Microplastics exacerbate virus-mediated mortality in fish.

Microplastics are a persistent and increasing environmental hazard. They have been reported to interact with a variety of biotic and abiotic environmental stressors, but the ramifications of such interactions are largely unknown. We investigated virus-induced mortalities in a commercially important salmonid following exposure to microplastics, plastic microfibers, and natural (non-plastic) microparticles. Microplastics or microparticles alone were not lethal. Mortality increased significantly when fish were co-exposed to virus and microplastics, particularly microfibers, compared to virus alone. This presents the unique finding that microplastics (not natural microparticulate matter) may have a significant impact on population health when presented with another stressor. Further, we found that mortality correlated with host viral load, mild gill inflammation, immune responses, and transmission potential. We hypothesize that microplastics can compromise host tissues, allowing pathogens to bypass defenses. Further research regarding this mechanism and the interplay between microplastics and infectious disease are paramount, considering microplastics increasing environmental burden.

Polystyrene microplastics reduce abundance of developing B cells in rainbow trout (Oncorhynchus mykiss) primary cultures.

Environmental microplastic pollution (including polystyrene, PS) may have detrimental effects on the health of aquatic organisms. Accumulation of PS microplastics has been reported to affect innate immune cells and inflammatory responses in fish. To date, knowledge on effects of microplastics on the antibody response is still very limited. Here, we investigated effects of small (0.8-20 µm) PS microplastics on the abundance of B lineage cells in primary cultures of developing immune cells from the anterior kidney of rainbow trout. Both purchased PS microbeads and PS microparticles generated from consumer products were used as microplastic sources. We first show that rainbow trout phagocytic B cells efficiently took up small (0.83-3.1 µm) PS microbeads within hours of exposure. In addition, our data revealed that PS microplastic exposure most significantly decreased the abundance of a population of non-phagocytic developing B cells, using both flow cytometry and RT-qPCR. PS microplastics-induced loss of developing B cells further correlated with reduced gene expression of RAG1 and the membrane form of immunoglobulin heavy chains mu and tau. Based on the induced loss of developing B cells observed in our in vitro studies, we speculate that in vivo, chronic PS microplastic-exposure may lead to suboptimal IgM/IgT levels in response to pathogens in teleost species. Considering the highly conserved nature of vertebrate B lymphopoiesis it is likely that PS microplastics will similarly reduce antibody responses in higher vertebrate species, including humans. Further, RAG1 provides an effective biomarker to determine effects of PS microplastics on B cell development in teleost species.

A BCWD-Resistant line of rainbow trout is less sensitive to cortisol implant-induced changes in IgM response as compared to a susceptible (control) line.

In salmonids, stress responses increase cortisol levels and disease susceptibility, including to Flavobacterium psychrophilum (Fp), the causative agent of BCWD. A BCWD-resistant line (R-line) of rainbow trout was used here to investigate potential differences in immunoglobulin response after a combined treatment of cortisol and Fp, as compared to a susceptible (S-line) control line. Expression of membrane and secreted immunoglobulin heavy chains mu and tau were determined by RT-qPCR in spleen and anterior kidney. Cortisol treatment did not affect B cell development in the anterior kidney, but delayed IgM responses at the early stage of infection in the spleen of both lines. An earlier IgM response was a determining factor in differential disease progression between resistant- and susceptible fish after Fp-challenge. S-line fish had a delayed and exacerbated IgM response after cortisol implant indicative of a detrimental cycle of sustained IgM responses and high pathogen loads. In contrast, R-line fish had delayed but milder, and protective IgM responses and cleared pathogen successfully. Fp challenge after cortisol implant increased serum cortisol levels on days 3 and 5 compared to mock treatments in S-line fish, but only on day 3 in R-line. Hence, combined cortisol treatment and Fp challenge differentially modulated B cell activation and Fp loads in BCWD-resistant and susceptible lines of rainbow trout. We propose that under conditions of increased stress, BCWD-resistant fish may remain immunologically better equipped to respond to infections compared to BCWD susceptible fish.

Transient increase in abundance of B lineage but not myeloid-lineage cells in anterior kidney of sockeye salmon during return migration to the natal grounds.

As anadromous fish, sockeye salmon undergo complex endocrine changes when they return to their natal grounds to spawn. This is correlated with major immunological changes that will affect their response to pathogens. In spite of these challenges, salmon need to maintain sufficiently robust immunity to survive until spawning is complete, but the nature of immune adaptations during the spawning stage remains poorly understood. Our central question is to determine if sockeye salmon stimulate their immune system during the return migration and if so, whether this is a protective response. To begin answering this question, here we characterized the nature and timing of potential changes in anterior kidney immune fingerprints between salmon collected from seven different sites along the Kenai river, including the mouth of the river and two spawning sites. Our results revealed significant changes in abundance of B lineage, but not myeloid lineage cells during the spawning journey. This included early, transient and significant increases in abundance of both IgM+ and IgT+ B cells soon after fish entered the river, followed by a transient, significant increase in abundance of IgM++ secreting cells in fish caught mid-river, and ending with a return to base levels of both cell populations in fish caught at spawning sites. Further, males appeared to have higher immune activation than females, as reflected by higher abundance of IgM++ secreting cells, higher spleen index, and higher titers of serum IgM. Although roles for these newly generated IgM++ secreting cells remain unclear at this time, the data complement our previous work which supported roles for long-lived plasma cells to protect returning salmon from pathogens at their natal grounds. We conclude that sockeye salmon are capable of inducing B cell responses during their spawning journey, with males having stronger responses compared to females. B cell activation during the return journey may provide returning adults with additional protection against pathogens not encountered as juveniles.

Innate immune cell signatures in a BCWD-Resistant line of rainbow trout before and after in vivo challenge with Flavobacterium psychrophilum.

Phenotypes of myeloid-lineage cells remain poorly understood in the rainbow trout, and were the focus of this study, including effects of in vivo challenge to Flavobacterium psychrophilum (Fp), the cause of Bacterial Cold Water Disease (BCWD). A genetic line was used that is highly resistant to BCWD (R-line) as well as a susceptible control line (S-line). Using flow cytometry, we describe two Pax5-negative, myeloid-lineage populations: Population 1 consisted of small cells with high SSC and strong staining for Q4E, MPO, Pu1, EBF, and IL- 1ß, which we named "neutrophil-like" cell. Population 2 had high Q4E, but weaker MPO, Pu1, EBF, and IL-1ß staining. Five days after Fp-challenge, both genetic lines had a reduced abundance of neutrophil-like cells in anterior kidney, PBL, and spleen. Pop. 2 abundance was reduced in anterior kidney, and increased in spleen. S-line fish responded more strongly to Fp-challenge compared to R-line fish. Challenged fish with a higher abundance of neutrophil-like cells had significantly lower Fp-loads after challenge, suggesting that these cells aid in the resistance to BCWD.

The humoral immune system of anadromous fish.

The immune system of anadromous fish is extremely complex, a direct consequence of their diadromous nature. Hormone levels fluctuate widely throughout their life cycle, as fish move between fresh and salt water. This poses major challenges to the physiology of anadromous fish, including adaptation to very different saline environments, distinct pathogen fingerprints, and different environmental stressors. Elevated cortisol and sex hormone levels inhibit B lymphopoiesis and IgM+ antibody responses, while catecholamines, growth hormones and thyroid hormones are generally stimulatory and enhance the humoral immune response. Immunological memory in the form of long-lived plasma cells likely plays important roles in health and survival during the life cycle of anadromous fishes. This review discusses some of the complex immune-endocrine pathways in anadromous fish, focusing on essential roles for B lineage cells in the successful completion of their life cycle. A discussion is included on potential differences in immuno-competence between wild and hatchery-raised fish.

Sockeye salmon immunoglobulin VH usage and pathogen loads differ between spawning sites.

The Immunological Imprinting Hypothesis proposes that juvenile anadromous fish respond to the pathogen fingerprint specific to their natal site by producing protective long lived plasma cells (LLPCs) that constitutively produce antibodies against those pathogens. Hence, fish returning to their natal streams have immunological protection from pathogens at that specific location. Here, we tested the hypothesis through analysis of antibody composition and usage in sockeye salmon populations in Alaska. Spleen and anterior kidney were sampled from salmon from six sites, and relative usage levels of six different Immunoglobulin VH gene families determined using RT-qPCR. Additionally, prevalence and pathogen loads were measured in each fish for Renibacterium salmoninarum, Flavobacterium psychrophilum, and Infectious Hematopoietic Necrosis Virus. Results revealed differences in VH usage, pathogen loads, and infection rates between spawning sites, while probability of infection was dependent on location for each pathogen analyzed. Further, several negative correlations between specific VH usage patterns and pathogen loads were uncovered. Greater understanding of site-dependent VH usage in spawning fish potentially suggests a method of natural immunization against common fish pathogens and thus protection of both farmed and wild populations.

A BCWD-resistant line of rainbow trout exhibits higher abundance of IgT+ B cells and heavy chain tau transcripts compared to a susceptible line following challenge with Flavobacterium psychrophilum.

Bacterial Cold Water Disease (BCWD) is a common, chronic disease in rainbow trout, and is caused by the gram-negative bacterium Flavobacterium psychrophilum (Fp). Through selective breeding, the National Center for Cool and Cold Water Aquaculture has generated a genetic line that is highly resistant to Fp challenge, designated ARS-Fp-R (or R-line), as well as a susceptible "control" line, ARS-Fp-S (S-line). In previous studies, resistance to Fp had been shown to correlate with naive animal spleen size, and further, naïve R-line trout had been shown to have a lower abundance of IgM+ and IgM++ cells compared to S-line fish. Here we wished to first determine whether the abundance of IgT+ and/or IgT++ cells differed between the two lines in naïve fish, and if so, how these patterns differed after in vivo challenge with Fp. Fp challenge was by intramuscular injection of live Fp and tissue collections were on days 5, 6, and/or 28 post-challenge, in two independent challenge experiments. Flow cytometric and gene expression analyses revealed that naïve R-line fish had a higher abundance of IgT+ B cells in their anterior kidney, spleen, and blood, compared to S line fish. Further, that after Fp challenge, this difference was maintained between the two lines. Lastly, abundance of IgT+ B cells and expression of secHCtau correlated with lower Fp pathogen loads in challenged fish. In the anterior kidney, IgM+ B cell abundance correlated with increased Fp loads. Together, these results suggest that IgT+ B lineage cells may have a protective function in the immune response to Fp.

Tissue Phthalate Levels Correlate With Changes in Immune Gene Expression in a Population of Juvenile Wild Salmon.

Phthalates have detrimental effects on health and have been shown to dysregulate the immune system of mammals, birds, and fish. We recently reported that di(2-ethylhexyl) phthalate exposure reduces the abundance and inhibits the proliferation of rainbow trout (Oncorhynchus mykiss) IgM(+) B lymphocytes and expression of secreted immunoglobulin heavy-chain mu transcripts in an in vitro culture system. We proposed that phthalates act as immunomodulators by modifying the normal B cell-activation pathways by accelerating B cell differentiation while suppressing plasmablast expansion, thus resulting in fewer IgM-secreting plasma cells. This hypothesis was tested here in an in vivo field study of juvenile Dolly Varden (Salvelinus malma) from a plastic-polluted lake in the Gulf of Alaska. Fish tissues were analyzed for both phthalate levels using liquid chromatography-coupled tandem mass spectrometry and for changes in immune gene expression using reverse transcriptase-real time polymerase chain reaction. Results showed that fish with higher tissue levels of di(2-ethylhexyl) phthalate, di(n-butyl) phthalate, and/or dimethyl phthalate expressed significantly fewer secreted and membrane-bound immunoglobulin heavy-chain mu and Blimp1 transcripts in their hematopoietic tissue. This suggests that in vivo uptake of phthalates in fish changes the expression of B cell-specific genes. Chronic exposure to phthalates likely dysregulates normal B-lymphoid development and antibody responses in salmonids and may increase susceptibility to infection. Given the conserved nature of B-lineage cells in vertebrate animals, other marine species may be similarly affected by chronic phthalate exposure.

Di(2-ethylhexyl) phthalate inhibits B cell proliferation and reduces the abundance of IgM-secreting cells in cultured immune tissues of the rainbow trout.

Plasticizer di(2-ethylhexyl) phthalate (DEHP) and its active metabolite MEHP have important immunotoxic effects in mammalian species, including inhibition of cell proliferation, inflammation inhibition, lowering of the antibody response, and apoptosis. Virtually nothing is known about the potential detrimental effects of DEHP/MEHP on the teleost immune system, although phthalates are a likely threat to fish health. Here we investigated whether short-term in vitro DEHP exposure would affect B lineage cells in the rainbow trout, using cultured immune tissues. Cell culture conditions, evidence of cellular incorporation of DEHP, and possible effects of DEHP on immune genes were first established using the mouse pre-B cell line PD31 and data confirmed a dose-dependent cellular uptake of DEHP using liquid chromatography-coupled ion trap mass spectrometry. Effects of in vitro DEHP exposure on trout B cell proliferation were tested by flow cytometry. Significant, dose-dependent inhibition was evident in both anterior and posterior kidney cultures after 24 h exposure to ≥4 µM DEHP. DEHP-induced cell death was not significant for the range of DEHP tested. Further, the abundance of IgM-secreting plasmablasts and plasma cells was significantly reduced after in vitro exposure of ≥16 µM DEHP for 2 or 7 days. Finally, in vitro DEHP exposure significantly lowered the levels of secreted HCmu transcripts in a dose-dependent manner. B lineage cells from posterior kidney were more sensitive to effects of in vitro DEHP exposure than those from anterior kidney. Together, the data support a model where DEHP modifies the normal B cell activation pathways in rainbow trout, promoting B cell differentiation while suppressing plasmablast expansion, resulting in fewer IgM-secreting plasma cells. Insufficient production of protective antibody make fish more susceptible to infection, and increases their risk for disease and mortality in polluted waters.

B cell signatures of BCWD-resistant and susceptible lines of rainbow trout: a shift towards more EBF-expressing progenitors and fewer mature B cells in resistant animals.

Bacterial cold water disease (BCWD) is a chronic disease of rainbow trout, and is caused by the Gram-negative bacterium Flavobacterium psychrophilum (Fp), a common aquaculture pathogen. The National Center for Cool and Cold Water Aquaculture has bred two genetic lines of rainbow trout: a line of Fp-resistant trout (ARS-Fp-R or R-line trout) and a line of susceptible trout (ARS-Fp-S, or S-line). Little is known about how phenotypic selection alters immune response parameters or how such changes relate to genetic disease resistance. Herein, we quantify interindividual variation in the distribution and abundance of B cell populations (B cell signatures) and examine differences between genetic lines of naive animals. There are limited trout-specific cell surface markers currently available to resolve B cell subpopulations and thus we developed an alternative approach based on detection of differentially expressed transcription factors and intracellular cytokines. B cell signatures were compared between R-line and S-line trout by flow cytometry using antibodies against transcription factors early B cell factor-1 (EBF1) and paired domain box protein Pax5, the pro-inflammatory cytokine IL-1ß, and the immunoglobulin heavy chain mu. R-line trout had higher percentages of EBF(+) B myeloid/ progenitor and pre-B cells in PBL, anterior and posterior kidney tissues compared to S-line trout. The opposite pattern was detected in more mature B cell populations: R-line trout had lower percentages of both IgM(+) mature B cells and IgM-secreting cells in anterior kidney and PBL compared to S-line trout. In vitro LPS-activation studies of PBL and spleen cell cultures revealed no significant induction differences between R-line and S-line trout. Together, our findings suggest that selective resistance to BCWD may be associated with shifts in naive animal developmental lineage commitment that result in decreased B lymphopoiesis and increased myelopoiesis in BCWD resistant trout relative to susceptible trout.

Reduction of rainbow trout spleen size by splenectomy does not alter resistance against bacterial cold water disease.

In lower vertebrates, the contribution of the spleen to anti-bacterial immunity is poorly understood. We have previously reported a phenotypic and genetic correlation between resistance to Flavobacterium psychrophilum, the causative agent of bacterial cold water disease (BCWD) and spleen somatic index (spleen weight normalized to body weight, SI). Fish families with larger pre-challenge SI values were found to have greater BCWD survival (resistance) following intraperitoneal injection of a lethal dose of F. psychrophilum. Since the mammalian spleen is known to be crucial for capture and destruction of encapsulated bacteria, we tested the hypothesis that reduction of spleen size, by surgical splenectomy, should reduce the survival advantage of the larger-spleen, disease-resistant fish. Experiments were performed using two separate lines of fish that had previously been selected either based on BCWD survival (resistant and susceptible), or selected based on spleen size (high and low SI). Following 65 to 81 days post-surgical recovery, fish were challenged with F. psychrophilum and mortality monitored for a minimum of 21 days. No significant difference in the relative survival was detected between splenectomized or sham-operated groups, while SI of splenectomized fish was reduced to an average of 8-12% of control animals. A positive correlation was observed between the SI, measured at the time of splenectomy, and time-to-death post-challenge. In summary, these experiments argue that larger spleen size alone is not sufficient for greater BCWD resistance, but rather it is an indirect indicator of immunological status.

Alternative splicing of the trout Pax5 gene and identification of novel B cell populations using Pax5 signatures.

Pax5 is an alternatively spliced transcription factor that regulates B cell development and activation. The function of specific Pax5 isoforms is unknown. Here we report the existence of seven alternatively spliced isoforms of Pax5 in the rainbow trout. We hypothesized that B cells differentially express specific Pax5 isoforms as a means of modulating Pax5 activity during cell maturation. Flow cytometric analyses using Pax5-specific antibodies recognizing the paired domain, a central (exon 6-encoding) domain, or the C-terminus, revealed the existence of distinct Pax5-expressing cell populations in trout immune tissues. Additionally, using the transcription factor EBF, we show that Pax5 isoforms lacking a paired domain are already expressed at the earliest stages of trout (B) lymphopoiesis, and unexpectedly, that minor populations of such cells reside in blood and spleen. These data support use of differentially expressed Pax5 isoforms to identify novel B cell subsets in the form of Pax5 tissue signatures, and as such, provides new biomarkers for malignancy, infectious disease, and disease resistance in trout and humans.

Sockeye salmon retain immunoglobulin-secreting plasma cells throughout their spawning journey and post-spawning.

Antibody-producing plasma cells are a major source of protective immunity in vertebrates, including salmon. During the spawning phase, salmon undergo drastic, hormonally driven changes in their physiology, including elevated levels of cortisol, which are known to suppress the immune system. As adult fish need to survive their long journey to the spawning grounds, we hypothesized that humoral immunity, in the form of IgM-secreting plasma cells, remains functional until post-spawning. This was investigated by measuring changes in membrane and secreted immunoglobulin heavy chain mu and Pax5 transcripts in spleen and kidney from migrating sockeye salmon, using real-time qPCR. As an additional measurement, the abundance of developing B, mature B, and plasma cells was determined in spawning fish, using flow cytometry. Immune tissue samples were collected from fish from the Kenai River drainage and Main Bay, Prince William Sound. Our results reveal that spawning fish express high levels of secreted heavy chain mu transcripts in their spleen and anterior kidney throughout the spawning journey. Furthermore, we show that IgM-secreting PCs (HCmu++/Pax5-) remain abundant in anterior kidney and spleen of post-spawning sockeye salmon, with a concomitant loss in developing B cells (HCmu-/Pax5+). This suggests that successful spawners retain their PCs throughout the spawning journey and post-spawning.

Decreased immune response in zebra finches exposed to sublethal doses of mercury.

Mercury (Hg) is a ubiquitous contaminant with deleterious effects on many wildlife species. Most studies to date have focused on fish-eating birds and mammals because much historical Hg pollution is aquatic. Recently, however, comparable blood-Hg levels have been found in terrestrial insectivorous songbirds. As a result, research is needed to clarify the effects of Hg exposure on songbirds. One fundamental end point that is still poorly understood is the effect of Hg on the songbird immune system. If Hg affects the functioning of the immune system, exposed songbirds may be less able to mount an appropriate immune response against invading pathogens. To gain insight into how Hg affects songbird immune function on a cellular level, a flow cytometric assay was developed to measure lipopolysaccharide-induced B-lymphocyte proliferation in zebra finches (Taeniopygia guttata). This is the first experimental (dosing) study of the potential effect of Hg on songbird immune system functioning. Decreased B cell proliferation was observed after lipopolysaccharide exposure in individuals with greater concentrations of Hg in their blood and tissues. In addition, these individuals had decreased ratios of proliferating-to-resting B cells. This decrease in lymphocyte proliferation in response to an effective mitogen suggests that environmental exposure to sublethal levels of Hg may inhibit or delay B cell proliferation in songbirds, potentially increasing susceptibility to disease and decreasing survivorship.

Why spawning salmon return to their natal stream: the immunological imprinting hypothesis.

The immune system of salmonids is remarkably similar to that of mammals, including the presence of B and T lymphocytes and a highly diverse antibody repertoire. However, fish lack bone marrow. Instead, development of immune cells takes place in the anterior kidney, which also houses long-lived, immunoglobulin-secreting plasma cells (LLPCs). These LLPCs should protect adult salmon against pathogens that are encountered upon return to their natal grounds for spawning. Here I present a hypothesis which links immune memory in the form of LLPCs to chemical imprinting and the highly accurate return rates of adults to their natal streams.

Defining terminally differentiating B cell populations in rainbow trout immune tissues using the transcription factor XbpI.

The nature of antibody-secreting cells in the rainbow trout is poorly defined. Here we describe a flow cytometric approach to help differentiate between four major trout B cell subsets present during terminal B cell differentiation: resting B cells, activated B cells, plasmablasts, and plasma cells. To aid in the identification of B cell subsets, the LPS-inducible transcription factor XbpI-S was used as a marker. An antibody specific to the stable form of inducible transcription factor X-box protein I (XbpI) was generated, which detects XbpI-S protein expression for species within the Oncorhyncus genus, including rainbow trout. Combinatorial expression patterns, or B cell signatures, were established using antibodies to XbpI-S, Pax5, and IgM in combination with a proliferation marker. We show that XbpI-S induction in trout splenic B cells increases throughout a 10-day in vitro LPS-induction period and that increased XbpI-S expression correlates with increased HCmu expression in the cell. PBLs displayed a lower level of XbpI-S induction during this incubation period, compared to spleen. We conclude that trout B cells follow a highly conserved B cell activation pathway, albeit slower than what has been observed in mammalian species. The use of XbpI-S as an activation marker for trout humoral immune activation promises to be useful for future in vivo studies, and can be applied to a broad range of teleost species.

Dissecting teleost B cell differentiation using transcription factors.

B cell developmental pathways in teleost fishes are poorly understood. In the absence of serological reagents, an alternative approach to dissecting teleost B cell development is to use transcription factors that are differentially expressed during B cell development. This review discusses the structure and function of six transcription factors that play essential roles during teleost B cell development: Ikaros, E2A, EBF, Pax5, Blimp1, and XbpI. Research on alternative splicing of both the Ikaros and Pax5 genes in rainbow trout is presented, including their functional significance. An application is discussed that should aid in elucidating teleost B cell development and activation, by using transcription factors as developmental markers in flow cytometric analysis. Possible future studies in teleost B cell development are suggested in the context of gene regulation. Lastly, broader impacts and practical applications are discussed.

Comparative analyses of B cell populations in trout kidney and mouse bone marrow: establishing "B cell signatures".

This study aimed to identify the frequency and distribution of developing B cell populations in the kidney of the rainbow trout, using four molecular B cell markers that are highly conserved between species, including two transcription factors, Pax5 and EBF1, recombination-activating gene RAG1, and the immunoglobulin heavy chain mu. Three distinct B cell stages were defined: early developing B cells (CLP, pro-B, and early pre-B cells), late developing B cell (late pre-B, immature B, and mature B cells), and IgM-secreting cells. Developmental stage-specific, combinatorial expression of Pax5, EBF1, RAG1 and immunoglobulin mu was determined in trout anterior kidney cells by flow cytometry. Trout staining patterns were compared to a well-defined primary immune tissue, mouse bone marrow, and using mouse surface markers B220 and CD43. A remarkable level of similarity was uncovered between the primary immune tissues of both species. Subsequent analysis of the entire trout kidney, divided into five contiguous segments K1-K5, revealed a complex pattern of early developing, late developing, and IgM-secreting B cells. Patterns in anterior kidney segment K1 were most similar to those of mouse bone marrow, while the most posterior part of the kidney, K5, had many IgM-secreting cells, but lacked early developing B cells. A potential second B lymphopoiesis site was uncovered in segment K4 of the kidney. The B cell patterns, or "B cell signatures" described here provide information on the relative abundance of distinct developing B cell populations in the trout kidney, and can be used in future studies on B cell development in other vertebrate species.

Molecular and cellular analysis of B-cell populations in the rainbow trout using Pax5 and immunoglobulin markers.

To date, the trout B-cell is poorly defined, as many essential molecular markers are not yet available for this species. In mammalian systems, the transcription factor Pax5, expressed from pre-B through plasmablast stages, provides an important marker for B-cell differentiation. In a previous study we showed that Pax5 is expressed in the trout. Here we identify trout B-cell populations that vary in expression of Pax5, membrane and secreted Ig. Immune tissues were separated based on concentration of surface IgM, and analyzed by qPCR and flow cytometry. Results suggest that spleen and PBL contain mostly resting B cells, which lack secreted Ig. While the great majority of splenic B cells become strongly activated upon LPS stimulation, PBLs do not. Additionally, anterior kidney contains both developing B and Ig-secreting B-cell populations, but few resting, mature B cells. Lastly, posterior kidney contains multiple B-cell populations in various states of activation. We conclude that trout immune tissues contain multiple, developmentally diverse and tissue-specific B-cell populations as defined by their relative expression of Pax5, surface IgM, and secreted IgM.