CERATONOVA SHASTA MONITORING STUDIES IN THE KLAMATH RIVER

Life cycle of Ceratonova shastaCeratonova 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 polychaete worms infect salmonid fishes and develop into myxospores which then infect polychaetes (see life cycle on 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, polychaete 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.

 

 

Klamath River Index Sites with site abbreviations and river kilometers (Rkm).

 

 

 

 

 

 

 Sentinel Fish Exposures

Barth Monitoring fish cagesSentinel fish cages
in the Klamath River
Barth Monitoring trout frySusceptible
out- of-basin
rainbow trout

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 the lab (AAHL) and monitored for infection (~ 60 days). Prevalence of infection and severity of infection (percent morbidity and mean days to morbidity) are recorded through visual observations and molecular assay (PCR).

The first fish exposure in 2016 occurred April 21 at Beaver Creek (KBC) and Seiad Valley (KSV).

The second fish exposure occurred May 16 at all index sites.

The third sentinel fish exposure occurred June 21 at all index sites.

The fourth and final exposure occurred September 17 at Beaver Creek (KBC), Seiad Valley (KSV) and Orleans (KOR).

DATA UPDATES: For updates on sentinel fish exposures, click the following links as they become available:  April      May     June   September  (last update November 8).

 

 

Water Samples

Barth Monitoring filtering
 Barth Monitoring filter paper

 Filtering a water sample
using a vacuum pump

 Folding the filter paper
with captured material

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. Each 1L sample is filtered through a nitrocellulose membrane using a vacuum pump, and any captured DNA is extracted using a kit. 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 ISCO water samples. Water samples are also taken in conjunction with the sentinel fish exposures; manual samples are collected on the first and last day of the exposure.

DATA UPDATES: The density of C. shasta (average number of spores in 3 x 1L river water samples) in samples collected up to October 24, 2016 at Klamath River monitoring index sites is shown in the graphs below.

Density of Ceratonova shasta in Klamath River water samples in 2016

Density (spores per liter) of Ceratonova shasta in water samples collected at index sites in 2016 with Iron Gate Dam discharge (data points are the average of three 1 liter water samples)

Density of Ceratonova shasta in Klamath River water samples in 2016
Density (spores per liter) of Ceratonova shasta in water samples collected at two mainstem index sites, near Beaver Creek (KBC) and Seiad Valley (KSV), in 2016 with 2014 for comparison (data points are the average of three 1 liter water samples).

 

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 O. 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. 

DATA UPDATES: Genotyping in 2016:

April 4: All sites where any spores were detected (KMN, KSV, KOR) were 100% genotype I (Chinook).

April 11: KBC 100% genotype I.

April 18: KBC 100% genotype I.

April 25: KBC 100% genotype I. At the current total spore level of 13-30 spores/L, we would detect 1 spore/L of type II, if it was present.

May 2: KBC 93% type I, 7% type 0 (about 30 spores/L type I, 3 spore/L type 0); no type II detected.

May 9: Genotype I (Chinook) continues to dominate at KBC, but genotype O (Steelhead) is significantly higher this week at 22-50% of the signal (4-5 spores per liter). Trace amounts (~5%, <1 spore per liter) of genotype II (coho) are also present.

May 16: There is insufficient target DNA for genotyping this week.

May 23: KBC continued to be dominated by genotype I (Chinook) with 5-25% genotype O (steelhead). Trace amounts (<1 spore per liter) of genotype II (coho) detected.

May 31: KBC samples were predominantly type I with 0 spores/L, <1 spore/L and 1 spore/L type II detected in the three replicate water samples.

June 6: All three KBC samples were dominated by genotype I. Any type II or 0 was less than 2% i.e. < 1 spore/L. Two water samples from this week and the previous week from KOR were genotyped and only type I was detected.

June 13: KBC is 90-99% type I, 1-10% type II   (which means 1-5 spores per liter, therefore with an average of 50 spores per liter total, this means type II is just under 5 spores per liter, so we are near the threshold for the first time this year). Because spore levels were highest at KOR (>100 spores per liter total), those samples were also genotyped; KOR was 95% I, 5% II, thus type II was > 5 spores per liter.

June 20: at KBC >90% type 1 detected, <10% type 0; no type II detected.

 

Polychaete Sampling

BARTH polychaete monitoring 

BARTH polychaete monitoring 2

Close up photo showing polychaetes in tubes comprised of mucus and fine sediment grains. Polychaetes are suspension feeders that consume small particles transported by river currents. Ceratonova shasta myxpospores are likely consumed by filter feeding polychaetes. Divers observing polychaetes on boulder substrate in the Klamath River.

To monitor abundance and prevalence of infection in the invertebrate host of C. shasta, polychaete samples are collected annually at seven sites in the Klamath River spanning a discharge gradient. Sites are located in the upper basin downstream from Keno dam, 3 sites are located in the middle basin downstream from Iron Gate Dam, and 2 sites are located in the lower basin downstream from the Scott and Salmon Rivers. Samples are collected once each in fall, winter, spring, and summer, and more frequently if flooding or pulse flow events are scheduled to occur. Samples are processed to determine density, simple demographics, and prevalence of C. shasta infection.

 

 

 

DATA UPDATES: Polychaete samples were collected March 13-15, 2016 prior to the peak of the March 15 discharge event. Samples were again collected April 23-27. Visual detection of polychaetes was more difficult in April (2/7 sites) than in March (6/7 sites), and we noted evidence of sediment (sand and gravel) movement at all of our monitoring sites. Density assays are in progress and will be updated ASAP.

Polychaete sampling is again planned for May and we have plans with the FWS to collect data this summer to update the polychaete predictive model.

 

ANNUAL REPORTS

Bureau of Reclamation Report for 2008

Bureau of Reclamation Report for 2009 

Bureau of Reclamation Report for 2010

Bureau of Reclamation Report for 2011

Bureau of Reclamation Report for 2012

Bureau of Reclamation Report for 2013

Bureau of Reclamation Report for 2014

Bureau of Reclamation Report for 2015

B

 '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

Monitoring Studies are Primarily Supported by the Bureau of Reclamation.