Transcriptome Analysis Elucidates the Potential Key Genes Involved in Rib Development in bmp6-Deficient Silver Carp (Hypophthalmichthys molitrix).

Bone morphogenetic protein 6 (BMP-6) is a constituent of the TGF-ß superfamily, known for its ability to stimulate bone and cartilage formation. The investigation of bmp6's involvement in the formation of intermuscular bones in fish has garnered significant attention in recent years. The rib cage is an important skeletal structure that plays a protective function for internal organs in fish. However, there has been limited research conducted on the effects of the bmp6 gene on rib development. Silver carp is one of four major fish in China, favoured for its affordability and tender muscle. Nevertheless, the presence of numerous intermuscular bones in silver carp significantly hinders the advancement of its palatability and suitability for processing. This study showcases the effective utilisation of CRISPR/Cas9 technology for the purpose of disrupting the bmp6 gene in silver carp, leading to the creation of chimeras in the P0 generation, marking the first instance of such an achievement. The chimeras exhibited complete viability, normal appearance, and partial intermuscular bones loss, with approximately 30% of them displaying rib bifurcation or bending. Subsequently, a transcriptome analysis on ribs of P0 chimeras and wild-type silver carp was conducted, leading to the identification of 934 genes exhibiting differential expression, of which 483 were found to be up-regulated and 451 were found to be down-regulated. The results of the KEGG analysis revealed that the "NF-kappa B signalling pathway", "Hippo signalling pathway", "osteoclast differentiation", and "haematopoietic cell lineage" exhibited enrichment and displayed a significant correlation with bone development. The up-regulated genes such as tnfα, fos, and ctgf in pathways may facilitate the proliferation and differentiation of osteoclasts, whereas the down-regulation of genes such as tgfb2 and tgfbr1 in pathways may hinder the formation and specialisation of osteoblasts, ultimately resulting in rib abnormalities. This study presents novel findings on the impact of bmp6 gene deletion on the rib development of silver carp, while simultaneously investigating the previously unexplored molecular mechanisms underlying rib defects in fish.

Roles of estrogen receptors during sexual reversal in Pelodiscus sinensis.

BACKGROUND: The Chinese soft-shelled turtle, Pelodiscus sinensis, exhibits distinct sexual dimorphism, with the males growing faster and larger than the females. During breeding, all-male offspring can be obtained using 17ß-estradiol (E2). However, the molecular mechanisms underlying E2-induced sexual reversal have not yet been elucidated. Previous studies have investigated the molecular sequence and expression characteristics of estrogen receptors (ERs). METHODS AND RESULTS: In this study, primary liver cells and embryos of P. sinensis were treated with ER agonists or inhibitors. Cell incubation experiments revealed that nuclear ERs (nERs) were the main pathway for the transmission of estrogen signals. Our results showed that ERα agonist (ERα-ag) upregulated the expression of Rspo1, whereas ERα inhibitor (ERα-Inh) downregulated its expression. The expression of Dmrt1 was enhanced after ERα-Inh + G-ag treatment, indicating that the regulation of male genes may not act through a single estrogen receptor, but a combination of ERs. In embryos, only the ERα-ag remarkably promoted the expression levels of Rspo1, Wnt4, and ß-catenin, whereas the ERα-Inh had a suppressive effect. Additionally, Dmrt1, Amh, and Sox9 expression levels were downregulated after ERß inhibitor (ERß-Inh) treatment. GPER agonist (G-ag) has a significant promotion effect on Rspo1, Wnt4, and ß-catenin, while the inhibitor G-Inh does not affect male-related genes. CONCLUSIONS: Overall, these results suggest that ERs play different roles during sexual reversal in P. sinensis and ERα may be the main carrier of estrogen-induced sexual reversal in P. sinensis. Further studies need to be performed to analyze the mechanism of ER action.

Comparison of Body Characteristics, Carotenoid Composition, and Nutritional Quality of Chinese Mitten Crab (Eriocheir sinensis) with Different Hepatopancreas Redness.

In this study, we investigated the body characteristics, carotenoid composition, and nutritional quality of Eriocheir sinensis with different hepatopancreas redness (a*). We distributed the crabs into two groups based on the hepatopancreas a* values and compared their body characteristics, chroma, carotenoid composition, and protein, lipid, total sugar, amino acid, and fatty acid content via paired t-test. The results revealed that the relationships between hepatopancreas a* values and crab quality are sex specific. In female crabs, the differences in nutritional characteristics were evident mainly in the hepatopancreases and ovaries. In the redder hepatopancreases, the content of zeaxanthin and ß-carotene increased, and the levels of C22:6n3 and C20:5n3 decreased (p < 0.05). In the ovaries, the content of astaxanthin, canthaxanthin, ß-carotene, umami, and sweet amino acids were lower in the redder hepatopancreas crabs (p < 0.05). In male crabs, there were positive relationships between hepatopancreas a* and amino acid and fatty acid content. The content of leucine, arginine, and total umami amino acids in muscles and of unsaturated fatty acids and n-6 polyunsaturated fatty acids in hepatopancreases and testicles increased with increasing hepatopancreas a* values (p < 0.05). Therefore, the redder the hepatopancreas, the higher the nutritional quality of male crabs.

Single-Molecule Real-Time Sequencing for Identifying Sexual-Dimorphism-Related Transcriptomes and Genes in the Chinese Soft-Shelled Turtle (Pelodiscus sinensis).

The Chinese soft-shelled turtle (Pelodiscus sinensis), an economically important aquatic species in China, displays considerable sexual dimorphism: the male P. sinensis is larger and, thus, more popular in the market. In this study, we obtained the full-length (FL) transcriptome data of P. sinensis by using Pacific Biosciences (PacBio)'s isoform sequencing and analyzed the transcriptome structure. In total, 1,536,849 high-quality FL transcripts were obtained through single-molecule real-time (SMRT) sequencing, which were then corrected using Illumina sequencing data. Next, 89,666 nonredundant FL transcripts were generated after mapping to the reference genome of P. sinensis; 291 fusion genes and 17,366 novel isoforms were successfully annotated using data from the nonredundant protein sequence database (NR), eukaryotic orthology groups (KOG), the Gene Ontology (GO) project, and the KEGG Orthology (KO) database. Additionally, 19,324 alternative polyadenylation sites, 101,625 alternative splicing events, 12,392 long noncoding RNAs, and 5916 transcription factors were identified. Smad4, Wif1, and 17-ß-hsd were identified as female-biased genes, while Nkd2 and Prp18 held a higher expression level in males than females. In summary, we found differences between male and female P. sinensis individuals in AS, lncRNA, genes, and transcripts, which relate to the Wnt pathway, oocyte meiosis, and the TGF-ß pathway. Female-biased genes such as Smad4, Wif1, and 17-ß-hsd and male-biased genes such as Nkd2 and Prp18 played important roles in the sex determination of P. sinensis. FL transcripts are a precious resource for characterizing the transcriptome of P. sinensis, laying the foundation for further research on the sex-determination mechanisms of P. sinensis.

Effect of Exogenous Hormone on R-Spondin 2 (Rspo2) and R-Spondin 3 (Rspo3) Gene Expression and Embryo Development in Chinese Soft-Shelled Turtle (Pelodiscus sinensis).

The Chinese soft-shelled turtle, Pelodiscus sinensis, is an important aquaculture species in China that exhibits distinct sexual dimorphism; male individuals are economically more valuable than females. In vertebrates, several R-spondin family proteins have been associated with sex differentiation mechanisms; however, their involvement in P. sinensis sex differentiation is unclear. Exogenous hormones such as estradiol (E2) also influence the sex differentiation of P. sinensis and induce sexual reversal. In the present study, we investigated the effects of E2 on the embryonic development of P. sinensis and the expression of R-spondin 2 (Rspo2) and R-spondin 3 (Rspo3). We amplified P. sinensis Rspo2 and Rspo3 and analyzed their expression patterns in different tissues. Comparative analyses with protein sequences from other species elucidated that P. sinensis RSPO2 and RSPO3 sequences were conserved. Moreover, phylogenetic analysis revealed that P. sinensis RSPO2 and RSPO3 were closely related to these two proteins from other turtle species. Furthermore, Rspo2 and Rspo3 were highly expressed in the brain and gonads of adult turtles, with significantly higher expression in the ovaries than in the testes (p < 0.05). We also evaluated the expression of Rspo2 and Rspo3 after the administration of three concentrations of E2 (1.0, 5.0, and 10.0 mg/mL) to turtle eggs during embryonic development. The results revealed that E2 upregulated Rspo2 and Rspo3, and the expression trends varied during different embryonic developmental stages (stages 13-20). These findings lay the groundwork for future investigations into the molecular mechanisms involved in the sex differentiation of Chinese soft-shelled turtles.

Identification and Expression Analysis of Wnt2 Gene in the Sex Differentiation of the Chinese Soft-Shelled Turtle (Pelodiscus sinensis).

The Chinese soft-shelled turtle (Pelodiscus sinensis) is an important freshwater aquaculture animal in China. The Wnt gene family plays important regulatory roles in the development and growth of mammals. However, the precise function of these family genes has not been well understood in the sex differentiation of Chinese soft-shelled turtles. Here, we cloned a member of the Wnt family, Wnt2, which obtained a 1077 bp open reading frame that encoded a 358-aa protein. The putative amino acid sequences of proteins are exceeded 80% identical to other turtles. The expression level of Wnt2 peaked at the 14th stage both in female and male embryos during the early gonadal differentiation period of Chinese soft-shelled turtles, which occurred before gonadal differentiation. Wnt2 mRNA was expressed at higher levels in the brains and gonads of mature P. sinensis females compared with those in mature males. Wnt agonists significantly affected the expression level of Wnt2 during the gonadal differentiation period. After Wnt agonists (1.0 µg/µL, 2.5 µg/µL, 5.0 µg/µL) treatment, the expression level of the Wnt2 generally appeared to have an inverted-V trend over time in female embryonic gonads. The results suggested that Wnt2 may participate in the regulation of gonad development in P. sinensis during the early embryonic stages. These results could provide a theoretical basis for the reproduction process of the Chinese soft-shelled turtle.

Role of Sox3 in Estradiol-Induced Sex Reversal in Pelodiscus sinensis.

The Chinese soft-shelled turtle Pelodiscus sinensis, an economically important species in China, exhibits significant sexual dimorphism. Males are more valuable than females owing to their wider calipash and faster growth. Estradiol (E2)-induced sex reversal is used to achieve all-male breeding of turtles; however, the mechanism of this sex reversal remains unclear. In this study, we characterized the Sox3 gene, whose expression level was high in the gonads and brain and exhibited significant sexual dimorphism in the ovary. During embryonic development, Sox3 was highly expressed at the initiation of ovarian differentiation. E2 and Sox3-RNAi treatment before sexual differentiation led to 1352, 908, 990, 1011, and 975 differentially expressed genes in five developmental stages, respectively, compared with only E2 treatment. The differentially expressed genes were clustered into 20 classes. The continuously downregulated and upregulated genes during gonadal differentiation were categorized into Class 0 (n = 271) and Class 19 (n = 606), respectively. KEGG enrichment analysis showed that Sox3 significantly affected sexual differentiation via the Wnt, TGF-ß, and TNF signaling pathways and mRNA surveillance pathway. The expression of genes involved in these signaling pathways, such as Dkk4, Nog, Msi1, and Krt14, changed significantly during gonadal differentiation. In conclusion, the deletion of Sox3 may lead to significant upregulation of the mRNA surveillance pathway and TNF and Ras signaling pathways and downregulation of the Wnt and TGF-ß signaling pathways, inhibiting E2-induced sex reversal. These findings suggest that Sox3 may play a certain promoting effect during E2-induced sex reversal in P. sinensis.

Tandem Mass Tag-Based Quantitative Proteomics Analysis of Gonads Reveals New Insight into Sexual Reversal Mechanism in Chinese Soft-Shelled Turtles.

Chinese soft-shelled turtles display obvious sex dimorphism. The exogenous application of hormones (estradiol and methyltestosterone) can change the direction of gonadal differentiation of P. sinensis to produce sex reversed individuals. However, the molecular mechanism remains unclear. In this study, TMT-based quantitative proteomics analysis of four types of P. sinensis (female, male, pseudo-female, and pseudo-male) gonads were compared. Quantitative analysis of 6107 labeled proteins in the four types of P. sinensis gonads was performed. We identified 440 downregulated and 423 upregulated proteins between pseudo-females and males, as well as 394 downregulated and 959 upregulated proteins between pseudo-males and females. In the two comparisons, the differentially expressed proteins, including K7FKG1, K7GIQ2, COL4A6, K7F2U2, and K7FF80, were enriched in some important pathways, such as focal adhesion, endocytosis, apoptosis, extracellular matrix-receptor interaction, and the regulation of actin cytoskeleton, which were upregulated in pseudo-female vs. male and downregulated in pseudo-male vs. female. In pathways such as ribosome and spliceosome, the levels of RPL28, SRSF3, SNRNP40, and HNRNPK were increased from male to pseudo-female, while they decreased from female to pseudo-male. All differentially expressed proteins after sexual reversal were divided into six clusters, according to their altered levels in the four types of P. sinensis, and associated with cellular processes, such as embryonic development and catabolic process, that were closely related to sexual reversal. These data will provide clues for the sexual reversal mechanism in P. sinensis.

Microsatellite Characteristics of Silver Carp (Hypophthalmichthysmolitrix) Genome and Genetic Diversity Analysis in Four Cultured Populations.

Hypophthalmichthys molitrix is one of the four most important fish in China and has high breeding potential. However, simple sequence repeat (SSR) markers developed on H. molitrix genome level for genetic diversity analysis are limited. In this study, the distribution characteristics of SSRs in the assembled H. molitrix genome were analyzed, and new markers were developed to preliminarily evaluate the genetic diversity of the four breeding populations. A total of 368,572 SSRs were identified from the H. molitrix genome. The total length of SSRs was 6,492,076 bp, accounting for 0.77% of the total length of the genome sequence. The total frequency and total density were 437.73 loci/Mb and 7713.16 bp/Mb, respectively. Among the 2-6 different nucleotide repeat types, SSRs were dominated by di-nucleotide repeats (204,873, 55.59%), and AC/GT was the most abundant motif. The number of SSRs on each chromosome was positively correlated with the length. The 13 pairs of markers developed were used to analyze the genetic diversity of four cultivated populations in Hubei Province. The results showed that the genetic diversity of the four populations was low, and the ranges of alleles (Na), effective alleles (Ne), observed heterozygosity (Ho), and Shannon's index information (I) were 3.538-4.462, 2.045-2.461, 0.392-0.450, and 0.879-0.954, respectively. Genetic variation occurs mainly among individuals within populations (95.35%). UPGMA tree and Bayesian analysis showed that four populations could be divided into two different branches. Therefore, the genome-wide SSRs were effectively in genetic diversity analysis on H. molitrix.

Direct Full-Length RNA Sequencing Reveals an Important Role of Epigenetics During Sexual Reversal in Chinese Soft-Shelled Turtle.

Sex dimorphism is a key feature of Chinese soft-shelled turtle (Pelodiscus sinensis). The males (M) have higher econosmic value than females (F) due to wider calipash and faster growth. Exogenous hormones like estradiol and methyltestosterone can induce sexual reversal to form new phenotypes (pseudo-female, PF; pseudo-male, PM) without changing the genotype. The possibility of inducing sexual reversal is particularly important in aquaculture breeding, but the underlying biological mechanisms remain unclear. Here we applied a direct RNA sequencing method with ultralong reads using Oxford Nanopore Technologies to study the transcriptome complexity in P. sinensis. Nanopore sequencing of the four gender types (M, F, PF, and PM) showed that the distribution of read length and gene expression was more similar between same-sex phenotypes than same-sex genotypes. Compared to turtles with an M phenotype, alternative splicing was more pronounced in F turtles, especially at alternative 3' splice sites, alternative 5' splice sites, and alternative first exons. Furthermore, the two RNA methylation modifications m5C and m6A were differentially distributed across gender phenotypes, with the M type having more modification sites in coding sequence regions, but fewer modification sites in 3'UTR regions. Quantitative analysis of enriched m6A RNAs revealed that the N6-methylated levels of Odf2, Pacs2, and Ak1 were significantly higher in M phenotype individuals, while the N6-methylated levels of Ube2o were reduced after sexual reversal from both M and F phenotypes. Taken together, these findings reveal an important role of epigenetics during sexual reversal in Chinese soft-shelled turtles.

Tandem Mass Tagging-Based Quantitative Proteomics Analysis Reveals Damage to the Liver and Brain of Hypophthalmichthys molitrix Exposed to Acute Hypoxia and Reoxygenation.

Aquaculture environments frequently experience hypoxia and subsequent reoxygenation conditions, which have significant effects on hypoxia-sensitive fish populations. In this study, hepatic biochemical activity indices in serum and the content of major neurotransmitters in the brain were altered markedly after acute hypoxia and reoxygenation exposure in silver carp (Hypophthalmichthys molitrix). Proteomics analysis of the liver showed that a number of immune-related and cytoskeletal organization-related proteins were downregulated, the ferroptosis pathway was activated, and several antioxidant molecules and detoxifying enzymes were upregulated. Proteomics analysis of the brain showed that somatostatin-1A (SST1A) was upregulated, dopamine-degrading enzyme catechol O methyltransferase (COMT) and ferritin, heavy subunit (FerH) were downregulated, and the levels of proteins involved in the nervous system were changed in different ways. In conclusion, these findings highlight that hypoxia-reoxygenation has potential adverse effects on growth, locomotion, immunity, and reproduction of silver carp, and represents a serious threat to liver and brain function, possibly via ferroptosis, oxidative stress, and cytoskeleton destruction in the liver, and abnormal expression of susceptibility genes for neurodegenerative disorders in the brain. Our present findings provide clues to the mechanisms of hypoxia and reoxygenation damage in the brain and liver of hypoxia-sensitive fish. They could also be used to develop methods to reduce hypoxia or reoxygenation injury to fish.

The Important Role of Sex-Related Sox Family Genes in the Sex Reversal of the Chinese Soft-Shelled Turtle (Pelodiscus sinensis).

The Chinese soft-shelled turtle Pelodiscus sinensis shows obvious sexual dimorphism. The economic and nutrition value of male individuals are significantly higher than those of female individuals. Pseudo-females which are base to all-male breeding have been obtained by estrogen induction, while the gene function and molecular mechanism of sex reversal remain unclear in P. sinensis. Here, comparative transcriptome analyses of female, male, and pseudo-female gonads were performed, and 14,430 genes differentially expressed were identified in the pairwise comparison of three groups. GO and KEGG analyses were performed on the differentially expressed genes (DEGs), which mainly concentrated on steroid hormone synthesis. Furthermore, the results of gonadal transcriptome analysis revealed that 10 sex-related sox genes were differentially expressed in males vs. female, male vs. pseudo-female, and female vs. pseudo-female. Through the differential expression analysis of these 10 sox genes in mature gonads, six sox genes related to sex reversal were further screened. The molecular mechanism of the six sox genes in the embryo were analyzed during sex reversal after E2 treatment. In mature gonads, some sox family genes, such as sox9sox12, and sox30 were highly expressed in the testis, while sox1, sox3, sox6, sox11, and sox17 were lowly expressed. In the male embryos, exogenous estrogen can activate the expression of sox3 and inhibit the expression of sox8, sox9, and sox11. In summary, sox3 may have a role in the process of sex reversal from male to pseudo-female, when sox8 and sox9 are inhibited. Sox family genes affect both female and male pathways in the process of sex reversal, which provides a new insight for the all-male breeding of the Chinese soft-shelled turtle.

Molecular Characterization and Response of Prolyl Hydroxylase Domain (PHD) Genes to Hypoxia Stress in Hypophthalmichthys molitrix.

As an economically and ecologically important freshwater fish, silver carp (Hypophthalmichthys molitrix) is sensitive to low oxygen tension. Prolyl hydroxylase domain (PHD) proteins are critical regulators of adaptive responses to hypoxia for their function of regulating the hypoxia inducible factor-1 alpha subunit (HIF-1α) stability via hydroxylation reaction. In the present study, three PHD genes were cloned from H. molitrix by rapid amplification of cDNA ends (RACE). The total length of HmPHD1, HmPHD2, and HmPHD3 were 2981, 1954, and 1847 base pair (bp), and contained 1449, 1080, and 738 bp open reading frames (ORFs) that encoded 482, 359, and 245 amino acids (aa), respectively. Amino acid sequence analysis showed that HmPHD1, HmPHD2, and HmPHD3 had the conserved prolyl 4-hydroxylase alpha subunit homolog domains at their C-termini. Meanwhile, the evaluation of phylogeny revealed PHD2 and PHD3 of H. molitrix were more closely related as they belonged to sister clades, whereas the clade of PHD1 was relatively distant from these two. The transcripts of PHD genes are ubiquitously distributed in H. molitrix tissues, with the highest expressional level of HmPHD1 and HmPHD3 in liver, and HmPHD2 in muscle. After acute hypoxic treatment for 0.5 h, PHD genes of H. molitrix were induced mainly in liver and brain, and different from HmPHD1 and HmPHD2, the expression of HmPHD3 showed no overt tissue specificity. Furthermore, under continued hypoxic condition, PHD genes exhibited an obviously rapid but gradually attenuated response from 3 h to 24 h, and upon reoxygenation, the transcriptional expression of PHD genes showed a decreasing trend in most of the tissues. These results indicate that the PHD genes of H. molitrix are involved in the early response to hypoxic stress, and they show tissue-specific transcript expression when performing physiological regulation functions. This study is of great relevance for advancing our understanding of how PHD genes are regulated when addressing the hypoxic challenge and provides a reference for the subsequent research of the molecular mechanisms underlying hypoxia adaptation in silver carp.

Comparative transcriptome analysis provides novel insights into the molecular mechanism of the silver carp (Hypophthalmichthys molitrix) brain in response to hypoxia stress.

The brain of fish plays an important role in regulating growth and adapting to environmental changes. However, few studies have been performed to address the changes in gene expression profiles in fish brains under hypoxic stress. In the present study, silver carp (Hypophthalmichthys molitrix) were kept under hypoxic experimental conditions by using the method of natural oxygen consumption, which resulted in a significant decrease in malondialdehyde (MDA) and glutathione (GSH) content and superoxide dismutase (SOD) activity in the brain. In addition, RNA sequencing (RNA-Seq) was performed to analyze transcriptional regulation in the brains of silver carp under normoxia (control group), hypoxia, semi-asphyxia, and asphyxia conditions. The results of KEGG enrichment pathway analysis showed that the immune system, such as antigen processing and presentation, natural killer cell-mediated cytotoxicity, was enriched in the hypoxia group; the nervous system (e.g., "glutamatergic synapse"), signal transduction (e.g., "calcium signaling pathway"; "foxo signaling pathway"), and signaling molecules and interactions (e.g., "neuroactive ligand-receptor interaction") were enriched in the semi-asphyxia group; and signaling molecules and interactions (e.g., "neuroactive ligand-receptor interaction") were enriched in the asphyxia group. These results provide novel insights into the molecular regulatory mechanism of the fish brain coping with hypoxia stress.

MicroRNAs May Play an Important Role in Sexual Reversal Process of Chinese Soft-Shelled Turtle, Pelodiscus sinensis.

The Chinese soft-shelled (Pelodiscus sinensis) turtle exhibits obvious sex dimorphism, which leads to the higher economic and nutritional value of male individuals. Exogenous hormones can cause the transformation from male to female phenotype during gonadal differentiation. However, the molecular mechanism related to the sexual reversal process is unclear. In this study, we compared the difference between the small RNAs of male, female, and pseudo-female turtles by small RNA-seq to understand the sexual reversal process of Chinese soft-shelled turtles. A certain dose of estrogen can cause the transformation of Chinese soft-shelled turtles from male to female, which are called pseudo-female individuals. The result of small RNA-seq has revealed that the characteristics of pseudo-females are very similar to females, but are strikingly different from males. The number of the microRNAs (miRNAs) of male individuals was significantly less than the number of female individuals or pseudo-female individuals, while the expression level of miRNAs of male individuals were significantly higher than the other two types. Furthermore, we found 533 differentially expressed miRNAs, including 173 up-regulated miRNAs and 360 down-regulated miRNAs, in the process of transformation from male to female phenotype. Cluster analysis of the total 602 differential miRNAs among females, males, and pseudo-females showed that miRNAs played a crucial role during the sexual differentiation. Among these differential miRNAs, we found 12 miRNAs related to gonadal development and verified their expression by qPCR. The TR-qPCR results confirmed the differential expression of 6 of the 12 miRNAs: miR-26a-5p, miR-212-5p, miR-202-5p, miR-301a, miR-181b-3p and miR-96-5p were involved in sexual reversal process, which was consistent with the results of omics. Using these six miRNAs and some of their target genes, we constructed a network diagram related to gonadal development. We suggest that these miRNAs may play an important role in the process of effective sex reversal, which would contribute to the breeding of all male strains of Chinese soft-shelled turtles.

Identification of microRNAs in Silver Carp (Hypophthalmichthys molitrix) Response to Hypoxia Stress.

Hypoxia is one of the serious stresses in fish culture, which can lead to physical and morphological changes, and cause injury and even death to fish. Silver carp (Hypophthalmichthys molitrix) is an important economic fish and widely distributed in China. MicroRNA is a kind of endogenous non-coding single-stranded small RNA, which is involved in cell development, and immune response and gene expression regulation. In this study, silver carp were kept in the closed containers for hypoxia treatment by spontaneous oxygen consumption. The samples of heart, brain, liver and gill were collected, and the total RNAs extracted separately from the four tissues were mixed in equal amounts according to the concentration. Afterwards, the RNA pool was constructed for high-throughput sequencing, and based on the small RNA sequencing, the differentially expressed microRNAs were identified. Furthermore, their target gene prediction and enrichment analyses were carried out. The results showed that a total of 229 known miRNAs and 391 putative novel miRNAs were identified, which provided valuable resources for further study on the regulatory mechanism of miRNAs in silver carp under hypoxia stress. The authors verified 16 differentially expressed miRNAs by qRT-PCR, and the results were consistent with small RNA sequencing (sRNA-seq). The predicted target genes number of differentially expressed miRNAs was 25,146. GO and KEGG functional enrichment analysis showed that these target genes were mainly involved in the adaption of hypoxia stress in silver carp through biological regulation, catalytic activity and apoptosis. This study provides references for further study of interaction between miRNAs and target genes, and the basic data for the response mechanism under hypoxia stress in silver carp.

Physiological responses and molecular strategies in heart of silver carp (Hypophthalmichthys molitrix) under hypoxia and reoxygenation.

A sufficient oxygen concentration is essential for fish growth, reproduction, and metabolism. Silver carp (Hypophthalmichthys molitrix) is sometimes challenged by hypoxia during intensive aquaculture or because of environmental changes. However, the response to hypoxic stress in the heart of silver carp remains relatively unknown. In the present study, we reported the effects of hypoxia on histological structures, enzyme activities, and gene expression in the heart of silver carp. Hematoxylin and eosin (H&E) staining of heart sections showed that the myocardial fibers gradually became disordered, swollen, and even ruptured during hypoxic treatment. These phenotypes were also supported by increased activities of injury-related enzymes. Moreover, the transcriptome was analyzed to determine the molecular strategies of hypoxia adaptation in the heart. PI3K-Akt signaling pathway, FoxO signaling pathway, and JAK-STAT signaling pathway were the most prominent pathways activated by hypoxia. Twenty significantly differentially expressed genes were selected to create a network diagram related to cell proliferation, carbohydrate metabolism, oxidative stress, and angiogenesis. Additionally, reoxygenation could ameliorate cardiac injury and eliminate the effects of hypoxia on gene expression. This was the first comparative transcriptomic study to explore the molecular mechanism of the response to hypoxia and reoxygenation in the heart of silver carp. Our results provide a theoretical basis for cultivating hypoxia-tolerant carp varieties in the future.

Genetic Characteristic and RNA-Seq Analysis in Transparent Mutant of Carp-Goldfish Nucleocytoplasmic Hybrid.

In teleost, pigment in the skin and scales played important roles in various biological processes. Iridophores, one of the main pigment cells in teleost, could produce silver pigments to reflect light. However, the specific mechanism of the formation of silver pigments is still unclear. In our previous study, some transparent mutant individuals were found in the carp-goldfish nucleocytoplasmic hybrid (CyCa hybrid) population. In the present study, using transparent mutants (TM) and wild type (WT) of the CyCa hybrid as a model, firstly, microscopic observations showed that the silver pigments and melanin were both lost in the scales of transparent mutants compared to that in wild types. Secondly, genetic study demonstrated that the transparent trait in the CyCa hybrid was recessively inherent, and controlled by an allele in line with Mendelism. Thirdly, RNA-Seq analysis showed that differential expression genes (DEGs) between wild type and transparent mutants were mainly enriched in the metabolism of guanine, such as hydrolase, guanyl nucleotide binding, guanyl ribonucleotide binding, and GTPase activity. Among the DEGs, purine nucleoside phosphorylase 4a (pnp4a) and endothelin receptor B (ednrb) were more highly expressed in the wild type compared to the transparent mutant (p < 0.05). Finally, miRNA-Seq analysis showed that miRNA-146a and miR-153b were both more highly expressed in the transparent mutant compared to that in wild type (p < 0.05). Interaction analysis between miRNAs and mRNAs indicated that miRNA-146a was associated with six DEGs (MGAT5B, MFAP4, GP2, htt, Sema6b, Obscn) that might be involved in silver pigmentation.

Effect of exogenous hormones on R-spondin 1 (RSPO1) gene expression and embryo development in Pelodiscus sinensis.

Little is known about sex determination and differentiation in the Chinese soft-shelled turtle Pelodiscus sinensis. R-Spondin 1 (RSPO1), a candidate sex-determining gene, is an important regulator of ovarian differentiation in animals. Exogenous drugs can affect sex differentiation. In this study we cloned the RSPO1 gene from P. sinensis (psRSPO1) and analysed its expression profile. The psRSPO1 gene exhibited sequence identity with RSPO1 genes from other species. RSPO1 protein-based phylogenetic analysis showed that psRSPO1 in P. sinensis is closely related to RSPO1 proteins from other turtles. psRSPO1 showed abundant expression in adult brain and gonads, with higher levels in females than males. We also evaluated the effects of three finaconcentration of 2.5, 5.0 and 10mgmL-1 exogenous oestradiol (E2) and aromatase inhibitor (letrozole) on the expression of psRSPO1, external embryo morphology, growth status of embryos and the sex ratio when the drugs were injected to eggs during incubation. The expression of psRSPO1 was upregulated and downregulated by exogenous oestradiol and letrozole respectively, despite inconsistent expression trends at different embryo development times. External embryo morphology, growth status and sex ratio were affected by both exogenous oestradiol and the aromatase inhibitor. Feminisation was induced by oestradiol, but inhibited by letrozole. These results will contribute to studies of the potential molecular mechanisms underlying sex differentiation and sex control in the Chinese soft-shelled turtle.