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To monitor physiological and toxicological conditions in an endangered species, noninvasive to minimally invasive sampling methods are needed. We analyzed peripheral blood cells to determine if we could monitor some physiological responses of the American alligator following capture stress. Juvenile American alligators were restrained for 16 h to examine the stress response in plasma and blood cells. Plasma corticosterone concentrations were increased by restraint as were plasma concentrations of aspartate aminotransferase (AST), creatine kinase (CK), uric acid, and glucose; a sexually dimorphic response was seen in AST and CK concentrations. The lapse time of restraint was associated with altered messenger RNA (mRNA) levels of the glucocorticoid receptor (GCR) in red blood cells and JUN proto-oncogene in both white and red blood cells. A two-way cluster analysis revealed that two major clusters of factors were associated with the responses seen: (a) mRNA levels of GCR and heat-shock proteins in both blood cells were associated with plasma corticosterone concentration, whereas (b) androgen receptors and JUN mRNA levels in both blood cells were associated with cloacal temperature and body composition. Blood cells appear to be an excellent source to examine the cellular stress response to steroid hormone signals in mRNA levels. We propose that this approach, using blood cells, could provide essential insights into the molecular responses associated with stress in reptiles as well as many other nontraditional model species, including endangered species.

Leucism (white skin, dark eyes) is a rare color disorder occurring in a range of invertebrates and vertebrates, and as a result, relatively few reports exist of leucistic individuals in the wild. In March 2014, we found 6 leucistic Alligator mississippiensis (American Alligator) hatchlings in coastal South Carolina. All individuals were basking under cool, cloudy conditions within ∼15 m of the den, appeared moderately emaciated, and were somewhat lethargic upon capture. The animals were removed from the field and treated for malnutrition under veterinary supervision. Three Alligators died within 6 days of collection, and the remaining 3 individuals were transferred to different institutions for long-term care and display. These animals also eventually died after surviving in captivity for ∼4.5–45 months. Leucistic Alligators are known to suffer from a variety of health problems, and the mortalities and associated causes of death in the animals we describe here were consistent with previous reports of other leucistic Alligators. The incidence of leucism among wild crocodilians is very low, and disease, increased susceptibility to predation, and collection by humans further exacerbate its rarity.

Estrogens regulate key aspects of sexual determination and differentiation, and exposure to exogenous estrogens can alter ovarian development. Alligators inhabiting Lake Apopka, FL, are historically exposed to estrogenic endocrine disrupting contaminants and are characterized by a suite of reproductive abnormalities, including altered ovarian gene expression and abated transcriptional responses to follicle stimulating hormone. Here, we test the hypothesis that disrupting estrogen signaling during gonadal differentiation results in persistent alterations to ovarian gene expression that mirror alterations observed in alligators from Lake Apopka. Alligator embryos collected from a reference site lacking environmental contamination were exposed to estradiol-17 beta or a nonaromatizable androgen in ovo and raised to the juvenile stage. Changes in basal and gonadotropin-challenged ovarian gene expression were then compared to Apopka juveniles raised under identical conditions. Assessing basal transcription in untreated reference and Apopka animals revealed a consistent pattern of differential expression of key ovarian genes. For each gene where basal expression differed across sites, in ovo estradiol treatment in reference individuals recapitulated patterns observed in Apopka alligators. Among those genes affected by site and estradiol treatment were three aryl hydrocarbon receptor (AHR) isoforms, suggesting that developmental estrogen signaling might program sensitivity to AHR ligands later in life. Treatment with gonadotropins stimulated strong ovarian transcriptional responses; however, the magnitude of responses was not strongly affected by steroid hormone treatment. Collectively, these findings demonstrate that precocious estrogen signaling in the developing ovary likely underlies altered transcriptional profiles observed in a natural population exposed to endocrine disrupting contaminants.

Type 1 diabetes (T1D) is a chronic autoimmune disease that affects 1 in 300 children by age 18. T1D is caused by inflammation-induced loss of insulin-producing pancreatic beta cells, leading to high blood glucose and a host of downstream complications. Although multiple genes are associated with T1D risk, only 5% of genetically susceptible individuals actually develop clinical disease. Moreover, a growing number of T1D cases occur in geographic clusters and among children with low risk genotypes. These observations suggest that environmental factors contribute to T1D etiology. One potential factor, supported primarily by epidemiological studies, is the presence of nitrate and nitrite in drinking water. To test this hypothesis, female hatchling alligators were exposed to environmentally relevant concentrations of nitrate in their tank water (reference, 10mg/L, or 100mg/L NO3-N) from hatch through 5 weeks or 5 months of age. At each time point, endpoints related to T1D were investigated: plasma levels of glucose, triglycerides, testosterone, estradiol, and thyroxine; pancreas, fat body, and thyroid weights; weight gain or loss; presence of immune cells in the pancreas; and pancreatic beta cell number, assessed by antibody staining of nkx6.1 protein. Internal dosing of nitrate was confirmed by measuring plasma and urine nitrate levels and whole blood methemoglobin. Cluster analysis indicated that high nitrate exposure (most animals exposed to 100mg/L NO3-N and one alligator exposed to 10mg/L NO3-N) induced a profile of endpoints consistent with early T1D that could be detected after 5 weeks and was more strongly present after 5 months. Our study supports epidemiological data correlating elevated nitrate with T1D onset in humans, and highlights nitrate as a possible environmental contributor to the etiology of T1D, possibly through its role as a nitric oxide precursor.

Across diverse taxa, germ cell development is controlled by an intricate cascade of processes that are tightly controlled by the hypothalamic-pituitary-gonadal axis. Endocrine disturbances, such as those induced by endocrine disrupting chemicals (EDCs) can negatively affect spermatogenesis. Here, we investigate whether spermatogenesis is altered in the giant toad, Rhinella marina, living in agricultural areas where EDCs are used relative to suburban areas. We also ask if reductions in spermatogenesis were associated with developmental gonadal abnormalities (intersex) found in the same frogs. We found that toads in agricultural areas exhibited reduced spermatogenesis relative to non-agricultural animals, and that those reductions were not associated with gross gonadal abnormalities. All toads living in agricultural areas had reduced spermatogenesis relative to those living in non-agricultural areas regardless of whether they had gonadal abnormalities originating during development. Similarities in reproductive dysfunction among diverse taxa living in agricultural areas, including humans, suggest that many vertebrate taxa living in agricultural areas around the globe are likely experiencing some level of reproductive dysfunction.

Endocrine disrupting contaminants, in combination with other environmental variables, are associated with altered reproductive health. Assisted reproductive technology (ART) procedures offer valuable opportunities to explore the connections between environmental contaminants in the ovarian microenvironment and measures of fertility, including impaired responsiveness to gonadotropins. Here, we investigate an emerging class of environmental contaminants, the perfluorinated alkyl acids (PFAAs), to determine whether ovarian contaminant levels are associated with measures of ovarian responsiveness and fertility outcomes in a South Carolina population of women undergoing ART. Levels of PFAAs in plasma and follicular fluid samples collected from women undergoing ovarian stimulation were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Six PFAAs were detected in both plasma and follicular fluid. PFAA concentrations in plasma correlate strongly to those detected in ovary and, with the exception of one compound, remain stable throughout ovarian stimulation. The concentration of PFHxS in follicular fluid inversely relates to baseline follicle counts. While no significant relationships were detected between ovarian response measures and PFAA concentrations, we identified a negative relationship between follicular fluid PFDA and PFuNA and blastocyst conversion rates. Our assessments indicate that plasma levels of PFAAs serve as a sound proxy of those in the ovarian compartment and that follicular fluid levels of specific PFAA compounds are inversely related to important clinical measures of reproductive health including baseline follicle count and post-fertilization success.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that initiates a transcriptional pathway responsible for the expression of CYP1A subfamily members, key to the metabolism of xenobiotic compounds. Toxic planar halogenated aromatic hydrocarbons, including dioxin and PCBs, are capable of activating the AHR, and while dioxin and PCB inputs into the environment have been dramatically curbed following strict regulatory efforts in the United States, they persist in the environment and exposures remain relevant today. Little is known regarding the effects that long-term chronic exposures to dioxin or dioxin-like compounds might have on the development and subsequent health of offspring from exposed individuals, nor is much known regarding AHR expression in reptilians. Here, we characterize AHR and CYP1A gene expression in embryonic and juvenile specimen of a long-lived, apex predator, the American alligator (Alligator mississippiensis), and investigate variation in gene expression profiles in offspring collected from sites conveying differential exposures to environmental contaminants. Both age- and tissue-dependent patterning of AHR isoform expression are detected. We characterize two downstream transcriptional targets of the AHR, CYP1A1 and CYP1A2, and describe conserved elements of their genomic architecture. When comparisons across different sites are made, hepatic expression of CYP1A2, a direct target of the AHR, appears elevated in embryos from a site associated with a dioxin point source and previously characterized PCB contamination. Elevated CYP1A2 expression is not persistent, as site-specific variation was absent in juveniles originating from field-collected eggs but reared under lab conditions. Our results illustrate the patterning of AHR gene expression in a long-lived environmental model species, and indicate a potential contemporary influence of historical contamination. This research presents a novel opportunity to link contamination events to critical genetic pathways during embryonic development, and carries significant potential to inform our understanding of potential health effects in wildlife and humans.

Environmental contamination resulting from the production or release of harmful chemicals can lead to negative consequences for wildlife and human health. Perfluorinated alkyl acids (PFAAs) were historically produced as protective coatings for many household items and currently persist in the environment, wildlife, and humans. PFAAs have been linked to immune suppression, endocrine disruption, and developmental toxicity in wildlife and laboratory studies. This study examines the American alligator, Alligator mississippiensis, as an important indicator of ecosystem contamination and a potential pathway for PFAA exposure in humans. Alligator meat harvested in the 2015 South Carolina (SC) public hunt season and prepared for human consumption was collected and analyzed for PFAAs to determine meat concentrations and relationships with animal body size (total length), sex, and location of harvest. Of the 15 PFAAs analyzed, perfluorooctane sulfonate (PFOS) was found in all alligator meat samples and at the highest concentrations (median 6.73 ng/g). No relationship was found between PFAA concentrations and total length or sex. Concentrations of one or all compounds varied significantly across sampling locations, with alligators harvested in the Middle Coastal hunt unit having the highest PFOS concentrations (median 16.0 ng/g; p = 0.0001). Alligators harvested specifically from Berkley County, SC (located in the Middle Coastal hunt unit) had the highest PFOS concentrations and the greatest number of PFAAs detected (p < 0.0001). The site-specific nature of PFAA concentrations in alligator meat observed in this study suggests a source of PFAA contamination in Berkley County, SC.

Routine monitoring of contaminant levels in wildlife is important for understanding chemical exposure and ultimately the link to ecosystem and human health. This is particularly important when the monitored species is recreationally hunted for human consumption. In the southeastern United States, recreational alligator harvesting takes place annually and in locations that are known to be contaminated with environmental pollutants. In this study, we investigated the biodistribution of trace elements in the American alligator (Alligator mississippiensis) from five sites in Florida, USA. These sites are locations where annual recreational alligator harvesting is permitted and two of the sites are identified as having high mercury contamination with human consumption advisories in effect. We utilized routinely collected monitoring samples (blood and scute), a commonly consumed tissue (muscle), and a classically analyzed tissue for environmental contaminants (liver) to demonstrate how the trace elements were distributed within the American alligator. We describe elemental tissue compartmentalization in an apex predator and investigate if noninvasive samples (blood and scute) can be used to estimate muscle tissue concentrations for a subset of elements measured. We found significant correlations for Hg, Rb, Se, Zn and Pb between noninvasive samples and consumed tissue and also found that Hg was the only trace metal of concern for this population of alligators. This study fills a gap in trace elemental analysis for reptilian apex predators in contaminated environments. Additionally, comprehensive elemental analysis of routinely collected samples can inform biomonitoring efforts and consumption advisories.

The American alligator, Alligator mississippiensis, like all crocodilians, has temperature-dependent sex determination, in which the sex of an embryo is determined by the incubation temperature of the egg during a critical period of development. The lack of genetic differences between male and female alligators leaves open the question of how the genes responsible for sex determination and differentiation are regulated. One insight into this question comes from the fact that exposing an embryo incubated at male-producing temperature to estrogen causes it to develop ovaries. Because estrogen response elements are known to regulate genes over long distances, a contiguous genome assembly is crucial for predicting and understanding its impact.We present an improved assembly of the American alligator genome, scaffolded with in vitro proximity ligation (Chicago) data. We use this assembly to scaffold two other crocodilian genomes based on synteny. We perform RNA sequencing of tissues from American alligator embryos to find genes that are differentially expressed between embryos incubated at male-versus female-producing temperature. Finally, we use the improved contiguity of our assembly along with the current model of CTCF-mediated chromatin looping to predict regions of the genome likely to contain estrogen-responsive genes. We find that these regions are significantly enriched for genes with female-biased expression in developing gonads after the critical period during which sex is determined by incubation temperature. We thus conclude that estrogen signaling is a major driver of female-biased gene expression in the post-temperature sensitive period gonads.

American alligators are exposed to mercury (Hg) throughout their natural range and may maternally transfer Hg into their eggs. Wildlife species are highly sensitive to Hg toxicity during embryonic development and neonatal life, and information on Hg transfer into eggs is critical when attempting to understand the effects of Hg exposure on developing oviparous organisms. To examine Hg transfer in alligators, the objectives of the present study were to 1) determine Hg concentrations in yolk (embryonic and neonatal food source) from wild alligator eggs collected from three locations - Yawkey Wildlife Center SC (YWC), Lake Apopka FL (LA), and Lake Woodruff FL (LW); 2) examine the relationship between THg concentrations in wild alligator nest material and egg yolk at Merritt Island National Wildlife Refuge, FL; 3) examine the Hg concentrations in wild maternal female alligators (blood) and the THg in corresponding egg yolks and embryos across three nesting seasons at a single location (YWC), and evaluate the relationship between nesting female THg concentrations (blood) and their estimated age and number of nesting years (YWC); and 4) assess the transfer of biologically-relevant Hg concentrations (based on Hg measured in maternal female blood) into embryos using an egg-dosing experiment. Mean total Hg (THg) concentrations observed at each site were 26.3 ng/g ± 11.0 ng/g (YWC), 8.8 ng/g ± 5.1 ng/g (LA), and 22.6 ng/g ± 6.3 ng/g (LW). No relationship was observed between THg in alligator nest material and corresponding yolk samples, nor between THg in maternal alligator blood and estimated age and number of nesting years of these animals. However, significant positive relationships were observed between THg in blood of nesting female alligators and THg in their corresponding egg yolk. We observed that 12.8% of the maternal blood THg is found in the corresponding egg yolk, and a highly significant correlation was observed between the two sample types (r = 0.66; p 0.0001). The egg dosing experiment revealed that Hg did not transfer through the eggshell at developmental stage 19. Overall, this study provides new information regarding Hg transfer in American alligators which can improve biomonitoring efforts and may inform ecotoxicological investigations and population management programs in areas of high Hg contamination.

Mercury is a widespread, naturally occurring contaminant that biomagnifies in wetlands due to the methylation of this element by sulfate-reducing bacteria. Species that feed at the top trophic level within wetlands are predicted to have higher mercury loads compared to species feeding at lower trophic levels and are therefore often used for mercury biomonitoring. However, mechanisms for mercury bioaccumulation in sentinel species are often poorly understood, due to a lack of long-term studies or an inability to differentiate between confounding variables. We examined mercury bioaccumulation patterns in the whole blood of American alligators (Alligator mississippiensis) from a long-term mark-recapture study (1979–2017) in South Carolina, USA. Using a growth model and auxiliary information on predicted age at first capture, we differentiated between age- and size-related variation in mercury bioaccumulation, which are often confounded in alligators due to their determinate growth pattern. Contrary to predictions that the oldest or largest individuals were likely to have the highest mercury concentrations, our best-supported model indicated a peak in mercury concentration at 30–40 years of age, depending on the sex, and lower concentrations in the youngest and oldest animals. To evaluate the robustness of our findings, we re-analyzed data from a previously published study of mercury in alligators sampled at Merritt Island National Wildlife Refuge in Florida. Unlike the South Carolina data, the data from Florida contained minimal auxiliary information regarding age, yet the best supported model similarly indicated a peaked rather than increasing relationship between mercury and body size, a less-precise indicator of age. These findings highlight how long-term monitoring can differentiate between confounding variables (e.g., age and size) to better elucidate complex relationships between contaminant exposure and demographic factors in sentinel species.

Steroid hormones are a key regulator of reproductive biology in vertebrates, and are largely regulated via nuclear receptor families. Estrogen signaling is regulated by two estrogen receptor (ER) subtypes alpha and beta in the nucleus. In order to understand the role of estrogen in vertebrates, these ER from various species have been isolated and were functionally analyzed using luciferase reporter gene assays. Interestingly, species difference in estrogen sensitivity has been noted in the past, and it was reported that snake ER displayed highest estrogen sensitivity. Here, we isolated additional ER from three lizards: chameleon (Bradypodion pumilum), skink (Plestiodon finitimus), and gecko (Gekko japonicus). We have performed functional characterization of these ERs using reporter gene assay system, and found high estrogen sensitivity in all three species. Furthermore, comparison with results from other tetrapod ER revealed a seemingly uniform gradual pattern of ligand sensitivity evolution. In silico 3D homology modeling of the ligand-binding domain revealed structural variation at three sites, helix 2, and juncture between helices 8 and 9, and caudal region of helix 10/11. Docking simulations indicated that predicted ligand-receptor interaction also correlated with the reporter assay results, and overall squamates displayed highest stabilized interactions. The assay system and homology modeling system provides tool for in-depth comparative analysis of estrogen function, and provides insight toward the evolution of ER among vertebrates.

Incubation temperatures experienced by developing embryos exert powerful influences over gonadal sex determination and differentiation in many species. However, the molecular mechanisms controlling these impacts remain largely unknown. We utilize the American alligator to investigate the sensitivity of the reproductive system to thermal signals experienced during development and ask specifically whether individuals of the same sex, yet derived from different incubation temperatures display persistent variation in the expression patterns of sex biased transcripts and plasma sex hormones. Our analysis focuses on assessments of circulating sex steroids and transcript abundance in brain and gonad, two tissues that display sexually dimorphic gene expression and directly contribute to diverse sexually dimorphic phenotypes. Whereas our results identify sexually dimorphic patterns for several target gonadal genes in postnatal alligators, sex linked variation in circulating 17β-estradiol, testosterone, and expression of two brain transcripts (aromatase and gonadotropin releasing hormone) was not observed. Regarding intrasexual variation, we found that AMH transcript abundance in hatchling testes is positively correlated with temperatures experienced during sexual differentiation. We also describe highly variable patterns of gene expression and circulating hormones within each sex that are not explained by the intensity of embryonic incubation temperatures. The magnitude of sexually dimorphic gene expression, however, is directly associated with temperature for SOX9 and AMH, two transcripts with upstream roles in Sertoli cell differentiation. Collectively, our findings regarding temperature linked variation provide new insights regarding the connections between embryonic environment and persistent impacts on sexual differentiation in a reptile species that displays temperature dependent sex determination.

Eicosanoids are signaling lipids known to regulate several physiological processes in the mammalian placenta, including the initiation of parturition. Though all amniotes construct similar extraembryonic membranes during development, the composition and function of eicosanoids in extraembryonic membranes of oviparous reptiles is largely unknown. The majority of effort placed in eicosanoid investigations is typically targeted toward defining the role of specific compounds in disease etiology; however, comprehensive characterization of several pathways in eicosanoid synthesis during development is also needed to better understand the complex role of these lipids in comparative species. To this end, we have examined the chorioallantoic membrane (CAM) of the American alligator (Alligator mississippiensis) and domestic chicken (Gallus gallus) during development. Previously, our lab has demonstrated that the CAM of several oviparous species shared conserved steroidogenic activity, a feature originally attributed to mammalian amniotes. To further explore this, we have developed a liquid chromatography/tandem mass spectrometry method that is used here to quantify multiple eicosanoids in the CAM of two oviparous species at different stages of development. We identified 18 eicosanoids in the alligator CAM; the cyclooxygenase (COX) pathway showed the largest increase from early development to later development in the alligator CAM. Similarly, the chicken CAM had an increase in COX products and COX activity, which supports the LC-MS/MS analyses. Jointly, our findings indicate that the CAM tissue of an oviparous species is capable of eicosanoid synthesis, which expands our knowledge of placental evolution and introduces the possibility of future comparative models of placental function.

Obesity has reached pandemic proportions, and there is mounting evidence that environmental exposures to endocrine disrupting chemicals known as "obesogens" may contribute to obesity and associated medical conditions. The Deepwater Horizon (DWH) oil spill resulted in a massive environmental release of crude oil and remediation efforts applied large quantities of Corexit dispersants to the oil spill. The Corexit-enhanced Water Accommodated Fraction (CWAF) of DWH crude oil contains PPARγ transactivation activity, which is attributed to dioctyl sodium sulfosuccinate (DOSS), a probable obesogen. In addition to its use in oil dispersants, DOSS is commonly used as a stool softener and food additive. Because PPARγ functions as a heterodimer with RXRα to transcriptionally regulate adipogenesis we investigated the potential of CWAF to transactivate RXRα and herein demonstrated that the Corexit component Span 80 has RXRα transactivation activity. Span 80 bound to RXRα in the low micromolar range and promoted adipocyte differentiation of 3T3-L1 preadipocytes. Further, the combination of DOSS and Span 80 increased 3T3-L1 adipocyte differentiation substantially more than treatment with either chemical individually, likely increasing the obesogenic potential of Corexit dispersants. From a public health standpoint, the use of DOSS and Span 80 as food additives heightens concerns regarding their use and mandates further investigations.

Mercury is a widespread environmental contaminant with exposures eliciting a well-documented catalog of adverse effects. Yet, knowledge regarding the underlying mechanisms by which mercury exposures are translated into biological effects remains incomplete. DNA methylation is an epigenetic modification that is sensitive to environmental cues, and alterations in DNA methylation at the global level are associated with a variety of diseases. Using a liquid chromatography tandem mass spectrometry-based (LC-MS/MS) approach, global DNA methylation levels were measured in red blood cells of 144 wild American alligators (Alligator mississippiensis) from 6 sites with variable levels of mercury contamination across Florida’s north-south axis. Variation in mercury concentrations measured in whole blood was highly associated with location, allowing the comparison of global DNA methylation levels across different “treatments” of mercury. Global DNA methylation in alligators across all locations was weakly associated with increased mercury exposure. However, a much more robust relationship was observed in those animals sampled from locations more highly contaminated with mercury. Also, similar to other vertebrates, global DNA methylation appears to decline with age in alligators. The relationship between age-associated loss of global DNA methylation and varying mercury exposures was examined to reveal a potential interaction. These findings demonstrate that global DNA methylation levels are associated with mercury exposure, and give insights into interactions between contaminants, aging, and epigenetics.

Phthalates have been shown to cause endocrine disruption in laboratory animals and are associated with altered development of the reproductive system in humans. Further, human have significant exposure to phthalates. However, little is known concerning the exposure of wildlife to phthalates. We report urinary phthalate metabolite concentrations from fifty juvenile alligators from three Florida lakes and a site in the Everglades. Urinary phthalate monoester concentrations varied widely among alligators from the different sites but also among alligators from the same site. Mono-2-ethylhexy phthalate and monobutyl phthalate were found in most samples of alligator urine with maximums of 35,700 ng/mL and 193 ng/mL, respectively. Monobenzyl phthalate was found in 5 alligators with a maximum of 66.7 ng/mL. Other monoesters were found in only one or two alligator urine samples. The wide variation within and among sites, in addition to the high levels of mEHP, mBP and mBzP, is consistent with exposure arising from the intermittent spraying of herbicide formulations to control invasive aquatic plants in Florida freshwater sites. Phthalate diesters are used as adjuvants in many of these formulations.

BackgroundThe obesity pandemic is associated with multiple major health concerns. In addition to diet and lifestyle, there is increasing evidence that environmental exposures to chemicals known as ...

Species displaying temperature-dependent sex determination (TSD) are especially vulnerable to the effects of a rapidly changing global climate due to their profound sensitivity to thermal cues during development. Predicting the consequences of climate change for these species, including skewed offspring sex ratios, depends on understanding how climatic factors interface with features of maternal nesting behaviour to shape the developmental environment. Here, we measure thermal profiles in 86 nests at two geographically distinct sites in the northern and southern regions of the American alligator's (Alligator mississippiensis) geographical range, and examine the influence of both climatic factors and maternally driven nest characteristics on nest temperature variation. Changes in daily maximum air temperatures drive annual trends in nest temperatures, while variation in individual nest temperatures is also related to local habitat factors and microclimate characteristics. Without any compensatory nesting behaviours, nest temperatures are projected to increase by 1.6–3.7°C by the year 2100, and these changes are predicted to have dramatic consequences for offspring sex ratios. Exact sex ratio outcomes vary widely depending on site and emission scenario as a function of the unique temperature-by-sex reaction norm exhibited by all crocodilians. By revealing the ecological drivers of nest temperature variation in the American alligator, this study provides important insights into the potential consequences of climate change for crocodilian species, many of which are already threatened by extinction.

Toxic trace element exposure occurs through release of the ubiquitous and naturally occurring elements arsenic (As), cadmium (Cd), lead (Pb), and mercury (Hg). The unique environmental conditions of the wetland ecosystems along the southeastern Atlantic coast of the United States lead to the accumulation of Hg which is greater than in most other ecosystems in the country. There are also point sources of As, Cd, and Pb in this region. To effectively monitor trace element concentrations, and consequently the potential human exposure, accessible local sentinel species are needed. In this study, concentrations of As, Cd, Pb, Hg and six other trace elements (Al, Ni, Cu, Zn, Se, Mo) were examined in American alligators (Alligator mississippiensis) from seven wetland sites in South Carolina and Florida and assessed for their utility as a sentinel species for human trace element exposure. Alligators were chosen as a potential sentinel as they share a common exposure with the local human population through their aquatic diet, and they are directly consumed commercially and through recreation hunting in this region. Sex was significantly related to the concentration of Zn, Mo, and Al, but not As, Pb, Hg, Cd, Se, or Cu. Site specific differences in element concentrations were observed for As, Pb, Hg, Cd, Se, Zn, and Mo. Size/age was significantly related to the element Hg and Pb concentrations observed. The observed concentration ranges for the four toxic elements, As (6–156 ng/g), Cd (0.3–1.3 ng/g), Pb (3–4872 ng/g), and Hg (39–2765 ng/g), were comparable to those previously reported in diverse human populations. In this region alligators are hunted recreationally and consumed by the local community, making them a vehicle of direct human toxic element exposure. We propose that the similarity in As, Cd, Pb, and Hg concentrations between alligators observed in this study and humans underscores how alligators can serve as a useful sentinel species for toxic element exposure.

Sceloporus aeneus exhibits reproductive bimodality. That is, one taxon (Sceloporus aeneus bicanthalis) is viviparous whereas the other (Sceloporus aeneus aeneus) is oviparous. Morphological differences in luteal and oviductal structure were examined. Oviparous and viviparous females have distinct corpora lutea that form immediately after ovulation and remain active until just prior to oviposition or parturition. Luteal activity is correlated positively with follicular atresia. The oviduct of both subspecies is divided into three distinct morphological regions: an anterior infundibulum, a median uterus, and a posterior vagina. The infundibulum and vagina of females exhibiting either parity type are similar, whereas distinct differences in utering morphology are apparent. Primarily, these differences include the loss of uterine glands and a reduction in epithelial cell height in the viviparous form. Moreover, viviparous females possess a simple but well-developed chorioallantoic placenta and a simple choriovitelline placenta. Chorioallantoic placentation is associated with a significant increase in uterine vascularity, indicating a role in gas and/or water exchange. The evolution of viviparity and placentation are discussed in relation to these observations.

The thyroid gland is sensitive to steroid hormone signaling, and many thyroid disrupting contaminants also disrupt steroid hormone homeostasis, presenting the possibility that thyroid disruption may occur through altered steroid hormone signaling. To examine this possibility, we studied short-term and persistent impacts of embryonic sex steroid exposure on thyroid physiology in the American alligator. Alligators from a lake contaminated with endocrine disrupting contaminants (Lake Apopka, FL, USA) have been shown to display characteristics of thyroid and steroid hormone disruption. Previous studies suggest these alterations arise during development and raise the possibility that exposure to maternally deposited contaminants might underlie persistent organizational changes in both thyroidal and reproductive function. Thus, this population provides a system to investigate contaminant-mediated organizational thyroid disruption in an environmentally-relevant context. We assess the developmental expression of genetic pathways involved in thyroid hormone biosynthesis and find that expression of these genes increases prior to hatching. Further, we show that nuclear steroid hormone receptors are also expressed during this period, indicating the developing thyroid is potentially responsive to steroid hormone signaling. We then explore functional roles of steroid signaling during development on subsequent thyroid function in juvenile alligators. We exposed alligator eggs collected from both Lake Apopka and a reference site to 17β-estradiol and a non-aromatizable androgen during embryonic development, and investigated effects of exposure on hatchling morphometrics and thyroidal gene expression profiles at 5 months of age. Steroid hormone treatment did not impact the timing of hatching or hatchling size. Furthermore, treatment with steroid hormones did not result in detectable impacts on thyroid transcriptional programs, suggesting that precocious or excess estrogen and androgen exposure does not influence immediate or long-term thyroidal physiology.

The structure and seasonal changes of the oviductal-cloacal junction remain poorly understood in most squamates. This study was undertaken to describe the histology of the oviductal-cloaca junction of a female viviparous snake Toluca lineata, during gestation, previtellogenesis, and vitellogenesis. The oviductal-cloacal junction exhibits a wider lumen and thicker layers of connective tissue, smooth muscle layers, and total wall width compared to the posterior vagina. The lining is characterized by thick, short longitudinal mucosal folds. The luminal epithelia differ morphologically from anterior to posterior portions of the oviductal-cloacal junction. The anterior portion is lined with a simple columnar epithelium composed of nonciliated cells. The middle portion is lined with stratified epithelium that contains an apical columnar cell layer that undergoes morphological changes coincident with the reproductive cycle. The posterior portion is lined with a stratified squamous epithelium. The connective tissue underlying the epithelium contains numerous ovoid cells having abundant acidophilic cytoplasmic granules-eosinophils. Copulation occurs during the previtellogenic stage, as evidenced by the presence of abundant spermatozoa in the lumen of the anterior portion and of a copulatory plug in the middle and posterior portion of the oviductal-cloacal junction. J. Morphol. 237:91-100, 1998. © 1998 Wiley-Liss, Inc.

The ovarian follicles of Ctenosaura pectinata exhibit a clear seasonal cycle in morphology. Early in development, each oocyte is surrounded by a granulosa composed of a single layer of cuboidal or squamous cells and thin thecal layers. As folliculogenesis progresses, the granulosa becomes multilayered and composed of three distinct cell types. After vitellogenesis begins and active sequestration of yolk into the ooplasm is initiated, the granulosa is reduced to a single cell type. We observed a striking change in the appearance of the ooplasm during folliculogenesis. Early ovarian development is characterized by an ooplasm with homogeneously distributed fine fibrils, but as development progresses, the ooplasm contains dense clumps of fibers aggregated into distinctive bundles. The ooplasm displays further complexity in morphology as previtellogenic growth continues and as different regions exhibit various combinations of fibers and vacuoles. Yolk platelet formation is complex, with distinctive stages generating platelets with varying morphologies. © 1996 Wiley-Liss, Inc.

Tuatara (Sphenodon spp.) are rare reptiles, members of the reptilian order Sphenodontida, inhabiting small offshore islands of New Zealand. Females usually require about three years to yolk a clutch of eggs followed by an 8-month period of in utero egg shelling. As in other vertebrates, the post-ovulatory follicle forms a transitory endocrine structure, the corpus luteum. The tuatara Sphenodon punctatus exhibits a corpus luteum having several unusual morphological features as compared to turtles and squamate reptiles. Like the crocodilians, the tuatara has a corpus luteum in which the luteal cell mass never fills the central cavity and in which the thecal fibroblasts do not close the ovulation aperture. As in all oviparous reptiles examined, however, the corpus luteum appears to persist throughout gravidity based on its histological appearance. During gravidity, plasma progesterone concentrations are detectable, even though gravidity lasts an exceptionally long time (8 months) for an oviparous species. Luteolysis is initiated within two months following oviposition. The initial stages of luteolysis appear rapid, but luteal scar tissue is apparent in the ovaries of all adult females we examined and probably persists for many years post-oviposition. J Morphol 232:79-91, 1997. © 1997 Wiley-Liss, Inc.

Scanning electron microscopy (SEM) was used to examine the process of shell formation in tuatara. Tuatara carry eggs in the oviducts for ∼ 7-8 mo before nesting, a period of gravidity more than three times as long as in any other oviparous reptile. Our aim was to determine whether shell formation occurred rapidly after ovulation, or whether it occurred gradually throughout gravidity. Eggs were obtained from females in early gravidity (May, ∼ 1 mo after ovulation), midgravidity (August and September, 4-5 mo after ovulation), and late gravidity, immediately prior to nesting (December, 8 mo after ovulation). The shell membrane (fibrous layer) was well formed by May, but calcification of the outer surface had only just begun. Vertical columns of calcium carbonate were embedded in the shell membrane and appeared to erupt through the outer surface between early and midgravidity. Changes in the appearance of the outer calcareous layer were evident as gravidity progressed. In all shells, calcium carbonate was present as calcite. The appearance of the inner boundary (innermost layer of eggshell) was variable; some shells had a smooth and amorphous inner boundary as previously reported for tuatara and other reptiles, whereas other shells had an inner boundary composed of small spherical granules on the inner surface of which small calcareous spicules were scattered. A previously published model of the process of shell formation in tuatara eggshells is refined in light of our observations. We interpret the ability of female tuatara to shell their eggs gradually during winter as further evidence of their unusual physiological tolerance of cold conditions. © 1996 Wiley-Liss, Inc.

Despite the widespread occurrence of environmental sex determination (ESD) among vertebrates, our knowledge of the temporal dynamics by which environmental factors act on this process remains limited. In many reptiles, incubation temperature determines sex during a discrete developmental window just prior to and coincident with the differentiation of the gonads. Yet, there is substantial variation in sex ratios among different clutches of eggs incubated at identical temperatures during this period. Here, we test the hypothesis that temperatures experienced prior to the reported thermosensitive period for alligators (Alligator mississippiensis) can impact how the sex determination system responds to thermal cues later in development. Temperature shift experiments on eggs collected from the field within 24 h of oviposition were employed to decouple various maternal influences from thermal effects, and results demonstrate a previously undefined window of thermosensitivity occurring by stage 15 of embryonic development, six stages earlier than previously reported. We also examine the intrasexual expression of several male- and female-biased genes and show that while male-biased genes display no intrasexual differences, ovarianCYP19A1(aromatase) transcript abundance differs by approximately twofold depending on thermal exposures experienced at early stages of embryonic development. These findings expand our understanding of the ESD in the alligator and provide the rationale for reevaluation of the temporal dynamics of sex determination in other crocodilians.

Androgens are essential for the development, reproduction, and health throughout the life span of vertebrates, particularly during the initiation and maintenance of male sexual characteristics. Androgen signaling is mediated by the androgen receptor (AR), a member of the steroid nuclear receptor superfamily. Mounting evidence suggests that environmental factors, such as exogenous hormones or contaminants that mimic hormones, can disrupt endocrine signaling and function. The American alligator (Alligator mississippiensis), a unique model for ecological research in that it exhibits environment-dependent sex determination, is oviparous and long lived. Alligators from a contaminated environment exhibit low reproductive success and morphological disorders of the testis and phallus in neonates and juveniles, both associated with androgen signaling; thus, the alterations are hypothesized to be related to disrupted androgen signaling. However, this line of research has been limited because of a lack of information on the alligator AR gene. Here, we isolated A mississippiensis AR homologs (AmAR) and evaluated receptor-hormone/chemical interactions using a transactivation assay. We showed that AmAR responded to all natural androgens and their effects were inhibited by cotreatment with antiandrogens, such as flutamide, p,p'-dichlorodiphenyldichloroethylene, and vinclozolin. Intriguingly, we found a spliced form of the AR from alligator cDNA, which lacks seven amino acids within the ligand-binding domain that shows no response to androgens. Finally, we have initial data on a possible dominant-negative function of the spliced form of the AR against androgen-induced AmAR.

While no pansteatitis-related large-scale mortality events have occurred since 2008, the current status of pansteatitis (presence and pervasiveness) in the Olifants River system and other regions of South Africa remain largely unknown. In part, this is due to both a lack of known biological markers of pansteatitis and a lack of suitable non-invasive assays capable of rapidly classifying the disease. Here, we propose the application of a point-of-care (POC) device using lipid-based test strips (total cholesterol (TC) and total triglyceride (TG)), for classifying pansteatitis status in the whole blood of pre-spawning Mozambique tilapia (Oreochromis mossambicus). Using the TC strips, the POC device was able to non-lethally classify the tilapia as either healthy or pansteatitis-affected; the sexes were examined independently because sexual dimorphism was observed for TC (males p = 0.0364, females χ2 = 0.0007). No significant difference between diseased and pansteatitis-affected tilapia was observed using the TG strips. This is one of the first described applications of using POC devices for on-site environmental disease state testing. A discussion on the merits of using portable lipid-based analyzers as an in-field disease-state diagnostic tool is provided.

Concentrations of mercury (Hg) are not well studied in free-ranging wildlife. Atmospheric deposition patterns of Hg have been studied in detail and have been modeled for both global and specific locations and often correlate to environmental impact. However, monitoring the impact of Hg deposition in wildlife is complicated due to local environmental conditions that can affect the transformation of atmospheric Hg to the biologically available forms (e.g., rainfall, humidity, pH, the ability of the environment to methylate Hg), as well as affect the accessibility to organisms for sampling. In this study, Hg concentrations in blood samples from a population of American alligators (Alligator mississippiensis) at Merritt Island National Wildlife Refuge (MINWR), FL, USA, over a seven-year period (2007 to 2014; n=174 individuals) were examined to assess Hg variation in the population, as well as the difference in Hg concentration as a function of health status. While most of this population is healthy, 18 individuals with low body mass indices (BMI, defined in this study) were captured throughout the sampling period. These alligators exhibited significantly elevated Hg concentrations compared to their age/sex/season matched counterparts with normal BMI, suggesting that health status should be taken into account when examining Hg concentrations and effects. Alligator blood Hg concentrations were related to the interaction of age/size, sex, and season. This study illustrates the value of a routinely monitored population of large predators in a unique coastal wetland ecosystem, and illuminates the value of long-term environmental exposure assessment.

Concentrations of 28 trace elements (Li, Mg, Al, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, As, Se, Rb, Sr, Mo, Ag, Cd, In, Sn, Sb, Cs, Tl, Hg, Pb, and Bi) in the livers of juvenile and adult American alligators inhabiting two central Florida lakes, Lake Apopka (LA), and Lake Woodruff National Wildlife Refuge (LW) and one lagoon population located in Merritt Island National Wildlife Refuge (MINWR; NASA), were determined. In juveniles from MINWR, concentrations of nine elements (Li, Fe, Ni, Sr, In, Sb, Hg, Pb and Bi) were significantly higher, whereas six elements (V, Fe, As, Sr, Hg and Bi) were elevated in adults (p0.05) obtained from MINWR. Significant enrichment of some trace elements in adults, relative to juveniles, was observed at all three sampling areas. Specifically, Fe, Pb and Hg were significantly elevated in adults when compared to juveniles, suggesting age-dependent accumulation of these elements. Further, As, Se and Sn showed the same trend but only in animals collected from MINWR. Mean Fe concentrations in the livers of adults from LA, LW and MINWR were 1770 μg g(-1) DW, 3690 μg g(-1) DW and 5250 μg g(-1) DW, respectively. More than half of the adult specimens from LW and MINWR exhibited elevated hepatic Fe concentrations that exceed the threshold value for toxic effects in donkey, red deer and human. These results prompted us to express our concern on possible exposure and health effects in American alligators by some trace elements derived from NASA activities.

Comparatively, little data are available detailing the geographic variation that exists in the reproductive endocrinology of adult alligators, especially those living in barrier islands. The Merritt Island National Wildlife Refuge (MI) is a unique barrier island environment and home to the Kennedy Space Center (FL, USA). Seasonal patterns of sex steroids were assessed in adult female American alligators from MI monthly from 2008 to 2009, with additional samples collected at more random intervals in 2006, 2007, and 2010. Plasma 17β-estradiol and vitellogenin concentrations peaked in April, coincident with courtship and mating, and showed patterns similar to those observed in adult female alligators in other regions. Plasma concentrations of progesterone, however, showed patterns distinctly different than those reported for alligator populations in other regions and remained relatively constant throughout the year. Plasma DHEA peaked in July around the time of oviposition, decreased in August, and then remained constant for the remaining months, except for a moderate increase in October. Circulating concentrations of DHEA have not been previously assessed in a female crocodilian, and plasma concentrations coincident with reproductive activity suggest a reproductive and/or behavioral role. Interestingly, plasma testosterone concentrations peaked in May of 2008, as has been shown in female alligator populations in other regions, but showed no peak in 2009, demonstrating dramatic variability from year to year. Surveys showed 2009 to be particularly depauperate of alligator nests in MI, and it is possible that testosterone could serve as a strong indicator of breeding success.

Nonlethal methods are needed to assess the health of wild fish and quantify the robustness of the broader population. Results could be used to indicate exposure to various stressors, such as contaminants, infectious disease, external parasite loads, and fishing pressure, to monitor changes in fish population health over time. The wild Red Drum Sciaenops ocellatus population in the Kennedy Space Center Reserve of Merritt Island National Wildlife Refuge was used to develop a protocol to define the health of free-ranging fish using nonlethal techniques. This health index incorporated morphometric measurements, weight, an evaluation for external parasite fauna, notation of physical deformities, and the presence of lesions. A total of 126 adult Red Drum were collected using hook-and-line angling during prespawning (May), spawning (September and October), and postspawning (December) periods. All fish were released alive back into their environment. The nonlethal health assessment scored fish in the "healthy" range of the health index during the prespawning and spawning periods. Fish caught during the postspawning period scored slightly below this range. Parasite load contributed to the depressed score during the postspawning period. Fish collected in all sampling periods were rated on average as "excellent" for condition factor, which suggests that the sampled population in the reserve were thriving.

Epigenetic modifications are key mediators of the interactions between the environment and an organism's genome. DNA methylation represents the best-studied epigenetic modification to date and is known to play key roles in regulating transcriptional activity and promoting chromosome stability. Our laboratory has previously demonstrated the utility of the American alligator (Alligator mississippiensis) as a sentinel species to investigate the persistent effects of environmental contaminant exposure on reproductive health. Here, we incorporate a liquid chromatography–tandem mass spectrometry method to directly measure the total (global) proportion of 5-methyl-2′-deoxycytidine (5mdC) in ovarian and whole blood DNA from alligators. Global DNA methylation in ovaries was significantly elevated in comparison with that of whole blood. However, DNA methylation appeared similar in juvenile alligators reared under controlled laboratory conditions but originating from three sites with dissimilar environmental qualities, indicating an absence of detectable site-of-origin effects on persistent levels of global 5mdC content. Analyses of tissues across individuals revealed a surprising lack of correlation between global methylation levels in blood and ovary. In addition, global DNA methylation in blood samples from juvenile alligators was elevated compared with those from adults, suggesting that age, as observed in mammals, may negatively influence global DNA methylation levels in alligators. To our knowledge, this is the first study examining global levels of DNA methylation in the American alligator and provides a reference point for future studies examining the interplay of epigenetics and environmental factors in a long-lived sentinel species.

The oil spill resulting from the explosion of the Deepwater Horizon drilling platform initiated immediate concern for marine wildlife, including common bottlenose dolphins in sensitive coastal habitats. To evaluate potential sublethal effects on dolphins, health assessments were conducted in Barataria Bay, Louisiana, an area that received heavy and prolonged oiling, and in a reference site, Sarasota Bay, Florida, where oil was not observed. Dolphins were temporarily captured, received a veterinary examination, and were then released. Dolphins sampled in Barataria Bay showed evidence of hypoadrenocorticism, consistent with adrenal toxicity as previously reported for laboratory mammals exposed to oil. Barataria Bay dolphins were 5 times more likely to have moderate–severe lung disease, generally characterized by significant alveolar interstitial syndrome, lung masses, and pulmonary consolidation. Of 29 dolphins evaluated from Barataria Bay, 48% were given a guarded or worse prognosis, and 17% were considere...

Sixteen monthly collections of adult male Gambusia holbrooki (eastern mosquitofish) were obtained from two lakes in central Florida, USA. Lake Woodruff and Lake Apopka are 36 miles apart, but differ in several environmental parameters. Compared with Lake Woodruff, Lake Apopka is warmer, more shallow in sampling areas (particularly during drought conditions; approximately 15-90 cm in Lake Apopka versus 60-120 cm in Lake Woodruff), more turbid, and more heavily contaminated with nutrients and industrial and agricultural chemicals. Here, we present detailed information on seasonal reproduction patterns in mosquitofish in their native range and compare patterns between fish from the two lakes. Male mosquitofish were reproductively active from spring through fall. Spermatogenesis, which is regulated in part by 11-ketotestosterone, ceased in October, and fish stored spermatozoa through the winter for immediate fertilization of offspring in the spring. Compared with Lake Woodruff, fish from Lake Apopka tended to be larger and have longer gonopodia and greater gonado- and hepato-somatic indices (GSI and HSI). High GSI in Apopka fish correlated with greater spermatid production, but fewer mature spermatozoa and either the same or lower sperm counts and sperm viability. Taken together, these observations suggest that differentiation of spermatids to spermatozoa is disrupted in Apopka fish, leading to reductions in fertility in some months. Delivery of sperm to females could also be affected in Apopka fish, which exhibit lower prevalence of efferent duct tissue in the testes during the summer.

Steroid hormones are essential for health in vertebrates. Corticosteroids, for example, have a regulatory role in many physiological functions, such as osmoregulation, respiration, immune responses, stress responses, reproduction, growth, and metabolism. Although extensively studied in mammals and some non-mammalian species, the molecular mechanisms of corticosteroid hormone (glucocorticoids and mineralocorticoids) action are poorly understood in reptiles. Here, we have evaluated hormone receptor-ligand interactions in the American alligator (Alligator mississippiensis), following the isolation of cDNAs encoding a glucocorticoid receptor (GR) and a mineralocorticoid receptor (MR). The full-length alligator GR (aGR) and aMR cDNAs were obtained using 5' and 3' rapid amplification cDNA ends (RACE). The deduced amino acid sequences exhibited high identity to the chicken orthologs (aGR: 83%; aMR: 90%). Using transient transfection assays of mammalian cells, both aGR and aMR proteins displayed corticosteroid-dependent activation of transcription from keto-steroid hormone responsive, murine mammary tumor virus promoters. We further compared the ligand-specifity of human, chicken, Xenopus, and zebrafish GR and MR. We found that the alligator and chicken GR/MR have very similar amino acid sequences, and this translates to very similar ligand specificity. This is the first report of the full-coding regions of a reptilian GR and MR, and the examination of their transactivation by steroid hormones.

This study aimed to quantify concentrations of fifteen perfluoroalkyl acids (PFAAs) in the plasma of American alligators (Alligator mississippiensis) inhabiting wetlands surrounding the Kennedy Space Center (KSC) in Florida, USA located at Merritt Island National Wildlife Refuge (MINWR). Approximately 10 male and 10 female alligators (ntotal = 229) were sampled each month during 2008 and 2009 to determine if seasonal or spatial trends existed with PFAA burden. PFOS represented the highest plasma burden (median 185 ng/g) and PFHxS the second highest (median 7.96 ng/g). While no significant seasonal trends were observed, unique spatial trends emerged. Many of the measured PFAAs co-varied strongly together and similar trends were observed for PFOS, PFDA, PFUnA, and PFDoA, as well as for PFOA, PFHxS, PFNA, PFTriA, and PFTA, suggesting more than one source of PFAAs at MINWR. Higher concentrations of PFOS and the PFAAs that co-varied with PFOS were collected from animals around sites that included the Shuttle Landing Facility (SLF) fire house and the Neil Armstrong Operations and Checkout (O&C) retention pond, while higher concentrations of PFOA and the PFAA that co-varied with PFOA were sampled from animals near the gun range and the old fire training facility. Sex-based differences and snout-vent length (SVL) correlations with PFAA burden were also investigated.

Within the past few decades, the concept of endocrine-disrupting chemicals (EDCs) has risen from a position of total obscurity to become a focus of dialogue, debate, and concern among scientists, physicians, regulators, and the public. The emergence and development of this field of study has not always followed a smooth path, and researchers continue to wrestle with questions about the low-dose effects and nonmonotonic dose responses seen with EDCs, their biological mechanisms of action, the true pervasiveness of these chemicals in our environment and in our bodies, and the extent of their effects on human and wildlife health. This review chronicles the development of the unique, multidisciplinary field of endocrine disruption, highlighting what we have learned about the threat of EDCs and lessons that could be relevant to other fields. It also offers perspectives on the future of the field and opportunities to better protect human health.

Sub-individual biomarkers are sub-lethal biological responses commonly used in the assessment of wildlife exposure to environmental contaminants. In this study, we examined the activity of glutathione-s-transferase (GST) and lactate dehydrogenase (LDH), and metallothionein (MT) concentrations among captive-raised alligator hatchlings, wild-caught juveniles, and wild-caught adults. Juveniles and adults were collected from three locations in Florida (USA) with varying degrees of contamination (i.e. Lake Apopka (organochlorine polluted site), Merritt Island National Wildlife Refuge (NWR) (metal polluted site), and Lake Woodruff NWR (reference site)). We examined whether changes in the response of these three biomarkers were age and sex dependent or reflected site-specific variations of environmental contaminants. Juvenile alligators from Merritt Island NWR had higher MT concentrations and lower GST activity compared to those from the other two sites. This outcome was consistent with higher metal pollution at this location. Sexually dimorphic patterns of MT and GST (F > M) were observed in juvenile alligators from all sites, although this pattern was not observed in adults. GST activity was lower in captive-raised alligators from Lake Apopka and Merritt Island NWR as compared to animals from Lake Woodruff NWR, suggesting a possible developmental modulator at these sites. No clear patterns were observed in LDH activity. We concluded that GST and MT demonstrate age and sex specific patterns in the alligators inhabiting these study sites and that the observed variation among sites could be due to differences in contaminant exposure.

The present study aimed to quantitate 15 perfluoroalkyl acids (PFAAs) in 125 adult American alligators at 12 sites across the southeastern United States. Of those 15 PFAAs, 9 were detected in 65% to 100% of samples: perfluorooctanoic acid, perfluorononanoic acid, perfluorodecanoic acid (PFDA), perfluoroundecanoic acid (PFUnA), perfluorododecanoic acid, perfluorotridecanoic acid (PFTriA), perfluorotetradecanoic acid, perfluorohexanesulfonic acid (PFHxS), and perfluorooctane sulfonate (PFOS). Males (across all sites) showed significantly higher concentrations of 4 PFAAs: PFOS (p = 0.01), PFDA (p = 0.0003), PFUnA (p = 0.021), and PFTriA (p = 0.021). Concentrations of PFOS, PFHxS, and PFDA in plasma were significantly different among the sites in each sex. Alligators at both Merritt Island National Wildlife Refuge (FL, USA) and Kiawah Nature Conservancy (SC, USA) exhibited some of the highest PFOS concentrations (medians of 99.5 ng/g and 55.8 ng/g, respectively) in plasma measured to date in a crocodilian species. A number of positive correlations between PFAAs and snout-vent length were observed in both sexes, suggesting that PFAA body burdens increase with increasing size. In addition, several significant correlations among PFAAs in alligator plasma may suggest conserved sources of PFAAs at each site throughout the greater study area. The present study is the first to report PFAAs in American alligators, to reveal potential PFAA hot spots in Florida and South Carolina, and to provide a contaminant of concern when assessing anthropogenic impacts on ecosystem health. Environ Toxicol Chem 2017;36:917-925. Published 2016 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.