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Simple Summary: Gonadal cell lines possess the abilities of self-renewal and differentiation, being used as an efficient tool to analyzing the genes' functions involved in sex differentiation and gametogenesis. Although some significant achievements have been obtained in the gonadal cells' culture or manipulation across multiple phyla including teleost and mammals, there is limited study on gonadal cell manipulation in turtles. In this study, we isolated an ovarian cell line (fGSCs), from the juvenile Asian yellow pond turtle in vitro. The cultured fGSCs were characterized with germline stem cell specific markers' expression using transcriptome sequencing on Illumina platform, reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence (IF). Our study aims to provide a valuable tool to elaborate the molecular mechanisms behind germ cells development, differentiation and oogenesis in the turtle even in reptiles, and also, the established cell line has potential applications to enrich the conservation strategy of aquatic animal germplasm resources. Gonadal cell lines possess the abilities of self-renewal and differentiation, being used as an efficient tool to analyzing the genes' functions involved in sex differentiation and gametogenesis. Although some significant achievements have been obtained in the gonadal cells' culture or manipulation across multiple phyla including teleost and mammals, there is limited study on gonadal cell manipulation in turtles. In this study, we established a new ovarian cell line from the young Asian yellow pond turtle (Mauremys mutica), which exhibited a normal diploid karyotype with high alkaline phosphatase activity. The cell line, designated as YTO2, was then characterized through the analysis of gene expression profiles. The transcriptome analysis and the reverse transcription polymerase chain reaction (RT-PCR) showed that the cells expressed germline genes such as tdrd7, nanos1, klf5, igtb1, hsd17b4 and rad51. Moreover, the immunostaining showed that the germ cell markers, Tdrd7 and Rad51 proteins, were detected predominant in cytoplasm of perinuclear region, while proliferation marker, PCNA, was dominantly observed in the nuclei of cultured cells. Intriguingly, the cells could respond to the retinoic acid induction with significantly increasing the expression level of some meiosis genes, including vasa, dazl, figla, and dmc1. Furthermore, YTO2 cells could be efficiently transfected with the pHBAd-BHG-EGFP adenovirus and properly expressed the exogenous genes. To sum up, an ovarian cell line of the Asian yellow pond turtle had been established and could be stably propagated under in vitro culture condition, as well as being capable of efficiently expressing the exogenous gene tdrd7. This cell line would provide a valuable tool to elaborate the molecular mechanisms behind germ cells development, differentiation and oogenesis in the turtle, even in reptiles. [ABSTRACT FROM AUTHOR]

Gonadal cell lines possess the abilities of self-renewal and differentiation, being used as an efficient tool to analyzing the genes’ functions involved in sex differentiation and gametogenesis. Although some significant achievements have been obtained in the gonadal cells’ culture or manipulation across multiple phyla including teleost and mammals, there is limited study on gonadal cell manipulation in turtles. In this study, we established a new ovarian cell line from the young Asian yellow pond turtle (Mauremys mutica), which exhibited a normal diploid karyotype with high alkaline phosphatase activity. The cell line, designated as YTO2, was then characterized through the analysis of gene expression profiles. The transcriptome analysis and the reverse transcription polymerase chain reaction (RT-PCR) showed that the cells expressed germline genes such as tdrd7, nanos1, klf5, igtb1, hsd17b4 and rad51. Moreover, the immunostaining showed that the germ cell markers, Tdrd7 and Rad51 proteins, were detected predominant in cytoplasm of perinuclear region, while proliferation marker, PCNA, was dominantly observed in the nuclei of cultured cells. Intriguingly, the cells could respond to the retinoic acid induction with significantly increasing the expression level of some meiosis genes, including vasa, dazl, figla, and dmc1. Furthermore, YTO2 cells could be efficiently transfected with the pHBAd-BHG-EGFP adenovirus and properly expressed the exogenous genes. To sum up, an ovarian cell line of the Asian yellow pond turtle had been established and could be stably propagated under in vitro culture condition, as well as being capable of efficiently expressing the exogenous gene tdrd7. This cell line would provide a valuable tool to elaborate the molecular mechanisms behind germ cells development, differentiation and oogenesis in the turtle, even in reptiles.

Asian yellow pond turtle (Mauremys mutica) is listed as an endangered species on the IUCN Red List and recorded in the CITES Appendix II. The wild populations of M. mutica are extremely rare in China, while the main living resources are captive populations. The development of the molecular markers is very important for population identification, population conservation and scientific management of M. mutica. In this study, 27 novel SNP markers for M. mutica were discovered and validated. The observed and expected heterozygosities ranged from 0.0361 to 0.9277 and 0.0588 to 0.5004, respectively. Seven loci showed significant deviation from HWE and no significant linkage disequilibrium was found. These markers would be used in genetic studies helping conservation and management of M. mutica. [ABSTRACT FROM AUTHOR]

Asian yellow pond turtle ( Mauremys mutica) is an endangered species, being listed in the IUCN red list of threatened species. In China, most living resources are kept in turtle farms. Insufficient molecular markers have hindered the effective management and artificial breeding programs of this important species. In this study, we discovered and validated 43 novel polymorphic bi-allelic SNP markers for M. mutica from the transcriptome of brain and gonad tissue. The observed and expected heterozygosity ranged from 0.0303 to 0.5758 and 0.0303-0.5072, respectively. These markers would be used in genetic studies helping conservation and management of M. mutica. [ABSTRACT FROM AUTHOR]

V-set and immunoglobulin domain-containing protein 4 (VSIG4), a member of the immunoglobulin superfamily, plays an important role in the immune system. This study isolated and characterized a cDNA encoding VSIG4 ( MaVSIG4) from the Asian yellow pond turtle ( Mauremys mutica). The MaVSIG4 cDNA is 1840 bp long and contains an open reading frame of 1,182 bp that encodes a polypeptide of 372 amino acids. The genomic sequence of MaVSIG4 spans 7,682 bp, with six exons and five introns. The phylogenetic tree shows that MaVSIG4 is most closely related to Gallus gallus VSIG4. The expression analysis by real-time PCR reveals that MaVSIG4 is ubiquitously expressed in various healthy tissues, with a higher expression level in the liver. After immune stimulation, the expression level of MaVSIG4 sharply decreased in the liver, heart, and kidney at 12 h ( P < 0.01). These results provide a basis for further study of the function of MaVSIG4 in the turtle's immune system. [ABSTRACT FROM AUTHOR]

Abstract: The Asian yellow pond turtle, Mauremys mutica (Cantor), is a potential aquaculture target in China owing to the higher values for food and remedy than other species of turtle. In this study, color and morphological changes of fertilized eggs were observed during embryogenesis, and the effects of incubation temperature on embryonic development were analyzed. Both calcium layer and membrane layer are thicker in the middle portion of egg-shell than that in the terminal portion, and become thinner after embryo hatching than before embryonic development. Significant change in the white spot and subsequent white ring on the egg-shell occurs during embryonic development. Of five different incubation temperatures used to investigate the effects of incubation temperatures on embryonic development, 29.0±0.5 °C was optimal for embryo survival and development. Moreover, the incubation temperature of 33.0±0.5 °C was harmful effect to embryonic development. The data provide important and useful information for husbandry and management of the Asian yellow pond turtle. [Copyright &y& Elsevier]

Asian yellow pond turtle ( Mauremys mutica) is an endangered species. In this study, we developed and characterized 19 novel polymorphic microsatellite markers for M. mutica through the Restriction-site Associated DNA tags sequencing (RAD-seq). 60 individuals were used to assess the level of polymorphism of each locus. The number of alleles ranged from 4 to 25, the observed and expected heterozygosity ranged from 0.40000 to 0.90000 and 0.39572 to 0.93201, respectively. These markers would be used in genetic studies helping conservation and management of M. mutica. [ABSTRACT FROM AUTHOR]

Growth rate variation of organisms might stem from a series of physiological changes induced by different environmental conditions. Hatchling turtles from different incubation temperatures frequently exhibited diverse growth rates. However, the physiological mechanisms underlying this variation remain largely unclear. Here, we investigated the metabolic rate and feeding performance of hatchling Asian yellow pond turtles (Mauremys mutica) from eggs incubated at two different temperatures (26 and 30 °C) to evaluate the role of differences in metabolic expenditure, food intake and digestive efficiency on growth rate variation. Hatchlings from the cooler temperature had a greater growth rate and metabolic rate, tended to eat more food but showed a lower digestive efficiency of energy (DEE) than those from the warmer temperature. Accordingly, the difference in energy acquisition acts as a potential source for growth rate variation of hatchling turtles from different incubation temperatures, and increased energy intake (rather than enhanced digestive efficiency and reduced metabolic expenditure of other physiological processes) might be associated with the higher growth rate of cooler-incubated hatchling M. mutica. • Hatchling Mauremys mutica from lower incubation temperature (26°C) grew faster than those from higher temperature (30 °C). • 26°C-incubated turtles had higher metabolic rate and food intake, but lower digestive efficiency than 30°C-incubated ones. • More energy intake (not higher digestive efficiency, lower metabolic cost) caused faster growth of cooler-incubated turtles. [ABSTRACT FROM AUTHOR]

The complete mitochondrial genomes of three lineages (N, TW and S) ofMauremys muticaare determined in this study. The total lengths of the mitogenomes were 16,758 bp for N, 16 500bp for TW, and 16 494bp for S. The nucleotide composition was 26.3–27% for T, 26.2–26.8% for C, and 33.8–33.9% for A. The genomes encoded 37 genes typically found in other vertebrates. Three CSBs were identified, and the CSB1 were variable. A long tandem repeats of (TTATTATA) 30 were found in the control region of N mitogenome, but none in TW and S lineage. These sequences would be useful for the phylogenetic and conservation studies of Asian endangered turtles. [ABSTRACT FROM AUTHOR]