Teleost leukocyte immune-type receptors (LITRs) are a large family of immunoregulatory receptor types that share distant evolutionary relationships with various mammalian immunoregulatory receptor types, including members of the Fc receptor-like family. Carassius auratus (Ca)-LITR3 is a putative stimulatory LITR-type expressed by goldfish immune cells. To investigate the regulatory functions of this putative activating receptor in goldfish, a polyclonal antibody (anti-CaL3.D1 pAb) was generated, which targets an epitope within the D1 domain of CaLITR3. Previous research showed that anti-CaL3.D1 pAb strongly stained the surface of goldfish primary kidney neutrophils (PKNs) and crosslinking of CaLITR3 with anti-CaL3.D1 pAb activated ERK1/2 and p38 in the mitogen-activated protein kinase (MAPK) pathway in PKNs. In the present study, we show that activation of CaLITR3 significantly upregulates transcription of the pro-inflammatory cytokine gene il1β in goldfish PKNs, indicating that beyond ERK1/2 and p38 signaling, CaLITR3 may induce functional effector responses in goldfish PKNs. To further investigate the roles of CaLITR3 as an activator of PKN antimicrobial responses, we examined its ability to trigger NETosis. This process represents a critical antimicrobial mechanism of neutrophils, where activated neutrophils release neutrophil extracellular traps (NETs) composed of decondensed chromatin mixed with granular enzymes to immobilize and kill invading microbes. Since triggering of CaLITR3 with anti-CaL3.D1 pAb activated MAPK pathway proteins in goldfish PKNs, we hypothesized that CaLITR3 is an inducer of NETosis formation in goldfish PKNs. To specifically examine goldfish PKN NETosis, we optimized NETosis assays for the quantitative and visual detection of NET formation in goldfish. Our data showed that goldfish PKNs release NETs in response to both receptor-independent (PMA, calcium ionophore) and receptor-mediated (Zymosan A, heat-killed Aeromonas salmonicida) stimuli. We also showed that CaLITR3 engagement triggers goldfish PKN NET release, implicating this LITR-type as an activator of neutrophil antimicrobial responses in fish.

This study evaluated whether polyethylene glycol (PEG) formulation could prolong the biological efficacy of dietary tyramine (TA) and provide sustained oral immunostimulation in Litopenaeus vannamei by examining growth performance, immune responses, disease resistance, and biogenic amine regulation during a 120-day feeding trial. Shrimp were assigned to four dietary treatments: basal diet (BD), TA, PEG, and TA+PEG. Results showed that TA significantly enhanced growth performance, exhibiting higher weight gain than all other treatment groups at 56 days and improved feed efficiency at 56 and 120 days. At 120 days, both TA and TA+PEG groups showed significantly greater weight gain than the BD group. Immune analyses revealed that TA induced rapid activation of hemocyte parameters, phenoloxidase activity, and respiratory burst during the early feeding period (7-28 days), whereas TA+PEG maintained elevated immune responses at later stages (56-120 days), indicating prolonged immunostimulation. In disease challenge tests against Vibrio alginolyticus, both TA and TA+PEG improved survival at 28 days, but only TA+PEG maintained significantly higher survival at 56 days compared with all other groups, demonstrating superior long-term protection. Biogenic amine analysis showed that TA-fed shrimp had significantly lower tyramine levels than the BD group at 28 days, whereas the TA+PEG group exhibited significantly higher tyramine levels than the BD group, while octopamine levels were consistently elevated in both TA and TA+PEG groups at 28 and 56 days. Overall, PEG formulation prolonged the oral biological efficacy of dietary tyramine, resulting in sustained immune activation and enhanced disease resistance against to V. alginolyticus.

The mandarin fish (Siniperca chuatsi) is an economically important freshwater species in China, with an optimal growth temperature ranging from 18 to 25°C. Fluctuations in water temperature, however, can increase its susceptibility to disease. To compare the immunoregulatory response characteristics of mandarin fish under high- and low-temperature conditions and to elucidate their shared and distinct mechanisms, 20°C was used as the control, and the temperature was gradually adjusted from 20°C to 10°C and 30°C at a rate of 2°C/day; after reaching the target temperatures, fish were maintained for an additional 7 days as experimental groups. Liver transcriptomic profiles were analyzed, key genes involved in significantly enriched pathways were examined. KEGG enrichment analysis showed that both high- and low-temperature treatments commonly affected the Cytokine-cytokine receptor interaction pathway, but exhibited distinct regulatory characteristics. High temperature mainly induced upregulation of chemokines (CCL, CXCL) and pro-inflammatory cytokines (e.g., il1β, tnf-α), whereas these responses were attenuated under low temperature. In addition, several cytokine receptor genes (including IL and TNF receptor family members) exhibited opposite expression trends between treatments. RT-qPCR validation confirmed the transcriptomic patterns, with significant upregulation of tnf-α, il1β, and il10 under high temperature, while no marked changes were observed under low temperature. Furthermore, high temperature significantly enriched IL-17 signaling and leukocyte chemotaxis pathways, indicating enhanced inflammatory activation and immune cell recruitment, whereas low temperature was mainly associated with monocyte migration and metabolic adjustment. PPI network analysis further supported distinct regulatory architectures of cytokine signaling under different thermal conditions. Overall, high temperature (30°C) predominantly induces activation of inflammatory responses, whereas low temperature (10°C) tends to regulate metabolism and maintain immune homeostasis. This study provides new insights into the molecular mechanisms underlying immune responses of fish adapting to different environmental temperatures.

The interleukin-1 (IL-1) family is a central regulator of vertebrate inflammation. While eleven IL-1 family members have been identified in mammals, only three members, IL-1β, IL-18, and a teleost-specific novel IL-1 family member (nIL-1F), have been reported in fish to date. Here, we report a previously uncharacterized IL-1 family cytokine (termed nIL-1Fb) in cyprinid fish. The gene encoding nIL-1Fb is tandemly linked with the nIl-1f gene and is phylogenetically related to the nIl-1f, suggesting their common ancestral origin. We characterized the expression and function of nIL-1Fb in grass carp (Ctenopharyngodon idella), a major aquaculture species in Asia. Following infection with grass carp reovirus type II (GCRV-II), the nIl-1fb expression was markedly upregulated in the gills and hindgut. Immunofluorescence analyses revealed that nIL-1Fb was produced by virus infected cells and upregulated in infected tissues. Furthermore, nIL-1Fb co-localized with macrophage colony-stimulating factor receptor (MCSFR), indicating that monocytes/macrophages (Mo/Mφ) are the major source of nIL-1Fb. Functionally, nIL-1Fb not only induced the expression of pro-inflammatory genes (Tnf-α3, Il-8, and Inos) but also enhanced the phagocytic activity of Mo/Mφ. However, nIL-1Fb exhibited a much weaker pro-inflammatory activity than IL-1β. Taken together, our findings identify nIL-1Fb as a cyprinid-specific pro-inflammatory cytokine, offering novel insights into the evolution and functional diversification of the IL-1 family in teleost fish.

Antiviral immunity is a conserved biological process that is critical for host survival, although its strategies vary among taxa. The silkworm, Bombyx mori, serves as a unique invertebrate model for studying both conserved and species-specific antiviral mechanisms, with implications for fundamental biology and sericulture. Here, we integrate and compare classical signalling pathways, epigenetic regulation, and RNA-mediated defenses to demonstrate how these mechanisms coordinate to regulate antiviral immune responses. Comparative analyses reveal conserved processes such as RNA interference, alongside insect-specific adaptations, such as melanization, which differ from vertebrate interferon responses. We also discuss emerging technologies, including live-cell imaging, single-cell and spatial transcriptomics, and CRISPR-based functional genomics, which are revolutionizing the study of host-virus interactions at unprecedented resolution. Translational opportunities consist of engineering virus-resistant silkworm strains and developing RNA-based antiviral strategies. In conclusion, we present an integrated network model linking signalling, RNA, and epigenetic regulation across species, providing a conceptual framework to guide mechanistic studies, cross-species comparisons, and the development of next-generation antiviral strategies.

Cetaceans are mammals that have adapted to aquatic environments, but their immunological characteristics are not well understood. In this study, we performed comparative transcriptomic analyses of major immune-related tissues (thymus, spleen, liver, and kidney) from post-mortem samples of stranded cetaceans in Korea, including the finless porpoise, fetal minke whale and Risso's dolphin. For cross-taxa comparisons, we retrieved publicly available transcriptomic datasets from terrestrial mammals (human and mouse), teleosts (zebrafish, common carp, and turbot), birds (chicken), reptiles (green sea turtle and loggerhead sea turtle), and cartilaginous fish (shark). We analyzed immune-related gene expression patterns using a unified KEGG-based annotation framework. Despite the post-mortem nature of the samples, tissue-specific transcriptional characteristics were maintained, with the thymus showing the widest variety of immune-related genes. Consequently, our comparative transcriptomic analyses focused on thymic tissues across the different animal taxa. We found that pathways related to antigen processing and presentation, as well as T cell receptor signaling, were relatively downregulated. In contrast, the complement/coagulation cascades and platelet activation pathways exhibited more prominent transcriptional representation. Within the complement system, components of the lectin and alternative pathways were more pronounced than those of the classical pathway, which may be associated with constant exposure to diverse waterborne microorganisms inherent to mammalian life in aquatic environments. These findings highlight immune-physiological features relevant to mammalian life in aquatic environments and lay the groundwork for future standardized comparative studies.

Extracellular vesicles (EVs), as antimicrobial factors, load a variety of immune-related molecules, play a complex and critical role in host-pathogen interactions. The regulatory mechanism of how EVs respond to external stimuli by regulating immune regulatory elements in the innate immune response of marine invertebrates remains unresolved. To address this, we conducted a global transcriptomic analysis of hemocytes in Chlamys farreri stimulated by EVs derived from different sources. Transcriptomic analysis identified 892 specifically upregulated and 246 specifically downregulated genes in the group of scallops injected with EVs obtained after LPS stimulation (group S) relative to the group of scallops injected with PBS buffer (group P). Functional annotation analysis revealed that the differentially expressed genes (DEGs) participate in critical immune signaling pathways, including the NOD-like receptor, RIG-I-like receptor, and HIF-1 signaling pathways. Among the DEGs unique to the group S, we identified canonical genes linked to autophagy, apoptosis, and immune responses, such as cell division control protein 42 homolog (CDC42), dual specificity protein phosphatase 1 (DUSP1), TNF receptor-associated factor 3 (TRAF3), Caspase 3 (CASP3), serine/threonine-protein kinase ULK3 (ULK3), and Myeloid differentiation primary response protein MyD88-3 (MyD88-3). Protein-protein interaction (PPI) network analysis pointed to ADP-ribosylation factor (CDKN2A), phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase (PTEN), CDC42, CASP3, and the downregulated protein heat shock protein 90 (HSP90) as prominent hub DEGs involved in the regulation of innate immunity. Temporal analysis of selected immune DEGs further demonstrated the immunomodulatory role of EVs in Chlamys farreri. Our study establishes that EVs are essential for regulating innate immunity in marine invertebrates, offering novel insights for shellfish disease prevention and control.

This study evaluated the effects of dietary supplementation with Trachyspermum ammi (ajwain) seed extract on growth performance, immune response, physiological status, and disease resistance in the giant freshwater prawn (Macrobrachium rosenbergii) against Vibrio parahaemolyticus. Juvenile prawns (2.1 ± 0.25 g) were fed diets supplemented with three levels of T. ammi extract (0%, 0.025%, and 0.05%) for 8 weeks, followed by a bacterial challenge with V. parahaemolyticus (10[6] CFU/mL) and a 14-day observation period. Prawns receiving ajwain-supplemented diets showed improved growth performance, including higher final weight, weight gain, and specific growth rate, together with improved feed utilization efficiency. Immune-related parameters, including total hemocyte count, hemolymph clotting time, prophenoloxidase activity, respiratory burst activity, superoxide anion production, and superoxide dismutase activity, were significantly enhanced compared to the control group, although responses varied among parameters and sampling periods. Dietary inclusion of T. ammi extract modified whole body composition with increased protein content in both supplemented groups compared with the control, while the lower dose showed the highest protein deposition. The higher dose resulted in greater lipid and ash accumulation, suggesting dose-dependent differences in nutrient allocation. Digestive enzyme activities (protease and amylase) were also significantly stimulated. Following the V. parahaemolyticus challenge, extract-treated groups showed improved survival, with the higher inclusion levels demonstrating the greatest resistance to infection. These findings indicate that T. ammi seed extract has potential as an effective natural feed additive to enhance growth, immunity, and disease resistance in M. rosenbergii; however, further molecular investigations are required to clarify the underlying mechanisms.

The CXC chemokine system is fundamental to vertebrate immune defense, yet its evolutionary diversity and functional adaptations in non-model teleost species facing complex environmental stressors are largely unexplored. Here, we present a genome-wide identification of the CXC chemokine system in the economically important yellow catfish (Pelteobagrus fulvidraco), revealing 15 CXCL and 9 CXCR genes. Evolutionary analyses revealed these genes are under strong purifying selection yet exhibit lineage-specific expansion, including teleost-specific duplicates. They displayed spatially divergent expression patterns across healthy tissues, with enrichment in immune organs such as spleen, kidney, gill, and intestine. Furthermore, these genes showed dynamic, tissue-specific transcriptional responses to hypoxia and bacterial challenge including Aeromonas hydrophila and Aeromonas veronii, highlighting their role in environmental adaptation. Most importantly, functional investigation of a novel gene, CXCL18b, unveiled its essential apoptotic role during hypoxic stress. siRNA-mediated knockdown of CXCL18b led to the altered expression of apoptosis-related genes (including MyD88) and decreased hepatocyte apoptosis, suggesting a potential transcriptional link between CXCL18b and the apoptotic program. This discovery not only deepens our understanding of the molecular mechanisms underlying hypoxic tolerance in fish but also provides a promising target for genetic improvement of stress-resistant aquaculture strains. Our study provides the first comprehensive resource on the CXC chemokine system in yellow catfish and establishes CXCL18b as a crucial mediator of hypoxic tolerance, offering new insights for breeding stress-resilient aquaculture species.

Photobacterium damselae subsp. Piscicida (Pdp) is characterized by its broad distribution and is capable of infecting numerous cultured fish species. A previous study revealed the epidemic of Pdp in Lateolabrax maculatus with high mortality. In the current study, comparative transcriptome analysis was carried out on the spleens of L. maculatus infected with Pdp at 6, 12, and 24 h post infection (hpi). The results demonstrated that compared with unchallenged individuals, Pdp infection resulted in 1631, 2085 and 2085 differentially expressed genes (DEGs) being detected at 6, 12 and 24 hpi, respectively. Among these, immune-related DEGs exhibited a trend of first increasing and then decreasing, particularly those involved in pattern recognition receptors, antigen processing and presentation, and inflammatory factors. In addition to terms related to immune system processes, GO enrichment analysis also identified terms related to signal transduction, metabolic processes, catalytic activity, and molecular function regulation. In KEGG enrichment analysis, numerous immune-related pathways were significantly enriched, such as the TNF signaling, NF-κB signaling, cytokine-cytokine receptor interaction, pattern recognition receptor, and natural killer cell mediated cytotoxicity. Subsequent weighted gene co-expression network analysis (WGCNA) uncovered immune-related pathways that were positively correlated at 6 and 12 hpi, and negatively correlated at 24 hpi, especially identified transcriptional trends suggesting Pdp may interfere with phagosome maturation and reduce endocytosis/autophagy-related gene expression at late infection stage. This study identified multiple immune-related genes and pathways that are dynamically regulated and potentially regulated by Pdp, which may contribute to its immune evasion and colonization in spotted sea bass. These findings provide new insights into the molecular immune mechanisms against Pdp infection and establish a basis for further studies on antibacterial defense in L. maculatus.

Limited information is available on how seasonal changes affect host immunity, hemato-biochemical status, antioxidant responses, and parasite infection in cultured fish. Therefore, this study evaluated the associations among seasonal changes, parasitic infections (Cichlidogyrus tilapiae and Centrocestus formosanus), and hemato-biochemical, antioxidant, and immune responses in Nile tilapia (Oreochromis niloticus). Fish (n = 593) and water samples were collected seasonally from Abbasa Fish Farm, El-Sharkia Governorate, Egypt. Parasite prevalence was highest in summer and lowest in winter. Infected fish showed marked hemato-biochemical changes, including reduced RBCs, hemoglobin (Hb), total protein, albumin, and globulin, whereas WBCs increased in spring, summer, and autumn but declined in winter; uric acid, glucose, ALT, and AST activities increased, particularly in winter. Antioxidant enzymes in gills and liver (SOD, CAT, and GPx) increased in spring, declined in summer, partially recovered in autumn, and reached their lowest values in winter, while malondialdehyde (MDA) levels increased in summer and winter. Immune responses, including lysozyme activity, respiratory burst activity, phagocytic activity, complement C3, and IgM, were enhanced in summer and reduced in winter. Seasonal changes also modulated the expression of C3, LZ, IL-1β, IL-8, IL-10, COX-2, TNF-α, and IFN-γ genes. Marked seasonal histological alterations were observed in the gills, liver, and spleen of infected fish, especially during winter. PCA indicated seasonal separation associated with water quality and immune variables. Generally, season-specific health management strategies are essential to reduce parasite-related losses and improve the sustainability of tilapia aquaculture.

Nervous necrosis virus (NNV) is a major viral pathogen that causes viral nervous necrosis in a wide range of fish species and poses a serious threat to global aquaculture. However, the antiviral responses of mandarin fish (Siniperca chuatsi) to NNV infection remain poorly understood. In this study, we investigated host antiviral responses using Chinese perch brain (CPB) cell line and in vivo infection models. Transcriptome sequencing of red-spotted grouper nervous necrosis virus (RGNNV)-infected CPB cells revealed extensive transcriptional reprogramming, with numerous interferon-stimulated genes (ISGs) significantly upregulated. Comparative transcriptomic analysis across multiple teleost species identified a conserved set of ISGs induced during RGNNV infection, including CMPK2, RSAD2, DHX58, IRF3, and EPSTI1. Among these genes, CMPK2 showed strong induction in both infected CPB cells and mandarin fish brain tissues. Functional assays further demonstrated that CMPK2 overexpression significantly inhibited RGNNV replication in CPB cells and was accompanied by increased expression of several interferon-related genes. These findings provide the first functional evidence that CMPK2 restricts RGNNV infection in S. chuatsi. Our study highlights a conserved interferon-responsive antiviral module in teleost fish and identifies CMPK2 as a potential antiviral effector involved in host defense against RGNNV infection.

Birnaviridae have a wide host range and can infect a variety of fish, causing significant economic losses to the aquaculture industry. Herein, we analyzed the amino acid sequence conservation and antigenic epitope distribution of the VP2 protein to evaluate its suitability as a vaccine antigen, and subsequently developed a full-length VP2-based DNA vaccine to prevent the virus. In detail, bioinformatics analyses revealed that VP2 is highly conserved and contains abundant predicted B-cell and T-cell epitopes. Variation analysis of the VP2 amino acid sequence showed that the homology of VP2 protein among largemouth bass birnavirus (LBBV) strains was 99.8%, indicating that the VP2 protein was relatively conserved. Furthermore, the VP2 gene was inserted into the pcDNA3.1(+) plasmid to construct a DNA vaccine, and the results showed that VP2 could be effectively expressed in CPB cells and fish. Importantly, the qPCR was used to detect MHCⅡ, TNF-a, Mx, and IgM -related immune factors. The results showed that the expression levels of these immune factors were significantly increased after DNA vaccine immunization. In addition, when the largemouth bass (Micropterus salmoides) were challenged with a virulent strain of LBBV, the relative immune protection rate (RPS) was 57.1%, indicating that the DNA vaccine could effectively prevent the LBBV virus. This study developed a reliable vaccine based on the conserved amino acid sequence of VP2 and its abundant antigenic epitopes, marking a significant advancement in the field.

In our previous study, broiler-type chicks exhibited greater susceptibility to infection with avian pathogenic Escherichia coli (APEC) than layer-type chicks under identical experimental conditions. To better understand the underlying mechanism, most likely attributed to the immune response, the present work aimed to investigate the humoral and cellular immune parameters in specific pathogen-free broiler-type and layer-type chicks using samples collected from that experiment. Briefly, thirty-two chicks of each bird type were equally divided into two groups. At 14 days of life, one group of broilers and layers were aerosolized with bioluminescent APEC, while the remaining groups received phosphate-buffered saline as negative controls. Birds were necropsied at 3, 7, 10, and 14 days post inoculation (dpi). Blood samples were collected prior to necropsy for serum IgY quantification using an in-house enzyme linked immunosorbent assay (ELISA) based upon the homologous strain. Lung and spleen samples were collected, and the frequencies of mononuclear cell subsets (CD45, CD4, CD8α, TCR-γδ, Bu-1, monocytes/macrophages) were determined by flow cytometry. Both infected groups demonstrated a significant rise in IgY levels post-infection, with antibody titers rising in infected broilers from 7 dpi and in infected layers from 10 dpi onward. In infected broilers, a significant increase of CD8α[+] cells in the lung and spleen was observed. Additionally, a significant decrease in CD4[+] cells in the lung and TCR-γδ[+] cells as well as Bu-1[+] cells in the spleen was detected. Infected layers showed a reduction of monocytes/macrophages in the lung compared to non-infected birds. Overall, the present investigation revealed an earlier onset of the humoral immune response in broilers being more severely affected than layers. Furthermore, APEC infection also induced marked alterations in cellular immune cell populations, particularly in broilers whereas layers responded with much less cellular changes.

This work investigates the potential protective effects of the diatom Amphora coffeaeformis as a feed additive against the deleterious effects (primarily on immune organs) of cypermethrin (CYP) in Nile tilapia, Oreochromis niloticus, with respect to hematology, immune/antioxidant parameters, biochemical indices, proinflammatory and apoptotic regulatory genes, and tissue architecture. For subacute exposure, a total of 240 healthy O. niloticus (30 ± 2.5 g) were allocated into four groups in triplicate (20 fish/replicate). The control and AMP groups were fed a basal diet and a 5% A. coffeaeformis supplement, respectively. The CYP and AMP + CYP groups were fed a basal diet and a 5% A. coffeaeformis supplement, respectively, and exposed to sublethal concentrations of CYP for 21 days. A substantial decrease in hematological indices (RBCs, Hb, PCV, and WBCs) and immune/antioxidant parameters (phagocytic and respiratory burst activities, GSH, TAC, SOD, and CAT) was observed with CYP exposure. Additionally, notable increases were observed in serum levels of hepatorenal indicators (ALT, AST, ALP, urea, creatinine, and uric acid), the lipid profile (cholesterol and triglycerides), the MDA lipid peroxidation biomarker, and pro-inflammatory IL1β and apoptotic Cas3 gene expression. Furthermore, hepatic and splenic tissues were severely affected by CYP exposure. Feeding CYP-exposed fish an A. coffeaeformis diet significantly improved hematological indices, immune/antioxidant status, hepatorenal function, and lipid profile, and decreased expression of the stress protein HSP70 and pro-inflammatory cytokines (IL1β, TNFα, and CC1- chemokine) and the apoptotic Cas3 gene compared with CYP-exposed fish. Additionally, tissue architecture improved following A. coffeaeformis supplementation. Overall, A. coffeaeformis diets could be enriched as a feed supplement in the O. niloticus diet to alleviate adverse effects on fish health and immune response during CYP exposure.