The intestine of living organisms harbors different microbiota associated with the biological functioning and health of the host and influences the process of ecological adaptation. Here, we studied the intestinal microbiota\'s composition and functional differences using 16S rRNA and metagenomic analysis in the wild, farm, and released Chinese three-keeled pond turtle (Mauremys reevesii). At the phylum level, Bacteroidota dominated, followed by Firmicutes, Fusobacteriota, and Actinobacteriota in the wild group, but Chloroflexi was more abundant in the farm and released groups. Moreover, Chryseobacterium, Acinetobacter, Comamonas, Sphingobacterium, and Rhodobacter were abundant in the released and farm cohorts, respectively. Cetobacterium, Paraclostridium, Lysobacter, and Leucobacter showed an abundance in the wild group. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database revealed that the relative abundance of most pathways was significantly higher in the wild turtles (carbohydrate metabolism, lipid metabolism, metabolism of cofactors, and vitamins). The comprehensive antibiotic resistance database (CARD) showed that the antibiotic resistance gene (ARG) subtype macB was the most abundant in the farm turtle group, while tetA was higher in the wild turtles, and srpYmcr was higher in the released group. Our findings shed light on the association between the intestinal microbiota of M. reevesii and its habitats and could be useful for tracking habitats to protect and conserve this endangered species.

In this study, we investigated the effects of Clostridium butyricum (group A), Bacillus subtilis (group B), and the immune enhancer algal β-1,3 glucan (group C) on the intestinal flora of Reeves\' turtle Mauremys reevesii and the effects of C. butyricum on the transcriptome of M. reevesii splenic immune tissues. Reeve\' turtles were assigned to four groups, each containing three replicates from 18 samples. Juvenile turtles with an initial weight of 106.35 ± 0.03 g were fed a basic diet containing no probiotics (group D), or a basic diet containing C. butyricum TF20201120, B.subtilis, or algal β-1,3 glucan supplement, respectively. After the turtles had been fed for 60, 90, and 120 d of the experimental period, high-throughput sequencing of the 16S rRNA gene revealed no significant difference in alpha diversity among the four groups at 60 days of feeding (P > 0.05), and at 90 days, the alpha diversity in group A was significantly different (P < 0.05), with an increase of 26.62% in the Shannon index and a decrease of 83.33% in the Simpson index; at 120 d, the alpha diversity (Shannon index) showed a decreasing trend in order for groups A, B, and C, At the phylum level, the abundance of Bacteroidetes, Proteobacteria, and Fusobacteria in group A increased significantly with increasing feeding time (P < 0.05), At the genus level, the abundance of Ruminococcaceae and Anaerotruncus in group A increased significantly compared with that in the other three groups (P < 0.05). Transcriptome analysis showed that 384 genes were differentially expressed in the spleen of M. reevesii, 195 genes were upregulated and 189 genes were downregulated, and C. butyricum TF201120 regulated the hematopoietic cell lineage signaling pathway in the spleen of M. reevesii (P < 0.05). The regulation of several identified immune-related genes was confirmed by qPCR. These results showed that C. butyricum, B. subtilis, and the immune enhancer algal β-1,3 glucan can improve the intestinal flora of M. reevesii, with C. butyricum TF20201120 being the most effective and significantly enhancing the immunity of M. reevesii.

The unique topological structure of a turtle shell, including the special ribs-scapula relationship, is an evolutionarily novelty of amniotes. The carapacial ridge is a key embryonic tissue for inducing turtle carapace morphologenesis. However, the gene expression profiles and molecular regulatory mechanisms that occur during carapacial ridge development, including the regulation mechanism of rib axis arrest, the development mechanism of the carapacial ridge, and the differentiation between soft-shell turtles and hard-shell turtles, are not fully understood. In this study, we obtained genome-wide gene expression profiles during the carapacial ridge development of Mauremys reevesii using RNA-sequencing by using carapacial ridge tissues from stage 14, 15 and 16 turtle embryos. In addition, a differentially expressed genes (DEGs) analysis and a gene set enrichment analysis (GSEA) of three comparison groups were performed. Furthermore, a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was used to analyze the pathway enrichment of the differentially expressed genes of the three comparative groups. The result displayed that the Wnt signaling pathway was substantially enriched in the CrTK14 vs. the CrTK15 comparison group, while the Hedgehog signaling pathway was significantly enriched in the CrTK15 vs. the CrTK16 group. Moreover, the regulatory network of the Wnt signaling pathway showed that Wnt signaling pathways might interact with Fgfs, Bmps, and Shh to form a regulatory network to regulate the carapacial ridge development. Next, WGCNA was used to cluster and analyze the expression genes during the carapacial ridge development of M. reevesii and P. sinensis. Further, a KEGG functional enrichment analysis of the carapacial ridge correlation gene modules was performed. Interesting, these results indicated that the Wnt signaling pathway and the MAPK signaling pathway were significantly enriched in the gene modules that were highly correlated with the stage 14 and stage 15 carapacial ridge samples of the two species. The Hedgehog signaling pathway was significantly enriched in the modules that were strongly correlated with the stage 16 carapacial ridge samples of M. reevesii, however, the PI3K-Akt signaling and the TGF-β signaling pathways were significantly enriched in the modules that were strongly correlated with the stage 16 carapacial ridge samples of P. sinensis. Furthermore, we found that those modules that were strongly correlated with the stage 14 carapacial ridge samples of M. reevesii and P. sinensis contained Wnts and Lef1. While the navajo white 3 module which was strongly correlated with the stage 16 carapacial ridge samples of M. reevesii contained Shh and Ptchs. The dark green module strongly correlated with the stage 16 carapacial ridge samples of P. sinensis which contained Col1a1, Col1a2, and Itga8. Consequently, this study systematically revealed the signaling pathways and genes that regulate the carapacial ridge development of M. reevesii and P. sinensis, which provides new insights for revealing the molecular mechanism that is underlying the turtle\'s body structure.

Hybridization induced by human activities, such as crossbreeding between invasive and native species, can adversely affect the natural biodiversity of an ecosystem. In Japan, the endemic turtle species Mauremys japonica is known to hybridize with the alien species Mauremys reevesii, and putative hybrids have been encountered in the wild. If M. japonica × M. reevesii hybrids can readily crossbreed with M. japonica, the hybridization with M. reevesii could lead to the extinction of pure M. japonica populations. However, information on the reproductive ability of M. japonica × M. reevesii hybrids is limited. In this study, we collected wild-caught hybrids from across western Japan to assess their reproductive ability. We investigated the nesting season timing, clutch size, embryonic development, hatching success, and sperm viability. The results showed that female hybrids nested during the same months as the parental species and had similar clutch sizes and hatching success. No embryonic development abnormalities were detected, and viable sperm were observed in all hybrid male semen samples. In conclusion, the fertility of M. japonica × M. reevesii hybrids appears to be similar to the fertilities of the parental species, posing a potential challenge for M. japonica conservation.

UNASSIGNED: Reeves\' Turtles (Mauremys reevesii) are economically important in aquaculture in China. Understanding the effects of incubation temperature and substrate moisture on embryos and hatchlings is of great significance for improving the artificial culture of M. reevesii. However, available studies have not yet determined the thermal and hydric optima for M. reevesii eggs, and the potential interaction between the two factors.
UNASSIGNED: In this study, eggs of M. reevesii were incubated at five temperature levels (23, 26, 29, 32 and 35 °C, fluctuation range ± 0.5 °C). In each temperature level, there were three substrate moisture levels (1:0.5, 1:0.9 and 1:1.2, weight ratio of vermiculite to water). Thus, a total of 15 combinations of temperature and moisture were used to examine the effects of incubation temperature and substrate moisture on incubation duration, hatching success, hatchling phenotypes, post-hatching growth and hatchling survival.
UNASSIGNED: Substrate moisture did not significantly affect most development parameters (except incubation duration and carapace width of hatchlings). Eggs incubated at low moisture level (1:0.5) had a longer incubation duration and produced hatchlings with smaller carapace widths than those incubated at medium (1:0.9) or high (1:1.2) moisture levels. Incubation temperature had a significant effect on incubation duration, hatching success, hatchling phenotypes and hatchling survival. Incubation duration decreased as incubation temperature increased. Eggs incubated at 23, 26 and 29 °C showed higher hatching success than those incubated at 32 and 35 °C. Hatchlings incubated at 32 °C were smaller in body size and mass than those incubated at 23, 26 and 29 °C. At 12 months of age, incubation temperature had no long-lasting effect on body mass, but hatchlings incubated at 23 and 35 °C had lower survival rates than those incubated at 26, 29 and 32 °C. For the development of embryos and hatchlings, the interaction between incubation temperature and substrate moisture was not significant.
UNASSIGNED: Our results indicate that incubation temperature has a significant influence on the development of embryos and hatchlings of M. reevesii, while substrate moisture only significantly affects the incubation duration and carapace width of hatchlings. The combination of an incubation temperature of 29 ± 0.5 °C and a substrate moisture level of 1:1.2 represented optimal incubation conditions in this experiment. Such incubation conditions are helpful in obtaining higher hatching success, shorter incubation duration and higher survival rates for this aquaculture species.

Some differentially expressed genes (DEGs) that encode key enzymes involved in steroidogenic biosynthesis (CYP19A1) and key molecules related to gonadal functions (DMRT1, SOX9, AMH, FOXL2, WNT4, RSPO2, and GDF9) have been identified in adult gonadal RNA-seq studies of Reeves\' pond turtle (Mauremys reevesii) with temperature-dependent sex determination (TSD). Gonadal functional maintenance and gametogenesis comprises a highly regulated and coordinated biological process, and increasing evidence indicates that microRNAs (miRNAs) may be involved in this dynamic program. However, it is not clear how the regulatory network comprising miRNAs changes the expression levels of these genes. In this study, miRNA sequencing of adult testis and ovary tissues from M. reevesii detected 25 known and 379 novel miRNAs, where 60 miRNAs were differentially expressed in the testis and ovary. A total of 1,477 target genes based on the differentially expressed miRNAs were predicted, where 221 target genes also exhibited differential expression. To verify the accuracy of the sequencing data, 10 differentially expressed miRNAs were validated by quantitative reverse transcription real-time PCR, and were found to be consistent with the transcriptome sequencing results. Moreover, several miRNA/target gene pairs, i.e., mre-let-7a-5p/mre-let-7e-5p and CYP19A1, mre-miR-200a-3p and DMRT1, mre-miR-101-3p and SOX9, and mre-miR-138-5p and AMH were identified. To explore the regulatory role of miRNAs, we conducted target gene enrichment analysis of the miRNAs and 221 target genes in the regulatory network. The signaling pathways related to gonadal functional maintenance and gametogenesis based on the DEGs and target genes were then compared. Our findings provide crucial information to facilitate further research into the regulatory mechanisms involving miRNAs in turtle species with TSD.

Mauremys reevesii is a classical organism with temperature-dependent sex determination (TSD). Gonad development in early life has recently received considerable attention but gonadal maintenance after sex differentiation in turtles with TSD remains a mystery. In this study, we sequenced the transcriptomes for the adult testis and ovary using RNA-seq, and 36,221 transcripts were identified. In total, 1,594 differentially expressed genes (DEGs) were identified where 756 DEGs were upregulated in the testis and 838 DEGs were upregulated in the ovary. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis suggested that the TGF-beta signaling pathway and Hedgehog signaling pathway have important roles in testis maintenance and spermatogenesis, whereas the Hippo signaling pathway and Wnt signaling pathway are likely to participate in ovary maintenance. We determined the existence of antagonistic networks containing significant specific-expressed genes and pathways related to gonadal maintenance and gametogenesis in the adult gonads of M. reevesii. The candidate gene Fibronectin type 3 and ankyrin repeat domains 1 (FANK1) might be involved with the regulation of testis spermatogenesis.

The thermal physiological performance of invasive species may play a crucial role in determining their invasion success. In this study, we acclimated two cohorts of hatchlings of freshwater turtles (native Mauremys reevesii and invasive Trachemys scripta elegans) from low and high-latitude collection sites, respectively, to different thermal conditions (20 and 30 °C) for 4 weeks, and then compared their thermal tolerance and locomotor performance. T. scripta elegans hatchlings could swim faster (but righted themselves more slowly), and tolerate a higher temperature and wider temperature range than M. reevesii hatchlings. Similarly, T. scripta elegans hatchlings had a greater maximal performance (Pmax) value for swimming speed (but a lower Pmax value for righting time) than M. reevesii hatchlings. Temperature acclimation had a significant impact on the thermal tolerance and locomotor ability of turtles, but the acclimation effect did not differ between the two species. T. scripta elegans hatchlings seemed to have a greater thermal plasticity than M. reevesii hatchlings. High-latitude individuals showed a greater low-temperature tolerance, but lower locomotor ability (longer righting time) than low-latitude ones. However, the thermal plasticity did not differ between latitudinal cohorts. Our results indicated that T. scripta elegans performed better than M. reevesii, which might contribute to its range expansion and invasive success.

Mauremys reevesii (Geoemydidae) is one of the most common and widespread semi-aquatic turtles in East Asia. The unusually long lifespan of some individuals makes this turtle species a potentially useful model organism for studying the molecular basis of longevity. In this study, pooled total RNA extracted from liver, spleen and skeletal-muscle of three adult individuals were sequenced using Illumina Hiseq 2500 platform. A set of telomere-related genes were found in the transcriptome, including tert, tep1, and six shelterin complex proteins coding genes (trf1, trf2, tpp1, pot1, tin2 and rap1). These genes products protect chromosome ends from deterioration and therefore significantly contribute to turtle longevity. The transcriptome data generated in this study provides a comprehensive reference for future molecular studies in the turtle.

The thermal acclimatory capacity of a particular species may determine its resilience to environmental change. Evaluating the physiological acclimatory responses of economically important species is useful for determining their optimal culture conditions. Here, juvenile Chinese three-keeled pond turtles (Mauremys reevesii) were acclimated to one of three different temperatures (17, 25 or 33°C) for four weeks to assess the effects of thermal acclimation on some physiological traits. Thermal acclimation significantly affected thermal resistance, but not thermal preference, of juvenile M. reevesii. Turtles acclimated to 17°C were less resistant to high temperatures than those acclimated to 25°C and 33°C. However, turtles increased resistance to low temperatures with decreasing acclimation temperature. The acclimation response ratio of the critical thermal minimum (CTMin) was lower than that of the critical thermal maximum (CTMax) for acclimation temperatures between 17 and 25°C, but slightly higher between 25 and 33°C. The thermal resistance range (i.e., the difference between CTMax and CTMin) was widest in turtles acclimated to the intermediate temperature (25°C), and narrowest in those acclimated to low temperature (17°C). The standard metabolic rate increased as body temperature and acclimation temperature increased, and the temperature quotient (Q10) between acclimation temperatures 17 and 25°C was higher than the Q10 between 25 and 33°C. Our results suggest that juvenile M. reevesii may have a greater resistance under mild thermal conditions resembling natural environments, and better physiological performance at relatively warm temperatures.