Innate immunity is vital to vertebrates as first line of defence during infections but more critical for fish species that rely on innate immune system when they are infected. Unfortunately, innate immune pathways, including PRRs and associated cytokines, have not been characterized in African catfish. In this study, PCR and qPCR techniques were employed to assess immune gene expression in both untreated and vaccinated African catfish. Transcripts of nod1, nod2, tlr3, mhcii, il1β, cox2 and mpo genes were identified, cloned, and characterized. mRNA levels of nod1, nod2, tlr3 and mhcii genes were further compared in two fish groups distinguished by body weight. Following vaccination, expression profile of nod1 and nod2 was assessed in 72 fish. In healthy fish, transcripts of the genes were widely expressed across tested tissues. mRNA expression levels varied significantly among tissues. Between the two fish groups, expression levels of nod1 and nod2 differed in spleen, whereas tlr3 expression varied significantly in gill and liver. After vaccination, expression of nod1 and nod2 in spleen was significantly altered across treatment groups at 24 h and 7 days post-treatment. Highest and lowest expression levels were observed in vaccine group at 24 h and bacterin group at 7 days, respectively. In contrast, all treatments triggered continuously increased expression of both receptors in gill, reaching a peak level at 34 days in bacterin group. Whereas expression was clearly downregulated at 7 days in vaccine group. Furthermore, at 34 days, nod1 expression in gills of vaccine group was significantly lower compared to other treatment groups, whereas nod2 expression in bacterin group was greater than that in vaccine group. Overall, the findings demonstrate that African catfish express immune genes much like other teleost species, and that both heat-killed whole-cell A. hydrophila vaccines and mineral oil-based adjuvants can stimulate African catfish innate immune system.

Human epithelial ovarian cancer (EOC) is characterized by profound immune dysregulation with a tumor microenvironment (TME) dominated by immunosuppressive populations such as regulatory T-cells that actively limit anti-tumor responses. Spontaneous ovarian tumors in older laying hens (Gallus gallus domesticus) closely resemble human EOC development and histopathological features, making them an ideal model to study human EOC. Here, we examined pathological and immunological changes in ovarian TME from hens with spontaneous ovarian tumors (OT). Histopathological characterization was consistent with the endometrioid subtype of ovarian adenocarcinoma, while the immunohistochemistry analysis showed reduced intratumoral T cell infiltration compared to control. The OT tissue gene expression analysis showed significant upregulation of FoxP3, CD25, CD40, PD-1, LAG-3 and GITR genes, accompanied by an increased IL-10 transcription compared to control, indicating regulatory T cell expansion and effector T cell exhaustion. Furthermore, significant reduction in the IL-1β, chTNFα and iNOS transcription suggested suppressed cellular inflammatory activation. These findings demonstrate that ovarian tumors in laying hens undergo a transition from an inflammatory state to an immunoregulatory, checkpoint-dominant TME, an observation resembling human EOC. This work supports the laying hen as a translational model for studying human ovarian cancer and developing diagnostic and therapeutic strategies.

Pebrine disease, caused by Nosema bombycis, threatens global sericulture due to the lack of green and efficient control agents. Zeolitic imidazolate frameworks (ZIFs) are promising antimicrobial nanomaterials, but their efficacy and mechanism against microsporidia in insects remain unexplored. In this study, we synthesized and characterized ZIF-8 and ZIF-67 nanomaterials to evaluate their protective effects against N. bombycis infection in silkworms and to investigate the underlying mechanisms. Results showed that both materials, administered at 150 μg/g body weight, significantly suppressed the proliferation of N. bombycis and markedly improved the survival rate of infected silkworms, with ZIF-67 showing superior efficacy. Mechanistically, ZIF-67 directly damaged spore structure, while both materials alleviated host oxidative stress by reducing reactive oxygen species levels and upregulating antioxidant enzyme genes (GSTs1, CAT, Aldh), and activated the Toll signaling pathway to increase the expression of antimicrobial peptides genes (gloverin2, Attacin1). In conclusion, ZIF-8 and ZIF-67, particularly ZIF-67, protect silkworms through a multi-mechanistic strategy involving direct pathogen damage, enhanced host antioxidant capacity, and innate immune activation. Our work not only proposes novel nano-candidates for pebrine control but also provides insights for sustainable agricultural applications.

This study evaluated the immunostimulatory effects of passion fruit peel extract (PPE) in Litopenaeus vannamei through injection and its potential application as a functional feed additive. Shrimp were injected with different PPE dosages including 10 μg shrimp[-1] PPE extract (PPE10), 20 μg shrimp[-1] PPE extract (PPE20), and 40 μg shrimp[-1] PPE extract (PPE40), and immune parameters were assessed, including total haemocyte count (THC), differential haemocyte counts, phenoloxidase (PO) activity, respiratory bursts (RBs) activity, phagocytic activity, clearance efficiency, and lysozyme activity. The results showed that PPE, particularly at the higher dose (PPE40), significantly enhanced THC, hyaline cells, and semigranular cells at later stages post-injection, while granular cells remained unchanged. PPE40 also significantly increased PO activity and RBs, indicating activation of the proPO system and oxidative defense. Additionally, phagocytic activity, clearance efficiency, and lysozyme activity were significantly elevated, demonstrating enhancement of both cellular and humoral immune responses. No significant differences in haemolymph glucose and lactate levels were observed among treatments, indicating that PPE did not induce physiological stress. The enhanced immune responses were further confirmed by a Vibrio alginolyticus challenge test, in which PPE40 exhibited significantly higher survival rates compared to other groups, demonstrating improved disease resistance.

Sex-dependent variation in immune responses in arthropods is well established in cases of microbial infection or parasitism but remains less explored under environmental stress. In this study we examined whether the sex of terrestrial isopods Porcellio laevis affects their immune response to soil microplastic (MP) exposure. Animals were exposed for 14 days to polypropylene MPs at 0.05%, 0.5%, and 5% (w/w soil), and immune parameters, including total haemocyte count, haemocyte viability, phenoloxidase (PO)-like activity, and haemolymph protein content, were assessed alongside survival and growth. Overall, MP exposure induced sex-dependent alterations in immune status, with males and females exhibiting distinct response patterns: females exhibited reduced haemocyte-related parameters at specific MP concentrations, whereas males showed higher haemolymph protein levels and increased growth at the highest concentration. PO-like activity was consistently higher in females. Although not all responses were statistically significant, effect size analysis indicated medium to large sex-related differences across multiple endpoints. Most of the effects were related to the dose of MP, with changes detectable already at lower concentrations. These findings highlight the importance of incorporating sex as a biological factor in ecotoxicological studies and support the use of immune markers as sensitive indicators of sublethal environmental stress.

Adjuvants play a key role in boosting vaccine effectiveness. Adjuvant type and composition influence immune response, which can vary across fish species and populations. This study investigates the innate immune response of lumpfish (Cyclopterus lumpus) individuals collected from Newfoundland (NL), Canada, and Iceland (IC) to oil-based and water-soluble adjuvants, respectively. Fish were intraperitoneally injected with Carbigen (a carbomer-based adjuvant), Freund's Incomplete Adjuvant (FIA), or Phosphate-Buffered Saline (PBS). Head kidney samples were collected at 24 h post-injection (hpi) for RNA sequencing and qPCR analysis. At 24 hpi, Carbigen triggered a significantly higher number of differentially expressed genes (DEGs 594) compared to FIA-treated fish (DEGs 14). Gene Ontology enrichment analysis indicated dysregulation of cytokine signaling and pathogen recognition. The genes ccl25a, il34, il10, vwal1, and serpinf2b for Carbigen treatments, and ccl20b, cd22, il6r, atp2a1l, and il8b for FIA treatments were validated by quantitative polymerase chain reaction (qPCR). These genes were either up- or downregulated in both Carbigen and FIA treatments at 24 hpi, compared to the PBS control. The gene expression pattern was different between NL and IC lumpfish. NL lumpfish showed a stronger transcript level response to adjuvants than IC. This suggests underlying immunogenetic variation, possibly shaped by distinct environmental pressures in their native habitats. Overall, the study highlights the potential of a water-soluble vs oil-based adjuvant to effectively stimulate innate immune responses in teleost fish for aquaculture applications.

Streptococcosis and motile Aeromonad septicemia (MAS) are major tilapia diseases causing significant economic losses. Feed-based vaccination offers a sustainable control strategy. However, the efficacy of oral vaccines in tilapia is largely constrained by inadequate adjuvant performance and associated side effects, while the safety, antigen interaction, and immunomodulatory potential of mycogenic metal oxide nanoparticles (MONPs) as a vaccine adjuvant remain insufficiently understood. This study evaluated the safety and immune responses of a novel feed-based bivalent vaccine (FBBV) containing formalin-killed Streptococcus agalactiae and Aeromonas hydrophila, enriched with mycogenic zinc oxide (ZnO-NPs) or aluminium oxide nanoparticles (Al2O3-NPs), in red hybrid tilapia. Four hundred and eighty red hybrid tilapia (10.0 ± 1.5 g) were randomly assigned to four groups (control, AquaBooster, FBBV/ZnO-NPs, and FBBV/Al2O3-NPs) and orally immunized at weeks 0, 2, and 6. Fourier transform infrared (FTIR) spectroscopy and transmission electron microscopy (TEM) analyses suggested interactions between mycogenic MONPs and formalin-killed whole cells (FKWC). Furthermore, spray-coating onto feed maintained the estimated DNA abundance of formalin-killed S. agalactiae while reducing that of A. hydrophila by approximately three-fold. Haematological parameters were largely stable, though white blood cell (WBC) and lymphocytes increased in all vaccinated groups. Histopathological examination revealed generally mild alterations in the control, AquaBooster, and FBBV/Al2O3-NPs groups, whereas the FBBV/ZnO-NP group exhibited marked renal mineralization. Meanwhile, spleen histology was comparable across groups. Moreover, early IgM induction occurred in FBBV/ZnO-NPs, while sustained IgM levels were seen in FBBV/Al2O3-NPs and AquaBooster groups. Gut-associated lymphoid tissue (GALT) area was significantly increased in all vaccinated groups compared with the control. Hindgut qPCR demonstrated comparable estimated DNA abundance of FKWC among the vaccinated groups, whereas hindgut RT-qPCR analysis revealed upregulation of innate (IL-1β, TNF-α) and adaptive (IgM, IgT) immune markers. Overall, both MONP formulations stimulated immune responses; however, their safety profiles differed. The Al2O3-NPs formulation exhibited minimal histopathological alterations, whereas the ZnO-NP formulation induced notable renal mineralization, indicating that this formulation warrants further dose optimization and safety evaluation.

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.