Molecular characterization of a fish-specific toll-like receptor 22 (TLR22) gene from common carp (Cyprinus carpio L.): Evolutionary relationship and induced expression upon immune stimulants.

In the host innate immune system, various pattern recognition receptors (PRRs) recognize conserved pathogens-associated molecular patterns (PAMPs), and represent an efficient first line of defense against invading pathogens. TLR22 is one of the fish-specific Toll-like receptors (TLRs), identified in a variety of fish species. In this study, we report the cloning and identification of a TLR22 cDNA from the gills of common carp (Cyprinus carpio L.). The full-length CcTLR22 cDNA was 3301 bp long, including a 32 bp 5'-untranslated region (UTR), an open reading frame (ORF) of 2838 bp and a 432 bp 3'-UTR.The CcTLR22 protein was found to comprise a signal peptide, 16 LRR domains, a LRRCT domain in the extracellular region and a TIR domain in the cytoplasmic region, which fits with the characteristic TLR domain architecture. The genomic organization of CcTLR22 was identified, which was encoded by an uninterrupted exon. Sequence alignment and phylogenetic analysis showed that all known teleost TLR22 members were clustered into an independent clade of the TLR22 family, and showed high amino acid identities with other fish TLRs. Real-time PCR assay showed that CcTLR22 mRNA was expressed in almost all tissues examined, while the levels obviously varied among different tissues. When challenged with poly(I:C) (a viral model) or A. hydrophila bacteria, the expression level of CcTLR22 was up-regulated in a variety of common carp tissues. These results indicate that CcTLR22 plays a significant role in systemic as well as mucosal defence after viral or bacterial stimulation or infection.

Immune-relevant thrombocytes of common carp undergo parasite-induced nitric oxide-mediated apoptosis.

Common carp thrombocytes account for 30-40% of peripheral blood leukocytes and are abundant in the healthy animals' spleen, the thrombopoietic organ. We show that, ex vivo, thrombocytes from healthy carp express a large number of immune-relevant genes, among which several cytokines and Toll-like receptors, clearly pointing at immune functions of carp thrombocytes. Few studies have described the role of fish thrombocytes during infection. Carp are natural host to two different but related protozoan parasites, Trypanoplasma borreli and Trypanosoma carassii, which reside in the blood and tissue fluids. We used the two parasites to undertake controlled studies on the role of fish thrombocytes during these infections. In vivo, but only during infection with T. borreli, thrombocytes were massively depleted from the blood and spleen leading to severe thrombocytopenia. Ex vivo, addition of nitric oxide induced a clear and rapid apoptosis of thrombocytes from healthy carp, supporting a role for nitric oxide-mediated control of immune-relevant thrombocytes during infection with T. borreli. The potential advantage for parasites to selectively deplete the host of thrombocytes via nitric oxide-induced apoptosis is discussed.

Adaptive immune responses at mucosal surfaces of teleost fish.

This review describes the extant knowledge on the teleostean mucosal adaptive immune mechanisms, which is relevant for the development of oral or mucosal vaccines. In the last decade, a number of studies have shed light on the presence of new key components of mucosal immunity: a distinct immunoglobulin class (IgT or IgZ) and the polymeric Ig receptor (pIgR). In addition, intestinal T cells and their putative functions, antigen uptake mechanisms at mucosal surfaces and new mucosal vaccination strategies have been reported. New information on pIgR of Atlantic cod and common carp and comparison of natural and specific cell-mediated cytotoxicity in the gut of common carp and European seabass, is also included in this review. Based on the known facts about intestinal immunology and mucosal vaccination, suggestions are made for the advancement of fish vaccines.

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.

Molecular characterization of LEAP-2 cDNA in common carp (Cyprinus carpio L.) and the differential expression upon a Vibrio anguillarum stimulus; indications for a significant immune role in skin.

LEAP-2 is a cysteine-rich cationic antimicrobial peptide (AMP) playing an important role in host innate immune system. LEAP-2 genes have been identified from higher vertebrates and several fish species. Here we report the cloning and identification of two LEAP-2 cDNA sequences from the liver of common carp (Cyprinus carpio L.). The LEAP-2A cDNA was 1325 bp long and contained an ORF of 279 bp encoding a protein of 92 amino acids. The LEAP-2B cDNA was 608 bp long and contained an ORF of 276 bp encoding a protein of 91 amino acids. Both LEAP-2 proteins consisted of 41 amino acid residues and shared four cysteines at the conserved positions in the predicted mature peptides, highly similar to LEAP-2 of other species. Sequence alignment showed that LEAP-2 amino acid sequences were well conserved in different species, and the phylogenetic relation of LEAP-2 was coincident with evolution of biological species. Expression analysis data revealed that LEAP-2A and LEAP-2B mRNAs were expressed in a wide range of common carp tissues including liver, spleen, head kidney, skin, gills, hindgut and foregut. When injected intraperitoneally with Vibrio anguillarum, the expression level of common carp LEAP-2A was quickly up-regulated in liver, spleen, head kidney, skin, gills, foregut and hindgut, however, the expression level of LEAP-2B was similarly up-regulated in spleen, skin, gills and hindgut but not in liver, head kidney and foregut. Our results showed that the LEAP-2A had a markedly high constitutive expression in skin, and the LEAP-2A and the LEAP-2B had a significantly high up-regulated expression after stimulus in skin. This differential expression of LEAP-2 in common carp suggests that it may play a key role in immune responses against invading pathogens and both LEAP-2 molecules may be involved in mucosal immunity.

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.

Molecular cloning and expression of two ß-defensin and two mucin genes in common carp (Cyprinus carpio L.) and their up-regulation after ß-glucan feeding.

In this study, we described the partial structure, mRNA tissue distribution and regulation of two carp mucin and two ß-defensin genes. This is the first description of these genes in fish. The genes might provide relevant tools to monitor feed-related improvements of fish health under aquaculture conditions. Carp mucin 2 and mucin 5B genes show a high similarity to their mammalian and avian counterparts. The carp ß-defensin 1 and ß-defensin 2 genes cluster together well with their piscine family members. The influence of a ß-glucan immunomodulant on the expression of these genes in mucosal tissues could be confirmed for the first time. Muc5B expression was significantly increased in the skin. For Muc2 no significant up- or down-regulation could be observed. Significantly higher expression levels of ß-defensin 2 in gills and both ß-defensin genes in skin were found. Thus, the mucosal system can be influenced by the addition of ß-glucans to the food.

Proteome reference map of the skin mucus of Atlantic cod (Gadus morhua) revealing immune competent molecules.

The skin mucosal proteome of Atlantic cod (Gadus morhua) was mapped using a 2D PAGE, LC-MS/MS coupled approach. Mucosal proteins from naive fish were identified primarily by similarity searches across various cod EST databases. The identified proteins were clustered into 8 groups based on gene ontology classification for biological process. Most of the proteins identified from the gel are hitherto unreported for cod. Galectin-1, mannan binding lectin (MBL), serpins, cystatin B, cyclophilin A, FK-506 binding protein, proteasome subunits (alpha-3 and -7), ubiquitin, and g-type lysozyme are considered immune competent molecules. Five of the aforementioned proteins were cloned and their tissue distribution was analysed by RT-PCR.

Differential expression of immune and stress genes in the skin of Atlantic cod (Gadus morhua).

The present study describes the transcriptional profiles of selected immune and stress genes with putative important roles in the cutaneous immune defense of Atlantic cod (Gadus morhua). In addition it shows differential expression of many genes at the dorsal and ventral sides of fish, in general having the highest expression at the latter side. Genes related to antibacterial activity, antiviral response, cytokine production, glucose transport, stress response and anti-apoptotic activity were monitored and bactericidal/permeability-increasing protein/lipopolysaccharide-binding protein (BPI-LBP), g-type lysozyme, transferrin, metallothionein, fortilin, interferon regulatory factor-1 (IRF-1), a CC chemokine isoform, interleukin-8 (IL-8), glucose transport (GLUT)-1, -3 and -4, Cu,Zn-superoxide dismutase (Cu,Zn-SOD), catalase and hsp 70 showed significantly higher expression at the ventral side. Further g-type lysozyme, metallothionein, fortilin, IRF-1, interferon γ, interleukin-1ß (IL-1ß), GLUT-3 and -4, catalase and anti apoptotic gene Bcl-X1 were highly expressed in adult cod skin. Therefore fish skin can be considered an immunological active site, especially at the ventral side of Atlantic cod.

Teleost intestinal immunology.

Teleosts clearly have a more diffuse gut associated lymphoid system, which is morphological and functional clearly different from the mammalian GALT. All immune cells necessary for a local immune response are abundantly present in the gut mucosa of the species studied and local immune responses can be monitored after intestinal immunization. Fish do not produce IgA, but a special mucosal IgM isotype seems to be secreted and may (partly) be the recently described IgZ/IgT. Fish produce a pIgR in their mucosal tissues but it is smaller (2 ILD) than the 4-5 ILD pIgR of higher vertebrates. Whether teleost pIgR is transcytosed and cleaved off in the same way needs further investigation, especially because a secretory component (SC) is only reported in one species. Teleosts also have high numbers of IEL, most of them are CD3-ɛ+/CD8-α+ and have cytotoxic and/or regulatory function. Possibly many of these cells are TCRγδ cells and they may be involved in the oral tolerance induction observed in fish. Innate immune cells can be observed in the teleost gut from first feeding onwards, but B cells appear much later in mucosal compartments compared to systemic sites. Conspicuous is the very early presence of putative T cells or their precursors in the fish gut, which together with the rag-1 expression of intestinal lymphoid cells may be an indication for an extra-thymic development of certain T cells. Teleosts can develop enteritis in their antigen transporting second gut segment and epithelial cells, IEL and eosinophils/basophils seem to play a crucial role in this intestinal inflammation model. Teleost intestine can be exploited for oral vaccination strategies and probiotic immune stimulation. A variety of encapsulation methods, to protect vaccines against degradation in the foregut, are reported with promising results but in most cases they appear not to be cost effective yet. Microbiota in fish are clearly different from terrestrial animals. In the past decade a fast increasing number of papers is dedicated to the oral administration of a variety of probiotics that can have a strong health beneficial effect, but much more attention has to be paid to the immune mechanisms behind these effects. The recent development of gnotobiotic fish models may be very helpful to study the immune effects of microbiota and probiotics in teleosts.

Common carp have two subclasses of bonyfish specific antibody IgZ showing differential expression in response to infection.

Immunoglobulin heavy chains identified in bony fish are broadly classified into three classes namely IgM, IgD and IgZ. The most recently described isotype is IgZ, a teleosts-fish specific isotype that shows variations in gene structure across teleosts. In this study we have identified two IgZ subclasses in common carp. IgZ1 is a four constant heavy chain domains containing antibody isolated across teleosts and IgZ2 is a two constant domains containing heavy chain chimera with a µ1 and ζ4 domain. Sequence analyses suggest that these subtypes are expressed from two separate genomic loci. Expression analyses show that IgZ1 is more abundant in systemic organs and IgZ2 chimera is preferentially expressed at mucosal sites. The basal expression level of IgM in fish is much higher than of the other isotypes. We show that IgZ1 expression in systemic and mucosal organs is responsive to blood parasites, while mucosal parasite infection induces IgM and IgZ2 gene expression. This report is the first to show differential expression of the IgZ variants in response to pathogens and suggests that the IgZ subtypes in carps may have mutually exclusive humoral functions.

Immunological differences in intestine and rectum of Atlantic cod (Gadus morhua L.).

The defence system of the distal gut (hindgut and rectum) of Atlantic cod, (Gadus morhua L.) was studied using (immuno)histochemical, electron microscopical and real-time quantitative PCR techniques. The uptake and transport of macromolecules in the intestinal epithelium was also investigated. In this study we observed that cod has many and large goblet cells in its intestinal epithelium and that IgM(+) cells are present in the lamina propria and their number is considerably higher in the rectum than in the intestine. Myeloperoxidase staining revealed low numbers of granulocytes in and under the epithelium of the distal intestine, whereas high numbers were found clustered in the submucosa of the rectum. Electron microscopy not only confirmed these observations, but also revealed the presence of lymphoid cells and macrophages within the intestinal epithelium. Acid phosphatase staining demonstrated more positive macrophage-like cells in the rectum than in the distal intestine. Antigen uptake studies showed a diffused absorption of horse radish peroxidase (HRP) and LTB-GFP, whereas ferritin uptake could not be detected. Basal gene expression of cytokines (IL-1beta, IL-8 and IL-10) and immune relevant molecules (hepcidin and BPI/LPB) were compared in both the intestine and rectum and revealed approximately 2-9 times higher expression in the rectum, of which IL-1beta expression showed the most prominent difference. The present results clearly indicate that intestinal immunity is very prominent in the rectum of cod.

Differential contribution of neutrophilic granulocytes and macrophages to nitrosative stress in a host-parasite animal model.

Tyrosine nitration is a hallmark for nitrosative stress caused by the release of reactive oxygen and nitrogen species by activated macrophages and neutrophilic granulocytes at sites of inflammation and infection. In the first part of the study, we used an informative host-parasite animal model to describe the differential contribution of macrophages and neutrophilic granulocytes to in vivo tissue nitration. To this purpose common carp (Cyprinus carpio) were infected with the extracellular blood parasite Trypanoplasma borreli (Kinetoplastida). After infection, serum nitrite levels significantly increased concurrently to the upregulation of inducible nitric oxide synthase (iNOS) gene expression. Tyrosine nitration, as measured by immunohistochemistry using an anti-nitrotyrosine antibody, dramatically increased in tissues from parasite-infected fish, demonstrating that elevated NO production during T. borreli infection coincides with nitrosative stress in immunologically active tissues. The combined use of an anti-nitrotyrosine antibody with a panel of monoclonal antibodies specific for several carp leukocytes, revealed that fish neutrophilic granulocytes strongly contribute to in vivo tissue nitration most likely through both, a peroxynitrite- and an MPO-mediated mechanism. Conversely, fish macrophages, by restricting the presence of radicals and enzymes to their intraphagosomal compartment, contribute to a much lesser extent to in vivo tissue nitration. In the second part of the study, we examined the effects of nitrosative stress on the parasite itself. Peroxynitrite, but not NO donor substances, exerted strong cytotoxicity on the parasite in vitro. In vivo, however, nitration of T. borreli was limited if not absent despite the presence of parasites in highly nitrated tissue areas. Further, we investigated parasite susceptibility to the human anti-trypanosome drug Melarsoprol (Arsobal), which directly interferes with the parasite-specific trypanothione anti-oxidant system. Arsobal treatment strongly decreased T. borreli viability both, in vitro and in vivo. All together, our data suggest an evolutionary conservation in modern bony fish of the function of neutrophilic granulocytes and macrophages in the nitration process and support the common carp as a suitable animal model for investigations on nitrosative stress in host-parasite interactions. The potential of T. borreli to serve as an alternative tool for pharmacological studies on human anti-trypanosome drugs is discussed.

Haemocyte reactions in WSSV immersion infected Penaeus monodon.

White spot syndrome virus (WSSV) has been a major cause of shrimp mortality in aquaculture worldwide in the past decades. In this study, WSSV infection (by immersion) and behaviour recruitment of haemocytes is investigated in gills and midgut, using an antiserum against the viral protein VP28 and a monoclonal antibody recognising haemocytes (WSH8) in a double immunohistochemical staining and in addition transmission electron microscopy was applied. More WSH 8(+) haemocytes were detected at 48 and 72 h post-infection in the gills of infected shrimp compared to uninfected animals. Haemocytes in the gills and midgut were not associated with VP28-immunoreactivity. In the gills many other cells showed virus replication in their nuclei, while infected nuclei in the gut cells were rare. Nevertheless, the epithelial cells in the midgut showed a clear uptake of VP28 and accumulation in supranuclear vacuoles (SNV) at 8h post-infection. However, epithelial nuclei were never VP28-immunoreactive and electron microscopy study suggests degradation of viral-like particles in the SNV. In contrast to the gills, the midgut connective tissue shows a clear increase in degranulation of haemocytes, resulting in the appearance of WSH8-immunoreactive thread-like material at 48 and 72 h post-infection. These results indicate recruitment of haemocytes upon immersion infection in the gills and degranulation of haemocytes in less infected organs, like the midgut.

The effect of oral immuno-stimulation in juvenile carp (Cyprinus carpio L.).

The effect of a 2-week period of oral immuno-stimulation from the age of 2 or 6 weeks post-fertilisation (wpf; before and after reaching the ability to produce antibodies) onwards was investigated on various immune functions of the common carp, Cyprinus carpio. The immuno-stimulants Aeromonas salmonicida lipopolysaccharide, Yeast DNA (containing unmethylated CpG motifs) or high-M alginate (an extract of algae containing poly-mannuronic acid) were used. The effect of this treatment was studied on the kinetics of B cells in head kidney and peripheral blood leucocytes using flow cytometry, on the total plasma IgM level using ELISA, on cytokine and inducible nitric oxide synthase (iNOS) expression in the intestine, and acute phase protein expression in the liver, using real time quantitative PCR, and on exposure to Vibrio anguillarum. Oral administration of immuno-stimulants from 6 wpf resulted in decreased WCI12(+) (B) cell percentages in PBL (only after administration of LPS) and head kidney (all test groups), and a decreased total IgM level in plasma, suggesting that suppressive effects are strongly indicative of oral or juvenile tolerance. After administration from 2 wpf, the effects on WCI12(+) (B) cell percentages were less pronounced: the group fed with Yeast DNA showed higher percentages compared to the control group at 6 wpf, but lower percentages at 8 wpf. No changes were observed in the cytokine or iNOS expression levels in the intestine or acute phase protein expression in the liver. A challenge with V. anguillarum resulted in an initially higher cumulative mortality in the group fed with LPS, but lower mortality in the groups fed with Yeast DNA or high-M alginate compared to the control group, providing a provisional warning especially for the use of pathogen-derived immuno-stimulants, such as A. salmonicida LPS, in larval and juvenile fish.

Ontogeny of the common carp (Cyprinus carpio L.) innate immune system.

The ontogeny of the teleost innate immune system was studied in carp using cellular, histological and quantitative molecular techniques. Carp myeloid cells first appeared ventro-lateral of the aorta at 2 days post fertilization (the start of hatching), and subsequently around the sinuses of the vena cardinalis (or posterior blood islet), head kidney and trunk kidney. In addition, the hematopoietic tissue around the sinuses of the vena cardinalis transformed into that of the trunk kidney, which is the first description of the ontogeny of the trunk kidney hematopoietic tissue in teleosts. The mAb's used in this study reacted with carp myeloid surface molecules that are already transcribed and processed from the first appearance of myeloid cells, and thus serve a significant role in unraveling ontogenetic processes of teleost immunology. Finally, this study associated the first appearance of myeloid cells with an immune response on the molecular level: 2 days post fertilization embryos responded to LPS injection with upregulation of interleukin 1-beta, inducible nitric oxide synthase and serum amyloid A, and down-regulation of complement factor 3 and alpha2-macroglobulin, implying a functional embryonic innate defense system.

Carp (Cyprinus carpio L.) innate immune factors are present before hatching.

Expression of the innate immune factors, complement factor 3 (C3), alpha2-macroglobulin (alpha2M), serum amyloid A (SAA) and a complement factor 1 r/s--mannose binding lectin associated serine protease-like molecule (C1/MASP2), was determined with Real Time Quantitative-PCR in carp (Cyprinus carpio L.) ontogeny around hatching. Furthermore, the expression of C3 mRNA and the presence of C3 protein were studied in carp embryos and larvae using In Situ Hybridisation, Western Blotting and Immunohistochemistry. C3, alpha2M, SAA and C1/MASP2 mRNA were produced by embryos from 12 h post-fertilisation, which is relatively long before hatching (2 days post-fertilisation (dpf)), indicating either involvement of these factors in development itself or more probably a preparation of the immune system for the post-hatching period. In addition, maternal mRNA of the aforementioned innate immune factors and maternal C3- and immunoglobulin protein was present in unfertilised eggs. Furthermore, C3 mRNA production was situated in the yolk syncytial layer in embryos from 24 h post-fertilisation to 5 dpf, followed by the liver in larvae, providing a new sequence of C3 production in teleost development.

The ontogeny of mucosal immune cells in common carp (Cyprinus carpio L.).

The ontogeny of carp (Cyprinus carpio L.) immune cells was studied in mucosal organs (intestine, gills and skin) using the monoclonal antibodies WCL38 (intraepithelial lymphocytes), WCL15 (monocytes/macrophages) and WCI12 (B cells). In addition, recombination activating gene 1 expression was examined in the intestine with real time quantitative PCR and in situ hybridization to investigate extrathymic generation of lymphocytes. WCL38(+) intraepithelial lymphocytes (putative T cells) appeared in the intestine at 3 days post-fertilization (dpf), which is shortly after hatching but before feeding, implying an important function at early age. These lymphoid cells appear in the intestine before the observation of the first thymocytes at 3-4 dpf, and together with the expression of recombination activating gene 1 in the intestine, suggests that similar to mammals at least part of these cells are generated in the intestine. WCL15(+)monocytes/macrophages appeared in the lamina propria of the intestine at 7 dpf, but considerably later in the epithelium, while WCI12(+) (B) cells appeared in intestine and gills at 6-7 weeks. From these results it can be concluded that putative T cells occur much earlier than B cells, and that B cells appear much later in the mucosae than in other internal lymphoid organs (2 wpf).