Soft-shelled Turtle Research Articles

The Infection Properties of Trionyx sinensis Hemorrhagic Syndrome Virus and the Antiviral Effect of Curcumin In Vivo

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.

Comparative analysis of whole-transcriptome RNA expression of lung tissue of Chinese soft-shell turtle infected by Trionyx sinensis Hemorrhagic Syndrome Virus

Trionyx sinensis Hemorrhagic Syndrome Virus (TSHSV), the first aquatic arterivirus identified in China, causes severe mortality to T. sinensis. In this study, we sought to determine the functions of T. sinensis mRNAs and non-coding RNAs (ncRNAs) that were differentially expressed (DE) over different periods of TSHSV infection of T. sinensis lung. We used RT-qPCR to validate the sequencing results of select RNAs, confirming their reliable and referable nature. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were used to predict multiple biological functions and signaling pathways in various comparison groups (1-day versus mock, 3-day versus 1-day, and 5-day versus 3-day). Multiple types of differentially expressed RNA, including mRNA, lncRNA, circRNA, and miRNA, were associated with cardiac dysfunction, coagulation abnormalities, and arachidonic acid metabolism at day 1. Pre-inflammatory cytokines and inflammatory factors such as PLA2G4A, cPLA2, γ-GGT1, TNFRSF14, TCP11L2, PTER CYP2J2 and LTC4S, were noticeably regulated at the same time. On day 3, multiple GO terms and KEGG pathways were implicated, including those related to virus defense, apoptosis, pyroptosis, and inflammatory response. Notably, key genes such as RSAD2, TRIM39, STAT4, CASP1, CASP14, MYD88, CXCL3, CARD11, ZBP1, and ROBO4 exhibited significant regulation. The lncRNAs and circRNAs that targeted the genes involved in viral recognition (TLR5), apoptosis (CARD11), pyroptosis (ZBP1), inflammatory processes (NEK7, RASGRP4, and SELE) and angiogenesis (ROBO4) exhibited significant regulation. Significantly regulated miRNAs were primarily linked to genes involved in apoptosis (Let-7f-3p, miR-1260a, miR-455–3p), and inflammation (miR-146a, miR-125a, miR-17a, miR-301b, and miR-30a-3p). The findings could advance our understanding of the host immunological response to TSHSV and offer new ideas for developing effective strategies to prevent infection of T. sinensis.

Correction to "The impact of aquaculture system on the microbiome and gut metabolome of juvenile Chinese softshell turtle (Pelodiscus sinensis)".

[This corrects the article DOI: 10.1002/imt2.17.].

Comparative genomic survey and functional analysis of DKKL1 during spermatogenesis in the Chinese soft-shelled turtle (Pelodiscus sinensis)

A feature of the Chinese soft-shelled turtle (Pelodiscus sinensis) is seasonal spermatogenesis; however, the underlying molecular mechanism is not well clarified. Here, we firstly cloned and characterized P. sinensis DKKL1, and then performed comparative genomic studies, expression analysis, and functional validation. P. sinensis DKKL1 had 2 putative N-glycosylation sites and 16 phosphorylation sites. DKKL1 also had classic transmembrane structures that were extracellularly localized. DKKL1's genetic distance was close to turtles, followed by amphibians and mammals, but its genetic distance was far from fishes. DKKL1 genes from different species shared distinct genomic characteristics. Meanwhile, they were also relatively conserved among themselves, at least from the perspective of classes. Notably, the transcription factors associated with spermatogenesis were also identified, containing CTCF, EWSR1, and FOXL2. DKKL1 exhibited sexually dimorphic expression only in adult gonads, which was significantly higher than that in other somatic tissues (P < 0.001), and was barely expressed in embryonic gonads. DKKL1 transcripts showed a strong signal in sperm, while faint signals were detected in other male germ cells. DKKL1 in adult testes progressively increased per month (P < 0.05), displaying a seasonal expression trait. DKKL1 was significantly downregulated in testes cells after the sex hormones (17β-estradiol and 17α-methyltestosterone) and Wnt/β-catenin inhibitor treatment (P < 0.05). Likewise, the Wnt/β-catenin inhibitor treatment dramatically repressed CTCF, EWSR1, and FOXL2 expression. Conversely, they were markedly upregulated after the 17β-estradiol and 17α-methyltestosterone treatment, suggesting that the three transcription factors might bind to different promoter regions, thereby negatively regulating DKKL1 transcription in response to the changes in the estrogen and androgen pathways, and positively controlling DKKL1 transcription in answer to the alterations in the Wnt/β-catenin pathway. Knockdown of DKKL1 significantly reduced the relative expression of HMGB2 and SPATS1 (P < 0.01), suggesting that it may be involved in seasonal spermatogenesis of P. sinensis through a positive regulatory interaction with these two genes. Overall, our findings provide novel insights into the genome evolution and potential functions of seasonal spermatogenesis of P. sinensis DKKL1.

Effects of dietary chito-oligosaccharide and β-glucan on the water quality and gut microbiota, intestinal morphology, immune response, and meat quality of Chinese soft-shell turtle (Pelodiscus sinensis).

Chito-oligosaccharides (COS) and β-glucan are gradually being applied in aquaculture as antioxidants and immunomodulators. However, this study examined the effects of dietary supplementation of COS and β-glucan on the water quality, gut microbiota, intestinal morphology, non-specific immunity, and meat quality of Chinese soft-shell turtle. To investigate the possible mechanisms, 3-year-old turtles were fed basal diet (CK group) and 0.1%, 0.5%, and 1% COS or β-glucan supplemented diet for 4 weeks. Colon, liver, blood and muscle tissues, colon contents, water and sediment of paddy field samples were collected and analyzed after feeding 2 and 4 weeks. The results indicated that COS and β-glucan altered microbial community composition and diversity in Chinese soft-shell turtles. The relative abundance of Cellulosilyticum, Helicobacter and Solibacillus were increased after feeding COS, while Romboutsia, Akkermansia and Paraclostridium were increased after feeding β-glucan, whereas Cetobacterium, Vibrio and Edwardsiella were enriched in the control group. Furthermore, colon morphology analysis revealed that COS and β-glucan improved the length and number of intestinal villi, and the effect of 0.5% β-glucan was more obvious. Both β-glucan and COS significantly improved liver and serum lysozyme activity and antibacterial capacity. COS significantly increased the total antioxidant capacity in the liver. Further, 0.1% β-glucan significantly increased the activity of hepatic alkaline phosphatase, which closely related to the bacteria involved in lipid metabolism. Moreover, dietary supplementation with 1% COS and 1% β-glucan significantly enhanced the content of total amino acids, especially umami amino acids, in muscle tissue, with β-glucan exerting a stronger effect than COS. Additionally, these two prebiotics promoted the quality of culture water in paddy fields and reshaped the bacterial community composition of aquaculture environment. All these phenotypic changes were closely associated with the gut microbes regulated by these two prebiotics. In summary, the findings suggest that dietary supplementation with COS and β-glucan in Pelodiscus sinensis could modulate the gut microbiota, improve intestinal morphology, enhance non-specific immunity and antioxidant capacity of liver and serum, increase meat quality, and improve the culture water environment. This study provides new insights and a comprehensive understanding of the positive effects of COS and β-glucan on Pelodiscus sinensis.

Histomorphic analysis and expression of mRNA and miRNA in embryonic gonadal differentiation in Chinese soft-shelled turtle (Pelodiscus sinensis)

The Chinese soft-shelled turtle (Pelodiscus sinensis) is extensively cultured in Asia for its nutritional and medical value. Gonadal differentiation is fantastic in turtles, whereas morphologic, mRNA, and miRNA expressions were insufficient in the turtle. In this study, ovaries and testes histomorphology analysis of 14–23 stage embryos were performed, and mRNA and miRNA expression profiles were analyzed. Histomorphology analysis revealed that gonads were undifferentiated at embryonic stage 14. Ovarian morphological differentiation became evident from stage 15, which was characterized by the development of the cortical region and degeneration of the medullary region. Concurrently, testicular morphological differentiation was apparent from stage 15, marked by the development of the medullary region and degeneration of the cortical region. qRT-PCR results showed that Cyp19a1 and Foxl2 exhibited female-specific expression at stage 15 and the expression increased throughout most of the embryonic development. Dmrt1, Amh, and Sox9 displayed male-specific expression at stage 15 and tended to increase substantially at later developmental stages. The expression of miR-8356 and miR-3299 in ZZ gonads were significantly higher than that in ZW gonads at stage 15, 17 and 19, and they had the highest expression at stage 15. While the expression of miR-8085 and miR-7982 had the highest expression at stage 19. Furthermore, chromatin remodeler genes showed differential expression in female and male P. sinensis gonads. These results of master sex-differentiation genes and morphological characteristics would provide a reference for the research of sex differentiation and sex reversal in turtles. Additionally, the expression of chromatin remodeler genes indicated they might be involved in gonadal differentiation of P. sinensis.

Effects of dietary sodium butyrate on growth performance, immune function, and intestinal microflora of Chinese soft-shelled turtle (Pelodiscus sinensis).

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.

Morganella morganii, a bacterial pathogen in diseased Chinese soft-shelled turtles (Pelodiscus sinensis)

The Chinese Soft-Shelled Turtle (CSST) is a significant commercial aquatic animal in China. However, the increased frequency of diseases in the breeding process has hindered industrial development. A gram-negative bacterium, Z617 strain, was isolated from diseased CSST on a farm in Hubei Province, China. It was identified as Morganella morganii by 16S rDNA. The median lethal dose of Z617 strain was (8.32 ± 0.09) × 104 colony-forming units/g CSST weight. The histopathological changes of infected CSSTs, including hepatic sinus congestion, vacuolization of hepatocytes, ulceration of the intestinal mucosa, destruction of the intestinal villi, an unclear distinction between red and white pulp of the spleen, and swelling and necrosis of the glomerular of the kidney, were examined. The structure of the intestinal community of CSSTs was analyzed, and the distinction between richness and diversity was found. The intestinal microbiota community structure differed between the control and infected groups. The relative abundance of Cetobacterium, Lawsonia, and Bacteroides was increased in the infected group compared with the control group at the genus level. Z617 strain is susceptible to antibiotics such as Ceftazidime and Piperacillin. Moreover, it showed various degrees of drug resistance to Enrofloxacin, Doxycycline, Cefuroxim, Streptomycin, Midecamy Cinum, Minocycline, Gentamicin, Neomycin, Florfenicol, and Carbenicillin. This research indicated that M. morganii handled numerous CSSTs' death and clarified the biological profile and potential mechanisms of the Z617 strain. It throws some light on the disease diagnosis and may provide the referenced value for disease treatment.

Exploring the relationship between environmental DNA concentration and biomass in Asian giant softshell turtle (Pelochelys cantorii).

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.

From pets to plates: network analysis of trafficking in tortoises and freshwater turtles representing different types of demand

AbstractDespite being protected under the law, illegal trade in tortoises and freshwater turtles is common in India, with different species being trafficked for different markets. Indian species of tortoises and hard-shell turtles are predominantly trafficked for the pet trade and soft-shell turtles for the meat trade. Given their distinct markets, the operation of trade may vary between these different groups of tortoises and freshwater turtles, thereby necessitating different types of interventions. However, a systematic examination of illegal trade in tortoises and freshwater turtles that takes into account the differences between these markets is currently lacking. Here we compare the supply networks of tortoises/hard-shell turtles (in demand for pet trade) vs soft-shell turtles (meat trade), using information from 78 and 64 seizures, respectively, that were reported in the media during 2013–2019. We used social network analysis to compare the two networks and the role of individual nodes (defined as locations at the district or city scale) within these networks. We found that the tortoise/hard-shell turtle network had a larger geographical scale, with more international trafficking links, than the soft-shell turtle network. We recorded convoluted smuggling routes in tortoise/hard-shell turtle trafficking, whereas soft-shell turtle trafficking was uni-directional from source to destination. Within both networks, we found that a few nodes played disproportionately important roles as key exporting, importing or transit nodes. Our study provides insights into the similarities and differences in the illegal supply networks of different groups of tortoises and freshwater turtles, in demand for different markets. We highlight the need for intervention strategies tailored to address the illegal trade in each of these groups.

Variations in the Intestinal Microbiota of the Chinese Soft-Shelled Turtle (Trionyx sinensis) between Greenhouse and Pond Aquaculture.

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.

Development of an enzyme-linked immunosorbent assay for determining FSH plasma concentrations in green sea turtle (Chelonia mydas), using recombinant gonadotropins

Follicle-stimulating hormone (FSH) is involved in the regulation of essential reproductive processes such as gametogenesis and follicular growth. There are presently no immunoassays for measuring FSH in turtles. Recently we produced green sea turtle (Chelonia mydas) recombinant (r) FSH as a single-chain polypeptide using the methylotrophic yeast Pichia pastoris expression system, and polyclonal antibodies for the recombinant FSH. In this work we developed a competitive enzyme-linked immunosorbent assay (ELISA) for the measurement of FSH concentrations in plasma samples from green sea turtles. We used the rFSHβα for standard, rFSHβ for coating and a cmFSHβ antibody. The sensitivity of the assay was 0.13 ng/ml and the intra-assay and inter-assay coefficients of variation were 5.54% and 13.52% respectively. Parallelism was observed between the linearized FSH standard curves and the corresponding serial dilutions of green sea turtle plasma samples. We also observed parallelism between the linearized standard and serial dilutions of plasma samples from the loggerhead sea turtle (Caretta caretta), hawksbill sea turtle (Eretmochelys imbricate), and African softshell turtle (Trionyx triunguis). The ELISA was used to study the FSH plasma concentrations during the reproductive cycles and was compared to hormonal steroid concentrations (Testosterone, Estradiol and Progesterone). This revealed a positive correlation between FSH and estradiol concentrations in females; estradiol concentrations were increased immediately after FSH elevation. In addition, nested females presented an increase in FSH concentrations prior to progesterone elevation in January to April, slightly before egg laying. This ELISA will increase our understanding of gonadotropin functions, and their effects on reproduction in the green sea turtle.

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

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

Molecular cloning and expression analysis of rad51 gene associated with gametogenesis in Chinese soft-shell turtle (Pelodiscus sinensis)

Rad51 is a recA-like recombinase that plays a crucial role in repairing DNA double-strand breaks through homologous recombination during mitosis and meiosis in mammals and other organisms. However, its role in reptiles remains largely unclear. In this study, we aimed to investigate the physiological role of the rad51 gene in reptiles, particularly in Pelodiscus sinensis. Firstly, the cDNA of rad51 gene was cloned and analyzed in P. sinensis. The cloned cDNA contained an open reading frame (ORF) of 1020 bp and encodeed a peptide of 339 amino acids. The multiple alignments and phylogenetic tree analysis of Rad51 showed that P. sinensis shares the high identity with Chelonia mydas (97.95%) and Mus musculus (95.89%). Secondly, reverse transcription-polymerase chain reaction (RT-PCR) and real-time quantitative polymerase chain reaction (RT-qPCR) analysis showed that rad51 mRNA was highly expressed in both ovary and testis, while being weak in the somatic tissues examined in this study. Furthermore, chemical in situ hybridization (CISH) was performed to examine the expression profile of rad51 mRNA in germ cells at different stages. In the testis, rad51 mRNA expression was found to be stronger in the germ cells at early stages, specifically in spermatogonia and spermatocytes, but it was undetectable in spermatids. In the ovary, rad51 mRNA exhibited a uniform distribution in the cytoplasm of oocytes at early stages. The signal intensity of rad51 mRNA was highest in primary oocytes and gradually declined during oogenesis as the oocytes developed. These results suggest that rad51 plays a vital role in the development of germ cells, particularly during the early stages of gametogenesis in P. sinensis. The dynamic expression pattern of rad51 mRNA provides insights into the mechanisms underlying germ cell development and differentiation into gametes in turtles, even in reptiles.

Dietary Curcumin Supplementation Could Improve Muscle Quality, Antioxidant Enzyme Activities and the Gut Microbiota Structure of Pelodiscus sinensis.

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.

Reutilization of Pelodiscus sinensis (Chinese Soft-Shelled Turtle) Visceral Waste for cosmeceutical Applications

Pelodiscus sinensis, commonly known as the Chinese soft-shelled turtle, possesses abundant collagen, proteins, and amino acids in its shell and muscle. Pelodiscus sinensis has been historically utilized as a valuable traditional Chinese medicine. While the Chinese consume the meat and shell of the turtle, the internal visceral organs are typically discarded. The visceral organs contain valuable components, including 8%-15% collagen and 8% blood. This study focused on extracting collagen from the internal visceral organs, while the remaining turtle parts were condensed into a refined product referred to as "Turtle refined," for cosmeceutical applications. We explored the acid solubilization (ASC) and pepsin solubilization (PSC) techniques to extract collagen. Preliminary findings indicate that the PSC method outperformed the ASC method in terms of collagen extraction. To characterize the collagen derived from the visceral organs and blood, SDS-PAGE, FTIR and DSC analyses were employed. Furthermore, we assessed the antioxidant property of the Turtle refined product, utilizing the DPPH radical scavenging activity test.

Redescription of the soft-shell turtle Rafetus bohemicus (Testudines, Trionychidae) from the Early Miocene of Czechia.

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.

Transcriptiomics analysis reveals immue regulation and cell death regulation of IFNg in Pelodiscus sinensis STA cells

Our previous study showed that overexpression interferon gamma (IFNg) in soft-shelled turtle (Pelodiscus sinensis) artery cell line (STA cells) could induce soft-shelled turtle iridovirus (STIV)-infected cell death and inhibit STIV replication. To understand the molecular basis, four samples (named as Ctrl, Ctrl_STIV, IFNg and IFNg_STIV) including cells treatment with recombinant IFNg (rIFNg) or not, and infected or mock-infected with STIV were used to transcriptome analysis. A total of 77.18 Gb clean data were obtained and the clean data of each sample was over 5.97 Gb. Compared to the Ctrl group, there are 2172, 1192 and 2638 DEGs were detected, with 1127, 580 and 1349 up-regulated genes and 1045, 612, and 1259 down-regulated genes, in STA cells after treatment with IFNg, Ctrl_STIV and IFNg_STIV respectively. Subsequently, DEGs were annotated and enriched to GO items and the KEGG pathway, the result showed that immune activation of rIFNg in STA cells including Jak-STAT signaling activation, pattern recognition receptor (PRR) mediated innate immunity activation and cell death regulation etc. The activation of PRR mediated innate immunity may contribute to antiviral activity of IFNg, and it was not affected by STIV infection. From the aspect of cell death, rIFNg could regulated a series of cell death associated Go items including positive regulation and negative regulation, however some GO items including negative regulation of cell death were no longer enriched after STIV infection. The enriched death-related KEGG pathways including “Apoptosis” and “Necroptosis” were enriched in STA cells after rIFNg treatment, and however, besides “Necroptosis”, “Cellular senescence”, “p53 signaling pathway” were enriched after STIV infection. All of these indicate that rIFNg could build an antiviral activity in STA cells and may induce STIV-infected cell death through limiting the negative regulation of cell death and inducing necroptosis, apoptosis and cellular senescence.

Pathological characteristics of Chinese soft-shelled turtle (Pelodiscus sinensis) with white abdominal disease

White abdominal disease is one of the major diseases in Chinese soft-shelled turtle (Pelodiscus sinensis, SST) aquaculture. To clarify the pathological characteristics of white abdominal disease, 24 SSTs with typical white abdominal symptoms were collected from a farm in Hubei Province, China. First, the pathogens of the SSTs were detected, including viruses and bacteria. No virus was detected. A variety of bacteria have been isolated. The pathological characteristics of the diseased SSTs were then studied using hematological analysis, histopathological observation, intestinal microbial analysis, and immunity-related gene expression. Compared with the healthy SST, the number of red blood cells reduced from 1.2 ± 0.5 × 1013 /L to 1.5 ± 0.2 × 1012 /L in the diseased SSTs. Meanwhile, the number of white blood cells increased by nearly four-fold (from 1.8 ± 0.5 × 1010 /L to 7.1 ± 3.5 × 1010 /L). In the diseased SSTs, the liver displayed several abnormalities, such as hepatocyte vacuolation and changes in nonparenchymal tissue. Some glomeruli of the kidney showed necrosis and disintegration. Cavities and loose muscles were observed in the gastric muscle layer. The blurred boundary between the red pulp and white pulp of the spleen was accompanied by massive invasion of inflammatory cells. Significant differences were detected in the relative abundances of Proteobacteria (from 29% to 7%) and Fusobacteria (from 12% to 28%) between the diseased and healthy SSTs. Compared with those in the healthy SSTs, the gene expression levels of toll-like receptor 2(TLR), TLR3, and myeloid differentiation primary response 88 (MyD88) were increased, by more than 4-fold, 7-fold, and 5-fold, respectively (P < 0.01) in the diseased SST liver. The TLR2, TLR4, and MyD88 expression levels increased significantly in the diseased SST kidney. These results indicated that the diseased SSTs experienced anemia, visceral tissue lesions, reduction of intestinal microbial species, and innate immune activation.

Effect of Exogenous Hormone on R-Spondin 2 (Rspo2) and R-Spondin 3 (Rspo3) Gene Expression and Embryo Development in Chinese Soft-Shelled Turtle (Pelodiscus sinensis).

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.