Fish & shellfish immunology最新文献

Pinctada fucata martensii is the primary pearl-producing bivalve species in China. However, immune rejection following transplantation reduces pearl formation rates and yields. Previous acetylome analysis revealed elevated acetylation levels at lysine residues K91 and K159 of the rhamnose-binding lectin (PmRBL) in gill tissue after implantation. To investigate its immunoregulatory role, a recombinant mutant protein (rPmKQL), mimicking acetylation at these sites, was generated through site-directed mutagenesis. Both wild-type rPmRBL and rPmKQL were injected into nucleus-transplanted P. f. martensii, followed by assessment of immune-related gene expression, immune enzyme activities, and gill cell apoptosis. In vitro, we evaluated the antibacterial and agglutination activities of rPmKQL. The acetylation mimic significantly reduced PmRBL transcript levels and altered the time-dependent expression of NF-κB pathway genes (IRAK-1, TRAF-3, NF-κB, and IKK) and inflammatory cytokines (TNF-α and IL-17). Furthermore, rPmKQL significantly enhanced the activities of superoxide dismutase (SOD), catalase (CAT), acid phosphatase (ACP), alkaline phosphatase (AKP), and total antioxidant capacity (T-AOC) at 12 h post-injection, and increased AKP activity at later stages, suggesting improved antioxidant and immune capabilities. Both proteins induced gill cell apoptosis, with no significant difference between the two groups. In vitro, rPmKQL exhibited enhanced antibacterial activity against Staphylococcus aureus and stronger agglutination activity against Pseudomonas aeruginosa and S. aureus, but reduced activity against certain Gram-negative bacteria. Collectively, these results suggest that acetylation at K91/K159 modulates PmRBL function in P. f. martensii, providing mechanistic insight into immune regulation and informing strategies for breeding disease-resistant bivalves.

Viral RNA diseases, notably Viral Nervous Necrosis (VNN), routinely produce very high losses (≈80-100% mortality) in cultured groupers (Epinephelus sp.), driving major economic impacts across Asia. Conventional vaccination has been unreliable, largely because of antigen instability and suboptimal, short-lived immunity. Nanovaccine platforms-here, Chlorella-derived proteins augmented with silver nanoparticles (AgNPs)-offer a route to better antigen delivery and immune modulation in aquatic hosts. This study evaluated the efficacy of an AgNPs- Chlorella vulgaris protein nanovaccine in VNN-challenged groupers to enhance growth, hematology, survival, and tissue integrity. Eight cohorts were examined: two controls (K-, K+) and six dosing regimens (T1-T6). Fish received the nanovaccine by injection, followed by viral challenge. Key endpoints included hematological indices (RBC, WBC, hemoglobin, hematocrit, mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC)), histopathology of intestine and kidney, and flow-cytometric profiling of CD4, CD8, IgM, growth (length, weight), survival rate (SR), and relative percent survival (RPS). Nanovaccination significantly enhanced performance and immunity. The T3 dose yielded the best outcomes, with body length 12.73 ± 0.87 cm and weight 18.00 ± 1.50 g, SR = 90% and RPS = 82%. Hematology improved (RBC 2.52 × 10⁶ cells/μL, WBC 1.76 × 10³ cells/μL, hemoglobin 7.4 g/dL, MCV 102 μm³, MCHC 26.4%). Immune readouts showed CD4⁺ 13.44%, CD8⁺ 8.48%, and IgM 4.25%. Fish tissue showed milder lesions (≤25% damage) compared to those infected with severe injury virus (>45%). The Nanovaccine supports its potential as environmentally friendly and long-lasting means of protecting marine aquaculture from disease. This work supports the objectives of SDG 14, emphasizing the conservation of life below water.

Glutathione S-transferases (GSTs) constitute a superfamily of multifunctional detoxification isoenzymes that play essential roles in innate immunity. In this study, a μ-class GST gene was identified from Scapharca broughtonii (designated SbGSTμ) using the RACE approach. The full-length cDNA of SbGSTμ is 1040 bp and encodes a cytosolic protein of 215 amino acids. Sequence analysis revealed that SbGSTμ contains conserved structural features characteristic of the μ-class GST family, including an N-terminal thioredoxin-like domain with glutathione (GSH)-binding sites and a C-terminal domain harboring substrate-binding sites. Tissue distribution analysis showed that SbGSTμ is ubiquitously expressed across all examined tissues, with the highest expression level detected in the foot. Upon challenge with Staphylococcus aureus or Vibrio anguillarum, or exposure to Cu²⁺, SbGSTμ mRNA expression was significantly upregulated compared to controls. Recombinant SbGSTμ protein was successfully expressed in Escherichia coli, purified, and functionally characterized. The enzyme exhibited optimal activity at temperatures between 30 °C and 40 °C and at pH 7.4. Furthermore, benzo [a]pyrene exposure assays demonstrated the detoxification capacity of SbGSTμ through a significant reduction in 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels. Collectively, these findings support the classical role of GSTs in xenobiotic detoxification while providing evidence for their involvement in immunological host defense mechanisms in the ark shell S. broughtonii.

Bacterial disease is the principal threat to farmed Litopenaeus vannamei, and outbreaks inflict heavy economic losses. Understanding the immune response mechanisms triggered by distinct pathogen-associated molecular patterns (PAMPs) will furnish a theoretical foundation and technical support for the precise prevention and control of bacterial diseases. In this study, three experimental groups were established and injected with lipopolysaccharide (LPS), peptidoglycan (PGN), or phosphate-buffered saline (PBS), respectively. The results showed that both the two PAMPs damaged the hepatopancreas, midgut, muscle, and gills, with the hepatopancreas suffering more severe lesions in the LPS group. In hepatopancreas homogenates, LPS stimulation significantly increased alkaline-phosphatase (AKP) and lysozyme (LZM) activities and significantly reduced total antioxidant capacity (T-AOC), whereas PGN stimulation significantly elevated superoxide dismutase (SOD) activity. In the hepatopancreas, 1253 and 182 differentially expressed genes (DEGs) were detected post LPS and PGN stimulation, respectively. Further analysis on DEGs showed that LPS mainly activated innate immune defenses, whereas PGN tended to regulate metabolism and cellular repair to avoid excessive immunity. Overall, histopathological observations in the hepatopancreas, midgut, muscle, and gills, together with hepatopancreatic immune-enzyme activities and hepatopancreas transcriptomic profiling, indicate that LPS strongly induces innate immunity and oxidative stress, causing more severe tissue damage, while PGN elicits a more conservative immune modulation that balances the immune response and tissue protection, thereby lessening overall injury.

Carp edema virus (CEV) causes koi sleepy disease (KSD), a severe disease targeting the gills of common carp, which is associated with gills dysfunction and mortality. In this study, we employed a combination of proteomics and clinical, histopathological, physiological, and transcriptional analyses to characterize the alterations induced by the virus in the gills proteome of experimentally infected common carp. Gills collected at the peak of the disease showed high viral loads and marked epithelial hyperplasia with interlamellar occlusion, leading to severe hyponatraemia and hyperammonaemia. Two-dimensional differential in-gel electrophoresis (2D-DIGE) revealed 91 differentially abundant protein spots, 85 of which were identified by mass spectrometry. Interferon-stimulated and innate effector proteins, including Mx, Mx2, Gig1, Trim21, lysozyme C and apolipoprotein A1, were strongly upregulated, together with heat shock proteins and chaperonins, which are indicative of cellular stress and enhanced protein folding. Furthermore, the upregulation of Mmp13 and Tgm1 may be linked to abnormal cell proliferation within the gills lamellae and contribute to the characteristic tissue occlusion. In contrast, antioxidant enzymes, cytoskeletal regulators and metabolic enzymes involved in energy and xenobiotic metabolism were suppressed. Ingenuity Pathway Analysis revealed networks associated with protein quality control, inflammation, nucleic acid metabolism, and impaired chemotaxis. These molecular changes are consistent with gills hyperplasia and interlamellar space occlusion, providing a mechanistic link between viral infection and tissue dysfunction. Overall, our results define a CEV-specific gills response that explains the characteristic pathology and functional impairment of the gills observed in KSD.

Hepcidins, a family of cysteine-rich antimicrobial peptides, play a pivotal role in the innate immune defense of teleosts. This study aimed to characterize the antibacterial activity, mechanism of action, and in vitro/in vivo protective efficacy of the mature peptide mSshep 1 derived from hepcidin 1 of Sebastes schlegelii (Sshepcidin 1), and to verify the functional role of Sshepcidin 1 via RNA interference (RNAi). Time-kill kinetic assay showed that mSshep 1 exhibited rapid and broad-spectrum bactericidal activity against Staphylococcus aureus, Edwardsiella piscicida, Listonella anguillarum, and Aeromonas hydrophila, with complete eradication of all tested strains within 120 min. Thermal stability analysis revealed that mSshep 1 retained significant antibacterial activity against most pathogens even after treatment at 100 °C for 30 min, and circular dichroism spectroscopy confirmed only minor conformational changes without substantial secondary structure alterations. Mechanistically, mSshep 1 exerted bactericidal effects by disrupting bacterial membrane integrity, as evidenced by increased propidium iodide penetration, elevated intracellular reactive oxygen species levels, and obvious morphological damage observed via scanning and transmission electron microscopy. In vitro, mSshep 1 showed no cytotoxicity to HEK293T, EPC, and SSI cells at concentrations up to 128 μM, and effectively eliminated intracellular E. piscicida in SSI cells while upregulating the expression of immune-related genes. In vivo experiments demonstrated that pre-injection of mSshep 1 (1, 2, 5 μg/g body weight) significantly improved the survival rate of S. schlegelii challenged with E. piscicida and reduced bacterial loads in the liver, spleen, and head kidney. RNAi analysis showed that si-Sshep1-1 (2 μg/g body weight) stably silenced Sshepcidin 1 expression in multiple tissues, and Sshepcidin 1 knockdown significantly increased bacterial loads in E. piscicida-challenged fish and disrupted the expression balance of immune-related genes. Collectively, these findings confirm that mSshep 1 is a promising candidate for the development of novel antibacterial agents in aquaculture, and Sshepcidin 1 plays a crucial role in the innate immune defense of S. schlegelii against bacterial infections.

Chemokine ligand 20 (CCL20) is a key mediator of inflammatory and homeostatic responses in mammal, acting through its receptor CC chemokine receptor 6 (CCR6). However, its functional roles in teleost fish remain poorly defined. In this study, we identified two CCL20-like genes (PaCCL20l1 and PaCCL20l2) from ayu (Plecoglossus altivelis). Phylogenetic analysis confirmed their classification within the CCL20 subgroup. Tissue distribution analysis revealed ubiquitous expression of both genes, which were significantly upregulated in multiple tissues (liver, spleen, head kidney, gill, skin, and intestine) following infection with Vibrio anguillarum, albeit with distinct expression profiles. We produced the recombinant mature peptide of PaCCL20ls (rPaCCL20l1 and rPaCCL20l2) and generated specific antibodies. Functional assays demonstrated that both rPaCCL20l1 and rPaCCL20l2 exhibit chemotactic, and anti-apoptosis activities, but with differing cell specificities. rPaCCL20l1 attracted lymphocytes and neutrophils and inhibited lymphocyte apoptosis. In contrast, rPaCCL20l2 attracted monocytes/macrophages (MO/MΦ), lymphocytes, and neutrophils, and suppressed apoptosis in both MO/MΦ and lymphocytes. Crucially, neutralizing PaCCR6 completely abolished all effects of both rPaCCL20l1 and rPaCCL20l2. In conclusion, our findings demonstrate that PaCCL20l1 and PaCCL20l2 play pivotal but distinct roles in ayu immunity by modulating leukocyte recruitment and survival via PaCCR6 pathway.

In recent years, multiple pathogenic organisms have been isolated from diseased fish, highlighting the need for combination vaccines to prevent infections caused by various pathogens. This study presents a comparative evaluation of heat- and formalin-inactivated bivalent vaccines targeting Aeromonas jandaei and Edwardsiella tarda tested in Oscar fish (Astronotus ocellatus). Formalin and heat-inactivated vaccines were prepared after optimizing the inactivation conditions. A concentration of 0.3% formalin applied overnight was optimal for formalin inactivation, while heating at 70 °C for 1 h was suitable for heat inactivation. After a combined bacterial challenge 12 weeks from the primary dose, the relative percentage survival (RPS) was 92.6% for the formalin-inactivated vaccine (VFI) and 82.5% for the heat-inactivated vaccine (VHI), indicating strong protective efficacy. Biochemical analysis showed that the vaccinated groups had significantly lower glutamic oxaloacetic transaminase (GOT) and glutamate pyruvic transaminase (GPT) levels post-infection, while oxidative stress assessment revealed enhanced antioxidant defence in the vaccinated fish, with catalase activity peaking on the 2nd dpc in the VFI group. Immune-related gene expression analysis (IgM-H, IL-8, MHC-IIα, and CC-1) revealed significantly higher expression levels in the vaccinated groups than in the control group. Additionally, specific serum antibody against both A. jandaei and E. tarda were significantly elevated in the vaccinated groups compared to those in the controls. The highest anti-A. jandaei antibodies occurred in week 5 after the final booster in both the VFI and VHI groups, whereas anti-E. tarda antibodies peaked in week 4 after the final booster, underscoring the vaccine's robust humoral immune response. Overall, this study demonstrated that both inactivated bivalent vaccines (VFI and VHI) effectively stimulated immune responses in Oscar fish. However, VFI exhibited a slightly superior performance compared to VHI in terms of survival, protective efficacy, and enhanced immune responses against both A. jandaei and E. tarda.

The survival ability of artificially selected ark shells (Scapharca subcrenata) and wild populations was assessed under different combinations of temperatures (25, 27, 29, and 31 °C) and Vibrio anguillarum immersion concentrations (0, 3 × 10⁸, 6 × 10⁸, 9 × 10⁸, 12 × 10⁸, 15 × 10⁸, and 18 × 10⁸ CFU/mL). Significant differences in mortality rates between the two ark shell populations were observed across most temperature and immersion concentration combinations, with only a few exceptions. The gene expression profiles of selected and wild ark shells were analyzed through transcriptome sequencing following exposed to 1 × 10⁸ CFU/mL of V. anguillarum strain PL2. Gene Ontology (GO) functional classification of the 1558 differentially expressed genes (DEGs) common to the three comparisons revealed that 'cellular process', 'membrane' and 'binding' were the most enriched terms across the biological process, cellular component, and molecular function categories in the D (treated wild ark shells) _vs_C (Untreated wild ark shells were used as the control group) and KD (treated selectively bred ark shells) _vs_D groups. COG analysis reveals that the 'Energy Production and Conversion' category is significantly downregulated in the KD_vs_D comparison compared to the D_vs_C and C_vs_KD comparisons. Heatmap visualization of DEGs across C, D, and KD groups showed numerous genes exhibiting inverse expression trends among the groups. Among the top 10 KEGG pathways identified across the three comparison groups, no pathways were shared between the KD_vs_D group and the other two comparisons, suggesting that these pathways may be related to the resistance of ark shells to V. anguillarum. Our study indicates that ark shell populations selected under high-intensity environmental stress exhibit greater tolerance to elevated temperatures and V. anguillarum. This further demonstrates that selecting ark shells under stress conditions may enhance their survival rates in aquaculture. These findings will deepen the understanding of bivalve adaptability to environmental changes and aid in the advancement of shellfish aquaculture practices.