Neobenedenia melleni-specific antibodies are associated with protection after continuous exposure in Mozambique tilapia.

Neobenedenia melleni is a significant monogenean pathogen of fish in aquaculture facilities and public aquaria. Immunity after exposure to live N. melleni is well established, but the mechanisms of immunity remain unclear. In this study, tilapia (Oreochromis mossambicus) were continuously exposed to N. melleni over a four-month period and assessed for immunity as determined by a reduction in the number of parasites dislodged from the experimental animals during freshwater immersion. Specific mucosal and systemic antibody levels were by determined via enzyme-linked immunosorbent assay. At 45 days postexposure (DPE), fish displayed high parasite loads and baseline levels of mucosal antibodies. At 102 and 120 DPE parasite loads were significantly decreased, and antibody levels were significantly increased for mucus and plasma samples. The correlation between immunity (reduction in parasite load) and an increased humoral antibody response suggests a key role of antibody in the immune response. This is the first report of immunity against N. melleni that is associated with specific mucosal or systemic antibodies.

The founding charter of the Genomic Observatories Network.

The co-authors of this paper hereby state their intention to work together to launch the Genomic Observatories Network (GOs Network) for which this document will serve as its Founding Charter. We define a Genomic Observatory as an ecosystem and/or site subject to long-term scientific research, including (but not limited to) the sustained study of genomic biodiversity from single-celled microbes to multicellular organisms.An international group of 64 scientists first published the call for a global network of Genomic Observatories in January 2012. The vision for such a network was expanded in a subsequent paper and developed over a series of meetings in Bremen (Germany), Shenzhen (China), Moorea (French Polynesia), Oxford (UK), Pacific Grove (California, USA), Washington (DC, USA), and London (UK). While this community-building process continues, here we express our mutual intent to establish the GOs Network formally, and to describe our shared vision for its future. The views expressed here are ours alone as individual scientists, and do not necessarily represent those of the institutions with which we are affiliated.

Production of homozygous transgenic rainbow trout with enhanced disease resistance.

Previous studies conducted in our laboratory showed that transgenic medaka expressing cecropin B transgenes exhibited resistant characteristic to fish bacterial pathogens, Pseudomonas fluorescens and Vibrio anguillarum. To confirm whether antimicrobial peptide gene will also exhibit anti-bacterial and anti-viral characteristics in aquaculture important fish species, we produced transgenic rainbow trout expressing cecropin P1 or a synthetic cecropin B analog, CF-17, transgene by sperm-mediated gene transfer method. About 30 % of fish recovered from electroporation were shown to carry the transgene as determined by polymerase chain reaction (PCR) amplification assay. Positive P1 transgenic fish were crossed to non-transgenic fish to establish F1 transgenic founder families, and subsequently generating F2, and F3 progeny. Expression of cecropin P1 and CF-17 transgenes was detected in transgenic fish by reverse transcription (RT)-PCR analysis. The distribution of body sizes among F1 transgenic fish were not significantly different from those of non-transgenic fish. Results of challenge studies revealed that many families of F2 and F3 transgenic fish exhibited resistance to infection by Aeromonas salmonicida and infectious hematopoietic necrosis virus (IHNV). All-male homozygous cecropin P1 transgenic families were produced by androgenesis from sperm of F3 heterozygous transgenic fish in one generation. The resistant characteristic to A. salmonicida was confirmed in progeny derived from the outcross of all-male fish to non-transgenic females. Results of our current studies confirmed the possibility of producing disease-resistant homozygous rainbow trout strains by transgenesis of cecropin P1 or CF-17 gene and followed by androgenesis.

DNA vaccines against viral diseases of farmed fish.

Immunization by an antigen-encoding DNA was approved for commercial sale in Canada against a Novirhabdovirus infection in fish. DNA vaccines have been particularly successful against the Novirhabdoviruses while there are reports on the efficacy against viral pathogens like infectious pancreatic necrosis virus, infectious salmon anemia virus, and lymphocystis disease virus and these are inferior to what has been attained for the novirhabdoviruses. Most recently, DNA vaccination of Penaeus monodon against white spot syndrome virus was reported. Research efforts are now focused on the development of more effective vectors for DNA vaccines, improvement of vaccine efficacy against various viral diseases of fish for which there is currently no vaccines available and provision of co-expression of viral antigen and immunomodulatory compounds. Scientists are also in the process of developing new delivery methods. While a DNA vaccine has been approved for commercial use in farmed salmon in Canada, it is foreseen that it is still a long way to go before a DNA vaccine is approved for use in farmed fish in Europe.

Licensed DNA Vaccines against Infectious Hematopoietic Necrosis Virus (IHNV).

This article reviews some of the recent patents on DNA vaccines against fish viruses, in particular against the novirhabdovirus infectious hematopoitic necrosis virus (IHNV). Although very effective in protecting fish against IHNV, only one DNA vaccine has been approved to date for use in Canada. In Europe and in US, its commercialization is restricted due to safety concerns.

The genome of Chelonid herpesvirus 5 harbors atypical genes.

The Chelonid fibropapilloma-associated herpesvirus (CFPHV; ChHV5) is believed to be the causative agent of fibropapillomatosis (FP), a neoplastic disease of marine turtles. While clinical signs and pathology of FP are well known, research on ChHV5 has been impeded because no cell culture system for its propagation exists. We have cloned a BAC containing ChHV5 in pTARBAC2.1 and determined its nucleotide sequence. Accordingly, ChHV5 has a type D genome and its predominant gene order is typical for the varicellovirus genus within the alphaherpesvirinae. However, at least four genes that are atypical for an alphaherpesvirus genome were also detected, i.e. two members of the C-type lectin-like domain superfamily (F-lec1, F-lec2), an orthologue to the mouse cytomegalovirus M04 (F-M04) and a viral sialyltransferase (F-sial). Four lines of evidence suggest that these atypical genes are truly part of the ChHV5 genome: (1) the pTARBAC insertion interrupted the UL52 ORF, leaving parts of the gene to either side of the insertion and suggesting that an intact molecule had been cloned. (2) Using FP-associated UL52 (F-UL52) as an anchor and the BAC-derived sequences as a means to generate primers, overlapping PCR was performed with tumor-derived DNA as template, which confirmed the presence of the same stretch of "atypical" DNA in independent FP cases. (3) Pyrosequencing of DNA from independent tumors did not reveal previously undetected viral sequences, suggesting that no apparent loss of viral sequence had happened due to the cloning strategy. (4) The simultaneous presence of previously known ChHV5 sequences and F-sial as well as F-M04 sequences was also confirmed in geographically distinct Australian cases of FP. Finally, transcripts of F-sial and F-M04 but not transcripts of lytic viral genes were detected in tumors from Hawaiian FP-cases. Therefore, we suggest that F-sial and F-M04 may play a role in FP pathogenesis.

Preserving and using germplasm and dissociated embryonic cells for conserving Caribbean and Pacific coral.

Coral reefs are experiencing unprecedented degradation due to human activities, and protecting specific reef habitats may not stop this decline, because the most serious threats are global (i.e., climate change), not local. However, ex situ preservation practices can provide safeguards for coral reef conservation. Specifically, modern advances in cryobiology and genome banking could secure existing species and genetic diversity until genotypes can be introduced into rehabilitated habitats. We assessed the feasibility of recovering viable sperm and embryonic cells post-thaw from two coral species, Acropora palmata and Fungia scutaria that have diffferent evolutionary histories, ecological niches and reproductive strategies. In vitro fertilization (IVF) of conspecific eggs using fresh (control) spermatozoa revealed high levels of fertilization (>90% in A. palmata; >84% in F. scutaria; P>0.05) that were unaffected by tested sperm concentrations. A solution of 10% dimethyl sulfoxide (DMSO) at cooling rates of 20 to 30°C/min most successfully cryopreserved both A. palmata and F. scutaria spermatozoa and allowed producing developing larvae in vitro. IVF success under these conditions was 65% in A. palmata and 53% in F. scutaria on particular nights; however, on subsequent nights, the same process resulted in little or no IVF success. Thus, the window for optimal freezing of high quality spermatozoa was short (∼5 h for one night each spawning cycle). Additionally, cryopreserved F. scutaria embryonic cells had∼50% post-thaw viability as measured by intact membranes. Thus, despite some differences between species, coral spermatozoa and embryonic cells are viable after low temperature (-196°C) storage, preservation and thawing. Based on these results, we have begun systematically banking coral spermatozoa and embryonic cells on a large-scale as a support approach for preserving existing bio- and genetic diversity found in reef systems.

Immune protection of Mozambique tilapia (Oreochromis mossambicus) exposed to different infectious doses of ectoparasite (Cryptocaryon irritans).

The objectives of the present study were to standardize a reproducible infection procedure with Cryptocaryon irritans and to examine the effects of infectious dose level on the immune protection in Mozambique tilapia (Oreochromis mossambicus). This study demonstrated that direct enumeration of trophonts on the pectoral fin was useful to quantitatively assess immune protection against C. irritans. The number of trophonts on a pectoral fin was positively correlated with infectious dose of live theronts. Fish immunized by direct exposure under controlled laboratory conditions allowed for in depth examination of the effects of the degree of infectious dose on immune response. There was no significant positive correlation between the initial infectious dose and degree of immune responses. Mozambique tilapia initiated a strong immune protection by direct exposure with even a small number of parasites (e.g. 300 theronts per fish). Moreover, as the result of the protein analysis, we identified 28 kD proteins that could be responsible for the immobilizing antigen.

Elicited cross-protection and specific antibodies in Mozambique tilapia (Oreochromis mossambicus) against two different immobilization serotypes of Cryptocaryon irritans isolated in Hawaii.

The objective of this study was to determine whether immunization of Mozambique tilapia with different Cryptocaryon irritans i-antigen serotypes elicited cross-protection against challenge infection by both serotypes. Fish were directly exposed to live theronts of isolate W1 or isolate K1, that express different surface i-antigens. There was no significant difference in the number of trophonts infecting the fish between the two isolates, W1 and K1, following primary exposure. Serum from immunized fish exposed to live theronts showed higher immobilization titres and ELISA values against homologous isolates than to heterologous isolates after the primary exposure. However, mucus antibody did not immobilize theronts although the ELISA results clearly indicated that mucus antibodies recognizing C. irritans were generated. In a study with Western blot analyses, serum antibodies recognized only an antigen of the corresponding serotype and no proteins common to both serotypes were identified. Sequence analyses of 754 bases of rDNA nucleotide sequence including complete nuclear ribosomal ITS-1-5.8S rDNA-ITS-2 region were conducted and found to be identical for W1- and K1-isolates. These findings confirmed that both isolates were members of the species, C. irritans, and that rDNA analysis would not distinguish the two isolates. In conclusion, despite the fact that the immobilization assays and ELISA detected two serotypes in vitro, challenge assays provided evidence for only one type of C. irritans.

Development of a suicidal DNA vaccine for infectious hematopoietic necrosis virus (IHNV).

We developed a suicidal DNA vaccine (pIRF1A-G-pMT-M) for salmonid fish susceptible to Infectious Hematopoietic Necrosis Virus (IHNV). The suicidal vaccine consists of two operons: i) an inducible fish promoter, the interferon regulatory factor 1A promoter (pIRF1A), driving the expression of the IHNV viral glycoprotein (G) gene that induces protection, and ii) a ZnCl(2) inducible fish promoter, the metallothionein promoter (pMT), driving the expression of the IHNV matrix (M) protein that induces apoptosis. The vaccine induces an immune response to the G protein and then induces the cell to undergo apoptosis to eliminate the DNA vaccine-containing cell. Also developed is another suicidal construct (pCMV-luc-pMT-M) for monitoring the persistence of luciferase (luc) expression after induction of apoptosis. In this study, we evaluated the inducibility of the MT promoter with ZnCl(2) and the capacity of cells transfected with the suicidal vector pCMV-luc-pMT-M to undergo apoptosis after ZnCl(2) addition. We also demonstrated the protective immunity elicited by the suicidal DNA vaccine pIRF1A-G-pMT-M, the survival of fish after treatment with ZnCl(2), and the elimination of the suicidal vector in fish after ZnCl(2) treatment.

An overview of marine biodiversity in United States waters.

Marine biodiversity of the United States (U.S.) is extensively documented, but data assembled by the United States National Committee for the Census of Marine Life demonstrate that even the most complete taxonomic inventories are based on records scattered in space and time. The best-known taxa are those of commercial importance. Body size is directly correlated with knowledge of a species, and knowledge also diminishes with distance from shore and depth. Measures of biodiversity other than species diversity, such as ecosystem and genetic diversity, are poorly documented. Threats to marine biodiversity in the U.S. are the same as those for most of the world: overexploitation of living resources; reduced water quality; coastal development; shipping; invasive species; rising temperature and concentrations of carbon dioxide in the surface ocean, and other changes that may be consequences of global change, including shifting currents; increased number and size of hypoxic or anoxic areas; and increased number and duration of harmful algal blooms. More information must be obtained through field and laboratory research and monitoring that involve innovative sampling techniques (such as genetics and acoustics), but data that already exist must be made accessible. And all data must have a temporal component so trends can be identified. As data are compiled, techniques must be developed to make certain that scales are compatible, to combine and reconcile data collected for various purposes with disparate gear, and to automate taxonomic changes. Information on biotic and abiotic elements of the environment must be interactively linked. Impediments to assembling existing data and collecting new data on marine biodiversity include logistical problems as well as shortages in finances and taxonomic expertise.

Analysis of internal osmolality in developing coral larvae, Fungia scutaria.

Coral species throughout the world are facing severe local and global environmental pressures. Because of the pressing conservation need, we are studying the reproduction, physiology, and cryobiology of coral larvae with the future goal of cryopreserving and maintaining these organisms in a genome resource bank. Effective cryopreservation involves several steps, including the loading and unloading of cells with cryoprotectant and the avoidance of osmotic shock. In this study, during the time course of coral larvae development of the mushroom coral Fungia scutaria, we examined several physiologic factors, including internal osmolality, percent osmotically active water, formation of mucus cells, and intracellular organic osmolytes. The osmotically inactive components of the cell, V(b), declined 33% during development from the oocyte to day 5. In contrast, measurements of the internal osmolality of coral larvae indicated that the internal osmolality was increasing from day 1 to day 5, probably as a result of the development of mucus cells that bind ions. Because of this, we conclude that coral larvae are osmoconformers with an internal osmolality of about 1,000 mOsm. Glycine betaine, comprising more than 90% of the organic osmolytes, was found to be the major organic osmolyte in the larvae. Glycerol was found in only small quantities in larvae that had been infected with zooxanthellae, suggesting that this solute did not play a significant role in the osmotic balance of this larval coral. We were interested in changes in cellular characteristics and osmolytes that might suggest solutes to test as cryoprotectants in order to assist in the successful cryopreservation of the larvae. More importantly, these data begin to reveal the basic physiological events that underlie the move from autonomous living to symbiosis.

In vitro biology of fibropapilloma-associated turtle herpesvirus and host cells in Hawaiian green turtles (Chelonia mydas).

Fibropapillomatosis (FP) of green turtles has a global distribution and causes debilitating tumours of the skin and internal organs in several species of marine turtles. FP is associated with a presently non-cultivable alphaherpesvirus Chelonid fibropapilloma-associated herpesvirus (CFPHV). Our aims were to employ quantitative PCR targeted to pol DNA of CFPHV to determine (i) if DNA sequesters by tumour size and/or cell type, (ii) whether subculturing of cells is a viable strategy for isolating CFPHV and (iii) whether CFPHV can be induced to a lytic growth cycle in vitro using chemical modulators of replication (CMRs), temperature variation or co-cultivation. Additional objectives included determining whether non-tumour and tumour cells behave differently in vitro and confirming the phenotype of cultured cells using cell-type-specific antigens. CFPHV pol DNA was preferentially concentrated in dermal fibroblasts of skin tumours and the amount of viral DNA per cell was independent of tumour size. Copy number of CFPHV pol DNA per cell rapidly decreased with cell doubling of tumour-derived fibroblasts in culture. Attempts to induce viral replication in known CFPHV-DNA-positive cells using temperature or CMR failed. No significant differences were seen in in vitro morphology or growth characteristics of fibroblasts from tumour cells and paired normal skin, nor from CFPHV pol-DNA-positive intestinal tumour cells. Tumour cells were confirmed as fibroblasts or keratinocytes by positive staining with anti-vimentin and anti-pancytokeratin antibodies, respectively. CFPHV continues to be refractory to in vitro cultivation.

The effect of in vitro exposure to tributyltin on the immune competence of Chinook salmon (Oncorhynchus tshawytscha) leukocytes.

We evaluated the direct effects of in vitro exposures to tributyltin (TBT), a widely used biocide, on the cell-mediated immune system of Chinook salmon (Oncorhynchus tshawytscha). Splenic and pronephric leukocytes isolated from juvenile Chinook salmon were exposed to TBT (0, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mg/l) in cell cultures for 24 h. Effects of TBT on cell viability, induction of apoptosis, and mitogenic responses were measured by flow cytometry. Splenic and pronephric leukocytes in the presence of TBT experienced a concentration-dependent decrease in viability in cell cultures. Apoptosis was detected as one of the mechanisms of cell death after TBT exposure. In addition, pronephric lymphocytes exhibited a greater sensitivity to TBT exposure than pronephric granulocytes. The functional ability of splenic B-cells to undergo blastogenesis upon lipopolysaccharide stimulation was also significantly inhibited in the presence of 0.05, 0.07, or 0.10 mg/l of TBT in the cell cultures. Flow cytometric assay using a fluorescent conjugated monoclonal antibody against salmon surface immunoglobulin was employed for the conclusive identification of B-cells in the Chinook salmon leukocytes. Our findings suggest that adverse effects of TBT on the function or development of fish immune systems could lead to an increase in disease susceptibility and its subsequent ecological implications.

Induction of antiviral genes, Mx and vig-1, by dsRNA and Chum salmon reovirus in rainbow trout monocyte/macrophage and fibroblast cell lines.

The expression of potential antiviral genes, Mx1, Mx2, Mx3 and vig-1, was studied in two rainbow trout cell lines: monocyte/macrophage RTS11 and fibroblast-like RTG-2. Transcripts were monitored by RT-PCR; Mx protein by Western blotting. In unstimulated cultures Mx1 and vig-1 transcripts were seen occasionally in RTS11 but rarely in RTG-2. A low level of Mx protein was seen in unstimulated RTS11 but not in RTG-2. In both cell lines, Mx and vig-1 transcripts were induced by a dsRNA, poly inosinic: poly cytidylic acid (poly IC), and by Chum salmon reovirus (CSV). Medium conditioned by cells previously exposed to poly IC or CSV and assumed to contain interferon (IFN) induced the antiviral genes in RTS11. However, RTG-2 responded only to medium conditioned by RTG-2 exposed previously to CSV. In both cell lines, poly IC and CSV induced Mx transcripts in the presence of cycloheximide, suggesting a direct induction mechanism, independent of IFN, was also possible. For CSV, ribavirin blocked induction in RTS11 but not in RTG-2, suggesting viral RNA synthesis was required for induction only in RTS11. In both RTS11 and RTG-2 cultures, Mx protein showed enhanced accumulation by 24h after exposure to poly IC and CSV, but subsequently Mx protein levels declined back to control levels in RTS11 but not in RTG-2. These results suggest that Mx can be regulated differently in macrophages and fibroblasts.

Molecular and expression analysis of an interferon-gamma-inducible guanylate-binding protein from rainbow trout (Oncorhynchus mykiss).

Guanylate-binding proteins (GBPs) are some of the most abundant proteins accumulating in mammalian cells in response to interferon-gamma (IFN-gamma). GBPs have been suggested to function in antiviral activity, macrophage activation, fibroblast proliferation and inhibition of endothelial cell proliferation and invasiveness. Here we confirm that IFN-gamma-inducible GBP also exist in fish. A 2 kb GBP cDNA was cloned from head kidney of rainbow trout treated with an IFN-inducing compound. The open reading frame predicts a 635 amino acid protein (rbtGBP) of 72.7 kDa possessing a tripartite GTP binding motif and a secondary structure similar to human GBP1. Like most mammalian GBPs, rbtGBP possesses an isoprenylation motif at the C-terminal end. The overall amino acid sequence identity between rbtGBP and mammalian GBPs is only 41-47%, however. The rainbow trout macrophage cell line RTS11 showed a dose-dependent increase in rbtGBP transcripts in response to IFN-gamma after 6h of stimulation, with rbtGBP being undetectable in non-treated RTS11 cells. Moreover, polyinosinic polycytidylic acid (poly I:C) induced increased GBP transcript levels in RTS11 and RTG2 cells after 4-6 h of stimulation, and in head kidney and liver of live fish after 24 h. These studies suggest that rbtGBP is an early response gene in rainbow trout, which may have similar functions in IFN-gamma mediated responses as mammalian GBPs.

p,p'-DDE depresses the immune competence of chinook salmon (Oncorhynchus tshawytscha) leukocytes.

p,p'-DDE, the main metabolite of DDT, is still detected in aquatic environments throughout the world. Here, the effects and mechanisms by which p,p'-DDE exposure might affect the immune system of chinook salmon (Oncorhynchus tshawytscha) was studied. Isolated salmon splenic and pronephric leukocytes were incubated with different concentrations of p,p'-DDE, and cell viability, induction of apoptosis, and mitogenic responses were measured by flow cytometry and Alamar Blue assay. p,p'-DDE significantly reduced cell viability and proliferation and increased apoptosis. The effect of p,p'-DDE on pronephric leukocytes was more severe than on splenic leukocytes, likely because pronephric leukocytes had a higher proportion of granulocytes, cells that appear more sensitive to p,p'-DDE. The effect of p,p'-DDE on leukocytes appeared to vary between developmental stages or seasonal differences. The mitogenic response of leukocytes of chinook salmon exposed to p,p'-DDE in vivo exhibited a biphasic dose-response relationship. Only leukocytes isolated from salmon treated with 59 ppm p,p'-DDE had a significantly lower percentage of Ig+ blasting cells than controls, although the response was biphasic. These results support the theory that exposure to chemical contaminants could lead to an increase in disease susceptibility and mortality of fish due to immune suppression.

Investigator profile. An interview with Jo-Ann Leong, Ph.D. Interview by Vicki Glaser.

Jo-Ann Leong, Ph.D., is Director of the Hawaii Institute of Marine Biology. She received her Bachelor's degree in Zoology from the University of California at Berkeley and her Ph.D. in Microbiology from the University of California School of Medicine. Dr. Leong's research interests focus on virology, including the study of rhabdoviruses and birnaviruses, fish vaccines, and, specifically, genetic vaccination and aquaculture and fisheries.

The NV gene of snakehead rhabdovirus (SHRV) is not required for pathogenesis, and a heterologous glycoprotein can be incorporated into the SHRV envelope.

Snakehead rhabdovirus (SHRV) affects warm-water fish in Southeast Asia and belongs to the genus Novirhabdovirus by virtue of its "nonvirion" (NV) gene. To examine the function of the NV gene, we used a recently developed reverse genetic system to produce a viable recombinant SHRV carrying an NV gene deletion. The recombinant virus was produced at the same rate and same final concentrations as the wild-type virus in cultured fish cells in spite of the NV gene deletion. The role of the NV protein in fish pathogenesis was also investigated. Zebra fish (Danio rerio) were infected with the NV deletion mutant or with a recombinant virus containing a copy of the SHRV genome, and similar mortality rates as well as final mortalities were recorded, suggesting no apparent role for the NV protein in fish pathogenesis. Interestingly, the unsuccessful rescue of fully viable recombinants with genomes containing deletions in the G/NV gene junction suggested a role for the gene junction in virus transcription and replication. Finally, we demonstrated that the SHRV glycoprotein can be replaced by the glycoprotein of infectious hematopoietic necrosis virus (IHNV) or by a hybrid protein composed of SHRV and IHNV sequences.

In vitro detection of functional humoral immunocompetence in juvenile chinook salmon (Oncorhynchus tshawytscha) using flow cytometry.

A flow cytometric (FCM) assay for detection of immunomodulatory effects of environmental factors on the humoral response of chinook salmon (Oncorhynchus tshawytscha) is described and validated. This technique combines exposure of whole animals or leucocyte cultures to immunomodulatory agents/conditions with in vitro mitogenic activation of B-lymphocytes. The proportion of leucocytes undergoing blastogenesis following in vitro stimulation with lipopolysaccharide (LPS) is quantified by FCM analysis of forward and side scatter properties. In addition, binding of a fluorescein isothiocyanate labelled anti-rainbow trout immunoglobulin M monoclonal antibody (anti-RBT SIgM-FITC), quantified by FCM analysis, is used to determine the ability of the lymphoblasts to express surface immunoglobulin M (SIgM). Through a series of calibration steps, it was confirmed that anti-RBT IgM-FITC was specific for B-lymphocyte SIgM in chinook salmon. Binding of anti-RBT IgM-FITC to chinook salmon SIgM positive leucocytes was effectively blocked with salmon serum and an isotype control was established. B-lymphocytes were partially removed from a population of leucocytes through adherence to a nylon wool column, which then demonstrated a consequent reduction in anti-RBT IgM-FITC binding. Using anti-RBT IgM-FITC as a marker, the distribution of resting lymphocytes expressing SIgM in lymphoid tissues of juvenile chinook salmon was described. The mean percentage of SIgM positive cells in spleen, pronephros and blood were found to be 62.1 (+/-2.82), 34.8 (+/-1.86) and 56.7% (+/-4.7) of all viable leucocytes, respectively. In a time-course experiment for optimal in vitro activation of leucocytes for this assay, blastogenesis and up-regulation of SIgM expression of splenic leucocytes were observed through FCM by 4 days post in vitro stimulation with LPS, continued through 7 days, but was no longer visible by 10 days post stimulation. Using this assay, reduced expression of SIgM in splenic and pronephric B-lymphocytes was detected following in vitro exposure to physiologically relevant stress concentrations of cortisol in conjunction with mitogenic stimulation. This technique will be a useful addition to the assays already available in the rapidly growing field of fish immunology.