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Viral haemorrhagic septicaemia virus (VHSV) is one of the highly pathogenic virus, which causes viral haemorrhagic septicaemia disease in both marine and freshwater fish. Micro RNA-155 (miRNA-155) is a multifunctional small non-coding RNA and it involves regulation of immune responses during viral infection. In this study, dre-miR-155 mimics were encapsulated into chitosan nanoparticles (CNPs). Resulted encapsulated product (miR-155-CNPs) was investigated for its immunomodulation role in zebrafish during experimentally challenged VHSV infection. Successful encapsulation of dre-miR-155 mimics into CNPs was confirmed through average nanoparticle (NPs) size (341.45 ± 10.00 nm), increased encapsulation efficiency percentage (98.80%), bound dre-miR-155 with chitosan, sustained release in vitro (up to 40%), and the integrity of RNA. Overexpressed miR-155 was observed in gills, muscle, and kidney tissues (5.42, 19.62, and 140.72-folds, respectively) after intraperitoneal delivery of miR-155-CNPs into zebrafish upon VHSV infection (miR-155-CNPs + VHSV). The miR-155-CNPs + VHSV infected fish had the highest cumulative survival (85%), which was associated with low viral copy numbers. The miR-155-overexpressing fish showed significantly decreased expression of ifnγ, irf2bpl, irf9, socs1a, il10, and caspase3, compared to that of the miR-155 inhibitor + VHSV infected fish group. In contrast, il1β, tnfα, il6, cd8a, and p53 expressions were upregulated in miR-155-overexpressed zebrafish compared to that of the control. The overall findings indicate the successful delivery of dre-miR-155 through miR-155-CNPs that enabled restriction of VHSV infection in zebrafish presumably by modulating immune gene expression., Competing Interests: Declaration of competing interest The authors declared that no conflicts of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Streptococcus parauberis is a pathogenic gram-positive bacterium that causes streptococcosis infection in fish. Since S. parauberis is becoming resistant to multiple antibiotics, the development of alternatives, such as antimicrobial peptides, has gained great attention. Octominin, derived from the defense protein of Octopus minor, showed a significant antimicrobial activity against multidrug resistance S. parauberis, with a minimum inhibitory concentration (MIC) and a minimum bactericidal concentration (MBC) of 50 and 100 μg/mL, respectively. Furthermore, time-kill kinetics, agar diffusion, and bacterial viability assays confirmed the concentration-dependent antibacterial activity of Octominin against S. parauberis. Field emission scanning electron microscopy analysis showed morphological and ultra-structural changes in S. parauberis upon Octominin treatment. Moreover, Octominin treatment demonstrated changes in membrane permeability, induced reactive oxygen species (ROS), and its binding ability to genomic DNA, suggesting its strong bactericidal activity with multiple modes of action. We confirmed the inhibition of biofilm formation and the eradication of existing biofilms in a concentration-dependent manner. Additionally, Octominin on S. parauberis at transcriptional level exhibited downregulation of membrane formation (pgsA and cds1), DNA repairing (recF), biofilm formation (pgaB and epsF) genes, while upregulation of ROS detoxification (sodA) and DNA protecting (ahpF) related genes. An in vivo study confirmed a significantly (P < 0.05) higher relative percentage survival in Octominin-treated larval zebrafish exposed to S. parauberis (93.3%) compared to the control group (20.0%). Collectively, our results confirm that Octominin could be a potential antibacterial and anti-biofilm agent against S. parauberis., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

MicroRNAs (miRNAs) constitute a group of small non-coding RNAs (~22 nucleotides) and one of their main functions is to regulate the immune responses. Gram-positive bacterium, Streptococcus parauberis is the main causative agent of "Streptococcosis" in wide range of fish species. In this study, we performed high throughput sequencing analysis to identify the miRNA profile against S. parauberis infection in the spleen of zebrafish (Danio rerio). Overall, 349 known and 151 novel miRNAs were discovered. Among them, 12 known miRNAs (dre-miR-34b, dre-miR-135a, dre-miR-200b-5p, dre-miR-146b, dre-miR-31, dre-miR-17a-3p, dre-miR-222a-3p, dre-miR-731, dre-miR-301b-3p and dre-miR-30a-3p) and 9 novel miRNAs were differentially expressed (DE) in the spleen of S. parauberis challenged zebrafish. The identified 12 DE miRNAs were predicted to regulate 721 target genes. We confirmed the miRNA expression results by validating selected known and novel DE miRNAs using qRT-PCR. Gene Ontology (GO), Kyoto Encyclopedia of Genes (KEGG) pathway analysis and miRNA-mRNA interactions implies that specific target genes of DE miRNAs are associated with immune responses. The enriched pathways included Toll-like receptor (TLR), C-type lectin, NOD-like receptor, and RIG-I-like receptor signaling pathways, etc. Especially, dre-miR-200b-5p, dre-miR-146b, dre-miR-731, dre-miR-222a-3p, and dre-miR-34b were able to target potential immune-related genes such as il10, irak1, traf6, hspa8 and ikbke upon S. parauberis challenge. Thus, overall results could lay a foundation to understand the underlying immune regulatory role of miRNAs in response to pathogenic S. parauberis infection in teleosts., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

This study was aimed to understand the expression of miR-146a in zebrafish (Danio rerio) and its role in regulating immune responses during Aeromonas hydrophila and Edwardsiella piscicida infections. The miR-146a expression was observed from the 1-h post fertilization (hpf) stage and gradually increased up to the early larval stage of zebrafish. The ubiquitous expression of miR-146a was detected in all tested tissues, with the highest level in gills. The expression of miR-146a was significantly increased in larvae when exposed to E. piscicida infection at 24 and 48 h post exposure (hpe). Intraperitoneally (i.p.) injected A. hydrophila and E. piscicida into adult zebrafish showed significant upregulation of miR-146a in gills. Furthermore, immune-related genes, toll-like receptor, tlr-4, transducing signaling pathway molecules, traf-6 and myd88 (bacteria-infected larvae and adults), transcription factor relA and mcp-1b (bacteria-infected adults), pro-inflammatory, il-6 (A. hydrophila-exposed larvae) and mmp-9 (bacteria-exposed larvae) were significantly repressed. In contrast, il-1β, tnf-α, cxcl-18b, and ccl-34a.4 were induced in both bacteria-challenged larvae and adults. Based on the results, it is suggested that endogenous miR-146a could act as an infection inducible miRNA in zebrafish upon A. hydrophila and E. piscicida infections; also, it could potentially regulate the immune responses in zebrafish.

Purified porcine gastric mucin (PGM) is an alternative biomaterial to native mucin which displays multifunctional properties for exploring a wide range of biomedical applications. The present study evaluated the in vitro (RAW 264.7 macrophage cells) and in vivo (zebrafish embryos and larvae) bioactivities of PGM. The median lethal concentration (LC 50 ) of PGM was 197.9 µg/mL for embryos, while it was non-toxic to RAW 264.7 cells, even at 500 µg/mL. Following PGM exposure (100 µg/mL), a higher embryo hatching rate (59.9%) was observed at 48 h post fertilization, compared to the control (30.6%). Protective effects of PGM from pathogenic Aeromonas hydrophila were demonstrated by high larvae survival rates of 85.0% and 94.0% at 50 and 100 μg/mL of PGM exposure, respectively. Heat tolerance effect of PGM (50 and 100 µg/mL) on larvae (40 °C for 48 h) was confirmed by 75% and 100% of survival rates, respectively. Additionally, PGM reduced the A. hydrophila -induced reactive oxygen species (ROS) generation in larvae. The qRT-PCR results in PGM exposed larvae exhibited induction of immune-related genes ( tlr5a and tlr5b , myd88 , c-rel , il1β, tnf-α , il6, il10 , cxcl18b , ccl34a.4 , defbl1 , hamp , ctsd, muc2.1 , muc5.1 , muc5.2 , and muc5.3 ), stress response ( hsp70 , hsp90aa1.1 , and hsp90ab1 ), and antioxidant genes ( cat and sod1 ). Moreover, our results revealed that PGM involved in the regulation of transcriptional gene induction increases Hsp90 protein in the zebrafish larvae. Furthermore, upregulation of Il6, Il10, Tnfα, Ccl3, Defa-rs2, Defa21 and Camp and antioxidant genes ( Sod2 and Cat ) were observed in PGM-exposed RAW 264.7 cells. Overall findings confirmed the activation of immune responses, disease resistance against pathogenic bacteria, heat tolerance, and ROS-scavenging properties by PGM, which may provide insights into new applications for PGM as a multifunctional immunomodulator.

This study evaluated the modulation of gut microbiota, immune responses, and gut morphometry in C57BL/6 mice, upon oral administration of S. maxima -derived modified pectin (SmP, 7.5 mg/mL) and pectin nanoparticles (SmPNPs; 7.5 mg/mL). Metagenomics analysis was conducted using fecal samples, and mice duodenum and jejunum were used for analyzing the immune response and gut morphometry, respectively. The results of metagenomics analysis revealed that the abundance of Bacteroidetes in the gut increased in response to both modified SmP and SmPNPs (75%) as compared with that in the control group (66%), while that of Firmicutes decreased in (20%) as compared with that in the control group (30%). The mRNA levels of mucin, antimicrobial peptide, and antiviral and gut permeability-related genes in the duodenum were significantly ( p < 0.05) upregulated (> 2-fold) upon modified SmP and SmPNPs feeding. Protein level of intestinal alkaline phosphatase was increased (1.9-fold) in the duodenum of modified SmPNPs feeding, evidenced by significantly increased goblet cell density (0.5 ± 0.03 cells/1000 µm 2 ) and villi height (352 ± 10 µm). Our results suggest that both modified SmP and SmPNPs have the potential to modulate gut microbial community, enhance the expression of immune related genes, and improve gut morphology., Competing Interests: The authors declare that they have no conflict of interest.

In this study, we demonstrate the enhanced disease resistance and positive immunomodulation of novel pectin isolated from Spirulina maxima (SmP) in zebrafish model. Zebrafish larvae exposed to SmP had significantly (p < 0.05) higher cumulative percent survival (CPS) at 25 (44.0%) and 50 μg/mL (67.0%) against Edwardsiella piscicida compared to the control. However, upon Aeromonas hydrophila challenge, SmP exposed larvae at 50 μg/mL had slightly higher CPS (33.3%) compared to control group (26.7%). SmP supplemented zebrafish exhibited the higher CPS against E. piscicida (93.3%) and A. hydrophila (60.0%) during the early stage of post-infection (<18 hpi). qRT-PCR results demonstrated that exposing (larvae) and feeding (adults) of SmP, drive the modulation of a wide array of immune response genes. In SmP exposed larvae, up-regulation of the antimicrobial enzyme (lyz: 3.5-fold), mucin (muc5.1: 2.84, muc5.2: 2.11 and muc5.3: 2.40-fold), pro-inflammatory cytokines (il1β: 1.79-fold) and anti-oxidants (cat: 2.87 and sod1: 1.82-fold) were identified. In SmP fed adult zebrafish (gut) showed >2-fold induced pro-inflammatory cytokine (il1β) and chemokines (cxcl18b, ccl34a.4 and ccl34b.4). Overall results confirmed the positive modulation of innate immune responses in larval stage and it could be the main reason for developing disease resistance against E. piscicida and A. hydrophila. Thus, non-toxic, natural and biodegradable SmP could be considered as the potential immunomodulatory agent for sustainable aquaculture., (Copyright © 2019 Elsevier Ltd. All rights reserved.)