Antigen presentation genes in gadoid species (haddock: Melanogrammus aeglefinus and Atlantic cod: Gadus morhua) raise questions about cross-presentation pathways and glycosylated beta-2-microglobulin.

The Atlantic cod immune system deviates from antigen presentation processes seen in other vertebrates in that it lacks the necessary genes for exogenous antigen presentation (i.e., MHC-II and li) and a key MHC-II interacting molecule necessary for T-helper cell function (i.e., CD4), while possessing an expanded repertoire of MHC-I genes that facilitate endogenous antigen presentation. These observations, combined with the identification of putative endosomal sorting signals in MHC-I cytoplasmic tails, have led to speculation that cod rely on cross-presentation of exogenous antigens to elicit cytotoxic T-lymphocyte responses against extracellular threats. In light of this suggestion, we investigated MHC-I transcriptional profiles and endosomal sorting signals in a closely related gadoid species, the haddock. Analysis of transcripts from one individual identified 13 unique MHC-I molecules, including two non-classical molecules as determined by the level of conservation at their peptide anchoring sites. This suggests that like the cod, the haddock has an expanded MHC-I repertoire. Analysis of haddock MHC-I cytoplasmic tail sequences revealed that the dileucine- and tyrosine-based endosomal signaling motifs, that are suggested to facilitate cross-presentation in cod, were absent. Closer examination of the cod signaling motifs, including their relative position in the cytoplasmic tail region, indicates that these motifs might be non-functional, further supporting the need for functional studies to assess cross-presentation. Finally, in silico analysis and in vitro N-type de-glycosylation experiments demonstrate that haddock and cod beta-2-microglobulin (ß2M) are glycosylated at the same NQT sequon. Interestingly, whole genome tBLASTn searches also revealed that putative ß2 M glycosylation sites appear frequently within the Gadiformes lineage, as the predictive NQT and other N-X-S/T sequons were located in ß2M orthologues from 19 of the 25 additional gadoid genomes analyzed. Though the exact significance of ß2M glycosylation has yet to be elucidated, phylogenetic comparisons predict that the same NQT glycosylation sequence occurs in 13 additional species comprising four different orders of Actinopterygii (Gymnotiformes, Esociformes, Beryciformes and Perciformes). This suggests either that this feature has arisen independently in multiple lineages or that it comes from a common ancestor and has been lost or modified in many species. Together, the analysis of gadoid MHC-I genes and ß2M molecules highlights the challenges in generalizing immune system paradigms across the most diverse vertebrate lineage (i.e., fish) and between fish and more well-studied mammals.

Innovation in Nucleotide-Binding Oligomerization-Like Receptor and Toll-Like Receptor Sensing Drives the Major Histocompatibility Complex-II Free Atlantic Cod Immune System.

The absence of MHC class II antigen presentation and multiple pathogen recognition receptors in the Atlantic cod has not impaired its immune response however how underlying mechanisms have adapted remains largely unknown. In this study, ex vivo cod macrophages were challenged with various bacterial and viral microbe-associated molecular patterns (MAMP) to identify major response pathways. Cytosolic MAMP-PRR pathways based upon the NOD-like receptors (NLRs) and RIG-I-like receptors (RLRs) were identified as the critical response pathways. Our analyses suggest that internalization of exogenous ligands through scavenger receptors drives both pathways activating transcription factors like NF-kB (Nuclear factor-kappa B) and interferon regulatory factors (IRFs). Further, ligand-dependent differential expression of a unique TLR25 isoform and multiple NLR paralogues suggests (sub)neofunctionalization toward specific immune defensive strategies. Our results further demonstrate that the unique immune system of the Atlantic cod provides an unprecedented opportunity to explore the evolutionary history of PRR-based signaling in vertebrate immunity.

T-box transcription factor eomesodermin/Tbr2 in Atlantic cod (Gadus morhua L.): Molecular characterization, promoter structure and function analysis.

Eomesodermin (Eomes) is a member of T-box transcription factor family and plays an important role in the regulation of a wide variety of developmental processes and immune response in animals. Here we report cloning and characterization of the full-length cDNA of Atlantic cod Eomes (GmEomes), which possesses a TBOX_3 domain similar to its counterpart in mammals. The regulated expression was observed in head kidney and spleen in response to live Vibrio anguillarum infection in vivo, and spleen leukocytes in vitro after PMA and poly I:C stimulation. Furthermore, we determined a 694 bp sequence, upstream of the transcriptional start site (TSS), to contain a number of sequence motifs that matched known transcription factor-binding sites. Activities of the presumptive regulatory gene were assessed by transfecting different 5'-deletion constructs in CHSE-214 cells. The results showed that the basal promoters and positive transcriptional regulator activities of GmEomes were dependent by sequences located from -694 to -376 bp upstream of TSS. Furthermore, we found that some Eomes binding sites were present in the 5'-flanking regions of the cod IFNγ gene predicted by bioinformatics. However, Co-transfection of eomesodermin overexpression plasmids with INFγ reporter vector into CHSE-214 cells determined that Atlantic cod eomesodermin played a minor role in activation of the INFγ promoter.

Complement component C4-like protein in Atlantic cod (Gadus morhua L.) - Detection in ontogeny and identification of post-translational deimination in serum and extracellular vesicles.

The complement system is a critical part of teleost immune defences, with complement component C4 forming part of the classical and lectin pathways. Cod C4-like protein was isolated from plasma, specific antibodies generated and C4-like protein was assessed in cod sera, mucus and in extracellular vesicles (EVs) from serum and mucus. Higher levels of C4-like protein were detected in serum- than mucus-derived EVs. Post-translational deimination, caused by conversion of arginine into citrulline, can affect protein structure and function. Here we detected deiminated forms of C4-like protein in cod serum and at lower levels in mucus. C4-like protein was also found in deiminated form at low levels in EVs from both serum and mucus. C4-like protein was assessed by immunohistochemistry in cod larvae and detected in a range of organs including in liver, kidney, gut, muscle, skin and mucus, as well as in neuronal tissues of the brain, spinal cord and eye. This abundance of C4-like protein during early development may indicate roles in tissue remodelling, in addition to immune defences. The presence of deiminated C4-like protein in serum and mucosa, as well as in EVs, may suggest C4 protein moonlighting via post-translational deimination.

Whole transcriptome analysis of the Atlantic cod vaccine response reveals subtle changes in adaptive immunity.

Atlantic cod has lost the Major Histocompatibility complex class II pathway - central to pathogen presentation, humoral response and immunity. Here, we investigate the immunological response of Atlantic cod subsequent to dip vaccination with Vibrioanguillarum bacterin using transcriptome sequencing. The experiment was conducted on siblings from an Atlantic cod family found to be highly susceptible towards vibriosis where vaccination has demonstrated improved pathogen resistance. Gene expression analyses at 2, 4, 21 and 42 days post vaccination revealed GO-term enrichment for muscle, neuron and metabolism-related pathways. In-depth characterization of immune-related GO terms demonstrated down-regulation of MHCI antigen presentation, C-type lectin receptor signaling and granulocyte activation over time. Phagocytosis, interferon-gamma signaling and negative regulation of innate immunity were increasingly up-regulated over time. Individual differentially expressed immune genes implies weak initiation of acute phase proteins with little or no inflammation. Furthermore, gene expression indicates presence of T-cells, NK-like cells, B-cells and monocytes/macrophages. Three MHCI transcripts were up-regulated with B2M and SEC61. Overall, we find no clear immune-related transcriptomic response which could be attributed to Atlantic cod's alternative immune system. However, we cannot rule out that this response is related to vaccination protocol/sampling strategy. Earlier functional studies demonstrate significant memory in Atlantic cod post dip vaccination and combined with our results indicate the presence of other adaptive immunity mechanisms. In particular, we suggest that further investigations should look into CD8+ memory T-cells, γδ T-cells, T-cell independent memory or memory induced through NK-like/other lymphoid cells locally in the mucosal lining for this particular vaccination strategy.

Aeromonas salmonicida subsp. salmonicida Early Infection and Immune Response of Atlantic Cod (Gadus morhua L.) Primary Macrophages.

In contrast to other teleosts, Atlantic cod (Gadus morhua) has an expanded repertoire of MHC-I and TLR components, but lacks the MHC-II, the invariant chain/CD74, and CD4+ T cell response, essential for production of antibodies and prevention of bacterial infectious diseases. The mechanisms by which G. morhua fight bacterial infections are not well understood. Aeromonas salmonicida subsp. salmonicida is a recurrent pathogen in cultured and wild fish, and has been reported in Atlantic cod. Macrophages are some of the first responders to bacterial infection and the link between innate and adaptive immune response. Here, we evaluated the viability, reactive oxygen species (ROS) production, cell morphology, and gene expression of cod primary macrophages in response to A. salmonicida infection. We found that A. salmonicida infects cod primary macrophages without killing the cod cells. Likewise, infected Atlantic cod macrophages up-regulated key genes involved in the inflammatory response (e.g., IL-1ß and IL-8) and bacterial recognition (e.g., BPI/LBP). Nevertheless, our results showed a down-regulation of genes related to antimicrobial peptide and ROS production, suggesting that A. salmonicida utilizes its virulence mechanisms to control and prevent macrophage anti-bacterial activity. Our results also indicate that Atlantic cod has a basal ROS production in non-infected cells, and this was not increased after contact with A. salmonicida. Transmission electron microscopy results showed that A. salmonicida was able to infect the macrophages in a high number, and release outer membrane vesicles (OMV) during intracellular infection. These results suggest that Atlantic cod macrophage innate immunity is able to detect A. salmonicida and trigger an anti-inflammatory response, however A. salmonicida controls the cell immune response to prevent bacterial clearance, during early infection.

Disentangling the immune response and host-pathogen interactions in Francisella noatunensis infected Atlantic cod.

The genetic repertoire underlying teleost immunity has been shown to be highly variable. A rare example is Atlantic cod and its relatives Gadiformes that lacks a hallmark of vertebrate immunity: Major Histocompatibility Complex class II. No immunological studies so far have fully unraveled the functionality of this particular immune system. Through global transcriptomic profiling, we investigate the immune response and host-pathogen interaction of Atlantic cod infected with the facultative intracellular bacterium Francisella noatunensis. We find that Atlantic cod displays an overall classic innate immune response with inflammation, acute-phase proteins and cell recruitment through up-regulation of e.g. IL1B, fibrinogen, cathelicidin, hepcidin and several chemotactic cytokines such as the neutrophil attractants CXCL1 and CXCL8. In terms of adaptive immunity, we observe up-regulation of interferon gamma followed by up-regulation of several MHCI transcripts and genes related to antigen transport and loading. Finally, we find up-regulation of immunoglobulins and down-regulation of T-cell and NK-like cell markers. Our analyses also uncover some contradictory transcriptional findings such as up-regulation of anti-inflammatory IL10 as well as down-regulation of the NADPH oxidase complex and myeloperoxidase. This we interpret as the result of host-pathogen interactions where F. noatunensis modulates the immune response. In summary, our results suggest that Atlantic cod mounts a classic innate immune response as well as a neutrophil-driven response. In terms of adaptive immunity, both endogenous and exogenous antigens are being presented on MHCI and antibody production is likely enabled through direct B-cell stimulation with possible neutrophil help. Collectively, we have obtained novel insight in the orchestration of the Atlantic cod immune system and determined likely targets of F. noatunensis host-pathogen interactions.

Extracellular vesicles from cod (Gadus morhua L.) mucus contain innate immune factors and deiminated protein cargo.

Extracellular vesicles are released from cells and participate in cell communication via transfer of protein and genetic cargo derived from the parent cells. EVs play roles in normal physiology and immunity and are also linked to various pathological processes. Peptidylarginine deiminases (PADs) are phylogenetically conserved enzymes with physiological and pathophysiological roles. PADs cause post-translational protein deimination, resulting in structural and, in some cases, functional changes in target proteins and are also linked to EV biogenesis. This study describes for the first time EVs isolated from cod mucosa. Mucosal EVs were characterised by electron microscopy, nanoparticle tracking analysis and EV-specific surface markers. Cod mucosal EVs were found to carry PAD, complement component C3 and C-reactive proteins. C3 was found to be deiminated in both whole mucus and mucosal EVs, with some differences, and further 6 deiminated immune and cytoskeletal proteins were identified in EVs by LC-MS/MS analysis. As mucosal surfaces of teleost fish reflect human mucosal surfaces, these findings may provide useful insights into roles of EVs in mucosal immunity throughout phylogeny.

Serum IgE cross-reactivity between fish and chicken meats in dogs.

BACKGROUND: In humans, a cross-reactive clinical allergy has been reported between three chicken and fish meat proteins: beta-enolase, aldolase A and parvalbumin. OBJECTIVE: To evaluate if IgE cross-reactivity between chicken and fish also existed in the dog. ANIMALS: Sera from dogs with suspected allergic skin disease and with IgE against chicken and fish. METHODS AND MATERIALS: Sera were analysed by ELISA and immunoblotting with chicken, white fish (haddock and cod) and salmon extracts. Reciprocal inhibition ELISAs and inhibition immunoblots were then performed. Protein sequencing of bands identified on multiple extracts was determined by mass spectrometry. RESULTS: Out of 53 archived canine sera tested by ELISA against chicken, white fish or salmon, 15 (28%), 12 (23%) and 26 (49%), respectively, had elevated IgE against one, two or all three of these extracts. Seven of the triple-reactive sera were subjected to reciprocal inhibition ELISAs. A >50% inhibition was found between chicken-fish, chicken-salmon and fish-salmon in seven, four and five of seven dogs, respectively. Immunoblotting identified multiple IgE-binding proteins of identical molecular weights in the three extracts; these were partially to fully cross-reactive by inhibition immunoblotting. Mass spectrometry identified nine cross-reactive proteins as: pyruvate kinase, creatine kinase, alpha-actin, glyceraldehyde-3-phosphate dehydrogenase, beta-enolase, aldolase, malate dehydrogenase, lactate dehydrogenase and triose-phosphate isomerase 1. All of these have been reported previously as fish, shellfish and/or chicken allergens for humans. CONCLUSIONS AND CLINICAL IMPORTANCE: Whether any of these newly identified IgE cross-reactive chicken-fish allergens is the cause of clinical allergy needs to be determined in dogs reacting to at least two of these common food sources.

A method to detect allergenic fish, specifically cod and pollock, using quantitative real-time PCR and COI DNA barcoding sequences.

BACKGROUND: Fish are one of eight major allergens defined in the US Food Allergen Labeling and Consumer Protection Act, and cod and pollock are two of the major fish allergens. This paper describes development and validation of a method to detect cod and pollock in complex food matrices using real-time polymerase chain reaction (PCR). Mitochondrial cytochrome oxidase I (COI) sequences obtained through DNA barcoding were used to design a single set of primers and probe which detected three species in the genus Gadus: Atlantic cod, Pacific cod, and walleye pollock. RESULTS: Cod spiked into three different food matrices (cooking oil, clam chowder, and hushpuppy mix) yielded high linearity, dynamic range spanning six orders of magnitude, and lower limits of detection at 1-10 ppm (ppm; mg kg-1 ). Frying had an adverse effect on the lower limit of detection, but not on linearity. CONCLUSIONS: This work shows that COI DNA barcoding sequences can be used to effectively design real-time PCR assays for detection of food allergens in complex matrices. While full-length DNA barcodes distinguish individual species, the PCR assay designed here detected three different species. This is likely because real-time PCR assays are tolerant to basepair mismatches and do not utilize the full length of the DNA barcode. Published 2018. This article is a U.S. Government work and is in the public domain in the USA.

Pentraxins CRP-I and CRP-II are post-translationally deiminated and differ in tissue specificity in cod (Gadus morhua L.) ontogeny.

Pentraxins are fluid phase pattern recognition molecules that form an important part of the innate immune defence and are conserved between fish and human. In Atlantic cod (Gadus morhua L.), two pentraxin-like proteins have been described, CRP-I and CRP-II. Here we show for the first time that these two CRP forms are post-translationally deiminated (an irreversible conversion of arginine to citrulline) and differ with respect to tissue specific localisation in cod ontogeny from 3 to 84 days post hatching. While both forms are expressed in liver, albeit at temporally differing levels, CRP-I shows a strong association with nervous tissue while CRP-II is strongly associated to mucosal tissues of gut and skin. This indicates differing roles for the two pentraxin types in immune responses and tissue remodelling, also elucidating novel roles for CRP-I in the nervous system. The presence of deimination positive bands for cod CRPs varied somewhat between mucus and serum, possibly facilitating CRP protein moonlighting, allowing the same protein to exhibit a range of biological functions and thus meeting different functional requirements in different tissues. The presented findings may further current understanding of the diverse roles of pentraxins in teleost immune defences and tissue remodelling, as well as in various human pathologies, including autoimmune diseases, amyloidosis and cancer.

Discovery of microRNAs associated with the antiviral immune response of Atlantic cod macrophages.

MicroRNAs (miRNAs) are known to play important immunoregulatory roles in teleosts, although miRNAs involved in the antiviral immune response of Atlantic cod (Gadus morhua) were previously uncharacterised. Using deep sequencing and qPCR, the present study was conducted to identify miRNAs responsive to the viral mimic, polyriboinosinic polyribocytidylic acid (pIC) in Atlantic cod macrophages. Macrophage samples isolated from Atlantic cod (n=3) and treated with pIC or phosphate buffered saline (PBS control) for 24 and 72h were used for miRNA profiling. Following deep sequencing, DESeq2 analyses identified four (miR-731-3p, miR-125b-3-3p, miR-150-3p and miR-462-3p) and two (miR-2188-3p and miR-462-3p) significantly differentially expressed miRNAs at 24 and 72h post-stimulation (HPS), respectively. Sequencing-identified miRNAs were subjected to qPCR validation using a larger number of biological replicates (n=6) exposed to pIC or PBS over time (i.e. 12, 24, 48 and 72 HPS). As in sequencing, miR-731-3p, miR-462-3p and miR-2188-3p showed significant up-regulation by pIC. The sequencing results were not qPCR-validated for miR-125b-3-3p and miR-150-3p as up- and down-regulated miRNAs at 24 HPS, respectively; however, qPCR results showed significant up-regulation in response to pIC stimulation at later time points (i.e. 48 and/or 72 HPS). We also used qPCR to assess the expression of other miRNAs that were previously shown as immune responsive in other vertebrates. qPCR results at 48 and/or 72 HPS revealed that miR-128-3-5p, miR-214-1-5p and miR-451-3p were induced by pIC, whereas miR-30b-3p and miR-199-1-3p expression were repressed in response to pIC. The present study identified ten pIC-stimulated miRNAs, suggesting them as important in antiviral immune responses of Atlantic cod macrophages. Some pIC-responsive miRNAs identified in this study were predicted to target putative immune-related genes of Atlantic cod (e.g. miR-30b-3p targeting herc4), although the regulatory functions of these miRNAs need to be validated by future studies.

Francisella noatunensis subspecies noatunensis clpB deletion mutant impairs development of francisellosis in a zebrafish model.

BACKGROUND: Francisella noatunensis ssp. noatunensis (F.n.n.) is the causative agent of francisellosis in Atlantic cod and constitutes one of the main challenges for future aquaculture on this species. A facultative intracellular bacterium like F.n.n. exert an immunologic challenge against which live attenuated vaccines in general are most effective. Thus, we constructed a deletion in the F.n.n. clpB gene as ΔclpB mutants are among the most promising vaccine candidates in human pathogenic Francisella. PURPOSE: Characterization of F.n.n. ΔclpB using primary Atlantic cod head kidney leukocytes, the zebrafish embryo and adult zebrafish model with focus on potential attenuation, relevant immune responses and immunogenic potential. MAIN RESULTS: Interleukin 1 beta transcription in Atlantic cod leukocytes was significantly elevated from 24 to 96 h post infection with F.n.n. ΔclpB compared to F.n.n. wild-type (wt). Growth attenuation of the deletion mutant in zebrafish embryos was observed by fluorescence microscopy and confirmed by genome quantification by qPCR. In the immunization experiment, adult zebrafish were immunized with 7 × 106 CFU of F.n.n. ΔclpB before challenge four weeks later with 6 × 108 CFU of F.n.n. wt. One day after challenge, immunized zebrafish responded with significantly lower interleukin 8 levels compared to the non-immunized control. Immunized fish were protected against the acute mortality observed in non-immunized zebrafish after challenge and bacterial genomes quantified by qPCR were reduced to a minimum 28 days post challenge, indicating protective immunity stimulated by F.n.n. ΔclpB. CONCLUSION: Deletion mutation of clpB in F.n.n. causes in vitro and in vivo attenuation and elicits a protective immune response in adult zebrafish against a lethal dose of F.n.n. wt. Taken together, the results presented increases the knowledge on protective immune responses against F.n.n.

Immunomodulatory effects of excretory/secretory compounds from Contracaecum osculatum larvae in a zebrafish inflammation model.

Excretory/secretory (ES) compounds isolated from third-stage larvae of the anisakid nematode Contracaecum osculatum parasitizing liver of Baltic cod were investigated for effects on immune gene expression in a zebrafish LPS-induced inflammation model. ES products containing a series of proteins, of which some had enzymatic activity, were injected solely or with LPS. ES proteins alone induced up-regulation of a number of immune-related genes, but generally to a lower degree compared to LPS. When co-injected with LPS, the worm products exacerbated merely expression of five genes affecting Th1, Th2, Th17 and innate responses compared to the LPS-injected group. However, the level of overexpression decreased in an inverse dose-dependent manner. The immune regulating action of C. osculatum ES products is interpreted as an important evolutionary ability of larval parasites in the transport host which makes it less susceptible to host immune responses whereby the probability of reaching the final host is increased.

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.

How the codfish changed its immune system.

A common ancestor of the modern codfish acquired a set of mutations that eliminated a major arm of the adaptive immune system-the MHC II pathway of antigen presentation to CD4(+) T cells. Subsequent to this event, there was a radiation of these fish in which the number and diversity of MHC I genes increased in species-specific ways.

Transcriptome profiling of the antiviral immune response in Atlantic cod macrophages.

A study was conducted to determine the transcriptome response of Atlantic cod (Gadus morhua) macrophages to the viral mimic, polyriboinosinic polyribocytidylic acid (pIC), using a 20K Atlantic cod microarray platform and qPCR. We identified 285 significantly up-regulated and 161 significantly down-regulated probes in cod macrophages 24 h after pIC stimulation. A subset of 26 microarray-identified transcripts was subjected to qPCR validation using samples treated with pIC or phosphate-buffered saline (control) over time (3, 6, 12, 24, 48 h), and 77% of them showed a significant response to pIC. The microarray and qPCR analyses in this study showed that pIC induced the expression of cod macrophage transcripts involved in RLR- and TLR-dependent pathogen recognition (e.g. tlr3, tlr7, mda5 and lgp2), as well as signal transducers (e.g. stat1 and nfkbia) and transcription activators (e.g. irf7 and irf10) in the MyD88-independent and dependent signalling pathways. Several immune effectors (e.g. isg15s, viperin, herc4, mip2 and ccl13) were significantly up-regulated in pIC-stimulated cod macrophages. The expression of some transcripts (e.g. irf7, irf10, viperin) was significantly up-regulated by pIC as early as 12 h. All pIC-induced transcripts had peak expression at either 24 h (e.g. tlr7, irf7, mip2) or 48 h (e.g. tlr3, lgp2, stat1). This study suggests possible roles of both vertebrate-conserved (e.g. tlr3 as an up-regulated gene) and fish-specific (tlr22g as a down-regulated gene) receptors in dsRNA recognition, and the importance of conserved and potentially fish-specific interferon stimulated genes in cod macrophages.

Multiple specialised goose-type lysozymes potentially compensate for an exceptional lack of chicken-type lysozymes in Atlantic cod.

Previous analyses of the Atlantic cod genome showed unique combinations of lacking and expanded number of genes for the immune system. The present study examined lysozyme activity, lysozyme gene distribution and expression in cod. Enzymatic assays employing specific bacterial lysozyme inhibitors provided evidence for presence of g-type, but unexpectedly not for c-type lysozyme activity. Database homology searches failed to identify any c-type lysozyme gene in the cod genome or in expressed sequence tags from cod. In contrast, we identified four g-type lysozyme genes (LygF1a-d) constitutively expressed, although differentially, in all cod organs examined. The active site glutamate residue is replaced by alanine in LygF1a, thus making it enzymatic inactive, while LygF1d was found in two active site variants carrying alanine or glutamate, respectively. In vitro and in vivo infection by the intracellular bacterium Francisella noatunensis gave a significantly reduced LygF1a and b expression but increased expression of the LygF1c and d genes as did also the interferon gamma (IFNγ) cytokine. These results demonstrate a lack of c-type lysozyme that is unprecedented among vertebrates. Our results further indicate that serial gene duplications have produced multiple differentially regulated cod g-type lysozymes with specialised functions potentially compensating for the lack of c-type lysozymes.

ß-naphthoflavone interferes with cyp1c1, cox2 and IL-8 gene transcription and leukotriene B4 secretion in Atlantic cod (Gadus morhua) head kidney cells during inflammation.

The objective of this study was to evaluate how ß-naphthoflavone interacts with lipopolysaccharide (LPS) and polyinosinic acid: polycytidylic acid (poly I: C) induced innate immune parameters as well as phase I and phase II detoxification enzymes in head kidney cells isolated from Atlantic cod. ß-naphthoflavone is a pure agonist of aryl hydrocarbon receptor (AhR) while LPS and poly I: C are not. ß-naphthoflavone was added to head kidney leukocytes alone or together with LPS or poly I: C and the responses were evaluated in terms of protein and gene expression. The results showed that ß-naphthoflavone (25 nM), with and without LPS, significantly induced cytochrome P450 (cyp1c) transcription in cod head kidney cells. ß-naphthoflavone (100 nM) in the presence of the virus mimic, poly I: C, also increased cyp1c1transcription. LPS induced cyp1c1, cyclooxygenase 2 (cox2), interleukin 1ß (IL-1ß), interleukin 6 (IL-6) and interleukin 8 (IL-8) transcription, genes that were not affected by the tested ß-naphthoflavone concentrations alone. However, ß-naphthoflavone (25 and 50 nM) strengthened LPS induced cox2 and IL-8 transcription. Cod head kidney cells exposed to ß-naphthoflavone concentrations ranging from 25 to 100 nM, with and without LPS or poly I: C, expressed AhR protein. LPS or ß-naphthoflavone (5-50 nM) significantly induced leukotriene B4 (LTB4) secretion compared to control. In conclusion, this study suggests that ß-naphthoflavone could interfere with LPS induced immune cell signaling in cod head kidney cells.