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Mycobacterium marinum is a major pathogen in aquaculture, posing a substantial threat to the health and sustainability of tongue sole (Cynoglossus semilaevis) farming. This study investigated the genetic basis of immune response in tongue sole by comparing transcriptome profiles of liver and spleen tissues from symptomatic (susceptible) and healthy (resistant) individuals during a natural M. marinum outbreak. Transcriptomic analyses identified differentially expressed genes and enriched pathways related to immune responses. Key genes, including atp6ap1, gpi, and idh3a, were found to be crucial in immune response to M. marinum infection, involved in immune processes such as signal transduction, antigen processing, and metabolic pathways. Protein-protein interaction networks highlighted central hub genes such as nedd8, jun and junb, which play pivotal roles in immune regulation. These findings provide insights into the orchestrated immune responses to mycobacteriosis, which can inform selective breeding strategies for disease-resistant tongue sole strains. This is the first comprehensive transcriptome analysis of M. marinum natural infection in tongue sole, offering valuable data for future research and disease management in aquaculture., Competing Interests: Declaration of interest statement The authors declare no conflicts of interest., (Copyright © 2025. Published by Elsevier Ltd.)

Frequent outbreaks of infectious diseases in aquaculture have led to significant economic losses. The leopard coral grouper (Plectropomus leopardus) often suffers from vibriosis. Improving host immunity presents a superior strategy for disease control, with minimal side effects compared to the use of antibiotics, highlighting the necessity of exploring the mechanisms underlying the fish's response to pathogen infections. Here, we conducted a comparative metabolomic analysis on the livers of the P. leopardus infected with Vibrio harveyi. A total of 1124 differential metabolites (DMs) were identified, with 190, 218, 359, and 353 DMs being identified at 6, 12, 24, and 48 h post-infection (hpi), respectively. Then, based on the time series analysis, we found that the lipid metabolism pathways were modulated in response to the Vibrio infection, with an increase in the quantity of eicosanoids and gycerophospholipids (GPLs), as well as a decrease in the quantity of bile acids (BAs), vitamin D, and sex hormones. Furthermore, 13 enriched pathways involving 31 DMs were identified through KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analyses. We identified histamine, 15(S)-HpETE, and anandamide in the transient receptor potential (TRP) channels pathway, as well as (7S,8S)-DiHODE, 5S,8R-DiHODE, and 13(S)-HpODE in the linoleic acid (LA) metabolism pathway. The DM levels increased, which may be attributed to inflammation. The DMs in the thyroid hormone synthesis pathway were identified, and the contents of nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione (GSH) decreased, which may be crucial in antioxidants. Our findings highlighted the dynamic adjustments in lipid metabolism and the response to inflammation and oxidative stress during the infection of V. harveyi in P. leopardus. This study not only deepens our understanding of the metabolic underpinnings of fish immune responses but also lays the groundwork for research into functional metabolomics and mechanisms of disease resistance., (© 2024 Fisheries Society of the British Isles.)

Hepcidin belongs to a class of small cationic antimicrobial peptides rich in cysteine. It is synthesized by liver and is widely involved in host antimicrobial, antiviral and other immune responses. We identified and characterized three hepcidin genes (OpHep1, OpHep2 and OpHep3) in spotted knifejaw. All the OpHeps shared high identities with hepcidins in other teleost, containing alpha helix and β-sheets. Three OpHeps were all detected in healthy tissues, with the abundant expression in liver. They were significantly increased after Vibrio harveyi infection in the six immune-relevant tissues (liver, kidney, spleen, gill, skin and intestine). OpHeps knockdown in spotted knifejaw liver cells affected the mRNA levels of inflammation-related genes, including il1β, il6, il8, and nfκb. Further, the recombinant hepcidin proteins were effective in suppressing the growth of both Gram-negative and Gram-positive bacteria. To identify the function of OpHeps in vivo, we performed the overexpression of three OpHeps in zebrafish, and found OpHeps could significantly induce immune-related genes expression in transgenic zebrafish, including myd88, il10, il21, il16, tlr1, tlr3 and lysozyme. When infected with V. harveyi, OpHeps transgenic zebrafishes had a higher survival rate than wild-type zebrafishes. The expression of myd88, il10, il8, il1β, nfκb and lysozyme were all significantly up-regulated in transgenic fishes during bacterial infection. In summary, these results indicated that hepcidin could protect fish fight against pathogen through TLR/NFκB signaling cascade and Lysozyme. Three OpHeps would be potential targets for prevention of bacterial infections in aquaculture industry of spotted knifejaw, which provided a new idea for the molecular breeding of fish disease resistance., Competing Interests: Declaration of competing interest The authors read and approved this version of the final manuscript, confirmed the integrity of this work and declared no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Leopard coral grouper ( Plectropomus leopardus ) is one of the most important cultured fish in the Pacific and Indian oceans. Vibrio harveyi is a serious pathogen causing serious skin ulceration and high mortality in P. leopardus . To gain more insight into the tissue-specific and dynamic immune regulation process of P. leopardus in response to V. harveyi infection, RNA sequencing (RNA-seq) was used to examine the transcriptome profiles in the spleen and liver at 0, 6, 12, 24, 48, and 72 h post-infection. The upregulated differentially expressed genes (DEGs) were predominantly involved in the immune response in the spleen and liver at the early infection stage (6-12 h), and downregulated DEGs were mainly involved in metabolic processes in the liver at the early and middle infection stage (6-48 h). Moreover, an overview of the immune response of P. leopardus against V. harveyi was exhibited including innate and adaptive immune-related pathways. Afterwards, the results of WGCNA analysis in the spleen indicated that TAP2, IRF1, SOCS1, and CFLAR were the hub genes closely involved in immune regulation in the gene co-expression network. This study provides a global picture of V. harveyi -induced gene expression profiles of P. leopardus at the transcriptome level and uncovers a set of key immune pathways and genes closely linked to V. harveyi infection, which will lay a foundation for further study the immune regulation of bacterial diseases in P. leopardus ., Competing Interests: Author BZ was employed by the company Wanning Linlan Aquaculture Co., LTD. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Liu, Lu, Guo, Wang, Hu, Zhou and Chen.)

Ras-related associated with diabetes (RRAD) is a member of the Ras GTPase superfamily that plays a role in several cellular functions, such as cell proliferation and differentiation. In particular, the superfamily acts as an NF-κB signaling pathway inhibitor and calcium regulator to participate in the immune response pathway. A recent transcriptome study revealed that rrad was expressed in the spleen of disease-resistant Japanese flounder ( Paralichthys olivaceus ) individuals compared with disease-susceptible individuals, and the results were also verified by qPCR. Thus, the present study aimed to explore how rrad regulates antimicrobial immunity via the NF-κB pathway. First, the coding sequence of P. olivaceus rrad was identified. The sequence was 1092 bp in length, encoding 364 amino acids. Based on phylogenetic and structural relationship analyses, P. olivaceus rrad appeared to be more closely related to teleosts. Next, rrad expression differences between disease-resistant and disease-susceptible individuals in immune-related tissues were evaluated, and the results revealed that rrad was expressed preferentially in the spleen of disease-resistant individuals. In response to Edwardsiella piscicida infection, rrad expression in the spleen changed. In vitro, co-culture was carried out to assess the hypo-methylated levels of the rrad promoter in the disease-resistant spleen, which was consistent with the high mRNA expression. The siRNA-mediated knockdown of rrad performed with the gill cell line of P. olivaceus affected many rrad -network-related genes, i.e., dcp1b , amagt , rus1 , rapgef1 , ralbp1 , plce1 , rasal1 , nckipsd , prkab2 , cytbc-1 , sh3 , and others, as well as some inflammation-related genes, such as bal2 and Il-1β . In addition, flow cytometry analysis showed that rrad overexpression was more likely to induce cell apoptosis, with establishing a link between rrad 's function and its potential roles in regulating the NF-κB pathway. Thus,. the current study provided some clarity in terms of understanding the immune response about rrad gene differences between disease-resistant and disease-susceptible P. olivaceus individuals. This study provides a molecular basis for fish rrad gene functional analysis and may serve as a reference for in-depth of bacterial disease resistance of teleost.

Complement factor H-related protein (CFHR) plays an important role in regulating complement activation and defensive responses. The function of CFHR2 (complement factor H related 2), a member of the CFHR family, in fish remains unclear. Here, we report the genetic relationship, expression characteristics and regulatory mechanism of cfhl5 (complement factor H like 5) gene, which encodes CFHR2 in Chinese tongue sole. We observed that the cfhl5 gene was widely expressed in several tissues, such as brain, heart and immune organs, and was most abundantly expressed in liver. After injection with Vibrio harveyi, the expression of cfhl5 was up-regulated significantly in liver, spleen and kidney at 12 or 24 hours post infection (hpi), suggesting an involvement of this gene in the acute immune response. Knockdown of cfhl5 in liver cells significantly up-regulated the expression of the pro-inflammatory cytokines tnf-α (tumor necrosis factor-alpha) and il1β (interleukin-1beta), the immunomodulatory factor il10 (interleukin-10) and the lectin complement pathway gene masp1 (MBL-associated serine protease 1), and down-regulated the expression of complement components c3 (complement 3) and cfi (complement factor I). In our previous work, we found that cfhl5 gene was significantly higher methylated and lower expressed in the resistant family compared with the susceptible family. Therefore, we used dual-luciferase reporter system to determine the effect of DNA methylation on this gene and found that DNA methylation could inhibit the promoter activity to reduce its expression. These results demonstrated that the expression of cfhl5 is regulated by DNA methylation, and this gene might play an important role in the immune response by regulating the expression of cytokines and complement components genes in Chinese tongue sole., Competing Interests: Declaration of competing interest All the authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Vibrio spp. are major pathogens responsible for mortality and disease in various marine aquaculture organisms. Effective disease control and genetic breeding strategies rely heavily on understanding host vibriosis resistance mechanisms. The Chinese tongue sole (Cynoglossus semilaevis) is economically vital but suffers from substantial mortalities due to vibriosis. Through continuous selective breeding, we have successfully obtained vibriosis-resistant families of this species. In this study, we conducted RNA-seq analysis on three organs, including liver, spleen and intestine from selected resistant and susceptible tongue soles. Additionally, we integrated these data with our previously published RNA-seq datasets of skin and gill, enabling the construction of organ-specific transcriptional profiles and a comprehensive gene co-expression network elucidating the differences in vibriosis resistance. Furthermore, we identified 12 modules with organ-specific functional implications. Overall, our findings provide a valuable resource for investigating the molecular basis of vibriosis resistance in fish, offering insights into target genes and pathways essential for molecular selection and genetic manipulation to enhance vibriosis resistance in fish breeding programs., (© 2024. The Author(s).)

Vibriosis, caused by Vibrio , seriously affects the health of fish, shellfish, and shrimps, causing large economic losses. Teleosts are represent the first bony vertebrates with both innate and adaptive immune responses against pathogens. Aquatic animals encounter hydraulic pressure and more pathogens, compared to terrestrial animals. The skin is the first line of defense in fish, constituting the skin-associated lymphoid tissue (SALT), which belongs to the main mucosa-associated lymphoid tissues (MALT). However, little is known about the function of immunity related proteins in fish. Therefore, this study used iTRAQ (isobaric tags for relative and absolute quantitation) to compare the skin proteome between the resistant and susceptible families of Cynoglossus semilaevis . The protein integrin beta-2, the alpha-enolase isoform X1, subunit B of V-type proton ATPase, eukaryotic translation initiation factor 6, and ubiquitin-like protein ISG15, were highly expressed in the resistant family. The 16S sequencing of the skin tissues of the resistant and susceptible families showed significant differences in the microbial communities of the two families. The protein-microbial interaction identified ten proteins associated with skin microbes, including immunoglobulin heavy chain gene (IGH), B-cell lymphoma/leukemia 10 (BCL10) and pre-B-cell leukemia transcription factor 1 isoform X2 (PBX2). This study highlights the interaction between skin proteins and the microbial compositions of C. semilaevis and provides new insights into understanding aquaculture breeding research., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Wang, Tian and Chen.)

The Chinese tongue sole (Cynoglossus semilaevis) holds significant economic importance within the fishing industry along the eastern coasts of China. In recent years, the frequent outbreaks of bacterial diseases have become a common concern as the aquaculture scale expands. The majority of the diseased fish exhibit symptoms such as skin congestion, damage and skin ulceration. As the skin serves as the first line of defense against bacterial infections, establishing a skin cell line for immunological research on Chinese tongue sole's response to bacterial infection is of utmost importance. In this study, a cell line named CSS (derived from the skin of the Chinese tongue sole) was successfully established. The cells have demonstrated stability during passages and exhibit a multipolar fibroblast-like morphology. They were cultured in L-15 medium with 20% serum and have been successfully passed through 60 passages over a period of 20 months. The identification of the mitochondrial CO1 gene confirmed that the cell originated from Chinese tongue sole. The karyotype detection revealed that the cell had a chromosome number of 2n = 42. After being stored in liquid nitrogen for 15 months, the cells can maintain more than 75% viability upon recovery. After transfecting with cy3-labeled scramble siRNA and pEGFP-N3 plasmid, clear fluorescence was observed in the transfected cells. We observed that lipopolysaccharide (LPS) from Escherichia coli significantly upregulate the gene expression of various immune-related pathways at 2 h in the CSS cell line. Additionally, the differentially expressed genes showed a higher enrichment in immune-related pathways at 2 and 6 h after stimulation compared to the 24 h point. Moreover, we identified 347 genes that exhibited a gradual increase in expression during the 0-24 h stimulation period. These genes were primarily enriched in pathways related to Autophagy, GABAergic synapse, Apelin signaling and Ferroptosis. In general, the CSS cell line established in this study exhibits stable growth and can serve as a valuable tool for in vitro studies of immunology and other basic biologies of Chinese tongue sole., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The tumor necrosis factor receptor-associated factor 6 (TRAF6) can act as a fundamental adaptor protein in a chain reaction of signal transduction and cascade events to finish off immune defenses. However, immunomodulatory research on TRAF6 gene is still limited in fish. In this study, a novel miRNA, Cse-miR-33 was identified from the whole genome of Chinese tongue sole (Cynoglossus semilaevis). After separate infections with three different Vibrio strains (V. harveyi, V. anguillarum, V. parahemolyticus) and one virus (nervous necrosis virus, NNV), the expressions of CsTRAF6 and Cse-miR-33 displayed significant time-dependent changes in immune related tissues and the trends were opposite in general. Through target gene prediction and dual luciferase reporter assay, Cse-miR-33 was proven to regulate CsTRAF6 by combining with 3'-UTR sequence of the gene. The results of qRT-PCR and western blotting (WB) analyses showed that Cse-miR-33 blocked the translation of CsTRAF6 protein at post-transcriptional level, rather than degrading the target mRNA. Further experiment indicated that Cse-miR-33 inhibitor largely reduced the death rate of Chinese tongue sole caused by V. harveyi and NNV. The expressions of CsTRAF6-associated immune genes (such as CsIL-1R, CsMYD88, CsIRAK1, CsTNFα, CsIL6 and CsIL8) were also significantly changed in response to Cse-miR-33 agomir and inhibitor. The study suggested that Cse-miR-33 affected the immune response via targeting CsTRAF6 in C. semilaevis, which would provide us deep insights into miRNA-mediated regulatory network and help improve the immunity in fish., Competing Interests: Declaration of competing interest The authors read and approved this version of the final manuscript, confirmed the integrity of this work and declared no conflicts of interest., (Copyright © 2023. Published by Elsevier Ltd.)

As one of the most valuable maricultured species, spotted knifejaw (Oplegnathus punctatus) has high popularity in eastern Asia. In recent years, diseases caused by Vibrio harveyi have brought huge economic losses in spotted knifejaw industry. To better understand the molecular mechanisms of immune response about V. harveyi resistance in spotted knifejaw, a comparative transcriptome analysis was performed on spleen tissues at five different time points post-infection (0, 12, 24, 48 and 72 hpi). A total of 4279 differentially expressed genes (DEGs) were identified. KEGG pathways analysis showed that multiple immune-related pathways were significant regulated, including Toll-like receptor signaling pathway, ECM-receptor interaction pathway, cytokine-cytokine receptor interaction pathway and hematopoietic cell lineage pathway. Weighted gene co-expression network analysis showed that several immune-related pathways of the highest correlation with 12 hpi (cor = 0.89, P = 7e-06) were significantly enriched. In addition, 12 hpi was a turning point for 7 gene clusters out of 9 that were divided according to gene expression patterns. Therefore, we speculated that 12 hpi might be a very critical time point for spotted knifejaw against V. harveyi infection. Additionally, qRT-PCR was carried out to validate the expressions of 12 DEGs. This study provided the first systematical transcriptome analysis of spotted knifejaw against V. harveyi. The results could help us better understand the dynamic immune responses of spotted knifejaw against bacterial infection, and provide useful information for antibacterial defense in spotted knifejaw industry as well., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Interleukin 10 (IL-10), a pleiotropic cytokine, plays an essential role in multiple immunity responses. In the current study, the sequences of IL-10 family were identified from spotted knifejaw (Oplegnathus punctatus) whole genome, and O. punctatus IL-10 (OpIL-10) was cloned and characterized. OpIL-10 encodes 187 amino acids with a typical IL-10 family signature motif and predicted α-helices. It shared high identities with Notolabrus celidotus IL-10 and Epinephelus Lanceolatus IL-10. OpIL-10 was widely detected in healthy tissues, with the abundant expression in liver and skin. It was significantly up-regulated in the six immune-related tissues (liver, spleen, kidney, intestine, gill and skin) after infection against Vibrio harveyi and spotted knifejaw iridovirus (SKIV). Dual-luciferase analysis showed that OpIL-10 overexpression could suppress the activity of NF-κB. Meanwhile, OpIL-10 knockdown caused the down-regulation of five immune-related genes in JAK2/STAT3 signaling pathway and NF-κB signaling pathway, including IL-10R2, TYK2, STAT3, NOD2, and IκB. In addition, LPS and poly I:C stimulated expression of pro-inflammatory cytokines, including IL-6, IL-1β, IL-8, and IL-12, were lower with recombinant OpIL-10 (rOp IL-10) than the control group, indicating the anti-inflammatory roles of rOpIL-10. Taken together, these results indicated OpIL-10 as a negative regulator in the inflammatory responses of spotted knifejaw against bacterial and viral infection, which would help us better understand the role of IL-10 in teleost immunity., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

IgT is a specific Ig isotype in teleosts, which plays extremely important roles in the mucosal immunity of fish. In the present study, the membrane-bound and secretory IgT of the half-smooth tongue sole (Cynoglossus semilaevis) were identified for the first time. The V-D-J-C structure of two forms of csIgT are translated by the same Cτ gene, and the secretory tail and transmembrane domain were encoded through alternative splicing at the 3' end of the Cτ4. The CH regions of csIgT had high similarity with that of other flatfish (P. olivaceus and S. maximus). In healthy C. semilaevis, sIgT and mIgT were mainly expressed in mucus related tissues such as skin, intestine and gill. The transcript levels of sIgT and mIgT mRNA showed a significant induction in the immune-related tissues upon Vibrio Harveyi infection. A polyclonal rabbit anti-csIgT was successfully prepared using the csIgT heavy chain recombinant protein. Using this antibody, we detected the native IgT with the molecular mass at 220 kDa in skin total protein under non-reducing SDS-PAGE condition. Immunofluorescence analysis indicated that IgT + B lymphocytes were intensively located in the skin, gill, intestine, and head kidney of C. semilaevis. These results suggest that IgT may participate in the immune response of C. semilaevis, which will facilitate the investigations of the immunoglobulins of marine fish., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., (© 2021 Fisheries Society of the British Isles.)

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-1β, 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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Based on 1572 re-sequenced Chinese tongue sole (Cynoglossus semilaevis), we investigated the accuracy of four genomic methods at predicting genomic estimated breeding values (GEBVs) of Vibrio harveyi resistance in C. semilaevis when SNPs varying from 500 to 500 k. All methods outperformed the pedigree-based best linear unbiased prediction when SNPs reached 50 k or more. Then, we developed an SNP array "Solechip No.1" for C. semilaevis breeding using the Affymetrix Axiom technology. This array contains 38,295 SNPs with an average of 10.5 kb inter-spacing between two adjacent SNPs. We selected 44 candidates as the parents of 23 families and genotyped them by the array. The challenge survival rates of offspring families had a correlation of 0.706 with the mid-parental GEBVs. This SNP array is a convenient and reliable tool in genotyping, which could be used for improving V. harveyi resistance in C. semilaevis coupled with the genomic selection methods., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Edwardsiella tarda causes acute symptoms with ascites in Japanese flounder (Paralichthys olivaceus) and is a major problem for China's aquaculture sector. Genomic selection (GS) has been widely adopted in breeding industries because it shortens generation intervals and results in the selection of individuals that have great breeding potential with high accuracy. Based on an artificial challenge test and re-sequenced data of 1099 flounders, the aims of this study were to estimate the genetic parameters of resistance to E. tarda in Japanese flounder and to evaluate the accuracy of single-step GBLUP (ssGBLUP), weighted ssGBLUP (WssGBLUP), and BayesB for improving resistance to E. tarda by using three subsets of pre-selected single nucleotide polymorphisms (SNPs). In addition, SNPs that are associated with this trait were identified using a single-SNP genome-wide association study (GWAS) and WssGBLUP., Results: We estimated a heritability of 0.13 ± 0.02 for resistance to E. tarda in Japanese flounder. One million SNPs at fixed intervals were selected from 4,978,724 SNPs that passed quality controls. GWAS identified significant SNPs on chromosomes 14 and 24. WssGBLUP revealed that the putative quantitative trait loci on chromosomes 1 and 14 contained SNPs that explained more than 1% of the genetic variance. Three 50 k-SNP subsets were pre-selected based on different criteria. Compared with pedigree-based prediction (ABLUP), the three genomic methods evaluated resulted in at least 7.7% greater accuracy of predictions. The accuracy of these genomic prediction methods was almost unchanged when pre-selected trait-related SNPs were used for prediction., Conclusions: Resistance to E. tarda in Japanese flounder has a low heritability. GWAS and WssGBLUP revealed that the genetic architecture of this trait is polygenic. Genomic prediction of breeding values performed better than ABLUP. It is feasible to implement genomic selection to increase resistance to E. tarda in Japanese flounder with 50 k SNPs. Based on the criteria used here, pre-selection of SNPs was not beneficial and other criteria for pre-selection should be considered.

Vibrio harveyi is a major bacterial pathogen that causes fatal vibriosis in Chinese tongue sole (Cynoglossus semilaevis), resulting in massive mortality in the farming industry. However, the molecular mechanisms of C. semilaevis response to V. harveyi infection are poorly understood. Here, we performed transcriptomic analysis of C. semilaevis, comparing resistant and susceptible families in response to V. harveyi challenge (CsRC and CsSC) and control conditions (CsRU and CsSU). RNA libraries were constructed using 12 RNA samples isolated from three biological replicates of the four groups. We performed transcriptome sequencing on an Illumina HiSeq platform, and generating a total of 1,095 million paired-end reads, with the number of clean reads per library ranging from 75.27 M to 99.97 M. Through pairwise comparisons among the four groups, we identified 713 genes exhibiting significant differences at the transcript level. Furthermore, the expression levels were validated by real-time qPCR. Our results provide a valuable resource and new insights into the immune response to V. harveyi infection.

Rac1 and Rac2, belonging to the small Rho GTPase family, play an important role during the immune responses. In this study, a Rac1 homolog (CsRac1) and a Rac2 homolog (CsRac2) were cloned from the Cynoglossus semilaevis. The full-length of CsRac1 and CsRac2 cDNA was 1219 bp and 1047 bp, respectively. Both CsRac1 and CsRac2 contain a 579 bp open reading frame (ORF) which encoding a 192 amino acids putative protein. The predicted molecular weight of CsRac1 and CsRac2 was 21.41 kDa and 21.35 kDa, and their theoretical pI was 8.50 and 7.91, respectively. Sequence analysis showed that the conserved RHO domain was detected both from amino acid of CsRac1 and CsRac2. Homologous analysis showed that CsRac1 and CsRac2 share high conservation with other counterparts from different species. The CsRac1 and CsRac2 transcript showed wide tissue distribution, in which CsRac1 and CsRac2 exhibit the highest expression level in liver and gill, respectively. The expression level of CsRac1 and CsRac2 fluctuated in the liver and gill tissues at different time points after challenged by Vibrio harveyi. Specifically, CsRac1 and CsRac2 were significantly up-regulated at 48 h and 96 h post injection. Moreover, the knocking down of CsRac1 and CsRac2 in cell line (TSHKC) reduced the expression of CsPAK1, CsIL1-β and CsTNF-α. The present data suggests that CsRac1 and CsRac2 might play important roles in the innate immunity of half-smooth tongue sole., (Copyright © 2018. Published by Elsevier Ltd.)

Receptor interacting protein kinase 3 (RIP3) has emerged as an essential molecular regulator in human inflammation with accumulating evidence in vertebrates. However, the structure and functions of RIP3 in teleosts remains elusive. Here we identified one RIP3 homologue from half-smooth tongue sole Cynoglossus semilaevis, designated CsRIP3. The open reading frame (ORF) of CsRIP3 contained 1491 nucleotides and encoded a protein with 496 amino acids (Mw = 55.44 kDa). CsRIP3 expression was detected in various tissues in half-smooth tongue soles. CsRIP3 expression was up-regulated after pathogens i.e. Vibrio and poly (i:c) treatment in vivo, indicating its possible role in teleost immune response. Further analysis using human cells demonstrated that CsRIP3 exhibited highly conserved pro-apoptotic and pro-necroptotic functions in comparison with human RIP3. In conclusion, for the first time we reported the homologous structure and functions of CsRIP3 in inflammatory cell death, which provides novel perspectives on fish immunity studies in future., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The Japanese flounder is one of the most widely farmed economic flatfish species throughout eastern Asia including China, Korea, and Japan. Edwardsiella tarda is a major species of pathogenic bacteria that causes ascites disease and, consequently, a huge economy loss for Japanese flounder farming. After generation selection, traditional breeding methods can hardly improve the E. tarda resistance effectively. Genomic selection is an effective way to predict the breeding potential of parents and has rarely been used in aquatic breeding. In this study, we chose 931 individuals from 90 families, challenged by E. tarda from 2013 to 2015 as a reference population and 71 parents of these families as selection candidates. 1,934,475 markers were detected via genome sequencing and applied in this study. Two different methods, BayesCπ and GBLUP, were used for genomic prediction. In the reference population, two methods led to the same accuracy (0.946) and Pearson's correlation results between phenotype and genomic estimated breeding value (GEBV) of BayesCπ and GBLUP were 0.912 and 0.761, respectively. In selection candidates, GEBVs from two methods were highly similar (0.980). A comparison of GEBV with the survival rate of families that were structured by selection candidates showed correlations of 0.662 and 0.665, respectively. This study established a genomic selection method for the Japanese flounder and for the first time applied this to E. tarda resistance breeding.

The liver is an important organ for bacterial pathogen attack in fish. The differential proteomic response of the Japanese flounder liver to Edwardsiella tarda infection was examined using isobaric tags for relative and absolute quantitation (iTRAQ) labeling followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 3290 proteins were identified and classified into categories related to biological process (51.4%), molecular function (63.6%), and cellular component (57.7%). KEGG enrichment analysis indicated the complement and coagulation cascade pathways and the mineral absorption pathway were significantly enriched. Among the differentially expressed proteins, those involved in mediating complement cascade (e.g. complement component C7, C8, C9, complement factor H, complement factor Bf/C2) and mineral absorption (e.g. ferritin, STEAP-4) were most significantly upregulated during infection. Subsequently, five significantly upregulated (C4, C8beta, ferritin middle subunit, PRDX4-like and KRT18) and one significantly downregulated (transferrin) candidate immune proteins were validated by multiple reaction monitoring (MRM) assays. Furthermore, changes in expression of 15 proteins in the complement cascade and mineral absorption pathways were validated at the transcriptional level using quantitative real-time PCR (qPCR). The transcriptional levels of four transcription factors (p21Ras, Rab-31-like, NF-κB, STAT3) were also investigated by qPCR following infection with E. tarda. This study contributes to understanding the defense mechanisms of the liver in fish., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In the present study, the peripheral blood cells of half smooth tongue sole (Cynoglossus semilaevis) were examined by blood smear under the light microscopy. The proportion of main types of blood cells are as following: erythrocyte occupied the majority (92.3%), followed by thrombocyte (4.15%), granulocyte (1.7%), lymphocyte (1.5%) and monocyte (0.3%), respectively. Meanwhile, the isolation method of monocytes was established, by density gradient centrifugation to isolate mononuclear leukocytes of peripheral blood. In primary culture, the monocytes were adhered to the bottom of the flask without feeder cells and separated easily with suspended leukocytes in the medium in 3 h. After suspended leukocytes were removed, the monocytes multiplied rapidly with the two doubly during the 24 h, then the cells proliferated and kept stable until 48 h. When co-cultured with suspended leukocytes after three days, the monocytes could derive to typical macrophages, of which the size enlarged significantly and showed various forms such as like fried eggs, and giant irregular shape with pseudopod because cells fusion or deformation occurred until macrophages died in about two weeks. Monocytes showed strong respiratory burst activity after treated with Phorbol ester PMA and challenged by bacteria respectively. In addition, macrophage of half smooth tongue sole had typical macrophage features such as phagocytic capability, positive esterase activity, and the considerable expression of M-CSFR, MHC-II, IL-6, IL-10, TNF and arginase genes. That arginase expression in macrophages (3d and 5d after differentiation) was upregulated fluctuant suggest that the cultivation was mixture of alternatively activated type macrophage (M2) in the majority while the classically activated type (M1) win the minority. Furthermore, MHC-Ⅱ, M-CSFR and IL-6 were significantly induced following LPS challenge. Collectively, the present study will be useful for the study on half smooth tongue sole immune systems and immune function., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The complement system is important in the innate immune response. C1q-domain-containing proteins have multiple functions and occur extensively in invertebrates and vertebrates. In this study, PoC1ql3 encoding a C1q-domain-containing protein in the Japanese flounder was identified. The 266-amino-acid polypeptide encoded, PoC1ql3, shares high sequence and structural similarity with orthologues in other fish and mammals. PoC1ql3 is abundantly expressed in the brain, but less in the blood, gills, and liver. Transcripts of PoC1ql3 were down-regulated in the spleen and liver 6-24 h after bacterial infection, but were significantly up-regulated after 48 h. Full-length PoC1ql3 (C1ql3-full) and its gC1q domain (C1ql3-part) were both exerted anti-Edwardsiella tarda activity. C1ql3-part bound to lipopolysaccharide and peptidoglycan, and exerted antibacterial effects against E. tarda in vivo, suggesting that C1ql3 functions as a pathogen-recognition receptor. Therefore, PoC1ql3 functions in the innate immune system, which would facilitate the investigation of the immune system in Japanese flounder., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Half-smooth tongue sole (Cynoglossus semilaevis) is an important marine commercial fish species in China, which suffers from widespread disease outbreaks. Recently, in this regard, our group identified immune-related microRNAs (miRNAs) of C. semilaevis following Vibrio anguillarum infection. Furthermore, miRNA microarray was utilized to characterize the immune roles of important miRNA candidates in response to bacterial infection. Therefore, in the present study, we characterized miR-221 and miR-222 and profiled their expression after challenge. Here, miR-221 and miR-222 precursors were predicted to have a typical hairpin structure. Both miRNAs were expressed in a broad range of tissues in C. semilaevis, while miR-221 and miR-222 were significantly differentially expressed in the immune tissues of C. semilaevis among three small RNA libraries [control group (CG), bacteria-challenged fish without obvious symptoms of infection (NOSG), and bacteria-challenged fish with obvious symptoms of infection (HOSG)]. In order to further characterize and understand the immune response of miR-221 and miR-222, therefore, we profiled miR-221 and miR-222 expression in selected immune tissues after challenge with V. anguillarum. Both miR-221 and miR-222 were upregulated in the liver and spleen, while different expression patterns were observed in the head kidney. In addition, in half-smooth tongue sole head kidney cell line after challenge with lipopolysaccharide (LPS), polyinosinic:polycytidylic acid (poly I:C), peptidoglycan (PGN), and red-spotted grouper nervous necrosis virus (RGNNV), both miR-221 and miR-222 showed significant difference in expression response to pathogen. Meanwhile, the target gene of miR-221 and miR-222 was predicted, which indicated that tumor necrosis factor receptor-associated factor 6 (TRAF6) and interleukin-1 beta (IL-1β) were the target genes of miR-221 and miR-222, respectively. Collectively, these findings indicated that miR-221 and miR-222 have putative roles in innate immune response during C. semilaevis exposure to pathogens. Our findings could expand the knowledge of immune function of C. semilaevis miRNA and guide future studies on C. semilaevis immunity.

Complement factor I (Cfi) is a soluble serine protease which plays a crucial role in the modulation of complement cascades. In the presence of substrate modulating cofactors (such as complement factor H, C4bp, CR1, etc), Cfi cleaves and inactivates C3b and C4b, thereby controlling the complement-mediated processes. In this study, we sequenced and characterized Cfi gene from Cynoglossus Semilaevis (designated as CsCfi) for the first time. The full-length cDNA of CsCfi was 2230 bp in length, including a 98 bp 5'-untranslated region (UTR), a 164 bp 3'-UTR and a 1968 bp open reading frame (ORF). It encoded a polypeptide of 656 amino acids, with a molecular mass of 72.28 kDa and an isoelectric point of 7.71. A signal peptide was defined at N-terminus, resulting in a 626-residue mature protein. Multiple sequence alignment revealed that Cfi proteins were well conserved with the typical modular architecture and identical active sites throughout the vertebrates, which suggested the conserved function of Cfi. Phylogenetic analysis indicated that CsCfi and the homologous Cfi sequences from teleosts clustered into a clade, separating from another clade from the cartilaginous fish and other vertebrates. Tissue expression profile analysis by quantitative real-time PCR (qRT-PCR) showed that CsCfi mRNA constitutively expressed in all tested tissues, with the predominant expression in liver and the lowest in stomach. Temporal expression levels of CsCfi after challenging with Vibrio anguillarum showed different expression patterns in intestine, spleen, skin, blood, head kidney and liver. The recombinant CsCfi (rCsCfi) protein showed broad-spectrum antimicrobial activities against the Gram-positive bacteria Staphylococcus aureus and the Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa and Shewanella putrefaciens. The research revealed that CsCfi plays an important role in C. Semilaevis immunity., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

To investigate the roles of microRNAs (miRNA) of Cynoglossus semilaevis in response to Vibrio anguillarum that were previously identified using high-throughput sequencing, microarray analyses was performed on three small RNA libraries (CG, NOSG, and HOSG) prepared from C. semilaevis immune tissues. In total, of 1279 designed probes, 739 (57.78 %) were detectable. The expression levels of these miRNAs were analyzed using pairwise comparisons among the three libraries, and a total of 99 miRNAs were observed to be significantly differentially expressed. The expression patterns of 10 differentially expressed miRNAs were validated by real-time quantitative PCR (RT-qPCR). In addition, expression of miR-142-5p, miR-223, and miR-181a in response to V. anguillarum at numerous time-points in four tissues, as well as the responses to lipopolysaccharide (LPS), polyinosinic:polycytidylic acid (poly I:C), peptidoglycan (PGN), and red-spotted grouper nervous necrosis virus (RGNNV) in head kidney cells, were studied by qRT-PCR. Taken together, all of the expression profiles showed significant differences compared to the control group; both similarities and differences in the expression responses to the same pathogen were observed. Collectively, these findings highlighted the putative roles for miRNAs in the context of the innate immune response of C. semilaevis exposing to pathogens and that further studies are needed to understand the molecular mechanisms of miRNA regulation in C. semilaevis host-pathogen interactions.

High-resolution genetic maps are essential for fine mapping of complex traits, genome assembly, and comparative genomic analysis. Single-nucleotide polymorphisms (SNPs) are the primary molecular markers used for genetic map construction. In this study, we identified 13,362 SNPs evenly distributed across the Japanese flounder (Paralichthys olivaceus) genome. Of these SNPs, 12,712 high-confidence SNPs were subjected to high-throughput genotyping and assigned to 24 consensus linkage groups (LGs). The total length of the genetic linkage map was 3,497.29 cM with an average distance of 0.47 cM between loci, thereby representing the densest genetic map currently reported for Japanese flounder. Nine positive quantitative trait loci (QTLs) forming two main clusters for Vibrio anguillarum disease resistance were detected. All QTLs could explain 5.1-8.38% of the total phenotypic variation. Synteny analysis of the QTL regions on the genome assembly revealed 12 immune-related genes, among them 4 genes strongly associated with V. anguillarum disease resistance. In addition, 246 genome assembly scaffolds with an average size of 21.79 Mb were anchored onto the LGs; these scaffolds, comprising 522.99 Mb, represented 95.78% of assembled genomic sequences. The mapped assembly scaffolds in Japanese flounder were used for genome synteny analyses against zebrafish (Danio rerio) and medaka (Oryzias latipes). Flounder and medaka were found to possess almost one-to-one synteny, whereas flounder and zebrafish exhibited a multi-syntenic correspondence. The newly developed high-resolution genetic map, which will facilitate QTL mapping, scaffold assembly, and genome synteny analysis of Japanese flounder, marks a milestone in the ongoing genome project for this species., (© The Author 2015. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.)

Half-smooth tongue sole (Cynoglossus semilaevis) is one of the most valuable marine aquatic species in Northern China. Given to the rapid development of aquaculture industry, the C. semilaevis was subjected to disease-causing bacteria Vibrio anguillarum. It therefore is indispensable and urgent to understand the mechanism of C. semilaevis host defense against V. anguillarum infection. In the present study, the extensively analysis at the transcriptome level for V. Anguillarum disease in tongue sole was carried out. In total, 94,716 high quality contigs were generated from 75,884,572 clean reads in three libraries (HOSG, NOSG, and CG). 22,746 unigenes were identified when compared with SwissProt, an NR protein database and NT nucleotide database. 954 genes exhibiting the differentially expression at least one pair of comparison in all three libraries were identified. GO enrichment for these genes revealed gene response to biotic stimulus, immune system regulation, and immune response and cytokine production. Further, the pathways such as complement and coagulation cascades and Vibrio cholerae infection pathways were enriched in defensing of pathogen. Besides, 13,428 SSRs and 118,239 SNPs were detected in tongue sole, providing further support for genetic variation and marker-assisted selection in future. In summary, this study identifies several putative immune pathways and candidate genes deserving further investigation in the context of development of therapeutic regimens and lays the foundation for selecting resistant lines of C. semilaevis against V. anguillarum., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The C1q family includes many proteins that contain a globular (gC1q) domain, and this family is widely conserved from bacteria to mammals. The family is divided into three subgroups: C1q, C1q-like and ghC1q. In this study, a novel C1q family member, sghC1q, was cloned and identified from Cynoglossus semilaevis (named CssghC1q). The full-length CssghC1q cDNA spans 905 bp, including an open reading frame (ORF) of 768 bp, a 5'-untranslated region (UTR) of 25 bp and a 3'-UTR of 112 bp. The ORF encodes a putative protein of 255 amino acids (aa) with a deduced molecular weight of 28 kDa. The predicted protein contains a signal peptide (aa 1-19), a coiled-coil region (aa 61-102) and a globular C1q (gC1q) domain (aa 117-255). Protein sequence alignment indicated that the C-terminus of CssghC1q is highly conserved across several species. Phylogenetic analysis indicated that CssghC1q is most closely related to Maylandia zebra C1q-like-2-like. The CssghC1q genomic sequence spanned 1562 bp, with three exons and two introns. CssghC1q is constitutively expressed in all evaluated tissues, with the highest expression in the liver and the weakest in the heart. After a challenge with Vibrio anguillarum, CssghC1q transcript levels exhibited distinct time-dependent response patterns in the blood, head kidney, skin, spleen, intestine and liver. Recombinant CssghC1q protein exhibited antimicrobial activities against Gram-negative bacteria, Gram-positive bacteria and viruses. The minimum inhibitory concentration (MIC) values against Vibrio harveyi, Vibrio anguillarum, Pseudomonas aeruginosa and Staphylococcus aureus were 0.043 mg/mL, 0.087 mg/mL, 0.174 mg/mL and 0.025 mg/mL, respectively. A low concentration (0.06 mg/mL) of CssghC1q showed significant antiviral activity in vitro against nervous necrosis virus (NNV). These results suggest that CssghC1q plays a vital role in immune defense against bacteria and viruses., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

A recent genetic linkage map was employed to detect quantitative trait loci (QTLs) associated with Vibrio anguillarum resistance in Japanese flounder. An F1 family established and challenged with V. anguillarum in 2009 was used for QTL mapping. Of the 221 simple sequence repeat (SSR) markers used to detect polymorphisms in the parents of F1, 170 were confirmed to be polymorphic. The average distance between the markers was 10.6 cM. Equal amounts of genomic DNA from 15 fry that died early and from 15 survivors were pooled separately to constitute susceptible bulk and resistance bulk DNA. Bulked segregant analysis and QTL mapping were combined to detect candidate SSR markers and regions associated with the disease. A genome scan identified four polymorphic SSR markers, two of which were significantly different between susceptible and resistance bulk (P=0.008). These two markers were located in linkage group (LG) 7; therefore, all the SSR markers in LG7 were genotyped in all the challenged fry by single marker analysis. Using two different models, 11-17 SSR markers were detected with different levels of significance. To confirm the associations of these markers with the disease, composite interval mapping was employed to genotype all the challenged individuals. One and three QTLs, which explained more than 60 % of the phenotypic variance, were detected by the two models. Two of the QTLs were located at 48.6 cM. The common QTL may therefore be a major candidate region for disease resistance against V. anguillarum infection.

Background: Infectious diseases have caused huge economic loss and food security issues in fish aquaculture. Current management and breeding strategies heavily rely on the knowledge of regulative mechanisms underlying disease resistance. Though the intestinal microbial community was linked with disease infection, there is little knowledge about the roles of intestinal microbes in fish disease resistance. Cynoglossus semilaevis is an economically important and widely cultivated flatfish species in China. However, it suffers from outbreaks of vibriosis, which results in huge mortalities and economic loss. Results: Here, we used C. semilaevis as a research model to investigate the host-microbiome interactions in regulating vibriosis resistance. The resistance to vibriosis was reflected in intestinal microbiome on both taxonomic and functional levels. Such differences also influenced the host gene expressions in the resistant family. Moreover, the intestinal microbiome might control the host immunological homeostasis and inflammation to enhance vibriosis resistance through the microbe-intestine-immunity axis. For example, Phaeobacter regulated its hdhA gene and host cyp27a1 gene up-expressed in bile acid biosynthesis pathways, but regulated its trxA gene and host akt gene down-expressed in proinflammatory cytokines biosynthesis pathways, to reduce inflammation and resist disease infection in the resistant family. Furthermore, the combination of intestinal microbes and host genes as biomarkers could accurately differentiate resistant family from susceptible family. Conclusion: Our study uncovered the regulatory patterns of the microbe-intestine-immunity axis that may contribute to vibriosis resistance in C. semilaevis. These findings could facilitate the disease control and selective breeding of superior germplasm with high disease resistance in fish aquaculture. 8F1qVDdq8rkntrj1jj62Gc Video Abstract [ABSTRACT FROM AUTHOR]