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Simple Summary: 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 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 parent–offspring relationships of all offspring in four consecutive years were confirmed using sixteen microsatellite loci. We also 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. This study 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. 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. [ABSTRACT FROM AUTHOR]

Biotic factors (e.g., maternal age) and abiotic factors (e.g., weather) play vital roles in reproduction. However, there is little information about the combined effects of maternal age and weather on the reproductive output of cultured Asian yellow pond turtles, Mauremys mutica. To address this issue, we surveyed and compared the reproductive output of 13-year-old and 25-year-old female turtles on non-rainy and rainy days. The results showed that older females produced larger eggs than younger females regardless of the weather conditions. Females laid larger clutch sizes (number of eggs per nest) under non-rainy conditions compared with those under rainy conditions. However, the variation in clutch frequency (number of clutches per day) was independent of maternal age and weather. There was no effect of an interaction between maternal age and weather on egg mass, clutch size or clutch frequency. Our results suggest that maternal age and weather had significant effects on egg mass and clutch size, respectively. These results imply that maternal age, together with weather conditions (mainly conditions of rain), can affect the reproductive output of M. mutica. Our results provide useful information for the artificial breeding and stock management of M. mutica. [ABSTRACT FROM AUTHOR]

The mitochondrial DNA cytochrome c oxidase subunit I gene (COI) has been used as an efficient barcoding tool for species identification of animals. In this study, the barcoding sequences were used to assess the genetic diversity and relationship ofMauremy muticaandM. annamensis. Four currently recognized groups ofM. muticawere classified into two groups in this study, with 6% intergroup distances, the S group and the N group, consistent to the calling of “southern turtle” and “northern turtle” in folk of China. The north population and Taiwan population formed the N group, and further, the Taiwan population was differentiated as a monophyly originated from the north population, consistent to the calling of “big green head” for the Taiwan population and “small green head” for the north population. The Vietnam, Hainan population, andM. annamensisformed the S group, and the barcoding sequences could not distinguish them from each other. Based on the molecular data and phenotypes of existing hybrids, hybrid origin ofM. annamensismay be another possibility. [ABSTRACT FROM AUTHOR]

The crucial roles of miRNAs in regulating animal growth, development, and disease resistance have been extensively reported, but their roles in relation to the reproductive capacity of aquatic animals (numbers of eggs laid and hatchlings), especially reptiles, remain unclear. In this study, high-throughput sequencing technology was used to screen miRNAs related to reproductive capacity based on the construction of a cDNA library of ovaries from higher-fecundity (HF) and lower-fecundity (LF) M. mutica. The results showed that 15,767,494 (93.98%) and 14,137,621 (94.17%) high-quality reads were obtained from the HF and LF groups, respectively. We screened 131 miRNAs that were differentially expressed between the HF and LF groups, of which 78 were upregulated and 53 were downregulated compared with the M. mutica reference genome. GO and KEGG pathway enrichment analyses of the target genes of differentially expressed miRNAs revealed significant differences in the enrichment frequencies of genes associated with ATP binding and proteolysis between the HF and LF groups, while the tricarboxylic acid cycle, glucagon signaling pathway and vitamin B6 metabolic pathway were shown to potentially help determine reproductive capacity. Ten miRNAs were verified by qRT-PCR to confirm the reliability and accuracy of the sequencing results, and a miRNA-mRNA target gene interaction network was constructed. These results will further our understanding of the regulatory mechanism of miRNAs in regards to turtle reproductive capacity. [Display omitted] • 131 differentially expressed miRNAs were detected between higher fecundity group and lower fecundity group. • There were 10 differentially expressed miRNAs validated by using qRT-PCR. • A miRNA-mRNA target gene interaction network was constructed. [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]

Mitochondria function as an energy transfer organelle for cell metabolism with the energy being used for processes such as reproduction. To investigate whether mutations and copy number variations in mitochondrial genes are related to the reproductive capacity of the Asian yellow pond turtle, Mauremys mutica , there was exploration of the distribution frequency of 129 Single Nucleotide Polymorphism loci of six mitochondrial genes, ND1 , ND2 , COX2 , ND4 , Cytb andD- loop , of turtles from a relatively greater and lesser fecundity group by direct sequencing. The validation results for five candidate SNP loci in 83 female turtles indicated that only three SNP loci (C119T, A320G and A417C) in ND1 were positively correlated with reproductive capacity in M. mutica (P < 0.05). In addition, by constructing linear regression equations of the copy numbers of ND1 , ND4 , Cytb , D-loop , COX3 , and ATP6 (log10 transformed) genes and the mean offspring number of different female turtles during a 4-year period, the copy numbers of ND4 and ATP6 (log10 transformed) genes were positively correlated (P < 0.05) with the fecundity of female turtles. Results from the present study may provide useful genetic markers for breeding M. mutica with greater reproductive capacity. [ABSTRACT FROM AUTHOR]

In order to understand the endocrine mechanism associated with fecundity of seasonally breeding animals, we investigated the plasma reproductive hormones levels and detected the differences in steroidogenic enzymes and sex hormone receptor mRNA levels in female Mauremys mutica. These turtles were divided into higher fecundity (HF) group than those in lower fecundity (LF) group based on paternity identification in our previous research. The plasma estrogen (E2), testosterone (T) and progesterone (P4) levels were significantly higher in pre-breeding season (PBS) than those in non-breeding season (NBS) and were markedly higher in the HF group than those in LF group. In the hypothalamus, there was significantly higher mRNA abundance of P450-cholesterol side-chain cleavage enzyme (P450Scc) encoded by Cyp11α1, aromatase (Cyp19α1) and 5-reductase (5α-R), but significantly lower mRNA levels of follicular stimulating hormone receptor (FSHR) and progesterone receptor (PR) detected in PBS than those in NBS. The pituitary steroidogenic acute regulatory protein (StAR), cytochrome P450-17alpha-hydroxylase (Cyp17α1), 3-hydroxy-steroid dehydrogenase (3βHSD), 17-hydroxy-steroid dehydrogenase 3 (17βHSD3), Cyp19α1, 5α-R, FSHR, estrogen receptor 1 (ESR1), androgen receptor (AR) and PR transcriptional levels in HF group were up-regulated significantly compared with the LF group. In the ovary, Cyp17α1 and 17βHSD3 transcriptional levels were markedly higher in PBS than those in NBS. We detected significantly increased expression levels of all steroidogenic enzymes, but notably lower mRNA levels of FSHR and PR in uterus during the PBS, and the HF group has significantly higher expression levels of StAR, Cyp17α1, 5α-R and AR than LF group. Our work reveals seasonal variations in hormone regulation as well as gene regulation in turtles, providing reliable information to understand the mechanisms underlying the different reproductive capacity of reptiles. [Display omitted] • Plasma T level can be an indicator for screening high-fecundity females. • Steroidogenic enzymes in uterus were significantly higher in PBS than those in NBS. • Pituitary sex hormone receptors expressed significantly more in HF than that in LF. • Uterus plays a critical role in regulating turtle's reproduction. [ABSTRACT FROM AUTHOR]

• We identified 8,237 DE mRNAs, 9,573 DE lncRNAs, 84 DE circRNAs and 665 DE miRNAs. • We identified DE mRNAs majorly involved in reproduction, reproductive process. • dmrt3a etc were closely associated with the levels of their corresponding ncRNA. Noncoding RNAs (ncRNAs), including long ncRNAs (lncRNAs), circular RNAs (circRNAs) and microRNAs (miRNAs) have been extensively studied in biological processes such as disease development, cell proliferation but remained unclear in sex differentiation in organisms. In this study, the transcriptome profiles were comparatively analyzed between male and female gonads in Mauremys mutica. A total of 8237 differentially expressed genes (DEGs), 9573 DE lncRNAs, 84 DE circRNAs and 665 DE miRNAs were identified between male and female gonads. Through gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) of the DE RNAs, it was revealed that the DE mRNAs were majorly involved in GO terms, such as 'reproduction', 'reproductive process' and the pathways of 'focal adhesion' and 'oocyte meiosis'. In addition, a co-expression network showed that the expression of gametogenesis and sex differentiation related genes, including dmrt3a , tdrd7 , sox14 , etc were closely associated with the levels of their corresponding ncRNAs. Intriguingly, the dmrt1 circRNA and its target mRNA were detected upregulated both in adult testis and male producing temperature (MPT) embryos. Our findings demonstrated the sexually dimorphic expression profiles of mRNAs and ncRNAs in turtle gonads, which will provide the index to find out the molecular mechanisms behind the sex differentiation in turtles, even in other environmental sex determination (ESD) species. [ABSTRACT FROM AUTHOR]