Progress 06/01/07 to 09/30/08
Outputs
OUTPUTS: To determine if the observed differences in mortality of rainbow trout and Chinook salmon in the upper Klamath River (UKR) is a result of genetic differences in the parasite Ceratomyxa shasta, we collected samples of infected hosts from the upper and lower Klamath River (LKR) basin as well as from locations where the parasite is endemic throughout the Pacific Northwest. Forty five isolates were obtained, and a PCR primer pair was designed to amplify the most variable region of the parasite's small subunit ribosomal RNA gene (ssrRNA). This region of the gene has been successful for identifying differences in other myxozoan species. Sequences were obtained from 37 of the isolates, including 4 UKR, 10 LKR, 3 Columbia River basin and 1 coastal Oregon river, and spanned 4 fish species and the invertebrate polychaete host. We also analyzed the parasite's Internal Transcribed Spacer 1 region (ITS1) which is regarded as more highly variable than the ssrRNA. For this analysis, 177 sequences were obtained from a wider range of samples which included Chinook salmon, hatchery rainbow trout, infected polychaetes and water samples from both the UKR and LKR as well as from wild rainbow trout, steelhead and coho salmon from the LKR. The results of these analyses were presented to the Oregon Department of Fish and Wildlife Restoration Enhancement Board, resulting in additional funding to test the study hypothesis in vivo. This study also resulted in expanded funding through the Bureau of Reclamation to further develop the parasite-specific quantitative PCR assay to have the capability of recognizing different genetic isolates of the parasite. In addition, preliminary results of the study were presented at the annual Klamath River Fish Health Workshop and will be shared at the upcoming annual meeting. This meeting is attended by other researchers working on the Klamath River, resource managers, fishermen and members of the public. This research is a major chapter of a graduate student thesis and will be published upon completion. PARTICIPANTS: Jerri Bartholomew, Principal investigator, was responsible for project design and coordination. Collaborators: Research samples were supplied by collaborators from the US Fish and Wildlife Service, California Department of Fish and Game, Oregon Department of Fish and Wildlife and the Department of Fisheries and Oceans, Canada. Training opportunities for students: Two PhD candidates worked on this project. One designed and supervised the sequence analysis as part of his thesis research and a second was trained in sequence analysis on this project as part of his rotation experience in the laboratory. In addition, an undergraduate student was trained in sequence analysis and research design while working as an undergraduate researcher in the laboratory. TARGET AUDIENCES: The target audience for this study includes agency and tribal resource managers and researchers. These include the US Fish and Wildlife Service, National Marine Fisheries Service, Bureau of Reclamation, Oregon Department of Fish and Wildlife, California Department of Fish and Game, and the Karuk and Yurok tribes. In addition, commercial fishermen have been informed of project results as part of our efforts to provide information on factors that affect health of the salmon fishery. Efforts have included presentation of preliminary results at the annual Klamath River Fish Health Workshop. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The ssrRNA genes from 37 parasite isolates throughout the region were >99.9% similar. This result was in contrast to the high variability seen in Parvicapsula minibicornis, a similar myxozoan that shares both vertebrate and invertebrates hosts with C. shasta. This lead us to examine the more variable ITS1 region which is next to the ssrRNA. Sequence analysis of 177 isolates from the UKR and LKR showed 94% sequence similarity. The sequences could be grouped into at least 8 genotypes: Oa, Oc, Oe, Ia, Ic, IIa, IIIa, IIIb. These were defined primarily on the number of ATC repeats (0, 1, 2 or 3) at a specific locus, in addition to a small number of insertions and substitutions across the other 330nt of the ITS1. Several strong correlations were apparent between genotype, fish host and geographic location. Genotype O was only found in native rainbow trout and steelhead in both the UKR and LKR, and did not cause mortalities. Genotype I was only found in Chinook, and only in the LKR. It appeared to cause mortalities in the Chinook. Genotype II was found in both UKR and LKR, in Chinook, coho and hatchery rainbow trout. Genotype II appeared to cause significant mortalities in the hatchery rainbow trout, but not in Chinook. It appeared to cause mortalities in coho at high doses. Genotype III was only detected rarely, but was found in Chinook, coho and hatchery rainbow trout in both the UKR and LKR. The absence of genotype I from the UKR is likely a result of the exclusion of anadromous salmonids from that portion of the river, and may explain why Chinook held in the UKR did not develop fatal disease, whereas hatchery rainbow trout did. These findings have significantly modified our understanding of this parasite and addressed research gaps identified by Klamath Basin co-managers. Subsequent research will test the species-specificity of the different genotypes in challenge experiments using Klamath River Chinook and coho salmon and steelhead, and further development of our molecular assays will enable us to monitor parasite populations in the upper Klamath River basin as salmonids are reintroduced, to determine if foreign parasite genotypes are also introduced.
Publications
- No publications reported this period
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: To test the hypothesis that Chinook salmon do not become infected by the parasite Ceratomyxa shasta in the upper Klamath River (UKR) because of genetic differences in the parasite we collected isolates of C. shasta from the upper and lower Klamath River (LKR) basin as well as from locations where the parasite is endemic throughout the Pacific Northwest. Forty five isolates were obtained, and variable regions of the 18S ribosomal DNA gene (rDNA) have been sequenced from 22 of these isolates, including 4 UKR, 10 LKR, 3 Columbia River basin and 1 coastal Oregon river isolate. These isolates also spanned 4 fish hosts and the invertebrate polychaete host. This region of the gene has been successful for identifying differences in other myxozoan species, but in C. shasta the sequence for all of the 22 isolates was identical. We have started to look at another gene, the ribosomal ITS region, which is regarded as more highly variable. Initial results from sequence of 6 isolates (4
LKR, 1 Columbia River basin, 1 Fraser River) show some degree of genetic diversity. Three genotypes were identified: genotype 1 having a 3 base pair (bp) insertion, genotype 2 with no insertion and genotype 3 with a 5 bp difference from type 2. Genotype 1 was not represented in the isolates obtained from Chinook salmon (2), whereas this genotype was present in rainbow trout. This may indicate that we can differentiate species-specific forms of the parasite using this method. This information will be disseminated at the annual Klamath River Fish Health Conference.
PARTICIPANTS: Jerri Bartholomew, principal investigator, coordinated this project. Sequencing was supervised by a doctoral student from the University of Queensland, Australia and performed by undergraduate students as training. Samples were provided by collaborating agencies, including Oregon Department of Fish and Wildlife, California Department of Fish and Game, the US Fish and Wildlife Service, and the Department of Fisheries and Oceans, Canada.
TARGET AUDIENCES: The target audience for this project includes researchers in the field of myxozoan parasites, who will benefit from the information on parasite diversity. It also includes state and federal agencies and tribes who are trying to understand the effects of disease impacts in the Klamath River and how these will change with proposed fish passage efforts.
Impacts
Although results are preliminary, they indicate that it may be possible to identify species-specific parasite strains. This will enable us to monitor parasite populations in the upper Klamath River basin as salmonids are introduced to determine if new parasite genotypes are also introduced.
Publications
- No publications reported this period
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