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Pacific lamprey (Entosphenus tridentatus) is a First Food of Native Americans. Declines in lamprey abundance due to dam construction and habitat loss have curtailed traditional harvest opportunities. Artificial propagation is an emerging recovery tool for this imperiled species, and other lamprey species in Finland and Japan. To manage disease risk at prospective lamprey hatcheries, it is critical to understand the pathogens and parasites most likely to affect lamprey and other fish, both in culture and in the wild. We assembled data from regional fish health laboratories to assess pathogen prevalence in Pacific lamprey sampled from various sources (518 adults, 275 larvae, and one juvenile). In adults, Aeromonas salmonicida was identified using standard health screening methods in 0–69% of dead lamprey samples submitted, but no other pathogens were typically observed. All larvae were negative for bacterial and viral infections, and larvae experimentally exposed to A. salmonicida showed no infection or mortality. Conspicuously absent were detailed health assessments for metamorphosed larvae (juveniles). However, external examinations of over 20,000 juveniles captured at dams on the lower Columbia River indicated that they are susceptible to fungal infection in warm water conditions (> 12 °C), which can lead to mortality. To fully evaluate the disease risk associated with lamprey culture, directed research is needed along with standardized health screenings of lamprey at all life stages. Control of A. salmonicida, the etiological agent for furunculosis, will be a top priority for Pacific lamprey use, as this was the only pathogen regularly identified in this species.

All cities face complex challenges managing urban stormwater while also protecting urban water bodies. Green stormwater infrastructure and process-based restoration offer alternative strategies that prioritize watershed connectivity. We report on a new urban floodplain restoration technique being tested in the City of Seattle, USA: an engineered hyporheic zone. The hyporheic zone has long been an overlooked component in floodplain restoration. Yet this subsurface area offers enormous potential for stormwater amelioration and is a critical component of healthy streams. From 2014 to 2017, we measured hyporheic temperature, nutrients, and microbial and invertebrate communities at three paired stream reaches with and without hyporheic restoration. At two of the three pairs, water temperature was significantly lower at the restored reach, while dissolved organic carbon and microbial metabolism were higher. Hyporheic invertebrate density and taxa richness were significantly higher across all three restored reaches. These are some of the first quantified responses of hyporheic biological communities to restoration. Our results complement earlier reports of enhanced hydrologic and chemical functioning of the engineered hyporheic zone. Together, this research demonstrates that incorporation of hyporheic design elements in floodplain restoration can enhance temperature moderation, habitat diversity, contaminant filtration, and the biological health of urban streams.

Primary fungal diseases in marine mammals are rare. Mucormycosis, a disease caused by fungi of the order Mucorales, has been documented in few cetaceans and pinnipeds. In 2012, the first case of mucormycosis in the Pacific Northwest was documented in a dead stranded harbor porpoise (Phocoena phocoena) in Washington state. Since then, mucormycosis has been detected in a total of 21 marine mammals; fifteen harbor porpoises, five harbor seals (Phoca vitulina), and one southern resident killer whale (Orcinus orca). Infected animals were predominately found in the inland waters of Washington and British Columbia, and one harbor seal was recovered in northern Oregon. Fungal hyphae were detected histologically in a variety of tissues, including brain, lung, spleen, pancreas, kidneys, muscle, lymph nodes, and skin. Three fungal species were identified from seven cases by PCR screening or fungal culture; Rhizomucor pusillus (four cases), Lichtheimia corymbifera (two cases), and Cunninghamella bertholletiae. Underlying conditions such as emaciation, current or recent pregnancy, multisystemic parasitism, protozoal infection, and herpesvirus were found in several affected animals. Reasons for the appearance and subsequent increase of these fungal infections in marine mammals are unknown. The emergence of this disease as a source of marine mammal mortality in the Pacific Northwest is of particular concern for endangered southern resident killer whales that spend time in this region. Current population-level stressors such as insufficient prey, high levels of contaminants, and noise pollution, could predispose them to these fatal infections.

In the Salish Sea, the endangered Southern Resident Killer Whale (SRKW) is a high trophic indicator of ecosystem health. Three major threats have been identified for this population: reduced prey availability, anthropogenic contaminants, and marine vessel disturbances. These perturbations can culminate in significant morbidity and mortality, usually associated with secondary infections that have a predilection to the respiratory system. To characterize the composition of the respiratory microbiota and identify recognized pathogens of SRKW, exhaled breath samples were collected between 2006–2009 and analyzed for bacteria, fungi and viruses using (1) culture-dependent, targeted PCR-based methodologies and (2) taxonomically broad, non-culture dependent PCR-based methodologies. Results were compared with sea surface microlayer (SML) samples to characterize the respective microbial constituents. An array of bacteria and fungi in breath and SML samples were identified, as well as microorganisms that exhibited resistance to multiple antimicrobial agents. The SML microbes and respiratory microbiota carry a pathogenic risk which we propose as an additional, fourth putative stressor (pathogens), which may adversely impact the endangered SRKW population.

Sablefish ( Anoplopoma fimbria ) is a highly desired seafood product, which encourages development of sustainable aquaculture methods for this marine fish. Conventional marine fish-based feeds provide essential nutrients including long chain fatty acids for piscivorous species such as sablefish. Alternative terrestrial ingredients could reduce fishing pressures on pelagic species that are the source of fish meal and fish oil, and improve source sustainability. Using juvenile sablefish, we compared the effects of a standard fish-based diet to two diets that contained primarily terrestrial plant ingredients with flaxseed or corn oil replacing the added fish oil. After an 8-week trial feeding period, there were striking differences attributable to diet. Fish receiving the alternative feeds had lower weight gain and shorter length than fish receiving the fish-based feed, suggesting sablefish obtained lower nutrients from the alternative feeds. Among the histological differences, the intestinal mucosa was significantly less vacuolated and the frequency of intestinal mucous cells was reduced in alternative feed fish. The most dramatic lesions were observed in the liver, where severe bile duct hyperplasia (53%, flaxseed oil diet; 33%, corn oil diet), and hepatocellular lesions (nuclear pleomorphism/megalocytosis, regeneration, hypertrophy, clear cell foci) occurred in only alternative feed fish. The hepatic and biliary lesions indicate the alternative diets may be deficient or possibly harmful to sablefish. The bacterial community structures from corn oil fish showed much less diversity than those for the other diets, and the microbiome structures from the three diets were distinctly different from each other. The intestinal microbiome for the fish-based diet included the largest number of families (68), and these fish also had the largest number of unique bacterial families (11) compared to those for corn oil (two) or flaxseed oil (one) fish. Regardless of diet, the stomach and intestinal microbiomes differed significantly from each other, and the feed microbiome differed from all gastrointestinal communities, suggesting that feed is not a significant source of gut bacterial diversity. Similar to other teleosts, the sablefish gastrointestinal microbiome is dominated by Bacteroidetes, Firmicutes, and Proteobacteria. These results suggest that diet-induced shifts in microbiome can occur relatively quickly in sablefish, but the shifts may not be sufficiently adaptive or cannot overcome nutrient deficiencies. This multidisciplinary study demonstrates the utility of histology and microbiology in characterizing dietary effects for novel aquaculture species. Statement of relevance Physiological assessments such as gastrointestinal microbiomes and histopathology can provide a near-term evaluation of the nutritional adequacy of alternative feeds, such as plant-based lipid diets for marine carnivorous finfish. This is the first examination of the microbiome of sablefish, a marine species under development for sustainable aquaculture.

Identifying causes of structural ecosystem shifts often requires understanding trophic structure, an important determinant of energy flow in ecological communities. In coastal pelagic ecosystems worldwide, increasing jellyfish (Cnidaria and Ctenophora) at the expense of small fish has been linked to anthropogenic alteration of basal trophic pathways. However, this hypothesis remains untested in part because baseline description of fish–jellyfish trophic dynamics, and the environmental features that influence them are lacking. Using stable isotopes of carbon (δ13C) and nitrogen (δ15N), we examined spatiotemporal patterns of fish and jellyfish trophic structure in greater Puget Sound, an urbanizing fjord estuary in the NW United States. We quantified niche positions of constituent species, niche widths and trophic overlap between fish and jellyfish assemblages, and several community‐level trophic diversity metrics (resource diversity, trophic length, and niche widths) of fish and jellyfish combined. We then related assemblage‐ and community‐level measures to landscape gradients of terrestrial–marine connectivity and anthropogenic influence in adjacent catchments. Relative niche positions among species varied considerably and displayed no clear pattern except that fish generally had higher δ15N and lower δ13C relative to jellyfish, which resulted in low assemblage‐level trophic overlap. Fish assemblages had larger niche widths than jellyfish in most cases and, along with whole community trophic diversity, exhibited contrasting seasonal patterns across oceanographic basins, which was positively correlated to landscape variation in terrestrial connectivity. In contrast, jellyfish niche widths were unrelated to terrestrial connectivity, but weakly negatively correlated to urban land use in adjacent catchments. Our results indicate that fish–jellyfish trophic structure is highly heterogeneous and that disparate processes may underlie the trophic ecology of these taxa; consequently, they may respond divergently to environmental change. In addition, spatiotemporal variation in ecosystem connectivity, in this case through freshwater influence, may influence trophic structure across heterogeneous landscapes.

Rising populations around coastal systems are increasing the threats to marine water quality. To assess anthropogenic fecal influence, subtidal waters were examined monthly for human- and ruminant-sourced Bacteroidales markers at 80 sites across six oceanographic basins of the Salish Sea (Washington State) from April through October, 2011. In the basins containing cities with individual populations >190,000, >50% of sites were positive for the human marker, while in the basins with high densities of dairy and cattle operations, ∼30% of sites were positive for the ruminant marker. Marker prevalence was elevated in spring (April and May) and fall (October) and reduced during summer (June through September), corresponding with seasonal precipitation. By logistic regression, the odds of human marker detection increased with percentage of adjacent catchment impervious surface, dissolved nitrate concentration, and abundance of low nucleic acid bacteria, but decreased with salinity and chlorophyll fluorescence. The odds of ruminant marker detection increased with dissolved ammonium concentration, mean flow rate for the nearest river, and adjacent shoreline length. These relationships are consistent with terrestrial to marine water flow as a transport mechanism. Thus, Bacteroidales markers traditionally used for identifying nearby sources can be used for assessing anthropogenic fecal inputs to regional marine ecosystems.

As in marine regions throughout other areas of the world, fishes in the Salish Sea serve as hosts for many pathogens, including nematodes, trematodes, protozoans, protists, bacteria, viruses, and crustaceans. Here, we review some of the better-documented infectious diseases that likely contribute to significant losses among free-ranging fishes in the Salish Sea and discuss the environmental and ecological factors that may affect the population-level impacts of disease. Demonstration of these diseases and their impacts to critical and endangered resources provides justification to expand pathogen surveillance efforts and to incorporate disease forecasting and mitigation tools into ecosystem restoration efforts.

Renibacterium salmoninarum is the causative agent of bacterial kidney disease, an important disease of farmed and wild salmonid fish worldwide. Despite the wide spatiotemporal distribution of this disease and habitat pressures ranging from the natural environment to aquaculture and rivers to marine environments, little variation has been observed in the R. salmoninarum genome. Here we use the coverage depth from genomic sequencing corroborated by real-time quantitative PCR to detect copy number variation (CNV) among the genes of R. salmoninarum. CNV was primarily limited to the known dominant virulence factors msa and p22. Among 68 isolates representing the UK, Norway and North America, the msa gene ranged from two to five identical copies and the p22 gene ranged from one to five copies. CNV for these two genes co-occurred, suggesting they may be functionally linked. Isolates carrying CNV were phylogenetically restricted and originated predominantly from sites in North America, rather than the UK or Norway. Although both phylogenetic relationship and geographical origin were found to correlate with CNV status, geographical origin was a much stronger predictor than phylogeny, suggesting a role for local selection pressures in the repeated emergence and maintenance of this trait.

Renibacterium salmoninarum is the causative agent of bacterial kidney disease, an important disease of farmed and wild salmonid fish worldwide. Despite the wide spatiotemporal distribution of this disease and habitat pressures ranging from the natural environment to aquaculture and rivers to marine environments, little variation has been observed in the R. salmoninarum genome. Here we use the coverage depth from genomic sequencing corroborated by real-time quantitative PCR to detect copy number variation (CNV) among the genes of R. salmoninarum. CNV was primarily limited to the known dominant virulence factors msa and p22. Among 68 isolates representing the UK, Norway and North America, the msa gene ranged from two to five identical copies and the p22 gene ranged from one to five copies. CNV for these two genes co-occurred, suggesting they may be functionally linked. Isolates carrying CNV were phylogenetically restricted and originated predominantly from sites in North America, rather than the UK or Norway. Although both phylogenetic relationship and geographical origin were found to correlate with CNV status, geographical origin was a much stronger predictor than phylogeny, suggesting a role for local selection pressures in the repeated emergence and maintenance of this trait.

Disease epidemiology requires information about ecological and environmental condi- tions to identify factors that can influence disease progression. Bacterial kidney disease (BKD) is an endemic disease among Pacific Northwest salmonids that causes significant morbidity and mortality in artificially propagated stocks, but risk factors for infection among free-living salmon are unknown. We evaluated infection by the causative agent of BKD, Renibacterium salmoninarum, in 1752 fish across 52 sampling sites monthly from May to November 2003 as a component of a broader study of neritic habitat use in Puget Sound by juvenile Chinook salmon Oncorhynchus tshawytscha. Infection intensity was ≤10 cells per slide for 77% of the fish. Correlations between the density of Chinook salmon with infection prevalence and with infection intensity were observed across multiple spatial scales. Capture location was a stronger predictor of infection than fish origin (based on coded wire tags) or genetic stock. Influential risk factors by logistic regression were temperature, densities of marked and unmarked Chinook salmon, and density of river lamprey Lampetra ayresis. Renibac- terium salmoninarum were found in gut contents and kidney of river lamprey, suggesting this species may be a transmission vector. The low infection intensity, lack of an effect of fish origin, effect of capture bay, and strong associations with Chinook salmon density are consistent with horizontal transmission of R. salmoninarum during the juvenile neritic phase, posing a potential for infectious interaction between sympatric hatchery and wild fish.

An effective host response to Renibacterium salmoninarum, the etiologic agent of bacterial kidney disease, is poorly characterized. Using suppression subtractive hybridization, we exploited the difference in early host response in the pronephros of fish challenged by an attenuated strain (MT239) or a virulent strain (ATCC 33209) of R. salmoninarum. Among the 132 expressed sequence tag (EST) clones that were sequenced, 20 were selected for expression analysis at 24 and 72h after challenge. ESTs matching two interferon inducible genes (IFN-inducible GBP and VLIG1), the ligand GAS6, and the kinase VRK2 were upregulated in fish exposed to MT239, but downregulated or unchanged in fish exposed to 33209. A second group of ESTs matching genes involved in apoptosis (caspase 8) and immune function (IkappaBalpha, p47(phoX), EMR/CD97) were more slowly upregulated in fish exposed to 33209 compared to fish exposed to MT239. The ESTs displaying elevated expression in MT239-exposed fish may represent important cellular processes to bacterial challenge, and may be useful indicators of an effective host response to R. salmoninarum infection.

Renibacterium salmoninarum , a gram-positive diplococcobacillus that causes bacterial kidney disease among salmon and trout, has two chromosomal loci encoding the major soluble antigen ( msa ) gene. Because the MSA protein is widely suspected to be an important virulence factor, we used insertion-duplication mutagenesis to generate disruptions of either the msa1 or msa2 gene. Surprisingly, expression of MSA protein in broth cultures appeared unaffected. However, the virulence of either mutant in juvenile chinook salmon ( Oncorhynchus tshawytscha ) by intraperitoneal challenge was severely attenuated, suggesting that disruption of the msa1 or msa2 gene affected in vivo expression.

Renibacterium salmoninarum causes bacterial kidney disease (BKD), a chronic and sometimes fatal disease of salmon and trout that could lower fitness in populations with high prevalences of infection. Prevalence of R. salmoninarum infection among juvenile Chinook salmon Oncorhynchus tshawytscha inhabiting neritic marine habitats in North Puget Sound, Washington, USA, was assessed in 2002 and 2003. Fish were collected by monthly surface trawl at 32 sites within 4 bays, and kidney infections were detected by a quantitative fluorescent antibody technique (qFAT). The sensitivity of the qFAT was within an order of magnitude of the quantitative real-time PCR (qPCR) sensitivity. Prevalence of infection was classified by fish origin (marked/hatchery vs. unmarked/likely natural spawn), month of capture, capture location and stock origin. The highest percentages of infected fish (63.5 to 63.8%) and the greatest infection severity were observed for fish collected in Bellingham Bay. The lowest percentages were found in Skagit Bay (11.4 to 13.5%); however, there was no difference in prevalence between marked and unmarked fish among the capture locations. The optimal logistic regression model of infection probabilities identified the capture location of Bellingham Bay as the strongest effect, and analysis of coded wire tagged (CWT) fish revealed that prevalence of infection was associated with the capture location and not with the originating stock. These results suggest that infections can occur during the early marine life stages of Chinook salmon that may be due to common reservoirs of infection or horizontal transmission among fish stocks.

Renibacterium salmoninarum , a gram-positive diplococcobacillus, causes bacterial kidney disease, a condition that can result in extensive morbidity and mortality among stocks of fish. An immunodominant extracellular protein, called major soluble antigen (MSA), is encoded by two identical genes, msa1 and msa2 . We found evidence for a third msa gene, msa3 , which appears to be a duplication of msa1 . Unlike msa1 and msa2 , msa3 is not present in all isolates of R. salmoninarum . The presence of the msa3 locus does not affect total MSA production in culture conditions. In a challenge study, isolates possessing the msa3 locus reduced median survival in juvenile chinook salmon ( Oncorhynchus tshawytscha ) by an average of 34% at doses of ≤10 5 cells per fish compared to isolates lacking the msa3 locus. In contrast, no difference in survival was observed at the highest dose, 10 6 cells per fish. The phenotype associated with the msa3 locus and its nonuniform distribution may contribute to observed differences in virulence among R. salmoninarum isolates.

Epizootiological studies of gonadal tumors of Maine soft-shell clams (Mya arenaria) have shown prevalences of over 20% in certain populations in eastern Maine. The etiology of these tumors is unknown, and both environmental as well as genetic factors may play a role. In order to investigate the effect of exposure to environmental contaminants, we initiated a short-term exposure study of M. arenaria to [3H]tetrachlorodibenzo-p-dioxin ([3H]TCDD). Gill, digestive gland, foot and gonad were sampled up to two weeks after a 24-h exposure to a low dose (10 parts per trillion or pptr) or high dose (2000pptr) of [3H]TCDD in the water. In the gill, peak concentrations occurred at the beginning of depuration, whereas peak concentrations in the digestive gland and foot were found 12–24 h after exposure. In contrast, tissue concentrations in the gonad increased through the post-exposure period; at two weeks after exposure, [3H]TCDD concentrations were highest in the gonad. Examination of gill and gonad by differential display polymerase chain reaction (ddPCR) identified potential alterations in expression of genes that may be associated with increased cell cycling (heparan sulfate proteoglycan, E3 ubiquinating enzyme) or translation initiation (p68 RNA helicase/initiation factor eIEF-4A). Histopathological analysis indicated no observable alterations in gonadal or gill tissue from exposed animals.

A short-term, waterborne exposure and depuration study of Mya arenaria to [ 3 H]tetrachlorodibenzo-p-dioxin([ 3 H]TCDD) was performed. Gill, digestive gland, foot, and gonad were sampled up to 2 weeks after a 24-h exposure to a low dose (10 parts per trillion [pptr]) or high dose (2,000 pptr) of [ 3 H]TCDD in the water. In the gill, peak concentrations occurred at the beginning of depuration (406 and 8,658 pg/g wet weight, low- and high-dose groups, respectively), whereas peak concentrations in the digestive gland and foot were found 12 to 24 h after exposure (digestive gland, 539 and 9,369 pg/g wet weight; foot, 146 and 2,718 pg/g wet weight; low- and high-dose groups, respectively). In contrast, tissue concentrations in the gonad increased through the post-exposure period; at 2 weeks after exposure, tissue concentrations were highest in the gonad (243 and 3,012 pg/g wet weight, low-and high-dose groups, respectively). Examination of gill and gonad by differential display polymerase chain reaction identified potential alterations in expression of genes that may be associated with increased cell cycling or translation initiation. This study shows that M. arenaria can accumulate TCDD in the gonad from an acute exposure, and it identifies several M. arenaria genes whose expression may be altered by TCDD exposure.

Ribosomal proteins contribute to the regulation and activity of ribosomes, and hence, the translational activity of the cell. Aberrant expression of ribosomal proteins has been linked to certain pathological conditions such as neoplasms. We have isolated and characterized a cDNA for the ribosomal protein (rp) S19 from a marine bivalve, the soft-shell clam ( Mya arenaria ), and we have examined its pattern of mRNA expression in the ovary and testis. The S19 cDNA contains a 450 nucleotide (nt) open reading frame (ORF), flanked by 89 nt and 26 nt of 5′ and 3′ untranslated regions, respectively. Probes synthesized from the S19 cDNA recognize a single transcript of approximately 550 nt in four different tissues. The predicted amino acid sequence from the ORF exhibits 58% identity with human and rat S19. Southern analysis of genomic DNA suggests that M . arenaria may have multiple copies of S19 , a feature that is more similar to vertebrate than invertebrate rp genes. Expression of S19 mRNA in both ovary and testis was elevated throughout gametogenesis until after spawning, when a decrease in S19 message was observed. A comparison of S19 mRNA levels in post-spawn animals revealed a trend of elevated expression in ovaries and testes affected by a gonadal neoplasm, indicating that S19 may be a useful molecular marker for the pathological condition.

Pathological conditions, especially neoplasms, in feral animals have been shown to be indicators of environmental degradation by pollutants. A gonadal neoplasm in soft shell clam (Mya arenaria) has been identified among certain populations of clams in Maine, but the etiology of these neoplasms is unknown. We have undertaken a study of these neoplasms to determine whether they may be a result of exposure to contaminants. One approach we have taken is to identify genes that are differentially expressed by neoplastic cells. Such genes may be part of the molecular pathway that is responsible for the neoplastic phenotype, and their identification could shed light on the etiology of the neoplasm. By using differential display polymerase chain reaction (ddPCR), we found at least four genes that are involved in biosynthetic activities related to cell growth and proliferation. These findings demonstrate that ddPCR can be a useful tool to study neoplasms in feral animals, especially for species that have not been genetically well characterized.

Several Seattle-area streams in Puget Sound were the focus of habitat restoration projects in the 1990s. Post-project effectiveness monitoring surveys revealed anomalous behaviors among adult coho salmon returning to spawn in restored reaches. These included erratic surface swimming, gaping, fin splaying, and loss of orientation and equilibrium. Affected fish died within hours, and female carcasses generally showed high rates (>90%) of egg retention. Beginning in the fall of 2002, systematic spawner surveys were conducted to 1) assess the severity of the adult die-offs, 2) compare spawner mortality in urban vs. non-urban streams, and 3) identify water quality and spawner condition factors that might be associated with the recurrent fish kills. The forensic investigation focused on conventional water quality parameters (e.g., dissolved oxygen, temperature, ammonia), fish condition, pathogen exposure and disease status, and exposures to metals, polycyclic aromatic hydrocarbons, and current use pesticides. Daily surveys of a representative urban stream (Longfellow Creek) from 2002-2009 revealed premature spawner mortality rates that ranged from 60-100% of each fall run. The comparable rate in a non-urban stream was

Although there are a variety of methods available for the detection of Renibacterium salmoninarum, the causative agent of bacterial kidney disease in salmon and trout, the enzyme-linked immunosorbent assay (ELISA) is probably the most widely used method. However, ELISA measures bacterial antigen, which does not necessarily reflect the number of cells present. We hypothesized that dual analysis of kidney tissue by ELISA and a quantitative real-time polymerase chain reaction assay (qPCR) would provide complementary information about antigen level and the number of bacterial genomes. We found that DNA extracted from the insoluble fraction of the ELISA tissue preparation produced the same qPCR result as DNA extracted directly from frozen tissue, permitting true dual analysis of the same tissue sample. We examined kidney tissue in this manner from individual free-ranging juvenile Chinook salmon and antibiotic-treated captive subadult Chinook salmon and observed 3 different patterns of results. Among the majority of fish, there was a strong correlation between the ELISA value and the qPCR value. However, subsets of fish exhibited either low ELISA values with elevated qPCR values or higher ELISA values with very low qPCR values. These observations suggest a conceptual model that allows inferences about the state of infection of individual fish based on dual ELISA/qPCR results. Although this model requires further assessment through experimental infections and treatments, it may have utility in broodstock selection programs that currently apply egg-culling practices based on ELISA alone.

Ovarian development was monitored in female English sole Parophrys vetulus Girard from three sites in Puget Sound: Sinclair Inlet and Port Susan, where fish are normally resident, and University Point, a known spawning area for English sole. Histological changes in ovary and liver as well as changes in plasma estradiol and vitellogenin levels, gonadosomatic index, hepatosomatic index, and calcium, phosphate, magnesium, glucose, triglyceridesm total protein, and albumin concentrations in the plasma over the reproductive cycle were assessed. Vitellogenesis in female English sole began in the early fall, and swapping activity reached its peak in February. Oocyte development was group synchronous with two distinct clutches of oocytes in the maturing ovary. In general, the cytological and physiological changes which English sole underwent during the reproductive cycle were similar to those observed in other teleost species. However, certain distinctive features were observed, including a high prevalence of atresia of nonyolked oocytes.

The Oct-2 gene appears to encode a developmental regulator of immunoglobulin gene transcription. We demonstrate that the Oct-2 gene is expressed at low levels in a variety of transformed pre-B-cell lines and is induced specifically in these cells by lipopolysaccharide signalling. This work extends an earlier observation in the pre-B-cell line 70Z/3 and therefore suggests that the inducible expression of the Oct-2 gene, like that of the kappa gene, is a characteristic feature of the pre-B stage of B-cell development. In 70Z/3 cells, the lymphokine interleukin-1 also induces the expression of the Oct-2 and kappa loci. Interestingly, expression of the Oct-2 gene is rapidly induced at the transcriptional level and may not require de novo protein synthesis. Since the changes in the activity of the Oct-2 locus completely correlate with the changes of the activity of the kappa locus, the two genes may be transcriptionally regulated by a common trans-acting factor. In 70Z/3 cells, transforming growth factor beta, an inhibitor of kappa-gene induction, blocks the upregulation of Oct-2 but not the activation of NF-kappa B. These results suggest that the combinatorial action of increased levels of Oct-2 and activated NF-kappa B may be necessary for the proper stage-specific expression of the kappa locus.

Vitellogenic female English sole were sampled from four areas in Puget Sound that varied in the nature and degree of chemical contamination. The fish were then injected with an analogue of Luteinizing Hormone Releasing Hormone (LHRH) in the laboratory to induce spawning. Ability to spawn, time to spawn, larval viability, and initial concentrations of plasma estradiol and vitellogenin [measured as alkaline-labile protein associated phosphate (ALP)] were assessed. In general, low initial plasma estradiol and ALP concentrations and subsequent reproductive impairment were most common in English sole from sites where levels of sediment contaminants [e.g. polycyclic aromatic hydrocarbons and polychlorinated biphenyls (PCB)] and measures of contaminant exposure in fish (hepatic PCB concentrations and aryl hydrocarbon hydroxylase activity) were highest, and where pollution-associated liver lesions in fish (i.e. neoplasms, foci of cellular alterations, specific degeneration/necrosis, and storage disorders) were most prevalent. Additionally, the time to spawn was found to be inversely correlated with initial plasma estradiol concentrations. Spawning success was found to be positively correlated with initial plasma estradiol and ALP concentrations as determined by logistic regression analysis; nearly 62% of the variability in the spawning response could be accounted for by these factors. Fertilization success was also found to be positively correlated with initial ALP concentrations, whereas fish captured from contaminated sites produced lower proportions of normal larvae. Overall, these findings suggest that contaminant exposure may result in poor reproductive success of female English sole. This may be related to a hormone imbalance or to slower ovarian development (non-synchronous timing) of female English sole from contaminated sites.

Three cohorts of juvenile and subadult Chinook salmon Oncorhynchus tshawytscha received multiple treatments with macrolide antibiotics for bacterial kidney disease (BKD) during rearing in a captive broodstock program. A total of 77 mortalities among the cohorts were screened for Renibacterium salmoninarum, the etiologic agent of BKD, by agar culture from kidney, and isolates from 7 fish were suitable for growth testing in the presence of macrolide antibiotics. The minimum inhibitory concentration (MIC) of erythromycin and azithromycin was determined by a modification of the standardized broth assay using defined medium. The American Type Culture Collection (ATCC) type strain 33209 exhibited a MIC of 0.008 microg m(-1) to either erythromycin or azithromycin. Isolates from 3 fish displayed MICs identical to the MICs for the ATCC type strain 33209. In contrast, isolates from 4 fish exhibited higher MICs, ranging between 0.125 and 0.250 microg ml(-1) for erythromycin and between 0.016 and 0.031 microg ml(-1) for azithromycin. Sequence analysis of the mutational hotspots for macrolide resistance in the 23S rDNA gene and the open reading frames of ribosomal proteins L4 and L22 found identical sequences among all isolates, indicating that the phenotype was not due to mutations associated with the drug-binding site of 23S rRNA. These results are the first report of R. salmoninarum with reduced susceptibility to macrolide antibiotics isolated from fish receiving multiple antibiotic treatments.

Renibacterium salmoninarum is the causative agent of bacterial kidney disease and a significant threat to healthy and sustainable production of salmonid fish worldwide. This pathogen is difficult to culture in vitro, genetic manipulation is challenging, and current therapies and preventative strategies are only marginally effective in preventing disease. The complete genome of R. salmoninarum ATCC 33209 was sequenced and shown to be a 3,155,250-bp circular chromosome that is predicted to contain 3,507 open-reading frames (ORFs). A total of 80 copies of three different insertion sequence elements are interspersed throughout the genome. Approximately 21% of the predicted ORFs have been inactivated via frameshifts, point mutations, insertion sequences, and putative deletions. The R. salmoninarum genome has extended regions of synteny to the Arthrobacter sp. strain FB24 and Arthrobacter aurescens TC1 genomes, but it is approximately 1.9 Mb smaller than both Arthrobacter genomes and has a lower G+C content, suggesting that significant genome reduction has occurred since divergence from the last common ancestor. A limited set of putative virulence factors appear to have been acquired via horizontal transmission after divergence of the species; these factors include capsular polysaccharides, heme sequestration molecules, and the major secreted cell surface antigen p57 (also known as major soluble antigen). Examination of the genome revealed a number of ORFs homologous to antibiotic resistance genes, including genes encoding β-lactamases, efflux proteins, macrolide glycosyltransferases, and rRNA methyltransferases. The genome sequence provides new insights into R. salmoninarum evolution and may facilitate identification of chemotherapeutic targets and vaccine candidates that can be used for prevention and treatment of infections in cultured salmonids.

A consistent and statistically significant association between prevalence of hepatic neoplasms in free-living sole (Parophrys vetulus) and levels of anthropogenic polycyclic aromatic hydrocarbons in bottom sediment from sites of fish capture was documented in a series of studies conducted over a period of 7 years in Puget Sound, Washington. This result strengthens the evidence supporting a causal relationship between exposure to sediment-associated hydrocarbons and development of hepatic neoplasms in this bottom-dwelling marine fish species. Prevalences of two other distinct categories of idiopathic hepatic lesions-megalocytic hepatosis and steatosis/hemosiderosis-also showed consistent, statistically significant associations with polycylic aromatic hydrocarbon concentrations in bottom sediment, and association with prevalence of a third category (putatively preneoplastic foci of cellular alteration) approached statistical significance. On the basis of other studies, megalocytic hepatosis and foci of cellular alteration are both considered to be important precursor lesions in the stepwise histogenesis of hepatic neoplasms.

DNA adjuvants and whole bacterial cell vaccines against bacterial kidney disease (BKD) were tested in juvenile chinook salmon. Whole cell vaccines of either a nonpathogenic Arthrobacter spp. or an attenuated Renibacterium salmoninarum strain provided limited prophylactic protection against acute intraperitoneal challenge with virulent R. salmoninarum, and the addition of either synthetic oligodeoxynucleotides or purified R. salmoninarum genomic DNA as adjuvants did not increase protection. However, a combination of both whole cell vaccines significantly increased survival among fish naturally infected with R. salmoninarum, and the surviving fish treated with the combination vaccine exhibited reduced levels of bacterial antigens in the kidney. This is the first demonstration of a potential therapeutic effect of a whole cell vaccine against BKD.

Renibacterium salmoninarum is a gram-positive bacterium responsible for bacterial kidney disease of salmon and trout. R . salmoninarum has two identical copies of the gene encoding major soluble antigen (MSA), an immunodominant, extracellular protein. To determine whether one or both copies of msa are expressed, reporter plasmids encoding a fusion of MSA and green fluorescent protein controlled by 0.6 kb of promoter region from msa1 or msa2 were constructed and introduced into R . salmoninarum . Single copies of the reporter plasmids integrated into the chromosome by homologous recombination. Expression of mRNA and protein from the integrated plasmids was detected, and transformed cells were fluorescent, demonstrating that both msa1 and msa2 are expressed under in vitro conditions. This is the first report of successful transformation and homologous recombination in R . salmoninarum .

A full-length cDNA clone of the medaka ( Oryzias latipes ) p53 tumor suppressor gene was isolated from a cDNA library from adult liver tissue, sequenced and characterized. Sequence analysis revealed a high degree of homology between putative functional domains of medaka p53 and p53 genes from other vertebrate taxa including rainbow trout ( Oncorhynchus mykiss ), frog ( Xenopus laevis ), chicken ( Gallus gallus ), rat ( Rattus norvegicus ), mouse ( Mus musculus ), hamster ( Mesocricetus auratus ), green monkey ( Ceropithecus aethiops ) and human ( Homo sapiens ). A single 1.9-kb p53 mRNA is expressed at a very low level in normal adult liver tissue. This transcript is similar in size to transcripts of p53 genes from other species. Preliminary screening of six MNNG-induced tumors in four adult medaka revealed no mutations within characteristic mutational hotspots encompassing conserved domains IV and V. © 1997 Elsevier Science B.V. All rights reserved.

The kappa immunoglobulin (Igk) light chain locus is transcriptionally silent in the mouse B-cell lymphoma 70Z/3. However, exposure to lipopolysaccharide (LPS) or interferon-gamma (IFN) causes a marked increase in Igk transcription. By immunoselection, we isolated two variants that are nonresponsive to IFN. One variant, AT7.2, has retained its response to LPS (IFN-LPS+), whereas the other, AT3.3, is also nonresponsive to LPS (IFN-LPS-). Stable transfection of an intact Igk gene does not rescue the phenotype of either variant. Both variants have intact Igk genes and neither is deficient in the binding or uptake of IFN. Nuclear extracts from LPS-treated wild-type 70Z/3 cells show strong increases in three transcription factors: OTF-2, NF-kappa B, and kBF-A. Remarkably, when the IFN-LPS- variant is treated with LPS, all three transcription factors are still observed in the nuclear extracts. Treatment of wild-type cells with either LPS or IFN also causes a decrease in nuclear complexes that bind to two other regions of the Igk intron enhancer, the octenh and the E kappa MHCIC regions. Both of these changes are also observed after LPS or IFN treatment of the IFN-LPS- variant. Thus, this variant transduces the IFN and LPS signals at least into the nuclear compartment, but still fails to activate Igk transcription. In contrast, the IFN-LPS+ variant decreases neither the octenh nor the E kappa MHCIC binding complexes in response to IFN. This variant may be defective in transducing the IFN signal to the nucleus. These variants will be useful in studying the activation of Igk transcription and the IFN signaling pathway in B cells.

Interferon-gamma (IFNg) is a central cytokine in modulating innate and adaptive immunity in a wide range of vertebrates. A study of genes associated with an effective host response by Chinook salmon (Oncorhynchus tshawytscha) to a persistent Gram-positive bacterial pathogen (Renibacterium salmoninarum) suggested that an interferon-inducible signal was abrogated during early stages of exposure. Splenic DNA was amplified with IFNg primers from a related species, rainbow trout (Oncorhynchus mykiss), and multiple clones from individual fish were sequenced. Up to five genotypes per fish were found, and sequence variations included microsatellites within introns and single nucleotide polymorphisms (SNPs) within exons. Southern hybridizations supported the prediction that Chinook salmon possess at least three related IFNg loci. Multiple clones of cDNA, synthesized from RNA isolated from pronephros of fish exposed to an attenuated strain of R. salmoninarum, displayed a surprising abundance of SNPs. These findings are consistent with the phenomenon of genome duplication known to have occurred in teleosts. Although the presence of multiple loci complicates the analysis of IFNg gene expression, it may also offer a mode of immune modulation not found among mammals. Research supported by National Marine Fisheries Service, NOAA Fisheries, U.S. Department of Commerce.

Primary cultures of myogenic cells from progressively older embryonic and adult chickens were incubated in medium containing Merocyanine 540 (MC540) and were exposed to white light during the incubation period. After exposure, the cultures were followed to determine cell survival and differentiation. MC540 attached to the surface membranes of all cells. In cultures from 10-day embryos (E10 cells), concentrations of MC540 greater than or equal to 60 micrograms/ml resulted in death of nearly all myogenic cells upon exposure to light, but non-myogenic cells survived and replicated. Below 60 micrograms/ml, there was a dose-dependent reduction in muscle differentiation. At concentrations less than 40 micrograms/ml, there was no effect on myogenesis. Cultures of cells from 18-day (E18) embryos (myogenic stem cells) and from adult muscle (satellite cells) were resistant to doses of MC540 that killed E10 cells. E14 myogenic cell populations contained both resistant and sensitive sub-populations. Terminally differentiated muscle cells were more sensitive to MC540 than precursor cells from any age embryo. Progeny of E18 cells acquired sensitivity to MC540 as differentiation proceeded. In clonal cultures, cells that normally give rise to small muscle clones (committed cells) were selectively destroyed by exposure to the dye. These observations demonstrate that an MC540-resistant myogenic population is present in low numbers in 10-day embryonic pectoral muscle. As development proceeds, this population increases such that, by 18 days of gestation, most of the myogenic cells are resistant to MC540. The results also suggest that embryonic chick myogenic stem cells and adult satellite cells have surface membrane properties which differ from those of their committed progeny.

Investigation of the interactive effects of representatives of three classes of compounds was performed, using English sole (Parophrys vetulus) as the test organism, juvenile fish were exposed orally to cadmium chloride and Aroclor 1254 (PCB), either independently or simultaneously, for a 4-wk period, followed by exposure to seawater-accommodated No. 2 diesel fuel for 2 wk. Blood was collected for analysis of serum constituents, and tissue samples were examined for histological changes. Hepatocellular necrosis, regeneration, and karyomegaly were observed. Differential lesion prevalences were observed among the exposure groups, with high proportions among cadmium-exposed fish, low proportions among PCB-exposed fish, and intermediate proportions in combination cadmium- plus PCB-exposed fish. Levels of aspartate aminotransferase activity and magnesium in the sera of these groups exhibited similar patterns. Depressed serum calcium levels in both PCB-exposed and cadmium- plus PCB-exposed groups were found after the first week, and lower serum albumin concentrations occurred in all cadmium- and PCB-exposed groups following the third week. Subsequent exposure to No. 2 diesel fuel produced few effects in any of the exposure groups. The observed antagonistic effect of Aroclor 1254 against cadmium toxicity emphasizes the importance of employing multiple as well as single contaminant exposure in toxicity studies.

Statistical comparisons were made between the relative mean concentrations of metabolites of aromatic compounds in bile (as estimated by a high-performance liquid chromatography/fluorescence detection method) and idiopathic liver lesions in English sole (Parophrys vetulus) from eleven Puget Sound sites. A significant positive correlation was found between prevalences of neoplasms, foci of cellular alteration, megalocytic hepatosis and total hepatic lesions and the concentrations of metabolites of aromatic compounds. These findings provide further evidence of the putative relationship between aromatic compounds and serious idiopathic liver diseases in bottom-dwelling fish.

English sole, Parophrys vetulus, from eight locations within Puget Sound, U.S.A. were examined for histological abnormalities of the liver and kidney. A total of 1083 fish were collected between February 1979 and June 1984 in nine separate sampling periods. Idiopathic lesions, i.e. lesions of unknown cause, were classified into eight hepatic and four renal lesion categories. The significance of certain potential risk factors associated with lesion prevalences, including age, gender, site of capture, season of capture and year of capture, were assessed by logistic regression. The odds ratios for the significant risk factors were also calculated. Site of capture and age of the examined fish were found to be the most important factors for most of the observed lesion types, e.g., hepatic neoplasms and foci of cellular alteration, whereas year and season of capture were significant influences for only a few lesion types, e.g., specific and nonspecific degenerative/necrotic lesions of the liver. Gender of examined fish was not found to be a significant factor for any of the lesion types. These results emphasize the importance of including certain potential influences, such as age and season of capture, in the analysis of field-collected organ pathology data.

A 4-year multidisciplinary study was conducted on relationships between pollutants and diseases of fish in Puget Sound, WA. In this study, high concentrations of a large number of anthropogenic chemicals such as petroleum hydrocarbons, polychlorinated biphenyls (PCBs) and other chlorinated compounds, pesticides, and metals were found in certain Puget Sound sediments. For example, over 900 individual organic compounds were detected in sediment from one urban bay (Commencement Bay), and evidence was obtained for the presence of numerous additional compounds. Many of the chemicals accumulated in bottom-dwelling fish, and high levels of certain toxic chemicals in the urban bays were linked to serious diseases (e.g., liver carcinomas) of English sole (Parophrys vetulus) and other demersal fish species. It was concluded that application of the approach used would reveal comparable serious pollution-related health problems in marine biota in other urban coastal areas of the world.

High prevalences of idiopathic hepatic lesions, including neoplasms (e.g., hepatocellular carcinomas, cholangiocellular carcinomas) (27%, 20 of 75 fish) and foci of cellular alteration (putative preneoplastic lesions) (44%, 33 of 75 fish), were found in English sole (Parophrys vetulus) exposed to creosote-contaminated sediments in Eagle Harbor, Puget Sound, WA. Sediments from the contaminated region of the harbor contained particularly high concentrations of aromatic hydrocarbons (e.g., benzo(a)pyrene and benz(a)anthracene), and a variety of nitrogen-containing aromatic compounds (e.g., carbazole and acridine). The composition of the aromatic compounds was characteristic of creosote. Dramatically lower concentrations of aromatic compounds were found in sediments from a reference site in which the bottom-dwelling fish examined were free of detectable neoplastic or preneoplastic hepatic lesions. Food organisms in the stomachs of the English sole from Eagle Harbor contained substantially higher concentrations of aromatic hydrocarbons than comparable organisms from the reference site. The concentrations of individual aromatic hydrocarbons in muscle and liver from the Eagle Harbor fish were low; however, high concentrations of metabolites of aromatic compounds were present in the bile. The findings strongly suggest an association between exposure to creosote and the prevalence of hepatic lesions, including neoplasms, in the bottom-dwelling fish, and furthermore support the putativemore » role of aromatic hydrocarbons in liver carcinogenesis in fish.« less

High prevalences of idiopathic liver lesions, including 7.5% hepatic neoplasms (e.g., hepatocellular and cholangiocellular carcinomas) and 16.7% foci of cellular alteration (putative preneoplastic hepatic lesions), were found in English sole (Parophrys vetulus) from waters near Mukilteo, a small community on Puget Sound in Washington State. Sediments from the sampling sites contained particularly high concentrations of aromatic hydrocarbons. Substantially lower concentrations of these compounds were found in sediments from another Puget Sound (reference) site in which fish showed no evidence of hepatic neoplasms or foci of cellular alteration. Stomach contents from the fish at Mukilteo contained substantially higher concentrations of the chemicals than did stomach contents of fish from the relatively uncontaminated site. High concentrations of metabolites of aromatic compounds were measured in the bile of fish from Mukilteo. These findings support previously observed relationships between sediment chemicals (e.g., aromatic hydrocarbons) and high prevalences of liver lesions in English sole from Puget Sound. In addition, a dietary route of uptake by English sole of environmental chemicals, including known carcinogens, was documented.

A comprehensive description of the histopathologic characteristics of a spectrum of idiopathic lesions in feral English sole (Parophrys vetulus), a bottom-dwelling flatfish, from Puget Sound, Washington State, is presented. Among these lesions are unique degenerative conditions, regeneration, storage disorders, foci of hepatocellular alteration (putative preneoplastic lesions), hepatocellular and biliary neoplasms, and nonneoplastic proliferative conditions, all of which morphologically resemble the lesions induced by various hepatocarcinogens-hepatotoxins in experimental exposures of fish and/or rodents. Results from a statistical analysis of the patterns of co-occurrence of these lesions in English sole are consistent with the concept, developed from experimental studies of liver carcinogenesis in rodents, that there are morphologically identifiable steps representing progression toward hepatic neoplasms. This is the first study in which it has been possible to demonstrate a close morphological congruity between a set of idiopathic hepatic lesions in any feral population and an established series of hepatic lesions inducible in rodents by certain hepatocarcinogens under laboratory conditions. Since sediments from the habitats occupied by the fish in this study have been shown to contain multiple hepatocarcinogens, the findings strengthen cumulative evidence that English sole are useful as indicators of exposure to hepatocarcinogens in aquatic environments. 156 references.

The cellular and subcellular morphology of hepatocellular carcinomas (HCs) from feral English sole ( Parophrys vetulus) living in contaminated waterways of Puget Sound was compared with normal-appearing liver from English sole inhabiting reference sites in Puget Sound. The most distinctive feature of English sole HCs was the occurrence of large dilations of RER containing densely packed microfilamentous-like material that was often arranged in complex branching arrays that nearly filled the cytoplasm of affected hepatocytes. These arrays have not previously been reported in HCs of any other fishes. Several other characteristics also distinguished HCs from reference liver, including: the apparent proliferation of Golgi complexes and mitochondria; occurrence of torus-shaped mitochondria; elongation and/or proliferation of perisinusoidal cells; and inclusion of large intrahepatocellular vacuoles.

Relationships between selected serum components and microscopically detected idiopathic liver and kidney lesions were evaluated for English sole, Parophrys vetulus, sampled from urban and non-urban areas of Puget Sound, Washington. The normal range for each serum chemistry parameter was based on English sole with no lesions, or sole with minor parasitic conditions or with lesions in tissues other than liver and kidney which did not significantly affect serum chemistry values. Generally, sole with idiopathic liver lesions had significantly different haematocrit values and levels of serum albumin, calcium, and bilirubin when compared to sole which were free of idiopathic liver and/or kidney lesions. Serum aspartate aminotransferase and alanine aminotransferase activity and serum levels of glucose, total protein, phosphates and magnesium were also abnormal, but appeared to be associated only with certain types of liver lesions. No association between kidney lesions and abnormal serum chemistry values was identified. In addition, English sole sampled from some urban areas of Puget Sound had particular serum chemistry values and liver lesion prevalences which were significantly different from those of sole sampled from non-urban areas. These findings support the hypothesis that sole near highly urbanized environments may display other signs of impaired health in addition to high prevalences of tissue lesions. Thus, measurements of serum chemistry parameters, in conjunction with histopathological examination, appear to be useful techniques for evaluating the health of English sole.