Diversification of Toll-like receptor 1 in swamp eel (Monopterus albus).

Toll-like receptor 1 (TLR1) is a pattern recognition receptor that plays critical roles in triggering immune activation via detecting bacterial lipoproteins and lipopeptides. In this study, the genetic characteristic of TLR1 was studied for an important aquaculture fish, swamp eel Monopterus albus. The eel has been seriously threatened by infectious diseases. However, a low level of genetic heterogeneity in the fish that has resulted from a demographic bottleneck presents further challenges in breeding for disease resistance. A comparison with the homologue of closely related species M. javanensis revealed that amino acid replacement (nonsynonymous) but not silent (synonymous) differences have accumulated nonrandomly over the coding sequences of the receptors at the early stage of their phylogenetic split. The combined results from comparative analyses of nonsynonymous-to-synonymous polymorphisms showed that the receptor has undergone significant diversification in M. albus driven by adaptive selection likely after the genetic bottleneck. Some of the changes reported here have taken place in the structures mediating heterodimerization with co-receptor TLR2, ligand recognition, and/or formation of active signaling complex with adaptor, which highlighted key structural elements and strategies of TLR1 in arms race against exogenous challenges. The findings of this study will add to the knowledge base of genetic engineering and breeding for disease resistance in the eel.

Population-level immunologic variation in wild threespine stickleback (Gasterosteusaculeatus).

Wild organisms are regularly exposed to a wide range of parasites, requiring the management of an effective immune response while avoiding immunopathology. Currently, our knowledge of immunoparasitology primarily derives from controlled laboratory studies, neglecting the genetic and environmental diversity that contribute to immune phenotypes observed in wild populations. To gain insight into the immunologic variability in natural settings, we examined differences in immune gene expression of two Alaskan stickleback (Gasterosteus aculeatus) populations with varying susceptibility to infection by the cestode Schistocephalus solidus. Between these two populations, we found distinct immune gene expression patterns at the population level in response to infection with fish from the high-infection population displaying signs of parasite-driven immune manipulation. Further, we found significant differences in baseline immune gene profiles between the populations, with uninfected low-infection population fish showing signatures of inflammation compared to uninfected high-infection population fish. These results shed light on divergent responses of wild populations to the same parasite, providing valuable insights into host-parasite interactions in natural ecosystems.

Galectin-8-like isoform X1 mediates antibacterial, antiviral, and antioxidant responses in red-lip mullet (Planiliza haematocheilus) through positive modulation of pro-inflammatory cytokine, chemokine, and enzymatic antioxidant activity.

Galectin 8 belongs to the tandem repeat subclass of the galectin superfamily. It possesses two homologous carbohydrate recognition domains linked by a short peptide and preferentially binds to ß-galactoside-containing glycol-conjugates in a calcium-independent manner. This study identified Galectin-8-like isoform X1 (PhGal8X1) from red-lip mullet (Planiliza haematocheilus) and investigated its role in regulating fish immunity. The open reading frame of PhGal8X1 was 918bp, encoding a soluble protein of 305 amino acids. The protein had a theoretical isoelectric (pI) point of 7.7 and an estimated molecular weight of 34.078 kDa. PhGal8X1 was expressed in various tissues of the fish, with prominent levels in the brain, stomach, and intestine. PhGal8X1 expression was significantly (p < 0.05) induced in the blood and spleen upon challenge with different immune stimuli, including polyinosinic:polycytidylic acid, lipopolysaccharide, and Lactococcus garvieae. The recombinant PhGal8X1 protein demonstrated agglutination activity towards various bacterial pathogens at a minimum effective concentration of 50 µg/mL or 100 µg/mL. Subcellular localization observations revealed that PhGal8X1 was primarily localized in the cytoplasm. PhGal8X1 overexpression in fathead minnow cells significantly (p < 0.05) inhibited viral hemorrhagic septicemia virus (VHSV) replication. The expression levels of four proinflammatory cytokines and two chemokines were significantly (p < 0.05) upregulated in PhGal8X1 overexpressing cells in response to VHSV infection. Furthermore, overexpression of PhGal8X1 exhibited protective effects against oxidative stress induced by H2O2 through the upregulation of antioxidant enzymes. Taken together, these findings provide compelling evidence that PhGal8X1 plays a crucial role in enhancing innate immunity and promoting cell survival through effective regulation of antibacterial, antiviral, and antioxidant defense mechanisms in red-lip mullet.

Fish-specific Toll-like receptor 14 (TLR14) from Asian swamp eel (Monopterus albus) is involved in immune response to bacterial infection.

Toll-like receptors (TLRs) are a class of pattern recognition receptors (PRRs) that play a critical role in innate immune responses against pathogens. In the present study, a fish-specific TLR14 was identified and characterized from Monopterus albus (named MaTLR14), which consisted of a 2658 bp open reading frame encoding a protein of 885 amino acids. Phylogenetic analysis revealed that MaTLR14 belong to the TLR1 subfamily and shared the highest similarity to Paralichthys olivaceus TLR14. Immunohistochemistry assay showed that MaTLR14 mainly located in intestinal epithelial cells of hindgut. Immunofluorescence revealed that MaTLR14 largely localized to the intracellular region and partially co-localized with cell membrane of HeLa cells. The expression levels of MaTLR14 were upregulated in the liver, spleen, foregut and hindgut post infection with Aeromonas hydrophila. When stimulated with LPS and Flagellin, the MaTLR14 expression was elevated in isolated peripheral blood leukocytes. Further studies showed that recombinant MaTLR14-LRR could bind to both the gram-negative and gram-positive bacteria and cause agglutination. Subsequently, the signaling pathway of MaTLR14 was investigated. Confocal microscopy and co-immunoprecipitation assay demonstrated that MaTLR14 recruited MyD88 as adaptor. When overexpressed, MaTLR14 augmented the expression of TRAF6 and phosphorylation of ERK and p65, activated NF-κB and AP-1 and elicited the expression of il-6 and tnf-α. Collectively, MaTLR14 plays an important role in the microorganism recognition and signaling transduction.

Characterization of reference genes for qRT-PCR normalization in rice-field eel (Monopterus albus) to assess differences in embryonic developmental stages, the early development of immune organs, and cells infected with rhabdovirus.

Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) has become a popular technique to assess gene expression. Suitable reference genes are normally identified first to ensure accurate normalization. The aim of the present study was to select the most stable genes in embryonic developmental stages, the early development of immune organs, and cells infected with Chinese rice-field eel rhabdovirus (CrERV) of the rice-field eel (Monopterus albus). Four reference genes, including those encoding 18S ribosomal RNA (18SrRNA), beta actin (ß-actin), elongation factor 1 alpha (EF1ɑ), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were assessed using geNorm, NormFinder, BestKeeper, and RefFinder software. Analyses indicated the stability ranking was 18SrRNA > ß-actin > GAPDH > EF1α in the embryonic stage, with 18SrRNA as the most stable reference gene. For immunity-related organs at different developmental stages, the order in the thymus was ß-actin > GAPDH > EF1α > 18SrRNA, with ß-actin as the most stable gene. In both spleen and kidney tissues, the rank order was EF1ɑ > GAPDH > ß-actin > 18SrRNA, with EF1α as the most stable gene. Furthermore, in rice-field eel kidney (CrE-K) cells infected with CrERV, the ranking was EF1ɑ > ß-actin > GAPDH > 18SrRNA, with EF1α as the most stable gene. The results for cells infected with CrERV were verified by testing signaling pathway genes catenin beta 1 (CTNNB1) and NOTCH1 based on the above four genes after virus infection in CrE-K cells. This study laid the foundation for choosing suitable reference genes for immunity-related gene expression analysis in rice-field eel.

Immune Gene Expression Covaries with Gut Microbiome Composition in Stickleback.

Commensal microbial communities have immense effects on their vertebrate hosts, contributing to a number of physiological functions, as well as host fitness. In particular, host immunity is strongly linked to microbiota composition through poorly understood bi-directional links. Gene expression may be a potential mediator of these links between microbial communities and host function. However, few studies have investigated connections between microbiota composition and expression of host immune genes in complex systems. Here, we leverage a large study of laboratory-raised fish from the species Gasterosteus aculeatus (three-spined stickleback) to document correlations between gene expression and microbiome composition. First, we examined correlations between microbiome alpha diversity and gene expression. Our results demonstrate robust positive associations between microbial alpha diversity and expression of host immune genes. Next, we examined correlations between host gene expression and abundance of microbial taxa. We identified 15 microbial families that were highly correlated with host gene expression. These families were all tightly correlated with host expression of immune genes and processes, falling into one of three categories-those positively correlated, negatively correlated, and neutrally related to immune processes. Furthermore, we highlight several important immune processes that are commonly associated with the abundance of these taxa, including both macrophage and B cell functions. Further functional characterization of microbial taxa will help disentangle the mechanisms of the correlations described here. In sum, our study supports prevailing hypotheses of intimate links between host immunity and gut microbiome composition.IMPORTANCE Here, we document associations between host gene expression and gut microbiome composition in a nonmammalian vertebrate species. We highlight associations between expression of immune genes and both microbiome diversity and abundance of specific microbial taxa. These findings support other findings from model systems which have suggested that gut microbiome composition and host immunity are intimately linked. Furthermore, we demonstrate that these correlations are truly systemic; the gene expression detailed here was collected from an important fish immune organ (the head kidney) that is anatomically distant from the gut. This emphasizes the systemic impact of connections between gut microbiota and host immune function. Our work is a significant advancement in the understanding of immune-microbiome links in nonmodel, natural systems.

Molecular characterization, immune and xenobiotic responses of glutathione S-transferase omega 1 from the big-belly seahorse: Novel insights into antiviral defense.

Glutathione S-transferases (GSTs) are important enzymes involved in phase II detoxification and function by conjugating with the thiol group of glutathione. In this study, we isolated an omega class GST from the big-belly seahorse (Hippocampus abdominalis; HaGSTO1) to study the putative xenobiotic responses and defense ability against viral and bacterial infections in this animal. The isolated HaGSTO1 gene, with a cording sequence of 720 bp, encodes a peptide of 239 amino acids. The predicted molecular mass and theoretical isoelectric point of HaGSTO1 was 27.47 kDa and 8.13, respectively. In-silico analysis of HaGSTO1 revealed a characteristic N-terminal thioredoxin-like domain and a C-terminal domain. Unlike other GSTs, the C-terminal of HaGSTO1 reached up to the N-terminal, and the N-terminal functional group was cysteine rather than tyrosine or serine, as observed in other GSTs. Phylogenetic analysis showed the evolutionary proximity of HaGSTO1 with other identified vertebrate and invertebrate GST orthologs. For the first time, we demonstrated the viral defense capability of HaGSTO1 against viral hemorrhagic septicemia virus (VHSV) infection. All six nucleoproteins of VHSV were significantly downregulated in HaGSTO1-overexpressing FHM cells at 24 h after infection compared with those in the control. Moreover, arsenic toxicity was significantly reduced in HaGSTO1-overexpressing FHM cells, and cell viability increased. Real-time polymerase chain reaction analysis showed that HaGSTO1 transcripts were highly expressed in the pouch and gill when compared with those in other tissues. Blood HaGSTO1 transcripts were significantly upregulated after Edwardsiella tarda, Streptococcus iniae, lipopolysaccharide, and polyinosinic:polycytidylic acid challenge experiments. Collectively, these findings suggest the involvement of HaGSTO1 in the host defense mechanism of seahorses.

A newly discovered teleost disulfide isomerase, thioredoxin domain containing 5 (TXNDC5), from big-belly seahorse (Hippocampus abdominalis): Insights into its molecular and functional properties and immune regulatory functions.

The thioredoxin domain containing 5 (TXNDC5) is a recently discovered member of the protein disulfide isomerase family (PDI), which is mainly involved in the proper folding of and the correct formation of disulfide bonds in newly synthesized proteins via its disulfide isomerase and chaperone activities. Although the structural and functional features of mammalian TXNDC5 have been explored in previous studies, no studies have reported the functional characteristics of TXNDC5 in teleost fish. In this study, we report the identification and characterization of TXNDC5 from big-belly seahorse (Hippocampus abdominalis) (ShTXNDC5) accompanied by functional studies. The in-silico analysis revealed that the gene encodes a 433 amino acid (aa) long polypeptide chain with a predicted molecular weight of 49.3 kDa. According to homology analysis, ShTXNDC5 shares more than 55% sequence similarity with other teleost TXNDC5 proteins, and the alignment of the gene sequence convincingly reflects the accepted phylogeny of teleost. Analysis of the spatial distribution of ShTXNDC5 expression showed that its highest expression was observed in the ovary, gill, and pouch of seahorses. Moreover, significant upregulation of ShTXNDC5 transcription was noted in seahorse blood and kidney tissues in a time-dependent manner upon viral and bacterial immune challenges. Furthermore, considerable NADPH turnover, insulin reduction ability and significant cell survival effects of ShTXNDC5 were determined by the functional assay, revealing its capability to overcome cellular oxidative stress. Altogether, these findings expand our understanding of TXNDC5 at the molecular and functional levels, and its putative role in seahorse immunity.

Marine-Derived Chitosan Nanoparticles Improved the Intestinal Histo-Morphometrical Features in Association with the Health and Immune Response of Grey Mullet (Liza ramada).

Marine-derived substances are known for their beneficial influences on aquatic animals' performances and are recommended to improve intestinal health, immunity, and anti-oxidative status. The present study investigates the role of chitosan nanoparticles on the intestinal histo-morphometrical features in association with the health and immune response of Grey Mullet (Liza ramada). Chitosan nanoparticles are included in the diets at 0, 0.5, 1, and 2 g/kg and introduced to fish in a successive feeding trial for eight weeks. The final body weight (FBW), weight gain (WG), and specific growth rate (SGR) parameters are significantly increased while feed conversion ratio (FCR) decreases by chitosan nanoparticles compared to the control (p < 0.05). The morphometric analysis of the intestines reveals a significant improvement in villus height, villus width, and the number of goblet cells in chitosan-treated groups in a dose-dependent manner. Additionally, there is a positive correlation between the thickness of the enterocyte brush border and the chitosan dose, referring to an increasing absorptive activity. Histologically, the intestinal wall of Grey Mullet consists of four layers; mucosa, sub-mucosa, tunica muscularis (muscular layers), and serosa. The histological examination of the L. ramada intestine shows a normal histo-morphology. The epithelial layer of intestinal mucosa is thrown into elongated finger-like projections, the intestinal villi. The values of hemoglobin, hematocrit, red blood cells (RBCs), total protein (TP), albumin, and globulin are significantly increased in fish fed 1, and 2 g/kg of chitosan nanoparticles compared to fish fed 0 and 0.5 g/kg (p < 0.05). The highest levels of TP and albumin are observed in fish fed 1 g/kg diet (p < 0.05). The lysozyme activity and phagocytic index are significantly enhanced by feeding chitosan nanoparticles at 0.5, 1, and 2 g/kg, whereas the phagocytic activity is improved in fish fed 1 and 2 g/kg (p < 0.05). The highest lysozyme activity and phagocytic index are observed in fish fed 1 g/kg. SOD is significantly activated by feeding chitosan nanoparticles at 1 g/kg. Simultaneously, glutathione peroxidase (GPx) and catalase (CAT) activities also are enhanced by feeding chitosan at 1 and 2 g/kg, compared to fish fed 0 and 0.5 g/kg (p < 0.05). The highest GPx and CAT activities are observed in fish fed 1 g/kg (p < 0.05). Conversely, the malondialdehyde (MDA) levels are decreased by feeding chitosan at 1 and 2 g/kg, with the lowest being in fish fed 1 g/kg (p < 0.05). To summarize, the results elucidate that L. ramada fed dietary chitosan nanoparticles have a marked growth rate, immune response, and anti-oxidative response. These improvements are attributed to the potential role of chitosan nanoparticles in enhancing intestinal histo-morphometry and intestinal health. These results soundly support the possibility of using chitosan nanoparticles at 1-2 g/kg as a feasible functional supplement for aquatic animals.

ß-hydroxybutyrate (ß-HB) exerts anti-inflammatory and antioxidant effects in lipopolysaccharide (LPS)-stimulated macrophages in Liza haematocheila.

Poly-ß-hydroxybutyrate (PHB) can be hydrolyzed to ß-hydroxybutyrate (ß-HB) in the intestinal tract of animals, and dietary PHB supplementation could enhance the immunity and disease resistance of aquatic animals. Antioxidant system is responsive to PHB stimuli via MAPK/PI3K-Akt/TNF/NF-κB/TCR/TLR signaling pathways. However, the precise immunopotentiation mechanism needs further study. In this study, macrophages from spleen in Liza haematocheila was used to study the effect of ß-HB on cell viability and antioxidant function to illustrate the immunopotentiation mechanism of PHB. The results showed that ß-HB (100 µg/mL) promoted the viability of macrophages and balanced the production of reactive oxygen species, but inhibited the excessive production of intracellular nitric oxide. In order to further explore the immunopotentiation mechanism of ß-HB, LPS (100 µg/mL) was used to induce the inflammation and investigated the inhibitory effect of ß-HB on inflammation. The results showed that LPS could induce inflammation successfully, and ß-HB exerted anti-inflammatory and antioxidant effects in LPS-stimulated macrophages. Compared with LPS stimuli alone, the expression of anti-inflammatory genes NF-κBIA, MAP3K8 and TLR5 in ß-HB pretreatment group was up-regulated, and the expression of pro-inflammatory genes TNFSF6, TNF-α, PI3K, NF-κB and TLR1 down-regulated. It suggested that ß-HB inhibited the inflammatory response by up-regulation of anti-inflammatory genes such as NF-κBIA, thereby enhancing the immunity of the body.

Structural analysis of toll-like receptor 18 from soiny mullet (Liza haematocheila): Giving insights on the ligand binding mechanism of fish specific TLRs.

Toll-like receptors (TLRs) are important pattern recognition receptors (PRRs) of innate immune system, playing crucial roles in immune defense against pathogens. TLR18, a member of TLR1 family, is fish-specific TLR and involves in the immune response against bacterial infection. Currently, the structural biology of fish TLR18 is poorly elaborated. In this study, the structure and ligand binding of TLR18 (smTLR18) of soiny mullet (Liza haematocheila), an economically valuable aquaculture mugilid species, were analyzed. The extracellular domain (ECD) of smTLR18 formed an open-loop horseshoe-shaped structure with the concave surfaces made up of 19 parallel ß-strands (LRR1-LRR19), lacking Z-loop that seen in human TLR9. The intracellular Toll/interleukin (IL)-1 (TIR) domain contained a central 4-parallel ß-sheet (ßA-ßD) surrounded by 5 α-helices (αA-αE). Molecular docking analysis revealed that both ECD domain and TIR domain of smTLR18 could form homodimers. For the ECD homodimer, the main residues involved in dimer formation were located from LRR10 to LRR14. For the TIR homodimer, the residues involved in dimer formation were located in BB loop, αB helix, αC helix and DD loop. Ligand binding analyses revealed that peptidoglycans (PGNs) and lipopolysaccharides (LPS), two main bacterial pathogen-associated molecular patterns (PAMPs), were the potential ligands of smTLR18. The van der Waals and Coulombic interactions contributed to the interactions between smTLR18 and PGNs, while only van der Waals dominated the interactions between smTLR18 and LPS. The residues involved in ligands binding were located from LRR9 to LRR13. Our results provided the structural bases for elucidate the ligand binding of fish TLR18.

Efficacy of a formalin-inactivated Lactococcus garvieae vaccine in farmed grey mullet (Mugil cephalus).

Lactococcosis [Lactococcus garvieae (LG)] is one of the most prevalent bacterial diseases affecting grey mullet (Mugil cephalus) aquaculture. Therefore, the present research evaluated the efficacy of formalin-killed LG vaccine with an oil-based adjuvant in grey mullet under laboratory and field trials. The laboratory evaluation for LG vaccine and its cross-protection upon challenge in grey mullet found that single-dose immunization of formalin-killed LG with adjuvant resulted in 91.4% and 100% relative per cent survival (RPS) when challenged with homologous and heterologous strains. The levels of specific antibody titre and lysozyme activity increased significantly in the vaccinated group. Immune gene expression at 24 hr after challenge showed an increase in levels of pro-inflammatory and anti-inflammatory cytokines. A parallel field trial experiment was conducted to investigate the long-term effectiveness of the LG vaccine. Results demonstrated that at one month and three months post-immunization with heterologous strain, 100% RPS was recorded in the vaccinated group. The findings suggested that the formalin-inactivated LG vaccine strain (S3) protected grey mullet against LG infection for a period of three months.

Identification, molecular characterization, expression analysis and wound-healing ability of multifunctional calreticulin from big-belly seahorse Hippocampus abdominalis.

Calreticulin (CRT) is a multifunctional ubiquitous protein that is widely presented in all cells in eukaryotes except erythrocytes. CRT is well known for diverse cellular functions such as endoplasmic reticulum (ER)-specialized protein quality control during protein synthesis and folding, in-vivo Ca2+ homeostasis, antigen presentation, phagocytosis, wound-healing, proliferation, adhesion, and migration of cells. In the current study, we identified CRT from Hippocampus abdominalis (HaCRT) and analyzed expression profiles and functional properties. The cDNA sequence of HaCRT was identified with an open reading frame of 1226 bp. The molecular weight of HaCRT was estimated as 49 kDa. The in-silico study revealed conserved sequence arrangements such as two CRT signature motifs (5'-KHEQSIDCGGGYVKVF-3' and 5'-LMFGPDICG-3'), triplicate repeats (5'-IKDPEAKKPEDWD-3', 5'-IPDPDDTKPEDWD-3', 5'-IPDPDAKKPDDWD-3'), signal peptide and an ER-targeting 5'-KDEL-3' sequence of HaCRT. Close sequence similarity of HaCRT was observed with Hippocampus comes from phylogenetic analysis and pairwise sequence comparison. From quantitative polymerase chain reaction (qPCR) results, HaCRT was ubiquitously distributed in all tested tissues and expression levels of HaCRT were significantly modulated in blood, liver and gill tissues after stimulation with Streptococcus iniae, Edwardsiella tarda, polyinosinic:polycytidylic acid, and lipopolysaccharides. Bacterial- and pathogen-associated molecular patterns-binding activities were observed with recombinant HaCRT (rHaCRT). The treatment of murine macrophages with rHaCRT induced the expression of immune genes, such as tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), inducible nitric oxide synthase (iNOS), and interleukin-1ß (IL-1ß). Furthermore, rHaCRT exhibited wound-healing ability. Based on the results from the above study, we suggest that HaCRT play an indispensable role in the immunity of big-belly seahorses by recognition and elimination of pathogens as well as the tissue repairing process.

Molecular and functional characterization of a mitochondrial glutathione reductase homolog from redlip mullet (Liza haematocheila): Disclosing its antioxidant properties in the fish immune response mechanism.

Glutathione reductase (GSHR) is a biologically important enzyme involved in the conversion of oxidized glutathione (GSSG) into its reduced form, reduced glutathione (GSH), with the catalytic activity of NADPH. Most animals and aquatic organisms, including fish, possess high levels of this enzyme system to neutralize oxidative stress in cells. The current study was conducted to broaden our knowledge of GSHR in fish by identifying a mitochondrial isoform of this enzyme (LhGSHRm) in redlip mullet, Liza haematocheila, and clarifying its structure and function. The complete open reading frame of LhGSHRm consists of 1527 base pairs, encoding 508 amino acids, with a predicted molecular weight of 55.43 kDa. Multiple sequence alignment revealed the conservation of important amino acids in this fish. Phylogenetic analysis demonstrated the closest evolutionary relationship between LhGSHRm and other fish GSHRm counterparts. In tissue distribution analysis, the highest mRNA expression of LhGSHRm was observed in the gill tissue under normal physiological conditions. Following pathogenic challenges, the LhGSHRm transcription level was upregulated in a time-dependent manner in the gill and liver tissues, which may modulate the immune reaction against pathogens. rLhGSHRm showed considerable glutathione reductase activity in an enzyme assay. Further, the biological activity of rLhGSHRm in balancing cellular oxidative stress was observed in both disk diffusion and DPPH assays. Collectively, these results support that LhGSHRm has profound effects on modulating the immune reaction in fish to sustain precise redox homeostasis.

TLR13, TLR22, TRAF6, and TAK1 in the soiny mullet (Liza haematocheila): Molecular characterization and expression profiling analysis.

Toll-like receptors (TLRs) and their associated signaling pathways play pivotal roles in the immune response to invading pathogens. Here, TLR13, TLR22, tumor necrosis factor receptor-associated factor 6 (TRAF6), and transforming growth factor-ß-activated kinase1 (TAK1) were characterized in the soiny mullet (Liza haematocheila), representative mugilid species that is widely cultured in Asia. The four mullet genes, which shared characteristic features with their counterparts in other teleosts, were ubiquitously expressed in all of the examined tissues, albeit with different expression patterns. Following Streptococcus dysgalactiae infection, the four genes were upregulated to different degrees in various mullet tissues. These results indicated that the four genes were involved in the mullet immune response to bacterial infection. To the best of our knowledge, this is the first characterization of these four genes in mullet. Our results provide a basis for future studies of TLR signaling pathways in mullet, as well as for similar studies in other mugilids.

Prior exposure to long-day photoperiods alters immune responses and increases susceptibility to parasitic infection in stickleback.

Seasonal disease and parasitic infection are common across organisms, including humans, and there is increasing evidence for intrinsic seasonal variation in immune systems. Changes are orchestrated through organisms' physiological clocks using cues such as day length. Ample research in diverse taxa has demonstrated multiple immune responses are modulated by photoperiod, but to date, there have been few experimental demonstrations that photoperiod cues alter susceptibility to infection. We investigated the interactions among photoperiod history, immunity and susceptibility in laboratory-bred three-spined stickleback (a long-day breeding fish) and its external, directly reproducing monogenean parasite Gyrodactylus gasterostei. We demonstrate that previous exposure to long-day photoperiods (PLD) increases susceptibility to infection relative to previous exposure to short days (PSD), and modifies the response to infection for the mucin gene muc2 and Treg cytokine foxp3a in skin tissues in an intermediate 12 L : 12 D photoperiod experimental trial. Expression of skin muc2 is reduced in PLD fish, and negatively associated with parasite abundance. We also observe inflammatory gene expression variation associated with natural inter-population variation in resistance, but find that photoperiod modulation of susceptibility is consistent across host populations. Thus, photoperiod modulation of the response to infection is important for host susceptibility, highlighting new mechanisms affecting seasonality of host-parasite interactions.

Immune defense parameters of wild fish as sensitive biomarkers for ecological risk assessment in shallow sea ecosystems: A case study with wild mullet (Liza haematocheila) in Liaodong Bay.

Environmental monitoring is important to the health management of an ecosystem. Biomarkers are particularly relevant because they are direct indicators of any toxic effects on organisms and are cheaper to use compared with chemical indicators, especially for extremely low-level organic contaminants. Fish can be significantly affected by pollutants, given their high trophic levels in aquatic food chains. Their immune function is closely related to their survival. The present study compared immune function-related parameters of wild mullet (Liza haematocheila) samples from low (Jinzhou) and high (Yingkou) polluted sites during the pre-winter (PW) and pre-breeding (PB) periods in Liaodong Bay, to evaluate the effect of water pollution on fish health and to explore potential biomarkers of coast water pollution. Compared with Jinzhou mullet, there was a significantly higher level of hematocrit in Yingkou mullet, but a significantly lower serum lysozyme level (P < 0.001), indicating that these fish were immunosuppressed. Significant differences occurred in the spleen between the two site populations. The abnormal: normal fish ratio in Yingkou L. haematochila was significantly higher than that of Jinzhou L. haematochila (2.5 times of that of Jinzhou during PB and nine times during PW). The splenic index of male Yingkou L. haematochila was 47.2% higher than that of Jinzhou L. haematochila in PW (P = 0.001). Moreover, histological observations showed that the spleen of the former was more congestive, with increased numbers (39.6% more) of melanomacrophage centers (MMCs) and changes in pigments (hemosiderin 8.3% higher and melanin 29.4% higher), compared with the latter. The splenic MMC area of Yingkou L. haematochila was significantly smaller than that of Jinzhou L. haematochila (P < 0.05) in PB, but showed no clear difference in PW (P > 0.05). Splenic MMC number was significantly higher in individual Yingkou L. haematochila with abnormal livers compared with normal Yingkou L. haematochila during both sampling periods. The splenic MMC area in abnormal livers was approximately four times those of normal individuals during PB in Yingkou L. haematochila. The number of splenic melanomacrophages (MM) in abnormal livers was approximately nine times those of the normal livers during PW. There were also differences in pigments in normal Yingkou individuals compared with normal Jinzhou samples during PW (melanin 29.4% higher and hemosiderin 8.3% higher). Based on these results, we suggest that serum lysozyme activity, splenic MM number and MMC (both number and area), and melanin of local fish have potential as sensitive biomarkers for the assessment of coastal water pollution.

Molecular and functional insights into a novel teleost malectin from big-belly seahorse Hippocampus abdominalis.

Malectin is a carbohydrate-binding lectin protein found in the endoplasmic reticulum (ER). It selectivity binds to Glc2-N-glycan and is involved in a glycoprotein quality control mechanism. Even though malectin may play a role in immunity, its role in innate immunity is not fully known. In the present study, we identified and characterized the malectin gene from Hippocampus abdominalis (HaMLEC). We analyzed sequence features, spatial expression levels, temporal expression profiles upon immune responses, bacterial and carbohydrate binding abilities and anti-viral properties to investigate the potential role of HaMLEC in innate immunity. The molecular weight and isoelectric point (pI) were estimated to be 31.99 kDa and 5.17, respectively. The N-terminal signal peptide, malectin superfamily domain and C-terminal transmembrane region were identified from the amino acid sequence of HaMLEC. The close evolutionary relationship of HaMLEC with other teleosts was identified by phylogenetic analysis. According to quantitative PCR (qPCR) results, HaMLEC expression was observed in all the examined tissues and high expression was observed in the ovary and brain, compared to other tested tissues. Temporal expression of HaMLEC in liver and blood tissues were significant modulated upon exposure to immunogens Edwardasiella tarda, Streptococcus iniae, polyinosinic:polycytidylic and lipopolysaccharide. The presence of carbohydrate binding modules (CBMs) of bacterial glycosyl hydrolases were functionally confirmed by a bacterial binding assay. Anti-viral activity significantly reduced viral hemorrhagic septicemia virus (VHSV) replication in cells overexpressing HaMLEC. The observed results suggested that HaMLEC may have a significant role in innate immunity in Hippocampus abdominalis.

Identification, expression profiling and functional characterization of interleukin 11a ortholog from redlip mullet Liza haematocheila: Insight into its roles in the inflammation and apoptosis regulation.

Interleukin 11 (IL-11) is a secretory cytokine with pleotropic properties, including anti-inflammatory and anti-apoptotic functions. This study aimed to functionally characterize a teleostean IL-11a ortholog from redlip mullet (LhIL-11a) through bioinformatic analysis, transcriptional expression profiling and protein function assays. The deduced LhIL-11a protein sequence is 200 amino acids long, with a predicted molecular weight of 23.168 kDa. Multiple sequence alignment indicates that LhIL-11a has a typical four-bundle architecture of α-helixes as observed in other IL-11s. The identity-similarity matrix show a higher identity between LhIL-11a and other fish IL-11a sequences. Phylogenetic analysis demonstrated that LhIL-11a falls within a clade including other fish counterparts. In the tissue distribution analysis, the highest constitutive expression of LhIL-11a mRNA was observed in the mullet gastrointestinal tract and brain tissues. Following the challenges with LPS, poly I:C and Lactococcus garvie, the transcription levels of LhIL-11a were significantly upregulated in both PBCs and liver. In the biological functional assay, recombinant LhIL-11a protein showed strong activities of suppressing pro-inflammatory cytokines and apoptotic gene expression in mullet kidney cells and reducing LPS stimulated NO production in murine macrophage cells. Overall, the findings in this study provide the experimental clues to understanding the functional roles of fish IL-11a in inflammation and apoptosis regulation during host defense against invading microbial pathogens.

Molecular and functional explication of thioredoxin mitochondrial-like protein (Trx-2) from big-belly seahorse (Hippocampus abdominalis) and expression upon immune provocation.

Thioredoxin (Trx) is a small ubiquitous multifunctional protein with a characteristic WCGPC thiol-disulfide active site that is conserved through evolution. Trx plays a crucial role in the antioxidant defense system. Further, it is involved in a variety of biological functions including gene expression, apoptosis, and growth regulation. Trx exists in several forms, with the cytosolic (Trx-1) and mitochondrial (Trx-2) forms being the most predominant. In this study, the mitochondrial Trx protein (HaTrx-2), from the big-belly seahorse (Hippocampus abdominalis) was characterized, and its molecular features and functional properties were investigated. The cDNA sequence of HaTrx-2 consists of a 519 bp ORF, and it encodes a polypeptide of 172 amino acids. This protein has a calculated molecular mass of 18.8 kDa and a calculated isoelectric point (pI) of 7.80. The highest values of identity (78.7%) and similarity (86.2%) were observed with Fundulus heteroclitus Trx-2 from the pairwise alignment results. The phylogenetic analysis revealed that HaTrx-2 is closely clustered with teleost fishes. The qPCR results showed that HaTrx-2 was prevalently expressed at various levels in all the tissues examined. The ovary showed the highest expression, followed by the brain and kidney. HaTrx-2 showed varying mRNA expression levels during the immune challenge experiment, depending on the type of tissue and the time interval. Our results confirmed the antioxidant property of HaTrx-2 by performing the MCO assay, DPPH radical scavenging activity, and cell viability assays. Further, an insulin disulfide reduction assay revealed the dithiol remove the enzymatic activity of HaTrx-2. Altogether these results indicate that HaTrx-2 plays indispensable roles in the regulation of oxidative stress and immune response in the seahorse.