Amphibian malformation syndromes significantly impact both conservation efforts and aquaculture, yet their underlying systemic pathophysiological mechanisms remain poorly characterized. This study comprehensively examines the multi-level pathological processes associated with the "short-leg" malformation syndrome in the black-spotted frog (Pelophylax nigromaculatus) using an integrated methodology, encompassing morphological, histopathological, gut microbiome, and hepatic transcriptomic analyses. Affected frogs demonstrated shortened limbs, impaired motor function, and a distinctive metabolic phenotype, including increased body weight despite a shorter body length, accumulation of visceral fat, and shortened intestines. Gut microbiota analysis identified significant compositional shifts, characterized by a decreased Firmicutes-to-Bacteroidota ratio, expansion of pro-inflammatory Proteobacteria, and reduction in beneficial Actinobacteriota, suggesting microbial niche restructuring that likely promotes metabolic and inflammatory disorders. Hepatic transcriptome profiling revealed 2617 differentially expressed genes, demonstrating a clear molecular dichotomy with concurrent up-regulation of immune-related pathways (e.g., neutrophil extracellular trap formation, complement cascades, and inflammatory signaling) and broad suppression of metabolic pathways (e.g., lipid oxidation, nutrient absorption, and PPAR and renin-angiotensin systems). This integrated analysis illustrates that the malformation syndrome represents a systemic pathophysiological state involving dysfunction of the gut-liver axis, characterized by the coexistence of gut microbiota alterations, hepatic metabolic suppression, and immune activation. These findings provide a framework for understanding amphibian malformations and suggest potential strategies to improve health outcomes in aquaculture.

MicroRNA(miRNAs) are key post-transcriptional regulators of gene expression and play a role in body color formation in aquatic animals. However, their functional roles in body color formation in Chinese soft-shelled turtle (Pelodiscus sinensis) remain largely uncharacterized. Here, we combined in vitro and in vivo approaches to dissect the regulatory mechanisms of miR-18a-3p_3 and miR-221 on pigmentation-related genes. Dual-luciferase reporter assays confirmed direct binding between miR-18a-3p_3 and the 3’UTR of Sox10, as well as between miR-221 and Mitf. Mutations in the predicted binding sites abolished these interactions, validating the specificity of targeting. Subsequently, we successfully isolated and cultured melanocytes from P. sinensis embryos using a specialized medium, enabling functional validation of miRNA effects on pigmentation gene expression. Transfection of miR-18a-3p_3 antagomir and miR-221 agomir into primary melanocytes modulated the expression of Sox10 and Mitf, respectively, and altered the transcription of downstream melanogenic genes including Tyr, Tyrp1, and Gpnmb. In vivo administration of miRNA modulators (antagomir/agomir) confirmed these regulatory patterns in turtle tissues, though tissue-specific expression differences were less pronounced than in cultured cells, suggesting context-dependent regulatory complexity. Collectively, we speculated that miR-18a-3p_3 might be negatively modulating Sox10, while miR-221 positively regulated Mitf, thereby coordinating melanin synthesis in P. sinensis. These findings provide novel insights into the molecular basis of body color variation in turtles and expand our understanding of miRNA-mediated pigmentation regulation in aquatic vertebrates.

Pelodiscus sinensis (Chinese soft-shelled turtle) is a freshwater species with economic and biomedical value. Here, we report a near-telomere-to-telomere (near-T2T) chromosome-level genome assembly. Using a combination of PacBio HiFi, Oxford Nanopore ultra-long reads, and Hi-C data, we assembled a 2.27 Gb genome (contig N50 = 132.52 Mb). Hi-C contact maps guided scaffolding and curation, anchoring 40 contigs to 34 chromosomes and resolving telomeres for 61 out of the 68 chromosomal ends. Gap filling and polishing yielded a chromosome-level assembly with no ambiguous bases (QV = 41.19; GC = 45.8%). BUSCO analysis indicated 97.9% completeness. Mapping rates for BGI short reads (99.4%), ONT long reads (100%), and HiFi reads (99.98%) confirm high accuracy across platforms. We annotated 21,124 protein-coding genes (97.77% functionally annotated) and 12,868 non-coding RNAs, with support from RNA-seq and full-length transcript data. Sex chromosomes (chrZ and chrW) were inferred from male-to-female depth ratios and validated using two known sex-linked SNP markers. This high-quality genome provides an essential resource for studying reptilian chromosome evolution, sex determination, and molecular breeding.

The development of sex markers is crucial for addressing monosexual breeding in aquaculture species and for identifying traits that are sexually inherited, especially for elucidating the mechanisms of sex determination in amphibians. In aquaculture, comprehending sex determination is especially vital because the market value of animal products frequently depends on their sex. Quasipaa spinosa (Anura, Dicroglossidea) is a valuable frog species in the aquaculture industry of China and southeast Asia, yet there exists limited genomic information regarding this organism. Current data indicates that the adoption of all-male breeding techniques in Q. spinosa could substantially benefit the Chinese aquaculture industry, both by augmenting its economic prospects and by ensuring the effectiveness of wildlife reintroduction efforts. The growth rate, adult size, disease resistance, and other traits of male Q. spinosa surpass those of females, making the development of all-male breeding a significant focus in the Q. spinosa aquaculture industry. Therefore, it is imperative to establish a marker specific to males. In this research, we used the male Q. spinosa genome as reference and performed whole-genome resequencing on 30 males and 30 females. Subsequently, we exhibited evident sexual differentiation on chromosome 3 and primers were designed for PCR detection of the identified candidate male INDEL loci. Ultimately, two sex-associated INDELs that could be effectively detected were obtained and validated on the samples collected from the remaining three locations, thereby confirming the robustness of these two INDELs for sex identification in Q. spinosa.

Hybridization is an effective method for the genetic improvement of breeding varieties, while establishing standardized management systems and identification methods is an important foundation for obtaining stable hybrid new varieties with desirable traits. This study conducted measurements and analyses of eight morphological indicators of three Chinese soft‐shelled turtle ( Pelodiscus sinensis ) populations: the Huangsha turtle ( P. sinensis Huangsha, HS), Japanese turtle ( P . sinensis Japanese strains, JP), and their hybrid F1 generation (HS ♂ × JP ♀, HJ). Based on several important indicators, a discriminant function was established, and after cross‐validation, the comprehensive discriminant accuracy was 86.4%. In addition, we used super‐genotyping‐by‐sequencing (GBS) sequencing technology to analyze the genetic structure and genetic diversity of the three populations. The results showed that the HJ population had a closer genetic relationship with the JP population as the maternal parent and a farther genetic relationship with the HS population. The genetic diversity was between the HS and JP populations, and the genetic distance between the HS and JP populations was far. Through fingerprint analysis technology, core SNP markers for germplasm identification were screened, and a total of 148 core SNP marker loci were obtained. One SNP marker that could distinguish the HJ population from the HS population was developed. This study will provide reference for the hybrid breeding and genetic management of Chinese soft‐shelled turtle.

Background/Objectives: The Chinese soft-shelled turtle (Pelodiscus sinensis) is an important species in freshwater aquaculture. Genetic admixture and degradation due to rapid industry expansion threaten sustainable development. This study aims to assess the genetic diversity and structure of six P. sinensis populations for better management. Methods: We combined morphological analysis and microsatellite markers to evaluate the genetic diversity of six populations. A discriminant function based on morphology was developed, achieving 71.4% classification accuracy. Two SSR markers were identified to specifically distinguish the HS population. Results: The six populations were classified into three subgroups. Frequent gene flow was observed among the CY, W, and DT populations, with most genetic variation occurring within individuals. However, significant genetic differentiation was detected between populations. While gene flow enhanced diversity, it suppressed differentiation. Conclusions: This study provides insights into the genetic structure and diversity of six P. sinensis populations. The discriminant function and SSR markers offer a basis for germplasm conservation and management, supporting sustainable aquaculture development.

Cyprinus carpio var. Xiangxi is a regionally farmed rice-field carp endemic to the Xiangxi region of Hunan Province, China. To adapt to the rice-fish farming environment, this variety has developed a range of genetic and physiological adaptations. However, genomic research on this strain remains limited. Here, we assembled a high-quality chromosome-level genome of C. carpio var. Xiangxi to fill this knowledge gap. Using PacBio HiFi, DNBSEQ, and Hi-C technology, We generated a 1.54 Gb genome with a contig N50 of 28.12 Mb. Using Hi-C data, 99.38% of the assembly (1,535.34 Mb) was anchored to 50 pseudochromosomes, yielding a scaffold N50 of 30.26 Mb. The quality value and Benchmarking Universal Single-Copy Ortholog score were 62.32 and 97.4%, respectively, indicating that our genome sequence is of high quality and completeness. A total of 46,362 protein-coding genes were predicted, of which 45,409 (97.94%) were functionally annotated. This study provides a valuable genomic resource for the conservation of C. carpio var. Xiangxi and offers new insights into the evolutionary history of common carp.

Amphibian aquaculture has rapidly expanded due to the increasing global demand for animal proteins. Among cultured species, black-spotted frog (Pelophylax nigromaculatus) has gained attention because of its high economic and market value. However, frequent disease outbreaks under intensive farming conditions considerably hinder its sustainable growth. Recently, plant-derived functional additives have emerged as promising alternatives to enhance disease resistance in aquaculture due to their immunostimulatory, antioxidative, and gut microbiota-modulating activities. This study evaluated the effects of different concentrations of a herbal supplement (HS) containing 0.32 % ginsenosides and 20 % ginseng polysaccharides on P. nigromaculatus growth performance and disease resistance over a 60-day feeding trial. Compared to the controls, frogs fed HS-supplemented diets showed significantly higher specific growth and weight gain rates (P < 0.05). Serum antioxidant enzyme activity was significantly elevated in the HS groups (P < 0.05). Intestinal barrier-related gene (zona occludens 1 and 2 and claudin 3 and 5) levels were upregulated, and antimicrobial peptide gene OL-CATH1 levels were significantly increased, whereas pro-inflammatory cytokine gene (interleukin-10 and -12) levels were markedly downregulated (P < 0.05), suggesting enhanced innate immunity without systemic inflammation. Moreover, 16S rRNA sequencing revealed a notable increase in beneficial gut bacteria abundance and reduction in pathogenic species abundance in HS-treated frogs, indicating a favourable shift in intestinal microbiota composition. In the pathogen challenge test with Aeromonas hydrophila, HS-fed frogs exhibited significantly higher survival rates than the controls (P < 0.05). Validation trial revealed that Lactococcus lactis dietary supplementation improved the frog growth performance and gut microbial stability. Overall, HS enhanced the growth performance and disease resistance by modulating the gut microbiota, boosting the antioxidant capacity, and promoting the innate immune responses of P. nigromaculatus. Our findings suggest 0.04 % HS as a safe, eco-friendly, and sustainable functional additive for amphibian aquaculture.

Trichosporon asahii, a fungal pathogen predominantly associated with immunocompromised humans, has now been identified as a novel pathogen in amphibian aquaculture. During a 2023 outbreak on a 300,000-individual bullfrog farm in Hunan Province, China, we documented 113,000 mortalities (37.7 % mortality rate) within 60 days, marking the first large-scale epidemic of putrid skin disease in commercial amphibians. Through Koch's postulate fulfillment - isolating seven T. asahii strains from lesions and reproducing disease in laboratoryinfected frogs - we confirmed its causative role. Notably, BALB/c murine models revealed systemic infections, demonstrating mammalian virulence potential. These findings redefine T. asahii as a transboundary pathogen requiring coordinated veterinary-medical interventions and provide epidemiological benchmarks for developing biosecurity protocols in intensive frog farming systems.

The Chinese soft-shelled turtle (Pelodiscus sinensis) is an important aquaculture animal in China and exhibits growth dimorphism. Single-male cultures are often selected for higher economic efficiency. However, the mechanism of sex differentiation in P. sinensis is not well-known. In this study, a comparative transcriptome analysis of male (ZZ)- and 17β-oestradiol (E2)-induced pseudo-female (ZZ + E2)-stage embryonic gonads of P. sinensis was performed. A total of 420 differentially expressed genes (DEGs), which included 271 upregulated genes and 149 downregulated genes, were identified. These DEGs were mainly involved in several sex-related pathways, such as “ovarian steroidogenesis”, “steroid hormone biosynthesis”, “PPAR signalling pathway”, and “metabolism of xenobiotics by cytochrome P450”. In addition, 50 known and novel candidate genes involved in sex differentiation, such as the male-biased genes AMH, DMRT1, TBX1, and CYP26A1 and the female-biased genes CYP1A1, RASD1, and SOX17, were investigated and identified. For further verification, the full-length cDNAs of SOX17 and CYP26A1 were obtained. SOX17 contains a 1218-bp ORF and encodes 405 amino acids containing an HMG functional domain unique to the Sox superfamily. CYP26A1 contains a 1485-bp ORF and encodes 494 amino acids. Different expression levels of SOX17 and CYP26A1 could be detected in all the tested tissues of males and females. Notably, the expression of CYP26A1 was markedly greater in the gonads of male embryos (P < 0.05) than in those of female embryos, whereas the expression of SOX17 showed the opposite trend (P < 0.05). Taken together, the RNA-seq and qRT‒PCR results suggested potential roles for SOX17 and CYP26A1 in promoting female and male gonadal development, respectively, in P. sinensis. Our results provide new evidence for the mechanism of sex differentiation in P. 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/β/γ β and γ (Rorβ/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, β and γ (Pparα/β/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 Rorβ/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.

Pelophylax nigromaculatus is an important aquaculture economic animal in China, with an annual output of more than 80,000 tons in recent years. P. nigromaculatus demonstrates distinct sexual dimorphism, characterized by females being substantially larger than males. Consequently, the cultivation of female populations presents higher economic value. The RNA-Seq technology is harnessed in this study to delve into the sexual differentiation and reproductive mechanisms of these frogs. By analyzing the transcriptomes of mature testes and ovaries, 17.24 Gbp and 23.71 Gbp of clean data were obtained respectively. This analysis yielded 107,897 annotated genes, among which 8099 were differentially expressed; 4370 were upregulated and 3729 were downregulated in ovaries. GO and KEGG analyses highlighted 70 genes exhibiting sexual dimorphism. For validation, 13 differentially expressed genes were subjected to qRT-PCR, corroborating the results of transcriptome sequencing. Furthermore, the transcriptome analysis of P. nigromaculatus’ sexual glands revealed 1,062,663 SNPs, with 676,172 (63.63

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 Ps TRIM 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 Ps TRIMs were found across 43 distinct scaffolds, and phylogenetic analyses classified them into three principal clades. The Ps TRIMs 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 Ps TRIMs across various P. sinensis tissues. Notably, seven Ps TRIMs 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 Ps TRIM14 and Ps TRIM28 as key players in host defense against bacterial invasion. Real-time quantitative PCR results indicated that Ps TRIM1, Ps TRIM2, Ps TRIM14, and Ps TRIM28 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.

Background: Toll-like receptor (TLR), as an important pattern recognition receptor, is a bridge between non-specific immunity and specific immunity, and plays a vital role in the disease resistance of aquatic animals. However, the function of TLR in Pelodiscus sinensis is still unclear. Methods and Results: The sequence characteristics and homology of three TLRs (PsTLR2, PsTLR3 and PsTLR5) were determined in this investigation. Their annotation and orthologies were supported by phylogenetic analysis, functional domain prediction, and sequence similarity analysis. qPCR showed that the identified TLRs were expressed in all tissues, among the high expression of PsTLR5 in the brain and liver and the high expression of PsTLR2 and PsTLR3 in the liver. PsTLR2 mRNA expression increased 6.7-fold in the liver 12 h after Aeromonas hydrophila infection, while the mRNA expression of PsTLR3 was down-regulated by 0.29 times in liver and 0.31 times in spleen. The mRNA expression of PsTLR5 was significantly up-regulated in four immune tissues, and it was up-regulated by 122.8 times in the spleen after 72 h infection. Finally, the recombinant proteins of extracellular LRR domains of these three TLRs were obtained by prokaryotic expression technology, and the binding tests were performed to discover their ability of binding pathogenic microorganisms. Microbial binding test showed that rPsTLR2, rPsTLR3 and rPsTLR5 can combine A. hydrophila, Edwardsiella tarda, Vibrio parahaemolyticus, Staphylococcus aureus, Streptococcus agalactiae and Candida albicans, while rPsTLR3 can bind A. hydrophila, E. tarda, V. parahaemolyticus and C. albicans. Conclusions: Our findings suggested that TLRs may be crucial to turtles’ innate immune response against microbes.

Chinese soft-shelled turtle (Pelodiscus sinensis) is an important aquaculture species in China. However, with the continuous development of P. sinensis cultures, problems related to germplasm degradation and population mixing have become increasingly prominent. This has seriously affected the protection and utilization of germplasm resources by P. sinensis. To broaden the understanding of the germplasm resources of P. sinensis and improve the efficiency of germplasm identification, Super-GBS sequencing technology was used to conduct population genetic analysis of P. sinensis samples from six different populations distributed in five provinces. As a result, 1464,488 SNPs were identified. Genetic structure analysis revealed that the six P. sinensis populations could be divided into three sub-populations. The genetic differentiation coefficient of the DT and CY populations was very low (Fst=0.0088), indicating that differentiation had not yet occurred. Additionally, 15 KASP core markers for the identification of P. sinensis populations were developed using KASP typing technique for the first time. Genetic information can lay a foundation for the protection and utilization of germplasm resources by P. sinensis, and the fingerprinting database developed in this study provides a practical tool for authenticating P. sinensis.

The skin microbiota plays an essential role in helping the host adapt to different environments and maintain health. By examining the characteristics of amphibian skin flora alongside ontogenetic traits, we can gain insights into the adaptation mechanisms of amphibian skin flora to environmental changes during development. In this study, we analyzed the skin microbiota of Quasipaa spinosa during metamorphosis using Illumina sequencing. Venn diagrams and UpSet analysis revealed that the LTS (hindlimb tadpoles’ skin, aquatic habitat) and FTS (forelimb tadpoles’ skin, shift from aquatic to amphibious habitats) groups exhibited a higher number of unique amplicon sequence variants (ASVs), while the TS (tadpoles’ skin, aquatic habitat) and LFS (land frogs’ skin, amphibious habitats) groups displayed a lower abundance of ASVs. Diversity analysis indicated similarities in the microorganisms between the LTS and the FTS groups, with higher microbial diversity compared to the TS and the LFS groups. Additionally, microbial co-occurrence network analysis indicated a more stable microecology in the LTS group and FTS group. Proteobacteria, Firmicutes, and Bacteroidota were identified as the dominant phyla, although their relative abundances varied widely among groups. LEfSe (Linear discriminant analysis effect size) showed significant enrichment of beneficial bacteria at various developmental stages, including Bacteroides, Bacillus, and Lactobacillus. Functional prediction analysis shows significant differences in skin microorganism functions across various developmental stages, with a primary focus on metabolic functions. This study provides valuable insights into the compositional dynamics of skin microbiota in Q. spinosa at various developmental stages.

Background: As intensive aquaculture practices have progressed, the prevalence of bacterial diseases in the Chinese soft-shell turtle (Pelodiscus sinensis) has escalated, particularly infections caused by Aeromonas hydrophila, such as ulcerative dermatitis and abscess disease. Despite this, little is known about their immune defenses against this pathogen. Methods: Our study pioneers an integrated analysis of transcriptomics and proteomics to investigate the immune responses of Chinese soft-shelled turtles to A. hydrophila infection. Results: The investigation revealed significant differences in immune-related pathways between groups susceptible and resistant to A. hydrophila infection after 4 days. A total of 4667 and 3417 differentially expressed genes (DEGs), 763 and 568 differentially expressed proteins (DEPs), and 13 and 5 correlated differentially expressed genes and proteins (cor-DEGs-DEPs) were identified in susceptible and resistant Chinese soft-shelled turtles, respectively. In the resistant group, upregulation of immune-related genes, such as CD3ε and CD45, enhanced T-cell activation and the immune response. The proteomic analysis indicated that immune proteins, such as NF-κB1, were significantly upregulated in the resistant group. The correlation analysis between transcriptomics and proteomics demonstrated that the CD40 gene and protein, differentially expressed in the resistant group compared to the control group, were commonly upregulated within the Toll-like receptor signaling pathway. Conclusions: The transcriptomic and proteomic data obtained from this study provide a scientific foundation for understanding the immune mechanisms that enable the Chinese soft-shelled turtle to resist A. hydrophila infection.

Amphibians are an essential class in the maintenance of global ecosystem equilibrium, but they face serious extinction risks driven by climate change and infectious diseases. Unfortunately, the virus diversity harbored by these creatures has been rarely investigated. By profiling the virus flora residing in different tissues of 100 farmed black-spotted frogs (Rana nigromaculata) using a combination of DNA and RNA viromic methods, we captured 28 high-quality viral sequences covering at least 11 viral families. Most of these sequences were remarkably divergent, adding at least 10 new species and 4 new genera within the families Orthomyxoviridae, Adenoviridae, Nodaviridae, Phenuiviridae, and Picornaviridae. We recovered five orthomyxovirus segments, with three distantly neighboring two Chinese fish-related viruses. The recombination event of frog virus 3 occurred among the frog and turtle strains. The relative abundance and molecular detection revealed different tissue tropisms of these viruses, with the orthomyxovirus and adenoviruses being enteric and probably also neurotropic, but the new astrovirus and picornavirus being hepatophilic. These results expand the spectrum of viruses harbored by anurans, highlighting the necessity to continuously monitor these viruses and to investigate the virus diversity in a broader area with more diverse amphibian species.

Tapeworms are infamous and highly successful invasive parasites in aquatic animals, especially in fish. However, tapeworm-caused diseases in soft-shelled turtles are rarely discovered. In this study, a case of cultured Chinese soft-shelled turtle (Pelodiscus sinensis) infected with tapeworm in Changde city of China was reported, and the taxonomic identification of the tapeworm and the effects of tapeworm on the Chinese soft-shelled turtle were revealed. The 28S rRNA sequences of the tapeworm from P. sinensis had the highest similarity (99.82%) with Testudotaenia testudo of Proteocephalidea (Eucestoda), suggesting they are identical species. Infected turtles showed typical clinical symptoms of cestodiasis, which included nutrition loss, and histopathological lesions in the liver and intestine. The serum of infected turtles had significantly lower (P < 0.05) concentrations of glucose, creatinine, total protein, and low-density lipoprotein, and significantly higher (P < 0.05) alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), superoxide dismutase (SOD) activities. Trancriptional analyses showed that 158 differentially expressed genes (DEGs) were found in liver of infected turtles compared with uninfected groups. These DEGs mainly involved in metabolism, including "Arachidonic acid metabolism", "Linoleic acid metabolism", "Metabolism of xenobiotics by cytochrome P450", "alpha-Linolenic acid metabolism", "Ether lipid metabolism", "Retinol metabolism" and "Glycerophospholipid metabolism", which suggested that the tapeworm infection seriously affected the nutrient metabolism of P. sinensis. Besides, 12 immune-related DEGs were discovered and 7 of them were down-regulated, which inferred that tapeworm may regulate immune responses of host. Gut microflora analysis showed that there were no significant differences in richness and diversity between the infected and uninfected groups. Nevertheless, some opportunistic pathogens such as Citrobacter, Edwardsiella, and Lawsonia were significant higher (P < 0.05) in infected turtles, and the intestinal microflora in infected turtles were associated with "Bacterial invasion of epithelial cells" based on functional prediction. Overall, our results suggested that the tapeworm infection caused mechanical damage in the intestine, metabolism disorder in liver, and increased abundance of pathogenic bacteria in gut microflora. Our results provided a first insight into the pathogenesis of tapeworm infection in the Chinese soft-shelled turtle.