Screening of temperature-responsive signalling molecules during sex differentiation in Asian yellow pond turtle (Mauremys mutica).

BACKGROUND: The Asian yellow pond turtle (Mauremys mutica) is an important commercial freshwater aquaculture species in China. This species is a highly sexually dimorphic species, with males growing at a faster rate than females and exhibits temperature-dependent sex determination (TSD), in which the incubation temperature during embryonic development determines the sexual fate. However, the mechanisms of the sex determination or sex differentiation in the Asian yellow pond turtle are remain a mystery. RESULTS: Temperature-specific gonadal transcriptomics of the Asian yellow pond turtle were performed during the thermosensitive period (stage 15) using RNA-seq technology to identify candidate genes that initiate gonadal differentiation. We uncovered candidates that were the first to respond to temperature. These candidates were sexually dimorphic in expression, reflecting differences in gonadal (Cirbp, Runx1) and germline differentiation (Vasa, Nanos1, Piwil2), gametogenesis (Hmgb3, Zar1, Ovoinhibitor-like, Kif4), steroid hormone biosynthesis (Hsd17b5, Hsd17b6), heat shock (Dnajb6, Hsp90b1, Hsp90aa1) and transient receptor potential channel genes (Trpm1, Trpm4, Trpm6, Trpv1). CONCLUSIONS: Our work will provide important genetic information to elucidate the mechanisms of sex control in the Asian yellow pond turtles, and will contribute important genetic resources for further studies of temperature-dependent sex determination in turtles.

Anaerobic fungi in the tortoise alimentary tract illuminate early stages of host-fungal symbiosis and Neocallimastigomycota evolution.

Anaerobic gut fungi (AGF, Neocallimastigomycota) reside in the alimentary tract of herbivores. While their presence in mammals is well documented, evidence for their occurrence in non-mammalian hosts is currently sparse. Culture-independent surveys of AGF in tortoises identified a unique community, with three novel deep-branching genera representing >90% of sequences in most samples. Representatives of all genera were successfully isolated under strict anaerobic conditions. Transcriptomics-enabled phylogenomic and molecular dating analyses indicated an ancient, deep-branching position in the AGF tree for these genera, with an evolutionary divergence time estimate of 104-112 million years ago (Mya). Such estimates push the establishment of animal-Neocallimastigomycota symbiosis from the late to the early Cretaceous. Further, tortoise-associated isolates (T-AGF) exhibited limited capacity for plant polysaccharides metabolism and lacked genes encoding several carbohydrate-active enzyme (CAZyme) families. Finally, we demonstrate that the observed curtailed degradation capacities and reduced CAZyme repertoire is driven by the paucity of horizontal gene transfer (HGT) in T-AGF genomes, compared to their mammalian counterparts. This reduced capacity was reflected in an altered cellulosomal production capacity in T-AGF. Our findings provide insights into the phylogenetic diversity, ecological distribution, evolutionary history, evolution of fungal-host nutritional symbiosis, and dynamics of genes acquisition in Neocallimastigomycota.

A chromosome-level genome assembly of the pig-nosed turtle (Carettochelys insculpta).

The pig-nosed turtle (Carettochelys insculpta) represents the only extant species within the Carettochelyidae family, is a unique Trionychia member fully adapted to aquatic life and currently facing endangerment. To enhance our understanding of this species and contribute to its conservation efforts, we employed high-fidelity (HiFi) and Hi-C sequencing technology to generate its genome assembly at the chromosome level. The assembly result spans 2.18 Gb, with a contig N50 of 126 Mb, encompassing 34 chromosomes that account for 99.6% of the genome. The assembly has a BUSCO score above 95% with different databases and strong collinearity with Yangtze giant softshell turtles (Rafetus swinhoei), indicating its completeness and continuity. A total of 19,175 genes and 46.86% repetitive sequences were annotated. The availability of this chromosome-scale genome represents a valuable resource for the pig-nosed turtle, providing insights into its aquatic adaptation and serving as a foundation for future turtle research.

Embryonic development and transcriptomic analysis in red-eared slider Trachemys scripta elegans under salinity stress.

The elevated salinity in freshwater causes a serious threat to the survival and reproduction of freshwater organisms. The effect of salinity on embryonic development of freshwater turtles is little known. In this study, we investigated the embryonic morphology and underlining mechanism of red-eared slider (Trachemys scripta elegans) in different salinities incubated environment (2.5 ppt and 5 ppt). Results showed that salinity caused various forms of malformed embryos, including brain hypoplasia, eye defects, skeletal dysplasia, deformities of carapace, plastron, limb in the embryo. Severely, salinity could lead to embryos decease. Transcriptome analysis showed that differentially expressed genes induced by salinity primarily enriched in development pathways, metabolism pathways, disease pathways as well as cell processes through KEGG enrichment analysis. In addition, in early and middle embryonic developmental stages, the mRNA expression of apoptotic genes (p38 and bax) significantly increased, whereas anti-apoptotic gene bcl-2 decreased in salinities incubated environment. These findings demonstrated that salinity inhibited the process of embryonic development and damaged organogenesis of turtles through promoting apoptotic pathways.

Population genetics of the critically endangered three-striped turtle, Batagur dhongoka, from the Ganga river system using mitochondrial DNA and microsatellite analysis.

The three-striped roofed (Batagur dhongoka) is a semi-aquatic turtle that belongs to family Geoemydidae. Due to anthropogenic pressure, it has been facing an intense decline of over 80% in its distribution range in the past 50 years. It is considered as 'Critically Endangered' so effective conservation strategies are needed to protect the species by determining their genetic diversity and population genetic structure. This study investigates the genetic diversity, population structure and demographic pattern of B. dhongoka from two Turtle Rescue and Rehabilitation Centre established near Ganga river using mitochondrial cytochrome b (Cyt b: 1140 bp) ; control region (CR: 451 bp) and ten nuclear microsatellite loci. mtDNA results show low levels of nucleotide diversity (π = 0.0022) in B. dhongoka haplotypes and provide evidence for a low substitution rate. The demographic pattern estimated by the Bayesian skyline plot (BSP) analysis indicates historical stability followed by growth in the effective population size, with a recent reduction in population size from ~ 2 thousand years ago. The microsatellite findings show a moderate level of observed heterozygosity (Ho: 0.49). Bayesian-based clustering analysis revealed weak genetic structures in B. dhongoka and presence of admixed assignations suggesting close genetic relationships. These findings shed light on B. dhongoka's genetic status and underline the necessity of comprehensive rehabilitation and relocation programs and conservation and management techniques to ensure the species' long-term survival. In order to ensure the effective protection and conservation of B. dhongoka, the Government of India has taken a proactive measure by incorporating it into Schedule I of the Wildlife (Protection) Act, 1972, as amended in 2022.

Circadian Rhythm Alteration of the Core Clock Genes and the Lipid Metabolism Genes Induced by High-Fat Diet (HFD) in the Liver Tissue of the Chinese Soft-Shelled Turtle (Trionyx sinensis).

Physiology disorders of the liver, as it is an important tissue in lipid metabolism, can cause fatty liver disease. The mechanism might be regulated by 17 circadian clock genes and 18 fat metabolism genes, together with a high-fat diet (HFD). Due to their rich nutritional and medicinal value, Chinese soft-shelled turtles (Trionyx sinensis) are very popular among the Chinese people. In the study, we aimed to investigate the influence of an HFD on the daily expression of both the core clock genes and the lipid metabolism genes in the liver tissue of the turtles. The two diets were formulated with 7.98% lipid (the CON group) and 13.86% lipid (the HFD group) to feed 180 juvenile turtles, which were randomly divided into two groups with three replicates per group and 30 turtles in each replicate for six weeks, and the diet experiment was administrated with a photophase regimen of a 24 h light/dark (12L:12D) cycle. At the end of the experiment, the liver tissue samples were collected from nine turtles per group every 3 h (zeitgeber time: ZT 0, 3, 6, 9, 12, 15, 18, 21 and 24) for 24 h to investigate the daily expression and correlation analysis of these genes. The results showed that 11 core clock genes [i.e., circadian locomotor output cycles kaput (Clock), brain and muscle arnt-like protein 1 and 2 (Bmal1/2), timeless (Tim), cryptochrome 1 (Cry2), period2 (Per2), nuclear factor IL-3 gene (Nfil3), nuclear receptor subfamily 1, treatment D, member 1 and 2 (Nr1d1/2) and retinoic acid related orphan receptor α/ß/γ ß and γ (Rorß/γ)] exhibited circadian oscillation, but 6 genes did not, including neuronal PAS domain protein 2 (Npas2), Per1, Cry1, basic helix-loop-helix family, member E40 (Bhlhe40), Rorα and D-binding protein (Dbp), and 16 lipid metabolism genes including fatty acid synthase (Fas), diacylglycerol acyltransferase 1 (Dgat1), 3-hydroxy-3-methylglutaryl-CoA reductase (Hmgcr), Low-density lipoprotein receptor-related protein 1-like (Ldlr1), Lipin 1 (Lipin1), Carnitine palmitoyltransferase 1A (Cpt1a), Peroxisome proliferator activation receptor α, ß and γ (Pparα/ß/γ), Sirtuin 1 (Sirt1), Apoa (Apoa1), Apolipoprotein B (Apob), Pyruvate Dehydrogenase kinase 4 (Pdk4), Acyl-CoA synthase long-chain1 (Acsl1), Liver X receptors α (Lxrα) and Retinoid X receptor, α (Rxra) also demonstrated circadian oscillations, but 2 genes did not, Scd and Acaca, in the liver tissues of the CON group. However, in the HFD group, the circadian rhythms' expressional patterns were disrupted for the eight core clock genes, Clock, Cry2, Per2, Nfil3, Nr1d1/2 and Rorß/γ, and the peak expression of Bmal1/2 and Tim showed delayed or advanced phases. Furthermore, four genes (Cry1, Per1, Dbp and Rorα) displayed no diurnal rhythm in the CON group; instead, significant circadian rhythms appeared in the HFD group. Meanwhile, the HFD disrupted the circadian rhythm expressions of seven fat metabolism genes (Fas, Cpt1a, Sirt1, Apoa1, Apob, Pdk4 and Acsl1). Meanwhile, the other nine genes in the HFD group also showed advanced or delayed expression peaks compared to the CON group. Most importantly of all, there were remarkably positive or negative correlations between the core clock genes and the lipid metabolism genes, and their correlation relationships were altered by the HFD. To sum up, circadian rhythm alterations of the core clock genes and the lipid metabolism genes were induced by the high-fat diet (HFD) in the liver tissues of T. sinensis. This result provides experimental and theoretical data for the mass breeding and production of T. sinensis in our country.

Effects of butyl paraben on behavior and molecular mechanism of Chinese striped-necked turtle (Mauremys sinensis).

Butyl paraben (BuP) is widely used in cosmetics, drugs, and food preservation. Recently it is an identified new pollutant that affects various aspects of reproduction, lipid metabolism, and nervous system. Behavioral activity serves as a pre-warning biomarker for predicting water quality. So, in this study, the changes in some behaviors and its neurotransmitters and cell apoptosis in the brain of Chinese striped-necked turtles (Mauremys sinensis) were studied when the turtles were exposed to BuP concentrations of 0, 5, 50, 500, and 5000 µg/L for 21 weeks. The results showed that, the basking time and altering scores to external stimuli in the groups of 50, 500, and 5000 µg/L were significantly reduced, while the time for body-righting was significantly increased, compared with the control (0 µg/L), indicating that the turtles exhibited depression and inactive behavior. The analysis of neurotransmitter in the brain showed that 5-hydroxytryptamine (5-HT) contents in the groups of 500 and 5000 µg/L were significantly higher than the other groups, which was due to an increase in the mRNA relative expression levels of the 5-HT receptor gene (5-HTR), neurotransmitter transporter genes (Drd4, Slc6a4), and neurotransmitter synthase tryptophan hydroxylase (TPH). Furthermore, GABA transaminase (GABA-T) activity increased in the 500 and 5000 µg/L groups, and tyrosine hydroxylase (TH) activity increased dramatically in the 5000 µg/L group. However, acetyl-CoA (AChE) activity was significantly reduced in these four BuP exposure groups. These changes could be attributed to decreased movement velocity and increased inactivity. Meanwhile, the mRNA expression level of BAX, Bcl-2, caspase-9 and TUNEL assay indicated the occurrence of cell apoptosis in the brains of the higher BuP exposed groups, which may play an important role in neuronal death inducing behavior change. In summary, these findings offer fundamental insights into turtle ecotoxicology and serve as a foundation for a comprehensive assessment of the ecological and health risks associated with BuP.

Genomic and transcriptome insight into the structure and immunity role of TRIM proteins in Chinese soft-shelled turtles (Pelodiscus sinensis) after Aeromonas hydrophila infection.

BACKGROUND: TRIM proteins, recognized as a class of E3 ubiquitin ligases, are increasingly acknowledged for their antipathogen immune functions in mammals and fish. In the Chinese soft-shelled turtle (Pelodiscus sinensis), a secondary aquatic reptile that occupies a unique evolutionary position, the TRIM gene has rarely been reported. METHODS AND RESULTS: In the present study, 48 PsTRIM proteins were identified from the genome of Pelodiscus sinensis via Hidden Markov Model (HMM) searches and Signal Transduction ATPases with Numerous Domains (SMART) analysis. These PsTRIMs were found across 43 distinct scaffolds, and phylogenetic analyses classified them into three principal clades. The PsTRIMs feature a conserved assembly of either RING-B-box-coiled-coil (RBCC) or B-box-coiled-coil (BBC) domains at the N-terminus, in addition to eight unique domains at the C-terminus, including the B30.2 domain, 19 of which were identified. Expression profiling revealed ubiquitous expression of the 48 PsTRIMs across various P. sinensis tissues. Notably, seven PsTRIMs exhibited significant differential expression in liver transcriptomes following infection with Aeromonas hydrophila. Weighted gene coexpression network analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis implicated PsTRIM14 and PsTRIM28 as key players in host defense against bacterial invasion. Real-time quantitative PCR results indicated that PsTRIM1, PsTRIM2, PsTRIM14, and PsTRIM28 experienced marked upregulation in P. sinensis livers at 12 h post-infection with A. hydrophila. CONCLUSIONS: Our study is the first to comprehensively identify and analyze the functions of TRIM genes in P. sinensis, unveiling their considerable diversity and potential roles in modulating immune responses.

HAT: de novo variant calling for highly accurate short-read and long-read sequencing data.

MOTIVATION: de novo variants (DNVs) are variants that are present in offspring but not in their parents. DNVs are both important for examining mutation rates as well as in the identification of disease-related variation. While efforts have been made to call DNVs, calling of DNVs is still challenging from parent-child sequenced trio data. We developed Hare And Tortoise (HAT) as an automated DNV detection workflow for highly accurate short-read and long-read sequencing data. Reliable detection of DNVs is important for human genomics and HAT addresses this need. RESULTS: HAT is a computational workflow that begins with aligned read data (i.e. CRAM or BAM) from a parent-child sequenced trio and outputs DNVs. HAT detects high-quality DNVs from Illumina short-read whole-exome sequencing, Illumina short-read whole-genome sequencing, and highly accurate PacBio HiFi long-read whole-genome sequencing data. The quality of these DNVs is high based on a series of quality metrics including number of DNVs per individual, percent of DNVs at CpG sites, and percent of DNVs phased to the paternal chromosome of origin. AVAILABILITY AND IMPLEMENTATION: https://github.com/TNTurnerLab/HAT.

New colonisers drive the increase of the emerging loggerhead turtle nesting in Western Mediterranean.

The loggerhead sea turtle (Caretta caretta) is sensitive to climate change and is responding by colonising the Western Mediterranean. To understand the rapid nesting increase in recent years in Spain, we sampled 45 hatchlings from 8 nests between 2016 and 2019. We sequenced a mtDNA D-loop region, genotyped 2291 SNPs using 2bRAD and collected data on clutch size, hatching success, and incubation duration. We confirmed that the colonisation has a Mediterranean and Atlantic mixed origin and we detected that these nests were laid by different females, except for two nests within the same season. Our results suggest that the recent increase in nesting is due to an increase in the number of colonising individuals rather than females born in the same area returning to breed. We hypothesize that this increase in the number of colonisers results from successful conservation efforts, feminisation of the populations of origin and earlier sexual maturation. However, the percentage of offspring females produced in Spain suggests that future returning individuals will aid to the settlement of the new population. These results allow defining the current status of this colonisation although future efforts are needed to detect remigrants to confirm the establishment of a resident population.

Identification of Sex-Specific Markers and Candidate Genes Using WGS Sequencing Reveals a ZW-Type Sex-Determination System in the Chinese Soft-Shell Turtle (Pelodiscus sinensis).

Male and female Chinese soft-shelled turtles (Pelodiscus sinensis) have sex-dimorphic growth patterns, and males have higher commercial value because of their larger size and thicker calipash. Thus, developing sex-specific markers is beneficial to studies on all-male breeding in P. sinensis. Here, we developed an accurate and efficient workflow for the screening of sex-specific sequences with ZW or XY sex determination systems. Based on this workflow, female and male P. sinensis reference genomes of 2.23 Gb and 2.26 Gb were obtained using de novo assembly. After aligning and filtering, 4.01 Mb female-specific sequences were finally identified. Subsequently, the seven developed sex-specific primer pairs were 100% accurate in preliminary, population, and embryonic validation. The presence and absence of bands for the primers of P44, P45, P66, P67, P68, and P69, as well as two and one bands for the PB1 primer, indicate that the embryos are genetically female and male, respectively. NR and functional annotations identified several sex-determining candidate genes and related pathways, including Ran, Eif4et, and Crkl genes, and the insulin signaling pathway and the cAMP signaling pathway, respectively. Collectively, our results reveal that a ZW-type sex-determination system is present in P. sinensis and provide novel insights for the screening of sex-specific markers, sex-control breeding, and the studies of the sex determination mechanism of P. sinensis.

Isolation, identification and characterization of Aeromonas jandaei from diseased Chinese soft-shell turtles.

Aeromonas jandaei is a gram-negative bacterium commonly found in aquatic environments and can induce illnesses in amphibians, reptiles and aquatic animals. In this study, a strain of bacteria was isolated from the diseased Chinese soft-shell turtle (Pelodiscus sinensis), then named strain JDP-FX. This isolate was identified as A. jandaei after analysis of morphological, physiological and biochemical characteristics, as well as 16S rRNA and gyrB gene sequences. Virulence genetic testing further detected temperature-sensitive protease (eprCAI), type III secretion system (TTSS) (ascv), nuclease (nuc), cytotonic enterotoxin (alt) and serine proteinase (ser) in JDP-FX. Compared with healthy Chinese soft-shell turtle, the serum levels of total protein (TP), albumin (ALB) and globulin (GLB) were significantly decreased in the diseased Chinese soft-shell turtle, while, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were significantly increased. Histopathological observations showed that multiple tissues, including intestinal mucosa, liver and kidney, were severely damaged in the diseased Chinese soft-shell turtle. Moreover, the diseased Chinese soft-shell turtle had significant cell degeneration, necrosis, sloughing and interstitial inflammatory cell infiltration. The pathogenicity of JDP-FX was tested via artificial infection. The median lethal dosage (LD50 ) of the strain was 1.05 × 105 colony forming units (CFU/g) per weight of Chinese soft-shell turtle. Drug susceptibility analysis revealed that JDP-FX was susceptible to ceftazidime, minocycline, cefoperazone, ceftriaxone and piperacillin. In addition, JDP-FX was resistant to doxycycline, florfenicol, sulfonamides, gentamicin, ampicillin and neomycin. Therefore, this study may provide guidance for further research into the diagnosis, prevention and treatment of JDP-FX infection.

Comparative genomic survey and functional analysis of DKKL1 during spermatogenesis in the Chinese soft-shelled turtle (Pelodiscus sinensis).

A feature of the Chinese soft-shelled turtle (Pelodiscus sinensis) is seasonal spermatogenesis; however, the underlying molecular mechanism is not well clarified. Here, we firstly cloned and characterized P. sinensis DKKL1, and then performed comparative genomic studies, expression analysis, and functional validation. P. sinensis DKKL1 had 2 putative N-glycosylation sites and 16 phosphorylation sites. DKKL1 also had classic transmembrane structures that were extracellularly localized. DKKL1's genetic distance was close to turtles, followed by amphibians and mammals, but its genetic distance was far from fishes. DKKL1 genes from different species shared distinct genomic characteristics. Meanwhile, they were also relatively conserved among themselves, at least from the perspective of classes. Notably, the transcription factors associated with spermatogenesis were also identified, containing CTCF, EWSR1, and FOXL2. DKKL1 exhibited sexually dimorphic expression only in adult gonads, which was significantly higher than that in other somatic tissues (P < 0.001), and was barely expressed in embryonic gonads. DKKL1 transcripts showed a strong signal in sperm, while faint signals were detected in other male germ cells. DKKL1 in adult testes progressively increased per month (P < 0.05), displaying a seasonal expression trait. DKKL1 was significantly downregulated in testes cells after the sex hormones (17ß-estradiol and 17α-methyltestosterone) and Wnt/ß-catenin inhibitor treatment (P < 0.05). Likewise, the Wnt/ß-catenin inhibitor treatment dramatically repressed CTCF, EWSR1, and FOXL2 expression. Conversely, they were markedly upregulated after the 17ß-estradiol and 17α-methyltestosterone treatment, suggesting that the three transcription factors might bind to different promoter regions, thereby negatively regulating DKKL1 transcription in response to the changes in the estrogen and androgen pathways, and positively controlling DKKL1 transcription in answer to the alterations in the Wnt/ß-catenin pathway. Knockdown of DKKL1 significantly reduced the relative expression of HMGB2 and SPATS1 (P < 0.01), suggesting that it may be involved in seasonal spermatogenesis of P. sinensis through a positive regulatory interaction with these two genes. Overall, our findings provide novel insights into the genome evolution and potential functions of seasonal spermatogenesis of P. sinensis DKKL1.

Effects of blood mercury accumulation on DNA methylation levels in the Khorat snail-eating turtle (Malayemys khoratensis).

Mercury (Hg) has adverse effects on humans and wildlife. Hg exposure can cause significant alterations in DNA methylation, an epigenetic modification that causes various illnesses. Hg accumulation in the blood of the Khorat snail-eating turtle (Malayemys khoratensis) from northeastern Thailand was previously reported. Thus, this study aimed to assess total mercury (THg) levels in M. khoratensis blood and to examine the impact of these concentrations on DNA methylation (5-methylcytosine, 5-mC) levels. We divided turtles based on morphological characteristics into two groups, normal and deformed, and then the levels of each variable in both groups were assessed. The deformed group presented higher mean THg concentration and DNA methylation levels compared to the normal group; however, the differences were not significant. Additionally, we found no correlation between DNA methylation levels and THg concentrations in both groups. This study is the first attempt to investigate the relationship between mercury accumulation and DNA methylation in the blood of deformed freshwater turtles.

Comparative analysis of whole-transcriptome RNA expression of lung tissue of Chinese soft-shell turtle infected by Trionyx sinensis Hemorrhagic Syndrome Virus.

Trionyx sinensis Hemorrhagic Syndrome Virus (TSHSV), the first aquatic arterivirus identified in China, causes severe mortality to T. sinensis. In this study, we sought to determine the functions of T. sinensis mRNAs and non-coding RNAs (ncRNAs) that were differentially expressed (DE) over different periods of TSHSV infection of T. sinensis lung. We used RT-qPCR to validate the sequencing results of select RNAs, confirming their reliable and referable nature. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were used to predict multiple biological functions and signaling pathways in various comparison groups (1-day versus mock, 3-day versus 1-day, and 5-day versus 3-day). Multiple types of differentially expressed RNA, including mRNA, lncRNA, circRNA, and miRNA, were associated with cardiac dysfunction, coagulation abnormalities, and arachidonic acid metabolism at day 1. Pre-inflammatory cytokines and inflammatory factors such as PLA2G4A, cPLA2, γ-GGT1, TNFRSF14, TCP11L2, PTER CYP2J2 and LTC4S, were noticeably regulated at the same time. On day 3, multiple GO terms and KEGG pathways were implicated, including those related to virus defense, apoptosis, pyroptosis, and inflammatory response. Notably, key genes such as RSAD2, TRIM39, STAT4, CASP1, CASP14, MYD88, CXCL3, CARD11, ZBP1, and ROBO4 exhibited significant regulation. The lncRNAs and circRNAs that targeted the genes involved in viral recognition (TLR5), apoptosis (CARD11), pyroptosis (ZBP1), inflammatory processes (NEK7, RASGRP4, and SELE) and angiogenesis (ROBO4) exhibited significant regulation. Significantly regulated miRNAs were primarily linked to genes involved in apoptosis (Let-7f-3p, miR-1260a, miR-455-3p), and inflammation (miR-146a, miR-125a, miR-17a, miR-301b, and miR-30a-3p). The findings could advance our understanding of the host immunological response to TSHSV and offer new ideas for developing effective strategies to prevent infection of T. sinensis.

Molecular cloning, characterization and expression analysis of the Chinese soft-shelled turtle (Pelodiscus sinensis) chemokine CXCL11.

Chemokines are small, secreted proteins with chemoattractive properties, which play an important role in the recruitment and activation of immune cells. CXCL11 is a CXC chemokine specific for the CXCR3 receptors, which has been shown to mediate the generation of Th1-type immune responses and have bactericidal effects similar to defensins. Herein, we cloned the full-length cDNA of Chinese soft-shelled turtle (Pelodiscus sinensis) CXCL11, designated as PsCXCL11, which consist of an open reading frame (ORF) of 282 bp encoding 93 amino acids, with estimated molecular weight of 10.055 kDa and isoelectric point of 10.37. The deduced PsCXCL11 sequence had a signal peptide, a highly conserved family-specific small cytokine (SCY) domain, one putative N-glycosylation site and ten potential phosphorylation sites. Phylogenetic analysis showed a close relationship between P. sinensis and Chelydra Serpentina CXCL11. P. sinensis CXCL11 basal expression levels were higher in heart, kidney and spleen than in other organs of health turtles. Infections of Aeromonas hydrophila and Staphylococcus aureus led to significant upregulation of P. sinensis CXCL11 in the blood, while significant upregulation of PsCXCL11 were observed in liver and spleen after infection of A. hydrophila, but not S. aureus. PsCXCL11 recombinant protein with His-tag was successfully expressed by an auto-inducible expression system, and purified by Ni-NTA affinity chromatography. These findings laid a solid foundation for further research towards development of the Chinese soft-shelled turtle as a model for the role of CXCL11 in regulating inflammatory responses to stimulation by invading pathogens.

Isolation and identification of Chryseobacterium indologenes and its pathological changes in Pelodiscus sinensis.

The Chinese revered a species of aquatic reptile known as Pelodiscus sinensis as both an edible and medicinal species. When artificially breeding, many deaths occurred at the farmed P. sinensis, mainly due to excessive breeding density, water contamination, and turtles biting each other secondary to bacterial infections. In this study, an isolate of gram-negative bacteria WH0623 was isolated from the liver and kidney of diseased P. sinensis to trace the potential pathogen of this disease. Based on biochemical characteristics and 16S rRNA gene sequencing analyses, this isolated strain of WH0623 was identified as Chryseobacterium indologenes. The strain's median lethal dose (LD50 ) was 3.3 × 105 colony-forming units (CFU)/g per fish weight tested using artificial infection. Histopathological analysis revealed pathological changes, including cell swelling, hyperaemia, and necrosis in many tissues. Antibiotic susceptibility tests revealed that the bacteria WH0623 was susceptible to doxycycline, sulphonamides, ceftazidime, norfloxacin, and ciprofloxacin. These antibiotics could treat the disease. In conclusion, the pathogen causing the death of farmed P. sinensis was isolated and identified, and a drug-sensitive test was conducted. Our findings contribute to the future diagnosis and treatment of the disease.

Morphological and transcriptomic analyses of embryonic development of red-eared slider Trachemys scripta elegans.

Embryology provides an understanding of individual's origin and developmental patterns. Turtles are among the oldest living reptiles and have unique body structure. However, the morphogenesis and mechanisms of turtles are not fully understood. In this study, we focused on the embryonic development of red-eared slider (Trachemys scripta elegans) which widely distributes in the world. At an incubation temperature of 28 °C, the turtle eggs had a 61-day incubation cycle, and the entire embryonic development process was divided into 27 stages and 3 phases according to variations in age, body size, and morphological characteristics. The early phase of embryonic development (the first 12 stages) were characterized by embryo growth, and the appearance of internal organ precursors. The middle phase (stages 13-20) involved prominent heart division at stage 13 and the appearance of carapace and plastron at stages 14 and 17, respectively. In the later phase (stages 21-27), the hatchlings formed, and the carapace and plastron thickened. Transcriptome analysis of embryos showed enrichment of the differential genes in pathways related to development, metabolism, disease, and cellular processes. The Kyoto Encyclopedia of Genes and Genomes enrichment (KEGG) analysis implied the crucial regulatory role of the axon guidance pathway. Real-time fluorescence quantitative PCR indicated upregulated expression of wnt5a and bmp7 in stages 7 and 16 compared to that in stage 12. This study revealed the development process of red-eared slider embryo and the dynamics of the signaling pathway affecting its development, which supplemented the theory of embryo development, and provided new ideas for the molecular mechanism of turtle embryo development.

Histomorphic analysis and expression of mRNA and miRNA in embryonic gonadal differentiation in Chinese soft-shelled turtle (Pelodiscus sinensis).

The Chinese soft-shelled turtle (Pelodiscus sinensis) is extensively cultured in Asia for its nutritional and medical value. Gonadal differentiation is fantastic in turtles, whereas morphologic, mRNA, and miRNA expressions were insufficient in the turtle. In this study, ovaries and testes histomorphology analysis of 14-23 stage embryos were performed, and mRNA and miRNA expression profiles were analyzed. Histomorphology analysis revealed that gonads were undifferentiated at embryonic stage 14. Ovarian morphological differentiation became evident from stage 15, which was characterized by the development of the cortical region and degeneration of the medullary region. Concurrently, testicular morphological differentiation was apparent from stage 15, marked by the development of the medullary region and degeneration of the cortical region. qRT-PCR results showed that Cyp19a1 and Foxl2 exhibited female-specific expression at stage 15 and the expression increased throughout most of the embryonic development. Dmrt1, Amh, and Sox9 displayed male-specific expression at stage 15 and tended to increase substantially at later developmental stages. The expression of miR-8356 and miR-3299 in ZZ gonads were significantly higher than that in ZW gonads at stage 15, 17 and 19, and they had the highest expression at stage 15. While the expression of miR-8085 and miR-7982 had the highest expression at stage 19. Furthermore, chromatin remodeler genes showed differential expression in female and male P. sinensis gonads. These results of master sex-differentiation genes and morphological characteristics would provide a reference for the research of sex differentiation and sex reversal in turtles. Additionally, the expression of chromatin remodeler genes indicated they might be involved in gonadal differentiation of P. sinensis.

A chromosome-level genome assembly of the Asian giant softshell turtle Pelochelys cantorii.

The Asian giant softshell turtle Pelochelys cantorii is one of the largest aquatic turtles in China and has been designated a First Grade Protected Animal in China. To advance conservation research, a combination of Illumina short-read, PacBio long-read, and Hi-C scaffolding technologies was used to develop a high-quality chromosome-level genome assembly for P. cantorii. A total of 262.77 Gb of clean data were produced (121.6 × depth) and then the genome was assembled into 2.16 Gb with a contig N50 of 41.44 Mb and scaffold N50 length of 120.17 Mb, respectively. Moreover, about 99.98% assembly genome sequences were clustered and ordered onto 33 pseudochromosomes. Genome annotation revealed that 21,833 protein-coding genes were predicted, and 96.40% of them were annotated. This new chromosome-level assembly will be an enabling resource for genetic and genomic studies to support fundamental insight into P. cantorii biology.