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Developmental environmental stressors can have instructive effects on an organism's phenotype. This developmental plasticity can prepare organisms for potentially stressful future environments, circumventing detrimental effects on fitness. However, the physiological mechanisms underlying such adaptive plasticity are understudied, especially in vertebrates. We hypothesized that captive male zebra finches (Taeniopygia castanotis) exposed to a mild heat conditioning during development would acquire a persisting thermotolerance, and exhibit increased heat-shock protein (HSP) levels associated with a decrease in oxidative damage when exposed to a high-intensity stressor in adulthood. To test this, we exposed male finches to a prolonged mild heat conditioning (38°C) or control (22°C) treatment as juveniles. Then in a 2 × 2 factorial manner, these finches were exposed to a high heat stressor (42°C) or control (22°C) treatment as adults. Following the adult treatment, we collected testes and liver tissue and measured HSP70, HSP90, and HSP60 protein levels. In the testes, finches exhibited lower levels of HSP90 and HSP60 when exposed to the high heat stressor in adulthood if they were exposed to the mild heat conditioning as juveniles. In the liver, finches exposed to the high heat stressor in adulthood had reduced HSP90 and HSP60 levels, regardless of whether they were conditioned as juveniles. In some cases, elevated testes HSP60 levels were associated with increased liver oxidative damage and diminishment of a condition-dependent trait, indicating potential stress-induced tradeoffs. Our results indicate that a mild conditioning during development can have persisting effects on HSP expression and acquired thermotolerance., (© 2024 Wiley Periodicals LLC.)

AbstractAt certain intensities and durations, environmental stressors during development can result in changes in physiology that prepare organisms for future stressful conditions. Such plasticity can allow organisms to maintain good condition when confronted with a poor environment, potentially conferring an advantage in fitness. However, the physiological changes underlying these adaptive phenotypic adjustments are understudied. Using captive male zebra finches ( Taeniopygia castanotis ), we tested whether exposure to a prolonged mild stressor during development would adaptively modify their antioxidant enzyme expression, reducing oxidative damage when exposed to a high-intensity stressor in adulthood and allowing the maintenance of a secondary sexual trait. To do this, we exposed juvenile finches to either a prolonged mild heat stressor treatment (38°C) or a control temperature treatment (22°C). As adults, these finches were then exposed to either an acute high-intensity heat stressor treatment (42°C) or control temperature treatment (22°C). The beak color of males-a sexually selected trait-was quantified, as were oxidative stress parameters in the testes and liver tissues. We saw that the mild-heat-conditioned males had beaks with higher saturation and lower brightness at baseline in adulthood but that the changes in beak color in response to the high heat stressor varied. After exposure to the high heat stressor as adults, finches had higher levels of superoxide dismutase 1 and 2 in the testes and lower levels of lipid damage in the liver if they were also exposed to the mild heat conditioning as juveniles, indicating an adaptive phenotypic change.

Human activity has increased concentrations of endocrine-disrupting contaminants (EDCs) in many ecological systems. Many EDCs are xenoestrogens, which imitate naturally derived estrogen like estradiol 17-β (E2). These pollutants can critically affect a broad range of biological functions, particularly in organisms inhabiting aquatic environments. E2 and associated receptors are involved in regulating innate immune responses, where documentation of exogenous E2 on immune parameters is important for understanding health consequences. In this study, we explore the impact of environmentally relevant concentrations of E2 on circulating glucocorticoid levels and several innate immune parameters in hatchling American alligators (Alligator mississippiensis). Twenty-three hatchling alligators were randomly placed in one of three groups that differed in dietary E2 concentration: control (no E2 exposure), low E2 (0.5 μg/kg E2), or high E2 (1 μg/kg E2) for 10 weeks. Following this period, several biomarkers were quantified to monitor the impact of E2: growth, change in body condition, white blood cell (WBC) counts, glucocorticoid levels, and general antibody response. Blood E2 concentrations were greater in individuals exposed to E2, but plasma corticosterone levels were reduced among the experimental groups. Morphology, growth, and immune parameters of E2 exposed animals did not differ from controls. These results suggest that acute exposure to increased environmental estrogen concentrations may alter plasma hormone concentrations but have little to no impact on immediate morphology or immune responses. Future studies may expand on this by monitoring biomarkers in wild populations across time, which will provide insight into how different ontogenetic stages are impacted by environmental contaminants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Environmental oestrogens pose serious concerns for ecosystems through their effects on organismal survival and physiology. The gut microbiome is highly vulnerable to environmental influence, yet the effects of oestrogens on gut homeostasis are unknown because they are poorly studied in wildlife populations. To determine the influence of environmental oestrogens (i.e., xenoestrogens) on the diversity and abundance of gut microbiota, we randomly assigned 23 hatchling American alligators (Alligator mississippiensis) to three ecologically relevant treatments (control, low, and high oestrogen concentrations) for 10 weeks. We predicted that xenoestrogen exposure would decrease microbial diversity and abundance within the digestive tract and that this effect would be dose-dependent. Microbial samples were collected following diet treatments and microbial diversity was determined using 16S rRNA gene-sequencing. Individuals in oestrogen-treatment groups had decreased microbial diversity, but a greater relative abundance of operational taxonomic units than those in the control group. In addition, this effect was dose-dependent; as individuals were exposed to more oestrogen, their microbiome became less diverse, less rich and less even. Findings from this study suggest that oestrogen contamination can influence wildlife populations at the internal microbial-level, which may lead to future deleterious health effects., (© 2022 Society for Applied Microbiology and John Wiley & Sons Ltd.)

In complex terrestrial environments, chemical signals can be the most important sensory modality for locating conspecifics for potential mating opportunities, especially in spatially segregated populations or habitats. Organisms must evolve chemical signals to maximize the efficacy of conveying information, particularly in creating trails or mate-choice cues. Long-distance transmission of chemical signals may be an increasingly important management concern for small and fractured populations or potentially threatened species, such as gopher tortoises in the southeastern U.S. Mental gland secretions have been shown to have pheromonal function in gopher tortoises, suggesting a potential role as trail or marking pheromones, allowing males to track females or other males to find females. In this study, male gopher tortoises were given paired presentations of a negative control (distilled water) with serial dilutions (1:4, 1:20, 1:100, and 1:500) of male mental gland secretions. Male tortoises were able to discern treatment differences up to 1:20 diluted secretions, responding with an array of social behaviors (e.g. for the 1:20 dilution trial, carapace alignment and head bobbing occurred more frequently for the mental gland secretion relative to the control; p < 0.01). Multivariate principal components analysis yielded PC1 (including, approach, carapace alignment, head bobbing, tasting air, sniffing, and doubleback) that differed by treatment (p = 0.0007) and also was higher for the 1:20 diluted presentation relative to the 1:500 diluted presentation (p = 0.04). This study provides insight into gopher tortoise ecology, mate-choice, and the utility of environmentally diluted mental gland secretions in the external environment when seeking mating opportunities., Competing Interests: Conflict of Interest The authors declare that there is no conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Total mercury (THg) concentrations were measured in wild alligators inhabiting a coastal marsh in southern Louisiana, to determine the tissue distribution of THg among various body organs and tissue compartments. Concentrations of THg in claws and dermal tail scutes were compared to those in blood, brain, gonad, heart, kidney, liver, and skeletal muscle to determine if the former tissues, commonly available by non-lethal sampling, could be used as measures of body burdens in various internal organs. Mercury was found in all body organs and tissue compartments. However, overall, THg concentrations measured in alligators were below the FDA action level for fish consumption and were comparable to previous data reported from southwestern Louisiana. Our results suggest consumption of meat from alligators found in this region may be of little public health concern. However, the extended period of time between sampling (in this study) and the present-day highlight the need for continuous, additional, and more recent sampling to ensure consumer safety. Total mercury concentrations were highest in the kidney (3.18 ± 0.69 mg/kg dw) and liver (3.12 ± 0.76 mg/kg dw). THg levels in non-lethal samples (blood, claws, and dermal tail scutes) were positively correlated with all tissue THg concentrations (blood: R 2  = 0.513-0.988; claw: R 2  = 0.347-0.637, scutes: R 2  = 0.333-0.649). Because THg concentrations from blood, claws, and scutes were correlated with those of the internal organs, non-lethal sampling methods may be a viable method of estimating levels of THg in other body tissues., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The prevalence of antibiotic resistance genes (ARGs) can be driven by direct selection from antibiotic use and indirect selection from substances such as heavy metals (HMs). While significant progress has been made to characterize the influence of HMs on the enrichment and dissemination of ARGs in the environment, there is still much we do not know. To fill this knowledge gap, we present a comprehensive analysis of gut bacteria associated with wild cotton mice (Peromyscus gossypinus) trapped from several areas affected by legacies of HM and radionuclide contamination. We explore how these contaminants affect gut microbial community (GMC) composition and diversity and the enrichment of antibiotic, biocide, and metal resistance genes. Although we were able to identify that a myriad of co-occurring antimicrobial and HM resistance genes appear in mice from all areas, including those without a history of contamination, the proportions of co-occurring ARGs and metal resistance genes (MRGs) are higher in sites with radionuclide contamination. These results support those from several previous studies and enhance our understanding of the coselection process, while providing new insights into the ubiquity of antimicrobial resistance in the resistome of wild animals. IMPORTANCE Antimicrobial resistance is a serious global public health concern because of its prevalence and ubiquitous distribution. The rapid dissemination of antibiotic resistance genes is thought to be the result of the massive overuse of antibiotics in agriculture and therapeutics. However, previous studies have demonstrated that the spread of antibiotic resistance genes can also be influenced by heavy metal contamination. This coselection phenomenon, whereby different resistance determinants are genetically linked on the same genetic element (coresistance) or a single genetic element provides resistance to multiple antimicrobial agents (cross-resistance), has profound clinical and environmental implications. In contrast to antibiotics, heavy metals can persist in the environment as a selection pressure for long periods of time. Thus, it is important to understand how antibiotic resistance genes are distributed in the environment and to what extent heavy metal contaminants may be driving their selection, which we have done in one environmental setting.

Chemical communication is important for mate choice, especially at long distances in fragmented populations. The gopher tortoise is a social species that is threatened in the southeast U.S. due to habitat fragmentation and decline. One consequence of habitat loss is reduced mating opportunities, yet chemical signalling in gopher tortoises is relatively under-studied. Here, we investigated chemoreception of tortoise discrimination of chin secretions, or mental gland (MG) secretions. To assess conspecific recognition of male MG secretions, we conducted two paired-choice experiments: one with a neutral odorant control (NC; distilled water) and one with a pungent odorant control (PC; acetone) vs. male MG secretions. Behaviours were defined a priori, and their durations were quantified relative to treatments. Each sex spent significantly more time with MG secretions vs. acetone control during the PC study (p= 0.001). Each sex also sniffed MG swabs more frequently in both studies (PC study: p=0.0003; NC study: p=0.001). A principal components analysis of behavioural durations from the PC study identified one component with a significant treatment effect performed to MG secretions (p=0.0003), including the behaviours sniffing, head bobbing, biting, and eating near a swab. Our study provides the first chemical-behavioural bioassay of MG secretions from male gopher tortoises, suggesting MG secretions may be a source of pheromones., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Selenium represents an essential trace nutrient that is necessary for biological functions. Deficiencies can induce disease, but excess can induce toxicity. Selenium deficiency is a major concern in underdeveloped countries, while also posing as a toxic pollutant in waterways surrounding landfills, agricultural areas, and fossil fuel production sites. We examined the microbiome of selenomethionine (SeMet) fed American alligators (Alligator mississippiensis) at the beginning and end of a 7-week exposure experiment. Alligators were randomly divided into three groups: control and 1000 or 2000 ppm SeMet. DNA from before exposure (oral and cloaca swabs) and post-exposure (oral, cloaca, small & large intestines) sampling were extracted and amplified for bacterial 16 s rRNA. While treatment did not seem to have much effect, we observed a predominance of Fusobacteriaceae and Porpyromonodaceae across all tissue types. Cetobacterium and Clostridium are the most abundant genera as potential indicators of the aquatic and carrion feeding lifestyle of alligators.

MicroRNAs (miRNAs) are 18-24 nucleotide regulatory RNAs. They are involved in the regulation of genetic and biological pathways through post transcriptional gene silencing and/or translational repression. Data suggests a slow evolutionary rate for the saltwater crocodile (Crocodylus porosus) over the past several million years when compared to birds, the closest extant relatives of crocodilians. Understanding gene regulation in the saltwater crocodile in the context of relatively slow genomic change thus holds potential for the investigation of genomics, evolution, and adaptation. Utilizing eleven tissue types and sixteen small RNA libraries, we report 644 miRNAs in the saltwater crocodile with >78% of miRNAs being novel to crocodilians. We also identified potential targets for the miRNAs and analyzed the relationship of the miRNA repertoire to transposable elements (TEs). Results suggest an increased association of DNA transposons with miRNAs when compared to retrotransposons. This work reports the first comprehensive analysis of miRNAs in Crocodylus porosus and addresses the potential impacts of miRNAs in regulating the genome in the saltwater crocodile. In addition, the data suggests a supporting role of TEs as a source for miRNAs, adding to the increasing evidence that TEs play a significant role in the evolution of gene regulation., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Contaminants such as heavy metals may contribute to the dissemination of antimicrobial resistance (AMR) by enriching resistance gene determinants via co-selection mechanisms. In the present study, a survey was performed on soils collected from four areas at the Savannah River Site (SRS), South Carolina, USA, with varying contaminant profiles: relatively pristine (Upper Three Runs), heavy metals (Ash Basins), radionuclides (Pond B) and heavy metal and radionuclides (Tim's Branch). Using 16S rRNA gene amplicon sequencing, we explored the structure and diversity of soil bacterial communities. Sites with legacies of metal and/or radionuclide contamination displayed significantly lower bacterial diversity compared to the reference site. Metagenomic analysis indicated that multidrug and vancomycin antibiotic resistance genes (ARGs) and metal resistance genes (MRGs) including those associated with copper, arsenic, iron, nickel and zinc were prominent in all soils including the reference site. However, significant differences were found in the relative abundance and diversity of certain ARGs and MRGs in soils with metal/radionuclide contaminated soils compared to the reference site. Co-occurrence patterns revealed significant ARG/MRG subtypes in predominant soil taxa including Acidobacteriaceae, Bradyrhizobium, Mycobacterium, Streptomyces, Verrumicrobium, Actinomadura and Solirubacterales. Overall, the study emphasizes the potential risk of human activities on the dissemination of AMR in the environment., (© 2020 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Environmental contaminants, such as the trace element selenium (Se), are a continuing concern to species worldwide due to their potential pathophysiological effects, including their influence on the stress response mediated through glucocorticoids (GCs; stress hormones). Environmental concentrations of Se are increasing due to anthropogenic activities, including the incomplete combustion of coal and subsequent disposal of coal combustion wastes. However, most studies examining how Se affects GCs have been focused on lower trophic organisms. The objectives of this study were to investigate the effects of long-term Se exposure on traditionally used stress parameters and to identify which of these parameters best indicate Se accumulation in liver and kidney of the American alligator (Alligator mississippiensis), a top trophic carnivore found in the southeastern United States and known to inhabit Se-containing areas. Alligators were divided into three dietary treatments and fed prey spiked with 1000 or 2000 ppm of selenomethionine (SeMet) or deionized water (control treatment) for 7 weeks. Following the 7-week treatment protocol, blood and tissue samples were obtained to measure plasma corticosterone (CORT; the main crocodilian GC), tail scute CORT, the ratio of peripheral blood heterophils (H) to lymphocytes (L) as H/L ratio, and body condition. To evaluate which parameter best indicated Se accumulation in the liver and kidney, principal component and discriminant analyses were performed. The only parameter significantly correlated with liver and kidney Se concentrations was scute CORT. Our results suggest that measurement of CORT in tail scutes compared with plasma CORT, H/L ratios, and body condition is the best indicator of Se-exposure and accumulation in crocodilians.

Saltwater crocodiles (Crocodylus porosus) across three size categories (hatchlings, grower and harvest-size) were repeatedly blood sampled on two farms in the Northern Territory, Australia to determine reference plasma corticosterone (CORT; crocodilian stress hormone) levels. The mean CORT values for hatchlings (<1 year old), growers (1-3 years) and harvest-size individuals (2 + years) were 1.65 ± 0.15 ng/ml, 2.73 ± 0.21 ng/ml and 2.19 ± 0.16 ng/ml, respectively. No inter-farm differences within the hatchling or harvest-size crocodiles were detected, but growers on Farm 2 had significantly lower plasma CORT than those on Farm 1. However, the grower growth rate coefficients were the same across both farms so the repeated blood sampling design most likely contributed to the difference in CORT values rather than any management procedures. Plasma corticosterone levels significantly increased with time of day. Substantial variation in plasma CORT was observed at each sampling which is not unprecedented in the literature but requires further elucidation. Irrespective, as CORT values were generally low, our results suggest that the farming environment and husbandry practices, as implemented under the Australian industry Code of Practice, are effective as baseline animal welfare measures although they should be viewed as a foundation for further welfare research and not considered static., (Copyright © 2018. Published by Elsevier Inc.)

Baseline plasma corticosterone (CORT) concentrations have been widely used to investigate the effects of stressors in wild and captive crocodilians. However, collecting baseline plasma CORT samples from wild crocodilians may be particularly difficult due to the capture and handling protocols used for large individuals. Thus, it may prove beneficial to use recently modified techniques for extracting CORT deposited in keratinized and non-keratinized tissues to better quantify the effects of long-term stress in crocodilians. In this study, we investigated the feasibility of using American alligator (Alligator mississippiensis) tail scute tissues to quantify CORT by collecting blood and tail scutes from 40 alligators before and after a short-term handling stressor. The objective of the current study was to better understand CORT deposition in crocodilian scutes and whether short-term increases in CORT could be detected. We found that CORT can be reliably extracted from alligator scute tissue and quantified using a commercially available enzyme immunoassay. However, there was a significant increase in scute CORT concentrations following an alligator being exposed to a short-term stressor (p = 0.017), although the magnitude of change was less than observed in plasma samples from the same individuals (p = 0.002). Furthermore, our results indicate that there was a significant effect of body condition on an alligator's post-stressor CORT concentration (p = 0.02). While our study is among the first to experimentally examine the usefulness of tissue CORT in crocodilians, a combination of field and laboratory experiments are needed to better understand deposition rates of CORT in scute tissues and to further validate the usefulness of tissue glucocorticoids for evaluating the effects of stress., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In oviparous species, the embryonic environment-particularly temperature-can alter phenotype and survival of an individual by affecting its size as well as its metabolic rate. Previous studies have shown that incubation temperatures can affect sex ratio in birds; specifically, low incubation temperatures were shown to produce a male-biased sex ratio in zebra finches (Taeniopygia guttata) possibly because of a higher pre- or postnatal mortality rate in females. We hypothesized that sexes respond differently to suboptimal incubation temperature, leading to a male-biased sex ratio. To test this hypothesis, zebra finch eggs were incubated at 36.1°, 37.5°, or 38.5°C and hatching success, hatchling mass, residual yolk mass, and pectoralis mass were measured. We found that while hatchling mass was similar between the sexes at 37.5°C, female hatchlings were heavier at 36.1°C, and male hatchlings were heavier at 38.5°C. Pectoralis muscle mass was similar between the sexes at 36.1°C; however, at 37.5°C, female pectoralis mass was heavier at hatching than that of males. Females at 37.5°C also had lower residual yolk at hatching compared with males, reflecting a higher use of energy by female embryos compared with male embryos at this temperature. In contrast, residual yolk was similar between the sexes at 36.1° and 38.5°C. Our results suggest that there are sex differences in how incubation temperature alters organ mass and yolk energy reserve; this can lead to a difference in survival at different incubation temperatures between the sexes. Taken together with previous studies showing that females alter incubation behavior with ambient temperature, rising ambient temperatures could impact phenotype and survival of avian offspring in a sex-specific manner.

Selenium (Se) is an essential nutrient which in excess causes toxicity. The disposal of incompletely combusted coal, which often is rich in Se, into aquatic settling basins is increasing the risk of Se exposure worldwide. However, very few studies have looked at the physiological effects of Se exposure on long-lived, top trophic vertebrates, such as the American alligator (Alligator mississippiensis). During a 7-week period, alligators were fed one of three dietary treatments: mice injected with deionized water or mice injected with water containing 1000 or 2000 ppm selenomethionine (SeMet). One week after the last feeding alligators were bled within 3 min of capture for plasma corticosterone (CORT). A few days later, all alligators were euthanized and whole blood and tail tissue were harvested to measure oxidative damage, an antioxidant-associated transcription factor, and antioxidant enzymes [glutathione peroxidase-1 (GPX1), superoxide dismutase-1 (SOD1), and SOD2] by Western blotting. There was a dose-dependent increase in baseline CORT levels in alligators administered SeMet. Except for blood SOD2 levels, SeMet treatment had no effect (p > 0.05 for all) on oxidative status: oxidative damage, GPX1, SOD1, and muscle SOD2 levels were similar among treatments. Our results illustrate that high levels of Se may act as a stressor to crocodilians. Future studies should investigate further the physiological effects of Se accumulation in long-lived, top-trophic vertebrates.

Developmental stressors can have strong effects that persist well into adulthood, and are generally seen as detrimental. However, recent work suggests that a mild developmental stressor can have beneficial effects by preparing the organism to better withstand negative impacts when exposed to high levels of the stressor later in life, also known as a conditioning hormesis. Still, little is known about the influence of such hormetic effects on fitness-related measures. We hypothesized that exposure to a mild stressor during development will protect individuals later in life from the negative effects of a high heat stressor on immune function and reproduction. To test this hypothesis, we subjected zebra finches (Taeniopygia guttata) to a repeated mild heat stressor (38°C) as juveniles for 28 days. As adults, the birds were then exposed to a high heat stressor (42°C) for 3 consecutive days and we examined the effects on immune function via wound healing, and on female reproductive output. We found that females given the mild heat stressor as juveniles healed wounds marginally slower, but also had higher clutch viability than controls. For the adult treatment, we saw that high heat had a stimulatory effect on clutch viability as well. Our findings point toward the occurrence of trade-offs between immune function and reproduction due to a cost of hormetic priming when the adult environment does not match that of early life., (© 2018 Wiley Periodicals, Inc.)

Songbirds exposed to methylmercury (MeHg) often exhibit reduced reproductive success and cognitive abilities. To better understand whether oxidative stress plays a role, we dosed zebra finches (Taeniopygia guttata) with a contaminated (1.2 ppm MeHg-cysteine) or control diet for their entire lives, including during development in the egg. Levels of antioxidant enzymes [superoxide dismutase (SOD1 and SOD2)], oxidative damage (4-hydroxynonenal; 4-HNE), and antioxidant transcription factors [nuclear factor (erythroid-derived 2)-like 2; Nrf2] were measured in the liver and pectoralis muscle of adults. MeHg treatment did not affect levels of 4-HNE or liver SOD2 or Nrf2. Birds in the MeHg treatment differed significantly from controls in pectoralis SOD1 and Nrf2, and tended to differ in liver SOD1 and pectoralis SOD2; however, we detected no overall pattern of effect of MeHg on oxidative status in dosed finches. We suspect that this is a consequence of the differential survival of MeHg-tolerant birds.

Selenium (Se) is a naturally occurring essential element that can be toxic to vertebrates at high concentrations. Despite studies that have documented that wild reptile species can accumulate copious amounts of Se, little is known regarding specific toxicologic effects of Se. In this study, 70 juvenile yellow-bellied sliders (Trachemys scripta scripta) were exposed to one of three seleno-L-methionine (SetMet) treatments (control, n = 24; 15 mg/kg, n = 23; and 30 mg/kg, n = 23) via weekly oral gavage for 5 weeks. At the conclusion of the experiment, kidney, liver, muscle, and blood samples were collected for quantitative Se analysis. Turtles in the SeMet treatment groups accumulated significantly higher amounts of Se in all tissue types relative to controls (all p < 0.001). Turtles in the 30 mg/kg SeMet group also accumulated significantly higher amounts of Se compared to the 15 mg/kg group (all p < 0.001). Although toxicity thresholds for reptiles have not been established, Se concentrations in liver tissue from both SeMet treatment groups exceeded reported avian toxicity thresholds for liver tissue. Neither oxygen consumption nor innate bactericidal capacity were impacted by SeMet exposure. However, turtles in the 30 mg/kg SeMet group exhibited anemia, which has been reported in other vertebrates exposed to Se. Furthermore, juvenile T. s. scripta in the 30 mg/kg SeMet group experienced 17% mortality compared to 0% in the 15 mg/kg treatment and control groups. To our knowledge, this study is the first to report dose-dependent Se-associated anemia and mortality in a chelonian species.

Anthropogenic activities such as industrial processes often produce copious amounts of contaminants that have the potential to negatively impact growth, survival, and reproduction of exposed wildlife. Coal combustion residues (CCRs) represent a major source of pollutants globally, resulting in the release of potentially harmful trace elements such as arsenic (As), cadmium (Cd), and selenium (Se) into the environment. In the United States, CCRs are typically stored in aquatic settling basins that may become attractive nuisances to wildlife. Trace element contaminants, such as CCRs, may pose a threat to biota yet little is known about their sublethal effects on reptiles. To assess the effects of CCR exposure in turtles, we sampled 81 yellow-bellied sliders (Trachemys scripta scripta) in 2014-2015 from CCR-contaminated and uncontaminated reference wetlands located on the Savannah River Site (Aiken, SC, USA). Specific aims were to (1) compare the accumulation of trace elements in T. s. scripta claw and blood samples between reference and CCR-contaminated site types, (2) evaluate potential immunological effects of CCRs via bacterial killing assays and phytohaemagglutinin (PHA) assays, and (3) quantify differences in hemogregarine parasite loads between site types. Claw As, Cd, copper (Cu), and Se (all p ≤ 0.001) and blood As, Cu, Se, and strontium (Sr; p ≤ 0.015) were significantly elevated in turtles from CCR-contaminated wetlands compared to turtles from reference wetlands. Turtles from reference wetlands exhibited lower bacterial killing (p = 0.015) abilities than individuals from contaminated sites but neither PHA responses (p = 0.566) nor parasite loads (p = 0.980) differed by site type. Despite relatively high CCR body burdens, sliders did not exhibit apparent impairment of immunological response or parasite load. In addition, the high correlation between claw and blood concentrations within individuals suggests that nonlethal tissue sampling may be useful for monitoring CCR exposure in turtles., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Selenium (Se) is an essential trace nutrient, but in excess, it can induce toxicity. Incomplete combustion of coal produces coal combustion wastes, which are enriched in Se and often disposed of in aquatic basins. While a multitude of studies have investigated Se accumulation in vertebrates, few studies have examined its effects on longer-lived top trophic carnivores, such as the American alligator (Alligator mississippiensis). In this study, alligators were fed one of three Dietary Treatments: mice injected with water (controls) or water supplemented with 1000 or 2000 ppm selenomethionine (SeMet). Dietary Treatment significantly affected Se levels in both the liver (p < 0.0001; raw mean ± SE: 1000 ppm group, 35.20 ± 6.32 ppm; 2000 ppm group, 49.97 ± 4.00 ppm) and kidney (p < 0.0001; raw mean ± SE: 1000 ppm group, 101.60 ± 8.64 ppm; 2000 ppm, 96.38 ± 5.81 ppm), which were significantly higher in alligators fed SeMet than in controls. Post-treatment head length, used to control for size variation, was negatively related to both kidney (p = 0.0142) and liver (p = 0.0010) Se concentrations. Dietary treatment with SeMet significantly reduced body condition (1000 ppm, p < 0.0029; 2000 ppm, p = 0.0075), but it significantly increased growth (1000 ppm, p < 0.0001; 2000 ppm, p = 0.0316). Body condition and growth remained unchanged in control alligators (p > 0.05). Our results demonstrate alligators are capable of accumulating high levels of Se through trophic transfer. The positive effects of accumulation on growth may demonstrate Se essentiality, whereas the negative effects on condition may demonstrate toxicity. Accumulation also was associated with mortality, further demonstrating toxicity. Future studies should further investigate the physiological effects of Se accumulation in long-lived, top-trophic carnivores.

Coal-burning power plants supply approximately 37 % of the electricity in the United States. However, incomplete combustion produces ash wastes enriched with toxic trace elements that have historically been disposed of in aquatic basins. Organisms inhabiting such habitats may accumulate these trace elements; however, studies investigating the effects on biota have been primarily restricted to shorter-lived, lower-trophic organisms. The American alligator (Alligator mississippiensis), a long-lived, top-trophic carnivore, has been observed inhabiting these basins, yet the health or immune effects of chronic exposure and possible accumulation remains unknown. In this study, we investigated how chronic dietary ingestion of prey contaminated with coal combustion wastes (CCWs) for 25 months, and subsequent accumulation of trace elements present in CCWs, affected juvenile alligator immune function and health. Alligators were assigned to one of four dietary-treatment groups including controls and those fed prey contaminated with CCWs for one, two, or three times a week. However, no effect of Dietary Treatment (p > 0.05) was observed on any immune parameter or hematological or plasma analyte we tested. Our results suggest that neither exposure to nor accumulation of low doses of CCWs had a negative effect on certain aspects of the immune and hematological system. However, future studies are required to elucidate this further.

Little is known about the propensity of crocodilians to bioaccumulate trace elements as a result of chronic dietary exposure. We exposed 36 juvenile alligators (Alligator mississippiensis) to one of four dietary treatments that varied in the relative frequency of meals containing prey from coal combustion waste (CCW)-contaminated habitats vs. prey from uncontaminated sites, and evaluated tissue residues and growth rates after 12 mo and 25 mo of exposure. Hepatic and renal concentrations of arsenic (As), cadmium (Cd) and selenium (Se) varied significantly among dietary treatment groups in a dose-dependent manner and were higher in kidneys than in livers. Exposure period did not affect Se or As levels but Cd levels were significantly higher after 25 mo than 12 mo of exposure. Kidney As and Se levels were negatively correlated with body size but neither growth rates nor body condition varied significantly among dietary treatment groups. Our study is among the first to experimentally examine bioaccumulation of trace element contaminants in crocodilians as a result of chronic dietary exposure. A combination of field surveys and laboratory experiments will be required to understand the effects of different exposure scenarios on tissue residues, and ultimately link these concentrations with effects on individual health., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

: American alligators ( Alligator mississippiensis ) are one of the most studied crocodilian species in the world, yet blood and plasma biochemistry information is limited for juvenile alligators in their northern range, where individuals may be exposed to extreme abiotic and biotic stressors. We collected blood samples over a 2-yr period from 37 juvenile alligators in May, June, and July to establish reference intervals for 22 blood and plasma analytes. We observed no effect of either sex or blood collection time on any analyte investigated. However, our results indicate a significant correlation between a calculated body condition index and aspartate aminotransferase and creatine kinase. Glucose, total protein, and potassium varied significantly between sampling sessions. In addition, glucose and potassium were highly correlated between the two point-of-care devices used, although they were significantly lower with the i-STAT 1 CG8+ cartridge than with the Vetscan VS2 Avian/Reptile Rotor. The reference intervals presented herein should provide baseline data for evaluating wild juvenile alligators in the northern portion of their range.

Agricultural production of the saltwater crocodile (Crocodylus porosus) is an emergent industry in northern Australia with many of the factors affecting production remaining unknown. In this study, we sought to expand upon our previous findings of reference corticosterone and immune function by reporting baseline sex hormone levels [testosterone (TEST) and estradiol (ESTR)] and their association with growth. This was achieved by sampling 253 hatchling crocodiles repeatedly at 3, 6, and 9months of age. Sampling age had a significant effect on both TEST (p<0.001) and ESTR (p<0.001) suggesting climatic/abiotic factors have an influence even in prepubescent crocodiles. Stress, as measured by plasma corticosterone, had no detectable effect on plasma ESTR or TEST levels. Unexpectedly however, TEST was higher in slower-growing crocodiles, which is contrary to what has been reported for the American alligator. ESTR was not associated with growth., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The assessment of wildlife health has been enhanced by the ability of point-of-care (POC) blood analysers to provide biochemical analyses of non-domesticated animals in the field. However, environmental limitations (e.g. temperature, atmospheric humidity and rain) and lack of reference values may inhibit researchers from using such a device with certain wildlife species. Evaluating the use of alternative sample types, such as plasma, in a POC device may afford researchers the opportunity to delay sample analysis and the ability to use banked samples. In this study, we examined fresh whole blood, fresh plasma and frozen plasma (sample type) pH, partial pressure of carbon dioxide (PCO2), bicarbonate (HCO3 (-)), total carbon dioxide (TCO2), base excess (BE), partial pressure of oxygen (PO2), oxygen saturation (sO2) and lactate concentrations in 23 juvenile American alligators (Alligator mississippiensis) using an i-STAT CG4+ cartridge. Our results indicate that sample type had no effect on lactate concentration values (F 2,65 = 0.37, P = 0.963), suggesting that the i-STAT analyser can be used reliably to quantify lactate concentrations in fresh and frozen plasma samples. In contrast, the other seven blood parameters measured by the CG4+ cartridge were significantly affected by sample type. Lastly, we were able to collect blood samples from all alligators within 2 min of capture to establish preliminary reference ranges for juvenile alligators based on values obtained using fresh whole blood.

To determine reference levels for on-farm stressors on immune responsiveness and growth rate, 253 hatchling crocodiles from 11 known breeding pairs were repeatedly measured and blood sampled during their first year. Plasma corticosterone (CORT) was used to quantify baseline stress levels in captive animals and were found to be lower (mean 1.83±SE 0.16 ng/mL) than previously reported in saltwater crocodile hatchlings. Two tests of immune function were also conducted. Innate constitutive immunity was assessed using bacterial killing assays (BKA) against two bacterial species: Escherichia coli and Providencia rettgeri, whereby the latter causes considerable economic loss to industry from septicaemic mortalities. Although the bactericidal capabilities were different at approximately 4 months old (32±3% for E. coli and 16±4% for P. rettgeri), the differences had disappeared by approximately 9 months old (58±2% and 68±6%, respectively). To assess immune responsiveness to a novel antigen, the inflammatory swelling response caused by phytohaemagglutinin (PHA) injection was assessed but was only significantly different between Samplings 1 and 3 (5% LSD). There were no significant clutch effects for CORT or PHA but there were for both BKA traits. CORT was not significantly associated with growth (head length) or the immune parameters except for P. rettgeri BKA where higher CORT levels were associated with better bactericidal capability. As such, these results suggest that the crocodiles in this study are not stressed, therefore endorsing the management strategies adopted within the Australian industry Code of Practice., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Crocodilians are important keystone species and indicators of environmental health. Much remains unknown, however regarding utility of field-collected crocodilian blood samples for ecologic assessments. Field sampling sites are also often distant to analysis centers, necessitating development of new techniques and panels of assays that will yield environmentally relevant data. Stability and viability of hematological and immunological indices have been of particular interest for linking ecosystem health to biomarkers in resident species. In this study, we investigated the effect of time at analysis post-blood sampling at 4 and 24 hr on a panel of potential biomarkers in alligator blood. Our results suggest alligator blood samples can be reliably evaluated for both hematologic and immunologic profile 24 hr after sampling.

Avian influenza has emerged as one of the most ubiquitous viruses within our biosphere. Wild aquatic birds are believed to be the primary reservoir of all influenza viruses; however, the spillover of H5N1 highly pathogenic avian influenza (HPAI) and the recent swine-origin pandemic H1N1 viruses have sparked increased interest in identifying and understanding which and how many species can be infected. Moreover, novel influenza virus sequences were recently isolated from New World bats. Crocodilians have a slow rate of molecular evolution and are the sister group to birds; thus they are a logical reptilian group to explore susceptibility to influenza virus infection and they provide a link between birds and mammals. A primary American alligator (Alligator mississippiensis) cell line, and embryos, were infected with four, low pathogenic avian influenza (LPAI) strains to assess susceptibility to infection. Embryonated alligator eggs supported virus replication, as evidenced by the influenza virus M gene and infectious virus detected in allantoic fluid and by virus antigen staining in embryo tissues. Primary alligator cells were also inoculated with the LPAI viruses and showed susceptibility based upon antigen staining; however, the requirement for trypsin to support replication in cell culture limited replication. To assess influenza virus replication in culture, primary alligator cells were inoculated with H1N1 human influenza or H5N1 HPAI viruses that replicate independent of trypsin. Both viruses replicated efficiently in culture, even at the 30 C temperature preferred by the alligator cells. This research demonstrates the ability of wild-type influenza viruses to infect and replicate within two crocodilian substrates and suggests the need for further research to assess crocodilians as a species potentially susceptible to influenza virus infection.

To provide context for the diversification of archosaurs--the group that includes crocodilians, dinosaurs, and birds--we generated draft genomes of three crocodilians: Alligator mississippiensis (the American alligator), Crocodylus porosus (the saltwater crocodile), and Gavialis gangeticus (the Indian gharial). We observed an exceptionally slow rate of genome evolution within crocodilians at all levels, including nucleotide substitutions, indels, transposable element content and movement, gene family evolution, and chromosomal synteny. When placed within the context of related taxa including birds and turtles, this suggests that the common ancestor of all of these taxa also exhibited slow genome evolution and that the comparatively rapid evolution is derived in birds. The data also provided the opportunity to analyze heterozygosity in crocodilians, which indicates a likely reduction in population size for all three taxa through the Pleistocene. Finally, these data combined with newly published bird genomes allowed us to reconstruct the partial genome of the common ancestor of archosaurs, thereby providing a tool to investigate the genetic starting material of crocodilians, birds, and dinosaurs., (Copyright © 2014, American Association for the Advancement of Science.)

Endocrine-disrupting chemicals (EDCs) alter cellular and organ system homeostasis by interfering with the body's normal physiologic processes. Numerous studies have identified environmental estrogens as modulators of EDC-related processes in crocodilians, notably in sex determination. Other broader studies have shown that environmental estrogens dysregulate normal immune function in mammals, birds, turtles, lizards, fish, and invertebrates; however, the effects of such estrogenic exposures on alligator immune function have not been elucidated. Alligators occupy a top trophic status, which may give them untapped utility as indicators of environmental quality. Environmental estrogens are also prevalent in the waters they occupy. Understanding the effects of these EDCs on alligator immunity is critical for managing and assessing changes in their health and is thus the focus of this review.