Evolution and expression analysis of STAT family members in blunt snout bream (Megalobrama amblycephala).

The Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway is involved in regulating the body's immunity, cell proliferation, differentiation, and apoptosis. Members of the STAT family have been extensively studied in different mammalian species. However, there are few studies on the STAT family genes in farmed economic fish. In this study, eight STAT genes including STAT1a, STAT1b, STAT2, STAT3, STAT4, STAT5a, STAT5b and STAT6, in blunt snout bream (Megalobrama amblycephala), an economically important fish in China, were identified and characterized. Analyses of gene location, phylogeny and conserved synteny were conducted to infer the evolutionary origin of these STAT family genes. Furthermore, the evolutionary origin model of STATs was constructed based on the 2R hypothesis and teleost genome duplication (TGD) hypothesis, which clarified the evolutionary origin of the eight STATs in blunt snout bream. Besides, expression of the eight STATs was detected in 10 tissues of healthy blunt snout bream, which showed different expression patterns, and all had the highest level in the blood. In addition, expression of the STATs was significantly induced in the spleen, liver, and kidney after infection of Aeromonas hydrophila, suggesting that they play an important role in protecting the host from pathogens. In general, the evolution of cytokine-related genes parallels that of the immune system, which has likely been a main evolutionary driver. Therefore, the evolutionary model of STAT genes, constructed in the non-model organism pioneeringly, may provide some enlightenment for the evolution of the fish STAT family genes and their involvement in the immune function.

Dietary watermelon residue influencing the nonspecific immunity of juvenile Pseudorasbora parva.

The study explored the improvement of disease resistance, non-specific immunity and anti-oxidation reactions for Pseudorasbora parva (PP) using dietary watermelon residue. The cumulative PP mortality and the pathogenic bacteria number in 15-45% groups reduced relative to those in control group (CK). Under 15-45% groups, AKP, ACP activities and akp, acp genes expression levels were increased markedly in nonspecific immunity system. Similarly, antioxidant response (SOD, CAT activities) and their genes was promoted also at 15-45% groups. Organic matter (vitamin and polyphenols) in watermelon residue improved AKP, ACP, SOD, CAT activities by increasing corresponding gene expressions. Theoretically, they could also function as stimulus signal, active center or composition to modulate enzyme activities and gene expressions. Besides, watermelon residue ameliorated NF-kB, mTOR responses pathway, and consequently suppressed Aeromonas hydrophila which augmented disease resistance.

An effective live attenuated vaccine against Aeromonas veronii infection in the loach (Misgurnus anguillicaudatus).

Aeromonas veronii is a major pathogenic bacterium in humans and animals. When it causes outbreaks, there are enormous economic losses to the aquaculture industry. An effective live attenuated vaccine strain, ΔhisJ, was obtained in our previous studies by gene knockout in Aeromonas veronii TH0426 using the suicide vector pRE112. Here, we evaluated whether the live attenuated vaccine ΔhisJ was suitable for prevention of Aeromonas veronii infection by injection and immersion in loaches. Compared with that of the TH0426 wild-type strain, the virulence of the live vaccine was significantly weakened. Vaccine safety assessment results also indicated that 1 × 107 CFU/mL live vaccine was safe and did not induce clinical symptoms or obvious pathological changes. Additionally, after challenging loaches with Aeromonas veronii TH0426, the relative percent survival of the IN3 injection group was 65.66%, and that of the IM group was 50.78%. Our data show that the live attenuated vaccine ΔhisJ can improve the immune protection rate of loaches. Furthermore, increased enzyme activity parameters (SOD, LZM, ACP, and AKP) in the skin mucus, increased enzyme activity parameters (SOD, LZM, ACP, AKP, and GPx) in the serum, increased specific IgM antibodies and cytokine IL-1ß contents in the serum, and increased cytokine (IL-15, pIgR, IL-1ß, and TNF-α) expression in the liver and spleen were observed. These data are the first to indicate that the live attenuated vaccine ΔhisJ is suitable for the development of a safe and effective vaccine against Aeromonas veronii infection in loach aquaculture.

A triple WAP domain containing protein acts in antibacterial immunity of weather loach, Misgurnus anguillicaudatus.

Whey acidic protein domain (WAPD) occurs in a variety of proteins in animals and many of WAPD-containing proteins are involved in immunity. In the present study, a novel protein containing three WAPDs was identified from the weather loach, Misgurnus anguillicaudatus, designated as MaTWD. MaTWD share high identity with TWDs from fish but low identity with TWDs from other animal phyla. MaTWD transcripts mainly distributed in gills and head kidney responded to bacterial challenge with significant upregulation. In vitro assay with recombinant MaTWD protein revealed that MaTWD had antiprotease activity against bacterial proteases. Moreover, MaTWD exhibited bacterial binding capacity and antimicrobial activity. Most importantly, exogenous MaTWD protected loach against bacterial infection by reducing loach mortality. We infer that MaTWD participates in the antibacterial immunity of loach via its antiprotease and antimicrobial activities.

A C-type lectin with antibacterial activity in weather loach, Misgurnus anguillicaudatus.

C-type lectins are carbohydrate-binding proteins that play important roles in immunity by serving as pattern recognition receptors. In the present study, a novel nattectin-like C-type lectin was obtained from the weather loach, Misgurnus anguillicaudatus, designated as MaCTL. MaCTL encodes a peptide with 165 amino acids, with a signal peptide and a single C-type lectin domain (CTLD), containing a galactose-specific QPD motif and a conserved Ca2+ -binding site. Transcripts of MaCTL were significantly upregulated after immune challenge with its pathogen A. hydrophila. In vitro assays with recombinant MaCTL protein revealed that it exhibited hemagglutinating and bacterial agglutinating activities, in a Ca2+ -dependent manner. MaCTL was found to bind to a wide range of bacteria, as well as bind to bacterial polysaccharides LPS and PGN. Moreover, MaCTL displayed antimicrobial activity by inhibiting the growth of bacteria. These results collectively suggest that MaCTL is involved in the antibacterial defence of weather loach.

Effect of acclimated temperature on thermal tolerance, immune response and expression of HSP genes in Labeo rohita, Catla catla and their intergeneric hybrids.

The ability of a species and population to respond to a decrease or an increase in temperature depends on their adaptive potential. Here, the critical thermal tolerance (CTmax and CTmin) of four populations: Labeo rohita, Catla catla, and their reciprocal hybrids L. rohitaâ™€× C. catla♂ (RC) and C. catla♀ × L. rohita♂ (CR) being acclimatized at four acclimation temperatures (22, 26, 30 and 34 °C) were determined. All populations indicated substantial variations (P < 0.05) in CTmax and CTmin values. L. rohita displayed, comparatively the highest CTmax with largest total and intrinsic polygon zones as well as the upper and lower acquired thermal tolerance zones followed by RC and CR hybrids, while C. catla showed significantly the highest CTmin value and the smallest intrinsic and acquired thermal tolerance zones. Both hybrids illustrated low parent heterosis (≤11%). Additionally, the highest expression of Hsp70 and Hsp90 (heat shock proteins) genes, serum lysozyme level, respiratory burst activity and lowest lipid peroxidation level under lower and higher temperature shock further illustrated strong physiological mechanism of L. rohita in contrast to C. catla, to deal with acute temperature, while hybrids, especially F1 RC hybrid appeared as a good option to replace C. catla in relatively higher and lower temperature areas.

Molecular cloning and expression analysis of myd88 from oriental weatherfish (Misgurnus anguillicaudatus) in response to bacterial challenge.

Myeloid differentiation factor 88 (Myd88) plays an important role in both innate and adaptive immune response. In this study, the full-length complementary DNA (cDNA) of myd88 from Misgurnus anguillicaudatus was characterized. The myd88 cDNA is 1333 bp in length and contains an 855 bp open reading frame encoding a predicted protein of 284 amino acids. The predicted protein possesses typical Myd88 domain structural features including a death domain in the N-terminus, and box 1, 2, and 3 motifs of the Toll/IL-1 receptor domain in the C-terminus. Quantitative real-time PCR (qRT-PCR) revealed that myd88 messenger RNA (mRNA) was ubiquitously expressed in all examined tissues, especially highly in brain, kidney, blood, intestines and liver. qRT-PCR and western blotting were used to determine the mRNA and protein levels of Myd88 after Aeromonas veronii challenge, respectively. The Myd88 was remarkably upregulated in response to infection of A. veronii. These results suggested that Myd88 may play a vital role during the immune response of M. anguillicaudatus against bacterial infection.

Transcriptome Analysis of Maternal Gene Transcripts in Unfertilized Eggs of Misgurnus anguillicaudatus and Identification of Immune-Related Maternal Genes.

Maternal genes are important in directing early development and determining egg quality in fish. We here report the de novo transcriptome from four tissue libraries of the cyprinid loach, Misgurnus anguillicaudatus, and for the first time identified maternal gene transcripts in unfertilized eggs and suggest their immune system involvement. Expression profiles and functional enrichment revealed a total 24,116 transcripts were expressed as maternal transcripts in unfertilized eggs, which were involved in a wide range of biological functions and pathways. Comparison expression profiles and analysis of tissue specificity revealed that the large numbers of maternal transcripts were stored in unfertilized eggs near the late phase of ovarian maturation and before ovulation. Functional classification showed a total of 279 maternal immune-related transcripts classified with immune system process GO term and immune system KEGG pathway. qPCR analysis showed that transcript levels of identified maternal immune-related candidate genes were dynamically modulated during development and early ontogeny of M. anguillicaudatus. Taken together, this study could not only provide knowledge on the protective roles of maternal immune-related genes during early life stage of M. anguillicaudatus but could also be a valuable transcriptomic/genomic resource for further analysis of maternally provisioned genes in M. anguillicaudatus and other related teleost fishes.

Combined effects of temperature increase and immune challenge in two wild gudgeon populations.

In the context of global changes, aquatic ecosystems are increasingly exposed to multiple stressors that can have unexpected interactive effects on aquatic organisms. Among these stressors, the occurrence of heat waves and pathogens is changing rapidly in freshwater rivers, but their combined effects on fish health are still understudied. In this study, we experimentally tested the crossed effects of increased temperature (mimicking a heat wave) and a standardized immune challenge (mimicking a parasite attack) on wild gudgeon (Gobio occitaniae) physiology and behaviour across biological levels from molecules to the whole individual. We also investigated the potential variation of sensitivity among populations by comparing two wild populations from contrasted thermal regimes. Combined stressors (i.e. temperature increase and immune challenge) had contrasted effects on fish physiology and behaviour compared to single stressors, but only at the individual level. In particular, the immune challenge inhibited the effect of the temperature on fish behaviour (activity, exploration and foraging) but amplified the negative effect of temperature on fish survival. No interactions were found at other biological levels. This study thus shows that it is essential to consider biotic stressors such as pathogens to better anticipate the effects of global changes on aquatic organisms. In addition, there was a high variability of response between the two gudgeon populations, suggesting that future studies should take into account population variability to better predict the responses of aquatic wildlife to current and future stressors.

Dietary chitosan oligosaccharides modulate the growth, intestine digestive enzymes, body composition and nonspecific immunity of loach Paramisgurnus dabryanus.

Three test diets containing three different levels (1, 3, and 5 g kg-1) of dietary chitosan oligosaccharides (COs) were formulated and used to test the growth performance, body composition, intestine digestive enzymes, antioxidant responses and resistance to Aeromonas hydrophila of loach Paramisgurnus dabryanus. A basal diet without any COs served as the control. After 60 days of feeding, the growth performance, intestine digestive-enzyme activities, body protein content and total polyunsaturated fatty acids, antioxidant responses, and resistance to A. hydrophila of loach P. dabryanus were higher than those of the control when the loach P. dabryanus was fed with CO-containing diets. The optimum dose of dietary COs required for the maximum growth of loach was 3 g kg-1 of the diet. Results indicated that dietary COs can improve the growth performance, body composition, intestine digestive enzymes, antioxidant responses, and resistance to A. hydrophila of loach P. dabryanus and can thus be used as a diet supplement for them.

Expression analysis of the heat shock protein genes and cellular reaction in dojo loach (Misgurnus anguillicaudatus) under the different pathogenic invasion.

As molecular chaperones, heat shock proteins (HSPs) play essential roles in cells in response to stress conditions. Recent studies about immune functions of HSPs in fish have also been reported. In this study, based on the reported cDNA sequences of the four HSP genes, HSP70, HSC70, HSP90α and HSP90ß, the temporal expression patterns of the four genes during embryonic development of dojo loach(Misgurnus anguillicaudatus) was assayed with qRT-PCR. All of the four genes were ubiquitously expressed in all detected embryonic developmental stages. Among of them, HSP70, HSC70 and HSP90ß were highly expressed in the organ formation stage, while HSP90α was the highest expressed in myotome formation stage. Further, the immune responses of the four HSP genes were assayed when loach were infected with three different pathogens, bacterium (Flavobacterium cloumnare G4), parasite (Ichthyophthirius multifiliis) and fungus (Saprolegnia). All of the four genes were differentially expressed in four tissues such as skin, gills, spleen and kidney in response to the pathogenic invasion, but both HSP70 and HSP90α expressions were dramatically up-regulated. Further, the cellular responses of the loach skinand gill tissues were observed, in which the number of the skin goblet cells were significantly increased, and the gill lamellae became shorter and wider after infected. Thus, our work indicated that the HSPs may directly or indirectly involved in immune defense in fish, at least in the loach.

Immunity augmentation in Botia dario (Hamilton, 1822) through carotenoid: a dietary approach.

The immunity and health status of ornamental fish is an important aspect, as they are kept in a confined environment and various stressful conditions which lead to depletion of overall colourful appearance and mortality. The carotenoids can act as immunity boosters in captive aquarium system and may be supplemented in the feed as aquarium fish have no access to natural carotenoids. The study aimed to assess the role of carotenoid on the immunity of B. dario. Marigold petal meal is an important source of carotenoids and used in experimental diets. Four immunogenes namely IL20, TLR9, TRAIL, and Nramp in B. dario were characterized and also studied for their relative expression in the kidney after feeding the fish with marigold petal meal supplemented diet. The expression pattern of the genes was compared with the fish of nature. The IL20 and Nramp gene were upregulated significantly (p < 0.05) in the fish of nature as compared to the experimental fish at the 60th day of feeding carotenoid-rich diet. But the TLR9 and TRAIL gene was upregulated significantly (p < 0.05) in experimental fish as compared to nature. The haematological parameters of fish after feeding with the experimental enriched diet for 60 days also confirmed the role of carotenoids in immunity.

Polymeric immunoglobulin receptor in dojo loach (Misgurnus anguillicaudatus): Molecular characterization and expression analysis in response to bacterial and parasitic challenge.

The polymeric immunoglobulin receptor (pIgR) is an essential component of the mucosal immune system in jawed vertebrates including teleost fish, which mediate transepithelial transport of secretory immunoglobulins (sIgs) to protect organisms against environmental pathogens. In this study, we firstly cloned and identified the pIgR from dojo loach (Misgurnus anguillicaudatus). The full-length cDNA of Ma-pIgR was of 1145 bp, containing an open reading frame (ORF) of 1101 bp encoded a predicted protein of 336 amino acids. The structure of Ma-pIgR is comprised of a signal peptide, a transmembrane region, an intracellular region and an extracellular region with two Ig-like domains (ILDs), which are similar to their counterparts described in other teleosts. Multiple sequence alignment and phylogenetic analysis showed the dojo loach is closely related to the fish family Cyprinidae. The transcriptional level of Ma-pIgR was detected by quantitative real-time PCR (qRT-PCR) in different tissues and high expression was found in liver, skin, kidney, eye, fin and gills. Two infection models of the loach with bacteria (Aeromonas hydrophila) and parasite (Ichthyophthirius multifiliis) were constructed for the first time. Histological studies showed the goblet cells in skin significantly increased and the ratio of gill length to width also significantly changed after challenged with A.hydrophila. Both challenge experiments resulted in the significant up-regulated expression of Ma-pIgR not only in kidney and spleen, but also in skin and gills. Our results suggest that pIgR may play an important role in skin and gill mucosal immunity to protect the loach against bacteria and parasite.

Molecular characterization and expression analysis of complement component 3 in dojo loach (Misgurnus anguillicaudatus).

The complement component 3 (C3) is a central component of complement system. All three pathways converge at formation of C3 convertases and share the terminal pathways of membrane attack complex (MAC) formation. In this study, three isoforms of C3 were discovered in Misgurnus anguillicaudatus, named "C3-1", "C3-2" and "C3-3", respectively. The full-length of C3-1 cDNA sequence was firstly identified and analyzed from dojo loach (Misgurnus anguillicaudatus). The Ma-C3-1 cDNA sequence comprised of 4509 bp encoding 1454 amino acids with a putative signal peptide of 20 amino acid residues. The deduced amino acid sequence showed that Ma-C3-1 has conserved residues and domain, which are known to be crucial for C3 function. Interestingly, an amino acid substitution of the highly conserved GCGEQ was discovered in Ma-C3-1. Phylogenetic analysis showed that Ma-C3-1 was closely related to Cyprinidae. The mRNA expression levels of three isoforms of C3 were detected in kidney, eye, spleen, gonad, heart, fin ray, gut, muscle, brain, gill, skin, blood and liver. The expression of Ma-C3-1 and Ma-C3-3 were mainly detected in liver, followed by spleen, gonad. However, the high expression of Ma-C3-2 was found in kidney, followed by blood and gonad. The morphological changes of gill and skin, and the expression pattern of these three isoforms C3 molecular following the infection with Aeromonas hydrophila were investigated. The mRNA expression levels of three C3 isoforms were up-regulated in the gill, skin, liver and spleen after infection with A.hydrophila. Similarly, challenge experiments resulted in significant up-regulated expression of other complement-relevant genes in gill, liver and skin, such as C4, C5, C8b, especially at 24 h and 36 h. These results suggest that complement system might play an important role not only in liver, but also in the mucosal tissues as gill and skin of teleost fish.

Identification and molecular characterization of an Akirin2 homolog in Chinese loach (Paramisgurnus dabryanus).

Akirin is a nuclear factor involved in innate immune responses of arthropods and mammals. In this study we have cloned an Akirin2 gene, pdakirin2, from freshwater Chinese loach (Paramisgurnus dabryanus) and characterized its biological functions. Phylogenetic analysis revealed deduced PdAkirin2 had high sequence identities to Akirin2 homologs from fish and mammals (70-91%), it contained two conserved nuclear localization signals (NLSs) with verified sub-cellular localization. Quantitative real-time (qRT)-PCR analysis indicated that PdAkirin2 was present in a wide range of loach tissues and showed up-regulation with challenges of Aeromonas hydrophila NJ-1, LPS and poly I:C. PdAkirin2 as an immune factor had significant effects on the expression of cytokines (TNFα, IFN-α, IFN-γ, IL-4 and IL-1ß) and transcription factor NF-κB. This study provides insights into the potential role of PdAkirin2 in the innate immune system.

cDNA cloning and expression characterization of serum transferrin gene from oriental weatherfish Misgurnus anguillicaudatus.

In this study, the cDNA coding serum transferrin (stf) of Misgurnus anguillicaudatus (mastf) was cloned. mastf cDNA is composed of 2326 bp with a 2007 bp open reading frame encoding 668 amino acids. The deduced Mastf protein consists of a signal peptide, two lobes (N and C-lobes) and signature motifs of transferrin (Tf) family. The results of tissue distribution indicated that mastf mRNA was predominantly expressed in the liver. The results indicate that the mastf expression increased significantly in liver, blood, spleen and head kidney after the challenge with Aeromonas sobria, acting as a positive acute protein, suggesting that mastf is related to the immune response. The cloning and expression analysis of mastf further demonstrates the evolutionary conservation of Stf and immune function in vertebrates.

Identification, expression and bioactivity of Paramisgurnus dabryanus ß-defensin that might be involved in immune defense against bacterial infection.

ß-defensins are a large family of multi-disulfide-bonded peptides with broad-spectrum antimicrobial activities that contribute to innate host defense in many organisms, but little information is available about ß-defensins produced by freshwater fish lacking scales. We therefore cloned and identified a ß-defensin gene from Chinese loach (Paramisgurnus dabryanus) by designing degenerate primers and using thermal asymmetric interlaced PCR. This gene is the first defensin gene ever identified in a non-scaled freshwater fish. Annotation of the protein domain architecture showed that the putative Chinese loach ß-defensin contains the signature motif of six conserved cysteines within the mature peptide, an aspect similar to ß-defensins of other marine fish. We also used quantitative real-time PCR to investigate the expression pattern of the Chinese loach ß-defensin gene, mRNA of which could be observed in various tissues. After challenge with the pathogenic bacterium Aeromonas hydrophila, ß-defensin expression was induced in the eye, gill, skin, and spleen of the adult loach. The bioactivity of the recombinant P. dabryanus ß-defensin was examined against pathogenic bacteria, and the results suggest that this class 2 ß-defensin has potential applications for treatment of bacterial infections.

The transcriptome sequencing analysis reveals immune mechanisms of soybean fermented powder on the loach (Misgurnus anguillicaudatus) in response to Lipopolysaccharide (LPS) infection.

The loach (Misgurnus anguillicaudatus), a small commercial fish that is widely cultivated for its high-quality protein, vitamins, minerals, and essential amino acid, is a member of the genus Misgurnus and the family Cyprinidae. In this study, we gave the LPS-injected loach fermented soybean meal and used transcriptome sequencing to investigate the impact of the fermented soybean powder on the loach's immune system. 3384 up-regulated genes and 12116 down-regulated genes were found among the 15500 differentially expressed genes, according to the results. The differentially expressed genes were shown to be involved in cellular processes, metabolic processes, cellular anatomical entities, and binding, according to the Go functional annotation. Meanwhile, the KEGG enrichment analysis indicated that the soybean fermented powder treated groups showed significant differences in DNA replication, Nucleotide excision repair, Fanconi anemia pathway, and Base excision repair pathways, suggesting that these pathways are closely related to the enhancement of the immune function of loach by soybean fermented powder. The particular conclusions not exclusively can provide a new conception for the rational utilization of soybean fermented powder but also can provide theoretical guidance for the subsequent healthy breeding of loach.

Development of a quantitative assay to measure expression of transforming growth factor ß (TGF-ß) in Lost River sucker (Deltistes luxatus) and shortnose sucker (Chasmistes brevirostris) and evaluation of potential pitfalls in use with field-collected samples.

The Nature Conservancy is in the process of restoring the Williamson River Delta in an attempt to recreate important juvenile habitat for the endangered shortnose sucker Chasmistes brevirostris and the endangered Lost River sucker Deltistes luxatus. Measurement of TGF-ß mRNA expression level was one of the indicators chosen to evaluate juvenile sucker health during the restoration process. TGF-ß mRNA expression level has been correlated with disease status in several laboratory studies and TGF-ß mRNA expression level has been used as a species-specific indicator of immune status in field-based fish health assessments. We describe here the identification of TGF-ß and a possible splice variant from shortnose sucker and from Lost River sucker. The performance of a quantitative RT-PCR assay to measure TGF-ß mRNA expression level was evaluated in field-collected spleen and kidney tissue samples. The quality of extracted RNA was higher in tissues harvested in September compared to July and higher in tissues harvested at lower temperature compared to higher temperature. In addition, the expression level of both TGF-ß and 18S as assessed by qRT-PCR was higher in samples with higher quality RNA. TGF-ß mRNA expression was lower in kidney than in spleen in both Lost River sucker and shortnose sucker.

Molecular characterization of hepcidin gene from mud loach (Misgurnus mizolepis; Cypriniformes).

The gene encoding hepcidin, an antimicrobial peptide, was isolated and characterized in the mud loach Misgurnus mizolepis (Cypriniformes). Mud loach hepcidin shows a considerable degree of structural homology to other vertebrate hamp1 orthologues at both the gene and protein levels, particularly with respect to its tripartite genomic organization, typical transcription-factor-binding motifs in its promoter, and conserved cysteine residues in the mature cationic peptide. The mud loach possesses at least two allelic forms of hamp1, which are expected to be translated into the same hepcidin preproprotein. The two alleles are transmitted from parental fish to offspring with a Mendelian inheritance pattern, as demonstrated with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) genotyping. Southern blot hybridization analysis showed a high degree of polymorphisms in the restriction patterns of individuals. Mud loach hamp1 mRNA is predominantly expressed in the liver, although many other tissues showed detectable levels of hamp1 transcripts in RT-PCR assay. Lipopolysaccharide and bacterial challenges induced significant hamp1 expression, whereas hamp1 was not clearly stimulated by polyinosinic:polycytidylic acid [poly(I:C)] injection. Iron overload and Cu exposure also elevated hamp1 transcripts in various tissues. The transcriptional activation of mud loach hamp1 in response to these stimuli varied among tissue types, and the liver appears predominantly involved in hepcidin-mediated iron regulation. However, hepcidin expression in the kidney and spleen was preferentially modulated by inflammation-mediated signals produced by immune challenges. Our results suggest that mud loach hepcidin has two basic functions, in iron regulation and antimicrobial activity, and that its transcription is also modulated by other environmental perturbations, including heavy metal exposure.