Distribution and habitat characteristics of Manayunkia speciosa and infection prevalence with the parasite Ceratomyxa shasta in the Klamath River, Oregon-California

A survey for Manayunkia speciosa, the freshwater polychaete host for the myxozoan parasite Ceratomyxa shasta, was conducted from 2003 to 2005 as part of an integrated study of the epizootiology of ceratomyxosis in Klamath River salmonids. Substrata samples (n = 257) were collected in a variety of habitats from Klamath Lake to the mouth of the Klamath River to document occurrence and relative abundance of the polychaete by habitat type and to estimate the prevalence of C. shasta within selected polychaete populations. Populations of M. specios a were identified throughout the Klamath River within pools (51.6%), eddy-pools (47.0%), and runs (40.0%). Large populations of M. speciosa were consistently found at the inflow to the main-stem reservoirs where densities were correlated with distance from the inflow into the reservoir. Using polymerase chain reaction assay and composite samples, 12 of 71 populations identified were tested for C. shasta, revealing a mean infection prevalence of 0.27%. An area of elevated infection prevalence (4.9 and 8.3%) was identified with 2 populations below a barrier to salmonid migration, which explains the high infectious spore densities demonstrated in concurrent studies and observations of C. shasta-induced mortality in Klamath River fall Chinook salmon (Oncorhynchus tshawytscha).     

Mapping multiple genetic loci associated with Ceratomyxa shasta resistance in Oncorhynchus mykiss

Ceratomyxa shasta is enzootic to some rivers of the Pacific Northwest and California, and has caused extensive mortalities of salmonids, the only known host of the actinosporean stage of the pathogen. Mortalities from C. shasta are induced by severe inflammation and necrosis of the intestinal tract, and no therapeutants have been discovered for the treatment of ceratomyxosis. Inheritance studies by previous investigators suggested that C. shasta resistance is genetically controlled, but none have revealed the number and locations of gene regions associated with resistance. Here, we characterized differences in resistance among clonal lines, and used line crosses between clonal lines with resistance differences for a genetic analysis of C. shasta resistance in Oncorhynchus mykiss, following a short term in situ exposure to the pathogen. We found that Oregon State University (OSU) x Arlee (ARL) clonal hybrids were highly susceptible, while OSU x Clearwater (CW) hybrids were highly resistant to C. shasta. Doubled haploids, produced by androgenesis from an F1 hybrid between the OSU and CW lines, were evaluated for the co-segregation of molecular markers and resistance phenotype. Amplified fragment length polymorphic (AFLP) markers, used to construct a genetic linkage map in this cross, were tested for associations with resistance expressed as absolute survival (binary trait) and days to death (survival analysis). Segregation of the resistance phenotype suggested that the resistance was inherited as a single Mendelian locus, but the binary trait failed to map to a single genomic location. Markers identified in single-marker analyses were used in multiple-regression model selection, within both the linear regression and Cox proportional-hazards survival analysis frameworks, to determine the number and potential location of loci simultaneously contributing to variation in the survival phenotypes of doubled-haploid progeny. We found multiple genomic loci associated with C. shasta resistance through these models, confirming studies by previous investigators that suggested C. shasta resistance is polygenic.     

Density of the waterborne parasite Ceratomyxa shasta and its biological effects on salmon

The myxozoan parasite Ceratomyxa shasta is a significant pathogen of juvenile salmonids in the Pacific Northwest of North America and is limiting recovery of Chinook (Oncorhynchus tshawytscha) and coho (O. kisutch) salmon populations in the Klamath River. We conducted a 5-year monitoring program that comprised concurrent sentinel fish exposures and water sampling across 212 river kilometers of the Klamath River. We used percent mortality and degree-days to death to measure disease severity in fish. We analyzed water samples using quantitative PCR and Sanger sequencing, to determine total parasite density and relative abundance of C. shasta genotypes, which differ in their pathogenicity to salmonids. We detected the parasite throughout the study zone, but parasite density and genetic composition fluctuated spatially and temporally. Chinook and coho mortality increased with density of their specific parasite genotype, but mortality-density thresholds and time to death differed. A lethality threshold of 40% mortality was reached with 10 spores liter(-1) for Chinook but only 5 spores liter(-1) for coho. Parasite density did not affect degree-days to death for Chinook but was negatively correlated for coho, and there was wider variation among coho individuals. These differences likely reflect the different life histories and genetic heterogeneity of the salmon populations. Direct quantification of the density of host-specific parasite genotypes in water samples offers a management tool for predicting host population-level impacts.     

Mortality threshold for juvenile Chinook salmon Oncorhynchus tshawytscha in an epidemiological model of Ceratomyxa shasta

The myxozoan parasite Ceratomyxa shasta is a significant pathogen of juvenile Chinook salmon Oncorhynchus tshawytscha in the Klamath River, California, USA. This parasite requires 2 hosts to complete its life cycle: a freshwater polychaete (Manayunkia speciosa) and a salmonid. The complex life cycle and large geographic area where infection occurs make it difficult to monitor and manage ceratomyxosis. We present a model for ceratomyxosis-induced mortality in O. tshawytscha, from which parameters important to the persistence of C. shasta are identified. We also experimentally quantify specific parameters from the model and identify a mortality threshold (a critical parameter), by naturally exposing native O. tshawytscha to C. shasta in the Klamath River. The average percent mortality that resulted from these experimental challenges ranged from 2.5 to 98.5% over an exposure dose of 4.4 to 612 x 10(6) parasites. This experiment identified a non-linear mortality threshold of 7.7 +/- 2.1 x 10(4) actinospores fish(-1) for Chinook salmon from the Iron Gate Hatchery on the Klamath River. Below this threshold no mortality occurred and above it mortality increased dramatically, thus providing a target by which to reduce parasitism in emigrating juvenile O. tshawytscha.     

Development, Characterization, and Use of Monoclonal and Polyclonal Antibodies against the Myxosporean, Ceratomyxa shasta

Both monoclonal and polyclonal antisera were produced against Ceratomyxa shasta. Ascites containing trophozoites of the parasite was collected from infected fish and used as antigen for immunization of mice. The resulting monoclonal antibodies reacted specifically with trophozoite and sporoblast stages but did not react with C. shasta spores by either indirect fluorescent antibody techniques or in Western blots. This indicates that some C. shasta antigens are specific to certain life stages of the parasite. Polyclonal antiserum was produced in a rabbit by injecting a spore protein electro-eluted from an SDS-polyacrylamide gel. This antiserum reacted with both trophozoites and spores by indirect fluorescent antibody techniques and in Western blots. All antisera were tested for cross-reactivity to trout white blood cells, a contaminant of the ascites, and to other myxosporea. Two monoclonal antibodies reacted with white blood cells and myxosporea of the genera Sphaerospora and Myxobilatus. One hybridoma produced antibodies of high specificity for C. shasta pre-spore stages. This is the first report of a monoclonal antibody produced against a myxosporean parasite.     

Monitoring Ceratomyxa shasta infection during a hatchery rearing cycle: comparison of molecular, serological and histological methods

The prevalence of Ceratomyxa shasta infection in production stocks of steelhead Oncorhynchus mykiss and cutthroat trout O. clarki was monitored using a parasite-specific polymerase chain reaction (PCR) assay. For all 4 stocks of fish followed through their 1 yr rearing cycle, C. shasta infection was detected despite their genetic resistance to the disease and the treatment of the incoming water with ozone. Infection was confirmed using serological methods and standard histological procedures, except when prevalence was low (<10%). This suggests that at the lowest infection levels PCR is more sensitive than other methodologies, and can be used as an early indicator of infection. Results of the PCR assay continued to correlate with histological and serological detection as the numbers of parasites and the lesion severity increased over the rearing cycle. For both steelhead and cutthroat trout, early infections were characterized by large numbers of parasites on the epithelial surface, but with little associated inflammation. At release as yearlings, the infection prevalence in all stocks was greater than 90 % and the inflammatory response in many fish was extensive, with tissue necrosis and mucosal damage. Although C. shasta infections no longer result in high mortality at this facility, results of this study indicate that the parasite remains a contributor to low condition indices in these fish, despite their genetic resistance and ozone disinfection of the water supply.     

Application of a real-time PCR assay to detect and quantify the myxozoan parasite Ceratomyxa shasta in river water samples

Ceratomyxa shasta is a virulent pathogen of salmonid fishes that is enzootic in the Pacific Northwest of North America. Current parasite detection methods involve sentinel fish exposures that are laborious and time-consuming. As a substitute, a filtering protocol and a quantitative real-time TaqMan polymerase chain reaction (QPCR) assay were developed to detect and enumerate parasite spores in river water. Fluorescence was detected from both the myxospore and actinospore stages of the parasite but not from the fish or polychaete hosts or from 9 other myxozoans tested. Less than 1/1000th of a spore was detected, indicating each had >1000 copies of the target 18S rRNA gene. The assay detected 1 spore in 1 l river water. Inhibition of the assay by some river samples was overcome by reducing the template volume and including bovine serum albumin in the reaction; occasionally a second purification step was required. The QPCR methodology was utilised to investigate the temporal and spatial distribution of C. shasta in the Klamath River, Oregon/ California. The parasite was detected throughout the river, and 2 of 5 tributaries tested contributed parasites to the mainstem. Correlation of QPCR cycle threshold values with a standard curve for known starting numbers of whole spores revealed several sites where parasite abundance was in excess of 20 spores l(-1). Although QPCR data corroborated results of sentinel fish exposures, spore numbers did not correlate consistently with mortality in the exposure groups. The water sampling and filtering protocol combined with the QPCR assay is a simple and relatively rapid method for detection and quantification of parasite levels in environmental water samples.     

Relationship between temperature and mortality in the city of Tunis: 2005-2007

The influence of thermal stress on health is proved. In the light of the current climate change this relationship should be extensively investigated. This study aims to describe the relationship between temperature and total mortality in the city of Tunis over a period of three years, from 2005 to 2007 using time series analysis. The form of the relation (crude and adjusted) between mortality and temperatures was investigated using Poisson generalized additive models (GAM). Confounders included in the models were pollutant, trend, calendar month, day of the week, the period of Ramadan, and holidays. The adjusted relationship between mortality and temperature was "V" shaped with a steeper slope for low temperatures than for high temperatures. The impact of heat on mortality will be further analyzed to refine our findings and to contribute to the elaboration of guidelines for preventive strategies.     

Hatchery practices may result in replacement of wild salmonids: adult trends in the Klamath basin,California

Appraisal of hatchery-related effects on Pacific salmonids (Oncorhynchus spp.) is a necessary component of species conservation. For example, hatchery supplementation can influence species viability by changing genetic, phenotypic and life-history diversity. We analyzed time series data for seven salmonid taxa from the Klamath River basin, California, to investigate trajectories of wild and hatchery adult populations. Linear regression coupled with randomized permutations (n?=?99,999), two- tailed t tests, and Bayesian change point analysis were used to detect trends over time. Cross correlation was also used to evaluate relationships between wild and hatchery populations. The taxa of interest were spring, fall, and late-fall Chinook Salmon (O. tshawytscha); Coho Salmon (O. kisutch); Coastal Cutthroat Trout (O. clarki clarki); and summer and hybrid Steelhead Trout (O. mykiss). Significant decreases were detected for summer and hybrid Steelhead Trout. The proportion of wild fall Chinook has also significantly decreased concurrently with increases in hatchery returns. In comparison, returns of most Chinook and coho runs to the hatcheries, and fall Chinook strays to wild spawning areas from Iron Gate Hatchery have significantly increased since the 1970s. Increases were also detected for wild late-fall Chinook and spring Chinook adults. However, both of these were significantly correlated with Chinook Salmon returns to Trinity River Hatchery, suggesting augmentation by hatchery strays. Changes in abundances appeared related to changing ocean habitat conditions and hatchery practices. Our results suggest that anadromous salmonid populations in the Klamath River basin are becoming increasingly dependent on hatchery propagation, a pattern that can threaten population persistence.     

Sub-alpine amphibian distributions related to species palatability to non-native salmonids in the Klamath mountains of northern California

The goal of this study was to examine how introduced trout influence the distributions and abundances of a sub‐alpine amphibian assemblage whose members display a variety of different life‐history and defence strategies. Our study was conducted in the sub‐alpine lentic habitats of three wilderness areas that form the core of the Klamath‐Siskiyou Bioregion of northern California, a biodiversity ‘hotspot’ that supports the highest diversity of sub‐alpine, lentic‐breeding amphibians in the western USA. These wilderness areas contain no native fishes, but all have been populated with non‐native trout for recreational fishing. Five of the eight amphibian species that occur in this region were sufficiently common to use in our study; these included one that breeds in both temporary and permanent waters and is palatable to fish (Pacific treefrog, Pseudacris regilla), two that breed primarily in permanent waters and are unpalatable to fish (western toad, Bufo boreas, and rough‐skinned newt, Taricha granulosa), and two that breed primarily in permanent waters and are palatable to fish (Cascades frog, Rana cascadae, and long‐toed salamander, Ambystoma macrodactylum). Based on life histories and predator defence strategies (i.e. palatable or not), we predicted that the three palatable species would likely be negatively correlated with introduced trout, but with P. regilla less impacted because of its use of both temporary and permanent waters. We predicted that B. boreas and T. granulosa would not be significantly correlated with introduced trout due to the lack of any predator/prey interactions between them. We surveyed 728 pond, lake, or wet meadow sites during the summers of 1999–2002, using timed gill‐net sets to measure trout occurrence and relative density, and visual encounter surveys to determine amphibian presence and abundance. We used semiparametric logistic regression models to quantify the effect of trout presence/absence and density on the probability of finding amphibian species in a water body while accounting for variation within and among the various lentic habitats sampled. The distributions of P. regilla, A. macrodactylum and R. cascadae were strongly negatively correlated with trout presence across all three wilderness areas. Ambystoma macrodactylum was 44 times more likely to be found in lakes without fish than in lakes with fish. Rana cascadae and P. regilla were 3.7 and 3.0 times more likely, respectively, to be found in fishless than fish‐containing waters. In contrast, the two unpalatable species were either uncorrelated (T. granulosa) or positively correlated (B. boreas) with fish presence. We found that the relative density of fish (catch per unit effort) was negatively correlated with the combined abundances of the three palatable amphibians, and also with both the length and the condition of the fish themselves. Our results are consistent with a compelling body of evidence that introduced fishes greatly alter the aquatic community structure of mountain lakes, ponds, and wet meadows.     

Nutrient limitation of Microcystis aeruginosa in northern California Klamath River reservoirs

Nutrient limitations were investigated in Copco and Iron Gate Reservoirs, on the Klamath River in California, where blooms of the toxin-producing cyanobacterium Microcystis aeruginosa were first reported in 2005. Nutrient enrichment experiments conducted in situ in June and August, 2007 and 2008, determined responses in phytoplankton biomass, Microcystis abundance and microcystin concentration to additions of phosphorus and different forms of nitrogen (NH₄⁺, NO₃, and urea). Microcystis abundance was determined using quantitative PCR targeting the phycocyanin intergenic spacer cpcBA.Total phytoplankton biomass increased with additions of N both before and during Microcystis blooms, with no primary effects from P, suggesting overall N limitation for phytoplankton growth during the summer season. NH₄⁺ generally produced the greatest response in phytoplankton growth, while Microcystis abundance increased in response to all forms of N. Microcystis doubling time in the in situ experiments was 1.24–1.39 days when N was not limiting growth. The results from this study suggest availability of N during the summer is a key growth-limiting factor for the initiation and maintenance of toxic Microcystis blooms in Copco and Iron Gate Reservoirs in the Klamath River.     

Geographic variation in vascular mortality in eurasia: Relationship between mortality variables and risk factors

This study was based on a sample of 68 populations from 42 countries of Eurasia. Most of the data were from the MONICA Project, while additional information was from the abundant literature. Mortality rates (from cardiovascular, cerebrovascular and ischaemic heart diseases, and death rate from all causes) and related to them potential risk factors (systolic and diastolic blood pressure, total serum cholesterol and body mass index) were described for men and women, separately for 5 age cohorts, ranging between 25 and 64 years. The results showed that, in contrast to the studies carried out on theintrapopulational level, in our work risk factors did not demonstrate any strong association with mortality rate variables. Moreover, it was found that the vascular mortality on theinterpopulational level explains between 57% and 71% in total mortality and could serve as a quite fair indicator of the populational well-being in general. Although mortality rate from vascular diseases in women was much lower than in man of equal ages, geographic variation of the above mentioned death rates has showed a very similar pattern in the two sexes: the death rate is high in women when it is high in men.     

Assessment of genetic variation between reproductive ecotypes of Klamath River steelhead reveals differentiation associated with different run-timings

Seven microsatellite DNA loci were optimized to assess genetic differentiation in coastal steelhead (Oncorhynchus mykiss irideus) sampling groups from the lower Klamath River (California, USA). Genetic relationships among three winter‐run and two summer‐run groups were investigated. The different groups displayed high levels of allelic variation. Pairwise F_ST comparisons and Nei's genetic distance supported low, yet significant, genetic differentiation between summer and winter run‐timings similar to other studies of temporal variation in salmonids. Analysis of molecular variance showed that most of the genetic variation was at the individual level (97.9%), although significant genetic variation existed between timing of runs (2.59%). Additionally, at least one locus in each group was out of Hardy–Weinberg equilibrium due to a deficiency in heterozygotes, and significant F_IS values were observed in three temporal collections. Together, these results suggest stock admixture, caused by multiple populations of origin in each sampling group, better known as the Wahlund Effect. These observations are preliminary evidence for isolation by time between Klamath River steelhead runs during distinct periods.     

Nutrient limitation of Microcystis aeruginosa in northern California Klamath River reservoirs

Nutrient limitations were investigated in Copco and Iron Gate Reservoirs, on the Klamath River in California, where blooms of the toxin-producing cyanobacterium Microcystis aeruginosa were first reported in 2005. Nutrient enrichment experiments conducted in situ in June and August, 2007 and 2008, determined responses in phytoplankton biomass, Microcystis abundance and microcystin concentration to additions of phosphorus and different forms of nitrogen (NH₄⁺, NO₃, and urea). Microcystis abundance was determined using quantitative PCR targeting the phycocyanin intergenic spacer cpcBA.Total phytoplankton biomass increased with additions of N both before and during Microcystis blooms, with no primary effects from P, suggesting overall N limitation for phytoplankton growth during the summer season. NH₄⁺ generally produced the greatest response in phytoplankton growth, while Microcystis abundance increased in response to all forms of N. Microcystis doubling time in the in situ experiments was 1.24–1.39 days when N was not limiting growth. The results from this study suggest availability of N during the summer is a key growth-limiting factor for the initiation and maintenance of toxic Microcystis blooms in Copco and Iron Gate Reservoirs in the Klamath River.     

Relationship between prostate-specific antigen levels and ambient temperature

We examined the association between prostate-specific antigen (PSA) and daily mean ambient temperature on the day of the test in healthy men who had three annual checkups. We investigated 9,694 men who visited a hospital for routine health checkups in 2007, 2008, and 2009. Although the means and medians of ambient temperature for the three years were similar, the mode in 2008 (15.8 °C) was very different from those in 2007 and 2009 (22.4 °C and 23.2 °C). After controlling for age, body mass index, and hematocrit, a multiple regression analysis revealed a U-shaped relationship between ambient temperature and PSA in 2007 and 2009 (P?<?0.001 and P?=?0.004, respectively), but not in 2008 (P?=?0.779). In 2007, PSA was 13.5 % higher at 5 °C and 10.0 % higher at 30 °C than that at 18.4 °C (nadir). In 2009, PSA was 7.3 % higher at 5 °C and 6.8 % at 30 °C compared with the level at 17.7 °C (nadir). In logistic regression analysis, a U-shaped relationship was found for the prevalence of a higher PSA (> 2.5 ng/mL) by ambient temperature, with the lowest likelihood of having a high PSA at 17.8 °C in 2007 (P?=?0.038) and 15.5 °C in 2009 (P?=?0.033). When tested at 30 °C, there was a 57 % excess risk of having a high PSA in 2007 and a 61 % higher risk in 2009 compared with those at each nadir temperature. We found a U-shaped relationship between PSA and ambient temperature with the lowest level of PSA at 15–20 °C.     

The relationship between smolt density and fry density in salmonids

If the Ricker stock–recruitment model describes the relationship between egg density and survivor density at different stages of the life cycle, then the relationship between smolt density and fry density is not simple. Using data from a long-term study (1966–1990) of a sea trout population Salmo trutta , the relationship between density of potential smolts and fry densities in late M ay/early June or late August/early September is shown to be a reflexed curve with zero origin, so that there are two smolt densities for each fry density. A linear relationship is obtained only when the exponential parameter in the Ricker model is constant for the different life Stages.     

Effect of acclimation temperature on routine metabolic rate in triploid salmonids

The objective of this research was to determine whether triploid fish differ from diploids in their routine metabolic rates across a range of acclimation temperatures. Sibling diploids and triploids were acclimated to 12, 15 and 18 degrees C (Atlantic salmon; Salmo salar) and to 9, 12 and 15 degrees C (brook charr; Salvelinus fontinalis) prior to experimentation. Routine metabolic rates were then determined three times over a two-month period. Triploids of both species had higher metabolic rates than diploids at lower temperatures, and lower metabolic rates than diploids at higher temperatures, demonstrating that triploids have different (i.e., lower) thermal optima than diploids. This likely explains prior observations of high mortality of triploids at chronically elevated, but sub-lethal, rearing temperatures for sibling diploids.