Chinese Soft-shelled Turtle Pelodiscus Sinensis Research Articles

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.

Multifaceted Hi-C benchmarking: what makes a difference in chromosome-scale genome scaffolding?

BackgroundHi-C is derived from chromosome conformation capture (3C) and targets chromatin contacts on a genomic scale. This method has also been used frequently in scaffolding nucleotide sequences obtained by de novo genome sequencing and assembly, in which the number of resultant sequences rarely converges to the chromosome number. Despite its prevalent use, the sample preparation methods for Hi-C have not been intensively discussed, especially from the standpoint of genome scaffolding.ResultsTo gain insight into the best practice of Hi-C scaffolding, we performed a multifaceted methodological comparison using vertebrate samples and optimized various factors during sample preparation, sequencing, and computation. As a result, we identified several key factors that helped improve Hi-C scaffolding, including the choice and preparation of tissues, library preparation conditions, the choice of restriction enzyme(s), and the choice of scaffolding program and its usage.ConclusionsThis study provides the first comparison of multiple sample preparation kits/protocols and computational programs for Hi-C scaffolding by an academic third party. We introduce a customized protocol designated “inexpensive and controllable Hi-C (iconHi-C) protocol,” which incorporates the optimal conditions identified in this study, and demonstrate this technique on chromosome-scale genome sequences of the Chinese softshell turtle Pelodiscus sinensis.

The Role of Anti-Müllerian Hormone in Testis Differentiation Reveals the Significance of the TGF-β Pathway in Reptilian Sex Determination.

Anti-Müllerian hormone (Amh, or Müllerian-inhibiting substance, Mis), a member of TGF-β superfamily, has been well documented in some vertebrates as initiator or key regulator in sexual development, and particularly in fish. However, its functional role has not yet been identified in reptiles. Here, we characterized the Amh gene in the Chinese soft-shelled turtle Pelodiscus sinensis, a typical reptilian species exhibiting ZZ/ZW sex chromosomes. The messenger RNA of Amh was initially expressed in male embryonic gonads by stage 15, preceding gonadal sex differentiation, and exhibited a male-specific expression pattern throughout embryogenesis. Moreover, Amh was rapidly upregulated during female-to-male sex reversal induced by aromatase inhibitor letrozole. Most importantly, Amh loss of function by RNA interference led to complete feminization of genetic male (ZZ) gonads, suppression of the testicular marker Sox9, and upregulation of the ovarian regulator Cyp19a1 Conversely, overexpression of Amh in ZW embryos resulted in female-to-male sex reversal, characterized by the formation of a testis structure, ectopic activation of Sox9, and a remarkable decline in Cyp19a1 Collectively, these findings provide the first solid evidence that Amh is both necessary and sufficient to drive testicular development in a reptilian species, P. sinensis, highlighting the significance of the TGF-β pathway in reptilian sex determination.

Characterization of a classical 2-cysteine peroxiredoxin1 gene from Chinese soft-shelled turtle Pelodiscus sinensis with its potent antioxidant properties and putative immune response

Peroxiredoxin family members could function in host defense against oxidative stress, and modulate immune response. In the present study, a 2-cysteine peroxiredoxin gene named PsPrx1 was isolated from Chinese soft-shelled turtle Pelodiscus sinensis. The PsPrx1 cDNA was composed of 1130 bp, consisted of 199 amino acid residues and included a Redoxin and AphC-TSA domain. As detected by qPCR, PsPrx1 was ubiquitously expressed in the examined tissues with the higher levels in liver and spleen. Upon the immune challenge with A. jandaei bacteria and oxidative stress with ammonia pressure, both mRNA and protein expression level in liver could be significantly enhanced. The results of immunohistochemical examinations showed PsPrx1 was mainly distributed at the junction between the hepatic cells. The general functional properties of PsPrx1 were confirmed using purified rPsPrx1 protein. From the results, rPsPrx1 protein was confirmed to exhibit antioxidant activity and antibacterial properties. The potential for scavenging extracellular H2O2 was evidenced by the purified rPsPrx1 protein in vitro system. In the mixed-function oxidase assay, rPsPrx1 also exhibited a dose-dependent inhibition of DNA damage. These results suggest that rPsPrx1 was implicated defense against microbial pathogens and oxidants, and would provide important information to further understand the functional mechanism of Prx1 in P. sinensis immunity.

Newly identified C‐type lysozyme in Chinese soft‐shelled turtle (Pelodiscus sinensis) exhibits potent antimicrobial activity

Lysozymes play vital roles in humoural immune response against bacterial invasion by its lytic activity. In the present study, a new C-type lysozyme was identified and characterized from Chinese soft-shelled turtle Pelodiscus sinensis. The full-length cDNA of PslysC was of 923 bp, encoding a polypeptide of 148 amino acid residues. The multiple alignments and phylogenetic relationship analysis revealed the highly enzyme-related conserved residues. The real-time PCR analysis suggested that PslysC was constitutively expressed in a wide range of tissues with highest level in blood cells and liver. The expression of PslysC could be significantly up-regulated under Aeromonas jandaei infection and ammonia exposure, while no significant changes were found under Poly I:C infection. The rPslysC protein was expressed in E. coli and purified by Ni-NTA. The optimal pH and temperature for rPslysC protein lytic activities were determined at pH 7 and 30℃. rPslysC can inhibit the growth of eight kinds of Gram-negative bacteria, and three kinds of Gram-positive bacteria. The binding activity of rPslysC to different microbial polysaccharides and microorganism was analysed. The results showed that rPslysC could bind to selected bacteria, and exhibit a strong binding activity to lipopolysaccharide and peptidoglycan, but a weak binding activity to β-glucan. This suggests that the binding activity might be the major mechanism of action to realize the antibacterial activity. The present study will provide helpful evidence to further understand the innate immunity of P. sinensis, and the interaction mechanisms of C-type lysozymes with bacterial membranes.

Genomewide identification and analysis of heat-shock proteins 70/110 to reveal their potential functions in Chinese soft-shelled turtle Pelodiscus sinensis.

Heat‐shock proteins 70/110 (Hsp70/110) are vital molecular chaperones and stress proteins whose expression and production are generally induced by extreme temperatures or external stresses. The Hsp70/110 family is largely conserved in diverse animals. Although many reports have studied and elaborated on the characteristics of Hsp70/110 in various species, the systematic identification and analysis of Hsp70/110 are still poor in turtles. In this study, a genomewide search was performed, and 18 candidate PsHSP70/110 family genes were identified in Chinese soft‐shelled turtle, Pelodiscus sinensis. These PsHSP70/110 proteins contained the conserved “heat shock protein 70” domain. Phylogenetic analysis of PsHSP70/110 and their homologs revealed evolutionary conservation of Hsp70/110 across different species. Tissue‐specific expression analysis showed that these PsHSP70/110 genes were differentially expressed in different tissues of P. sinensis. Furthermore, to examine the putative biological functions of PsHSP70/110, the dynamic expression of PsHSP70/110 genes was analyzed in the testis of P. sinensis during seasonal spermatogenesis following germ cell apoptosis. Notably, genes such as PsHSPA1B‐L, PsHSPA2, and PsHSPA8 were significantly upregulated in P. sinensis testes along with a seasonal decrease in apoptosis. Protein interaction prediction revealed that PsHSPA1B‐L, PsHSPA2, and PsHSPA8 may interact with each other and participate in the MAPK signaling pathway. Moreover, immunohistochemical analysis showed that PsHSPA1B‐L, PsHSPA2, and PsHSPA8 protein expression was associated with seasonal temperature variation. The expression profiling and interaction relationships of the PsHSPA1B‐L, PsHSPA2, and PsHSPA8 proteins implied their potential roles in inhibiting the apoptosis of germ cells in P. sinensis. These results provide insights into PsHSP70/110 functions and will serve as a rich resource for further investigation of HSP70/110 family genes in P. sinensis and other turtles.

Genome-wide identification of Toll-like receptors in the Chinese soft-shelled turtle Pelodiscus sinensis and expression analysis responding to Aeromonas hydrophila infection

Toll-like receptors (TLRs) recognizing specific pathogen-associated molecular patterns play crucial roles in immune defence against pathogen invasion. Although recent advances in many species have reported the characterization and functional roles of TLRs in innate immunity, systematic knowledge of TLRs is still lacking in the Chinese soft-shelled turtle Pelodiscus sinensis. In this study, a genome-wide search was performed and identified 15 candidate PsTLR family genes in P. sinensis. Protein structure analysis revealed the conserved domain arrangements for these PsTLR proteins. Phylogenetic analysis indicated the evolutionary conservation of TLRs among various species. Additionally, a putative interaction network among PsTLR proteins was proposed and several functional partner proteins involved in TLR signalling pathway were predicted in P. sinensis. Expression profiling showed that these PsTLRs exhibited constitutive expression patterns in different tissues of P. sinensis. Moreover, several genes were highly expressed in the major immune organ spleen. Remarkably, the mRNA levels of PsTLR2-1, PsTLR4 and several TLR signalling molecules were significantly up-regulated in the spleen after Aeromonas hydrophila infection, indicating that PsTLRs and these genes responded to bacterial stress. These results provide rich information for the functional exploration of PsTLRs and will facilitate uncovering the molecular mechanisms underlying immune regulation in P. sinensis.

Depletion Study of Oxolinic Acid in Freshwater Softshell Turtle (Pelodiscus sinensis) with Multiple-Dose Oral Administration

All drugs used in aquaculture must be approved by the government agency responsible for veterinary medicine; prudent use of antibiotics under veterinary supervision is critical in ensuring safety of aquaculture products. The international production of farmed turtle, considered a cultured aquatic species, has been growing continuously but control of bacteria proliferation in turtle farms requires frequent use of antimicrobials. In the present investigation, the residue depletion and withdrawal period of quinolone antibacterial oxolinic acid (OXO) was evaluated in Chinese softshell turtle Pelodiscus sinensis, after repeated (once daily for 5 days) oral doses of medicated feed containing OXO, at the dose of 30 and 60 mg/kg body weight (bw). Muscle and liver tissues were collected at 1, 2, 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, and 48 days posttreatment, and OXO was quantified using a validated high-performance liquid chromatography with diode array detector (HPLC-DAD) method. According to the test preparation record, the limit of detection (LOD) and limit of quantification (LOQ) for OXO were estimated at 0.03 and 0.1 µg/g, respectively. Considering a maximum residue level (MRL) of 0.1 µg/g for OXO in muscle in European Union (EU) legislative framework, the longest withdrawal period of 72 days was calculated on the basis of a safety span (corresponding to 50% of the time point when at which residues fall below the MRL) added to the depletion time (48 days). These results may be helpful not only for the establishment of public policies regarding the use of OXO for turtle farming but also to producers for proper handling to ensure safe consumption.

Effect of exogenous hormones on R-spondin 1 (RSPO1) gene expression and embryo development in Pelodiscus sinensis.

Little is known about sex determination and differentiation in the Chinese soft-shelled turtle Pelodiscus sinensis. R-Spondin 1 (RSPO1), a candidate sex-determining gene, is an important regulator of ovarian differentiation in animals. Exogenous drugs can affect sex differentiation. In this study we cloned the RSPO1 gene from P. sinensis (psRSPO1) and analysed its expression profile. The psRSPO1 gene exhibited sequence identity with RSPO1 genes from other species. RSPO1 protein-based phylogenetic analysis showed that psRSPO1 in P. sinensis is closely related to RSPO1 proteins from other turtles. psRSPO1 showed abundant expression in adult brain and gonads, with higher levels in females than males. We also evaluated the effects of three finaconcentration of 2.5, 5.0 and 10mgmL-1 exogenous oestradiol (E2) and aromatase inhibitor (letrozole) on the expression of psRSPO1, external embryo morphology, growth status of embryos and the sex ratio when the drugs were injected to eggs during incubation. The expression of psRSPO1 was upregulated and downregulated by exogenous oestradiol and letrozole respectively, despite inconsistent expression trends at different embryo development times. External embryo morphology, growth status and sex ratio were affected by both exogenous oestradiol and the aromatase inhibitor. Feminisation was induced by oestradiol, but inhibited by letrozole. These results will contribute to studies of the potential molecular mechanisms underlying sex differentiation and sex control in the Chinese soft-shelled turtle.

Lipophagy contributes to long-term storage of spermatozoa in the epididymis of the Chinese soft-shelled turtle Pelodiscus sinensis.

Spermatozoa are known to be stored in the epididymis of the Chinese soft-shelled turtle Pelodiscus sinensis for long periods after spermiation from the testes, but the molecular mechanisms underlying this storage are largely unknown. In this study, epididymal spermatozoa were investigated to determine the potential molecular mechanism for long-term sperm storage in P. sinensis. Transmission electron microscopy (TEM) and Oil red O staining indicated that unusually large cytoplasmic droplets containing lipid droplets (LDs) were attached to the epididymal spermatozoa. However, the content of LDs decreased gradually with the sperm storage. LDs were surrounded by autophagic vesicles and sequestered as degradative cargo within autophagosome. Immunofluorescence and western blotting demonstrated that autophagy in spermatozoa increased gradually with the storage time. Invitro studies found that spermatozoa obtained from soft-shelled turtles in January can survive more than 40 days at 4°C. Furthermore, immunofluorescence and TEM showed that autophagy was involved in the degradation of LDs with the extension of sperm incubation. Inhibition of autophagy with 3-methyladenine significantly suppressed LD degradation. Moreover, adipose triglyceride lipase was involved in the metabolism of LDs. These findings indicate that lipophagy was activated to maximise LD breakdown, which contributes to long-term sperm storage in the epididymis of P. sinensis.

Physiological responses in vitamin C system during hibernation in juvenile Chinese soft-shelled turtle Pelodiscus sinensis

Vitamin C (Vc) is an important antioxidant that helps turtles tolerating stressful environment. This work quantified changes in tissue Vc levels during winter hibernation in the soft-shelled turtle Pelodiscus sinensis, to reveal the stress response pattern of tissue Vc during hibernation and contribute basic data for turtle culture. We sampled juvenile soft-shelled turtles at pre-hibernation (17.0°C mud temperature; MT), during hibernation (5.8°C MT) and after arousal (20.1°C MT) in the field. The transcript levels of the gene encoding L-gulonolactone oxidase (GLO), the key enzyme for Vc synthesis, decreased significantly during hibernation. However, GLO activity did not match the GLO transcription patterns and remained stable during hibernation, and showed temperature-dependent kinetic characteristics. Vitamin C levels in the brain, liver, kidney, and spleen (but not muscle) all decreased significantly during hibernation, but recovered to pre-hibernation levels or even higher levels after arousal. The soft-shelled turtle endured 5 months of hibernation with no significant oxidative damage in most tissues, except in the spleen. Splenic Vc was nearly exhausted during hibernation, accompanied by a significantly elevated malonaldehyde (MDA) level. Although the high level of oxidative damage quickly decreased after arousal, the potential tissue damage in the spleen during hibernation might account for the weakened immune capacity of turtles after hibernation.

Effect of exogenous hydrogen peroxide on ROS balance and antioxidant response in Chinese soft-shelled turtle Pelodiscus sinensis

Chinese soft-shelled turtle, Pelodiscus sinensis, is an important farmed species in the Southeast Asia and often encounters adverse environmental stresses during cultivation. Knowledge about the stress response, especially antioxidant response, in P. sinensis would help to understand the effect of adverse stress on turtles and to reduce the production loss. In the present study, intraperitoneal injection of H2O2 (0%, 0.3%, 0.6%, and 2.4%) was employed as an exogenous oxidative stressor to directly challenge the antioxidant defense in Chinese soft-shelled turtle, Pelodiscus sinensis, and evaluate the antioxidant capacity and characteristics of antioxidant defense in P. sinensis. Level of reactive oxygen species (ROS), total antioxidant capacity (TAC), antioxidant enzyme activities, ascorbic acid (AA) content and malondialdehyde (MDA) level were measured in the brain, liver, kidney and spleen of P. sinensis at 2 h, 6 h and 24 h post-injection. In general, all cerebral and hepatic parameters did not change, although there were some variations in the liver after 2 h, which recovered after 6 h. In the kidney, only glutathione peroxidase (GPx) activity and AA level increased by 24 h post-injection. Splenic ROS level decreased after 6 h; TAC level and catalase (CAT) activity increased, whereas other antioxidant enzyme activities and AA decreased following by H2O2 injection. Results for MDA level and histology indicated transitory oxidative damage in the liver, but longstanding damage in the spleen. In summary, turtle antioxidant defense work effectively to decrease ROS level even when tissues are faced with intense oxidative stress. However, spleen should be paid more attention because it seemed to be more fragile to intense or long-term oxidative stress than other tissues. Specifically, AA level was important in antioxidant defense of P. sinensis.

Immunohistochemical analysis of the development of olfactory organs in two species of turtles Pelodiscus sinensis and Mauremys reevesii

The nasal cavity of turtles is composed of the upper and lower chambers, lined by the upper and lower chamber epithelia, respectively. In many turtles including the Reeve’s turtle Mauremys reevesii, the upper chamber epithelium contains ciliated olfactory receptor neurons (ORNs) and the lower chamber epithelium contains microvillous ORNs. However, in the olfactory organ of the Chinese soft-shelled turtle Pelodiscus sinensis, both the upper and lower chamber epithelia contain ciliated ORNs. In the present study, we immunohistochemically examined the developmental process of olfactory organs in soft-shelled turtle and the Reeve’s turtle to clarify the developmental origins of the lower chamber epithelium in these turtles. Obtained data indicate that olfactory organs of these turtles have identical origin and follow similar process of development, suggesting that, in the lower chamber epithelium of the nasal cavity, ciliated ORNs differentiate in soft-shelled turtle whereas microvillous ORNs differentiate in the Reeve’s turtle.

Growth performance, digestion and metabolism to fish meal replacement by rice protein concentrate in Chinese soft-shelled turtle Pelodiscus sinensis

An 8-week feeding trial was conducted to evaluate growth performance, whole-body composition, metabolism and digestion of Chinese soft-shelled turtle Pelodiscus sinensis fed with fish meal replacement by rice protein concentrate and squid paste supplementation diets. Three isonitrogenous and isoenergetic practical diets were formulated with the inclusion of 60% fishmeal (CT), 42% fish meal +18% rice protein concentrate (RP) and 42% fish meal +18% rice protein concentrate +1% squid paste (feeding attractant) (RPS), respectively. Microcapsule lysine was supplemented in RP and RPS diets to balance the amino acid profile. Turtles (initial weight 30.65 g) were fed thrice (6:00, 12:00 and 18:00) daily to apparent satiation. The results showed that growth performance in RP group showed no significant difference with that in CT group (P > .05). In addition, daily squid paste supplementation significantly improved growth performance, nutrients retention and relative feed intake (P < .05). Significantly increased activities of hepatic alanine aminotransferase as well as intestinal protease, γ-glutamyl transpeptidase, aminopeptidase N and carboxypeptidase A were also observed in RPS group (P < .05). The results suggested that rice protein concentrate could replace 18% dietary fish meal in Pelodiscus sinensis with microcapsule lysine supplementation. Besides, diet with squid paste inclusion could improve feed intake and growth performance of Pelodiscus sinensis.

Digital Gene Expression Profiling reveals transcriptional responses to acute cold stress in Chinese soft-shelled turtle Pelodiscus sinensis juveniles

Turtles are well known for their stress tolerance, including an ability to deal with temperature extremes or rapid thermal change. To know more about the comprehensive molecular basis of thermal stress responses in turtles, we assessed differentially expressed genes (DEGs) in the brain, liver and kidney of juvenile soft-shelled turtles, Pelodiscus sinensis, after acute cold stress (28 °C–8 °C acute transfer and held for 12 h) and following recovery (back to 28 °C and held for 24 h) by digital gene expression profiling. Selected DEGs were also validated via real-time PCR. We found the fewest DEGs in the brain, only one-tenth of the number seen in liver, indicating a tissue-specific gene expression pattern. The DEGs indicated the potential activation of several important functions in response to cold stress and recovery in P. sinensis. This included response to oxidative stress or regulation of reactive oxygen species metabolism in the brain and liver, cerebral inositol metabolism, hepatic monosaccharide metabolism, hepatic complement system, renal DNA repair mechanisms, and TNF and PI3K-Akt signaling pathways in the kidney. These functions likely responded to cold stress in different tissues of P. sinensis to help minimize or repair cell damage as well as enhance innate immunity. The outcomes of this study provide some fundamental insight into the tissue specific complex mechanisms underlining cold stress responses in the soft-shelled turtle P. sinensis.

Chromosomal Context Affects the Molecular Evolution of Sex-linked Genes and Their Autosomal Counterparts in Turtles and Other Vertebrates.

Sex chromosomes evolve differently from autosomes because natural selection acts distinctly on them given their reduced recombination and smaller population size. Various studies of sex-linked genes compared with different autosomal genes within species support these predictions. Here, we take a novel alternative approach by comparing the rate of evolution between subsets of genes that are sex-linked in selected reptiles/vertebrates and the same genes located in autosomes in other amniotes. We report for the first time the faster evolution of Z-linked genes in a turtle (the Chinese softshell turtle Pelodiscus sinensis) relative to autosomal orthologs in other taxa, including turtles with temperature-dependent sex determination (TSD). This faster rate was absent in its close relative, the spiny softshell turtle (Apalone spinifera), thus revealing important lineage effects, and was only surpassed by mammalian-X linked genes. In contrast, we found slower evolution of X-linked genes in the musk turtle Staurotypus triporcatus (XX/XY) and homologous Z-linked chicken genes. TSD lineages displayed overall faster sequence evolution than taxa with genotypic sex determination (GSD), ruling out global effects of GSD on molecular evolution beyond those by sex-linkage. Notably, results revealed a putative selective sweep around two turtle genes involved in vertebrate gonadogenesis (Pelodiscus-Z-linked Nf2 and Chrysemys-autosomal Tspan7). Our observations reveal important evolutionary changes at the gene level mediated by chromosomal context in turtles despite their low overall evolutionary rate and illuminate sex chromosome evolution by empirically testing expectations from theoretical models. Genome-wide analyses are warranted to test the generality and prevalence of the observed patterns.

Dmrt1 is required for primary male sexual differentiation in Chinese soft-shelled turtle Pelodiscus sinensis

In vertebrates, the primary sex-determining signals that initiate sexual development are remarkably diverse, ranging from complete genetic to environmental cues. However, no sex determination-related genes have been functionally identified in reptiles. Here, we characterized a conserved DM domain gene, Dmrt1, in Chinese soft-shelled turtle Pelodiscus sinensis (P. sinensis), which exhibits ZZ/ZW sex chromosomes. Dmrt1 exhibited early male-specific embryonic expression, preceding the onset of gonadal sex differentiation. The expression of Dmrt1 was induced in ZW embryonic gonads that were masculinized by aromatase inhibitor treatment. Dmrt1 knockdown in ZZ embryos by RNA interference resulted in male to female sex reversal, characterized by obvious feminization of gonads, significant down-regulation of testicular markers Amh and Sox9, and remarkable up-regulation of ovarian regulators, Cyp19a1 and Foxl2. Conversely, ectopic expression of Dmrt1 led to largely masculinized genetic females, production of Amh and Sox9, and a decline in Cyp19a1 and Foxl2. These findings demonstrate that Dmrt1 is both necessary and sufficient to initiate testicular development, thereby acting as an upstream regulator of the male pathway in P. sinensis.

Cellular Evidence of Exosomes in the Reproductive Tract of Chinese Soft-Shelled Turtle Pelodiscus sinensis.

The oviduct is a location of egg production, fertilization, and sperm storage. While its secretions have broadly attributes toward different physiological functions. We examined the ultrastructure of oviduct epithelium and glands in relation to the secretions, particularly with exosomes origin in Chinese soft-shelled turtle Pelodiscus sinensis using immunohistochemistry and transmission electron microscopy. The ciliated epithelial and gland cells were involved in the release of exosomes and secretions into lumen throughout the year. The exosomes were either released directly from epithelium or in relation with multivesicular body (MVB). The average size of the particles varies between 50 and 130 nm. These exosomes were also widely distributed in the epithelial ciliated cells and pericytoplasm of glands lumen. Intracellular MVB was characterized by membrane-bounded exosomes of different sizes. Exosomes were also found in close contact with the cilia and sperm membrane in the lumen, which is suggestive of their fusogenic properties. Immunohistochemistry results showed strong to moderate positive expression of exosomes, in ciliated and gland cells, during January, September, and December, as it is the time of sperm storage in this turtle, whereas they showed moderate to weak level of expression during breeding season (May). This is first study about identification of the exosomes in female turtles. Epithelial and glandular exosomes, intracellular MVB, secretions, and secretory vesicles give this turtle specie a unique secretory morphology and a potential model for investigating the secretory nature of the oviduct.

A comparative study on genetic characteristics of two new varieties of Pelodiscus sinensis and their hybrid.

Chinese soft-shelled turtle Pelodiscus sinensis has been an important aquaculture species in Southeast Asian countries. To breed a new variety of soft-shelled turtle with excellent properties and to evaluate the effect of hybridization of two turtle strains with a highly different trait phenotype, inheritance, microsatellite loci, and transcriptome analysis were studied in the hybrid turtles and their parents of P. sinensis Japanese strain and Qingxi black turtle. The genotypic characteristics and economic trait of the hybrid turtles were analyzed and compared to the two parents, showing significant growth vigor. The chromosome number of the hybrid turtle was diploid (2N = 66). The karyotype formulae were 8m+10sm+26t+22mc, with little differences between the two parents. Genotypic segregations of 241 microsatellite loci were screened in 3 populations including 90 species and showed that the specific allele numbers and polymorphic fragments increased in hybrid turtles indicating genetic diversity increased by hybridization. The liver transcriptome analysis of the hybrids and two parents showed similar distribution abundance in the parental and hybrid groups, but the transcripts with high abundance appeared in the hybrid group. There were 274 significant differentially expressed transcripts in the hybrid group compared to the two parental groups, among them 7 differentially expressed genes indicating super-parent expression, and only 2 genes showing low-parent expression. In the differentially expressed genes, expression changes were mainly contributed to regulatory region changes rather than coding region sequences. These results would be important for facilitating successful breeding strategies by hybridization in P. sinensis.

Antioxidant responses in hibernating Chinese soft-shelled turtle Pelodiscus sinensis hatchlings

The antioxidant defense system protects turtles from oxidative stress during hibernation. The present study examined changes of the antioxidant enzymes both on mRNA level and enzyme activity level during hibernation of Chinese soft-shelled turtle Pelodiscus sinensis hatchlings. The upstream regulator NF-E2 related factor 2 (Nrf2) mRNA was also measured. Samples were taken at pre-hibernation (17.0°C, Mud temperature (MT)), hibernation (5.8°C, MT) and arousal (20.1°C, MT). Nrf2 exhibited a tissue-specific pattern of expression with a decrease in the brain, slight increase in the liver and heart during hibernation, and significant increase during arousal in all the three tissues. Superoxide dismutase (SOD) mRNA, catalase (CAT) mRNA, and glutathione peroxidase 3 (GPx3) mRNA exhibited a similar pattern as Nrf2 in the brain and liver during the entire hibernation period. Hepatic GPx4 mRNA level increased during hibernation and decreased during arousal, whereas it did not change in the heart. Cerebral SOD and CAT activities kept stable during the experimental period, but GPx activity decreased significantly during hibernation and arousal. Hepatic GPx enzyme activity did not change, whereas those of SOD and CAT exhibited a notable decrease during arousal. Malondialdehyde concentration did not increase during the hibernation process, indicating an effective protection of the antioxidant defense system.