Search

AbstractIndirect genetic effects (IGEs) exist when there is heritable variation in one organism's ability to alter a second organism's traits. For example, parasites have antigens that can induce a host immune response, as well as disparate strategies to evade or suppress host immunity; among-parasite genetic variation in these antigens generates among-host variation in immune traits. Here, we experimentally show that the cestode parasite Schistocephalus solidus exerts an IGE on an immune trait (peritoneal fibrosis) in its threespine stickleback host: stickleback developed strong fibrosis after exposure to some parasite genotypes but not others. A complication arises during coinfection, when two or more parasite genotypes may impose conflicting IGEs on the same host trait. What parasite-controlled trait will the host express? Will the host trait reflect the more immune-stimulatory parasite genotype or the more immune-evasive genotype? These alternatives can be quantified by estimating the dominance coefficient, as if a coinfected host were a heterozygote. We experimentally estimated the dominance of S. solidus IGEs by coinjecting antigens from different parasite genotypes. Contrary to our a priori hypotheses, coinjected antigens induced an overdominant effect, stronger than either parasite's antigens alone. We present a mathematical model showing that the value of this IGE dominance is biologically important, affecting the evolutionary dynamics of parasites in a density- and frequency-dependent manner. The model indicates that overdominance would be detrimental to immigrants when resident prevalence is high. This combination of experimental data and modeling provides an example of a parasite IGE on host traits and the evolutionary significance of IGE dominance.

Galectin-4 belongs to the galactoside-binding protein family and is a type of tandem repeat galectin. Despite previous studies indicating its importance in fish immunology, a comprehensive investigation is necessary to fully understand its role in immunomodulatory functions and cellular dynamics. Therefore, this study aimed to explore the immunomodulatory functions of galectin-4 with a particular focus on its antimicrobial and cellular proliferative properties. The open reading frame of PhGal4 spans 1092 base pairs and encodes a soluble protein of 363 amino acids with a theoretical isoelectric point (IEP) of 6.39 and a molecular weight of 39.411 kDa. Spatial expression analysis under normal physiological conditions revealed ubiquitous expression of PhGal4 across all examined tissues, with the highest level observed in intestinal tissue. Upon stimulation with poly I:C, LPS, and L. garvieae, a significant increase (p < 0.05) in PhGal4 expression was observed in both blood and spleen tissues. Subsequent subcellular localization assay demonstrated that PhGal4 was predominantly localized in the cytoplasm. The recombinant PhGal4 (rPhGal4) exhibited specific binding capabilities to pathogen-associated molecular patterns (PAMPs), including LPS and peptidoglycan, but not poly I:C. The rPhGal4 negatively affected the bacterial growth kinetics. Additionally, rPhGal4 demonstrated complete hemagglutination of fish erythrocytes, which could be inhibited by the presence of D-galactose and α-lactose. The overexpression of PhGal4 in FHM epithelial cells demonstrated a significant suppression of viral replication during VHSV infection. Furthermore, the in vitro scratch assay and WST-1 assay demonstrated a wound healing effect of PhGal4 overexpression in FHM cells, potentially achieved through the promotion of cell proliferation by activating genes involved in cell cycle regulation. In conclusion, the responsive expression to immune stimuli, antimicrobial properties, and cell proliferation promotion of PhGal4 suggest that it plays a crucial role in immunomodulation and cellular dynamics of red-lip mullet. The findings in this study shed light on the multifunctional nature of galectin-4 in teleost fish., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

As an environmentally friendly alternative to antibiotics, bee venom holds promise for aquaculture due to its diverse health advantages, including immune-amplifying and anti-inflammatory features. This study investigated the effects of dietary bee venom (BV) on the growth and physiological performance of Thinlip mullet (Liza ramada) with an initial body weight of 40.04 ± 0.11 g for 60 days. Fish were distributed to five dietary treatments (0, 2, 4, 6, and 8 mg BV/kg diet) with three replicates. Growth traits, gut enzyme ability (lipase, protease, amylase), intestinal and liver histology, blood biochemistry, immune responses [lysozyme activity (LYZ), bactericidal activity (BA), nitroblue tetrazolium (NBT%)], and antioxidant status [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), malondialdehyde (MDA)] were evaluated. BV supplementation significantly improved growth performance, digestive enzyme activity, histological integrity of organs, immune responses (LYZ, BA), and antioxidant status (SOD, CAT, GPx), while declining MDA levels. Optimal BV levels were identified between 4.2 and 5.8 mg/kg diet for different parameters. Overall, the findings suggest that BV supplementation can enhance growth and physiological performance in Thinlip mullet, highlighting its potential as a beneficial dietary supplement for fish health and aquaculture management., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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 β-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 H 2 O 2 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., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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 (β-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 > β-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 β-actin > GAPDH > EF1α > 18SrRNA, with β-actin as the most stable gene. In both spleen and kidney tissues, the rank order was EF1ɑ > GAPDH > β-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 > β-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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2021 Fuess et al.)

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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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., (© 2020 John Wiley & Sons Ltd.)

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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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.

Poly-β-hydroxybutyrate (PHB) can be hydrolyzed to β-hydroxybutyrate (β-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 β-HB on cell viability and antioxidant function to illustrate the immunopotentiation mechanism of PHB. The results showed that β-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 β-HB, LPS (100 μg/mL) was used to induce the inflammation and investigated the inhibitory effect of β-HB on inflammation. The results showed that LPS could induce inflammation successfully, and β-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 β-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 β-HB inhibited the inflammatory response by up-regulation of anti-inflammatory genes such as NF-κBIA, thereby enhancing the immunity of the body., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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 β-strands (LRR1-LRR19), lacking Z-loop that seen in human TLR9. The intracellular Toll/interleukin (IL)-1 (TIR) domain contained a central 4-parallel β-sheet (βA-βD) 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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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-β-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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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 Ca 2+ 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-1β (IL-1β). 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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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.

Vertebrates' gut microbial communities can be altered by the hosts' parasites. Helminths inhabiting the gut lumen can interact directly with their host's microbiota via physical contact, chemical products, or competition for nutrients. Indirect interactions can also occur, for instance when helminths induce or suppress host immunity in ways that have collateral effects on the microbiota. If there is genetic variation in host immune responses to parasites, we would expect such indirect effects to be conditional on host genotype. To test for such genotype by infection interactions, we experimentally exposed Gasterosteus aculeatus to their naturally co-evolved parasite, Schistocephalus solidus. The host microbiota differed in response to parasite exposure, and between infected and uninfected fish. The magnitude and direction of microbial responses to infection differed between host sexes, and also differed between variants at autosomal quantitative trait loci. These results indicate that host genotype and sex regulate the effect of helminth infection on a vertebrate gut microbiota. If this result holds in other taxa, especially humans, then helminth-based therapeutics for dysbiosis might need to be tailored to host genotype and sex.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Malectin is a carbohydrate-binding lectin protein found in the endoplasmic reticulum (ER). It selectivity binds to Glc 2 -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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Suppressors of cytokine signaling (SOCS) family members have negative effects on cytokine signaling pathways involved in immunity, growth and development. Owing to their typical feature, they have been extensively studied in mammalians, but they have not offered systematic studies among teleosts. In the present study, nine SOCS family genes were identified in the swamp eel genome and analyzed regulation mechanisms of SOCS family members in swamp eels. The open reading frames of MaSOCS1a, MaSOCS1b, MaSOCS2, MaSOCS3a, MaSOCS3b, MaSOCS4, MaSOCS5, MaSOCS6 and MaSOCS7 were 663 bp, 603 bp, 717 bp, 618 bp, 645 bp, 1188 bp, 1488 bp, 1611 bp and 1998 bp and encoded 220, 238, 200, 205, 214, 395, 496, 536 and 655 amino acids, respectively. All SOCS proteins have no signal peptides. Multiple alignment revealed that MaSOCS family members possessed a typical conserved SOCS box and SH2 region. Phylogenetic analyses showed that all SOCS proteins were divided into two main clusters. Taken together with the similarity and identity of SOCS protein amino acids, these results indicated that MaSOCS family members shared conserved with other homologous genes, in which MaSOCS7 was more conserved. Further syntenic analysis confirmed the phylogenetic analysis results and annotation of SOCS protein, suggesting that MaSOCS5 shared a common ancestor gene with that of fish and humans. MaSOCS family members were constitutively expressed in a wide range of tissues with different levels. In particular, spleen and head kidneys play an important role in immune-related pathways. After Aeromonas veronii and polyinosinic-polycytidylic acid (poly I:C) challenge in the spleen and head kidney, MaSOCS family members exhibit different expression profiles. These expression patterns indicated that MaSOCS family members could make acute responses after pathogen invasion. Taken together, these results indicate that MaSOCS family members participate in the immune response against pathogens and offer a solid foundation for future studies of SOCS function., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The interferon-induced GTP-binding protein Mx is responsible for a specific antiviral state against a broad spectrum of viral infections that are induced by type-I interferons (IFN α/β) in different vertebrates. In this study, the Mx gene was isolated from the constructed mullet cDNA database. Structural features of mullet Mx (MuMx) were analyzed using different in-silico tools. The pairwise comparison revealed that the MuMx sequence was related to Stegastes partitus Mx with an 83.7% sequence identity, whereas MuMx was clustered into the teleost category in the phylogentic analysis. Sequence alignment showed that the dynamin-type guanine nucleotide-binding domain (G&#95;DYNAMIN&#95;2), central interactive domain (CID), and GTPase effector domain (GED) were conserved among Mx counterparts. The transcriptional expression of MuMx was the highest in blood cells from unchallenged fish. The temporal mRNA profile showed that MuMx expression was significantly elevated in all tissues, including blood, spleen, head kidney, liver, and gills after the injection of polyinosinic-polycytidylic acid (poly I:C) at many time points. Moreover, MuMx expression increased slightly, in the blood, spleen, and head kidney at a few time points after the injection of lipopolysaccharide (LPS) and Lactococcus garvieae (L. garvieae). Results of the subcellular localization analysis confirmed that the MuMx protein was highly expressed in the cytoplasm. The analysis of the gene expression of the viral hemorrhagic septicemia virus (VHSV) under conditions of MuMx overexpression confirmed the significant inhibition of viral transcripts. The cell viability (MTT) assay and VHSV titer quantification with the presence of MuMx indicated a significant reduction in virus replication. Collectively, these findings suggest that Mx is a specific immune-related gene that elicits crucial antiviral functions against viral antigens in the mullet fish., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

As a dietary supplement, poly-β-hydroxybutyrate (PHB) has been reported to positively influence growth, boost the immune system and enhance disease resistance in fish and shellfish. However, the protective mechanism is little known. Thus, the present study was conducted to evaluate the effect of PHB supplementation on immune-related enzyme activity and transcriptome-based gene expression in soiny mullet (Liza haematocheila). Results showed that dietary PHB supplementation could increase antioxidant enzyme activity, including total antioxidant capacity, catalase and superoxide dismutase. A total of 7,082,094,175 and 7,650,341,357 raw reads with mean length of 757 bp were obtained from control and PHB (dietary PHB supplementation at 2%) groups, respectively. There were 46,106 differentially expressed genes (DEGs) between control and PHB groups, including 21,828 upregulated and 24,278 downregulated DEGs. All the DEGs were classified into three gene ontology categories, and 312 DEGs related with immune system process and 760 with the response to a stimulus. Additionally, all DEGs were allocated to 261 Kyoto Encyclopedia of Gene and Genome pathways, and major immune-related pathways were detected, including MAPK/PI3K-Akt/TNF/NF-κB/TCR/TLR signaling pathways. Moreover, the regulation of several observed immune-related genes was confirmed by qRT-PCR. Altogether, this study suggests that antioxidant system is more effective for dietary PHB supplementation and lays the foundation for further study on the precise immunostimulatory mechanism of PHB. Hopefully, it provides insights into exploring biomarker for assessment of immunostimulants in fish culture., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Chemokines direct cell migration in development and immune defense, and bridge between innate and adaptive immune responses. The chemokine gene family has been rapidly evolving and has undergone species/lineage-specific expansion. Mammals possess inflammatory CXC chemokines CXCL1-8/15 and CXCL9-11 sub-groups, and homeostatic CXCL12-14, 16-17. Orthologues of mammalian CXCL12-14, three chemokines related to CXCL1-8/15 (CXCL8&#95;L1-3), two chemokines related to CXC9-11 (CXCL11&#95;L1-2), and five fish-specific chemokines (CXCL&#95;F1-5) have been described in teleosts. In this study, we reported three novel CXC chemokines in Asian swamp eel Monopterus albus, a commercially important freshwater fish species in China. Two of them belong to the fish-specific CXCL&#95;F2 group, named CXCL&#95;F2a/b, that share 89.5% amino acid identity. The other (CXCL11&#95;L3) belongs to a third CXCL11&#95;L related to the mammalian CXCL9-11 subfamily found only in percomorph fish species, and is the only CXCL9-11 related molecules in this lineage. Mammalian CXCL9-11 attract Th1 cells, and block the migration of Th2 cells in an immune response. This study suggests that all major lineages of teleosts have a CXCL9-11 related chemokine that will aid future functional investigation of CXCL11&#95;L in fish. Cxcl&#95;f2a is highly expressed constitutively in the skin of swamp eels that may attract immune cells to protect the skin in the absence of scales. Cxcl11&#95;l3 and cxcl&#95;f2b are highly expressed in immune tissues/organs and are up-regulated by the viral mimic poly I:C, but not bacterial infection in vivo, suggesting their role in anti-viral defense. The two cxcl&#95;f2 paralogues are differentially expressed and modulated, indicating sub- and/or neo-functionalization., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Glutaredoxins are oxidoreductases present in almost all living organisms. They belong to the thioredoxin superfamily and share the thioredoxin structure and catalytic motif. Glutaredoxin 2 has been identified as a mitochondrial protein in vertebrates. In this study, the sequence of Glutaredoxin 2 from Hippocampus abdominalis (HaGrx2) was analyzed by molecular, transcriptional, and functional assays. In-silico analysis revealed that HaGrx2 shows the highest homology with Hippocampus comes, while distinctly cluster with fish Grx2 orthologs. Tissue distribution analysis showed that HaGrx2 is ubiquitously expressed in all tissues tested, and the highest expression was observed in the brain and skin. Significant HaGrx2 transcript modulation was identified in blood and liver upon injecting bacterial and Pathogen Associated Molecular Patterns. The redox activity of HaGrx2 was revealed by Dehydroascorbic reduction and insulin disulfide reduction activity assays. Further, the deglutathionylation activity of 1 nM HaGrx2 was found to be equivalent to that of 0.84 nM HaGrx1. HaGrx2 exhibited antiapoptotic activity against H 2 O 2 -induced oxidative stress in FHM cells. Altogether, the results of this study suggest that HaGrx2 plays a role in redox homeostasis and innate immune responses in fish., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Glutathione S-transferases (GSTs) are essential enzymes for the bioactivation of xenobiotics through the conjugation of the thiol group of glutathione (GSH). In this study, a kappa class of GST was identified from the big belly seahorse (Hippocampus abdominalis) (HaGSTκ1) and its biochemical and functional properties were analyzed. HaGSTκ1 has 231 amino acids encoded by a 696 bp open reading frame (ORF). The protein has a predicted molecular mass of 26.04 kDa and theoretical isoelectric point (pI) of 8.28. It comprised a thioredoxin domain, disulfide bond formation protein A (DsbA) general fold, and Ser15 catalytic site as well as GSH-binding and polypeptide-binding sites. Phylogenetic analysis revealed that HaGSTκ1 is closely clustered with the kappa class of GSTs from teleost fishes. The recombinant (rHaGSTκ1) protein exhibited activity toward 1-chloro-2,4-dinitrobenzene (CDNB), 4-nitrobenzyl (4-NBC), and 4-nitrophenethyl bromide (4-NPB) but not 1,2-dichloro-4-nitrobenzene (DCNB). The optimum pH and temperature were 8 and 30 °C, respectively, for the catalysis of CDNB and the universal substrate of GSTs. The rHaGSTκ1 activity was efficiently inhibited in the presence of Cibacron blue (CB) as compared with hematin. Most prominent expression of HaGSTκ1 was observed in the liver and kidney among the fourteen different tissues of normal seahorse. After challenge with lipopolysaccharide (LPS), polyinosinic-polycytidylic (poly I:C), gram-negative Edwardsiella tarda, and gram-positive Streptococcus iniae, HaGSTκ1 expression was significantly modulated in the liver and blood tissues. Altogether, our study proposes the plausible important role of HaGSTκ1 in innate immunity and detoxification of harmful xenobiotics., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Soiny mullet (Liza haematocheila) is an important economic fish species in China, but stress and diseases have seriously restricted its culture. There are no effective methods including vaccines to prevent or control these diseases. Alternative methods should be employed, such as using novel immunostimulant poly-β-hydroxybutyrate (PHB). The present study aimed to evaluate effects of dietary PHB supplementation on the growth, antioxidant enzymes activity, immune-related genes expression and intestinal microbiota in soiny mullet. The fish was fed for 30 or 60 days with six diets at different PHB supplementation of 0, 0.5, 1, 2, 4 and 8%, named as groups P0, P0.5, P1, P2, P4 and P8. The results showed that the weight gain and specific growth rate of fish in P2 and P0.5 groups were significantly higher than those in control P0 group at 30 and 60 days, respectively (P < 0.05). The antioxidant enzymes activity of catalase and superoxide dismutase in serum were significantly increased in P0.5/P1/P2 groups after 30 days. The transcriptional levels of penicillin-binding protein A and interleukin-8 analyzed by qRT-PCR were significantly upregulated in P2 and P4 groups compared to those in P0/P0.5/P1/P8 groups at 30 days. The transcriptional level of major histocompatibility complex class II in P2 group was significantly upregulated, and aldehyde oxidase downregulated compared to P0 group. Intestinal microbiota analysis by Illumina high-throughput sequencing showed that the microbiota diversity was not changed significantly, but the microbiota structure shifted significantly post PHB treatment. At the phyla level, Firmicutes and Proteobacteria were predominant in both P0 and P2 groups. At the genus level, the relative abundance of Bacillus spp. in P2 group increased significantly, and abundance of Achromobacter spp. decreased significantly. KEGG pathway analysis by PICRUSt showed that oral administration PHB significantly upregulated abundances of genes responsible for 10 pathways and downregulated genes involved in 17 pathways. In conclusion, soiny mullet fed with 2% PHB supplemental diets for 30 days showed better growth performance, higher antioxidant enzymes activity and immune-related genes expression. Their regulation of growth and immunity might be related with the intestinal microbiota change post PHB supplementation. It will provide very useful basic information to study the regulation mechanism of PHB in aquatic animals, and provide good green method to prevent disease in soiny mullet., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Glutaredoxins (Grx) are redox enzymes conserved in viruses, eukaryotes, and prokaryotes. In this study, we characterized glutaredoxin 1 (HaGrx1) from big-belly seahorse, Hippocampus abdominalis. In-silico analysis showed that HaGrx1 contained the classical glutaredoxin 1 structure with a CSYC thioredoxin active site motif. According to multiple sequence alignment and phylogenetic reconstruction, HaGrx1 presented the highest homology to the Grx1 ortholog from Hippocampus comes. Transcriptional studies demonstrated the ubiquitous distribution of HaGrx1 transcripts in all the seahorse tissues tested. Significant modulation (p < 0.05) of HaGrx1 transcripts were observed in blood upon stimulation with pathogen-associated molecular patterns and live pathogens. The β-hydroxyethyl disulfide reduction assay confirmed the antioxidant activity of recombinant HaGrx1. Further, dehydroascorbate reduction and insulin disulfide reduction assays revealed the oxidoreductase activity of HaGrx1. HaGrx1 utilized 1,4-dithiothreitol, l-cysteine, 2-mercaptoethanol, and reduced l-glutathione as reducing agent with different dehydroascorbate reduction activity levels. Altogether, our results suggested a vital role of HaGrx1 in redox homeostasis as well as the host innate immune defense system., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Organisms possess a cellular antioxidant defense system inclusive of ROS scavengers to maintain the homeostasis of antioxidant levels. Catalase is a major ROS scavenger enzyme that plays a significant role in the antioxidant defense mechanism of organisms by reducing toxic hydrogen peroxide molecules into a nontoxic form of oxygen and water with a high turnover rate. In the present study, we performed molecular and functional characterization of the catalase homolog from Hippocampus abdominalis (HaCat). The HaCat cDNA sequence was identified as a 1578 bp ORF (open reading frame) that encodes a polypeptide of 526 amino acids with 59.33 kDa molecular weight. Its estimated pI value is 7.7, and it does not have any signal sequences. HaCat shared a conserved domain arrangement including the catalase proximal active site signature and heme ligand signature domain with the previously identified catalase counterparts. Phylogenetic analysis displayed close evolutionary relationships between HaCat and catalases from other teleost fish. According to our qPCR results, ubiquitous expression of HaCat transcripts were observed in all the tested tissues with high expression in the kidney followed by liver. Significant modulations of HaCat transcription were observed in blood, liver, and kidney tissues post-challenge with Streptococcus iniae, Edwardsiella tarda, poly I:C, and LPS. Peroxidase activity of recombinant HaCat (rHaCat) was evaluated using an ABTS assay and the ROS removal effect was further confirmed by oxidative DNA damage protection and cell viability assays. The rHaCat showed more than 97% activity over a temperature and pH range of 10 °C-40 °C and 5 to 6, respectively. The above results suggest that HaCat plays an indispensable role in the oxidative homeostasis of the seahorse during pathogenic attack., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

TLR7 subfamily members are important pattern recognition receptors participating in the recognition of pathogen-associated molecular patterns. In this study, we successfully identified 3 members of TLR7 subfamily from the spiny eel Mastacembelus armatus (MaTLR7, MaTLR8 and MaTLR9). The amino acid sequence identities of MaTLR7 and MaTLR8 with Monopterus albus TLR7 were 87.2% and 76.5%, respectively and the identity of MaTLR9 with Seriola lalandi TLR9 was 74.7%. The phylogenetic analysis revealed MaTLRs showed close relationship to other species in Synbranchiformes or Perciformes. Quantitative real-time PCR analysis revealed that they were expressed in all tested tissues and higher expression was found in spleen or gill. After infection with Aeromonas veronii, expression of MaTLR7, MaTLR8 and MaTLR9 were all significantly downregulated in spleen and kidney. Evolutionary analysis suggested that the ancestral lineages of teleost TLR8 and TLR9 had been subject to positive selection pressures and multiple Maximum likelihood methods recovered 3 positively selected sites in teleost TLR7, 4 in TLR8 and 8 in TLR9. Domain distribution revealed most positively selected sites were located in leucine-rich repeat domain. Our results will contribute to better understanding the antibacterial mechanism of TLRs and their co-evolution with pathogens., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Interleukin-17 (IL-17) is an important cytokine that plays a critical role in the inflammatory response and host defense against extracellular pathogens. In the present study, six novel IL-17 family genes (MaIL-17) were identified by analyzing Asian swamp eel (Monopterus albus) genome. Sequence analysis revealed that the MaIL-17 family genes shared similar features, comprising a signal peptide, an IL-17 superfamily region, and four conserved cysteines. Phylogenetic analysis showed that the MaIL-17 genes were clustered together with their corresponding IL-17 genes from other species. The similarity and identity of all IL-17 family genes indicated that the MaIL-17 genes are conserved among teleosts, while Ma-IL-17D is more conserved than the other Ma-IL-17s. Except for MaIL-17A/F3 and MaIL-17D, all MaIL-17s shared the same genomic structure as the genes from other fish, namely three exons and two introns. The MaIL-17s showed conserved synteny among fish, and we found that the MaIL-17D locus has a more conserved syntenic relationship with the loci from other fish and humans. These results demonstrated that MaIL-17D and human IL-17D might have evolved from a common ancestral gene and subsequently diverged. The analysis of swamp eel reference genes revealed that EEF1A1 (encoding eukaryotic translation elongation factor 1 alpha 1) was an ideal reference gene for accurate real-time qRT-PCR normalization in the swamp eel. The MaIL-17 genes are widely distributed throughout tissues, suggesting that MaIL-17s carry out their biological functions in immune and non-immune tissues compartments. The transcript of Ma-IL17s exhibited different fold changes in head kidney cells in response to Aeromonas veronii phorbol 12-myristate 13-acetate (PMA) and polyinosinic:polycytidylic acid (poly I:C) challenge, showing that MaIL-17A/F1 has stronger antiviral activities compared with other MaIL-17 family genes, and that MaIL-17A/F3 and MaIL-17A/F2 possess stronger effects against extracellular pathogens compared with the others; however, MaIL-17C2 and MaIL-17D may play vital roles during pathogen infection. The differential immune responses of these genes to Aeromonas veronii, PMA and poly I:C implied distinct mechanisms of host defense against extracellular pathogens., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Phospholipid scramblases (PLSCRs) are a family of transmembrane proteins known to be responsible for Ca 2+ -mediated bidirectional phospholipid translocation in the plasma membrane. Apart from the scrambling activity of PLSCRs, recent studies revealed their diverse other roles, including antiviral defense, tumorigenesis, protein-DNA interactions, apoptosis regulation, and cell activation. Nonetheless, the biological and transcriptional functions of PLSCRs in fish have not been discovered to date. Therefore, in this study, two new members related to the PLSCR1 family were identified in the red lip mullet (Liza haematocheila) as MuPLSCR1like-a and MuPLSCR1like-b, and their characteristics were studied at molecular and transcriptional levels. Sequence analysis revealed that MuPLSCR1like-a and MuPLSCR1like-b are composed of 245 and 228 amino acid residues (aa) with the predicted molecular weights of 27.82 and 25.74 kDa, respectively. A constructed phylogenetic tree showed that MuPLSCR1like-a and MuPLSCR1like-b are clustered together with other known PLSCR1 and -2 orthologues, thus pointing to the relatedness to both PLSCR1 and PLSCR2 families. Two-dimensional (2D) and 3D graphical representations illustrated the well-known 12-stranded β-barrel structure of MuPLSCR1like-a and MuPLSCR1like-b with transmembrane orientation toward the phospholipid bilayer. In analysis of tissue-specific expression, the highest expression of MuPLSCR1like-a was observed in the intestine, whereas MuPLSCR1like-b was highly expressed in the brain, indicating isoform specificity. Of note, we found that the transcription of MuPLSCR1like-a and MuPLSCR1like-b was significantly upregulated when the fish were stimulated with poly(I:C), suggesting that such immune responses target viral infections. Overall, this study provides the first experimental insight into the characteristics and immune-system relevance of PLSCR1-related genes in red lip mullets., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The majority of parasites have evolved strategies to evade the immune responses of their hosts. Neuroactive substances produced by cestodes are possible candidate molecules for regulating host immune responses. The neurons of helminths can synthesize a wide range of molecules that are identical to the ones functioning in their host organisms, and host lymphocytes have receptors for these neuroactive substances. We hypothesized that in teleost fish, antihelminthic immune responses are regulated via 5-hydroxytryptamine (5-HT, or serotonin) and γ-aminobutyric acid (GABA). In the present study, we investigated the in vitro influence of serotonin, GABA and Schistocephalus solidus (helminth) antigens on basic characteristics of the three-spined stickleback Schistocephalus solidus cellular immune response. Head kidney leucocytes (HKLs) were analysed by flow cytometry for cell viability and the frequency of leucocyte subsets (the granulocyte-to-lymphocyte ratio) and by a chemiluminescence assay for the production of reactive oxygen species (ROS). In short-term (2-h) HKL cultures, 5-HT did not change the total numbers of live HKLs, but the production of ROS decreased significantly with all 5-HT concentrations. In long-term (96-h) cultures, high 5-HT concentrations induced a decrease in leucocyte viability. This coincided with elevated ROS production in cultures with all 5-HT concentrations. In short-term (2-h) HKL cultures, GABA did not change the total numbers of live HKLs, but the production of ROS decreased significantly with high (100 nmol L -1 ) GABA concentrations. In long-term (96-h) cultures, high and medium concentrations of GABA (100 nmol L -1 and 10 nmol L -1 ) elevated the numbers of live HKLs compared to controls. The granulocyte-to-lymphocyte ratios generally increased upon exposure to GABA at all concentrations. All concentrations of GABA alone elevated the ROS production of HKLs compared to controls. In the present work, we showed that the neuroactive substances serotonin and GABA regulate the teleost immune system. Our study supports the hypothesis that these substances might be immunomodulators in tapeworm-fish parasite-host interactions., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Host genotype - parasite genotype co-evolutionary dynamics are influenced by local biotic and abiotic environmental conditions. This results in spatially heterogeneous selection among host populations. How such heterogeneous selection influences host resistance, parasite infectivity and virulence remains largely unknown. We hypothesized that different co-evolutionary trajectories of a vertebrate host-parasite association result in specific virulence patterns when assessed on a large geographic scale. We used two reference host populations of three-spined sticklebacks and nine strains of their specific cestode parasite Schistocephalus solidus from across the Northern Hemisphere for controlled infection experiments. Host and parasite effects on infection phenotypes including host immune gene expression were determined., Results: S. solidus strains grew generally larger in hosts coming from a population with high parasite diversity and low S. solidus prevalence (DE hosts). Hosts from a population with low parasite diversity and high S. solidus prevalence (NO hosts) were better able to control the parasite's growth, regardless of the origin of the parasite. Host condition and immunological parameters converged upon infection and parasite growth showed the same geographic pattern in both host types., Conclusion: Our results suggest that NO sticklebacks evolved resistance against a variety of S. solidus strains, whereas DE sticklebacks are less resistant against S. solidus. Our data provide evidence that differences in parasite prevalence can cause immunological heterogeneity and that parasite size, a proxy for virulence and resistance, is, on a geographic scale, determined by main effects of the host and the parasite and less by an interaction of both genotypes.

Both wild and aquacultured seahorses are currently under great threat from marine pollution, notably from the potent contaminant and carcinogen benzo[a]pyrene (BaP). However, very little data are available regarding the immunomodulating effects of BaP in seahorses. Therefore, in this study, we exposed lined seahorses (Hippocampus erectus) for 7 d to BaP at three dosages (0.5, 5, and 50 μg/L) to evaluate sexual dimorphism in immune response. We measured eight immune parameters in the blood, including respiratory burst (RB), phagocytic activity (PA), monocytes/leucocytes, immunoglobulin M, complement 3, complement, interferon-a, and interleukin-2. Male seahorses had significantly higher parameters than females, except in terms of monocytes/leucocytes (P < 0.05). Although flow cytometry showed that RB and PA variation per BaP dose were roughly similar across sexes, RB and PA exhibited distinct patterns. Additionally, fluorescence intensity and leucocyte percentage were positively correlated in PA but not RB for all treatment and sex combinations. Through ELISA, we showed that the other six parameters had complex responses that nevertheless varied in a BaP-dosage and sex-dependent manner. Overall, adult male seahorses had higher immunocompetence than females before BaP exposure, and sexual dimorphism continued to be apparent during BaP exposure. Furthermore, all eight parameters were sensitive to BaP. Based on these results, we highly recommend H. erectus as a sentinel species for crude contamination, whereas PA and RB are valuable bioindicators of marine contaminants such as BaP., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Viperin is recognized as an antiviral protein that is stimulated by interferon, viral exposures, and other pathogenic molecules in vertebrate. In this study, a viperin homolog in the Big-belly seahorse (Hippocampus abdominalis; HaVip) was functionally characterized to determine its subcellular localization, expression pattern, and antiviral activity in vitro. The HaVip coding sequence encodes a 348 amino acid polypeptide with predicted molecular weight of 38.48 kDa. Sequence analysis revealed that HaVip comprises three main domains: the N-terminal amphipathic α-helix, a radical S-adenosyl-l-methionine (SAM) domain, and a conserved C-terminal domain. Transfected GFP-tagged HaVip protein was found to localize to the endoplasmic reticulum (ER). Overexpressed-HaVip in FHM cells was found to significantly reduce viral capsid gene expression in VHSV infection in vitro. Under normal physiological conditions, HaVip expression was ubiquitously detected in all 14 examined tissues of the seahorse, with the highest expression observed in the heart, followed by skin and blood. In vivo studies showed that HaVip was rapidly and predominantly upregulated in blood, kidney, and intestinal tissue upon poly (I:C) stimulus. LPS and Streptococus iniae challenges caused a significant increase in expression of HaVip in all the analyzed tissues. The obtained results suggest that HaVip is involved in the immune system of the seahorse, triggering antiviral and antibacterial responses, upon viral and bacterial pathogenic infections., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Thioredoxin domain-containing protein 17 (TXNDC17) is a small protein (∼14 kDa) involved in maintaining cellular redox homeostasis via a thiol-disulfide reductase activity. In this study, TXNDC17 was identified and characterized from Hippocampus abdominalis. The open reading frame (ORF) consisted of 369 bp and 123 amino acids. Similar to the other thioredoxins, TXNDC17 contained a conserved WCXXC functional motif. The highest spatial mRNA expressions of HaTXNDC17 were observed in the muscle, brain, and intestine. Interestingly, the mRNA expression of HaTXNDC17 in blood showed significant upregulation at 48 h against all the pathogen associated molecular patterns (PAMPs) and bacteria. Further, HaTXNDC17 transcripts in the trunk kidney were significantly upregulated at 24-48 h by bacterial endotoxin lipopolysaccharides (LPS), viral mimic polyinosinic: polycytidylic acid (poly I:C), and gram-negative bacteria (Edwardsiella tarda). The DPPH assay showed that the radical scavenging activity varies in a concentration-dependent manner. The insulin reduction assay demonstrated a significant logarithmic relationship with the concentration of rHaTXNDC17. Moreover, FHM cells treated with recombinant HaTXNDC17 significantly enhanced cellular viability under oxidative stress. Together, these results show that HaTXNDC17 function is important for maintaining cellular redox homeostasis and that it is also involved in the immune mechanism in seahorses., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Arachidonate 5-lipoxygenase (ALOX5) is an essential enzyme for the biosynthesis of leukotrienes, which are pro-inflammatory and anti-inflammatory mediators. In this study, the ALOX5 paralog of the big-belly seahorse (Hippocampus abdominalis; HaALOX5) was identified from our transcriptome database, and then molecularly and functionally characterized to determine its oxygenation capability and expression under pathogenic stress. The coding sequence of HaALOX5 consisted of 2025 bp and encoded a protein of 674 amino acids in length. Sequence and phylogenetic tree analysis of HaALOX5 revealed a close relationship with its corresponding teleost HaALOX5 counterparts. Structure prediction detected an N-terminal regulatory C2-like domain and a C-terminal catalytic domain, which are the two main functional domains in ALOX5 enzymes. Quantitative PCR showed that HaALOX5 was expressed in all the analyzed tissues at different magnitudes. The highest expression was detected in the intestine and stomach. In blood cells, the liver and the intestine, HaALOX5 transcripts were significantly elevated at many post injection time points, when immune challenged with lipopolysaccharide, polyinosinic:polycytidylic acid, and Streptococcus iniae, indicating its contribution to post immune defense mechanisms in the seahorse., (Copyright © 2018. Published by Elsevier Ltd.)

Complement system orchestrates the innate and adaptive immunity via the activation, recruitment, and regulation of immune molecules to destroy pathogens. However, regulation of the complement is essential to avoid injuries to the autologous tissues. The present study unveils the characteristic features of an important complement component, anaphylatoxin inactivator from red lip mullet at its molecular and functional level. Mullet carboxypeptidase N1 (MuCPN1) cDNA sequence possessed an open reading frame of 1347 bp, which encoded a protein of 449 amino acids with a predicted molecular weight of 51 kDa. In silico analysis discovered two domains of PM14-Zn carboxypeptidase and a C-terminal domain of M14 N/E carboxypeptidase, two zinc-binding signature motifs, and an N-glycosylation site in the MuCPN1 sequence. Homology analysis revealed that most of the residues in the sequence are conserved among the other selected homologs. Phylogeny analysis showed that MuCPN1 closely cladded with the Maylandia zebra CPN1 and clustered together with the teleostean counterparts. A challenge experiment showed modulated expression of MuCPN1 upon polyinosinic:polycytidylic acid and Lactococcus garviae in head kidney, spleen, gill, and liver tissues. The highest upregulation of MuCPN1 was observed 24 h post infection against poly I:C in each tissue. Moreover, the highest relative expressions upon L. garviae challenge were observed at 24 h post infection in head kidney tissue and 48 h post infection in spleen, gill, and liver tissues. MuCPN1 transfected cells triggered a 2.2-fold increase of nitric oxide (NO) production upon LPS stimulation compared to the un-transfected controls suggesting that MuCPN1 is an active protease which releases arginine from complement C3a, C4a, and C5a. These results have driven certain way towards enhancing the understanding of immune role of MuCPN1 in the complement defense mechanism of red lip mullet., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The CXC chemokine receptors (CXCRs) play critical roles in innate and adaptive immune systems. In this study, six Asian swamp eel (Monopterus albus) CXCRs (MaCXCR1-4) were identified and their molecular characterization and expression patterns were analyzed. The open reading frames (ORFs) of MaCXCR1a, MaCXCR1b, MaCXCR2, MaCXCR3a, MaCXCR3b, and MaCXCR4 were 1074 bp (base pairs), 1080 bp, 1125 bp, 1146 bp, 1083 bp, and 1140 bp, and encoded proteins of 357 aa (amino acids), 359 aa, 374 aa, 381 aa, 360 aa, and 379 aa, respectively. All these CXCRs have seven conserved transmembrane domains and four cysteines (with the exception of MaCXCR3b). Multiple sequence alignment revealed that the MaCXCRs possess a typical G-protein receptor family 1 signature and a DRY motif. There are also one to four potential N-glycosylation sites in the extracellular regions of the MaCXCRs, mainly distributed in the N-terminus and extracellular hydrophilic loop (ECL) 2 region. Phylogenetic analysis demonstrated that the MaCXCRs were clustered together with homologous proteins from other fish. Taken together with the amino acid identity and similarity analysis, these results suggested that the MaCXCRs are conserved with other homologous genes, in which CXCR4 is more conserved than CXCR1-3. The MaCXCRs loci showed conserved synteny among teleost fish, and we found that human CXCR1 shares a common ancestor with fish CXCR1a. MaCXCRs were constitutively expressed in a wide range of tissues (especially in immune-related tissues) with different expression levels, suggesting that the MaCXCRs have different roles in un-stimulated tissues, and may play vital roles under normal conditions. MaCXCRs showed different fold changes in the spleen after Aeromonas veronii and polyinosinic-polycytidylic acid (poly I:C) challenge, which suggested that MaCXCR1a and MaCXCR3a have longer antiviral activities compared with their antibacterial functions, and that MaCXCR1b possesses stronger antiviral than antibacterial activity. MaCXCR4 may play vital roles during bacterial and viral infection; however, MaCXCR2 has relatively small effect in antibacterial and antiviral responses. The differential responses of these genes to bacteria and poly I:C implied the differences in the mechanisms of defense against viruses and bacteria., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The structural and evolutionary linkage between tumor necrosis factor (TNF) and the globular C1q (gC1q) domain defines the C1q and TNF-related proteins (CTRPs), which are involved in diverse functions such as immune defense, inflammation, apoptosis, autoimmunity, and cell differentiation. In this study, red-lip mullet (Liza haematocheila) CTRP4-like (MuCTRP4-like), CTRP5 (MuCTRP5), CTRP6 (MuCTRP6), and CTRP7 (MuCTRP7) were identified from the red-lip mullet transcriptome database and molecularly characterized. According to in silico analysis, coding sequences of MuCTRP4-like, MuCTRP5, MuCTRP6, and MuCTRP7 consisted of 1128, 753, 729, and 888 bp open reading frames (ORF), respectively and encoded 375, 250, 242, and 295 amino acids, respectively. All CTRPs possessed a putative C1q domain. Additionally, MuCTRP5, MuCTRP6, and MuCTRP7 consisted of a collagen region. Phylogenetic analysis exemplified that MuCTRPs were distinctly clustered with the respective CTRP orthologs. Tissue-specific expression analysis demonstrated that MuCTRP4-like was mostly expressed in the blood and intestine. Moreover, MuCTRP6 was highly expressed in the blood, whereas MuCTRP5 and MuCTRP7 were predominantly expressed in the muscle and stomach, respectively. According to the temporal expression in blood, all MuCTRPs exhibited significant modulations in response to polyinosinic:polycytidylic acid (poly I:C) and Lactococcus garvieae (L. garvieae). MuCTRP4-like, MuCTRP5, and MuCTRP6 showed significant upregulation in response to lipopolysaccharides (LPS). The results of this study suggest the potential involvement of Mullet CTRPs in post-immune responses., (Copyright © 2018. Published by Elsevier Ltd.)

During mate choice decisions, females of many vertebrates use male olfactory cues to achieve immunogenetic optimality of their offspring. Three-spined sticklebacks ( Gasterosteus aculeatus) populating habitats that differ in their parasite communities evolve locally adapted combinations of genetic variants encoded at the major histocompatibility complex (MHC). Such adaptation confers optimal resistance to the local parasite fauna. Immunogenetic signatures co-evolved with local parasites favour population-specific assortative mate choice behaviour. Previous studies have shown that female sticklebacks evaluate male MHC-associated olfactory cues during the process of mate choice, but how habitat-specific information is exchanged between males and females has remained elusive. Here, we directly demonstrate the molecular nature of the olfactory cue providing habitat-specific information. Under controlled laboratory conditions, females that are ready to mate prefer mixtures of synthetic MHC peptide ligands mimicking the optimal allele number of their original population. These results imply that female sticklebacks can determine the number of MHC alleles of their prospective mates, compare it to their own immunogenetic status, and, if optimal with respect to the immunogenetic complementarity, accept the male as mate. Our results suggest a potentially common mechanism of ecological speciation in vertebrates that is based on the olfactory assessment of habitat-specific immunogenetic diversity.

Females choose specific mates in order to produce fitter offspring. However, several factors interfere with females' control over fertilization of their eggs, including sneaker males and phenotypically unpredictable allele segregation during meiosis. Mate choice at the individual level thus provides only a poor approximation for obtaining the best genetic match. Consequently, postcopulatory sperm selection by female oocytes has been proposed as a mechanism to achieve complementary combinations of parental haplotypes. Here, using controlled in vitro fertilization of three-spined stickleback eggs, we find haplotype-specific fertilization bias toward gametes with complementary major histocompatibility complex (MHC) immunogenes. The resulting zygote (and thus offspring) genotypes exhibit an intermediate level of individual MHC diversity that was previously shown to confer highest pathogen resistance. Our finding of haplotype-specific gamete selection thus represents an intriguing mechanism for fine-tuned optimization of the offspring's immune gene composition and an evolutionary advantage in the Red Queen dynamics of host-parasite coevolution., (© 2018 The Author(s). Evolution published by Wiley Periodicals, Inc. on behalf of The Society for the Study of Evolution.)

The redlip mullet (Liza haematocheila) is one of the most economically important fish in Korea and other East Asian countries; it is susceptible to infections by pathogens such as Lactococcus garvieae, Argulus spp., Trichodina spp., and Vibrio spp. Learning about the mechanisms of the complement system of the innate immunity of redlip mullet is important for efforts towards eradicating pathogens. Here, we report a comprehensive study of the terminal complement complex (TCC) components that form the membrane attack complex (MAC) through in-silico characterization and comparative spatial and temporal expression profiling. Five conserved domains (TSP1, LDLa, MACPF, CCP, and FIMAC) were detected in the TCC components, but the CCP and FIMAC domains were absent in MuC8β and MuC9. Expression analysis of four TCC genes from healthy redlip mullets showed the highest expression levels in the liver, whereas limited expression was observed in other tissues; immune-induced expression in the head kidney and spleen revealed significant responses against Lactococcus garvieae and poly I:C injection, suggesting their involvement in MAC formation in response to harmful pathogenic infections. Furthermore, the response to poly I:C may suggest the role of TCC components in the breakdown of the membrane of enveloped viruses. These findings may help to elucidate the mechanisms behind the complement system of the teleosts innate immunity., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Thioredoxin is a highly conserved protein found in both prokaryotes and eukaryotes. Reactive oxygen species (ROS) are produced in response to metabolic processes, radiation, metal oxidation, and pathological infections. High levels of ROS lead to cell death via autophagy. However, thioredoxin acts as an active regulatory enzyme in response to excessive ROS. Here, we performed in-silico analysis, immune challenge experiments, and functional assays of seahorse thioredoxin-like protein 1 (ShTXNL1). Evolutionary identification showed that ShTXNL1 protein belongs to the thioredoxin superfamily comprising 289 amino acids. It possesses an N-terminal active thioredoxin domain and C-terminal proteasome-interacting thioredoxin domain (PITH) of ShTXNL1 which is a component of 26S proteasome and binds to the matrix or cell. Pairwise alignment results showed 99.0% identity and 99.7% similarity with the sequence of Hippocampus species. Conserved thiol-disulfide cysteine residue containing Cys-X-X-Cys motif may be found in the first few amino acids in the second beta sheet starting from the N-terminus. This motif can be discovered in ShTXNL1 as 14 CRPC 17 and comprised two N-linked glycosylation sites at 72 NISA 75 and 139 NESD 142 . According to the quantitative real-time polymerase chain reaction analysis from healthy seahorses, highest ShTXNL1 mRNA expression was observed in muscle, followed by ovary, brain, gill, and blood tissues. Moreover, significant temporal expression of ShTXNL1 was observed in gill and blood tissues after bacterial stimuli. Thus, the ShTXNL1 gene may be identified as an immunologically important gene in seahorse., (Copyright © 2018 Elsevier Ltd. All rights reserved.)