This study investigated the immunotoxic effects of chronic waterborne chlorpyrifos (CPO) exposure in Nile tilapia (Oreochromis niloticus) and evaluated the protective efficacy of dietary cinnamaldehyde (CA). Fish were exposed to 15 μg/L CPO for 60 days, while CIN was incorporated into the diet at 0.42 g/kg feed. Immune function was comprehensively assessed through hematological profiling, serum innate immune responses, splenic oxidative stress and apoptotic biomarkers, expression of immune- and cell survival-related genes, and histopathological examination of the spleen. Chronic CPO exposure caused marked hematological disturbances, characterized by significant reductions in erythrocyte count, hematocrit, hemoglobin concentration, and platelet count, together with pronounced leukocytosis and alterations in differential leukocyte populations. Moreover, CPO significantly suppressed innate immune responses, as proved by decreased serum lysozyme activity and nitric oxide production. Splenic oxidative injury was confirmed by elevated malondialdehyde, accompanied by marked depletion of reduced glutathione, catalase, and superoxide dismutase. At the molecular level, CPO markedly dysregulated the expression of akt-1, mtor, becn-1, jnk, bcl-2, and caspase-3. Histopathological examination further revealed severe splenic lesions, including lymphoid depletion, melanomacrophage center activation, vascular congestion, and disruption of normal splenic architecture. In contrast, dietary CA markedly alleviated CPO-induced immunotoxicity by restoring hematological indices, enhancing innate immune responses, reinforcing antioxidant defenses, and normalizing the expression of akt-1, mtor, becn-1, jnk, caspase-3, and bcl-2 toward physiological levels. Histological observations corroborated these biochemical and molecular findings, demonstrating substantial preservation of splenic architecture in CA-treated fish. Collectively, these findings indicate that dietary CA effectively protects Nile tilapia against CPO-induced immunotoxicity through enhancement of antioxidant capacity, restoration of innate immune competence, inhibition of apoptosis, modulation of autophagy, and regulation of the AKT/mTOR signaling pathway.

Extracellular vesicles (EVs) are nanoscale, lipid bilayer membrane-bound particles carry bioactive cellular cargos such as protein, lipids, nucleic acids. They are considered as key mediators of intercellular communication, potential biomarkers, and play role in numerous pathophysiological processes including modulating innate and adaptive immune responses. In this study, plasma-derived EVs were isolated from rainbow trout (Oncorhynchus mykiss) and determined the physicochemical-molecular properties to investigate their anti-inflammatory activities. Isolated EVs had inherent cup shaped morphology with an average particle diameter of 106.5 nm, and negative surface charge (zeta potential) of -24.2 mV. The purity of EVs was confirmed through immunoblot detection of EVs markers (ALIX, HSP90, HSP70, CD63, and TSG101), and highly enriched acetylcholinesterase (AChE). Transcriptional analysis demonstrated that EVs treatment up-regulated the Il-6 and Il-8 expression and down-regulation of Isg15, Isg20, Ifnβ1, and Tnf-α expression and in RAW 264.7 cells. Moreover, flow cytometry results revealed EVs treated RAW 264.7 cells have decreased the reactive oxygen species (ROS) generation upon lipopolysaccharide (LPS)-induction. Enzyme-linked immunosorbent assay results evidenced that EVs role on the suppression of Inf-α and Ifn-β, and induction of Il-6 proteins in LPS-induced RAW 264.7 cells. LPS-induced neuroinflammation was lowered through suppression of inflammatory (tnfα and il1β) genes and inducing pro-inflammatory (il6 and il8) genes in EVs treated zebrafish brain. Thus, we suggest that plasma-derived EVs from rainbow trout have potential to regulate inflammatory and antioxidant responses and may serve as anti-inflammatory agent to mitigate cellular damage associated with inflammatory conditions.

Fas-associated protein with death domain (FADD) is a crucial adaptor protein in the insect Immune Deficiency (IMD) signaling pathway. Although its role in regulating antimicrobial peptide (AMP) synthesis has been established in Drosophila, its function in Ostrinia furnacalis remains unclear. In this study, we identified a FADD (designated Of-FADD) with a full-length cDNA of 971 bp, containing a 666 bp open reading frame encoding a 221-residue protein with a characteristic death effector domain (DED) and a death domain (DD). Expression profiling showed the highest transcript levels in fifth-instar larvae, with significant enrichment in the midgut and hemocytes. Bacterial infection experiments demonstrated that both Escherichia coli and Staphylococcus aureus significantly induced Of-FADD expression, with peak upregulation observed at 12 h and 24 h post-infection, respectively. Concurrently, the expression of Relish and AMP genes was also significantly upregulated. RNA interference (RNAi) revealed that knockdown of Of-FADD significantly downregulated the expression of Relish and AMP genes. Following RNAi and subsequent bacterial infection, Of-FADD expression exhibited a compensatory response. Subcellular localization analysis indicated that Of-FADD is distributed in the cytoplasm. Of-FADD can form oligomers but does not assemble into amyloid fibril structures independently under the tested conditions. This study provides transcript-level evidence in a lepidopteran pest that Of-FADD positively modulates AMP-mediated immune responses, likely via the IMD signaling pathway, and that its signal transduction mechanism does not rely on amyloid fibril formation under our experimental conditions. These findings contribute to our understanding of immune mechanisms in lepidopteran pests and offer potential molecular targets for developing novel pest control strategies.

Little is known about how the Toll and Imd pathways are deployed across tissues and over time in insects outside the Drosophila lineage. To address this, we performed a systematic analysis of immune gene responses in Manduca sexta using deep RNA-seq of fat body, hemocytes, and midgut after aseptic wounding or microbial injection, identifying 1895 genes strongly induced by injury or infection. Immune challenge triggered extensive transcriptional reprogramming, including induction of pattern recognition receptors, serine protease cascades, cytokines, antimicrobial peptides (AMPs), and core immune signaling pathway components, whereas wounding elicited a more restricted immune response characterized primarily by AMP induction. Comparison among tissues revealed striking specialization of immune and physiological programs. Fat body and hemocytes exhibited coordinated immune activation accompanied by suppression of metabolic functions, whereas midgut displayed a distinct profile. To examine temporal patterns associated with Toll- and Imd-biased responses, we analyzed AMP induction kinetics after challenge with live or heat-killed bacteria and fungi. Strongly Toll-biased AMP genes showed delayed induction, whereas Imd-biased genes exhibited more heterogeneous temporal patterns. Moreover, Toll- and Imd-associated gene sets were enriched among early-induced genes in fat body and hemocytes but not in midgut, where early responses were dominated by Imd-associated genes. These results provide a tissue- and time-resolved analysis of Toll- and Imd-associated transcriptional responses in M. sexta and reveal substantial spatial and temporal diversification in the deployment of Toll- and Imd-associated immune programs under the conditions examined.

Leukotriene B4 (LTB4) is a pro-inflammatory lipid mediator that cooperates with CXC chemokine receptor type 4 (CXCR4) in regulating immune responses in vertebrates, but their function in bivalve innate immunity remains largely unknown. This study investigated the involvement of LTB4 and a CXCR4-like protein in extracellular traps (ET) formation in Ruditapes philippinarum. Metabolomics revealed that LTB4 was a key differential metabolite in gill tissues following LPS stimulation, and ELISA further confirmed that LTB4 levels were significantly elevated upon LPS challenge. A CXCR4 homolog designated RpCXCR4-like was further characterized, which possesses typical seven transmembrane (7-TM) and the DRY motif. In unstimulated clams, RpCXCR4-like transcripts were constitutively expressed in all examined tissues, especially in gills. After LPS stimulation, expression of RpCXCR4-like mRNA in hemocytes and gills was up-regulated. Furthermore, LTB4 treatment also induced RpCXCR4-like expression, whereas pretreatment with the LTB4 receptor antagonist docosatrienoic acid (DTA) significantly reduced this upregulation. Functional assays demonstrated that LTB4 and LPS significantly induced reactive oxygen species (ROS) production and ET formation in gill cells, whereas pretreatment with DTA or knockdown of RpCXCR4-like significantly attenuated these effects. Collectively, LTB4 and RpCXCR4-like promote ET formation in gills through ROS production, providing novel insights into the regulatory network between lipid mediators and innate immunity in clams.

Wolbachia can enhance dipteran resistance to fungal infections, yet the effects of this symbiont on antifungal defense mechanisms remain poorly understood. We studied immune parameters in male and female Drosophila melanogaster with (W+) and without (W-) Wolbachia. We assessed the constitutive expression of Toll pathway genes and several components of the melanization cascade, including expression of prophenoloxidase genes, phenoloxidase activity, levels of reactive oxygen species, and concentrations of tyrosine, dopamine, and N-acetyldopamine. In addition, we analyzed the induction of Toll signaling components and Toll-related antimicrobial peptides in males and females of the W+ and W- flies following natural infection with the fungus Beauveria bassiana. We show that increased female survival following infection is associated with a Wolbachia-dependent constitutive shift toward catecholamine-dependent melanization.In males, by contrast, the effect of the symbiont is primarily reflected in stronger infection-induced expression of Toll pathway components, particularly spaetzle, dorsal, and Drosomycin. A common feature in both sexes was constitutively elevated expression of Metchnikowin. Together, these results demonstrate that Wolbachia modifies antifungal immunity in a sex-specific manner and highlight new directions for investigating the molecular mechanisms by which Wolbachia influences immune-signaling cascades.

Black soldier fly larvae (BSFL) have emerged as a promising and sustainable source of ingredients for animal feed due to their rapid growth and ability to efficiently bioconvert different organic waste and by-products into high-nutritional-quality insect biomass. Although considerable research has focused on the optimization of larval rearing conditions to improve the chemical composition of the insect biomass, the interplay between diet composition and immune function in BSFL remains poorly understood. Herein, we investigated how two diets differing in their protein-to-carbohydrate (P:C) ratio affect larval performance and the immune response following bacterial challenge. Our results demonstrate that dietary P:C ratio influences both growth and immunity in BSFL. Specifically, larvae fed on the diet with lower P:C ratio (0.32) reached a higher body weight than those grown on the higher P:C ratio diet (1.28), while developmental time and adult emergence rate were unaffected. In contrast, BSFL reared on the higher P:C ratio diet exhibited an enhanced immune response, characterised by an increased number of circulating haemocytes and upregulated expression of antimicrobial peptides genes, resulting in a more rapid bacterial clearance. Collectively, our findings demonstrate that, under these two dietary conditions, macronutrient composition drives distinct trade-offs between growth and antibacterial immune response in BSFL. These results provide a foundation for future studies aimed at identifying nutritionally optimised feeding strategies that balance biomass production with disease resistance under industrial rearing conditions.

Peroxiredoxins (Prxs) are widely distributed thiol-dependent antioxidant enzymes that contribute to cellular redox homeostasis by reducing hydrogen peroxide, organic hydroperoxides, and peroxynitrite. Studies of molluscan Prxs remain relatively limited and are concentrated mainly in Bivalvia and Gastropoda, with Prx1, Prx2, Prx4, Prx5, and Prx6 currently reported. Notably, re-examination of the sequence previously designated AwPrx4A indicates that it is more consistent with a TXNL4/DIM1-family thioredoxin-like protein than with a member of the Prx family. This review summarizes current knowledge of the sequence characteristics, phylogenetic relationships, tissue-expression patterns, and biological responses of molluscan Prxs. Available evidence supports conserved peroxide-reducing and oxidative-protective activities for several recombinant Prxs, whereas pathogen- and environmental-stress studies are dominated by transcriptional responses whose direction and magnitude vary among species, tissues, Prx subtypes, and experimental conditions. Emerging pathway-level evidence, such as Nrf2/ARE-mediated regulation of CpPrx5, has begun to clarify upstream control of Prx transcription, but direct in vivo evidence linking Prx activity to pathogen resistance, stress tolerance, or other physiological outcomes remains scarce. Future studies should expand taxonomic coverage and combine Prx-specific functional manipulation with pathway analysis and physiological phenotyping to clarify both canonical antioxidant functions and potential noncanonical roles in molluscan biology.

Rainbow trout (Oncorhynchus mykiss) is a key aquaculture species in many countries, but the role of biological sex in shaping fish immune responses remains understudied. Characterizing sex-based immune variation is essential for optimizing disease management, vaccination strategies, and selective breeding programs. This study investigates the baseline innate immunity in sexually mature male and female rainbow trout (n = 23/sex) by examining both transcript and protein expression in immune relevant tissues, including head kidney, spleen, foregut, midgut, and hindgut. RT-qPCR was used to quantify the expression of key innate immune genes and hormone receptors, while ELISA measured cytokines and immunoglobulins (IgT and IgM) in head kidney and spleen. Results revealed statistically significant sex- and tissue-specific differences at the transcript level, with males exhibiting elevated expression of interferons (ifn-1 and ifn-γ) inflammatory and regulatory cytokines (il1β, il10, tgf-β), and interferon-stimulated genes (pkr) particularly in gut-mucosal tissues. Androgen receptor transcripts (adr-a and adr-b) were also significantly higher in males, whereas estrogen receptor expression showed a complex, tissues- and isoform-specific pattern. In contrast, protein-level differences were limited with only IgT showing higher expression in the female head kidney. These findings demonstrate that sex is a significant determinant of baseline innate immunity in rainbow trout with male-biased transcript level responses and limited protein-level regulation, which highlights the need to consider sex as a biological variable in fish immunology and aquaculture applications, especially when facilities use only one sex for production.

A 60-day trial was carried out to assess the effect of Kappaphycus alvarezii as a dietary feed supplement on the growth and disease resistance of Penaeus vannamei reared in RAS and biofloc systems against Vibrio parahaemolyticus. The experimental diets were formulated as isoproteic diets (35 ± 0.5% crude protein) and supplemented with K. alvarezii at three inclusion levels (5%, 10% and 15%), while an unsupplemented basal diet as the control. The findings showed that the addition of 5% of K.alvarezii to the diet significantly enhanced final weight, weight gain, Feed Conversion Ratio (FCR) and Feed Efficiency Ratio (FER) compared to the other groups. Diets for shrimp that included K.alvarezii resulted in a higher crude protein content in the whole body of shrimp compared to the control. After the feeding trial, P.vannamei was challenged with V.parahaemolyticus for 15 days to assess the immune response. The result of the immunological parameters was significantly higher than that of the control group. The maximum survival rate against V. parahaemolyticus was shown in K.alvarezii supplemented diets. The addition of K.alvarezii to shrimp diets improved the histological features of hepatopancreatic tissues, especially at 10% and 15% levels. The results showed that shrimp fed a diet containing K. alvarezii showed enhanced growth and immune response profiles. Overall, the study suggested that incorporating K.alvarezii into the feed of P.vannamei improves the growth and immune response against V.parahaemolyticus.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes mitochondrial dysfunction and induces mitophagy to facilitate virus replication. The regulator of calcineurin 1 (RCAN1) is a biomarker of oxidative stress caused by mitochondrial dysfunction. However, whether RCAN1 is involved in PRRSV pathogenesis remains unclear. In this study, we reveal that the glycoprotein 5 (GP5) of PRRSV induced the porcine RCAN1-1 L isoform through oxidative stress. Overexpression of RCAN1-1 L was found to significantly promote PRRSV proliferation in PK15[CD163] cells. Transcriptome analysis of PRRSV-infected PK15[CD163] cells transfected with siRNA targeting RCAN1-1 L identified 62 differentially expressed genes, most of which were associated with mitochondrial functions. Furthermore, RCAN1-1 L downregulated the expression of the mitochondrial outer membrane protein TOM20 while upregulating the mitophagy-associated proteins PINK1 and the autophagosome marker LC3II. The accumulation of lysosomes acidified mitochondria and enhanced mitophagic flux were observed following the overexpression of RCAN1-1 L during PRRSV infection. In addition, RCAN1-1 L decreases intracellular ROS levels and downregulates the expression of the apoptotic marker cleaved Caspase-3 in PRRSV infected cells. The findings demonstrate that RCAN1-1 L links mitophagy to PRRSV pathogenesis and serves as a potential antiviral therapeutic target.

Snails depend entirely on innate immunity, primarily mediated by hemolymph amoebocytes and hyalinocytes, which are responsible for pathogen recognition, phagocytosis and immune signaling. While amoebocyte functions are well established, the role of hyalinocytes and the origin of hemolymph cells remain unclear. Hyalinocytes proliferate rapidly during pathogenic infections associated with increased mortality and inflammation. This study investigated snail dysbiosis following oral administration and hemocoel injection of the intracellular Listeria monocytogenes SN3 and the extracellular Pseudomonas baetica 7 A snail pathogens and the symbiotic effect of the probiotic Lactiplantibacillus plantarum Sgs14. Chemotaxis and necrosis assays revealed that snail pathogens increased amoebocyte and hyalinocyte numbers and induced cell necrosis, whereas the probiotic elicited the so-called "normal" inflammatory response without cell necrosis. Histological analysis showed increased cell accumulation within hepatopancreatic cavities without significant connective tissue damage. Immunohistochemistry indicated elevated expression of the LGR5 receptor homologue in hepatopancreatic cavity cells following pathogenic treatments, while no expression was detected in hemolymph cells. Hemocoel injection of bacteria or hepatopancreatic cell suspensions altered hemolymph cell populations, causing short-term increase in amoebocytes, hyalinocytes, and dead cells. In vitro and in vivo assays demonstrated that L. monocytogenes SN3 strain-activated hyalinocytes adhered to infected amoebocytes and induced necrosis, highlighting their potential cytotoxic role. Flow cytometry and immunofluorescence analysis revealed upregulation of immune marker homologues (CD16, CD49b, IFN-γ, IL-17A), mainly in hyalinocytes, following pathogenic challenge. These findings identify the hepatopancreas as a primary hemopoietic organ in snails and demonstrate pathogen-specific immune modulation, elucidating the mechanisms underlying snail responses and the functional activation of hyalinocytes as cytotoxic effectors.

The tripartite motif 45 (TRIM45) protein belongs to the RING-type E3 ubiquitin ligases and exerts multiple biological functions in cell signaling and inflammation regulation by catalyzing ubiquitination of adaptor proteins in the corresponding cascades. In the present work, we obtained the porcine TRIM45 (porTRIM45) coding sequence and examined its contribution to type I IFN induction. The open reading frame (ORF) encodes a 580-amino-acid polypeptide that shares 84.7-89.7% sequence identity with orthologs from human, mouse, and monkey. porTRIM45 harbors a RING domain, two B-box domains, a coiled-coil domain, and a FIL domain, and exhibits broad tissue distribution across porcine tissues. Functional analyses showed that porTRIM45 participates in modulating type I IFN production upon poly(I:C) stimulation. Mechanistically, porTRIM45 associates with porcine IκB kinase epsilon (IKKε) and promotes its K48-linked polyubiquitination for proteasomal degradation, consequently dampening RLRs signaling pathway. Additionally, we verified that the RING and FIL domains are indispensable for the E3 ligase function of porTRIM45. Collectively, our findings underscore the crucial role of porTRIM45 in IKKε-mediated innate immune signaling in pigs.

The prophenoloxidase (PPO) activation pathway and the Toll-mediated antimicrobial peptide (AMP) synthesis pathway constitute critical immune responses within the insect immune system. The activation of these pathways is driven by extracellular serine protease cascades, a process counterbalanced by the negative regulation of serpins. In this study, we identified a typical serpin, BmSerpin-7, and characterized its function in the silkworm, Bombyx mori. BmSerpin-7 expression was up-regulated in the hemocytes but down-regulated in the fat body after Micrococcus luteus and Yersinia pseudotuberculosis challenge. Melanization, phenoloxidase (PO) activity and the activation of PPO were markedly suppressed by the recombinant BmSerpin-7. Meanwhile, the antibacterial activity of the hemolymph and bacteria-induced expression of AMP genes, including gloverin-2, gloverin-3, cecropin-D, and cecropin-E, were also remarkably decreased. Furthermore, based on sequence homology and established serpin-protease relationships in Manduca sexta, B. mori hemolymph protease 1 (BmHP1) and hemolymph protease 6 (BmHP6) are proposed as potential proteases involved in BmSerpin-7-mediated immune regulation. Together, our findings support BmSerpin-7 as a dual negative regulator of PPO activation and bacteria-induced AMP production, thereby providing insights into the regulation of immune responses in silkworm.

Viral hemorrhagic septicemia virus genotype II is generally considered less virulent than other genotypes. However, the molecular mechanisms underlying viral infection and its impact on host gene expression are poorly understood. The tumor necrosis factor receptor-associated factor 6 (TRAF6) is a pivotal molecule that is involved in different processes and plays an important role in the immune response during viral infection in fish. In this work, we investigated the effect of viral infection with VHSV genotype II on EPC and ΔTRAF6 cells at the gene expression level and on alternative splicing events (ASEs). Following VHSV genotype II infection, ΔTRAF6 cells exhibited more severe cytopathic effects accompanied by significantly higher viral copy numbers than EPC cells, indicating that TRAF6 plays a protective role in the antiviral response against VHSV genotype II. The transcriptomic analysis revealed that 1236 genes were differentially expressed in EPC cells, and 1372 in ΔTRAF6. Following viral infection, the immune-related pathways, including NOD-like receptor signaling, RIG-I-like receptor signaling, toll-like receptor signaling, cytokine-cytokine receptor signaling, C-type lectin receptor signaling pathway, and herpes simplex virus 1 infection, were activated in both cells, with lower gene ratios in ΔTRAF6 cells compared with EPC cells. Furthermore, TRAF6 influences alternative splicing in infected cells. Significant ASEs were observed in both cells, but were notably more frequent in ΔTRAF6 cells. Gene Ontology (GO) enrichment analysis and KEGG pathway enrichment analysis of differential alternative splicing genes showed enrichment in the mRNA surveillance and IgSF CAM signaling pathways. These results suggest that TRAF6 plays an important role in regulating the antiviral response at multiple levels, and that the absence of TRAF6 alters immune responses and signaling pathways, facilitating viral replication of VHSV genotype II and inducing cytopathic effects in cells. In addition, knocking out TRAF6 results in changes in alternative splicing correlating with weakened immune signaling and increased viral replication.