Chengqing Wei

The critically endangered Asian giant softshell turtle (Pelochelys cantorii) is a national first-class protected aquatic animal in China, and artificial breeding is vital for its conservation. Given the pivotal role of substrate in captive rearing, this study aimed to investigate the effects of different substrate types on the growth, serum biochemistry, and behavior of juvenile P. cantorii. A total of 45 8-month-old juveniles [(121.11 ± 0.65) g] were randomly allocated to three groups (fine sand [FS], pea gravel [PG], and no substrate [NS]) for an 18-day rearing trial. Results indicated that the FS and PG groups exhibited significantly higher weight gain and specific growth rates than the NS group (p < 0.01). Serum malondialdehyde (MDA) levels were lower in the FS and PG groups than in the NS group (p < 0.05), with no significant difference between FS and PG. Notably, three individuals in the NS group exhibited symptoms of skin ulceration. No significant intergroup differences were observed in glucose (GLU), triglyceride (TG), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), or cortisol (CORT) levels (p > 0.05). Behaviorally, the FS group demonstrated the highest hidden rest frequency and duration (p < 0.01) and significantly lower active avoidance behavior compared to PG and NS (p < 0.01). In conclusion, substrate type significantly influences captive juvenile P. cantorii, with fine sand being optimal as it enhances growth, alleviates oxidative stress, and reduces maladaptive behaviors.

The formation of the Chinese three-keeled pond turtle (Mauremys reevesii) carapace is a complex biological event involving developmental processes such as collagen deposition and ossification. As a key regulator of collagen deposition and ossification, Col10a1 may play a crucial role in carapace development, though its specific mechanism remains unclear. To investigate the role of Col10a1 during carapace development and its regulatory mechanisms, we cloned its cDNA sequence and performed bioinformatic analysis. This revealed that Col10a1 encodes a stable, hydrophilic, and basic protein, with phylogenetic analysis showing closest evolutionary relationships to other reptiles and the greatest divergence from fish. Further RT-qPCR analysis examined Col10a1 expression patterns in M. reevesii embryos at stages 14, 18, and 22, as well as in various tissues of adult males and females. Results indicated that during embryonic development, Col10a1 expression levels progressively increased alongside the progression of carapace ossification and collagen deposition, suggesting its involvement in regulating this process. In adult tissues, Col10a1 exhibited widespread expression, with particularly high levels in the brain, kidneys, and liver, suggesting potential specialized functions in these organs. Finally, in vitro experiments demonstrated that inhibition of the Wnt/β-catenin pathway with salinomycin sodium salt downregulated the expression of both its target genes (Sp5, Myc, Ccnd1) and Col10a1. In contrast, inhibition of the TGF-β/Smad pathway with oxymatrine suppressed its target genes (Serpine1, Cdkn1a) but concomitantly upregulated Col10a1. These results suggest that Col10a1 expression may be positively regulated by the Wnt/β-catenin pathway and negatively regulated by the TGF-β/Smad pathway. Our findings provide novel insights into the molecular mechanisms governing carapace development and collagen deposition in M. reevesii, laying a crucial foundation for further investigations into the regulatory networks involving Col10a1 during carapace formation.

Background/Objectives: The Sox transcription factor family is critical for gonadal development and sex differentiation in animals, yet its roles in chelonians, particularly in the Chinese soft-shelled turtle (Pelodiscus sinensis), have rarely been investigated. Methods: This study cloned and analyzed the cDNA sequences of Sox3 and Sox30 genes from P. sinensis, examining their amino acid sequences and structural properties. Real-time quantitative PCR (RT-qPCR) was used to assess the expression of these two genes in different adult tissues and at various stages of embryonic gonadal development. Additionally, the effects of exogenous hormones (17β-estradiol, E₂ and 17α-Methyltestosterone, MT) on the expression of Sox3 and Sox30 were also investigated. Results: The results indicated that Sox3 showed significantly elevated expression in female gonads, kidney, brain, liver, lung, spleen, and muscle relative to male counterparts, displaying a female-biased expression pattern. In contrast, Sox30 showed a male-biased pattern, with higher expression in male gonads, spleen, muscle, brain, and liver than in females, showing expression. Both genes were expressed at low levels. Exogenous hormone treatments revealed that MT significantly downregulated Sox3 expression in female embryos, whereas E₂ significantly enhanced Sox3 expression in male embryos. Furthermore, MT treatment significantly upregulated Sox30 expression in female embryos, and E₂ treatment also significantly increased Sox30 expression in male embryos. Conclusions: These findings suggest that Sox3 and Sox30 play crucial roles in the gonadal development of P. sinensis, with Sox3 potentially involved in ovarian development and Sox30 in testicular maturation. Both genes are regulated by exogenous hormones, highlighting their importance in sex differentiation and gonadal development. This study provides valuable theoretical insights for further exploration of the molecular mechanisms of sex regulation in reptiles.

In farmed animals, body color is not only an ecological trait but also an important trait that influences the commercial value of the animals. Melanin plays an important role in the formation of body color in animals, while the tyrosinase (TYR) gene family is a group of key enzymes that regulate melanogenesis. The Chinese soft-shelled turtle (Pelodiscus sinensis) is one of the most important reptiles in freshwater aquaculture. However, the potential role of the TYR gene family in the body color formation of P. sinensis remains unclear. This study aimed to investigate the expression and conservation of the TYR gene family in relation to body color variation in P. sinensis. Three core members of this gene family were identified from the P. sinensis genome. Following identification, the genomic features were analyzed. They shared similar physicochemical properties and conserved domains. Chromosome mapping showed that the three genes of P. sinensis were all located on the autosomes, while phylogenetic and collinearity analysis suggested that the protein functions of the three genes in the studied species were highly conserved. Amino acid sequence alignment indicated high conservation among the three TYR gene family proteins (TYR, TYRP1, and DCT) in multiple critical regions, particularly in their hydrophobic N-/C-terminal regions and cysteine/histidine-rich conserved domains. The qRT-PCR revealed that the TYR and DCT genes were highly expressed in the eyes of individuals with different body colors. The expression levels of TYR and TYRP1 genes in the skin were significantly higher in dark-colored individuals than in light-colored ones (p < 0.05). These results will lay the groundwork for functional studies and breeding programs targeting color traits in aquaculture.

The Chinese soft-shelled turtle (Pelodiscus sinensis) is an economically important species in aquaculture, and its growth pattern is characterized by significant sexual dimorphism. However, the underlying molecular mechanisms of this phenomenon have mostly been investigated in the gonadal tissues of P. sinensis, and there are no articles on sex differentiation from the brain of P. sinensis. Here, we analyzed transcriptomes of the brains of adult male and female P. sinensis using high-throughput Illumina sequencing technology, establishing a set of differential genes and differential transcription factors. The data showed that there were 908 genes with significant differences in expression, of which 357 genes were up-regulated and 551 genes were down-regulated. We annotated using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG), and screened some genes and pathways related to growth. There were 282 growth-related differential genes and 181 sex-related differential genes. We screened the genes’ growth hormone receptor (GHR) and vascular endothelial growth factor A (VEGFA), which may be related to the growth of P. sinensis. The pathways related to the growth and development of P. sinensis are the growth hormone synthesis, secretion, and action pathway; the MAPK (mitogen-activated protein kinase) pathway; and the calcium signaling pathway. In addition, through gene set enrichment analysis (GSEA), we screened out two genes, LIM homeobox protein 1 (LHX1) and fibroblast growth factor 7 (FGF7), which are related to both growth and sex differentiation, and through protein interaction analysis of these genes, we screened out eight genes, including LHX1, FGF7, GHR, fibroblast growth factor 4 (FGF4), EGFR, BMP3, GLI family zinc finger 2 (GLI2), and neuronal differentiation 1 (NEUROD1), and verified the expression levels of these eight genes in the brain of the P. sinensis by real-time quantitative PCR (qRT-PCR), which supported the reliability and accuracy of our transcriptome analysis. Our study provides a solid foundation for analyzing the mechanisms of sexual-dimorphic growth of P. sinensis and even other turtles.

The Asian yellow pond turtle (Mauremys mutica) is widely traded in China, and its artificial breeding has now become a major industry. However, the insufficient offspring supply and reproductive decline of farmed turtles make the wild turtles more vulnerable. The present study was mainly designed to quantify the fecundity of M. mutica and attempt to screen for good reproductive performance in females. The genetic variability of the population and its genetic structure were also analysed. The parent–offspring relationships of all offspring in four consecutive years were confirmed using sixteen microsatellite loci. The genetic variability between the parents and offspring was low, and offspring of different years also showed little variability. We summarised the reproductive results of all females and counted the annual number of offspring and the variation in the number of offspring. The females were then divided into three types (stable, undulating and levelling off) according to the continuity. We selected seven females with good reproductive ability, which provided 16.94% of the annual contributions, while there were two females that had no offspring in four years. We also analysed the possible reasons for this difference and the importance of carrying out a family survey. This research can provide the basis and materials for the creation of a good reproductive group and the study of the reproductive biology of turtles in M. mutica aquaculture.

Hibernation in turtle species is an adaptive survival strategy to colder winter conditions or food restrictions. However, the mechanisms underlying seasonal adaptions remain unclear. In the present study, we collected hemocytes from Pelochelys cantorii and compared the molecular signature of these cells between the active state and hibernation period based on single-cell RNA sequencing (scRNA-seq) analysis. We found six cell types and identified a list of new marker genes for each cell subpopulation. Moreover, several heat shock genes, including the Hsp40 family chaperone gene (DNAJ) and HSP temperature-responsive genes (HSPs), were upregulated during the hibernation period, which predicted these genes may play crucial roles in the stress response during hibernation. Additionally, compared to hemocytes in the active state, several upregulated differentially expressed immune-related genes, such as stat1, traf3, and socs6, were identified in hemocytes during the hibernation period, thus indicating the important immune function of hemocytes. Therefore, our findings provide a unified classification of P. cantorii hemocytes and identify the genes related to the stress response, thereby providing a better understanding of the adaptive mechanisms of hibernation.

Offspring size-number trade-off is a critical component of life-history theory and is important for further understanding the reproductive strategies of animals. The relationship between this trade-off and maternal size has been explored in several turtle species, except for the Asian yellow pond turtle, Mauremys mutica. To investigate how the maternal condition affects offspring size and number, we explored the relationships among the maternal body size and the number and size of cultured M. mutica hatchlings using a 4-year dataset. Our results showed that different females not only produced different sizes of offspring but also produced different numbers of offspring. No trade-off in egg size number was detected. According to regression analysis, we did not find that the maternal body size significantly influenced the offspring mass; however, we detected that the offspring size was significantly correlated with the clutch size and maternal age. The mean body mass of offspring increased with maternal age, and the clutch size varied significantly over four years, which was correlated with offspring size, maternal body size and age. However, the number of offspring per female increased with the maternal plastron length rather than age. Our results were inconsistent with the optimal offspring size theory in that females did not increase their offspring size but rather increased the offspring number to increase their fitness, which will also provide a basis for the efficient cultivation management of turtles.

The Asian giant soft-shelled turtle, Pelochelys cantorii (Trionychidae), is one of the largest aquatic turtles in China and was designated as a First-Grade Protected Animal in China in 1989. Previous investigation based on a combination of Illumina short-read, PacBio long-read and Hi-C scaffolding technologies acquired a high-quality chromosome-level genome of Pc. cantorii. In this study, comparative genomic analysis between Pc. cantorii and 16 other vertebrate genomes indicated that turtles separated from the ancestor of archosaurians approximately 256.6 (95% highest posterior density interval, 263.6–251.9) million years ago (Mya) (Upper Permian to Triassic) and that Pc. cantorii separated from the ancestor of Pd. sinensis and R. swinhoei approximately 59.3 (95% highest posterior density interval, 64.3–54.3) Mya. Moreover, several candidate genes, such as VWA5A, ABCG2, A2M and IGSF1, associated with tumor suppression, growth and age were expanded, implicating their potential roles in the exceptional longevity of turtles. This new chromosome-level assembly has important scientific value in the study of conservation of Pc. cantorii and also enriches the evolutionary investigation of turtle species.

The Asian giant softshell turtle, Pelochelys cantorii, is a Class I protected animal in China and a CITES Appendix II species. In this paper, the behavior of 15 cultivated 4-month-old Asian giant softshell turtles was recorded in 1 day by a surveillance camera. Focusing on the posture act environment (PAE) and based on ecological functions, a PAE behavior ethogram of juvenile P. cantorii was constructed and analyzed. The results showed that there were 10 postures of young turtles, including floating, hanging, swimming, and lying, and 48 postures of the head and neck, mouth, eyes and nose, etc. Forty-six kinds of behaviors, such as ingestion, elimination, exploration, locomotive, rest, amicable, group and conflict, were recorded under 8 environmental codes. These findings will be helpful for the study of the behavior and conservation of P. cantorii and can also provide a reference for the study of the behavior ethograms of other soft-shelled turtles.