Natural immunomodulatory compounds have emerged as vital tools in sustainable aquaculture for their capacity to boost growth and immunity. This study, for the first time, investigates the dietary effects of Lagenaria siceraria flower (LSF) in Nile tilapia (Oreochromis niloticus), specifically analyzing growth performance, metabolic profiles, intestinal integrity, and the expression of cytokine and immune-related genes. In a 70-day feeding trial, 240 Nile tilapia were divided into four groups (60 fish per group). Each group received a basal diet supplemented with varying concentrations of LSF: 0, 1, 2, or 3 g/kg (designated as LSF0, LSF1, LSF2, and LSF3, respectively). The HPLC analysis of LSF indicates that hesperidin (49.75%) was the primary constituent, followed by quercetin (11.6%), ellagic acid (11.56%), gallic acid (4.97%), and quercetin dihydrate (2.2%). Our results demonstrated that the supplementation of 2 or 3 g/kg LSF significantly improved growth performance indices (final body weight, weight gain, average weight gain, and specific growth rate) compared to the other groups (P < 0.05). Feed intake remained unaffected (P > 0.05), and feed conversion ratio (FCR) was significantly lower in the LSF2 and LSF3 groups (P < 0.05). Erythrocyte and leukogram indices were significantly improved (P < 0.05); however, the percentages of lymphocytes and basophils showed a notable decline in the LSF-supplemented groups (P < 0.05). LSF supplementation significantly increased serum protein levels and digestive enzymes (lipase and amylase), while reducing liver enzyme activities (AST and ALT) and nitrogenous metabolites (urea and creatinine) compared to the control group (P < 0.05). At a dosage of 3 g/kg, LSF supplementation effectively minimized lipid peroxidation (lowered MDA) and maximized the antioxidant defenses (SOD, CAT, and GPx) compared to the control (P < 0.05). Dietary LSF demonstrated significant immunomodulatory effects by increasing lysozyme activity, total immunoglobulin (Ig) levels, phagocytic activity, and the phagocytic index (P < 0.05) as well as preserved the gut integrity. Furthermore, LSF modulated immune-related genetic markers, as evidenced by the significantly higher expression of CXC chemokine, IL-1β, IL-8, IL-6, MHC-II, TNF-alpha, TLR9, and TLR3 in fish, with maximum values observed in the LSF3 group (P < 0.05). After a challenge with Aeromonas sobria, the survival rates significantly improved from 30% in the control group (LSF0) to 55%, 65%, and 70% in the LSF1, LSF2, and LSF3 groups, respectively (P < 0.05). Molecular docking analysis further supported these findings, showing that hesperidin and quercetin had strong binding affinities to GSH (ranging from -7.54 to -6.91 kcal/mol and -5.00 to -4.66 kcal/mol, respectively). Notably, hesperidin had a superior docking score with Keap1 (-10.53 kcal/mol) compared to quercetin (-6.13 to -5.95 kcal/mol). In conclusion, LSF supplementation significantly boosts the health and growth of Nile tilapia, specifically through improved gut health and antioxidant defenses. This study underscores the viability of repurposing agricultural waste into high-value natural compounds, paving the way for more sustainable and resilient aquaculture practices.
The development of functional feed additives represents a promising strategy to enhance shrimp performance and resilience in sustainable aquaculture. This study investigated the individual and combined effects of octacosanol and Saccharomyces cerevisiae on growth performance, physiological status, antioxidant capacity, immune response, and disease resistance in Litopenaeus vannamei. A total of 360 shrimp were randomly assigned to four dietary treatments in triplicate and fed for 60 days: a control diet, octacosanol (12 mg/kg), S. cerevisiae (4 g/kg), and a combined treatment (12 mg/kg octacosanol + 4 g/kg yeast). Disease resistance was further evaluated using a waterborne immersion challenge with the pathogenic Aeromonas hydrophila, with survival monitored over 10 days. All supplemented groups exhibited significantly improved growth performance, with OCTA×SC recording the highest final body weight, weight gain, and specific growth rate, alongside the lowest feed conversion ratio, relative to control (p < 0.05). Digestive enzyme activities, including lipase and protease, were significantly enhanced, particularly in the OCTA×SC group. Fatty acid profiling of both muscle and hepatopancreas revealed a significant reduction in saturated fatty acids and a concurrent increase in n-6 and n-3 polyunsaturated fatty acids, with DHA accumulation being most pronounced in OCTA×SC. Serum biochemical analysis demonstrated significant improvements in total protein, albumin, globulin, and HDL-cholesterol, alongside significant reductions in triglycerides and hepatic transaminase activities (AST and ALT) in supplemented groups. Antioxidant enzyme activities (CAT, SOD, GPx) and total antioxidant capacity were significantly upregulated, while lipid peroxidation (MDA) was significantly reduced, most markedly under combined supplementation. Transcriptional analysis confirmed significant (p < 0.05) upregulation of immune-related genes (HMC-Lv, Pen3-Lv, LZM-Lv, ProPO-Lv) and antioxidant genes (cMnSOD-Lv, CAT-Lv, GSH-Px-Lv) in all supplemented groups, with OCTA×SC consistently yielding the greatest induction. Following bacterial challenge, OCTA×SC exhibited the highest survival rate and relative percentage of survival (RPS = 43.48%), significantly exceeding single-supplement groups (RPS = 30.43% each). In conclusion, the synergistic application of octacosanol and S. cerevisiae (12 mg/kg octacosanol + 4 g/kg yeast) effectively enhances growth, physiological stability, and disease resistance in L. vannamei. These findings support the integrated use of octacosanol and S. cerevisiae as a synergistic nutritional strategy to enhance shrimp performance and disease resilience in intensive aquaculture systems. Future work should address mechanisms, dosage optimization, and long-term impacts on health and sustainability.
In addition to highly polymorphic classical MHC class I genes, teleost fish also possess variable numbers of non-classical MHC class I genes, the functions of which are largely unknown. This work focuses on genes belonging to the non-classical MHC class I U lineage revealing unique evolutionary and regulatory patterns across salmonids, indicative of sub-clade specific and diversified functional roles. Comparative analysis of a total of 175 salmonid U lineage genes supported a classification into seven subclades following one of two main evolutionary patterns indicative of different selective pressures. Genes belonging to the ULA, UC/DA, UFA, and UZA sub-clades exhibited variable degrees of species-specific diversification, characterized by gene duplications, gene loss and pseudogenization. In contrast, the UGA, UEA, and UHA sub-clades have remained conserved, with minimal sequence divergence across divergent species, suggesting functional constraints. Notably, the UGA subclade represents a striking example of conservation across divergent salmonid species. Promoter analyses of the U lineage subclades revealed two main regulatory architectures: i) overall conserved promoter elements similar to the UBA gene in ULA, UFA, and UZA, implying shared transcriptional regulation; and ii) divergent promoters in UGA, UEA, UC/DA and UHA suggesting diversified regulatory mechanisms. Expression profiling further supported functional diversity, revealing non-classical U lineage subclade specific expression patterns and variable expression levels. While non-classical U lineage genes, as a group, showed limited response to stimulation with viral mimics, subclade specific responses were observed following infection with salmon alpha virus 3 (SAV3) and in response to interferon stimulation. Similar to the classical UBA gene, UGA and, to a lesser extent, UHA transcript expression levels were upregulated in response to stimulation with either type I IFNa1 or type II IFNγ, as well as in response to SAV3 infection but not to IFNc stimulation. In contrast, UC/DA and UEA genes showed minimal response to interferon stimulation or SAV3 challenge. Combined, these findings underscore the adaptability and complexity of the salmonid MHC class I system and provide a comprehensive framework for understanding the diversification of salmonid MHC class I genes, offering valuable insights into the variation, regulation, and potential roles of these genes in salmonid immunity.
Interferon-gamma (IFN-γ) serves as an inflammatory cytokine essential for modulating innate and cell-mediated immune responses by associating with a receptor complex composed of IFNGR1 and IFNGR2. In this research, the entire cDNA of IFNGR2 from Nibea albiflora was cloned and functionally analyzed (referred to as NaIFNGR2), with the complete cDNA sequence measuring 924 bp and encoding 307 amino acids. The phylogenetic analysis and multiple sequence alignment revealed a significant similarity of NaIFNGR2 with homologous sequences found in other bony fish, especially within the FNⅢ domain and the transmembrane region. Real-time PCR analysis revealed that NaIFNGR2 was consistently expressed across all examined tissues, including the head-kidney, spleen, liver, kidney, gill, muscle, and blood, with the highest levels found in the gills. Following stimulation with Polyinosinic-polycytidylic acid (Poly (I:C)), Vibrio alginolyticus, or Vibrio parahaemolyticus, the mRNA levels of NaIFNGR2 showed an increase in a time-dependent manner. Subcellular localization studies indicated that NaIFNGR2 resided on the cell membrane and NaIFN-γ, once synthesized within the cell, was transported to the membrane to interact with NaIFNGR2. Additionally, NaIFNGR1 and NaIFNGR2 were completely co-localized on the cell membrane, which was consistent with the findings that NaIFNGR1 and NaIFNGR2 could form a heterodimeric complex. Being treated with the NaIFN-γ recombinant protein, both alone and in combination with LPS, various concentrations of NaIFN-γ were non-toxic to the growth of RAW 264.7 macrophages and significantly promoted their proliferation. The expression levels of IL-1β, IL-6, and TNF-α proteins were markedly upregulated in a concentration- and time-dependent manner following 24 and 48 h of combined stimulation. Furthermore, the secretion of nitric oxide (NO) was significantly increased after 36 and 48 h of stimulation, highlighting the regulatory effect of NaIFN-γ on macrophages and its influence on the inflammatory response. Collectively, the findings indicated that the NaIFN-γ ligand-receptor system was present in N. albiflora and played a crucial role in the immune response to pathogenic bacterial infections, enhancing our comprehension of the function of IFN-γ within the immunomodulatory processes of teleosts.
The thymus is vital for T cell development, yet avian thymocyte maturation remains poorly characterized. Here, we present a single-cell transcriptomic atlas of thymic development in Zhedong White geese across embryonic, gosling, juvenile, and adult stages. Morphological and histological analysis revealed a multilobulated, paired thymus with rapid post-hatch expansion and age-dependent involution. Single-cell RNA sequencing identified all major thymic cell types, with T cells predominant and γδ T cells unusually abundant compared to mammals. High-resolution profiling defined key T cell subsets and reconstructed sequential differentiation, showing DN cells transition from pre-commitment states marked by BCL11A, SPI1, and HHEX to committed states (BCL11B, TCF7) and β-selection gene activation. Thymic epithelial cells functioned as central signaling hubs, mediating microenvironmental regulation. Comparative analysis with human and mouse thymus revealed a conserved developmental framework, yet pronounced species-specific differences at the DP stage and in γδ T cell enrichment. These findings contribute foundational insights for avian immunology and poultry breeding.
Salinity fluctuations represent a major environmental stressor for freshwater bivalves; however, the regulatory interplay among osmoregulation, energy metabolism, and immune responses in Hyriopsis cumingii remains poorly understood. In this study, physiological and biochemical assays, histopathological examinations, and transcriptomic analyses were integrated to comprehensively characterize the responses of H. cumingii to increasing salinity stress. Elevated salinity significantly disrupted osmotic homeostasis, as evidenced by increased hemolymph osmolality, tissue dehydration, enhanced Na+/K+-ATPase activity, and the accumulation of alanine and glutamate. Salinity stress also induced oxidative stress, as indicated by increased catalase activity and salinity-dependent alterations in superoxide dismutase activity, accompanied by progressive histological damage to the gills and hepatopancreas. Transcriptomic analyses further revealed extensive molecular reprogramming in response to salinity stress, including the enrichment of pathways associated with energy metabolism, amino acid and lipid utilization, and innate immune regulation. Notably, immune-related signaling pathways, including MAPK and NF-κB, were significantly activated, suggesting that osmotic imbalance and metabolic adjustments were closely linked to inflammatory and stress-response signaling. Collectively, these findings indicate that the response of H. cumingii to elevated salinity involves short-term compensatory mechanisms, including osmotic adjustment, metabolic remodeling, antioxidant defense, and immune activation. However, the concurrent occurrence of tissue injury and stress-associated immune signaling suggests that these responses may not constitute stable adaptation to high-salinity conditions. This study provides species-specific insights into the integrated physiological and transcriptional mechanisms underlying salinity stress responses in freshwater pearl mussels and offers a theoretical foundation for stress assessment and aquaculture management.
This study examined the effects of sodium acetate (SA) on immunity, antioxidant status, intestinal barrier function, and disease resistance in juvenile common carp. A 1-week short-term gavage trial was first conducted using SA concentrations of 0, 0.25%, 0.5%, 1.0%, 1.5%, and 2.0%. SA at various doses enhanced immune and antioxidant responses, with optimal gavage ranges of 0.87%-1.04% and 0.92%-1.20%, respectively. Subsequently, an 8-week feeding trial was performed with five groups: two controls (CK, CK+, 0% SA) and three SA-supplemented groups (0.5%, 1.0%, 1.5%), with three replicates per group (n = 30). After feeding, serum D-LA, ET-1, 5-HT and DAO did not differ significantly (P > 0.05). However, SA supplementation significantly increased immune indices [TNF-α (1.0%), IL-1β (0.5-1.5%), IL-6 (1.0-1.5%), IL-10 (1.0-1.5%), TGF-β (1.0%), NO (1.0-1.5%), LZM (1.0-1.5%), AKP (1.0-1.5%)], hepatopancreatic antioxidant parameters [T-AOC (1.5%), SOD (0.5%), CAT (0.5-1.5%), GSH-Px (0.5-1.0%), GSH (1.0-1.5%)], and intestinal tight junction gene expression [zo-1 (0.5-1.0%), occluding (0.5-1.5%), claudin-1 (0.5%)], while decreasing MDA (0.5-1.0%) (P < 0.05). After infection, Aeromonas hydrophila triggered inflammatory responses and oxidative stress by significantly elevating the contents of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and NO, as well as the activities of LZM and AKP and related antioxidant indices (P < 0.05), down-regulating the expression of intestinal tight junction protein genes and increasing intestinal permeability, thereby impairing intestinal integrity and ultimately reducing the survival rate to 29.80% (P < 0.05). In contrast, dietary SA supplementation significantly reversed these changes: it reduced the levels or activities serum immune indicators, hepatopancreas antioxidant indices and intestinal permeability markers (P< 0.05). Moreover, the levels of anti-inflammatory cytokines (IL-10, TGF-β), the expression of intestinal tight junction proteins, and the survival rate were all significantly higher in the SA-supplemented group than in the infected control (CK+) group (P < 0.05). Collectively, these results demonstrate that dietary SA supplementation enhances innate immunity, antioxidant capacity, intestinal barrier function, and disease resistance in juvenile common carp, and the recommended dosage is 1.0%.
Multifunctional probiotics offer a promising strategy for sustainable aquaculture. This study evaluated Bacillus velezensis S141 by integrating in vitro and in vivo approaches to assess its effects on growth and immune responses in Nile tilapia. The strain produced hydrolytic enzymes (amylase, protease, and lipase), demonstrated tolerance to bile salts across a wide pH range and temperature spectrum, and exhibited antagonistic activity against Aeromonas veronii, Flavobacterium columnare, and Streptococcus iniae. It also showed catalase activity, non-hemolytic properties, and antibiotic susceptibility. A 90-day feeding trial was performed using Nile tilapia fed diets containing 0, 106, and 108 CFU/g B. velezensis S141. Supplementation at 106 and 108 CFU/g improved growth performance at days 60 and 90. Immune parameters (ACH50, total Ig, phagocytic activity) were significantly elevated at 108 CFU/g at days 30 and 90, whereas antioxidant activities (SOD, CAT, MDA, and TAC) were markedly improved at 108 CFU/g at day 90. Fish were challenged with A. veronii on days 30 and 90 to evaluate protective efficacy. The day 30 challenge revealed early immune priming, with upregulation of proinflammatory cytokines (on-cc1, on-cxc1, and il-1β) within 6-12 h, whereas tnfβ peaked at 48 h. Transcriptomic analysis revealed enrichment of immune-related pathways, including cytokine-cytokine receptor interaction and NOD-like receptor signaling, indicating enhanced antibacterial defense. On day 90, probiotic-treated groups exhibited higher survival, demonstrating sustained protection. Overall, B. velezensis S141 improved growth, immune response, antioxidant status, and resistance to A. veronii in Nile tilapia.
Cytopathic (CP) bovine viral diarrhea virus (BVDV) infection is a major cause of lymphocyte dysfunction and immunosuppression in cattle; however, the involvement of the immune checkpoint P-selectin glycoprotein ligand-1 (PSGL-1) in this process remains poorly defined. In this study, bovine peripheral blood lymphocytes (PBL) - rather than total peripheral blood mononuclear cells (PBMCs) - were infected with CP BVDV in vitro. PBL were used because they consist primarily of T cells, B cells, and NK cells without the interference of monocytes/macrophages, allowing a more specific assessment of lymphocyte-intrinsic responses to BVDV infection and antibody blockade. The dynamic expression of PSGL-1, programmed death-1 (PD-1), and their ligands was analyzed by quantitative real-time PCR and Western blotting. Functional antibody blockade was employed to evaluate the effects of individual and combined PSGL-1 and PD-1 inhibition on lymphocyte proliferation, apoptosis, cytokine secretion, and viral replication. We found that CP BVDV infection significantly upregulated PSGL-1 and PD-1 expression in PBL. While blockade of either PSGL-1 or PD-1 partially restored lymphocyte function, combined blockade produced a pronounced synergistic effect, characterized by enhanced proliferation, reduced apoptosis, increased interferon-γ (IFN-γ) and interleukin-2 (IL-2) secretion, and suppression of viral replication. Mechanistically, this synergistic restoration was associated with reactivation of the PI3K/AKT/mTOR and ERK signaling pathways. Collectively, these findings identify PSGL-1 as a critical immune checkpoint involved in BVDV-induced lymphocyte exhaustion and demonstrate that combined PSGL-1/PD-1 blockade effectively restores antiviral lymphocyte function. This study provides new insight into host immune regulation during BVDV infection and highlights a potential combinatorial immunotherapeutic strategy for viral immunosuppression.
The superworm Zophobas morio, a coleopteran beetle, is widely distributed, adapted to environments with decaying organic material and a well-known secondary pest of stored commodities. The larval stage is found to inhabit in putrid conditions, and so are expected to possess distinct immune adaptations vital for their survival against microbial infections in such environments. Using transcriptome analysis of the entire gut tissue of Z. morio, this work systematically surveyed distinct transcripts involved in various pathways of the innate immune system on a comparative account. This study profiled 113 transcripts in the whole gut tissue associated with the innate immune mechanism in insects. Among them, 39 transcripts were involved in the mitogen-activated protein kinase (MAPK) signalling pathway, 24 in the Toll signalling pathway, 12 in the immune deficiency (IMD) pathway, 4 in the Janus kinases activating signal transducers and activators of transcription (JAK/STAT) pathway, and 34 transcripts were involved in other immunity-related processes. Analysis of immune-related genes revealed that about one-fourth of the transcripts showed homology to related beetle groups and, to an extent, to human immunity among the functionally annotated transcripts assigned to various categories from the whole larval gut transcriptome of Z. morio. Further, identification of conserved domains in immune genes showing sequence similarity between Z. morio and other insects, with evidence for deep phylogenetic relationships and a shared common evolutionary ancestor through structural and functional homologies. Understanding the resemblances among the important functional genes involved in biological systems, such as immunity, definitely helps in using insects as excellent in vivo experimental model organisms.
This study aimed to investigate the effects of dietary supplementation with different levels of curcumin on the growth performance, digestive enzyme activity, hemolymph immune parameters, and hepatic antioxidant function of crayfish (Procambarus clarkii). A total of 300 healthy crayfish (10.5 ± 1.0 g) were randomly divided into 5 treatment groups with 3 replicates each: Group 1 fed a basal diet (0 mg/kg group), Group 2 fed basal diet + 50 mg/kg curcumin (50 mg/kg group), Group 3 fed basal diet + 100 mg/kg curcumin (100 mg/kg group), Group 4 fed basal diet + 200 mg/kg curcumin (200 mg/kg group), and Group 5 fed basal diet + 300 mg/kg curcumin (300 mg/kg group) for 8 weeks. The results indicated that dietary curcumin supplementation significantly affected the weight gain rate and feed conversion ratio of crayfish. Specifically, the weight gain rate in the 100 mg/kg group was significantly higher than other treatment groups. Furthermore, different levels of curcumin significantly influenced the activities of protease, lipase, and amylase. The 100 mg/kg group exhibited significantly higher activities of these digestive enzymes compared to the other groups. The acid phosphatase activity in the 100 mg/kg group was extremely significantly higher than that in the 0 mg/kg group (P < 0.01). The complement C3 level in the 100 mg/kg group was extremely significantly higher than in other groups (P < 0.01). The complement C4 levels in the 100 mg/kg and 200 mg/kg groups were significantly higher than those in the 0 mg/kg group (P < 0.01), while the lysozyme activity in the 100 mg/kg group was extremely significantly higher than in the 0 mg/kg group (P < 0.01) and significantly higher than in other groups (P < 0.05). With increasing dietary curcumin levels, the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and total antioxidant capacity (T-AOC) in the hepatopancreas of crayfish initially increased and then decreased, reaching their peak at 100 mg/kg curcumin supplementation, while the malondialdehyde (MDA) level in the hepatopancreas reached its minimum. Meanwhile, the activities of the antioxidant-related genes SOD, CAT, GPX, and the cytokines Keap1a and Nrf2 involved in the antioxidant response in the hepatopancreas of crayfish also showed an initial increase followed by a decrease, peaking at the supplementation level of 100 mg/kg curcumin. The study demonstrates that moderate dietary curcumin addition exerts multiple positive effects on crayfish: it not only significantly promotes growth and improves digestive efficiency but also effectively enhances antioxidant capacity and immune function. Among the tested levels, the 100 mg/kg supplementation yielded the most prominent improvements in antioxidant indices and immune parameters, indicating that this dosage is the optimal choice for promoting healthy crayfish cultivation and overall benefits.
Farmed Atlantic salmon (Salmo salar) experience stressors throughout their growth including handling, transportation, and exposure to a diversity of pathogens, which triggers the release of cortisol. Cortisol mediates the effects of stress on many physiological processes within Atlantic salmon, including the immune response. The binding of cortisol to corticosteroid receptors, namely glucocorticoid (GR) and mineralocorticoid (MR) receptors, leads to cortisol mediated transcriptional modulation. The Atlantic salmon genome has experienced recent whole genome duplication resulting in multiple corticosteroid receptor paralogues: four GR and two MR. Currently, it is not known whether there is structural and functional divergence between GR and MR paralogues in driving cortisol-mediated immune modulation. We have addressed this by investigating GR and MR sequence divergence and transcriptional modulation in Atlantic salmon tissues following stimulation with a viral mimic (poly I:C) or heat-killed Vibrio anguillarum. GR and MR paralogue-specific expression changes were found to be tissue-type and immune stimulant-dependent, suggesting GR and MR paralogues may have distinct roles in the crosstalk between stress and immune responses. These results further our understanding of the impacts of stress on the immune function of Atlantic salmon and demonstrate the importance of exploring receptor paralogues and their function in greater depth.
Chlamydia trachomatis (Ct), an obligate intracellular bacterium, is the primary infectious cause of blindness through trachoma. Ct undergoes a unique biphasic developmental cycle between infectious elementary bodies and replicating reticulate bodies, manipulating host cells via secreted effector proteins. Whilst previous studies have characterised host-pathogen interactions including transcriptomes of urogenital Ct genovars E and L2, limited studies exist on ocular Ct genovars. This study examined transcriptomic responses of human conjunctival epithelial (HCjE) cells and PMA-differentiated THP-1 macrophages to infection with ocular Ct strains A/2497 or B/Tunis864 (live or heat-inactivated) at 4 and 24 hours post-infection (hpi). Transcriptomic profiling was performed using RNA sequencing, with differential gene expression analysis. Pathway enrichment analyses were performed to identify strain-specific and cell type-specific transcriptional signatures. HCjE cells exhibited progressive transcriptional activation, with differentially expressed genes (DEGs) increasing from 4 to 24 hpi (144 to 259), whilst THP-1 macrophages showed temporal attenuation (391 to 154). B/Tunis864 consistently elicited stronger responses than A/2497 in HCjE cells at both time points (152 vs. 54 DEGs at 4 hpi; 259 vs. 83 at 24 hpi). Conversely, THP-1 macrophages showed stronger responses to A/2497 than B/Tunis864 at both time points (599 vs. 376 DEGs at 4 hpi; 166 vs. 114 at 24 hpi). HCjE cells demonstrated markedly higher proportions of strain-specific DEGs and pathways compared to macrophages. B/Tunis864 infection in HCjE cells induced pronounced interferon (IFN)-stimulated gene signatures, particularly at 24 hpi. This study revealed contrasting temporal and strain-specific patterns: THP-1 macrophages showed peak-then-decline, strain-invariant responses, whilst HCjE cells exhibited progressive activation with predominantly strain-specific programmes. Enhanced IFN-stimulated gene and inflammatory pathway activation by B/Tunis864 in HCjE cells may provide molecular insights into genovar B-associated trachoma severity.
This study evaluated the influence of dietary supplementation with prebiotics, probiotics and synbiotics on growth performance, immune response, antioxidant capacity, antimicrobial peptides (AMPs) expression and disease resistance in juvenile olive flounder (Paralichthys olivaceus) fed a low-fish meal (LFM, 45% fish meal) diet. Experimental diets were formulated by supplementing an LFM diet (Con) with 0.6% mannan oligosaccharides (Mos), Lactiplantibacillus plantarum (LP), Bacillus subtilis (BS), B. licheniformis (BL) and their combination (Syn; 0.15% Mos, 0.15% LP, 0.15% BS and 0.15% BL). Fish (initial body weight: 65.9 ± 0.09g) were randomly distributed into triplicate groups and fed the experimental diets for 11 weeks. After the feeding trial, growth performance, feed utilization efficiency, survival, condition factor and somatic indices showed no significant variation among dietary groups. Similarly, immune parameters and hematological indices remained comparable across all treatments. Aspartate aminotransferase and alanine aminotransferase levels were lower, whereas antioxidant capacity was higher in all supplemented groups. After the Edwardsiella piscicida challenge, cumulative survival, immune parameters and antioxidant capacity were significantly higher in probiotics and Syn groups than in Con group. The expression of AMPs (hepcidin, β-defensin, LEAP-2, NK-lysin and pleurocidin) was significantly upregulated in fish fed LP, BS, BL and Syn diets, whereas prebiotic supplementation showed a minimal effect on AMPs transcription. Therefore, these results demonstrate that dietary probiotics and synbiotics effectively enhance immunity, antioxidant defense and disease resistance of P. olivaceus when fed LFM diets without compromising growth performance. These findings support the use of such additives as sustainable alternatives to chemotherapeutic agents in intensive P. olivaceus aquaculture.
Metabolites synthesized by the gut microbial community act as critical mediators in shaping host-pathogen interactions by influencing host immunity, metabolism and behaviour. Previously, S. Typhi infection was reported to induce heritable adaptive avoidance behaviour mediated by dopaminergic signalling and insulin signalling in C. elegans. However, the role of pathogen-derived biomolecules which include metabolites that modulate neuronal function through the gut-brain axis, in regulating these heritable responses remain unexplored. In this study, S. Typhi metabolites were identified as potent modulators capable of inducing infection-like host responses even in the absence of live bacterial infection. Among five extraction methods tested, 80 % methanolic extracted metabolites significantly reduced lifespan (∼41.1 % reduction with p = 0.0409) and induced heritable avoidance response (p < 0.0001) similar to that of whole bacterial infection. LC-MS profiling revealed uniquely enriched and differentially enriched metabolites in S. Typhi M5 extract with 315 metabolites exclusively detected in S. Typhi and several redox-active and neuroactive active metabolites exhibiting higher abundance compared to control E. coli OP50 M5 extract. Pathway enrichment analysis indicated significant enrichment in D-amino acid, galactose, phenylalanine, tyrosine and tryptophan biosynthesis and glutathione metabolism pathways. Consistently, several redox active and neuroactive compounds, such as p-benzoquinone, harmane, and glutathione-related dipeptides, were abundant in S. Typhi metabolome. Exposure to these metabolites disrupted dopaminergic regulation, evidenced by altered DAT-1GFP localization and loss of avoidance behaviour in dat-1 mutants, corroborated by reduced dat-1 and cat-2 expression. Additionally, a significant upregulation of immune-related genes (tol-1, clec-67, skn-1) and activation of the DAF-2/DAF-16 insulin signalling pathway indicated immune activation and host defence responses. Collectively, these findings suggest that S. Typhi derived metabolites can reprogram host neuroimmune signalling through the gut-brain axis, contributing to metabolite-driven adaptive behavioural outcomes in C. elegans.
Naringenin (NAR), a citrus-derived flavanone, is a bioactive compound widely recognized for its potent antidiabetic, anti-inflammatory, and antioxidant properties. However, a significant gap remains in understanding its therapeutic potential against Aflatoxin B1 (AFB1)-induced male reproductive dysfunction. This study investigated the mitigating role of NAR against AFB1-induced reproductive toxicity in rabbit bucks, focusing on redox imbalance, the IL-27/NLRP3 Inflammasome axis, and pyroptosis pathways. Thirty-six mature male rabbits were randomly assigned to four equal groups (n = 9 per group) for two months: control (basal diet), AFB1 (0.3 mg/kg diet), NAR (100 mg/kg diet), and AFB1+NAR (0.3 mg AFB1 + 100 mg NAR/kg diet). Results showed that AFB1 significantly impaired systemic metabolic function, characterized by reduced renal efficiency, elevated hepatic enzymes, increased lipid accumulation, and a marked decline in serum protein concentrations (P < 0.05). Furthermore, AFB1 exposure led to significant deterioration in semen quality, diminished immunological and antioxidant biomarkers, and reduced testosterone levels (P < 0.05). At the cellular level, AFB1 toxicity triggered DNA damage, oxidative stress, adipokine dysregulation, and pyroptosis. Supplementing the diets of AFB1-challenged bucks with NAR substantially restored systemic metabolic function, as evidenced by improved hepatic and renal parameters and modulated lipid profiles (P < 0.01). Furthermore, NAR supplementation mitigated testicular damage, enhanced immunological biomarkers, and suppressed both oxidative stress and inflammation, ultimately rescuing semen quality. Notably, the protective effect may be associated with modulation of the IL-27/NLRP3 pathway. These findings suggest that the IL-27/NLRP3 inflammasome pathway serves as a crucial protective mechanism against AFB1-induced inflammation and pyroptosis. Ultimately, this study demonstrates that dietary NAR effectively mitigates AFB1-induced reproductive dysfunction in rabbits, highlighting its potential as a therapeutic agent for preserving reproductive health.
Brain infections are often life-threatening and have been linked to the development of neurodegenerative diseases. The fruit fly Drosophila melanogaster is a valuable experimental model to study immunity and the pathophysiology of brain infections. The exact cellular pathways through which brain-specific immune responses are mounted in Drosophila, however, remain poorly characterized. Here, we investigated how brain-specific or systemic infection with Micrococcus luteus and Escherichia coli bacteria activates the Drosophila NF-κB innate immune pathways Toll and immune deficiency (IMD) in the central nervous system of the fly. We tested the hypothesis that these pathways are acutely activated in the Drosophila brain, and that their activation persists over time, even if bacteria have been cleared. We demonstrate that in control genotypes, brain-specific bacterial infection leads to Drosomycin (Drs, Toll pathway) and Diptericin B (DiptB, IMD pathway) upregulation and that glia appear to be the primary cell type mounting this immune response at both early and later time points, although some activation is observed in neurons as well. We show that the upregulation of Drs and DiptB expression also depends on canonical components of the Toll and IMD pathways, respectively. Interestingly, we found that systemic infection with M. luteus leads to brain-specific Drs activation and that signals from the fat body and hemocytes can activate the Toll pathway in the brain, pointing to an inter-organ communication. Together, these results contribute to our understanding of how non-lethal bacterial infections result in activation of NF-κB immunity in the Drosophila brain that could potentially be targeted to prevent progression of neurodegeneration.
Type 2 immunity, characterized by eosinophil activation, is essential for pinnipeds to control helminth infections-one of the leading causes of pup mortality-yet the developmental drivers of eosinophilic responses in free-ranging mammals remain poorly understood. We evaluated tissue eosinophil infiltration in South American fur seal (Arctocephalus australis) pups on Guafo Island (northern Patagonia, Chile) following an inflammatory challenge with phytohemagglutinin (PHA). Physiological and immunological markers were measured at birth, six weeks, and ten weeks of age to assess how developmental stage influences later eosinophilic responsiveness. Neonatal cholesterol received the strongest relative support among candidate predictors, with lower plasma cholesterol at birth associated with stronger eosinophilic responses at ten weeks of age. In contrast, cholesterol measured at ten weeks showed a positive association with eosinophil infiltration, indicating a developmental shift in this relationship. Neonatal total globulin concentrations and ten-week creatinine levels were also associated with tissue eosinophil counts. Together, these findings indicate that both early-life metabolic context and contemporaneous physiological condition contribute to variation in eosinophil recruitment. Importantly, the results highlight the neonatal period-strongly influenced by maternal metabolic and inflammatory environment-as a critical window shaping later inflammatory capacity. By linking developmental metabolic markers with induced immune responses, this study advances understanding of how early-life conditions influence immune trajectories in free-ranging pinnipeds facing intense parasitic pressure.
Cathepsins are a major class of lysosomal proteases that play key roles in various physiological, immune, and pathological processes. Although cathepsins have been reported in some teleost fish, their inflammatory responses have not yet been characterized in Takifugu obscurus. In this study, 14 cathepsin genes were first systematically characterized in T. obscurus, which were further classified into cysteine and aspartic proteases. Phylogenetic analysis revealed that lineage-specific expansion (e.g., Cathepsin B, D, L1) and loss (e.g., Cathepsin A, E, W) might have occurred in the cathepsin family. Structural prediction showed that the cathepsin family has conserved function domains (e.g., inhibitor_I29 and peptidase_C1 domains) and a shared α/β-fold core in the tertiary structures. Tissue distribution analysis revealed high expression of cathepsins in immune-related tissues (liver, head kidney) and mucosal barriers (intestine, skin). Upon stimulation with bacteria (Edwardsiella tarda, Aeromonas hydrophila, Vibrio harveyi) and poly (I:C), cathepsins displayed dynamic and pathogen-specific expression patterns. Several members (e.g., Cathepsin B-like, S and Z) were consistently upregulated, suggesting their potential involvement in antibacterial and antiviral immunity. Collectively, our findings provide insights into the molecular mechanisms of cathepsins in T. obscurus and contribute to understanding the roles of cathepsins in teleost immunity.
Edwardsiella tarda is a severe aquaculture and zoonosis pathogen that infects a wide range of fish, especially the Oliver flounder fish. Investigation of its pathogenetic mechanisms is important for developing an effective protective agent, such as a vaccine or antibiotics. In this study, we characterized the involvement of two target genes, lon protease and tryptophan operon gene trpA, in the virulence and pathogenesis pathways of E. tarda. Deletion of the trpA gene induced tryptophan auxotrophy. This ensured clearance from the host and made the microbe more vulnerable to acidic and oxidant stress, which are the main antimicrobial implements of host innate immunity. Lon deletion caused impaired motility and intracellular invasion by downregulating the overall flagella assembly pathway genes, as revealed by a transcriptome analysis. Those biological features were in line with the in vivo challenge results. The groups challenged with the mutant strain showed significantly higher survival rates and delayed mortality onsets than the group challenged with the wild-type strain, which showed rapid and extensive mortality. Most importantly, the cytokine profiles of the fish after challenge contribute to understanding the mechanism of this enhanced survival. Instead of an uncontrolled inflammatory state, the mutant strain administration elicited distinct immune responses. The Δlon strain induced a robust IFN-γ-mediated response, whereas the ΔtrpA strain was characterized by the downregulation of pro-inflammatory cytokines; notably, both mutants contributed to restored IL-10 regulation. By identifying the target genes and their involvement in virulence pathways, this study provides insights for the development of effective vaccine candidates against edwardsiellosis in aquaculture and its surroundings.