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.

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.

Effects-Based Monitoring of Bioactive Chemicals Discharged to the Colorado River before and after a Municipal Wastewater Treatment Plant Replacement.

Monitoring of the Colorado River near the Moab, Utah, wastewater treatment plant (WWTP) outflow has detected pharmaceuticals, hormones, and estrogen-receptor (ER)-, glucocorticoid receptor (GR)-, and peroxisome proliferator-activated receptor-gamma (PPARγ)-mediated biological activities. The aim of the present multi-year study was to assess effects of a WWTP replacement on bioactive chemical (BC) concentrations. Water samples were collected bimonthly, pre- and post-replacement, at 11 sites along the Colorado River upstream and downstream of the WWTP and analyzed for in vitro bioactivities (e.g., agonism of ER, GR, and PPARγ) and BC concentrations; fathead minnows were cage deployed pre- and post-replacement at sites with varying proximities to the WWTP. Before the WWTP replacement, in vitro ER (24 ng 17ß-estradiol equivalents/L)-, GR (60 ng dexamethasone equivalents/L)-, and PPARγ-mediated activities were detected at the WWTP outflow but diminished downstream. In March 2018, the WWTP effluent was acutely toxic to the fish, likely due to elevated ammonia concentrations. Following the WWTP replacement, ER, GR, and PPARγ bioactivities were reduced by approximately 60-79%, no toxicity was observed in caged fish, and there were marked decreases in concentrations of many BCs. Results suggest that replacement of the Moab WWTP achieved a significant reduction in BC concentrations to the Colorado River.

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.

Estimating the effects of 17α-ethinylestradiol on stochastic population growth rate of fathead minnows: a population synthesis of empirically derived vital rates.

Urban freshwater streams in arid climates are wastewater effluent dominated ecosystems particularly impacted by bioactive chemicals including steroid estrogens that disrupt vertebrate reproduction. However, more understanding of the population and ecological consequences of exposure to wastewater effluent is needed. We used empirically derived vital rate estimates from a mesocosm study to develop a stochastic stage-structured population model and evaluated the effect of 17α-ethinylestradiol (EE2), the estrogen in human contraceptive pills, on fathead minnow Pimephales promelas stochastic population growth rate. Tested EE2 concentrations ranged from 3.2 to 10.9 ng L(-1) and produced stochastic population growth rates (λ S ) below 1 at the lowest concentration, indicating potential for population decline. Declines in λ S compared to controls were evident in treatments that were lethal to adult males despite statistically insignificant effects on egg production and juvenile recruitment. In fact, results indicated that λ S was most sensitive to the survival of juveniles and female egg production. More broadly, our results document that population model results may differ even when empirically derived estimates of vital rates are similar among experimental treatments, and demonstrate how population models integrate and project the effects of stressors throughout the life cycle. Thus, stochastic population models can more effectively evaluate the ecological consequences of experimentally derived vital rates.

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.

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.

Elevated Winter Stream Temperatures below Wastewater Treatment Plants Shift Reproductive Development of Female Johnny Darter Etheostoma nigrum: A Field and Histologic Approach.

River water temperatures are increasing globally, particularly in urban systems. In winter, wastewater treatment plant (WWTP) effluent inputs are of particular concern because they increase water temperatures from near freezing to ~7-15 °C. Recent laboratory studies suggest that warm overwinter temperatures impact the reproductive timing of some fishes. To evaluate winter water temperature's influence in the wild, we sampled Johnny Darter Etheostoma nigrum from three urban South Platte River tributaries in Colorado upstream and downstream of WWTP effluent discharge sites. Fish were collected weekly during the spring spawning season of 2021 and reproductive development was determined from histological analysis of the gonads. Winter water temperatures were approximately 5-10 °C greater ~300 m downstream of the WWTP effluent compared to upstream sites, and approximately 3°C warmer at sampling sites ~5000 m downstream of the effluent discharge. Females collected downstream of WWTP effluent experienced accelerated reproductive development compared to upstream by 1-2 weeks. Water quality, including total estrogenicity, and spring water temperatures did not appear to explain varying reproductive development. It appears that small increases in winter water temperature influence the reproductive timing in E. nigrum. Further investigations into how shifts in reproductive timing influence other population dynamics are warranted.

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.

Effects-based monitoring of bioactive compounds associated with municipal wastewater treatment plant effluent discharge to the South Platte River, Colorado, USA.

Previous studies have detected numerous organic contaminants and in vitro bioactivities in surface water from the South Platte River near Denver, Colorado, USA. To evaluate the temporal and spatial distribution of selected contaminants of emerging concern, water samples were collected throughout 2018 and 2019 at 11 sites within the S. Platte River and surrounding tributaries with varying proximities to a major wastewater treatment plant (WWTP). Water samples were analyzed for pharmaceuticals, pesticides, steroid hormones, and wastewater indicators and screened for in vitro biological activities. Multiplexed, in vitro assays that simultaneously screen for agonistic activity against 24 human nuclear receptors detected estrogen receptor (ER), peroxisome proliferator activated receptor-gamma (PPARγ), and glucocorticoid receptor (GR) bioactivities in water samples near the WWTP outflow. Targeted in vitro bioassays assessing ER, GR, and PPARγ agonism corroborated bioactivities for ER (up to 55 ± 9.7 ng/L 17ß-estradiol equivalents) and GR (up to 156 ± 28 ng/L dexamethasone equivalents), while PPARγ activity was not confirmed. To evaluate the potential in vivo significance of the bioactive contaminants, sexually-mature fathead minnows were caged at six locations upstream and downstream of the WWTP for 5 days after which targeted gene expression analyses were performed. Significant up-regulation of male hepatic vitellogenin was observed at sites with corresponding in vitro ER activity. No site-related differences in GR-related transcript abundance were detected in female adipose or male livers, suggesting observed environmental concentrations of GR-active contaminants do not induce a detectable in vivo response. In line with the lack of detectable targeted in vitro PPARÉ£ activity, there were no significant effects on PPARÉ£-related gene expression. Although the chemicals responsible for GR and PPAR-mediated bioactivities are unknown, results from the present study provide insights into the significance (or lack thereof) of these bioactivities relative to short-term in situ fish exposures.

Evaluation of targeted and untargeted effects-based monitoring tools to assess impacts of contaminants of emerging concern on fish in the South Platte River, CO.

Rivers in the arid Western United States face increasing influences from anthropogenic contaminants due to population growth, urbanization, and drought. To better understand and more effectively track the impacts of these contaminants, biologically-based monitoring tools are increasingly being used to complement routine chemical monitoring. This study was initiated to assess the ability of both targeted and untargeted biologically-based monitoring tools to discriminate impacts of two adjacent wastewater treatment plants (WWTPs) on Colorado's South Platte River. A cell-based estrogen assay (in vitro, targeted) determined that water samples collected downstream of the larger of the two WWTPs displayed considerable estrogenic activity in its two separate effluent streams. Hepatic vitellogenin mRNA expression (in vivo, targeted) and NMR-based metabolomic analyses (in vivo, untargeted) from caged male fathead minnows also suggested estrogenic activity downstream of the larger WWTP, but detected significant differences in responses from its two effluent streams. The metabolomic results suggested that these differences were associated with oxidative stress levels. Finally, partial least squares regression was used to explore linkages between the metabolomics responses and the chemical contaminants that were detected at the sites. This analysis, along with univariate statistical approaches, identified significant covariance between the biological endpoints and estrone concentrations, suggesting the importance of this contaminant and recommending increased focus on its presence in the environment. These results underscore the benefits of a combined targeted and untargeted biologically-based monitoring strategy when used alongside contaminant monitoring to more effectively assess ecological impacts of exposures to complex mixtures in surface waters.

Direct and indirect effects of predation on mosquitofish behavior and survival.

Predation can have strong direct and indirect effects on the behavior of prey. We investigated whether predation by chain pickerel (Esox niger) caused adult eastern mosquitofish (Gambusia holbrooki) to alter their habitat use and whether pickerel predation influenced survival of adult and neonate mosquitofish. The number of adult mosquitofish using the riskier of three habitats was lowest when two predators occupied the risky habitat, intermediate in the treatment with one predator, and highest when no predators occurred there. More mosquitofish neonates survived high predation treatments than treatments lacking pickerel. We conclude that pickerel predation causes adult mosquitofish to shift to refuge habitats. The pattern of neonate survival suggests that adult habitat use may create a refuge from cannibalism for neonate mosquitofish, resulting in higher neonate survival in treatments with more pickerel. Hence, pickerel predation has a direct effect on adult mosquitofish behavior and a strong indirect effect on neonate survival. Both interspecific and intraspecific predation can effect prey populations and can interact to produce important indirect effects.

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.

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.

Soil clay content underlies prion infection odds.

Environmental factors-especially soil properties-have been suggested as potentially important in the transmission of infectious prion diseases. Because binding to montmorillonite (an aluminosilicate clay mineral) or clay-enriched soils had been shown to enhance experimental prion transmissibility, we hypothesized that prion transmission among mule deer might also be enhanced in ranges with relatively high soil clay content. In this study, we report apparent influences of soil clay content on the odds of prion infection in free-ranging deer. Analysis of data from prion-infected deer herds in northern Colorado, USA, revealed that a 1% increase in the clay-sized particle content in soils within the approximate home range of an individual deer increased its odds of infection by up to 8.9%. Our findings suggest that soil clay content and related environmental properties deserve greater attention in assessing risks of prion disease outbreaks and prospects for their control in both natural and production settings.