Ecological Drivers and Species Interactions of Whirling Disease

University of Colorado, Boulder CU Scholar Undergraduate Honors Theses Honors Program Spring 2014 Ecological Drivers and Species Interactions of Whirling Disease Julie Byle University of Colorado Boulder Follow this and additional works at: http://scholar.colorado.edu/honr_theses Recommended Citation Byle, Julie, "Ecological Drivers and Species Interactions of Whirling Disease" (2014). Undergraduate Honors Theses. Paper 58. This Thesis is brought to you for free and open access by Honors Program at CU Scholar. It has been accepted for inclusion in Undergraduate Honors Theses by an authorized administrator of CU Scholar. For more information, please contact cuscholaradmin@colorado.edu.

Ecological Drivers and Species Interactions of Whirling Disease Julie Allyson Byle Department of Ecology and Evolutionary Biology University of Colorado Boulder April 7, 2014 Dr. Robert Guralnick of the Department of Ecology and Evolutionary Biology Dr. Barbara Demmig-Adams of the Department of Ecology and Evolutionary Biology Dr. Diane McKnight of the Department of Environmental Science, Civil, Environmental, and Architectural Engineering, and the Institute of Arctic and Alpine Research

Table of Contents Abstract..3 Introduction 4 Background...5 Whirling Disease....6 Myxobolus cerebralis lifecycle...7 Study System Species Didymosphenia geminata. 8 Tubifex tubifex....10 Trout 11 Species interactions 12 Materials and Methods...12 Patterns of oligochaete abundance and D. geminata cover...12 D. geminata as a refuge from predation for T. tubifex..13 D. geminata as a stream flow refuge for T. tubifex....14 Results 16 Oligochaete abundance sampling..16 Predator refuge experiment 18 Stream flow refuge experiment.19 Driver of disease table...20 Discussion..21 Acknowledgements 25 Literature Cited..26 2

Abstract Whirling disease is on the rise since its introduction in the United States in 1958 and is a health problem both in fisheries and in wild populations of salmonids. Prevalence of the disease is dependent on ecological context and interactions among multiple other species, including algal species such as the also invasive diatom Didymosphenia geminata, the oligochaete worm Tubifex tubifex, and the myxozoan parasite Myxobolus cerebralis that is the causative agent of whirling disease in salmonid fishes. D. geminata, a stalk-producing diatom has increased in frequency worldwide and is now invasive across the United States. The stalks of D. geminata create an environment suitable for oligochaetes such as T. tubifex. Based on existing data oligochaete abundance is higher in areas with higher percent cover of D. geminata, and whirling disease prevalence in trout is 3X higher in streams with blooms. My research further examined why oligochaetes are more abundant specifically in streams with D. geminata blooms focusing on the mechanisms that might promote T. tubifex density increases, including predator release. The results provide some new insights into the multifaceted and complex ecological interactions that can promote increased amounts of whirling disease in ecologically and economically important salmonid fishes. I attempt to place these results in the context of a larger collection of ecological drivers that explain interactions among all the actors involved. 3

Introduction In the summer of 2013, I conducted a research project on direct and indirect effects of algal blooms by the diatom, Didymosphenia geminata on the intermediate whirling disease host, Tubifex tubifex, a freshwater oligochaete worm, at the Rocky Mountain Biological Laboratory with Dr. Brad Taylor from Dartmouth College. To understand why oligochaetes are more abundant in streams with D. geminata blooms, I aimed to experimentally test several hypotheses. My first hypothesis was that the long filaments, or stalks, that D. geminata produces may be providing a refuge for oligochaetes against predatory invertebrates such as the stone fly Hesperopola pacifica. I hypothesized that oligochaete mortality from invertebrate predators would be lower when D. geminata stalks are more abundant. Second, since D. geminata stalks form dense, thick mats on the streambed, I aimed to test the hypothesis that D. geminata mats engineer an ecosystem that is more suitable for T. tubifex who prefer slow-moving backwaters habitats (Larned, 2011), which are rare habitats in most high-gradient Rocky Mountain streams. My experimental work provided a snapshot view of how substrate may directly or indirectly impact the abundance of T. tubifex, which appears to be a key regulator in whirling disease prevalence (McMurtry, 1983). In order to better understand how important the latter factor may be given the complex life-cycle of Myxobolus cerebralis and the actors with whom it interacts, I also looked more broadly at the whirling disease literature in order to properly frame my results. In particular, I performed a literature review on those other factors and assembled a conceptual model on what might drive increases in whirling disease, and where gaps in our knowledge exist of this system overall. This model could serve as a means to generate new hypotheses and to integrate both my current and previous work. 4

I have organized this work to first provide a brief background on the history of whirling disease and the methods of introduction of the parasite M. cerebralis into the United States. Next, the life cycles and habitat preferences of each main species involved in whirling disease are described and it is explained how each of these actors interact with each other in their environment to make this disease so successful. Relevant literature is incorporated on environmental conditions that influence availability of preferable habits, rates of distribution, establishment in these habitats, and the susceptibility of trout to infection with whirling disease in these habitats. Details on my experimental design are provided as well as results related to my hypotheses that the diatom blooms both provide predator refuge and engineer a more suitable habitat for T. tubifex, and then close by again broadly considering management implications for my work. Background Disease ecology is an interdisciplinary field that utilizes ecological theory and practice to understand living, biotic and non-living, abiotic drivers determining host and pathogen interactions and their ultimate impacts on human-relevant issues such as health. Living systems are dynamic and complex and their behavior may be hard to predict from the properties of individual parts, requiring an integrative discipline that can take a systems view of emerging diseases and their course. Studying the ecology of specific diseases and creating models to better understand interactions among wildlife hosts, vectors, and pathogens, can be a tool to help determine risk, manage, and possibly prevent disease. My research aim was to both better understand the interactions among the pathogens, hosts, and their environment; both in the broad picture, as well as perform a targeted set of field experiments. The interest here was meant to clarify a complex system and many strands and pieces of knowledge found in the literature, 5

informed by my own fieldwork. Below I provide an overview of that literature as background before describing my experimental work in the field. Whirling Disease Whirling disease is on the rise since its introduction in the United States in 1958 and is a health problem both in fisheries and in wild populations of salmonids (Gilbert, 2003). In Colorado alone, 14 out of 15 major drainages in Colorado tested positive for whirling disease (Nehring, 2003). Also in Colorado, recreational fishing generates millions of dollars in economic activity each year, and since whirling disease has been introduced, some locations in Colorado (e.g., Gunnison River) have experienced declines in salmonid density and biomass by as much as 90% (Nehring, 2003; Nehring, 2006). Whirling disease was first described in rainbow trout in Europe in 1893 when Bruno Hofer recorded signs and symptoms such as whirling behavior and a blackened caudal region, and detected microscopic parasitic spores that he named Myxobolous cerebralis (Spaulding, 2007). M. cerebralis, a myxozoan parasite, has been identified as the causative agent of whirling disease that requires a vertebrate and non-vertebrate host to complete its life cycle. M. cerebralis is likely to persist in North America and is found in new places every year (Elwell, 2009). When high numbers of parasites are around susceptible fish, there can be high mortality rates in native trout (Spaulding, 2007). Much is already know about M. cerebralis and its salmonid host, yet less is understood with regard to M. cerebralis and its T. tubifex host (Gilbert, 2003). T. tubifex is a common, native freshwater worm that serves as the intermediate host for the myxozoan parasite and thus 6

is required for the whirling disease parasite to complete its life cycle. Many environmental variables, including substrate properties, can influence habitat selection by tubificids, which has produced conflicting results (McMurtry, 1983). Changes that affect T. tubifex abundance and density thus have the potential to strongly affect downstream outcomes of whirling disease (Minchella, 1991). What is less well understood is how and why changes in substrate influence T. tubifex density. Myxobolus Cerebralis lifecycle and requirements Myxobolus has a two-stage life-cycle, consisting of triactinomyxons(tams) that infect salmonids and develop in T. tubifex, and myxospores that infect T. tubifex develop in salmonids (Wolf and Markiw, 1984). The life cycle of M. cerebralis starts when myxospores are released from infected fish (Figure 1). Next, myxospores must be ingested by T. tubifex. The germ cell in the myxospore then migrates to the intercellular space of the intestinal epithelium where it undergoes reproduction and development into a TAM over a (70-120 day period). TAMs are released from feces of T. tubifex and then enter the water column. TAMs are short-lived and attach to fish and penetrate the skin. Once in the fish, reproduction takes place in the epidermis and the parasites migrate through central nervous system to the associated cartridge where they mature to plasmodia containing vegetative nuclei and generative cells. Approximately 80 days after exposure to TAMs, the generative cells initiate sporogenesis to produce myxospores at which stage whirling disease is manifested. The only way fish can become infected is if T. tubifex ingest the myxospores and shed TAMs (Gilbert, 2003). 7

Figure 1: M. cerebralis lifecycle. Discus Club Romania, 2007 M. cerebralis infection timing is strongly tied to water temperature where disease outbreak is strongly related to summer increases in water temperature (Allen, 2002). The ability of M. cerebralis to complete its life-cycle in both hosts in order to produce both parasite spore stages is critical for the continuation of the disease ( El- Matbouli, 1999). Didymosphenia geminata Didymosphenia geminata, a stalk-producing diatom, has increased in frequency worldwide. Blooms of this algae have been reported in North America, Europe, Asia, and New Zealand (Figure 2) and the diatom is believed to be expanding its geographic range in North America where research is now just starting to be conducted (Spaulding, 2007). These singlecelled algal blooms result from excessive extracellular stalk production by individual cells that form a contiguous mat covering the stream bottom (Spaulding, 2007). D. geminata is now 8

causing concern because of the possible impacts on rivers where blooms occur and on the salmonid fisheries in these rivers; in particular, D. geminata blooms have the potential to alter stream ecosystems by impacting the ecosystem s metabolism, nutrient cycling, hydraulics and food web (Spaulding 2007). The thick mats (e.g., 2-5 cm) that can cover much of the streambed lead to changes in invertebrate species diversity and population sizes that may propagate up the food web to affect fish populations (Spaulding, 2007). D. geminata forms thick mats that cover >75% of the stream area and extend for 1 km, and may persist for several months of the year (Spaulding, 2007). D. geminata cells produce copious amounts of extracellular stalk material that form thick benthic mats, or blooms. D. geminata thrives in a wide range of conditions such as both low-nutrient and highnutrient lakes and slow-moving shallow water to waters with greater depth and increased flow. D. geminata mats alter the water velocity along the stream bottom in ways that may be important to invertebrates such as oligochaetes that prefer slow-moving water and areas with stable sediments (Larned, 2011). Hiner (2011) found that water flow rate has an effect on the propagation of M. cerebralis, and habitats with lower velocity were found to promote higher prevalence of infection and greater proliferation of the parasites invertebrate host T. tubifex leading to greater severity of infection of whirling disease in fish (Hallett, 2007). D. geminata may also provide a food source for T. tubifex by trapping particulate organic matter that is readily colonized by bacteria, and D. geminata mats may also provide refuge for T. tubifex by seeking protection from invertebrate hosts in the long stalks that D. geminata produces. D. geminata is a good resource for T. tubifex due to their preference for fine silt and clay substratum (i.e., depositional areas) that are areas with abundant microflora, which are a source of bacterial food for oligochaetes (Kreuger, 2006). 9

Figure 2: World-wide distribution of records for D. geminata. Past and recent records show the range expansion of D. geminata (Whitton, 2009). Tubifex tubifex T. tubifex is a habitat generalist and is extremely tolerant of a wide range of environmental conditions (Kerans, 2002). McMurtry (1983) found a significant correlation between abundance of heterotrophic aerobic bacteria in sediment and tubificid preference and believes worms were attracted to the leaves because of the microfloras associated and provided 10

bacterial food. T. tubifex prefers silt and clay substratum to coarser substratum and have been found to reproduce faster in silt sediment than other substrates (Arndt. Et al, 2002). Trout M. cerebralis affects several species of trout and salmon (Hoffman, 1990; Hedrick et al., 1998; Gilbert and Granath, 2003). The infection of M. cerebralis that gives rise to whirling disease has caused reductions in populations of rainbow trout, cutthroat trout, Yellowstone cutthroat trout, brook trout, Chinook salmon, and kokanee salmon (Hedrick et al., 1998, Macconnell and Vincent, 2002) The main sign of infection of whirling disease in salmonids is a tail chasing behavior that causes the infected fish to whirl. As the disease progresses in the fish skeletal deformation, misshaped heads, jaws and gill covers, and spinal curvature can develop (Hnath, 1993). The severity of the infection is evaluated by presence of clinical signs of whirling and or a darkened caudal region, prevalence of infection, severity of microscopic lesions, and spore counts 5 months after exposure (Hendrick, 1999). The development and severity of whirling disease is known to be dependent on the age of fish when first exposed to the infective triactinomyxon stage of Myxobolus cerebralis and the density of TAMs to which the fish are exposed (Ryce, 2005). The time at which a salmonid is infected with M. cerebralis is crucial in determining the likelihood and severity of infection. Ryce (2005) found that Rainbow trout must be both 9 week post-hatch or older and at least 40 mm in fork length at time of exposure to exhibit enhanced resistance to whirling disease. 11

Species Interactions This study involved interactions among the algae, D. geminata, the oligochaete worm T. tubifex, and the parasite M. cerebralis that is the causative agent of whirling disease in salmonid fishes. M. cerebralis has a complex life cycle with two hosts and two intermediary spore stages (Kerans, 2002). T. tubifex is one of the hosts in the two stage life cycle of Myxobolus cerebralis, a myxozoan parasite that causes whirling disease in salmonid populations in the United States (Gilbert, 2003). The myxozoan is spread by the death of infected salmonids near susceptible T. tubifex and salmonids and it has been found that the death of just one infected salmonid upstream is sufficient for establishment of M. cerebralis in a stream (Hallett, 2007). T. tubifex distribution is strongly correlated to the distribution of leaf litter (Lazim, 1987) and the deposition of fine particles (McMurtry et al, 1983; Bartholomew et al, 2005). Thus, D. geminata has the potential to increase the available habitat for oligochaetes such as T. Tubifex, and thereby increase the prevalence of M. cerebralis. Further, T. tubifex has been shown to itself to be more abundant in streams with D. geminata mats (Kilroy et al, 2009). Moreover, brook trout in streams with D. geminata blooms exhibit a higher M. cerebralis prevalence than brook trout in streams without blooms (B. Taylor unpublished data). Materials and Methods Patterns of oligochaete abundance and D. geminata cover To measure oligochaete abundance in relation to D. geminata cover in the East River of Gothic, Colorado (Figure 3), I measured the percentage of the stream covered by algae with a 12

square-meter quadrant placed over various sections of the river. Assessment of algal cover was made from each grid for a total percent of 0, 2, 5,35,55,90, and 95% D. geminata cover. Once the percentage of algal cover was determined, sections of the sediments in the river were placed into plastic bags and immediately filled with alcohol and red dye. Samples were then taken back to the lab to be processed. This process was done by pouring the sample into a sifter and finishing the collection and then placing the contents into a plastic tub to be sorted. Macroinvertebrates were picked out with tweezers and placed into glass jars filled with alcohol and labeled according the amount of D. geminata percentage cover that correlated with the collection. Oligochaetes were then counted from the collection macroinvertebrates. The relationship between the percent of D. geminata cover and the number of oligochaetes found along a D. geminata cover gradient was calculated via a regression analysis with oligochaete abundance as the response (e.g y-axis) variable and D.geminata cover as the predictor, in order to determine how well cover might explain variation in oligochaetes. D. geminata as a refuge from predation for T. tubifex To test the hypothesis that D. geminata mats provides a refuge from invertebrate predators for oligochaetes such as T. tubifex, I performed an experiment testing the effects of D. geminata and the predatory stonefly Hesperoperla pacifica on T. tubifex mortality in Gothic, Colorado on the upper East River near the Rocky Mountain Biological Lab. I used 80 streamside flow-through tanks where stream water was gravity fed into the tanks from a stream located 200 m upslope draining the side of Gothic Mountain into the tanks inside of a portable weatherport. I randomly assigned four treatments to each tank where twenty tanks received substrata covered with polyester pillow stuffing to mimic the structural properties of D. geminata mats and one H. 13

pacifica stonefly, twenty tanks received substrata without the mimic material and one H. pacifica stonefly, twenty tanks received substrata covered with the mimic material and no predators and the remaining twenty tanks received substrata without the mimic material and no predators, which served as controls for losses due to factors other than stonefly predation. Three T. tubifex collected from the East River were added to each tank in the morning of 29 July 2013 and H. pacifica collected from Avery Creek were added to specific predator assigned treatments three hours later. After ~18 hours, I removed the stoneflies and counted the number of T. tubifex remaining in each tank and whether they had been eaten, were dead, or were found to be bitten, which was indicated by a bite mark out of T. tubifex by the stonefly. Because no T. tubifex were dead or missing from the control treatments, and there was no significant block effect associated with tank arrangement, I used a t-test to test for differences in stonefly-induced mortality between treatments with and without the D. geminata mimic. D. geminata as a stream flow refuge for T. tubifex To test the degree to which D. geminata mats provide a refuge from high water velocity for oligochaetes such as T. tubifex, I covered individual substrata with an artificial mimic of D. geminata stalks and placed these substrata as well as substrata without the mimic in fast and slow water velocity areas of the stream. Substrata were deployed for 3-4 weeks with the aim that the number of oligochaetes colonizing would be quantified. Treatment rocks (8 mimic and 8 nonmimic covered) were placed along a water velocity gradient (16 sites), to test the hypothesis that diatom stalks provide a critical habitat or refuge especially at the fastest water velocities. I predicted that oligochaetes would be more abundant on rocks with the mimic material, and that the difference in number of oligochaetes between rocks with and without the mimic material would be greatest at the fastest water velocities. Although data is still be counted, I plan to use an 14

ANCOVA to test for differences in oligochaete abundance between substrates with and without mimic material along a water velocity gradient with water velocity as the covariate. A significant interaction between water velocity and substrate type would suggest that D. geminata is an important refuge or habitat for oligochaetes in fast flowing streams. Figure 3: East River and Copper Creek field sites at the Rocky Mountain Biological Laboratory in Gothic, Colorado. 15

Results Patterns of oligochaete abundance and D. geminata cover Oligochaete abundance increased along D. geminata cover gradient in the East River (Figure 4 and 5). A regression analysis was done to show the variation of invertebrates found in various samples, and the results showed that there is a relationship in the percentage of D. geminata and the oligochaetes found per square meter. Table 1: Number of oligochaetes found in relation to the percent of D. geminata cover. Percent of D. geminata cover N # oligochaetes found 0% cover 1 0 2% cover 1 0 5 % cover 1 2 35 % cover 1 1 55 % cover 1 3 90% cover 1 9 95% cover 2 1.5 16

Figure 5: Relationship between percent of D. geminata cover and number of oligochaetes found along a D. geminata cover gradient. (R^2=0.5321, P-value=0.0628). Given the order of magnitude differences in the response values, the y- axis was log 10 transformed to show that the oligochaetes found per square meter most closely follows an exponential increase with a linear increase in a percentage of D. geminata cover. The p-value is marginal, and although we cannot falsify the hypothesis of no relationship between cover and oligochaete abundance, we also lack power given our sampling. R^2=0.5321, P-value=0.0628 17

Figure 6: The stonefly H. pacifica consumed, killed, or injured nearly twice as few T. tubifex in treatments with the D. geminata mimic relative to treatments without D. geminata mimic (t38 = 3.19, P = 0.0014; Fig. 4). This data shows that the rocks that were covered in D. geminata mimic were beneficial to the survival of T. tubifex from the stonefly H. pacifica. The body size of H. pacifica was not different between treatments (t38 = 0.43, P = 0.6). T 38=3.19, P=0.0014 18

Table 2: East River Flow Refuge Experiment Table Multiple variables were measured in the East River for the flow refuge experiment. (Mean: Depth: 0.26m, Velocity0.25 m/s, Temperature: 15.32 c, Mimic rock: 208.4cm, Nonmimic rock: 200.93 cm) Rock Number Water Depth (Meters) Water Velocity (Meters per second) Water Temperature (Degrees Celsius) Distance from shore of mimic rock (centimeters) Distance from shore of nonmimic rock (centimeters) 1 0.21 m 0.0983 m/s 15.41 50 cm 69 cm 2 0.22 m 0.0575 m/s 15.53 70 cm 83 cm 3 0.21 m 0.0001 m/s 15.54 112 cm 135 cm 4 0.22 m 0.022 m/s 15.92 110 cm 100 cm 5 0.22 m 0.4354 m/s 15.48 197 cm 161 cm 6 0.21 m 0.4511 m/s 15.34 335 cm 301 cm 7 0.19 m 0.0195 m/s 15.26 70 cm 46 cm 8 0.22 m 0.3325 m/s 15.23 283 cm 306 cm 9 0.22 m 0.4749 m/s 15.18 225 cm 210 cm 10 0.2 m 0.3379 m/s 15.15 335 cm 305 cm 11 0.23 m 0.7513 m/s 15.1 423 cm 423 cm 12 0.2 m 0.0398 m/s 15.12 38 cm 65 cm 13 0.24 m 0.1399 m/s 15.13 585 cm 565 cm 14 0.24 m 0.2847 m/s 15.2 148 cm 133 cm 15 0.2 m 0.3048 m/s 15.22 145 cm 112 cm 19

Table 3: Summary of disease drivers in main species involved in whirling disease. This table lists species that play major roles in whirling disease, the top five ecological drivers that influence the success of each species, which in turn could contribute to the prevalence of whirling disease. D. geminata T. tubifex M. cerebralis Trout Habitat preference 1,2 Habitat preference 2,4,5,6,7,8 Water flow 9 Age and size of trout at time of infection 12,15 Season length 1,3 Range expansion 1,3,4 Stalk length 1 Susceptibility of infection 9 Presence of D. geminata 4 Water velocity 10 Lifespan of spores 6 Water temperature 12 Susceptible T. Tubifex 9 Prevalence of M. cerebralis is water 1,15, 16 Time and means of stocking 15,17 Severity of infection 18,19 Means of introduction 1,4 Heterotrophic bacteria in sediment 5,8 Actinospore- Triactinomyxon lifecycle 6,13,14 Drainage Location 20 1-20 Spaulding, 2007, Larned, 2011, Whitton, 2009, Kilroy, 2009, McMurty, 1983, Kerans, 2004, Lazim, 1987, Kruger, 2006, Hallett, 2007, Hiner, 2001, Hallett, 2007, Allen, 2002, Gilbert, 2003, El-Matbouli, 1999, Ryce, 2005, Hoffman, 1990, Schlister, 2002, Hendrick, 1999, Nickum, 1999, Nehring, 2006 20

Discussion The impact of an invasive diatom on oligochaetes and their predators This study focused on the effects of the diatom D. geminata on the intermediate host of whirling disease, T. tubifex. I asked two questions does D. geminata cover impact abundance of T. tubifex and if so, is part of the explanation for this relationship based on predator avoidance potential afforded by the diatom were tested experimentally at the Rocky Mountain Biological Lab. Percent of D. geminata cover present did have an effect on abundance of oligochaetes. This pattern of increased abundance of oligochaetes could be explained by the habitat preference of oligochaetes to fine particles that D. geminata mats trap (Larned, 2011). The predator refuge experiment data illustrates that treatments with the D. geminata mimic did provide a refuge for T. tubifex from the predatory stonefly H. pacifica. The higher mortality rate of T. tubifex in treatments without the D. geminata mimic shows that T. tubifex was found eaten, dead, or bitten more often due to a lack of protective environment in which to find refuge. The patterns observed in a controlled experimental setting illustrate the habitat refuge that D. geminata may be providing for oligochaetes in East River from predatory stoneflies. Multiple variables were measured in the East River for the flow refuge experiment. Based on my hypothesis that T. tubifex finds a refuge in D. geminata from fast moving streams, it was important to measure variables of the rock sites to make conclusions about which areas of the stream had a higher velocity and depth that could be contributing to the amount of variables that could be contributing to T. tubifex finding refuge in D. geminata. Final data on oligochaete count 21

is currently being processed by Dr. Brad Taylor. The predicted results are that there will be a higher number of oligochaetes found on mimic D. geminata rocks than non-mimic D. geminata rocks Prior to my work, and based on preliminary work in Rocky Mountain Biological Lab, it was known that the continued spread of D. geminata may impact abundances of T. tubifex, but it was not known if this was related to changing habitats, or reduced predation, or both. My work shows that both are likely to be happening, at least at the site I chose to examine. One outcome is that simple measures of cover percent in relation to oligochaete abundance was a somewhat weak result, perhaps dependent on sampling, compared to the predator experiments. More systematic experiments may be needed to properly determine how and how much oligochaete abundance changes are related to different predictors Ecological Drivers While M. cerebralis has been found in many watersheds in the US, distribution and severity of whirling disease on fish populations vary regionally and locally (Nickum, 1999). A main finding from my literature review related to disease prevalence and severity is that habitat factors act synergistically across different species warming, for example, may lead to both increased algal invasions and increased amount of D. geminata, while also benefiting the myxozoan parasite. How these timing events play out with regard to resistance of the trout remains one of many unanswered questions that deserve further study, perhaps using forecasting approaches based on a more formal model that can be constructed based on my initial assembly of drivers presented here. 22

The best current way to manage the disease is for fish to be raised in a M. cerebralis spore-free source of water, or in a farm setting, they need to be constantly monitored for the presence of spores (Hoffman, 1990). Another means of managing the disease is the elimination of susceptible T. tubifex, the parasite's alternate host, and their habitat, which could interrupt the parasite's life cycle and prevent fish infections. Each of the species that interact in this system play a key role in whirling disease. Further research on parasite, hosts, and habitats that support them will be necessary to better understand whirling disease outbreaks and to meet the goals of management or eradication of the disease. Also needed is further research on the genetics of M. cerebralis infection in relation to susceptible T. tubifex, and whether ecological or genetic variation within oligochaete host populations may be responsible for determining whirling disease risk in a body of water (Kerans, 2004). In addition, examination how D. geminata will expand its range with a changing climate and continue to alter ecosystem structure will be critical for forecasting how T. tubifex populations may grow. These efforts may provide a data and analysis driven basis for decision about how to manage watersheds. The Bigger Picture of Whirling Disease: Towards A More Conceptual Model Although I only covered a small piece of the larger puzzle related to whirling disease, part of my Honors work was developing a larger-picture of how the different players and their interactions may drive disease emergence and persistence and the directionality (positive or negative effects on the disease) with the goal of documenting best- case and worst-case scenarios for the spread of the disease. The ecological drivers above and some information about strength of interactions and impacts allow me to assemble some initial ideas about change dynamics. For 23

example, a worst-case scenario would involve warming temperatures that may allow D. geminata to continue blooming, with its filaments reaching their maximum length (Spaulding, 2007). Abundance of D. geminata would create a yet more suitable habitat for T. tubifex (Lazim and Learner, 1983). At the same time, warming temperatures would also benefit the myxozoan parasite given its preference for activity in warmer conditions (Allen, 2002). Management solutions that could implement best-case scenarios for avoiding whirling disease outbreak would involve partially controlling D. geminata in order to limit T. tubifex prevalence across sites. Further, year-round cooler water temperature would further limit both D. geminata and M. cerebralis, as would stocking streams at 9 weeks old or older so they would be less likely to be affected by M. cerebralis if there happened to be any left in the water (Ryce, 2005). 24

Acknowledgements This research was conducted first and foremost thanks to my parents William and Allyson Byle for introducing me to the Rocky Mountain Biological Lab. I would like to thank Dr. Brad Taylor from Dartmouth College for the intellectual design of the field component of this study at The Rocky Mountain Biological Laboratory. I would like to thank my primary advisor, Dr. Robert Guralnick at the University of Colorado Boulder for all of his assistance in the conceptual model of this project and his unconditional support during this process. I would like to thank Barbara Demmig-Adams for her support through the honors program and Dr. Diane Mcknight for serving on my honors committee. I would like to acknowledge Dr. Alexander Cruz and Dr. David Stock for their recommendations to the Rocky Mountain Biological Laboratory where the field component of this project took place. Thank you to Dr. Jennifer Reithel, Dr. Emily Mooney, Shannon Sprott, and Billy Barr for all of their support at The Rocky Mountain Biological Laboratory. Finally, last but certainty not least, a special thank you to the Rocky Mountain Biological Laboratory donors for their assistance in the funding of this project. 25

Literature Cited Allen, B., Bergersen, E. 2002. Factors influencing the distribution of Myxobolus cerebralis, the causative agent of whirling disease, in the Cache la Poudre River, Colorado. Diseases of Aquatic Organisms 49(1):51-60. Arndt, R. E., E. J. Wagner, Q. Cannon, and M. Smith. 2002. TAM production as related to rearing substrate and diel light cycle. Whirling disease: reviews and current topics. Pages 87-92. El-Matbouli, M. McDowell, TS., 1999. Effect of water temperature on the development, release and survival of the triactinomyxon stage of Myxobolus cerebralis in its oligochaete host. International Journal for Parasitology. 29(4):627 641. Gilbert, M and Granath, W. 2003. Whirling disease of salmonid fish: Life cycle, biology and disease. Journal of Parasitology. 89(4): 658-667. Gilbert M, and Granath W. Jr,. 2007. Epizootiology of Myxobolus cerebralis, the causative agent of salmonid whirling disease in the Rock Creek drainage of west-central Montana. J Parasitol. 93(1):104-19. Hallett, S., and Bartholomew, J. 2007. effects of water flow on the infection dynamics of myxobolus cerebralis. Parasitology 135:371-384. Hnath, JG. 1993. Whirling disease. Great Lakes fisheries comission. Chapter 27/ Fisheries Section. Michigan Department of Natural Resources. Mattawan. MI. Hiner, M., and C. M. Moffitt. 2001. Variation in Myxobolus cerebralis infections in fieldexposed cutthroat and rainbow trout in Idaho. Journal of Aquatic Animal Health 13:124-132. Hendrick.R, Mcdowell, T., 1999. Comparative susceptibility of rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta to Myxobolus cerebralis, the cause of salmonid whirling disease. Diseases of Aquatic Organisms. 37(3):173-183. Hoffman, G L 1990. Myxobolus cerebralis, a Worldwide Cause of Salmonid Whirling Disease Journal of Aquatic Animal Health. 2(1):30-37. Kerans, B. Zale, A. 2002. The Ecology of Myxobolus cerebralis. American Fisheries Society Symposium. 29:145-166. Kerans BL, Rasmussen C. 2004. Differential propagation of the metazoan parasite Myxobolus cerebralis by Limnodrilus hoffmeisteri, Ilyodrilus templetoni, and genetically distinct strains of Tubifex tubifex. Journal of parasitology. 90(6):1366-73. 26

Kilroy, C, Larned ST, and Biggs BJF. 2009. The non-indigenous diatom Didymosphenia geminata alters benthic communities in New Zealand rivers. Freshwater Biology 54: 1990-2002 Krueger, R., Kerans, 2006. Risk of Myxobolus Cerebralis Infection To Rainbow Trout In The Madison River, Montana, USA. Ecological Applications 16:770 783. Lazim, M., Learner,M. 1987 The influence of sediment composition and leaf litter on the distribution of tubificid worms (Oligochaeta) Oecologia.72(1):131-136. Larned, ST, Packman AI, Plew DR, and Vopel K. 2011. Interactions between the mat-forming alga Didymosphenia geminata and its hydrodynamic environment. Limnology & Oceanography: Fluids & Environments 1: 4-22. Lemmon, J. C., and B.L. Kerans. 2001. Extraction of whirling disease myxospores from sediments using the plankton centrifuge and sodium hexametaphosphate. Intermountain Journal of Sciences 7:57-62. McGinnis, S., and Kerans, B. 2013. Land use and host community characteristics as predictors of disease risk. Landscape Ecology. 28(1):29-44. McMurtry, M., Rapport, D. 1983. Substrate Selection by Tubificid Oligochaetes. Canadian Journal of Fisheries and Aquatic Sciences 40:1639-1646, 10.1139/f83-190. Minchella, D.J., and M. E. Scott. 1991. Parasitism: a cryptic detriment of animal community structure. Trends in Ecology and Evolution. 6: 250-254. Nehring, R.B. 2006. Colorado s cold water fisheries: whirling disease case histories and insights for risk management. Special Report Number 79. Colorado Division of Wildlife Nehring, R.B., and K.G. Thompson. 2003. Whirling disease investigations. Colorado Division of Wildlife, Final Report, Federal Aid Project F-237-R-10. Fort Collins. Nickum,D. 1999. Whirling disease in the United States: A summary of progress in research and management. Trout unlimited Report, Trout Unlimited, Arlington, Virginia, 36 p. Reynoldson, Trefor. 1987. The role of environmental factors in the ecology of tubificid oligochaetes- an experimental study. Holarctic Ecology 10: 241-248 Ryce, E, Zale, A..2005. Effects of fish age versus size on the development of whirling disease in rainbow trout. Aquatic Organisms. 63(1):69-76. Schisler,G., Bergersen, E. 2002. Evaluation of risk of high elevation Colorado waters to the establishment of Myxobolus cerebralis. American Fisheries Society Symposium 29:33-41. 27

Spaulding, S.A., Elwell, L. 2007. Increase in Nuisance Blooms and Geographic Expansion of the Freshwater Diatom Didymosphenia geminata. USGS. Whitton, B A. 2009. Biology of the freshwater diatom Didymosphenia: a review Journal: Hydrobiologia. 630(1) DOI: 10.1007/s10750-009-9753-5 Wolf,K., Markiw, M. 1984. Biology contravenes taxonomy in the myxozoa: new discoveries show alternation of invertebrate and vertebrate hosts. Science. 225(4669):1449-52. 28