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Freshwater turtles are often used as terrarium pets, especially juveniles of exotic species. At the adult stage they are often released by their owners into the wild despite their high invasion potential. In Europe these thermophilic potentially invasive alien species occupy the habitats of the native European pond turtle Emys orbicularis (Linnaeus, 1758), with new records from the wild being made specifically in Eastern Europe (Latvia and Ukraine) during recent decades. Assessing the potential of alien freshwater turtles to establish in new territories is of great concern for preventing invasion risks while preserving native biodiversity in the present context of climate change. We explored this issue by identifying the present and future (by 2050) suitable habitats of the European pond turtle and several potentially invasive alien species of freshwater turtle already settled in Europe, using a geographic information system (GIS) modelling approach based on datasets from CliMond for climate, Near-global environmental information (NGEI) for freshwater ecosystems (EarthEnv) and Maxent modelling using open-access databases, data from the literature and original field data. Modelling was performed for seven species of alien freshwater turtles occurring from the extreme northern to southern borders of the European range of E. orbicularis: the pond slider Trachemys scripta (Thunberg and Schoepff, 1792), the river cooter Pseudemys concinna (Le Conte, 1830), the Florida red-bellied cooter Pseudemys nelsoni (Carr, 1938), the false map turtle Graptemys pseudogeographica (Gray, 1831), the Chinese softshell turtle Pelodiscus sinensis (Wiegmann, 1835), the Caspian turtle Mauremys caspica (Gmelin, 1774) and the Balkan terrapin Mauremys rivulata (Valenciennes, 1833). In Ukraine, the most Eastern limit of E. orbicularis distribution, were previously reported northern American originated T. scripta, M. rivulata, M. caspica, whereas in Latvia, Emys' most northern limit, were additionally reported P. concinna, P. nelsoni, G. pseudogeographica and Asia originated P. sinensis. The resulting Species Distribution Models (SDM) were of excellent performance (AUC > 0.8). Of these alien species, the most potentially successful in terms of range expansion throughout Europe were T. scripta (34.3% of potential range expansion), G. pseudogeographica (24.1%), and M. caspica (8.9%) and M. rivulata (4.3%) mainly in Eastern Europe, especially in the south of Ukraine (Odesa, Kherson, Zaporizhzhia regions, and Crimean Peninsula). Correlation between the built SDMs for the native E. orbicularis and the invasive alien T. scripta was reliably high, confirming the highly likely competition between these two species in places they cooccur. Moreover, a Multiple Regression Analysis revealed that by 2050, in most of Europe (from the western countries to Ukraine), the territory overlap between E. orbicularis and potentially invasive alien species of freshwater turtles will increase by 1.2 times, confirming higher competition in the future. Importantly, by 2050, Eastern Europe and Ukraine are predicted to be the areas with most suitable habitats for the European pond turtle yet with most limited overlap with the invasive alien species. We conclude that Eastern Europe and Ukraine are the most relevant priority conservation areas for the European pond turtle where it is now necessary to take protective measures to ensure safe habitat for this native species on the long-term. [ABSTRACT FROM AUTHOR]

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To examine the regulatory mechanisms of oligopeptides derived from soft-shelled turtle protein on tubulin activity in tumor cells, this study employed three specific oligopeptides D-7-A, E-7-W, and G-8-V obtained from soft-shelled turtle protein. The objective was to assess their impact on tubulin polymerization and depolymerization, investigate their interaction with tubulin, and elucidate the mechanisms induce apoptosis in tumor cells. The results showed that the three oligopeptides exhibited similarities to vincristine and acted as inhibitors of tubulin polymerization, their respective IC50 values was 3.72, 5.01, and 5.95 µmol/L. Furthermore, the molecular docking analysis revealed that D-7-A interacted with the active center of α-tubulin 4X1I, specifically binding to Ser178 and Thr179. The active center of E-7-W was found to be bound by Gln15, Thr225, Tyr224, Tyr210 and Arg214. Similarly, G-8-V was observed to bind to Gln11, Ser178 and Asp329 at the active center, exerting a significant impact on α-tubulin. The results of cell experiments showed that D-7-A, E-7-W and G-8-V had dose-dependent inhibitory effects on A549 cells, with IC50 values of 2003±72, 1877±102 and 1789±137 μmol/L, respectively. The G-8-V exhibited the most potent inhibitory effect on A549 cells, leading to the induction of apoptosis. Moreover, the apoptosis rate demonstrated a significant increase with prolonged drug treatment time. The underlying mechanism of action involved the inhibition of tubulin polymerization and the induction of apoptosis by perturbing the valence bond of α-tubulin. [ABSTRACT FROM AUTHOR]

Light is one of the important factors in Chinese soft-shelled turtle culture. The study investigated the effects of light intensity on growth performance, stress, immune-related parameters, and antioxidant enzyme activities of Chinese soft-shelled turtles under the light environment of lighting the basking area in intensive farming. Four degrees of light intensities with three replicates were used, including LL (low light intensity, 6.56 ± 0.14 W/m2), ML (medium light intensity, 18.11 ± 0.54 W/m2), HL (high light intensity, 37.81 ± 2.85 W/m2), and control group (0 W/m2). The experimental photoperiod was fixed as 12 L: 12 D, and the water temperature was controlled at 30 ± 2 °C. After 152-day rearing, it was found that the growth performance of Chinese soft-shelled turtles in the LL group was significantly higher than that in the HL group. The serum cortisol level of turtles in the LL group was significantly lower than that in the HL group, indicating lower stress in the LL group compared to other groups. The content of lysozyme (LZM) was highest in the LL group, the content of acid phosphatase (ACP) in the LL group was significantly higher than that in the control group and the HL group, and the content of nitric oxide (NO) in the LL group was significantly higher than that in the HL group, implying that turtles in the LL group had the best immune performance. The superoxide dismutase (SOD) activity and glutathione peroxidase (GSH-PX) activity in the liver of turtles in the LL group were significantly lower than that in the control and HL groups. Further, the catalase (CAT) activity of turtles in the LL group was the lowest among the treatments. Malonaldehyde (MDA) content was significantly lower in the LL group than in the control and HL groups. Therefore, considering the growth performance and physiological status of Chinese soft-shelled turtles, it is recommended to provide a low intensity of light when lighting the basking area only, and the optimum light intensity for the growth of Chinese soft-shelled turtles is between 7.07 and 11.50 W/m2. [ABSTRACT FROM AUTHOR]

Sexually dimorphic hearing sensitivity has evolved in many vertebrate species, and the sex with a larger body size typically shows more sensitive hearing. However, generalizing this association is controversial. Research on sexually dimorphic hearing sensitivity contributes to an understanding of auditory sense functions, adaptations, and evolution among species. Therefore, the hypothesized association between body size and hearing needs further validation, especially in specific animal groups. In this study, we assessed hearing sensitivity by measuring auditory brainstem responses (ABRs) in both sexes of 3‐year‐old Chinese softshell turtles (Pelodiscus sinensis). In this species, male bodies are larger than those of female, and individuals spend most of their lives in the mud at the bottom of freshwater habitats. We found that for both sexes, the hearing sensitivity bandwidth was 0.2–0.9 kHz. Although males were significantly larger than females, no significant differences in ABR thresholds or latencies were found between males and females at the same stimulus frequency. These results indicate that P. sinensis hearing is only sensitive to low‐frequency (typically <0.9 kHz) sound signals and that sexually dimorphic hearing sensitivity is not a trait that has evolved in P. sinensis. Physiological and environmental reasons may account for P. sinensis acoustic communication via low‐frequency sound signals and the lack of sexually dimorphic hearing sensitivity in these benthic turtles. The results of this study refine our understanding of the adaptation and evolution of the vertebrate auditory system. [ABSTRACT FROM AUTHOR]

The present study aimed to investigate the transcriptomic response to virus simulant (polyinosinic-polycytidylic acid, poly I:C), and to determine if the aqueous leech (Whitmania pigra) extract has the function of interfering with the transcriptomic response in primary culture cells from the embryo of Chinese soft-shelled turtle (Pelodiscus sinensis). The results showed that the transfection with poly I:C and treatment with leech extract reduced cell viability. A total of 468 differentially expressed genes (DEGs) was induced by poly I:C, including 272 upregulated genes and 196 downregulated genes. A total of 965 DEGs were induced by leech extract, including 209 upregulated genes and 756 downregulated genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that 43 GO terms and 12 pathways were significantly enriched by poly I:C, including immune response and immune system processes, nucleotide-binding and oligomerization domain (NOD)–like receptor signaling pathway, RIG-I-like receptor signaling pathway, and Toll-like receptor signaling pathway. Leech extract significantly enriched 50 GO terms and 56 pathways, including translation, peptide biosynthetic process, ribosome, and oxidative phosphorylation pathway. In addition, qRT-PCR of seven DEGs from poly I:C transfection and four DEGs from leech extract treatment verified the reliability of transcriptome analysis. Immunofluorescence analysis indicated poly I:C transfection enhanced the generation of DDX58 protein in cytoplasm and the nuclear translocation of NF-κB(p65), while the leech extract inhibited above action to DDX58 and NF-κB(p65). In conclusion, this study improved the understanding of immune system response to the virus in primary culture cells of Chinese soft-shelled turtle, and suggested that aqueous leech extract from W. pigra may inhibit immune response genes induced by poly I:C. [ABSTRACT FROM AUTHOR]

Simple Summary: Carbohydrate is an important energy nutrient in the feed of aquatic animals. Generally, aquatic animals usually exhibit a varying efficiency in utilizing different carbohydrate sources. In order to understand the carbohydrate utilization efficiency, this study investigated the physiological responses of soft-shelled turtles (Pelodiscus sinensis) that were fed four types of carbohydrates with different complexities and configurations. The results indicated that the best growth performance and feed efficiency were found in the starch diet, followed by the glucose, fructose, and cellulose groups in sequence. Dietary starch demonstrated a robust lipogenic function by inducing the expression of the genes involved in glucolipid metabolism, with the results of elevated plasma triglyceride levels and an increased lipid content in both the whole body and the liver. Glucose and fructose diets caused postprandial hyperglycemia in P. sinensis due to the un-inhibited gluconeogenesis. P. sinensis that were fed a fructose diet did not exhibit a higher lipid deposition compared to the glucose diet, as seen from mammals. Cellulose was not a suitable energy source for P. sinensis. A 60 day feeding trial was conducted to evaluate the impacts of dietary carbohydrates with different complexities and configurations on the growth, plasma parameters, apparent digestibility, intestinal microbiota, glucose, and lipid metabolism of soft-shelled turtles (Pelodiscus sinensis). Four experimental diets were formulated by adding 170 g/kg glucose, fructose, α-starch, or cellulose, respectively. A total of 280 turtles (initial body weight 5.11 ± 0.21 g) were distributed into 28 tanks and were fed twice daily. The results showed that the best growth performance and apparent digestibility was observed in the α-starch group, followed by the glucose, fructose, and cellulose groups (p < 0.05). Monosaccharides (glucose and fructose) significantly enhanced the postprandial plasma glucose levels and hepatosomatic index compared to polysaccharides, due to the un-inhibited gluconeogenesis (p < 0.05). Starch significantly up-regulated the expression of the genes involved in glycolysis, pentose phosphate pathway, lipid anabolism and catabolism, and the transcriptional regulation factors of glycolipid metabolism (srebp and chrebp) (p < 0.05), resulting in higher plasma triglyceride levels and lipid contents in the liver and the whole body. The fructose group exhibited a lower lipid deposition compared with the glucose group, mainly by inhibiting the expression of srebp and chrebp. Cellulose enhanced the proportion of opportunistic pathogenic bacteria. In conclusion, P. sinensis utilized α-starch better than glucose, fructose, and cellulose. [ABSTRACT FROM AUTHOR]

The Euphrates soft-shell turtle Rafetus euphraticus is classified as endangered on the IUCN Red List and is thought to have recently undergone large population declines. Species information in Iraq is limited to a few rapid surveys with little detailed information on its breeding and distribution. Turtles have been recorded inside the water by setting nets in certain areas in the Central Marshes. Morphometric measurements were recorded in Euphrates soft-shelled turtles Rafetus euphraticus, sampled in the Central Marshes. According to nine morphometric ratios, the populations of R. euphraticus in the Central Marshes are smaller in size than previous specimens that have been recorded in neighboring countries. The carapaces length (CL) rang for current study was 70 to 560 mm, whereas it was 187 to 680 mm according to a previous study. The carapaces width (CW) rang for the current study was 63 to 420 mm, while in a previous study it was recorded to be 152 to 465 mm. [ABSTRACT FROM AUTHOR]

Organisms whose early life stages are environmentally sensitive produce offspring within a relatively narrow range of suitable abiotic conditions. In reptiles, development rate and survival are often maximized if incubation temperatures remain under 31°C, though this upper bound may vary within and among species. We addressed this expectation by comparing responses to egg incubation at 30°C versus 33°C in congeneric turtle species pairs with broad syntopic geographic distributions. In the two softshell turtles (Apalone spp.), the greatest changes in development rate and phenotypic variance were observed in the northernmost population, which had a low survival rate (40%) at 33°C. The presumably suboptimal temperature (33°C) for northern populations otherwise yielded 76%–93% survival rates and fast swimming speeds in more southern populations. Still, in one species, northern hatchlings incubated at 33°C matched the elevated speeds of their southern counterparts, revealing a countergradient response. In northern populations of the two map turtles (Graptemys spp.), survival was also reduced (28%–60%) at 33°C and the development rate (relative to 30°C) increased by up to 75%. Our experiments on divergent taxa with similar nesting ecologies substantiate that the optimal thermal range for offspring production is variable. These findings encourage further work on how population‐ and species‐level differences relate to local adaptation in widely distributed oviparous species. Research highlights: Geographic variation was evaluated in turtles.High egg incubation temperatures reduced offspring survival in the north, with a countergradient response in some populations.The optimal thermal regimes likely differ according to trait and population. [ABSTRACT FROM AUTHOR]

Hutchemys rememdium is a poorly understood softshell turtle (Trionychidae) from the mid Paleocene of the Williston Basin of North America previously known only from postcranial remains. A particularly rich collection of previously undescribed material from the Tiffanian 4 North American Land Mammal Age (NALMA) of North Dakota is here presented consisting of numerous shells that document new variation, some non-shell postcrania, and cranial remains, which are described based on 3D models extracted from micro-CT data. Although the observed shell variation weakens previously noted differences with the younger species Hutchemys arctochelys from the Clarkforkian NALMA, the two taxa are still recognized as distinct. Parsimony and Bayesian phylogenetic analyses reaffirm the previously challenged placement of Hutchemys rememdium within the clade Plastomenidae, mostly based on novel observations of cranial characters made possible by the new material and the micro-CT data. The new topology supports the notion that the well-ossified plastron of plastomenids originated twice in parallel near the Cretaceous/Paleogene boundary, once in the Hutchemys lineage and once in the Gilmoremys/Plastomenus lineage. Hutchemys rememdium is notable for being the only documented species of trionychid in the mid Paleocene of the Williston Basin. The presence of multiple individuals in a carbonaceous claystone indicates this taxon lived in swamps and lakes and its expanded triturating surface suggests it had a durophagous diet. [ABSTRACT FROM AUTHOR]

Introduction: Aeromonas hydrophila, a bacterium widely distributed in the natural environment, causes multiple diseases in various animals. Exploring the mechanism of the host defense against A. hydrophila can help develop efficient strategies against Aeromonas infection. Methods: Herein, we investigated the temporal influence of A. hydrophila on the Chinese soft-shelled turtle, an economically important species, at the biochemical, transcriptomic, and metabolomic levels. Plasma parameters were detected with the test kits. Transcriptome and metabolome were respectively applied to screen the differentially expressed genes and metabolites. Results: The contents or activities of these plasma parameters were significantly increased at 24 hpi and declined at 96 hpi, indicating that 24 and 96 hpi were two important time points during infection. Totals of 3121 and 274 differentially expressed genes (DEGs) from the transcriptome while 74 and 91 differentially abundant metabolites (DAMs) from the metabolome were detected at 24 and 96 hpi. The top DEGs at 24 hpi included Ccl2, Ccl3, Ccl4, Il1β, Il6, Il7, Il15, Tnf, and Tnfr1 while Zap70, Cd3g, Cd8a, Itk, Pik3r3, Cd247, Malt1, and Cd4 were the most abundant at 96 hpi. The predominant DAMs included O-phospho-L-serine, γ-Aminobutyric acid, orotate, L-tyrosine, and L-tryptophan at 24 hpi, as well as Lglutamic acid, L-arginine, glutathione, glutathione disulfide, and citric acid at 96 hpi. Discussion: The combined analysis of DEGs and DAMs revealed that tryptophan metabolism, nicotinate and nicotinamide metabolism, as well as starch and sucrose metabolism, were the most important signaling pathways at the early infective stage while tyrosine metabolism, pyrimidine metabolism, as well as alanine, aspartate and glutamate metabolism were the most crucial pathways at the later stage. In general, our results indicated that the Chinese softshelled turtle displays stage-specific physiological responses to resist A. hydrophila infection. [ABSTRACT FROM AUTHOR]

Frog virus 3 (FV3) in the genus Ranavirus of the family Iridoviridae causes mass mortality in both anurans and urodeles worldwide; however, the phylogenetic origin of FV3-like ranaviruses is not well established. In Asia, three FV3-like ranaviruses have been reported in farmed populations of amphibians and reptiles. Here, we report the first case of endemic FV3-like ranavirus infections in the Korean clawed salamander Onychodactylus koreanus, caught in wild mountain streams in the Republic of Korea (ROK), through whole-genome sequencing and phylogenetic analysis. Two isolated FV3-like ranaviruses (Onychodactylus koreanus ranavirus, OKRV1 and 2) showed high similarity with the Rana grylio virus (RGV, 91.5%) and Rana nigromaculata ranavirus (RNRV, 92.2%) but relatively low similarity with the soft-shelled turtle iridovirus (STIV, 84.2%) in open reading frame (ORF) comparisons. OKRV1 and 2 formed a monophyletic clade with previously known Asian FV3-like ranaviruses, a sister group of the New World FV3-like ranavirus clade. Our results suggest that OKRV1 and 2 are FV3-like ranaviruses endemic to the ROK, and RGV and RNRV might also be endemic strains in China, unlike previous speculation. Our data have great implications for the study of the phylogeny and spreading routes of FV3-like ranaviruses and suggest the need for additional detection and analysis of FV3-like ranaviruses in wild populations in Asian countries. [ABSTRACT FROM AUTHOR]

Simple Summary: In the sorting process of Chinese softshell turtles, it is necessary to classify them based on their weight and accurately identify their plastron and carapace. This process requires heavy manual labor and complex mechanical processing methods. To improve processing efficiency and reduce costs, this article introduces machine vision technology, and a new image processing method is proposed. This method can estimate the weight of Chinese softshell turtles and accurately locate the positions of their plastron and carapace. The automation level of aquaculture can be greatly enhanced, and hardware costs can be reduced through software optimization through this approach. With the rapid development of the turtle breeding industry in China, the demand for automated turtle sorting is increasing. The automatic sorting of Chinese softshell turtles mainly consists of three parts: visual recognition, weight prediction, and individual sorting. This paper focuses on two aspects, i.e., visual recognition and weight prediction, and a novel method for the object detection and weight prediction of Chinese softshell turtles is proposed. In the individual sorting process, computer vision technology is used to estimate the weight of Chinese softshell turtles and classify them by weight. For the visual recognition of the body parts of Chinese softshell turtles, a color space model is proposed in this paper to separate the turtles from the background effectively. By applying multiple linear regression analysis for modeling, the relationship between the weight and morphological parameters of Chinese softshell turtles is obtained, which can be used to estimate the weight of turtles well. An improved deep learning object detection network is used to extract the features of the plastron and carapace of the Chinese softshell turtles, achieving excellent detection results. The mAP of the improved network reached 96.23%, which can meet the requirements for the accurate identification of the body parts of Chinese softshell turtles. [ABSTRACT FROM AUTHOR]

The Critically Endangered Cantor's giant softshell turtle Pelochelys cantorii is a freshwater turtle found in South and Southeast Asia. Its population is declining because of habitat destruction and alteration, and hunting for its meat. Data on the species' ecology, behaviour, population size, distribution and threats are limited, and previous surveys undertaken in India have failed to detect individuals in the wild. This lack of data hinders strategic conservation planning. Ecologists and conservationists increasingly utilize local ecological knowledge to determine the status and gain insights into the ecology of threatened and rare species that are difficult to detect in field studies. To examine the historical and current occurrence of Cantor's giant softshell turtle, we conducted community interviews along the Chandragiri River in Kerala, India. With data from these interviews, we identified multiple sites where the species continues to occur. Older respondents and those that used the river for fishing and irrigation were more likely to report sightings of the species. Our findings also improved knowledge about the turtles' seasonal and diel activity patterns. A network of key informants identified through the interviews provided information on turtle bycatch, sightings and nesting. This network is being utilized for the continued monitoring of the species and could help in devising evidence-based management strategies for softshell turtles in India. These methods can also be adopted more widely for other threatened species of freshwater turtles globally. [ABSTRACT FROM AUTHOR]

The 'bigger is better' hypothesis (BIBH) predicts that fitness increases with body size. Eastern spiny softshell turtles (Apalone spinifera) exhibit sexual size dimorphism (SSD) at maturity, and the much larger female body size is attributed to selection favouring greater reproductive output. Hatchlings exhibit sexually dimorphic markings, but hatchling SSD has not been investigated. The BIBH predicts that hatchlings will not exhibit SSD because reproductive benefits to larger, mature females drive size dimorphism at maturity, but selection on hatchlings relates only to survival. We tested this prediction with A. spinifera hatchlings (N  = 2630) incubated in controlled conditions to avoid confounding environmental effects on phenotype. Hatchling mass was similar between sexes, suggesting similar nutritional allocation to male and female offspring. Hatchling size (carapace length) was also similar between sexes. Hatchling mass and body size varied among clutches and were positively related to maternal size, suggesting morphological constraints on egg size. Surprisingly, clutch size was related only weakly to maternal size. Taken together, our results suggest that hatchling size in A. spinifera is consistent with the BIBH and that offspring quality increases with maternal age, but that maternal effects drive a large proportion of the variation in clutch size and offspring quality. [ABSTRACT FROM AUTHOR]

The collection of blood from large softshell turtles, such as the threatened black softshell turtle (Nilssonia nigricans), Indian softshell turtle (Nilssonia gangetica), and the peacock softshell turtle (Nilssonia hurum), has been a challenge for researchers because of their aggressive nature and thick skin folds. In this pilot study, a technique was developed for blood collection from these turtles, which involves physical restraint and venipuncture of the brachial venous plexus. The method proved to be effective, efficient, and requires no anesthesia, making it suitable for field studies or on-site collection. Blood samples were successfully collected from 30 turtles captured, and basic hematology and biochemical examinations were performed. Based on our study, this method can be used for both large and small-sized animals and provides a reliable technique for routine health assessments in these species. [ABSTRACT FROM AUTHOR]

The pig-nosed turtle (Carettochelys insculpta) represents the only extant species within the Carettochelyidae family, is a unique Trionychia member fully adapted to aquatic life and currently facing endangerment. To enhance our understanding of this species and contribute to its conservation efforts, we employed high-fidelity (HiFi) and Hi-C sequencing technology to generate its genome assembly at the chromosome level. The assembly result spans 2.18 Gb, with a contig N50 of 126 Mb, encompassing 34 chromosomes that account for 99.6% of the genome. The assembly has a BUSCO score above 95% with different databases and strong collinearity with Yangtze giant softshell turtles (Rafetus swinhoei), indicating its completeness and continuity. A total of 19,175 genes and 46.86% repetitive sequences were annotated. The availability of this chromosome-scale genome represents a valuable resource for the pig-nosed turtle, providing insights into its aquatic adaptation and serving as a foundation for future turtle research. [ABSTRACT FROM AUTHOR]

Simple Summary: Different culturing modes have adopted different living spaces, foods, shelter, substrates, and surroundings for and human actions regarding animals. The Chinese soft-shelled turtle (Pelodiscus sinensis) is one of most economically important turtles, and healthier culturing modes should be applied for high-quality aquatic products to remit serious environmental and health problems. The differences in growth performance, physiological indices, and related gene expression were compared between two culturing modes in the present study. The results indicated that rice–turtle-co-cultured turtles with limited natural foods grew slower than pond-cultured turtles fed with commercial feeds. The creatinine, uric acid, blood urea nitrogen, alkaline phosphatase, acid phosphatase, glutamic oxaloacetic transaminase, and catalase were higher in co-cultured turtles than pond-cultured turtles. More differentially expressed genes were related to environmental information processing, metabolism, and human diseases when comparing pond-cultured turtles and co-cultured turtles. P. sinensis can adapt to different culture modes by adjusting their growth and physiology over a short culture period, and rice–turtle co-culturing proves to be a healthier culturing mode for turtles within the proper culturing strategies. The Chinese soft-shelled turtle (Pelodiscus sinensis) is an important freshwater aquaculture turtle due to its taste and nutritional and medicinal value. More ecological culturing modes, such as rice–turtle co-culture, should be developed to meet the ecological benefit demand. We compared growth, physiological parameters, and transcriptome data to detect the physiological responses and regulatory mechanisms of pond-cultured turtles as compared to co-cultured turtles. The co-cultured turtles grew slower than pond-cultured turtles. The gonadosomatic index of co-cultured male turtles was lower than that of pond-cultured male turtles, and both the mesenteric fat index and limb fat index were lower in co-cultured turtles than in pond-cultured turtles (p < 0.05). The blood GLU of the co-cultured turtles was significantly lower than the GLU of the pond-cultured turtles (p < 0.05), while the values of CRE, UA, BUN, AKP, ACP, GOT, and CAT were higher in the co-cultured turtles than in the pond-cultured turtles (p < 0.05). In total, 246 and 598 differentially expressed genes (DEGs) were identified in the brain and gut from turtles cultured in the two different modes, respectively. More DEGs were related to environmental information processing, metabolism, and human diseases. In the brain, the top enriched pathways of DEGs included the longevity regulating pathway, glycerolipid metabolism, cytokine–cytokine receptor interaction, Toll-like receptor signaling pathway, and PI3K-Akt signaling pathway, while in the gut, the top enriched pathways of DEGs included the cell cycle, DNA replication, cellular senescence, and p53 signaling pathway. The turtles acclimated to the different culturing conditions by adjusting their growth, physiological, and biochemical characteristics and related gene expression during a short culture period. [ABSTRACT FROM AUTHOR]

Physiology disorders of the liver, as it is an important tissue in lipid metabolism, can cause fatty liver disease. The mechanism might be regulated by 17 circadian clock genes and 18 fat metabolism genes, together with a high-fat diet (HFD). Due to their rich nutritional and medicinal value, Chinese soft-shelled turtles (Trionyx sinensis) are very popular among the Chinese people. In the study, we aimed to investigate the influence of an HFD on the daily expression of both the core clock genes and the lipid metabolism genes in the liver tissue of the turtles. The two diets were formulated with 7.98% lipid (the CON group) and 13.86% lipid (the HFD group) to feed 180 juvenile turtles, which were randomly divided into two groups with three replicates per group and 30 turtles in each replicate for six weeks, and the diet experiment was administrated with a photophase regimen of a 24 h light/dark (12L:12D) cycle. At the end of the experiment, the liver tissue samples were collected from nine turtles per group every 3 h (zeitgeber time: ZT 0, 3, 6, 9, 12, 15, 18, 21 and 24) for 24 h to investigate the daily expression and correlation analysis of these genes. The results showed that 11 core clock genes [i.e., circadian locomotor output cycles kaput (Clock), brain and muscle arnt-like protein 1 and 2 (Bmal1/2), timeless (Tim), cryptochrome 1 (Cry2), period2 (Per2), nuclear factor IL-3 gene (Nfil3), nuclear receptor subfamily 1, treatment D, member 1 and 2 (Nr1d1/2) and retinoic acid related orphan receptor α/β/γ β and γ (Rorβ/γ)] exhibited circadian oscillation, but 6 genes did not, including neuronal PAS domain protein 2 (Npas2), Per1, Cry1, basic helix-loop-helix family, member E40 (Bhlhe40), Rorα and D-binding protein (Dbp), and 16 lipid metabolism genes including fatty acid synthase (Fas), diacylglycerol acyltransferase 1 (Dgat1), 3-hydroxy-3-methylglutaryl-CoA reductase (Hmgcr), Low-density lipoprotein receptor-related protein 1-like (Ldlr1), Lipin 1 (Lipin1), Carnitine palmitoyltransferase 1A (Cpt1a), Peroxisome proliferator activation receptor α, β and γ (Pparα/β/γ), Sirtuin 1 (Sirt1), Apoa (Apoa1), Apolipoprotein B (Apob), Pyruvate Dehydrogenase kinase 4 (Pdk4), Acyl-CoA synthase long-chain1 (Acsl1), Liver X receptors α (Lxrα) and Retinoid X receptor, α (Rxra) also demonstrated circadian oscillations, but 2 genes did not, Scd and Acaca, in the liver tissues of the CON group. However, in the HFD group, the circadian rhythms' expressional patterns were disrupted for the eight core clock genes, Clock, Cry2, Per2, Nfil3, Nr1d1/2 and Rorβ/γ, and the peak expression of Bmal1/2 and Tim showed delayed or advanced phases. Furthermore, four genes (Cry1, Per1, Dbp and Rorα) displayed no diurnal rhythm in the CON group; instead, significant circadian rhythms appeared in the HFD group. Meanwhile, the HFD disrupted the circadian rhythm expressions of seven fat metabolism genes (Fas, Cpt1a, Sirt1, Apoa1, Apob, Pdk4 and Acsl1). Meanwhile, the other nine genes in the HFD group also showed advanced or delayed expression peaks compared to the CON group. Most importantly of all, there were remarkably positive or negative correlations between the core clock genes and the lipid metabolism genes, and their correlation relationships were altered by the HFD. To sum up, circadian rhythm alterations of the core clock genes and the lipid metabolism genes were induced by the high-fat diet (HFD) in the liver tissues of T. sinensis. This result provides experimental and theoretical data for the mass breeding and production of T. sinensis in our country. [ABSTRACT FROM AUTHOR]

Trionychid turtles are rare in the fossil record of the Eastern Mediterranean and therefore, knowledge about their presence is restricted both geographically and stratigraphically. In this work, the first record of a fossil trionychid from the Plio-Pleistocene of Kos Island, Greece, is presented, extending the known distribution of trionychids in Greece both geographically and chronologically. Together with a record from the Early Pleistocene of Italy, the new find from Kos represents the youngest occurrence of soft-shelled turtles from Europe. The occasional presence of the extant Trionyx triunguis in the area of south-eastern Aegean Islands is also discussed. [ABSTRACT FROM AUTHOR]

Male and female Chinese soft-shelled turtles (Pelodiscus sinensis) have sex-dimorphic growth patterns, and males have higher commercial value because of their larger size and thicker calipash. Thus, developing sex-specific markers is beneficial to studies on all-male breeding in P. sinensis. Here, we developed an accurate and efficient workflow for the screening of sex-specific sequences with ZW or XY sex determination systems. Based on this workflow, female and male P. sinensis reference genomes of 2.23 Gb and 2.26 Gb were obtained using de novo assembly. After aligning and filtering, 4.01 Mb female-specific sequences were finally identified. Subsequently, the seven developed sex-specific primer pairs were 100% accurate in preliminary, population, and embryonic validation. The presence and absence of bands for the primers of P44, P45, P66, P67, P68, and P69, as well as two and one bands for the PB1 primer, indicate that the embryos are genetically female and male, respectively. NR and functional annotations identified several sex-determining candidate genes and related pathways, including Ran, Eif4et, and Crkl genes, and the insulin signaling pathway and the cAMP signaling pathway, respectively. Collectively, our results reveal that a ZW-type sex-determination system is present in P. sinensis and provide novel insights for the screening of sex-specific markers, sex-control breeding, and the studies of the sex determination mechanism of P. sinensis. [ABSTRACT FROM AUTHOR]

Aquaculture has suffered significant financial losses as a result of the infection of zoonotic Aeromonas hydrophila, which has a high level of resistance to classic antibiotics. In this study, we isolated an A. hydrophila strain B3 from diseased soft-shelled turtle (Pelodiscus sinensis), which is one of the most commercially significant freshwater farmed reptiles in East Asia, and found that A. hydrophila was its dominant pathogen. To better understand the inhibition effect and action mechanism of Chinese herbs on A. hydrophila, we conducted Chinese herbs screening and found that Lonicera japonica had a significant antibacterial effect on A. hydrophila B3. Experimental therapeutics of L. japonica on soft-shelled turtle showed that the supplement of 1% L. japonica to diet could significantly upregulate the immunity-related gene expression of soft-shelled turtle and protect soft-shelled turtle against A. hydrophila infection. Histopathological section results validated the protective effect of L. japonica. As the major effective component of L. japonica, chlorogenic acid demonstrated significant inhibitory effect on the growth of A. hydrophila with MIC at 6.4 mg/mL. The in vitro assay suggested that chlorogenic acid could inhibit the hemolysin/protease production and biofilm formation of A. hydrophila and significantly decrease the expression of quorum sensing, biofilm formation, and hemolysin-related genes in A. hydrophila. Our results showed that the Chinese herb L. japonica would be a promising candidate for the treatment of A. hydrophila infections in aquaculture, and it not only improves the immune response of aquatic animals but also inhibits the virulence factor (such as biofilm formation) expression of A. hydrophila. Key points: • A. hydrophila was the dominant pathogen of the diseased soft-shelled turtle. • L. japonica can protect soft-shelled turtle against A. hydrophila infection. • Chlorogenic acid inhibits the growth and biofilm formation of A. hydrophila. [ABSTRACT FROM AUTHOR]

The Spotted Softshell Turtle (Pelodiscus variegatus) has been recognised since 2019 from Vietnam and Hainan Island, China, but little information about its population status and distribution range is currently available. The species has been provisionally listed as Critically Endangered by the Turtle and Tortoise Working Group, although the status has not been officially accepted by the IUCN, due to the threats the species is facing, including habitat loss and degradation, overexploitation for food, competition with other non-native softshell turtles and pollution. To identify conservation priority sites for P. variegatus in mainland Indochina, this study combines molecular analyses and species distribution modelling. Our results show that, in Vietnam, Phong Nha-Ke Bang National Park has the largest suitable area and high probability of species occurrence, followed by Vu Quang National Park and Song Thanh and Ke Go Nature Reserves. In addition, the central provinces, from Thanh Hoa to Thua Thien Hue in Vietnam, constitute a key part of the species distribution and should be prioritised for conservation actions. According to the study's findings, although P. variegatus is possibly found in Laos, the probability decreases sharply at the border between both countries and there is also a gap in the occurrence of wetlands, arguing for strong natural barriers. Unfortunately, to date, only part of the species potential distribution is protected, while no records are known from protected areas, highlighting the need for extended or even new reserves. To recover natural populations of the species and following the IUCN's One Plan Approach to Conservation, breeding programmes have been established in Vietnam with a potential to expand to other facilities in the country and abroad. Once suitable sites are identified, offspring can be released into the protected areas to improve the current conservation status of this highly-threatened softshell turtle. [ABSTRACT FROM AUTHOR]

Simple Summary: The Chinese soft-shelled turtle shows obvious sexual dimorphism. The male P. sinensis is larger and, thus, more popular in the market. We obtained full-length (FL) transcriptome data and compared the difference between male and female P. sinensis. Compared to female individuals, there were 5050 upregulated genes and 3590 downregulated genes in male individuals. Female-biased genes such as Smad4, Wif1, Cypj2, and 17β-hsd and male-biased genes such as Nkd2 and Prp18 were identified by omics and quantitative analysis. The results may facilitate future comparative studies on the transcriptome and gene function related to sex determination in P. sinensis. The Chinese soft-shelled turtle (Pelodiscus sinensis), an economically important aquatic species in China, displays considerable sexual dimorphism: the male P. sinensis is larger and, thus, more popular in the market. In this study, we obtained the full-length (FL) transcriptome data of P. sinensis by using Pacific Biosciences (PacBio)'s isoform sequencing and analyzed the transcriptome structure. In total, 1,536,849 high-quality FL transcripts were obtained through single-molecule real-time (SMRT) sequencing, which were then corrected using Illumina sequencing data. Next, 89,666 nonredundant FL transcripts were generated after mapping to the reference genome of P. sinensis; 291 fusion genes and 17,366 novel isoforms were successfully annotated using data from the nonredundant protein sequence database (NR), eukaryotic orthology groups (KOG), the Gene Ontology (GO) project, and the KEGG Orthology (KO) database. Additionally, 19,324 alternative polyadenylation sites, 101,625 alternative splicing events, 12,392 long noncoding RNAs, and 5916 transcription factors were identified. Smad4, Wif1, and 17-β-hsd were identified as female-biased genes, while Nkd2 and Prp18 held a higher expression level in males than females. In summary, we found differences between male and female P. sinensis individuals in AS, lncRNA, genes, and transcripts, which relate to the Wnt pathway, oocyte meiosis, and the TGF-β pathway. Female-biased genes such as Smad4, Wif1, and 17-β-hsd and male-biased genes such as Nkd2 and Prp18 played important roles in the sex determination of P. sinensis. FL transcripts are a precious resource for characterizing the transcriptome of P. sinensis, laying the foundation for further research on the sex-determination mechanisms of P. sinensis. [ABSTRACT FROM AUTHOR]

Simple Summary: Trionyx sinensis (Chinese soft-shelled turtle) is one of the main economic animals in the aquaculture industry due to its nutritional value and medicinal value. However, the farming industry is facing various challenges, including water quality, stocking density, and viral disease outbreaks. Trionyx sinensis hemorrhagic syndrome virus has high lethality to Trionyx sinensis, but the detailed pathogenic characteristics of the virus are unclear, and there is no available treatment for the virus. After experiments, we revealed the pathogenic characteristics of the virus at the cellular and tissue levels and found a material for the culture of the virus. Curcumin is a natural herbal extract that has antiviral, antifungal, and antibacterial properties and acts as an immunomodulator. In vivo tests have shown that curcumin is effective in preventing viral infection and improving the immune response. These results demonstrate that curcumin is a promising strategy for preventing viral disease. Trionyx sinensis hemorrhagic syndrome virus (TSHSV) is an aquatic arterivirus causing a high mortality rate for T. sinensis (Chinese soft-shelled turtle), but the detailed infection properties of TSHSV are unclear, and no effective treatment is available. In this study, cell culture and histopathology were performed to elucidate the infection properties of TSHSV. Furthermore, the anti-TSHSV and immune-enhancing effects of curcumin were evaluated using survival statistics, qPCR, and tissue immunofluorescence. The results demonstrated that TSHSV could proliferate in the spleen cell line of T. sinensis, leading to cytopathic effects. TSHSV damaged the livers, kidneys, and lungs, characterized by cell disintegration and hyperemia. Curcumin at 250 mg/kg improved the survival of T. sinensis, and significantly reduced the viral load in the spleens, kidneys, and lungs. Moreover, curcumin inhibited the mRNA expression of immune-related genes, RSAD2, IFN-γ, and TNF-α (p < 0.05). In conclusion, these results imply that TSHSV is pathogenic to the spleen cell line, liver, spleen, kidney, and lung of T. sinensis. Curcumin effectively inhibits TSHSV and modulates the immune function of T. sinensis, so it holds promise as a means to prevent TSHSV. [ABSTRACT FROM AUTHOR]

The Asian giant softshell turtle Pelochelys cantorii is one of the largest aquatic turtles in China and has been designated a First Grade Protected Animal in China. To advance conservation research, a combination of Illumina short-read, PacBio long-read, and Hi-C scaffolding technologies was used to develop a high-quality chromosome-level genome assembly for P. cantorii. A total of 262.77 Gb of clean data were produced (121.6 × depth) and then the genome was assembled into 2.16 Gb with a contig N50 of 41.44 Mb and scaffold N50 length of 120.17 Mb, respectively. Moreover, about 99.98% assembly genome sequences were clustered and ordered onto 33 pseudochromosomes. Genome annotation revealed that 21,833 protein-coding genes were predicted, and 96.40% of them were annotated. This new chromosome-level assembly will be an enabling resource for genetic and genomic studies to support fundamental insight into P. cantorii biology. [ABSTRACT FROM AUTHOR]

Exotic species introductions are a major threat to biodiversity. The first step to their control involves identifying their presence, distribution, and impacts on native species and their habitats, especially in highly diverse regions. We update and document new records of exotic freshwater fauna from the Baja California Mediterranean region. We found 157 locality records of 17 exotic aquatic species; 16 genera and 15 families were recorded. Five species are new records for the region: Malaysian trumpet snail (Melanoides tuberculata), black bullhead catfish (Ameiurus melas), common carp (Cyprinus carpio), spiny softshell turtle (Apalone spinifera), and red-eared slider (Trachemys scripta). The catfish, carp, and softshell turtle have been previously reported from Baja California, but only from the Colorado River and adjacent Mexicali Valley, while the snail and slider represent new state records. Of the 157 locality records, 71 were found in published literature and 86 represent new records from field observations, collected specimens and citizen science observations. Based on discussions with local ranchers and landowners, some exotic species were introduced for food or aesthetic purposes, emphasizing the need for environmental programs to stop these actions. [ABSTRACT FROM AUTHOR]

The Chinese soft-shelled turtle (Pelodiscus sinensis) has become increasingly susceptible to frequent diseases with the intensification of farming, which severely impacts the development of the aquaculture industry. Sodium butyrate (SB) is widely used as a feed additive due to its promotion of growth, enhancement of immune function, and antioxidative properties. This study aimed to investigate the effects of dietary SB on the growth performance, immune function, and intestinal microflora of Chinese soft-shelled turtles. A total of 300 Chinese soft-shelled turtles (mean weight: 11.36 ± 0.21g) were randomly divided into four groups with three parallel sets in each group. Each group was fed a diet supplemented with 0%, 0.005%, 0.01%, or 0.02% SB for 60 days. The results demonstrated an upward trend in weight gain rate (WGR) and specific growth rate (SGR) with increasing SB supplementation, and the experimental group fed with 0.02% SB showed a significant increase in WGR and SGR compared to other groups (P< 0.05). These levels of SB also decreased the levels of feed conversion ratio (FCR) and the total cholesterol (TC) content of Chinese soft-shelled turtles, and the 0.02% SB was significantly lower than that of other groups (P< 0.05). The activity of complement protein in vivo increased with increases in SB content, and the activities of complement C3 and C4 reached the highest level with 0.02% SB. The species abundance of the experimental group D fed with 0.02% SB was significantly higher than that of other groups (P< 0.05). Furthermore, the relative abundance of Clostridium sensu stricto 1 was significantly increased with 0.02% SB (P< 0.05). In conclusion, adding 0.02% SB to the diet improves the growth performance, feed digestion ability, and intestinal microbiota of Chinese soft-shelled turtles. [ABSTRACT FROM AUTHOR]

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In recent years, environmental DNA (eDNA) technology has become an accepted approach for investigating rare and endangered species because of its economic efficiency, high sensitivity, and non-invasiveness. The Asian giant softshell turtle (Pelochelys cantorii) is a first-class protected aquatic animal in China, and traditional resource survey methods have not identified its natural populations for many years. In this study, primers and a TaqMan probe targeting ND5 were designed, reaction conditions were optimized, a standard curve was constructed using synthetic DNA, and an eDNA quantitative PCR (qPCR) detection method was established. The eDNA detection technology for P. cantorii revealed that the number of species in the experimental pools showed a significant linear relationship with the eDNA concentration (p < 0.05). The eDNA concentration was negatively correlated with the length of time after the removal of P. cantorii and retention in the water body for 9 days. The qPCR detection method for P. cantorii eDNA established in this study can be applied to the qualitative detection of P. cantorii in water bodies, as well as to preliminary evaluation of its relative biomass. This can serve as a baseline for the investigation of natural P. cantorii population and the evaluation of its wild release effects. [ABSTRACT FROM AUTHOR]

Simple Summary: An intuitive understanding of microbial diversity and abundances of Chinese soft-shelled turtle aquaculture is crucial to comprehending these ecosystems. Herein, the evolutionary characteristics of the bacterial communities in the Chinese soft-shelled turtle and its aquaculture water systems were investigated using Illumina MiSeq sequencing. The sequencing results revealed significant differences in the microflora compositions at the phylum and genus levels in both the intestine and aquaculture water of SSTs in greenhouse and pond culture environments. The results showed that the bacterial diversity and richness compositions in the intestinal tract and aquaculture water were the same. However, the relative abundances of bacterial communities varied. The results of this study are of great significance in understanding how the environment affects SST cultures. These data may provide valuable instructions for Chinese soft-shelled turtle aquaculture management. The microbial community structure in aquaculture water plays an important role in the intestinal microbial diversity of aquatic animals. The Chinese soft-shelled turtle (SST) (Trionyx sinensis) is an important aquaculture species of high economic value in the Asia-Pacific region. An intuitive understanding of the microbial diversity and abundances of SST aquaculture is crucial for comprehending these ecosystems. Herein, the evolutionary characteristics of the bacterial communities in the SST and its aquaculture water systems were investigated using Illumina MiSeq sequencing. This experiment sampled nine SSTs from a pond outside a greenhouse and was repeated three times. The sequencing results revealed significant differences in the microflora composition at the phylum and genus levels in both the intestine and aquaculture water of the SSTs in the greenhouse and pond aquaculture environments. A total of 1039 genera belonging to 65 phyla were identified. At the phylum level, the relative abundances of Chloroflexi (24%), Acidobacteria (5%), and Nitrospira (3%) were higher in the greenhouse water than in the pond water. The relative abundances of Bacteroidetes (35%), Actinobacteria (8%), and Cyanobacteria (4%) were higher in the pond water than in the greenhouse water. The intestinal microorganisms in the SSTs experienced significant changes after the SSTs were transferred from a greenhouse culture to a pond culture environment for 28 days. After the SSTs were cultured in the ponds, we observed decreases in the relative abundances of Actinobacteria (39% to 25%), Cyanobacteria (24% to 0.8%), Chlorobacteria (9% to 3%), and Firmicutes (5.5% to 0.8%. However, we observed increases in the relative abundances of Bacteroidetes (2% to 35%) and Acidobacteria (0.3% to 25%). These results showed that the bacterial diversity and richness compositions in the intestinal tract and aquaculture water were the same. However, the relative abundances of bacterial communities varied. The results of this study are of great significance in understanding how the environment affects SST cultures. These data may provide valuable instructions for Chinese soft-shelled turtle aquaculture management. [ABSTRACT FROM AUTHOR]

Climate warming and biological invasion can interact to affect ecosystems, and the results of such interactions depend on whether invaders show physiological advantages in their responses to warming environments as compared with native species after the establishment and local adaptation. However, it has rarely been evaluated.The present study compared the heat tolerance of a globally invasive turtle, the red‐eared slider Trachemys scripta elegans, and two co‐occurring native turtle species in China, the Chinese soft‐shelled turtle Pelodiscus sinensis and Chinese pond turtle Mauremys reevesii, after acclimation in the same thermal environment. The expression patterns of heat shock factor 1 (HSF1), multiple heat shock proteins (HSPs) and telomere reverse transcriptase (TERT), and changes in relative telomere length (RTL) were then evaluated in liver and muscle of turtles under thermal stress.The results showed that T. scripta elegans had greater physiological resistance to heat than the co‐occurring native turtle species, and that this higher tolerance was accompanied by a higher temperature induction of the peak expression of inducible HSPs in liver and a higher onset temperature for the main inducible HSPs in muscle. During thermal stress, T. scripta elegans showed a better ability to maintain the expression levels of TERT under extremely hot conditions, whereas high temperatures inhibited TERT in muscles of native turtle species. Meanwhile, thermal stress did not cause telomere erosion in any of the three turtle species and some treatments can lead to slightly longer telomeres in T. scripta elegans or M. reevesii.Overall, the invasive turtle species showed higher heat tolerance and physiological advantages in enduring hot weather in comparison to the co‐occurring native turtle species. This may favour these invaders in situations of climate warming. Our study highlights the role of physiological adaptations displayed by invaders in achieving advantages over native species under climate change. [ABSTRACT FROM AUTHOR]

Biological invasions are among the major global causes of biodiversity loss. The pet trade is the main pathway by which reptiles are introduced into new environments. After the EU import ban on the pond slider (Trachemys scripta), other species of pet-traded freshwater turtles are more often reported from the wild. Here we report the first and genetically verified record of Chinese soft-shelled turtle (Pelodiscus sinensis) from the eastern part of the Czech Republic. The genus Pelodiscus has been reported from the Czech Republic and neighbouring countries; however, only as a visual record, which is insufficient for identification due to the complexity of the taxonomic status of this species. We physically captured the turtle and used genetic data (sequences of 16S rRNA and cytochrome b) to confirm the species affiliation as P. sinsensis. The studied individual represents a lineage of P. sinensis that is widely bred on farms with a native range in North Vietnam, South and south-east China. [ABSTRACT FROM AUTHOR]

Reptile embryos can move inside eggs to seek optimal thermal conditions, falsifying the traditional assumption that embryos are simply passive occupants within their eggs. However, the adaptive significance of this thermoregulatory behavior remains a contentious topic. Here we demonstrate that behavioral thermoregulation by turtle embryos shortened incubation periods which may reduce the duration of exposure to dangerous environments, decreased egg mortality imposed by lethally high temperatures, and synchronized hatching which reduces predation risk. Our study provides empirical evidence that behavioral thermoregulation by turtle embryos is adaptive. Studies of soft-shelled turtles show that behavioral thermoregulation decreases egg mortality and synchronizes hatching. [ABSTRACT FROM AUTHOR]

Simple Summary: Curcumin (CM) is a hydrophobic polyphenolic compound derived from the rhizome of turmeric (Curcuma longa Linn.). It has been proven to have antioxidant, anti-inflammatory, and nutritional metabolic regulatory functions in mammals. Currently, little is known about its effects on reptiles. In this study, dietary supplementation of 2–4 g/kg curcumin improved the antioxidant enzyme activities and muscle quality of Chinese soft-shelled turtles (Pelodiscus sinensis). Moreover, dietary curcumin supplementation increased the abundance of antioxidant bacteria (Lactobacillus and Flavobacterium) in the gut of turtles and affected hepatic metabolism-related pathways, reducing the crude lipid content of the body. These data could guide curcumin application to promote the health status of turtles. This experiment aimed to assess the impact of different dietary curcumin (CM) levels on growth, muscle quality, serum-biochemical parameters, antioxidant-enzyme activities, gut microbiome, and liver transcriptome in Chinese soft-shelled turtles (Pelodiscus sinensis). Five experimental diets were formulated to include graded levels of curcumin at 0 (control, CM0), 0.5 (CM0.5), 1 (CM1), 2 (CM2) and 4 g/kg (CM4). Each diet was randomly distributed to quadruplicate groups of turtles (164.33 ± 5.5 g) for 6 weeks. Our findings indicated that dietary curcumin supplementation did not have a significant influence on growth performance (p > 0.05); however, it significantly improved the muscular texture profiles (p < 0.05). Serum total superoxide dismutase (SOD), liver catalase (CAT), and total antioxidant capacity (T-AOC) activities increased significantly as dietary curcumin levels rose from 0.5 to 4 g/kg (p < 0.05). Dietary curcumin supplementation improved gut microbiota composition, as evidenced by an increase in the proportion of dominant bacteria such as Lactobacillus and Flavobacterium. Liver transcriptome analysis revealed that curcumin altered metabolic pathways in the liver. In conclusion, based on the evaluation of the activities of SOD in serum and CAT in liver under current experimental design, it was determined that the appropriate dietary curcumin supplementation for Chinese soft-shelled turtles is approximately 3.9 g/kg. [ABSTRACT FROM AUTHOR]

We review in detail the published fossil record of turtles from Sardinia and, in addition, we document previously undescribed specimens for the first time. Among these undescribed specimens, is the oldest occurrence of Testudo hermanni on the island, from the Early Pleistocene of Monte Tuttavista. The turtle fossil record in Sardinia goes back to the Eocene and comprises 18 different taxa, pertaining to 6 lineages: Podocnemididae, Cheloniidae, Emydidae, Geoemydidae, Testudinidae, and Trionychidae. Remarkable is the occurrence of Eocene pleurodires, whose presence is in agreement with the Oligo-Miocene rifting of the Corso-Sardinian block. Interestingly, the fossil record provides evidence for the presence in the island of both Testudo hermanni and Emys orbicularis during the Pleistocene although according to molecular data the extant populations of these two taxa were introduced in recent times. Finally, a large ungual phalanx from the Middle–Late Pleistocene Monte San Giovanni bone breccia testifies the occurrence of a giant tortoise in the Quaternary terrestrial ecosystem of Sardinia. [ABSTRACT FROM AUTHOR]

Simple Summary: Hibernation in turtle species is an adaptive survival strategy to colder winter conditions or food restrictions. However, the mechanisms underlying seasonal adaptions remain unclear. In this study, we isolated and performed scRNA-seq analysis on P. cantorii hemocytes in the active state and during hibernation to classify the hemocyte types and obtain the relevant cell maps. We identified the marker genes of each cell population and the differentiation pathways associated with their maturation. We also discovered different immune-related genes that might play crucial roles in hemocyte differentiation before and after hibernation. Our study aims to provide a reference dataset for the unified classification of P. cantorii hemocytes and improve our understanding of the immune system of P. cantorii. Hibernation in turtle species is an adaptive survival strategy to colder winter conditions or food restrictions. However, the mechanisms underlying seasonal adaptions remain unclear. In the present study, we collected hemocytes from Pelochelys cantorii and compared the molecular signature of these cells between the active state and hibernation period based on single-cell RNA sequencing (scRNA-seq) analysis. We found six cell types and identified a list of new marker genes for each cell subpopulation. Moreover, several heat shock genes, including the Hsp40 family chaperone gene (DNAJ) and HSP temperature-responsive genes (HSPs), were upregulated during the hibernation period, which predicted these genes may play crucial roles in the stress response during hibernation. Additionally, compared to hemocytes in the active state, several upregulated differentially expressed immune-related genes, such as stat1, traf3, and socs6, were identified in hemocytes during the hibernation period, thus indicating the important immune function of hemocytes. Therefore, our findings provide a unified classification of P. cantorii hemocytes and identify the genes related to the stress response, thereby providing a better understanding of the adaptive mechanisms of hibernation. [ABSTRACT FROM AUTHOR]

Simple Summary: The Asian giant soft-shelled turtle (Pelochelys cantorii), belonging to the order Testudines (family Trionychidae, genus Pelochelys), is one of the largest inland aquatic turtle species. However, due to excessive economic development, Pc. cantorii is critically endangered and rarely seen in the wild. As early as 1989, China listed the turtle as a key aquatic wildlife protection animal at the national level, but the conservation biology of Pc. cantorii has not been fully elucidated due to a lack of reference genomes. Here, based on a high-quality chromosome-level genome for Pc. cantorii, acquired by a combination of Illumina short-read, PacBio long-read and Hi-C scaffolding technologies in a previous study, we analyzed the evolutionary state of Pc. cantorii. Moreover, we found that several candidate genes associated with tumor suppression, growth and age were expanded, implicating their potential roles in the exceptional longevity of turtles. These findings will be an enabling resource for genetic and genomic studies to support fundamental insights into Pc. cantorii conservation. The Asian giant soft-shelled turtle, Pelochelys cantorii (Trionychidae), is one of the largest aquatic turtles in China and was designated as a First-Grade Protected Animal in China in 1989. Previous investigation based on a combination of Illumina short-read, PacBio long-read and Hi-C scaffolding technologies acquired a high-quality chromosome-level genome of Pc. cantorii. In this study, comparative genomic analysis between Pc. cantorii and 16 other vertebrate genomes indicated that turtles separated from the ancestor of archosaurians approximately 256.6 (95% highest posterior density interval, 263.6–251.9) million years ago (Mya) (Upper Permian to Triassic) and that Pc. cantorii separated from the ancestor of Pd. sinensis and R. swinhoei approximately 59.3 (95% highest posterior density interval, 64.3–54.3) Mya. Moreover, several candidate genes, such as VWA5A, ABCG2, A2M and IGSF1, associated with tumor suppression, growth and age were expanded, implicating their potential roles in the exceptional longevity of turtles. This new chromosome-level assembly has important scientific value in the study of conservation of Pc. cantorii and also enriches the evolutionary investigation of turtle species. [ABSTRACT FROM AUTHOR]

With his Inventaire Général de l’Indochine. La Faune de l’Indochine. Vertébrés (1927) RENÉ BOURRET provided the first extensive overview over the vertebrates of Indochina. He had a personal copy of this book bound in a special manner, interleaved, and made many corrections, additions, and amendments therein over at least 14 years. It is interesting to note that RENÉ BOURRET believed, based on a publication by CHABANAUD (1923) at some stage, as can be seen from his handwritten comments, that Chamaeleonidae occur in Indochina, but corrected this opinion later. From personal talks of the author of this article with the professors ĐÀO VĂN TIẾN and VÕ QUÝ some anecdotal information is reported on the giant softshell turtles of Hanoi (today Rafetus swinhoei) and why Bourrets works are so scarce. Furthermore, the differences between the two editions of the „Serpents marins de l‘Indochine“ and the „Batraciens de l‘Indochine“.are presented. an order form for the “Serpents de l‘Indochine” allows insight into the distribution of this work. [ABSTRACT FROM AUTHOR]

The Chinese soft-shelled turtle, Pelodiscus sinensis, is an important aquaculture species in China that exhibits distinct sexual dimorphism; male individuals are economically more valuable than females. In vertebrates, several R-spondin family proteins have been associated with sex differentiation mechanisms; however, their involvement in P. sinensis sex differentiation is unclear. Exogenous hormones such as estradiol (E2) also influence the sex differentiation of P. sinensis and induce sexual reversal. In the present study, we investigated the effects of E2 on the embryonic development of P. sinensis and the expression of R-spondin 2 (Rspo2) and R-spondin 3 (Rspo3). We amplified P. sinensis Rspo2 and Rspo3 and analyzed their expression patterns in different tissues. Comparative analyses with protein sequences from other species elucidated that P. sinensis RSPO2 and RSPO3 sequences were conserved. Moreover, phylogenetic analysis revealed that P. sinensis RSPO2 and RSPO3 were closely related to these two proteins from other turtle species. Furthermore, Rspo2 and Rspo3 were highly expressed in the brain and gonads of adult turtles, with significantly higher expression in the ovaries than in the testes (p < 0.05). We also evaluated the expression of Rspo2 and Rspo3 after the administration of three concentrations of E2 (1.0, 5.0, and 10.0 mg/mL) to turtle eggs during embryonic development. The results revealed that E2 upregulated Rspo2 and Rspo3, and the expression trends varied during different embryonic developmental stages (stages 13–20). These findings lay the groundwork for future investigations into the molecular mechanisms involved in the sex differentiation of Chinese soft-shelled turtles. [ABSTRACT FROM AUTHOR]

The taxonomy of the soft-shell turtle Rafetus bohemicus (Liebus, 1930), family Trionychidae, subfamily Trionychinae, is revised based on new and previously mentioned material (including the type material) from the Early Miocene (Burdigalian, MN 3) sites of the Most Basin, Czechia. Given that the diagnosis was so far based only on plastral elements, here we focused on the cranial material and combined our study with previously published data on postcranial elements. 3D models of the skulls derived from CT scans allow us to provide the first complete skull description of R. bohemicus, including several new cranial diagnostic characters of the species. Our results not only enable the distinction of the trionychid genera Trionyx and Rafetus, both recorded from Central Europe during the Early Miocene, but further allow us to provide an emended diagnosis for R. bohemicus. We confirm the conclusions of a previous study according to which Trionyx pontanus, T. preschenensis, T. aspidiformis, and T. elongatus are nomina dubia. R. bohemicus from Břešt'any (MN 3) represents the oldest record of this genus in Europe as well as the oldest occurrence of the genus. [ABSTRACT FROM AUTHOR]

The paper describes a series of turtle remains from the Early Late Miocene of Grytsiv (Khmelnytskyi Oblast, Ukraine). This fossil assemblage was quite diverse and included representatives of four families (Emydidae, Geoemydidae, Testudinidae, and Trionychidae), most of which are described only to the family level due to incompleteness. The discovery of shell fragments, identified here as Testudo chernovi, is the earliest record of this species on the territory of Ukraine. Scratches on the nuchal of T. chernovi are interpreted as traces of gnawing or rasping and are described as a new ichnospecies, Machichnus inrosus isp. n. These traces are identified as gnawing marks of the rodent Anomalomys grytsivensis, as evidenced by the width of the lower incisors and the presence of two characteristic longitudinal ridges on their labial surface. [ABSTRACT FROM AUTHOR]

The Chinese softshell turtle (CST; Pelodiscus sinensis) is a freshwater aquaculture species of substantial economic importance that is commercially farmed across Asia, particularly in Taiwan. Although diseases caused by the Bacillus cereus group (Bcg) pose a major threat to commercial CST farming systems, information regarding its pathogenicity and genome remains limited. Here, we investigated the pathogenicity of Bcg strains isolated in a previous study and performed whole-genome sequencing. Pathogenicity analysis indicated that QF108-045 isolated from CSTs caused the highest mortality rate, and whole-genome sequencing revealed that it was an independent group distinct from other known Bcg genospecies. The average nucleotide identity compared to other known Bcg genospecies was below 95%, suggesting that QF108-045 belongs to a new genospecies, which we named Bacillus shihchuchen. Furthermore, genes annotation revealed the presence of anthrax toxins, such as edema factor and protective antigen, in QF108-045. Therefore, the biovar anthracis was assigned, and the full name of QF108-045 was Bacillus shihchuchen biovar anthracis. In addition to possessing multiple drug-resistant genes, QF108-045 demonstrated resistance to various types of antibiotics, including penicillins (amoxicillin and ampicillin), cephalosporins (ceftifour, cephalexin, and cephazolin), and polypeptides, such as vancomycin. [ABSTRACT FROM AUTHOR]

Ten nests recorded on the banks of the Seyhan River were transferred to artificial nests with the same specifications as the original nests (depth and diameter) on Akyatan Beach due to the high predation rate along the banks of the Seyhan River. The mean clutch size in the relocated nests was 36.6 ±13.2 eggs, nest depth 43.4±5.4 cm, and nest diameter 17.0±2.1 cm. The mean diameter of the eggs was 33.6 ±1.4 mm, and the weight was 21.5 ±2.7 g. The nests differed significantly in terms of egg diameter and weight. A positive correlation was found between nest depth and clutch size. A very strong positive correlation was found between egg diameter and egg weight. Of the total 366 eggs, 7.65% were empty shells/hatched, 0.82% were not hatched, and 91.53% (82.79% early, 6.28% middle, and 2.46% late stage) remained in the embryonic stage. The mean carapace length, carapace width, plastron length, and plastron width of the 28 hatchlings were 43.2±1.9 mm, 42.2±2.2 mm, 33.6±1.6 mm, and 33.8±1.7 mm, respectively. Significant differences were detected for seven morphological characteristics of the hatchlings that emerged from the two nests. Also, significant differences were found for seven morphometric characteristics when hatchlings from this study were compared with the western Anatolian population. [ABSTRACT FROM AUTHOR]

The dietary effects of replacing fish meal with enzymatic cottonseed protein (ECP) on the growth performance, immunity, antioxidant, and intestinal health of Chinese soft-shelled turtles have not been explored. An eight-week feeding trial was conducted with a quadruplicated group of turtles (3.44 ± 0.01 g) that were randomly assigned to 16 cages (0.6 m × 0.6 m × 0.6 m) with 30 turtles that were stocked in each cage. Four dietary groups were fed with diets supplemented with 0, 2%, 4%, and 6% (ECP0 group (control group), ECP2 group, ECP4 group, ECP6 group) of enzymatic cottonseed protein replacing fishmeal. The present study illustrated that the final weight and WG in the ECP2 and ECP4 groups were significantly increased (P < 0.05) compared with the control group. The ECP2, ECP4, and ECP6 groups significantly reduced the feed coefficient (P < 0.05) and significantly increased the SGR (P < 0.05). The serum TP and ALB of the ECP4 group were significantly increased (P < 0.05). The ECP2, ECP4, and ECP6 groups significantly increased the activity of intestinal pepsin (P < 0.05), and the activity of intestinal lipase of the EPC4 group was significantly increased (P < 0.05). The intestinal villus height of the EPC4 group and EPC6 group, the villus width of the EPC2 group and EPC4 group, and the intestinal muscle thickness of the EPC4 group were significantly increased (P < 0.05). At the same time, replacing fishmeal with enzymatic cottonseed protein also affected the intestinal inflammation-related genes compared with the control group. Besides that, the expression of the IL-10 gene in the experimental group was significantly upregulated (P < 0.05). Nevertheless, the expression of TNF-α and IL-8 genes in the ECP2 group and TNF-α and IL-1β genes in the ECP4 group was significantly downregulated (P < 0.05). In summary, replacing fish meal with enzymatic cottonseed protein positively affects the growth, immunity, and intestinal health of Chinese soft-shelled turtles. The appropriate proportion of enzymatic cottonseed protein to replace fish meal in turtle feed is 4%. [ABSTRACT FROM AUTHOR]

[Display omitted] • Oculotrema hippopotami is the single monogenean platyhelminth infecting a mammal. • Molecular phylogenetic relationships place it within the chelonian parasites. • Results clearly illustrate host-switching between freshwater turtles and hippopotamuses. • This parasite could have originated from an ancestral African trionychid in the Miocene. • The proximity in the ecological habitat of parasites could enhance speciation and diversification. While monogenean worms are mainly parasites of the gills and skin of fish, and to a lesser extent parasites of the oral cavity, urinary bladder, and/or conjunctival sacs of amphibians and freshwater turtles, Oculotrema hippopotami Stunkard, 1924 is the single monogenean polystome reported from a mammal, the common hippopotamus (Hippopotamus amphibius Linnaeus). Several hypotheses have been suggested in the last decade to explain the origin of this enigmatic parasite which infects the conjunctival sacs of H. amphibius. Based on a molecular phylogeny inferred from nuclear (28S and 18S) and mitochondrial (12S and COI) sequences of O. hippopotami and chelonian polystomes, we found a sister group relationship between O. hippopotami and Apaloneotrema moleri (Du Preez & Morrison, 2012). This result suggests lateral parasite transfer between freshwater turtles and hippopotamuses, thus likely reflecting one of the most exceptional known examples of host-switching in the course of vertebrate evolution. It also demonstrates that the proximity in the ecological habitat of parasites within host species is an important feature for their speciation and diversification. Because A. moleri and its host, the Florida softshell turtle (Apalone ferox (Schneider)), are restricted to the USA, we suggest that an ancestral stock of parasites may have been isolated on primitive African trionychids after they diverged from their American relatives, and then switched to hippopotamuses or anthracotheres in Africa. [ABSTRACT FROM AUTHOR]

Citizen science is emerging as a powerful tool for the early detection of biological invasions. The exotic pet trade has been deemed as the most significant cause of establishment for reptiles, and among them, turtles have the highest number of introduction events. In Europe, at least 13 species of alien freshwater turtles have been recorded in the wild. In Greece, only two species of alien turtles have been reported, namely the American pond slider Trachemys scripta and the Florida cooter Pseudemys floridana. In this study, we provide an updated checklist of the alien freshwater turtles recorded in Greece, using citizen science, personal observations, and literature. Our results provide the first records for the country of five species, namely the Florida red-bellied cooter Pseudemys nelsoni, the Eastern river cooter Pseudemys concinna, the Chinese stripe-necked turtle Mauremys sinensis, the Chinese soft-shelled turtle Pelodiscus sinensis, and the Common snapping turtle Chelydra serpentina and new localities for T. scripta. Similar to the myth of the Lernaean Hydra (i.e., for every head chopped off, more would regrow), the pet turtle trade has been proven to have multiple heads. [ABSTRACT FROM AUTHOR]

Bacillus cereus is ubiquitous in the environment and a well-known causative agent of foodborne disease. Surprisingly, more and more emerging strains of atypical B. cereus have been identified and related to severe disease in humans and mammals such as chimpanzees, apes, and bovine. Recently, the atypical B. cereus isolates, which mainly derive from North America and Africa, have drawn great attention due to the potential risk of zoonosis. The cluster of B. cereus carries several anthrax-like virulent genes that are implicated in lethal disease. However, in non-mammals, the distribution of atypical B. cereus is still unknown. In this study, we conducted a retrospective screening of the 32 isolates of Bacillus spp. from diseased Chinese soft-shelled turtles from 2016 to 2020. To recognize the causative agent, we used various methods, such as sequencing analysis using PCR-amplification of the 16S rRNA gene, multiplex PCR for discriminating, and colony morphology by following previous studies. Furthermore, the digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values were calculated, respectively, below the 70 and 96% cutoff to define species boundaries. According to the summarized results, the pathogen is taxonomically classified as Bacillus tropicus str. JMT (previous atypical Bacillus cereus). Subsequently, analyses such as targeting the unique genes using PCR and visual observation of the bacteria under various staining techniques were implemented in our study. Our findings show that all (32/32, 100%) isolates in this retrospective screening share similar phenotypical properties and carry the protective antigen (PA), edema factor (EF), hyaluronic acid (HA), and exopolysaccharide (Bps) genes on their plasmids. In this study, the results indicate that the geographic distribution and host range of B. tropicus were previously underestimated. [ABSTRACT FROM AUTHOR]

Aeromonas veronii is widespread in aquatic environments and is capable of infecting various aquatic organisms. A. veronii infection is lethal for Chinese soft-shelled turtles (Trionyx sinensis, CSST). We isolated a gram-negative bacterium from the liver of diseased CSSTs, which was named XC-1908. This isolate was identified as A. veronii based on its morphological and biochemical characteristics, and 16S rRNA gene sequence analysis. A. veronii was pathogenic for CSSTs with an LD50 of 4.17 × 105 CFU/g. The symptoms of CSSTs artificially infected with isolate XC-1908 were consistent with those of the naturally infected CSSTs. The levels of total protein, albumin, and white globule in the serum samples of the diseased turtles were decreased, whereas those of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase were elevated. Moreover, the diseased CSSTs exhibited the following histopathological changes: the liver contained numerous melanomacrophage centers, renal glomerulus were edematous, intestinal villi were shed, and in oocytes, the number of vacuoles increased and red-rounded particles were observed. Antibiotic sensitivity tests revealed that the bacterium was sensitive to ceftriaxone, doxycycline, florfenicol, cefradine, and gentamicin, and resistant to sulfanilamide, carbenicillin, benzathine, clindamycin, erythromycin, and streptomycin. This study provides control strategies to prevent outbreaks of A. veronii infection in CSSTs. [ABSTRACT FROM AUTHOR]

Nest site selection differs among species based on a characteristic crucial to the embryo's survival. The identified nest-site characteristics of the Euphrates softshell turtle highlight that these turtles, like their other soft-shelled relatives, highly prefer the sandy substrate, even pure sand, to nest. However, alteration and loss of natural habitats for many decades limited the suitable oviposition sites along the Euphrates River. This study presents the uncharacteristic nest-site choice and the nesting time of Euphrates softshell turtle for the first time. I discovered four nests, as well as six unhatched eggs in a nest on 20 August 2017. This discovery extends the known nesting season-end from early June to at least mid-August for the Euphrates River. The eggs were spherical, and their size was 28.38 (±0.2, 28.1-28.6) mm on average. The nesting substrate, consisting of only 51.8% sand, was substantially dissimilar to what was previously reported. Apart from the soil texture, the nest and its site characteristics concerning nest dimension, clutch size, egg size, and distance to the shoreline concord with what was formerly reported. I conclude that many anthropogenic alterations, such as sand mining and damming and hence the scarcity of suitable oviposition sites, may have caused location and time shift of breeding and forced the turtles to nest in these uncommon soil textures. [ABSTRACT FROM AUTHOR]