Morphological variations and demographic responses of the Mediterranean pond turtle Mauremys leprosa to heterogeneous aquatic habitats.

Human activities affect terrestrial and aquatic habitats leading to changes at both individual and population levels in wild animal species. In this study, we investigated the phenotype and demographics of the Mediterranean pond turtle Mauremys leprosa (Schweigger, 1812) in contrasted environments of Southern France: two peri-urban rivers receiving effluents from wastewater treatment plants (WWTP), and another one without sewage treatment plant. Our findings revealed the presence of pesticides and pharmaceuticals in the three rivers of investigation, the highest diversities and concentrations of pollutants being found in the river subsections impacted by WWTP effluents. Principal component analysis and hierarchical clustering identified three levels of habitat quality, with different pollutant concentrations, thermal conditions, nutrient, and organic matter levels. The highest turtle densities, growth rates, and body sizes were estimated in the most disturbed habitats, suggesting potential adult benefits derived from harsh environmental conditions induced by pollution and eutrophication. Conversely, juveniles were the most abundant in the least polluted habitats, suggesting adverse effects of pollution on juvenile survival or adult reproduction. This study suggests that turtles living in polluted habitats may benefit from enhanced growth and body size, at the expense of reproductive success.

Alfaxalone is an effective anesthetic for the electrophysiological study of anoxia-tolerance mechanisms in western painted turtle pyramidal neurons.

Anoxia in the mammalian brain leads to hyper-excitability and cell death; however, this cascade of events does not occur in the anoxia-tolerant brain of the western painted turtle, Chrysemys picta belli. The painted turtle has become an important anoxia-tolerant model to study brain, heart, and liver function in the absence of oxygen, but being anoxia-tolerant likely means that decapitation alone is not a suitable method of euthanasia. Many anesthetics have long-term effects on ion channels and are not appropriate for same day experimentation. Using whole-cell electrophysiological techniques, we examine the effects of the anesthetic, Alfaxalone, on pyramidal cell action potential amplitude, threshold, rise and decay time, width, frequency, whole cell conductance, and evoked GABAA receptors currents to determine if any of these characteristics are altered with the use of Alfaxalone for animal sedation. We find that Alfaxalone has no long-term impact on action potential parameters or whole-cell conductance. When acutely applied to naïve tissue, Alfaxalone did lengthen GABAA receptor current decay rates by 1.5-fold. Following whole-animal sedation with Alfaxalone, evoked whole cell GABAA receptor current decay rates displayed an increasing trend with 1 and 2 hours after brain sheet preparation, but showed no significant change after a 3-hour washout period. Therefore, we conclude that Alfaxalone is a suitable anesthetic for same day use in electrophysiological studies in western painted turtle brain tissue.

Trained quantity discrimination in invasive red-eared slider and a comparison with the native stripe-necked turtle.

Little is known about the behavioral and cognitive traits that best predict invasion success. Evidence is mounting that cognitive performance correlates with survival and fecundity, two pivotal factors for the successful establishment of invasive populations. We assessed the quantity discrimination ability of the globally invasive red-eared slider (Trachemys scripta elegans). We further compared it to that of the native stripe-necked turtle (Mauremys sinensis), which has been previously evaluated for its superior quantity discrimination ability. Specifically, our experimental designs aimed to quantify the learning ability as numerosity pairs increased in difficulty (termed fixed numerosity tests), and the immediate response when turtles were presented with varied challenges concurrently in the same tests (termed mixed numerosity tests). Our findings reaffirm the remarkable ability of freshwater turtles to discern numerical differences as close as 9 vs 10 (ratio = 0.9), which was comparable to the stripe-necked turtle's performance. However, the red-eared slider exhibited a moderate decrease in performance in high ratio tests, indicating a potentially enhanced cognitive capacity to adapt to novel challenges. Our experimental design is repeatable and is adaptable to a range of freshwater turtles. These findings emphasize the potential importance of cognitive research to the underlying mechanisms of successful species invasions.

Establishment and characterization of turtle liver organoids provides a potential model to decode their unique adaptations.

Painted turtles are remarkable for their freeze tolerance and supercooling ability along with their associated resilience to hypoxia/anoxia and oxidative stress, rendering them an ideal biomedical model for hypoxia-induced injuries (including strokes), tissue cooling during surgeries, and organ cryopreservation. Yet, such research is hindered by their seasonal reproduction and slow maturation. Here we developed and characterized adult stem cell-derived turtle liver organoids (3D self-assembled in vitro structures) from painted, snapping, and spiny softshell turtles spanning ~175My of evolution, with a subset cryopreserved. This development is, to the best of our knowledge, a first for this vertebrate Order, and complements the only other non-avian reptile organoids from snake venom glands. Preliminary characterization, including morphological, transcriptomic, and proteomic analyses, revealed organoids enriched in cholangiocytes. Deriving organoids from distant turtles and life stages demonstrates that our techniques are broadly applicable to chelonians, permitting the development of functional genomic tools currently lacking in herpetological research. Such platform could potentially support studies including genome-to-phenome mapping, gene function, genome architecture, and adaptive responses to climate change, with implications for ecological, evolutionary, and biomedical research.

Experimental study on color and texture as cues for plastic debris ingestion by captive sea turtles.

Sea turtles face considerable risks from ingesting marine debris. They are primarily visual feeders, so color may be important for identifying food suitability or enhancing prey detection. Here, we investigated the impacts of color and texture on foraging behavior in relation to plastic consumption. We experimentally assessed the influences of color and texture as attractors for sea turtles using edible jellyfish. The findings showed that the colors of objects significantly affected selective preferences, as evidenced by different behaviors by sea turtles in response to different colors. They exhibited diet-related selectivity toward colors similar to common aquarium food, and texture had a significant impact on complete ingestion. The results suggest that plastic resembling natural prey is more likely ingested. Also, sea turtles were attracted by the color yellow, suggesting that visually distinctive items can attract them. Our results provide fundamental knowledge, helping mitigate the effects of plastic pollution on wildlife.

Adaptation of sea turtles to climate warming: Will phenological responses be sufficient to counteract changes in reproductive output?

Sea turtles are vulnerable to climate change since their reproductive output is influenced by incubating temperatures, with warmer temperatures causing lower hatching success and increased feminization of embryos. Their ability to cope with projected increases in ambient temperatures will depend on their capacity to adapt to shifts in climatic regimes. Here, we assessed the extent to which phenological shifts could mitigate impacts from increases in ambient temperatures (from 1.5 to 3°C in air temperatures and from 1.4 to 2.3°C in sea surface temperatures by 2100 at our sites) on four species of sea turtles, under a "middle of the road" scenario (SSP2-4.5). Sand temperatures at sea turtle nesting sites are projected to increase from 0.58 to 4.17°C by 2100 and expected shifts in nesting of 26-43 days earlier will not be sufficient to maintain current incubation temperatures at 7 (29%) of our sites, hatching success rates at 10 (42%) of our sites, with current trends in hatchling sex ratio being able to be maintained at half of the sites. We also calculated the phenological shifts that would be required (both backward for an earlier shift in nesting and forward for a later shift) to keep up with present-day incubation temperatures, hatching success rates, and sex ratios. The required shifts backward in nesting for incubation temperatures ranged from -20 to -191 days, whereas the required shifts forward ranged from +54 to +180 days. However, for half of the sites, no matter the shift the median incubation temperature will always be warmer than the 75th percentile of current ranges. Given that phenological shifts will not be able to ameliorate predicted changes in temperature, hatching success and sex ratio at most sites, turtles may need to use other adaptive responses and/or there is the need to enhance sea turtle resilience to climate warming.

Anthropogenic noise predicts sea turtle behavioural responses.

Anthropogenic noise is a pollutant of global concern. While the effects of underwater noise pollution have been frequently studied in fish and mammals, our understanding of how this anthropogenic stressor affects marine reptiles is scant. Using a multichannel data logger equipped with a camera and hydrophone, we quantified behavioural responses of a free-ranging green turtle (Chelonia mydas) to vessel noise in the Galapagos Archipelago, an important nesting site in the eastern Pacific. We found that while travelling the turtle increased its vigilance with increasing vessel noise. However, when on the seabed the turtle did not increase its vigilance with increasing noise levels. Our findings illustrate that noise pollution has the potential to alter overall time budgets of animals. Identifying real-time responses of wild animals illustrate how in situ approaches allow to assess the effects of human activities on marine systems.

Morphological Analysis of the Olfactory System of the Pig-Nosed Turtle, Carettochelys insculpta.

The turtle olfactory organ consists of the upper (UCE) and lower (LCE) chamber epithelium, projecting to the ventral and dorsal parts of the olfactory bulbs, respectively. The UCE is associated with glands, contains ciliated olfactory receptor neurons, and is assumed to detect odorants primarily in air, while the LCE is devoid of glands, contains microvillous olfactory receptor neurons, and is assumed to detect odorants primarily in water. Examining the olfactory system of the pig-nosed turtle, Carettochelys insculpta, this study found that both the upper and lower chambers of the nasal cavity were lined with sensory epithelium devoid of associated glands and contained ciliated olfactory receptor neurons. Moreover, the olfactory bulbs were not divided into dorsal and ventral parts. These results suggest that the olfactory system of the pig-nosed turtle is a single system specialized for detecting odorants in water.

Daily, seasonal, and long-distance movements inferred from Fastloc-GPS telemetry of immature green turtles (Chelonia mydas) at a high-latitude, mid-ocean developmental site.

To characterize the movements and habitat use of juvenile green turtles (Chelonia mydas) in benthic developmental habitat, we deployed Fastloc-GPS-enabled satellite transmitters on 16 individuals captured as part of a multi-decade study of green turtles on the Bermuda Platform. We characterized residence areas, distinct use areas within them, and seasonal movements based on an average of 562 Fastloc-GPS positions and 284 tracking days per turtle. We estimated residence area sizes using traditional home range methods, e.g., 90% utilization distribution (UD) (mean 2.29 ±2.71 km2) and 50% UD (mean 0.54 ±0.69 km2). Total residence area size increased significantly over the 8-year study, from <1 km2 before 2013 to ≥3 km2 in 2018 (R2 = 0.51, F1,14 = 14.55, p = 0.0019), corresponding to a period of decline in seagrass habitat and suggesting increased foraging effort. We identified three types of distinct use areas within residence areas where tracked turtles typically exhibited behavioral fidelity: foraging, resting, and cool weather refugia. These distinct use areas were smaller than high-use areas from previous studies; e.g., seagrass meadow foraging areas averaged 0.05 km2. Most turtles made daily transits between foraging and resting sites; for some individuals, these involved crossing frequently used vessel navigation channels. Seasonal variation in behavior suggested that the overwintering strategy for green turtles on the Bermuda Platform involves "optional dormancy," during which turtles spent less time on seagrass meadows and made brief excursions to distinct deeper habitats. Four individuals made directed (mean path straightness = 0.93 ±0.02 SD) developmental migrations away from Bermuda toward known adult foraging range. Results of our study further knowledge of the green turtle life cycle at a high-latitude site; they demonstrate that green turtles show fidelity to distinct use areas within developmental habitats over many years and exhibit seasonal movements.

Effects of temperature heterogeneity on freshwater turtle habitat selection at their northern range limit.

Environmental temperature is a crucial resource for ectotherms, affecting their physiology, behaviour and fitness. To maintain body temperatures within a suitable performance range, ectotherms select thermally-favourable locations, but this selection may be challenging in environments with high spatio-temporal heterogeneity. We assessed thermal habitat selection in two freshwater turtles (Emydoidea blandingii; Chrysemys picta) within a thermally heterogeneous environment at two spatial scales (selection of home ranges within the landscape, selection of locations within home ranges) and across seasons, by comparing temperatures at turtle locations vs. those available in the environment. Turtles selected warmer locations compared to those available in aquatic and terrestrial habitats only within home ranges, but did not show any temperature preferences when selecting home ranges at the larger scale. Turtles selected locations that were less thermally-variable than their surroundings, both at the home range scale and within home ranges. Thermal habitat selection was strongest during colder and more thermally-variable pre-nesting season compared to later periods. Despite differences in thermal mass between species, both species responded similarly to temperature variation. We conclude that freshwater turtles at their northern range margin select suitable microclimates within the suite of conditions that are naturally available.

Incubation in shaded hatcheries biases sex-determination but preserves Lepidochelys olivacea hatchling physiology.

Some studies have associated ex situ conservation with cerebral and gonadal developmental delay, as well as decreased motor performance in Lepidochelys olivacea offspring. Ex situ management is also related to a more mature spleen and a differential leukocyte count in newly emerged Lepidochelys olivacea hatchlings. The physiological relevance of a more mature spleen is unknown in sea turtles, but studies in birds suggest an increased immune response. Because egg relocation to hatcheries is a common conservation practice, it is imperative to know its impact on hatchling physiology. Herein, plasma activity of superoxide dismutase, alkaline phosphatase and the alternative complement pathway, as well as total antioxidant capacity and hydrogen peroxide concentrations were quantified in hatchlings from in situ and ex situ nests under basal conditions at nest emergence. Toll-like receptor 4 (tlr4), heat shock proteins (hsp) 70 and hsp90 expression were quantified in the spleen and liver of the hatchlings. Hepatocyte density and nuclear area were quantified in histological sections of the liver and all turtles were sexed by histological sectioning of the gonads. Total antioxidant capacity and hydrogen peroxide concentrations in plasma were lower in turtles from ex situ nests, while tlr4 and hsp70 mRNA expression was higher in the spleen but not in the liver. Ex situ incubation produced 98% male hatchlings, whereas in situ incubation produced 100% females. There were no other differences in the attributes sampled between hatchlings emerging from ex situ and in situ treatments. The results suggest that ex situ relocated turtles may be less prone to oxidative stress than in situ incubated hatchlings and could have more mature splenic function. Together, the data suggest that ex situ relocation to shaded hatcheries biased sex determination but preserved the general physiological condition of sea turtle hatchlings.

Short-term resilience to climate-induced temperature increases for equatorial sea turtle populations.

Projection models are being increasingly used to manage threatened taxa by estimating their responses to climate change. Sea turtles are particularly susceptible to climate change as they have temperature-dependent sex determination and increased sand temperatures on nesting beaches could result in the 'feminisation' of hatchling sex ratios for some populations. This study modelled likely long-term trends in sand temperatures and hatchling sex ratios at an equatorial nesting site for endangered green turtles (Chelonia mydas) and critically endangered hawksbill turtles (Eretmochelys imbricata). A total of 1078 days of sand temperature data were collected from 28 logger deployments at nest depth between 2018 and 2022 in Papua New Guinea (PNG). Long-term trends in sand temperature were generated from a model using air temperature as an environmental proxy. The influence of rainfall and seasonal variation on sand temperature was also investigated. Between 1960 and 2019, we estimated that sand temperature increased by ~0.6°C and the average hatchling sex ratio was relatively balanced (46.2% female, SD = 10.7). No trends were observed in historical rainfall anomalies and projections indicated no further changes to rainfall until 2100. Therefore, the sex ratio models were unlikely to be influenced by changing rainfall patterns. A relatively balanced sex ratio such as this is starkly different to the extremely female-skewed hatchling sex ratio (>99% female) reported for another Coral Sea nesting site, Raine Island (~850 km West). This PNG nesting site is likely rare in the global context, as it is less threatened by climate-induced feminisation. Although there is no current need for 'cooling' interventions, the mean projected sex ratios for 2020-2100 were estimated 76%-87% female, so future interventions may be required to increase male production. Our use of long-term sand temperature and rainfall trends has advanced our understanding of climate change impacts on sea turtles.

Experiencing short heat waves early in development changes thermal responsiveness of turtle embryos to later heat waves.

Although physiological responses to the thermal environment are most frequently investigated using constant temperatures, the incorporation of thermal variability can allow for a more accurate prediction of how thermally sensitive species respond to a rapidly changing climate. In species with temperature-dependent sex determination (TSD), developmental responses to incubation temperature are mediated by several genes involved in gonadal differentiation. Kdm6b and Dmrt1 respond to cool incubation temperatures and are associated with testis development, while FoxL2 and Cyp19A1 respond to warm incubation temperatures and are associated with ovary development. Using fluctuating incubation temperatures, we designed two studies, one investigating how conflicting thermal cues affect the timing of commitment to gonadal development, and another investigating the rapid molecular responses to conflicting thermal cues in the red-eared slider turtle (Trachemys scripta). Using gene expression as a proxy of timing of commitment to gonadal fate, results from the first study show that exposure to high amounts of conflicting thermal cues during development delays commitment to gonadal fate. Results from the second study show that Kdm6b splice variants exhibit differential responses to early heat wave exposure, but rapidly (within 2 days) recover to pre-exposure levels after the heat wave. Despite changes in the expression of Kdm6b splice variants, there was no effect on Dmrt1 expression. Collectively, these findings demonstrate how short exposures to heat early in development can change how embryos respond to heat later in development.

Effects of postovipositional hypoxia and hyperoxia on leatherback turtle reproductive success and hatchling performance.

Leatherback egg clutches typically experience lower hatching success (~50%) than those of other sea turtle species (>70%). The majority of embryonic death (>50%) occurs at early stages of development, possibly because embryos fail to break preovipositional embryonic arrest after oviposition. The embryonic arrest is maintained by hypoxia in the oviduct and following oviposition increased availability of oxygen is the trigger that breaks arrest in all turtle species studied to date. We conducted an ex situ incubator experiment and an in situ hatchery experiment to examine the influence of oxygen availability on hatching success and hatchling traits in leatherbacks. After oviposition, eggs (n = 1005) were incubated in either normoxia (21% O2 ), hyperoxia (32%-42% O2 ) for 5 days, or hypoxia (1% O2 ) for 3 or 5 days. As with other turtles, hypoxic incubation maintained embryos in arrest, equivalent to the time spent in hypoxia. However, extending arrest for 5 days resulted in greater early-stage death and a significant decrease in hatching success (4% 5-day hypoxia vs. 72% normoxia). Eggs placed in incubators had greater hatching success than those placed into hatchery nests (67% vs. 47%, respectively). We found no impact of hyperoxia on the stage of embryonic death, hatching success, hatchling phenotype, exercise performance, or early dispersal. Our findings indicate that delayed nesting and the subsequent extension of embryonic arrest may negatively impact embryonic development and therefore the reproductive success of leatherbacks. They also indicate that incubation under hyperoxic conditions is unlikely to be a useful method to improve hatching success in this species.

DETERMINATION OF ECHOCARDIOGRAPHIC TECHNIQUES FOR CLINICAL ASSESSMENT OF GALAPAGOS (CHELONOIDIS NIGRA COMPLEX) AND ALDABRA (ALDABRACHELYS GIGANTEA) TORTOISES.

The first objective of this study was to establish clinically relevant techniques for cardiac echocardiography in nonanesthetized Galapagos (Chelonoidis nigra complex) and Aldabra (Aldabrachelys gigantea) tortoises. A second objective was to establish guidelines for determining normal echocardiographic anatomy and function in both species. Select echocardiographic reference values were defined for 17 healthy Galapagos tortoises and 27 healthy Aldabra tortoises. Tortoises were either placed in ventral recumbency on an elevated surface or allowed to stand in a natural position by using food distraction. An ultrasound probe was applied in the left or right cervicobrachial window and was positioned in two long axis views to evaluate the three chambers of the heart and the associated great vessels, the presence of pericardial effusion, the atrioventricular inflow velocities, and pulmonic and aortic outflow velocities. The heart rate was 28 ± 12 (median ± SD) bpm, and the ejection fraction was 60.5 ± 10%. Thirty-four of 44 tortoises had identifiable physiologic pericardial effusion. All tortoises were successfully imaged using the techniques described, with consistent identification of cardiac structure and assessment of function. This study provides echocardiographic reference intervals for the clinical evaluation of suspected cardiac disease in captive-managed Galapagos and Aldabra tortoises.

Quantum Mechanics/Molecular mechanics calculations predict A1, not A2, is present in melanopsin (Opn4m) of red-eared slider turtles (Trachemys scripta elegans).

Melanopsin is a photopigment that plays a role in non-visual, light-driven, cellular processes such as modulation of circadian rhythms, retinal vascular development, and the pupillary light reflex (PLR). In this study, computational methods were used to understand which chromophore is harbored by melanopsin in red-eared slider turtles (Trachemys scripta elegans). In mammals, the vitamin A derivative 11-cis-retinal (A1) is the chromophore, which provides functionality for melanopsin. However, in red-eared slider turtles, a member of the reptilian class, the identity of the chromophore remains unclear. Red-eared slider turtles, similar to other freshwater vertebrates, possess visual pigments that harbor a different vitamin A derivative, 11-cis-3,4-didehydroretinal (A2), making their pigments more sensitive to red-light than blue-light, therefore, suggesting the chromophore to be the A2 derivative instead of the A1. To help resolve the chromophore identity, in this work, computational homology models of melanopsin in red-eared slider turtles were first constructed. Next, quantum mechanics/molecular mechanics (QM/MM) calculations were carried out to compare how A1 and A2 derivatives bind to melanopsin. Time dependent density functional theory (TDDFT) calculations were then used to determine the excitation energy of the pigments. Lastly, calculated excitation energies were compared to experimental spectral sensitivity data from responses by the irises of red-eared sliders. Contrary to what was expected, our results suggest that melanopsin in red-eared slider turtles is more likely to harbor the A1 chromophore than the A2. Furthermore, a glutamine (Q622.56) and tyrosine (Y853.28) residue in the chromophore binding pocket are shown to play a role in the spectral tuning of the chromophore.

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.

Embryonic development, hatchling performance and metabolic profile after egg exposure to environmentally relevant levels of chlorpyrifos in an aquatic turtle.

The extensive use of organophosphorus insecticides poses a threat to the survival of non-target organisms. Ecotoxicological outcomes of embryonic exposure to insecticides are rarely evaluated in various oviparous species. In this study, soft-shelled turtle (Pelodiscus sinensis) eggs were incubated in moist substrate containing different levels (0, 2, 20 and 200 µg/kg) of chlorpyrifos to investigate its toxic effects on embryonic development and survival, and hatchling physiological performance. Chlorpyrifos exposure had no significant impacts on embryonic development rate and egg survival in P. sinensis. Similarly, embryonic chlorpyrifos exposure neither obviously affected the size and locomotor performance of hatchlings, nor changed the activities of superoxide dismutase and catalase, and content of malondialdehyde in their erythrocytes. Based on liquid chromatography-mass spectrometry analysis, minor metabolic perturbations related to amino acid, lipid and energy metabolism in hatchlings after embryonic chlorpyrifos exposure were revealed by hepatic metabolite profiling. Overall, our results suggested that embryonic exposure to environmentally relevant levels of chlorpyrifos had only a limited impact on physiological performances of hatchlings, although it would result in a potential risk of hepatotoxicity in P. sinensis.

Effect of Purslane (Portulaca oleracea L.) on Intestinal Morphology, Digestion Activity and Microbiome of Chinese Pond Turtle (Mauremys reevesii) during Aeromonas hydrophila Infection.

Large-scale mortality due to Aeromonas hydrophila (A. hydrophila) infection has considerably decreased the yield of the Chinese pond turtle (Mauremys reevesii). Purslane is a naturally active substance with a wide range of pharmacological functions, but its antibacterial effect on Chinese pond turtles infected by A. hydrophila infection is still unknown. In this study, we investigated the effect of purslane on intestinal morphology, digestion activity, and microbiome of Chinese pond turtles during A. hydrophila infection. The results showed that purslane promoted epidermal neogenesis of the limbs and increased the survival and feeding rates of Chinese pond turtles during A. hydrophila infection. Histopathological observation and enzyme activity assay indicated that purslane improved the intestinal morphology and digestive enzyme (α-amylase, lipase and pepsin) activities of Chinese pond turtle during A. hydrophila infection. Microbiome analysis revealed that purslane increased the diversity of intestinal microbiota with a significant decrease in the proportion of potentially pathogenic bacteria (such as Citrobacter freundii, Eimeria praecox, and Salmonella enterica) and an increase in the abundance of probiotics (such as uncultured Lactobacillus). In conclusion, our study uncovers that purslane improves intestinal health to protect Chinese pond turtles against A. hydrophila infection.

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.