ceRNA crosstalk mediated by ncRNAs is a novel regulatory mechanism in fish sex determination and differentiation.

Competing endogenous RNAs (ceRNAs) are vital regulators of gene networks in mammals. The involvement of noncoding RNAs (ncRNAs) as ceRNA in genotypic sex determination (GSD) and environmental sex determination (ESD) in fish is unknown. The Chinese tongue sole, which has both GSD and ESD mechanisms, was used to map the dynamic expression pattern of ncRNAs and mRNA in gonads during sex determination and differentiation. Transcript expression patterns shift during the sex differentiation phase, and ceRNA modulation occurs through crosstalk of differentially expressed long ncRNAs (lncRNAs), circular RNAs (circRNAs), microRNAs (miRNAs), and sex-related genes in fish. Of note was the significant up-regulation of a circRNA from the sex-determining gene dmrt1 (circular RNA dmrt1) and a lncRNA, called AMSDT (which stands for associated with male sex differentiation of tongue sole) in Chinese tongue sole testis. These two ncRNAs both share the same miRNA response elements with gsdf, which has an up-regulated expression when they bind to miRNA cse-miR-196 and concurrent down-regulated female sex-related genes to facilitate testis differentiation. This is the first demonstration in fish that ceRNA crosstalk mediated by ncRNAs modulates sexual development and unveils a novel regulatory mechanism for sex determination and differentiation.

Differential expression of PLAC1 and Netrin-1 in liver metastasis of colorectal cancer and its predictive value.

OBJECTIVE: To explore the differential expression of placental specific gene 1 (PLAC1) and neurite guidance factor 1 (netrin-1) in colorectal cancer (CRC) liver metastasis and its predictive value. METHODS: Paraffin specimens of primary CRC were selected, including 60 simple colorectal cancer specimens and 47 liver metastasis specimens. At the same time, 40 cases of normal colorectal mucosa were taken as the control group. The expression of PLAC1 and Netrin-1 in tissue was detected by immunohistochemistry (IHC). The correlation between PLAC1 and Netrin-1 expression and clinicopathological characteristics of patients with CRC liver metastases was analyzed. Logistic analysis was adopted to analyze the influencing factors of liver metastasis in CRC. A prediction model was established and ROC curve was used to detect the discrimination of the prediction model. The clinical value of PLAC1 and netrin-1 in predicting liver metastasis of CRC was analyzed using ROC curve. The relationship between the expression of PLAC1 and netrin-1 and the prognosis of CRC patients with liver metastasis was analyzed using Kaplan Meier survival curve. RESULTS: The positive staining of PLAC1 and netrin-1 was mainly located in the cytoplasm by IHC detection. Positive expression of PLAC1 and netrin-1 in CRC tissues was markedly higher than that in normal colorectal mucosal epithelium (P < 0.05). Positive expression of PLAC1 in metastatic group was higher than that in non-metastatic group without significant difference (P > 0.05). The metastasis group had much higher positive expression of netrin-1 than the non-metastasis group (P < 0.05). The content of PLAC1 in the tissues of CRC with liver metastasis had a close relationship with differentiation degree and lymph node metastasis (P < 0.05). The expression of Netrin-1 in the tissues of CRC with liver metastasis was associated with Dukes stage, differentiation degree and lymph node metastasis (P < 0.05). Logistic regression analysis showed that Dukes stage, differentiation, lymph node metastasis, CEA, Alb and D-dimer were the independent risk factors for liver metastasis of CRC (P < 0.05). The model was constructed according to the regression coefficients and constant terms, and the discrimination of the prediction model was evaluated using ROC curve, with the AUC of 0.903 (95% CI 0.831 ~ 0.975), the sensitivity of 93.80%, the specificity of 80.00%, and the Jordan index of 0.738. The AUC of PLAC1 and netrin-1 alone and combined detection to predict liver metastasis of CRC were 0.805, 0.793 and 0.921, respectively. The survival time of patients with positive PLAC1 and netrin-1 expression were sharply shorter than that of the patients with negative expression (P < 0.05). CONCLUSIONS: The expression of PLAC1 and netrin-1 was strongly increased in CRC with liver metastasis, which had a certain clinical value in predicting liver metastasis of CRC. Dukes stage, differentiation degree, lymph node metastasis, CEA, Alb and D-dimer were independent risk factors for liver metastasis of CRC, and the model based on these indicators had good discrimination for effectively evaluating the risk of liver metastasis in CRC.

METTL3/YTHDC1-medicated m6A modification of circRNA3634 regulates the proliferation and differentiation of antler chondrocytes by miR-124486-5-MAPK1 axis.

BACKGROUND: The deer antler, a remarkable mammalian appendage, has a growth rate surpassing that of any other known osseous organ. Emerging evidence indicates that circRNA and MAPK1 play critical roles in chondrocytes. Thus, exploration of their functions in antler chondrocytes will help us to understand the mechanism regulating the rapid antler growth. METHODS: qRT-PCR, western blot, and immunohistochemistry were used to assess the expression of mRNAs and proteins. CCK-8, EdU, Cell migration, ALP activity detection, and ALP staining examined the effects of MAPK1 in antler chondrocytes. FISH, RIP, and luciferase assays were performed to evaluate the interactions among circRNA3634/MAPK1 and miR-124486-5. RIP and RAP assays proved the binding interaction between circRNA3634 and RBPs. Me-RIP was used to determine the m6A methylation modification of circRNA3634. RESULTS: This study revealed high MAPK1 expression in antler cartilage tissue. Overexpression of MAPK1 promoted the proliferation, migration, and differentiation of antler chondrocytes and increased the expression of MAPK3, RAF1, MEK1, RUNX2, and SOX9. The silencing of MAPK1 had the opposite effect. CircRNA3634 was found to act as a molecular sponge for miR-124486-5, leading to increased MAPK1 expression and enhanced proliferation and migration of antler chondrocytes through competitive miR-124486-5 binding. We discovered that METTL3 mediates m6A modification near the splicing site of circRNA3634 and is involved in the proliferation and differentiation of antler chondrocytes. The m6A reader YTHDC1 facilitated the nuclear export of circRNA3634 in an m6A-dependent manner. Our results indicate that m6A-modified circRNA3634 promotes the proliferation of antler chondrocytes by targeting MAPK1 and show that the nuclear export of circRNA3634 is related to the expression of YTHDC1, suggesting that circRNA3634 could represent a critical regeneration marker for the antler. CONCLUSIONS: Our results revealed a novel m6A-modified circRNA3634 promoted the proliferation and differentiation of antler chondrocytes by regulating MAPK1. The nuclear export of circRNA3634 was related to the expression of YTHDC1.

Genome-Wide Identification and Involvement in Response to Biotic and Abiotic Stresses of lncRNAs in Turbot (Scophthalmus maximus).

Long non-coding RNAs (lncRNAs) play crucial roles in a variety of biological processes, including stress response. However, the number, characteristics and stress-related expression of lncRNAs in turbot are still largely unknown. In this study, a total of 12,999 lncRNAs were identified at the genome-wide level of turbot for the first time using 24 RNA-seq datasets. Sequence characteristic analyses of transcripts showed that lncRNA transcripts were shorter in average length, lower in average GC content and in average expression level as compared to the coding genes. Expression pattern analyses of lncRNAs in 12 distinct tissues showed that lncRNAs, especially lincRNA, exhibited stronger tissue-specific expression than coding genes. Moreover, 612, 1351, 1060, 875, 420 and 1689 differentially expressed (DE) lncRNAs under Vibrio anguillarum, Enteromyxum scophthalmi, and Megalocytivirus infection and heat, oxygen, and salinity stress conditions were identified, respectively. Among them, 151 and 62 lncRNAs showed differential expression under various abiotic and biotic stresses, respectively, and 11 lncRNAs differentially expressed under both abiotic and biotic stresses were selected as comprehensive stress-responsive lncRNA candidates. Furthermore, expression pattern analysis and qPCR validation both verified the comprehensive stress-responsive functions of these 11 lncRNAs. In addition, 497 significantly co-expressed target genes (correlation coefficient (R) > 0.7 and q-value < 0.05) for these 11 comprehensive stress-responsive lncRNA candidates were identified. Finally, GO and KEGG enrichment analyses indicated that these target genes were enriched mainly in molecular function, such as cytokine activity and active transmembrane transporter activity, in biological processes, such as response to stimulus and immune response, and in pathways, such as protein families: signaling and cellular processes, transporters and metabolism. These findings not only provide valuable reference resources for further research on the molecular basis and function of lncRNAs in turbot but also help to accelerate the progress of molecularly selective breeding of stress-resistant turbot strains or varieties.

Genome-Wide Identification, Molecular Characterization, and Involvement in Response to Abiotic and Biotic Stresses of the HSP70 Gene Family in Turbot (Scophthalmus maximus).

Heat shock proteins 70 (HSP70s) are known to play essential roles in organisms' response mechanisms to various environmental stresses. However, no systematic identification and functional analysis has been conducted for HSP70s in the turbot (Scophthalmus maximus), a commercially important worldwide flatfish. Herein, 16 HSP70 genes unevenly distributed on nine chromosomes were identified in the turbot at the genome-wide level. Analyses of gene structure, motif composition, and phylogenetic relationships provided valuable data on the HSP70s regarding their evolution, classification, and functional diversity. Expression profiles of the HSP70 genes under five different stresses were investigated by examining multiple RNA-seq datasets. Results showed that 10, 6, 8, 10, and 9 HSP70 genes showed significantly up- or downregulated expression after heat-induced, salinity-induced, and Enteromyxum scophthalmi, Vibrio anguillarum, and Megalocytivirus infection-induced stress, respectively. Among them, hsp70 (hspa1a), hspa1b, and hspa5 showed significant responses to each kind of induced stress, and qPCR analyses further validated their involvement in comprehensive anti-stress, indicating their involvement in organisms' anti-stress mechanisms. These findings not only provide new insights into the biological function of HSP70s in turbot adapting to various environmental stresses, but also contribute to the development of molecular-based selective breeding programs for the production of stress-resistant turbot strains in the aquaculture industry.

Transcriptome analysis and protein-protein interaction in resistant and susceptible families of Japanese flounder (Paralichthys olivaceus) to understand the mechanism against Edwardsiella tarda.

Edwardsiella tarda is one of the most harmful bacterial pathogens for aquaculture flatfish. After artificial infection of 47 Japanese flounder (Paralichthys olivaceus) families, resistant and susceptible families were identified in this study. High-throughput sequencing was performed on the liver transcriptome of uninfected groups (PoRU and PoSU) and infected groups (PoRC and PoSC). Through assembly and annotation, a total of 3012 and 1386 differentially expressed genes (DEGs) were identified in PoRU vs. PoSU and PoRC vs. PoSC. The significant enrichment pathways between PoRU and PoSU were mainly in metabolic and biosynthesis pathways. A total of thirty dominant enrichment pathways between PoRC and PoSC mainly focused on some immune-related pathways, including the hematopoietic cell lineage, cytokine-cytokine receptor interaction, complement and coagulation cascades, antigen processing and presentation, the intestinal immune network for immunoglobulin A (IgA) production and T/B cell receptor signaling pathway. Under the protein-protein interaction (PPI) analysis, hub genes, including CD molecules, complement component factors and chemokines, were identified in the network, and 16 core genes were differentially expressed in resistant and sustainable families in quantitative polymerase chain reaction (qPCR) validation. This study represents the first transcriptome analysis based on resistant and susceptible families and provides resistant genes to understand the potential molecular mechanisms of antibacterial function in marine fish. The results obtained in this study provide crucial information on gene markers for resistant breeding of Japanese flounder.

A genome-wide survey of NOD-like receptors in Chinese tongue sole (Cynoglossus semilaevis): Identification, characterization and expression analysis in response to bacterial infection.

As intracellular pathogen recognition receptors (PRRs), nucleotide-binding domain, leucine-rich repeat containing receptors (NLRs, NOD-like receptors) are involved in innate immune responses in vertebrates. However, there is no systemic study on NLRs in Chinese tongue sole (Cynoglossus semilaevis), a popular maricultured fish in China. In the present study, a genome-wide survey of NLRs was performed in C. semilaevis, with the identification of 29 NLRs, including five genes from the NLR-A subfamily (referred to as CsNOD1-5), two genes from the NLR-B subfamily, 18 genes from the NLR-C subfamily (referred to as CsNLR-C1 to 18) and four other NLR genes. Phylogenetic analysis implied that CsNOD1-5 contained conserved functional domains and had orthologous relationships with human NOD1-5. Moreover, CsNLR-C genes all possessed the FISHNA domain, which is a fish-specific NACHT subdomain. Expression analysis showed that CsNOD1-5 and CsNLR-C1/2 were ubiquitously expressed in various normal tissues. Bacterial infection with Vibro harveyi revealed distinct expression patterns of all the tested CsNLRs in gill, intestine, trunk kidney, liver and spleen. In particular, CsNOD1-4 and CsNLR-C2 were significantly upregulated in gills at 48 h post bacterial infection. In addition, CsNOD3 and CsNOD4 were significantly elevated in infectious intestine, trunk kidney, liver and spleen, revealing that their expressions were more sensitive to bacterial infection than other CsNLRs. Together with the computational protein-protein interaction network of CsNLRs, it was suggested that individual NLR genes had different roles in the innate immune cascades of C. semilaevi against bacterial infection. This study provides valuable information for further studies on CsNLR immune function.

Evolutionary dynamics of lincRNA transcription in nine citrus species.

Long intergenic non-coding RNAs (lincRNAs) play important roles in various biological processes in plants. However, little information is known about the evolutionary characteristics of lincRNAs among closely related plant species. Here, we present a large-scale comparative study of lincRNA transcription patterns in nine citrus species. By strand-specific RNA-sequencing, we identified 18 075 lincRNAs (14 575 lincRNA loci) from 34 tissue samples. The results indicated that the evolution of lincRNA transcription is more rapid than that of mRNAs. In total, 82.8-97.6% of sweet orange (Citrus sinensis) lincRNA genes were shown to have homologous sequences in other citrus genomes. However, only 15.5-28.8% of these genes had transcribed homologous lincRNAs in these citrus species, presenting a strong contrast to the high conservation of mRNA transcription (81.6-84.7%). Moreover, primitive and modern citrus lincRNAs were preferentially expressed in reproductive and vegetative organs, respectively. Evolutionarily conserved lincRNAs showed higher expression levels and lower tissue specificity than species-specific lincRNAs. Notably, we observed a similar tissue expression pattern of homologous lincRNAs in sweet orange and pummelo (Citrus grandis), suggesting that these lincRNAs may be functionally conserved and selectively maintained. We also identified and validated a lincRNA with the highest expression in fruit that acts as an endogenous target mimic (eTM) of csi-miR166c, and two lincRNAs that act as a precursor and target of csi-miR166c, respectively. These lincRNAs together with csi-miR166c could form an eTM166-miR166c-targeted lincRNA regulatory network that possibly affects citrus fruit development.

Genome-wide identification, characterization, and expression analysis of the TRAF gene family in Chinese tongue sole (Cynoglossus semilaevis).

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) play crucial roles as signaling mediators for the TNF receptor (TNFR) superfamily and the interleukin-1 receptor/Toll-like receptor (IL-1R/TLR) superfamily. TRAFs collectively play important roles in multiple biological processes and organismal immunity. However, systematic identification of the TRAF gene family in teleost fish has not yet been reported, and there is little available information about its roles in innate immunity in Chinese tongue sole (Cynoglossus semilaevis), an aquaculture fish of high economic value. In the present study, we identified and characterized seven TRAF genes, namely, CsTRAF2a, CsTRAF2b, CsTRAF3, CsTRAF4, CsTRAF5, CsTRAF6 and CsTRAF7, in Chinese tongue sole, and the complete ORFs of the CsTRAFs were cloned. Sequence analysis revealed various genomic structures of the CsTRAFs and showed that they contain typical conserved domains compared with mammalian TRAFs. Phylogenetic analysis indicated the evolutionary relationships of TRAF family members in teleost fish and revealed an absence of TRAF1 in most species and TRAF5 in some species of teleosts. Analysis of the gene structures and motifs showed the diversity and distribution of exon-intron structures and conserved motifs in Chinese tongue sole and several other teleost species. Real-time quantitative PCR was used to investigate the expression patterns of CsTRAF genes in tissues of healthy fish and in the gills, livers and spleens of fish after bacterial infection with Vibrio harveyi. The results indicate that only CsTRAF2a is relatively highly expressed in the brain and that the other CsTRAFs are highly expressed in immune-related tissues and may participate in the immune response after infection with pathogenic bacteria. Functional analysis of CsTRAF3, CsTRAF4 and CsTRAF6 revealed that only CsTRAF6 could strongly activate the NF-кB pathway after overexpression of CsTRAF3, CsTRAF4 and CsTRAF6 in HEK-293T cells. This systematic analysis provided valuable information about the diverse roles of TRAFs in the innate immune response to pathogenic bacterial infection in teleost fish and will contribute to the functional characterization of CsTRAF genes in further research.

Seasonal Pattern of Nutrient Limitation in a Eutrophic Lake and Quantitative Analysis of the Impacts from Internal Nutrient Cycling.

Nutrient dynamics in lakes are determined by the external anthropogenic discharges and unobserved internal cycling processes. In this work, a decadal nutrient data set from the eutrophic Lake Taihu, China, revealed a strong seasonal pattern of nutrient concentration and limitation. A nutrient-driven dynamic eutrophication model based on a Bayesian hierarchical framework was established to quantify the relative contributions to temporal variations from external discharges and internal processes. Results showed that after years of efforts on nutrient reduction, external discharges were relatively small and fluctuated less between seasons compared to the internal processes. A quantitative relationship between monthly nutrient concentration and corresponding internal loading was observed. Lake sediment could transform from a source of phosphorus in overlying water in summer and autumn to a sink in winter and spring. Together with temporal variations in nitrification and denitrification, seasonal transformation from the limitation of phosphorus induced colimitation of nitrogen and phosphorus. Understanding the potential impact of internal nutrient cycling on a seasonal pattern of nutrient concentration and limitation, the growth of phytoplankton, and, possibly, phytoplankton community composition should be emphasized, given the change in the relative importance of external discharges and internal loading in the process of lake restoration.

The status quo of short video as sources of health information on gastroesophageal reflux disease in China: a cross-sectional study.

Background: Positive lifestyle adjustments have become effective methods in treating gastroesophageal reflux disease (GERD). Utilizing short video platforms to encourage GERD patients for effective self-disease management is a convenient and cost-effective approach. However, the quality of GERD-related videos on short video platforms is yet to be determined, and these videos may contain misinformation that patients cannot recognize. This study aims to assess the information quality of GERD-related short videos on TikTok and Bilibili in China. Methods: Search and filter the top 100 GERD-related videos on TikTok and Bilibili based on comprehensive rankings. Two independent gastroenterologists conducted a comprehensive evaluation of the video quality using the Global Quality Score and the modified DISCERN tool. Simultaneously, the content of the videos was analyzed across six aspects: definition, symptoms, risk factors, diagnosis, treatment, and outcomes. Results: A total of 164 GERD-related videos were collected in this study, and videos from non-gastrointestinal health professionals constitute the majority (56.71%), with only 28.66% originating from gastroenterology health professionals. The overall quality and reliability of the videos were relatively low, with DISCERN and GQS scores of 2 (IQR: 2-3) and 3 (IQR: 2-3), respectively. Relatively speaking, videos from gastrointestinal health professionals exhibit the highest reliability and quality, with DISCERN scores of 3 (IQR: 3-4) and GQS scores of 3 (IQR: 3-4), respectively. Conclusion: Overall, the information content and quality of GERD-related videos still need improvement. In the future, health professionals are required to provide high-quality videos to facilitate effective self-disease management for GERD patients.

Genome-wide analysis of BMP/GDF family and DAP-seq of YY1 suggest their roles in Cynoglossus semilaevis sexual size dimorphism.

Sexual size dimorphism (SSD) characterized by different body size between females and males have been reported in various animals. Gonadectomy experiments have implied important regulatory roles of the gonad in SSD. Among multiple factors from the gonad, TGF-ß superfamily (especially BMP/GDF family) attracted our interest due to its pleiotropy in growth and reproduction regulations. Thus, whether BMP/GDF family members serve as crucial regulators for SSD was studied in a typically female-biased SSD flatfish named Chinese tongue sole (Cynoglossus semilaevis). Firstly, a total of 26 BMP/GDF family members were identified. The PPI network analysis showed that they may interact with ACVR2a, ACVR2b, ACVR1, BMPR2, SMAD3, BMPR1a, and other proteins. Subsequently, DAP-seq was employed to reveal the binding sites for yin yang 1 (yy1), a transcription factor involved in gonad function and cell growth partly by regulating TGF-ß superfamily. The results revealed that two yy1 homologues yy1a and yy1b in C. semilaevis could regulate Hippo signaling pathway, mTOR signaling pathway, and AMPK signaling pathway. Moreover, BMP/GDF family genes including bmp2, bmp4, bmp5, gdf6a, and gdf6b were important components of Hippo pathway. In future, the crosstalk among yy1a, yy1b, and TGF-ß family would provide more insight into sexual size dimorphism in C. semilaevis.

Assembly of the poorly differentiated Verasper variegatus W chromosome by different sequencing technologies.

The assembly of W and Y chromosomes poses significant challenges in vertebrate genome sequencing and assembly. Here, we successfully assembled the W chromosome of Verasper variegatus with a length of 20.48 Mb by combining population and PacBio HiFi sequencing data. It was identified as a young sex chromosome and showed signs of expansion in repetitive sequences. The major component of the expansion was Ty3/Gypsy. The ancestral Osteichthyes karyotype consists of 24 protochromosomes. The sex chromosomes in four Pleuronectiformes species derived from a pair of homologous protochromosomes resulting from a whole-genome duplication event in teleost fish, yet with different sex-determination systems. V. variegatus and Cynoglossus semilaevis adhere to the ZZ/ZW system, while Hippoglossus stenolepis and H. hippoglossus follow the XX/XY system. Interestingly, V. variegatus and H. hippoglossus derived from one protochromosome, while C. semilaevis and H. stenolepis derived from another protochromosome. Our study provides valuable insights into the evolution of sex chromosomes in flatfish and sheds light on the important role of whole-genome duplication in shaping the evolution of sex chromosomes.

High-quality Japanese flounder genome aids in identifying stress-related genes using gene coexpression network.

The Japanese flounder is one of the most economically important marine flatfish. However, due to the increased frequency of extreme weather events and high-density industrial farming, an increasing number of environmental stresses have become severe threats to the healthy development of the Japanese flounder culture industry. Herein, we produced a high-quality chromosome-scale Japanese flounder genome using PacBio Circular Consensus Sequencing technologies. The assembled Japanese flounder genome spanned 588.22 Mb with a contig N50 size of 24.35 Mb. In total, 105.89 Mb of repetitive sequences and 22,565 protein-coding genes were identified by genome annotation. In addition, 67 candidate genes responding to distinct stresses were identified by gene coexpression network analysis based on 16 published stress-related RNA-seq datasets encompassing 198 samples. A high-quality chromosome-scale Japanese flounder genome and candidate stress-related gene set will not only serve as key resources for genomics studies and further research on the underlying stress responsive molecular mechanisms in Japanese flounder but will also advance the progress of genetic improvement and comprehensive stress-resistant molecular breeding of Japanese flounder.

Identification of stress-related genes by co-expression network analysis based on the improved turbot genome.

Turbot (Scophthalmus maximus), commercially important flatfish species, is widely cultivated in Europe and China. With the continuous expansion of the intensive breeding scale, turbot is exposed to various stresses, which greatly impedes the healthy development of turbot industry. Here, we present an improved high-quality chromosome-scale genome assembly of turbot using a combination of PacBio long-read and Illumina short-read sequencing technologies. The genome assembly spans 538.22 Mb comprising 27 contigs with a contig N50 size of 25.76 Mb. Annotation of the genome assembly identified 104.45 Mb repetitive sequences, 22,442 protein-coding genes and 3,345 ncRNAs. Moreover, a total of 345 stress responsive candidate genes were identified by gene co-expression network analysis based on 14 published stress-related RNA-seq datasets consisting of 165 samples. Significantly improved genome assembly and stress-related candidate gene pool will provide valuable resources for further research on turbot functional genome and stress response mechanism, as well as theoretical support for the development of molecular breeding technology for resistant turbot varieties.

The chromosome-level genome assembly of the Japanese yellowtail jack Seriola aureovittata provides insights into genome evolution and efficient oxygen transport.

Fishes of the genus Seriola are widely farmed and highly valued in global aquaculture production. To further understand their economically important traits and help improve aquaculture product quality and sustainability, we performed a chromosome-level genome construction for Seriola aureovittata. Combining two technologies, PacBio and BGISEQ-500, we assembled 649.86 Mb S. aureovittata genome sequences with a contig N50 of 22.21 Mb, and 98% of BUSCO genes were detected in total. The initial assembly was then further scaffolded into 24 pseudochromosomes using Hi-C data, indicating the high quality of the genome. Genome evolution analysis showed that many genes related to fatty acid metabolism and oxygen binding, or transport were expanded, which provided insights into the metabolic characteristics of fatty acids and efficient oxygen transport. Based on the genome data, we confirmed the evolutionary relationship of S. aureovittata, S. dorsalis and S. lalandi and identified chr12 as the putative sex chromosome of S. aureovittata. Our chromosome-level genome assembly provides a genetic foundation for the phylogenetic and taxonomic investigation of different Seriola species. Moreover, the genome will provide an important genomic resource for further biological and aquaculture studies of S. aureovittata.

Transcriptomic Analysis Revealed Candidate Genes Involved in Pseudomale Sperm Abnormalities in Chinese Tongue Sole (Cynoglossus semilaevis).

Chinese tongue sole (Cynoglossus semilaevis) has a ZZ/ZW sex determination system, but the genotypic female (ZW) can be sex-reversed into phenotypic males, namely, pseudomales. Pseudomale fish can produce only Z-type sperm but not W sperm. However, the molecular mechanism is unclear. To screen the key genes involved in pseudomale sperm abnormalities, we analysed the transcriptomic profiles of pseudomale and male sperm. In comparison to male sperm, 592 differentially expressed genes (DEGs) were identified in pseudomale sperm, including 499 upregulated and 93 downregulated genes. KEGG analysis indicated that the FoxO signalling pathway, especially the foxo3a and foxo6-like genes, may play an important role in spermatogenesis. The DEGs were mainly distributed on sex chromosomes, with 158 downregulated genes on the Z chromosome and 41 upregulated genes on the W chromosome. A specific area (14-15 M) on the Z chromosome was identified, which enriched eight DEGs inside the ~1 M region. In addition, there were five gene alleles on the sex chromosomes, which showed the opposite transcription pattern (upregulated for the W allele, downregulated for the Z allele). This study has provided valuable data for screening candidate genes involved in the pseudomale sperm abnormality.

Molecular cloning and immune characterization of CIITA in artificially challenged Chinese tongue sole (Cynoglossus semilaevis) with Vibrio harveyi.

In mammals, Class II, major histocompatibility complex (MHC II) transactivator (CIITA) recognizes microbial pathogens and triggers immune responses. In Chinese tongue sole Cynoglossus semilaevis, Cs-CIITA was prevalently expressed in various tissues. Cs-CIITA, Cs-MHC IIA and Cs-MHC IIB were expressed significantly higher in skin in susceptible families infected with Vibrio harveyi, while higher expression of Cs-CIITA and Cs-MHC IIB was examined in liver in resistant families. In addition, the three genes were up-regulated in gill, skin, intestine, liver, spleen and kidney at 48 h or 72 h after V. harveyi infection. Furthermore, the three genes were co-expressed in the epithelial mucous cells of gill, skin, and intestine. Knockdown of Cs-CIITA regulates the expression of other inflammation-related genes, including CD40, IL-1ß, IL-8, RelB, NFκB, and Myd88. These results suggest that CIITA functions in the inflammatory responses of C. semilaevis against V. harveyi, via MHC II transcriptional regulation.

Transcriptomic analysis revealed gene expression profiles during the sex differentiation of Chinese tongue sole (Cynoglossus semilaevis).

Sex differentiation in aquatic fish is important both for theoretical study and practical production, as growth dimorphism frequently appears in different sexes, especially in marine fish. The deciphered genome, identification of the male-determining gene dmrt1 and established genotypic sex screening method make Chinese tongue sole (Cynoglossus semilaevis) an ideal model to study sex differentiation in fish. In this study, comparative gonadal transcriptomic analyses were conducted for genetic females and males at 48, 68, and 108 days post hatching (dph), representing pre-, during- and post-gonadal differentiation stages, although the gonad is not completely differentiated and isolable in 48 and 68 dph individuals, while it is in 108 dph individuals. Altogether, 28 libraries were constructed, and a mean of 46.64 M clean reads was obtained. Differentially expressed gene (DEG) analysis revealed that 179 genes had similar expression patterns in males and females in all three stages. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis suggested that the enriched pathways included ubiquitin-mediated proteolysis, lysosomes, and RNA degradation. Moreover, weighted gene coexpression network analyses (WGCNA) identified 14 modules, one of which was closely correlated with female differentiation, exhibiting female-biased expression in all three stages (48, 68, 108 dph). An illustrated core gene interaction network of this module identified 50 genes, most of which are on W chromosomes. Six genes, including two ubiquitin conjugating enzymes, were selected for further investigation, and their female-biased expression was confirmed in even earlier stages, at 10 and 30 dph. These data facilitate our understanding of sex differentiation in fish and provide a genomic rationale for screening candidate genes (preferentially W-linked genes) that could be involved in the female differentiation process.