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.

A 60 day feeding trial was conducted to evaluate the impacts of dietary carbohydrates with different complexities and configurations on the growth, plasma parameters, apparent digestibility, intestinal microbiota, glucose, and lipid metabolism of soft-shelled turtles (Pelodiscus sinensis). Four experimental diets were formulated by adding 170 g/kg glucose, fructose, α-starch, or cellulose, respectively. A total of 280 turtles (initial body weight 5.11 ± 0.21 g) were distributed into 28 tanks and were fed twice daily. The results showed that the best growth performance and apparent digestibility was observed in the α-starch group, followed by the glucose, fructose, and cellulose groups (p < 0.05). Monosaccharides (glucose and fructose) significantly enhanced the postprandial plasma glucose levels and hepatosomatic index compared to polysaccharides, due to the un-inhibited gluconeogenesis (p < 0.05). Starch significantly up-regulated the expression of the genes involved in glycolysis, pentose phosphate pathway, lipid anabolism and catabolism, and the transcriptional regulation factors of glycolipid metabolism (srebp and chrebp) (p < 0.05), resulting in higher plasma triglyceride levels and lipid contents in the liver and the whole body. The fructose group exhibited a lower lipid deposition compared with the glucose group, mainly by inhibiting the expression of srebp and chrebp. Cellulose enhanced the proportion of opportunistic pathogenic bacteria. In conclusion, P. sinensis utilized α-starch better than glucose, fructose, and cellulose.

The DKK family is a canonical small family of WNT antagonists. Though recent studies have suggested that the DKK gene family may be involved in sex differentiation in Pelodiscus sinensis, there are still a lot of things about the DKK gene family that we do not know. In this study, we used bioinformatics methods to identify members of the DKK gene family in P. sinensis and analyzed their phylogeny, covariance, gene structure, structural domains, promoter conserved sites, signal peptides, gonadal transcription factors, transcriptional profiles, and tissue expression profiles. Additionally, qRT-PCR results were utilized for the validation and preliminary investigation of the function of the DKK gene family in P. sinensis. The results showed that the DKK gene family is divided into six subfamilies, distributed on six different chromosomal scaffolds containing different gene structures and conserved motifs with the same structural domains, and all of the members were secreted proteins. Our transcriptional profiling and embryonic expression analysis showed that DKKL1 and DKK4 were significantly expressed in the testes, whereas DKK1 and DKK3 were significantly upregulated in the ovaries. This suggests a potential function in sex differentiation in P. sinensis. Our results may provide a basic theoretical basis for the sex differentiation process in P. sinensis.

Chinese soft-shelled turtle (Pelodiscus sinensis) hibernates without eating and drinking when the ambient temperature is very low. To better understand the characteristics of energy utilization during hibernation, the turtles in the physiological phases of summer active (SA), Pre-Hibernation (Pre-H), Mid-Hibernation (Mid-H) and early arousal (EA) were sampled. The results showed that the levels of serum triglyceride and hepatic lipid droplet were markedly increased in Pre-H and decreased in Mid-H compared with that in SA, indicating that P. sinensis experiences lipid accumulation in Pre-H and lipid is the predominant energy reserve during hibernation. The mRNA expression levels of genes (FABP and CPT-2) involved in lipolysis and lipid oxidation were up-regulated in Mid-H, while the genes related to lipid synthesis (FAS, ACSL-1, ACC, elovl5, and SCD1) were inhibited in Mid-H. Meanwhile, the mRNA expression levels of endoplasmic reticulum stress marker gene Bip and key genes (ATF4, ATF6, and IRE1α) involving the unfolded protein response were significantly increased in Mid-H and EA. Also, the expression levels of genes (ASK1, JNK1, and Bax) associated with cell apoptosis increased in Mid-H and EA, however, the expression of Bcl2 was inhibited in Mid-H. Therefore, hibernation can cause endoplasmic reticulum stress and apoptosis. The findings will provide a theoretical framework for an animal\'s cold adaptation and offer insights into preventing and managing metabolic syndrome.

BACKGROUND: TRIM proteins, recognized as a class of E3 ubiquitin ligases, are increasingly acknowledged for their antipathogen immune functions in mammals and fish. In the Chinese soft-shelled turtle (Pelodiscus sinensis), a secondary aquatic reptile that occupies a unique evolutionary position, the TRIM gene has rarely been reported.
RESULTS: In the present study, 48 PsTRIM proteins were identified from the genome of Pelodiscus sinensis via Hidden Markov Model (HMM) searches and Signal Transduction ATPases with Numerous Domains (SMART) analysis. These PsTRIMs were found across 43 distinct scaffolds, and phylogenetic analyses classified them into three principal clades. The PsTRIMs feature a conserved assembly of either RING-B-box-coiled-coil (RBCC) or B-box-coiled-coil (BBC) domains at the N-terminus, in addition to eight unique domains at the C-terminus, including the B30.2 domain, 19 of which were identified. Expression profiling revealed ubiquitous expression of the 48 PsTRIMs across various P. sinensis tissues. Notably, seven PsTRIMs exhibited significant differential expression in liver transcriptomes following infection with Aeromonas hydrophila. Weighted gene coexpression network analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis implicated PsTRIM14 and PsTRIM28 as key players in host defense against bacterial invasion. Real-time quantitative PCR results indicated that PsTRIM1, PsTRIM2, PsTRIM14, and PsTRIM28 experienced marked upregulation in P. sinensis livers at 12 h post-infection with A. hydrophila.
CONCLUSIONS: Our study is the first to comprehensively identify and analyze the functions of TRIM genes in P. sinensis, unveiling their considerable diversity and potential roles in modulating immune responses.

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

The Chinese soft-shelled turtle Pelodiscus sinensis, an economically important species in China, exhibits significant sexual dimorphism. Males are more valuable than females owing to their wider calipash and faster growth. Estradiol (E2)-induced sex reversal is used to achieve all-male breeding of turtles; however, the mechanism of this sex reversal remains unclear. In this study, we characterized the Sox3 gene, whose expression level was high in the gonads and brain and exhibited significant sexual dimorphism in the ovary. During embryonic development, Sox3 was highly expressed at the initiation of ovarian differentiation. E2 and Sox3-RNAi treatment before sexual differentiation led to 1352, 908, 990, 1011, and 975 differentially expressed genes in five developmental stages, respectively, compared with only E2 treatment. The differentially expressed genes were clustered into 20 classes. The continuously downregulated and upregulated genes during gonadal differentiation were categorized into Class 0 (n = 271) and Class 19 (n = 606), respectively. KEGG enrichment analysis showed that Sox3 significantly affected sexual differentiation via the Wnt, TGF-β, and TNF signaling pathways and mRNA surveillance pathway. The expression of genes involved in these signaling pathways, such as Dkk4, Nog, Msi1, and Krt14, changed significantly during gonadal differentiation. In conclusion, the deletion of Sox3 may lead to significant upregulation of the mRNA surveillance pathway and TNF and Ras signaling pathways and downregulation of the Wnt and TGF-β signaling pathways, inhibiting E2-induced sex reversal. These findings suggest that Sox3 may play a certain promoting effect during E2-induced sex reversal in P. sinensis.

The aim of this study was to evaluate the effect of brown fishmeal in replacement of white fishmeal in the diet of Chinese soft-shelled turtles and to find the optimal amount of brown fishmeal to add. Five experimental groups were set up and fed to animals, and they were composed by different proportions of white and brown fishmeal: G1 (30% white and 25% brown fishmeal), G2 (25% white and 30% brown fishmeal), G3 (20% white and 35% brown fishmeal), G4 (15% white and 40% brown fishmeal), G5 (10% white and 45% brown fishmeal). G1 is regarded as the control group. Turtles were randomly divided into five experimental groups with four replicates each. The experiment lasted 72 days. The results showed that the WGR, SGR, FCR, and HSI of the G3 group were not significantly different from those of the control group (P > 0.05). In addition, brown fishmeal can increase the crude protein content in the muscles of them. Among the serum biochemical indices, there was no significant difference between the G3 group and the G1 group, except for the level of TG (P > 0.05). Meanwhile, the activities of AST, ALT, and CAT in the liver of the G3 group did not differ significantly from those of the G1 group (P > 0.05). However, the activities of ACP, AKP, and T-AOC were significantly decreased in the G3 group (P < 0.05). In addition, the alteration of fishmeal did not affect the digestive enzyme activities in the stomach, liver, and intestine, and there is no significant difference (P > 0.05). Importantly, with increasing brown fishmeal addition, the expression of Fas, Pparγ, Scd, and Stat3 showed a significant increase, while the expression of Bmp4 decreased significantly (P < 0.05). In this study, the addition of 20% white fishmeal and 35% brown fishmeal to the diet of Chinese soft-shelled turtles did not adversely affect growth performance. Therefore, 20% white fishmeal and 35% brown fishmeal are the most practical feed formulations for Chinese soft-shelled turtles in this study.

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.

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.