OREGON STATE UNIVERSITY
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Ceratonova shasta is a freshwater, myxozoan parasite that is native to the Pacific North West of North America. It causes enteronecrosis in juvenile salmonids and is associated with population-level impacts in the Klamath River. Transmission occurs through waterborne stages: actinospores released from annelids infect salmonid fishes and in the fish's intestine develop into myxospores which then infect annelids (see the life cycle on the right). The parasite proliferates in each host.
In response to the high prevalence and severity of C. shasta-infection in Klamath salmonids, we developed a parasite monitoring program to track the spatial and temporal abundance of C. shasta. The three main approaches are based on the parasite's life cycle and include sentinel fish exposures, annelid host sampling and molecular quantification of parasite DNA in water samples. These are described in more detail below. Monitoring occurs at established index sites which are shown on the following map.
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Klamath River Index Sites with site abbreviations and river kilometers (Rkm). Iron Gate Dam (Rkm 306) blocks anadromous salmonid migration. |
Sentinel Fish Exposures
| Sentinel fish cages in the Klamath River |
Susceptible out- of-basin rainbow trout |
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In sentinel fish exposures, fish highly susceptible to the parasite (out-of-basin rainbow trout) are placed in cages alongside fish of interest such as in-basin Chinook and coho salmon at index sites along the river for a three day exposure. All fishes are transported to OSU's John L Fryer Aquatic Animal Health Lab and monitored for infection (~ 60 days). Severity of infection (percent morbidity and mean days to morbidity) are recorded through visual observations and molecular assay (PCR).
2022 DATA UPDATES:
An April sentinel fish exposure occurred 04/18 - 04/21 at KBC and KSV with IGH Fall Chinook and out of basin rainbow trout.
A second exposure is scheduled to occur in May.
A third sentinel fish exposure is scheduled to occur in June.
A September exposure is also planned.
Water Samples
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Filtering a water sample |
Folding the filter paper |
To detect and quantify waterborne stages of C. shasta, river water samples are collected at five mainstem index sites; once a week all year round at two sites (KBC and KSV) and once per week from April through October at three other sites. Solar-powered automatic samplers (ISCOs) collect 1L water every 2 hours for 24 hours, from which 4 1L samples are manually taken (see photos below). Each 1L sample is filtered through a nitrocellulose membrane using a vacuum pump, and any captured DNA in 3 of the replicate samples is extracted using a kit (see images on right). A quantitative PCR (qPCR) specific for C. shasta is used to detect and quantify any parasite DNA present. Cq values generated by the qPCR are converted to numbers of parasite spores per liter of water using reference samples with known quantities of spores. The Karuk and Yurok tribal biologists are integral to the collection and filtration of the water samples. Water samples are also taken in conjunction with the sentinel fish exposures; manual 'grab' samples are collected on the first and last day of the exposure. Data are presented as the average spores per liter of three replicate 1 liter water samples collected at each site and time.
2022 DATA UPDATES:
During Winter, sampling occurs only at two index sites, KBC and KSV. Spatial sampling will increase to six sites in Spring.
This was the first week of the 2022 season when all sites were sampled. Samples were taken either by ISCO automatic sampler "i" and are given as the average density of C. shasta spores per liter of river water (from 3x 1-L subsamples of a 24-hour composite sample, Sunday-Monday March 27-28), or taken manually "g" (3 x 1-L samples taken Monday March 28).
C. shasta DNA was detected at 5 of the 6 index sites, with density increasing downstream to up to an average of 5 spores per liter at the lowermost site, KTC. Full sequencing (all genotypes via Sanger sequencing) of subsamples of the lowermost sites is underway and genotype II analysis will commence this week.
KI5 0 i
KBC <1 i
KMN 1 i
KSV 2 i
KOR 4 i
KTC 5 g
4/04/2022
This was the second week of the 2022 season when all sites were sampled. Due to a shipping misalignment, KTC data will be delayed to next week. Samples were taken either by ISCO automatic sampler "i" and are given as the average density of total C. shasta spores per liter of river water (from 3x 1-L subsamples of a 24-hour composite sample, Sunday-Monday April 3-4), or taken manually "g" (3 x 1-L samples taken Monday April 4).
This week, C. shasta DNA was detected at all sites measured. Density at all sites increased over the past week – it more than doubled at all sites. Densities ranged from 3-20 spores per liter. The highest density was at the most downstream sites, with more than 10 spores per liter measured at both KSV and KOR (but note that KSV were grab samples).
KI5 3 i
KBC 7 i
KMN 6 i
KSV 20 g
KOR 12 i
KTC 30g
A second qPCR assay was run on the samples to determine the density of C. shasta genotype II (relevant to coho salmon). All samples tested negative for genotype II.
04/11/2022
This was the third week of the 2022 season when all sites were sampled.
Samples were taken either by ISCO automatic sampler "i" and are given as the average density of total C. shasta spores per liter of river water (from 3x 1-L subsamples of a 24-hour composite sample, Sunday-Monday April 10-11), or taken manually "g" (3 x 1-L samples taken Monday April 11). This week's data also include last week's KTC samples that were not processed earlier due to a shipping misalignment.
Spore density was at or above 10 spores per liter at all six index sites. Similar to last week, spore denstiues were lowest at the upstream sites and highest at the downstream sites. Last week's KTC samples had the highest spore density of the year so far @ 30sp/L, decreasing to 23 this week (highlighted). Densities at the uppermost three sites and KOR were 2-3 times higher this week. Densities at KSV were similar bewteen the two weeks
KI5 10 i
KBC 15 i
KMN 19 i
KSV 22 g
KOR 23 i
KTC 23 g
04/18/2022
This was the fourth week of the 2022 season when all sites were sampled. This week's sampling was during the prescribed surface flushing flow event.
Only two sites were sampled by ISCO automatic sampler "i" and are given as the average density of total C. shasta spores per liter of river water (from 3x 1-L subsamples of a 24-hour composite sample, Sunday-Monday April 17-18), the rest were taken manually "g" (3 x 1-L samples taken Monday April 18).
Densities were lower than the previous week, except at the lowermost site KTC, and remained above 10 spores per liter at 5 of the 6 sites (exception KI5). Again, densities were lowest at the uppermost site KI5 with 4 spores per liter measured then increased downstream to the lowermost site KTC with 26 spores per liter detected.
Coho-relevant Cs genotype data in spores per liter are also given: levels remain <1 sp per liter or not detectable (n.d.).
KI5 4 g n.d.
KBC 11 i n.d.
KMN 12 i n.d.
KSV 12 g <<1
KOR 17 g <1
KTC 26 g <1
04/25/2022
This was the fifth week of the 2022 season when all sites were sampled. This week's sampling was the first after the prescribed surface flushing flow event.
Three sites were sampled by ISCO automatic sampler "i" and are given as the average density of total C. shasta spores per liter of river water (from 3x 1-L subsamples of a 24-hour composite sample, Sunday-Monday April 24-25), the rest were taken manually "g" (3 x 1-L samples taken Monday April 25). Site KTC had both automatic and manual samples taken.
Spore densities were above 10 spores per liter at 5 of the 6 sites sampled this week (all except KTC). Density increased at the three uppermost sites and decreased at the lowermost sites, compared to the previous week. The uppermost site increased three fold whereas the lowermost site decreased three fold. Density decreased as sampling progressed downstream, with the highest densities measured at KI5, KBC & KMN (14-15 sp/L) and the lowest at KTC (7 sp/L).
Coho-relevant Cs genotype data in spores per liter are also given: for the first time this year this genotype was detected at all sites, however the levels at the upmost three sites are only just detectable. The genotype was detected at lower river sites at levels up to 1 spore per liter (a maximum of about 10% of the total C. shasta spore density, at KSV).
KI5 15 g <<1
KBC 14 i <<1
KMN 15 i <<1
KSV 11 g 1
KOR 11 g <1
KTC 7 g <1
KTC 7 i <<1
05/02/2022
This was the seventh week of the 2022 season when all sites were sampled.
Almost all sites were sampled by ISCO automatic sampler "i" and are given as the average density of total C. shasta spores per liter of river water (from 3x 1-L subsamples of a 24-hour composite sample, Sunday-Monday May 8-9), site KTC (like usual) was taken manually "g" (3 x 1-L samples taken Monday May 9).
The density of C. shasta continued to decrease over the past week with an average of 1 or fewer spores per liter measured throughout the lower basin. All sites have returned to an early spring pattern of very low levels of C. shasta. Interestingly, a similar decrease in spore levels happened in almost the same week in 2020 and 2021.
KI5 1 i
KBC <1 i
KMN <1 i
KSV <1 i
KOR 1 i
KTC <1 g
05/16/2022
Four of the six index sites were sampled by ISCO automatic sampler "i" and are given as the average density of total C. shasta spores per liter of river water (from 3x 1-L subsamples of a 24-hour composite sample, Sunday-Monday May 15-16), sites KI5 and KTC (like usual) were taken manually "g" (3 x 1-L samples taken Monday May 16).
The density of C. shasta remained low at all 6 sites, although more C. shasta was measured at the three lower-river sites than the three upper river sites (1-2sp/L versus <1 sp/L).
Coho-relevant C. shasta genotype data will be forthcoming, but will not exceed that of total spore density (i.e. <2 sp/L).
KI5 <1 g
KBC <1 i
KMN <1 i
KSV 1 i
KOR 2 i
KTC 1 g
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| Density (average spores per liter) of Ceratonova shasta in 24-hour composite water samples collected at the mainstem long term index site, near Beaver Creek (KBC), in 2022 (blue data points) compared to 2015 (gray shading), 2020 (pink shading), and 2021 (green shading). |
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Density (average spores per liter) of Ceratonova shasta in 24-hour composite water samples collected at the mainstem index sites in 2022. Note that KMN is sampled only during salmonid outmigration, KBC and KSV year round and remaining sites April through October. KBC = near Beaver Creek, KSV = Seiad Valley, KI5 = near I5 bridge, KTC = Tully Creek, KMN = Kinsman Fish Trap, KOR = Orleans. The line denotes 10 spores per liter which corresponds with 40% mortality threhold in Chinook salmon. |
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Density (average spores per liter) of Ceratonova shasta in 24-hour composite water samples collected at the mainstem index sites in 2021. Note that KMN is sampled only during salmonid outmigration, KBC and KSV year round and remaining sites April through October. KBC = near Beaver Creek, KSV = Seiad Valley, KI5 = near I5 bridge, KTC = Tully Creek, KMN = Kinsman Fish Trap, KOR = Orleans. The line denotes 10 spores per liter which corresponds with 40% mortality threhold in Chinook salmon. |
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Manual water sampling by Karuk Tribal Biologist Larry Alameda at index site K-I5 |
Solar powered automatic sampler at the Kinsman index site. An ISCO is used at all index sites but manual sampling provides a backup. | Collection of 4 1L technical replicate water samples from the larger 12L composite collected over 24 hours at KSV |
Manual water sampling at Orleans index site. Photos courtesy of Karuk Tribal Biologist Larry Alameda |
Genotyping: There are multiple genetic types or genotypes of C. shasta simultaneously present in the Klamath River. These differentially disease the various salmonid species. For example, type I causes mortality in Chinook salmon whereas type II can be fatal for coho salmon. Type 0 is found in sympatric Oncorhynchus mykiss (steelhead and redband rainbow trout).
Therefore, we also genotype each water sample using a qPCR that amplifies the variable ITS1 region and then we sequence that amplicon. From the sequencing chromatogram, we can determine the proportion of each genotype present in a sample. We use the total spore density to then determine the number of spores of each genotype in a sample.
2022 DATA UPDATES
04/04/2022
Ceratonova shasta DNA was detected at all sites, however, genotype II was detected only at the lowermost site, KTC with a density of <1 spore per liter.
04/11/2022
Low densities of genotype II was detected at the three lowermost sites (densities in spores per liter):
KI5 0
KBC 0
KMN 0
KSV <1
KOR <1
KTC <1
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| Density (average spores per liter) of Ceratonova shasta genotype II in 24-hour composite water samples collected at the mainstem index sites in 2021. Note that KMN is sampled only during salmonid outmigration, KBC and KSV year round and remaining sites April through October. KBC = near Beaver Creek, KSV = Seiad Valley, KI5 = near I5 bridge, KTC = Tully Creek, KMN = Kinsman Fish Trap, KOR = Orleans. The line denotes 5 spores per liter which corresponds with a 40% mortality threhold in coho salmon. |
Annelid Sampling
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| Manayunkia occidentalis tubes from the image on right | A high density assemblage sample at TOH |
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Diver collecting a benthic sample from boulder substrate at the TOH reach. We use a modified Hess sampler fitted with 80um Nytex mesh netting and a 500mL Nalgene collection bottle. Samples are preserved in ethanol until processing. |
Benthic samples are fractioned prior to sorting.
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Dr. Alexander photographing annelids on riprap substrate at the KBC monitoring site. |
Annelid sampling at the KI5 reach. Foreground diver measures depth and velocity, while background diver measures substrate grain sizes. |
To monitor abundance and prevalence of C. shasta infection in the invertebrate annelid host, Manayunkia occidentalis, benthic samples are collected annually at seven sites in the Klamath River. Sites span a discharge gradient; 2 are located in the upper basin downstream from Keno Dam, 3 are located in the middle basin downstream from Iron Gate Dam, and 2 are located in the lower basin downstream from the Scott River. Samples are routinely collected in fall, winter, spring, and summer, and are scheduled to occur more frequently if flooding or pulse flow events are planned. Samples are processed to determine density, simple demographics, and prevalence of C. shasta infection.
2021 DATA UPDATES:
Annelid sampling occurred in March, April, May, July, and October. In the middle river basin (the infectious zone) in March, the prevalence of C. shasta infection ranged from 3.25-6.8%. This was concerning because densities of M. occidentalis (annelid hosts) were above average. Benthic samples collected from sites located in the infectious zone in spring (April, May), summer and fall also returned above averages densities of M. occidentalis. Infection prevalence data are in progress.
The next sample collection is planned for March 2022.
ANNUAL REPORTS
Annual reports for Bureau of Reclamation funded studies for 2015 onwards are available. Please contact Sascha Hallett (hallettsAToregonstate.edu). Annual reports are submitted June 1 the year after the research year.
ANNUAL KLAMATH RIVER FISH HEALTH WORKSHOP
The latest workshop was held virtually via Zoom on Tuesday March 16th, 2021.
Graphical abstracts from the workshop.
The next workshop is being planned for summer 2022.
Redescription of the invertebrate host:
For a lyrical rendition with more information, enjoy: The Ballad of Ceratonova shasta
'Tully Creek' Longitudinal Water Sampling
Parasite levels at our lowermost index site, Tully Creek (at ~60 Rkm), have increased over the past several years to surpass those detected at previously high sites, such as Beaver Creek (KBC). To investigate the extent of the high parasite densities at the Tully Creek site, we sampled water downstrea m (beginning at the lowermost road-accessible location, ~38.4Rkm) and upstream of the index site at Orleans (~90Rkm). Link to report.
Data shared here are preliminary and subject to modification.
Page photo credits: S Atkinson, S Hallett & J Alexander
Monitoring Studies are Primarily Supported by the Bureau of Reclamation.
