Concentration, Life Stage, Feeding, Density, Flow, and Strain Effects on Formalin Sensitivity in Rainbow Trout (Oncorhynchus mykiss).

Formalin is one of the most widely used and effective chemotherapeutic compounds for treatment of fungal infections and external parasites of fish eggs and fish. However, exposure to formalin can cause mortality in eggs and fingerlings, dependent upon the concentration used and the rearing conditions in which fish are treated. Additionally, strains within a species can exhibit differential susceptibility to formalin. Four experiments were conducted to evaluate the differential sensitivity to formalin of four rainbow trout (Oncorhynchus mykiss) strains in both the egg and fingerling life stages. Eggs were exposed to concentrations of 1667, 2000, and 5000 ppm formalin, and sensitivity differed among the strains when formalin concentration exceeded 2000 ppm. Exposure to higher formalin concentrations (i.e., 5000 ppm) as eggs did not increase mortality when fish were re-exposed to concentrations of 0, 167, 250, or 500 ppm formalin at 77 mm total length (TL). Fish size affected formalin sensitivity, with larger fish (128 mm TL) exhibiting higher rates of mortality than fish ≤ 77 mm TL when exposed to 250 ppm formalin. The effects of crowding, feeding, flow, and density on the formalin sensitivity of 77 mm TL fish were also investigated. Mortality increased in fish crowded away from the inflow to prevent contact with formalin as it entered the tank, potentially the result of an increase in density index within the crowded tanks. Feeding fish on the day they were treated caused mortality to increase by 5.4 to 8.8% in fish exposed to 167 and 250 ppm formalin, respectively, and mortality differed by strain. Reducing flows by half resulted in doubled to quadrupled mortality, and increased densities resulted in increased mortality in some strains but not others. Hatchery managers should consider what effect rearing conditions and formalin concentrations might have on the strain of fish being treated, prior to large-scale treatment.

Vertical transmission of Renibacterium salmoninarum in cutthroat trout (Oncorhynchus clarkii).

Vertical transmission of Renibacterium salmoninarum has been well-documented in anadromous salmonids but not in hatchery-reared inland trout. We assessed whether the bacterium is vertically transmitted in cutthroat trout (Oncorhynchus clarkii) from a Colorado, USA hatchery, and assessed the rate of transmission from male and female brood fish. Adult brood fish were killed, tested for R. salmoninarum in kidney, liver, spleen, ovarian fluid, blood and mucus samples, then stripped of gametes to create 32 families with four infection treatments (MNFN, MNFP, MPFN, MPFP; M: male, F: female, P: positive, N: negative). Progeny from each treatment was sampled at 6 and 12 months to test for the presence of R. salmoninarum with an enzyme-linked immunosorbent assay and quantitative polymerase chain reaction. Our study indicated that vertical transmission was high and occurred among 60% of families across all infection treatments. However, the average proportion of infected progeny from individual families was low, ranging from 1% (MNFP, MPFN and MPFP treatments) up to 21% (MPFP treatment). Hatcheries rearing inland salmonids would be well suited to limit vertical transmission through practices such as lethal culling because any amount of transmission can perpetuate the infection throughout fish on a hatchery.

Factors Affecting Post-Challenge Survival of Flavobacterium psychrophilum in Susceptible Rainbow Trout from the Literature.

Infectious bacterial pathogens are a concern for aquaculture as estimates suggest that billions of US dollars are lost annually in aquaculture due to disease. One of the most prevalent salmonid pathogens is the bacterium Flavobacterium psychrophilum that causes bacterial coldwater disease. We reviewed the published F. psychrophilum literature and conducted a Bayesian analysis to examine large-scale patterns in rainbow trout (Oncorhynchus mykiss) mortality associated with laboratory challenge. We incorporated factors that were common across a majority of the laboratory exposure studies and these included bacterial dose, culture time, exposure method, bacterial isolate, experimental duration, and fish weight. The comparison showed that injection as the exposure method produced higher mortality than bath immersion, bacterial isolates differed in their effect on mortality, and bacterial dose has an interactive effect with fish weight and exposure method. Our comparison allows for inference on factors affecting rainbow trout mortality due to exposure to F. psychrophilum and suggests avenues to further optimize research protocols to better reach study goals.

Dual resistance to Flavobacterium psychrophilum and Myxobolus cerebralis in rainbow trout (Oncorhynchus mykiss, Walbaum).

Aquatic pathogens are a major concern for fish hatchery production, fisheries management, and conservation, and disease control needs to be addressed. Two important salmonid pathogens are Myxobolus cerebralis and Flavobacterium psychrophilum that cause whirling disease and bacterial coldwater disease (BCWD), respectively. Innate disease resistance is a potential option for reducing disease-related mortality in hatchery-reared rainbow trout (Oncorhynchus mykiss, Walbaum). Two experiments were conducted to assess pathogen resistance of first-generation (F1) rainbow trout created by crossing M. cerebralis- and F. psychrophilum-resistant strains. In the first experiment, we exposed two rainbow trout strains and one F1 cross to six treatments: control (no exposure), mock injection, F. psychrophilum only, M. cerebralis only, F. psychrophilum then M. cerebralis, and M. cerebralis then F. psychrophilum. Results indicated that the F1 cross was not resistant to either pathogen. In the second experiment, we exposed five rainbow trout strains and four rainbow trout crosses to F. psychrophilum. The second experiment indicated that at least one rainbow trout cross was F. psychrophilum-resistant. Achieving dual resistance may be possible using selective breeding but only some multigenerational strains are suitable candidates for further evaluation.

Habitat associations of rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta fry.

Habitat restoration activities continue to increase in large rivers, but many of these projects focus on improving juvenile or adult habitats. Incorporating the habitat associations of fry into restoration designs will allow for broader successes from restoration for all life stages and may be useful for either multispecies or specific-species management. This study investigated the habitat associations of rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta fry in the upper Colorado River, focusing on the mean substrate size (D50 ), velocity (m s-1 ), depth (m) and presence of wood in near-shore habitats. S. trutta and O. mykiss were found in higher numbers in fry sites with a D50 of 151 mm (ranging from 96 to 206 mm), velocities ranging from 0.20 to 0.23 m s-1 and depths ranging from 0.17 to 0.18 m. Although there was considerable overlap in habitat associations between the two species, there may be opportunities for single-species management, if this is a goal of such restoration activities, by adjusting design criteria based on differing habitat associations. In addition, the results suggest that including larger particle sizes in near-shore habitats and upstream of fry sites could decrease Tubifex tubifex habitat and thereby fry infection severity by reducing exposure to Myxobolus cerebralis. Stocking, interspecific competition and/or the presence of pathogens can affect fry habitat associations and cause deviations from demonstrated suitability indices. As such, evaluating system-specific differences in habitat associations may allow future habitat restoration activities to be more effective.

Evidence for the Use of Mucus Swabs to Detect Renibacterium salmoninarum in Brook Trout.

Efforts to advance fish health diagnostics have been highlighted in many studies to improve the detection of pathogens in aquaculture facilities and wild fish populations. Typically, the detection of a pathogen has required sacrificing fish; however, many hatcheries have valuable and sometimes irreplaceable broodstocks, and lethal sampling is undesirable. Therefore, the development of non-lethal detection methods is a high priority. The goal of our study was to compare non-lethal sampling methods with standardized lethal kidney tissue sampling that is used to detect Renibacterium salmoninarum infections in salmonids. We collected anal, buccal, and mucus swabs (non-lethal qPCR) and kidney tissue samples (lethal DFAT) from 72 adult brook trout (Salvelinus fontinalis) reared at the Colorado Parks and Wildlife Pitkin Brood Unit and tested each sample to assess R. salmoninarum infections. Standard kidney tissue detected R. salmoninarum 1.59 times more often than mucus swabs, compared to 10.43 and 13.16 times more often than buccal or anal swabs, respectively, indicating mucus swabs were the most effective and may be a useful non-lethal method. Our study highlights the potential of non-lethal mucus swabs to sample for R. salmoninarum and suggests future studies are needed to refine this technique for use in aquaculture facilities and wild populations of inland salmonids.

Efficacy of Erymicin 200 Injections for Reducing Renibacterium salmoninarum and Controlling Vertical Transmission in an Inland Rainbow Trout Brood Stock.

Bacterial Kidney Disease, caused by Renibacterium salmoninarum (Rs), is widespread and can cause significant mortality at most life stages in infected salmonids. Rs is commonly found in inland trout, which can be carriers of the bacterium. Lethal spawns can be used to control vertical transmission to progeny through the culling of eggs from infected parents, but can be costly, time-consuming, and can negatively impact important and rare brood stocks. Erymicin 200 is an Investigational New Animal Drug (INAD) intended to reduce Rs levels in hatchery brood stocks and control vertical transmission to progeny. We tested the efficacy of Erymicin 200 injections in a positive, hatchery-resident rainbow trout (Oncorhynchus mykiss) brood stock in Colorado, USA. Brood fish age two and three were injected with 25 mg per kg of body weight Erymicin 200 three times prior to spawning. Erymicin 200 was effective in reducing Rs to below detectable levels in treated fish. However, both negative treated and control brood fish produced positive progeny, suggesting that Erymicin 200 did not prevent the vertical transmission of Rs.

Survival of Whirling-Disease-Resistant Rainbow Trout Fry in the Wild: A Comparison of Two Strains.

Introduced pathogens can affect fish populations, and three main factors affect disease occurrence: the environment, host, and pathogen. Manipulating at least one of these factors is necessary for controlling disease. Myxobolus cerebralis, the parasite responsible for salmonid whirling disease, became established in Colorado during the 1990s and caused significant declines in wild Rainbow Trout Oncorhynchus mykiss populations. Attempts to re-establish Rainbow Trout have focused on manipulating salmonid host resistance. A Rainbow Trout strain known as GR × CRR was developed for stocking in Colorado by crossing a whirling-disease-resistant strain known as the German Rainbow Trout (GR) with the Colorado River Rainbow Trout (CRR). The GR × CRR fish exhibit resistance similar to that shown by GR, and survival and reproduction were expected to be similar to those of CRR. One disadvantage of stocking GR × CRR is that outcrossing and backcrossing could decrease resistance, and laboratory studies have indicated that this can occur. A potential disadvantage of stocking pure GR is lower survival due to domestication. To compare fry survival between the strains, a field experiment was conducted in 1.6-km reaches of nine Colorado streams. Each stream was stocked in August 2014 with 5,000 GR × CRR and 5,000 GR individuals. In October 2014, April 2015, and August 2015, apparent survival was assessed. Two laboratory predation experiments were also conducted. The field experiment revealed that short-term apparent survival was influenced by stream, and growth rate was influenced by strain and stream. However, after 12 months, there was no difference in apparent survival or growth rate between the GR and GR × CRR strains. Laboratory experiments showed that survival did not differ between the strains when confronted with Brown Trout Salmo trutta predation. Our results indicate that the GR strain is a viable option for stocking in streams where M. cerebralis is enzootic. Further evaluation is needed to determine whether GR fish will survive to maturity and reproduce.

Survival and reproduction of Myxobolus cerebralis-resistant rainbow trout introduced to the Colorado river and increased resistance of age-0 progeny.

Myxobolus cerebralis caused severe declines in rainbow trout populations across Colorado following its introduction in the 1980s. One promising approach for the recovery of Colorado's rainbow trout populations has been the production of rainbow trout that are genetically resistant to the parasite. We introduced one of these resistant crosses, known as the GR×CRR (cross between the German Rainbow [GR] and Colorado River Rainbow [CRR] trout strains), to the upper Colorado River. The abundance, survival, and growth of the stocked GR×CRR population was examined to determine if GR×CRRs had contributed offspring to the age-0 population, and determine whether these offspring displayed increased resistance and survival characteristics compared to their wild CRR counterparts. Apparent survival of the introduced GR×CRR over the entire study period was estimated to be 0.007 (±0.001). Despite low survival of the GR×CRRs, age-0 progeny of the GR×CRR were encountered in years 2008 through 2011. Genetic assignments revealed a shift in the genetic composition of the rainbow trout fry population over time, with CRR fish comprising the entirety of the fry population in 2007, and GR-cross fish comprising nearly 80% of the fry population in 2011. A decrease in average infection severity (myxospores fish-1) was observed concurrent with the shift in the genetic composition of the rainbow trout fry population, decreasing from an average of 47,708 (±8,950) myxospores fish-1 in 2009 to 2,672 (±4,379) myxospores fish-1 in 2011. Results from this experiment suggest that the GR×CRR can survive and reproduce in rivers with a high prevalence of M. cerebralis. In addition, reduced myxospore burdens in age-0 fish indicated that stocking this cross may ultimately lead to an overall reduction in infection prevalence and severity in the salmonid populations of the upper Colorado River.

Genetic basis of differences in myxospore count between whirling disease-resistant and susceptible strains of rainbow trout.

We used a quantitative genetics approach and estimated broad sense heritability (h2b) of myxospore count and the number of genes involved in myxospore formation to gain a better understanding of how resistance to Myxobolus cerebralis, the parasite responsible for whirling disease, is inherited in rainbow trout Oncorhynchus mykiss. An M. cerebralis-resistant strain of rainbow trout, the German Rainbow (GR), and a wild, susceptible strain of rainbow trout, the Colorado River Rainbow (CRR), were spawned to create 3 intermediate crossed populations (an F1 cross, F2 intercross, and a B2 backcross between the F1 and the CRR). Within each strain or cross, h2b was estimated from the between-family variance of myxospore counts using full-sibling families. Estimates of h2b and average myxospore counts were lowest in the GR strain, F1 cross, and F2 intercross (h2b = 0.34, 0.42, and 0.34; myxospores fish-1 = 275, 9566, and 45780, respectively), and highest in the B2 backcross and CRR strain (h2b = 0.93 and 0.89; myxospores fish-1 = 97865 and 187595, respectively). Comparison of means and a joint-scaling test suggest that resistance alleles arising from the GR strain are dominant to susceptible alleles from the CRR strain. Resistance was retained in the intermediate crosses but decreased as filial generation number increased (F2) or backcrossing occurred (B2). The estimated number of segregating loci responsible for differences in myxospore count in the parental strains was 9 ± 5. Our results indicate that resistance to M. cerebralis is a heritable trait within these populations and would respond to either artificial selection in hatcheries or natural selection in the wild.

The effects of Myxobolus cerebralis on the physiological performance of whirling disease resistant and susceptible strains of rainbow trout.

The development of rainbow trout Oncorhynchus mykiss strains that are resistant to whirling disease has shown promise as a management tool for populations in areas where Myxobolus cerebralis is present. However, the physiological effects of the disease on characteristics necessary for fish survival in natural river conditions have not been tested in many of these strains. Five rainbow trout strains were evaluated for their swimming ability and growth characteristics in relation to M. cerebralis exposure: the resistant German rainbow trout (GR) strain (Hofer strain), the susceptible Colorado River rainbow trout (CRR) strain, and three intermediate (hybrid) strains (F1 = GR x CRR; F2 = F1 x F1; B2 = backcross of F1 x CRR). Three broad response patterns among strain and exposure were evident in our study. First, exposure metrics, growth performance, and swimming ability differed among strains. Second, exposure to the parasite did not necessarily produce differences in growth or swimming ability. Exposure to M. cerebralis did not affect batch weight for any strain, and critical swimming velocity did not differ between exposed and unexposed families. Third, although exposure did not necessarily affect growth or swimming ability, individuals that exhibited clinical deformities did show reduced growth and swimming performance; fish with clinical deformities were significantly smaller and had lower critical swimming velocities than exposed fish without clinical deformities. Research and management have focused on GR x CRR hybrid strains; however, given the performance of the GR strain in our study, it should not be discounted as a potential broodstock. Additional field trials comparing the GR and F1 strains should be conducted before wholesale adoption of the GR strain to reestablish rainbow trout populations in Colorado.