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The caspase, functioning as a proteinase, plays a crucial role in eukaryotic cell apoptosis, regulation of apoptosis, cellular growth, differentiation, and immunity. The identification of caspase gene family in Sebastes schlegelii is of great help to understand its antimicrobial research. In S. schlegelii , we totally identified nine caspase genes, including four apoptosis initiator caspases (caspase 2, caspase 8, caspase 9 and caspase 10), four apoptosis executioners (caspase 3a, caspase 3b, caspase 6 , and caspase 7) and one inflammatory executioner (caspase 1). The duplication of caspase 3 genes on chr3 and chr8 may have been facilitated by whole genome duplication (WGD) events or other complex evolutionary processes. In general, the number of caspase genes relatively conserved in high vertebrates, while exhibiting variation in teleosts. Furthermore, syntenic analysis and phylogenetic relationships analysis supported the correct classification of these caspase gene family in S. schlegelii , especially for genes with duplicated copies. Additionally, the expression patterns of these caspase genes in different tissues of S. schlegelii under healthy conditions were assessed. The results revealed that the expression levels of most caspase genes were significantly elevated in the intestine, spleen, and liver. To further investigate the potential immune functions of these caspase genes in S. schlegelii , we challenged individuals with A. salmonicida and V. anguillarum, respectively. After infection with A. salmonicida , the expression levels of caspase 1 in the liver and spleen of S. schlegelii remained consistently elevated throughout the infection time points. The expression levels of most caspase family members in the intestine exhibited significant divergence following V. anguillarum infection. This study provides a comprehensive understanding of the caspase gene families in S. schlegelii , thereby establishing a solid foundation for further investigations into the functional roles of these caspase genes. [Display omitted] • Nine caspase family members were identified in S. schlegelii. • The caspase genes including 4 executioner genes, 4 initiator genes, and 1 inflammatory gene. • Most caspase genes showed high expression levels in intestine, spleen, and liver. • Most caspase genes in liver and spleen were significantly induced by A. salmonicida infection. • The expression pattern of caspase gene was different after the infection of V. anguillarum. [ABSTRACT FROM AUTHOR]

Aquaculture plays important roles in global food security. Prized as a valuable commercial species, the aquaculture industry of half-smooth tongue sole (Cynoglossus semilaevis) has faced unprecedented challenge due to bacterial diseases, including vibriosis caused by Vibrio anguillarum. Alternative splicing (AS) is an important layer of post-transcriptional regulation, being involved in almost all biological processes, including development, reproduction, and immunity. However, its roles in immunity in half-smooth tongue sole remain elusive. In this study, therefore, we profiled the changes of alternative splicing in half-smooth tongue sole after infection with different concentrations of V. anguillarum , and investigated their functions by both enrichment analysis and targeted gene transcript overexpression analysis with half-smooth tongue sole macrophages. Intriguingly, differentially alternatively spliced (DAS) genes were found to be involved in RNA splicing for each bacterial treatment and for the shared gene set, consistent with previous studies that splicing factor themselves often underwent alternative splicing. A set of 12 immune-related shared DAS genes were identified, including hp , c5 , and traf2b. Targeted gene analysis using the shared DAS gene, haptoglobin (hp), showed that alternative splicing resulted in changes of protein domains, and overexpression analysis demonstrated that two transcripts of hp exhibited contrary roles in stimulating or reducing the expression of immune factors in response to lipopolysaccharides (LPS), reflecting the vital roles of alternative splicing in immunity of tongue sole. DAS events were further verified by qRT-PCR (20 primer pairs for 10 DAS events) to confirm the reliability of our bioinformatics analysis. This study, for the first time, revealed the involvement of alternative splicing in immune responses of half-smooth tongue sole using both omics data and targeted gene functional analysis, setting a solid foundation for further analysis and providing clues for potential usage in controlling bacterial diseases in aquaculture. • RNA splicing was enriched for DAS genes in each comparison and the shared gene set. • A set of 12 immune-related genes were identified in 104 shared DAS genes. • Two hp splicing isoforms exhibited contrary roles in stimulation of immune factors. [ABSTRACT FROM AUTHOR]

MicroRNAs play crucial roles in various biological processes, including but not limited to differentiation, development, disease, and immunity. However, their immunoregulatory roles in half-smooth tongue sole are lacking. Our previous studies indicated that miR-722 could target C5aR1 to modulate the complement pathway to alleviate inflammatory response and even affect the mortality after the bacterial infection with Vibrio anguillarum. Driven by the purpose of revealing the underlying mechanisms, in this study, we investigated the effects of miR-722 on the gene expression and alternative splicing (AS) in the liver of half-smooth tongue sole after Vibrio anguillarum infection, with the approach of miR-722 overexpression/silencing and subsequent RNA-seq. Among the different comparisons, the I group (miR-722 inhibitor and V. anguillarum) versus blank control (PBS) exhibited the highest number of differentially expressed genes (DEGs), suggesting that the immune response was overactivated after inhibiting the miR-722. In addition, enrichment analyses were performed to reveal the functions of DEGs and differential AS (DAS) genes, reflecting the enrichment of RNA splicing and immune-related pathways including NF-κB and T cell receptor signaling pathway. Comparing the M group (miR-722 mimic and V. anguillarum) with the negative control (random sequence and V. anguillarum), two immune-related genes, cd48 and mapk8 , were differentially expressed, of which mapk8 was also differentially spliced, indicating their importance in the immune response. Furthermore, representative gene analysis was performed, suggesting their corresponding functional changes due to AS. To verify the RNA-seq data, quantitative real-time PCR was employed with twenty pairs of primers for DEGs and DAS events. Overall, our results demonstrated that miR-722 could mediate the transcriptome-wide changes of gene expression and AS in half-smooth tongue sole, and provided insights into the regulatory role of miR-722 in immune responses, laying the foundation for further functional analyses and practical applications in aquaculture. • miR-722 mediated the transcriptome-wide changes of gene expression and AS in tongue sole. • DAS contributed significantly to the immune response after the bacterial infection. • mapk8 was the only gene that underwent both differential expression and DAS in M vs N. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are small non-coding RNAs involved in various biological processes, including immunity. Previously, we investigated the miRNAs of half-smooth tongue sole (Cynoglossus semilaevis) and found that miR-722 (designated Cse -miR-722) was significantly differentially expressed after infection with Vibrio anguillarum , reflecting its importance in immune response. Our preliminary bioinformatic analysis suggested that Cse -miR-722 could target C5aR1 (designated CsC5aR1), which was known to play crucial roles in complement activation and inflammatory response, as a receptor of C5a. However, the underlying mechanisms of their interactions and specific functions in inflammatory and immune response are still enigmas. In this study, we successfully cloned the precursor sequence of Cse -miR-722 (94 bp) and the full length of CsC5aR1 (1541 bp, protein molecular weight 39 kDa). The target gene of Cse -miR-722 was verified as CsC5aR1 by a dual luciferase reporter assay, and Cse -miR-722 was confirmed to regulate CsC5aR1 at the protein level using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. The expression of CsC5aR1 and Cse -miR-722 in liver cells and four immune tissues of half-smooth tongue sole changed significantly after LPS stimulation and infection with V. anguillarum. To explore the functional role of Cse -miR-722 in half-smooth tongue sole, we performed both in vitro and in vivo experiments. Cse -miR-722 was observed to affect phagocytosis and respiratory burst activity of macrophages by regulating CsC5aR1 in half-smooth tongue sole. Furthermore, we found that Cse -miR-722 regulated the expression of CsC5aR1 , CsC5a , and the inflammatory factors CsIL1-β , CsIL6 , CsIL8 , and CsTNF-α both in vitro and in vivo. In addition, Cse -miR-722 reduced mortality and pathological damage. This study clarified the regulatory mechanism of Cse- miR-722 on CsC5aR1 and provided insight into the regulatory roles of Cse- miR-722 in immune responses, laying a theoretical foundation for the feasibility of using miR-722 to prevent and control bacterial diseases in teleost. • Cse -miR-722 targeted CsC5aR1 and silenced CsC5aR1 at the translation level. • Cse -miR-722 affected the phagocytic activity and respiratory burst of macrophages. • Cse -miR-722 regulated immune responses through the C5a/C5aR1 complement pathway. • Cse -miR-722 reduced mortality and body damage after Vibrio anguillarum infection. [ABSTRACT FROM AUTHOR]