Phenotypic and Genetic Characterization of Flavobacterium psychrophilum Recovered from Diseased Salmonids in China.

Flavobacterium psychrophilum, the etiological agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome, causes great economic losses in salmonid aquaculture worldwide. Recent molecular studies have uncovered important epidemiological and ecological aspects of this pathogen; however, such data are lacking for F. psychrophilum populations affecting aquaculture in China. Herein, F. psychrophilum phenotype, genotype, and virulence were characterized for isolates recovered from epizootics in multiple salmonid aquaculture facilities across China. Thirty-one F. psychrophilum isolates, originating from four provinces and three host fish species, were predominantly homogeneous biochemically but represented 5 sequence types (STs) according to multilocus sequence typing (MLST) that belonged to clonal complex CC-ST10 or 3 newly recognized singleton STs. PCR-based serotyping classified 19 and 12 F. psychrophilum isolates into molecular serotypes 1 and 0, respectively, showing an obvious relationship with host species. Antimicrobial susceptibility analysis via broth microdilution revealed reduced susceptibility to enrofloxacin, flumequine, and oxolinic acid, moderate susceptibility to gentamicin, erythromycin, and florfenicol, and variable susceptibility to ampicillin and oxytetracycline. In vivo challenge experiments confirmed the ability of two representative Chinese F. psychrophilum isolates to induce typical signs of BCWD and mortality in 1-year-old rainbow trout (Oncorhynchus mykiss). Findings collectively demonstrate (i) that BCWD outbreaks in China studied thus far are caused by F. psychrophilum lineages that are common on other continents (e.g., CC-ST10) and others that have not been reported elsewhere (e.g., ST355, ST356, ST357), (ii) that F. psychrophilum molecular serotypes distinguish isolates from different host fish species, even within STs, and (iii) reduced F. psychrophilum antimicrobial susceptibility against compounds used for BCWD control in China. IMPORTANCE Flavobacterium psychrophilum causes substantial economic losses in salmonid aquaculture worldwide. Although this bacterium is also believed to be a disease source in China, published reports of its presence do not yet exist. Herein, F. psychrophilum was linked to multiple disease outbreaks in several salmonid aquaculture facilities within four Chinese provinces, and polyphasic characterization revealed that most isolates were genetically distinct from strains recovered on other continents. Analyses further revealed the predominating molecular serotypes, antimicrobial susceptibility profiles, and pathogenic potential of two representative recovered isolates. Collectively, the results presented here provide important data on the epidemiology and disease ecology of F. psychrophilum in China and pave the way for targeted prevention and control methods to be pursued in the future.

Assessment of spawning site fidelity in interior Fraser River Coho salmon Oncorhynchus kisutch using otolith microchemistry, in British Columbia, Canada.

Coho Salmon Oncorhynchus kisutch show fidelity to natal spawning watersheds. Fine-scale homing, however, within rivers is not well understood. Interior Fraser Coho (IFC) salmon eggs were incubated at known spawning locations in the Coldwater River, two main stem sites and one-off channel pond site, providing otolith reference data for comparison to otolith signatures for returning adults using laser ablation inductively coupled plasma mass spectrometry. Elemental ratios for Ba:Ca and Sr:Ca in otoliths of juvenile O. kisutch differed significantly among the spawning locations examined. Juvenile otolith data were used to conduct a linear discriminant analysis to assess fine-scale homing in adults. Juvenile data were all assigned to the location where they had been incubated, producing a robust data set used to compare adult otoliths and define natal locations based on elemental signatures in otoliths of adult spawners. Homing and straying were apparent at the reach level; 57.1% of adults returned to their natal spawning locations, while 42.9% strayed to other spawning sites within the Coldwater River. Straying to novel incubation sites at the reach scale demonstrated plasticity in homing within a watershed.

Assessment of genetic diversity in Coho salmon (Oncorhynchus kisutch) populations with no family records using ddRAD-seq.

OBJECTIVE: Selective breeding for desirable traits is becoming popular in aquaculture. In Miyagi prefecture, Japan, a selectively bred population of Coho salmon (Oncorhynchus kisutch) has been established with the original, randomly breeding population maintained separately. Since they have been bred without family records, the genetic diversity within these populations remains unknown. In this study, we estimated the genetic diversity and key quantitative genetic parameters such as heritability and genomic breeding value for body size traits by means of genomic best linear unbiased prediction to assess the genetic health of these populations. RESULTS: Ninety-nine and 83 females from the selective and random groups, respectively, were genotyped at 2350 putative SNPs by means of double digest restriction associated DNA sequencing. The genetic diversity in the selectively bred group was low, as were the estimated heritability and prediction accuracy for length and weight (h2 = 0.26-0.28; accuracy = 0.34), compared to the randomly bred group (h2 = 0.50-0.60; accuracy = 0.51-0.54). Although the tested sample size was small, these results suggest that further selection is difficult for the selectively bred population, while there is some potential for the randomly bred group, especially with the aid of genomic information.

Genetic assessment of the effects of streamscape succession on coho salmon Oncorhynchus kisutch colonization in recently deglaciated streams.

Measures of genetic diversity within and among populations and historical geomorphological data on stream landscapes were used in model simulations based on approximate Bayesian computation (ABC) to examine hypotheses of the relative importance of stream features (geomorphology and age) associated with colonization events and gene flow for coho salmon Oncorhynchus kisutch breeding in recently deglaciated streams (50-240 years b.p.) in Glacier Bay National Park (GBNP), Alaska. Population estimates of genetic diversity including heterozygosity and allelic richness declined significantly and monotonically from the oldest and largest to youngest and smallest GBNP streams. Interpopulation variance in allele frequency increased with increasing distance between streams (r = 0·435, P < 0·01) and was inversely related to stream age (r = -0·281, P < 0·01). The most supported model of colonization involved ongoing or recent (<10 generations before sampling) colonization originating from large populations outside Glacier Bay proper into all other GBNP streams sampled. Results here show that sustained gene flow from large source populations is important to recently established O. kisutch metapopulations. Studies that document how genetic and demographic characteristics of newly founded populations vary associated with successional changes in stream habitat are of particular importance to and have significant implications for, restoration of declining or repatriation of extirpated populations in other regions of the species' native range.

Growth-Enhanced Transgenic Coho Salmon (Oncorhynchus kisutch) Strains Have Varied Success in Simulated Streams: Implications for Risk Assessment.

Growth hormone (GH) transgenic fish have accelerated growth and could improve production efficiency in aquaculture. However, concern exists regarding potential environmental risks of GH transgenic fish should they escape rearing facilities. While environmental effects have been examined in some GH transgenic models, there is a lack of information on whether effects differ among different constructs or strains of transgenic fish. We compared growth and survival of wild-type coho salmon (Oncorhynchus kisutch) fry, a fast-growing GH transgenic strain containing a metallothionein promoter (TMT), and three lines/strains containing a reportedly weaker histone-3 promoter (TH3) in hatchery conditions and semi-natural stream tanks with varying levels of natural food and predators. Rank order of genotype size and survival differed with varying environmental conditions, both within and among experiments. Despite accelerated growth in hatchery conditions, TMT fry gained little or no growth enhancement in stream conditions, had enhanced survival when food was limiting, and inconsistent survival under other conditions. Rank growth was inconsistent in TH3 strains, with one strain having highest, and two strains having the lowest growth in stream conditions, although all TH3 strains had consistently poor survival. These studies demonstrate the importance of determining risk estimates for each unique transgenic model independent of other models.

Muscle fibre size optimisation provides flexibility for energy budgeting in calorie-restricted coho salmon transgenic for growth hormone.

Coho salmon (Oncorhynchus kisutch) transgenic for growth hormone (GH) show substantially faster growth than wild-type (WT) fish. We fed GH-transgenic salmon either to satiation (1 year; TF) or the same smaller ration of wild-type fish (2 years; TR), resulting in groups matched for body size to WT salmon. The myotomes of TF and WT fish had the same number and size distribution of muscle fibres, indicating a twofold higher rate of fibre recruitment in the GH transgenics. Unexpectedly, calorie restriction was found to decrease the rate of fibre production in transgenics, resulting in a 20% increase in average fibre size and reduced costs of ionic homeostasis. Genes for myotube formation were downregulated in TR relative to TF and WT fish. We suggest that muscle fibre size optimisation allows the reallocation of energy from maintenance to locomotion, explaining the observation that calorie-restricted transgenics grow at the same rate as WT fish whilst exhibiting markedly higher foraging activity.

Development of 54 novel single-nucleotide polymorphism (SNP) assays for sockeye and coho salmon and assessment of available SNPs to differentiate stocks within the Columbia River.

Single-nucleotide polymorphisms (SNPs) have potential for broad application in population and conservation genetics, but availability of these markers is limited in many nonmodel species. In this study, genomic and expressed sequence tagged (EST) sequences from closely related salmonids (Chinook salmon and rainbow trout) were used to design primers for amplification and sequencing of sockeye (Oncorhynchus nerka) and coho (Oncorhynchus kisutch) salmon DNA for SNP discovery. One hundred and six primer sets were designed and tested for amplification in each species. An ascertainment panel of 32 diverse individuals from each species was used as template for PCR amplification and Sanger sequencing. In total, 21,647 bases of consensus sequence were screened in sockeye salmon and 20,784 bases in coho salmon with 93 and 149 SNP sites identified, respectively. Sixty-four SNP sites were chosen for assay development, and 54 of the assays were validated by comparison with genotype and sequence data (O. nerka = 23; O. kisutch = 31). These validated SNP assays along with 142 other available SNP assays [O. nerka = 103 (126 total); O. kisutch = 30 (61 total)] were used to genotype collections of O. nerka (N = 5) and O. kisutch (N = 4) from various sites in the Columbia River to evaluate the utility of these markers in this region. Results from factorial correspondence analysis indicate that these SNP markers are capable of distinguishing O. nerka populations, but O. kisutch collections were less distinct because of their common ancestry.

The synergistic toxicity of pesticide mixtures: implications for risk assessment and the conservation of endangered Pacific salmon.

BACKGROUND: Mixtures of organophosphate and carbamate pesticides are commonly detected in freshwater habitats that support threatened and endangered species of Pacific salmon (Oncorhynchus sp.). These pesticides inhibit the activity of acetylcholinesterase (AChE) and thus have potential to interfere with behaviors that may be essential for salmon survival. Although the effects of individual anticholin-esterase insecticides on aquatic species have been studied for decades, the neurotoxicity of mixtures is still poorly understood. OBJECTIVES: We assessed whether chemicals in a mixture act in isolation (resulting in additive AChE inhibition) or whether components interact to produce either antagonistic or synergistic toxicity. METHODS: We measured brain AChE inhibition in juvenile coho salmon (Oncorhynchus kisutch) exposed to sublethal concentrations of the organophosphates diazinon, malathion, and chlorpyrifos, as well as the carbamates carbaryl and carbofuran. Concentrations of individual chemicals were normalized to their respective median effective concentrations (EC50) and collectively fit to a nonlinear regression. We used this curve to determine whether toxicologic responses to binary mixtures were additive, antagonistic, or synergistic. RESULTS: We observed addition and synergism, with a greater degree of synergism at higher exposure concentrations. Several combinations of organophosphates were lethal at concentrations that were sublethal in single-chemical trials. CONCLUSION: Single-chemical risk assessments are likely to underestimate the impacts of these insecticides on salmon in river systems where mixtures occur. Moreover, mixtures of pesticides that have been commonly reported in salmon habitats may pose a more important challenge for species recovery than previously anticipated.

Acute toxicity and hazard assessment of spinosad and R-11 to three cladoceran species and Coho salmon.

Acute toxicity data and hazard assessments were developed for three cladoceran species, Ceriodaphnia dubia, Daphnia pulex, and Daphnia magna, and juvenile Coho salmon, Oncorhynchus kisutch after exposure to the insecticide, spinosad and adjuvant R-11. The effect of a mixture of these compounds was also determined with C. dubia and O. kisutch. Spinosad was virtually non-toxic to O. kisutch. Hazard assessments indicated that R-11 posed no hazard to any of the species tested while spinosad only posed a hazard to C. dubia. Mixture studies indicated that spinosad and R-11 may interact synergistically in C. dubia.

Assessment of harbor seal predation on adult salmonids in a Pacific Northwest estuary.

The populations of many native species have increased or expanded in distribution in recent decades, sometimes with negative consequences to sympatric native species that are rarer or less adaptable to anthropogenic changes to the environment. An example of this phenomenon from the Pacific Northwest is predation by locally abundant pinnipeds (seals and sea lions) on threatened, endangered, or otherwise depleted salmonid (Oncorhynchus spp.) populations. We used survey sampling methodology, acoustic telemetry, and molecular genetics to quantify the amount of harbor seal (Phoca vitulina) predation on a depressed run of coho salmon (O. kisutch) and to determine whether some seals consumed a disproportionately higher number of salmonids than others. Based on a probability sample totaling 759.5 h of observation, we estimated that seals consumed 1161 adult salmonids (95% CI = 503-1818 salmonids) during daylight hours over an 18.9-km estuarine study area in Oregon during an 84-d period in fall 2002. Simultaneous tracking of 56 seals via an acoustic telemetry array indicated that a small proportion of marked seals (12.5%) exhibited behavior that was consistent with specialization on salmonids. These seals spent the majority of their time in the riverine portion of the study area and did so disproportionately more at night than day. Genetic analysis of 116 salmonid structures recovered from 11 seal fecal samples suggested that coho salmon accounted for approximately one-half of total salmonid consumption. Though subject to considerable uncertainty, the combined results lead us to infer that seals consumed 21% (range = 3-63%) of the estimated prespawning population of coho salmon. We speculate that the majority of the predation occurred upriver, at night, and was done by a relatively small proportion of the local seal population. Understanding the extent and nature of pinniped predation can provide important inputs into risk assessments and other modeling efforts designed to aid the conservation and recovery of salmonids in the Pacific Northwest. Such understanding may also help inform management actions designed to reduce the impact of pinniped predation on salmonids, which potentially range from short-term lethal removal programs to long-term ecosystem restoration and protection efforts.

Global assessment of organic contaminants in farmed salmon.

The annual global production of farmed salmon has increased by a factor of 40 during the past two decades. Salmon from farms in northern Europe, North America, and Chile are now available widely year-round at relatively low prices. Salmon farms have been criticized for their ecological effects, but the potential human health risks of farmed salmon consumption have not been examined rigorously. Having analyzed over 2 metric tons of farmed and wild salmon from around the world for organochlorine contaminants, we show that concentrations of these contaminants are significantly higher in farmed salmon than in wild. European-raised salmon have significantly greater contaminant loads than those raised in North and South America, indicating the need for further investigation into the sources of contamination. Risk analysis indicates that consumption of farmed Atlantic salmon may pose health risks that detract from the beneficial effects of fish consumption.

Valuing freshwater salmon habitat on the west coast of Canada.

Changes in land use can potentially reduce the quality of fish habitat and affect the economic value of commercial and sport fisheries that rely on the affected stocks. Parks and protected areas that restrict land-use activities provide benefits, such as ecosystem services, in addition to recreation and preservation of wildlife. Placing values on these other benefits of protected areas poses a major challenge for land-use planning. In this paper, we present a framework for valuing benefits for fisheries from protecting areas from degradation, using the example of the Strait of Georgia coho salmon fishery in southern British Columbia, Canada. Our study improves upon previous methods used to value fish habitat in two major respects. First, we use a bioeconomic model of the coho fishery to derive estimates of value that are consistent with economic theory. Second, we estimate the value of changing the quality of fish habitat by using empirical analyses to link fish population dynamics with indices of land use in surrounding watersheds. In our example, we estimated that the value of protecting habitat ecosystem services is C$0.93 to C$2.63 per ha of drainage basin or about C$1322 to C$7010 per km of salmon stream length (C$1.00=US$0.71). Sensitivity analyses suggest that these values are relatively robust to different assumptions, and if anything, are likely to be minimum estimates. Thus, when comparing alternative uses of land, managers should consider ecosystem services from maintaining habitat for productive fish populations along with other benefits of protected areas.