Non-coding RNAs (ncRNAs) have emerged as critical regulators of host immune responses against pathogen infection in aquatic animals, yet their roles in reptiles remain largely unexplored. Herein, we investigated the splenic ncRNA expression profiles of Chinese soft-shelled turtles (Pelodiscus sinensis) following Aeromonas hydrophila infection. Histopathological analysis at 24 hours post infection revealed severe splenic lesions, including the obscure red-white pulp demarcation, lymphocyte infiltration, necrosis, and increased melano-macrophages. Transcriptome sequencing identified 843 differentially expressed (DE) mRNAs (DEmRNAs), 108 DE microRNA, 87 DE lncRNAs, and 341 DE circRNAs. Competing endogenous RNA (ceRNA) networks were constructed with these DE RNAs, yielding 253 lncRNA-miRNA-mRNA and 436 circRNA-miRNA-mRNA triplets. Hub nodes showed mRNA LOC102455872 (degree = 10), lncRNAs MSTRG.3526.7 and MSTRG.8091.1 (degree = 10), circRNA novel-circ-026391 (degree = 12), and miRNA novel-m0923-3p (degree = 7) had the large connectivity of network. Functional enrichment revealed four pathways commonly involved in both networks, including MAPK signaling, cell adhesion molecules, notch signaling, and tryptophan metabolism. Notably, tryptophan metabolism and MAPK signaling contained the large hub RNAs, suggesting they served as critical bridges of ceRNA networks. This study described the first comprehensive ceRNA networks in a reptile following bacterial infection and identified novel ncRNAs serving as critical regulators in turtles defending against A. hydrophila.
Largemouth bass virus (LMBV) is the most prevalent and threatening virus in largemouth bass aquaculture. To establish effective control measures of LMBV, this study vaccinated broodstock with an LMBV MCP subunit vaccine to assess whether the subunit vaccine could activate maternal immunity to block vertical transmission of LMBV. Fourteen days post-immunization, significant upregulation of IgM, IgT, IL-6, TNF-α, and CD4 gene expression was observed in the relevant tissues of broodstocks. Serum titers reached their peak at 21 days post-immunization as detected by ELISA within 28 days. After challenge, the subunit vaccine achieved a relative protection rate of 87.4%. The MCP + LMBV group showed a significant reduction in viral load in the relevant tissues and a marked alleviation of histopathological damage compared to the Control + LMBV group. Compared to the Control group, vaccinated broodstocks exhibited normal spawning behavior, with no significant differences in spawning trends or total egg production, while egg production in the Control + LMBV group was significantly reduced. The fertilization and hatching rates in the MCP + LMBV group were 94.06% and 94.50%, respectively, with the hatching rate 27.77% higher than the Control + LMBV group. In the MCP + LMBV group, the virus positivity rate for both gonads and fertilized eggs were 0.0%. This study confirms that LMBV can be transmitted vertically from infected broodstock to their offspring, however, the MCP subunit vaccine can activate maternal immunity to block vertical transmission of LMBV, which provides a new approach to protect largemouth bass offspring from LMBV infection.
It remains unknown whether vitamin E (VE), a fat-soluble vitamin known for its potent antioxidant effects, alleviates the stress caused by ammonia-N exposure in post-larval Pacific white shrimp (Penaeus vannamei). Five isonitrogenous (crude protein: 32.5%) and isolipidic (crude lipid: 9.22%) diets were formulated containing DL-α-tocopheryl acetate at concentrations of 0, 40, 80, 120 and 160 mg/kg (E13.1, E50.6, E90.5, E130.6 and E163.9, respectively). Post-larval P. vannamei individuals (3.95 ± 0.21 mg) were randomly assigned to 20 tanks and fed the experimental diets for 34 days. After the feeding trial, the shrimp were challenged with ammonia-N for 48 h to evaluate their stress tolerance and immune responses. The shrimp groups fed diet supplemented with VE exhibited significantly improved growth performance and feed utilization compared to the E13.1 group (P < 0.05). However, dietary VE levels had no significant effect on survival (P > 0.05). Dietary VE supplementation increased LT50 from 36 h in the E13.1 group to 48 h in the E90.5 group. Dietary VE supplementation significantly upregulated the expression of antioxidant-related (SOD, CAT and GPx) and immune-related (Crustin, proPO, HSP70 and Penaeidin-3a) genes both after the feeding trial (0 h) and after ammonia-N exposure (48 h) (P < 0.05). Optimal dietary VE levels estimated using broken-line and second-order polynomial regression analyses ranged from 89.3 to 104.8 mg/kg for growth and 94.9 to 113.9 mg/kg for ammonia-N stress resistance in post-larval P. vannamei. Overall, these findings indicate that dietary VE mitigates acute ammonia-N induced damage in shrimp by enhancing antioxidant defense and immune responses.
NOD-like receptors (NLRs) are cytosolic pattern-recognition receptors that detect pathogen-associated and damage-associated molecular patterns and mediate innate immune signaling in vertebrates. However, the genomic repertoire, evolutionary diversification, and infection-associated expression of NLR genes remain poorly defined in non-model amphibians. In this study, 66 NLR genes were identified from the Chinese spiny frog (Quasipaa spinosa) genome and designated as QsNLR1-QsNLR66. These genes were unevenly distributed across chromosomes and were classified into three phylogenetic groups, with most members exhibiting conserved motif architectures. Gene duplication analysis indicated that dispersed duplication was the main contributor to QsNLR expansion. Synteny analysis detected five conserved orthologous gene pairs between Q. spinosa and Pelophylax nigromaculatus, suggesting partial conservation of NLR genomic organization between the two amphibians. Ka/Ks analysis showed that several duplicated gene pairs, including NLRC3-like/QsNLR36 and NLRC3-like/QsNLR50, exhibited Ka/Ks ratios greater than one, suggesting potential sequence divergence after duplication. Spleen RNA sequencing (RNA-seq) after Aeromonas hydrophila challenge revealed enrichment of immune-related Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Weighted gene co-expression network analysis linked several QsNLRs to infection-associated modules, among which QsNLR57 was co-expressed with CYBB, ADAM17, SPI1, and HK2. RT-qPCR using time-matched phosphate-buffered saline (PBS) controls showed distinct temporal patterns, with stronger induction of QsNLR29, QsNLR57, and QsNLR66 and weaker or delayed responses of QsNLR50 and QsNLR56. These results characterize the NLR repertoire of Q. spinosa and identify infection-associated QsNLR candidates for future studies of antibacterial immunity in amphibians.
Type I interferons (IFNs) are a class of pleiotropic cytokines that play a pivotal role in host defense against pathogenic invaders by binding with specific heterodimeric receptors. Among teleosts, these receptors consist of diverse subunits, including CRFB1, CRFB2, and CRFB5, whereas their pairing patterns and structural basis of interactions remain ill-defined. In this study, we identified and characterized three type I IFN receptor genes (CaCRFB1, CaCRFB2, and CaCRFB5) in the humpback grouper (Cromileptes altivelis), all sharing a common architecture of two extracellular FNIII domains. Furthermore, all three CaCRFB genes exhibited not only constitutive expression across all examined tissues but also significant transcriptional upregulation in response to poly I:C, LPS and pathogenic challenges (Vibrio harveyi and nervous necrosis virus), highlighting their active involvement in innate immunity. To elucidate their assembly mechanisms, we integrated molecular docking with co-immunoprecipitation (Co-IP) assays targeting their extracellular domains. Our results validated the canonical heterodimerization of the short-chain CaCRFB5 with both long-chain subunits (CaCRFB1 and CaCRFB2) as well as a noncanonical interaction of CaCRFB1/CaCRFB2, suggesting a more intricate receptor pairing in teleosts. Structural interface analysis revealed that these interactions are principally driven by electrostatic forces, including hydrogen bonds and salt bridges. Intriguingly, while the membrane-proximal FNIII domain II mediates canonical interactions (CaCRFB1/CaCRFB5 and CaCRFB2/CaCRFB5), it is the membrane-distal FNIII domain I that orchestrates the noncanonical CaCRFB1/CaCRFB2 pairing. These findings shed light on the structural basis of receptor heterodimerization, enriching the understanding of the teleost type I IFN system.
As important members of the ETS superfamily, the E74-like factor (ELF) transcription factor family regulates gene transcription through a conserved ETS domain and plays critical roles in immune regulation. However, the evolutionary characteristics and functions of this family in lampreys (Lethenteron reissneri) remain unclear. In this study, the ELF gene family of lampreys (Lr-ELF1, Lr-ELF2, Lr-ELF3, and Lr-ELF5) was systematically identified, and their molecular evolutionary features and immune response functions were investigated. Phylogenetic analysis revealed evolutionary characteristics reflecting the transition from jawless to jawed vertebrates. Domain architecture, gene structure, and three-dimensional structural analyses indicated that these genes appear to be conserved among vertebrates, with their three-dimensional structures showing high similarity to the core secondary structural elements of human homologous proteins. Synteny analysis demonstrated significant differences in the genomic neighborhoods of ELF genes between lampreys and jawed vertebrates. Quantitative real-time PCR (qRT-PCR) was performed with three biological and three technical replicates; relative expression levels were calculated using the ΔCt method, and statistical analysis was carried out with GraphPad Prism 9. Quantitative real-time PCR (qRT-PCR) results suggested that the ELF gene family may be involved in immune defense. This study not only enriches our understanding of the evolution of ELF genes but also provides new insights into the roles of lamprey ELFs in immune defense.
Schistosomiasis is a serious public health concern, with transmission facilitated by a small number of freshwater snail intermediate host species. Infection outcomes vary greatly across the primary vector genera, Biomphalaria (for Schistosoma mansoni), Bulinus (for S. haematobium), and Oncomelania (for S. japonicum), even within species, ranging from full resistance to high compatibility. Omics methods have altered this field by correlating host genotype, baseline immunological status, and time-resolved responses to whether invading miracidia are eliminated or develop sporocysts. Evidence from genomes, transcriptomics, proteomics, and epigenomics suggests that resistance is frequently primed prior to exposure. However, the clearest divergence between resistant and susceptible trajectories occurs during a small early window (<12-48 h) after penetration. During this time, recognition, hemocyte recruitment, and soluble effector deployment either come together quickly or are delayed and guided by parasite-derived modulators. Established infections cause the host to adapt to chronic conditions through immune regulation, metabolic reprogramming, tissue and neuroendocrine remodeling, microbiome modification, and parasite castration. Comparative genomics reveals that each vector genus has evolved its own immunogenomic profile, which includes lineage-specific expansions of recognition and effector gene families. Together, these findings can help with field surveillance and intervention by providing molecular compatibility markers, functional tools for testing candidate genes, and tactics that target parasite-derived immune modulators. Integrated multi-omics approaches are a top priority, yet they are still limited in snail vectors compared to other disease vector systems.
The red swamp crayfish (Procambarus clarkii) is an economically important freshwater crustacean in China. However, the harvest of farmed crayfish is often reduced because of high mortality caused by hypoxic stress. In this study, three single nucleotide polymorphisms in the hypoxia-inducible factor-1α gene (HIF1α) were identified in the investigated crayfish germplasm populations, and these variants were associated with the hypoxia tolerance of crayfish. Further association analysis based on three different crayfish populations identified the most favorable HIF1α haplotype (CC-CC-CC) for hypoxia tolerance. Furthermore, to verify the function of HIF1α, RNA interference, transcriptomic analysis, and biochemical assays were employed to investigate the mechanisms underlying the role of this gene in the hypoxic adaptation process of crayfish. The results showed that the inhibition of HIF1α significantly impaired hypoxic adaptation at the metabolic and physiological levels in crayfish, as evidenced by blocked anaerobic respiration, disrupted energy metabolism, and compensatory increase in antioxidant capacity. Collectively, these results provide new insights into the molecular mechanisms underlying hypoxic adaptation in crayfish and lay a theoretical foundation for the cultivation of hypoxia-tolerant crayfish varieties.
Malaria, a vector-borne disease caused by Plasmodium and transmitted by Anopheles mosquitoes, remains a major health burden in tropical regions. During blood feeding, the mosquito midgut plays a key role in blood digestion and parasite development, where immune responses against the parasite occur. This study employed a stepwise immunoinformatics approach to design a conserved multiepitope vaccine based on the midgut-specific SGU protein of Anopheles. B and T-cell epitopes were selected based on antigenicity and non-allergenicity, linked using AAY and GPGPG linkers, and combined with β-defensin as an adjuvant to enhance immunogenicity. Physicochemical analysis indicated vaccine stability, and codon optimization supported efficient expression in E. coli BL21. Docking confirmed strong interaction with TLR3, and the expressed recombinant protein (∼16 kDa) successfully elicited an immune response. Further studies are needed to assess its potential to block SGU function and inhibit Plasmodium development in the mosquito midgut.
Chitooligosaccharides (COS) has shown promising potential in enhancing the immunoprotective effects of Apostichopus japonicus. However, its intracellular distribution in within the coelomocytes of this species remains unclear. In this study, using fluorescein isothiocyanate (FITC) labeled COS technology combined with fluorescence microscopy and a microplate detection system, the uptake kinetics and subcellular distribution of FITC-COS in five types of coelomocytes (amoebocytes, fusiform cells, lymphocytes, hyaline cells and spherulocytes) were analyzed. The results indicate that the cellular uptake of FITC-COS was time- and dose-dependent, and exhibits distinct distribution patterns across various cell types: diffuse distribution in spherulocyte, polar distribution in fusiform cell, and early perinuclear punctate aggregation in lymphocytes. In all cell types, FITC-COS does not enter the nucleus. This study reveals the dynamic distribution patterns of COS in the immune cells of Apostichopus japonicus, providing cytological evidence for elucidating its immune-enhancing mechanisms and theoretical support for the development of COS as an aquatic immunostimulant. However, this study did not conduct co-localization experiments of COS with specific organelles, and its exact intracellular target remains to be further verified.
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.
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.
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.
