A longitudinal study of endocrinology and foraging ecology of subadult gray whales prior to death based on baleen analysis.

Individual-level assessments of wild animal health, vital rates, and foraging ecology are critical for understanding population-wide impacts of exposure to stressors. Large whales face multiple stressors, including, but not limited to, ocean noise, pollution, and ship strikes. Because baleen is a continuously growing keratinized structure, serial extraction, and quantification of hormones and stable isotopes along the length of baleen provide a historical record of whale physiology and foraging ecology. Furthermore, baleen analysis enables the investigation of dead specimens, even decades later, allowing comparisons between historic and modern populations. Here, we examined baleen of five sub-adult gray whales and observed distinct patterns of oscillations in δ15N values along the length of their baleen plates which enabled estimation of baleen growth rates and differentiation of isotopic niche widths of the whales during wintering and summer foraging. In contrast, no regular patterns were apparent in δ13C values. Prolonged elevation of cortisol in four individuals before death indicates that chronic stress may have impacted their health and survival. Triiodothyronine (T3) increased over months in the whales with unknown causes of death, simultaneous with elevations in cortisol, but both hormones remained stable in the one case of acute death attributed to killer whale predation. This parallel elevation of cortisol and T3 challenges the classic understanding of their interaction and might relate to increased energetic demands during exposure to stressors. Reproductive hormone profiles in subadults did not show cyclical trends, suggesting they had not yet reached sexual maturity. This study highlights the potential of baleen analysis to retrospectively assess gray whales' physiological status, exposure to stressors, reproductive status, and foraging ecology in the months or years leading up to their death, which can be a useful tool for conservation diagnostics to mitigate unusual mortality events.

Can spines tell a story? Investigation of echidna spines as a novel sample type for hormone analysis in monotremes.

The short-beaked echidna (Tachyglossus aculeatus) is a monotreme endemic to Australia and New Guinea, and is the most widespread native mammal in Australia. Despite its abundance, there are considerable gaps in our understanding of echidna life history such as reproductive cycles in both sexes, patterns of stress physiology, and possible seasonal changes in metabolism. Slow-growing integumentary sample types comprised of keratin (hair, claw, etc.) have been used in other wildlife to assess these questions via analysis of longitudinal patterns in steroid and thyroid hormones that are deposited in these tissues as they grow. Hairs and spines comprise the pelage of echidnas, the spines being keratinized structures homologous to hair. Thus, echidna spines could be a viable sample type for hormone analysis contributing to a better understanding of the biology of echidnas. The aim of this work was to determine whether steroid hormones are detectable in echidna spines, to perform assay validations, and to establish a protocol for extracting and quantifying hormones in echidna spines using commercially available assay kits. We also inspected cross-sectioned spines using light and electron microscopy for any evidence of annual growth markers that might enable inferences about spine growth rate. Corticosterone, progesterone, estradiol, and testosterone were detectable in all samples, and echidna spine extract passed standard assay validations (parallelism and accuracy), indicating that commercially available assay kits can quantify hormones accurately in this sample type. No visible growth marks were identified in the spines and thus spine growth rate is currently unknown. Echidna spines show promise as a novel matrix from which hormones can be quantified; next steps should involve determination of spine annual growth rate, possible seasonal changes in growth rate, and persistence of spines over time in order to perform physiological validations, i.e., relationship between physiological status and hormone concentrations in spines.

Optimizing hormone extraction protocols for whale baleen: Tackling questions of solvent:sample ratio and variation.

Obtaining endocrine data from alternative sample types such as baleen and other keratinized tissues has proven a valuable tool to investigate reproductive and stress physiology via steroid hormone quantification, and metabolic stress via thyroid hormone quantification in whales and other vertebrates. These alternative sample types provide an integrated measure of plasma levels over the period that the structure was growing, thus capturing months or even years of an individual's endocrine history. Additionally, their robust and stable keratin matrix allows such samples to be stored for years to decades, enabling the analysis and comparison of endocrine patterns from past and modern populations. However, the extraction and analysis of hormones from baleen and other keratinized tissues remains novel and requires both biological and analytical validations to ensure the method fulfills the requirements for its intended use. We utilized baleen recovered at necropsy from southern right whales (Eubalaena australis) that died at Península Valdés, Argentina, using a commercially available progesterone enzyme immunoassay (EIA) to address two methodological questions: 1) what is the minimum sample mass required to reliably quantify hormone content of baleen samples analyzed with commercially available EIAs, and 2) what is the optimal ratio of solvent volume to sample mass, i.e., the ratio that yields the maximum amount of hormone with high accuracy and low variability between replicates. We concluded that masses of at least 20 mg should be used whenever possible, and extraction is best performed using an 80:1 ratio of solvent to sample (volume of solvent to sample mass; µl:mg). These results can help researchers to make informed methodological decisions when using a destructive extraction method with rare or unique specimens.

Investigation of keratinase digestion to improve steroid hormone extraction from diverse keratinous tissues.

Monitoring the physiology of wild populations presents many technical challenges. Blood samples, long the gold standard of wildlife endocrinology studies, cannot always be obtained. The validation and use of non-plasma samples to obtain hormone data have greatly improved access to more integrated information about an organism's physiological state. Keratinous tissues like skin, hair, nails, feathers, or baleen store steroid hormones in physiologically relevant concentrations, are stable across decades, and can be used to retrospectively infer physiological state at prior points in time. Most protocols for steroid extraction employ physical pulverization or cutting of the sample, followed by mixing with a solvent. Such methods do produce repeatable and useful data, but low hormone yield and detectability issues can complicate research on small or rare samples. We investigated the use of keratinase, an enzyme that breaks down keratin, to improve the extraction and yield of corticosterone from vertebrate keratin tissues. Corticosterone content of keratinase-digested extracts were compared to non-keratinase extracts for baleen from three species of whale (blue, Balaenoptera musculus; bowhead, Balaena mysticetus; southern right, SRW; Eubalaena australis), shed skin from two reptiles (tegu lizard, Salvator merianae; narrow-headed garter snake, Thamnophis rufipunctatus), hair from arctic ground squirrel (AGS; Urocitellus parryii), feathers from Purple Martins (PUMA; Progne subis), and spines from the short-beaked echidna (Tachyglossus aculeatus). We tested four starting masses (10, 25, 50, 100 mg) for each sample; digestion was most complete in the 10 and 25 mg samples. A corticosterone enzyme immunoassay (EIA) was validated for all keratinase-digested extracts. In all sample types except shed skin from reptiles, keratinase digestion improved hormone yield, with PUMA feathers and blue whale baleen having the greatest increase in apparent corticosterone content (100% and 66% more hormone, respectively). The reptilian shed skin samples did not benefit from keratinase digestion, actually yielding less hormone than controls. With further optimization and refinement, keratinase digestion could greatly improve yield of steroid hormones from various wildlife epidermal tissue types, allowing more efficient use of samples and ultimately improving understanding of the endocrine physiology of wild populations.

Hormonal correlates of the annual cycle of activity and body temperature in the South-American tegu lizard (Salvator merianae).

Life history transitions and hormones are known to interact and influence many aspects of animal physiology and behavior. The South-American tegu lizard (Salvator merianae) exhibits a profound seasonal shift in metabolism and body temperature, characterized by high daily activity during warmer months, including reproductive endothermy in spring, and metabolic suppression during hibernation in winter. This makes S. merianae an interesting subject for studies of interrelationships between endocrinology and seasonal changes in physiology/behavior. We investigated how plasma concentrations of hormones involved in regulation of energy metabolism (thyroid hormones T4 and T3; corticosterone) and reproduction (testosterone in males and estrogen/progesterone in females) correlate with activity and body temperature (Tb) across the annual cycle of captive held S. merianae in semi-natural conditions. In our initial model, thyroid hormones and corticosterone showed a positive relationship with activity and Tb with independent of sex: T3 positively correlated with activity and Tb, while T4 and corticosterone correlated positively with changes in Tb only. This suggests that thyroid hormones and glucocorticoids may be involved in metabolic transitions of annual cycle events. When accounting for sex-steroid hormones, our sex separated models showed a positive relationship between testosterone and Tb in males and progesterone and activity in females. Coupling seasonal endocrine measures with activity and Tb may expand our understanding of the relationship between animal's physiology and its environment. Manipulative experiments are required in order to unveil the directionality of influences existing among abiotic factors and the hormonal signaling of annual cyclicity in physiology/behavior.

Retrospective analysis of the lifetime endocrine response of southern right whale calves to gull wounding and harassment: A baleen hormone approach.

Physiological measurements are informative in assessing the relative importance of stressors that potentially impact the health of wildlife. Kelp Gulls, Larus dominicanus (KG), resident to the region of Península Valdés, Argentina, have developed a unique behavior of landing on the backs of southern right whale adults and calves, Eubalaena australis (SRW), where they feed on their skin and blubber. This parasitic behavior results in large open wounds on the dorsal surface of the whale. Coincidently, the SRW population off the coast of Península Valdés has experienced elevated calf mortality. We quantified levels of glucocorticoids and thyroid hormone extracted from baleen of dead calves to evaluate, retrospectively, the endocrine response of whale calves to gull wounding and harassment. Baleen accumulates hormones as it grows, allowing evaluation of long-term trends in physiological condition. While glucocorticoids (GCs) are known to increase in response to stressors such as disturbance, the metabolic hormone triiodothyronine (T3) has been shown to decrease under sustained food deprivation but is largely unaffected by disturbance stress. We quantified lifetime patterns of GCs and T3 in baleen recovered at necropsy from 36 southern right whale calves with varying severity of wounding from KGs. GC levels in baleen correlated positively with the degree of wounding, while T3 levels remained stable irrespective of the severity of the wounding. Our results suggest no evidence of malnutrition in low vs. severely wounded whales. However, the positive correlation of GCs with wound severity indicates that heavily wounded calves are suffering high levels of physiological stress before they die. This suggests that KG wounding may have contributed to the high southern right whale calf mortality observed in the Península Valdés region of Argentina.

Seasonal changes in plasma concentrations of the thyroid, glucocorticoid and reproductive hormones in the tegu lizard Salvator merianae.

The tegu lizard Salvator merianae is a large, widely distributed teiid lizard endemic to South America that exhibits annual cycles of high activity during the spring and summer, and hibernation during winter. This pattern of activity and hibernation is accompanied by profound seasonal changes in physiology and behavior, including endothermy during the austral spring. The unusual combination of seasonal endothermy, hibernation and oviparity, in a non-avian, non-mammalian species, makes S. merianae an interesting subject for study of comparative aspects of endocrine regulation of seasonal changes in physiology. In the present study, we first validated commercially available immunoassay kits for quantification of hormone concentrations of the reproductive (testosterone, estradiol and progesterone), adrenal (corticosterone), and thyroid [thyroxine (T4) and triiodothyronine (T3)] axes in plasma of an outdoor, captive adult male and female S. merianae in southeastern Brazil. All assays exhibited parallelism and accuracy with S. merianae plasma. We next assessed patterns of concentration of these hormones across the annual cycle of S. merianae. Testosterone in males and estradiol in females peaked in spring coincident with the peak in reproductive behavior. Progesterone in females was significantly elevated in October coincident with putative ovulation when gravid females build nests. Thyroid hormones, known for regulating energy metabolism, varied seasonally with some sex-dependent differences. T4 gradually increased from an annual nadir during pre-hibernation and hibernation to high concentrations during spring in both sexes. In contrast, T3 did not vary seasonally in males, but females showed a two-fold increase in T3 during the spring reproductive season. T3 may be involved in energy investment during the seasonal production of large clutches of eggs. Corticosterone was significantly elevated during the active season in both sexes, suggesting its involvement in mobilization of energy stores and modulation of behavior (territoriality) and physiology. Ours is the first investigation of concurrent changes in reproductive, thyroid and adrenal hormone concentrations in this endemic and physiologically unique South American lizard. Our findings set the stage for future investigations to determine the extent to which these hormones influence activity and thermoregulation in S. merianae.

Gonadal soma controls ovarian follicle proliferation through Gsdf in zebrafish.

BACKGROUND: Aberrant signaling between germ cells and somatic cells can lead to reproductive disease and depends on diffusible signals, including transforming growth factor-beta (TGFB) -family proteins. The TGFB-family protein Gsdf (gonadal soma derived factor) controls sex determination in some fish and is a candidate for mediating germ cell/soma signaling. RESULTS: Zebrafish expressed gsdf in somatic cells of bipotential gonads and expression continued in ovarian granulosa cells and testicular Sertoli cells. Homozygous gsdf knockout mutants delayed leaving the bipotential gonad state, but then became a male or a female. Mutant females ovulated a few oocytes, then became sterile, accumulating immature follicles. Female mutants stored excess lipid and down-regulated aromatase, gata4, insulin receptor, estrogen receptor, and genes for lipid metabolism, vitellogenin, and steroid biosynthesis. Mutant females contained less estrogen and more androgen than wild-types. Mutant males were fertile. Genomic analysis suggests that Gsdf, Bmp15, and Gdf9, originated as paralogs in vertebrate genome duplication events. CONCLUSIONS: In zebrafish, gsdf regulates ovarian follicle maturation and expression of genes for steroid biosynthesis, obesity, diabetes, and female fertility, leading to ovarian and extra-ovarian phenotypes that mimic human polycystic ovarian syndrome (PCOS), suggesting a role for a related TGFB signaling molecule in the etiology of PCOS. Developmental Dynamics 246:925-945, 2017. © 2017 Wiley Periodicals, Inc.

Exogenous iodide ameliorates perchlorate-induced thyroid phenotypes in threespine stickleback.

Perchlorate is a ubiquitous environmental contaminant that has widespread endocrine disrupting effects in vertebrates, including threespine stickleback (Gasterosteus aculeatus). The target of perchlorate is thyroid tissue where it induces changes in the organization, activation, and morphology of thyroid follicles and surrounding tissues. To test the hypothesis that some phenotypes of perchlorate toxicity are not mediated by thyroid hormone, we chronically exposed stickleback beginning at fertilization to perchlorate (10, 30, 100ppm) or control water with and without supplementation of either iodide or thyroxine (T4). Stickleback were sampled across a one-year timespan to identify potential differences in responses to treatment combinations before and after sexual maturation. We found that most thyroid histomorphological phenotypes induced by perchlorate (follicle proliferation, reduced follicle area (adults only), colloid depletion, thyrocyte hypertrophy (subadults only)) were significantly ameliorated by exogenous iodide supplementation. In contrast, treatment with exogenous T4 did not correct any of the thyroid-specific histopathologies induced by perchlorate. Whole-body thyroid hormone concentrations were not significantly affected by perchlorate exposure; however, supplementation with iodide and T4 significantly increased T4 concentrations. This study also revealed an increased erythrocyte area in the thyroid region of perchlorate-exposed adults, while lipid droplet number increased in perchlorate-exposed subadults. Increased erythrocyte area was ameliorated by both iodide and T4, while neither supplement was able to correct lipid droplet number. Our finding on lipid droplets indicates that exposure to perchlorate in early development may have obesogenic effects.

Perchlorate disrupts embryonic androgen synthesis and reproductive development in threespine stickleback without changing whole-body levels of thyroid hormone.

Perchlorate, an environmental contaminant, disrupts normal functioning of the thyroid. We previously showed that perchlorate disrupts behavior and gonad development, and induces external morphological changes in a vertebrate model organism, the threespine stickleback. Whether perchlorate alters these phenotypes via a thyroid-mediated mechanism, and the extent to which the effects depend on dose, are unknown. To address these questions, we chronically exposed stickleback to control conditions and to three concentrations of perchlorate (10, 30 and 100ppm) at various developmental stages from fertilization to reproductive maturity. Adults chronically exposed to perchlorate had increased numbers of thyroid follicles and decreased numbers of thyrocytes. Surprisingly, T4 and T3 levels in larval, juvenile, and adult whole fish chronically exposed to perchlorate did not differ from controls, except at the lowest perchlorate dose, suggesting a non-monotonic dose response curve. We found no detectable abnormalities in external phenotype at any dose of perchlorate, indicating that the increased number of thyroid follicles compensated for the disruptive effects of these doses. In contrast to external morphology, gonadal development was altered substantially, with the highest dose of perchlorate causing the largest effects. Perchlorate increased the number both of early stage ovarian follicles in females and of advanced spermatogenic stages in males. Perchlorate also disrupted embryonic androgen levels. We conclude that chronic perchlorate exposure may not result in lasting adult gross morphological changes but can produce lasting modifications to gonads when compensation of T3 and T4 levels occurs by thyroid follicle hyperplasia. Perchlorate may therefore affect vertebrate development via both thyroidal and non-thyroidal mechanisms.

Perchlorate exposure does not modulate temporal variation of whole-body thyroid and androgen hormone content in threespine stickleback.

Previously we showed that exposure of threespine stickleback (Gasterosteus aculeatus) to the endocrine disruptor perchlorate results in pronounced structural changes in thyroid and gonad, while surprisingly, whole-body thyroid hormone concentrations remain unaffected. To test for hormone titer variations on a finer scale, we evaluated the interactive effects of time (diel and reproductive season) and perchlorate exposure on whole-body contents of triiodothyronine (T3), thyroxine (T4), and 11-ketotestosterone (11-KT) in captive stickleback. Adult stickleback were exposed to 100ppm perchlorate or control water and sampled at 4-h intervals across the 24-hday and at one time-point (1100h) weekly across the reproductive season (May-July). Neither whole-body T3 nor T4 concentration significantly differed across the day in control or perchlorate treated stickleback. Across the reproductive season, whole-body T3 concentration remained stable while T4 significantly increased. However, neither hormone concentration was significantly affected by perchlorate, verifying our previous studies. The concentration of whole-body 11-KT, a major fish androgen, displayed significant diel variation and also steadily declined across the reproductive season in untreated males; perchlorate exposure did not influence the concentration of 11-KT in either diel or reproductive season schedules. Diel and reproductive season variations in 11-KT content in male stickleback are likely related to reproductive physiology and behavior. The observed increase in T4 content across the reproductive season may be reflective of increased energy investment in reproduction near the end of the life cycle.

Manganese Exacerbates Seasonal Health Declines in a Suicidally Breeding Mammal.

Reproductive costs must be balanced with survival to maximize lifetime reproductive rates; however, some organisms invest in a single, suicidal bout of breeding known as semelparity. The northern quoll (Dasyurus hallucatus) is an endangered marsupial in which males, but not females, are semelparous. Northern quolls living near mining sites on Groote Eylandt, Northern Territory, Australia, accumulate manganese (Mn) in their brains, testes, and hair, and elevated Mn impacts motor performance. Whether Mn is associated with other health declines is yet unknown. In the present study we show that male and female northern quolls with higher Mn accumulation had a 20% reduction in immune function and a trend toward reduced cortisol concentrations in hair. The telomere lengths of male quolls did not change pre- to postbreeding, but those with higher Mn levels had longer telomeres; in contrast, the telomeres of females shortened during the breeding season but recovered between the first year and second year of breeding. In addition, the telomeres of quolls that were recaptured declined at significantly higher rates in quolls with higher Mn between prebreeding, breeding, and/or postbreeding seasons. Future research should determine whether changes in cortisol, immune function, or telomere length affect reproductive output or survival-particularly for semelparous males. Environ Toxicol Chem 2024;43:74-86. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Differential gene expression and developmental pathologies associated with persistent organic pollutants in sentinel fish in Troutman Lake, Sivuqaq, Alaska.

Persistent organic pollutants (POPs) are lipophilic compounds that bioaccumulate in animals and biomagnify within food webs. Many POPs are endocrine disrupting compounds that impact vertebrate development. POPs accumulate in the Arctic via global distillation and thereby impact high trophic level vertebrates as well as people who live a subsistence lifestyle. The Arctic also contains thousands of point sources of pollution, such as formerly used defense (FUD) sites. Sivuqaq (St. Lawrence Island), Alaska was used by the U.S. military during the Cold War and FUD sites on the island remain point sources of POP contamination. We examined the effects of POP exposure on ninespine stickleback (Pungitius pungitius) collected from Troutman Lake in the village of Gambell as a model for human exposure and disease. During the Cold War, Troutman Lake was used as a dump site by the U.S. military. We found that PCB concentrations in stickleback exceeded the U.S. Environmental Protection Agency's guideline for unlimited consumption despite these fish being low trophic level organisms. We examined effects at three levels of biological organization: gene expression, endocrinology, and histomorphology. We found that ninespine stickleback from Troutman Lake exhibited suppressed gonadal development compared to threespine stickleback (Gasterosteus aculeatus) studied elsewhere. Troutman Lake stickleback also displayed two distinct hepatic phenotypes, one with lipid accumulation and one with glycogen-type vacuolation. We compared the transcriptomic profiles of these liver phenotypes using RNA sequencing and found significant upregulation of genes involved in ribosomal and metabolic pathways in the lipid accumulation group. Additionally, stickleback displaying liver lipid accumulation had significantly fewer thyroid follicles than the vacuolated phenotype. Our study and previous work highlight health concerns for people and wildlife due to pollution hotspots in the Arctic, and the need for health-protective remediation.

Transcriptomic and developmental effects of persistent organic pollutants in sentinel fishes collected near an arctic formerly used defense site.

Alaska contains over 600 formerly used defense (FUD) sites, many of which serve as point sources of pollution. These sites are often co-located with rural communities that depend upon traditional subsistence foods, especially lipid-rich animals that bioaccumulate and biomagnify persistent organic pollutants (POPs). Many POPs are carcinogenic and endocrine-disrupting compounds that are associated with adverse health outcomes. Therefore, elevated exposure to POPs from point sources of pollution may contribute to disproportionate incidence of disease in arctic communities. We investigated PCB concentrations and the health implications of POP exposure in sentinel fishes collected near the Northeast Cape FUD site on Sivuqaq (St. Lawrence Island), Alaska. Sivuqaq residents are almost exclusively Yupik and rely on subsistence foods. At the request of the Sivuqaq community, we examined differential gene expression and developmental pathologies associated with exposure to POPs originating at the Northeast Cape FUD site. We found significantly higher levels of PCBs in Alaska blackfish (Dallia pectoralis) collected from contaminated sites downstream of the FUD site compared to fish collected from upstream reference sites. We compared transcriptomic profiles and histopathologies of these same blackfish. Blackfish from contaminated sites overexpressed genes involved in ribosomal and FoxO signaling pathways compared to blackfish from reference sites. Contaminated blackfish also had significantly fewer thyroid follicles and smaller pigmented macrophage aggregates. Conversely, we found that ninespine stickleback (Pungitius pungitius) from contaminated sites exhibited thyroid follicle hyperplasia. Despite our previous research reporting transcriptomic and endocrine differences in stickleback from contaminated vs. reference sites, we did not find significant differences in kidney or gonadal histomorphologies. Our results demonstrate that contaminants from the Northeast Cape FUD site are associated with altered gene expression and thyroid development in native fishes. These results are consistent with our prior work demonstrating disruption of the thyroid hormone axis in Sivuqaq residents.

A novel method for extraction and quantification of feather triiodothyronine (T3) and application to ecotoxicology of Purple Martin (Progne subis).

Seventy-three percent of aerial insectivore species of birds breeding in North America have declined in the past five years. This decline is even greater in migratory insectivorous species, which face stressors in both their breeding and non-breeding ranges. The Purple Martin (Progne subis) is an aerial insectivore swallow that overwinters in South America and migrates to North America to breed. Purple Martin populations have declined by an estimated 25% since 1966. The eastern subspecies (P. subis subis) has declined the most and overwinters in the Amazon Basin, a region rich in environmental mercury (Hg) contamination. Previous studies reported elevated levels of Hg in feathers of this subspecies, which correlated negatively with body mass and fat reserves. Given the propensity of Hg to disrupt the endocrine system, and the role of thyroid hormones in regulating fat metabolism, this study quantifies concentrations of Hg and the thyroid hormone triiodothyronine (T3) in the feathers of P. subis subis. To our knowledge, this is the first study to extract and quantify T3 in feathers; thus, we developed, tested, and optimized a method for extracting T3 from feather tissue and validated an enzyme immunoassay (EIA) to quantify T3 in Purple Martin feathers. The developed method yielded acceptable results for both parallelism and accuracy. The observed T3 concentrations were statistically modeled along with total Hg (THg) concentrations, but these variables were not significantly correlated. This suggests that the observed variation in THg concentration may be insufficient to cause a discernible change in T3 concentration. Furthermore, the observed effect of breeding location on feather T3 concentration might have obscured any effect of Hg.

Seasonal changes in steroid and thyroid hormone content in shed skins of the tegu lizard Salvator merianae.

Sampling blood for endocrine analysis from some species may not be practical or ethical. Quantification of hormones extracted from nontypical sample types, such as keratinized tissues, offers a less invasive alternative to the traditional collection and analysis of blood. Here, we aimed to validate assays by using parallelism and accuracy tests for quantification of testosterone, corticosterone, progesterone, and triiodothyronine (T3) in shed skins of tegu lizards. We assessed whether hormone content of sheds varied across one year similar to what was previously detected in plasma samples. In addition, we aimed to identify the phase relationship between hormone levels of shed skin and plasma levels obtained from the same animals. High frequency of shedding occurred during the active season for tegus (spring/summer), while shedding ceased during hibernation (winter). All hormones measured in shed skins exhibited seasonal changes in concentration. Levels of testosterone in shed skins of male tegus correlated positively with plasma testosterone levels, while corticosterone in both males and females exhibited an inverse relationship between sample types for the same month of collection. An inverse relationship was found when accounting for a lag time of 3 and 4 months between sheds and plasma testosterone. These results indicate that endocrine content of sheds may be confounded by factors (i.e., seasons, environmental temperature, thermoregulatory behavior, among others) that affect frequency of molting, skin blood perfusion, and therefore hormone transfer from the bloodstream and deposition in sheds of squamates.

Elevated mercury and PCB concentrations in Dolly Varden (Salvelinus malma) collected near a formerly used defense site on Sivuqaq, Alaska.

Environmental pollution causes adverse health effects in many organisms and contributes to health disparities for Arctic communities that depend on subsistence foods, including the Yupik residents of Sivuqaq (St. Lawrence Island), Alaska. Sivuqaq's proximity to Russia made it a strategic location for U.S. military defense sites during the Cold War. Two radar surveillance stations were installed on Sivuqaq, including at the Northeast Cape. High levels of persistent organic pollutants and toxic metals continue to leach from the Northeast Cape formerly used defense (FUD) site despite remediation efforts. We quantified total mercury (Hg) and polychlorinated biphenyl (PCB) concentrations, and carbon and nitrogen stable isotope signatures, in skin and muscle samples from Dolly Varden (Salvelinus malma), an important subsistence species. We found that Hg and PCB concentrations significantly differed across locations, with the highest concentrations found in fish collected near the FUD site. We found that 89% of fish collected from near the FUD site had Hg concentrations that exceeded the U.S. Environmental Protection Agency's (EPA) unlimited Hg-contaminated fish consumption screening level for subsistence fishers (0.049 µg/g). All fish sampled near the FUD site exceeded the EPA's PCB guidelines for cancer risk for unrestricted human consumption (0.0015 µg/g ww). Both Hg and PCB concentrations had a significant negative correlation with δ13C when sites receiving input from the FUD site were included in the analysis, but these relationships were insignificant when input sites were excluded. δ15N had a significant negative correlation with Hg concentration, but not with PCB concentration. These results suggest that the Northeast Cape FUD site remains a point source of Hg and PCB pollution and contributes to higher concentrations in resident fish, including subsistence species. Moreover, elevated Hg and PCB levels in fish near the FUD site may pose a health risk for Sivuqaq residents.

Interrelationships among feather mercury content, body condition and feather corticosterone in a Neotropical migratory bird, the Purple Martin (Progne subis subis).

Purple Martins (Progne subis) are migratory birds that breed in North America and overwinter and complete their molt in South America. Many of the breeding populations are declining. The eastern North American subspecies of Purple Martin (P. subis subis) comprises >90% of all Purple Martins. This subspecies overwinters and molts in the Amazon Basin, a region that is high in mercury (Hg) contamination, which raises the possibility that observed declines in Purple Martins could be linked to Hg exposure. Exposure to Hg results in numerous and systemic negative health outcomes, including endocrine disruption. Corticosterone (CORT) is a primary modulator of the stress and metabolic axes of vertebrates; thus, it is important in meeting metabolic and other challenges of migration. Because feathers accumulate Hg and hormones while growing, quantification of Hg and CORT in feathers provides an opportunity to retrospectively assess Hg exposure and adrenal activity of birds using minimally invasive methods. We evaluated interrelationships among concentrations of total Hg (THg) and CORT in feathers that grew in the Amazon Basin and body condition (mass, fat score) of these birds in North America. Concentrations of THg in Purple Martin feathers ranged from 1.103 to 8.740 µg/g dw, levels associated with negative physiological impacts in other avian species. Concentrations of CORT did not correlate with THg concentration at the time of feather growth. However, we found evidence that THg concentration may negatively impact the ability of Purple Martins to accumulate fat, which could impair migratory performance and survivorship due to the high energy requirements of migration. This finding suggests potential carryover effects of Hg contamination at the wintering grounds in the Amazon to the summer breeding grounds in North America.

Who Rules Over Immunology? Seasonal Variation in Body Temperature, Steroid Hormones, and Immune Variables in a Tegu Lizard.

Multiple factors can influence the immune response of ectothermic vertebrates, including body temperature (Tb), gonadal steroids, and seasonality, in ways that are thought to reflect trade-offs between energetic investment in immunity versus reproduction. Hibernating tegu lizards (Salvator merianae) are a unique model to investigate how immunocompetence might be influenced by different factors during their annual cycle. We assessed immunological measures (plasma bacterial killing ability, total and differential leukocyte count), plasma hormone levels (testosterone in males, estradiol and progesterone in females, and corticosterone [CORT] in both sexes), Tb, and body condition from adult tegus during each stage of their annual cycle: reproduction, post-reproduction/preparation for hibernation, and hibernation. Our hypothesis that immune traits present higher values during the reproductive phase, and a sharp decrease during hibernation, was partially supported. Immune variables did not change between life history stages, except for total number of leukocytes, which was higher at the beginning of the reproductive season (September) in both males and females. Average Tb of the week prior to sampling was positively correlated with number of eosinophils, basophils, monocytes, and azurophils, corroborating other studies showing that when animals maintain a high Tb, there is an increase in immune activity. Surprisingly, no clear relationship between immune traits and gonadal steroids or CORT levels was observed, even when including life history stage in the model. When gonadal hormones peaked in males and females, heterophil: lymphocyte ratio (which often elevates during physiological stress) also increased. Additionally, we did not observe any trade-off between reproduction and immunity traits, sex differences in immune traits, or a correlation between body condition and immune response. Our results suggest that variation in patterns of immune response and correlations with body condition and hormone secretion across the year can depend upon the specific hormone and immune trait, and that experienced Tb is an important variable determining immune response in ectotherms.

A fish with no sex: gonadal and adrenal functions partition between zebrafish NR5A1 co-orthologs.

People with NR5A1 mutations experience testicular dysgenesis, ovotestes, or adrenal insufficiency, but we do not completely understand the origin of this phenotypic diversity. NR5A1 is expressed in gonadal soma precursor cells before expression of the sex-determining gene SRY. Many fish have two co-orthologs of NR5A1 that likely partitioned ancestral gene subfunctions between them. To explore ancestral roles of NR5A1, we knocked out nr5a1a and nr5a1b in zebrafish. Single-cell RNA-seq identified nr5a1a-expressing cells that co-expressed genes for steroid biosynthesis and the chemokine receptor Cxcl12a in 1-day postfertilization (dpf) embryos, as does the mammalian adrenal-gonadal (interrenal-gonadal) primordium. In 2dpf embryos, nr5a1a was expressed stronger in the interrenal-gonadal primordium than in the early hypothalamus but nr5a1b showed the reverse. Adult Leydig cells expressed both ohnologs and granulosa cells expressed nr5a1a stronger than nr5a1b. Mutants for nr5a1a lacked the interrenal, formed incompletely differentiated testes, had no Leydig cells, and grew far larger than normal fish. Mutants for nr5a1b formed a disorganized interrenal and their gonads completely disappeared. All homozygous mutant genotypes lacked secondary sex characteristics, including male breeding tubercles and female sex papillae, and had exceedingly low levels of estradiol, 11-ketotestosterone, and cortisol. RNA-seq showed that at 21dpf, some animals were developing as females and others were not, independent of nr5a1 genotype. By 35dpf, all mutant genotypes greatly under-expressed ovary-biased genes. Because adult nr5a1a mutants form gonads but lack an interrenal and conversely, adult nr5a1b mutants lack a gonad but have an interrenal, the adrenal, and gonadal functions of the ancestral nr5a1 gene partitioned between ohnologs after the teleost genome duplication, likely owing to reciprocal loss of ancestral tissue-specific regulatory elements. Identifying such elements could provide hints to otherwise unexplained cases of Differences in Sex Development.