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Humans produce 350 million metric tons of plastic waste per year, leading to microplastic pollution and widespread environmental contamination, particularly in aquatic environments. This subsequently impacts aquatic organisms in myriad ways, yet the vast majority of research is conducted in marine, rather than freshwater systems. In this study, we exposed eggs and hatchlings of the Chinese soft-shelled turtle (Pelodiscus sinensis) to 80-nm polystyrene nanoplastics (PS-NPs) and monitored the impacts on development, behavior and the gut microbiome. We demonstrate that 80-nm PS-NPs can penetrate the eggshell and move into developing embryos. This led to metabolic impairments, as evidenced by bradycardia (a decreased heart rate), which persisted until hatching. We found no evidence that nanoplastic exposure affected hatchling morphology, growth rates, or levels of boldness and exploration, yet we discuss some potential caveats here. Exposure to nanoplastics reduced the diversity and homogeneity of gut microbiota in P. sinensis, with the level of disruption correlating to the length of environmental exposure (during incubation only or post-hatching also). Thirteen core genera (with an initial abundance >1 %) shifted after nanoplastic treatment: pathogenic bacteria increased, beneficial probiotic bacteria decreased, and there was an increase in the proportion of negative correlations between bacterial genera. These changes could have profound impacts on the viability of turtles throughout their lives. Our study highlights the toxicity of environmental NPs to the embryonic development and survival of freshwater turtles. We provide insights about population trends of P. sinensis in the wild, and future directions for research.

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Wetlands are among the most threatened ecosystems on the planet and pollution is a major factor causing the decline of wetland biodiversity. Despite the increasing use of pesticides, their fate and effects on freshwater reptiles remain largely unknown. We studied the European pond turtle (Emys orbicularis), a long-lived species at risk with a high exposure potential to pesticides. Between 2018 and 2020, we measured 29 pesticides and metabolites in 408 blood samples of turtles from two populations in the Camargue wetland (France). We were able to quantify 24 compounds and at least one pesticide or one degradation product in 62.5% of samples. Pesticide occurrences and concentrations were low, except for a herbicide widely used in rice cultivation and locally detected in water: bentazone that reached high blood concentrations in E. orbicularis. The occurrence and the concentration of pesticides in E. orbicularis blood depended mainly on the site and the sampling date in relation to pesticide application. Individual characteristics (sex, age, body condition) did not explain the occurrence or the concentration of pesticides found in turtle blood. Assessing the exposure of aquatic wildlife to a cocktail of currently-used pesticides is a first and crucial step before studying their effects at the individual and population levels.

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Understanding the microhabitat requirements of an animal is vital for ensuring the success of targeted conservation and microhabitat restoration measures. The big-headed turtle (Platysternon megacephalum) is a freshwater species that is distributed across Southeast Asia. Owing to the human threats posed by illegal pet trade and overharvesting for food and medicinal purposes, the species has undergone rapid decline. However, in Hainan, their microhabitat characteristics are still unknown, which is neither conducive to the conservation of the species nor to the establishment of the Hainan Tropical Rainforest National Park. This study examined the microhabitat characteristics of P. megacephalum using sample plot methods in the Diaoluo Mountain area of Hainan Tropical Rainforest National Park. Our results indicated that P. megacephalum prefers stream microhabitats with rocky substrates, several caves, and a high diversity of food sources. Microhabitat characteristics did not differ significantly between adults and juveniles. Our results suggest that protecting microhabitats and main food sources is important for the conservation of P. megacephalum. Our findings provide a reference for the protection of this species in Jianfeng Ridge, Yingge Ridge, and other areas in Hainan Tropical Rainforest National Park.

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The coincidence of hatching and emergence events with favorable conditions is crucial for turtle survival. Nocturnal emergence has been widely documented across marine and freshwater turtles, and has long been suggested as an adaptive behavior that reduces risks of heat stress and predation. To our knowledge, however, studies related to nocturnal emergence have mainly focused on the post-hatching behaviors of turtles, and very few experimental studies have been performed to investigate the effects of hatching time on the distribution of emergence times over the course of a day. Here, we visually monitored the activity of the Chinese softshell turtle (Pelodiscus sinensis)-a shallow-nesting freshwater turtle-from hatching to emergence. Our study provides evidence for the novel finding that (i) the timing of synchronous hatching events in P. sinensis coincides with the time of day when nest temperatures decrease, (ii) the synchrony between hatching and emergence may further facilitate their nocturnal emergence, and (iii) synchronous behaviors of hatchlings in the nest may be effective in reducing the risk of hatchling predation, and predation is more likely to occur in the asynchronous hatching groups. This study suggests that the hatching of shallow-nesting P. sinensis in response to temperature changes in the nest might be an adaptive nocturnal emergence strategy.

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Background: To determine species distributions and the factors underlying them, reliable occurrence data are crucial. Assembling such data can be challenging for species with cryptic life histories or that occur at low densities. Methods: We developed species-specific eDNA protocols, from sampling through data interpretation, to detect the common musk turtle (Sternotherus odoratus) and tested whether eDNA occurrences change our understanding of the species distribution and the factors that shape its northern range limit. We used Species Distribution Models (SDMs) with full parameter optimization on citizen science observations of S. odoratus in Southern Ontario alone and together with eDNA occurrences. Results: Our eDNA protocol was robust and sensitive. SDMs built from traditional observations and those supplemented with eDNA detections were comparable in prediction accuracy. However, models with eDNA detections suggested that the distribution of S. odoratus in Southern Ontario is underestimated, especially near its northern range limit, and that it is shaped by thermal conditions, hydrology, and elevation. Our study underscores the promise of eDNA for surveying cryptic aquatic organisms in undocumented areas, and how such insights can help us to improve our understanding of species distributions.

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Objective: The Balkan terrapin, Mauremys rivulata, is a freshwater turtle. This reptile is exposed to many environmental pollutants and some infectious agents, including Haemogregarina stepanowi parasite. This study was conducted to determine the microscopic prevalence of haemogregarine infection in M. rivulata caught in three different localities (Bozcaada, Gökçeada, and Dardanos) in Çanakkale province of Turkey, and assessment of some risk factors. Methods: Twenty-four blood samples were collected, thin blood smears were prepared, and the presence of haemogregarine parasites microscopically was screened. Water samples were also taken from the habitats, and these samples were analyzed physiochemically and microbiologically. Results: Morphological identification was made by detecting the sausage-shaped intra-cytoplasmic developmental stages of H. stepanowi, and thirteen of twenty-four turtles (54.2%) were found to be infected. The prevalence of H. stepanowi was detected as the highest (90.0%) in the Gökçeada district, where the water pollution is higher than in the other localities. A statistically significant relationship was observed between the distribution of the infection and the gender of the turtles, the temperature of the water, the number of faecal coliforms in water and the amount of dissolved oxygen in the water. A statistically significant difference was found between the localities in terms of the prevalence of H. stepanowi infection, and the infection was primarily detected in the Gökçeada district. Conclusion: This study has significance in providing information regarding haemoparasitic diseases of freshwater turtle, M. rivulata, in Turkey.

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Sex-biased dispersal is common in many animals, with male-biased dispersal often found in studies of mammals and reptiles, including interpretations of spatial genetic structure, ostensibly as a result of male-male competition and a lack of male parental care. Few studies of sex-biased dispersal have been conducted in turtles, but a handful of studies, in saltwater turtles and in terrestrial turtles, have detected male-biased dispersal as expected. We tested for sex-biased dispersal in the endangered freshwater turtle, the spotted turtle (Clemmys guttata) by investigating fine-scale genetic spatial structure of males and females. We found significant spatial genetic structure in both sexes, but the patterns mimicked each other. Both males and females typically had higher than expected relatedness at distances <25 km, and in many distance classes greater than 25 km, less than expected relatedness. Similar patterns were apparent whether we used only loci in Hardy-Weinberg equilibrium (n = 7) or also included loci with potential null alleles (n = 5). We conclude that, contrary to expectations, sex-biased dispersal is not occurring in this species, possibly related to the reverse sexual dimorphism in this species, with females having brighter colors. We did, however, detect significant spatial genetic structure in males and females, separate and combined, showing philopatry within a genetic patch size of <25 km in C. guttata, which is concerning for an endangered species whose populations are often separated by distances greater than the genetic patch size.

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Ammonia is a common pollutant in aquaculture system, and toxic to all aquatic animals. However, different aquatic animals exhibit diverse physiological responses to high-level ammonia exposure, potentially indicating their divergent resistance to ammonia stress. In this study, juveniles of three freshwater turtles (Mauremys reevesii, Pseudemys nelsoni and Trachemys scripta elegans) were exposed to different concentrations of ammonia (0, 0.3 and 3.0 mg/L) for 30 days, and their swimming, growth performance, gut microbiota, and hepatic metabolites were measured to evaluate the interspecific difference in physiological responses to ammonia stress. Despite no differences in swimming ability, growth rate, and gut microbial diversity, observable changes in microbial community composition and hepatic metabolite profiles were shown in ammonia-exposed turtles. A relatively higher abundance of potentially pathogenic bacteria was found in M. reevesii than in the other two species. Moreover, microbial compositions and metabolic responses differed significantly among the three species. M. reevesii was, out of the three tested species, the one in which exposure to ammonia had the greatest effect on changes in bacterial genera and hepatic metabolites. Conversely, only a few metabolites were significantly changed in T. scripta elegans. Integrating these findings, we speculated that native M. reevesii should be more vulnerable to ammonia stress compared to the invasive turtle species. Our results plausibly reflected divergent potential resistance to ammonia among these turtles, in view of differential physiological responses to ammonia exposure at environmentally relevant concentrations.

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PURPOSE: We evaluated how constant incubation temperatures affect life-history traits pre-hatching and post-hatching of the six-tubercled Amazon River turtle, Podocnemis sextuberculata. METHODS: We incubated eggs from natural nests at ten semi-constant temperatures between 22.26 ± 1.01 °C and 37.37 ± 0.38°C (2013) and at six temperatures between 25.75 ± 0.22 °C and 36.17 ± 0.15°C (2016). In 2013, we raised hatchling for 90 days to evaluate effects of temperature on early hatchling growth. We evaluated maternal effects in 2016. RESULTS: P. sextuberculata displays temperature-dependent sex determination and produces males at colder and females at warmer temperatures (TSD Ia). The estimated pivotal temperature was 33.73 ± 0.15 °C and the transitional range of temperatures (TRT) 1.16 ± 0.59 °C. Semi-constant temperatures below 26 °C and above 38 °C were lethal. Intermediate temperatures (32.25 °C and 31.5 °C, respectively) were optimal for hatching success and produced larger hatchlings that grew slower early in life compared to colder or warmer conditions, which produced smaller hatchlings. Warmer incubation temperatures within the optimal range (28°C-37 °C) accelerated embryonic development. In contrast, comparisons of 30, 60 and 90 days-old suggests that warmer incubation temperatures reduced growth and mass gain rates post-hatching, such that incubation temperature effects on body size at emergence disappeared by 3 months of age. CONCLUSIONS: Six-tubercled Amazon River turtles showed the highest pivotal temperature reported for any turtle. The relatively narrow TRT may limit the evolutionary potential of this vulnerable turtle in the face of global warming. Future incubation experiments at a finer scale (33°C-36 °C) are warranted to refine the sex-ratio reaction norm. Field studies that monitor natural nests are imperative to evaluate conservation measures and the effect of female-biased illegal hunting and climate change. By providing data about the thermal biology of an understudied lineage of non-model species, our study helps fill gaps in our understanding of the evolution of vertebrate sex determination and its potential adaptive value.

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Emys orbicularis is one of the freshwater turtles occurring in Europe, Middle East and Western Asia, but also occur in Caucasus region as Georgia, Armenia, Azerbaijan and Southern Caspian Sea region (northern Iran provinces as Guilan, Mazandaran, Golestan). In this study, seventeen localities in three Iranian provinces were investigated to study the response of this species to abiotic factors (water quality parameters) and road mortality. To test for differences in occurrence of E. orbicularis between sites that could be explained by variation in abiotic factors, one-way ANOVA test and Tukey's HSD post hoc tests were conducted. These showed significant differences in conductivity (CON) and Total Dissolved Solids (TDS) between locations in the three provinces of Golestan, Mazandaran and Gilan [CON: (F (2, 13) = 9.824, p = 0.003); TDS: (F (2, 13) = 13.957, p = 0.001)]. Differences in four other abiotic variables-water temperature, pH, salinity and dissolved oxygen (DO)-were not significant between provinces, although there were differences. These findings suggested that the species can occur in ponds with the following features: temperature 21-28 °C; pH 7-8; salinity > 750 mg/L; DO > 30%. Road mortality might be another factor that could contribute to the decline of this turtle's population in northern Iran, noting that the roads were created by human within the species' habitat and increased the mortality rate. Roads should be monitored in order to evaluate their effect on freshwater turtle mortality. Overall, our results demonstrated the advantage of using analysis of abiotic variables as a valid approach for defining species-responses to abiotic factors in E. orbicularis and recommended monitoring roads to find high-risk mortality of this species and create under road tunnels for moving turtles without any risk to reduce high rate of mortality.

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In the present study, the cadmium, lead, and zinc levels in the blood and shell of Caspian pond turtles (Mauremys caspica) were investigated at five stations in Zarivar International Wetland in Kurdistan Province. All specimens were released at their capture locations within 2 h of capture. Water samples were collected at each station. Heavy metal concentrations were determined using an atomic absorption spectrometer. The mean cadmium, lead, and zinc concentrations were 0.04, 32.10, and 11.45 mg/l in blood samples; 1.82, 16.91, and 89.22 mg/l in shell samples; 0.005, 1.30, and 0.07 mg/l in water samples, respectively. In this study, the highest metal adsorption was zinc and was observed in shell. According to the results of this study, the shell of the Caspian Pond Turtle can be used to estimate the concentration of heavy metals. Our results suggest that Caspian pond turtle can be used as a biological indicator to estimate heavy metals.

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Turtle leeches have not been recorded in Korea, although they occur in geographically adjacent countries including China and Japan. This study describes a turtle leech, Ozobranchus jantseanus (Clitellata: Ozobranchidae), found from Reeve's turtle (Mauremys reevesii) in Korea. Of the 143 Reeve's turtles collected from the freshwater reservoir in Jinju City, Gyeongsangnam-do, Korea, 95 unidentified leeches were found in 8 (5.6%) individuals. The leeches had 22 somites with 11 pairs of externally exposed branchiae, body-sized posterior suckers, and spines on the dorsal surface. We identified these leeches as Ozobranchus jantseanus Oka, 1912 (Clitellata: Ozobranchidae). This species of turtle leeches found in Korea may fill the gap in the biodiversity of East Asian annulus.

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During the last few decades, fungal pathogens have caused devastating population declines across a broad range of taxa. A newly emerging fungal disease, sea turtle egg fusariosis, caused by members of the Fusarium solani species complex (FSSC), has been reported to be responsible for hatching failure in sea turtles worldwide. However, this has not been detected in fresh water turtle species. Here, using relocated clutches and artificial incubation, we report high hatching failure in eggs symptomatic of fusariosis in the yellow-spotted Amazon River turtle (Podocnemis unifilis) inhabiting a pristine environment in the Ecuadorian Amazon. In 2020, we screened 680 eggs of the yellow-spotted Amazon River turtle, relocated from wild nesting areas to artificial nests, for visual symptoms of fusariosis and to estimate hatchability despite infection. We selected 68 eggs sampled in 2019 to confirm Fusarium infection by PCR amplification of the TEF-1α gene and sequenced seven of those amplicons on an Illumina Miseq to assess FSSC membership. We observed fusariosis symptoms in 42% of the 680 eggs. The proportion of symptomatic eggs within nests was negatively linked to the proportion of eggs that hatched. Hatchability was 8% for symptomatic eggs compared with 72% of asymptomatic eggs. Through PCR testing, 58% of symptomatic and 8% of asymptomatic eggs sampled in 2019 tested positive for Fusarium spp., and sequencing revealed that nine sequence variants from three asymptomatic and four symptomatic eggs corresponded to F. keratoplasticum, F. solani and F. falciforme, the three major FSSC pathogens reported in sea turtle egg fusariosis. Our study suggests that hatching failure in eggs linked to symptoms of fusariosis appears to be partially caused by Fusarium pathogens within FSSC in a freshwater turtle. Thus, fusariosis is more widespread among the Testudines than previously reported and is not limited to sea environments, findings of particular conservation concern.

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Numerous cases and a shortage of resources usually limit wild animal rescue. New technology might save these severely injured wild animals from euthanasia by easing the requirement of intensive medication. Three-dimensional (3D) technologies provide precise and accurate results that improve the quality of medical applications. These 3D tools have become relatively low-cost and accessible in recent years. In the medical field of exotic animals, turtle shell defects are highly challenging because of inevitable water immersion. This report is the first attempt to apply the combination of 3D scanning, computer-aided design (CAD), and 3D printing to make a device that protects the wound from exposure to water or infection sources. The presented techniques successfully extricate a wild freshwater turtle from an extensive shell defect within a short period. Integration of multiple sciences to 3D technology can provide a facile model for veterinary medical applications.

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Southern River Terrapin, Batagur affinis, is a freshwater turtle listed as critically endangered on the IUCN Red List since 2000. Many studies suggest that faecal DNA metabarcoding can shield light on the host-associated microbial communities that play important roles in host health. Thus, this study aimed to characterise and compare the faecal bacterial community between captive and wild B. affinis using metabarcoding approaches. A total of seven faeces samples were collected from captive (N = 5) and wild (N = 2) adult B. affinis aseptically, crossing the East and West coast of peninsular Malaysia. The DNA was extracted from the faeces samples, and the 16S rRNA gene (V3-V4 region) was amplified using polymerase chain reaction (PCR). The amplicon was further analysed using SILVA and DADA2 pipelines. In total, 297 bacterial communities taxonomic profile (phylum to genus) were determined. Three phyla were found in high abundance in all faeces samples, namely Firmicutes (38.69%), Bacteroidetes (24.52%), and Fusobacteria (6.95%). Proteobacteria were detected in all faeces samples (39.63%), except the wild sample, KBW3. Under genus level, Cetobacteriumwas found as the most abundant genus (67.79%), followed by Bacteroides (24.56%) and Parabacteroides (21.78%). The uncultured genus had the highest abundance (88.51%) even though not detected in the BK31 and KBW2 samples. The potential probiotic genera (75.00%) were discovered to be more dominant in B. affinis faeces samples. Results demonstrated that the captive B. affinis faeces samples have a greater bacterial variety and richness than wild B. affinis faeces samples. This study has established a starting point for future investigation of the gut microbiota of B. affinis.

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Infections with intravascular digenean trematodes of the Spirorchiidae family (spirorchiidoses) are of great conservation concern both in marine and freshwater turtles due to their pathogenic potential. Between 2014 and 2021, Spirorchis sp. infections associated with granulomatous inflammation and sudden death were detected in European pond turtles (Emys orbicularis) from three conservation breeding facilities in Switzerland. Blood fluke eggs associated with lesions were found in the intestine, spleen, testis, skeletal musculature, heart, kidneys, stomach, pancreas, liver, lung, and meninges from nine pond turtles submitted for necropsy and in the intestinal content from five of these animals. Two novel polymerase chain reactions (PCRs) targeting the 28S ribosomal RNA gene and the ITS2 region and subsequent sequencing revealed 100% nucleotide identity with a Spirorchis sp. previously isolated from an Escambia map turtle (Graptemys ernsti) in the USA. Our findings suggest a spill-over event secondary to direct or indirect contact with invasive North American turtle species in Switzerland. We describe the clinical, haematological, ultrasonographical, endoscopical, parasitological, pathological, and molecular findings associated with spirorchiid blood fluke infections of the Spirorchis genus in E. orbicularis, as well as the biosecurity measures that were developed to prevent the spread of this parasite among breeding and highly endangered free-ranging E. orbicularis populations in Switzerland.

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The objective of this study was to determine the structure of the helminth fauna and identify the macroscopic and histopathological alterations associated with parasitic infections in Phrynops geoffroanus. Freshwater turtles of both sexes were captured during the dry and rainy seasons in four municipalities along the Capibaribe River. The study included 63 animals, of which 79.37% (50/63) were parasitized by one or more helminths. In total, 933 helminths of seven taxa were recovered: Serpinema monospiculatus, Spiroxys figueiredoi, Nematophila grandis, Polystomoides brasiliensis, Cheloniodiplostomum testudinis, Telorchis birabeni, and Prionosomoides scalaris. Monogeneans and digenetic trematodes were more sensitive to environmental pressures, since the prevalences varied significantly between areas. Nematodes proved to be more resistant to environmental pressure and caused severe injuries to their hosts: nodules in the stomach and small intestine, adhesions in the liver capsule, and pulmonary emphysema. Parasitic granulomas were recorded at the infection sites and in the lungs and liver, the latter caused by migration of S. figueiredoi larvae. This is the first record of P. brasiliensis, N. grandis, C. testudinis, and T. birabeni parasitizing P. geoffroanus in the state of Pernambuco. Histopathology proved to be an important tool for studies on the impact of parasites at the individual, population, and ecosystem levels. Considering the use of the Capibaribe River for public water supply, fishing, and other activities, within the One Health perspective, this study demonstrates that the anthropogenic impact affects parasites and their hosts, in addition to the human population that uses this ecosystem.

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The Mesoamerican slider Trachemysvenusta is endemic to Central America and Southern Mexico. Several human-mediated disturbances, including habitat degradation and illegal hunting for food, have impacted its populations along the Usumacinta river basin. The extent to which these disturbances have affected the genetic diversity and population structure of T. venusta inhabiting the basin remains unresolved. To this end, we analyzed eight microsatellite markers in five wild populations of T. venusta from the middle and lower reaches of the basin as well as one captive population. Our results show high levels of genetic diversity for all analyzed populations, low F ST values, high gene flow and no genetic structure, indicating an absence of genetic differentiation across sites and, thus, a single panmictic population for the basin. Evidence of a genetic bottleneck was observed in two of the wild populations (and the captive one), indicating some impact from disturbances, whether from poaching or habitat fragmentation, despite the seemingly high connectivity of most populations. Results are discussed in terms of the relative importance of genetic parameters for the conservation of T. venusta, particularly in light of the importance of demographic stochasticity in local conditions undergoing rapid changes.

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Community-based conservation and resource management (CBCRM) programs often incorporate the dual goals of poverty alleviation and conservation. However, robust assessments of CBCRM program outcomes are relatively scarce. This study uses a multidisciplinary, systems approach to assess the ecological and social dimensions of success of an internationally acclaimed CBCRM program. This program, located in one of the largest protected areas in the Peruvian Amazon (Pacaya-Samiria National Reserve), strives for the sustainable harvest and trade of a turtle species (Podocnemis unifilis). We used mixed methods analysis, including interviews and population viability modeling, to understand three elements: how local perceptions of changes in the managed population compare to changes inferred by ecological analyses, the indicators stakeholders use to measure success, and the barriers to long-term program success and social-ecological system sustainability. We find that stakeholders perceive a growth trend in the managed turtle population, but this perception may diverge from our ecological understanding of the system under current management. Population viability analyses with a 1:1 sex ratio suggested population size will decline under two of three management scenarios (different degrees of harvest). Yet this and similar studies are plagued by a lack of species- and site-specific population parameters that could improve understanding of the system. Significant vulnerabilities exist for system sustainability, notably the recent decrease in foreign demand for the traded resource. Identifying a sustainable species-specific harvest rate, developing locally-grounded ecological and social indicators, and focusing on data-driven adaptive management will facilitate the identification of key leverage points for future management interventions.

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The snail-eating turtle, Malayemys macrocephala, is a common freshwater turtle that can be used as an animal model for developmental biology. However, a thorough investigation of its development is needed before this species can be used as a model. Thus, this study aimed to examine the gonadal development of M. macrocephala. Turtle eggs were collected from rice fields in Phra Nakhon Si Ayutthaya Province, Thailand, and transported to the laboratory. Eggs were incubated in microprocessor-controlled incubators and randomly dissected on a weekly basis to reveal the developing embryos, then their developmental stage was identified according to Yntema (1968). Primordial germ cells and gonad structure were processed through the paraffin method. Moreover, the dynamics of germ cell proliferation and apoptosis were examined by immunohistochemical detection of proliferating cell nuclear antigen (PCNA) and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), respectively. Examination of the gonad revealed four main stages of gonadal development: (i) germ cell migration, (ii) genital ridge appearance, (iii) testicular formation, and (iv) ovarian formation. In the male turtle (incubated at 26°C), gonad developed into the testis with medullary sex cords starting at Yntema stage 17. In the female turtle (incubated at 32°C), these sex cords then degenerated, followed by cortical development into an ovarian structure starting at Yntema stage 19. Subsequently, testicular and ovarian development occurred independently, and distinct sex organs were apparent at Yntema stage 25. In addition, the presumptive testis showed germ cell proliferation in the medulla at Yntema stages 17, 19, and 25 and germ cell apoptosis in the cortex at Yntema stages 19 and 25. The presumptive ovary showed germ cell proliferation in the cortex at Yntema stages 19 and 25, and germ cell apoptosis in the medulla at Yntema stages 19 and 25.