Soft-shelled Turtle Research Articles

Analysis of Lipid Metabolism in Adipose Tissue and Liver of Chinese Soft-Shelled Turtle Pelodiscus sinensis During Hibernation

Hibernation serves as an energy-conserving strategy that enables animals to withstand harsh environments by reducing their metabolic rate significantly. However, the mechanisms underlying energy adaptation in hibernating ectotherms, such as Pelodiscus sinensis, remain contentious. This paper first reports the decrease in lipid levels and the expression of metabolism-related genes in P. sinensis during hibernation. The results of physiological and biochemical analysis showed that adipocyte cell size was reduced and liver lipid droplet (LD) contents were decreased during hibernation in P. sinensis. Concurrently, serum levels of triglycerides (TGs), total cholesterol (TC), non-esterified fatty acids (NEFAs), high-density lipoprotein cholesterol (HDLC), and low-density lipoprotein cholesterol (LDLC) were diminished (n = 8, p < 0.01), while an increase in serum glucose (Glu) (n = 8, p < 0.01) was noted among hibernating P. sinensis. These observations suggest a shift in energy metabolism during hibernation. To gain insights into the molecular mechanisms, we performed integrated transcriptomic and lipidomic analyses of adipose tissue and livers from summer-active versus overwintering P. sinensis, which revealed downregulation of free fatty acids (FFAs), triglycerides (TGs), diglycerides (DGs), and ceramides (Cers) during hibernation. The results of GSEA analysis showed that metabolic pathways associated with lipid metabolism, including glycerolipid metabolism and regulation of lipolysis in adipocytes, were suppressed significantly. Notably, acute cold exposure induced significant downregulation of genes related to lipolysis such as PNPLA2, ABHD5, LPL, CPT1A, and PPARα. The results indicate that lipolysis is suppressed during hibernation in P. sinensis. Collectively, these findings deepen our understanding of survival mechanisms and elucidate the unique energy adaptation strategies employed by hibernating ectotherms. Future research should explore the implications of these findings for the conservation of ectotherms and the applications for artificially inducing hibernation.

Shell Constraints on Evolutionary Body Size–Limb Size Allometry Can Explain Morphological Conservatism in the Turtle Body Plan

ABSTRACTTurtles are a small clade of vertebrates despite having existed since the Late Triassic. Turtles have a conservative body plan relative to other amniotes, characterized by the presence of a shell and quadrupedality. This morphology is even retained in strong ecological specialists, such as sea turtles, which are secondarily adapted to marine locomotion by strong allometric scaling in their hands. It is possible that the body plan of turtles is strongly influenced by the presence of the shell, acting as a constraint to achieving greater diversity of body forms. Here, we explore the evolutionary allometric relationships of fore‐ and hindlimb stylopodia (i.e., humerus and femur) with one another as well as their relationship with shell size (carapace length) to assess evidence of constraint. All turtles, including Triassic shelled stem turtles, have near‐isometric relationships that do not vary strongly between clades, and evolve at slow evolutionary rates. This indeed indicates that body proportions of turtles are constrained to a narrow range of possibilities. Minor allometric deviations are seen in highly aquatic sea turtles and softshell turtles, which modified their shells by bone losses. Our allometric regressions allow accurate body size estimations for fossils. Several independent sea turtle lineages converged on maximum sizes of 2.2 m of shell length, which may be a biological maximum for the group.

Broad-scale predictions of herpetofauna occupancy and colonization in an agriculturally dominated landscape.

Predictions of species occurrence allow land managers to focus conservation efforts on locations where species are most likely to occur. Such analyses are rare for herpetofauna compared to other taxa, despite increasing evidence that herptile populations are declining because of landcover change and habitat fragmentation. Our objective was to create predictions of occupancy and colonization probabilities for 15 herptiles of greatest conservation need in Iowa. From 2006-2014, we surveyed 295 properties throughout Iowa for herptile presence using timed visual-encounter surveys, coverboards, and aquatic traps. Data were analyzed using robust design occupancy modeling with landscape-level covariates. Occupancy ranged from 0.01 (95% CI = -0.01, 0.03) for prairie ringneck snake (Diadophis punctatus arnyi) to 0.90 (95% CI = 0.898, 0.904) for northern leopard frog (Lithobates pipiens). Occupancy for most species correlated to landscape features at the 1-km scale. General patterns of species' occupancy included negative effects of agricultural features and positive effects of water features on turtles and frogs. Colonization probabilities ranged from 0.007 (95% CI = 0.006, 0.008) for spiny softshell turtle (Apalone spinifera) to 0.82 (95% CI = 0.62, 1.0) for western fox snake (Pantherophis ramspotti). Colonization probabilities for most species were best explained by effects of water and grassland landscape features. Predictive models had strong support (AUC > 0.70) for six out of 15 species (40%), including all three turtles studied. Our results provide estimates of occupancy and colonization probabilities and spatial predictions of occurrence for herptiles of greatest conservation need across the state of Iowa.

Reclassification of the first Bacillus tropicus phage calls for reclassification of other Bacillus temperate phages previously designated as plasmids

Bacillus tropicus is a recently identified subspecies of the Bacillus cereus group of bacteria that have been shown to possess genes associated with antimicrobial resistance (AMR) and identified as the causative agent for anthrax-like disease in Chinese soft-shelled turtles. In addition, B. tropicus has demonstrated great potential in the fields of bioremediation and bioconversion. This article describes the comparative genomics of a Bacillus phage vB_Btc-RBClinn15 (referred to as RBClin15) infecting the recently identified B. tropicus AOA-CPS1. RBClin15 is a temperate phage with a putative parABS partitioning system as well as an arbitrium system, which are presumed to enable extrachromosomal genome maintenance and regulate the lysis/lysogeny switch, respectively. The temperate phage RBClin15 has been sequenced however, was erroneously deposited as a plasmid in the NCBI GenBank database. A BLASTn search against the GenBank database using the whole genome sequence of RBClin15 revealed seven other putative temperate phages that were also deposited as plasmids in the database. Comparative genomic analyses shows that RBClin15 shares between 87 and 92% average nucleotide identity (ANI) with the seven temperate phages from the GenBank database. All together RBClin15 and the seven putative temperate phages share common genome arrangements and < 29% protein homologs with the closest phages, including 0105phi7-2. A phylogenomic tree and proteome-based phylogenetic tree analysis showed that RBClin15 and the seven temperate phages formed a separate branch from the closest phage, 0105phi7-2. In addition, the intergenomic similarity between RBClin15 and its closely related phages ranged between 0.3 and 47.7%. Collectively, based on the phylogenetic, and comparative genomic analyses, we propose three new species which will include RBClin15 and the seven temperate phages in the newly proposed genus Theosmithvirus under Caudoviricetes.

Replacing sea fish with black soldier fly fermentation homogenate in diet for Pelodiscus sinensis: growth, intestinal development and hepatic health

Abstract Black soldier fly larvae (BSFL) meal has the potential to partially replace fish meal in the diet of various aquatic animals. The fermentation treatment of BSFL can retain more nutrients and reduce processing and transportation costs. This study investigated the effects of partially replacing fish meal with Black Soldier Fly Fermentation Homogenate (BSFFH) on the growth performance and health of Chinese soft-shelled (Pelodiscus sinensis) turtle. The experiment was divided into 4 different groups of juvenile P. sinensis diets. The control diet included 60% compound feed and 40% sea fish. The 34%, 61%, and 82% substitution groups were fed mixtures, and varying percentages of sea fish were replaced with BSFFH. The results indicate that replacing 34% and 61% sea fish with BSFFH significantly improved growth (P &lt; 0.05). The 61% substitution significantly decreased the muscle fiber size while the 82% substitution significantly upregulated the expression of myogenic genes MYF5 and MRF4 (P &lt; 0.05). Furthermore, replacing sea fish with BSFFH significantly increased hepatic TG content (P &lt; 0.05) and induced mild liver fibrosis, while it reduced the TG content in skeletal (P &lt; 0.05). However, BSFFH did not affect subcutaneous fat weight (P &gt; 0.05). Lastly, the 34% substitution significantly increased the intestine villus height and the ratio of villus height/crypt depth, while higher substitution reduced both values. In addition, the 61% substitution increased the mucus secreted by the intestine goblet cells. In conclusion, partially replacing sea fish with BSFFH improves the growth performance and intestine structure of Chinese soft-shelled turtles. However, a high level of BSFFH could negatively affect growth performance, intestinal structure, and hepatic health.

Transcriptomic and Proteomic Analyses of the Immune Mechanism in Pathogenetic and Resistant Chinese Soft-Shelled Turtle (Pelodiscus sinensis) Infected with Aeromonas hydrophila

Background: As intensive aquaculture practices have progressed, the prevalence of bacterial diseases in the Chinese soft-shell turtle (Pelodiscus sinensis) has escalated, particularly infections caused by Aeromonas hydrophila, such as ulcerative dermatitis and abscess disease. Despite this, little is known about their immune defenses against this pathogen. Methods: Our study pioneers an integrated analysis of transcriptomics and proteomics to investigate the immune responses of Chinese soft-shelled turtles to A. hydrophila infection. Results: The investigation revealed significant differences in immune-related pathways between groups susceptible and resistant to A. hydrophila infection after 4 days. A total of 4667 and 3417 differentially expressed genes (DEGs), 763 and 568 differentially expressed proteins (DEPs), and 13 and 5 correlated differentially expressed genes and proteins (cor-DEGs-DEPs) were identified in susceptible and resistant Chinese soft-shelled turtles, respectively. In the resistant group, upregulation of immune-related genes, such as CD3ε and CD45, enhanced T-cell activation and the immune response. The proteomic analysis indicated that immune proteins, such as NF-κB1, were significantly upregulated in the resistant group. The correlation analysis between transcriptomics and proteomics demonstrated that the CD40 gene and protein, differentially expressed in the resistant group compared to the control group, were commonly upregulated within the Toll-like receptor signaling pathway. Conclusions: The transcriptomic and proteomic data obtained from this study provide a scientific foundation for understanding the immune mechanisms that enable the Chinese soft-shelled turtle to resist A. hydrophila infection.

Establishment and population genetic analysis of SNP fingerprinting of Chinese soft-shelled turtle (Pelodiscus sinensis)

Chinese soft-shelled turtle (Pelodiscus sinensis) is an important aquaculture species in China. However, with the continuous development of P. sinensis cultures, problems related to germplasm degradation and population mixing have become increasingly prominent. This has seriously affected the protection and utilization of germplasm resources by P. sinensis. To broaden the understanding of the germplasm resources of P. sinensis and improve the efficiency of germplasm identification, Super-GBS sequencing technology was used to conduct population genetic analysis of P. sinensis samples from six different populations distributed in five provinces. As a result, 1464,488 SNPs were identified. Genetic structure analysis revealed that the six P. sinensis populations could be divided into three sub-populations. The genetic differentiation coefficient of the DT and CY populations was very low (Fst=0.0088), indicating that differentiation had not yet occurred. Additionally, 15 KASP core markers for the identification of P. sinensis populations were developed using KASP typing technique for the first time. Genetic information can lay a foundation for the protection and utilization of germplasm resources by P. sinensis, and the fingerprinting database developed in this study provides a practical tool for authenticating P. sinensis.

Present and future distribution of the European pond turtle versus seven exotic freshwater turtles, with a focus on Eastern Europe

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.

The Youngest European Record of the Chelonian Family Trionychidae (Calabrian, Central Italy) Offers New Clues on the Quaternary Extirpation History of the Softshell Turtles

We report on the geologically youngest occurrence of the softshell turtle family Trionychidae in Europe, from middle Calabrian (Emilian) strata cropping out at Montalto, Pisa Province (Tuscany, central Italy). This record indicates that the softshell turtles survived well past the glacial pulse at ca. 1.8 Ma. That the most recent finds of Trionychidae all over Europe come from mainland Italy further evokes the role of the Apennine peninsula as a refugium for humid-dwelling herpetofaunas through most of the Plio-Pleistocene. Reduced humidity associated with the intensified cool stages at the beginning of the Early–Middle Pleistocene Transition may have been behind the demise of the last European softshell turtles.

Same but Different: Intersexual and Age‐Related Habitat Use of the Threatened West African Trionyx triunguis Clade—A Four‐Country Comparative Study

ABSTRACTBiological communities are structured by a variety of biotic and abiotic relationships, whose understanding forms the basis for effective conservation. Among the myriad factors influencing community dynamics, resource partitioning stands out, potentially enriching ecosystem complexity but also carrying risks for conservation if disregarded. In this study, we investigated intersexual and ontogenetic niche differences among populations of the West African softshell turtle Trionyx triunguis in Liberia, Cote d'Ivoire, Ghana and Nigeria. We observed that these distinct populations exhibited similar habitat preferences, favouring large rivers with abundant riverbank and aquatic vegetation, and muddy substrates. Disparities in habitat preferences were evident among populations and across various life stages, indicating the adaptability of the species. We found that the species was active throughout the year, but most of our sightings and captures occurred during the wet season. In light of the species' vulnerability to overhunting, our research findings provide important data on the ecology of this species that can assist in its effective conservation across all life stages.

Development and Application of a Portable Environmental DNA Test for the Detection of Rafetus swinhoei in Viet Nam

ABSTRACTSwinhoe's (or Yangtze) giant softshell turtle (Rafetus swinhoei) is a large, critically endangered freshwater turtle and considered among the rarest species in the world. As of 2024, only two individuals have been confirmed to remain alive, one at the Suzhou Zoo in China and one in Xuan Khanh Lake, Viet Nam. The only hope for the long‐term survival of R. swinhoei is finding additional, as yet undiscovered, animals that have thus far eluded detection by traditional survey methods. In recent years, numerous studies have been published on the use of environmental DNA (eDNA) for species detection and monitoring. This method takes advantage of the persistence of DNA in the environment, such as in water, soil, and air. An organism's DNA is shed into the environment through urine, feces, and the sloughing of skin. Species‐specific quantitative polymerase chain reaction (qPCR) testing can be used to detect eDNA in samples collected from the environment. eDNA trials to detect R. swinhoei were initiated by the Asian Turtle Program and Washington State University in 2013. To expand the use of eDNA for species detection by conservationists, we developed and validated a first‐of‐its‐kind innovative point‐of‐detection (POD) qPCR platform for the rapid, onsite detection of R. swinhoei from water samples. Here we show that the portable eDNA test kit can be used for the successful detection of R. swinhoei in a large body of water and that pooling filters may be a useful strategy to reduce test costs and improve detection efficiency. Use of this test can expand the search for R. swinhoei in unexplored and understudied lakes, reservoirs, and other bodies of water where this species may be present and could inform field surveys utilizing eDNA for other threatened species that are rare in nature.

Identification and expression analysis of NOD-like receptor gene family in response to Aeromonas hydrophila and poly I:C challenge in Chinese soft-shelled turtle (Pelodiscus sinensis)

Nucleotide-binding domain (NOD)-like receptors (NLRs), a type of pattern recognition receptor (PRR), specifically recognize pathogen-associated molecular patterns and damage-associated molecular patterns, playing crucial roles in the immune system. Herein, the composition, expression pattern, and co-expressed pathways of NLRs in Pelodiscus sinensis (PsNLRs) were analyzed. Eight PsNLRs, located on seven chromosomes, were first identified from the genome. Phylogenetic analysis revealed that PsNLRs were most closely related to the NLRs of the western painted turtle and the green sea turtle, and could be clustered into four subfamilies. Besides, the expression profile of PsNLRs exhibited tissue-specificity after infection with Aeromonas hydrophila or poly I:C. Nonetheless, PsCIITA and PsNLRP3.2 displayed infection-specific expression patterns, whereas other PsNLRs showed similar expression patterns during A. hydrophila and poly I:C infections. Weighted gene co-expression network analysis found that six PsNLRs were generated in the turquoise module, in which several immune-related signaling pathways were enriched, including “NOD-like receptor signaling pathway”, “NF-κB signaling pathway”, “PI3K-Akt signaling pathway”, “Chemokine signaling pathway”, “Antigen processing and presentation”, and ”Toll-like receptor signaling pathway”. Additionally, three immune-related genes, NF-κB1, TRAF3, and PIK3CG, were identified as hub genes. These results indicated that PsNLRs may play key roles in regulating immune responses in P. sinensis. Overall, an in-depth study of the NLR gene family in P. sinensis would yield insights into developing strategies to combat bacterial and viral infections.

Identification and expression analysis of dmc1 in Chinese soft-shell turtle (Pelodiscus sinensis) during gametogenesis

DNA meiotic recombinase 1 (disrupted meiotic cDNA, Dmc1) protein is homologous to the Escherichia coli RecA protein, was first identified in Saccharomyces cerevisiae. This gene has been well studied as an essential role in meiosis in many species. However, studies on the dmc1 gene in reptiles are limited. In this study, a cDNA fragment of 1,111 bp was obtained from the gonadal tissues of the Chinese soft-shell turtle via RT-PCR, containing a 60 bp 3′ UTR, a 22 bp 5′ UTR, and an ORF of 1,029 bp encoding 342 amino acids, named Psdmc1. Multiple sequence alignments showed that the deduced protein has high similarity (>95 %) to tetrapod Dmc1 proteins, while being slightly lower (86–88 %) to fish species.Phylogenetic tree analysis showed that PsDmc1 was clustered with the other turtles’ Dmc1 and close to the reptiles’, but far away from the teleost’s. RT-PCR and RT-qPCR analyses showed that the Psdmc1 gene was specifically expressed in the gonads, and much higher in testis than the ovary, especially highest in one year-old testis. In situ hybridization results showed that the Psdmc1 was mainly expressed in the perinuclear cytoplasm of primary and secondary spermatocytes, weakly in spermatogonia of the testes. These results indicated that dmc1 would be majorly involved in the developing testis, and play an essential role in the germ cells’ meiosis. The findings of this study will provide a basis for further investigations on the mechanisms behind the germ cells’ development and differentiation in Chinese soft-shell turtles, even in the reptiles.

Effects of a Chinese herbal medicine mixture on growth performance, immune response, and gene expression in Chinese soft-shelled turtles (Pelodiscus sinensis)

Chinese herbal medicines (CHMs), known for their safety, low toxicity, and minimal impact on the environment, are often used as a natural feed additive in aquaculture to prevent or control diseases of aquatic animals. However, their effect on cell responses of Chinese soft-shelled turtle (Pelodiscus sinensis) remains unexplored. In this study, the extract mixed with four Chinese herbs of Astragalus membranaceus, Lycium barbarum, Rhizoma polygonati and Codonopsis pilosula in proportions (7:8:3:2) was used as a feed additive for 60 days to study their effects on the growth performance and immune response of P. sinensis in greenhouse mode. Results showed that the weight gain rate and the specific growth rate in the CHMs group were 46.63 % and 0.64 %, which were 1.13- and 1.15-fold of that in the control group, respectively. Besides, CHMs could significantly improve the survival rate by 76.94 % after artificial infection of Aeromonas hydrophila. The CHMs group also exhibited significant increases in the activities of total superoxide dismutase (27.76 %), catalase (41.28 %), glutathione peroxidase (14.62 %), and glutathione sulfur transferase (36.75 %) compared to the control group. Conversely, malondialdehyde concentration was reduced to 67.82 % of that in the control group. Moreover, the concentration of lysozyme and complement C3, as well as the activity of acid phosphatase, rose by 16.43 %, 24.30 %, and 27.68 %, respectively. Comparative transcriptome analysis revealed that growth, immunity, and lipid metabolism-related pathways are involved in the response to CHMs. Among these, the two pathways of the cell cycle and the cytokine-cytokine receptor interaction may play important roles in the growth and immunity of P. sinensis. SMOX, STAT3, and HMGCS1 may serve as key genes in CHMs-stimulated immune response in P. sinensis; GADD45B, ARG1, ACVR1C, and BMP7 may be crucial genes in promoting the growth of P. sinensis. This study provides a theoretical and practical basis for the green and efficient culture of P. sinensis.

Transcriptome and pathophysiological analysis during Bacillus cereus group infection in Pelodiscus sinensis uncovered the importance of iron and toll like receptor pathway

The Chinese softshell turtle (CST; Pelodiscus sinensis) is an important freshwater aquaculture species with high economic value. CST is farmed throughout Asia, especially in China and Taiwan. However, the presence of diseases, particularly those caused by the Bacillus cereus group, poses significant threats to commercial CST farming. A recent study has unveiled new genospecies within the B. cereus group, Bacillus shihchuchen. Within this group, strain QF108–045 stands out for its high virulence, notably leading to severe septicemia in CST. This study aimed to explore the transcriptome and pathophysiological changes in CSTs infected with QF108–045. Upon infection with QF108–045, turtles exhibited upregulation of Toll-like receptor (TLR) 2, 4, and 5, among which, TLR5 was significantly upregulated. Activation of TLR signaling leading to elevated levels of IL-1β, TNFα, and Th17 cytokines, which subsequently activate nuclear factor kappa B (NF-κB). CSTs challenged with QF108–045 exhibited a substantial decrease packed cell volume, indicating severe hemolysis. The notable upregulation of transferrin, ZIP8/14, and HO-1 indicates an active transfer of iron or heme into cells, and melanomacrophages in the spleen showed a positive signal in Perl's Prussian blue staining, implying their importance as essential iron storage cells. However, despite the hemolysis, ferroptosis was inhibited by the upregulation of system Xc− related genes. Additionally, our study revealed a notable increase in TLR5 expression following immunization with recombinant flagellin from QF108–045, coupled with analysis of protein tertiary docking modules. These findings advance our understanding of defense mechanisms in CSTs during QF108–045 infection, providing valuable insights for future vaccine development efforts.

Adaptation and feeding preference of Chinese soft-shelled turtle (Pelodiscus sinensis) to different feeding levels in rice–turtle co-culture system

Rice–turtle co-culture is an ecological method involving a low exogenous input of chemicals and excessive feeding might increase the cost and reduce the mutual beneficial effect. To determine the optimal feeding level for co-cultured P. sinensis and for its adaptation, the turtles (878.0±10.3 g) were fed at feeding levels of 0.7 %, 1.4 % and 2.1 % for 90 days. The observation of feeding behavior showed that a low feeding level prompted the turtles to move into field paddies and prolonged their stay. The specific growth rate increased with increasing feeding level, but did not further increase when it exceeded 1.4 %, and the feed conversion ratio increased with increasing feeding level from 0.7 % to 2.1 %. The activities of digestive enzymes were higher in the group with a low feeding level. The plasma triglyceride and total cholesterol levels increased with increasing feeding level, while the creatinine level was significantly lower at a high feeding level of 2.1 %. The trend analysis of gene expression showed that the expression of most of differentially expressed genes (DEGs) in the brain decreased with increasing feeding levels from 0.7 % to 1.4 % and did not further decrease at a feeding level of 2.1 %, while in the gut, most of the genes were significantly downregulated or upregulated at a feeding level of 1.4 %. In the brain, the pathways associated with the DEGs were involved in the relaxin signaling pathway, melanogenesis and the age–rage signaling pathway in diabetic complications. In the gut, the DEGs were most associated with pancreatic secretion, protein digestion and absorption. The related genes expression was consistent with feeding behavior, growth and physiological metabolism. These findings suggest that co-cultured turtles can adapt to different feeding levels, that the optimal feeding level for co-cultured turtles is 1.4 %, and that additional feeding reduced the economic and ecological benefits of co-culture.

Peripheral hearing sensitivity is similar between the sexes in a benthic turtle species despite the larger body size of males.

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.

Integrative transcriptomics and metabolomics analysis provide insight into the formation of skin pigmentation diversity in Chinese soft-shelled turtle (Pelodiscus sinensis)

Chinese soft-shelled turtle (Pelodiscus sinensis) is an important freshwater breeding species in East and Southeast Asia. The most external differences among different strains are skin pigmentation and blotches, but the mechanism of their formation is unknown. To investigate the mechanism of skin pigmentation diversity in Chinese soft-shelled turtle, three unique phenotypes (Golden strain, Japanese strain and Qingxi black strain) were selected to observe the chromatophores, and the transcriptome and metabolome were compared. The results showed the presence of melanophores, xanthophores and iridophores in the skin of all three strains. However, there were differences in the composition and organelles of chromatophores. The upregulation of gene asip and elevated accumulation of glutathione and cysteine in Golden strain resulted in the production of pheomelanin, leading to yellow pigmentation in the skin. The upregulation of gene dct and elevated accumulation of indole-5, 6-quinone in Qingxi black strain resulted in the production of eumelanin, which made the skin appear black-brown. Japanese strain produced eumelanin and pheomelanin, however, the presence of melanosomes resulted in skin darkening and appeared dark green pigmentation. The white blotches of Gloden strain and Japanese strains were formed through increased production of iridophores, while the black blotches of Qingxi black strain were formed by inducing migration and apoptosis of melanophores within iridophores. In conclusion, the distinctive characteristics of the skin in three strains resulted from combination of melanin type, abundance of melanosomes and interaction between melanophores and iridophores. These results provided valuable reference for investigating the mechanisms underlying skin pigmentation diversity in Chinese soft-shelled turtle and other vertebrates.

Comparative transcriptome sequencing and weighted coexpression network analysis reveal growth-related hub genes and key pathways in the Chinese soft-shelled turtle (Pelodiscus sinensis)

The Chinese soft-shelled turtle (Pelodiscus sinensis) exhibits sexual dimorphism. Compared with females, males are considered to have higher economic value due to their accelerated growth, greater body mass, and longer skirt width. Studies focused on these sex disparities have largely neglected potential sex differences in growth. Here, we performed RNA sequencing of muscle tissue components from 1-year-old specimens to reveal gene expression patterns in P. sinensis. In our male and female cohorts, our analysis revealed, respectively, 388 and 526 upregulated differentially expressed genes (DEGs) and 1129 and 635 downregulated DEGs. Through weighted coexpression network analysis (WGCNA) and integration with phenotype data, we established two main gene modules: a light yellow module encompassing 191 genes (e.g., ACACB, CTH, HADHA, and CTNNB) that demonstrated a positive correlation with population traits, and a black module comprising 298 genes (e.g., CAV3, PIK3CD, SMAD3, and VEGFA) that demonstrated a negative correlation with population traits. We also performed a DEG evaluation and gene set enrichment analysis across individuals of different sizes and noted that pathways such as protein digestion and assimilation (ko04974), were substantially augmented in larger specimens. In these pathways, the collagen (COL) and solute carrier (SLC) gene families were noted to be crucial for sustaining body structure and facilitating nutrient and metabolite transportation. In conclusion, we elucidated the essential modules, pivotal genes, and pathways involved in gene expression differences among various P. sinensis size groupings. Our results provide novel insights for future studies on growth discrepancies in P. sinensis.

Morphometric Measurements of the Euphrates Soft-shelled Turtle, Rafetus euphraticus,, in the Central Marshes, Iraq

Morphometric Measurements of the Euphrates Soft-shelled Turtle, Rafetus euphraticus,, in the Central Marshes, Iraq