ODFW Fellowship Report

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1 ODFW Fellowship Report Myxozoan Disease Risks in the Willamette and Deschutes Rivers Introduction Salmonid species in both the Willamette and Deschutes Rivers are infected by the myxozoan parasite Ceratonova shasta as outmigrating juveniles and as adults returning to spawn. Ceratonova shasta has a complex life cycle - fish become infected when they encounter the actinospore stages of the parasite which are released into the water from the invertebrate host, a freshwater polychaete worm Manayukia speciosa; myxospore stages then develop in the salmonid host and are released and consumed by the invertebrate host (Figure 1). The purpose of this study was to determine the temporal and spatial dynamics of C. shasta in the Willamette and Deschutes Rivers, describe disease risks for the salmonid species in these rivers, and use polychaete habitat suitability models to identify high risk areas for management. Figure 1. Life cycle of the myxozoan parasite Ceratonova shasta. For the Willamette River, the main concern for parasite infections that result in high mortality of juveniles and pre-spawning adults is when the temperatures are high and the water flows are low. As for the Deschutes River, concern is centered on the Pelton Dam facility and cultured juvenile salmon mortalities and the mortality of returning adult salmonids to the Round Butte and Warm Springs Hatcheries. There is addition concern regarding the effects of parasite distribution and disease severity associated with fish passage over the Round Butte Dam. To obtain information about disease dynamics in each river, three main approaches were used molecular analysis of water samples to quantify waterborne parasite stages (Willamette and Deschutes), sentinel fish exposures to determine disease severity in different fish stocks and river sites (Deschutes), and polychaete prospecting (Willamette). Abundance of Ceratonova shasta in River Water Samples Methods: Information about the temporal and spatial distribution of C. shasta in each river was obtained primarily by establishing monitoring index sites with automated water sampling units (ISCOs). On the Deschutes River, this regular monitoring was supplemented with exploratory grab samples at various sites and times throughout the basin deemed informative by local biologists, as well as high resolution spatial sampling referred to as longitudinal sampling. Monitoring at index sites: ISCOs were established at 3 mainstem sites on each of the Willamette and Deschutes Rivers. Temperature loggers (Hobos) were attached to each machine and the temperature data downloaded 1

2 when ISCO sampling finished each field year. Waterborne levels of Ceratonova shasta were assessed by filtration of 1L water samples and molecular quantification of captured C. shasta. The ISCO collected 1L every 2h for 24h, from which triplicate 1L subsamples were taken, vacuum-filtered and the retentate frozen (Deschutes samples collected by non-osu staff were posted frozen to OSU). At OSU, the DNA retentate was extracted and C. shasta enumerated using quantitative polymerase chain reaction (qpcr) (per Hallett and Bartholomew 2006, Hallett et al. 2012). The monitoring sites and respective water sampling schedules for each river were as follows. Deschutes River: Three water sampling units (ISCOs) and water temperature data loggers (Hobos) were supplied by OSU for use at the two live-box exposure sites, Pelton Trap (downstream of the re-regulating dam) and Oak Springs (Figure 2). In 2015, water samples were collected approximately weekly starting March 24, 2015 at Oak Springs and bi-weekly starting April 14 at Pelton Dam, through November. In addition, sampling occurred briefly at a third site, Steelhead Falls, from April 6 th through June 17 th, 2015; sampling stopped prematurely because ISCO parts were stolen. In 2016, sampling at Pelton Trap occurred every other week starting March 8 th through July 26 th and sampling at Oak Springs occurred weekly starting March 9 th and continued through September. Figure 2. Deschutes River study sites. Red stars represent sites at which ISCO water sampling and sentinel fish exposures were conducted, black bars represent dams. 2

3 Willamette River: An ISCO supplied with a water temperature data logger was placed at each of three sites on the Willamette River: Willamette Falls (near Oregon City inside the PGE (Portland General Electric) power plant), in Albany near Bryant Park (=Blueberry Farm), and one in the North Albany area off of Springhill Road (=Weber Farm)(Figure 3). In 2015, samples were collected weekly from start date (below) until September 8 th, after which samples were taken every other week. In 2016, sampling occurred weekly to every other week through September 23 rd. Sampling dates in each year were as follows: Willamette Falls April 28 th Nov 17 th, 2015; March 24 th - Sept 23 rd 2016, samples every week. Weber Farm - June 9 th Nov 17 th, 2015; April 14 th Sept 23 rd 2016, samples every week. Blueberry Farm - June 9 th Nov 17 th, = ISCO Water Sampling Index site Figure 3. Willamette River ISCO Index sites for water sampling for C. shasta. The red stars denote ISCO index sites and the flags represent hatcheries. 3

4 Results: Deschutes River Monitoring: (Figures 4-6) Ceratonova shasta was more abundant at Oak Springs than Pelton Trap, with levels generally an order of magnitude higher (Figures 4-6; Based on studies in the Klamath River, abundance of C. shasta is considered to be high above 10 spores/l ). At Oak Springs, in 2015 levels quickly increased in spring, and peaked mid-season (at >300 spores/l)(figures 4, 6). Although water temperature continued to increase in spring, parasite levels did not. There was a second period of relatively high spore levels mid-late summer (~200 spores/l), after which levels decreased rapidly in the fall. In 2016, levels again increased in spring but rose more gradually, generally following the rise in water temperature (Figures 4, 6). At Pelton Trap, the trend was that spore density increased with water temperature, but there was a sudden drop in spore density in both 2015 and 2016 mid-july. In contrast to Oak Springs, levels at Pelton Trap were highest in late spring (< 20 spores/l) and there was no second peak (Figures 5-6). 4

5 Figure 4. Density of Ceratonova shasta in 1L water samples collected at Deschutes River mainstem index site, Oak Springs and water temperature in 2015 and Three replicate water samples were taken at each time point (dots); shading represents mean of the three samples. Figure 5. Density of Ceratonova shasta in 1L water samples collected at Deschutes River mainstem site, Pelton Trap and water temperature in 2015 and Three replicate water samples were taken at each time point (dots); shading represents mean of the three samples. Note that the scale differs to Oak Springs (maximum of only ~20 spores/l at Pelton compared to 400 spores/l at Oak Springs). 5

6 Figure 6. Relative levels of Ceratonova shasta in 1L water samples collected at Deschutes River mainstem index sites, Pelton Trap and Oak Springs in 2015 (upper graph) and 2016 (lower graph). Three replicate 1L water samples (dots) were taken at each time; shading represents mean of the three replicate samples. 6

7 Table 1. Density of Ceratonova shasta in water samples from Deschutes River mainstem monitoring site, Steelhead Falls. Spore values are the average of 3 x 1 liter water samples. date locality average spores/l 4/6/2015 Deschutes Steelhead Falls 6 4/20/ /4/ /19/ /18/ Grab Samples: Replicate water samples were also collected manually ( grab samples ) by ODFW and LCRFHC staff at various sites and times throughout the basin in 2015 and less often in Generally, the density of C. shasta was low in the grab samples tested, with trace amounts detected in Bakeoven Creek and fewer than 10 spores/l measured in Buck Hollow Creek, Warm Springs River, and Steelhead Falls (with the exception of > 10 spores/l in April 2015) (Table 1, 2). In contrast, high levels of C. shasta (> 100 spores/l) were measured at Heritage Park at the confluence of the Deschutes and Columbia Rivers in May and June Table 2. Water 'grab' samples from assorted Deschutes River mainstem, tributaries and Columbia River sites. Spore values are the average of 3 x 1 liter water samples. date locality average spores/l (range) 3/31/2015 Deschutes Mouth - Heritage Park 7 (1-17) 4/7/ (3-17) 5/5/ ( ) 5/19/ ( ) 6/1/ ( ) 3/31/2015 Buck Hollow Creek (tributary site) 0 (0-0) 5/5/ (0-10) 6/1/ (0-3) 7/16/ (0-0) 7/22/ (0-9) 4/13/2016 <1 (0-0.2) 3/31/2015 Bakeoven Creek (tributary site) 0 (0-0) 5/5/ (0-0) 6/1/ (0-0) 7/16/2015 <1 (0-0.8) 4/13/2016 <1 (0-0.2) 7/28/2015 Trout Creek (tributary site) 0 (0-0) 8/17/ (0-0) 4/7/2015 Warm Springs River (tributary site) <1 (0-1) 6/19/2015 <1 (0-1) 7/16/ (0-2) 7/28/ (1-1) 7

8 Longitudinal sampling: (Table 3, Figure 7) A collaborative longitudinal sampling effort occurred on August 17 th 2015 to examine the spatial distribution of the parasite throughout the basin ranging from the Columbia River to the Bend area (Table 3). Three replicate 1L water samples were collected manually by multiple teams at approximately 1pm for 26 sites. Samples were kept chilled and handed to OSU for filtration and molecular analysis. A second and third longitudinal sampling effort occurred at a subset of original sites (fewer tributaries) but with additional mainstem sites included in the lower basin to fill in several gaps (based on first effort results), on June 7 th and August 30 th Sampling occurred in an upstream direction from the Columbia River confluence to the Redmond area throughout one day, commencing at daybreak. 2015: Ceratonova shasta was detected throughout the Deschutes system, including in the Crooked and Metolius Rivers and Whychus Creek in the upper basin, and Buckhollow Creek in the lower basin (Figure 7). Parasite levels were highest in the lower basin mainstem (>300 spores/l). Despite levels of around 100 spores/l in the upper basin, few spores were detected immediately below the dams, which suggest that the reservoirs act as a spore sink. Intriguingly, high numbers of spores (>100 spores/l) were detected in the irrigation canal samples. Spore concentration decreased downstream from >300 spores/l to ~50 spores/l at the confluence with the Columbia River (Heritage Park site, Rkm 0.4) in August. However, considerably higher levels of the parasite ( spores/l) were detected at the confluence when it was sampled in May and June (Table 2; note that parasite levels increased 10-fold from April to May at this site). Downstream of the Deschutes River confluence with the Columbia River at Columbia River mile 201, 31.9 spores/l were detected whereas levels were lower upstream of the confluence at Columbia River mile 208, 7.2 spores/l. 2016: Waterborne C. shasta was high (>100 spores/l) immediately below RBD (at Round Butte), but decreased considerably below Pelton Dam. In June, levels increased downstream from Pelton Dam to peak at ~200 spores/l at Rkm 52, then decreased towards the mouth to ~50 spores/l (at Heritage Park). This gradual decrease is consistent with no further contribution of spores below Rkm 45. This pattern was similar in August, except that the highest levels downstream of Pelton Dam were detected at Rkm 84 (mainstem above Warm Springs River confluence). In June 2016 (but not August), spore levels in Columbia River water samples collected either side of the Deschutes River confluence indicate that the Deschutes River contributes spores to the Columbia; average spore levels upstream of the confluence at Columbia River mile 208, were 2.6 spores/l (0.0 spores/l in August) whereas levels downstream of the confluence at Columbia River mile 201 were higher at 11.9 spores/l (0.1 spores/l in August). 8

9 9

10 Figure 7. Series of graphs showing comparative densities of Ceratonova shasta in 1L water samples collected at multiple Deschutes River mainstem sites (blue dots) and tributaries (triangles and circled) in 2015 and Locations are displayed relative to Deschutes River miles from mouth. Table 3. All Deschutes basin sample localities, including longtitudinal surveys, index sites and grab sample sites, with river miles and latitude/longitude coordinates where available. Locality River mile Lat/Lon coordinates (where available) Columbia Celilo Park Columbia R N, W Columbia Junction Columbia R N, W Deschutes Mouth - Heritage Park N, W Macks Canyon boat ramp N, W Rattlesnake Canyon campground N, W Twin Springs Campground N, W Deschutes Buck Hollow boat ramp 42.8 Buck Hollow Creek 43 White River (tributary site) 46.5 Oak Springs FH 47.6 Deschutes Maupin City Park N, W Bakeoven Creek (tributary site) 51.4 Locked Gate N, W 4 miles from Locked Gate bathroom N, W 8 miles from Locked Gate bathroom N, W Railroad Bridge/ North Junction N, W Deschutes R below Warm Springs R N, W 10

11 Warm Springs River (tributary site) N, W Deschutes R above Warm Springs R N, W Trout Creek (tributary site) 87.8 N , E Deschutes Trout Ck boat ramp 88.3 N , E Warm Springs boat ramp 97.7 Round Butte 110 Pelton Dam index site ("Pelton Trap") Lake Stimtustus 104 Crooked R (mouth - for reference) Crooked R - 0 Metolius R (mouth - for reference) Metolius R - 0 Crooked Opal Springs Crooked R Whychus Creek N, W Deschutes Steelhead Falls PGE facility 128 North Unit Irrigation Ditch Crooked R N, W Metolius Candle Ck Campground Metolius R N, W Crooked R Forest Crossing / Pone Pine Crooked R N, W Cline Falls park N, W Metolius Camp Sherman Br Metolius R N, W Metolius Lake Ck Metolius R N, W Metolius R above Lake Ck Metolius R N, W Crooked Elliot Lane Bridge Crooked R Crooked Hancock Bridge Crooked R N, W Crooked R - Library Crooked R - 48 Bend 167 Willamette River Monitoring: (Figures 8-12) Waterborne Ceratonova shasta was more abundant at the lower mainstem site, Willamette Falls than the upper sites Weber Farm or Blueberry Farm, and in 2015 than in 2016 (Figures 8-12). In 2015, at Willamette Falls, levels were already high at the commencement of sampling in May and peaked this month (at ~80 spores/l) (Figure 8). Levels fluctuated throughout the collection period but generally decreased through summer with a small rise in fall. In 2016, levels again fluctuated week to week, peaking in spring (at ~40 spores/l) and then decreasing in summer. At Weber Farm, levels fluctuated with highest levels (~25 spores/l) detected in summer (Figure 9). At Blueberry Farm, C. shasta was detected throughout the sampling period with levels highest in summer (at ~25 spores/ L) (Figure 10). 11

12 Figure 8. Density of Ceratonova shasta in 1L water samples collected at Willamette River mainstem index site, Willamette Falls in 2015 and Three replicate water samples were taken at each time point (dots). 12

13 Figure 9. Density of Ceratonova shasta in 1L water samples collected at Willamette River mainstem site, Weber Farm in 2015 (upper graph) and 2016 (lower graph). Three replicate water samples were taken at each time point (dots). 13

14 Figure 10. Density of Ceratonova shasta in 1L water samples collected at Willamette River mainstem site, Blueberry Farm in Three replicate water samples were taken at each time point (dots). Note that the scale differs to Willamette Falls. Figure 11. Temporal and spatial variation in density of Ceratonova shasta in 1L water samples collected at three Willamette River mainstem sites in Three replicate water samples were taken at each time point (dots). 14

15 Figure 12. Temporal and spatial variation in density of Ceratonova shasta in 1L water samples collected at two Willamette River mainstem sites in Three replicate water samples were taken at each time point (dots). Sentinel Fish Exposures Methods: Field Sentinel Exposures: Groups of Spring Chinook salmon were placed in wire cages and exposed to river water for 72-hr from March 31-April 3 and May 5-8 at two mainstem locations: one upper river site (near the Pelton- Round Butte Dam Complex; site identifier = Pelton Dam, 30 RBH SCS) and one lower Deschutes River site (situated below the confluence of the Warm Springs and Deschutes Rivers by Oak Springs Hatchery; site identifier =Oak Springs, 30 RBH SCS and 30 WSNFH SCS) (see Fig. 2) by LCRFHC and ODFW-Fish Health staff. Timing of exposures was based on the release timing of hatchery fish at WSNFH and RBH as well as the optimal temperature and water sampling tests for C. shasta. After exposure, fish were treated for ectoparasites and bacterial infections then held for 90 days at the J.L. Fryer Aquatic Animal Health Laboratory (AAHL) at 13.8 C in flow-through well water. The fish were monitored daily for disease signs (e.g. inappetence, distended abdomen). Fish that displayed disease signs were euthanized with an overdose of MS222 (= mortality, fish that died before termination date ) and necropsied with microscopic examination of intestinal and kidney tissue. Mortalities that were visually negative for C. shasta (no myxospores observed) were tested by specific PCR. Fish kidney samples of the mortalities were frozen. The April exposures were terminated July 6 and the May exposures were terminated August 5. Surviving fish were euthanized and necropsied as above (hereafter 15

16 referred to as terminated ). Gut samples were collected from all terminated fish that were visually negative, to assay for C. shasta DNA by PCR. Lab Necropsy: Sentinel fish (morts and terminated fish) were first observed for any ectoparasites or fungus (on the skin, mucus, or external region of the fish) and for signs of disease such as a swollen gut. A flamed sterile loop was then inserted into the ventral hole to obtain a wetmount sample of the lower gut to observe visually for C. shasta myxospores. After wetmount observation by use of a microscope, further necropsy procedure consisted of cutting open the body wall taking care to not puncture any internal organs. Internal organs were inspected for any clinical disease signs, taking special care to observe the kidney for BKD (bacterial kidney disease, Renibacterium salmoninarum, and the intestinal gut for ascites, swollenness, and/or hemorrhaging, which are all characteristic of C. shasta. Abnormalities or clinical signs of disease in any internal organs were noted and recorded. If the wetmount observed was visually negative, a gut sample was obtained by use of sterile razors and toothpicks into a 2mL tubes for PCR analyzation. A wetmount of the kidney by use of microscope was recorded and if visually negative, a kidney sample was placed into a 2mL tube for PCR analyzation. Results: (Table 4, Figures 13-17) A summary of the sentinel fish exposure results is provided in Table 4. Results are also displayed graphically in Figures Three pathogens were identified in diseased fish C. shasta, Renibacterium salmoninarum (bacterial kidney disease, BKD) and a fungus. Fish that died within 5 days post-exposure were not included in the C. shasta analyses. Almost one third of the May Oak Springs WSNFH died in the first 5 days post exposure in association with a fungal infection. Overall mortality was higher in fish exposed in May than in April. Similarly, prevalence of infection of C. shasta was also higher in May than in April and at Oak Springs than at Pelton Trap. At both sites in April and at Pelton Trap in May, some fish survived infection with C. shasta, however none of the fish infected in May at Oak Springs survived. Of the two stocks of SCS exposed at Oak Springs, almost twice as many RBH salmon than WSNFH salmon died following exposure in April whereas mortality was similar in May. In RBH salmon, C. shasta-infection ranged from 7.7% at Pelton Trap in April, to 83.3% at Oak Springs in May. In WSNFH salmon, C. shasta-infection ranged from 15.6% in April to 87% in May. C. shasta was detected in 3.1% of the RBH control fish, but none of the WSNFH controls. 16

17 Table 4. Total mortality (any pathogen) and prevalence of infection of Ceratonova shasta in sentinel Spring Chinook exposed for 72h from March 31-April 3 (April) and May 5-8 (May) 2015 at one upper river (Pelton Trap) and one lower Deschutes River site (Oak Springs). Fish that died within 5 days post-exposure were not included in the C. shasta totals. Round Butte Hatchery juvenile Spring Chinook = RBH SCS, Warm Springs National Fish Hatchery juvenile Spring Chinook = WSH SCS. Exposure Date Site Fish Stock Total Mortalities (total fish in group surviving >5d) Prevalence of Infection Ceratonova shasta In Morts In Survivors In group April Pelton Trap RBH SCS 17.9% (39) 28.6% 3.1% 7.7% Oak Springs WSNFH SCS 12.5% (32) 100% 3.6% 15.6% RBH SCS 22.9% (35) 87.5% 11.1% 28.6% May Pelton Trap RBH SCS 38.6% (44) 52.9% 14.8% 29.5% Oak Springs WSNFH SCS 95.7% (23) 90.9% 0% 87.0% RBH SCS 96.7% (30) 86.2% 0% 83.3% Non-exposed controls WSNFH SCS 18.9% (37) 0% 0% 0% RBH SCS 28.1% (32) 11.1% 0% 3.1% 17

18 Figure 13. Total mortality (%) and associated pathogens in sentinel Spring Chinook exposed for 72-hr from March 31-April 3 (Apr) and May 5-8 (May) at one upper river (Pelton Trap) and one lower Deschutes River site (Oak Springs) by LCRFHC and ODFW-Fish Health staff in Note that pathogens caused mortality in the control fish. Numbers within the individual bars represent the number of fish that died from that particular pathogen or combination of pathogens. At least 30 fish were in each group (exact number above each bar; *excludes mortalities that occurred 0-5 days post-exposure; see Figure 14 for mortalities during days 0-5). May Oak Springs WSNFH and May Oak Springs RBH were both exposed in the river when levels were >100 spores/l (see graphs below). Cs = Ceratonova shasta, BKD = Bacterial Kidney Disease. 18

19 Figure 14. Total mortality (%) during days 0 5 post-exposure in sentinel Spring Chinook exposed for 72-hr from March 31- April 3 (Apr) and May 5-8 (May) 2015 at one upper river (Pelton Trap, P) and one lower Deschutes River site (Oak Springs, OS). Numbers within the individual bars represent the number of fish that died. At least 30 fish were in each group (see above bar for exact number). 19

20 Figure 15. Prevalence of infection of Ceratonova shasta in sentinel Spring Chinook that died (=mort) or survived (=terminated) the rearing period. Chinook were exposed for 72-hr from March 31-April 3 (Apr) and May 5-8 (May) 2015 at one upper river (Pelton Trap, P) and one lower Deschutes River site (Oak Springs, OS). Note that mortality occurred in the control fish (Ctr). Numbers within the individual bars represent the number of fish. May OS WSNFH and May OS RBH were both exposed in the river when levels were >100 spores/l (see water graphs below). 20

21 Figure 16. Total mortality* (%) and associated pathogens in sentinel Spring Chinook exposed for 72-hr from March 31-April at one upper river (Pelton Trap) and one lower Deschutes River site (Oak Springs). Note that pathogens caused mortality in the control fish. Numbers within the individual bars represent the number of fish that died from that particular pathogen or combination of pathogens. At least 30 fish were in each group (exact number above each bar; *excludes mortalities that occurred 0-5 days post-exposure). Cs = Ceratonova shasta, BKD = Bacterial Kidney Disease. 21

22 Figure 17. Total mortality* (%) and associated pathogens in sentinel Spring Chinook exposed for 72-hr from May at one upper river (Pelton Trap) and one lower Deschutes River site (Oak Springs). Note that pathogens caused mortality in the control fish. Numbers within the individual bars represent the number of fish that died from that particular pathogen or combination of pathogens. At least 30 fish were in each group (exact number above each bar; *excludes mortalities that occurred 0-5 days post-exposure). May Oak Springs WSNFH and RBH were both exposed in the river when levels were >100 spores/l (see graphs below). Cs = Ceratonova shasta, BKD = Bacterial Kidney Disease. Polychaete host habitat suitability model The aim of this aspect of the research was to apply the polychaete predictive model developed for the Klamath River to habitat in the Willamette River. The modeling approach for predicting the distribution of M. speciosa developed for the Klamath River (Alexander et al. 2016, Wright et al. 2014) involves the use of two-dimensional hydraulic models (2DHMs) to describe hydraulic variation during peak discharge coupled with a statistical model that delineates the relationship between physical habitat during peak discharge and M. speciosa distribution. One of the best fitting predictive models for the Klamath River demonstrates that summer M. speciosa distribution is associated with substrate, depth, and velocity conditions during peak discharge. The flow regime in the Willamette River is even more dynamic than that of the Klamath, which should result in lower probabilities of polychaete presence. In contrast, the flow regime of the Deschutes River is relatively stable, which should result in higher probabilities of polychaete presence. Thus we hypothesized that polychaete densities would be low in the Willamette River and high in the Deschutes River, relative to the Klamath River. 22

23 To test the predictive model in the Willamette and Deschutes Rivers, we planned to collect benthic samples targeting polychaete habitat at sites within area(s) found through water sampling (see above) to support high densities of C. shasta. We also planned to collect habitat data to further guide sample site selection. However, slow processing of water samples delayed site selection and habitat data were not collected in either river. Consequently, 2 sites were selected proximal to the Willamette Falls ISCO (the area C. shasta levels were highest), including the PGE channel in West Linn (PGE), upstream of the Willamette Falls near 1 st and Miller St. in West Linn (AWF), and the two Albany ISCO sites; Weber Farm (WF) and Blueberry Farm (BF). Methods: Field Polychaete collections: Polychaete samples were collected once (October 30, 2015) in the Willamette River. Samples were collected by Ponar Grab (presence/absence) and modified Hess (density) samplers. Three samples were collected at each site to determine the presence/absence and density (modified Hess samples only) of M. speciosa. Collections were made by snorkelers using a modified Hess sampler (constructed of lightweight PVC and fitted with 83µm Nytex mesh, Alexander et al. 2016) at all sites, except the PGE channel in West Linn, OR which required the use of Ponar Grab due to unsafe swimming conditions (Table 5). Ponar grab samples were obtained by scooping sediment at 3 randomly selected locations in the PGE channel. Hess samples were collected by a snorkeler at 3 randomly selected locations at AWF, BBF, and WF (Table 5). Samples were placed on ice and transferred into 95% ethanol within 24 hrs of collection. Ethanol was changed every 2 weeks post-collection. Laboratory Polychaete sample processing: Each sample was examined under a dissecting scope (10-50x magnification). The absence/ presence of polychaetes was recorded along with the number found in each sample, if present. Density of M. speciosa per square meter was calculated as total number of polychaetes per sample/area of Hess sampler and extrapolated to the area of a square meter. Results: Polychaetes were observed in samples collected at AWF, and WF sites but not in samples collected at PGE or BF sites. Numbers ranged between 0 7 polychaetes per sample ( M. speciosa per m 2 ). Weber Farm had a larger range with 5-10 polychaetes per sample ( M. speciosa per m 2 ) (Table 5). Discussion: Our aim was to apply the polychaete predictive model developed in the Klamath River to habitat in the Willamette River. Although we were unable to obtain two dimensional hydraulic model data for the study area s sections of the Willamette River, we identified segments and collected polychaetes. Four sites were selected based on proximity to water sampling locations and access. Benthic samples targeting polychaete habitat showed that polychaetes were present, but at low densities at 2 of the 4 sites. As expected, densities were low relative to samples collected at a similar time period in the Klamath River (Jordan 2012). One explanation for why polychaetes were not detected at all sites, particularly the PGE site, which is located within the area of high C. shasta densities, see above, is that sample collection was delayed until late fall. In other systems, polychaete densities peak with temperature (in summer), thus polychaetes may have been present at the other sites earlier in the water year. 23

24 Table 5. Results of October 31, 2015 polychaete collections at 4 sites on the Willamette River including the PGE channel in West Linn (PGE), above and upstream of the Willamette Falls near 1 st and Miller St. in West Linn (AWF Above Willamette Falls), and the two Albany ISCO sites (Weber Farm and Blueberry Farm). Collections were done on snorkel while using a modified Hess sampler with the exception of PGE, which required Ponar Grab sampling due to safety concerns. Three samples were collected at each site and were processed to determine presence or absence of M. speciosa and density when possible (Hess samples only). PG= Ponar Grab, H=modified Hess, SD=1 standard deviation. Site Latitude/longitude Sample Sample Polychaetes Count Density type present? (# per sample) (# per m2) PGE 1 PG No n/a n/a PG No n/a n/a PG No n/a n/a H Yes AWF H Yes H Yes 0 0 WF 1 H Yes H Yes H Yes BF 1 H No H No H No 0 0 Mean density per site (# per m 2 )+ SD n/a Acknowledgements: Funds were also provided by US Fish & Wildlife Service, Lower Columbia River Fish Health Center contract #F15PX00870 for sentinel fish exposures and water sampling in the Deschutes River in Patrick Ball, OSU Cascades campus, assisted with Steelhead Falls water sampling. Many people assisted with the longitudinal water sampling, including: Terry Shrader, Gary Lytle, Rick Stocking, Nate Dachtler, Tim Porter, Opal Springs volunteers, Bend ODFW Office Host volunteers, volunteer anglers, and other ODFW staff. The following Bartholomew Lab members assisted with research or preparation of this report: Sarah Vojnovich, Sascha Hallett, Stephen Atkinson, Julie Alexander, Rich Holt, Ryan Craig, Damien Barrett, Jamie Graen, Kate Threlfall, Nathan Clark, and Jerri Bartholomew. References Alexander, J, K Wright, N Som, N Hetrick, J Bartholomew (2016) Integrating models to predict distribution of the invertebrate host of myxosporean parasites. Freshwater Science 35(4): DOI: / Hallett SL, Bartholomew JL (2006) Application of a real-time PCR assay to detect and quantify the myxozoan parasite Ceratomyxa shasta in river water samples. Diseases of Aquatic Organisms 71:109. Hallett SL, Ray RA, Hurst CN, et al. (2012) Density of the waterborne parasite Ceratomyxa shasta and its biological effects on salmon. Applied and Environmental Microbiology 78: Wright, K.A., D.H.Goodman, N.A.Som, and T.B. Hardy Development of two-dimensional hydraulic models to predict distribution of Manayunkia speciosa in the Klamath River. U.S. Fish and Wildlife Service. Arcata Fish and Wildlife Office, Arcata Fisheries Technical Report Number TR , Arcata, California. These data are preliminary and subject to change. 24