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 2017 occured: April 20 - 23 at Beaver Creek (KBC) and Seiad Valley (KSV).

The second fish exposure in 2017 occured: May 25 - 28 at sites in the upper and lower basin.

The third fish exposure in 2017 occured: June 24 - 27 at sites in both the upper and lower Klamath River.

The fourth and final fish exposure occured: September 14-17 at Beaver Creek, Seiad Valley and Orleans.

 

DATA UPDATES: summaries of sentinel fish exposures are as follows:  April    May     June   September.

 

 

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 in 3 of the replicate samples 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 'grab' samples are collected on the first and last day of the exposure.

2017 DATA UPDATES: Expedited processing of water samples occurred April through June. Ceratonova shasta-DNA was first detected April 10th at 3 of 4 index sites. Abundance of waterborne C. shasta was relatively low in 2017. The highest levels detected were ~ 5 spores/L at KOR April 24 (genotype I) and 6 spores/L at KBC June 12 (80% I, 10% II, 10% 0). 

Density of waterborne Ceratonova shasta at Klamath River index sites
Density (spores per liter) of Ceratonova shasta in water samples collected at the mainstem longterm index site, near Beaver Creek (KBC), in 2017 (blue data points) compared to 2016 (shaded). The Arcata Fish and Wildlife Office Fish and Aquatic Habitat Conservation Program's predictive tool estimated that 80% of the natural juvenile Chinook Salmon had migrated downstream of the Kinsman trap site on the Klamath River sample week May 7-13; therefore, accounting for the court order's seven additional calendar days, the 80% outmigration 2017 date was May 20 (red line).

 

 

 Density of waterborne Ceratonova shasta at Klamath River index sites

Density (spores per liter) of Ceratonova shasta in water samples collected at the mainstem index sites in 2017. Note that KMN is sampled only during salmonid outmigration, KBC and KSV year round and remaining sites March through October. KBC = near Beaver Creek, KSV = Seiad Valley, KI5 = near I5 bridge, KTC = Tully Creek, KMN = Kinsman Fish Trap, KOR = Orleans. 

 

 

 Comparison of density of C. shasta at Orleans index site in 2016 and 2017

Comparison of density of waterborne Ceratonova shasta at mainstem index site Orleans in 2016 and 2017. Each data point is 1L river water; 3 are tested at each time point.

 

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. 

2017 DATA UPDATES:

Only samples with 1 spore or greater per liter are genotyped. Genotype I was dominant; type II was only detected May 1 at KSV (1 spore/L), June 5 + 12 + 19 + 26 at KBC (1 spore/L).

 

 Polychaete Sampling

many polychaetes in tubes in left photo compared to few worms 5 years later in right hand side image 

diver taking photo of polychaete population on boulder

Comparison of 2012 and 2017 polychaete populations
Scientific diver taking photos at one of the few locations that polychaetes were observed in 2017

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 June 17-19 from all sites except Orleans (flows still too high). Tubes were observed at Keno (many), JC Boyle (very few), and I5 (very few). No tubes were observed at TOH, KBC or KSV. The summer sampling consisted of predictive model data collection and monitoring. We completed surveys in the polychaete predictive modeling reaches in early July which included reaches adjacent the Tree of Heaven Campground, Fisher’s RV Park upstream of the mainstem’s confluence with Beaver Creek, and adjacent the Klamath Community Center (this project is a collaboration with the AFWO). We observed very few polychaetes in 2017. Polychaetes were observed primarily in flow refugia on large, stable substrate (boulders and bedrock). We didn’t observe high densities of tubes as in previous years, the majority of assemblages were sparse. In addition, the tubes we did observe were shorter than in previous years, which may be indicative of a delayed population expansion, likely a result of cool spring water temperatures and flow related disturbance. There were so many particulates in the water this year that visibility was poor and polychaetes were often found under deposited fines (5-15 mm deep).

We also completed monitoring in July and August, which includes sites from the upper river all the way downstream to Orleans, to overlap with our fish disease monitoring locations. Polychaete assemblages did not reach peak densities until August, in contrast to previous years when they reached peak densities by May (2015), June (2014), or even July (2016). Assays to determine prevalence of C. shasta infection are underway. We completed fall monitoring last weekend, and observed few polychaetes at all but the Keno and I5 sites.

 

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

Bureau of Reclamation Report for 2016

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 & J Alexander

Monitoring Studies are Primarily Supported by the Bureau of Reclamation.