L. Ma

Krüppel-like factors (KLFs) are transcription factors that bind DNA via three tandem zinc fingers, critically regulating cellular processes like proliferation, differentiation, and apoptosis. Among KLFs, KLF9 is particularly notable for its immunological roles in mammalian immune systems, where it regulates tumor growth, modulates interferon-related genes and inflammatory cytokines. However, the potential role of klf9 in fish immune responses remains poorly understood. In this study, we characterized turbot (Scophthalmus maximus L.) klf9 (Sm_klf9) and explored its role against bacterial infection. In our results, the full length of Sm_klf9 was 3,372 bp, including an open reading frame (ORF) of 684 bp. In phylogenetic analyses, Sm_klf9 grouped closely with the klf9 genes of Japanese flounder and Atlantic halibut, consistent with the broader phylogenetic relationships among flatfish species. Sm_klf9 exhibited widespread expression across all tested tissues in turbot with the highest expression level in blood. The expression of Sm_klf9 was significantly induced in a time-dependent manner in turbot intestine after bacterial infection (dramatic upregulation with 2.78-fold at 2 h, followed by significant downregulations with 2.29- and 2.74-fold at 12 h and 24 h, respectively), and the urgently significant induction at 2h suggested rapid response measures against bacterial infection at the early stages. The overexpression of Sm_klf9 up-regulated the key immune-related genes in the NF-κB signaling pathway, such as NF-κB2 and Myd88. Meanwhile, the overexpression of Sm_klf9 was found to accelerate the invasion of Edwardsiella tarda and promote the cell apoptosis in turbot intestine cells. Overall, the findings in this study indicated the important roles of Sm_klf9 in turbot immune system during pathogenic infection, which could provide a theoretical basis for investigating the key roles of Sm_klf9 in bacteriostatic ability and inflammatory regulations in turbot during bacterial infection.

Growth arrest and DNA damage-inducible 45 alpha, b gene (gadd45ab) is a key homologue of gadd45a subfamily, which participates in several biological processes, such as inflammation and apoptosis. Black rockfish (Sebastes schlegelii) is an important commercial fish species in China. In current study, black rockfish gadd45ab (Ss_gadd45ab) was characterized as a target gene of miR-20b-5p. The results indicated that high evolutionary conservations were found in the sequence and structure of Ss_gadd45ab, which was also detected to locate on the cytomembrane in the black rockfish intestine. The expressions of Ss_gadd45ab and diverse cytokines related to immune response were significantly induced with different patterns by the infection of E. tarda. Moreover, all tested immune genes were dramatically up-regulated when Ss_gadd45ab was overexpressed in the intestine cells, and significant up-regulations and down-regulations of almost these immune genes were observed in the overexpressed cells during the LPS stimulation at 12 h and 24 h, respectively, which implied the potential regulations of Ss_gadd45ab in the immune system. Meanwhile, the overexpression of Ss_gadd45ab was found to prevent the invasion of E. tarda and promote the cell apoptosis in the intestine cells of black rockfish. Therefore, a profile of a novel regulatory mechanism of Ss_gadd45ab targeted by miR-20b-5p in the inflammatory and apoptotic responses of black rockfish during pathogenic infections was hypothesized. Even though the potential functions of Ss_gadd45ab on the inflammation and apoptosis in the black rockfish intestine during pathogenic infections have been described preliminarily in this study, more efforts are still urgent to be done for the further verification of this regulatory mechanism in black rockfish.

Recently, Vibrio anguillarum, a Gram-negative pathogenic bacterium, has been becoming a major constraint on the development of the turbot aquaculture industry because of its characteristics of worldwide distribution, broad host range and potentially devastating impacts. Although the functions of protein-coding mRNAs in the immune response against bacterial infection have been reported, as well as several non-coding RNAs (ncRNAs), such as circular RNAs (circRNAs) and microRNAs (miRNAs), the relationships between mRNAs and ncRNAs in the immune system of turbot liver are still limited during bacterial infection. In present study, the comprehensive analyses of whole-transcriptome sequencing were conducted in turbot liver infected by V. anguillarum. The differential expression was analyzed in the data of circRNAs, miRNAs, and mRNAs. The interactions of miRNA-circRNA pairs and miRNA-mRNA pairs were predicted basing on the negative regulatory relationships between miRNAs and their target circRNAs\mRNAs. The circRNA-related ceRNA regulatory networks were constructed for the analyses of regulated mechanism in turbot immune system. Subsequently, the RT-qPCR was carried out to verify the results of sequencing. Finally, we identified 31 circRNAs, 53 miRNAs and 948 mRNAs with differential expression. Gene set enrichment analyses using Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways showed that innate immunity was principally activated at the early stages of infection, while adaptive immunity was activated after 24 h. Finally, 65 circRNA-miRNA-mRNA pathways were constructed, based on the hypothesis of ceRNA regulatory networks. In conclusion, our findings provide new insights on the underlying immune response to bacterial infection and identify novel target genes for the prevention and control of disease in turbot.

In teleost, the immune roles of the gut-associated lymphoid tissue (GALT) have been well recognized. The antibacterial functions of the spleen have been also documented in various fish species. However, the mechanism of fish intestine-spleen immunity during bacterial infection still remains largely unknown. In this study, an integrative analysis was conducted in the black rockfish intestine and spleen after Aeromonas salmonicida and Edwardsiella tarda infection on the transcriptomic level. The immune gene libraries were constructed in the intestine and spleen. A total of 211 and 253 differentially expressed genes were observed in the immune gene library of spleen and intestine, respectively. One more category of immune genes was found in the intestine (9 categories) than in the spleen (8 categories). Potential evidence of the cooperation in the intestine-spleen axis was obtained based on the functional annotation and enrichment analyses of joint immune genes between intestine and spleen. In addition, bioinformatic and ceRNA analyses for transcriptomic data were performed. Finally, two key immune genes (CO3 and MRC1) and their related non-coding RNAs were observed in the functions of bacterial recognition and binding. According to the pathway “intestinal immune network for IgA production” predicted in the splenic transcriptome by functional enrichment, a novel regulatory pathway of the immunity of the intestine-spleen axis was hypothesized in black rockfish during bacterial infection. Moreover, the regulatory relationships between the immune transcripts in this pathway were verified preliminarily by RT-qPCR. Dual-luciferase reporter assays were performed to determine the regulations between the ceRNAs of MRC1/miR-23b-5p/LNC_00057980 and CO3/miR-214/LNC_00096415 axis. Overall, our findings demonstrate the key pathways of MRC1/miR-23b-5p/LNC_00057980 and CO3/miR-214/LNC_00096415 in the regulation mechanism of intestine-spleen axis during two different Gram-negative bacteria infection in black rockfish.

Background
With more than 36,000 valid fish species, teleost fishes constitute the most species-rich vertebrate clade and exhibit extensive genetic and phenotypic variation, including diverse immune defense strategies. NLRC3 subfamily genes, which are specific to fishes, play vital roles in the immune system of teleosts. The evolution of teleosts has been impacted by several whole-genome duplication (WGD) events, which might be a key reason for the expansions of the NLRC3 subfamily, but detailed knowledge of NLRC3 subfamily evolution in the family Sebastidae is still limited.

Results
Phylogenetic inference of NLRC3 subfamily protein sequences were conducted to evaluate the orthology of NLRC3 subfamily genes in black rockfish (Sebastes schlegilii), 13 other fish species from the families Sebastidae, Serranidae, Gasterosteidae and Cyclopteridae, and three species of high vertebrates (bird, reptile and amphibian). WGD analyses were used to estimate expansions and contractions of the NLRC3 subfamily, and patterns of expression of NLRC3 subfamily genes in black rockfish following bacterial infections were used to investigate the functional roles of these genes in the traditional and mucosal immune system of the Sebastidae. Different patterns of gene expansions and contractions were observed in 17 fish and other species examined, and one and two whole-genome duplication events were observed in two members of family Sebastidae (black rockfish and honeycomb rockfish, Sebastes umbrosus), respectively. Subsequently, 179 copy numbers of NLRC3 genes were found in black rockfish and 166 in honeycomb rockfish. Phylogenetic analyses corroborated the conservation and evolution of NLRC3 orthologues between Sebastidae and other fish species. Finally, differential expression analyses provided evidence of the immune roles of NLRC3 genes in black rockfish during bacterial infections and gene ontology analysis also indicated other functional roles.

Conclusions
We hypothesize that NLRC3 genes have evolved a variety of different functions, in addition to their role in the immune response, as a result of whole genome duplication events during teleost diversification. Importantly, this study had underscored the importance of sampling across taxonomic groups, to better understand the evolutionary patterns of the innate immunity system on which complex immunological novelties arose. Moreover, the results in this study could extend current knowledge of the plasticity of the immune system.

Occludin (OCLD), as a vital member of TJ superfamily, plays important regulatory roles in the processes of proliferation, repair and survival of epithelial cells following various stresses. In teleost, OCLD gene was demonstrated to regulate the permeability near epithelial cells to be involved in the mucosal immune response. Even though the immune functions of OCLD genes were investigated in mammals and a part of fish species, no relevant studies have been done on the potential mechanism of the anti-inflammatory regulations of OCLD in the turbot mucosal immunity during bacterial infection, moreover, with the regulation of miRNAs to turbot OCLD. In this study, the full-length sequence of OCLD nucleotide was cloned and confirmed by PCR amplification reaction. Following bacterial infections, the mRNA expressions of OCLD and genes related to OCLD were detected in the turbot intestinal tissues and cells following bacterial infections. Afterward, knockdown of OCLD in turbot intestine cells (SMI cells) was used to confirm the key roles of OCLD in the mucosal immune system, as well as cell apoptosis. The promotions of cell proliferation and migration of turbot OCLD were confirmed in the SMI cells for wound healing. The target regulationship between miR-140-3P and turbot OCLD was predicted and verified by luciferase reporter assay. Finally, the anti-inflammatory functions of turbot OCLD were investigated in the OCLD knockdown SMI cells (in vitro), which was further verified in zebrafish model with DSS-induced colitis (in vivo) through recombinant turbot OCLD protein. Therefore, we hypothesized that turbot OCLD gene could be regulated by miR-140-3p to attenuate the apoptosis of SMI cells, thereby suppressing the inflammation in the intestine via the regulations of the NF-κB signaling pathway. This study contributes to the fundamental and advanced understanding of the immune regulatory functions of turbot OCLD in the intestinal inflammation, which will also provide the new sights for the studies on the prevention and treatment of fish diseases during bacterial invasion on the mucosal system.

Aeromonas salmonicides is a type of Gram-negative bacteria and has become the main fish pathogen in aquaculture because of its characteristics of worldwide distribution, broad host range and potentially devastating impacts. In the past years, studies have been focused to explore the regulatory roles of circRNA-miRNA-mRNA network in fish diseases. However, there are only few systematic studies linked to the anti-bacterial roles of circRNA-related ceRNA networks in the spleen immune system of black rockfish (Sebastes schlegelii). In this study, the whole-transcriptome sequencing (RNA-seq) was conducted in the black rockfish spleen with A. salmonicida challenging. The differentially expressed (DE) circRNAs were identified comprehensively for the following enrichment analysis. Interactions of miRNA-circRNA pairs and miRNA-mRNA pairs were predicted for the construction of circRNA-related ceRNA regulatory networks. Then, protein-protein interaction (PPI) analysis of mRNAs from these ceRNA networks were conducted. Finally, a total number of 39 circRNAs exhibited significantly differential expressions during A. salmonicida infection in the black rockfish spleen in 4338 identified circRNAs from 12 samples in 4 libraries. Functional enrichment analysis suggested that they were significantly enriched in several immune-related pathways, including Endocytosis, FoxO signaling pathway, Jak-STST signaling pathway, Herpes simplex infection, etc. Subsequently, 290 circRNA-miRNA-mRNA pathways (91 at 2 h, 142 at 12 h and 65 at 24 h) were constructed including 31 circRNAs, 50 miRNAs, and 156 mRNAs. In conclusion, the circRNA-related ceRNA networks were established, which will provide some novel insights in molecular mechanistic investigations of anti-bacterial immune response in teleost. Also, these findings will propose significant predictive values for the development of methods of treatment and prevention in black rockfish after bacterial infection in the future.

TNF alpha, as a pro-inflammatory cytokine, plays an important role in inflammation and immune homeostasis maintaining. However, the knowledge about the immune functions of teleost TNF alpha against bacterial infections is still limited. In this study, the TNF alpha was characterized from black rockfish (Sebastes schlegelii). The bioinformatics analyses showed the evolutionary conservations in sequence and structure. The expression levels of Ss_TNF alpha mRNA were significantly up-regulated in the spleen and intestine after Aeromonas salmonicides and Edwardsiella tarda infections, and dramatically down-regulated in PBLs after LPS and poly I:C stimulations. Meanwhile, the extremely up-regulated expressions of other inflammatory cytokines (especially for IL-1 beta and IL17C) were observed in the intestine and spleen after bacterial infection and down-regulations were obtained in PBLs. The significant regulation with expression patterns of Ss_TNF alpha and other inflammatory cytokine mRNAs illustrated the variations of immunity in different tissues and cells of black rockfish. The regulated functions of Ss_TNF alpha in the up/downstream signaling pathways were preliminarily verified on the transcription and translation levels. Subsequently, in vitro knockdown of Ss_TNF alpha in the intestine cells of black rockfish confirmed the important immune roles of Ss_TNF alpha. Finally, the apoptotic analyses were conducted in PBLs and intestine cells of black rockfish. The rapid increases of the apoptotic rates were obtained in both PBLs and intestine cells after treatment with rSs_TNF alpha, but distinct apoptotic rates at the early and late stages of apoptosis were observed between these two types of cells. The results of apoptotic analyses suggested that Ss_TNF alpha could trigger apoptosis of different cells in different strategies in black rockfish. Overall, the findings in this study indicated the important roles of Ss_TNF alpha in the immune system of black rockfish during pathogenic infection, as well as the potential function on biomarker for monitoring the health status.

Vibriosis, most commonly caused by the bacterium Vibrio anguillarum, is one of the most important diseases affecting the marine aquaculture industry throughout the world. The intestine is the main entry portal of V. anguillarum into host fishes. Although there has been extensive research on the immunity of the gut-associated lymphoid tissue (GALT) in teleost, the mechanisms underlying the immunity of the intestine-liver during bacterial infections are still not well understood. This study conducted an integrative analysis of the transcriptome in turbot (Scopthalmus maximus L.) intestine and liver after V. anguillarum infection. To provide evidence of immunological collaboration in the turbot intestine-liver axis, we constructed the immune gene libraries, and conducted the functional annotation and enrichment analyses of immune genes that were jointly expressed in both turbot intestine and liver. Additionally, we administered the bioinformatic analyses of transcriptomic data to investigate regulatory relationships between non-coding RNAs (ncRNAs) and their target immune genes, based on the ceRNA theory. The recognition and binding abilities to bacteria were activated by the Toll-like receptor (TLR) signaling pathway. Subsequently, NF-κB was activated by the TLR/MyD88 signaling, and its target genes were induced to up-regulate in intestine and associated tissues to the digestive tract to enhance disease resistance through the production of inflammation. Histology of the intestine and liver of infected fish showed pathological changes consistent with inflammation. Bioinformatic analysis suggested that ceRNA regulatory networks play a pivotal role in the regulation of TLR genes, and RT-qPCR analyses and dual-luciferase reporter assays showed confirmed evidence to verify these hypothesized regulatory relationships. Overall, the results in this study demonstrated that a lncRNA (LNC_002468) activated the TLR signaling pathway by upregulating the TLR9 gene through sponging miRNA (novel_709). This study provides a strong indication that ncRNAs play regulatory roles in the antibacterial immune response of teleost fishes. However, further functional verification is required of the key immune genes and their regulatory relationship with miRNAs and lncRNAs.

Tight junction proteins (TJs) are important component proteins that maintaining the structure and function of TJs, connecting to each other to form a TJ complex between cells, maintaining the biological homeostasis of the internal environment. In this study, a total of 103 TJ genes were identified in turbot according to our whole-transcriptome database. Transmembrane TJs were divided into seven subfamilies, including claudin (CLDN), occludin (OCLD), tricellulin (MARVELD2), MARVEL domain containing 3 (MARVELD3), junctional adhesion molecules (JAM), immunoglobulin superfamily member 5 (IGSF5/JAM4), blood vessel epicardial substance (BVEs). Moreover, the majority of homologous pairs of TJ genes showed highly conserved alongside length, exon/intron number and motifs. As for phylogenetic analysis for 103 TJ genes, eight of them have undergone a positive selection and JAMB-like has undergone the most neutral evolution. The expression patterns of several TJ genes showed the lowest expression levels in blood, while the highest expression levels were detected in intestine, gill and skin, which all belong to mucosal tissues. Meanwhile, most examined TJ genes showed down-regulated expression patterns during bacterial infection, while several TJ genes exhibited up-regulated expression patterns at a later stage (24 h). At the same time, several potential candidate genes (such as CLDN-15, CLDN-3, CLDN-12, CLDN-5 and OCLD) were significantly down-regulated, which may indicate their important functions that involved in the regulation of bacterial infection. Currently, there is little research on CLDN5 in the intestine, but it is highly expressed in the intestine and has significant changes in intestinal expression after bacterial infection. Thus, we knocked down CLDN5 by the method of lentiviral infection. The result showed CLDN5 was related to cell migration (wound healing) and apoptosis, and the method of dualluciferasereporterassay showed that the functions of CLDN5 could be regulated by miR-24. The study of TJs may lead to a better understanding of the function of TJs in teleost.