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Type I interferons (IFNs) are pleiotropic cytokines widely recognized for their roles in the innate immunity of vertebrates, from fish to mammals. Previously, seven type I IFNs were identified in the order of Cypriniformes, the largest group of freshwater fish. Using whole genome analysis, we have identified the IFNf from this order, expanding the number of type I IFNs in this order to eight. IFNf exhibited conserved synteny across 25 Cypriniformes genomes and phylogenetically predated the divergence of group 1 and group 2 type I IFNs. Despite low sequence identity at both the nucleotide and protein levels, IFNf retained a conserved gene structure and three-dimensional conformation essential for its function. Expression analysis revealed drastic upregulation of IFNf in grass carp upon infection with hemorrhagic virus (GCRV-II), with the highest level of activation among all the eight IFNs. Additionally, four chemically synthesized α-helix peptides derived from both grass carp and zebrafish IFNfs acted as antimicrobial peptides (AMPs), displaying direct inhibitory activity against a variety of microorganisms. Thus, this study establishes IFNf as an important component of the immune system in Cypriniformes.

Traditional Chinese medicines (TCMs) have a wide variety of chemical components and biological activities, which are applied in multiple fields such as medicine, agriculture and food. ( ), known as \"ChouMuDan\" in Chinese, is a traditional herb belonged to the shrub of the genus and used to treat various diseases. Previous studies indicated had certain activity in antimicrobial aspects, therefore, the present study focus on exploring its antibacterial effect on loaches infected with and the mechanism. This study first prepared the -butanol extract of , using relux extraction and liquid-liquid organic partition method. Then, the loach was randomly divided into two groups. After the experiment, the livers of loaches from two different groups were dissected for microRNA sequencing. The samples were sequenced in Hiseq Single-End mode to obtain the original data. The Unique Reads were aligned with the non-coding RNA sequences in the Rfam 13 database using Blast to screen out the non-coding RNAs and identify their types and numbers for further analysis of their functions and regulatory mechanisms. The Reads Count value of miRNA was calculated, and the expression data of the conserved miRNA in each sample were sorted out. Finally, seven differentially expressed miRNA that regulate immunity, were selected to verify the credibility of miRNA expression identified by sequencing. Two miRNA libraries of mCK1 and mC4 were constructed, using the Hiseq Single-End mode, to study the effect of -butanol extract of . (BECB) on loach infected with . Differentially expressed miRNAs were identified, among which 9 were up-regulated and 17 were down-regulated. The cluster analysis of differentially expressed miRNAs showed that the expression pattern of miRNAs changed significantly after BECB treatment. KEGG enrichment analysis was carried out on target genes, and the top 30 most significantly enriched pathways were selected by p-value. The result suggested the immune-related pathways mTOR signaling pathway and RIG-I-like receptor signaling pathway may critical for resistance to infection in loach after BECB treatment, together with RT-qPCR results. In this study, pathways related to carbohydrate and lipid metabolism were enriched after infection with for 24 h, which may be because the proliferation and differentiation of immune cells require a large number of nutrients to provide energy. This indicates that pathogens did not appear in the loach until exposure to for 24 h. After infection, both autophagy and mTOR signaling pathway are activated to promote the proliferation and differentiation of immune cells, induce the production of type I IFN and inflammatory factors, and then trigger innate and specific immunity. These findings could provide a basis for the research and development of antibacterial agents based on and the application of biopesticides in aquaculture, especially in loach farming.

Vine tea ( Ampelopsis grossedentata ) is a widely used Chinese herb with a long history of application in southern China, known for its notable anti-infective, antibacterial and immunomodulatory pharmacological properties. It has potential for application in aquaculture as an inexpensive and readily available dietary supplement, promoting growth, antioxidant activity, and immune regulation in fish. However, there have been very few studies investigating the effects of vine tea on fish miRNAs. Loach is an economically important freshwater fish species, highly valued for its delicious flavor, but research on its miRNA relatively limited. To examine immune-related miRNAs in loach and to further determine the antibacterial immune mechanisms of vine tea, we performed small RNA sequencing analysis of loach liver tissue before and after treatment with vine tea extract. The results showed that vine tea could affect the antibacterial immune activity of loach through the miRNA regulation. A total of 25 differentially expressed miRNAs were identified in liver tissues, and KEGG pathway analysis revealed that most of these miRNAs were involved in innate immune responses such as autophagy, lysosomes, endocytosis, and pattern receptor signaling pathways. To the best of our knowledge, this is the first study to profile miRNA expression in loach after treatment with vine tea extract. This work deepens the understanding of the role of loach miRNAs in the immune system and opens new prospects for application of vine tea in aquaculture.

This study investigates the immunomodulatory effects of Lonicera japonica Thunb. extract, a perennial semi-evergreen vine of the Caprifoliaceae family, on lipopolysaccharide (LPS)-induced immune responses in loach ( Misgurnus anguillicaudatus ) through miRNA regulatory mechanisms. Small RNA libraries constructed from hepatic tissues of LPS-challenged (CK) and Lonicera japonica-treated (LJ) groups yielded 139.6 million clean reads with characteristic 21–23 nucleotide length distribution. Abundance analysis revealed significant differential expression patterns within the let-7 family: miR-let-7-9, miR-let-7-6, and miR-let-7–18 exhibited higher abundance in the infection group, whereas miR-let-7-1, miR-let-7-17, and miR-let-7–16 showed elevated abundance in the treatment group. Comparative profiling identified 55 differentially expressed miRNAs (41 upregulated, 14 downregulated), with animal-undef-351, animal-mir-21-6, and animal-undef-603 demonstrating the most significant expression differences (P<0.01). KEGG enrichment analysis highlighted predominant involvement in sphingolipid signaling pathway, glycerophospholipid metabolism, T cell receptor signaling pathway, and TNF signaling pathway. GO analysis revealed significant enrichment in biological processes related to glycosylation, cellular components of transcription regulator complexes, and molecular functions associated with double-stranded DNA binding. These findings demonstrate that L. japonica alleviates LPS-induced inflammation by modulating miRNA expression networks, particularly through sphingolipid metabolism and TNF signaling pathways, providing novel molecular evolutionary insights into the immunoregulatory mechanisms of traditional Chinese medicine.

The loach ( Misgurnus anguillicaudatus ), a small commercial fish that is widely cultivated for its high-quality protein, vitamins, minerals, and essential amino acid, is a member of the genus Misgurnus and the family Cyprinidae. In this study, we gave the LPS-injected loach fermented soybean meal and used transcriptome sequencing to investigate the impact of the fermented soybean powder on the loach’s immune system. 3384 up-regulated genes and 12116 down-regulated genes were found among the 15500 differentially expressed genes, according to the results. The differentially expressed genes were shown to be involved in cellular processes, metabolic processes, cellular anatomical entities, and binding, according to the Go functional annotation. Meanwhile, the KEGG enrichment analysis indicated that the soybean fermented powder treated groups showed significant differences in DNA replication, Nucleotide excision repair, Fanconi anemia pathway, and Base excision repair pathways, suggesting that these pathways are closely related to the enhancement of the immune function of loach by soybean fermented powder. The particular conclusions not exclusively can provide a new conception for the rational utilization of soybean fermented powder but also can provide theoretical guidance for the subsequent healthy breeding of loach.

Maternal genes are important in directing early development and determining egg quality in fish. We here report the de novo transcriptome from four tissue libraries of the cyprinid loach, Misgurnus anguillicaudatus, and for the first time identified maternal gene transcripts in unfertilized eggs and suggest their immune system involvement. Expression profiles and functional enrichment revealed a total 24,116 transcripts were expressed as maternal transcripts in unfertilized eggs, which were involved in a wide range of biological functions and pathways. Comparison expression profiles and analysis of tissue specificity revealed that the large numbers of maternal transcripts were stored in unfertilized eggs near the late phase of ovarian maturation and before ovulation. Functional classification showed a total of 279 maternal immune-related transcripts classified with immune system process GO term and immune system KEGG pathway. qPCR analysis showed that transcript levels of identified maternal immune-related candidate genes were dynamically modulated during development and early ontogeny of M. anguillicaudatus. Taken together, this study could not only provide knowledge on the protective roles of maternal immune-related genes during early life stage of M. anguillicaudatus but could also be a valuable transcriptomic/genomic resource for further analysis of maternally provisioned genes in M. anguillicaudatus and other related teleost fishes.

Fish skin serves as the first line of defense against a wide variety of chemical, physical and biological stressors. Secretion of mucus is among the most prominent characteristics of fish skin and numerous innate immune factors have been identified in the epidermal mucus. However, molecular mechanisms underlying the mucus secretion and immune activities of fish skin remain largely unclear due to the lack of genomic and transcriptomic data for most economically important fish species. In this study, we characterized the skin transcriptome of mud loach using Illumia paired-end sequencing. A total of 40364 unigenes were assembled from 86.6 million (3.07 gigabases) filtered reads. The mean length, N50 size and maximum length of assembled transcripts were 387, 611 and 8670 bp, respectively. A total of 17336 (43.76%) unigenes were annotated by blast searches against the NCBI non-redundant protein database. Gene ontology mapping assigned a total of 108513 GO terms to 15369 (38.08%) unigenes. KEGG orthology mapping annotated 9337 (23.23%) unigenes. Among the identified KO categories, immune system is the largest category that contains various components of multiple immune pathways such as chemokine signaling, leukocyte transendothelial migration and T cell receptor signaling, suggesting the complexity of immune mechanisms in fish skin. As for mucin biosynthesis, 37 unigenes were mapped to 7 enzymes of the mucin type O-glycan biosynthesis pathway and 8 members of the polypeptide N-acetylgalactosaminyltransferase family were identified. Additionally, 38 unigenes were mapped to 23 factors of the SNARE interactions in vesicular transport pathway, indicating that the activity of this pathway is required for the processes of epidermal mucus storage and release. Moreover, 1754 simple sequence repeats (SSRs) were detected in 1564 unigenes and dinucleotide repeats represented the most abundant type. These findings have laid the foundation for further understanding the secretary processes and immune functions of loach skin mucus.

The immunity and health status of ornamental fish is an important aspect, as they are kept in a confined environment and various stressful conditions which lead to depletion of overall colourful appearance and mortality. The carotenoids can act as immunity boosters in captive aquarium system and may be supplemented in the feed as aquarium fish have no access to natural carotenoids. The study aimed to assess the role of carotenoid on the immunity of B. dario. Marigold petal meal is an important source of carotenoids and used in experimental diets. Four immunogenes namely IL20, TLR9, TRAIL, and Nramp in B. dario were characterized and also studied for their relative expression in the kidney after feeding the fish with marigold petal meal supplemented diet. The expression pattern of the genes was compared with the fish of nature. The IL20 and Nramp gene were upregulated significantly (p < 0.05) in the fish of nature as compared to the experimental fish at the 60th day of feeding carotenoid-rich diet. But the TLR9 and TRAIL gene was upregulated significantly (p < 0.05) in experimental fish as compared to nature. The haematological parameters of fish after feeding with the experimental enriched diet for 60 days also confirmed the role of carotenoids in immunity.

Aeromonas hydrophila is a kind of zoonotic pathogen, which can cause bacterial septicemia in fish and bring huge economic losses to global aquaculture. Outer membrane proteins (Omps) are conserved antigens of Aeromonas hydrophila , which can be developed as subunit vaccines. To evaluate the protective efficacy of inactivated vaccine and recombinant outer membrane protein A (OmpA) subunit vaccine against A. hydrophila in juvenile Megalobrama amblycephala , the present study investigated the immunogenicity and protective effects of both vaccines, as well as the non-specific and specific immune response of M. amblycephala . Compared with the non-vaccinated group, both inactivated and OmpA subunit vaccines improved the survival rate of M. amblycephala upon infection. The protective effects of OmpA vaccine groups were better than that of the inactivated vaccine groups, which should be attributed to the reduced bacterial load and enhanced host immunity in the vaccinated fish. ELISA assay showed that the titer of serum immunoglobulin M (IgM) specific to A. hydrophila up-regulated significantly in the OmpA subunit vaccine groups at 14 d post infection (dpi), which should contribute to better immune protective effects. In addition, vaccination enhanced host bactericidal abilities might also attribute to the regulation of the activities of hepatic and serum antimicrobial enzymes. Moreover, the expression of immune-related genes ( SAA, iNOS, IL-1 β, IL-6, IL-10, TNF α, C3, MHC I, MHC II, CD4, CD8, TCR α, IgM, IgD and IgZ ) increased in all groups post infection, which was more significant in the vaccinated groups. Furthermore, the number of immunopositive cells exhibiting different epitopes (CD8, IgM, IgD and IgZ) that were detected by immunohistochemical assay had increased in the vaccinated groups post infection. These results show that vaccination effectively stimulated host immune response (especially OmpA vaccine groups). In conclusion, these results indicated that both the inactivated vaccine and OmpA subunit vaccine could protect juvenile M. amblycephala against A. hydrophila infection, of which OmpA subunit vaccine provided more effective immune protection and can be used as an ideal candidate for the A. hydrophila vaccine.

Control and prevention of disease is a high priority in aquaculture, and vaccination is important to prevent outbreaks. Here, poly(d,l-lactide-co-glycolic acid) (PLGA) microparticles (MPs) approximately 36μm in diameter were used to encapsulate and deliver Aeromonas hydrophila formalin-killed cells (FKC) as an antigen, and the innate and adaptive immune responses of cyprinid loaches and common carp were assessed following vaccination. The antigen was confirmed to be well encapsulated by scanning electron microscopy analysis of PLGA MP sections. Blood and head kidney specimens were collected and analyzed for bacterial agglutination activity and relative mRNA expression of immune-related genes (IL-1β, IL-10, TNF-α, lysozyme C, TGF-β, and IgM) at 2, 4, 6, and 8weeks post vaccination (wpv). For both fish species, the curve of antibody titer over time was shallower in the PLGA group than the FKC group. These titers in loaches and carp were very similar in the two vaccination groups until 8 and 6 wpv, respectively, but differences were subsequently noted in both species until the end of experiment. Loaches and carp were then challenged with A. hydrophila at 12 and 20 wpv, and 10 and 14 wpv, respectively, and relative survival rates were calculated. For both species, the PLGA groups demonstrated higher survival rates at all time points. Relative expression of IL-1β and TNF-α mRNA was significantly upregulated in the PLGA group at 2 and 4 wpv. Moreover, PLGA-MP vaccination increased relative mRNA levels of lysozyme C and IgM, which were significantly higher than those observed with FKC treatment at 2 wpv and 4, 6, and 8 wpv, respectively. In conclusion, PLGA-MP vaccines have the potential to induce longer and more potent immune responses than FKCs alone, and protect both cyprinid loaches and common carp with greater efficiency.