Chinese Soft-shelled Turtle Pelodiscus Sinensis Research Articles

Aeromonas dhakensis, a pathogen associated with red abdominal shell disease in Chinese soft-shelled turtle Pelodiscus sinensis

Red abdominal shell disease is a major disease that has caused significant economic losses in Chinese soft-shelled turtle Pelodiscus sinensis aquaculture. Yet scarce information is available on Aeromonas dhakensis as a causal agent of red abdominal shell disease in P. sinensis. In the present study, a haemolytic and proteolytic A. dhakensis isolate (F04) was demonstrated as a causative agent of red abdominal shell disease in cultured P. sinensis. It carried virulent aerolysin, heat-labile cytotoxic enterotoxin, polar flagella, and lipase-encoding genes aer, alt, flaA, and lip, exhibited the mean lethal dose (LD50) value of 4.27×105 CFU/ml in P. sinensis, and developed multiple resistances against aminoglycosides, chloramphenicols, cephalosporins, lincosamides, macrolides, penicillins, peptides, sulfonamides, and tetracyclines antimicrobials. To our knowledge, this is the first study to identify a multiply resistant A. dhakensis strain as a pathogen of red abdominal shell disease in P. sinensis, and the findings of this study can also provide valuable insights into the virulence and antimicrobial resistance in Chinese soft-shelled turtle-pathogenic A. dhakensis.

Role of Sox3 in Estradiol-Induced Sex Reversal in Pelodiscus 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.

Alien Freshwater Turtles in Greece: Citizen Science Reveals the Hydra-Headed Issue of the Pet Turtle Trade

Citizen science is emerging as a powerful tool for the early detection of biological invasions. The exotic pet trade has been deemed as the most significant cause of establishment for reptiles, and among them, turtles have the highest number of introduction events. In Europe, at least 13 species of alien freshwater turtles have been recorded in the wild. In Greece, only two species of alien turtles have been reported, namely the American pond slider Trachemys scripta and the Florida cooter Pseudemys floridana. In this study, we provide an updated checklist of the alien freshwater turtles recorded in Greece, using citizen science, personal observations, and literature. Our results provide the first records for the country of five species, namely the Florida red-bellied cooter Pseudemys nelsoni, the Eastern river cooter Pseudemys concinna, the Chinese stripe-necked turtle Mauremys sinensis, the Chinese soft-shelled turtle Pelodiscus sinensis, and the Common snapping turtle Chelydra serpentina and new localities for T. scripta. Similar to the myth of the Lernaean Hydra (i.e., for every head chopped off, more would regrow), the pet turtle trade has been proven to have multiple heads.

The anti-lipopolysaccharide effect of glycine via fibroblastic cell culture from the embryonic calipash tissue of Chinese soft-shelled turtle Pelodiscus sinensis

As a representative kind of soft-shelled turtles, Pelodiscus sinensis has soft apron termed calipash around its pliable carapace as the most distinctive trait, which is functionally important in turtle defense. It is of great significance to explore the physiological function of stress resistance and the possible regulation of anti-stress nutrition for the survival of aquaculture. To address this issue, a fibroblast cell line from the embryonic calipash tissue of P. sinensis (PSCF) was established from the perspective of ethics, repeatability, and convenience. Effect of glycine, the main amino acid component in calipash extracellular matrix, on the proliferation of PSCF treated with 160 μg/mL lipopolysaccharide (LPS) and the underlying mechanisms were also studied. The results showed that adding 100 μg/mL glycine significantly improved the fibroblast survival, increased the mRNA levels of collagen biosynthesis genes, prolyl hydroxylase and collagen degradation genes, and inhibited reactive oxygen species (ROS) production. It suggested that glycine shows positive effects on the cell proliferation of PSCF, and could regulate the oxidative stress and collagen metabolism. To our knowledge, this report is the first to describe cell cultures established at specific calipash tissue-forming stages during the embryonic development in P. sinensis and to reveal the anti-stress effect of glycine in turtles. The results will contribute to the application of functional calipash tissue, or its manipulation by nutrition additives, such as glycine, to prevent or ameliorate stress in turtle industry.

SOX8 is essential for male sexual differentiation in the Chinese soft-shelled turtle Pelodiscus sinensis‡.

SOX8, which belongs to SOXE transcription factor subfamily together with SOX9, participates in sex differentiation and testicular development by enhancing the function of SOX9 in mammals. However, the functional role of SOX8 in sex differentiation has not yet been identified in any non-mammalian vertebrates. Here, we found in the Chinese soft-shelled turtle Pelodiscus sinensis that SOX8 exhibited male-specific higher expression from stage 14 to 18, the critical period of sex determination, prior to the onset of gonadal differentiation. In addition, SOX8 was rapidly down-regulated during male-to-female sex reversal induced by estradiol. Moreover, knockdown of SOX8 led to complete feminization of ZZ P. sinensis, determined by gonadal morphology and distribution of germ cells, as well as the down-regulation of testicular marker DMRT1 and the up-regulation of ovarian regulator FOXL2. In conclusion, this study provides evidence that SOX8 is a key regulator of early male differentiation in P. sinensis, highlighting the significance of the SOX family in reptile sex determination.

The forkhead factor Foxl2 participates in the ovarian differentiation of Chinese soft-shelled turtle Pelodiscus sinensis

The forkhead transcription factor Foxl2 plays a major role in ovarian development and function in mice and fish, and acts as a female sex-determining gene in goat. Its functional role in the sex determination and gonadal differentiation has not yet been investigated in reptiles. Here, we characterized Foxl2 gene in Chinese soft-shelled turtle Pelodiscus sinensis, exhibiting ZZ/ZW sex chromosomes. Foxl2 exhibited a female-specific embryonic expression pattern throughout the critical sex determination periods in P. sinensis. The expression of Foxl2 was induced at early stage in ZZ embryonic gonads that were feminized by estrogen treatment. Most importantly, Foxl2 knockdown in ZW embryos by RNA interference resulted in female-to-male sex reversal, characterized by obvious masculinization of gonads, significant up-regulation of testicular markers Dmrt1 and Sox9, and remarkable down-regulation of ovarian regulator Cyp19a1. Conversely, gain-of-function study showed that overexpression of Foxl2 in ZZ embryos led to largely feminized genetic males, production of Cyp19a1, and a decline in Dmrt1 and Sox9. These findings demonstrate that Foxl2 is both necessary and sufficient to initiate ovarian differentiation in P. sinensis, thereby acting as a key upstream regulator of the female pathway in a reptilian species.

Gene synteny, evolution and antiviral activity of type I IFNs in a reptile species, the Chinese soft-shelled turtle Pelodiscus sinensis

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.

Sex-relevant genes in the embryo stage of Chinese soft-shelled turtles as revealed by RNA-Seq analysis

Abstract The Chinese soft-shelled turtle (Pelodiscus sinensis) is an economically important and rare freshwater aquaculture species in China. The male turtles have better economic benefits than females due to their faster growth speed. In order to explore the mechanism of sex differentiation and determination of P. sinensis, transcriptome analysis was carried out using embryo samples from the sex determination, sex differentiation and shelling stages, respectively. The gender types of embryos were identified by genotyping and histological analysis. In all, 277,230 and 273,859 genes were identified from embryos of male and female turtles, respectively. Cluster analysis of gene expression patterns exhibited trends for all differentially expressed genes (DEGs), which can be classified into three subclusters. In the sex determination, sex differentiation, and shelling stages 975, 43, and 300 DEGs, respectively, were identified by comparing the male and female groups. KEGG analysis was used to explore the function of the DEGs. Fgf9, foxl2, mex3b, sox7,tgfβ-3, wnt4, wnt7a, and wt1 were upregulated in ovary development stages and chfr, ampk, aldh2, dao, glt1d1, hvcn1, psmd9, srsf9 and ubc were upregulated in testis development stages, indicating that these genes play important roles in the sex development of P. sinensis. The results of this study provide preliminary insights into the sex determination and differentiation of Chinese soft-shelled turtles.

Texture quality, histological and extracellular matrix‐related molecular changes of the calipash of Chinese soft‐shelled turtle Pelodiscus sinensis in response to Aeromonas hydrophila challenge

Chinese soft-shelled turtle Pelodiscus sinensis possesses a distinctively soft and pliable apron around its dorsal carapace termed calipash, which is of great importance to evaluate the nutritional quality in turtle industry. As bacterial infection such as Aeromonas hydrophila often brings severe economic losses to P. sinensis culturing in China, it is necessary to study the response of this most distinctive calipash tissue to bacterial infection, which has not been reported. In this study, the texture quality, histology and extracellular matrix-related molecules of calipash in P. sinensis after A. hydrophila challenge were evaluated. After A. hydrophila infection, calipash tissue samples were subjected to biological evaluation, histological analyses using hematoxylin and eosin staining, Masson trichrome staining, scanning electron microscopy and real-time polymerase chain reaction for measuring mRNA expression of collagen biosynthesis as well as collagen degradation-related genes. Picrosirius red staining was used to reveal the type I collagen distribution. The results showed histologically observable connective tissue disorganization with a notable increase in collagen-characteristic amino acid hydroxyproline level, greater collagen I density and more collagen content in infected turtles when compared to the control. For the infected, significantly decreased mRNA levels of type I collagen and proline-4-hydroxylase coding genes, increased matrix metalloproteinases and lowered tissue inhibitor of metalloproteinase expression were observed. These results suggest that collagen metabolism or mobilization characterized using collagen degradation may play an important role in the maintenance of calipash in response to the A. hydrophila challenge.

The Important Role of Sex-Related Sox Family Genes in the Sex Reversal of the Chinese Soft-Shelled Turtle (Pelodiscus sinensis)

Simple SummaryPelodiscus sinensis is an important aquatic economic species in China with sexual dimorphism. All-male breeding is becoming a research hotspot. Here, comparative transcriptome analyses of female, male, and pseudo-female gonads were performed. We found that the differences between males and pseudo-females were mainly related to steroid hormone synthesis at the transcriptome level. When it comes to the sox family genes, sox3 may have a role in the process of sex reversal from male to pseudo-female, when sox8 and sox9 were inhibited by exogenous estrogen.The Chinese soft-shelled turtle Pelodiscus sinensis shows obvious sexual dimorphism. The economic and nutrition value of male individuals are significantly higher than those of female individuals. Pseudo-females which are base to all-male breeding have been obtained by estrogen induction, while the gene function and molecular mechanism of sex reversal remain unclear in P. sinensis. Here, comparative transcriptome analyses of female, male, and pseudo-female gonads were performed, and 14,430 genes differentially expressed were identified in the pairwise comparison of three groups. GO and KEGG analyses were performed on the differentially expressed genes (DEGs), which mainly concentrated on steroid hormone synthesis. Furthermore, the results of gonadal transcriptome analysis revealed that 10 sex-related sox genes were differentially expressed in males vs. female, male vs. pseudo-female, and female vs. pseudo-female. Through the differential expression analysis of these 10 sox genes in mature gonads, six sox genes related to sex reversal were further screened. The molecular mechanism of the six sox genes in the embryo were analyzed during sex reversal after E2 treatment. In mature gonads, some sox family genes, such as sox9 sox12, and sox30 were highly expressed in the testis, while sox1, sox3, sox6, sox11, and sox17 were lowly expressed. In the male embryos, exogenous estrogen can activate the expression of sox3 and inhibit the expression of sox8, sox9, and sox11. In summary, sox3 may have a role in the process of sex reversal from male to pseudo-female, when sox8 and sox9 are inhibited. Sox family genes affect both female and male pathways in the process of sex reversal, which provides a new insight for the all-male breeding of the Chinese soft-shelled turtle.

Characterization and tissue expression analysis of mitochondrial creatine kinases (types I and II) from Pelodiscus sinensis

The aim of this study was to characterize the functions of the mitochondrial creatine kinases in the Chinese soft-shelled turtle Pelodiscus sinensis (PSCK-MT1 and PSCK-MT2) to characterize function in relation to hibernation. Computational prediction via molecular dynamics simulations showed that PSCK-MT1 had stronger kinase- and creatine-binding affinity than PSCK-MT2. We measured PSCK-MT1 and PSCK-MT2 levels in the myocardium, liver, spleen, lung, kidney, and ovary of P. sinensis before and after hibernation and found that the expression of these enzymes was the most significantly upregulated in the ovary. We enumerated the ovarian follicles and evaluated the physiological indices of P. sinensis and discovered that fat was the main form of energy storage in P. sinensis. Moreover, both PSCK-MTs promoted follicular development during hibernation. Immunohistochemistry was used to study follicular development and revealed that both PSCK-MTs were expressed primarily in the follicular fluid and granulosa layer before and after hibernation. We found that PSCK-MT1 and PSCK-MT2 could play important roles in ovarian follicular development under hibernation. Hence, both PSCK-MTs probably function effectively under the conditions of low temperature and oxygen during hibernation. Communicated by Ramaswamy H. Sarma

Knockdown of R-spondin1 leads to partial sex reversal in genetic female Chinese soft-shelled turtle Pelodiscus sinensis

Chinese soft-shelled turtle Pelodiscus sinensis is an important aquaculture species in China, the male individual being more valuable in aquaculture because of its larger body size, higher growth rate and less fat compared with females. Understanding the mechanism of ovarian differentiation and development is crucial for the production of mono-sex male offspring. However, little is known about the molecular mechanism underlying turtle ovarian differentiation. Here, we characterized the Rspo1 gene, an upstream regulator of vertebrate female sexual differentiation, in P. sinensis. The messenger RNA of Rspo1 was initially expressed at stage 14, preceding gonadal sex differentiation, and exhibited a sexually dimorphic expression pattern throughout the sex determination and gonadal differentiation periods. Rspo1 was rapidly downregulated during aromatase inhibitor-induced female-to-male sex reversal, which occurred prior to gonadal differentiation. Rspo1 loss of function by RNA interference led to partial female-to-male sex reversal, with masculinized changes in the phenotype of gonads, the distribution of germ cells and the expression of testicular regulators. Collectively, these findings suggest that Rspo1 is necessary for primary female sexual differentiation in P. sinensis. This study demonstrates for the first time the functional role of Rspo1 in reptilian sex determination, and is of fundamental significance for the production of fertile pseudo-female parents and mono-sex male offspring of P.sinensis.

Molecular and Cellular Mechanisms of Lipid Droplet Breakdown in the Liver of Chinese Soft-Shelled Turtle (Pelodiscus sinensis)

The Chinese soft-shelled turtle (Pelodiscus sinensis) is among the most primitive amphibians and reptiles in nature. On account of its environmental suitability and unique hibernation habit, the peculiar physiological phenomenon in P. sinensis attracted the attention of researchers in the field of marine science. The present study aimed to investigate the molecular mechanisms underlying the periodic variation of lipid droplet (LD) in the liver of P. sinensis. Histological results indicated that accumulated LD in the liver of P. sinensis during non-hibernation was gradually consumed during hibernation. RNA-Seq results revealed that genes responsible for carbohydrate catabolism were down-regulated during hibernation, while genes involved in lipid oxidation were up-regulated. These results suggest that energy metabolism in the liver of P. sinensis changes during hibernation, i.e., the energy generation mode shifted from carbohydrate catabolism to lipid oxidation. Further analysis of RNA-Seq results indicated that both lipolysis and autophagy could promote the degradation of hepatic LD during hibernation. To further determine the relationship between lipolysis and autophagy in the process of LD breakdown, we applied the inhibitors of lipolysis and autophagy (diethylumbelliferyl phosphate and 3-Methyladenine) in cultured primary hepatocytes of P. sinensis. The results indicated that lipolysis is the main way for LD degradation in the hepatocyte of P. sinensis. These data provide clear evidence about the seasonal changes in hepatocytes, corresponding with the different energy generation mode in the liver of P. sinensis.

Antioxidant defense response during hibernation and arousal in Chinese soft-shelled turtle Pelodiscus sinensis juveniles

Antioxidant defense is essential for animals to cope with homeostasis disruption during hibernation. The present study aimed to investigate the antioxidant defense response of juvenile soft-shelled turtle Pelodiscus sinensis during hibernation and following arousal. Turtle brain, liver, and kidney samples were collected at pre-hibernation (17 °C mud temperature; MT), during hibernation (5.8 °C MT) and after arousal (20.1 °C MT) in the field. Transcript levels of NF-E2-related factor 2 (Nrf2) decreased significantly during hibernation and recovered after arousal in all tissues. Cerebral and nephric copper-zinc superoxide dismutase (Cu/Zn SOD), catalase (CAT), glutathione peroxidase 3 (GPx3) and nephric GPx4 mRNA showed similar changing patterns as Nrf2. Cerebral Mn SOD, GPx1 and nephric GPx1 up-regulated after arousal. Hepatic Cu/Zn SOD, GPx1 and GPx3 mRNA kept stable, except hepatic GPx4 increased during hibernation. Hepatic Mn SOD and CAT increased after arousal. In the GSH system, mRNA levels of glutathione synthetases (GSs) kept stable during hibernation and up-regulated after arousal in most tissues except nephric GS2 mRNA remained unchanged. Gene expressions of glutathione reductase (GR) exhibited a tissue specific changing pattern, while those of glutathione-S-Transferase (GST) shared a similar pattern among tissues: remained stable or down-regulated during hibernation then recovered in arousal. In contrast to these diverse responses in gene expressions, most of the antioxidant enzyme activities maintained high and stable. Overall, no preparation for oxidative stress (POS) strategy was found in enzymatic antioxidant system in P. sinensis juveniles during hibernation, the Chinese soft-shelled turtles were able to stay safe from potential oxidative stress during hibernation by maintaining high level activities/concentrations of the antioxidant enzymes/antioxidants.

Extracellular vesicles in the male reproductive tract of the softshell turtle.

Extracellular vesicles (EVs) are a heterogeneous group of cell-derived membranous structures comprising exosomes and microvesicles that originate from the endosomal system or are shed from the plasma membrane respectively. As mediators of cell communication, EVs are present in biological fluids and are involved in many physiological and pathological processes. The role of EVs has been extensively investigated in the mammalian male reproductive tract, but the characteristics and identification of EVs in reptiles are still largely unknown. In this review we focus our attention on EVs and their distribution in the male reproductive tract of the Chinese softshell turtle Pelodiscus sinensis , mainly discussing the potential roles of EVs in intercellular communication during different phases of the reproductive process. In softshell turtles, Sertoli-germ cell communication via multivesicular bodies can serve as a source of EVs during spermatogenesis, and these EVs interact with epithelia of the ductuli efferentes and the principal cells of the epididymal epithelium. These EVs are involved in sperm maturation, transport and storage. EVs are also shed by telocytes, which contact and exchange information with other, as well as distant interstitial cells. Overall, EVs play an indispensable role in the normal reproductive function of P. sinensis and can be used as an excellent biomarker for understanding male fertility.

Molecular and functional identification of a short-type peptidoglycan recognition protein, PGRP-S, in the Chinese soft-shelled turtle Pelodiscus sinensis

Peptidoglycan recognition proteins (PGRPs), which are discovered in invertebrates and vertebrates, play an important role in antibacterial immunity. However, the function of PGRPs is largely uninvestigated in reptiles. In the present study, a short-type PGRP gene, designed as C-turtle-PGRP-S, was identified in the Chinese soft-shelled turtle, Pelodiscus sinensis. The C-turtle-PGRP-S contains a highly conserved PGRP domain and has close relationship with PGRP-S orthologues in other species according to sequence and phylogenetic analyses. C-turtle-PGRP-S gene was constitutively expressed in all detected tissues and was induced by Edwardsiella tarda. Additionally, recombinant C-turtle-PGRP-S showed PGN binding activity and antibacterial function against E. tarda. Therefore, it is suggested that the function of PGRP-S is likely to be conserved in reptile vertebrates, as observed in other vertebrates, shedding light on the evolutionary conservation of PGRPs.

Regulation of the limb shape during the development of the Chinese softshell turtles.

Interdigital cell death is an important mechanism employed by amniotes to shape their limbs; inhibiting this process leads to the formation of webbed fingers, as seen in bats and ducks. The Chinese softshell turtle Pelodiscus sinensis (Reptilia: Testudines: Trionychidae) has a distinctive limb morphology: the anterior side of the limbs has partially webbed fingers with claw‐like protrusions, while the posterior fingers are completely enclosed in webbings. Here, P. sinensis embryos were investigated to gain insights on the evolution of limb‐shaping mechanisms in amniotes. We found cell death and cell senescence in their interdigital webbings. Spatial or temporal modulation of these processes were correlated with the appearance of indentations in the webbings, but not a complete regression of this tissue. No differences in interdigital cell proliferation were found. In subsequent stages, differential growth of the finger cartilages led to a major difference in limb shape. While no asymmetry in bone morphogenetic protein signaling was evident during interdigital cell death stages, some components of this pathway were expressed exclusively in the clawed digit tips, which also had earlier ossification. In addition, a delay and/or truncation in the chondrogenesis of the posterior digits was found in comparison with the anterior digits of P. sinensis, and also when compared with the previously published pattern of digit skeletogenesis of turtles without posterior webbings. In conclusion, modulation of cell death, as well as a heterochrony in digit chondrogenesis, may contribute to the formation of the unique limbs of the Chinese softshell turtles.

Incubation temperature affects the development of calipash related traits and collagen deposition in embryos of soft-shelled turtle Pelodiscus sinensis

Chinese soft-shelled turtle Pelodiscus sinensis possesses a distinctive soft and pliable apron around its dorsal carapace termed calipash, of which the formation and its relation to extracellular matrix remain largely unclear. Herein, we investigated calipash quality traits and collagen deposition of turtle embryos from eggs incubated at two different temperatures (26 °C and 32 °C) to evaluate the involvement of collagen in calipash development during embryogenesis. We found that the calipash related traits including embryo mass, embryo length and calipash mass showed an overall increasing tendency as embryos developed. The positive correlation between collagen content and the above morphologic traits in calipash was also found. Embryos incubated at 32 °C incubation temperature had significant bigger embryo size, heavier calipash and higher collagen content at most developmental stages as compared with those embryos incubated at 26 °C. Strong birefringence color was observed from the starting of developmental stage 23 and there were much more collagen fibers presented polarized orange or red colors from embryos incubating at 32 °C. The percentage of type I collagen areas in turtle calipash tissue was found to increase overall and with significantly higher value in embryos incubated at 32 °C. The observed promoted mRNA level of type I collagen gene of turtle eggs incubated at 32 °C in developmental stage 23 indicated that the biosynthesis of collagen in P. sinensis embryos might be induced. The present work indicated that 32 °C incubation temperature is conducive to more collagen deposition and related more preferable calipash traits during the embryonic development of P. sinensis. The data provided the possible manipulation of calipash trait development by incubation temperature changing. High incubation temperature is suggested when attempting to develop large calipash tissue in P. sinensis aquaculture.

Dietary squid paste supplementation promotes feed intake via brain-gut dynamic response in Chinese soft-shelled turtle Pelodiscus sinensis.

BackgroundAs the primary source of protein for aquaculture, fishmeal has reached the extremity of sustainable development, our previous studies have proven that rice protein concentrate and squid paste are outstanding protein source and stimulant for Pelodiscus sinensis. However, little attention has been given to the molecular mechanism of the appetite modulated by the dietary nutrient factor, especially for a reptile. Thus, the present study aimed to evaluate feed intake and brain-gut dynamic responses to dietary rice protein concentrate and squid paste in Chinese soft-shelled turtle Pelodiscus sinensis.MethodsThree isonitrogenous and isoenergetic practical diets were formulated including 60% fishmeal (CT), 42% fishmeal + 18% rice protein concentrate (RP) and 42% fishmeal + 18% rice protein concentrate + 1% squid paste (RPS), respectively. Microcapsule lysine was supplemented in RP and RPS diets to balance the amino acid profile. Turtles (initial weight 30.65 ± 0.97 g) were fed three times daily to apparent satiation. After the 8-week feeding trial, the turtles were exposed to 48h food deprivation, then the dynamic expression of the orexigenic and anorexigenic peptides were measured.ResultsThe results showed that no significant effect was observed on feed intake when fishmeal was replaced by rice protein concentrate (P = 0.421), while significantly improved feed intake was found by squid paste supplemented (P = 0.02). The mRNA expression of anorexigenic peptides, such as leptin receptor, insulin receptor, pro-opiomelanocortin, cocaine and amphetamine-regulated transcript, cholecystokinin (and its receptor) and glucagon-like peptide-1 receptor in the brain increased significantly at 3 h past feeding (P < 0.05), and then decreased. Nevertheless, neuropeptide Y and peptide YY mRNA expression showed the valley at 3h and peak at 12h past feeding. Intestinal cholecystokinin receptor and glucagon-like peptide-1 receptor mRNA expression showed no difference during the postprandial time (P > 0.05). The results suggested that squid paste is an outstanding stimulant for Pelodiscus sinensis. Furthermore, the orexigenic and anorexigenic peptides evaluated here might play an essential role in short-term fasting to this species, of which the dynamic expression levels were regulated by squid paste.

Genome-wide characterization of TNF receptor-associated factors in the Chinese soft-shelled turtle Pelodiscus sinensis and their expression profiling in response to Aeromonas hydrophila challenge

Tumor necrosis factor receptor (TNFR)-associated factors (TRAFs) are a family of crucial signaling molecules that mediate the signal transduction of various immune signaling pathways. Extensive studies have demonstrated that TRAFs play vital roles in regulating cellular immune responses. However, the biological functions and expression profiling of TRAFs in Chinese soft-shelled turtle (Pelodiscus sinensis) remain unclear. In this study, the genes of the PsTRAF family at the genome-wide level were identified in P. sinensis, revealing six PsTRAF members that contained the conserved TRAF domain in the C-terminal regions. Molecular evolutionary analysis showed that PsTRAFs shared close evolutionary relationships and similar protein crystal structures with the TRAF homologs from other turtles, indicating the evolutionary conservation of PsTRAFs. Further expression analysis revealed the tissue-specific expression of PsTRAF genes. Obvious variations in the expression of PsTRAF genes were observed in the spleen in response to Aeromonas hydrophila infection. Three PsTRAF genes, PsTRAF2, PsTRAF3, and PsTRAF6, were significantly upregulated at the mRNA and protein levels post-infection, indicating their potential function in the immune response. Moreover, the protein-protein associations of PsTRAFs with several signaling receptors were predicted in P. sinensis. These results provide a basis for the investigation of the functional roles of PsTRAFs in immune defense against bacterial infection.