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 annelids infect salmonid fishes and develop into myxospores which then infect annelids (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, 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.

 

Map of monitoring index sites on the Klamath River

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
Barth Monitoring fish cages Barth Monitoring trout fry

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).

DATA UPDATES: Due to COVID-restrictions, the usual April fish exposure at Beaver Creek (KBC) and Seiad Valley (KSV) did not occur. However, two sentinel fish exposures were conducted to monitor the surface flushing flow event and Chinook salmon were exposed at KBC and KI5 April 18-21 & May 2-5 (pre-flow & post-flow exposure; held at 13C). The highest Fall Chinook mortality to date is 15% in the Beaver Creek group (held at 13C).

The May fish exposure for 2020 occurred May 18-21 at all index sites. The highest Fall Chinook mortality to date is 12.5% in the Beaver Creek group (held at 16C).

The June sentinel fish exposure occurred June 10 -13 at all index sites. The highest Fall Chinook mortality to date is 2.5% in each of the I5 Bridge and Beaver Creek groups (held at 18C).

The final exposure will occur in September at Beaver Creek (KBC) and Seiad Valley (KSV).

developmental stages of Ceratonova shasta in an intestinal smear from a fall Chinook exposed at KSV in April 2019
Developmental stages of Ceratonova shasta in an intestinal smear from a fall Chinook exposed at KSV in April 2019. Note the pseudopodia extending from the pansporocyst and the cells within cells. Two myxospores will develop within this 'disporoblast' stage. Developmental stages of Ceratonova shasta, including an immature myxospore, in a kidney smear from a fall Chinook exposed at KSV in April 2019. The infection was fatal.

 

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.

2020 DATA UPDATES:

OSU has received and processed water samples from index sites KBC and KSV for dates 01/06, 01/13, 02/03, 02/10, 02/17, 02/24, 03/02. Both sites at all dates are negative for C. shasta spores. Samples were also received for site KMN 03/02 (the first sampling date at that site this season). KMN was also negative for C. shasta.

03/09: Samples collected at three sites: KBC, KMN and KSV. No C. shasta DNA was detected at any site.

03/16: Samples were collected at 5 sites and density of C. shasta ranged from undetected to less than 1 spore/L: KI5 <1 spore/L, KBC <1 spore/L, KMN 0 spores/L, KSV 0 spores/L, KOR <1 spore/L.

03/24: Travel restrictions meant that regular ISCO samples were not able to be collected on 3/23 but grab samples were obtained 3/24. Processing these is in progress.

03/30: First sampling date to include all sites: ISCO samples from KBC, KSV, KMN, KI5 and KOR, plus the first samples from near Tully Creek (KTC - manual grab samples). All sites were positive for C. shasta-DNA, most at multiple spores per liter, showing a dramatic rise since the first detections (<1 sp/L) 2 weeks ago. Site-by-site mean spore levels: KI5: 1, KBC: 18, KMN: 35, KSV: 14, KOR: 5,   KTC: 8

04/06: All sites were positive 04/06 for C. shasta-DNA, all at multiple spores per liter, most doubling in quantity since last week (except KTC which remained about 7 sp/L). All samples were collected with an ISCO, except those at KTC. Site-by-site mean spore levels: KI5: 3, KBC: 30, KMN: 55, KSV: 27, KOR: 12, KTC: 7

04/13: All sites now have C. shasta-DNA levels at an equivalent of above 20 spores per liter. Density at all sites more than doubled since last week. Notably, KI5 and KTC had the highest inter-week increases. Site-by-site calculated mean spore densities: KI5: 70, KBC: 95, KMN: 180, KSV: 65, KOR: 35, KTC: 25

04/20: All sites now have C. shasta-DNA densities at an equivalent of above 30 spores per liter. Levels at most sites remained high; site KI5 was the only site to double in spore densitiy since last week. All samples were collected with an ISCO, except those at KTC. Site-by-site calculated mean spore densities: KI5: 139, KBC: 104, KMN: 158, KSV: 54, KOR: 31, KTC: 32

04/27: C. shasta densities at all sites dropped significantly from last week to this week. Levels at all sites except KI5 remained above 10 spores per liter; KI5 had the largest inter-week drop from 139 to 8 spores per liter. Site-by-site calculated mean spore levels 04/27: KI5: 8, KBC: 19, KMN: 37, KSV: 18, KOR: 14, KTC: 25 (regular grab sample, versus ISCO 12).

05/04: C. shasta-DNA densities at all sites remained lower than before the surface flushing flow commenced. At the three upriver sites (KI5, KBC, KMN), parasite densities increased to 30-50 spores per liter compared with last week, while the three downstream sites (KSV, KOR, KTC) remained lower at about 10 spores per liter. Note that site KI5 had the largest inter-week increase, but that some of this gain might be due to measurement variation as it was a manual grab sample this week and reported values are probably higher than would have been obtained with a 24-hr composite automatic sample.

Site-by-site calculated mean spore levels 05/04: KI5: 49 (grab sample), KBC: 31, KMN: 39, KSV: 10, KOR: 8, KTC: 10

05/11: C. shasta-DNA densities at all sites are much lower this week, with all except KI5 below an equivalent of 10 spores per liter.

Site-by-site calculated mean spore densities: KI5: 12, KBC: 9, KMN: 7, KSV: 3, KOR: 2, KTC: <1

05/18: Once again, there was a clear distinction in C. shasta-spore densities between the three uppermost sites (KI5, KBC, KMN) and lowermost sites (KSV, KOR and KTC). Densities remained low downstream this week (1-4 spores per liter), but at upstream sites densities were 2-3 times higher than last week (20-48 spores per liter).

Site-by-site calculated mean spore densities: KI5: 48 (grab), KBC: 20, KMN: 27, KSV: 4, KOR: 2, KTC: 1 (grab)

05/26: At the three upper sites (KI5, KBC, KMN), C. shasta-DNA levels were lower (~half), whereas at the three lower sites (KSV, KOR and KTC) levels were higher (~double).

Site-by-site calculated mean spore densities: KI5: 27 (ISCO), 22 (grab); KBC: 12; KMN: 13; KSV: 7; KOR: 4; KTC: 2

06/01: C. shasta-DNA densities decreased at all sites except Kinsman (KMN) and Tully Creek (KTC) where they remained about the same. Again this week, densities were highest at the upper three sites, upstream of Scott River.

Site-by-site calculated mean spore densities 06/01 (all ISCO-collected): KI5: 5, KBC: 7, KMN: 15, KSV: 2, KOR: 1, KTC: 2

06/08: The highest C. shasta-DNA densities were measured at KBC & KMN, with densities there having increased 2-3 times from the previous week (KBC from 7 to 24 and KMN from 15 to 32 spores per liter). In contrast, levels at KI5 dropped from 5 to <1 spores per liter.

Site-by-site calculated mean spore levels 06/08 (all ISCO-collected): KI5: <1, KBC: 24, KMN: 32, KSV: 3, KOR: 4, KTC: 1

06/15: Densities at the lowermost sites were similar or higher than last week, and remain relatively low compared to densities at the uppermost sites. Densities at KI5 were higher than last week whereas they were lower at KBC.

Site-by-site calculated C. shasta mean spore densities (spores per liter) 06/15 (all ISCO-collected):
   KI5: 24
   KBC: 6
   KMN: 24
   KSV: 3
   KOR: 6
   KTC: 4

Expedited processing of samples is now complete for the season. Data will be available within two weeks of collection.

06/22: Density of waterborne C. shasta varied throughout the basin. Levels at KBC and KOR were similar.

Site-by-site calculated C. shasta mean spore densities (spores per liter) 06/22:
   KI5: 38 (manual grab sample)
   KBC: 11
   KMN: 20 (manual grab sample)
   KSV: 1
   KOR: 10
   KTC: 3

 Density of Ceratonova shasta in water samples collected at Beaver Creek index site on the Klamath River 2020
Density (average spores per liter) of Ceratonova shasta in 24-hour composite water samples collected at the mainstem longterm index site, near Beaver Creek (KBC), in 2020 (blue data points) compared to 2019 (light blue shading), 2018 (yellow shading) and 2017 (gray shading).

 

 Density of Ceratonova shasta in water samples collected at index sites on the Klamath River 2020

Density (average spores per liter) of Ceratonova shasta in 24-hour composite water samples collected at the mainstem index sites in 2020. 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. 

 

Index site KI5 near I5 bridge on the Klamath River on May 11 Index site at Kinsman on the Klamath River on May 11 Index site at Seiad Valley on the Klamath River on May 11  Index site at Orleans on the Klamath River on May 11 

Klamath River Index site KI5
on May 11, 2020

Photos courtesy of Karuk Tribal Biologist Larry Alameda

KMN - Kinsman Trap KSV - Seiad Valley  KOR - Orleans

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. 

2020 DATA UPDATES:

Water samples from 03/30 and 04/06 were assayed also for presence of the specific C. shasta genotype relevant for coho salmon infection (genotype II). All samples tested negative for this genotype, except 1/3 subsamples from site KTC that had <1 spore per liter (trace) levels of genotype II on both 03/30 and 04/06.

04/13: Assay for C. shasta "coho" genotype II showed KMN and KSV had trace amounts (<1 sp/L average), all other sites were negative. Full genotyping by Sanger sequencing revealed that genotype I comprised the remainder of a sample.

04/20: Assay for C. shasta "coho" genotype II showed an increase in detections of this genotype, but all levels remain low:

KI5: trace amount (<1 sp/L average)
KBC: trace amount (<1 sp/L average)
KMN: trace amount (<1 sp/L average)
KSV: ~1 sp/L
KOR: ~1 sp/L
KTC: ~2 sp/L

04/27 Assay for C. shasta "coho" genotype II showed a decrease in detections of this genotype, with some sites below the level of detection.

KI5: 0
KBC: trace amount (<1 sp/L average)
KMN: trace amount (<1 sp/L average)
KSV: 0
KOR: ~1 sp/L
KTC: 0

05/04The assay for C. shasta "coho" genotype II showed an increase in detections from last week with the genotype present at all sites. Highest proportions were at the up-river sites (which correlates with the higher total C. shasta densities at these three sites).

KI5: 2
KBC: 1
KMN: 2
KSV: <1
KOR: 1
KTC: <1

05/11: The assay for C. shasta "coho" genotype II detected only low levels of the genotype at all sites, except KOR where none was detected:
KI5: <1
KBC: <1
KMN: 1
KSV: 1
KOR: none detected
KTC: <1

05/18: C. shasta "coho" genotype II was detected at all sites this week, with increased levels of this genotype in proportion with the increases in total spores at the upper river sites:

KI5: 3
KBC: 2
KMN: 1
KSV: 1
KOR: <1
KTC: <1

05/26: C. shasta "coho" genotype II was detected at all sites this week, with highest level of 6 spores/L at KI5:

KI5: 6 (ISCO), 2 (grab)
KBC: 1
KMN: 2
KSV: 2
KOR: 1
KTC: <1

06/01: C. shasta "coho" genotype II was detected at all sites except KOR, but levels have decreased compared with last week (except KMN, which remained the same, as it was for total C. shasta).

KI5: 1
KBC: <1
KMN: 3
KSV: <1
KOR: 0
KTC: <1

06/08: C. shasta-coho genotype II was detected at all sites except KI5.
KI5: 0
KBC: 3 (about 12% of total)
KMN: 3 (about 10% of total)
KSV: <1
KOR: <1
KTC: <1

06/15: C. shasta "coho" genotype II was detected at all sites except KTC.

KI5: 3 (about 8%)
KBC: <1
KMN: 2 (about 8%)
KSV: <1
KOR: <1
KTC: 0

06/22:C. shasta "coho" genotype II was detected at all sites.

KI5: 5 (about 12%)
KBC: 2 (about 10%)
KMN: 1 (about 5%)
KSV: <1
KOR: 1 (about 10%)
KTC: <1

 Annelid Sampling

Polychaete sampling

 

Polychaete sampling

Manayunkia occidentalis tubes from the image on right A high density assemblage sample at TOH

To monitor abundance and prevalence of infection in the invertebrate host of C. shasta, Manayunkia occidentalis, annelid 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: 

Annelid sampling at our Boyle Bypass and Keno Eddy index sites occurred in February 2020. Sampling at the other sites downstream occurred mid-March.

 

Annelid sampling can be challenging during high flows.
OSU diver Dr. Julie Alexander navigates high water conditions to get to sample sites near Seiad Valley, CA, in April 2019.

 HSU diver Dr. Eve Robinson prepares to take substrate data during annelid model project, July 2019

 

 

 

 

 

 

 OSU diver, Dr. Julie Alexander, prepares to collect annelids
from the benthos during polychaete model project, July 2019
 Water quality was poor in July 2019; algae limited visibility

ANNUAL REPORTS

Annual reports for Bureau of Reclamation funded studies for 2008 onwards are available. Please contact Sascha Hallett (hallettsAToregonstate.edu).

 

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.