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Pelodiscus sinensis is an aquatic product with a long growth cycle in pond culture and high nutritional value meat. The flavor compounds, nutrients, and lipidome were investigated to explore the edible value changes of turtle meat aged 3 to 6 years (Y3 to Y6). Typically, P. sinensis meat is rich in high-quality protein (EAAI ≥81.22, AAS ≥86.47). Y6 has the highest level of Se, protein, amino acids, and high unsaturated fatty acids, including EPA + DHA. Y5 has the most delicious amino acids, polyunsaturated fatty acids, and key odorant content. The stronger flavor of Y5 may be mainly related to C18:2n6t and C18:2n6c. Further, triacylglycerols (TAG) and phosphatidylcholine (PC) were significant changes in Y5. Additionally, PI (16:0/18:1) was identified as the potential biomarker. These results provided available information on P. sinensis marketing age and revealed the potential impact of nutrients on the formation of VOCs.

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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, Il1ß, 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 L-glutamic 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 soft-shelled turtle displays stage-specific physiological responses to resist A. hydrophila infection.

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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.

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Chemokines are small, secreted proteins with chemoattractive properties, which play an important role in the recruitment and activation of immune cells. CXCL11 is a CXC chemokine specific for the CXCR3 receptors, which has been shown to mediate the generation of Th1-type immune responses and have bactericidal effects similar to defensins. Herein, we cloned the full-length cDNA of Chinese soft-shelled turtle (Pelodiscus sinensis) CXCL11, designated as PsCXCL11, which consist of an open reading frame (ORF) of 282 bp encoding 93 amino acids, with estimated molecular weight of 10.055 kDa and isoelectric point of 10.37. The deduced PsCXCL11 sequence had a signal peptide, a highly conserved family-specific small cytokine (SCY) domain, one putative N-glycosylation site and ten potential phosphorylation sites. Phylogenetic analysis showed a close relationship between P. sinensis and Chelydra Serpentina CXCL11. P. sinensis CXCL11 basal expression levels were higher in heart, kidney and spleen than in other organs of health turtles. Infections of Aeromonas hydrophila and Staphylococcus aureus led to significant upregulation of P. sinensis CXCL11 in the blood, while significant upregulation of PsCXCL11 were observed in liver and spleen after infection of A. hydrophila, but not S. aureus. PsCXCL11 recombinant protein with His-tag was successfully expressed by an auto-inducible expression system, and purified by Ni-NTA affinity chromatography. These findings laid a solid foundation for further research towards development of the Chinese soft-shelled turtle as a model for the role of CXCL11 in regulating inflammatory responses to stimulation by invading pathogens.

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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-ß-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-ß pathway. Female-biased genes such as Smad4, Wif1, and 17-ß-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.

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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.

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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.

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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.

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OBJECTIVE To optimize the preparation process of Soft-shelled turtle blood lyophilized powder （STBLP）， and to provide a reference for improving the availability and quality stability of soft-shelled turtle blood （STB）. METHODS STBLP was prepared with vacuum freeze-drying. Taking the solubility as the index， the preparation process parameters of STBLP were optimized by single factor experiment and Box-Behnken response surface method. RESULTS The optimal freeze-drying process for STBLP was obtained： pre-freezing time of 4 h， total drying time of 13 h （before at 0 ℃）， and resolution drying temperature of 25 ℃. The average solubility of 3 batches of STBLP prepared according to the optimal process was 95.72% （RSD＝0.68%， n＝3）， the relative error of which was －0.97% to the theoretical solubility （96.66%）. CONCLUSIONS Optimized lyophilization process in this study are stable and feasible， the solubility of the prepared sample is high.

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The digestive tract development of the Pelodiscus sinensis embryo is described through the observation of the embryonic morphology on hematoxylin and eosin stained tissue sections. During the first 9 days of embryonic development, the anterior intestine of the embryo divides into the oral cavity, pharyngeal cavity, esophagus, stomach, and small intestine, while the caudal intestine differentiates into the cloaca, the anterior and caudal tubes of the large intestine. Between days 10-24, the wall of the digestive tract forms a two-layer structure consisting of mucosa and submucosa. The endoderm evolves into epithelial tissue in each part of the digestive tract, the mesoderm goes from a dense cluster of cells to looser mesenchymal tissue then divides into loose connective tissue, mesothelium, and muscle tissue. There is no clear temporal boundary between development of mesenchymal tissue and the early loose connective tissue, which is a gradual process.

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In traditional Chinese medicine, soft-shelled turtle protein and peptides serve as a nutraceutical for prolonging the lifespan. However, their effects on anti-aging have not been clarified scientifically in vivo. This study aimed to determine whether soft-shelled turtle peptides (STP) could promote the lifespan and healthspan in Drosophila melanogaster and the underlying molecular mechanisms. Herein, STP supplementation prolonged the mean lifespan by 20.23% and 9.04% in males and females, respectively, delaying the aging accompanied by climbing ability decline, enhanced gut barrier integrity, and improved anti-oxidation, starvation, and heat stress abilities, while it did not change the daily food intake. Mechanistically, STP enhanced autophagy and decreased oxidative stress by downregulating the target of rapamycin (TOR) signaling pathway. In addition, 95.18% of peptides from the identified sequences in STP could exert potential inhibitory effects on TOR through hydrogen bonds, van der Walls, hydrophobic interactions, and electrostatic interactions. The current study could provide a theoretical basis for the full exploitation of soft-shelled turtle aging prevention.

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Constant darkness and constant light exposure often disturb the circadian rhythm in the behavior and energy metabolism of vertebrates. Melatonin is known as the hormonal mediator of photoperiodic information to the central nervous system and plays a key role in food intake and energy balance regulation in vertebrates. The popularly cultured soft-shelled turtle Pelodiscus sinensis has been reported to grow better under constant darkness; however, the underlying physiological mechanism by which darkness benefits turtle growth is not clear yet. We hypothesized that increased melatonin levels induced by darkness would increase appetite and energy metabolism and thus promote growth in P. sinensis. In addition, in order to elucidate the interaction of photoperiod and density, juvenile turtles were treated under three photoperiods (light/dark cycle: 24L:0D, 12L:12D, 0L:24D, light density 900 lux) and two stocking densities (high density: 38.10 ind./m2, low density: 6.35 ind./m2) for 4 weeks, and then the blood and brain tissues of turtles were collected during the day (11:00-13:00) and at night (23:00-1:00) after 2 days of fasting. We examined changes in plasma melatonin levels, food intake (FI), and appetite-related hormones (plasma ghrelin and leptin), as well as growth and energy metabolism parameters such as specific growth rate (SGR), standard metabolic rate (SMR), plasma growth hormone (GH), and thyroid hormone/enzyme activity (plasma triiodothyronine T3, thyroxine T4, and T45'-deiodinase activity). Moreover, we also assessed the responses of mRNA expression levels of food intake-related genes (kisspeptin 1 (Kiss1); cocaine amphetamine-regulated transcript (CART); neuropeptide Y (NPY)) in the brain. The results showed that under high density, SGR was the lowest in 24L:0D and the highest in 0L:24D. FI was the highest in 0L:24D regardless of density. Plasma melatonin was the highest in 0L:24D under high density at night. SMR increased with decreasing light time regardless of density. Most expressions of the measured appetite-related genes (Kiss1, CART, and NPY) were not affected by photoperiod, nor were the related hormone levels, such as plasma leptin, ghrelin, and GH. However, thyroid hormones were clearly affected by photoperiod. T3 level in 0L:24D under high density during the day was the highest among all treatment groups. T4 in 24L:0D under high density during the day and T45'-deiodinase activity in 24L:0D under low density at night were significantly reduced compared with the control. Furthermore, the energy metabolism-related hormone levels were higher under higher density, especially during the day. Together, melatonin secretion is not only modulated by light but also likely to be regulated by unknown endogenous factors and density. Altered plasma melatonin induced by constant darkness and density seems to be involved in the modulation of energy metabolism rather than appetite in the soft-shelled turtle.

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Chinese soft-shelled turtles display obvious sex dimorphism. The exogenous application of hormones (estradiol and methyltestosterone) can change the direction of gonadal differentiation of P. sinensis to produce sex reversed individuals. However, the molecular mechanism remains unclear. In this study, TMT-based quantitative proteomics analysis of four types of P. sinensis (female, male, pseudo-female, and pseudo-male) gonads were compared. Quantitative analysis of 6107 labeled proteins in the four types of P. sinensis gonads was performed. We identified 440 downregulated and 423 upregulated proteins between pseudo-females and males, as well as 394 downregulated and 959 upregulated proteins between pseudo-males and females. In the two comparisons, the differentially expressed proteins, including K7FKG1, K7GIQ2, COL4A6, K7F2U2, and K7FF80, were enriched in some important pathways, such as focal adhesion, endocytosis, apoptosis, extracellular matrix-receptor interaction, and the regulation of actin cytoskeleton, which were upregulated in pseudo-female vs. male and downregulated in pseudo-male vs. female. In pathways such as ribosome and spliceosome, the levels of RPL28, SRSF3, SNRNP40, and HNRNPK were increased from male to pseudo-female, while they decreased from female to pseudo-male. All differentially expressed proteins after sexual reversal were divided into six clusters, according to their altered levels in the four types of P. sinensis, and associated with cellular processes, such as embryonic development and catabolic process, that were closely related to sexual reversal. These data will provide clues for the sexual reversal mechanism in P. sinensis.

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Peptides derived from egg protein hydrolysate have potential hypoglycemic benefits by inhibiting α-glucosidase. Herein, fluorescence spectroscopy and molecular docking were performed to investigate the α-glucosidase inhibitory mechanism of the oligopeptides ARDASVLK and HNKPEVEVR from soft-shelled turtle eggs. Gastrointestinal digestion characteristics of the two oligopeptides were further determined by LC-QTOF-MS/MS. The static fluorescence quenching of α-glucosidase by ARDASVLK and HNKPEVEVR indicated the formation of a stable α-glucosidase-peptide complex, mainly driven by hydrogen bonds and hydrophobic interactions. ARDASVLK and HNKPEVEVR could easily insert into the active pocket of α-glucosidase (docking scores of -157.1 and -158.4, respectively), thereby inhibiting enzyme activity by preventing substrate binding and inducing enzymatic conformation change. After gastrointestinal digestion, 14.3% and 30.4% of ARDASVLK and HNKPEVEVR were maintained intact, respectively, and their digestive products (mainly DASVLK and HNKPEVEV) still showed high inhibitory effects on α-glucosidase (about 35% inhibition). This study sheds light on the mechanism of oligopeptides derived from soft-shelled turtle eggs as a novel α-glucosidase inhibitor for diabetes. PRACTICAL APPLICATIONS: Oligopeptides from egg protein hydrolysate have potential hypoglycemic properties by inhibiting α-glucosidase. This study has provided insights into the inhibitory mechanism of oligopeptides from soft-shelled turtle egg on α-glucosidase. Interestingly, despite the fact that the oligopeptides are largely degraded during gastrointestinal digestion, their digestive metabolites displayed strong α-glucosidase inhibitory activities. Because α-glucosidase is highly expressed in small intestine brush border, our findings support the possibility of these oligopeptides as an attractive health-benefit compound to control glucose without being absorbed by intestinal epithelial cells, unlike other enzyme inhibitors such as ACE inhibitors, which have poor oral bioavailability. This study may facilitate the applications of oligopeptides from soft-shelled turtle egg as α-glucosidase inhibitors and food functional ingredients for the therapy of diabetes.

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Type I interferons (IFNs) are critical cytokines for the establishment of antiviral status in fish, amphibian, avian and mammal, but the knowledge on type I IFNs is rather limited in reptile. In this study, seven type I IFN genes, designed as IFN1 to IFN7, were identified from a reptile species, the Chinese soft-shelled turtle (Pelodiscus sinensis). These identified type I IFNs have relatively low protein identity, when compared with those in human and chicken; but they possess conserved cysteines, predicted multi-helix structure and N-terminal signal peptide. The Chinese soft-shelled turtle IFN1 to IFN5 have two exons and one intron, but IFN6 and IFN7 are the single-exon genes. Chinese soft-shelled turtle type I IFNs are located respectively on the two conserved reptile-bird loci, named as Locus a and Locus c, and are clustered into the four of the five reptile-bird groups (named as Groups I-V) based on phylogenetic evidence, due to the lack of IFNK in the turtle. Moreover, the Chinese soft-shelled turtle type I IFNs can be induced by soft-shelled turtle iridovirus (STIV) infection and show antiviral activity in soft-shelled turtle artery (STA) cells, except IFN6. In addition, due to the difference in genome organizations, such as the number of exons and introns of type I IFN genes from fish to mammal, the definition and evolution of 'intronless' type I IFN genes were discussed in lineages of vertebrates. Thus, the finding of type I IFNs on two different loci in P. sinensis sheds light on the evolution of type I IFN genes in vertebrates.

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Exogenous estrogen have shown their feminization abilities during the specific sex differentiation period in several reptiles. However, the specific regulatory mechanism and downstream regulatory genes of estrogen remain elusive. In the present study, 17ß-estradiol (E2), as well as drugs of specific antagonists and/or agonists of estrogen receptors, were employed to figure out the molecular pathway involved in the E2-induced feminization in Chinese soft-shelled turtles, an important aquaculture species in China. E2 treatment led to typical female characteristics in the gonads of ZZ individuals, including thickened outer cortex containing a number of germ cells and degenerated medullary cords, as well as the disappearance of male marker SOX9, and the ectopic expression of ovarian regulator FOXL2 at the embryonic developmental stage 27 and 1 month after hatching. The specific ESR1 antagonist or a combination of three estrogen receptor antagonists could block the sex reversal of ZZ individuals induced by estrogen. In addition, specific activation of ESR1 by agonist also led to the feminization of ZZ gonads, which was similar to the effect of estrogen treatment. Furthermore, transcriptome data showed that the expression level of FOXL2 was significantly upregulated, whereas mRNA levels of DMRT1, SOX9, and AMH were downregulated after estrogen treatment. Taken together, our results indicated that E2 induced the feminization of ZZ Chinese soft-shelled turtles via ESR1, and decrease of male genes DMRT1, SOX9, and AMH and increase of ovarian development regulator FOXL2 might be responsible for the initiation of E2-induced feminization.

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Sex dimorphism is a key feature of Chinese soft-shelled turtle (Pelodiscus sinensis). The males (M) have higher econosmic value than females (F) due to wider calipash and faster growth. Exogenous hormones like estradiol and methyltestosterone can induce sexual reversal to form new phenotypes (pseudo-female, PF; pseudo-male, PM) without changing the genotype. The possibility of inducing sexual reversal is particularly important in aquaculture breeding, but the underlying biological mechanisms remain unclear. Here we applied a direct RNA sequencing method with ultralong reads using Oxford Nanopore Technologies to study the transcriptome complexity in P. sinensis. Nanopore sequencing of the four gender types (M, F, PF, and PM) showed that the distribution of read length and gene expression was more similar between same-sex phenotypes than same-sex genotypes. Compared to turtles with an M phenotype, alternative splicing was more pronounced in F turtles, especially at alternative 3' splice sites, alternative 5' splice sites, and alternative first exons. Furthermore, the two RNA methylation modifications m5C and m6A were differentially distributed across gender phenotypes, with the M type having more modification sites in coding sequence regions, but fewer modification sites in 3'UTR regions. Quantitative analysis of enriched m6A RNAs revealed that the N6-methylated levels of Odf2, Pacs2, and Ak1 were significantly higher in M phenotype individuals, while the N6-methylated levels of Ube2o were reduced after sexual reversal from both M and F phenotypes. Taken together, these findings reveal an important role of epigenetics during sexual reversal in Chinese soft-shelled turtles.

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In this study, we use a specimen from wild-caught individual to determine the complete mitochondrial genome of the Amur soft-shelled turtle (Pelodiscus maackii). The complete mitogenome of P. maackii has 16,258 bp in length and consists of 13 protein-coding genes (PCGs), 22 tRNAs, two rRNAs, and one control region. The arrangement of genes of P. maackii is identical with previously reported mitogenomes in the family Trionychoidea. According to our result, the ML tree for the phylogenetic reconstruction revealed that the individuals used in present study is closely related with the previously reported sequences of P. sinensis (AY962573 and MG431983) in p-distance 0.7% and 2.5%.

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Hibernation is a biological status during which hibernating animals acclimatize themselves to reduced energy consumption through extreme but governed decline in self-metabolism. The role of mitochondria (Mt) in metabolic suppression during hibernation has already been elaborated in different organs and species. Nonetheless, the concretely changing process of mitochondrial architecture and the mechanism underlying this transformation during hibernation remains unclear. Herein, the present study was aimed at clarifying the detailed alteration of mitochondrial morphology and its potential role in the Chinese soft-shelled turtle (Pelodiscus sinensis) during different stages of hibernation. Compared with the nonhibernation period, the mitochondrial architecture was changing from round to crescent, and lipid droplet (LD)/Mt interaction was enhanced during hibernation, as observed by transmission electron microscopy (TEM). Further ultrastructural analysis uncovered that mitochondrial fusion was promptly accelerated in the early stage of hibernation, followed by mitochondrial fission in the middle stage, and mitophagy was boosted in the late stage. Moreover, gene and protein expression related to mitochondrial fusion, fission, and mitophagy accorded closely with the mitochondrial ultrastructural changes in different stages of hibernation. Taken together, our results clarified that the transformation of mitochondrial architecture and mitochondrial dynamics are of vital importance in maintaining internal environment homeostasis of Pelodiscus sinensis.

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The potential of soft-shelled turtle peptides (STP) against fatigue was evaluated. Mice orally supplemented with STP significantly increased the swimming time until tiredness by 35.4-57.1%. Although not statistically significant, STP increased muscle and thymus mass. In addition, the serum lactate, ammonia, blood urea nitrogen content and creatine kinase activity in STP-fed mice were dramatically decreased when compared to the control group. Furthermore, STP supplementation increased the reserves of liver glycogen and muscle glycogen, thus improved the energy metabolism system of mice. STP treatment contributed to increased superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities as well as a decrease in malondialdehyde (MDA), indicating an improvement in oxidative stress protection. The Western blot (WB) results indicated that the STP supplement effectively altered the expression of oxidative stress-related protein by modulating the NRF2/KEAP1 pathway. In summary, STP affected NRF2/KEAP1 levels in skeletal muscle, leading to antioxidant activity and a slower time to exhaustion during exercise.