Possible transport routes of IgM to the gut of teleost fish.

Fish rely on mucosal surfaces as their first defence barrier against pathogens. Maintaining mucosal homeostasis is therefore crucial for their overall well-being, and it is likely that secreted immunoglobulins (sIg) play a pivotal role in sustaining this balance. In mammals, the poly-Ig receptor (pIgR) is an essential component responsible for transporting polymeric Igs across mucosal epithelia. In teleost fish, a counterpart of pIgR has been identified and characterized, exhibiting structural differences and broader mRNA expression patterns compared to mammals. Despite supporting evidence for the binding of Igs to recombinant pIgR proteins, the absence of a joining chain (J-chain) in teleosts challenges the conventional understanding of Ig transport mechanisms. The transport of IgM to the intestine via the hepatobiliary route is observed in vertebrates and has been proposed in a few teleosts. Investigations on the stomachless fish, ballan wrasse, revealed a significant role of the hepatobiliary route and interesting possibilities for alternative IgM transport routes that might include pancreatic tissue. These findings highlight the importance of gaining a thorough understanding of the mechanisms behind Ig transport to the gut in various teleosts. This review aims to gather existing information on pIgR-mediated transport across epithelial cells and immunoglobulin transport pathways to the gut lumen in teleost fish. It provides comparative insights into the hepatobiliary transport of Igs to the gut, emphasizing the current understanding in teleost fish while exploring potential alternative pathways for Ig transport to the gut lumen. Despite significant progress in understanding various aspects, there is still much to uncover, especially concerning the diversity of mechanisms across different teleost species.

The ontogeny of lymphoid organs and IgM+ B-cells in ballan wrasse (Labrus bergylta) reveals a potential site for extrarenal B-cell lymphopoiesis: The pancreas.

Vaccination of farmed fish is the most effective prophylactic measure against contagious diseases but requires specific knowledge on when the adaptive immune system is fully developed. The present work describes kidney and spleen morphogenesis as well as B-cell development in the ballan wrasse (Labrus bergylta). The kidney was present at hatching (0 days pot hatching, dph) but was not lymphoid before larvae was 50-60 dph (stage 5), containing abundant Igµ+ cells. The spleen anlage was first observed in larvae at 20-30 dph and was later populated with B-cells. Unexpectedly, we found strong RAG1 signal together with abundant Igµ+ and IgM + cells in the exocrine pancreas of larvae from when the kidney was lymphoid and onwards, suggesting that B-cell lymphopoiesis occurs not only in the head kidney (HK) but also in pancreatic tissue. In this agastric fish, the pancreas is diffused along the intestine and the early presence of IgM+ B-cells in pancreatic tissue might have a role in maintain immune homeostasis in the peritoneal cavity, making a substantial contribution to early protection. IgM-secreting cells in HK indicate the presence of systemic IgM at stage 5, before the first IgM+ cells were identified in mucosal sites. This work together with our previous study on T-cell development in this species indicates that although T- and B-cells start to develop around the same time, B-cells migrate to mucosal tissues ahead of T-cells. This early migration likely involves the production of natural antibodies, contributing significantly to early protection. Moreover, a diet composed of barnacle nauplii did not result in an earlier onset of B-cell lymphopoiesis, as seen in the previous study analysing T-cell development. Nevertheless, components for adaptive immunity indicating putative immunocompetence is likely achieved in early juveniles (>100 dph). Additionally, maternal transfer of IgM to the offspring is also described. These findings provide important insights into the development of the immune system in ballan wrasse and lay the foundation for optimizing prophylactic strategies in the future. Furthermore, this work adds valuable information to broaden the knowledge on the immune system in lower vertebrates.

Transcriptomic and targeted immune transcript analyses confirm localized skin immune responses in Atlantic salmon towards the salmon louse.

Atlantic salmon (Salmo salar) are highly susceptible to infestations with the ectoparasite Lepeophtheirus salmonis, the salmon louse. Infestations elicit an immune response in the fish, but the response does not lead to parasite clearance, nor does it protect against subsequent infestations. It is, however, not known why the immune response is not adequate, possibly because the local response directly underneath the louse has been poorly evaluated. The present study describes the transcriptomic response by RNA sequencing of skin at the site of copepodid attachment. Analysing differentially expressed genes, 2864 were higher and 1357 were lower expressed at the louse attachment site compared to uninfested sites in the louse infested fish, while gene expression at uninfested sites were similar to uninfested control fish. The transcriptional patterns of selected immune genes were further detailed in three skin compartments/types: Whole skin, scales only and fin tissue. The elevation of pro-inflammatory cytokines and immune cell marker transcripts observed in whole skin and scale samples were not induced in fin, and a higher cytokine transcript level in scale samples suggest it can be used as a nonlethal sampling method to enhance selective breeding trials. Furthermore, the immune response was followed in both skin and anterior kidney as the infestation developed. Here, newly moulted preadult 1 stage lice induced a higher immune response than chalimi and adult lice. Overall, infestation with salmon louse induce a modest but early immune response with an elevation of mainly innate immune transcripts, with the response primarily localized to the site of attachment.

Mining Lepeophtheirus salmonis RNA-Seq data for qPCR reference genes and their application in Caligus elongatus.

Lepeophtheirus salmonis and Caligus elongatus are two parasitic copepod species posing a significant threat to salmonid aquaculture. Consequently, several gene expression studies are executed each year to gain new knowledge and treatment strategies. Though, to enable accurate gene expression measurements by quantitative real time PCR, stable reference genes are needed. Previous studies have mainly focused on a few genes selected based on their function as housekeeping genes, as these are often stably expressed in various cells and tissues. In the present study, however, RNA-sequencing data from 127 L. salmonis samples from different life stages and diverse environmental conditions were used to identify new candidate reference genes displaying low variation. From this, six genes were selected, and the stability validated by qPCR on samples from different life stages. Since neither a genome nor comprehensive RNA sequencing data are available for C. elongatus, homologous genes to those identified for L. salmonis were identified within a C. elongatus transcriptome assembly and validated by qPCR in different life stages. Overall, the genes eukaryotic translation initiation factor 1A (EIF1A) and serine/threonine-protein phosphatase 1 (PP1) displayed the highest stability in L. salmonis, while the combination of PP1 and ribosomal protein S13 (RPS13) was found to have the highest stability in C. elongatus. These genes are well-suited reference genes for qPCR applications which allow for accurate normalization of target genes.

Rainbow trout Oncorhynchus mykiss skin responses to salmon louse Lepeophtheirus salmonis: From copepodid to adult stage.

The marine crustacean Lepeophtheirus salmonis (salmon louse) is a common ectoparasite of wild and farmed salmonids. The parasite has a complex ontogeny comprising eight instars. The planktonic copepodid stage settles on host skin and pass through five instars to reach the adult stage. The present study comprises an experimental infestation of Oncorhynchus mykiss (rainbow trout) with salmon lice and describes histopathology and host immune responses in skin beneath the louse at multiple time points encompassing all louse developmental stages. Each fish was exposed to 80 infective copepodids, a mean no. of 32 parasites reached the preadult I stage whereas a mean no. of 11 parasites reached the adult stage. A progression in the severity of cutaneous lesions was observed, and levels of immune gene transcripts at the attachment site revealed a dynamic response, initially related to innate immunity. Later, immune cells accumulated in the dermis concomitant with a moderate decrease in levels of transcripts characteristic of both innate and adaptive immune responses. The present study also demonstrates that the cutaneous immune response was mainly induced at lice affected sites, while non-affected skin resembled the skin of untreated control. This indicates that the skin cannot be regarded as a uniform organ and requires careful sampling at all salmon louse stages.

Genomic characterization, phylogenetic position and in situ localization of a novel putative mononegavirus in Lepeophtheirus salmonis.

The complete genome sequence of a novel mononegavirus, Lepeophtheirus salmonis negative-stranded RNA virus 1 (LsNSRV-1), obtained from a salmonid ectoparasite, Lepeophtheirus salmonis was determined. The viral genome contains five open reading frames encoding three unknown proteins (ORF I, II and III), a putative glycoprotein (G), and a large (L) protein. Phylogenetic analysis placed LsNSRV-1 in the recently established mononegaviral family Artoviridae. LsNSRV-1 showed a prevalence of around 97% and was detected in all L. salmonis developmental stages. Viral genomic and antigenomic RNA was localized to nerve tissue, connective tissue, epithelial cells of the gut, subepidermal tissue, exocrine and cement glands, as well as the testis, vas deferens and spermatophore sac of male L. salmonis and the ovaries and oocytes of females. Viral RNA was detected in both the cytoplasm and the nucleoli of infected cells, and putative nuclear export and localization signals were found within the ORF I, III and L proteins, suggesting nuclear replication of LsNSRV-1. RNA interference (RNAi) was induced twice during development by the introduction of a double-stranded RNA fragment of ORF I, resulting in a transient knockdown of viral RNA. A large variation in the knockdown level was seen in adult males and off springs of knockdown animals, whereas the RNA level was more stable in adult females. Together with the localization of viral RNA within the male spermatophore and female oocytes and the amplification of viral RNA in developing embryos, this suggests that LsNSRV-1 is transmitted both maternally and paternally. Small amounts of viral RNA were detected at the site where chalimi were attached to the skin of Atlantic salmon (Salmo salar). However, as the RNAi-mediated treatment did not result in LsNSRV-1-negative offspring and the virus failed to replicate in the tested fish cell cultures, it is difficult to investigate the influence of secreted LsNSRV-1 on the salmon immune response.

Molecular characterization and functional analysis of a salmon louse (Lepeophtheirus salmonis, Krøyer 1838) heme peroxidase with a potential role in extracellular matrixes.

Heme peroxidases are the most abundant type of peroxidase catalyzing a H2O2-dependent oxidation of a wide variety of substrates. They are involved in numerous processes like the innate immune response, hormone and prostaglandin synthesis and crosslinking of proteins within extracellular matrixes (ECM) as well as molecules within the cuticle and chorion of arthropods and nematodes. In the present study, a Lepeophtheirus salmonis heme peroxidase (LsHPX) 1 was characterized. Amino acids in the active site of heme peroxidases were conserved, and the predicted protein sequence showed the highest similarity to genes annotated as chorion peroxidases and genes suggested to be involved in cuticle hardening or adhesion. LsHPX1 exhibited a dynamic expression during ontogenesis and during the nauplius molting cycle. Transcripts were localized to muscle cells near the muscle-tendon junction, in nerve tissue especially at neuromuscular junctions, subcuticular epithelium, subepithelial cells facing the hemolymph, exocrine glands within the subepithelial tissue and in isolated cells within the testis. Knock-down of LsHPX1 in nauplius larvae decreased the swimming activity of emerging copepodids. Histological analysis of knock-down animals revealed increased spacing between myofibers and changes in subepithelial and exocrine gland tissue. Considering these results, the potential role of LsHPX1 in crosslinking molecules of salmon louse ECMs is discussed.

A new intracellular bacterium, Candidatus Similichlamydia labri sp. nov. (Chlamydiaceae) producing epitheliocysts in ballan wrasse, Labrus bergylta (Pisces, Labridae).

Certain wrasse species (Labridae) are used as cleaner fish in salmon farms on the Norwegian coast, reducing salmon louse intensities. The pathogen repertoire of wrasse in Norway is poorly known, and the objective of the present study is to describe a novel intracellular bacterium detected in Norwegian Labrus bergylta. Histological examination of gill tissues from ballan wrasse, L. bergylta, revealed epitheliocysts occurring basally to the secondary lamellae in the interlamellar epithelium. Ultrastructurally, these had bacteria-filled inclusions with thickened membranes and radiating ray-like structures (actinae). 16S rRNA gene sequences from the gill bacteria showed the highest (97.1 %) similarity to Candidatus Similichlamydia latridicola from the gills of the latrid marine fish Latris lineata in Australia and 94.9 % similarity to Candidatus Actinochlamydia clariae, causing epitheliocystis in the freshwater catfish Clarias gariepinus in Uganda. A total of 47 gill samples from L. bergylta from Western Norway were screened by real time RT-PCR with an assay targeting Candidatus Actinochlamydiaceae 16S rRNA. Prevalence was 100 %. We propose the name Candidatus Similichlamydia labri sp. nov. for this new agent producing gill epitheliocysts in L. bergylta.

Molecular characterization and knock-down of salmon louse (Lepeophtheirus salmonis) prostaglandin E synthase.

The salmon louse (Lepeophtheirus salmonis) is a major parasite of salmonid fish in the marine environment. The interaction between the parasite and the host upon infection is not completely understood. However, it is clear that the parasite influences the host and its immune system. Prostaglandins produced by parasites such as flatworms, roundworms and ticks are documented or assumed to play a role in immunomodulation of the host. In the salmon louse, the effect of prostaglandins on the host is assumed, but remains to be documented. In this study, a salmon louse prostaglandin E2 synthase (LsPGES2) is characterized. Ontogenetic analysis showed that LsPGES2 is relatively stable expressed during development. The highest level of expression was seen in the free living stages, although elevated levels of LsPGES2 were also found in adult females. In copepodids, LsPGES2 is found around muscle cells, while it is observed in the reproductive organs of adult female lice. LsPGES2 expression was knocked-down by RNA interference in nauplii, but emerging copepodids did not display any changes in morphology nor ability to infect and develop to adult stages on fish. Additional knock-down of LsPGES2 in adult female lice did not produce any characteristic changes in phenotype nor reproductive output. It is concluded that under these experimental conditions, knock-down of LsPGES2 did not affect any essential functions of the salmon louse, neither in the free-living nor the parasitic stages.

Identification and characterization of two salmon louse heme peroxidases and their potential as vaccine antigens.

Salmon louse, Lepeophtheirus salmonis, represents major challenge for salmon farming. Current treatments impose welfare issues and are costly, whereas prophylactic measures are unavailable. Two salmon louse heme peroxidases (LsPxtl-1 and LsPxtl-2) were tested for their importance for parasite development and as potential vaccine candidates. LsPxtl-1 possesses two heme peroxidase domains and is expressed in ovaries and gut, whereas LsPxtl-2 encodes one domain and contains N-terminal signal peptide and an Eph receptor ligand-binding domain. LsPxtl-1, but not LsPxtl-2, knockdown in nauplius II stage caused poor swimming and death, indicating its importance for parasite development. Immunizations using single DNA plasmid injection encoding the peroxidases or heterologous prime (DNA) and boost (recombinant LsPxtl-2 protein) gave non-significant reduction in lice numbers. Single injection gave low specific antibody levels compared with the prime-boost. The findings suggest LsPxtl-1 is important for parasite development but formulations and vaccination modalities used did not significantly reduce lice infestation.

The thymus and T-cell ontogeny in ballan wrasse (Labrus bergylta) is nutritionally modelled.

Marine fish larvae often experience high mortality unrelated to predation during early life stages, and farmed ballan wrasse (Labrus bergylta) is no exception. Knowing when the adaptive immune system is developed and fully functional, and how nutrition may modulate these processes is therefore of importance to establish effective prophylactic measures and will also extend the relatively limited knowledge on the immune system in lower vertebrates. The thymus anlage of ballan wrasse was found to be histologically visible for the first time at larval stage 3 (20-30 days post hatch, dph) and becomes lymphoid at stage 5 (50-60 dph) correlating with an increase of T-cell marker transcripts. At this stage, a clear zonation into a RAG1+ cortex and a RAG1- CD3ϵ+ medulla was distinguished, indicating that T-cell maturation processes in ballan wrasse are similar to other teleosts. The higher abundance of CD4-1+ compared to CD8ß+ cells in the thymus together with the apparent lack of CD8ß+ cells in gill, gut, and pharynx, where CD4-1+ cells were identified, indicates that helper T-cells have a more prominent role during larval development compared to cytotoxic T-cells. As ballan wrasse lacks a stomach but has an exceptionally high IgM expression in the hindgut, we hypothesize that helper T-cells are crucial for activation and recruitment of IgM+ B-cells and possibly other leukocytes to the gut during early development. Nutritional factors such as DHA/EPA, Zn and Se may lead to an earlier expression of certain T-cell markers as well as a larger size of the thymus, indicating an earlier onset of adaptive immunity. Including live feeds that supplies the larva with higher amounts of these nutrients can therefore be beneficial for ballan wrasse farming.

Salmon louse labial gland enzymes: implications for host settlement and immune modulation.

Salmon louse (Lepeophtheirus salmonis) is a skin- and blood-feeding ectoparasite, infesting salmonids. While feeding, labial gland proteins from the salmon louse may be deposited on the Atlantic salmon (Salmo salar) skin. Previously characterized labial gland proteins are involved in anti-coagulation and may contribute to inhibiting Atlantic salmon from mounting a sufficient immune response against the ectoparasite. As labial gland proteins seem to be important in the host-parasite interaction, we have, therefore, identified and characterized ten enzymes localized to the labial gland. They are a large group of astacins named L. salmonis labial gland astacin 1-8 (LsLGA 1-8), one serine protease named L. salmonis labial gland serine protease 1 (LsLGSP1), and one apyrase named L. salmonis labial gland apyrase 1 (LsLGAp1). Protein domain predictions showed that LsLGA proteins all have N-terminal ShK domains, which may bind to potassium channels targeting the astacins to its substrate. LsLGA1 and -4 are, in addition, expressed in another gland type, whose secrete also meets the host-parasite interface. This suggests that LsLGA proteins may have an anti-microbial function and may prevent secondary infections in the wounds. LsLGAp1 is predicted to hydrolyze ATP or AMP and is, thereby, suggested to have an immune dampening function. In a knockdown study targeting LsLGSP1, a significant increase in IL-8 and MMP13 at the skin infestation site was seen under LsLGSP1 knockdown salmon louse compared to the control, suggesting that LsLGSP1 may have an anti-inflammatory effect. Moreover, most of the identified labial gland proteins are expressed in mature copepodids prior to host settlement, are not regulated by starvation, and are expressed at similar or higher levels in lice infesting the salmon louse-resistant pink salmon (Oncorhynchus gorbuscha). This study, thereby, emphasizes the importance of labial gland proteins for host settlement and their immune dampening function. This work can further contribute to anti-salmon louse treatment such as vaccine development, functional feed, or gene-edited salmon louse-resistant Atlantic salmon.

Characterisation and expression analysis of the Atlantic halibut (Hippoglossus hippoglossus L.) cytokines: IL-1ß, IL-6, IL-11, IL-12ß and IFNγ.

Genes encoding the five Atlantic halibut (Hippoglossus hippoglossus L.) cytokines; interleukin (IL)-1ß, IL-6, IL-11b, IL-12ßc, and interferon (IFN) γ, were cloned and characterised at a molecular level. The genomic organisation of the halibut cytokine genes was similar to that seen in mammals and/or other fish species. Several mRNA instability motifs were found within the 3'-untranslated region (UTR) of all cytokine cDNA sequences. The putative cytokine protein sequences showed a low sequence identity with the corresponding homologues in mammals, avian and other fish species. Nevertheless, important structural features were presumably conserved such as the presence, or absence in the case of IL-1ß, of a signal peptide, secondary structure and family signature motifs. The relative expression pattern of the cytokine genes was analyzed in several halibut organs, revealing a constitutive expression in both lymphoid and non-lymphoid organs. Interestingly, the gills showed a relatively high expression of IL-1ß, IL-12ßc and IFNγ. The real time RT-PCR data also showed that the mRNA level of IL-1ß, IL-6, IL-12ßc and IFNγ was high in the thymus, while IL-11b was relatively highly expressed in the posterior kidney and posterior gut. Moreover, the halibut brain showed a relatively high level of IL-6 transcripts. Anterior kidney leucocytes in vitro stimulated with imiquimod showed a significant increase in mRNA level of the five halibut cytokine genes. The sequence and characterisation data presented here will be useful for further investigation of both innate and adaptive immune responses in halibut, and be helpful in the design of vaccines for the control of various infectious diseases.

Small, charged proteins in salmon louse (Lepeophtheirus salmonis) secretions modulate Atlantic salmon (Salmo salar) immune responses and coagulation.

Little is known about glandular proteins secreted from the skin- and blood-feeding ectoparasite salmon louse (Lepeophtheirus salmonis). The labial gland has ducts extending into the oral cavity of the lice, and the present study aimed to identify novel genes expressed by this gland type and to investigate their role in modulation of host parameters at the lice feeding site. Five genes associated with labial gland function were identified and named Lepeophteirus salmonis labial gland protein (LsLGP) 1-4 and 1 like (LsLGP1L). All LsLGPs were predicted to be small charged secreted proteins not encoding any known protein domains. Functional studies revealed that LsLGP1 and/or LsLGP1L regulated the expression of other labial gland genes. Immune dampening functions were indicated for LsLGP2 and 3. Whereas LsLGP2 was expressed throughout the parasitic life cycle and found to dampen inflammatory cytokines, LsLGP3 displayed an increased expression in mobile stages and appeared to dampen adaptive immune responses. Expression of LsLGP4 coincided with moulting to the mobile pre-adult I stage where hematophagous feeding is initiated, and synthetic LsLGP4 decreased the clotting time of Atlantic salmon plasma. Results from the present study confirm that the salmon louse secretes immune modulating and anti-coagulative proteins with a potential application in new immune based anti-salmon louse treatments.

Roles of three putative salmon louse (Lepeophtheirus salmonis) prostaglandin E2 synthases in physiology and host-parasite interactions.

BACKGROUND: The salmon louse (Lepeophtheirus salmonis) is a parasite of salmonid fish. Atlantic salmon (Salmo salar) exhibit only a limited and ineffective immune response when infested with this parasite. Prostaglandins (PGs) have many biological functions in both invertebrates and vertebrates, one of which is the regulation of immune responses. This has led to the suggestion that prostaglandin E2 (PGE2) is important in the salmon louse host-parasite interaction, although studies of a salmon louse prostaglandin E2 synthase (PGES) 2 gene have not enabled conformation of this hypothesis. The aim of the present study was, therefore, to characterize two additional PGES-like genes. METHODS: Lepeophtheirus salmonis microsomal glutathione S-transferase 1 like (LsMGST1L) and LsPGES3L were investigated by sequencing, phylogenetics, transcript localization and expression studies. Moreover, the function of these putative PGES genes in addition to the previously identified LsPGES2 gene was analyzed in double stranded (ds) RNA-mediated knockdown (KD) salmon louse. RESULTS: Analysis of the three putative LsPGES genes showed a rather constitutive transcript level throughout development from nauplius to the adult stages, and in a range of tissues, with the highest levels in the ovaries or gut. DsRNA-mediated KD of these transcripts did not produce any characteristic changes in phenotype, and KD animals displayed a normal reproductive output. The ability of the parasite to infect or modulate the immune response of the host fish was also not affected by KD. CONCLUSIONS: Salmon louse prostaglandins may play endogenous roles in the management of reproduction and oxidative stress and may be a product of salmon louse blood digestions.

Evaluation of potential reference genes for real time RT-PCR studies in Atlantic halibut (Hippoglossus Hippoglossus L.); during development, in tissues of healthy and NNV-injected fish, and in anterior kidney leucocytes.

BACKGROUND: Real time RT-PCR has become an important tool for analyzing gene expression in fish. Although several housekeeping genes have been evaluated in Atlantic halibut (Hippoglossus Hippoglossus L.), appropriate reference genes for low copy mRNA transcripts at the earliest developmental stages have not been identified. No attempts have been reported to identify suitable reference genes in halibut infected with NNV or in stimulated halibut leucocytes. In this study, beta-actin1 (ACTB1), elongation factor 1 alpha (EF1A1), hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1), ribosomal protein L7 (RPL7), tubulin beta 2C (Tubb2C), and ubiquitin-conjugating enzyme (UbcE) were evaluated as reference genes for normalization of real time RT-PCR data during Atlantic halibut development, in tissue of healthy and NNV-infected fish, and in in vivo and in vitro stimulated anterior kidney leucocytes. RESULTS: The expression of all six genes was relatively stable from the unfertilized egg until 12 day degrees post fertilization (ddpf). However, none of the selected genes were found to be stably expressed throughout halibut development. The mRNA levels of the six genes increased from 18 ddpf, when zygotic transcription is likely to be activated, and stabilized at different time points. The Excel-based software programs BestKeeper, geNorm, and NormFinder ranked EF1A1 and UbcE as the best candidate reference genes before activation of zygotic transcription, and RPL7 and EF1A1 as the best candidates after hatching. EF1A1 and RPL7 were also listed as the best reference genes when exploring the expression levels of the six genes in various halibut organs, both in non-injected fish and in mock- and NNV-injected fish. None of the reference genes were found optimal for normalization of real time RT-PCR data from in vitro stimulated anterior kidney leucocytes. CONCLUSION: Generally, it was found that EF1A1 and RPL7 were the genes that showed least variation, with HPRT1 and UbcE as intermediate genes, and ACTB1 and Tubb2C as the least stable ones. None of the six reference genes can be recommended as reference gene candidates in ConA-PMA stimulated leucocytes. However, UbcE can be a good candidate in other experimental setups. This study emphasizes the need for reference gene evaluation, as universal reference genes have not been identified.

Cloning, characterization, and expression pattern of Atlantic halibut (Hippoglossus hippoglossus L.) CD4-2, Lck, and ZAP-70.

As known from mammalia, the co-receptors CD4 or CD8 associate with a lymphocyte cell-specific kinase (Lck) upon T-cell activation. Lck phosphorylates tyrosine residues within the CD3 chains, providing docking sites for a 70 kDa zeta-associated-protein (ZAP-70), a tyrosine protein kinase important for T-cell signaling. The sequences of a CD4-like gene (CD4-2), Lck, and ZAP-70 were cloned, characterized, and the relative expression pattern was explored in several organs of Atlantic halibut (Hippoglossus hippoglossus L.). Important structural features, as a signal peptide, two Ig-like domains followed by a connecting peptide, a transmembrane region, and a CxC motif within the cytoplasmic tail were conserved within the predicted halibut CD4-2 protein. The deduced halibut Lck protein sequence was found to be composed of a N-terminal Src homology (SH) 4 domain, required for membrane attachment and CD4/CD8 binding, SH3 and SH2 adapter domains, and a SH1 domain followed by a regulatory C-terminal tail (COOH-domain). Tyrosine residues important in Lck activation were conserved within the SH1 and COOH-domain. Structural features of ZAP-70 as tandem SH2 domains and a C-terminal SH1 domain were predicted within the halibut ZAP-70 sequence, having the highest level of conservation within these regions. Several important phosphorylation sites found to play a critical role in T-cell antigen receptor signaling in mammalian were conserved. The overall expression pattern of the three genes was highly similar, showing the highest mRNA level of all three genes in thymus. Some expression was seen in spleen, anterior and posterior kidney, gills, and fin, as seen for other halibut T-cell markers. This study will enable further experiments on halibut T-cell signaling and activation, and enhance understanding about the development of immunological memory T-cells of halibut.

A CD4 homologue in Atlantic halibut (Hippoglossus hippoglossus): molecular cloning and characterisation.

CD4 is expressed mainly on the surface of T helper cells, where it functions as a co-receptor for the T-cell receptor (TCR) by binding to MHC class II proteins. In this study we describe the cloning and sequencing of a cDNA encoding a CD4 homologue from Atlantic halibut (Hippoglossus hippoglossus L.) and the subsequent characterisation of the CD4 genomic sequence. The predicted CD4 protein has four extracellular immunoglobulin-like (Ig-like) domains (D1-D4), and was structurally similar to other CD4 homologues previously described in fish and mammals. Real time RT-PCR analysis showed that the highest levels of CD4 mRNA were found in halibut thymus, while lower expression was also seen in the spleen, gills, anterior and posterior kidneys, pectoral fins, and skin. In situ hybridisation confirmed the relatively high expression of CD4 mRNA seen in thymus by real time RT-PCR analysis, and showed that CD4 expressing cells were localised mainly in the cortex region. Only moderate changes in the levels of CD4 mRNA expression were observed during the 48 h post-injection of either nodavirus or Vibrio anguillarum.

Cloning and expression analysis of Atlantic halibut (Hippoglossus hippoglossus) CD3 genes.

The CD3 complex is in higher vertebrates shown to be important for the activation of T-cells. The T-cell system in fish is believed to be similar to that in higher vertebrates, and the CD3 chains could therefore be an important marker for identification of T-cells in fish. Here, we report the cDNA and corresponding gene sequence of Atlantic halibut (Hippoglossus hippoglossus) CD3gammadelta, CD3varepsilon, and CD3zeta chains, and the tissue-specific expression pattern of CD3 and T- cell receptor (TCR) genes. Important structural characteristics defining the CD3 genes seemed to be conserved in the halibut CD3 chains, such as a signal peptide, an extracellular region, a transmembrane helix having a negatively charged residue, and an ITAM bearing cytoplasmic tail. The extracellular domain of halibut CD3gammadelta and CD3varepsilon included two cysteines presumably involved in Ig-fold stabilisation and the CxxCxE motif important for dimerization. A spliced variant of CD3varepsilon was identified, lacking the Ig-fold, but with the CxxCxE motif intact. The real time RT-PCR analysis revealed a highly similar expression pattern of the CD3 genes and the TCRalpha and TCRbeta genes, indicating that the functional relationship between the TCR and the CD3 genes are preserved in teleosts.

Characterization of three salmon louse (Lepeophtheirus salmonis) genes with fibronectin II domains expressed by tegumental type 1 glands.

The salmon louse, Lepeophtheirus salmonis (Copepoda: Caligidae), is currently the most significant pathogen affecting the salmon farming industry in the Northern Hemisphere. Exocrine glands of blood-feeding parasites are believed to be important for the host-parasite interaction, but also in the production of substances for integument lubrication and antifouling. In L. salmonis; however, we have limited knowledge about the exocrine glands. The aim of this study was therefore to examine three genes containing fibronectin type II (FNII) domains expressed in L. salmonis tegumental type 1 (teg 1) glands, namely LsFNII1, 2 and 3. LsFNII1, 2 and 3 contains four, three, and two FNII domains respectively. Sequence alignment of LsFNII domains showed conservation of amino acids that may indicate a possible involvement of LsFNII domains in collagen binding. Ontogenetic analysis of LsFNII1, 2 and 3 revealed highest expression in pre-adult and adult lice. Localization of LsFNII1, 2 and 3 transcripts showed expression in teg 1 glands only, which are the most abundant exocrine gland type in L. salmonis. LsFNII1, 2 and 3 were successfully knocked-down by RNAi, however, alteration in gland morphology was not detected between the knock-down and control groups. Overall, this study gives first insight into FNII domain containing proteins in L. salmonis.