Relationships between Spatial Biology and Physiological Ecology in the Gopher Tortoise, Gopherus polyphemus.

AbstractThe overlap between spatial and physiological ecology is generally understudied, yet both fields are fundamentally related in assessing how individuals balance limited resources. Herein, we quantified the relationships between spatial ecology using two parameters of home range (annual home range area and number of burrows used in 1 yr) and four measures of physiology that integrate stress and immunity (baseline plasma corticosterone [CORT] concentration, plasma lactate concentration, heterophil-to-lymphocyte [H∶L] ratio, and bactericidal ability [BA]) in a wild free-ranging population of the gopher tortoise (Gopherus polyphemus) to test the hypothesis that space usage is correlated with physiological state. We also used structural equation models (SEMs) to test for causative relationships between the spatial and physiological parameters. We predicted that larger home ranges would be negatively correlated with traditional biomarkers of stress and positively correlated with immunity, consistent with our hypothesis that home ranges are determined based on individual condition. Males had larger home ranges, used more burrows, and had higher baseline CORT than females. We found significant negative correlations between lactate and home range (r=-0.456, df=21, P=0.029). CORT was negatively correlated with the number of burrows used in both sexes (F=7.322, df=2,20, P=0.003, adjusted R2=0.383). No correlations were observed between space use and BA or, notably, H∶L ratio. SEMs suggested that variation in the number of burrows used was a result of variation in baseline CORT. The lack of a relationship between H∶L ratio and home range suggests that home range differences are not associated with differences in chronic stress, despite the pattern between baseline CORT and number of burrows used. Instead, this study indicates that animals balance trade-offs in energetics, likely by way of baseline corticosteroid, in such a way as to maintain function across continuously variable home range strategies.

Evidence of oxidative stress responses of green turtles (Chelonia mydas) to differential habitat conditions in the Mexican Caribbean.

Important foraging and nesting habitats for Caribbean green sea turtles (Chelonia mydas) exist within the Mesoamerican Reef System in the Mexican Caribbean. During the last 25 years, urban development and touristic activities have drastically increased in Quintana Roo, Mexico. Moreover, in the last decade, massive pelagic sargasso blooms have also afflicted this region; however, information about the biochemical responses of Caribbean green turtles to these inputs is absent. This study aimed to assess if the oxidative stress indicators in the red blood cells of green turtles are valuable biomarkers of the extent of the anthropic impact in this region. Persistent organic pollutants (POPs) were also measured in the plasma of free-living green turtles during 2015-2018 to characterize these habitats further. As biochemical biomarkers, the production rate of superoxide radical (O2•-), carbonylated protein content, and lipid peroxidation (TBARS) levels, and the activities of superoxide dismutase, glutathione S-transferase (GST), catalase, glutathione peroxidase were measured in erythrocytes. A 15 % occurrence of fibropapillomatosis (FP) was revealed, with tumor size being positively correlated with CAT activity in the affected individuals. A multivariate analysis embracing all oxidative stress markers discriminated green turtles between years of capture (p < 0.001), with those sampled during 2015 presenting the highest production of O2•- (p = 0.001), activities of GST (p < 0.001), levels of TBARS (p < 0.001) and carbonylated proteins (p = 0.02). These local and temporal biochemical responses coincided with the first massive Sargassum spp. bloom reported in the region. The results of this study corroborate the utility of the oxidative stress indicators as biomarkers of environmental conditions (sargasso blooms and POPs) in the green turtle as sentinel species.

Niclosamide exposure disrupts antioxidant defense, histology, and the liver and gut transcriptome of Chinese soft-shelled turtle (Pelodiscus sinensis).

Niclosamide (NIC) is the only commercially available molluscicide for controlling schistosomiasis, and its negative effects on aquatic animals had been frequently reported in recent years. However, the toxicity mechanism of NIC on the Chinese soft-shelled turtle (Pelodiscus sinensis) have not yet been investigated. Therefore, juvenile turtles were exposed to 0 (control group), 10 (low NIC, L), and 50 (high NIC, H) µg/L NIC for 120 h and our results demonstrated that NIC exposure induced severe pathological changes in the liver of P. sinensis. And the typical symptom included edema, nuclear migration and deformation, and vacuolization. Compared with the liver, the NIC exposure did not cause significant damage in the gut tissue. In addition, the DHE staining demonstrated that the ROS production of liver and gut increased with the increase in concentration of NIC. The activities of antioxidant enzymes including superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) was inhibited with increased malondialdehyde (MDA) content, indicating that the antioxidant defense was significantly perturbed. Further, the transcriptome sequencing and was applied to evaluate the underlying toxicity mechanisms of NIC exposure in liver and gut of P. sinensis. Pathway enrichment showed that the disorder of lipid metabolism and innate immune regulation, including Toll-like receptors (TLRs), tumor necrosis factor (TNF), lectins, and complement and coagulation cascades, were toxicological properties of NIC on P. sinensis. Overall, the current study provides valuable information to understand the toxic effect of NIC on Chinese soft-shelled turtle.

Seasonal spermatogenesis in the red-eared slider (Trachemys scripta elegans): The roles of GnRH, actin cytoskeleton, and MAPK.

Reproduction is the key to the ecological invasion of alien species. As an invasive species, the characteristic and regularity of red-eared slider (Trachemys scripta elegans) spermatogenesis is an index for evaluating reproduction and ecological adaptation. Here, we investigated the characteristics of spermatogenesis i.e., the gonadosomatic index (GSI), plasma reproductive hormone levels, and the histological structure of testes by HE and TUNEL staining, and then RNA-Seq in T. s. elegans. The histomorphological evidence confirmed that seasonal spermatogenesis in T. s. elegans has four successive phases: quiescence (December-May of the following year), early-stage (June-July), mid-stage (August-September), and late-stage (October-November). In contrast to 17ß-estradiol, testosterone levels were higher during quiescence (breeding season) compared to mid-stage (non-breeding season). Based on RNA-seq transcriptional analysis, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were used to analyze the testis in the quiescent and mid-stage. Our study found that circannual spermatogenesis is regulated by interactive networks including gonadotropin-releasing hormone (GnRH) secretion, regulation of actin cytoskeleton, and MAPK signaling pathways. Moreover, the number of genes associated with proliferation and differentiation (srf, nr4a1), cell cycle (ppard, ccnb2), and apoptosis (xiap) were up-regulated in the mid-stage. With the maximum energy saving, this seasonal pattern of T. s. elegans determines optimal reproductive success and thus adapts better to the environment. These results provide the basis for the invasion mechanism of T. s. elegans and lay the foundation for deeper insight into the molecular mechanism of seasonal spermatogenesis in reptiles.

Identification and application of species-specific peptide biomarkers from soft-shelled turtles (Pelodiscus sinensis) using post-translational modification detection-based peptidomics analysis.

The soft-shelled turtle is a commercially aquatic species in Asian countries, which serves as an important source of collagen with high nutritional and medicinal value, so it is of great significance to distinguish soft-shelled turtle derived collagen from others or adulterations. In this work, peptidomics analysis based on post-translational modification (PTM) assay was used to discover specific peptide biomarkers of soft-shelled turtle gelatin (STG). In total eight specific sequences and 74 peptides with different PTM types were screened out, and seven peptides with good signal responses and STG specificity were selected and validated as STG-specific peptide biomarkers. These peptide biomarkers could be used for distinguishing STG from other animal gelatins, and applied for ensuring the quality of collagens or gelatins from soft-shelled turtle with authenticity and traceability.

Oviductal sperm storage in the Chinese pond turtle, Mauremys reevesii, depends on androgen-based promotion of the BCL 2 anti-apoptotic pathway.

CONTEXT: Sperm storage is a complex and highly coordinated process that is regulated by a variety of factors. The BCL 2 protein family plays a key role in regulating apoptosis, and determines sperm survival. AIMS: The objective of this study was to explore the correlation between sperm storage and the BCL 2 protein family in the oviduct of Mauremys reevesii . METHODS: Hematoxylin-eosin (HE) staining, immunohistochemistry (IHC) and real-time quantitative polymerase chain reaction (RT-qPCR) techniques were used to investigate three parts of the reproductive tract (isthmus, uterus and vagina) of mated and unmated female M. reevesii . KEY RESULTS: Hematoxylin-eosin staining revealed many sperm stored in the oviduct. IHC showed positive immunostaining for the BCL 2 and BAX proteins in epithelial ciliated and glandular cells. RT-qPCR indicated that the mRNA expressions of anti-apoptotic genes (BCL 2 , MCL 1 , BCL- W , BCL-XL ) and the androgen receptor (AR) were significantly higher in mated turtles than unmated turtles. However, the expression of pro-apoptotic genes (BAX , BAD , BID and CASPASE 3 ) showed the opposite relationship. CONCLUSIONS: These results suggest that sperm entering the oviduct can promote the synthesis of anti-apoptotic genes to protect themselves from various degradation factors. IMPLICATIONS: These findings will help researchers understand the mechanisms of sperm storage.

Male gopher tortoise (Gopherus polyphemus) concentration-dependent social responses to diluted mental gland pheromones.

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.

Temperature fluctuations and estrone sulfate affect gene expression via different mechanisms to promote female development in a species with temperature-dependent sex determination.

Variation in developmental conditions can affect a variety of embryonic processes and shape a number of phenotypic characteristics that can affect offspring throughout their lives. This is particularly true of oviparous species where development typically occurs outside of the female, and studies have shown that traits such as survival and behavior can be altered by both temperature and exposure to steroid hormones during development. In species with temperature-dependent sex determination (TSD), the fate of gonadal development can be affected by temperature and by maternal estrogens present in the egg at oviposition, and there is evidence that these factors can affect gene expression patterns. Here, we explored how thermal fluctuations and exposure to an estrogen metabolite, estrone sulfate, affect the expression of several genes known to be involved in sexual differentiation: Kdm6b, Dmrt1, Sox9, FoxL2 and Cyp19A1. We found that most of the genes responded to both temperature and estrone sulfate exposure, but that the responses to these factors were not identical, in that estrone sulfate effects occur downstream of temperature effects. Our findings demonstrate that conjugated hormones such as estrone sulfate are capable of influencing temperature-dependent pathways to potentially alter how embryos respond to temperature, and highlight the importance of studying the interaction of maternal hormone and temperature effects.

Gene expression of hypoxia-inducible factor (HIF), HIF regulators, and putative HIF targets in ventricle and telencephalon of Trachemys scripta acclimated to 21 °C or 5 °C and exposed to normoxia, anoxia or reoxygenation.

In anoxia-sensitive mammals, hypoxia inducible factor (HIF) promotes cellular survival in hypoxia, but also tumorigenesis. By comparison, anoxia-tolerant vertebrates likely need to circumvent a prolonged upregulation of HIF to survive long-term anoxia, making them attractive biomedical models for investigating HIF regulation. To lend insight into the role of HIF in anoxic Trachemys scripta ventricle and telencephalon, 21 °C- and 5 °C-acclimated turtles were exposed to normoxia, anoxia (24 h at 21 °C; 24 h or 14 d at 5 °C) or anoxia + reoxygenation and the gene expression of HIF-1α (hif1a) and HIF-2α (hif2a), two regulators of HIF, and eleven putative downstream targets of HIF quantified by qPCR. Changes in gene expression with anoxia at 21 °C differentially aligned with a circumvention of HIF activity. Whereas hif1a and hif2a expression was unaffected in ventricle and telencephalon, and BCL2 interacting protein 3 gene expression reduced by 30% in telencephalon, gene expression of vascular endothelial growth factor-A increased in ventricle (4.5-fold) and telencephalon (1.5-fold), and hexokinase 1 (2-fold) and hexokinase 2 (3-fold) gene expression increased in ventricle. At 5 °C, the pattern of gene expression in ventricle or telencephalon was unaltered with oxygenation state. However, cold acclimation in normoxia induced downregulation of HIF-1α, HIF-2α, and HIF target gene expression in telencephalon. Overall, the findings lend support to the postulation that prolonged activation of HIF is counterproductive for long-term anoxia survival. Nevertheless, quantification of the effect of anoxia and acclimation temperature on HIF binding activity and regulation at the protein level are needed to provide a strong scientific framework whereby new strategies for oxygen related pathologies can be developed.

A subpopulation of green turtle suprabasal epidermal cells are Langerin+ and migrate under in vitro stimulation of the chemokine CCL21.

Dendritic cells form the link between the innate and adaptative immune response, particularly on mucosal and epidermal surfaces. The Langerhans, an epidermal dendritic cell subpopulation, play a key role in the skin immune response across several species. Scarse immune cell subpopulations, including Langerhans-like cells, have been identified in endangered green turtles thereby complicating the understanding of the pathogenesis of diseases such as fibropapillomatosis, which induces skin tumours in this species worldwide. In biopsies from green turtle skin, we demonstrated that the polyclonal anti-human Langerin antibodies strongly stained a Langerin+ cell population in epidermal sheets, the suprabasal layer of the epidermis in cryosections and in cells from cytospin preparation of migration assays. The morphology of these cells was round to amoeboid in normal skin; however, in skin with ulcerative dermatitis, Langerin+ cells aggregated around ulcers and adopted a more pleomorphic morphology. To our knowledge, this is the first identification of Langerin+ cells with a molecular marker in a reptile species.

On-board study of gas embolism in marine turtles caught in bottom trawl fisheries in the Atlantic Ocean.

Decompression sickness (DCS) was first diagnosed in marine turtles in 2014. After capture in net fisheries, animals typically start showing clinical evidence of DCS hours after being hauled on-board, often dying if untreated. These turtles are normally immediately released without any understanding of subsequent clinical problems or outcome. The objectives of this study were to describe early occurrence and severity of gaseous embolism (GE) and DCS in marine turtles after incidental capture in trawl gear, and to provide estimates of on-board and post-release mortality. Twenty-eight marine turtles were examined on-board fishing vessels. All 20 turtles assessed by ultrasound and/or post-mortem examination developed GE, independent of season, depth and duration of trawl and ascent speed. Gas emboli were obvious by ultrasound within 15 minutes after surfacing and worsened over the course of 2 hours. Blood data were consistent with extreme lactic acidosis, reduced glomerular filtration, and stress. Twelve of 28 (43%) animals died on-board, and 3 of 15 (20%) active turtles released with satellite tags died within 6 days. This is the first empirically-based estimate of on-board and post-release mortality of bycaught marine turtles that has until now been unaccounted for in trawl fisheries not equipped with turtle excluder devices.

Endoscopy gender determination and reproductive hormone profiles of Painted Terrapins (Batagur borneoensis) subjected to ex situ incubation.

Chelonian exhibit temperature dependent sex determination, and ex situ incubation of eggs in conservation hatcheries may render a gender bias. The gender of juvenile Painted terrapins (Batagur borneoensis) produced at a conservation hatchery in Malaysia was determined by endoscopy of the gonads. Circulating reproductive hormones (testosterone, progesterone and estradiol) were profiled for 31 juveniles and nine captive-reared non-breeding adult terrapins. Endoscopy revealed a gender bias of 96.8% (30/31) females. Testosterone levels in the juvenile females (2.49 ± 1.29) were significantly lower than that of the adult females (12.20 ± 4.29), and lower than values in the juvenile male (9.36) and adult males (27.60, 35.62). The progesterone levels in the juvenile females (107.12 ± 68.68) were significantly higher than that of the adult females (51.13 ± 24.67), but lower than values in the juvenile male (33.27) and adult males (3.43, 8.51). Estrogen levels were significantly lower in the juvenile females (1.57 ± 1.35) compared to the adult females (77.46 ± 53.45). Negative correlations were observed between levels of progesterone and testosterone, and progesterone and estrogen. A positive correlation was noted between estrogen and testosterone. The present study constitutes the first attempt to determine the gender and reproductive hormone profiles of juvenile Painted terrapins produced by ex situ incubation, and captive non-breeding adults. Endoscopy of the gonads is a useful techniques for gender determination among juvenile turtles, while the use of testosterone as a gender biomarker warrants further investigation.

Effects of cadmium on the gene transcription of the liver in the freshwater turtle (Chinemys reevesii).

This study investigated the related gene transcription of liver in freshwater turtle Chinemys reevesii exposed to cadmium (Cd). After acclimation, healthy turtles were selected for experiments. They were randomly divided into four experimental groups and each group had 5 animals. The turtles were treated with 0 mg/kg, 7.5 mg/kg, 15 mg/kg, and 30 mg/kg Cd chloride separately by intraperitoneal injection. Liver samples were collected for examination of the transcription of related genes at 2 weeks after Cd exposure. The transcription of mRNA of MT, SOD, CAT, PNKP, and GPX4 genes in turtle liver cells were analyzed. Results showed that Cd promoted MT mRNA transcription in turtle's liver at low dose (7.5 mg/kg) and inhibited MT mRNA transcription in turtle's liver at middle dose (15 mg/kg) and high dose (30 mg/kg). Cd inhibited the transcription of SOD, CAT, and PNKP mRNA in turtle's liver, and the inhibition was obvious at high dose (30 mg/kg). Cd promoted GPX4 mRNA transcription in turtle's liver, especially at low dose (7.5 mg/kg). In conclusion, Cd had different effects on the mRNA transcription of liver cells in the freshwater turtle Chinemys reevesii exposed to Cd.

Advances in understanding mechanisms of long-term sperm storage-the soft-shelled turtle model.

Long-term sperm storage is a special reproductive strategy, which can extend the time window between mating and fertilization in some animal species. Spermatozoa of the soft-shelled turtle, Pelodiscus sinensis, can be stored in the epididymis and oviduct for at least six months and one year, respectively. How spermatozoa can be stored in vivo for such a prolonged period is yet to be explained. We analyze the mechanisms that contribute to long-term sperm storage in P. sinensis, and compare them with other species from three different perspectives: the spermatozoon itself, the storage microenvironment and the interaction between the spermatozoon and microenvironment. Characteristics of soft-shelled turtle spermatozoa itself, such as the huge cytoplasmic droplet with its content of several large lipid droplets (LDs) and onion-like mitochondira, facilitate long-term sperm storage. The microenvironment of reproductive tract, involving in the secretions, structural barriers, exosomes, androgen receptors, Toll-like receptors and survival factor Bcl-2, are important for the maintenance of spermatozoa long-term storage. Sperm heads are always embedded among the oviductal cilia and even intercalate into the apical hollowness of the ciliated cells, indicating that the ciliated cells support the stored spermatozoa. RNA seq is firstly used to detect the molecular mechanism of sperm storage, which shows that autophagy, apoptosis and immune take part in the long-term sperm storage in this species.

Effect of seasonal variance on intestinal epithelial barriers and the associated innate immune response of the small intestine of the Chinese soft-shelled turtles.

It is conceivable that pathological conditions can cause intestinal barrier disruption and innate immune dysfunction. However, very limited information has been reported on the effect of seasonal variance on intestinal barriers and innate immunity. The present study was designed to investigate the seasonal variance in intestinal epithelial barriers and the associated innate immune response of turtle intestines during hibernation and nonhibernation periods. Goblet cells (GCs) demonstrated dynamic actions of the mucosal barrier with strong Muc2 protein expression during hibernation. However, weak Muc2 expression during nonhibernation was confirmed by immunohistochemistry, immunofluorescence and immunoblotting. Furthermore, light and transmission electron microscopy revealed that the hypertrophy of GCs resulted in the hypersecretion of mucus granules (MGs) and created a well-developed mucosal layer during hibernation. The absorptive cells (ACs), forming a physical barrier of tight junctions, and desmosomes were firmly anchored during hibernation. Conversely, during nonhibernation, the integrity of tight junctions, adherence junctions and desmosomes was noticeable expanded, causing increased paracellular permeability. As further confirmation, there was strong zonula occluden-1 (ZO-1) and connexins 43 (Cx43) protein expression during hibernation and weak ZO-1 and Cx43 expression during nonhibernation. Moreover, the expression level of the innate immune response proteins Toll-like receptors 2 and 4 (TLR2 and 4) were enhanced during hibernation and were reduced during nonhibernation. These results provide rich information about the seasonal fluctuations that interrupt intestinal epithelial barriers and innate immune response, which might be essential for protection and intestinal homeostasis.

Cellular Evidence of CD63-Enriched Exosomes and Multivesicular Bodies within the Seminiferous Tubule during the Spermatogenesis of Turtles.

The seminiferous tubule (ST) is the location of spermatogenesis, where mature spermatozoa are produced with the assistance of Sertoli cells. The role of extracellular vesicles in the direct communication between Sertoli-germ cells in the ST is still not fully understood. In this study, we reported multivesicular bodies (MVBs) and their source of CD63-enriched exosomes by light and ultrastructure microscopy during the reproductive phases of turtles. Strong CD63 immunopositivity was detected at the basal region in the early and luminal regions of the ST during late spermatogenesis by immunohistochemistry (IHC), immunofluorescence (IF), and western blot (WB) analysis. Labeling of CD63 was detected in the Sertoli cell cytoplasmic processes that surround the developing germ cells during early spermatogenesis and in the lumen of the ST with elongated spermatids during late spermatogenesis. Furthermore, ultrastructure analysis confirmed the existence of numerous MVBs in the Sertoli cell prolongations that surround the round and primary spermatogonia during acrosome biogenesis and with the embedded heads of spermatids in the cytoplasm of Sertoli cells. Additionally, in spermatids, Chrysanthemum flower centers (CFCs) generated isolated membranes involved in MVBs and autophagosome formation, and their fusion to form amphiosomes was also observed. Additionally, autophagy inhibition by 3-methyladenine (after 24 h) increased CD63 protein signals during late spermatogenesis, as detected by IF and WB. Collectively, our study found MVBs and CD63 rich exosomes within the Sertoli cells and their response to autophagy inhibition in the ST during the spermatogenesis in the turtle.

In Vivo Autophagy Up-Regulation of Small Intestine Enterocytes in Chinese Soft-Shelled Turtles during Hibernation.

Many studies have focused on how autophagy plays an important role in intestinal homeostasis under pathological conditions. However, its role in the intestine during hibernation remains unclear. In the current study, we characterized in vivo up-regulation of autophagy in enterocytes of the small intestine of Chinese soft-shelled turtles during hibernation. Autophagy-specific markers were used to confirm the existence of autophagy in enterocytes through immunohistochemistry (IHC), immunofluorescence (IF), and immunoblotting. IHC staining indicated strong, positive immunoreactivity of the autophagy-related gene (ATG7), microtubule-associated protein light chain (LC3), and lysosomal-associated membrane protein 1 (LAMP1) within the mucosal surface during hibernation and poor expression during nonhibernation. IF staining results showed the opposite tendency for ATG7, LC3, and sequestosome 1 (p62). During hibernation ATG7 and LC3 showed strong, positive immunosignaling within the mucosal surface, while p62 showed strong, positive immunosignaling during nonhibernation. Similar findings were confirmed by immunoblotting. Moreover, the ultrastructural components of autophagy in enterocytes were revealed by transmission electron microscopy (TEM). During hibernation, the cumulative formation of phagophores and autophagosomes were closely associated with well-developed rough endoplasmic reticulum in enterocytes. These autophagosomes overlapped with lysosomes, multivesicular bodies, and degraded mitochondria to facilitate the formation of autophagolysosome, amphisomes, and mitophagy in enterocytes. Immunoblotting showed the expression level of PTEN-induced kinase 1 (PINK1), and adenosine monophosphate-activated protein kinase (AMPK) was enhanced during hibernation. Furthermore, the exosome secretion pathway of early-late endosomes and multivesicular bodies were closely linked with autophagosomes in enterocytes during hibernation. These findings suggest that the entrance into hibernation is a main challenge for reptiles to maintain homeostasis and cellular quality control in the intestine.

Close-kin mating, but not inbred parents, reduces hatching rates and offspring quality in a threatened tortoise.

Inbreeding depression, the reduction in fitness due to mating of related individuals, is of particular conservation concern in species with small, isolated populations. Although inbreeding depression is widespread in natural populations, long-lived species may be buffered from its effects during population declines due to long generation times and thus are less likely to have evolved mechanisms of inbreeding avoidance than species with shorter generation times. However, empirical evidence of the consequences of inbreeding in threatened, long-lived species is limited. In this study, we leverage a well-studied population of gopher tortoises, Gopherus polyphemus, to examine the role of inbreeding depression and the potential for behavioural inbreeding avoidance in a natural population of a long-lived species. We tested the hypothesis that increased parental inbreeding leads to reduced hatching rates and offspring quality. Additionally, we tested for evidence of inbreeding avoidance. We found that high parental relatedness results in offspring with lower quality and that high parental relatedness is correlated with reduced hatching success. However, we found that hatching success and offspring quality increase with maternal inbreeding, likely due to highly inbred females mating with more distantly related males. We did not find evidence for inbreeding avoidance in males and outbred females, suggesting sex-specific evolutionary trade-offs may have driven the evolution of mating behaviour. Our results demonstrate inbreeding depression in a long-lived species and that the evolution of inbreeding avoidance is shaped by multiple selective forces.

Red-eared slider hatchlings (Trachemys scripta) show a seasonal shift in behavioral types.

Correlated and repeatable patterns of behavior, termed behavioral types, can affect individual fitness. The most advantageous behavioral type may differ across predictable environments (e.g., seasonally), and maternally mediated effects may match hatchling behavior to the environment. We measured righting response, an indicator of behavioral type, of juvenile red-eared slider turtles (Trachemys scripta) emerging from early and late season clutches to understand if the production of behavioral types differs across the nesting season. There was a significant effect of season, with early season hatchlings righting more quickly than late season hatchlings, and we explored two potential underlying mechanisms, maternal estrogens and maternal investment (e.g., yolk allocation). We dosed early season eggs with an estrogen mixture to mimic late season eggs and assayed hatchling righting response, but found no significant effect of this maternal effect. We assessed maternal investment by measuring egg, hatchling, and residual yolk masses. We found a seasonal pattern in yolk allocation, where early season eggs have more yolk than late season eggs. Early season hatchlings used more yolk for growth rather than maintenance of existing tissues, resulting in larger hatchlings. Interestingly, across both seasons, hatchlings that received less maternal yolk appeared to be more efficient at converting yolk to tissue, but we found no direct correlation with righting behavior. We demonstrate that the prevalence of behavioral types varies across the nesting season, creating correlated suites of seasonal phenotypes in turtle hatchlings, but it appears that neither maternal estrogens or investment in yolk directly underlie this shift in behavior.

Year-round plasma steroid hormone profiles and the reproductive ecology of gopher tortoises (Gopherus polyphemus) at the southernmost edge of their range.

Populations of wide ranging ectotherms often exhibit variation in traits that are influenced by local environmental conditions. Although the gopher tortoise, Gopherus polyphemus, is well studied in pine flatwoods habitats across their range, little attention has been given to coastal populations existing in the southern extreme portion of the range. We examined the reproductive physiology of a coastal dune population in southwest Florida to determine if reproductive cycles vary across populations. Here we present the first year-round sex hormone profiles for a wild population of gopher tortoises. Male testosterone concentrations varied across the year (F11,54 = 2.52, P = 0.015) with elevated values from September to December and minimal levels from April to July, with the exception of a secondary peak during the month of June. Female testosterone and estradiol concentrations varied across the sampling period (T: F11,66 = 8.54, P < 0.001, E: F11,66 = 4.57, P < 0.001) with highest values from August to February, and lowest levels from May to July. Female progesterone concentrations varied over the year (F11,64 = 3.29, P = 0.002) and increased in late fall with a peak in March. These data suggest this population has an extended breeding season from fall through spring with mating likely occurring from September through March, and nesting in winter through spring. This pattern is similar to reproductive patterns described for tropical and sub-tropical chelonians but differs from that of gopher tortoise populations in northern portions of the range where hibernation may last for five months and a single clutch of eggs are deposited in late spring.