It's important to note that all of the samples of alligator eggshells were from post-

hatching and viable neonates, whereas bird eggshells had to be cracked or show evidence

of housing a dead embryo to be included in analyses. It is possible that bird eggshells

were collected at different stages of embryonic development, and would therefore be

expected to have different amounts of calcium mobilized from the shell, thereby

influencing shell thickness. This could explain why not all species were seemingly

influenced by p,p'-DDE. There can also be interspecific and/or intraspecific variation in

when, gestationally, shells begin cracking. Perhaps species that incur comparatively

increased gestation times or larger clutches are more impacted by compromised calcium

transport then those with shorter gestation times and smaller clutches. The same could

occur within species, depending on the maturity and/or overall health of the female

parent. Some species shells' may be more prone to swelling (from ambient humidity) or

dessication, which influences embryonic viability and ultimately biases the sample

collected. To avoid these biases, future studies could focus on shells from post-hatching

and viable avian neonates. Lastly, hormones are known to disrupt transport channels

indirectly related to calcium mobilization and can be another source of altered calcium

transport. These potential calcium transport offenders may not be mutually exclusive of

one another, thus compounding calcium transport further.

Both bird and alligator eggshells could serve as potential biomarkers of healthy

neonates since the embryos completed development within them. Conversely, it would

be interesting to compare alligator shells from early incubation to bird shells of early

incubation. Shells sampled from relatively parallel incubation windows could be directly

compared to determine relative constituents, their mobilization, and the influence of









environmental factors on these parameters. Regardless if the shells are from early or late

incubation, it could be worthy to examine the effects of other abiotic factors such as

substrate, temperature, or altitude on eggshell integrity of DDE exposed and unexposed

species.


b
TI-


b

T


V\bodruff Apopka Griffin
Lake
Figure 2-1 The mean (+/- Is.e.) equatorial eggshell thickness among lakes and years
(1999,2001,2002,2003). (Different superscripts indicate significant
differences between lakes).


0.42-
0.41
0.40
0.39
0.38
0.37
0.36
0.35
0.34
0.33
0.32














a
tI


Apopka
Lake


a
-I -T -


Griffin


Figure 2-2 The mean (+/- Is.e.) number of eggshell pores among lakes in 2003. (similar
superscripts denote no significant difference among lakes).


40 Woodruff
35- Apopka
|35 a
30 Griffin
25
20

0
o, 15
U 10

WO
Lu 0




Figure 2-3 The mean (+/- Is.e.) number of alligator eggshell pores per clutch, per cm2,
among lakes in 2003. Note the variation about the means, particularly from
Lake Apopka.


CM
E 20


o0
Iw

-
10
S15-



inL


5


Woodruff


* *












a

T


Oxygen


_70

60
Co

50-
0) -

>30-
(U

S20-

E 10-
w


Figure 2-4 The mean (+/- Is.e.) percent of the three most abundant elements in alligator
eggshells detected by Energy Dispersive Analysis (EDS), (n=3 shells/clutch, 5
clutches/lake; Different superscripts denote significant differences among
lakes).




Woodruff
0 Apopka
E Griffin
3000 -
b
2500
a
2000 a
bb
1500 -
1000 -
500 -
_- 0
LJ
Aluminum Magnesium
Element

Figure 2-5 The mean (+/- Is.e.) percent of the three most abundant elements in alligator
eggshells detected by Energy Dispersive Analysis (EDS),(n=3 shells/clutch, 5
clutches/lake; Different superscripts denote significant differencesamong
lakes).


Wodrulff
DApopka

a Griffin
a

a






Calcium Carbon
Bemert








































figure 2-6 Scanning electron micrographs ot the inner surface ot Lake Woodrutt alligator eggshells
(250X). Note the variation in relative fiber presence, a) no fibers b) few c) intermediate d) several.






































Figure 2-7 Scanning electron micrographs of the relative amount of shell fibers (F) in Apopka (a.b)
and Griffin (c,d). Shells from both lakes displayed few to no fibers (250X, n = 3 shells/clutch,
5 clutches / lake), relative to some Woodruff shells.


























Figure 2-8 SEM images of shell membranes from a) Woodruff, b) Apopka, c) Griffin in 2003. Note the numerous fibers and
the relative size of the nodes (N) on Woodruff vs. Apopka and Griffin (8000x) (n= 3 shells/clutch, 5 clutches/lake).


































Figure 2-9 SEM images of eggshell pore cavities from 2003 eggshells. Woodruff (a,b,c), a= cross section (200x),
b= outer g shell surface (200x), c= internal view of pore. Apopka (d,e,f): d=cross section (200x),
e= outer shell surface (200x), f=internal view of pore (8000x). Griffin (g,h,i,): g=cross section (200x),
h=outer shell surface (200x), I=internal view of pore, (n= 3 shells/clutch, 5 clutches/lake).

























Figure 2-10 SEM Cross-sectional views at 5000X from lakes a) Woodruff, b) Apopka, c) Griffin (2003). Note the
smooth surface layer (a) versus jagged and multi-edged surfaces (b & c) (n= 3 shells/clutch, 5 clutches
per lake).

























Figure 2-11 SEM image of the calcium crystalline structure on the inner eggshell surface, a) Woodruff, b) Apopka, c) Griffin
4000X, n = 3 shells/clutch, 5 clutches/lake).















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BIOGRAPHICAL SKETCH

Teresa Bryan received her Bachelor of Science degree in wildlife ecology and

conservation from the University of Florida, May, 2000. She worked as a laboratory

technician in a wildlife reproductive biology lab for one year before being accepted into

graduate school at UF under Louis J. Guillette. After receiving her Master of Science

degree, she plans to pursue doctoral research in vertebrate reproduction.