Insect meal in aquafeeds: A sustainable path to enhanced mucosal immunity in fish.

The mucosal surfaces of fish, including their intestines, gills, and skin, are constantly exposed to various environmental threats, such as water quality fluctuations, pollutants, and pathogens. However, various cells and microbiota closely associated with these surfaces work in tandem to create a functional protective barrier against these conditions. Recent research has shown that incorporating specific feed ingredients into fish diets can significantly boost their mucosal and general immune response. Among the various ingredients being investigated, insect meal has emerged as one of the most promising options, owing to its high protein content and immunomodulatory properties. By positively influencing the structure and function of mucosal surfaces, insect meal (IM) has the potential to enhance the overall immune status of fish. This review provides a comprehensive overview of the potential benefits of incorporating IM into aquafeed as a feed ingredient for augmenting the mucosal immune response of fish.

Mucosal barrier status in Atlantic salmon fed rapeseed oil and Schizochytrium oil partly or fully replacing fish oil through winter depression.

The study was designed to investigate the effects of replacing fish oil by algal oil and rapeseed oil on histomorphology indices of the intestine, skin and gill, mucosal barrier status and immune-related genes of mucin and antimicrobial peptide (AMP) genes in Atlantic salmon (Salmo salar). For these purposes, Atlantic salmon smolts were fed three different diets. The first was a control diet containing fish oil but no Schizochytrium oil. In the second diet, almost 50 % of the fish oil was replaced with algal oil, and in the third diet, fish oil was replaced entirely with algal oil. The algal oil contained mostly docosahexaenoic acid (DHA) and some eicosapentaenoic acid (EPA). The study lasted for 49 days in freshwater (FW), after which some fish from each diet group were transferred to seawater (SW) for a 48-h challenge test at 33 ppt to test their ability to tolerate high salinity. Samples of skin, gills, and mid intestine [both distal (DI) and anterior (AI) portions of the mid intestine] were collected after the feeding trial in FW and after the SW-challenge test to assess the effects of the diets on the structure and immune functions of the mucosal surfaces. The results showed that the 50 % VMO (Veramaris® algal oil) dietary group had improved intestinal, skin, and gill structures. Principal component analysis (PCA) of the histomorphological parameters demonstrated a significant effect of the algal oil on the intestine, skin, and gills. In particular, the mucosal barrier function of the intestine, skin, and gills was enhanced in the VMO 50 % dietary group after the SW challenge, as evidenced by increased mucous cell density. Immunolabelling of heat shock protein 70 (HSP70) in the intestine (both DI and AI) revealed downregulation of the protein expression in the 50 % VMO group and a corresponding upregulation in the 100 % VMO group compared to 0 % VMO. The reactivity of HSP70 in the epithelial cells was higher after the SW challenge compared to the FW phase. Immune-related genes related to mucosal defense, such as mucin genes [muc2, muc5ac1 (DI), muc5ac1 (AI), muc5ac2, muc5b (skin), and muc5ac1 (gills)], and antimicrobial peptide genes [def3 (DI), def3 (AI), and cath1 (skin)] were significantly upregulated in the 50 % VMO group. PCA of gene expression demonstrated the positive influences on gene regulation in the 50 % VMO dietary group. In conclusion, this study demonstrated the positive effect of substituting 50 % of fish oil with algal oil in the diets of Atlantic salmon. The findings of histomorphometry, mucosal mapping, immunohistochemistry, and immune-related genes connected to mucosal responses all support this conclusion.

Chronic stress negatively impacts wound healing, welfare, and stress regulation in internally tagged Atlantic salmon (Salmo salar).

The desire to understand fish welfare better has led to the development of live monitoring sensor tags embedded within individuals for long periods. Improving and understanding welfare must not come at the cost of impaired welfare due to a tag's presence and implantation process. When welfare is compromised, the individual will experience negative emotions such as fear, pain, and distress, impacting the stress response. In this study, Atlantic salmon (Salmo salar) underwent surgical implantation of a dummy tag. Additionally, half of this group was introduced to daily crowding stress. Both groups and an untagged group were followed for 8 weeks using triplicate tanks per group. Sampling took place once a week, and where stress was given, it was conducted 24 h before sampling. Stress-related measurements were taken to understand if tagging caused chronic stress and explore the chronic stress response and its impact on wound healing. Primary stress response hormones measured included CRH, dopamine, adrenocorticotropic hormone, and cortisol. Secondary stress response parameters measured included glucose, lactate, magnesium, calcium, chloride, and osmolality. Tertiary stress response parameters measured included weight, length, and five fins for fin erosion. Wound healing was calculated by taking the incision length and width, the inflammation length and width, and the inside wound length and width. The wound healing process showed that stressed fish have a larger and longer-lasting inflammation period and a slower wound healing process, as seen from the inside wound. The tagging of Atlantic salmon did not cause chronic stress. In contrast, daily stress led to an allostatic overload type two response. ACTH was elevated in the plasma after 4 weeks, and cortisol followed elevation after 6 weeks, highlighting a breakdown of the stress regulation. Fin erosion was elevated alongside cortisol increase in the stressed group. This data suggests that tagging previously unstressed fish in a controlled environment does not negatively affect welfare regarding stress responses. It also indicates that stress delays wound healing and increases the inflammatory response, highlighting how continued stress causes a breakdown in some stress responses. Ultimately, the tagging of Atlantic salmon can be successful under certain conditions where proper healing is observed, tag retention is high, and chronic stress is not present, which could allow for the possible measurement of welfare indicators via smart-tags.

A Truncated Galectin-3 Isolated from Skin Mucus of Atlantic Salmon Salmo salar Binds to and Modulates the Proteome of the Gram-Negative Bacteria Moritella viscosa.

The mucus of fish skin plays a vital role in innate immune defense. Some mucus proteins have the potential to incapacitate pathogens and/or inhibit their passage through the skin. In this study the aim was to isolate and characterize galectin(s), ß-galactosides binding proteins, present in skin mucus. A novel short form of galectin-3 was isolated from Atlantic salmon skin mucus by α-lactose agarose based affinity chromatography followed by Sephadex G-15 gel filtration. Mass spectrometric analysis showed that the isolated protein was the C-terminal half of galectin-3 (galectin-3C). Galectin-3C showed calcium independent and lactose inhabitable hemagglutination, and agglutinated the Gram-negative pathogenic bacteria Moritella viscosa. Galectin-3 mRNA was highly expressed in skin and gill, followed by muscle, hindgut, spleen, stomach, foregut, head kidney, and liver. Moritella viscosa incubated with galectin-3C had a modified proteome. Proteins with changed abundance included multidrug transporter and three ribosomal proteins L7/12, S2, and S13. Overall, this study shows the isolation and characterization of a novel galectin-3 short form involved in pathogen recognition and modulation, and hence in immune defense of Atlantic salmon.

Proteomic and structural differences in lumpfish skin among the dorsal, caudal and ventral regions.

Fish skin is a vital organ that serves a multitude of functions including mechanical protection, homeostasis, osmoregulation and protection against diseases. The expression of skin proteins changes under different physiological conditions. However, little is known about differences in protein expression among various body sites in naïve fish. The objectives of this work is to study potential differences in protein and gene expression among dorsal, caudal and ventral regions of lumpfish skin employing 2D gel based proteomics and real-time PCR and to assess structural differences between these regions by using Alcian blue and Periodic acid Schiff stained skin sections. The proteins collagen alfa-1, collagen alfa-2, heat shock cognate 71 kDa, histone H4, parvalbumin, natterin-2, 40S ribosomal protein S12, topoisomerase A and topoisomerase B were differentially expressed among the three regions. mRNA expression of apoa1, hspa8 and hist1h2b showed significant differences between regions. Skin photomicrographs showed differences in epidermal thickness and goblet cell counts. The ventral region showed relatively high protein expression, goblet cell count and epidermal thickness compared to dorsal and caudal regions. Overall, this study provides an important benchmark for comparative analysis of skin proteins and structure between different parts of the lumpfish body.

The Role of Galectins as Modulators of Metabolism and Inflammation.

Galectins are ß-galcotosid-binding lectins. The function of galectins varies with their tissue-specific and subcellular location, and their binding to carbohydrates makes them key players in several intra- and extracellular processes where they bind to glycosylated proteins and lipids. In humans, there are 12 identified galectins, some with tissue-specific distribution. Galectins are found inside cells and in the nucleus, cytosol, and organelles, as well as extracellularly. Galectin-1, -2, -3, -4, -7, -8, -9, and -12 can all induce T-cell apoptosis and modulate inflammation. In the context of metabolic control and loss of the same in, for example, diabetes, galectin-1, -2, -3, -9, and -12 are especially interesting. This review presents information on galectins relevant to the control of inflammation and metabolism and the potential to target galectins for therapeutic purposes.

Functional Aspects of Fish Mucosal Lectins-Interaction with Non-Self.

Mucosal surfaces are of key importance in protecting animals against external threats including pathogens. In the mucosal surfaces, host molecules interact with non-self to prevent infection and disease. Interestingly, both inhibition and stimulation of uptake hinder infection. In this review, the current knowledgebase on teleost mucosal lectins' ability to interact with non-self is summarised with a focus on agglutination, growth inhibition, opsonisation, cell adhesion, and direct killing activities. Further research on lectins is essential, both to understand the immune system of fishes, since they rely more on the innate immune system than mammals, and also to explore these molecules' antibiotic and antiparasitic activities against veterinary and human pathogens.

Chronic wounds alter the proteome profile in skin mucus of farmed gilthead seabream.

BACKGROUND: Skin and its mucus are known to be the first barrier of defence against any external stressors. In fish, skin wounds frequently appear as a result of intensive culture and also some diseases have skin ulcers as external clinical signs. However, there is no information about the changes produced by the wounds in the mucosae. In the present paper, we have studied the alterations in the proteome map of skin mucus of gilthead seabream during healing of experimentally produced chronic wounds by 2-DE followed by LC-MS/MS. The corresponding gene expression changes of some identified skin proteins were also investigated through qPCR. RESULTS: Our study has successfully identified 21 differentially expressed proteins involved in immunity and stress processes as well as other metabolic and structural proteins and revealed, for the first time, that all are downregulated in the skin mucus of wounded seabream specimens. At transcript level, we found that four of nine markers (ighm, gst3, actb and krt1) were downregulated after causing the wounds while the rest of them remained unaltered in the wounded fish. Finally, ELISA analysis revealed that IgM levels were significantly lower in wounded fish compared to the control fish. CONCLUSIONS: Our study revealed a decreased-expression at protein and for some transcripts at mRNA levels in wounded fish, which could affect the functionality of these molecules, and therefore, delay the wound healing process and increase the susceptibility to any infection after wounds in the skin of gilthead seabream.

Skin mucus proteins of lumpsucker (Cyclopterus lumpus).

Fish skin mucus serves as a first line of defense against pathogens and external stressors. In this study the proteomic profile of lumpsucker skin mucus was characterized using 2D gels coupled with tandem mass spectrometry. Mucosal proteins were identified by homology searches across the databases SwissProt, NCBInr and vertebrate EST. The identified proteins were clustered into ten groups based on their gene ontology biological process in PANTHER (www.patherdb.org). Calmodulin, cystatin-B, histone H2B, peroxiredoxin1, apolipoprotein A1, natterin-2, 14-3-3 protein, alfa enolase, pentraxin, warm temperature acclimation 65 kDa (WAP65kDa) and heat shock proteins were identified. Several of the proteins are known to be involved in immune and/or stress responses. Proteomic profile established in this study could be a benchmark for differential proteomics studies.

Novel mannose binding natterin-like protein in the skin mucus of Atlantic cod (Gadus morhua).

This study presents the first report of purification of natterin-like protein (Nlp) in a non-venomous fish. The peptide identities of purified cod Nlp were confirmed through LC-MSMS and matched to a cod expressed sequence tag (EST). A partial cod nlp nucleotide sequence was amplified and sequenced based on this EST. Multiple sequence alignment of cod Nlp showed considerable homology with other teleost Nlps and the presence of an N-terminal jacalin-like lectin domain coupled with a C-terminal toxin domain. nlp expression was higher in skin, head kidney, liver and spleen than in other tissues studied. Hemaggluttination of horse red blood cells by Nlp was calcium dependent and inhibited by mannose. A Vibrio anguillarum bath challenge however, did not alter the expression of cod nlp transcripts in the skin and gills. Further functional characterization is required to establish the significance of this unique protein in Atlantic cod and other teleosts.

Immune relevant molecules identified in the skin mucus of fish using -omics technologies.

This review will give an overview of immune relevant molecules in fish skin mucus. The skin of fish is continuously exposed to a water environment, and unlike that of terrestrial vertebrates, it is a mucosal surface with a thin epidermis of live cells covered by a mucus layer. The mucosa plays an important role in maintaining the homeostasis of the fish and preventing the entry of invading pathogens. This review provides an overview of proteins, RNA, DNA, lipids and carbohydrates found in the skin mucus of studied species. Proteins such as actin, histones, lectins, lysozyme, mucin, and transferrin have extracellular immune relevant functions. Complement complement molecules, heat shock molecules and superoxide dismutase present in mucus show differential expression during pathogen challenge in some species, but their functions in mucus, if any, need to be shown. RNA, DNA, lipids, carbohydrates and metabolites in mucus have been studied to a limited extent in fish, the current knowledge is summarized and knowledge gaps are pointed out.

Differential proteome profile of skin mucus of gilthead seabream (Sparus aurata) after probiotic intake and/or overcrowding stress.

Gilthead seabream (Sparus aurata L.) is the major cultured fish species in the Mediterranean area. High density stocking causes stress and increases the impact of diseases leading to economic losses. Probiotics could represent a solution to prevent diseases through several mechanisms such as improving the immune status and/or mucosal microbiota or competing with pathogens. The probiotic Shewanella putrefaciens, also known as Pdp11, was firstly isolated from the skin of healthy gilthead seabream. Our study focuses on the skin mucus proteome after dietary probiotic Pdp11 intake in fish maintained under normal or overcrowding conditions. 2-DE of skin mucus followed by LC-MS/MS analysis was done for each experimental group and differentially expressed proteins were identified. The results showed differentially expressed proteins especially involved in immune processes, such as lysozyme, complement C3, natural killer cell enhancing factor and nonspecific cytotoxic cell receptor protein 1, whose transcript profiles were studied by qPCR. A consistency between lysozyme protein levels in the mucus and lysozyme mRNA levels in skin was found. Further research is necessary to unravel the implications of skin mucosal immunity on fish welfare and disease. BIOLOGICAL SIGNIFICANCE: The present work reveals the proteomic changes, which are taking place in the skin mucus of stressed and non-stressed gilthead seabream after Pdp11 probiotic intake. The study contributes to improving the knowledge on skin mucosal immunology of this relevant farmed fish species. Furthermore, the paper shows for the first time how a suitable proteomic methodology, in this case 2-DE followed by LC-MS/MS is useful to perform a comparative study with a non-invasive technique of skin mucus of gilthead seabream.

Skin mucus proteome map of European sea bass (Dicentrarchus labrax).

Skin mucus is the first barrier of fish defence. Proteins from skin mucus of European sea bass (Dicentrarchus labrax) were identified by 2DE followed by LC-MS/MS. From all the identified proteins in the proteome map, we focus on the proteins associated with several immune pathways in fish. Furthermore, the real-time PCR transcript levels in skin are shown. Proteins found include apolipoprotein A1, calmodulin, complement C3, fucose-binding lectin, lysozyme and several caspases. To our knowledge, this is the first skin mucus proteome study and further transcriptional profiling of the identified proteins done on this bony fish species. This not only contributes knowledge on the routes involved in mucosal innate immunity, but also establishes a non-invasive technique based on locating immune markers with a potential use for prevention and/or diagnosis of fish diseases.

Evaluation of immune and apoptosis related gene responses using an RNAi approach in vaccinated Penaeus monodon during oral WSSV infection.

In the present study RNA interference was used to elucidate the connection between two endogenous genes [Penaeus monodon Rab7 (PmRab7) or P. monodon inhibitor of apoptosis (PmIAP)], and selected immune/apoptosis-related genes in orally 'vaccinated' shrimp after white spot syndrome virus (WSSV) infection. P. monodon were vaccinated by feeding them with formalin inactivated WSSV-coated feed. Thereafter, PmRab7 or PmIAP genes were silenced by injecting the shrimps with their respective dsRNA. The resulting groups of shrimps, Rab7 and IAP, were orally infected with WSSV and the expression of three immune-relevant genes in Rab7 group and five apoptosis-related genes in IAP group was evaluated. In the Rab7 group, PmToll, PmPPAE 2 and Pm penaeidin genes were down-regulated. The IAP-silenced shrimps were characterized by down-regulation of Pm caspase, PmERp57, Pm14-3-3 ε, Pm ald, and up-regulation of PmSTAT. Thus, silencing of PmRab7/PmIAP has provided important clues on their relationship with selected immune/apoptosis genes in orally vaccinated P. monodon during WSSV infection.

Protein profiling in the gut of Penaeus monodon gavaged with oral WSSV-vaccines and live white spot syndrome virus.

White spot syndrome virus (WSSV) is a pathogen that causes considerable mortality of the farmed shrimp, Penaeus monodon. Candidate 'vaccines', WSSV envelope protein VP28 and formalin-inactivated WSSV, can provide short-lived protection against the virus. In this study, P. monodon was orally intubated with the aforementioned vaccine candidates, and protein expression in the gut of immunised shrimps was profiled. The alterations in protein profiles in shrimps infected orally with live-WSSV were also examined. Seventeen of the identified proteins in the vaccine and WSSV-intubated shrimps varied significantly compared to those in the control shrimps. These proteins, classified under exoskeletal, cytoskeletal, immune-related, intracellular organelle part, intracellular calcium-binding or energy metabolism, are thought to directly or indirectly affect shrimp's immunity. The changes in the expression levels of crustacyanin, serine proteases, myosin light chain, and ER protein 57 observed in orally vaccinated shrimp may probably be linked to immunoprotective responses. On the other hand, altered expression of proteins linked to exoskeleton, calcium regulation and energy metabolism in WSSV-intubated shrimps is likely to symbolise disturbances in calcium homeostasis and energy metabolism.

A novel beta-defensin antimicrobial peptide in Atlantic cod with stimulatory effect on phagocytic activity.

A novel defensin antimicrobial peptide gene was identified in Atlantic cod, Gadus morhua. This three exon/two intron defensin gene codes for a peptide precursor consisting of two domains: a signal peptide of 26 amino acids and a mature peptide of 40 residues. The mature cod defensin has six conserved cysteine residues that form 1-5, 2-4 and 3-6 disulphide bridges. This pattern is typical of beta-defensins and this gene was therefore named cod beta-defensin (defb). The tertiary structure of Defb exhibits an α/ß fold with one α helix and ß1ß2ß3 sheets. RT-PCR analysis indicated that defb transcripts were present mainly in the swim bladder and peritoneum wall but could also be detected at moderate to low levels in skin, head- and excretory kidneys. In situ hybridisation revealed that defb was specifically expressed by cells located in the swim bladder submucosa and the oocytes. During embryonic development, defb gene transcripts were detectable from the golden eye stage onwards and their expression was restricted to the swim bladder and retina. Defb was differentially expressed in several tissues following antigenic challenge with Vibrio anguillarum, being up-regulated up to 25-fold in head kidney. Recombinant Defb displayed antibacterial activity, with a minimal inhibitory concentration of 0.4-0.8 µM and 25-50 µM against the Gram-(+) bacteria Planococcus citreus and Micrococcus luteus, respectively. In addition, Defb stimulated phagocytic activity of cod head kidney leucocytes in vitro. These findings imply that beta-defensins may play an important role in the innate immune response of Atlantic cod.

Differentially expressed proteins in the skin mucus of Atlantic cod (Gadus morhua) upon natural infection with Vibrio anguillarum.

BACKGROUND: Vibriosis caused by V. anguillarum is a commonly encountered disease in Atlantic cod farms and several studies indicate that the initiation of infection occurs after the attachment of the pathogen to the mucosal surfaces (gut, skin and gills) of fish. Therefore it is necessary to investigate the role of different mucosal components in fish upon V. anguillarum infection. The present study has two parts; in the first part we analyzed the differential expression of skin mucus proteins from Atlantic cod naturally infected with V. anguillarum using two dimensional gel electrophoresis coupled with mass spectrometry. In the second part, a separate bath challenge experiment with V. anguillarum was conducted to assess the mRNA levels of the genes in skin tissue, corresponding to the selected proteins identified in the first part. RESULTS: Comparative proteome analysis of skin mucus of cod upon natural infection with V. anguillarum revealed key immune relevant proteins like calpain small subunit 1, glutathione-S-transferase omega 1, proteasome 26S subunit, 14-kDa apolipoprotein, beta 2-tubulin, cold inducible RNA binding protein, malate dehydrogenase 2 (mitochondrial) and type II keratin that exhibited significant differential expression. Additionally a number of protein spots which showed large variability amongst individual fish were also identified. Some of the proteins identified were mapped to the immunologically relevant JNK (c-Jun N-terminal kinases) signalling pathway that is connected to cellular events associated with pathogenesis. A bath challenge experiment with V. anguillarum showed differential expression of beta 2-tubulin, calpain small subunit 1, cold inducible RNA binding protein, flotillin1, and glutathione S-transferase omega 1 transcripts in the skin tissue of cod during early stages of infection. CONCLUSIONS: Differentially expressed proteins identified in the cod skin mucus point towards their possible involvement in V. anguillarum pathogenesis. The role of some of these proteins in vibriosis in cod described in this paper can be considered unconventional with respect to their established functions in higher vertebrates. Based on the differential expression of these proteins they are possibly important components of fish defence against bacteria and innate immunity at large. The feasibility of utilizing these proteins/genes as markers of bacterial infection or stress in cod needs to be explored further.

Localization and functional properties of two galectin-1 proteins in Atlantic cod (Gadus morhua) mucosal tissues.

Galectin-1 is a ß-galactoside binding lectin with multiple immune functions in higher vertebrates. We report the characterization of two galectin-1 proteins from Atlantic cod, with emphasis on mucosal tissues. Tissue distribution of these two ≈14kDa galectin-1 proteins (Codgal1-1 and Codgal1-2) was ascertained by western blotting of one dimensional (1D) and two dimensional (2DE) gels. The two galectin-1 proteins were differentially localized in the mucosal tissues of cod. Codgal1-1 was predominantly localized in the basal cells of skin and this protein was present in all the early developmental stages examined, indicating a likely involvement in developmental processes. The two lectins were also localized in the adherent macrophage-like cells (MLC) from cod head kidney and results gathered indicate their possible secretion during Francisella noatunensis infection, suggesting that they are active components of immune defence. Lactose affinity chromatography coupled with gel filtration co-purified the two cod galectin-1 proteins, which hemagglutinated horse red blood cells in a lactose inhibitable manner. They also could bind and agglutinate both Gram-positive and Gram-negative bacteria. This study suggests multiple functional roles for galectin-1, especially in development and innate immune response of Atlantic cod.

Transcriptional regulation of cytokines in the intestine of Atlantic cod fed yeast derived mannan oligosaccharide or ß-glucan and challenged with Vibrio anguillarum.

Immunomodulatory feed additives are expected to exert their primary influence at the intestinal level through the expression of cytokines, which in turn affect the immune responses in fish. In two separate experiments a yeast-derived mannan oligosaccharide product (YM) or a purified ß-glucan (BG) product were fed to Atlantic cod (Gadus morhua L.) for 5 weeks, after which they were bath-challenged with a bacterial pathogen--Vibrio anguillarum. The transcription of selected cytokines (proinflammatory--il1b, il8, ifng; anti-inflammatory--il10) in different intestinal segments was analysed using qPCR. In the case of YM study, the effect of the compound was observed in both the posterior intestine and rectum of Atlantic cod, upon challenge with the pathogen. iIl1b expression in the posterior intestine and rectum of post-challenge fish was significantly higher than that of pre-challenge fish. In the case of il8 the difference was confined to rectum. The expression of ifng was altered only in the anterior intestine upon YM feeding. In the BG trial, the additive had a differential effect on the expression of the cytokine genes. In anterior intestine and rectum, the purified ß-glucan additive significantly elevated the expression of il1b when challenged with V. anguillarum. An effect of BG on the anti-inflammatory cytokine il10 was visible in the rectum after the pathogen challenge. The differential responses of cytokines in the intestine of fish upon exposure to V. anguillarum suggest that both mannan oligosaccharides and ß-glucans impact the ability of Atlantic cod to respond to the pathogen.

Ubiquitous presence of piscidin-1 in Atlantic cod as evidenced by immunolocalisation.

BACKGROUND: Antimicrobial peptides (AMPs), the natural antibiotics bestowed upon all forms of life, consist of small molecular weight proteins with a broad spectrum antimicrobial activity against a variety of pathogenic microorganisms. Piscidins are one of the AMP families that are imperative for the innate defence mechanisms of teleosts. Atlantic cod, a basal fish belonging to the superorder Paracanthopterygii also possesses multiple piscidin peptides. Two piscidin paralogues (pis1 and pis2) and a novel alternative splice variant of pis2 of this fish were previously described by us. To shed light on other potent roles of these molecules, now we have mapped the distribution of piscidin 1 (Pis1), in different tissues and organs of cod through immunohistochemistry (IHC) employing an affinity purified polyclonal antibody specific to Pis1. RESULTS: Various cell types and tissues of Atlantic cod including those from the immune organs of naïve fish are armed with Pis1 peptide. Different types of the blood leucocytes and phagocytic cells among the leucocytes examined gave a relatively strong indication of Pis1 immunopositivity. In addition, other cell types such as hematopoietic cells, epithelial cells and multi-granular cells located in the mucosal and hematopoietic tissues were also Pis1-immunoreactive. More interestingly, chondrocytes appear to produce Pis1 and this is the first report on the presence of an AMP in cartilage tissue of fish. Furthermore, Pis1 immunopositivity was detected in other tissues and organs of naïve fish including neural tissues, exocrine and endocrine glands, compound gland cells, excretory kidney, intestinal and respiratory epithelial cells, swim bladder, skin and hypodermis layer, myosepta, liver, heart, eye and oocytes. CONCLUSIONS: Pis1 peptide is produced by various cell types located in different tissues and organs of Atlantic cod. It is present in all immune-related organs of naïve fish and the elevated peptide expression following phagocytosis strongly suggest their involvement in innate defence. Further, its widespread occurrence in non-immune tissues and organs of apparently healthy fish implies that piscidin may have other functions in addition to its role as an immune effector molecule.