AN ABSTRACT OF THE DISSERTATION OF

Jayde A. Ferguson for the degree of Doctor of Philosophy in Microbiology presented on December 10, 2010.

Title: Impacts of Multispecies Parasitism on Coho Salmon (Oncorhynchus kisutch) in Oregon

Abstract approved:

______Michael L. Kent

Diseases caused by parasites are recognized as significant sources of mortality in wild fish populations. I assessed the impacts of multispecies parasitism on a threatened stock of juvenile coho salmon (Oncorhynchus kisutch). A crucial prerequisite to this research was proper identification of parasites, which can be difficult for species lacking distinct morphological features. Such was the case when I attempted to identify larval Apophallus sp. (Digenea) and a myxozoan infecting peripheral nerves, which I described as Myxobolus fryeri. The second essential step was to document the geographic distribution of infections in Oregon coastal juvenile coho salmon. I found 21 different parasite species in underyearlings and smolts from

10 different rivers. Some parasites, such as Apophallus sp., were more common in underyearlings than smolts and had a more restricted geographic distribution.

Additionally, I empirically compared histology to the evaluation of wet preparations for parasite detection. The latter was more sensitive, but the former provided data on tissue level impacts. I then focused my research on parasitized coho salmon from one river, the West Fork Smith River (WFSR). The lower abundance of some parasites in smolts, compared to underyearlings suggested parasite associated mortality. Therefore,

I evaluated the persistence of these parasites, as this trend could also be explained by infection recovery. The parasites in my study persisted throughout the overwintering period of these coho salmon. I also conducted a field study involving both pseudo- longitudinal and retrospective approaches. Results indicated that up to 95% of parr from the lower mainstem of the WFSR had infections levels of Apophallus sp. that were associated with mortality. I also performed a laboratory study on wild fish from two consecutive year classes and fish experimentally infected with Nanophyetus salmincola. Parasite associations were evaluated for the following fish performances: size, growth, swimming stamina, and gill Na+,K+-ATPase activity. Parasites were most negatively associated with size and growth, which was remarkably consistent between study years and likely influenced swimming stamina and ATPase activity levels. Taken together, results from the population, individual, and tissue levels, all indicate that these parasites impact this threatened stock of juvenile coho salmon.

These results may have implications for fishery management, as it represents a previously unrecognized limiting factor for this recovering population.

©Copyright by Jayde A. Ferguson December 10, 2010 All Rights Reserved

Impacts of Multispecies Parasitism on Coho Salmon (Oncorhynchus kisutch) in Oregon

by Jayde A. Ferguson

A DISSERTATION

submitted to

Oregon State University

in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

Presented December 10, 2010 Commencement June 2011

Doctor of Philosophy dissertation of Jayde A. Ferguson presented on December 10, 2010.

APPROVED:

______Major Professor, representing Microbiology

______Chair of the Department of Microbiology

______Dean of the Graduate School

I understand that my dissertation will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my dissertation to any reader upon request.

______Jayde A. Ferguson, Author

ACKNOWLEDGMENTS

I would first like to thank my graduate committee: Michael Kent, Carl

Schreck, Kym Jacobson, and Jerry Heidel. Their expertise was invaluable and their mentorship allowed me to reach my goals.

I would also like to express sincere appreciation to the following people for their assistance that made much of this project possible. Research personnel at Oregon

Department of Fish and Wildlife (ODFW), in particular David Jepsen, Erik Suring,

Bruce Miller, Ron Constable, Chris Lorion, and the late Kevin Leader for coordinating and assisting with sampling of wild coho salmon from West Fork Smith River.

Anthony Ngo, Whitney Madigan, and Brittani Goettl for assistance with counting parasites in fish tissues. Justin Sanders for assistance with seining parr coho salmon at

West Fork Smith River and attempting to isolate rDNA sequence for Apophallus sp.,

Virginia Watral for general laboratory support, and Trace Peterson for fish husbandry during periods of my absence and guidance on describing pathological lesions. Rob

Chitwood and Don Stevens for technical support involving fish captivity experiments.

Wataru Koketsu and Ikuo Ninomiya for inventing the congestion rate model and allowing me to apply it to my data. Bill Font and Michelle Steinauer for parasitological support for the Apophallus sp. study. Allison Evans, Elizabeth Dewey,

Yun Fan, and Xuan Che for providing statistical advice and consultations. My committee members, along with Rob Adlard, David Cone, and Stephen Feist, for providing essential manuscript reviews.

I would also like to acknowledge the following agencies and organizations for funding and support. The majority of this research was funded by ODFW through a

Fish Health Graduate Research fellowship (agency grant 010-7032-IAA-FISH).

Additional funding came from an Oregon Sea Grant under the award

NA06OAR41700100 (project R/BT-45-PD) from the National Oceanic and

Atmospheric Administration’s National Sea Grant College Program, U.S. Department of Commerce, and by appropriations made by the Oregon state legislature. The John

L. Fryer Fellowship for fish disease research and the Oregon Lottery Graduate

Scholarship also provided financial support. The Department of Microbiology at

Oregon State University also funded some of this work through a teaching assistantship.

Finally, I am grateful for my family. I am thankful for my parents who have instilled in me the importance of hard work and a good education. I am also deeply indebted for the love and support of my wife, Chrisie. Surely without her sacrifice and support, I would not be where I am today.

CONTRIBUTION OF AUTHORS

Michael Kent contributed to the experimental design and editing of all chapters. Chris Whipps and Stephen Atkinson contributed to writing, data collection, and analysis for Chapter 2. Sophie St-Hilaire, Kenneth Rodnick, and Trace Peterson contributed to writing and the collection of data for Chapter 4. Carl Schreck and

Robert Chitwood contributed to the study design for Chapter 5. Phil Rossignol was involved in creating the congestion rate model used in Chapter 6 and contributed to the writing of this chapter and Kym Jacobson also contributed to writing of this chapter; Lisa Madsen and Jeannie Stifnos contributed to the analysis and interpretation of data for Chapter 7; Jeremy Romer and Michael Glynn contributed to data collection and Carl Schreck contributed to writing for this chapter.

.

TABLE OF CONTENTS

Page

Chapter 1: General Introduction ...... 1

COHO SALMON ...... 2

PARASITES ...... 3

EFFECTS OF PARASITISM ON WILD FISHES ...... 6

Tissue and individual level effects...... 6

Population level effects...... 8

SUMMARY...... 13

REFERENCES ...... 17

Chapter 2: Molecular and Morphological Analysis of Myxobolus spp. of Salmonid Fishes with the Description of a New Myxobolus Species ...... 24

ABSTRACT...... 25

INTRODUCTION ...... 25

MATERIALS AND METHODS...... 27

Fish samples...... 27

Histopathology...... 28

Spore preparation and photography ...... 28

Morphometrics...... 29

Molecular analyses...... 30

DESCRIPTION...... 32

TAXONOMIC SUMMARY ...... 33

REMARKS ...... 34

DISCUSSION ...... 36

TABLE OF CONTENTS (Continued)

Page

REFERENCES ...... 58

Chapter 3: Apophallus sp. Infecting Coho Salmon (Oncorhynchus kisutch) from West Fork Smith River, Oregon...... 65

ABSTRACT...... 66

INTRODUCTION ...... 67

MATERIALS AND METHODS...... 68

Fish samples...... 68

Experimentally infecting definitive hosts ...... 69

Whole mount preparations...... 69

Morphometrics...... 69

RESULTS AND DISCUSSION ...... 70

REFERENCES ...... 82

Chapter 4: Survey of Parasites in Threatened Stocks of Coho Salmon (Oncorhynchus kisutch) in Oregon by Examination of Wet Tissues and Histology...... 85

ABSTRACT...... 86

INTRODUCTION ...... 87

MATERIALS AND METHODS...... 89

Sampling fish ...... 89

Histology...... 89

Wet preparations ...... 90

Parasite identifications ...... 91

Diagnostic method comparison...... 92

RESULTS ...... 93

TABLE OF CONTENTS (Continued)

Page

Muscle parasites...... 93

Gill parasites ...... 95

Viscera and brain parasites by histology...... 98

Diagnostic method comparison...... 100

DISCUSSION ...... 101

Nanophyetus salmincola ...... 101

Apophallus sp...... 102

Unidentified gill metacercariae...... 103

Sanguinicola spp...... 104

Gastrointestinal helminths...... 105

Myxozoans...... 107

Gill protozoans...... 109

Parasite associated mortality...... 110

Method comparison...... 111

REFERENCES ...... 126

Chapter 5: Persistence of Infection by Metacercariae of Apophallus sp., Neascus sp., and Nanophyetus salmincola Plus Two Myxozoans (Myxobolus insidiosus and Myxobolus fryeri) in Coho Salmon Oncorhynchus kisutch ...... 134

ABSTRACT...... 135

INTRODUCTION ...... 135

MATERIALS AND METHODS...... 138

Field collection...... 138

Holding wild fish ...... 139

TABLE OF CONTENTS (Continued)

Page

Sample processing...... 140

Parasite evaluation ...... 141

Statistical analyses ...... 143

RESULTS ...... 145

Nanophyetus salmincola ...... 145

Apophallus sp...... 146

Myxobolus spp...... 147

Neascus ...... 148

Mortalities from lower mainstem group ...... 149

DISCUSSION ...... 150

REFERENCES ...... 162

Chapter 6: Mortality in Coho Salmon (Oncorhynchus kisutch) Associated with Burdens of Multiple Parasite Species ...... 167

ABSTRACT...... 168

INTRODUCTION ...... 169

METHODS ...... 172

Sampling fish ...... 172

Parasite evaluation ...... 172

Inferring parasite-associated mortality...... 173

RESULTS ...... 177

Prevalence ...... 177

Mean intensity...... 178

TABLE OF CONTENTS (Continued)

Page

Variance to mean ratio ...... 178

Truncation of negative binomial model...... 179

Truncation of congestion rate model ...... 180

DISCUSSION ...... 181

REFERENCES ...... 189

Chapter 7: Impacts of Multispecies Parasitism on Juvenile Coho Salmon (Oncorhynchus kisutch) in Oregon ...... 192

ABSTRACT...... 193

INTRODUCTION ...... 194

MATERIALS AND METHODS...... 196

Sampling wild fish ...... 196

Laboratory maintenance of fish ...... 198

Experimental infections ...... 198

Parasite evaluation ...... 199

Evaluating fish performances...... 200

RESULTS ...... 204

Wild fish- brood year 2007 ...... 205

Wild fish- brood year 2008 ...... 207

Hatchery fish...... 211

DISCUSSION ...... 212

Size...... 213

Swimming performance...... 215

TABLE OF CONTENTS (Continued)

Page

ATPase activity...... 218

Parasites ...... 219

Conclusion ...... 220

REFERENCES ...... 229

Chapter 8: General Conclusion...... 234

PARASITE IDENTIFICATION ...... 236

PARASITOLOGICAL SURVEY ...... 238

PERSISTENCE OF INFECTION ...... 242

EVALUATING PARASITE ASSOCIATED MORTALITY WITH FIELD DATA...... 249

LABORATORY STUDY...... 253

Size...... 253

Swimming performance...... 256

Gill ATPase activity...... 259

GENERAL CONCLUSION ...... 261

REFERENCES ...... 264

Bibliography...... 273

LIST OF FIGURES

Figure Page

Figure 2.1. Line drawing of Myxobolus fryeri n. sp...... 45

Figure 2.2. Wet mount preparations of Myxobolus species ...... 46

Figure 2.3. Histological sections of coho salmon (Oncorhynchus kisutch) infected with Myxobolus species ...... 47

Figure 2.4. Frequency distribution of spore lengths comparing Myxobolus species .. 48

Figure 2.5. Phylogenetic trees resulting from analysis of myxozoan small subunit ribosomal DNA ...... 49

Figure 3.1. Photomicrograph of adult Apophallus sp. obtained from domestic chick (Gallus domesticus)...... 78

Figure 4.1. Histology showing infection sites of two common digenean metacercariae and two uncommon adult helminths...... 116

Figure 4.2. Wet preparation examination of parasites...... 117

Figure 4.3. Histologic examination of parasites and the tissues they infect...... 118

Figure 5.1. Parasites detected from coho salmon (Oncorhynchus kisutch) smolt by squash preparations...... 157

Figure 5.2. Histological sections (H&E) of muscle parasites in coho salmon (Oncorhynchus kisutch) smolts...... 158

Figure 6.1. Map of West Fork Smith River showing sampling sites for parr from Brood Year (BY) 2007 and 2008 from the Upper Mainstem (UMS) and Lower Mainstem (LMS)...... 186

Figure 6.2. Frequency distribution of parasites infecting coho salmon (Oncorhynchus kisutch) smolts from West Fork Smith River, using either the negative binomial distribution or the congestion rate model...... 187

Figure 7.1. Example of parasitism negatively associated with fitness endpoints of coho salmon (Oncorhynchus kisutch) smolts...... 222

Figure 8.1. Simplified diagram of some complex interactions of parasites, fish performance, and environment, leading to mortality...... 254

LIST OF TABLES

Table Page

Table 2.1. Myxobolus spp. from Oncorhynchus spp. evaluated for spore morphology, tissue location, and rDNA sequence...... 50

Table 2.2. Synopsis of Myxobolus species from salmonid fishes...... 51

Table 3.1. Synopsis of Apophallus species...... 79

Table 4.1. Prevalence and mean density (in parentheses) of parasites in axial skeletal muscle of coho salmon (Oncorhynchus kisutch) by two different methods ...... 119

Table 4.2. Prevalence and mean density (in parentheses) of gill parasites of coho salmon (Oncorhynchus kisutch) by two different methods...... 121

Table 4.3. Prevalence and mean density (in parentheses) of viscera or brain parasites of coho salmon (Oncorhynchus kisutch) by histology for fry, parr, and smolts from brood year 2006 and parr from brood year 2007...... 123

Table 4.4. Sensitivity and specificity of histology, using evaluations of wet preparation as a standard, for infections in smolts from 10 rivers and parr from one river...... 125

Table 5.1. Summary of parasite prevalence and abundance for fish from the lower mainstem (top), tributaries (middle), or lower mainstem mortalities (bottom) groups from the West Fork Smith River...... 159

Table 5.2. Counts of neascus cysts in individual coho salmon (Oncorhynchus kisutch) parr and smolts from the lower mainstem of West Fork Smith River...... 161

Table 6.1. Prevalence (%), mean intensity, and overdispersion (S2/x) of Apophallus sp. Nanophyetus salmincola in muscle and kidney, and Myxobolus insidiosus in parr from the lower mainstem (LMS) and upper mainstem (UMS) of West Fork Smith River, Oregon for parr and smolt samples from brood years 2007 and 2008...... 188

Table 7.1. Summary of parasite burden and fitness of coho salmon (Oncorhynchus kisutch) smolts of brood years 2007 and 2008 from the lower mainstem and tributary of West Fork Smith River...... 223

Table 7.2. Correlation matrix of explanatory and response variables for linear regression modeling for brood year 2007 lower mainstem coho salmon (Oncorhynchus kisutch) smolts...... 224

LIST OF TABLES (Continued)

Table Page

Table 7.3. Correlation matrix of explanatory and response variables for linear regression modeling for brood year 2008 lower mainstem coho salmon (Oncorhynchus kisutch) smolts...... 225

Table 7.4. Linear regression models of parasite burden and fitness endpoints for wild coho salmon (Oncorhynchus kisutch) smolts, originating from the lower mainstem of West Fork Smith River, held in captivity from brood year 2007 and 2008...... 226

Table 7.5. Linear regression models of parasite burden and fitness endpoints for wild coho salmon (Oncorhynchus kisutch) smolts, from the tributary group of West Fork Smith River, held in captivity from brood year 2007 and 2008...... 228

Chapter 1: General Introduction

2 COHO SALMON

Coho salmon (Oncorhynchus kistuch) are anadromous salmonids native to the

North Pacific, with their southern distribution extending to rivers in Hokkaido, Japan and Baja California, Mexico. The typical life cycle of coho salmon, as described by

Groot and Margolis (1991), consists of hatching from eggs in gravel of streams in the spring, followed by rearing as fry, and then as parr, for about one year throughout the river and smaller tributaries. The following spring they undergo a metamorphosis

(smoltification) and migrate to the ocean as smolts, where they mature to adulthood and return about 18 mo later to natal streams to reproduce and die (semelparous).

Smolts are morphologically distinct from parr, having a more streamlined body and silver color.

Coho salmon are extremely important economically, valued both commercially and recreationally. Unfortunately, over the last half century wild populations have been declining at alarming rates (Lackey et al., 2006). Currently, Oregon coastal coho salmon (Oncorhynchus kistuch), an evolutionarily significant unit, are listed as threatened under the U.S. Endangered Species Act (ESA). In response, Oregon has implemented “the Oregon Plan for Salmon and Watersheds” to study and improve the status of these stocks by collaborating with many different entities, such as state, federal, and tribal governments, as well as private landowners. In support of this effort, we are conducting studies to elucidate the impacts of parasites on these ESA listed stocks of coho salmon.

3 Many studies have tried to reveal the limiting factors of survival for these stocks during their juvenile freshwater stages, which has resulted in the general consensus that loss of habitat plays the biggest role (NRC, 1996). In particular, the overwintering period represents a time of high mortality for coho salmon (Ebersole et al., 2006; Ebersole et al., 2009) as well as other salmon species (Hurst, 2008). The source of overwintering mortality is not always fully understood. For example,

Ebersole et al. (2006) used habitat models and found that coho salmon rearing in the lower mainstem of West Fork Smith River (WFSR) had actually less than expected overwinter survival. However, these models did not evaluate the role of parasitism, which I believe should be considered. For example, Rodnick et al. (2008) reported extremely high digenean and myxozoan parasite burdens in lower mainstem coho salmon parr from this river, and there are many reports demonstrating that parasites can be associated with mortality in wild fishes (e.g., Gordon and Rau, 1982; Kennedy,

1984; Lemly and Esch, 1984; Adlard and Lester, 1994; Kalbe et al., 2002; Bourque et al., 2006), including salmon in the PNW (Johnson et al., 1996; Vincent, 1996; Traxler et al., 1998; Kocan et al., 2004; Jacobson et al., 2008; Nichols et al., 2008).

PARASITES

A critical prerequisite to determining the impacts of parasites on hosts is the accurate identification of parasites in study. However, the taxonomy and identification of many parasites can be problematic, often requiring in depth studies to resolve such issues. Indeed, this has been the focus of my research and is presented in Chapters 2

4 and 3 of this dissertation. Juvenile coho salmon can become infected by essentially every type of parasite phylum (Love and Moser, 1983; McDonald and Margolis, 1995;

Hoffman, 1999). As I have found in my survey (Chapter 4), the most prevalent and abundant parasites infecting juvenile coho salmon in Oregon were digenean trematodes and myxozoans. Both of these types of parasites have complex life cycles involving more than one host.

Myxozoans, which were once thought to be protists, are multicellular, spore forming parasites with an indirect life cycle. For most species, fish are infected with actinospores released from annelid worms, whereas annelids become infected with myxospores from fish. The typical lifecycle (reviewed by Kent et al., 2001) is as follows. In brief, the actinospore attaches to the surface of fish with a polar filament and releases the infectious sporoplasm. The parasite migrates and multiplies in various tissues, depending on the species of myxozoan. Ultimately the parasite reaches a target organ, were it forms a multicellular stage called a plasmodium. The plasmodium continues to enlarge by internal budding and asexual replication, and ultimately spores are formed within this structures. Mature myxospores are released from fish, usually upon death of the host, into the environment where they are ingested by annelids.

Once inside the annelid, the parasite multiplies in the gut of the worm. Gametogony

(sexual reproduction) is followed by sporogony to produce mature multicellular actinospores, which are shed with the worm’s feces into the water to contact the fish host and complete the life cycle.

5 Digenean trematodes are flatworms (phylum Platyhelminthes), which usually require aquatic snails as a first intermediate host. Fish may act as a second intermediate host, in which piscivorous animals are the definitive hosts. The standard life cycle of digenean trematodes, using fish as second intermediate hosts, as described by Ginetsinskaya (1988) is as follows. Eggs are released into the water through the feces of definitive hosts, which then hatch into actively swimming ciliated larvae called miracidia. These miracidia enter aquatic snails either passively or actively by penetrating snail tissues. Once in snails, the parasite metamorphoses into a simple, sac-like stage, called a mother sporocyst. Mother sporocysts undergo a round of parthenogenic (asexual) reproduction, giving rise to either rediae or daughter sporocysts. These second generation parthenitae in turn undergo further asexual reproduction to yield numerous free-living cercariae. Cercariae emerge from snails and move through the aquatic environment, often by swimming with the aid of a tail or simply crawling on substrate, to contact the second intermediate fish host. Fish may become infected either passively (e.g., consuming cercariae) or actively by penetrating cercariae. Cercariae will then migrate to a specific tissue or organ and develop into a less active stage, called metacercariae, which is usually encysted in a cyst of host and parasite origin, or encapsulated by host tissue only. Transmission to the definitive host occurs when infected fish are eaten. Here, in the digestive tract, metacercariae excyst in response to a variety of physical and chemical signals, such as pH levels, digestive enzymes, temperature, etc. Once excysted, digeneans develop into adults, sometimes

6 after migrating to specific locations in the host, and sexually reproduce to release eggs back into the environment for the life cycle to repeat.

EFFECTS OF PARASITISM ON WILD FISHES

Evaluating the effects of parasitism on wild fishes often requires the study of many different biological levels. For example, data on histopathological changes due to parasitism may assist with studies involving associations with parasites and host population recruitment by determing the most pathogenic species infecting juveniles

(Fiest and Longshaw, 2008). Indeed, pathology data can contribute to the understanding of parasite impacts, as not all parasites elicit the same host response.

Tissue and individual level effects

The pathology of myxozoans can range from simple tissue occupancy or displacement, with relatively minimal adverse effects, to intense tissue destruction leading to host death. A general review of myxozoans causing disease is presented by

Lom and Dyková (1995), which I have summarized below. Coelozoic myxozoans reside in the lumina of organs, such as the gall bladder and kidney tubules, and usually are associated with less damage than histozoic species. Exceptions exist, notably

Chloromyxum truttae that infects the gallbladder in salmonids and causes serious chronic disease, presented clinically as anorexia, emaciation, and yellow coloring of skin, which can be fatal. Myxozoan muscle infections can cause disease and/or make fish unmarketable. For example, salmonids infected with Henneguya salminicola have

7 cysts containing spores in suspension that ooze when cut during filleting, termed

“milky condition” (Lom and Dyková, 1995).

Myxobolus insidiosus, a common parasite in our study, does not elicit an immune response when immature (Amandi et al., 1985). However, mature or ruptured plasmodia are destroyed and infiltrated by mesenchymal-like cells and fibroblasts

(Wyatt, 1971). This occurs similarly with Kudoa thyrsites, another intramuscular species, as a severe inflammatory response ensues following rupture of the pseudocysts and release of parasites from the myofibers (Moran et al., 1999).

Like myxozoans, digeneans also vary greatly in their ability to elicit a host immune response. Digeneans can also be site or organ specific and pathological changes are related to the organ they infect. The pathology of metacercarial stages infecting fish is reviewed by Paperna (1995), which I summarize below. Generally, initial infection by high numbers of penetrating cercariae can be fatal, especially with smaller fish because parasites penetrate and encyst deeper in tissues. As with myxozoans, the inflammatory response to encysted metacercariae usually is not as intense as that directed towards ruptured cysts or dying parasites. However, there are examples of marked tissue responses to encysted metacercariae. For example, cysts of some species of neascus and heterophyids infecting skin may provoke the accumulation of melanophores, a condition resulting in ‘black spot disease’. A more pronounced tissue change, potentially interfering with organ function, involves metacercariae of Centrocestus formosanus (heterophyid) infecting gills of blue tilapia

(Oreochromis aureus), which become surrounded by a cartilaginous capsule as a

8 result of hyperplasia of chondrocytes. Similarly, metacercarial cysts of Apophallus brevis infecting muscle of yellow perch are composed of ectopic bone formed in response to infection, and Apophallus sp. causes skeletal deformities in cyprinid fishes from the Willamette River, Oregon (Kent et al., 2004).

Although clinical effects of infection are not often apparent, some studies have been able to detect more subtle and indirect effects of infection. For example,

Lemly and Esch (1984) confirmed that bluegill sunfish (Lepomis macrochirus) heavily infected with Uvulifer ambloplitis (neascus) did not survive the winter because of depleted fat reserves, which was linked to oxygen consumption of encapsulating parasites.

Population level effects

The lack of knowledge on the impacts that parasites have on wild fish populations is partly due to the inherent limitations involved in accessing these hosts in the aquatic environment. Furthermore, the most impacted fish are likely to be quickly removed from the system (Bakke and Harris, 1998) and the effects of chronic, sublethal, disease can be difficult to measure (McCallum and Dobson, 1995).

Nevertheless, there have been methods devised to estimate parasite associated mortality (e.g., Crofton, 1971; Lester, 1984; Rousset et al., 1996; Marty et al., 2003), and these have been applied to fish populations (e.g., Gordon and Rau, 1982;

Kennedy, 1984; Adlard and Lester, 1994). Many of these methods rely on observations of temporal declines in parasite burdens within the host population. For

9 example, using parasite overdispersion (variance/mean ratios), Gordon and Rau (1982) demonstrated that parasite induced mortality occurred in a population of brook sticklebacks (Culaea inconstans) infected with the metacercariae of the trematode,

Apatemon gracilis. The inference from this study was well supported for two reasons.

First, infections by A. gracilis, like many digeneans persist for a long time, so the decline in parasites was not due to recovery of infection. Second, the fish were non migratory, therefore patterns observed were not due to different populations with different burdens.

Observing declines in parasite burdens over a given period of time to indicate parasite associated mortality can be problematic when applied to migratory fish species, such as salmon. This is because fish sampled from different time points may represent different host populations. Therefore, considerable attention in study design is required when applying this approach with salmon. This was exemplified by

Jacobson et al. (2008), who demonstrated that a common digenean, Nanophyetus salmincola, is associated with ocean mortality of coho salmon by using microsatellite

DNA tags to identify salmon stock origins. However, similar studies have not been conducted on the freshwater phase of these fish.

Studying the impacts of parasites on freshwater stages of juvenile coho salmon presents two special problems: 1) many stocks are threatened, so large sample sizes are usually not available, and 2) parr rear as separate, multiple sub-populations and may migrate to the ocean as a randomly mixed smolt population. The latter challenge limits the inference of parasite associated mortality because lower parasite burdens in

10 smolts compared to certain parr, could reflect a bias estimate of infection at the earlier time point (i.e., smolts may be overrepresented by a lightly infected parr population).

Another technique used in field studies is to conduct a retrospective analysis by predicting the parasite distribution in host populations based on observed data from lightly to moderately infected fish. The original concept was proposed by Crofton

(1971). He demonstrated how comparing observed and predicted parasite distributions can estimate mortality associated with macroparasitism. Crofton (1971) used the negative binomial distribution for this procedure, because parasites are highly aggregated among hosts. His method has become widely accepted and is used extensively in theoretical and empirical models (e.g., May and Anderson, 1979;

Lanciani and Boyett, 1980; Anderson and May, 1982; Dobson, 1988; Royce and

Rossignol, 1990; Scott and Smith, 1994; Galvani, 2003). Although Crofton’s techniques rely on an approximation of the distribution, the statistical assay has been proven to be reliable as a theory (Dobson and Carper, 1992).

The importance of studying parasite load (e.g., abundance), rather than prevalence alone, is that the impact of macroparasitism is density dependent (Brass,

1958; Crofton, 1971; May and Anderson, 1979; Dobson, 1988; Burgett et al., 1990;

Galvani, 2003; Holt et al., 2003; Scott and Smith, 1994; Shaw and Dobson, 1995).

Furthermore, knowing the distribution of burdens is essential, as the aggregated distribution of parasites means that most hosts harbor few or no parasites (Smith,

1994; Galvani, 2003). A corollary is that most parasites are in few hosts, many of which may have died and cannot be sampled. Consequently, standard cross-sectional

11 indices, notably prevalence, used for microparasites, yield at best only a weak assessment of macroparasite impact (Smith, 1994) and may be misleading (Dobson and Hudson, 1986). Therefore, it is best to either following parasite burdens in a cohort of hosts, or to perform a retrospective analysis, such as Crofton’s, for associating mortality to parasitism in wild populations under field conditions.

The above methods for assessing impacts are generally applied to macroparasites, such as digenean metacercariae in my studies. Anderson and May

(1979) dichotomized micro- and macro- parasites. Macroparasites are defined, in part, as large parasites that do not multiply in the host of interest. Microparasites are defined, in part, as small parasites (such as bacteria and protozoa) that multiply within the host of interest. To my knowledge, no one has assigned the Myxozoa to one of these categories, and they share characteristics of both. They multiply before sporogenesis within plasmodia, as some are quite small. In contrast, pseudocysts of some myxozoans are macroscopic, and plasmodia of histozooic species, like those of interest in my study, do not generally multiply in fish. Hence, I applied the above methods to Myxobolus spp. in my dissertation.

An alternative approach to studying the effects of parasitism is to conduct laboratory studies on infected fish. In general, most macroparasites do not cause acute disease, and thus effects are often chronic or sublethal. Hence, evaluating fitness correlates, such as growth and swimming performance, is useful in determining the sublethal effects of parasitism. Fish size can influence freshwater juvenile overwinter survival (Quinn and Phil, 1996) smolt success (Holtby et al., 1990) and even ocean

12 migration of salmonids (Beckman et al., 1998). Reduced swimming performance can affect fish survival either directly by fish being purged during heavy winter water flows (Lawson et al., 2004) or indirectly by decreasing predator avoidance (Taylor and

McPhail, 1985). Osmoregulation is vital to anadromous fish migrating, and hence is an important factor for smolt survival (Moser et al., 1991). Poor osmoregulation has also been linked to increased avian predation risk (Kennedy et al., 2007). Gill Na+,K+-

ATPase activity, a major component of osmoregulation (reviewed in McCormick,

2001). Therefore, a reduction in any of these performances, due to parasitism, could indirectly lead to mortality.

There have been several laboratory studies, conducted on either experimentally or naturally infected fish, which evaluate performance. Examples of such studies with salmonids include the following: N. salmincola (Digenea) reducing burst swimming speed (Butler and Milleman, 1971), and immune response (Jacobson et al., 2003);

Crepidostomum farionis (Digenea) reducing hemoglobin and hematocrit (Klein et al.,

1969); Sanguinicola klamathensis (Digenea) reducing growth (Evans, 1974);

Gyrodactylus spp. (Monogenea) inducing cortisol (Stoltze and Buchmann, 2001);

Eubothrium salvelini (Cestoda) reducing swimming stamina, growth, and survival

(Boyce, 1979), saltwater adaptation (Boyce and Clarke, 1983), and altering migration orientation (Garnick and Margolis, 1990); Myxobolus arcticus (Myxosporea) reducing swimming stamina (Moles and Heifetz, 1998); Parvicapsula minibicornis

(Myxosporea) reducing swimming recovery rates (Wagner et al., 2005); and sea lice,

Lepeophtheirus salmonis (Copepoda) reducing osmoregulation (Birkeland and

13 Jakobsen, 1997), and swimming and cardiovascular performance (Wagner et al.,

2003).

SUMMARY

The aim of my dissertation research was to elucidate the impacts of infection by multiple parasite species on Oregon coastal juvenile coho salmon. Prior to this research, no information was available on the effects, or potential effects, these parasites may have on the survival of these populations, as parr or smolts. Indeed, the role of parasitism as a limiting factor, for these threatened coho salmon stocks, has not been addressed by fishery management programs.

Although some studies have evaluated the effects of chronic parasitism on different juvenile coho salmon stocks (e.g., N. salmincola; Jacobson et al., 2008), few have examined interactions of multiple pathogens on salmon (Sandell, 2010).

Accordingly, I performed a set of studies to assess the impact of multiple parasites on these threatened stocks. The specific objectives of my dissertation were: 1) determine the identity, abundance, prevalence and geographic distribution of parasites infecting juvenile coho salmon within the Oregon Coastal Evolutionarily Significant Unit

(ESU) (Chapters 2-4), 2) evaluate parasite associated mortality of different parasite species in juvenile coho salmon in the field (Chapters 5 and 6), and 3) determine the associations of different parasite species with selected performance endpoints (Chapter

7). Objectives 2 and 3 were done using fish from the WFSR.

14 The first important step in this research was to accurately identify parasite species infecting coho salmon in our study. However, often this task requires in depth taxonomic investigations and even the description of a new parasite species. Indeed, in

Chapter 2, I describe a new myxozoan species, Myxobolus fryeri, infecting peripheral nerves of skeletal muscle. Similarly, in Chapter 3, I report results from an extensive study attempting to identify Apophallus sp., which involved many different life stages of the parasite.

After properly diagnosing infections, documenting the infection levels and geographic distribution of these parasites in these fish was the next crucial step. In

Chapter 4, I conducted a parasitological survey of coho salmon underyearling and smolts from 10 Oregon coastal watersheds. I present data on parasite identification, prevalence, density, and geographic distribution from both age groups of these fish.

Additionally, I compared two diagnostic methods used to detect parasites in this study, histology and the evaluation of wet preparations. Lastly, I described the histopathology of these infections because this provides data on impacts of parasites at the tissue and organism level (Feist and Longshaw, 2008). This study showed that fish from the lower mainstem of WFSR were particularly heavily infected with N. salmincola, Apophallus sp., neascus, M. insidiosus and M. fryeri, and these parasites in fish from this river became the focus for the remainder of my dissertation research.

Although a decline in parasite burden in host populations may infer parasite associated mortality (e.g., Lester, 1984), an alternative explanation could be that the host recovers from infection. Therefore, in Chapter 5, I evaluated whether or not the

15 parasites of interest in our study are capable of persistent infections. To assess this, I collected wild parr from WFSR in the fall of 2006, evaluated a subsample of fish for parasite burden, and then held the remaining fish in captivity until the following spring for later parasite evaluation.

To study the impacts of parasites on coho salmon at the population level, I conducted a field study at WFSR over 3 years to associate trends in parasite burden to host mortality. In Chapter 6, I used three main approaches: 1) comparing parasite burdens between parr and smolts, 2) comparing parasite overdispersion (variance to mean ratio) between parr and smolts, and 3) a retrospective analysis of parasite distribution in smolts. Although smolts represent a mixed population of parr from different river locations, I combined data from parr originating from the lower and upper mainstem (high and low parasite burden, respectively) to provide the best representation of the parr for this river. Many studies have used parasite overdispersion (variance to mean ratios) to evaluate parasite associated mortality in fishes (e.g., Gordon and Rau, 1982; Jacobson et al., 2008). However, this method requires the investigators to study the same host population over time. Therefore, a retrospective analysis is most useful for circumventing this problem with migratory fishes, as was the case in my study. This procedure involves using the negative binomial distribution to comparing the observed parasite distribution to a predicted one (Crofton, 1971). The difference between these two distributions indicates parasite associated mortality. I also used a similar, but more biologically meaningful model

16 recently developed by Dr. P. Rossignol, Oregon State University, and colleagues, in my retrospective analyses.

In Chapter 7, I related parasites in our study to measured values of performance endpoints with laboratory experiments. Here, I used linear regression to study the relationships of parasite burden and the following fitness endpoints: size, growth, swimming stamina, and Na+,K+-ATPase activity. I also compared the fitness responses of heavily infected fish, which came from the lower mainstem of the WFSR, to lightly infected fish, which originated from the upper mainstem of this river. Lastly,

I performed a controlled infection experiment on naive hatchery fish and compared their growth to uninfected controls to corroborate my studies with naturally infected fish.

Finally, in Chapter 8, I discuss the importance of my results. Here, I assemble all of my results from the different approaches used and connect these data with a discussion of the overall impact that parasites may have on Oregon coastal coho salmon. I also provide a general conclusion to the dissertation in this chapter.

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24 Chapter 2: Molecular and Morphological Analysis of Myxobolus spp. of Salmonid Fishes with the Description of a New Myxobolus Species

Jayde A. Ferguson1, Stephen D. Atkinson1, Christopher M. Whipps2, and Michael L.

Kent1

1 Department of Microbiology, Oregon State University, 220 Nash Hall, Corvallis,

Oregon, 97331

2 College of Environmental Science and Forestry, State University of New York, 1

Forestry Drive, Syracuse, New York 13210

Published in Journal of Parasitology

American Society of Parasitologists Inc., Lawrence, Kansas

Issue 94

25 ABSTRACT

While investigating the parasite fauna of wild coho salmon Oncorhynchus kisutch (Walbaum, 1792), histological examination provided evidence of a new species of Myxobolus (Myxozoa: Myxosporea) infecting nerves of skeletal muscle.

Spores were morphologically similar to those of the intramuscular Myxobolus insidiosus Wyatt and Pratt, 1963, both having pyriform spores with clavate polar capsules. However, the former developed exclusively in the nerves of skeletal muscle rather than in myocytes. We examined both species of Myxobolus derived from either coho salmon, Chinook salmon Oncorhynchus tshawytscha (Walbaum, 1792), cutthroat trout Oncorhynchus clarkii (Richardson, 1836), and rainbow trout Oncorhynchus mykiss (Walbaum, 1792) from freshwater in Oregon. Spore morphology, small subunit ribosomal RNA gene (rDNA) sequences, and site of infection were compared.

Myxobolus arcticus Pugachev and Khokhlov, 1979 has pyriform spores, infects the central nervous system of many salmonids, and is found in the Pacific Northwest. It was, therefore, included in the analyses to rule out conspecificity with the new species.

Together, these data clearly show that the Myxobolus sp. from peripheral nerves in the skeletal musculature of coho salmon, rainbow trout, and cutthroat trout is a new species, described herein as Myxobolus fryeri n. sp.

INTRODUCTION

We are conducting a study on the impacts of chronic infections by myxosporeans and digenean trematodes on wild coho salmon Oncorhynchus kisutch

26 (Walbaum, 1792) in Oregon (Rodnick et al., 2008). A critical prerequisite of this work is the accurate identification and classification of these parasites. Of the myxosporeans, species of Myxobolus are the most frequently encountered thus far, which is not surprising given that myxosporeans are common fish parasites and

Myxobolus is the most speciose genus, with some 800 species described (Eiras et al.,

2005; Lom and Dyková, 2006). Myxobolus spp. have been reported to infect neural tissues of salmonids in western North America, including Myxobolus arcticus

Pugachev and Khokhlov, 1979, Myxobolus cerebralis Hofer, 1903, Myxobolus kisutchi Yasutake and Wood, 1957, Myxobolus neurobius Schuberg and Schröder,

1905, and Myxobolus neurotropus Hogge, Campbell, and Johnson, 2008 (see Hogge et al., 2004, 2008; Kent et al., 1994; Andree et al., 1999). Skin-infecting Myxobolus squamalis (Iversen, 1949) and muscle-infecting Myxobolus insidiosus Wyatt and Pratt,

1963, are also common parasites of salmonids in this region. Myxobolus insidiosus infects skeletal myocytes of several salmonid fishes: Chinook salmon Oncorhynchus tshawytscha (Walbaum, 1792); coho salmon; cutthroat trout Oncorhynchus clarkii

(Richardson, 1836); and rainbow trout Oncorhynchus mykiss (Walbaum, 1792) (Wyatt and Pratt, 1963; Wood, 1968; Amandi et al., 1985). During our ongoing parasitological survey, a Myxobolus sp. was identified that did not correspond to any described species. Thus, based on analysis of tissue tropism, spore morphology and small subunit (SSU) ribosomal DNA (rDNA) sequence, we describe a new species of

Myxobolus infecting nerves in skeletal muscle of Oncorhynchus spp., which is most closely related to M. insidiosus.

27 MATERIALS AND METHODS

Fish samples

Rotary screw traps were used to collect 20 wild coho salmon yearlings from

Mill Creek (Ck.) of the Siletz River (R.) system (44º 44´ 44.89´´ N, 123º 47´ 35.72´´

W) and 20 coho salmon from the upper mainstem of Lobster Ck. of the Alsea R. system (44º 14´ 43.26´´ N, 123º 38´ 28.38´´ W) Oregon (Table 2.1). Fish were killed on site by blunt head trauma, weighed, measured, dissected, and samples of muscle were frozen on solid CO2. The remainder of each carcass was placed in 10% buffered formalin for histological examination. Five other infected fish were obtained from routine wild and hatchery fish surveys conducted by the Oregon Department of Fish and Wildlife (ODFW) (Table 2.1). These fish were initially diagnosed with M. insidiosus infection based on the examination of fresh tissue squashes by ODFW staff.

These samples comprised: wild coho salmon from Wolf Ck. of the Siuslaw R. system

(43° 48' 57.6" N, 123° 15' 17.99" W); wild Chinook salmon from Cougar Lake from the McKenzie R. system (44° 6' 54" N, 122° 14' 16.79 W"); Chinook salmon from

Leaburg State Fish Hatchery (SFH) on the McKenzie R. (44° 8' 8.15" N, 122° 36'

33.48" W; Stock176R); wild cutthroat trout from Fall Ck. from the McKenzie R. system (44° 57' 28.79" N, 122° 49' 4.79" W); rainbow trout from Alsea SFH on the

Alsea R. (44° 25' 22.08" N, 123° 33' 56.16" W) (Table 2.1). One sockeye salmon

Oncorhynchus nerka (Walbaum, 1792) infected with Myxobolus arcticus was collected from Vancouver Island in 1998 by M.K.

28 Histopathology

Skeletal muscle, visceral organs, brain, and gills were processed for histology and slides were stained with either hematoxylin and eosin, Luxol fast blue (for neural tissues), Giemsa (for myxosporean spores), or Masson’s trichrome (for connective tissue) at the Oregon State University Veterinary Diagnostic Laboratory based on standard methods (Carson, 1997).

Spore preparation and photography

Based on the histological findings, infections were categorized as either intramuscular or intermuscular (neural) (Table 2.1). Parasite spores from the Mill Ck. and Lobster Ck. samples were concentrated as follows: approximately 0.5 g muscle was placed into a 50 ml conical tube, thawed, and digested with pepsin (0.75% pepsin and 1.5% HCl in sterile water) for 7 h at 37 C with constant agitation in a orbital incubator shaker (Lab-Line Instruments, Melrose Park, Illinois). The digested material was centrifuged at 4,500 g for 20 min in an Allegra 6KR Centrifuge

(Beckman Coulter, Fullerton, California) and supernatant removed. Twenty µl of concentrated spore material was placed on a microscope slide for examination.

Additionally, concentrated spores from fish infected with the intermuscular (neural)

Myxobolus sp. were stained with Lugol’s iodine as described by Lom and Dyková

(1992) to detect the presence or absence of an iodinophilic vacuole.

Myxobolus arcticus spores had been previously collected from brain, purified, concentrated and frozen when originally sampled in 1998. The sample was thawed

29 and examined as above. For all other samples, photographs were made of unconcentrated spores in fresh tissue squashes (Table 2.1).

Both histological sections and fresh specimens were examined with a Leica

DMLB microscope and digital images were made using a SPOT Advanced digital camera and software (Diagnostic Instruments Inc., Sterling Heights, Michigan).

Morphometrics

Samples from 8 fish having infections categorized as either intramuscular (4) or intermuscular (4) (Table 2.1) were used for morphological analysis. Spore length and width were measured from 21 to 55 spores/fish to the nearest 0.1 µm. Whenever possible, the number of polar filament coils was counted, and the presence or absence of an intercapsular appendix was noted. Measurement data were analyzed using S-

PLUS® software (Insightful Corp., Seattle, Washington). A 1-way ANOVA was used to determine significant differences in mean measurements among groups and a

Bonferroni correction was made to P-values to account for family-wise error. Paired

Student’s 2-tailed t-tests were used on pooled data when mean measurements had no significant difference within each group. Mean measurements of spore length and width for spores of M. arcticus and that of M. insidiosus and the intermuscular

(neural) Myxobolus sp., were compared using a Kruskal-Wallis test because of the small sample size and the indication of an assumed lack of normality (based on graphical displays) for the M. arcticus data set. P-values < 0.05 were considered significant.

30 Molecular analyses

Approximately 25 mg of infected muscle tissue was aseptically excised from each infected fish. Purification of total DNA from fish tissues were performed using the Qiagen DNeasy Blood & Tissue Kit following the manufacture’s instructions

(Qiagen Inc., Valencia, California). Similarly, total DNA was extracted from 25 µl of solution containing purified spores of M. arcticus.

Polymerase Chain Reaction (PCR) was performed on all samples in 50 µl reaction volumes containing 1.5 mM MgCl2, 0.2 mM dNTP, 2.5 Units Taq polymerase, 0.25 µM of each primer and 4 uL of template DNA. A ~1000bp 5` fragment of rDNA was amplified with either ERIB1 (Barta et al., 1997) or 18E (Hillis and Dixon, 1991) together with ACT1r (Hallett and Diamant, 2001). An overlapping

~1200bp 3` fragment was amplified with Myxgen4F (Diamant et al., 2004) and 18R

(Whipps et al., 2003) or ERIB10 (Barta et al., 1997). Amplifications were performed on a MJ Research Peltier 200 Thermocycler with initial denaturation at 95 C for 3 min, followed by 35 cycles of 94 C for 30 sec, 55 C for 45 sec, 72 C for 1 min 15 sec, and a final extension at 72 C for 7 min. Amplicons were excised from an agarose gel and purified using the QIAquick Gel Extraction Kit, or purified directly from PCR products using a QIAquick PCR Purification Kit, both per manufacture’s instructions

(Qiagen, Valencia, California). DNA was quantified using a DNA spectrophotometer

(NanoDrop Technologies Wilmington, DE). Direct sequencing was performed using the primers listed above as well as 18I, 18J (Jirků et al., 2006) and ALL1f (Hallett et al., 2002) to complete the sequences where required. Reactions were carried out with

31 the ABI BigDye Terminator Cycle Sequencing Ready Reaction Kit v3.1, using the

ABI3730 DNA Analyzer (Applied Biosystems, Foster City, California).

Sequences were assembled manually using BioEdit (Hall, 1999) and verified as myxozoan by GenBank BLAST search (Altschul et al., 1997). Sequences from multiple hosts, and locations with both tissue tropisms were aligned and compared by visual inspection in BioEdit. Alignment of sequences from M. insidiosus and the intermuscular (neural) Myxobolus sp. allowed us to identify that the 3’ fragment contained the most variations and hence this region (ca 1,000 bp) was then amplified from 6 fish with intramuscular infections and 9 fish with neural infections to validate our morphological data (Table 2.1).

To estimate the phylogeny of salmonid-infecting species of Myxobolus using

SSU rDNA, a preliminary test analysis (not shown) was conducted which included

116 SSU sequences from all Myxobolus species available on GenBank and relevant members of other genera within the ‘Myxobolus clade’ as defined by Fiala (2006).

From this framework, the data set was reduced to 44 sequences to speed subsequent analyses by reducing non-relevant well defined clades to a few representative sequences and eliminating duplicate sequences for some species. Sequences were first aligned with Clustal X version 1.8 (Thompson et al., 1997) and edited manually to assure correct alignment of homologous regions and to remove regions with uncertain homology based on secondary structure models for myxosporean rRNA of Holzer et al. (2007). Phylogenetic analyses were carried out on this 1357 character alignment as follows. Parsimony analyses were conducted in PAUP*4.0b10 (Swofford, 2003),

32 employing a heuristic search with 10 repetitions of random sequence addition, and tree bisection and reconnection branch swapping. Bootstrap confidence values were calculated with 100 replicates. Bayesian analyses were conducted in MrBayes

(Ronquist and Huelsenbeck, 2003) under a general time reversible model, with 106 generations, tree sampling every 100 generations, with a burn-in of 100 trees.

DESCRIPTION

Myxobolus fryeri n. sp.

(Figures 2.1 - 2.4)

Diagnosis: Myxozoa; Myxobolidae. Presporogonic stage not observed.

Polysporous, ellipsoidal plasmodia, 35 µm by 25 µm, in peripheral nerves of skeletal musculature. Mature spores ovoid to pyriform in frontal view with attenuated apex.

Thick spore wall with intercapsular appendix approximately one-seventh of spore length (Figures 2.2 a-g). Spores pyriform in sutural view with one valve appearing slightly more concave (Figure 2.2 h). Prominent sutural ridge with indistinct suture.

Large iodinophilic vacuole present. Polar capsules clavate, one slightly longer than the other, occupying approximately three-fourths the spore length, with 8-10 polar filament coils (Figures 2.2 a-h). Mean fresh spore measurements in µm ± SD (n =

131): length, 12.9 ± 0.8; width 8.6 ± 0.7; thickness (n=8) 7.2 ± 0.4. Polar capsule measurements: (n = 104) 7.1 ± 0.6 x 3.0 ± 0.3, and (n=102) 7.2 ± 0.7 x 2.9 ± 0.4. See

Table 2.2 for range.

33 TAXONOMIC SUMMARY

Type host: Coho salmon Oncorhynchus kisutch (Walbaum, 1792).

Other hosts: Cutthroat trout Oncorhynchus clarkii (Richardson, 1836), rainbow trout

Oncorhynchus mykiss (Walbaum, 1792).

Type locality: Mill Creek of Siletz River, Lincoln County, Oregon (44º 44´ 44.89´´ N,

123º 47´ 35.72´´ W).

Other localities: Fall Creek, Lane County Oregon (44° 57' 28.79" N, 122° 49' 4.79"

W) for cutthroat trout and Alsea State Fish Hatchery, Lincoln County Oregon (44° 25'

22.08" N, 123° 33' 56.16" W) for rainbow trout.

Site of infection: Peripheral nerves of skeletal musculature.

Prevalence: Nine of 20 coho salmon from Mill Creek.

Prevalence in other hosts: Parasite detected in one cutthroat and one rainbow trout from diagnostic examinations of 10-20 fish.

Type specimens: Parasyntypes of 2 Giemsa-stained histological sections (G465048 and G465049) and 1 hematoxylin and eosin stained histological section (G465050) are deposited in the parasitology collection of the Queensland Museum, Brisbane,

Australia. Extra material (spores preserved in 95% ethanol) has also been deposited in the parasitology collection of the Queensland Museum, Brisbane, Australia.

Etymology: The species was named in honor of the late Dr. John L. Fryer, an eminent fish biologist and microbiologist.

34 REMARKS

Hematoxylin and eosin stained histological sections of skeletal musculature infected with M. fryeri n. sp. showed ellipsoidal, intermuscular, polysporic pseudocysts measuring up to 35 µm by 25 µm (Figure 2.3). Masson’s trichrome stained sections of infected musculature with M. fryeri n. sp. indicated the absence of the parasite in connective tissue and Luxol fast blue stained sections confirmed the presence of M. fryeri n. sp. in nervous tissues. In contrast, histological sections of muscle infected with M. insidiosus showed large polysporic pseudocysts that were intramuscular, not intermuscular. These were spindle shaped and measured up to 187

µm by 68 µm (Figure 2.3), consistent with the original description by Wyatt and Pratt

(1963).

There was not a significant difference of mean spore length measurements among M. insidiosus or M. fryeri n. sp. samples (corrected P = 0.06 and P = 0.31, respectively, ANOVA with Bonferroni corrections). This allowed us to pool the data and compare samples. The mean spore length of M. fryeri n. sp. (12.9 µm) was significantly shorter than the mean spore length of M. insidiosus (14.6 µm) (P <

0.00005, t-test) (Figure 2.4). There was also no significant difference of mean spore width measurements among M. insidiosus or M. fryeri n. sp. samples (corrected P =

0.35 and P = 0.50, respectively, ANOVA with Bonferroni corrections). However, there was a significant difference of spore width between pooled samples of each species (P < 0.00005, t-test); with M. insidiosus (avg. 9.4 µm) being wider than M. fryeri n. sp. (avg. 8.6 µm). Spores of both species were otherwise similar in shape,

35 with tapered anterior ends and clavate polar capsules exceeding half the length of the spores. Our spore measurements of M. insidiosus correlated with that of the type description by Wyatt and Pratt (1963) (Table 2.2).

Other than M. insidiosus, Myxobolus vartanyanae Landsberg and Lom, 1991 is the only other Myxobolus species known to infect muscle tissues of salmonids.

However, spores of M. vartanyanae are round to oval and smaller in all dimensions than M. fryeri n. sp. (Table 2.2). Spores of M. arcticus that we examined had clavate polar capsules. Average spore dimensions ± SD (n = 14) were 15.1 ± 0.6 µm by 9.7 ±

0.5 µm, which were significantly longer and wider than M. fryeri n. sp (P < 0.00005,

Kruskal-Wallis test). These dimensions were also significantly longer and wider than the spores of M. insidiosus (P = 0.02, Kruskal-Wallis test). Additionally, these measurements were slightly larger than those of the type description of M. arcticus made by Pugachev and Khokhlov (1979), although it was not reported whether these were based on fresh or fixed material (Table 2.2). Distinguishing M. fryeri n. sp., M. insidiosus, and M. arcticus by spore morphology alone was difficult because spore lengths of the 3 (although significantly different) showed considerable overlap

(Figures 2.2, 2.4).

The SSU rRNA genes of M. fryeri n. sp. and M. insidiosus were estimated to be 2120 bp, including primers ERIB1 and ERIB10. The 2 species differed by 0.5%

(10 substitutions over 2066 bp) and were most closely related to each other phylogenetically (Figure 2.5). The rDNA sequences of the M. fryeri n. sp., i.e., parasites from nerves, were identical to one another (EU346370-2) and distinct from

36 the sequences (EU346373-7) of the intramuscular M. insidiosus. These data suggested that each infection type represented a distinct species. The M. insidiosus sequence was only 73% similar to the existing sequence on GenBank (U96494, Andree et al., 1999).

The M. arcticus sequence (EU346378) was 89.2% and 89.4% similar to M. fryeri n. sp. and M. insidiosus, respectively. It was identical to M. arcticus from sockeye salmon from Japan (AB353129), but only 66.5% similar to the first M. arcticus sequence archived in GenBank some 10 yr ago (AB085176, Andree et al.,

1999).

Estimates of phylogeny of M. fryeri n. sp. based on SSU rDNA placed it as a sister taxon to M. insidiosus, within a group composed exclusively of 8 species of

Myxobolus from salmonids (Figure 2.5). SSU rDNA sequences from other salmon- infecting Myxobolus species, e.g., M. cerebralis, were positioned elsewhere in the tree, which included the previously generated sequences of M. insidiosus and M. arcticus by Andree et al. (1999). Bayesian analyses tended to yield fewer polytomies and higher support values for tree topology, but these may have been overestimates relative to bootstrap support values from parsimony analysis (Simmons et al., 2004).

Thus we treat this phylogenetic estimate with caution and consider our parsimony analysis a more conservative result (Figure 2.5).

DISCUSSION

Molecular and morphological data indicated that M. fryeri n. sp. is a distinct species, very closely related to M. insidiosus. These myxosporeans can be

37 distinguished from one another by site of infection, SSU rDNA sequence, and spore length. Myxobolus fryeri n. sp. was always found in the nerves and had significantly shorter spores than M. insidiosus, which was always found in myocytes. The latter was originally described from large, spindle-shaped cysts comprised of pyriform shaped spores in myocytes of Chinook salmon collected in Oregon (Wyatt and Pratt, 1963), whereas M. fryeri n. sp. forms smaller, ellipsoidal cysts in nerves.

Myxobolus insidiosus and M. fryeri n. sp. share the same vertebrate host species, site of infection at the organ level, and geographical region, which likely explains some of the variability attributed to M. insidiosus in earlier records. Wyatt

(1979) described a subspecies, Myxobolus insidiosus clarki, based on its occurrence in a new host, cutthroat trout, and indicated the site of infection as “connective tissue usually, rarely found inside bundle of muscle fibers.” Amandi et al. (1985) proposed that this subspecies, based on host specificity, was invalid because infections they identified as M. insidiosus were found in both rainbow and cutthroat trout. Further complicating the subspecies description, measurements of M. i. clarki were made from formalin-fixed spores, a method known to shrink spores up to 15% (Parker and

Warner, 1970; Hedrick et al., 1991). We suggest that Wyatt (1979) encountered a mixed infection of M. insidiosus and M. fryeri n. sp., which skewed the average measurements of “M. i. clarki” to be equal to or greater than fresh spore measurements of M. fryeri n. sp., as M. insidiosus spores are longer (Table 2.2).

The traditional approach of classifying myxosporeans uses phenotypic differences such as spore morphology, host specificity, and tissue tropism. Spore

38 morphology is useful because those of species of Myxobolus have fairly rigid spores and have little variability in size and other features. However, using spore morphology alone to separate species of Myxobolus can be particularly difficult, as many of the ~800 species in this genus have similar spores and a paucity of distinguishing characteristics at the light microscopy level. Indeed, separation of M. fryeri n. sp., M. insidiosus, and M. arcticus by spore morphology alone requires critical measurement of many spores.

Multiple species sometimes infect the same tissue within the same host. For example, in salmonids several morphologically similar species infect neural tissues, i.e., M. cerebralis, M. neurobius, M. kisutchi, and M. neurotropus (Table 2.2).

However, even morphologically similar species can be differentiated using molecular data (Hogge et al., 2004). The SSU rRNA gene is commonly used for molecular systematics in the Myxosporea, with sequences of about 170 species currently available in GenBank. This gene is particularly useful for elucidating relationships in this group because it is highly variable between very closely related species (Kent et al., 2001). This of course is based on our current understanding of the species level concept for this group. Within the genus Myxobolus interspecific differences may be as low as 0.2% (Kent et al., 2001; Bahri et al., 2003; Fiala, 2006) to greater than 30 %

(based on our conservative sequence alignment). Intraspecifically, Molnár et al. (2006) observed as much as 3.6% variability in sequence between replicates of Myxobolus dujardini, and 0.1 – 2.3 % in 5 other species of Myxobolus. Within M. cerebralis,

Andree et al. (1999) reported some SSU variation (0.8%) between geographic isolates,

39 Arsan et al. (2007) found rDNA sequence variation at a single location (Alaska), and

Whipps et al. (2004) reported sequence variability within a single spore. For other genera, intraspecific variation may range from <1% as in Kudoa thyrsites (Whipps and

Kent, 2006) to as much as 2.6% in Myxidium lieberkuehni (Schlegel et al. 1996).

In our study, although M. fryeri n. sp. and M. insidiosus SSU rDNA differed only by 0.5%, this variation occurred at fixed loci across 15 isolates and no other intra- specimen variation or noise was apparent in the sequences. Given the parasites had distinct tissue tropism and statistically longer spores, these data indicate the presence of 2 species of Myxobolus. Likewise, Easy et al. (2005) reported cryptic species of

Myxobolus in trout-perch Percopsis omiscomaycus (Walbaum, 1792); Myxobolus intramusculi was intramuscular, whereas Myxobolus procerus was found in connective tissue. The separate sites of infection correlated directly to statistical differences in spore length and consistent SSU rDNA differences (2%). Both this and the current study suggest that sympatric speciation may occur within the Myxosporea through occupation of distinct microhabitats within a single host species.

Phylogenetic analysis of sequences of Myxobolus spp. from salmonids and other closely related species showed that most of these form a clade, the exception being Myxobolus cerebralis (Figure 2.5). Similarly, most species of Myxobolus of cyprinids form a large cluster that includes a few from other host families (Fiala,

2006). Thus, although species of Myxobolus generally cluster by host family, there are some inconsistencies. Within certain lineages or genera, tissue specificity can correlate to estimates of phylogeny, but this is inconsistent across the Myxozoa (Blaylock et al.,

40 2004; Eszterbauer, 2004; Cone et al., 2005; Fiala, 2006; Molnár et al., 2006).

Salmonid-infecting species of Myxobolus are predominantly found in nervous tissue.

Muscle-specific M. insidiosus is an exception (Table 2.2), but is phylogenetically nested within the neurotropic species (Figure 2.5). Morphologically, M. fryeri n. sp. and M. insidiosus have very similar myxospores and are phylogenetic sister species.

However, M. arcticus (which infects brain), is also morphologically similar but is more closely related to species infecting brain. Thus, there is no phylogenetically consistent pattern congruent with host family, tissue tropism, or spore morphology.

The sequences we generated for M. insidiosus were 27% different from the sequence available in GenBank (number U96494, Andree et al., 1999). We are confident of the accuracy of our sequence because we sequenced several isolates and our spore measurements corresponded to the original description. Andree et al. (1999) did not provide spore measurements to verify species identity, and several lines of evidence suggest these authors may have generated a SSU sequence from an undescribed myxosporean. The M. cerebralis-like sequences of Andree et al. (1999)

(Figure 2.5 - those in parentheses) are very similar to each other, contain ambiguous nucleotides, have many differences in conserved helical regions of the SSU, and conversely there is little variation in the loop regions where most variation would be expected. Understandably, the pioneering efforts of Andree et al. (1999) to generate rDNA sequences from species of Myxobolus were done without the availability of numerous comparable sequences, a plethora of PCR primers, and SSU secondary structure models that we have today. A study by Molnár et al. (2006) that included

41 detailed spore measurements provides updated SSU sequences for Myxobolus bramae and Myxobolus ellipsoides that also differ from Andree et al. (1999). The sequence of

M. arcticus (AB085176) from British Columbian sockeye salmon (Andree et al. 1999) was dramatically different (33.5%) from 3 M. arcticus sequences (AB353128-30) from Japan. This discrepancy prompted us to sequence M. arcticus from British

Columbia, which proved identical to the Japanese sequence. Finally, the SSU sequence we generated for Myxobolus sandrae (EU346379) was only 65.9% similar to that of Andree et al. (1999). In light of these data, we suspect that the sequences of

Andree et al. (1999) represent that of a single undescribed species. This case highlights the importance of reporting spore measurements and including multiple isolates when generating DNA sequences from species for the first time.

Moreover, the GenBank sequence of M. neurobius (number AF085180,

Andree et al. 1999) is also likely to represent that of an undescribed species, as it was generated from rainbow trout from California and its spores appear more spherical than those described from the original host, brown trout Salmo trutta fario L., 1758 from Germany. The sequence is different from M. kisutchi (EF431919) and M. neurotropus (DQ846661) (Hogge et al., 2004; 2008). Interestingly, Holzer et al.

(2006) provided an 854 bp sequence (AM042702) of Myxobolus sp. from the brain of brown trout from Scotland and those spores in histological sections are remarkably similar to those of M. neurobius based on the original description by Schuberg and

Schröder (1905), with slightly tapered anterior ends. Holzer et al. (2006) did not assign their specimen to M. neurobius because the sequence was different from “M.

42 neurobius” in GenBank (AF085180). Yet, given the host and geographic location of the Myxobolus sp. described by Holzer et al. (2006), their species may actually be M. neurobius. Sequences of M. neurobius from brown trout from Germany and other questionable species are needed to resolve these issues, although we suspect that M. neurobius may not occur in North America at all.

In a report of myxosporeans of the USSR, Shulman (1966) reported that some spores of M. neurobius are pyriform with a distinctly narrow anterior end, contrary to the original description by Schuberg and Schröder (1905). Pugachev and Khokhlov

(1979) erected the species M. arcticus for these spores, which they described from the brain of Pacific salmon. They also provided a supplemental diagnosis of M. neurobius in the USSR, clearly separating the 2 species. Although differences between these species was clarified, it took years for this Russian-language manuscript to be recognized by English speaking scientists, hence there are several reports of “M. neurobius” with pyriform spores infecting the brains of Oncorhynchus spp. from the

Pacific Northwest (e.g., Bailey and Margolis, 1987; Quinn et al., 1989). Records of these infections were considered to be M. arcticus (see Urawa and Nagasawa, 1988;

Kent et al., 1994; Awakura et al., 1995; McDonald and Margolis, 1995).

The difficulties of relying on spore morphology for species identification have led authors to recommend that SSU rDNA sequence be included when new species are described (e.g., Kent et al., 2001; Lom and Dyková, 2006). Moreover, type host and locality should be used when assigning new sequences to existing species, as recently done by Hogge et al. (2008). A number of salmonid-infecting species of Myxobolus

43 remain to be unambiguously characterized. For example, Mo et al. (1992) described infections by Myxobolus aeglefini Auerbach, 1906, a neurotropic parasite of Atlantic cod Gadus morhua (L., 1758), in salt-water reared Atlantic salmon Salmo salar L.,

1758. They based their identification on the consistency of spore morphology and close proximity of the farm rearing pens. Because many species of Myxobolus are host family-specific, this is a significant finding that remains to be confirmed or refuted by genotypic characterization. Lom and Dyková (1992) proposed that Myxobolus farionis

Gonzalez-Lanza and Alvarez-Pellitero, 1984 is likely a synonym of M. neurobius, as both have similar spores and infect trout in Europe. Likewise, based on morphology and spore size, M. squamalis and Myxobolus soldatovi Akhmerov, 1960 might be synonymous, as both infect the integument of Pacific salmon (Table 2.2).

When assessing impacts of various parasites on wild coho salmon in Oregon, it is critical that individual species be identified lest the prevalence and influence of each be misinterpreted. Great care should be taken when using solely spore morphology to distinguish morphologically similar myxosporeans, such as M. fryeri n. sp., M. insidiosus, and M. arcticus. All 3 species have overlapping spore dimensions, making routine identification based on spore size impractical as many precise measurements are required for statistical analysis. To differentiate these 3 species, we recommend histology to determine tissue tropism or PCR based techniques.

44 Acknowledgments

This study was funded largely by ODFW through a grant to S. Hilaire and K.

Rodnick and an ODFW Fish Health Graduate Research fellowship to J.A.F. (agency grant 010 7032-IAA-FISH). This report was partially prepared by Oregon Sea Grant under award NA06OAR41700100 (project R/BT-45-PD) from the National Oceanic and Atmospheric Administration’s National Sea Grant College Program, U.S.

Department of Commerce, and by appropriations made by the Oregon state legislature.

The statements, findings, conclusions, and recommendations are those of the authors and do not necessarily reflect the views of these funders. Support to J.A.F. was also provided by the John L. Fryer Fellowship. C.M.W. is grateful for the services of

Nevada Genomics Center, which is supported in part by a grant from the Nevada

IDeA Network of Biomedical Research Excellence (2 P20 RR016463). Sequencing support to S.A. was provided by an Oregon State University General Research Fund grant. Thanks to D. Jepsen and C. Banner, Oregon Department of Fish and Wildlife for providing hatchery and wild fish; R. Adlard and D. Cone for manuscript review; and J. Richard, Fisheries and Oceans Canada, for providing M. arcticus material. S.A. was supported by a University of Queensland postgraduate research scholarship.

45

Figure 2.1. Line drawing of Myxobolus fryeri n. sp. in sutural view (a, c) and frontal view (b, d). Bar = 5 µm.

46

Figure 2.2. Wet mount preparations of Myxobolus species. Myxobolus fryeri n. sp. (a- h), Myxobolus insidiosus (i-l), and Myxobolus arcticus (m-p). Nomarski phase contrast. Bar = 5 µm. Spore in sutural view (h and l).

47

Figure 2.3. Histological sections of coho salmon (Oncorhynchus kisutch) infected with either Myxobolus fryeri n. sp. or Myxobolus insidiosus. Bar = 20 µm, v = vacuole. (a, b) Spores of M. fryeri n. sp. (arrows) in intermuscular nerves. Hematoxylin and eosin (a) and Giemsa (b). (c) Plasmodium replete with M. insidiosus spores in myocyte. Hematoxylin and eosin.

48

Figure 2.4. Frequency distribution of spore lengths comparing Myxobolus species. Myxobolus fryeri n. sp. (a), Myxobolus insidiosus (b), and Myxobolus arcticus (c).

49

Figure 2.5. Phylogenetic trees resulting from analysis of myxozoan small subunit ribosomal DNA. Fifty percent majority rule consensus tree resulting from 472 most parsimonious trees (left) reveals a polytomy of the five main clades. Bootstrap support is indicated next to nodes. Bayesian consensus tree (right) yielded similar results to parsimony with greater resolution of tree topology. Posterior clade confidence values were 100% unless otherwise noted at nodes. Names in bold represent species with DNA sequences generated for this study. Shaded taxa indicate salmonid host origin. Unitalicized names in parentheses represent DNA sequences available on GenBank that are inconsistent with the species to which they are assigned.

50 above above above above above above above GenBank GenBank Accession # 1546 Identical as 1546 Identical as 1334 Identical 1374 Identical as 1374 Identical as 1535 Identical Bp sequenced ssue location, and rDNA sequence. N = ssue location, and rDNA

# Spores # Spores measured of infection 1998 Wet mount/brain 14 1201 EU346378 1201 14 mount/brain 1998 Wet spp. evaluated for spore morphology, ti Oncorhynchus Alsea SFH Nov 03, 2005 Histology/N 22 1051 EU346371 Island, Canada Canada Island, M = myocyte, NA not available. SFH Leaburg Cougar Lake 2004 25, Oct 2005 May 12, squash/M tissue Histology/M 32 37 1946 1914 EU346374 EU346375 Mill Ck 200715, Apr Mill Ck Histology/N 200715, Apr NA Histology/N NA Mill Ck Mill Ck 200715, Apr Mill Ck 200715, Apr Histology/N Mill Ck 200715, Apr Histology/N 21 200715, Apr Histology/N Mill Ck 55 Histology/N 26 2066 200715, Apr NA 1411 Histology/N EU346370 1379 Identical as NA Identical as Mill Ck 200715, Apr Histology/M NA 1559 Identical as Wolf Ck 2007 May 17, Mill Ck tissue squash/M 200715, Apr Lobster Ck 20 Histology/M 2007 Apr14, Histology/M 2120 31 NA EU346373 2075 1416 EU346376 EU346377 Fall Ck SFH Apr200404, tissue squash/NA NA 2048 EU346372 Vancouver Vancouver spp. from spp. from kisutch kisutch kisutch kisutch kisutch kisutch kisutch clarkii kisutch kisutch kisutch kisutch nerka

tshawytscha tshawytscha

. . . O. O. O. O. O. O O O. O. O. O. / a / b / c / e / f

Myxobolus / d / O. O. O / a O. / a / b / c / e / f / i / d / g / Table 2.1. musculature, nerves of skeletal Species / isolate Host M. fryeri Type material M. fryeri Location M. fryeri M. fryeri Collectiondate Examination/ site M. fryeri M. fryeri M. fryeri h M. fryeri / M. fryeri M. insidiosus mykiss O. M. insidiosus M. insidiosus M. insidiosus M. insidiosus M. insidiosus M. arcticus 51 accession # EU346378 NR , T. = 1999 AF085176 Salmo

, S. = Reference Sequence Sequence Reference Khokhlov, 1979 1979 Khokhlov, Salvelinus Material measured Fresh Fresh study Andree et al., Present Fresh NR Pugachev & NR Pugachev , Sa. = Polar capsule width & length L: NR NR W: L: NR NR W: L: 6.6-9.0 6.6-9.0 L: W: 2.5-3.5 Oncorhynchus Spore shape, Length Length shape, Spore (L),Width (W), & (T) Thickness Round-ovoid, L: NR NR W: T: NR Pyriform, (14.1-16.1) 15.1 L: W: 9.7 (8.3-10.2) T: NR Oval, L: 14.3-16.5 W: 7.6-7.7 T: NR water, FW = freshwater, SW = saltwater. All measurements in µm. in µm. = saltwater. All measurements = freshwater, SW water, FW Infection Infection site Hind- brain Hind- brain spinal cord species from salmonid fishes. O. = salmonid species from location & habitat Columbia, FW Columbia, FW Siberia, FW Brain & Myxobolus

British British kisutch, nerka, nerka,

O. O. nerka O. nerka O. malma Sa. malma, neiva Sa. , NR = not reported, BW = brackish , NR = not reported, BW Species Host Geographic M. arcticus M. arcticus M. arcticus Thymallus of Table 2.2. Synopsis 52 accession # NR

Reference Sequence Sequence Reference 1906 Material measured Stained Mo et al., 1992 NR Fresh Andree et al., 1999 U96492 NR Hofer, 1903 1903 NR NR Hofer, Fresh Hedrick1991 et al., NR Polar capsule width & length L: 4.5-5.0 4.5-5.0 L: Fresh 3.5-4.0 L: Auerbach, W: 2.5-3.0 L: NR NR W: L: 4.5 4.5 L: NR W: 5.4 L: 3.3 W: Spore shape, Length Length shape, Spore (L),Width (W), & (T) Thickness Round-elliptical, L: 10.8-11.7 W: 9.9-10.4 7.2-9.0 T: Oval-circular, L: 8.5-10.5 W: 8.5-10.5 T: NR Round-ovoid, L : NR NR W : T : NR Ovoid Ovoid 9.0 L: 7.2 W: T: NR Oval, 9.9 L: 8.4 W: 6.5 T: Infection Infection site Head cartilage Head cartilage Brain cartilage Brain cartilage Brain cartilage location & habitat Germany, Germany, SW Norway, SW West Virginia, FW FW FW Germany, Germany, California, mykiss mykiss

Gadus aeglefinus S. salar O. O. mykiss O.

* M. aeglefini M. aeglefini M. cerebralis M. cerebralis M. cerebralis Species Host Geographic Table 2.2. Continued. 53 accession # NR NR Reference Sequence Sequence Reference Alvarez-Pellitero, Alvarez-Pellitero, 1984 Pellitero, Alvarez- 1984 Material measured Fresh Gonzalez-Lanza, Gonzalez-Lanza, Fresh Gonzalez-Lanza, Fresh Fresh Present study EU346370 Fresh Present Polar capsule width & length L: 4.9 4.9 L: (4.5-5.5) W: 2.4 (2-2.8) 5.0 L: (4.0-6.0) W: 2.7 (2.2-3.5) L: 6.9 6.9 L: (5.9-8.1) W: 2.8 (2.0-3.3) Spore shape, Length Length shape, Spore (L),Width (W), & (T) Thickness Oviform-pyriform, Oviform-pyriform, (8.5-10) 9.2 L: W: 6.7 (6.0-7.5) (4.5-5.0) 4.7 T: Ellipsodial- subspherical, (9.0-11.0) 10.0 L : W: 8.6 (8.0-9.5) (6.0-7.0) 6.6 T: Ovoid-pyriform, Ovoid-pyriform, (11.1-14.8) 12.9 L: W: 8.6 (7.2-10.1) (6.4-7.7) 7.2 T: Infection Infection site spinal cord spleen, liver, ureter nerves of of nerves skeletal muscle location & habitat Spain, FW Brain & Spain, FW Kidney, Oregon, FW Oregon, Peripheral

kisutch, kisutch, clarkii mykiss,

O. fario O. fario O.

n.

sp Species Host Geographic M. farionis S. trutta M. fryeri M. ibericus S. trutta Table 2.2. Continued. 54 accession # NR Reference Sequence Sequence Reference 1963 Material measured Fresh Wyatt and Pratt, Fresh study Andree et al., 1999 EU346373 U96494 Fresh Present Fixed Wyatt, 1979 1979 NR Wyatt, Fixed Polar capsule width & length L: 8.8 8.8 L: (7-10.2) W: 3.3 (2.6-4.5) L: NR NR W: 7.7 L: (5.3-9.3) W: 3.2 (1.9-3.9) L: 8.3 8.3 L: (7.5-9.5) W: 2.9 (2.5-3.5) Spore shape, Length Length shape, Spore (L),Width (W), & (T) Thickness Oval (12.8-17.3) 15.0 L: W: 10.3(9-11.5) (6.4-9) 7.5 T: Round-ovoid, L: NR NR W: T: NR Pyriform, (13.3-15.9) 14.7 L: W: 9.4 (7.9-10.5) (6.8-8.3). 7.4 T: Oval, (11.5-13.5) 12.5 L: W: 8.4 (7.5-9.0) (7.5-8.4) 7.3 T: Infection Infection site muscles muscles Muscle muscles muscle, connect- ive tissue location & habitat Oregon, FW Oregon, Skeletal FW Oregon, Skeletal FW Oregon, Skeletal Oregon, FW Somatic Somatic FW Oregon,

O.

clarkii

O. tshawy- tscha O. tshawy- tscha O. tshawy- tscha, kisutch O. Species Host Geographic M. insidiosus M. insidiosus M. insidiosus M. insidiosus clarki Table 2.2. Continued. 55 accession # NR NR NR ., 1999 AF08518 Reference Sequence Sequence Reference Yasutake & Wood, 1957 in Shulman, 1966 Shulman Schuberg & & Schuberg Schröder, 1905 Material measured Fresh Hogge et al., 2004 EF431919 Fixed, in sections & NR Konovalov Fresh Andree et al Fixed, in sections Polar capsule width & length L: NR NR W: L: (3.8-5.5) L: (3.8-5.5) NR W: 5.0-6.6 L: W: 2.5-4.0 L: NR NR W: L: 6.0-7.0 6.0-7.0 L: NR W: Spore shape, Length Length shape, Spore (L),Width (W), & (T) Thickness Oval, L: NR NR W: T: NR Subspherical-oval, Subspherical-oval, L: (7.0-8.5), W: (6.5-7.0) T: (3.5-3.8) Oval , 9.6-12 L: W: 7.5-10.5 6.6-6.9 T: Round-ovoid, L: NR NR W: T: NR Ovoid, Ovoid, L: 10.0-12.0 8.0 W: 6.0 T: Infection Infection site Mid-brain & Spinal cord Mid-brain & Spinal cord Abdom- inal serosa Neural tissue Nervous Nervous system location & habitat Washington, FW FW Kamchatka, Russia, SW California, FW Germany, Germany, FW Washington, Washington,

, mykiss

Sa. Sa. alpinus alpinus O. nerka T. thymallus O. Species Host Geographic M. kisutchi O. kisutch M. kisutchi O. kisutch M. krokhini M. neurobius M. neurobius Table 2.2. Continued. 56 accession # NR

Reference Sequence Sequence Reference Hoshina, 1949 NR Material measured Fresh Hogge et al., 2008 DQ846661 1954 Iversen, Fresh Fresh Andree et al., 1999 U96495 Formalin Formalin fixed NR Akhmerov, 1960 NR NR 1960 Akhmerov, NR Polar capsule width & length L: 5.8 5.8 L: (5.0-6.9) 3.7 W: 4.4 L: (3.9-5.1) W: 3.1 (2.6-3.9) L: NR NR W: L: 4.6 4.6 L: (3.7-5.8) W: 3.2 (2.1-3.5) L: 4.0-4.2 4.0-4.2 L: W: 2.0-2.2 Spore shape, Length Length shape, Spore (L),Width (W), & (T) Thickness Oval-circular, Oval-circular, (11.2-13) 11.8 L: W: 10.8(10.4-12.3) T: (8.4-9.1) 8.8 (8.1-9.9) 9.0 L: W: 8.6 (7.7-9.9) (5.6-7.7) 6.7 T: Round-ovoid, L: NR NR W: T: NR Subspherical, Subspherical, (8.2-10.6) 9.3 L: W: 8.6 (7.4-9.5) (5.5-8.3) 7.2 T: Round, 8.0-9.5 L: W: 8.0-9.5 T: NR Infection Infection site spinal cord Round-oblong, Scales Scale pocket surface of scales Skin, scales location & habitat California, California, FW Pacific Northwest, FW USA, Asia, BW Asia, BW Idaho, FW Brain & & FW Brain Idaho,

Russia, SW Under Under SW Russia, Amur basin, basin, Amur , , keta keta mykiss nerka mykiss

O. O. O. O. kisutch O. O. tshawy- tscha O. Species Host Geographic M. neuro- tropus M. salmonis M. soldatovi M. squamalis M. squamalis Table 2.2. Continued. 57 accession # NR NR

Reference Sequence Sequence Reference Shulman 1966 1966 Shulman NR 1973 Donec et al., Material measured NR Konovalov, in NR Konovalov, Fresh Holzer et al., 2006 AM043702 L: 4.5-6.0 4.5-6.0 L: W: 2.5-3.3 Polar capsule width & length L: 5.2-6.5 5.2-6.5 L: W: 2.7-3.6 4.0 L: (3.2–4.7) W: 2.6 (2.1–3.2) L: 9.5-12.3 9.5-12.3 L: W: 8.5-10.0 7.3-8.0 T: Spore shape, Length Length shape, Spore (L),Width (W), & (T) Thickness Oval, L: 9-11 W: 8.0-10.5 5.9-7.2 T: end (7.8–9.2) 8.8 L: W: 6.4 (5.8–7.0) (4.7–5.5) 5.1 T: Infection Infection site Gall bladder Brain anterior Oval, tapered location & habitat Russia, FW Muscles Spherical-oval, Kamchatka, Kamchatka, Russia, FW Scotland, FW S. ischchan T. arcticus T. arcticus arcticus S. trutta trutta

Species Host Geographic M. thymalli M. vartanyane † Myxobolus sp. Table 2.2. Continued. 58

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65 Chapter 3: Apophallus sp. Infecting Coho Salmon (Oncorhynchus kisutch) from West Fork Smith River, Oregon

Jayde A. Ferguson1 and Michael L. Kent1*

1 Department of Microbiology, Oregon State University, 220 Nash Hall, Corvallis,

OR 97331 USA

* [email protected]

In preparation for Diseases of Aquatic Organisms

Inter-Research Science Center Oldendorf/Luhe, Germany

66 ABSTRACT

Diseases, including those caused by parasites, are recognized as significant sources of mortality in wild fish populations. We are studying the effects of multiple parasite infections on a threatened stock of juvenile coho salmon (Oncorhynchus kisutch) from the West Fork Smith River, Oregon. A crucial prerequisite to this research is the accurate identification of parasites, which can often be difficult for species lacking distinct morphological features or that require study of different life stages. These limitations occur when attempting to identify many species of larval digenean trematodes, which are commonly associated with mortality in fishes.

Apophallus species, like most heterophyids, are well known for causing disease in fishes, although there is a great deal of variability in these reports. We have identified metacercariae of Apophallus sp., which has been associated with host mortality in our studies. Given the importance of this parasite indicated by our research, it is imperative that a better understanding of identification be obtained, lest incorrect assessments may be made on a potentially cryptic species. Therefore, we sought to determine the true identity of this Apophallus sp. by infecting chicks with muscle tissue from coho salmon sampled from our study area to examine parasite morphology. Our results on the measurements of our speciemens indicated that this parasite may represent a new species. However, upon review of the literature, we concluded that criteria for species level identification are lacking and therefore could not assign a species name until further research, such as molecular typing, has resolved these issues.

67 INTRODUCTION

Diseases, including those caused by parasites, are recognized as significant sources of mortality in wild fish populations (Möller & Anders 1986, Adlard & Lester

1994, Bakke & Harris 1998, Jacobson et al. 2008). Specifically, digeneans infecting fish as metacercariae can cause disease (Paperna, 1995) and have been shown to be importantly associated with mortality (e.g., Gordon and Rau, 1982; Lemly and Esch,

1984; Lafferty and Morris, 1996; Jacobson et al., 2008). We are studying the effects of multispecies parasitism on a threatened stock of juvenile coho salmon (Oncorhynchus kisutch) from West Fork Smith (WFSR), Oregon.

The first critical step in evaluating the impacts of parasites is to accurately identify every parasite species within the host. This can often be difficult for species lacking definitive morphological characteristics or that may require further development to an identifiable life stage. These limitations occur when attempting to identify many species of larval digenean trematodes. We have identified metacercariae of Apophallus sp., which has been associated with host mortality in our studies

(Ferguson et al., 2010). As with other metacercariae, the proper species identification of this Apophallus sp. requires obtaining adult specimens from definitive hosts.

Heterophyids are notorious for causing pathology in fishes (Paperna, 1995).

With Apophallus species, there is a great deal of variability in the presentation of pathological changes exhibited with infections. For example, skin infections of

Apophallus brevis commonly cause the so-called ‘black spot disease’, but this same parasite causes ectopic bone formation in muscle of only yellow perch (Perca

68 flavescens) (Pike and Burt, 1983). Apophallus brevis is also associated with reduced growth and survival of yellow perch (Johnson and Dick, 2001). Similarly, apparently all species, except Apophallus venustus, are capable of causing black spot disease

(Cameron, 1936). It is often unclear if such differences in disease presentation represents different Apophallus species or if there are other factors involved, such as host species. However, such variation in tissue location and pathological changes stresses the importance of proper species identification, as these differences could reflect that certain species may impact coho salmon in our study more than others.

Therefore, we sought to determine the proper identification of Apophallus sp. infecting juvenile coho salmon from WFSR, Oregon, by obtaining adult worms fed experimentally to chicks. We found that the measurements of our adult worms did not closely correspond with any previously described Apophallus species. Furthermore, review of the literature showed that there is considerable confusion regarding the taxonomy and identification of members of the genus Apophallus.

MATERIALS AND METHODS

Fish samples

Ten coho salmon parr were captured by beach seine in the lower mainstem, near the smolt trap, of WFSR, Oregon in September of 2008. Fish were transported to

Oregon State University’s (OSU) Salmon Disease Laboratory. Fish were killed with an overdose of buffered tricaine methanesulfonate (MS-222) to study morphology of metacercariae and to infect chicks.

69 Experimentally infecting definitive hosts

Eight, 1-day old unfed domestic chicks (Gallus domesticus) were obtained from Featherland Farms, Oregon. Infected muscle tissue from 10 coho parr was made into slurry and concentrated by centrifugation at 4,500 g for 15 min in a 6KR

Centrifuge (Beckman Coulter). This concentrated slurry was force fed to four chicks with a pipette. Each chick received approximately 150 metacercariae. The other four chicks were untreated controls. Chicks were housed at OSU’s Laboratory Animal

Research Center. Chicks were examined for recovery of adult trematodes on 3 (n = 1),

6 (n= 1), and 8 (n = 2) days post infection. Chicks were euthanatized by CO2, necropsied, and intestines were opened in saline and examined under a stereoscope.

Worms were removed, heat fixed, and stored in Dietrich’s solution (30 ml 95% ethanol, 10 ml formaldehyde, 2 ml glacial acetic acid, and 58 ml distilled water) for later staining.

Whole mount preparations

Specimens were stained with Mayer's carmalum or Semichon's carmine and cleared in accordance with Pritchard and Kruse (1982) and mounted on microscope slides with Permount or Canada balsam (Fisher Scientific).

Morphometrics

Specimens were examined with a Leica DMLB microscope and digital images were made using a SPOT Advanced digital camera and software (Diagnostic

Instruments Inc.).

70 RESULTS AND DISCUSSION

Consistent with previous reports (Ferguson et al., 2010), fish were heavily infected, with several hundred heterophyid metacercariae in less than a gram of muscle tissue. For experimental infections, about 2-20 worms from each chick were recovered from the posterior 10-15 cm of the intestine. Immature worms were obtained from 3 day old post infected chicks, but sexually mature worms with eggs were recovered from 6 and 8 day post infected chicks.

Examination of adult Apophallus sp. whole mounts revealed few distinct morphological features. Worms had a spiny tegument, long esophagus, and pharynx close to the oral sucker (Figure 3.1), all features consistent for Apophallus species.

Although eggs often obscured other structures, the presence of oblique to tandem testes was observed (Figure 3.1). The cecum was bifurcated anterior to the acetabulum, and the egg follicles appeared to be disbursed, extending beyond the bifurcation (Figure 3.1). Our specimens measured (n = 20) 196.8 µm (150.1-230.7) in length and 57.2 µm (35.0-78.3) in maximum width. Eggs measured (n = 13) 23.1 µm

(20.1-26.2) in length and 12.0 µm (10.4-14.3) in maximum width. These worms were consistently smaller than any previously described Apophallus species (Table 3.1).

Apophallus sp. infecting coho salmon in our study may represent an undescribed species, as measurements of our worms did not approach the measurements of most species. Indeed, our specimens were almost 2-8 times shorter than other Apophallus species (Table 3.1). There may be many factors influencing the size variability of parasites, such as host type, extent of relaxation, or fixation method

71 (Meyer and Olsen, 1975). Lyster (1940) reported a wide range in body shape of worms between avian and mammalian hosts, despite handling specimens the same.

Although it is likely that the Apophallus sp. in question is a novel species, we do not assign the present worm to a new or existing species for two reasons. First, review of the literature shows great overlap in morphological features of Apophallus (Table 3.1), for example A. venustus and Apophallus similis have overlapping ranges of body and egg size, oblique testes, and vitellaria extending anterior to the ventral sucker.

Second, there is currently no consensus on criteria for differentiating Apophallus species.

The nomenclature and taxonomy of Apophallus species has been discussed previously (e.g., Lyster, 1940). In short, the genus, Apophallus Luhe, 1909, was created for a species infecting a European gull (Larus ridibundus) that was misidentified as Distomum lingua by Creplin in 1898, which was renamed Distomum mühlingi by Jagerskiold a year later. Also in 1899, Looss placed this species in the genus Tocotrema. Luhe decided to use this specimen as the type species for his new genus, naming it Apophallus mühlingi (Jagerskiold, 1899) Luhe, 1909. Later in 1919,

Skrjabin and Lindtrop made the genus Rossicotrema, with Rossicotrema donicum from Russian cats (Felis catus) and dogs (Canis lupus familiaris) as the type species, which he differentiated from Apophallus based on attributes of the vitellaria and testes.

A year later in America, Ransom, 1920, not having accessibility to Russian literature, created the genus Cotylophallus based on the same criteria. He described two species,

Cotylophallus venustus from cat, dog, and fox (Vulpes lagopus); and Cotylophallus

72 similis from harbor seal (Phoca vitulina). He also described A. brevis from a gull

(Larus delawarensis). Later in 1931, Price described Apophallus crami from a

California gull (Larus californicus) in Oregon and established that Tocotrema Loss

1899 is a synonym of Cryptocotyle, and that Rossicotrema and Cotylophallus are synonymous with Apophallus.

Much of the misunderstanding involved with Apophallus can be attributed to the confliction on the classification criteria of closely related genera used by earlier scientists. Witenberg (1929) regarded the relationship of the testes to each other to be the most important aspect. Whereas Price (1931) contended that the anterior or posterior position of the acetabulum, relative to the genital pores, was the most fundamental character. Lastly, Ciurea, (1924) considered both these criteria in conjunction with body shape and development to be important generic features. The most widely accepted classification is that of Price (1931).

The biggest challenge and thus confusion, lies at separating species within

Apophallus. This has resulted in the synonymization of many species. For example, A. donicus has been synonymized with three species (similis, venustus, and brevis) under various genera names (Table 3.1). There have been six or so keys proposed for

Apophallus species, none of which clearly demonstrate the criteria required for distinguishing species. This is because there is a great deal of similarity in described

“species”, which are based on characteristics that are highly variable. Cameron (1936) used three main features: body shape, juxtaposition of testes, and placement of yolk glands (vitellaria). He acknowledged the variability of the former two characters,

73 thereby placing much emphasis on the later. He divides A. mühlingi, A. donicus and A. brevis from A. venustus (= C. similis) based on yolk glands not reaching the level of the acetabulum for the former and these glands reaching the esophageal bifurcation in the latter. Within the first group, A. mühlingi is long and narrow with testes almost tandem; A. donicus is oval-pyriform with oblique testes; A. brevis is elongate-pyriform with oblique testes. He disagreed with Price 1931 in synonymizing A. venustus with A. donicus mainly based on minor differences in the testes and. Cameron (1937) also argues that these species differ for biological reasons based on host specificity and notes that A. mühlingi in the strict sense is a European parasite.

Lyster (1940) borrowed specimens from Cameron and concluded that the use of vitellaria is not sufficient enough to warrant differentiation of these species based on the variability between specimens. He proposed a new character, the “gonotyle”

(the genital sucker), in addition to the arrangement of the acetabulo-genital complex.

He noted two types of gonotyles: 1) “papillae-like, far removed from progenital type” and 2) “non papillae-like, showing affinity to a true genital sucker”. He assigned A. brevis to the former and A. venustus and A. donicus to the later, which could be separated further by the distinctness of these structures. He also noted that A. mühlingi and his newly described Apophallus imperator had similar gonotyls, but differed in size and shape. However, Miller, 1941) re-studied the morphology of A. imperator

Lyster, 1940, and A. brevis Ransom, 1920 and concluded that the use of the gonotyle was insufficient of specific importance, but was a valid for generic separation.

74 Therefore, it appears that there is currently no acceptable standard for separating

Apophallus species.

Further complicating maters is the description of two species based on larval forms. Apophallus americanus was described from the intestines of piscivorous fish by Van Cleave and Mueller (1932) using only two immature worms. Although, they felt this may have represented an accidental infection in aberrant hosts. Warren (1953) described metacercariae of Apophallus itascensis from yellow perch with an unusual cyst of ‘hard and glassy’ composition. There is little doubt that this represented the unique infection of A. brevis in yellow perch, which was further studied by Sinclair

(1972), Pike & Burt (1983), and Taylor et al., (1994). Further complicating the taxonomy of A. brevis is that Miller (1941) synonymized A. imperator, the causative agent of black spot in speckled trout (Salvelinus fontanalis), based on life cycle studies. However, Sinclair (1972) provided evidence that these two Apophallus were separate species. Although adult worms of both species are nearly identical morphologically, he showed that there were biological differences in metacercariae

(host specificity, sexual development, cyst type and location) and adult (maturation time and longevity) stages. Thus, A. brevis is most likely synonymous with two species and confused with a third.

Generally, metacercariae infecting fishes exhibit low host specificity, although there may be differences in host response to the same digeneans (Paperna, 1995).

However, there are some reports, particularly in the early literature, claiming that digenean species within the same genus are host-specific at the family level for fishes.

75 For example, Cameron (Cameron, 1945) contended that A. brevis was specific to speckled trout and that even rainbow trout (Oncorhynchus mykiss) were refractory to infection. However, earlier (Cameron, 1937) he also stated earlier that A. venustus infects 9 different families of fish from 6 different orders. Host specificity of larval stages in the molluskan hosts tend to be more restricted (Paperna, 1995). Apophallus venustus utilizes the pleuroceriid snail Goniobasis livescens (Cameron, 1937), whereas

A. brevis (and A. imperator) have been shown to use the hydrobiid snails, Amnicola limosa (Cameron, 1945). Apophallus sp. from our studies have been found in snails from both these families (Chapter 7) and Malek (1980) states that heterophyids are somewhat unusual in displaying plasticity in use of molluskan hosts.

Reports on the pathological changes elicited by different Apophallus species also vary greatly. For example, even though A. brevis infects and causes black spot disease in many fish species, this parasite causes ectopic bone formation only when infecting yellow perch (Pike and Burt, 1983; Taylor, et al., 1994). Another unusual lesion associated with Apophallus sp. is skeletal deformities in infected cyprinids from the Willamette River, Oregon (Kent, et al., 2004). Interestingly, these lesions were reported to be more severe in younger fish, which is consistent with limb malformations due to Ribeiroia ondatrae infections in frogs (Romansic, 2007).

Regarding black spot disease, Cameron (1937; 1945) argued that A. venustus predominantly infects musculature, rarely skin, and is not associated with forming black spots. He also asserted that A. venustus and A. brevis did not share fish hosts, but perhaps this observation could have actually been due to differences in response to

76 infection. Indeed, it may take a considerable amount of time for black spots to form, which may be influenced by water temperature (Ferguson et al., 2010). Apparently

Cameron’s observations are not completely substantiated, as A. venustus has been synomynized by many authors (Table 3.1). The infections in our coho salmon samples occur most often in skeletal muscle along the myomere (Ferguson, et al., 2010).

However, occasionally gill and skin infections, associated with black spots, have been observed (pers. obs.). Niemi and Macy (1974) studied black spot disease in Oregon fishes and concluded that A. donicus was the culprit. They studied the life cycle of this parasite and infected coho salmon with cercariae released from Fluminicola spp., which killed small fish with as few as 35 cercariae.

We have found Apophallus sp. from our studies to be much more abundant in lower mainstem parr from the WFSR, Oregon, compared to parr rearing in the upper reaches of this river (Rodnick, et al., 2008). Furthermore, we have consistently observed that outmigrating coho salmon smolts from this river have substantially lower parasite burdens than lower mainstem parr from WFSR (Ferguson et al., 2010).

Therefore, we have proposed that this pattern is likely due to differential mortality of fish with high parasite burdens, which has been supported by both field and laboratory studies (Ferguson et al., submitted).

In conclusion, we were unable to confidently assign the Apophallus sp. in our studies to a species based on the lack of easily observable features and confusion in agreement of specific criteria. There is also great variability in the pathological changes associated with different Apophallus reports, which could be due to

77 differences in parasites, hosts, or both. The size of our worms is much smaller than any reported previously for Apophallus species in the literature. The smallest record is for A. donicus, which is still about twice as long as our speciems. However, the predominant site of infection (i.e., skeletal muscle) more closely resembles A. vensustus or A. mühlingi, the later supposedly being restricted to Europe. Previous reports of species from this genus occurring in Oregon include Apophallus sp. from cyprinid fishes (Kent et al., 2001), A. donicus from gulls and many fish species including coho salmon (Shaw, 1947; Niemi and Macy, 1974) and A. crami from a

California gull (Price, 1931). Apophallus crami has not been reported in the literature since the original description. Further morphological studies on Apophallus species in

Oregon and elsewhere are greatly needed to resolve the taxonomic quagmire of these parasites. Given the similar morphology of these worms, information on genotypic characters will probably be needed to ultimately resolve the taxonomy of members of this important genus affecting fishes.

Acknowledgments

This research was funded, in most part, by an ODFW Fish Health Graduate

Research fellowship (agency grant 010-7032-IAA-FISH) to J.A.F. We would also like to thank the research personnel at ODFW for providing samples and J. Sanders for attempting to isolate rDNA sequence of Apophallus sp. samples.

78 Figure 3.1. Photomicrograph of adult Apophallus sp. obtained from domestic chick (Gallus domesticus). OS = Oral Sucker, ST = Spiny Tegument, P = Pharynx, BC = Bifurcated cecum, VS = Ventral sucker, T = Testes, V = Vitellaria.

79 Fish muscle skin (BS) skin (BS) skin (BS) skin (BS) infections x 18.9 18.9 30 x 18 skin (BS) (16-22) (16-22) (30-31) x (36-40) x 37 (32-41) (32-41) 37 (16-21) 20 mostly mostly tandem tandem oblique oblique 25 x 33 NA tandem? tandem? tandem? tandem? (pictured) (pictured) oblique or or oblique sucker, NA = Not applicable NR VS VS VS VS? VS? do not not do extend anterior as far as as far posterior posterior posterior posterior anteriorly (pictured)

A. slender slender elongate gonotyl oblique elongate gonotyl elongate, elongate, anteriorly muehlingi attenuated same as same posteriorly but broader broader but type description. VS = Ventral

x NR x NR (120-260) (230-320) (520 -820) x (279-341) (600-900) x (1,500-1,900) (910-1,330) x A.

- syn - syn. - syn. x 150 600 NR

Miller 1941 Rossicotrema Rossicotrema donicum imperator species. Bold type refers to Witenberg (1929);

Apophallus 1963 1939) Apophallus and Callot, Price, 1931 sp. of Balozet Sinclair, 1972 Timon-David, Timon-David, Ransom, 1920 Morosov, 1952 (for (for

Price Price

1931 Species Reference Synonyms Size Shape Vitellaria Testes Egg Testes Species Reference Vitellaria Synonyms Size Shape A. brevis A. brevis A. brevis A. bacalloti A. bacalloti Ransom 1920 Morosov 1952 Morosov A. crami of Table 3.1. Synopsis = Not reported. 80 Fish muscle, muscle, skin (BS) skin (BS) skin (BS) skin (BS) skin (BS) skin (BS) skin (BS) skin (BS) (pictured) Skin (BS) Skin (BS) infections skin (BS)? skin (BS)? 32 32 NR x 18 x 18 32x18 32x18 (15-20) (15-20) (18-25) (18-25) (16-17) (16-17) 32 x 18 NA (49-50) x (21-33) x (28-34) x 33 (27-37) (27-37) 33 18 (17-20) (17-20) 18 usually tandem tandem tandem tandem oblique oblique oblique oblique tandem ? tandem tandem ? tandem (pictured) (pictured) oblique to to oblique oblique to to oblique oblique to to oblique forl forl fork fork up to to up up to to up end of gonotyl posterior posterior intestinal intestinal (pictured) (pictured) up to VS? VS? to up up to VS? VS? to up esophagus esophagus NR gonotyl oblique NR gonotyl Oval, Oval, biscuit biscuit biscuit biscuit shaped shaped shaped shaped discoid discoid pointed pointed rounded rounded pyriform- linguiform linguiform posteriorly linguiform, linguiform, pyriform, or or pyriform, anteriorly & oval, bluntly bluntly oval, 303) (157-287) (190-230) (190-230) (200-400) x 262 (186- x 262 (745-941) x (500-1150) x 311 (298-554) (298-554) 311 (1200-1600) x (1200-1600) x

-

syn syn syn ] ] – syn R. simile, A. major hlingi - hlingi

x 257 881 NR

ü R. donicum Witenberg (1929) R. venustus, A. brevis A. R. venustus, syn Witenberg (1929) Witenberg syn [for [for Rossicotrema donicum, donicum, Rossicotrema venustus C. similis- venustus similis- C. Price (1931); Distoma m Distoma (Luhe 1909); R. simile, Cotylophallus

1970 1899 (after (after 1919) Niemi & Niemi Odening, Odening, Lindtrop, Lindtrop, Skrjabin & Skrjabin Luhe, 1909 Macy, 1974 Jagerskiold, Jagerskiold, Dawes 1946 Lyster, 1940 NR Sinclair, 1972

hlingi hlingi

ü ü 1909 Species Reference Species Reference Egg Testes Synonyms Vitellaria Size Shape A. donicus A. donicus A. donicus Price, 1931 A. m A. m A. mühlingi 1899) Luhe, A. imperator (Skrjabin & (Skrjabin A. imperator Lyster, 1940 (Jagerskiold, Lindtrop, 1919) Table 3.1. Continued. 81 NA NA Fish muscle muscle infections x 12 x 12 33x18 NA 33x18 (18-22) (18-22) (16-20) (16-20) (15-20) (15-20) (10-14) (10-14) (26-32) x (30-35) x (25-35) x 23 (20-26) (20-26) 23 side side by by side oblique oblique adjacent adjacent tandem? tandem? tandem? tandem? (pictured) (pictured) oblique to to oblique oblique to to oblique oblique to to oblique to to VS VS fork fork fork fork fork) fork) VS (to anterior anterior Anterior Anterior intestinal intestinal intestinal NR NR or oval- or elongate elongate pyriform pyriform pyriform pyriform pyriform pyriform elongate- oval; few few oval; elongated elongated linguiform, linguiform, up to to up up to to up 435 x 435 (250-550) (220-390) (230-650) (215-263) x 57 (35-78) x 57 (35-78) up1,300 to x (950-1,400) x (500-1,140) x 197 (150-231) (150-231) 197

syn Price (1931) Price syn syn Price (1931) Price syn syn (Ciurea1924) Pricetrema zalophi - Pricetrema zalophi Cotylophallus similis- Cotylophallus venustus-

1920 1936 1920 Ransom, Ransom, Ransom, Ransom, Cameron, Cameron, Price, 1932 Present study

sp. sp. Price Price

1932 Species Reference Species Reference Egg Testes Synonyms Vitellaria Size Shape A. similis Price 1931 Price 1931 A. venustus A. venustus A. venustus A. venustus Apophallus A. zalophi A. zalophi (Ransom, 1920) (Ransom, 1920) Table 3.1. Continued. 82 REFERENCES

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Johnson, M. W., and T. A. Dick. 2001. Parasite effects on the survival, growth, and reproductive potential of yellow perch (Perca flavescens Mitchill) in Canadian Shield lakes. Canadian Journal of Fisheries and Aquatic Sciences 79: 1980-1992.

83 Kent, M. L., V. G. Watral, C. M. Whipps, M. E. Cunningham, C. D. Criscione, J. R. Heidel, L. R. Curtis, J. Spitsbergen, and D. F. Markle. 2004. A digenean metacercaria (Apophallus sp.) and a myxozoan (Myxobolus sp.) associated with vertebral deformities in cyprinid fishes from the Willamette River, Oregon. Journal Aquatic Animal Health 16: 116-129.

Lafferty, K. D., and A. K. Morris. 1996. Altered behavior of parasitized killifish increases susceptibility to predation by bird final hosts. Ecology 77: 1390-1397.

Lemly, A. D., and G. W. Esch. 1984. Effects of the trematode Uvulifer ambloplitis on juvenile bluegill sunfish, Lepomis macrochirus: ecological implications. The Journal of Parasitology 70: 475-492.

Lyster, L. L. 1940. Apophallus imperator sp. nov., a herterophyid encysted in trout, with a contribution of its life history. Canadian Journal of Research 18: 106-121.

Malek, E. A. 1980. Snail-Transmitted Parasitic Diseases. CRC Press, Boca Raton, Florida. p. 658.

Meyer, M. C., and O. W. Olsen. 1975. Essentials of Parasitology. W. C. Brown Co. Dubuque, Iowa. p. 303.

Miller, M. J. 1941. The life history of Apophallus brevis Ransom, 1920. Journal of Parasitology 27: 12.

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85 Chapter 4: Survey of Parasites in Threatened Stocks of Coho Salmon (Oncorhynchus kisutch) in Oregon by Examination of Wet Tissues and Histology

Jayde A. Ferguson1, Sophie St-Hilaire2, Tracy S. Peterson1, Kenneth Rodnick2, and

Michael L. Kent1

1Department of Microbiology, Oregon State University, 220 Nash Hall, Corvallis,

Oregon 97331

2 Department of Biological Sciences, Idaho State University, 650 Memorial Drive,

Pocatello, Idaho 83209

Submitted to Diseases of Aquatic Organisms

Inter-Research Science Center Oldendorf/Luhe, Germany

86

ABSTRACT

When conducting studies on the impacts of parasites, documenting the geographic distribution of infections is an essential first step. Thus, the objectives of the current study are: 1) identify parasite burdens and distributions in Oregon coastal juvenile coho salmon, 2) assess the pathology of these infections, and 3) determine the sensitivity and specificity of histology for detecting parasites compared to examining wet preparations. We examined a total of 576 coho salmon from fry, parr and smolt stages by histology. The muscle and gills of 219 of these fish were also examined by wet preparation. Fish were collected from 10 different locations in 2006 - 2007. We identified 21 different species of parasites in these fish. Some parasites, such as

Nanophyetus salmincola and Myxobolus insidiosus, were common across all fish life stages from most basins. Other parasites, such as Apophallus sp., were more common in underyearling fish than smolts and had a more restricted geographic distribution.

Additional parasites commonly observed were as follows: Sanguinicola sp.,

Trichodina truttae, Epistylus sp., Capriniana piscium, and an unidentified metacercariae in gills; Myxobolus sp.in brain; Myxidium salvelini and Chloromyxum majori in kidney; Pseudocapillaria salvelini and adult digenean spp. in intestine.

Only a few parasites, such as the unidentified gill metacercariae, elicted overt pathologic changes. Histology had generally poor sensitivity for detecting parasites; however, it had relatively good specificity. We recommend using both methods for

87 studies or monitoring programs requiring a comprehensive assessment of parasite identification, enumeration, and parasite related pathology.

INTRODUCTION

Over the last half century, populations of wild salmonids have been declining at alarming rates (Lackey et al., 2006). Currently, Oregon coastal coho salmon

(Oncorhynchus kistuch), an evolutionarily significant unit, are listed as threatened under the U.S. Endangered Species Act (ESA). Therefore, we are conducting studies to elucidate the impacts of parasites on these threatened populations of coho salmon.

As a first step, we sought to identify the species of parasites infecting Oregon coastal coho salmon, and document their prevalence, abundance, and geographic distributions.

Furthermore, we conducted a pathology assessment, which provides data on impacts of parasites at the tissue and organism level (Feist and Longshaw, 2008).

The methods used for surveying parasite infections in wild populations include: gross examination, microscopic evaluation of tissues in wet mounts, histology, serology, culture, and polymerase chain reaction (PCR) based techniques.

However, currently there are no specific PCR or serological tests for most parasites of wild fishes. Thus, most fish surveys rely on identification of parasites from wet tissues, as whole fresh parasites usually provide the best morphological information for identification (Hoffman, 1999; Ferguson, 2006) and permits enumeration of parasites (Hoffman, 1999; Jacobson et al., 2008; Ferguson et al., 2010). Examples of such surveys with salmonid fishes include Kent et al. (1998) and Arkoosh et al.

88 (2004). Histology, however, remains useful as a general tool for conducting initial fish health surveys because it provides the best representation of pathologic changes and allows for detection of a wide variety of pathogens, including viral and bacterial agents (Kent et al., 1998; Feist and Longshaw, 2008). Indeed, most well recognized or common parasites can be readily identified by histology (Gardiner et al., 1998;

Gardiner and Poynton, 1999; Bruno et al., 2006). However, to our knowledge it is uncommon for parasite surveys to utilize both histology and examination of wet material. The few exceptions to this include Eaton et al. (1991), Moran and Kent

(1999), and Kent et al. (2005).

The Oregon Department of Fish and Wildlife (ODFW) manages Life-cycle

Monitoring (LCM) sites for estimating fish survival as part of the state’s plan for assessing the status of threatened coastal stocks (Solazzi et al., 2000). We have previously shown that parr coho salmon from one of these LCM sites, West Fork

Smith River, have remarkably high digenean and myxozoan infections by histology

(Rodnick et al., 2008). Here we examined coho salmon from several LCM sites in western Oregon using histology, wet preparations, or both, from fry, parr, and smolt stages. The objectives of our study were to: 1) identify parasite burdens and distributions in juvenile coho salmon, 2) assess the pathology of these infections, and

3) determine the sensitivity and specificity of histology for detecting parasites in axial skeletal muscle and gills compared to examining wet preparations. Here we report our results from this survey, summarize histopathological findings, and compare the different methods used for detecting parasite infections.

89 MATERIALS AND METHODS

Sampling fish

Coho salmon, because of their threatened status, were opportunistically sampled by ODFW from their LCM sites. Fry and parr were caught by electrofishing pools during ODFW snorkeling surveys and smolts were captured in screw traps during outmigration. In May and June of 2006, fry were sampled from the following eight ODFW LCM sites: the lower mainstem of West Fork (WFSR), Mill Creek (Ck)

Siletz R, East Fork (EF) Trask R, Cascade Ck of Alsea R, Upper North Fork (UNF) and Lower North Fork (LNF) of Nehalem R, and the Upper Main Stem (UMS) and EF in the Lobster Ck sub-basin of Alsea R. In September of 2006, parr (same year class) were sampled from the same locations, excluding the Lobster Ck sites and including an additional site of Winchester Ck of the South Slough of Coos Bay. In April and

May of 2007, smolts (same year class) were sampled from all of the locations that parr and fry were sampled from previously, plus the additional site, Mill Ck of Yaquina R, was included. Finally, in September of 2007, parr (different year class) were sampled from the lower mainstem of WFSR. All fish were taken by ODFW under their permits.

Histology

Samples for histologic analysis were fixed in Dietrich’s solution (30 ml 95% ethanol, 10 ml formaldehyde, 2 ml glacial acetic acid, and 58 ml distilled water).

Samples were processed using standard histology techniques and stained with

90 hematoxylin and eosin or Giemsa. Sagittal sections were made from intact fry. Parr and smolts required dissection and removal of the following organs: liver, spleen, kidney, heart, intestine, pyloric cecae, brain, gonad, gill, and axial skeletal muscle

(henceforth referred to simply as muscle). Mean density of infection

(parasites/histological section of infected fish) was determined by counting all parasites in a section using standard light microscopy. For muscle, we determined the area of tissue using the SPOT Advanced digital camera and software (Diagnostic

Instruments Inc., Sterling Heights, Michigan). Then volume was determined by multiplying the area by the thickness of histological sections (5 µm). Myxozoans were enumerated by counting pseudocysts or spore aggregates rather than individual spores, as these all arose from a single progenitor. Only prevalence was recorded for capillarid nematodes in the gut because cross sections of single worms were possible and thus we could not determine if counts represented more than one worm.

Wet preparations

We were able to obtain fresh or fresh frozen material from smolts collected in

April 2007 (brood year 2006) from all sites and parr in October 2007 (brood year

2007) from WFSR. Gills were evaluated as warranted by standard practices and muscle was also included because we were particularly interested in abundance of muscle parasites given the severity of infections seen in our previous study (Rodnick et al., 2008).

91 For gill tissue, two to three gill arches (including gill rakers) were collected from each fish at field sites or the laboratory and examined in wet mounts for parasites. Photomicrographs of representative parasites were obtained from gill preparations from fish that were examined in the laboratory. A few metacercariae were excysted in the laboratory as described by Irwin (1983) to better study morphology. Fish from sampling sites that were processed in the laboratory included smolts from Cascade Ck and Mill Ck Yaquina and parr (brood year 2007) from

WFSR.

For muscle tissue, samples were prepared and evaluated in accordance with previously published methods by Ferguson et al. (2010). In short, parasites were enumerated from one freshly thawed and squashed fillet per fish, and recorded in terms of parasites/fillet. Also, a gross examination of all fresh or frozen fresh fish was performed to enumerate neascus (black spot trematode) infecting skin, and recorded in terms of parasites/fish. Prevalence (number of infected animals/total animals), intensity (number of parasites/infected animals), and density (number of parasites/quantity of infected tissue) of infections are reported in accordance with the definitions provided by Bush et al. (1997). Gill wet mounts and muscle squashes are both referred to as wet preparations hence forth.

Parasite identifications

Parasites were identified as specifically as possible by recording morphological characters, measurements and tissue distribution and referring to reviews and keys

92 including Love and Moser (1983), Lom and Dyková (1992), McDonald and Margolis

(1995), Gibson (1996), Hoffman (1999), and Moles (2007).

Diagnostic method comparison

We calculated the sensitivity and specificity of histology using results from wet tissue preparation evaluations as our selected gold standard. Gold standard is a specific term used in epidemiology that is established by researchers to indicate the most generally accepted and commonly used diagnostic test. Here we used wet preparations as the gold standard, as this is the most common method used by parasitologists to enumerate and identify parasites in tissues (Hoffman, 1999;

Ferguson, 2006). Sensitivity was calculated by dividing the number of fish found positive by both wet preparation and histology by the total number of positive fish by wet preparation. Specificity was calculated by dividing the number of fish found negative by both wet preparation and histology by the total number of wet mount- negative fish. The 95% confidence intervals for the sensitivity and specificity of histology were determined for each parasite using Clinical Calculator 1 (Lowry,

2010). Only smolts and parr from WFSR (brood year 2007) could be included in our comparison analysis of the diagnostic test methods because these were the only samples that were assessed by both techniques.

We also calculated the sensitivity of histology for each muscle parasite when only heavily infected fish (top 10% of infections) and negative fish were included in the study to assess the accuracy of this diagnostic technique for detecting heavily

93 parasitized fish. This was not performed on gill infections, as there were insignificant numbers of fish with heavy infections.

RESULTS

We evaluated 576 coho salmon (254 fry, 118 parr and 204 smolts) from 10 coastal rivers in Oregon from 2006-2007. All groups were evaluated by histology, but wet preparations of gill and muscle were also evaluated for all smolt groups and one parr group. Most fish were infected with several protozoan or helminth parasites

(Tables 4.1-4.3) as we described below.

Muscle parasites

Using both methods, N. salmincola sp. was observed in muscle of fish from at least one life stage at eight sites. Based on histology, prevalence of N. salmincola in muscle was similar in fry and parr, which was approximately twice that of smolts

(Table 4.1). Infection density was similar with all three fish life stages of the same year class. Based on this method, fry most heavily parasitized were from Mill Ck

Siletz, WFSR, and UMS Lobster Ck (Table 4.1). Mill Ck Siletz also contained parr with the heaviest infections. Amongst smolts, fish from the UNF Nehalem harbored the highest infections (Table 4.1).

Based on wet preparations, prevalence of N. salmincola in muscle was 100% from smolts from Mill Ck. Siletz, WFSR, and LNF Nehalem, with the highest density

(22 metacercariae/fillet) occurring in smolts from WFSR. We did not detect N. salmincola in any stage of fish from the EF Trask by either method, and it was only

94 detected at a low prevalence and density with wet preparations for smolts from EF

Lobster Ck. In histological sections, N. salmincola was observed throughout muscle, multiple visceral organs, and even gills of smolts (Figure 4.1b). The kidney was particularly heavily infected. Metacercariae were usually surrounded by mild, focal, chronic inflammation. The histologic features of these lesions were similar to those found in other studies (Rodnick et al. 2008, Ferguson et al. 2010).

Using both methods, Apophallus sp. was observed in muscle of fish from at least one life stage seven sites. Similar to N. salmincola, smolts had a much lower infection prevalence of Apophallus sp. than fry and parr based on histology, with prevalence in smolts almost 15 times less than that in younger life stages (Table 4.1).

Hence, using histology, the parasite was only detected in smolts at LNF Nehalem and

Cascade Ck (Table 4.1). Almost all of the metacercariae of this species were found in myoseptal connective tissue, and was not associated with a tissue reaction (Figure

4.1a). Using wet preparations, the most commonly and heavily infected fish were from WFSR and LNF Nehalem, with the former harboring over 500 worms/fillet in parr from the lower mainstem section of this river.

The two myxobolids infecting muscle were M. insidiosus within myocytes, and

M. fryeri in the peripheral nerves. Using histology, no fry were infected with either species, but parr and smolts from half the sample sites were infected with both species

(Table 4.1). Overall, prevalence and density of infections in smolts were comparable to those in parr. Parr from WFSR had the highest prevalence and density of M. insidiosus, whereas those from Mill Ck. Siletz had the highest prevalence and density

95 of M. fryeri infections (Table 4.1). Smolts from WFSR, Mill Ck Siletz, and UNF

Nehalem had the highest density of infections of both species. Smolts from Mill Ck

Siletz and UNF Nehalem had the highest prevalence (Table 4.1). Myxobolus fryeri was absent from fish sampled from EF Trask, Cascade Ck. and both Lobster sites.

These parasites did not elicit any inflammatory changes when plasmodia were intact; however, after plasmodia ruptured, a mild inflammatory response was noted.

The size of M. fryeri pseudocysts (ca 30 µm) are about 6 times smaller than those of M. insidiosus (Ferguson et al., 2008), and hence were too small to detect in wet preparation. Myxobolus insidiosus was detected in smolts from every site in wet material. Mill Ck Siletz and WFSR had the most heavily infected smolts with this parasite, with means of nearly 200 and 150 pseudocysts/fillet, respectively.

The presence of neascus in skin was only evaluated from smolts and the one parr group (Table 4.1) as these were the only samples that were available for fresh gross examination. This parasite occurred in fish from seven sites, and the highest prevalence and intensity of neascus in fish was from smolts sampled at Mill Ck

Yaquina, with nearly three quarters of fish infected and a mean intensity of about

15/fish (Table 4.1).

Gill parasites

Nanophyetus salmincola was observed in the gills of fish from eight sample sites by histology (Table 4.2), with prevalent infections in fry from Mill Ck Siletz, and

UMS Lobster Ck. Prevalent infections in parr were from UMS Lobster Ck and

96 WFSR. Smolts had about half the prevalence of fry and parr, but a similar infection density. The highest prevalence of this parasite in smolts was in fish sampled from

LNF Nehalem (Table 4.2). Nanophyetus salmincola within the gill filaments was easily recognized by wet preparations by the presence of a prominent posterior excretory bladder (Figure 4.3a). However, this structure was not always clearly visible in metacercariae deep in tissues within the base of the gills or gill rakers.

Metacercariae of Apophallus sp. and Echinochasmus milvi were identified in wet preparations of the gills (Figure 4.3c-e). One exhibited prominent collar spines, consistent with members of the family Echinostomidae, while another type was a small species (about 200 µm long) with a long pharynx, which exhibited features consistent with the genus Apophallus, including tandem to oblique testes, a pharynx close to the oral sucker, and a long esophagus. Many of these two metacercariae were indistinguishable from each other in histological sections, and hence we classified them as “unidentified gill metacercariae”. Fish from six sample sites were infected with these parasites using this method. Parr had about three times the infection prevalence of fry and smolts (Table 4.2), and the highest prevalence in both fry and parr occurred from fish sampled from Cascade Ck. For smolts, these metacercariae were most prevalent in fish from Mill Ck Yaquina. These unidentified metacercariae elicited the most prominent tissue changes. Located within the base and mid-level of filaments, individual and clustered (2-4 metacercariae) cysts often caused profound intrafilamental fibrosis and concurrent chondroid hyperplasia and metaplasia (Figure

4.3a-c). Cysts were surrounded by irregular, pleomorphic and occasionally

97 binucleated nascent chondrocytes, not directly associated or contiguous with pre- existing cartilage, within a background of dense fibrovascular tissue. The expanded intrafilamental stroma was infiltrated by chronic inflammatory cells, mostly lymphocytes, but also histiocytes and eosinophilic granular cells to a lesser extent.

Embryonated eggs of Sanguinicola sp. were detected in fish from six sample sites in histological sections (Table 4.2). Infections were light, and not associated with appreciable tissue damage or inflammation (Figure 4.3d). Numerous larvae of

Philonema sp. were observed in wet preparations of gill from one smolt from LNF

Nehalem (Figure 4.2b). A monogenetic trematode suggestive of Gyrodactylus sp. was also observed in wet material from a smolt from the WFSR.

The following protozoan parasites were detected in wet preparations of gills from smolts (Table 4.2): Capriniana piscium (Figure 4.2i, insert), Trichodina truttae

(Figure 4.2g), Epistylus sp. (Figure 4.2f), and Ichthyophtherius multifiliis.

Corresponding to the histologic observations, C. piscium was on the gills of most

(90%) smolts from Mill Ck Yaquina. These ciliates were observed at the surface of the gill and were not associated with either inflammation or necrosis (Figure 4.3e).

Loma salmonae (Microsporidia) was observed in two smolts by wet preparations

(Figure 4.2h, insert) and one by histology (Figure 4.3f) from Mill Ck Yaquina, with a mean density of 30 xenomas/gill in wet preparations. Intact xenomas of L. salmonae were observed in primary lamellae within major blood vessels. They were associated with minimal inflammation. Trichodina truttae occurred at the highest prevalence and

98 density in smolts from WFSR and Cascade Ck. Epistylus sp. and I. multifiliis infected smolts from two sites, with Mill Ck Siletz harboring the highest infections (Table 4.2).

Viscera and brain parasites by histology

We did not detect any evidence of viral or bacterial pathogens by histology, however, additional parasite species were detected when visceral organs and brain were examined by histology, and N. salmincola (which was common in the muscle) was found in all visceral organs and even brain and ovary in one fish. Including histologic examination of these organs often resulted in doubling the prevalence or mean density of infection compared to examination of gills or muscle only (Tables

4.1-4.3). All three fish life stages had similar infection levels of N. salmincola, with smolts having the highest infection prevalence and density (Table 4.3).

Aggregates of spores of a Myxobolus sp. were found in the brain of fish

(Figure 4.3o) from five sample sites. This parasite was most prevalent in parr samples

(Table 4.3). Fry from Cascade Ck had the highest prevalence, and the most prevalent infections in parr occurred at this site and at UNF Nehalem (Table 4.3).

Two kidney myxozoans were observed: Myxidium salvelini and Chloromyxum majori infected kidney tubules and glomeruli, respectively. The highest prevalence of

M. salvelini occurred in fry samples, whereas that of C. majori occurred in parr (Table

4.3). Myxidium salvelini was confined to the lumen of renal tubules and was not associated with tissue damage. Plasmodia had prominent, retractile granules in the cytoplasm, and also contained developing and mature spores (Figure 4.3k,l).

99 Myxospores of M. salvelini were readily visualized with Giemsa stain (Fig. 3l insert).

Parr at five sites and smolts at one site (Cascade Ck) were infected with C. majori

(Table 4.3). This myxozoan was observed in the glomeruli, and was identified by the presence of spherical spores with 4 polar capsules (Figure 4.3m,n). Affected glomeruli were mostly effaced and expanded by the spores, with significant disruption of normal histoarchitectural features and a narrowing to near-complete obliteration of the uriniferous space associated with attenuated parietal epithelium. In some instances, there was mild periglomerular fibrosis surrounding the affected glomeruli.

Four helminth species were found in the gastrointestinal tract by histology

Pseudocapillaria salvelini (nematode) was clearly identified as a capillarid due to the presence of stichosomes and eggs with bipolar plugs (Figure 4.3g). The capillarid was most prevalent in smolt samples, with Mill Ck Siletz and both forks of Nehalem being the locations with most infections (Table 4.3). Additionally, parr from WFSR (brood year 2007) had a high prevalence of almost 90%. Fish from all stages originating from EF Trask R, EF Lobster Ck, and UMS Lobster Ck were void of these worms.

This nematode occurred within the intestinal epithelium, but was not associated with obvious tissue damage or inflammation.

Unidentified adult digeneans were observed in the lumen of the pyloric cecae of a few fish (Table 4.3), and were not associated with significant histopathologic changes (Figure 4.3j.) An adult cestode was observed in the intestine (Figure 4.1c) of one fish from EF Lobster Ck and adult acanthacephala (Figure 4.1d) were observed in the intestine of 4 fish from Winchester Ck. Numerous larval nematodes belonging to

100 the genus Philonema were observed in the coelomic cavity and ovary of one fish from

LNF Nehalem (Figure 4.3h,i), which was the same fish we detected larval worms in gill wet preparations. Typically, larvae were surrounded by many intermixed inflammatory cells, comprised of lymphocytes, histiocytes, and eosinophilic granular cells, with fewer plasma cells being present. This was occasionally accompanied by mild hemorrhage. Serosal surfaces were lined by hypertrophied reactive serosal cells overlying modest fibroplasia, indicating a low grade and chronic serositis.

Diagnostic method comparison

The sensitivity of histology was very poor for all the muscle parasites and most gill parasites, using wet preparations as our gold standard (Table 4.4). The sensitivity was increased when we excluded muscle tissues with low density of infection (Table

4.4), but it was still never above 45%. In the case of gill parasites, some were not detected at all using histology (i.e., Sanguinicola sp., T. truttae, Epistylus sp., and I. multifiliis; Table 4.4). However, histology was good for detecting C. piscium infections, with approximately 95% sensitivity (Table 4.4). In fact, when these tests were applied to fish populations the prevalence of metacercarial gill parasites was consistently higher for several populations compared with evaluation of wet preparation of tissues (Table 4.3). With the exception of the gill metacercariae, the specificity for histology was high for all other parasites ranging from 91% to 100%

(Table 4.4).

101 DISCUSSION

Diseases, including those caused by parasites, may be significant contributors to mortality in wild fish populations (Möller and Anders, 1986; Adlard and Lester,

1994; Bakke and Harris, 1998; Jacobson et al., 2008). One of the first steps for assessing impacts of parasites on wild fish populations is to identify the species they are infected with, and document their prevalence, abundance, and geographic distribution. We had previously reported heavy helminth and myxozoan infections in coho salmon parr from WFSR (Rodnick et al., 2008), which was consistent with the current survey on fry, parr, and smolt coho salmon from 10 coastal rivers. All of these parasites found in the present study have been described previously from salmonids in the Nearctic, mostly in parasite surveys or taxonomic descriptions. Nevertheless, we feel it is warranted to review the importance of these infections as they relate to pathologic changes and how they may impact juvenile coho salmon. Regarding geographic distributions of the parasites, we could not conduct spatial cluster analyses on these data with a samples size of only 10 rivers. However, some general trends are noteworthy.

Nanophyetus salmincola

One of the most common parasites observed was metacercariae of N. salmincola. This parasite was found in all life stages of fish and occurred in hosts from nine of 10 rivers sampled. The intermediate host for this digenean is Juga silicula, which is widely spread in coastal streams from northern California through

102 Washington. This snail supposedly prefers slower moving water, with coarse substrates (Furnish, 1990), which is consistent with many locations in our study.

Prominent infections in the muscle were seen in wet preparations, and this worm was detected in essentially every organ by histology. As reported previously (Wood and

Yasutake, 1956; Ferguson et al., 2010), the metacercariae were associated with localized, chronic inflammation.

Apophallus sp.

Metacercariae of Apophallus sp. were found in fish from about half the sites, sometimes occurring in high numbers. Niemi and Macy (1974) identified Apophallus donicus in juvenile coho salmon from the Willamette River in Oregon, and we are investigating the identification of Apophallus sp. in our current studies. Apophallus sp. was abundant and prevalent in locations with little riparian canopy, as the surrounding lands have been either logged (e.g., WFSR, LNF Nehalem, Cascade Ck) or are used for agriculture (e.g., Mill Ck. Siletz) (Solazzi et al., 2000). These activities are associated with increased water temperatures in the associated rivers (Beschta and

Taylor, 1988), and perhaps this digenean species thrives better under these conditions.

For example, summer water temperatures in the lower mainstem of the WFSR has often exceed 20°C (Ebersole et al., 2006), and Rodnick et al. (2008) reported that

Apophallus metacercariae were more common in coho salmon parr in this stretch of the river compared to cooler, upstream tributaries. Neimi and May (1974) described infections by A. donicus in juvenile coho salmon from the Willamette River and

103 showed that the first intermediate hosts were freshwater snails from the genus

Fluminicola. Snails (Malek, 1980) and digeneans (e.g., Poulin, 2006; Poulin and

Mouritsen, 2007) increase their reproduction under warmer temperatures, and we previously reported a high prevalence of infections by an Apophallus sp. in cyprinids from warm areas of the Willamette River (Kent et al., 2004). Hawkins et al. (1982) also reported that rivers in Oregon with little riparian canopy, presumably resulting in higher temperatures, had more snails. Consistent with this trend, Cairns et al. (2005) observed a positive correlation of neascus with temperature in WFSR. Metacercariae of N. salmincola, neascus, and the unidentified spp. in the gills were also abundant at the same locations that had high levels of Apophallus.

In contrast to N. salmincola, we did not observe Apophallus sp. in visceral organs. Indeed, the infections in muscle occurred at a specific location, within the connective tissue of the myosepta. Other reports showed that metacercariae of

Apophallus species target muscle of catfish (Cameron, 1936) or bone of percids

(Taylor et al., 1994) and cyprinids (Kent et al., 2004). The latter two reports noted that the metacercariae were associated with osseous hyperplasia and metaplasia. This was consistent with gill infections in our study, where metacercariae in the gills were often directly associated with chondroid hyperplasia and metaplasia.

Unidentified gill metacercariae

Identification of the metacercariae, other than N. salmincola, in the gills was difficult by histology. However, in many occasions we detected a distinct spiny collar

104 indicative of an echinostomid in histological section. Some excysted worms from wet preparations closely resembled E. milvi, a common species found in gills of Oregon salmonids associated with similar lamellar damage (Hoffman, 1999). Olson and Pierce

(1997) described metacercariae from the gills of Oregon steelhead trout

(Oncorhynchus mykiss) that caused cartilage proliferation, and concluded that they were likely members of the family Heterophyidae. Previously, worms from both families have been noted to induce lamellar hyperplasia (Uzmann and Hayduk, 1964).

In our study, chondrocyte hyperplasia and metaplasia associated with metacercariae was similar to that described with gill metacercariae in freshwater tropical fishes

(Blazer and Gratzek, 1985). Disease produced by such infections can be serious. For example, gill-infecting Centrocestus formosanus (Heterophyidae) causes massive mortalities in cultured warm water fishes (Paperna, 1995). Chondroid metaplasia is not restricted to the gills, as Heidel et al. (2002) reported that cartilage metaplasia was associated with putative parasites in viscera of rockfish (Sebastes spp.).

Sanguinicola spp.

Another digenean found in the gills of juvenile coho salmon was the blood fluke Sanguinicola spp. It was not possible to identify this worm to the species level as only eggs and miracidia were observed, however two species infect salmonids in

Oregon, Sanguinicola klamathensis and Sanguinicola alseae (Hoffman, 1999).

Embryonated eggs of Sanguinicola spp. are found in blood vessels of well vascularized organs, particularly the gills and kidney. Typical of blood fluke eggs,

105 they elicit local inflammatory changes, and severe infections are associated with morbidity when miracidia escape from gills causing loss of blood and respiratory function (Schäperclaus, 1991). High intensities of Sanguinicola spp. eggs in gills are associated with mortality in several species of fish, including salmonids (reviewed by

Paperna, 1995). Experimentally infected cutthroat trout (Oncorhynchus clarki) exhibit significantly lower growth, packed cell volumes (hematocrit), and oxyhemoglobin levels (Evans, 1974). In our study, infections were at most moderate, and the majority of the gill tissue was not affected.

Gastrointestinal helminths

A capillarid nematode in the lower intestine of many parr and smolts was found at about half the sites. We are confident that these infections represent

Pseudocapillaria salvelini (syn. Capillaria salvelini, Ichthyocapillaria salvelini), as it has been reported from numerous salmonid fishes, including coho salmon from

Canada (Bell and Beverly-Burton, 1981). The only other record of a different species in salmonids was Capillaria catenata (Fritts, 1959), which typically infects percids and centrarchids. This single report has been regarded as suspect by Bell and

Beverley-Burton (1981), due to both its rarity and fact that the worm was found only in cutthroat trout and not in percids and centrarchids from the same location.

Capillarids are recognized as severe pathogens in fishes (Dick and Choudhury, 1995) as they penetrate tissues and are associated with severe tissue damage and inflammation. For example, Pseudocapillaria tomentosa causes severe inflammation

106 and are associated with emaciation and ultimately death in zebrafish (Danio rerio)

(Kent et al., 2002). In contrast, all worms seen in our study were confined to the epithelium and were associated with little or no tissue damage.

We did not determine identities of the adult digeneans observed in the gastrointestinal tract, as there are numerous genera and species infecting salmonids in the Pacific Northwest (Love and Moser, 1983; Hoffman, 1999) and we only observed them by histology. Adult intestinal trematodes are considered to be less pathogenic than metacercariae, as most are confined to the lumen of the gastrointestinal tract

(Paperna, 1995). Numerous genera and species of acanthocephalans and cestodes infect salmon and trout in the Pacific Northwest as adult stages (Love and Moser,

1983; Hoffman, 1999; McDonald and Margolis, 1995). We were not able to identify our specimens to the genus level based on the available material.

A severe infection by a Philonema sp. was observed in one smolt, with massive numbers of larvae in the viscera and gills. Unfortunately we were unable to collect adult worms, and thus were not able to determine the precise species of this worm. Two genera of philometrids are described from salmonids in the Pacific

Northwest, Philonema and Philometra (Hoffman, 1999). We identified our worms as

Philonema based on the tapered posterior end. There are numerous reports of

Philonema oncorhynchi in salmonids from the Pacific Northwest (Hoffman, 1999;

Moravec and Nagasawa, 1999), and thus this may be the species seen here. This parasite is recognized to cause disease in salmonids, including coho salmon

107 (Brocklebank et al., 1996), and severity of infection and the visceral changes seen in the one infected fish certainly would have compromised this individual.

Myxozoans

Myxozoans are commonly observed in wild salmonids, and we found five species in our survey. Chloromyxum majori in the kidney and Myxobolus sp. in the brain were common in fish from rivers with high levels of digenean infections. The life cycles of these myxozoans have not been resolved, but based on others they likely involve an oligochaete host. Many species of these worms thrive in relatively warm, oligotrophic or eutrophic environments, which could account for their distribution seen here. In contrast, Myxobolus insidiosus infections occurred at a high prevalence and similar density in all rivers, suggesting that the putative oligochaete host for this parasite is widespread. These are general observations, and further studies are required to connect these parasite distributions with ecological parameters and land use practices.

We observed renal infections by M. salvelini in all life stages of coho salmon in seven of the rivers. Two Myxidium spp. infect the kidneys of salmonids in the

Pacific Northwest, M. salvelini and Myxidium minteri. The former has been recorded from urinary bladder, ureter (Shul'man, 1988) and kidney (Shul'man, 1988; Kent et al.,

1994; McDonald and Margolis, 1995), whereas the latter has only been found in renal tubules (Yasutake and Wood, 1957; Sanders and Fryer, 1970). We identified the species seen here as M. salvelini based on the presence of more elongated spores and

108 pyriform polar capsules. In contrast, the spores of M. minteri are oval and polar capsules are subspherical. In addition, one of us (M.K.) has observed M. salvelini infections in many salmonids in Canada, and as seen here, the occurrence of distinctive retractile, brown to black, granules in the cytoplasm of the trophozoites is a consistent finding for this species. Renal tubule degeneration can occur and spores are liberated from tubules and enter into the renal interstitium with heavy infections of M. minteri (Yasutake and Wood, 1957), whereas M. salvelini is not recognized to be pathogenic (Kent et al., 1994). We did not observe appreciable damage to the infected tubules.

Chloromyxum majori infected kidney of fry, parr, or smolts from five of the rivers. This species is the only one in this genus that infects glomeruli of salmonids in this geographic region (Yasutake and Wood, 1957; Hoffman, 1999). We found this parasite to predominantly infect the glomerular visceral epithelium and occlude the uriniferous space, with large numbers completely effacing glomeruli. However, it can also cause degeneration of intra-renal hematopoetic tissue in heavy infections, which has been associated with salmon losses in hatcheries (Yasutake and Wood, 1957). The glomerular lesions observed here were consistent with previous descriptions, and infected glomeruli were essentially obliterated by the parasite and associated inflammation.

Several neurotropic Myxobolus species infect salmonids (see review by

Ferguson et al., 2008). The spores seen here in brains from juvenile coho salmon were either Myxobolus kisutchi or Myxobolus neurotropus as both form oval spores in the

109 brain and spinal cord of salmonids in this region. It is unlikely that these infections were Myxobolus arcticus, which has a more northerly range and has not been reported in parr or smolts in freshwater south of Washington State (Ferguson et al., 2008).

There was minimal tissue reaction to the brain myxobolid, and infections by M. kisutchi and M. neurotropus have not been associated with disease. However, Moles and Heifetz (1998) found that M. arcticus, which causes similar infections, is associated with reduced swimming ability of sockeye salmon (Oncorhynchus nerka).

Gill protozoans

The gills of wild fishes are often infected with a variety of parasites, both on the surface and deep within tissues. Several protozoans were observed in or on the gills of juvenile coho salmon, including: L. salmonae, T. truttae, Epistylus sp.,

Capriniana piscium (syn. Tricophyra piscium), and I. multifiliis. Trichodina truttae was the most prevalent ectoparasitic protist, detected in fish from half the rivers. This species has been described from salmonids in freshwater in Oregon (Mueller, 1937) and other regions in the Pacific Northwest (Arthur and Margolis, 1984), based on size and morphology. This parasite is most pathogenic when it occurs in high numbers, causing epithelial hyperplasia, decreased osmoregulatory ability and even death (Lom,

1995). We did not associate tissue damage with this ciliate.

Both Epistylus sp. (Esch et al., 1976) and C. piscium (Hofer et al., 2005), have been associated with fish held in water with high organic loads, and certain rivers where these were prevalent (e.g., Siltez R, Yaquina R) predominantly have agriculture

110 activity above the sites where fish were collected. The proper identification of epistylids in fishes from North America is lacking (Hoffman, 1999), thus we refer to the organism seen here as Epistylus sp., which is considered more of an ectocommensal than a parasite of both skin and gills. However, high numbers of this organism have been implicated in ‘red sore disease’ with other possible synergistic etiologies (Lom, 1995). We observed high numbers of C. piscium on the gills of smolts at two locations. Although infections were very heavy in some fish, with every filament infected with numerous parasites, they were not associated with histologic changes. This is consistent with other reports, where Capriniana spp. on salmonid gills and other fishes are considered commensal, rather than parasitic (Lom, 1995;

Hofer et al., 2005).

Ichthyophthirius multifiliis is recognized as a serious pathogen (Traxler et al.,

1998), but we detected this parasite in just one fish. We only found two fish infected with the microsporidium, L. salmonae, which is a well known pathogen in net pen aquaculture (Kent and Speare, 2005). Severity of tissue damage is more related to the reaction associated with destruction of xenomas rather than density of infection

(Speare et al., 1998). With infections in freshwater, intact xenomas, as seen here, are associated with little damage to host tissues (Magor, 1987).

Parasite associated mortality

Fry and parr were generally more heavily infected by most parasites than smolts. There are many examples of temporal declines in parasite burdens of host

111 populations being associated with parasite associated mortality, often involving fishes

(reviewed by Anderson and Gordon, 1982; Lester, 1984; Hudson and Dobson, 1995;

Rousset et al., 1996). Recently Jacobson et al. (2008) showed this phenomenon to occur with N. salmincola infections in coho salmon off the coast of Oregon and

Washington. Temporal declines in parasite burdens can also be explained by recovery from infections or from non representative samples of the population from different time points (i.e. smolts are a mixed population of many parr sub-populations). We recently showed that the muscle parasites like those in our study can persist from parr to smolt stage in coho salmon (Ferguson et al., 2010), and thus parasite death between parr and smolt stage is not a plausible explanation for the differences seen between these fish life stages in the present study. High mortality often occurs in coho salmon during the winter preceding smoltification (Ebersole et al., 2006; Hurst, 2007) and pre- overwintering fish from rivers such as WFSR have substantially high parasite infections (Rodnick et al., 2008). Therefore, it is possible that the dramatic declines in parasite burdens in fish from parr to smolt stages in this study could be related to parasite associated mortality.

Method comparison

Diagnostic methods for identifying parasites include serology, non-lethal blood and fecal exams, molecular methods, biopsies, and necropsy with examination of tissues by wet mount or histology (Hendrix and Robinson, 2006). With fish, serological or molecular tests have been developed for some pathogens (Cunningham,

112 2002; AFS-FHS, 2007), yet there are few that have been developed or verified for helminth parasites. Concerning the parasites in our study, M. fryeri and M. insidiosus are the only parasites for which DNA sequences are available. Therefore, the most practical methods for examining fish for a variety of parasites were by histology and wet preparations. Surveys for parasites of wild fishes have been most often based on necropsies and examination of wet tissues (Arthur et al., 1976; Blaylock et al., 1998;

Arkoosh et al., 2004; Butorina et al., 2008; Jacobson et al., 2008). However, some studies have relied on or included data obtained by histology (e.g., Kent et al., 1998;

Longshaw et al., 2004; Stentiford and Feist, 2005).

We employed two diagnostic methods, histology and wet tissue examinations, and compared the sensitivity and specificity of histology using the latter as a gold standard. Although others have used both methods together in parasites surveys (e.g.,

Eaton et al., 1991; Moran and Kent, 1999; Kent et al., 2005), we are unaware of any studies in which both methods were compared empirically. Wet preparations consistently detected more parasites. On average, only 15% of Apophallus sp. was correctly detected by histology (Table 4.4). When histology was applied to determine prevalence of gill and muscle parasites in population surveys, it had an effect on the prevalence of infection. For example, when using histologic evaluation, Apophallus sp. was only detected in two smolt populations screened, but was found in six sites using wet preparations. Likewise, the use of only histology would have resulted in missed M. insidiosus infections in smolts from four sites, and the prevalence of

113 infection would have been greatly underestimated for most parasite infections except infections with gill trematodes (Tables 4.2 and 4.3).

The sensitivity of histology improved when we excluded lightly infected fish

(Table 4.4). However, this increase was still below 50% for all parasites examined, so less than 50% of heavily parasitized fish would be detected by histologic evaluation alone. Clearly, using wet preparations was better for detecting parasites than histology, most likely because more tissue is examined. Our calculations suggest that wet tissue examination, using 1 g of muscle, represents almost 2,400 times more volume than the average amount of muscle examined by histology.

Histology is also relatively poor for evaluating external gill parasites (Table

4.4) such as ciliates, as they often detach when gills are placed in fixatives, and thus data obtained by histology for these parasites are generally underestimated. For example, the reason why C. piscium was the only external ciliate that was detected by histology was probably because of the severity of infection by this parasite. In contrast, although L. salmonae infections were relatively light, this parasite was detected by both methods as this parasite is intracellular and thus would not be removed from the tissues during processing. Wet tissues also provide another advantage in that whole parasites are examined and thus important morphologic features are better visualized.

Histology, however, provides certain advantages. With the exception of the gill metacercariae, the specificity for histology was high for all other parasites (91 to

100%), thus histology is not more accurate at detecting infections than wet preparation

114 evaluation. Embryonated eggs of the Sanguinicola sp. were more often detected in histology compared to wet tissue examination (Table 4.2; note low specificity Table

4.4). These eggs are deep within blood vessels, are small and not motile, and thus are probably easier to detect by histology. Therefore, the better resolution provided by histology likely accounts for this difference, more than compensating for the less volume that is examined by this method compared to wet tissue. Although histology had poor specificity for gill trematodes in our analysis, this was actually due to the choice of wet preparations for the gold standard. Furthermore, detection of N. salmincola in gills appeared to be easier by histology, which could have been due to the difficulty of detecting this worm deep in thicker wet preparations of gill.

The taxonomic identity of parasites can often be determined from histologic sections for well recognized species (Gardiner et al., 1998; Gardiner and Poynton,

1999; Bruno et al., 2006). In addition, this method provides data on pathologic changes associated with parasites that cannot be obtained with wet tissue examination.

It was also easier to examine multiple organs on one slide, and thus several parasites were observed in organs that were not examined in wet preparations. Most of these parasites could have been identified by wet preparations, but it would have been very labor intensive to dissect certain organs (e.g., brain of very small fish). Furthermore, some tissues are difficult to squash thin enough to visualize parasites (e.g., heart) in wet preparations. Finally, histology is a broad based diagnostic method, allowing for detection of a variety of etiological agents beyond parasites.

115 In conclusion, we detected a wide variety of parasites in fish from some sites, and few were associated with prominent histologic changes. We found that parasites were more often detected in wet preparations. However, histology provides more information on pathology and may be more amendable for detecting parasites in certain tissues (e.g., heart and brain). We propose the use of both methods when possible to provide the most accurate identification, enumeration, and determination of impact of parasites. This is particularly important when sample availability is limited.

Acknowledgments

This research was funded, in most part, by an ODFW Fish Health Graduate

Research fellowship (agency grant 010-7032-IAA-FISH) to J.A.F. An NIH NCRR training grant (T32 RR023917) funded T.P. We are also indebted to the research personnel at ODFW and EPA for providing samples. Thank you to S. Fiest, J. Heidel,

K. Jacobson, and C. Schreck for manuscript review.

116

Figure 4.1. Histology showing infection sites of two common digenean metacercariae and two uncommon adult helminths. a) Apophallus sp. in myosepta (arrow), transverse section of axial skelatal muscle. Bar = 200 µm. b) Nanophyetus salmincola in gill arch, note prominent excretory bladder (B). Bar = 100 µm. c) Adult tapeworm. Bar = 100 µm. d) Adult acanthocephalan, note proboscis (arrow). Bar = 500 µm.

117

Figure 4.2. Wet preparation examination of parasites. Brightfield or Nomarski’s phase contrast. a) Metacercariae of Nanophyetus salmincola (arrows) in gill tissue at low magnification. Bar = 500 µm. b) Philonema sp. larvae (arrow) in gill tissue. Bar = 250 µm. c, d) Echinochasmus milvi metacercariae from gills. Arrows = collar spines. Nomarski’s. Bars = 25 µm. e) excysted metacercariae of Apophallus sp. from gill. Nomarski’s. Bar = 100 µm. f) Epistylus sp. from gills. Normaski’s. Bar = 25 µm. g) Trichodina truttae from gills. Nomarski’s. Bar = 50 µm. h) Loma salmonae xenomas (arrows) in gills at low maginifcation. Brightfield. Bar = 250 µm. Insert shows spores at high magnification of xenoma (X) with released spores (arrows). Normaski’s. Bar = 10 µm. i) Capriania piscium (arrows) on gill. Brightfield. Bar = 500 µm. Insert shows magnification with characteristic tentacles. Nomraski’s. Bar = 10 µm.

118

Figure 4.3. Histologic examination of parasites and the tissues they infect. H&E, unless indicated otherwise. a, b) Gill metacercariae (m) associated with hyperplasia. Bars = 50 µm. c) Metacercaria of Echinchasmus milvi, note spines in collar (arrows). Bar = 20µm. d) Embryonated eggs of Sangunicola sp. (arrow) in gills. Bar = 50µm. e) Capriania piscium on gills (arrows). Bar = 50 µm. f) Loma salmonae in gills (L). Bar = 50 µm. g) Pseudocapillaria salvelini in intestine. Bar = 100 µm. h, i) Philonema sp. in ovarian tissue. Bars = 100 µm. j) adult digenean in pyloric ceaca. Bar = 100 µm. k – o) Myxozoans in tissue sections. Bars =25 µm. k, l) Myxidium salvelini in kidney tubule. Arrows = spores. Giemsa stained insert showing dark-staining polar capsule. m, n) Chloromyxum majori in kidney glomerulus, n) shows polar capsules (arrow) with Giemsa stain. o) Myxobolus sp. in brain, Giemsa stained.

119

0 NA NA NA NA NA NA NA NA 24 (7) (7) 24 4 (15) (3) 20 15 (4) (4) 15 Gross Neascus Neascus Oncorhynchus Oncorhynchus

= Wet preperations, = Wet

0 0 0 0 0 0 4 (8) 4 (8) 5 (3) 5 (3) fryeri Histo 43 (5) (5) 43 (6) 10 40 (7) (7) 40 30 (10) (10) 30 (15) 45

rs to the mean number of rs to the mean

NA NA NA NA NA NA NA NA Wet year 2006), whereas histology and wet nstiy (in parentheses) refers to mean nstiy (in parentheses) refers to mean 75 (9) (9) 75 90 (10) (10) 90 100 (21) (21) 100 87 (147) (147) 87 100 (199) (199) 100 sto = Histology, Wet sto = Histology, Wet

= Upper North Fork, LNF = Lower North

0 0 0 0 0 0 0 5 (1) 5 (1) Histo 19 (7) (7) 19 (4) 29 (5) 25 60 (26) (26) 60 (30) 91

Myxobolus insidiosus Myxobolus Myxobolus insidiosus Myxobolus

0 NA NA NA NA NA NA NA NA sp. Wet 86 (4) (4) 86 (2) 45 95 (24) (24) 95 100 (522) (522) 100 ology or wet preparations refe parasites in axial skeletal muscle of coho salmon ( parasites in axial skeletal muscle of coho salmon

0 0 0 0 0 0 Apophallus 80 (1) (1) 80 (3) 20 (3) 79 (2) 40 (2) 47 (2) 35 100 (6) (6) 100 ogy was available for fry and parr (brood

0 s, Gross = gross examination and mean inte and mean = gross examination s, Gross NA NA NA NA NA NA NA NA 80 (7) (7) 80 100 (17) (17) 100 (22) 100 (28) 100 year 2006) and parr (brood 2007). Hi k, WFSR = West Fork Smith River, UNF Fork Smith = West k, WFSR salmincola Nanophyetus Nanophyetus

0 0 0 5 (1) 5 (1) 93 (3) (3) 93 (6) 85 (3) 59 (3) 60 (1) 17 (4) 91 (1) 13 (2) 55 (5) 60 Histo Wet ) by two different methods). Only histol ) by two different methods). Location and fish stage stage fish Location and EF Trask R R EF Trask Fry (n = 35) Parr (n = 20) 20) (n = Smolts Mill Ck, Siletz R Fry (n = 30) 20) = (n Parr 21) (n = Smolts WFSR Fry (n = 29) 10) = (n Parr 24) (n = Smolts 15) (n = 2007 Parr, R Nehalem UNF Fry (n = 30) 20) = (n Parr 20) (n = Smolts EF = East Fork, R River, Ck Cree hist Mean density data from Fork, UMS = Upper Main Stem. For neascu sampled. tissue parasites/infected of parasites/infected fish. number tissues were evaluated for smolts (brood tissues were evaluated for smolts density (in parentheses) of and mean Table 4.1. Prevalence kisutch 120

0 0 NA NA NA NA NA NA NA 40 (7) (7) 40 (1) 15 (8) 18 Gross 70 (16) (16) 70 Neascus Neascus

0 0 0 0 0 0 0 0 5 (5) 5 (5) 8 (6) Histo 20 (7) (7) 20 (5) 10 (6) 11

NA NA NA NA NA NA NA Wet 80(6) 80(6) 89 (42) (42) 89 90 (23) (23) 90 79 (14) (14) 79 80 (12) (12) 80 100 (56) (56) 100

0 0 0 0 0 0 0 5 (1) 5 (1) Histo 10 (4) (4) 10 10 (2) (2) 10 (5) 10 16 (12) (12) 16 50 (10) (10) 50

insidiosus Myxobolus fryeri Myxobolus

0 0 0 NA NA NA NA NA NA NA sp. Wet 20 (2) (2) 20 60 (20) (20) 60 (13) 32

0 0 0 0 0 0 Apophallus 2 (2) 2 (2) 37 (6) (6) 37 (2) 39 (2) 10 (2) 10 (3) 33 (3) 31

NA NA NA NA NA NA NA 5 (2) 5 (2) 60 (2) (2) 60 80 (2) (2) 80 (9) 67 79 (10) (10) 79 100 (11) (11) 100 salmincola Nanophyetus Nanophyetus

0 0 0 58 (2) (2) 58 (2) 28 (2) 30 (3) 83 (1) 42 (2) 10 (1) 15 (2) 45 (3) 40 (2) 19 Histo Wet Location and fish stage stage fish Location and LNF Nehalem R LNF Nehalem Fry (n = 60) 18) = (n Parr 20) (n = Smolts EF Lobster Ck, Alsea R Fry (n = 30) 20) (n = Smolts R Alsea Ck, Lobster UMS Fry (n = 30) 19) (n = Smolts Coos Bay Ck, Winchester 20) = (n Parr 20) (n = Smolts Mill Ck, YaquinaR 20) (n = Smolts (mean) Data Combined Fry254) (n = 118) = (n Parr 204) (n = Smolts Table 4.1. Continued.

121

0 0 0 0 0 NA NA NA NA NA NA NA NA NA NA Wet 15 (2) (2) 15 therius

multifiliis 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Ichthyoph- to His ) by two

0 0 0 0 0 0 NA NA NA NA NA NA NA NA NA NA Wet ations, EF = East Fork, piscium piscium

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Histo 10 (13) (13) 10 Capriniana Capriniana

0 0 0 0 0 sp. NA NA NA NA NA NA NA NA NA NA Wet Oncorhynchus kisutch 20 (11) (11) 20 mber of parasites/infected tissue sampled. mber ereas histology and wet tissues were

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Epistylus Histo

0 0 0 NA NA NA NA NA NA NA NA NA NA Wet 5 (3) 5 (3) 5 (1) 5 (1) 5 (1) 5 (1) = Histology, Wet = Wet prepar = Wet = Histology, Wet truttae

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Trichodina Trichodina Histo

0 0 0 0 0 NA NA NA NA NA NA NA NA NA NA Wet 5 (2) 5 (2)

sp.

0 0 0 0 0 0 0 0 0 0 Sanguinicola Sanguinicola 5 (1) 5 (1) Histo 10 (3) (3) 10 11 (2) (2) 11 (7) 10 15 (3) (3) 15 (5) 10 ntheses) of gill parasites of coho salmon ( of gill parasites ntheses) or wet preparations refers to the mean nu or wet preparations refers to the mean

0 0 NA NA NA NA NA NA NA NA NA NA Wet 5 (5) 5 (5) parr (brood year 2007). Histo 35 (2) (2) 35 (6) 92 10 (2) (2) 10 lable for fry and parr (brood year 2006), wh rk Smith River, UNF = Upper North Fork, LNF Lower UMS River, UNF rk Smith

0 0 0 0 metacercariae 7 (1) 7 (1) Histo 75 (4) (4) 75 (2) 19 10 (1) (1) 10 (3) 40 (1) 38 (4) 91 (6) 74 (3) 25 24 (1) (1) 24 (7) 40 (2) 40 Unidentified gill

0 0 NA NA NA NA NA NA NA NA NA NA Wet 20 (3) (3) 20 (4) 23 (3) 20 15 (1) (1) 15

0 0 0 salmincola Nanophyetus Nanophyetus 93 (3 (3 93 60(3) 60(3) Histo 85 (6) (6) 85 (2) 43 (3) 59 (3) 60 (1) 33 (2) 27 (1) 13 (2) 55 (2) 58 (2) 28 (3) 65 stage Location and fish R EF Trask Fry (n = 35) Parr (n = 20) 20) (n = Smolts Mill Ck, Siletz R Fry (n = 30) 20) = (n Parr 21) (n = Smolts WFSR Fry (n = 29) 10) = (n Parr 24) (n = Smolts 15) (n = 2007 Parr, R Nehalem UNF Fry (n = 30) 20) = (n Parr 20) (n = Smolts R LNF Nehalem Fry (n = 60) 18) = (n Parr 20) (n = Smolts density (in pare mean and Table 4.2. Prevalence avai Only histology was different methods. = West Fo R = River, Ck Creek, WFSR histology Mean density data from Main Stem. evaluated for smolts (brood year 2006) and evaluated for smolts 122

0 0 0 0 NA NA NA NA NA NA Wet 2 (4) 2 (4) therius

multifiliis 0 0 0 0 0 0 0 0 0 0 0 Ichthyoph- to

0 0 0 90 90 NA NA NA NA NA NA (56) (56) 9 (56) 9 (56) piscium piscium

0 0 0 0 0 0 0 0 0 95 (40) (40) 95 11 (27) (27) 11 Capriniana Capriniana

0 0 0 sp. NA NA NA NA NA NA 3 (14) 3 (14) 10 (17) (17) 10

0 0 0 0 0 0 0 0 0 0 0 Epistylus

0 0 0 NA NA NA NA NA NA 5 (3) 5 (3) 3 (2) 3 (2) truttae

sto Wet Histo Wet His Wet Histo Wet sto 0 0 0 0 0 0 0 0 0 0 0 Trichodina Trichodina

sp. 0 0 0 0 (0) (0) 0.5 0.5 NA NA NA NA NA NA

0 0 0 0 0 9 (7) 9 (7) 60 (7) (7) 60 3 (11) (3) 15 20 (11) (11) 20 (11) 10 Sanguinicola Sanguinicola

0 0 0 0 NA NA NA NA NA NA 5 (3) 5 (3)

0 0 0 0 metacercariae 55 (4) (4) 55 40 (5) (5) 40 (2) 10 (7) 46 (4) 20 90 (14) (14) 90 (12) 25 Unidentified gill

0 0 NA NA NA NA NA NA 5 (1) 5 (1) 5 (1) 7 (4)

0 0 salmincola Nanophyetus Nanophyetus 5 (1) 5 (1) Histo Wet Histo Wet Histo Wet Hi Wet Wet Histo Histo 50 (1) (1) 50 (2) 40 (2) 10 (3) 83 (1) 20 (2) 45 (3) 40 (2) 24 fish stage fish stage Location and Ck, Cascade R Alsea Fry (n = 10) 10) = (n Parr 20) (n = Smolts Ck, EF Lobster R Alsea Fry (n = 30) 20) (n = Smolts UMS Lobster R Alsea Ck, Fry (n = 30) 19) (n = Smolts Mill Ck, R Yaquina 20) (n = Smolts Combined (mean) Data Fry254) (n = 118) = (n Parr Smolts 204) = (n Table 4.2. Continued. 123 Table 4.3. Prevalence and mean density (in parentheses) of viscera or brain parasites of coho salmon(Oncorhynchus kisutch) by histology for fry, parr, and smolts from brood year 2006 and parr from brood year 2007. For Pseudocapillaria salvelini infections, only prevelence was recorded because cross sections could represent either single or multiple worms. EF = East Fork, R = River, Ck = Creek, WFSR = West Fork Smith River, UNF = Upper North Fork, LNF = Lower North Fork, UMS = Upper Main Stem. Mean density data refers to the mean number of parasites/infected tissue sampled. Location and fish Nano- Myxobolus Myxidium Chloro- Pseudo- Adult stage phyetus sp. salvelini myxum capillaria digenean salmincola majori salvelini spp. EF Trask R Fry (n = 35) 0 0 6 (9) 0 0 17 (1) Parr (n = 20) 0 25 (2) 0 0 0 5 (1) Smolts (n = 20) 0 0 0 0 0 5 (1) Mill Ck, Siletz R Fry (n = 30) 97 (5) 0 67 (4) 0 0 80 (3) Parr (n = 20) 100 (10) 0 0 5 (3) 25 50 (2) Smolts (n = 21) 86 (13) 0 0 0 50 24 (1) WFSR

Fry (n = 29) 76 (4) 0 45 (4) 0 0 76 (4) Parr (n = 10) 80 (6) 40 (10) 0 0 10 50 (1) Smolts (n = 24) 96 (9) 50 (27) 0 0 33 42 (2) Parr, 2007 82 (25) 45 (11) 0 0 85 10 (1) (n = 15) UNF Nehalem R Fry (n = 30) 33 (2) 0 60 (5) 0 0 67 (3) Parr (n = 20) 95 (1) 85 (11) 0 15 (11) 0 15 (1) Smolts (n = 20) 90 (9) 30 (12) 0 0 50 65 (5) LNF Nehalem R Fry (n = 60) 77 (4) 2 (1) 52 (3) 23 (8) 0 57(3) Parr (n = 18) 28 (5) 50 (6) 0 95 (11) 0 15 (1) Smolts (n = 20) 95 (42) 60 (6) 0 0 50 60 (3) Cascade Ck,

Alsea R 50 (3) 56 (13) 0 30 (3) 0 0 Fry (n = 10) 100 (9) 80 (3) 0 10 (3) 30 0 Parr (n = 10) 90 (8) 35 (2) 30 (5) 10 (4) 0 75 (3) Smolts (n = 20) EF Lobster Ck,

Alsea R 0 0 43 (4) 0 0 23 (5) Fry (n = 30) 0 0 0 0 0 15 (1) Smolts (n = 20) UMS Lobster

Ck, Alsea R 63 (4) 0 0 0 0 37 (4) Fry (n = 30) 95 (6) 0 0 0 0 11 (1) Smolts (n = 19)

124

Table 4.3. Continued.

Location and fish Nano- Myxobolus Myxidium Chloro- Pseudo- Adult stage phyetus sp. salvelini myxum capillaria digenean salmincola majori salvelini spp. Winchester Ck,

Coos Bay 30 (2) 0 0 20 (4) 25 0 Parr (n = 20) 65 (2) 0 0 0 30 0 Smolts (n = 20) Mill Ck, Yaquina

R 80 (4) 0 0 0 15 25 (3) Smolts (n = 20) Combined Data (mean) Fry (n = 254) 50 (4) 7 (4) 34 (5) 7 (5) 0 45 (3) Parr (n = 118) 62 (6) 40 (6) 0 21(6) 13 19 (1) Smolts 70 (12) 1 (8) 3 (5) 1(4) 23 32 (2) (n = 204)

125 Table 4.4. Sensitivity and specificity of histology, using evaluations of wet preparation as a standard, for infections in smolts from 10 rivers and parr from one river. 95% confidence intervals in parentheses. Heavy = Highest 10% density based on wet preparation results for parasites in axial skeletal muscle. The mean density and (range) of these heavy infections were as follows: Apophallus sp. = 309 (16-1,034), Nanophyetus salmincola = 39 (22-61), Myxobolus insidiosus = 262 (116-570). Specificity for heavy infections is not presented because they were the same as those that included all data.

Sensitivity Specificity Muscle parasites Apophallus sp. 0.15 (0.08-0.25) 0.99 (0.95-1.0) Apophallus sp., heavy 0.45 (0.25-0.67) -- Nanophyetus salmincola 0.26 (0.19-0.34) 0.91 (0.80-0.96) N. salmincola, heavy 0.32 (0.15-0.55) -- Myxobolus insidiosus 0.13 (0.09-0.19) 1.0 (0.83-1.0) M. insidiosus, heavy 0.41 (0.21-63) --

Gill parasites Gill metacercariae 0.31 (0.12-0.59) 0.80 (0.74-0.86) N. salmincola 0.44 (0.21-0.69) 0.77 (0.71-0.83) Sanguinicola sp. 0 (0-0.37) 0.91 (0.86-0.95) Trichodina truttae 0 (0-0.54) 1 (0.98-1.0) Epistylus sp. 0 (0-0.48) 1 (0.98-1.0) Ichthyophtherius multifiliis 0 (0-0.60) 1 (0.98-1.0) Capriniana piscium 0.95 (0.72-0.99) 0.98 (0.95-0.99)

126

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134 Chapter 5: Persistence of Infection by Metacercariae of Apophallus sp., Neascus sp., and Nanophyetus salmincola Plus Two Myxozoans (Myxobolus insidiosus and Myxobolus fryeri) in Coho Salmon Oncorhynchus kisutch

Jayde A. Ferguson1, Carl B. Schreck2, Robert Chitwood2, and Michael L. Kent1

1 Department of Microbiology, Oregon State University, 220 Nash Hall, Corvallis

Oregon 97331

2 Department of Fisheries and Wildlife, Oregon State University, and Oregon

Cooperative Fish and Wildlife Research Unit, U.S. Geological Survey, 104 Nash Hall,

Corvallis, Oregon 97331

Published in Journal of Parasitology

American Society of Parasitologists Inc., Lawrence, Kansas

Issue 96

135 ABSTRACT

We evaluated the ability of 5 muscle- or skin-dwelling parasites to persist in naturally infected coho salmon, Oncorhynchus kisutch, from the West Fork Smith

River, Oregon, by holding them in captivity from late summer to early spring (parr stage to the typical time of smoltification). These parasites included metacercariae of 3 digeneans, Nanophyetus salmincola, Apophallus sp., and neascus sp., and 2 myxozoans, Myxobolus insidiosus and Myxobolus fryeri. Two groups of wild caught fish were evaluated in the laboratory, i.e., heavily infected fish from the lower mainstem and less severely infected fish collected from tributaries of this river. All parasites survived in these fish for the 7-mo experiment. Only 2 parasites had a statistically significant lower median abundance between host life stages. The mean abundance of N. salmincola declined 45% in the tributary fish and Apophallus sp. declined 43% in the lower mainstem fish. However, more than 50% of each species persisted until the end of the study, with smolts still harboring relatively high infections.

INTRODUCTION

Evaluating the effects of heavy parasite infections on wild fish populations is difficult because these fish may die or become selectively removed by predators prior to sampling efforts (Gordon and Rau, 1982; Bakke and Harris, 1998). Methods have been devised for estimating mortality associated with parasitism in wild fishes

(Anderson and Gordon, 1982; Lester, 1984), which have been tested theoretically

136 (Rousset et al., 1996; Duerr et al., 2003) and under natural conditions (Kennedy, 1984;

Kalbe et al., 2002; Bourque et al., 2006). Evaluating fluctuations of parasites that persist for a long time is an ideal method for studying impacts of parasites in wild fishes, as large declines in parasite burdens could be explained by parasite-associated mortality (Lester, 1984). Pertinent to the present study, Jacobson et al. (2008) evaluated temporal changes in parasite dispersion (mean abundance to variance ratio) in coho salmon (Oncorhynchus kisutch) populations in Oregon and found that early marine resident mortality was associated with infections by metacercariae of

Nanophyetus salmincola (Nanophyetidae).

It is generally accepted that metacercariae of digenetic trematodes persist in their hosts for very long times. For example, Lo et al. (1985) conducted a pathology and ultrastructure study on Clinostomum complanatum (Clinostomatidae; “yellow grub”) from the muscle and parietal peritoneum of loach, Misgurnus anguillicaudatus, using fish that had been infected 6 mo prior. Also, many studies have shown that neascus-type metacercariae such as Uvulifer ambloplitis and neascus spp.

(Diplostomatidae), which infect skin and cause “black spot disease” in juvenile centrarchid fishes, can survive from late fall to early spring (Fischthal, 1949; Lemly and Esch, 1983; 1984), or even up to 4.5 yr (Hoffman and Putz, 1965). Farrell et al.

(1964) showed that metacercariae of N. salmincola acquired from freshwater habitats persist throughout the adult stage of coho salmon, even under conditions of high ocean salinity.

137 Myxozoans infecting solid tissues (histozooic species) also persist, often through the entire life of the fish host (Lom and Dyková, 1992). For example, neurotropic Myxobolus arcticus and Myxobolus neurobius have been used as biological tags to identify stocks of marine salmon because they infect juvenile salmonids in freshwater and persist throughout the saltwater phase of host development (e.g., Urawa and Nagasawa, 1989). Likewise, muscle-infecting

Myxobolus insidiosus and Henneguya salminicola are capable of similar persistent infections (Kent et al., 1994).

We are investigating the impacts of parasites on the survival of parr to smolt stage coho populations from the West Fork Smith River (WFSR) in Oregon. The life cycle of coho salmon consists of hatching from eggs in gravel of streams in the spring, followed by rearing as fry, and then as parr, for about one year throughout the river and its smaller tributaries. The following spring they undergo a metamorphosis

(smoltification) and migrate to the ocean as smolts, where they mature to adulthood and return about 18 mo later to natal streams to reproduce and die (Groot and

Margolis, 1991). Smolts are also morphologically distinct from parr, having a more streamlined body and silver color (Groot and Margolis, 1991)

Ebersole et al. (2006) conducted an extensive tagging study at WFSR and showed that coho salmon from the lower reach of the mainstem had lower than expected overwintering survival based on fisheries prediction models. They also found that underyearling fish from the lower mainstem had reduced over winter survival compared to fish from upper reaches of the mainstem and tributaries, which was an

138 opposite result predicted by fisheries models based on habitat. Rodnick et al. (2008) found higher prevalences and abundances of certain parasites in coho salmon, particularly black spot metacercariae and Apophallus sp. (Heterophyidae), in the lower mainstem compared to the upper reach of this system. Cairns et al. (2005) reported a positive correlation between high temperatures, often exceeding 20 C in the lower mainstem, and black spot infections in this river. As part of an ongoing field study, we have consistently observed that outmigrating coho salmon smolts from this river have strikingly lower parasite burdens than the parr from the lower mainstem. This could be due to differential mortality of fish with high parasite burdens or to removal of parasite infections by the hosts’ immune response. Therefore, we evaluated the latter by determining the ability of survival of 5 parasite species (N. salmincola, Apophallus sp., neascus sp., M. insidiosus, and Myxobolus fryeri) from WFSR naturally-infected coho salmon held in captivity from parr to smolt stage (from late summer to early spring).

MATERIALS AND METHODS

Field collection

Coho salmon parr were collected on 17 September 2007 from the WFSR. A total of 76 parr originating from 2 tributaries, Moore and Coon Creeks, were collected by electrofishing pools. Fish from these 2 streams were mixed together for transportation and are referred to hereafter as the tributaries group. Fifty-seven parr from the lower mainstem were collected similarly, but kept separately from the

139 tributary fish. Fish were transported in tanks supplied with pressure regulated O2 to the

Oregon State University’s Fish Performance and Genetics Laboratory to hold in captivity.

Holding wild fish

Eleven parr (74 mm mean fork length) from the lower mainstem and 14 parr

(63 mm mean fork length) from the tributaries were killed and necropsied on the first day of the captive study to obtain initial parasite abundance data. The remaining 46 lower mainstem and 62 tributary parr were placed in 2 separate outdoor flow-through

200-L tanks, respectively, covered with plastic mesh lids and supplied with well water at relatively constant temperature (13-16 C). Five days after transportation, all fish received a prophylactic formalin treatment (200 ppm, 1 hr static treatment supplied with O2) to remove external parasites such as ciliates and monogenetic trematodes because these parasites have direct life cycles and could potentially pose health problems to captive fish. Fish were fed to satiation twice daily with a mixture of 1.5 mm commercial pellets (Bio-Oregon Inc., Warrenton, Oregon) and freeze-dried brine shrimp and krill (Argent Laboratories, Redmond, Washington). Artificial plastic plants were placed in tanks to provide cover. Tanks were monitored for dead and moribund fish daily and the debris from tanks was siphoned weekly. Fish were reared to the approximate time of smoltification (21 April 2008) and lethally sampled to obtain parasite counts to compare to parr fish. At the end of the study, the mean fork length of lower mainstem fish was 140 mm and that of tributary fish was 135 mm.

140 Although we regarded all fish at the end of the study to be smolts for analysis purposes, there was variability among fish to their degree of smoltification, as development can vary in populations.

Eight days after transport, fish from the lower mainstem group were screened for obvious neascus infections to follow individual worms on individually marked fish. This was the only group initially screened because previous reports had shown that fish from the upper tributaries had fewer infections of neascus (Rodnick et al.,

2008). These neascus-infected fish were individually anesthetized with MS-222, marked by clipping pelvic or adipose fins, and neascus cysts on their skin were enumerated macroscopically. These fish were then held together in a separate 100-L tank in conditions as described above. The location and color of each cyst from both sides of the marked fish were recorded on a drawing of a typical juvenile coho salmon and repeated 7 mo later at the end of the study.

Sample processing

All fish were examined for the presence of black spots (neascus) on the skin, weighed, and measured. The entire left side of each fish was filleted and frozen to later count parasites in the skeletal muscle. The remaining carcasses were fixed in

Dietrich’s solution (30 mL 95% ethanol, 10 mL formaldehyde, 2 mL glacial acetic acid, 58 mL distilled water) for histological processing to assess pathological changes associated with parasite infections and any coincident acute infections, e.g., viral or bacterial.

141 Parasite evaluation

Prior to parasite enumeration, all frozen fillets were thawed and weighed to the nearest 0.01 g. Wet mounts were made by squashing each fillet between 2, 15- x

30-cm plexiglass plates with approximately 12 kg of weight applied. Parasites were enumerated by examining the entire fillet (wet mount) using a compound microscope at x50 and x100 magnification. Parasite counts from the fillets were multiplied by 2 in order to represent the number of parasites per fish in the skeletal muscle, as it was assumed that the parasites did not preferentially infect one side of the fish. We accounted for the increase in fish size from parr to smolt stage by counting parasites from the entire fillet of each fish. There were 20 captive smolts that had fillets too large to examine completely so these they were sub-sampled by randomly selecting and examining pieces of muscle from different locations of the fillet, totaling 5 g from the same fish. Total parasite counts were then estimated based on the total weight of the fillet. Prevalence (number of infected fish/total fish assayed), mean abundance

(number of parasites/total fish assayed), and mean density (number of parasites/sample weight of total fish assayed) of infections are reported in accordance with the definitions provided by Margolis et al. (1982) and that of Bush et al. (1997).

The parasites that were enumerated in the skeletal muscle included 2 metacercariae of digenean trematodes, i.e., Apophallus sp. and N. salmincola, and 2 myxozoan parasites, i.e., M. insidiosus and M. fryeri. The 2 Myxobolus species could only be differentiated by histological sections. However, M. fryeri pseudocysts are much smaller (maximum length 35 µm) than M. insidiosus pseudocysts (maximum

142 length 187 µm) (Ferguson et al., 2008), so we assumed that the pseudocysts detected by wet mounts (x100 magnification) represented the latter. Thus, wet mount counts from myxozoan infections are reported as M. insidiosus from the skeletal muscle. Data from enumerating Myxobolus species in wet mounts are expressed in terms of pseudocysts/fish. The identification of N. salmincola was easily determined based on observing the hallmark morphological feature of a distinct excretory bladder. This appears white by reflected light and black by transmitted light (Millemann and Knapp,

1970). Apophallus sp. were characterized by distinctive oval cysts, which contrasted to the spherical to sub-spherical cysts of N. salmincola. Moreover, the metacercaria within each cyst was folded approximately in half and had an obvious spiny tegument.

The identity of Apophallus sp. to the generic level was confirmed by excysting metacercariae resembling those above, as described by Irwin (1983), and matching the morphological features with descriptions provided by Schell (1985), Hoffman (1999), and Niemi and Macy (1974). It is important to note that some, but not all, species of

Apophallus and neascus are capable of causing black spot disease in fishes (Warren,

1953; Niemi and Macy, 1974; Schell, 1985; Hoffman, 1999). Infections by neascus, which is a larval genus of certain diplostomatids, were clearly differentiated from

Apophallus sp. in our study by having: (1) a pronounced distance between the parasite cyst and host encapsulating tissue; (2) the metacercaria having a distinct fore and hind body; and (3) prominent calcareous granules within the parasite. In contrast, the oval cysts of Apophallus sp. were nearly half the size of neascus and lacked the associated host response around it.

143 Histology was used to assess viability of metacercariae. Necrotic worms were characterized by lack of distinguishable organs and the presence of pycnotic nuclei.

Histology was also used to differentiate M. fryeri from M. insidiosus, as the pseudocysts of M. fryeri were too small to detect in wet mounts at x100 magnification.

At the end of the study, 57 individual neascus cysts (varying from white to grey to black) were removed from 14 lower mainstem fish that were held in the larger

(200 L) tank. These cysts were examined in wet mounts at x100 magnification with a

Leica DMLB compound microscope to determine viability. Viability was determined by observing movement of intact metacercariae within cysts, sometimes requiring prodding of the cyst with a probe pressed on the overlying coverslip.

Statistical analyses

® All statistical comparisons were conducted with either S-PLUS version 8.0 software (Insightful Corp., Seattle, Washington) or R, version 2.7.2 (R Development

Core Team, Vienna, Austria; 2008). Evaluation of normality was performed using the

® Kolmogorov-Smirnov Goodness of Fit test with S-PLUS , and the Levene’s test for homogeneity was used to assess constant variance with the statistical program R, which was also used for calculating 95% confidence limits. Median abundance of

Apophallus sp. and N. salmincola between parr and smolt stages were compared within each group using an Exact Wilcoxon rank-sum test, due to the non-normal distribution of data. Student’s t-tests were used to compare mean abundance of M. insidiosus between parr and smolt stages for both groups, as these data did conform to

144 the assumptions of parametric analysis. One-tailed tests were used in lieu of 2-tailed tests for analyzing metacercariae counts because fish were held in tanks free of infective intermediate hosts, and thus it was impossible for new infections to occur in these fish. However, a 2-tailed test was used for analyzing the myxozoan infections because these parasites are more similar to microparasites, as they asexually reproduce in the fish host and thus smaller pseudocysts may not have been detected initially but grew to detectable size by the end of the study. For 1-tailed tests, only upper confidence limits are presented to indicate the proximity of our estimate to zero, i.e., no difference. Statistical significance was set at P < 0.025 for 1-tailed tests and P <

0.05 for 2-tailed tests.

The mean neascus counts of both parr and smolt stages of the 6 individual lower mainstem fish held in a separate tank were compared using a 1-sided Bootstrap test with R software. This non-parametric test was used because the population parameters for this sample were unknown and inspection of the data indicated skewness. Additionally, it is more appropriate for small sample sizes (Ramsey and

Schafer, 2002).

Statistical analyses were not performed on fish that died during the experiment because the sample size was not large enough to be considered a representative population. Furthermore, parr that were sampled at the beginning of the study with comparable parasite burdens as the dead fish could have died and thus contributed to the population of dead fish if they had not been sampled initially. We, therefore, provide data on mean abundance and density for this group without statistical analysis.

145 RESULTS

Nanophyetus salmincola

Wet mounts: Almost all the parr and smolts from the lower mainstem were infected with N. salmincola. Within the lower mainstem, both fish stages had a similar mean abundance of this parasite (Table 5.1) based on wet mount observations (Figure

5.1d). There was not a significantly lower median abundance of N. salmincola in smolts compared to parr from the lower mainstem (P = 0.034, Exact Wilcoxon rank- sum test). The mean density of infection for parr from this group was 63 metacercariae/g (range: 28-175), and that of smolts was 5 metacercariae/g (range: 0-

17).

Prevalence of N. salmincola in the fish from the tributaries was also essentially

100% (Table 5.1). Parr and smolts from this location had a similar mean abundance of

N. salmincola, which was less than that of lower mainstem fish (Table 5.1). Smolts from the tributaries had a statistically significant lower median abundance than parr from this group (P = 0.001, Exact Wilcoxon rank-sum test). It was estimated that smolts had a median abundance of 19.6 N. salmincola/fish less than parr (95% upper confidence limit = 7.68 metacercariae/fish). Parr from this group had a mean density of infection of 182 metacercariae/g (range: 13-846), whereas smolts had 3 metacercariae/g (range: 0-14).

Histology: Histological evaluations revealed that all metacercariae were alive at the time of fixation, as cells were intact and exhibited no necrotic changes. Processing and sectioning artifacts caused post-mortem disruption of worm tissues. Mild, chronic

146 inflammation composed of macrophages laden with necrotic debris and mild fibroplasia surrounded each cyst (Figure 5.2e).

Apophallus sp.

Wet mounts: Several cysts (Figure 5.1a) were collected from infected coho salmon and excysted to reveal metacercariae morphology. Metacercariae had a spiny tegument and exhibited features consistent with other species of Apophallus sp., including tandem to oblique testis, a pharynx close to the oral sucker, and a long esophagus (Figures 5.1b, c). Every fish from the lower mainstem was infected with

Apophallus sp. and some were very heavily infected, with abundances ranging over

2,000 metacercariae/fish (Table 5.1). Smolts had about a 40% lower mean abundance of Apophallus sp. than parr for the lower mainstem group, and the median abundance of this worm in smolts was significantly lower than that of parr from this group (P <

0.001, Exact Wilcoxon rank-sum test). Smolts had a median abundance of 489.6

Apophallus sp./fish less than parr (95% upper confidence limit = 294.5 metacercariae/fish). The mean density of infection for parr from this group was 1,380 metacercariae/g (range: 748-2,068), and that of smolts was 78 metacercariae/g (range:

16-186).

Infections by Apophallus sp. in fish from the tributaries were much lighter than those of lower mainstem fish (Table 5.1). Tributary fish had a lower prevalence of

Apophallus sp. than lower mainstem fish, with parr from the former exhibiting approximately 44 times less mean abundance than that of parr from the latter (Table

147 5.1). The prevalence in the parr from the tributaries was higher than smolts from the same group. Median abundance of Apophallus sp. was not significantly lower in the smolts compared to the parr for the tributaries group (P = 0.032, Exact Wilcoxon rank- sum test). Parr from this group had a mean density of infection of 42 metacercariae/g

(range: 0-474), whereas smolts had 1 metacercariae/g (range: 0-47).

Histology: Histological evaluations consistently revealed Apophallus sp. in the myoseptal connective tissue between myomeres (Figures 5.2a-d). Essentially no tissue reaction was associated with the encysted metacercariae and all were judged to be viable at the time of fixation. Many metacercariae were dislodged during tissue processing and thus only empty spaces corresponding to the shape of Apophallus sp. were seen (Figure 5.2d).

Myxobolus spp.

Wet mounts: Pseudocysts of M. insidiosus were frequently observed and were readily detected in squashed tissue (Figure 5.1f). All the parr and about 80% of the smolts from the lower mainstem were infected with M. insidiosus (Table 5.1). There was not a statistically lower or higher mean abundance of M. insidiosus in smolts compared to parr for fish from this location (P = 0.409, 2-tailed Student’s t-test)

(Table 5.1). The mean density of infection for parr from this group was 400 pseudocysts/g (range: 23-1,084), and that of smolts was 56 pseudocysts/g (range: 0-

468).

148 Prevalence of M. insidiosus in the tributaries group was similar to that of the lower mainstem group, with about a 60% lower mean abundance compared to the latter (Table 5.1). Although smolts from the tributaries did not have a significantly lower mean abundance of M. insidiosus compared to parr, they actually had a significantly higher mean abundance (P = 0.007, 2-tailed Student’s t-test) (Table 5.1).

Parr from this group had a mean density of infection of 276 pseudocysts/g (range: 16-

743), whereas smolts had 37 pseudocysts/g (range: 0-302).

Histology: Histological evaluations revealed that all pseudocysts of M. insidiosus appeared intact within muscle fibers and were not associated with inflammatory changes (Figure 5.2g). Pseudocysts of M. fryeri were detected in peripheral nerves of the muscle in 9% (1/11) of parr and 8% (3/36) of smolts from the lower mainstem, and 7% (1/14) of parr and 24% (15/62) in smolts from the tributary fish. It was not associated with tissue damage or significant inflammation (Figure

5.2f). All but 1 fish with M. fryeri infections were also infected with M. insidiosus.

Neascus

Differentiation between encysted metacercariae of this black spot trematode and that of Apophallus sp. was very straightforward; the former was much larger and incited production of a thick host capsule (Figure 5.1e). Tracking individual neascus infections in the 6 individual lower mainstem fish held in a separate tank showed that infections generally persisted throughout the 7-mo study. Although the overall counts from parr to smolts were similar, 3 individuals had fewer cysts, while 2 fish had higher

149 counts after 7 mo (Table 5.2). The mean number of neascus cysts from the parr at the start of the study was not significantly different from the mean number of cysts from these same fish at the smolt stage 7 mo later (P = 0.307, two-sample Bootstrap test).

We noted that some of the cysts began as white or grey in color and then progressed to black by the end of the study. Additionally, some cysts that were grey in color at the end of the study were also circumscribed by a yellow to orange rim.

No neascus infections were detected in the tributary fish at the termination of the study. Of the 57 cysts examined from 14 lower mainstem fish, only 1 cyst contained a metacercaria that did not exhibit movement. This corresponds to a survival of 98.2%. We also detected 1 small black cyst that contained an Apophallus sp. metacercaria, which appeared intact.

Mortalities from lower mainstem group

Four fish died 1-2 mo after the beginning of the study, all of which were from the lower mainstem group. These fish were emaciated at the time of death, and had a mean condition factor of 0.74 ([g/cm3] x 100]) versus 0.95 ([g/cm3) x 100]) for lower mainstem parr initially sampled. Mean abundance of N. salmincola was nearly identical in lower mainstem fish from both the initially sampled parr and the 4 fish that died (Table 5.1), which was also similar to that of the smolts (Table 5.1). However, these four fish had a higher mean density of infection (215 metacercariae/g; range =

61-418), due to their reduced condition factor. Likewise, these dead fish displayed a similar mean abundance of Apophallus sp. as the smolts (Table 5.1). Parr sampled

150 showed a higher mean abundance of Apophallus sp. compared to the dead fish (Table

5.1). However, the latter were more emaciated and thus they had a greater mean density of infection (1,930 metacercariae/g; range = 950-3,410) than initially sampled parr. Finally, dead fish had a much lower mean abundance of M. insidiosus than parr and smolts and a lower mean density of infection (63 pseudocysts/g; range = 0-246) than both groups.

DISCUSSION

We found that metacercariae from all 3 trematodes and pseudocysts of the 2

Myxobolus species persisted at similar prevalences and abundances in coho salmon held from late summer parr stage to spring smolts (a 7-mo period). Consistent with other studies, only a small percentage of the metacercariae of the species studied were lost during over winter time periods (late fall to early spring) (Fischthal, 1949; Evans and Mackiewicz, 1958; Lemly and Esch, 1983, 1984).

Our findings that N. salmincola persists are consistent with those of other studies. Farrell et al. (1964) was able to induce clinical signs of salmon poisoning in dogs fed adult coho salmon infected with N. salmincola 33.5 mo prior. In our study, N. salmincola showed no statistically significant decline in infections in lower mainstem fish. However, a significantly lower median abundance of N. salmincola in muscle was detected in fish from the tributaries. Although this decrease was statistically significant, more than half of the metacercariae in the muscle persisted. Nanophyetus

151 salmincola elicited minimal tissue response and inflammation, which was also consistent with the previous histopathology studies by Wood and Yasutake (1956).

Apophallus sp. is of particular interest because we have detected dramatic declines of over 95% in abundance between parr and smolts in wild coho salmon populations from the lower mainstem of WFSR (unpubl. obs.), and thus we hypothesize that this parasite may be associated with over winter mortality. In contrast to the declines in parasites observed in the wild, lower mainstem fish held in captivity had only a 43% decline in mean abundance of Apophallus sp. over the same time period.

Metacercariae of other heterophyid trematodes have been shown to persist in fish (Sommerville, 1982a; Lysne et al., 1994) Similar to our study with Apophallus sp., Lemly and Esch (1983) observed a 30% reduction of Uvulifer ambloplitis (black- spot neascus) in centrarchid fishes from late fall to early spring. Kennedy (1987) showed that the greatest decline of metacercariae occurred when infections were most abundant and was hardly detectable at a low abundance, which agreed with our findings. We did not detect any significant pathological changes associated with encysted worms, which was consistent with the evaluation of Haplorchis pumilio in tilapia by Sommerville (1982b).

We are confident in our identification of Apophallus sp. to the generic level, as excysted metacercariae were consistent with the description from taxonomic keys by

Schell (1985) and Hoffman (1999). Plans for resolving the species identity of this

152 trematode and neascus are currently underway, which includes obtaining adult worms by feeding chicks metacercariae from coho salmon at our field sites.

Myxobolus insidiosus, the intramuscular myxozoan, actually increased in mean abundance between parr and smolt stages in the tributary group. The increase may be explained by the differences in the development of myxozoans and digeneans. Unlike metacercariae, pseudocysts (plasmodia) of myxozoans are initially very small and continue to grow in size by asexual reproduction and sporogenesis within the host.

The lower counts in parr may be reflected by very small pseudocysts not being detected in wet mounts. Myxobolus fryeri only infects nerves of the somatic muscle, and pseudocysts are much smaller than those of M. insidiosus (cf. Ferguson et al.,

2008). Therefore, we assumed that the counts from squash preparations were all of M. insidiosus. As reported by Amandi et al. (1985), no inflammatory response was seen while M. insidiosus pseudocysts were immature and intact in muscle. This phenomenon occurs with other intramuscular myxozoans, for example, with K. thyrsites a severe inflammatory response ensues following rupture of the pseudocysts and release of parasites from the myofibers (Moran et al., 1999).

The neascus metacercariae in our study has been detected previously from the same location of the WFSR. Cairns et al. (2005) reported a higher prevalence of black spot in coho salmon from the lower mainstem compared to those from the tributaries and Rodnick et al. (2008) reported similar findings. We did not observe a significant change in mean neascus counts from parr to smolt stage, and essentially all metacercariae were viable at the end of the study. Black spot is an ideal model for

153 evaluating persistence as the melanized cysts can be enumerated grossly without lethally sampling the fish. However, evaluation of these parasites by macroscopically counting black spots has limitations because the tissue reaction to the parasite, rather than the actual worm, is counted. Lemly and Esch (1983) compared black-spot neascus infections (U. ambloplitis) in skin of centrarchid fishes and determined that up to 30% of these cysts did not contain live metacercariae after a 7-mo time period, indicating that previous studies may have overestimated prevalence and intensity data of this parasite in fish populations. By the end of our study, all but 1 of the black spots contained live metacercariae (neascus-type). Indeed, we actually saw an increase in intensity in a few fish. New infections of these fish did not occur because snails were absent in our laboratory and the replacement water comes from a well that is sterilized.

The increased intensity was most likely due to our failure to recognize early infections in parr in which the host had not yet produced a significant inflammatory response or pigmentation. This stresses the importance of using techniques more sensitive than inspecting fish with the unaided human eye. We suggest using a dissecting microscope or magnifying glass for examinations.

The pathology associated with neascus (black spot) in the skin of coho salmon in our study involved obvious fibroplasia with melanosis around the parasite cyst. The yellow/orange pigmentation circumscribing the fibrosis was most likely due to xanthophore accumulation instead of melanophores, as was noted by Hoffman (1956) with 3 yr old infections of Crassiphiala bulboglossa (Diplostomatidae, black-spot

154 neascus) of fathead minnows, Pimephales promelas. This was also comparable to the earlier findings of Chapman and Hunter (1954).

The lack of tissue reaction, particularly to Apophallus sp., is commonly seen with digenetic trematodes that persist in tissues. This is caused by evasion of the host response, either by molecular mimicry or sequestration in thick-walled cysts that are not penetrated by host immune cells (Graczyk, 1997). Survival of neascus (black spot) in the skin clearly involves the latter.

Although metacercarial infections in fish persist, some reduction in overall abundance often generally occurs (Hoffman, 1958a). It is not entirely clear how encysted metacercariae actually die, as they are greatly inaccessible to host immune cells. However, Hoffman (1958b) showed that some cysts are expelled by the host. In addition to host response to the parasite, some worms simply die due to senescence

(Faliex, 1991). Lemly and Esch (1983) speculated that the reduction of viable U. ambloplitis in centrarchids from early fall to early spring may be due to lower temperatures or simply attrition, as metacercariae may depend on their own energy reserves for survival (Hunter and Hamilton, 1941).

The findings of Ebersole et al. (2006) that smolts from the lower mainstem of

WFSR had an unexpectedly lower overwintering survival, in conjunction with the findings of Rodnick et al. (2008) of higher prevalences and abundances of certain parasites only in the lower mainstem, suggest that heavily parasitized coho may not survive as well over the winter. A decline in burden of long-lived parasites within the same host population indicates parasite-induced mortality (Lester, 1984). Our study

155 showed that the parasites we are investigating are indeed long lived. However, our field observations have shown that lower mainstem parr have about 95% more mean abundance of Apophallus sp. compared with smolts of the same year class for 2 consecutive yr (unpubl. obs.). This suggests that these lightly infected smolts are tributary fish or the few lightly infected surviving fish from the lower mainstem.

Ebersole et al. (2006) proposed that poor over winter survival in the lower mainstem was due to differences in habitat, hydrology, and water quality parameters, e.g., the lower mainstem temperature often exceeds 20 C. It is plausible that life cycle dynamics of certain parasites also differ in these 2 stream localities in response to the respective environmental conditions. Indeed, Cairns et al. (2005) showed a positive correlation of black spot with temperature in the WFSR. It is possible that these abiotic factors, e.g., water temperature, also indirectly impact fish by enhancing snail and parasite production (Poulin, 2006), and thus parasites may also contribute to the observed poor survival.

In conclusion, the only significantly lower median parasite abundance between parr and smolt stages detected was with Apophallus sp. from lower mainstem fish and

N. salmincola from tributary fish, but still the majority of these parasites persisted from parr to smolt stage in both of these groups. Therefore, we have demonstrated that the muscle parasites examined in our wild coho salmon studies are long lived and most survive from parr to smolt stage. This provides another line of support for our hypothesis that heavily parasitized fish are associated with poor over wintering

156 survival. However, to elucidate a more concrete link, further investigations are required, and will be pursued.

Acknowledgments

This research was funded in most part by an ODFW Fish Health Graduate

Research fellowship (agency grant 010-7032-IAA-FISH) to J.A.F. We also graciously acknowledge K. Jacobson and J. Heidel for manuscript review.

157

Figure 5.1. Parasites detected from coho salmon (Oncorhynchus kisutch) smolt by squash preparations. Apophallus sp. metacercariae: (A) Encysted worms in muscle, scale bar = 100 µm. (B, C) Excysted metacercaria,(B) flattened to show internal organs, scale bar = 50 µm. T = testis, E = esophagus, V = ventral sucker, P = pharynx (C) same worm active and elongated (D) Nanophyetus salmincola metacercaria, EB = Excretory bladder. (E) Neascus from skin. (F) Myxobolus insidiosus pseudocysts, scale bar = 100 µm. Inset = high magnification showing spores of M. insidiosus.

158

Figure 5.2. Histological sections (H&E) of muscle parasites in coho salmon (Oncorhynchus kisutch) smolts. Scale bar = 50 µm. (A-D) Apophallus sp. in connective tissue of myosepta. (D) Empty space due to parasite loss during processing. (E) Nanophyetus salmincola; note mild, chronic inflammation surrounding cysts. (F) Myxobolus fryeri infecting somatic nerve; inset = high magnification showing spores of M. fryeri. (G) Myxobolus insidiosus in myocyte.

159 Table 5.1. Summary of parasite prevalence and abundance for fish from the lower mainstem (top), tributaries (middle), or lower mainstem mortalities (bottom) groups from the West Fork Smith River. Mortalities were not statistically analyzed due to small sample size. SE = standard error of the mean. Abundance is measured as metacercariae/fish for trematode infections and pseudocyst/fish for Myxobolus insidiosus. The median abundance was tested for metacercariae due to the non-normal distribution of data, which were 1-sided tests because of further infections were prevented under conditions void of intermediate hosts. The mean abundance was tested for M. insidiosus because these data did conform to the assumptions of parametric analysis, which were 2-sided because myxozoans are similar to microparasites in that they asexually reproduce in fish results in a delay of increased detection of pseudocysts.

Location and parasites Prevalence (%) Mean abundance (±SE), Median range abundance Lower mainstem Nanophyetus salmincola Parr 11/11 (100%) 52.5 (7.1) 46.0 28-96 Smolts 35/36 (97.2%) 44.9 (7.0) 33.4 0-181 Apophallus sp. Parr 11/11 (100%) 1,202.9 (133.5) 1022.0 692-2,068 Smolts 36/36 (100%) 682.6 (60.3) 574.8* 248-1,814 Myxobolus insidiosus Parr 11/11 (100%) 341.1 (100.2) 258.0 16-1,012 Smolts 30/36 (83.3%) 498.0 (159.4) 94.5 0-4,856 Tributary N. salmincola Parr 14/14 (100%) 39.7 (6.0) 36.0 10-88 Smolts 61/62 (98.4%) 21.6 (2.2) 14.3* 0-78 Apophallus sp. Parr 9/14 (64.3%) 26.7 (24.3) 2.0 0-342 Smolts 18/62 (29.0%) 10.5 (8.5) 0.0 0-529 M. insidiosus Parr 14/14 (100%) 109.4 (32.9) 80.0 4-464 Smolts 51/62 (82.3%) 289.4 (55.8) † 93.8 0-2,046

160 Table 5.1. Continued.

Location and parasites Prevalence (%) Mean abundance (±SE), Median range abundance Lower mainstem mortalities

67.0 (42.2) 71.0 N. salmincola 4/4 (100%) 22-104

Apophallus sp. 4/4 (100%) 605.0 (113.4) 624.0 340-832 M. insidiosus 2/4 (50%) 15.6 (14.8) 1.1 0-60

* = significantly lower median abundance of a parasite from parr to smolt stage within each location and parasite group. † = significantly different mean abundance of Myxobolus insidiosus from parr to smolt stage within each location.

161 Table 5.2. Counts of neascus cysts in individual coho salmon (Oncorhynchus kisutch) parr and smolts from the lower mainstem of West Fork Smith River. Counts of cysts are shown for each fish from when they were parr versus from when they were smolts. Average counts for parr, smolts, and difference between the two life stages are also presented. SE = standard error of the mean.

Fish No. Parr Smolt Difference

1 5 4 -1

2 15 18 3

3 11 29 18

4 5 3 -2

5 12 5 -6 6 14 14 0 Mean (±SE) 10.33 (1.78) 12.17 (4.17) 1.84 (2.42)

* = significant difference in cyst counts between parr and smolts.

162

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167 Chapter 6: Mortality in Coho Salmon (Oncorhynchus kisutch) Associated with Burdens of Multiple Parasite Species

Jayde A. Ferguson1, Philippe A. Rossignol2, Kym C. Jacobson3, Michael L. Kent1

1 Department of Microbiology, Oregon State University, 220 Nash Hall, Corvallis,

Oregon 97331 USA

2 Department of Fisheries and Wildlife, Oregon State University, Nash Hall, Corvallis,

Oregon 97331 USA

3 NOAA Fisheries, Northwest Fisheries Science Center, Hatfield Marine Science

Center, 2030 South Marine Science Drive, Newport, Oregon 97365 USA

In preparation for International Journal of Parasitology

Elsevier Science B.V., New York, New York

168 ABSTRACT

Assessing the impact of macroparasitism on wild host populations is difficult because effects depend on parasite burden more than prevalence, and because the natural distribution of parasites is highly aggregated. Therefore, the few hosts that are heavily infected and most impacted may not be detected by sampling efforts. We evaluated the impact of parasite burden of multiple parasite species in juvenile coho salmon (Oncorhynchus kisutch) from the West Fork Smith River, Oregon. These fish are listed as threatened under the US Endangered Species Act. We found heavy parasite infections of Myxobolus insidiosus (Myxozoa) and metacercariae of

Nanophyetus salmincola and Apophallus sp. in parr from the lower mainstem Smolts however harbor fewer parasites. Here, we test the hypothesis that mortality is a function of parasite burden with field studies involving multiple approaches on coho salmon sampled over 4 years. We compared infection prevalence, intensity, and overdispersion (variance to mean ratios) between sampled parr and smolts.

Additionally, we used a retrospective analysis on smolt data, involving the traditional negative binomial truncation technique, to estimate the threshold for parasite associated mortality. Lastly, we propose a parsimonious mathematical representation of macroparasite distribution that simplifies analysis and may have more direct biological meaning. We conclude that the parasites, especially Apophallus sp. and

Myxobolus insidiosus, were associated with coho salmon overwintering mortality. The estimated threshold level for mortality involving Apophallus sp. was about 200 or 500 parasites/fish, depending on the method employed. Most of the lower mainstem parr

169 (60-95%) had infections above this level, suggesting that the high overwintering mortality previously reported for these fish rearing in this area may be attributable to parasitism.

INTRODUCTION

Assessing the impact of macroparasitism on wild populations presents significant challenges because it is a function of parasite load rather than prevalence alone (Brass, 1958; Crofton, 1971; May and Anderson, 1979; Dobson, 1988; Burgett et al., 1990; Scott and Smith, 1994; Shaw and Dobson, 1995; Galvani, 2003; Holt et al., 2003). Furthermore, estimates of effects are complicated by the aggregated distribution of parasites, as most hosts harbor few or no parasites (Smith, 1994;

Galvani 2003). A corollary is that heavy infections occur in few hosts, many of which may have died and cannot be sampled. Consequently, standard cross-sectional indices, notably prevalence, as used for microparasites, yield at best only a weak assessment of macroparasite impact (Smith 1994) and may be misleading (Dobson and Hudson,

1986).

Lester (1984) reviewed the common methods used for estimating parasite associated mortality in wild fishes, many of which require temporal observations on the same host populations (i.e., longitudinal studies). However, there are practical limitations involved in the study of hosts in an aquatic environment. For example, fish are often inaccessible and the most impacted fish are likely to have died prior to sampling (Bakke and Harris, 1998). Nevertheless, studies suggest that fish with

170 heavier parasite burdens have a higher mortality rate than lightly infected fish. For example, Gordon and Rau (1982) demonstrated parasite-induced mortality to occur in a fixed lake population of brook sticklebacks (Culaea inconstans) infected with the metacercariae of the trematode, Apatemon gracilis, by evaluating parasite overdispersion (variance/mean ratios).

We conducted a long term study on the impacts of multiple parasite species infections on Oregon coastal juvenile coho salmon (Oncorhynchus kisutch). This species is listed as threatened under the Endangered Species Act (NRC, 1996). We found high parasite loads in parr from the lower reaches of West Fork Smith River

(WFSR), Oregon (Rodnick et al., 2008) while smolts from this river have low burdens

(Ferguson et al., 2010). Furthermore, parr from the lower reaches of the river have greater than expected overwintering mortality, based on fisheries prediction models

(Ebersole et al., 2006; 2009). Therefore, we hypothesized that parasites may impact overwintering survival of these threatened coho salmon from West Fork Smith River.

However, studying this host-parasite system presents two specific problems: 1) many populations are listed as threatened, making it difficult to obtain large samples, and 2) parr rear as separate, multiple, sub-populations and migrate to the ocean as a randomly mixed population of smolts, making longitudinal studies problematic.

An alternative approach is to conduct a retrospective analysis by predicting the parasite distribution in host populations based on observed data from lightly infected fish, as originally proposed by Crofton (1971). He demonstrated how analyzing the negative binomial distribution can estimate mortality associated with macroparasitism.

171 His technique has become widely accepted and is used extensively in theoretical and empirical models, (e.g., May and Anderson, 1979; Lanciani and Boyett, 1980;

Anderson and May, 1982; Dobson, 1988; Royce and Rossignol, 1990; Galvani, 2003;

Scott and Smith, 1994). Crofton’s techniques rely on approximating the distribution.

Although the statistical assay has proven reliable as a theory (Dobson and Carper,

1992), it is descriptive, having at best indirect biological interpretation, and it is also somewhat arduous to perform.

Here, we present results comparing parasite burdens of different age classes

(parr and smolt) of coho salmon from the WFSR using four approaches: 1) comparison of parasite prevalence and intensity between life stages, 2) comparison of parasite overdispersion (variance/mean ratios) between life stages, 3) a retrospective analysis of smolt data using the negative binomial truncation technique developed by

Crofton (1971), and 4) our proposed parsimonious mathematical representation of macroparasite distribution that simplifies analysis and may have more direct biological meaning, which was first proposed by Koketsu (2004) in a master’s thesis. We conclude that parasitism by Myxobolus insidiosus (Myxozoa), and metacercariae of

Apophallus sp. and Nanophyetus salmincola (Digenea), has a significant impact on coho salmon. Results from all four approaches indicated that burdens of Apophallus sp. were most strongly associated with host mortality. We estimated the threshold for mortality involving Apophallus sp. to be between 200 and 500 metacercariae/fish.

Comparison of Apophallus sp. infections in parr showed over 60% of lower mainstem

172 parr were above this level. Therefore, this parasite, in concert with other species, may be limiting the success of this threatened coho salmon population.

METHODS

Sampling fish

Coho salmon parr were collected in fall of 2007-2009 from the upper mainstem and lower mainstem of West Fork Smith River (WFSR) (see Figure 6.1 for sample sizes and exact locations) by electrofishing. Fish were transported in tanks supplied with pressure regulated O2 to the Oregon State University’s Salmon Disease

Laboratory to hold in captivity for a different study. A few fish (< 20) were initially evaluated for parasites at the study onset and the remaining fish were either evaluated as moribund, dead, or in the following spring as part of an entirely separate study.

Note that fish held in captivity overwinter were still considered to be parr based on their parasite burden - i.e. parasite counts would not have changed in captivity because

1) infections are persistent (Ferguson et al., 2010) and 2) fish were held in parasite- free water. This strategy allowed for maximum use of these highly threatened fish.

Wild coho salmon smolts were captured in spring 2007-2010 in a rotary screw trap at the mouth of WFSR (Figure 6.1) and killed immediately for parasite evaluation.

Parasite evaluation

We were particularly interested in abundance of muscle parasites because severe infections seen in previous studies (Rodnick et al., 2008, Ferguson et al., 2010).

The method for evaluating parasite abundance is published elsewhere (Ferguson et al.,

173 2010). In short, it involves filleting one side of each fish, which is then skinned and weighed to the nearest 0.01 g. Tissue squashes were prepared by squashing fillets between two 15- x 30-cm plexiglass plates and parasites were identified and enumerated using a compound microscope at x50 and x100 magnification, which were then multiplied by two in order to represent the number of parasites per fish. Parasites in kidney were similarly evaluated, but counts were not multiplied to estimate the entire kidney because Nanophyetus salmincola targets posterior kidney via the renal portal system (Baldwin et al., 1967). Prevalence (number of infected animals/total animals), and intensity (number of parasites/infected animals) of infections are reported in accordance with the definitions provided by Bush et al. (1997).

Inferring parasite-associated mortality

Comparison of parasite burden

Mean intensities of parasites in parr were compared to those of smolts with a non-parametric bootstrap t-test with 100,000 replications, as data were not normally distributed. Fisher’s exact tests were used to test differences in prevalence of parasites between parr and smolts. We analyzed all data pooled and also data from each year class separately. We had matched samples of parr and smolts for fish from brood years

2007 and 2008 for this analysis. All statistical procedures were performed with QP

(Rozsa et al., 2000), significance was set at p < 0.05 and p-values are 2-tailed.

174 Comparison of parasite overdispersion

Overdispersion (variance to mean abundance ratios) of each parasite was calculated for comparison between the two fish life stages. We had matched samples of parr and smolts for fish from brood years 2007 and 2008 for this comparison, and thus we used these two year data sets for this evaluation. We present data separately for each year class and also as pooled.

Crofton’s truncation model of the negative binomial distribution

Crofton’s model has been widely accepted, so we only provide a brief overview of this technique. For further details, including mathematical equations, see Crofton

(1971). The truncation technique includes estimating an overall expected host distribution from the low frequency classes, where lethal effects are less likely significant. The truncated curve will fit the negative binomial distribution better than the observed curve because the observed data will be missing hosts from the high parasite load class owing to parasite-induced mortality, and the difference of fitness is considered as the parasite-induced host mortality (see Royce and Rossignol 1990).

The analysis of Crofton’s truncation of the negative binomial distribution was performed using the DOS based software, BASICA (Ludwig, 1983) and used data on smolts sampled from 2006-2009.

175 Congestion rate model

We developed an alternative model that is more biologically meaningful (see

Koketsu, 2004), which is analogous to a standard growth model. The relationship between parasite load and host frequency is expressed with the exponential function:

Y = ae - bx (1)

Where, Y = frequency of hosts (observed plots), x = number of parasites per host, a = constant parameter, b = constant parameter (named congestion rate) e = natural log.

The slope of the line, b, may represent the degree of utility that parasites could make of the host population or the degree of the host capacity for containing parasites.

Equation (1) was transformed into a differential equation,

dY = −bY (2) dx then divided on both sides by dt, and solved for b that we labeled “congestion rate”.

1 dY dx − = b (3) Y dt dt

Equation (3) represents the proportion of the ratio of host frequency reduction,

1 dY dx − to the ratio of parasite load class increment, . Because b is a constant, if Y dt dt dx 1 dY increases, then − decreases at a regular rate, and vice versa. Furthermore, dt Y dt both sides are multiplied by x:

176 1 dY 1 dx − = bx (4) Y dt x dt

1 dY 1 dx Equation (4) represents the ratio of − to in proportion to x, that is, Y dt x dt the negative per capita rate of host frequency change to the per capita rate of parasite load class change. In other words, when x is large, the influence or regulation of the

1 dY 1 dx parasite load class on both − and become strong. Y dt x dt

Congestion rate may indicate a speed of parasite aggregation that is accumulated through host frequency, or a tightness of regulation between host and parasite. Fitting this model to observed data, and comparing differences from predicted values, was performed in Excel (Microsoft Office Corp., 2003; Redmond, WA) and used data on smolts sampled from 2006-2009.

Model validation

Koketsu (2004) validated our model by re-analyzing the classical host-parasite dataset (Hynes and Nicholas, 1963) used by Crofton with his negative binomial analysis (1971). Briefly, from Crofton’s paper, we used two types of plots: negative binomial approximated plots and negative binomial truncated plots at third parasite load class. For six stations, he plotted the frequency of the host population on a log scale against the number of acanthocephalans that hosts contain. Congestion rate was computed for both approximated and truncated negative binomial plots, and our output

177 was compared with Crofton’s output. The results did not differ between the two models (see Koketsu, 2004).

RESULTS

Prevalence

Although prevalence may be a weak metric of infection for macroparasites, we compared prevalences to provide additional information on parasite burden in host populations. All parr originating from the lower mainstem were infected with

Apophallus sp., whereas a prevalence of only about 30% was found in upper mainstem fish (Table 6.1). Prevalence was approximately 60% when samples for parr from both upper and lower mainstem locations were pooled. Using these pooled data, prevalence in the parr was about 30% lower than that for smolts (Table 6.1), which was significantly different (p < 0.01; Fisher’s exact test). For N. salmincola, parr originating from both locations and smolts were all infected (Table 6.1). For brood year 2007, the prevalence of M. insidiosus in parr was about 85% in fish from both sites, which was similar for smolts (Table 6.1). However, in brood year 2008, upper mainstem parr had a lower prevalence of about 50% for this parasite. Combined data from all years indicated a similar prevalence of this myxozoan parasite in parr and smolts (Table 6.1). Overall prevalence was about the same between life stages; except for Apophallus sp. Smolts had a higher prevalence of this parasite.

178 Mean intensity

We compared mean intensities of infection in our samples to evaluate differences in burden. The mean intensity of Apophallus sp. in parr was always higher in fish originating from the lower mainstem (Table 6.1). For each year comparing combined parr to smolts, the mean intensity of Apophallus sp. in smolts was about 3-7 times lower than that of parr (Table 6.1), which was significantly less (p <0.01; bootstrap t-test). Although we observed some overlap in the range of infection between combined smolt (4-1,150 metacercariae/fish) and parr samples (66-4,666 metacercariae/fish), only 36% (21 of 59 smolts) had infection levels overlapping with lower mainstem parr. The mean intensity of N. salmincola was higher in lower mainstem parr than parr from the upper mainstem for brood year 2007, but was about the same for the following brood year (Table 6.1). Smolts had a higher mean intensity of N. salmincola, which was statistically significant for both brood year 2007 and combined brood year’s (p <0.02; bootstrap t-test). The mean intensity of M. insidiosus was higher in parr from the lower mainstem, and smolts had about the same infection level compared to combined parr (Table 6.1). Overall, the most prominent difference in parasite burden was with Apophallus sp., as smolts always had a lower mean intensity compared to parr, with lower mainstem fish harboring the most worms.

Variance to mean ratio

We evaluated overdispersion (variance to mean ratio) as another indicator of parasite associated mortality. We applied this approach to evaluate parasite associated

179 mortality with species in our study. The variance to mean abundance ratio of

Apophallus sp. in parr was approximately three times higher than that of smolts of the same brood year for both years and when data from years were combined (Table 6.1).

The overdispersion was influenced by infections in the lower mainstem parr because they had high mean intensities compared to the upper mainstem parr (Table 6.1), which increased the variance of the dataset. With N. salmincola, the variance to mean abundance ratio was lower in parr compared to smolts, and infections may have accumulated in these older hosts. Lastly, the variance to mean ratio of M. insidiosus was similar between parr and smolts (Table 6.1). Of all three parasites, the most dramatic difference between parr and smolts was with Apophallus sp. and the heavily infected lower mainstem fish were not detected in smolt samples for either year.

Truncation of negative binomial model

We applied a retrospective technique to compare observed and expected parasite distributions and thus estimate a ‘threshold’ of parasite associated mortality. We used data for hosts sampled prior to parasite associated mortality, which allowed us to assign estimates to the proportion of heavily infected fish linked to mortality by parasitism. For this analysis, we included data for all available smolts (Figure 6.1).

This approach increased our sample size and is permissible because we are not making direct comparisons to parr of the same year classes. Analysis of data from all three parasites predicted that the parasite distribution in smolts was truncated. Of all the

180 parasites in our study, the most dramatic difference between observed and predicted parasite distributions was with Apophallus sp. (Figure 6.2a)

Analysis of our data fitted to the negative binomial distribution indicated that the threshold for parasite-associated mortality began around 200 parasites/fish for metacercariae and 400 parasites/fish for pseudocysts (Figures 6.2a-f). For Apophallus sp., approximately 11% of smolts had burdens above this level. However, 95% of lower mainstem parr, but only 1% of upper mainstem parr, had infections greater than this value. In contrast, about 31% of smolts had N. salmincola above this threshold, whereas only 14% and 6% of lower mainstem and upper mainstem parr, respectively, were above this value. With M. insidiosus, about 18% of smolts were above the threshold and 22% and 10% of lower mainstem and upper mainstem parr, respectively, were above this level. Based on this analysis, Apophallus sp. was the parasite most closely associated with host mortality, as essentially all of the lower mainstem parr were above the predicted threshold and these high infections are consistently not detected in smolt samples over a four year period.

Truncation of congestion rate model

We include a similar retrospective technique to augment our results with this approach and to provide a simpler analysis and more biologically meaningful interpretation. Using the newly proposed congestion rate model provided similar results as that of using the negative binomial method. The threshold for parasite associated mortality for both N. salmincola and M. insidiosus were the same.

181 However, this value changed slightly for Apophallus sp. (ca 500 parasites/host)

(Figures 6.2a,b), a level that about 59% of lower mainstem parr exhibited. Only one of

176 (0.5%) parr from the upper mainstem had an infection above this level. Therefore, this newly proposed model provided essentially the same results, Apophallus sp. being the most associated with fish mortality, but was simpler to use.

DISCUSSION

We conclude that macroparasitism is an important cause of overwintering mortality of coho salmon. The digenean, Apophallus sp., and myxozoan, M. insidiosus, were the most strongly associated with this mortality, particularly with

Apophallus sp. infecting lower mainstem fish. Mortality was apparent using 4 analytical approaches (i.e., parasite burden, overdispersion, and the two retrospective analyses). The predicted threshold of parasite associated mortality for Apophallus sp. was approximately 200 or 500 parasites/fish, using Crofton’s method and our proposed congestion model, respectively. This level was low, and therefore parasite- associated mortality was high, compared to the observed burdens in lower mainstem parr where approximately 60 to 95% of these parr were above this level. In comparison, hardly any upper mainstem parr were above this level (only 1-2 fish of

176 sampled), suggesting that smolts are overrepresented by parr originating from the upper mainstem. Ebersole et al. (2006) reported poor survival (ca 2-3%) for fish from the lower mainstem of this river, but concluded this was due to abiotic factors, such as poor habitat. However, our analyses indicate that parasitism is also involved. The

182 importance of our study is two fold; one, poor survival of fish from this section of

WFSR is associated with severe parasitism, and two, we contribute a new technique for estimating host mortality, which is analogous to standard growth, and suggest that it is amendable for biological and evolutionary analyses.

Nanophyetus salmincola was the least linked to parasite associated mortality, mean intensity and overdispersion of this parasite actually increased from parr to smolt and the percent of parr above the predicted threshold level was low.

Interestingly, Jacobson et al. (2008) used a similar approach and showed that N. salmincola was associated with mortality in coho salmon during early ocean residency. These two studies together suggest that N. salmincola may not be associated with high overwinter mortality in freshwater and that the infection actually increases until seawater migration. Therefore, parasite associated mortality by this parasite in coho salmon occurs only in the ocean phase of this fish off the coast of

Oregon.

Anderson and May (1979) dichotomized micro- and macro- parasites. They stated that microparasites are small organisms (e.g., bacteria, protozoa, and viruses) that reproduce directly on hosts, usually induce immunity to reinfection, and typically have a short infection duration (compared to host’s lifespan); thus generally transient infections. This was contrasted to macroparasites that are larger organisms (e.g., metazoan) that do not reproduce directly on hosts, usually only elicit an immune response with high densities, and typically have a long duration of infection

(compared to host’s lifespan); hence generally persistent infections. Myxobolus

183 insidiosus is a metazoan parasite, but it has features of a microparasite in that asexual reproduction occurs in the fish host. However, this species is like a macroparasite in that increased plasmodial cysts (from single progenitor cell) comes from contact with an alternative host, thus although the number of spores increase within the host, the number of pseudocysts do not. Therefore, the models used to observe a decrease in parasite burden over time to infer parasite associated mortality could be applied here.

In this study, mean intensity decreased significantly between parr and smolts and overdispersion was less in older fish. The truncation models indicated parasite associated mortality occurred and about 20% of parr were above the predicted threshold level. To our knowledge, this is the first study to use myxozoans with these analytical macroparasite techniques.

Another unique aspect of our study, is that by sampling lower mainstem parr, we were able to actually examine heavily infected hosts above the threshold predicted with smolt data (i.e., sampled prior to parasite associated mortality). In contrast, most studies using retrospective approaches, such as Crofton’s technique (e.g., Lanciani and

Boyett, 1980; Burgett et al., 1990; Royce and Rossignol, 1990), only have a prediction on the percent of heavily infected animals that should have theoretically existed. An additional irregularity of our study was the occurrence of heavy infections in almost all the lower mainstem parr. Typically, natural wild populations have only a few host more heavily infected than the truncation point and hence parasite associated mortality seldom threatens an entire population. Most of the lower mainstem fish are above this level, thus indicating that few fish survive. Ebersole et al., (2006) concluded that the

184 poor survival of fish in this stretch of the river was due to poor habitat. However, this could in turn be related to the severe infection levels associated with mortality in our study, as the production of digenean trematodes and their intermediate host’s increases with elevated temperature (Poulin, 2006) and the lower mainstem of WFSR often exceeds 20° C in the summer (Ebersole et al., 2006).

Studying the impacts of parasites in wild populations is difficult due to the nature of chronic infections being sublethal (McCallum and Dobson, 1995). Such studies involving fish populations are often further complicated because of their inaccessibility, especially for migratory salmonids (Bakke and Harris, 1998).

Therefore, conducting longitudinal studies to infer parasite associated mortality on salmonids may be limited by some assumptions. We attempted to sample the most representative population of parr from the WRSR in terms of parasite burden. Prior knowledge of this system being dichotomized into areas with heavily and lightly infected rearing parr (Rodnick et al., 2008; Ferguson et al, 2010), aided in this feat.

However, due to the legal disposition of this stock being ESA listed as threatened, sample sizes were restricted. Therefore, we used multiple approaches to infer parasite associated mortality. Results from all of the different analyses indicated that the parasites in our study, especially Apophallus sp. do indeed impact coho salmon. Such negative effects could be exasperated in this population, as this recovering population is already dwindling due to other limiting factors. Therefore, we propose mining for the most information possible in these types of studies. This could include the use of many different analytical and theoretical modeling in field studies, such as done here

185 with longitudinal and retrospective analyses. Furthermore, the use of biologically meaningful models, such as our newly proposed congestion model, would be particularly important when trying to make predictions on managed fisheries that are threatened.

In conclusion, we have shown that metazoan parasites, such as Apophallus sp. and M. insidiosus, are associated with parasite associated mortality in juvenile coho salmon. Fish from the lower mainstem were severely parasitized, and the level of mortality associated with parasitism indicated in our analysis approximates previous reports of estimated mortality of these fish in this section of WFSR. This stresses the importance of examining parasitism as a potential limiting factor for threatened populations. Given these findings, fish, especially managed species, from other systems should also be evaluated for parasite associated mortality. We recommend this be done by using multiple types of analyses in field studies and our newly proposed congestion rate model, which is easy to use and may provide biological relevance.

Acknowledgments

Thanks to Dept. of Fisheries and Wildlife, Oregon State University faculty and staff for office support and technical assistance. The original concept of congestion rate was developed by I. Ninomiya, Faculty of Agriculture, Ehime University.

186

Figure 6.1. Map of West Fork Smith River showing sampling sites for parr from Brood Year (BY) 2007 and 2008 from the Upper Mainstem (UMS) and Lower Mainstem (LMS). Sample sizes for all parr samples, along with smolt samples, are also shown.

187

Figure 6.2. Frequency distribution of parasites infecting coho salmon (Oncorhynchus kisutch) smolts from West Fork Smith River, using either the negative binomial distribution or the congestion rate model. Apophallus sp. (a), Nanophyetus salmincola (c) and Myxobolus insidiosus (e) using the negative binomial distribution (NBD) truncation technique compared to the frequency distributions of Apophallus sp. (b), N. salmincola (d), and M. insidiosus (f) using the congestion model (Exponential). Triangle = observed, square = predicted.

188 /x

2 S in muscle and in muscle

(95% CI) (95% Mean intensity intensity Mean for each brood year separately, /x % /x %

2 S Nanophyetus salmincola sp.

em (LMS) and upper mainstem (UMS), and (LMS) and upper mainstem em (95% CI) (95% Apophallus Mean intensity intensity Mean /x) of /x) of 2 (LMS plus UMS) and smolts ood years 2007 and 2008. The mean intensity of infection, with ood years 2007 and 2008. The mean /x % /x %

2 S Nanophyetus salmincola Myxobolus insidiosus s). * = significant difference (p < 0.05). sp.

(95% CI) (95% Mean intensity intensity Mean Apophallus in coho salmon parr from the lower mainst parr from in coho salmon %

n 58 100 753 237 100 123 50 84 487 1481 1481 84 487 50 123 100 1096 (210-753) 237 753 100 143 79 409 (110-220)* 58 998 97 313 76 37 208 97 158 (42-138)* 45 103 100 37 77 100* 881 29 856 100 100 380 70 14 52 86 300 643 30 93* (115-329)* 191 415 100 59 (125-210) 161 97* 88 (88-209)* 133 369 63 98 (108-422) 221 (128-195)* 159 575 112 88 (200-530) 323 956 170 59 603 (465-786) 1367 99 113 (102-126) 61 68 241 (176-366) 1049 1049 (176-366) 61 134 68 241 (102-126) 64 99 113 1367 759 (465-786) (423-636) 30 520 1251 231 64 1112 100 91 386 (248-388) 647 13 48 59 603 112 100 68 170 99 93 54 99 100 59 75 310 (91-108) 608 809 100 291 124 33 24 100 100 (73-95) 83 70 47 1056 128 (481-671) 176 136 52 878 63 565 304 85 (291-535) 380 1113 Myxobolus insidiosus

Years Years Brood Year 2007 2007 Year Brood 2008 Year Brood Combined Brood Brood Combined LMS Parr UMS Parr UMS) + (LMS Parr Smolts LMS Parr UMS Parr UMS) + (LMS Parr Smolts LMS Parr UMS Parr (LMS+UMS) Parr Smolts kidney, and

and combined (pooled data from both brood year (pooled data from and combined outmigrant smolts of West Fork Smith River, Oregon from br River, Oregon from Fork Smith smolts of West outmigrant 95% confidence intervals in parenthses, was tested between parr intensity, and overdispersion (S (%), mean Table 6.1. Prevalence 189

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192 Chapter 7: Impacts of Multispecies Parasitism on Juvenile Coho Salmon (Oncorhynchus kisutch) in Oregon

Jayde A. Ferguson1, Jeremy Romer2, Jean C. Sifneos3, Lisa Madsen3, Carl B.

Schreck2, Michael Glynn4, and Michael L. Kent1*

1 Department of Microbiology, Oregon State University, 220 Nash Hall, Corvallis,

Oregon 97331 USA

2 Oregon Cooperative Fish and Wildlife Research Unit, U.S.G.S. Department of

Fisheries and Wildlife, Oregon State University, 104 Nash Hall, Corvallis, Oregon

97331 USA

3 Department of Statistics, Oregon State University, 44 Kidder Hall, Corvallis, Oregon

97331 USA

4 College of Veterinary Medicine, Oregon State University, 200 Magruder Hall,

Corvallis Oregon 97331 USA

Submitted to Aquaculture

Elsevier Science B.V., New York, New York

193 ABSTRACT

We have been studying the impacts of parasites on threatened stocks of Oregon coastal coho salmon (Oncorhynchus kisutch). In our previous studies, we have found high infections of digeneans and myxozoans in coho salmon parr from the lower mainstem of West Fork Smith River (WFSR), Oregon. In contrast, parr from tributaries of this river, and outmigrating smolts, harbor considerably less parasites.

Thus, we have hypothesized that heavy parasite burdens in parr from this river are associated with poor overwintering survival. The objective of the current study was to ascertain the possible effects these parasites have on smolt survival. We captured parr from the lower mainstem and tributaries of WFSR and held them in the laboratory to evaluate performance endpoints of smolts with varying degrees of infection by three digeneans (Nanophyetus salmincola, Apophallus sp., and neascus) and one myxozoan

(Myxobolus insidiosus). The parameters we assessed were weight, fork length, growth, swimming stamina, and gill Na+,K+-ATPase activity. We repeated our study on the subsequent year class and with hatchery reared coho salmon experimentally with N. salmincola. The most significant associations between parasites and these performance or fitness endpoints were observed in the heavily infected groups from both years. We found that all parasite species, except neascus, were negatively associated with fish fitness. This was corroborated for N. salmincola causing reduced growth with our experimental infection study. Parasites were most negatively associated with growth and size, and these parameters likely influenced the secondary findings with swimming stamina and ATPase activity levels.

194 INTRODUCTION

Parasite associated mortality has been documented in many wild populations of animals, including fishes (Lester, 1984). We are studying the impacts of parasites on Oregon coastal coho salmon (Oncorhynchus kisutch), which have been listed as threatened under the Endangered Species Act (ESA) (NRC, 1996). The general consensus is that habitat loss plays the most important role in their decline (NRC,

1996).

For juvenile coho salmon, the overwintering period is recognized as a time of high mortality (Ebersole et al., 2006; Ebersole et al., 2009). Ebersole et al. (2006) used habitat quality models to predict overwinter survival of coho salmon parr from the West Fork Smith River (WFSR) in Oregon, and compared model results to survival data observed from tagged fish. They found that parr from the lower mainstem of WFSR had reduced overwinter survival compared to parr from the upper reaches of this river, which was opposite to the model predictions. As a follow up to their study, we have found extremely high digenean and myxozoan parasite burdens in coho salmon parr from the lower mainstem of WFSR, compared to parr rearing in the upper portion of this river (Rodnick et al., 2008). Furthermore, outmigrating smolts in this basin consistently harbor dramatically fewer parasites than lower mainstem parr of the same cohort when sampled earlier in the year (Ferguson et al., 2010). Therefore, we have proposed that these heavily infected parr are either subjected to high overwinter mortality, or fail to migrate due to poor smoltification and subsequently die.

195 Coho salmon are infected by numerous parasite species (Love and Moser,

1983; McDonald and Margolis, 1995; Hoffman, 1999), and some studies have indicated that certain parasites can be linked to host mortality. Particularly pertinent to our study, Jacobson et al. (2008) demonstrated that a common digenean, Nanophyetus salmincola, is associated with early ocean mortality of coho salmon. However, similar studies have not been conducted on the coho salmon smolts, nor have they evaluated effects of multiple parasite infections.

There are some inherent limitations with studying effects of parasitism on wild fishes, as moribund or dead fish are likely removed from the system by predators, and sub-lethal effects of disease are hard to measure (Bakke and Harris, 1998). However, laboratory studies, conducted on either experimentally or naturally infected fish, can directly examine mechanisms that lead to mortalities in the wild. Examples of such studies with salmonids include the following: N. salmincola (Digenea) reducing burst swimming speed (Butler and Milleman, 1971), and immune response (Jacobson et al.,

2003); Crepidostomum farionis (Digenea) reducing hemoglobin and hematocrit (Klein et al., 1969); Sanguinicola klamathensis (Digenea) reducing growth (Evans, 1974);

Gyrodactylus spp. (Monogenea) inducing cortisol (Stoltze and Buchmann, 2001);

Eubothrium salvelini (Cestoda) reducing swimming stamina, growth, and survival

(Boyce, 1979), saltwater adaptation (Boyce and Clarke, 1983), and altering migration orientation (Garnick and Margolis, 1990); Myxobolus arcticus (Myxosporea) reducing swimming stamina (Moles and Heifetz, 1998); Parvicapsula minibicornis

(Myxosporea) reducing swimming recovery rates (Wagner et al., 2005); and sea lice,

196 Lepeophtheirus salmonis (Copepoda) reducing osmoregulation (Birkeland and

Jakobsen, 1997), and swimming and cardiovascular performance (Wagner et al.,

2003).

Here we present results of our laboratory studies using 1) coho salmon from two year classes captured in the wild harboring multiple parasite infections; and 2) coho salmon experimentally infected with N. salmincola. The responses we assessed in these fish were size, growth, swimming stamina, and gill Na+,K+-ATPase activity.

These represented fitness endpoints, as many of these are either performance metrics or other parameters that are indirectly linked to fitness. Fish size influences freshwater juvenile overwinter survival (Quinn and Peterson, 1996) and smolt success (Holtby et al., 1990). Reduced swimming performance can affect fish survival by decreasing predator avoidance (Taylor and McPhail, 1985). Osmoregulation is an important factor for smolt survival (Moser et al., 1991) and gill Na+,K+-ATPase activity is a major component of this process (reviewed in McCormick, 2001). We performed analyses to determine if lightly infected fish had lower fitness responses than more heavily infected fish, and if parasitism was negatively associated with these responses.

MATERIALS AND METHODS

Sampling wild fish

As we are particularly interested in overwinter mortality, we captured wild parr from WFSR in September to hold and monitor in the laboratory until the typical time of smoltification. Fish were provided by the Oregon Department of Fish and Wildlife

197 (ODFW) in conjunction with monitoring activities for their Life Cycle Monitoring

Project. In September 2007, parr were gathered from the lower mainstem near Crane

Creek (Ck) at River kilometer (Rkm) 4.8 (n = 46) and Rkm 2.0 of the tributary Moore

Ck (enters mainstem at Rkm 8.6; n = 53) of WFSR by beach seine. Similarly, in

October 2008 parr were gathered from the same area of the tributary Moore Ck (n =

88), the upper mainstem near Gold Ck (Rkm 17; n = 10), the lower mainstem near

Crane Ck (Rkm 4.8; n = 61), and the lower mainstem of the WFSR near the ODFW smolt trap (Rkm 1.6; n = 11).

Although there were multiple sampling locations, fish were considered to represent two main groups based on different river habits as described by Ebersole et al. (2006). Thus they are referred to as fish from the lower mainstem (Rkm 1.6 and

4.8 of the mainstem) and tributary (Rkm 17 of the mainstem and the tributary Moore

Ck), respectively. Captured parr were transferred to Oregon State University’s (OSU) facilities, where they were held as described by Ferguson et al. (2010) until late April, the typical time of smoltification (100-130 mm fork length). Coho salmon undergo smoltification in spring (Groot and Margolis, 1991), so we chose the end of April (i.e., about 1 mo. after the Spring Equinox) to represent the typical time of smoltification for fish in our study.

198 Laboratory maintenance of fish

Captured brood year 2007 parr

Lower mainstem and tributary fish were held separately in outside circular 0.6 m2 diameter tanks at OSU’s Fish Performance and Genetics Laboratory. Initial fish density was 0.68g/L and 0.76g/L for lower mainstem and tributary fish, respectively.

Flow-through, parasite free well water (12-13C) was supplied. Fish were fed a mixture of commercial feed (1.5 mm size; Bio-Oregon Inc.) and freeze-dried brine shrimp and krill (Argent Labs), to satiation for 5 min once or twice daily.

Captured brood year 2008 parr

Fish were also held outside in 0.6 m2 tanks at OSU’s Salmon Disease

Laboratory (SDL). Lower mainstem and tributary fish were tagged with 12 mm

Passive Integrated Transponder (PIT) tags (Biomark) and randomly mixed into six tanks, with approximately equal numbers of fish from each location in each tank.

Initial fish density was about 0.64g/L for each tank. Water supply, temperature, and feeding regime were the same as described above.

Experimental infections

In June of 2009, 120 hatchery-reared coho salmon parr were obtained from the

ODFW’s Oxbow Hatchery, Oregon. At the beginning of our study, six fish were examined for parasites, as described below, to determine if these fish were free of parasite infections. Likewise 30 fish from the negative control group were also evaluated for the presence of parasites. For this experiment, fish were divided into four

199 groups (ca 30 fish/group): Nanophyetus salmincola only, Apophallus sp. only, N. salmincola plus Apophallus sp., or no parasites. To obtain parasites for experimental infection, approximately 900 Fluminicola sp. and 600 Juga silicula snails, the intermediate hosts of these parasites, were gathered from the lower river, near the smolt trap in WFSR from June to August 2009. Snails were transported to the SDL where they were held in flow through tanks at 20° C under a 12 h photoperiod produced by 19 watt aquaria lamps placed approximately 15 cm from the water surface. Here, they were screened for cercarial shedding between 0800-1100 and

1800-2000 for several days under a dissecting microscope at X50 magnification.

Infected snails were removed and placed in flow through tanks with uninfected hatchery fish. Juga silicula were fed organic lettuce, and Fluminicola sp. were fed algae that was gathered from WFSR and maintained in the laboratory. Fish were exposed for 4 mo. (July-November 2009) then individually PIT-tagged and separated into two 1.9 m2 tanks outside at the SDL for a growth study, as described above for the captive fish from brood year 2008. Initial fish density was about 1.3g/L for tanks in this study. The water source and temperature were the same as described above for the captive fish. These fish were fed to satiation daily with only the commercial feed.

There was only a single mortality from the “Apophallus sp. only” group.

Parasite evaluation

For each study, fish were euthanized at the end of April with an overdose of MS-

222 (Argent Chemical Labs), carcasses were weighed, measured, subjectively

200 assessed for degree of silver color, and macroscopically examined for neascus (black spot trematode) metacercariae. Skeletal muscle was filleted from the left side of each fish and frozen for later enumeration of parasites by microscopy as described by

Ferguson et al. (2010). The posterior kidney of each fish was similarly sampled and evaluated for the presence of N. salmincola metacercariae. Standard histopathological techniques were used to evaluate the presence of any other parasites or pathogens in the experimentally infected and control fish. Mortalities that occurred throughout the study were noted and carcasses were processed for parasite evaluation. Prevalence

(number of infected animals/total animals), intensity (number of parasites/infected animals), and density (number of parasites/infected tissue sample) of infections are reported in accordance with the definitions provided by Bush et al. (1997).

Evaluating fish performances

Size and growth

All fish were assessed for size at the end of April. Size was recorded as fork length (mm) and weight, to the nearest 0.01g. Parr size (fork length and weight) was also recorded at the beginning of the study for wild fish from brood year 2008 and the hatchery fish.

Growth of individual PIT-tagged fish that were intermixed with fish from different parasitized groups was followed for each individual fish by weighing fish at

6 wk intervals in water throughout the experiment (four intervals total).

201 Swimming stamina

Fish captured from the WFSR from both years were subjected to swimming stamina tests. At the end of April, fish were evaluated for swimming stamina by determining exhaustion time of swimming at a constant water velocity. This was accomplished by acclimating individual fish in a clear polyvinyl chloride tube (7.6 cm diameter x 1.5 m long) at 10 cm/sec for 15 min and then swimming them at 65 cm/sec for fish from brood year 2007 and 82 cm/sec for those from brood year 2008. The increased velocity for the second year was to decrease the amount of time required to run the swimming test. Water velocity was manipulated using a 1200 watt rheostat

(Staco Inc.) and a 575 watt submersible centrifugal pump (Simer Pump Co.), which was recirculated with occasional fresh cold water added to maintain temperature.

Plastic mesh was attached to both ends of the tubes to prevent fish from leaving and plastic straws were inserted at the outflow end to maintain uniform flow in the tube.

When fish were exhausted, as indicated by their movement towards the outflow end of the tube and/or lack of swimming, tapping on the tube was applied to motivate them to continue swimming. If swimming was not continued after a series of three tapping cycles, of three taps each, then the time (to the nearest sec) was recorded from a stopwatch, and the fish was removed.

Gill Na+,K+-ATPase activity

Fish captured from the WFSR from both brood years were evaluated for gill

Na+,K+-ATPase activity. After each fish had undergone the swimming stamina

202 challenge, they were euthanized with an overdose of MS-222 and gill filaments were harvested and frozen at -80 C for later analysis. Enzyme activity was measured in accordance with the protocol described by McCormick (1993).

Statistical analysis

We analyzed the data from the two different year class of wild fish separately because of the differences in study design (e.g., different holding facility, fish densities, swimming velocities, and mixing or lack of mixing of fish subpopulations).

Likewise, data from the experimentally infected fish were analyzed separately. We made between group (i.e., heavily infected vs. lightly or uninfected smolts) comparisons of results from our endpoint measurements. Additionally, we performed within group analyses to determine the relationship of parasitism to the measured fitness endpoints.

Data were assessed for normality by visual inspection of scatterplots. With the exception of weight and fork length data from all studies, and gill Na+,K+-ATPase activity data from the 2007 study, all other fitness variables were determined to be non-normal. Therefore, we used a non-parametric bootstrap 2-sample t-test, replicated

100,000 times, for between group analyses.

For within group analyses, we used multiple linear regression to model the fitness responses as predicted by parasite infection. Response variables were: weight fork length, gill Na+,K+-ATPase activity, swimming stamina, and growth rate. Our explanatory variables were: counts of N. salmincola in muscle or kidney; Apophallus

203 sp.; neascus; Myxobolus insidiosus; and total metacercariae. The later variable was created by summing metacercariae from all digenean species (N. salmincola from muscle and kidney, Apophallus, and neascus). We also accounted for other pertinent explanatory variables, such as sex, tank, and size (fork length). Multicolinearity was assessed by visual inspection of correlation matrices (Tables 7.1 and 7.2) and normality was assessed by visual inspection of scatterplots. We log(ln) transformed the data for all three parasite species in order to improve linearity. Many of the response variables were also transformed to meet the assumption of normality, as these data were skewed. Response variables not transformed included fork length and weight from all three studies, and ATPase activity from the 2007 study, as these data appeared to be approximately normally distributed.

Our modeling selection strategy was to compare results from full models (with predictors and interactions) to more reduced models using a backwards elimination technique. We chose final models by examining residual plots to identify outliers, non-linearity, and heterogeneous variation; R2 values; number of variables in relation to sample size; and interpretability of model. To determine if certain trematode species were more strongly associated with decreased endpoints than the total number of trematodes, regardless of species, we evaluated two different types of parasite models separately; one with all trematode parasite species combined (total metacercariae) plus

M. insidiosus, and the other with individual parasite species as variables. To determine when growth may be most impacted by parasites, we considered models with two different types of responses for the growth studies; one with individual 6 wk growth

204 intervals (i.e., difference in weight between measured time interval), and the other with overall growth (i.e., difference in weight from start to end of the experiment). We also included parr fork length to account for initial size in the growth studies. Finally, we analyzed data from the swimming stamina tests both with and without accounting for fish size, because we hypothesized that parasites were correlated with size.

® All statistical analyses were conducted with S-PLUS version 8.0 software

(Insightful Corp.) or R, version 2.7.2 (R Development Core Team). Significance was set at p < 0.05 and p-values are 2-tailed. Mortalities from all studies were excluded from our analysis. There were nine mortalities from the lower mainstem fish of brood year 2007, eight from this population of brood year 2008, and 11 from the tributary group of brood year 2008. Therefore, final samples sizes of wild fish were as follows: brood year 2007 fish from the lower mainstem (n = 37) and tributary (n = 56), and brood year 2008 fish from the lower mainstem (n = 64) and tributary (n = 87).

RESULTS

Examination of 93 and 151 coho smolts from brood year 2007 and 2008, respectively, revealed high burdens of the parasites in study, particularly in fish from the lower mainstem (Table 7.1). Within group analyses consistently revealed that many of the parasite species of interest were indeed associated with reduced fitness

(Tables 7.2-7.5). In general, parasite burdens were most negatively correlated with size, particularly with the heavily infected populations (Figure 7.1). Reduced smolt weight was consistently associated with Apophallus sp. and muscle infections of N.

205 salmincola, with even the slopes and y-intercepts being nearly identical (Figure 7.1).

To account for infections by different parasite species and other variables that may influence fitness endpoints, we used multiple linear regression techniques. Here, parasite associations were most notable for the lower mainstem fish, as parasites were still associated with lower values of all fitness endpoints even after accounting for other variables (Table 7.4). For the tributary group, parasites were only associated with reduced size and growth (Table 7.5). In regards to analytical approaches, multiple linear regressions demonstrated that the total number of metacercariae, regardless of species, typically did not predict the fitness responses as well as individual trematode species (Tables 7.4 and 7.5). When comparing performance between groups, we found differences between lower mainstem and tributary groups for Na+,K+-ATPase activity and swimming stamina, but these results were not constant for both years (Table 7.1).

The following sections detail the analyses within and between groups further.

Wild fish- brood year 2007

Parasites

Consistent with our previous studies (Rodnick et al., 2008; Ferguson et al.,

2010), the lower mainstem fish were more heavily infected compared to tributary fish

(Table 7.1). The most dramatic difference was in the mean density of Apophallus sp., with the lower mainstem fish harboring approximately 80 times more metacercariae/g than fish from the tributary. The infection levels of N. salmincola in muscle and kidney were also significantly higher in lower mainstem fish. However, M. insidiosus

206 infections were not significantly different between groups (p = 0.47; bootstrap t-test).

There was no neascus detected in the tributary group, and the mean infection in fish from the lower mainstem was 3 parasites/fish (Table 7.1). The prevalence of these parasites was as follows: Apophallus sp. was 100% and 18% in lower mainstem and tributary smolts, respectively; N. salmincola was 100% in both the lower mainstem and tributary smolts, respectively; M. insidiosus was 82% and 84% in lower mainstem and tributary smolts, respectively; neascus was 74% in lower mainstem smolts.

Size

Within the lower mainstem group, parasitism was negatively correlated with smolt weight (Tables 7.2-7.3; Figure 7.1). Analysis incorporating multiple parasite species showed that Apophallus sp. and kidney N. salmincola were significant predictors of small fish, with almost 40% of the variation in smolt weight explained by parasites alone (Table 7.4). The same result occurred for smolt fork length, as

Apophallus sp. and kidney N. salmincola were again significant predictors of low size values (Table 7.4). There was no evidence that smolt weight between the two groups differed (p = 0.34; bootstrap t-test). However, there was suggestive, but inconclusive evidence for a difference in fork length (p = 0.06; bootstrap t-test; Table 7.1).

Swimming stamina.

Within group analysis of the lower mainstem smolts showed that parasites were negatively associated with fitness. Total metacercariae was a significant predictor of lower swimming stamina, but only when not accounting for fish size

207 (Table 7.4). This model explained more than 30% of the variation in swimming stamina for this group of fish (Table 7.4). We saw no evidence that swimming ability between the lower mainstem and tributary groups differed (p = 0.20; bootstrap t-test;

Table 7.1).

ATPase activity.

Within the lower mainstem group, parasites were again negatively associated with fitness when ATPase activity was used as an endpoint. Nanophyetus salmincola in the kidney was a significant predictor of low enzyme activity, even after accounting for smolt size. This parasite contributed to explaining 27% of the variation in ATPase activity in this group of fish (Table 7.4). There was a difference in gill ATPase activity between the lower mainstem and tributary smolts (Table 7.1). The fish from the lower mainstem are estimated to have a mean gill ATPase activity of 1.9 μmole/mg/hr (95% confidence interval: 1.3 to 2.7 μmole/mg/hr) less than that of tributary fish (p < 0.01; bootstrap t-test).

Wild fish- brood year 2008

Parasite evaluation

Consistent with the study from the previous year, fish from the lower mainstem were significantly more heavily infected compared to fish from the tributary group

(Table 7.1), with the biggest difference being in levels of Apophallus sp. The difference this year was even greater, as fish from the lower mainstem had a mean density of about 420 times more metacercariae/g than tributary fish (Table 7.1).

208 Density of N. salmincola in muscle was again significantly higher in lower mainstem fish; however, in 2008 lower mainstem fish actually had a lower infection of this parasite in kidney compared to tributary fish (Table 7.1). With M. insidiosus, again there was no difference in infection density between groups. In the 2008 study, neascus was detected in fish from the tributary group, although at a significantly lower level than that found in lower mainstem fish (Table 7.1). The prevalence of these parasites was as follows: Apophallus sp. was 100% and 2% in lower mainstem and tributary smolts, respectively; N. salmincola was 100% in both the lower mainstem and tributary smolts, respectively; M. insidiosus was 97% and 32% in lower mainstem and tributary smolts, respectively; neascus was 94% and 23% in lower mainstem and tributary smolts, respectively.

Size and growth.

As we found with the 2007 study, within the lower mainstem group, parasites were negatively associated with both smolt weight and fork length. Parasites alone explained 45-50% of the variation in size, respectively (Table 7.4). The same parasite species from the previous year were negatively associated with size. Additionally, M. insidiosus was also significantly predicted small values of both smolt weight and fork length (Table 7.4). In the 2008 study, parasites were also negatively associated with fish size for the fish from the tributary group (Table 7.5). Here, N. salmincola in both muscle and kidney were significant predictors of both reduced weight and fork length

209 (Table 7.5). These associations were not as strong as those found for the lower mainstem fish (Tables 7.4 and 7.5).

We found the most profound impact of parasitism on lower mainstem fish with overall growth, with Apophallus, sp. and M. insidiosus being the most negatively associated species involved in explaining over 60% of the variation in total growth, after accounting for parr size (Table 7.4). Additionally, N. salmincola, infecting kidney, was also a significant predictor of reduced growth. For the tributary fish, parasitism was also negatively associated with overall growth, with N. salmincola, in both muscle and kidney, being the only significant species (Table 7.5). Nanophyetus salmincola infections contributed to explaining almost 50% of the variation in growth of tributary fish (Table 7.5).

Table 7.1 summarizes the comparison of size and growth between the two infected groups of fish. There was no evidence that either mean fork length or weight of smolts differed between fish groups (p = 0.45 and 0.64, respectively; bootstrap t- test). When examining individual growth periods, there was suggestive, but inconclusive evidence that lower mainstem fish grew less during the second growth interval (from 6 to 12wk) of the study (p = 0.06; bootstrap t-test). However, there was no evidence of a difference in overall growth between the infected groups (p = 0.30; bootstrap t-test).

210 Swimming stamina.

Within group analysis of the lower mainstem group showed that parasites were negatively associated with swimming stamina, when not accounting for fish size

(Table 7.4). Apophallus sp., M. insidiosus and kidney N. salmincola were significant negative predictors of time to exhaust, which were involved in explaining 40% of the variation in swimming stamina (Table 7.4). There was evidence of a difference in swimming exhaustion time between the two groups of fish (Table 7.1). The mean time to exhaust swimming for the lower mainstem smolts was about 3.5 min (95% back transformed confidence intervals: 1.7 to 5.8 min) less than that of tributary smolts (p =

0.02; bootstrap t-test).

ATPase activity.

For the lower mainstem fish, Apophallus sp. was negatively associated with enzyme activity, after accounting for other parasites and smolt size, and contributed to explaining about 30% of the variation of ATPase activity in this group (Table 7.4).

Myxobolus insidiosus was positively associated, although the regression coefficient indicated the slope to be only slightly positive, after accounting for other variables, and that of Apophallus sp. to be more strongly negative. Moreover, the correlation coefficient of M. insidiosus was only around 0.1 (Table 7.3). There was no evidence of a difference in ATPase activity between the lower mainstem and tributary fish for the second year study (p = 0.31; bootstrap t-test; Table 7.1).

211 Hatchery fish

Experimental Infections

We experimentally infected hatchery fish to broaden the inference of the associations in our study. We were able to successfully infect fish with moderate to high levels of N. salmincola, but were unable to reproduce infection levels of

Apophallus sp. similar to those found in the field, as less than 20 worms were found in muscle. Therefore, this experimental group was removed from the study. We did however detect this species, albeit in low levels, in fish exposed to J. silicula snails

(intermediate host of N. salmincola), but not in our controls. Furthermore, we actually observed a few cercariae consistent with the morphology of Apophallus sp.

(pleurolophocercous cercariae) released from this snail species. We included counts of

Apophallus sp. in our regression models to account for the presence of this parasite.

The mean abundance (and range) of infections were as follows: 7,379 (291-27,900) metacercariae/g for N. salmincola in kidney, 1.5 (0-9.6) metacercariae/g for N. salmincola in muscle, and 0.7 (0-4.7) for Apophallus sp. Controls were found to be free of infection and histopathologic evaluation revealed no other parasites in either group.

Size and growth.

Within the infected group, total N. salmincola (counts from muscle and kidney combined) was negatively associated with growth (p = 0.04; multiple linear regression). Overall growth was modeled as follows: ln overall growth = – 0.12 (ln

212 total N. salmincola) – 0.02 (ln Apophallus) + 0.21 (parr fork length). This model explained 43% of the variation in overall growth. Because these data were from a controlled experiment, we present the following interpretation for this model: A doubling of N. salmincola causes a decrease in overall median growth by 8% (back transformed 95% confidence interval: 3% to 13%), after accounting for initial parr fork length and Apophallus sp.

There was no evidence of a difference in the mean fork length or weight of smolts between fish groups (p = 0.14 and 0.19, respectively; bootstrap t-test). When looking at individual growth intervals, there was suggestive, but inconclusive evidence, in a difference in mean growth between treated and untreated fish for the second growth period (p=0.06; bootstrap t-test). However, overall growth did not differ between groups (p = 0.28; bootstrap t-test).

DISCUSSION

We consistently showed that digenean and myxozoan parasites in our study were negatively associated with size, growth, ATPase activity, and swimming stamina. In addition to the importance in their own right, these performance traits are all associated with the parr-smolt transformation process. The most dramatically negative association with parasitism and these performances occurred with fish from the lower mainstem of WFSR, which are most heavily parasitized. Here, the most profound negative association with parasitism occurred with growth, and clearly size is strongly linked to swimming stamina and ATPase activity, and thus survival.

213 Moreover, the reproducibility was remarkable for the negative association between weight and parasites for both Apophallus sp. and muscle infections of N. salmincola between study years. Indeed, even the slopes and Y intercepts were almost exactly the same, suggesting that these associations have relatively low temporal variation. In biological terms, most fish within the top 50th percentile of infections were also in the bottom 25th percentile of size. The negative associations that parasites had on fish performance were multiplicative effects (as indicated by log transformations of both axes), meaning that very heavy infections were substantially more detrimental than light infections, functioning in a density dependent manner.

We also found differences in fitness between lower mainstem fish and fish from the tributary group of WFSR. Although these results were not constant between study years, it is difficult to make comparisons between wild fish populations, even from the same watershed. There are many other environmental and genetic factors that influence growth, swimming performance, and smoltification. In addition, the timing of smoltification is not necessarily consistent between years for any stock, and hence sampling time could also account for discrepancies.

Size

Reduced growth and size are frequently associated with chronic parasite infections in fishes (reviewed in Barber and Wright, 2005), and these growth-related endpoints were most consistently associated with parasite burdens in our study.

Indeed, Apophallus sp. and N. salmincola were most strongly associated with low

214 values of size and growth in both brood year groups of naturally infected fish, which was corroborated for N. salmincola in our study with the experimentally infected fish.

Myxobolus insidiosus was also negatively correlated with size and growth, although only in lower mainstem fish.

Our findings that the parasites in our study are associated with reduced fish size may be important for understanding indirect sources of salmon mortality. For example, smaller fish have reduced survival due to increased predation risk and reduced physiological fitness (Beamish and Mahnken, 2001). Furthermore, fish size may not only influence freshwater juvenile overwinter survival (Quinn and Peterson,

1996), but also successful smolt survival (Holtby et al., 1990) and ocean migration of salmonids (Beckman et al., 1998). This is particularly evident for overwinter survival in years with more severe winter conditions (Quinn and Peterson, 1996). Effects on size and growth have been evaluated on other host-parasite systems. Cutthroat trout

(Oncorhynchus clarkii clarkii) experimentally infected with Sanguinicola klamathensis had significantly less growth than uninfected fish (Evans, 1974), and E. salvelini causes reduced growth of sockeye salmon (O. nerka), even with light infections (Boyce, 1979). Particularly pertinent to our study, Johnson and Dick (2001) found that yellow perch (Perca flavescens) in four Canadian lakes heavily infected with Apophallus brevis had significantly lower growth in terms of total length and somatic mass than those lightly infected.

Reduced growth in parasitized fish most likely involves an excessive energy demand of infection. Parasites divert energy from their hosts to undergo growth and

215 development, and even seemingly quiescent parasites utilize host energy for basal maintenance (Barber et al., 2000). Furthermore, the accompanying immune response to infection requires additional energy expenditure (Barber et al., 2000). To compensate for the increased energetic cost of parasitism, fish behavior may be affected, such as devoting increased time to foraging. Changes in foraging time of hosts have been well studied in three-spined sticklebacks (Gasterosteus aculeatus) infected with plerocercoids of Schistocephalus solidus. In our study, we fed fish to satiation, so the impacts of these parasites under natural conditions could be even more dramatic, as fish would need to balance foraging for limited resources with exposure to predation. Consistent with our study, Ebersole et al. (2009) also found that neascus was not associated with fish size in coho salmon from the WFSR. However, they did not examine fish for internal parasites. This stresses the importance of evaluating the role of more than one parasite species on host fitness.

Swimming performance

Salmon live in fast moving streams and rivers during their freshwater phase, and thus their swimming ability is implicitly tied to their survival. Reduced swimming performance could lead to mortality either directly by smaller fish being purged from systems during heavy winter water flows (Pearsons et al., 1992) or indirectly by decreasing predator avoidance (Taylor and McPhail, 1985). We found reduced swimming stamina was negatively associated with N. salmincola and Apophallus sp. in fish from the lower mainstem of WFSR from both brood years. Boyce (1979)

216 demonstrated that sockeye salmon experimentally infected with E. salvelini had significantly reduced critical swimming speeds (Ucrit) compared to controls. Likewise, the Ucrit of Atlantic salmon (Salmo salar) is reduced when challenged with sea lice, which is likely due to the accompanied decrease in cardiac output (Wagner et al.,

2003). Butler and Millemann (1971) examined both burst and sustained swimming abilities of juvenile salmonids experimentally infected with N. salmincola and reported the biggest impairment to swimming occurred shortly after initial infection.

They concluded this was due to the migration of cercariae destroying tissue and that encysted metacercariae may impact swimming only when occurring in very high numbers.

We found a negative association with swimming and M. insidiosus in brood year 2008. The difference in our results between the two study years could have been due to differences in infection, as the maximum infection in fish from the second year was more than double the heaviest infection in fish from the first year. Moles and

Heifetz (1998) reared wild sockeye salmon to smoltification in the laboratory, and found that fish naturally infected with M. arcticus had significantly lower Ucrit than those without infections. Similarly, sockeye salmon experimentally infected with the myxozoan P. minibicornis have reduced swimming recovery rates (Wagner et al.,

2005).

Size is linked to swimming performance in fish (e.g., Taylor and McPhail,

1985; Ojanguren and Brana, 2003), and thus most studies investigating swimming performance with salmonids evaluate results compensating for size. We, therefore,

217 were interested in the association that parasites may had on both size/growth and swimming separately. Parasites and size/growth were both strongly associated with swimming, and thus compensating for size/growth in regards to swimming tended to negate the effects of parasitism. In other words, parasites most strongly affected swimming by their association with reduced size/growth. Ebersole et al. (2006) suggested that reduced overwinter survival in lower mainstem fish in the WFSR may be associated with fish being flushed out of the system during heavy overwinter flows due to the simplified substrate at this location. However, parasites could further exacerbate this situation due to reduced swimming ability.

Many studies have illustrated the importance of predation on salmon survival

(e.g., Ward and Larkin, 1964; Parker, 1968, 1971; Larsson, 1985), and both size

(Bams, 1967; Parker, 1971; Pattern, 1977; Beamish and Mahnken, 2001) and swimming performance (Bams, 1967; Taylor and McPhail, 1985) are principal factors influencing predation. Therefore, reduction of either fish size or swimming performance would be advantageous to parasites with complex life cycles involving piscivorous definitive hosts. There are many examples host manipulation by parasites with complex life cycles (referred to as Parasite Induced Trophic Transmission; PITT) involving fish (reviewed in Barber et al., 2000). The digenean species infecting fish in our study complete their life cycle in piscivorous avian and/or mammalian hosts.

Therefore, the reduction in swimming stamina and growth of fish associated with parasites in our study is consistent with the strategy of PITT. Although myxozoan parasites do not share a similar life cycle with digeneans, spores of Myxobolus

218 cerebralis can survive passing through the digestive tract of birds (Taylor and Lott,

1978; El-Matbouli et al., 1991), which may act as phoretic hosts by transmitting parasites to alternate oligochaete hosts in other watersheds.

ATPase activity

Another measure of fitness used in our study was Na+,K+-ATPase activity, a major component of osmoregulation (reviewed in McCormick, 2001). The ability for anadromous fish to osmoregulate is central to the physiological preparedness of salmon migrating to sea water, and hence is an important factor for survival (Moser et al., 1991). Aside from the direct mortality associated with poor osmoregulation, poor enzyme activity has also been linked to increased avian predation risk (Kennedy et al.,

2007).

Smoltification involves changes in energy storage and is thought to be a stressful transition for salmon (Hoar, 1988), and thus the effects of infectious agents could be amplified during this sensitive stage. For example, Price and Schreck (2003) showed that Chinook salmon (Oncorhynchus tshawytscha) infected with

Renibacterium salmoninarum, a chronic bacterial infection that causes bacterial kidney disease, had less preference for seawater than control fish. Parasites have also been associated with decreased osmoregulation of fishes. Boyce and Clarke (1983) showed that sockeye salmon infected with E. salvelini had significantly higher mortality and plasma sodium levels when challenged to saltwater than controls.

Interestingly, these investigators did not find fish size to be significantly correlated

219 with parasites, suggesting that the parasite effects on osmoregulation were direct.

Birkeland and Jakobsen (1997) showed that sea lice infected juvenile (post smolt) sea trout returned to the freshwater environment prematurely compared to uninfected control fish, when simultaneously released in the ocean. In our study, a decrease in

ATPase activity was associated with all parasites, except for neascus. Therefore, when these fish migrate with the rest of the population in the system, they are likely to either directly die due to poor saltwater adaptation or are subjected to greater predation when lingering in the estuary awaiting maturation (Schreck et al., 2006).

Parasites

Summarizing our findings by parasite species, the negative associations of parasitism with host fitness, within heavily infected groups, demonstrated that

Apophallus sp. had the strongest negative relationships with all performance endpoints. Nanophyetus salmincola was negatively associated mostly with size and swimming stamina. This species was also associated with decreased growth in both the naturally and experimentally infected fish. For the most part, M. insidiosus was an important species only in the second wild fish captive study, and was negatively associated with size, growth and swimming stamina. The weakly positive association this parasite had on ATPase likely would have been negated by the much stronger negative association of Apophallus sp., as suggested by the magnitude of the regression coefficients. The difference in associations of M. insidiosus between study years could be attributed to differences in the composition of parasite burden. For

220 example, kidney infections of N. salmincola were much less in the lower mainstem fish the second year. Perhaps this allowed M. insidiosus to have a stronger effect, which may be expected if parasite species compete. Neascus was never associated with the performance endpoints measured in our study, which could suggest that not all parasite species contribute equally to the reduction of certain aspects of host fitness.

It was difficult to separate the effects of each parasite species as the infections were correlated with each other. For example, N. salmincola and Apophallus species were positively correlated, share the same intermediate snail host (J. silicula) (present study; Malek, 1980), and utilize mammals and birds as definitive hosts (Schlegel et al.,

1968; Niemi and Macy, 1974). Although synergism between these two parasites was not statistically tested, such a relationship would be plausible, as these species were positively correlated to each other and both were negatively associated with coho salmon performance after accounting for the mixed infections. Perhaps the negative associations these two parasite species have on fish fitness may work together to facilitate the completion of their life cycle by increasing predation susceptibility to the same definitive hosts. This stresses the importance of studying more than one parasite, as fish are commonly infected by numerous species of parasites (Hoffman, 1999).

Conclusion

In conclusion, we have consistently shown that the parasites examined in our study (i.e., Apophallus sp., N. salmincola, and M. insidiosus) were negatively

221 associated with several fitness endpoints in both naturally and experimentally exposed coho salmon smolts. Reductions in growth, swimming stamina and ATPase activity are linked to overwintering and smolt mortality and thus parasites associated with critical negative effects on individual smolts could have negative effects at the population level. Therefore, the drastic differences observed in parasite burden between parr from the lower mainstem of WFSR and outmigrating smolts could indicate parasite associated mortality, which may be indirect via decrease in these performances. Understanding why certain salmon populations are heavily infected with these parasites, which likely are driven by landscape characteristics, could help in management or recovery planning, given that our data indicates that severity of these infections are associated with survival. Such understanding would help with life history based population viability models by identification of the importance of a heretofore unconsidered mortality factor.

Acknowledgments

This research was funded, in most part, by an ODFW Fish Health Graduate

Research fellowship (agency grant 010-7032-IAA-FISH) to J.A.F. We would also like to thank the research personnel at ODFW for providing samples.

222

Figure 7.1. Example of parasitism negatively associated with fitness endpoints of coho salmon (Oncorhynchus kisutch) smolts. Simple linear regressions are shown for the two parasites most negatively correlated with smolt weight for wild fish, brood years 2007 and 2008, gathered from the lower mainstem of West Fork Smith River. Parasite counts were natural log transformed to improve linearity and scale to graphing. Coefficient of determination (R2) values are shown.

223 Table 7.1. Summary of parasite burden and fitness of coho salmon (Oncorhynchus kisutch) smolts of brood years 2007 and 2008 from the lower mainstem and tributary of West Fork Smith River. Means (±95% confidence interval) are presented and were tested with a bootstrap t-test using 100,000 replications. ** = p < 0.01, * = 0.01 ≥ p < 0.05 for tests between smolts from the lower mainstem and tributary within a given brood year. --- indicates data not collected.

Tributary, Lower mainstem, Lower mainstem, Tributary, brood year brood year 2007 brood year 2008 brood year 2008 2007 Apophallus sp. 77 (64.1-92.7) 1 (0.2-3.7) ** 84 (62.6-117.8) 0.2 (0-0.6) ** (metacercariae/g) Nanophyetus salmincola in muscle 5 (4.0-7.2) 3 (2.4-3.6) * 5 (4.0-7.4) 3 (2.2-3.4) * (metacercariae/g) Nanophyetus 2,386 914 ** 711 1,180 ** salmincola in kidney (1,817.6-3,149.4) (754.8-1118.5) (583.7-860.5) (988.3-1,492.9) (metacercariae/g) Neascus 3 (1.6-8.7) 0 9 (8.0-11.1) 1 (0.6-1.7) ** (metacercariae/fish) Myxobolus insidiosus 51 (25.5-99.8) 36 (24.7-55.0) 47 (26.6-97.4) 5 (1.8-12.8) (pseudocysts/g)

Weight (g) 30 (27.8-34.8) 28 (26.1-30.3) 30 (27.6-32.4) 30 (28.9-32.9) Fork length (cm) 14 (13.7-14.5) 13 (13.2-13.9) 14 (13.6-14.4) 14 (14.0-14.5) Gill Na+,K+-ATPase activity (µmole/mg/hr) 4 (3.7-5.0) 6 (4.9-7.3) ** 3 (1.8-3.2) 2 (1.6-2.7) Swimming stamina (min to exhaust) 17 (14.2-21.6) 20 (17.8-23.8) 11 (9.8-13.8) 15 (13.5-17.5) * Growth (g/wk) 0-7wk ------1.2 (1.1-1.3) 1.1 (1.1-1.2) 7-12wk ------0.9 (0.8-1.1) 1.1 (1.0-1.2) 12-18wk ------0.8 (0.6-0.9) 0.9 (0.7-1.0) 18-24wk ------1.0 (0.8-1.1) 0.9 (0.8-1.1) 0-24 wk (overall) ------2.9 (2.6-3.1) 3.1 (2.9-3.3)

224 Table 7.2. Correlation matrix of explanatory and response variables for linear regression modeling for brood year 2007 lower mainstem coho salmon (Oncorhynchus kisutch) smolts. Values represent correlation coefficients. ln = natural log, FL = Fork Length, ATP = ATPase activity, Swim = swimming stamina, Apo = Apophallus sp., Nano = Nanophyetus salmincola, M = Muscle, K = Kidney, Myxo = Myxobolus insidiosus, totmeta = total metacercariae. All data for parasite burden were log (ln) transformed.

smolt smolt ln Nano ATP Apo Myxo Nano neascus totmeta weight FL Swim (M) (K) smolt weight 1 0.93 -0.09 0.47 -0.59 -0.59 0.20 -0.43 0.15 -0.45 smolt FL 1 -0.22 0.42 -0.68 -0.52 0.31 -0.32 0.20 -0.34

ATP 1 -0.07 0.11 -0.15 -0.27 -0.29 0.01 -0.28 ln Swim 1 -0.31 -0.44 0.42 -0.38 0.12 -0.39

Apo 1 0.40 -0.31 0.22 -0.20 0.25 Nano (M) 1 -0.47 0.72 -0.16 0.72

Myxo 1 -0.11 0.15 -0.11 Nano (K) 1 -0.07 0.99

neascus 1 -0.08

totmeta 1

225 Table 7.3. Correlation matrix of explanatory and response variables for linear regression modeling for brood year 2008 lower mainstem coho salmon (Oncorhynchus kisutch) smolts. Values represent correlation coefficients. ln = natural log, FL = Fork Length, ATP = ATPase activity, Swim = swimming stamina, Apo = Apophallus sp., Nano = Nanophyetus salmincola, M = Muscle, K = Kidney, Myxo = Myxobolus insidiosus, totmeta = total metacercariae. All data for parasite burden were log (ln) transformed.

smolt smolt ln ln Nano Nano Apo Myxo neascus totmeta weight FL ATP Swim (M) (K) smolt weight 1 0.94 0.31 0.61 -0.44 -0.52 -0.35 -0.26 -0.02 -0.34 smolt FL 1 0.35 0.61 -0.52 -0.52 -0.37 -0.30 -0.03 -0.40

ln ATP 1 0.08 -0.39 -0.22 0.12 -0.07 -0.01 -0.14 ln Swim 1 -0.46 -0.48 -0.48 -0.37 -0.06 -0.43

Apo 1 0.58 0.28 -0.06 0.20 0.08 Nano (M) 1 0.22 0.31 -0.06 0.43

Myxo 1 0.23 0.03 0.27 Nano (K) 1 -0.07 0.98 ln neascus 1 -0.08 ln totmeta 1

226 2 ) 0.62 0.51 , totmeta = total = total , totmeta < 0.1). Two models < 0.1). Two models 2 Oncorhynchus kisutch

– 0.37 (K Nano) – 0.37 (K Nano) was used for parasite burden. Apo = Myxobolus insidiosus species pooled (totmeta) and the other (totmeta) species pooled FL)** – 0.19 (totmeta)** – 0.09(Myxo)** – 0.09(Myxo)** – 0.19 (totmeta)** FL)** 0.50 – 0.07 (Myxo)** 0.10 (K Nano)* log (ln) transformed. – 0.65 (Apo)** – 0.31 (M Nano) – 0.24 (Myxo)* – 0.31 (M Nano) 0.24 – 0.65 (Apo)** 0.14 (parr FL)* – 0.15 (Apo)** – 0.06 (M Nano) Nano) – 0.06 (M – 0.15 (Apo)** FL)* 0.14 (parr Model R 2 0.22 – 2.05 (Myxo)** – 4.44 (totmeta)** 0.26 0.39 0.39 (Myxo)* – 1.34 3.43 (M Nano)** – – 2.32(Apo) 0.44 River, held in captivity from brood year 2007 and 2008. ** = River, held in captivity from Density of infection (parasites/g) (parasites/g) infection Density of yxo) 0.16 – 0.36 (Myxo)** – 0.79 (totmeta)** 0.30 r parasite burdens were , M = muscle, K = kidney, Myxo , M = muscle, burden and fitness endpoints for wild coho salmon ( burden and fitness endpoints for wild coho salmon 2008 counts of metacercariae from all digenean from counts of metacercariae Model R t collected. NS = Not Significant, indicating no associations found (R no associations t collected. NS = Not Significant, indicating – 0.48 (K Nano)* – 0.48 (K Nano)* – 4.16 (K Nano) * – 4.16 (K Nano) ------0.24 (parr (parr 0.24 ------Nanophyetus salmincola – 0.88 (Apo)* – 0.01 (M Nano) + 0.06(Myxo) 0.06(Myxo) + – 0.01 (M Nano) – 0.88 (Apo)* – 5.02 (Apo)* – 0.98 (M Nano) + 0.27(Myxo) 0.27(Myxo) + – 0.98 (M Nano) – 5.02 (Apo)* p < 0.05. ln = natural logarithm. ≥ sp., Nano = 2007 (g/wk) (g/wk) (g/wk) (g/wk) variable Weight (g) Weight Weight (g) Weight (Myxo) + 0.44 – 4.98 (totmeta)** Response Response ln overall growth ln overall growth ln overall growth ln overall growth Fork Length (cm) Fork Length (cm) Fork Length (M + 0.08 – 0.54 (totmeta)** smolts, originating from the lower mainstem of West Fork Smith the lower mainstem originating from smolts, p < 0.01, * = 0.01 are presented for each response, one with fo each species separate. All data with counts from metacercariae. --- indicates data no metacercariae. of parasite regression models Table 7.4. Linear Apophallus 227 2 0.40 0.29

otmeta) + 0.20 (Myxo)* otmeta) + 0.20 0.22 + 0.21 (Myxo)* + 0.21 (Myxo)* – 0.23 (K Nano)* – 0.23 (K Nano)* 0.31 (totmeta)** – 0.16 (Myxo)** 0.31 (totmeta)** 0.30 – 0.27 (Apo)* – 0.01 (M Nano) – 0.12 (Myxo)* (Myxo)* – 0.12 – 0.01 (M Nano) – 0.27 (Apo)* 0.21 (smolt FL)* – 0.38 (Apo)* + 0.08 (M Nano) + 0.08 (M Nano) – 0.38 (Apo)* 0.21 (smolt FL)* Model R 2 0.25 (t – 0.05 0.28 (smolt FL)** 0.27 2008 NS Nano) Nano) Model R 0.01(Myxo) – 0.12 (Myxo) – 1.33 (K Nano)* – 0.12 (Myxo) 1.33 (K Nano)* – 0.26 (totmeta)* + 0.08 (Myxo) (Myxo) + 0.08 – 0.26 (totmeta)* 0.31 – – 0.39 (smolt FL) – 1.26 (totmeta)* – (totmeta)* – 0.39 (smolt FL) 1.26 – 0.61 smolt FL – 0.74 (Apo) + 0.08 (M + 0.08 FL – 0.74 (Apo) – 0.61 smolt -ATPase -ATPase 2007 + + ,K ,K activity activity + + stamina stamina variable Response Response ln swimming ln swimming ln swimming (µmole/mg/hr) (µmole/mg/hr) (µmole/mg/hr) (min to exhaust) (min to exhaust) (min to exhaust) ln Na ln Na Table 7.4. Continued. 228

2 ) 0.47 sp., Apophallus Oncorhynchus kisutch Two models are presented for Two models

, totmeta = total metacercariae. --- metacercariae. = total , totmeta < 0.1). 2 )** – 0.65 (Myxo) – 3.78 (K Nano)** Nano)** – 3.78 (K )** – 0.65 (Myxo) 0.34 otmeta) and the other with counts from and the other with counts from otmeta) 0.22 (totmeta)** + 0.01(Myxo) + 0.01(Myxo) 0.22 (totmeta)** 0.38 ln = natural log, Apo Nano)* + 0.01 (Myxo) – 0.39 (K Nano)** Nano)** (Myxo) – 0.39 (K + 0.01 Nano)* 0.25 – 0.36 (K Nano)** – 0.04 (Myxo) – 0.04 – 0.36 (K Nano)** Myxobolus insidiosus 0.07 (Parr FL)** + 0.06 (Apo) – 0.18 (M Nano)** – 0.18 (M Nano)** (Apo) + 0.06 0.07 (Parr FL)** associations found (R ld in captivity from brood year 2007 and 2008. ** = p < 0.01, * ld in captivity from used for parasite burden. Model R 2 2008 burden and fitness endpoints for wild coho salmon ( burden and fitness endpoints for wild coho salmon ariae from all digenean species pooled (t all digenean ariae from ficant, indicating no burdens were log (ln) transformed. were log (ln) transformed. burdens ------– FL)* 0.07 (Parr , M = muscle, K = kidney, Myxo , M = muscle,

2007 Weight (g) Weight NS – 2.65 (M Nano 1.43 (Apo) Weight (g) Weight NS – 0.87 (Myxo) – 4.43 (totmeta)** 0.25 Fork Length (cm) Fork Length (cm) Fork Length NS NS – 0.33 (M 0.11 (Apo) – 0.03 (Myxo) – 0.47 (totmeta)** 0.17 Response variable variable Response Model R -ATPase activity (µmole/mg/hr) -ATPase activity (µmole/mg/hr) NS NS NS NS + + ln overall growth (g/wk) (g/wk) ln overall growth (g/wk) growth ln overall ------p < 0.05. Density of infection (parasites/g) was Nanophyetus salmincola ,K ,K ≥ + + ln swimming stamina (min to ln exhaust) swimming stamina (min to ln exhaust) swimming NS NS NS NS ln Na ln Na

smolts, from the tributary group of West Fork Smith River, he from the tributary group of West Fork Smith smolts, = 0.01 each response, one with counts of metacerc each response, separate. All data for parasite each species

indicates no data collected. NS = Not Signi Nano = of parasite regression models Table 7.5. Linear 229

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234 Chapter 8: General Conclusion

235 The overall aim of this dissertation was to reveal the impacts of multiple parasite species infection on juvenile coho salmon from Oregon coastal streams. This was addressed by accomplishing three objectives: 1) identifying the parasites infecting these fish and describing parasite burdens among geographic locations and fish life stages, 2) examining patterns of parasite burden in juvenile coho salmon from the

West Fork Smith River (WFSR) more extensively to evaluate parasite associated mortality, and 3) conducting laboratory experiments to link parasite associated mortality observed in field studies with decreases in specific fitness endpoints.

I identified 21 parasite species in underyearling and smolts from 10 coastal systems. Some were more abundant in fish from some locations, or in certain fish life stages. Metacercariae of Apophallus sp. were most notable for infecting parr more than smolts and were most abundant in fish from Lower North Fork (LNF) Nehalem and WFSR. Consequently, I focused much of my studies on parasite patterns in fish from WFSR, where I found that Apophallus sp. was most associated with host mortality. Here, 95% of lower mainstem parr had infections higher than the lethal threshold estimated with smolt data, whereas only 11% of smolts had infections above this level (Chapter 6). Lastly, results from my laboratory studies showed that parasites, especially Apophallus sp., are negatively associated with size, growth, swimming stamina and Na+,K+-ATPase activity. These findings, taken together, suggest that parasites can affect coho salmon survival and that fishery management programs should consider parasitism as an important limiting factor.

236 PARASITE IDENTIFICATION

The first step in assessing impacts of parasites on wild fish populations is to properly identify each parasite, which can often be difficult. This was the case with a few species in my study. For example, it was challenging to differentiate M. insidiosus and the new species I described, M. fryeri, based solely on spore morphology.

Although it was unclear whether either of these two species affected the host differently, there are similar situations that illustrate the importance of proper diagnosis. For example, spores of M. cerebralis are virtually indistinguishable from M. kisutchi, M. neurobius, and M. neurotropus, all of which infect neural tissues of salmonids (Ferguson et al., 2008). However, clearly infections of M. cerebralis are more of a concern, as it has decimated many trout populations (Vincent, 1996).

Regarding species identification of Apophallus sp., I believe there needs to be a revision of all species within the genus. The confusion within this taxon is not new, as there are three different scientists who have adopted separate generic criteria

(Cameron, 1936), although most accept the criteria proposed by Price (1931), which would place Apophallus as a senior synonym of Rossicotrema and Cotylophallus.

However, the biggest uncertainty arises when dealing with the species level of identification. The characteristics used to differentiate these species vary greatly, which has resulted in the synonymization of species by some authors, but not others

(Chapter 3). Nonetheless, I attempted to identify specimens in my study to the species level. Adult specimens recovered from experimentally infected chicks suggested that the Apophallus sp. in my studies is a novel species, based on size and egg to body ratio

237 (Chapter 3). However, I felt that repeating the experiment with a different host taxon

(i.e. mammals) would further support my conclusion. Furthermore, I did not want to create additional confusion for these parasites. Although identification was not made to the specific level, a discussion of the implications of unknown Apophallus spp. is warranted.

There are many described species of Apophallus infecting fishes (Chapter 3), many of which infect different host species or tissue types. For example, Cameron

(1945) believed that Apophallus brevis was specific to the skin of speckled trout

(Salvelinus fontanalis) and Apophallus venustus was muscle-specific for fish from 9 different families, excluding salmonidae. Host specificity of digenean metacercariae in fish is usually quite broad (Paperna, 1995). Therefore, it is plausible that these species occur in both hosts, although ecological or sampling factors may explain the lack of these findings. For example, N. salmincola (metacercariae) was initially thought to only naturally infect salmonids, but further sampling has shown that 16 fish species from 4 families, and even Pacific giant salamanders (Dicamptodon ensatus), are natural hosts (Gebhardt et al., 1966). Indeed, I have observed Apophallus sp. in one

Umpqua dace (Rhinichthys evermanni) sampled from my study site at WFSR (pers. obs.). Interestingly, the burden of metacercariae of a given parasite species may also vary with host species. For example, steelhead trout have been shown to harbor about

2-4 times heavier infections of N. salmincola than coho salmon collected in the same stretch of rivers (Millemann and Knapp, 1970), which was likely due to differences in exposure to fish species occupying different microhabitats, as riffles had more

238 cercariae than top and mid levels of pools based on sentinel fish studies (Millemann and Knapp, 1970).

The importance of differentiating Apophallus species is even more substantiated when considering the pathological effects they have on fish hosts, as only A. brevis causes ectopic bone formation in perch (Pike and Burt, 1983).

Conversely, most species cause ‘black spot disease’, with the exception of Apophallus venustus (Chapter 3). A particularly interesting pathological change is exhibited by

Apophallus sp. infecting cyprinids from the Willamette River, Oregon, as it infects vertebrae and has been associated with skeletal deformities (Kent et al., 2004). Such differences in tissue location and pathology would certainly influence the impact that a given species has on a host.

PARASITOLOGICAL SURVEY

Documenting the prevalence, abundance, and geographic distribution of parasites can provide useful information for studying effects of parasitism because certain patterns may indicate parasite associated mortality. For example, I consistently found very high infections of Apophallus sp., neascus, N. salmincola, and M. insidiosus, in underyearling coho salmon from WFSR, compared to smolts. This pattern provided the essential foundation to drive my subsequent studies. I also saw similar patterns in fish from other watersheds such as LNF Nehalem and Cascade Ck.

Although I did not focus further efforts on all of these systems, given my findings

239 from fish in the WFSR, similar studies should be undertaken with fish from those systems as well.

Results from my survey also showed that the geographic distribution and abundance of parasites differed, which may be driven by environmental factors and thus land use. For example, although N. salmincola was present in fish from most sites, abundance of this parasite was high in fish from certain locations (e.g., WFSR).

The intermediate host for this digenean is Juga silicula, which is wide spread in coastal streams in the Pacific Northwest. The optimal habitat for this snail supposedly includes slower moving water, with coarse substrates (Furnish 1990). Therefore, certain land uses, such as dams, may influence the distribution of this snail and in turn affect infection patterns of N. salmincola.

The biggest abiotic factor thought to influence digeneans is temperature. For example, a meta-analysis by Poulin (2006) showed that the reproduction of intra- molluskan stages of most digeneans increases with elevated temperatures, which may included both cercarial production and shedding. Additionally, the reproductive rates of the intermediate snail hosts may increase at higher temperatures (Malek, 1980).

Such increases in parasite stages and their intermediate hosts may represent a net result of massive infection for second intermediate fish hosts. Indeed, abundant infections of Apophallus were found in fish from sites with little riparian canopy (e.g.,

WFSR, LNF Nehalem, Cascade Ck). These sites have been subjected to heavy logging practices in the past (Solazzi, 2000), which results in increased water temperatures in the associated rivers (Beschta and Taylor, 1988). Consistent with these findings, Kent

240 et al. (2004) reported a high infection prevalence of Apophallus sp. in cyprinids from warm areas of the Willamette River. Similarly, Cairns et al (2006) found a positive correlation of black spot disease (neascus) in coho salmon and temperature in the

WFSR. The temperature in sections of the lower mainstem WFSR often exceeds 20°C in the summer (Ebersole et. al, 2006). This would not only stress coho salmon physiologically, but it may also expose them to more parasites. In my parasitological survey, I found that metacercariae of N. salmincola, neascus, and the unidentified spp. in the gills, were also abundant at the same locations that had high levels of

Apophallus. Therefore perturbations to systems may cause large-scale effects to the composition of parasite fauna.

Although not part of my research, but warranting discussion, is the influence of environmental factors on infections. It is often difficult to understand the affects of land use on parasite dynamics. For example, although agricultural activity may increase nutrients of systems, and thus increasing some parasites, the associated pesticide use may actually decrease many parasites (Marcogliese, 2005). Similarly, such complexities were shown by King et al. (2007), who studied the impacts of agriculture wetlands in urban areas on parasite composition of metamorphic northern leopard frogs (Rana pipiens). They found that frogs from these sites, compared to reference areas, were almost void of species that subsequently infect birds or mammals, suggesting that pollution effects may be masked by a strong land use component such as urbanization. In my survey, Mill Ck. Siletz is associated with agricultural activities (Solazzi et al., 2000), and I found fish heavily infected N.

241 salmincola, Apophallus sp., and neascus. Interestingly, fish from this site were also heavily parasitized with gill protozoans, such as Epistylus sp. and C. piscium. Heavy infections by both these species have been associated with systems having high organic loads (Esch et al., 1976; Hofer et al. 2005).

Many of the myxozoans in my study, such as kidney-infecting Chloromyxum majori and brain-infecting Myxobolus sp., were common in fish from the same rivers where high levels of digeneans were recorded to infect fish. Similar to digenean parasites, reproduction of myxozoans in their invertebrate hosts is also temperature dependent (e.g., M. cerebralis; El-Matbouli et al., 1999). Although the life cycles of the myxozoans in my study have not been resolved, they likely involve an oligochaete host based on other described life cycles. Many oligochaete species thrive in relatively warm, oligotrophic or eutrophic environments (Granath and Gilbert, 2002), which could account for their distribution recorded here. This is only a general observation, further studies, including discovery of the intermediate hosts, would be required to connect these parasite distributions with ecological parameters and land use practices.

Regardless of the environmental factors influencing these infection patterns, I have conclusively shown that there are differences in parasite geographic distribution and infection abundance. Given that effects of parasitism on hosts are density dependent (e.g., Crofton, 1971; May and Anderson, 1979; Dobson, 1988) and that my research has shown certain parasites are associated with fish mortality and decreased performance, this should be a concern for fishery management programs. This is because modifying environmental factors contributing to accelerated parasite

242 production, and their intermediate hosts, may be the only solution to managing these infections. For example, habitat restoration projects could be used to manipulate temperatures, substrate and flow to decrease infection dynamics.

PERSISTENCE OF INFECTION

An important prerequisite for evaluating parasite associated mortality is that hosts do not recover from the infection (see Crofton, 1971; Anderson and Gordon,

1982; Lester, 1984). This is because infection recovery could explain declines in parasite burden in the host population. Although it is generally accepted that metacercariae of digeneans persist in their hosts for a very long time, this should still be evaluated for the species in question. For example, immunological responses to infection, and thus degree of recovery, can vary between parasite species.

Here I expand on the discussion of persistence from my manuscript (Chapter

5) on this subject. One of the most well studied metacercarial infections is black spot disease caused by neascus, because cysts can be enumerated grossly without lethal sampling. However, Lemly and Esch (1983) pointed out that counting black spots macroscopically does not necessarily equate to counts of actual worms, as the melanization is simply a host reaction. Indeed, Lemly and Esch (1983) determined that up to 30% of cysts in overwintering centrarchid fishes did not contain viable parasites.

Conversely, gross counts at earlier time points may underestimate parasite abundance, as was the case in my study, which was likely due to my inability to detect early infections not yet melanized. This stresses the importance of using magnified optics

243 when examining fish. Nonetheless, various studies have shown that these parasites are capable of very persistent infections, some lasting almost 5 years (Hoffman and Putz,

1965).

Lesions associated with neascus (black spot) in the skin of coho salmon in my study involved obvious fibroplasia with melanosis around the parasite cyst. I also noted a yellow/orange pigmentation circumscribing the fibrosis on occasion. This was most likely due to xanthophores accumulation instead of melanophores, as was noted by Hoffman (1956) with infections of Crassiphiala bulboglossa (black-spot neascus) persisting up to 3 years in fathead minnows (Pimephales promelas).

Heterophyids can also cause black spot disease, and persistence has been evaluated for many of these species. For example, Lysne et al. (1994) showed that

Cryptocotyle lingua (black-spot heterophyid) infections in Atlantic cod (Gadus morhua) last 18 mo. I did not detect any significant histopathological changes associated with encysted Apophallus sp. in my study. However, penetrating cercariae can cause acute effects. Indeed, the heterophyid Haplorchis pumilio causes focal hemorrhage of muscle in cultured tilapia (Sommerville, 1982). It is common for migrating cercariae to induce more pathology than encysted metacercariae. However there are exceptions, many of which involve heterophyid infections. For example,

Centrocestus formosanus metacercariae infecting gills of tilapia elicit profound cartilaginous hyperplasia (Paperna, 1995). Particularly pertinent to my study,

Apophallus brevis causes ectopic bone formation in perch (Taylor et al., 1994) and

244 Apophallus sp. is associated with spinal deformities in cyprinids from the Willamette

River, Oregon (Kent et al., 2004).

Persistent infections of Nanophyetus salmincola have been evaluated previously. This fluke harbors the neorickettsial agent causing “salmon disease” in dogs. Farrell et al. (1965) fed adult salmon, which had been infected with N. salmincola 33.5 mo. prior, to dogs and induced clinical disease, thus demonstrating this parasite persists throughout the adult stage of coho salmon. This was consistent with the level of persistence seen in my study involving infections in juvenile fish.

Nanophyetus salmincola elicited minimal tissue response and inflammation, which was consistent with the previous histopathological studies by Wood and Yasutake

(1956). They also noted occasional fibroplasia around metacercarial cysts, whereas I detected mild fibroplasia associated with all metacercariae. Yasutake and Wood

(1956) speculated that the most damage caused by this parasite was mechanical damage, especially in the heart. This parasite infects essentially every tissue type and I even encountered this worm in ovary and brain, which were new records to my knowledge.

Some metacercariae remain unencysted in fish, but are still able to persist. For example, muscle-infecting Rhipidocotyle johnstonei (Bucephalidae) in plaice

(Pleuronectes platessa) produces progenetic metacercariae that release many eggs into surrounding tissue, which elicit an intense granulomatous response by the host.

However, Pulsford and Matthews (1984) showed that this parasite can still persist for nearly a year, even in the presence of a severe host response. These authors concluded

245 that the epidermis of R. johnstonei must convey protection against potentially deleterious effects by the host’s immune response, which is in stark contrast to encysted metacercariae, such as C. lingua, that do not induce an intense cellular response (McQueen et al., 1973) and infect a wider range of hosts for much longer periods of time. Other unencysted metacercariae, such as Diplostomum spathaceum, evade the host’s immune response by occupying immunologically privileged sites such as the eyes. However, rainbow trout (Oncorhynchus mykiss) do acquire adaptive immunity to new infections, which is likely directed towards migrating cercariae

(Karvonen et al., 2005). Survival of D. spathaceum as long as 3 years has been recorded in the lens (Hendrickson, 1978). Although these species differ from mine in having unencysted metacercariae, it is plausible that the innate response to initial infection by cercariae is shared by all these parasites.

A more in depth investigation of adaptive immunity was performed by Wood and Matthews (1996) on C. lingua, a black-spot heterophyid that infects thick-lipped grey mullet (Chelon labrosus) for at least 2 years. They tested antiserum recognition of antigens of the metacercarial stages of C. lingua and found that there were only 10 of 20 parasite proteins that were not recognized by the fish, of which only 1 protein was unique to the metacercarial stage of the trematode. They concluded that because some of the metacercarial proteins were recognized, then the cyst may be semi- permeable in nature, permitting release of proteins such as essential parasite waste products, which could in turn produce antigenic variation.

246 I detected little or no tissue reaction to metacercarial muscle infections in my study. These encysted worms are likely evading the host’s response, thus permitting persistence. This is a common strategy used by metacercariae, which can be accomplished either by molecular mimicry or simply by the parasite forming thick, impermeable, cysts (Graczyk, 1997). Survival of neascus (black spot) in the skin clearly involves the latter, as I observed prominent fibroplasia with melanosis around the parasite cyst, yet no inflammatory cells within it. Apophallus sp. and N. salmincola also apparently have protective cysts, although the limited tissue reaction and fibrosis may indicate these species also utilize molecular mimicry.

Although the exact immunological mechanisms of host removal of metacercariae in fish are largely unknown, some studies have made gross observations of recovery. For example, Hoffman (1958a,b) noted that cyprinids, with visceral infections of neascus, formed perforations in their body wall to allow parasites to fall out. This occurred several months after infection and the wounds were subsequently healed by fibrosis. Expulsion of parasites and inflammatory debris through the body wall occurs with other parasites of fishes, such as tapeworm metacestodes (Moser et al., 1984). In essence, this process is analogous to removal and healing of deep tissue that has been impaled by wooden splinters or other inanimate objects.

Even though encysted metacercariae are inaccessible to host immune cells, these parasites still die. Faliex (1991) showed that infections of Labratrema minimus

(Bucephalidae) metacercariae persist for about 8 mo. in Liza ramada. She concluded this was due to parasite senescence because she detected blood vessels in contact with

247 the cyst that could have transferred nutrients and wastes, yet the worms died.

Similarly, Lemly and Esch (1983) speculated that the reduction in U. ambloplitis infections in centrarchids may be due to simple attrition, as metacercariae may depend on their own energy reserves for survival (Hunter and Hamilton, 1941). Noteworthy is that once encysted metacercariae die, extensive cellular changes can occur (Paperna,

1995).

Myxozoans, particularly histozooic species, are also thought to generally persist throughout much of the infected fish’s life (Lom and Dyková, 1992). Indeed, many neurotropic species, such as Myxobolus arcticus, have been used as biological tags to identify stocks of marine salmon that had been infected as juveniles (e.g.,

Urawa and Nagasawa, 1989). Likewise, muscle-infecting M. insidiosus and

Henneguya salminicola are capable of similar persistent infections (Kent et al., 1994).

Thus, not surprisingly M. insidiosus persisted in my study.

However, there are unique aspects of myxozoan infections in fish that are worthy of discussion. For example, I actually detected an increase of M. insidiosus abundance in my study. This could be explained by the development of myxozoans, as pseudocysts (plasmodia) are initially small and continue to grow in size by asexual reproduction within the host. The lower counts in parr may be reflected by very small pseudocysts not being detected in wet mounts. However, an alternative explanation is that autoinfection occurred, as suggested for M. insidiosus by Amandi et al. (1985).

Autoinfection is the production of spores in situ or within the same host, which has not been adequately documented (Lom and Dyková, 1995). The uncertainty of this

248 phenomenon precludes much emphasis on this explanation. Of most significance is that there was no evidence of recovery of infection from this parasite in my study.

Consistent with Amandi et al. (1985), I also did not detect an inflammatory response while M. insidiosus pseudocysts were immature and intact in muscle. This also occurs with other intramuscular myxozoans, for example, K. thyrsites only elicits inflammation after pseudocyst rupture and release of parasites to surrounding tissue

(Moran, et al., 1999).

In conclusion regarding persistence, I have shown that Apophallus sp., N. salmincola, neascus, and M. insidiosus persistently infect coho salmon from WFSR throughout the overwintering period, as the majority of these infections still occurred in smolts when they were held in captivity. Thus, my hypothesis of parasite-associated mortality of lower mainstem parr from WFSR, as indicated by consistently detecting nearly 95% lower infection levels in outmigrating smolts, is supported. Besides parasite-associated mortality, there is one other alternative explanation for my observations regarding these persistent infections – i.e., the smolts that I examined do not represent the same population of fish that were reared from the lower mainstem, as smolts are a mixed population of parr sub-populations. Here, I evaluated the former and my field studies (discussed below) have evaluated the later.

249 EVALUATING PARASITE ASSOCIATED MORTALITY

WITH FIELD DATA

Once I had shown that the parasites of interest in the study were capable of persistent infection, I was able to better conduct field studies. As noted in Chapter 6, studying parasite burden rather than prevalence is more informative for macroparasite systems. Macroparasites are defined, in part, by being relatively large and not directly replicating within the host (Anderson and May, 1979). Myxozoans share both macro and micro parasite attributes, although, here I used macroparasite techniques to analyze data from these parasites. Many of these methods require longitudinal studies to show declines in parasite burden within a cohort of hosts. However, apart from the inaccessibility of fish, coho salmon present a special problem in that they are migratory. Thus, I used a retrospective approach analyzing data from smolts based on theoretical modeling, which was supported by empirical data on parr infections. I was able to show that the parasites in my study are indeed associated with host mortality of coho salmon in WFSR, which was consistent between study years. Of these associations, Apophallus sp. was the strongest, indicating that up to 95% of parr rearing from the lower mainstem were subject to parasite-induced mortality. This study reinforced my hypothesis that the detection of only lightly infected smolts is due to parasite associated mortality, as coho salmon that survive to smolt stage would have disproportionately lighter infections than lower mainstem parr. Below is a discussion of the implications of these findings.

250 The fact that most of the lower mainstem parr are heavily infected represents an irregularity, as most wild populations have only a few heavily infected hosts resulting in a highly aggregated distribution. Perhaps there has been an environmental perturbation in this area of the river that promotes the extremely high infections in these fish to occur. The overwinter survival estimates of fish from this location (ca.

2%) reported by Ebersole et al. (2006) reflect such high levels of morality estimated by my study. These authors concluded that such poor survival was likely due to environmental factors, as this area of the river has poor substrate due to historical logging practices (Ebersole et al., 2006). Perhaps the high mortality is due to both abiotic and biotic factors acting in concert. Indeed, this portion of WFSR exceeds temperatures of 20° C in the summer (Ebersole et. al, 2006), which would in turn promote increased parasite reproduction, as Poulin (2006) has shown that increased temperatures generally enhances cercarial production and release. Therefore, these fish are subjected to both abiotic stressors and parasite infections, which may have cumulative effects resulting in decreased performance (Schreck, 2000).

Given that the WFSR coho salmon population is ESA listed as threatened and that habitat is poor for these fish, the negative effects of parasitism could be exacerbated because the combination of such limiting factors may be too much for this already declining population to sustain. Although it is well known that predators can regulate populations, currently, there is no consensus regarding the ability of parasites to perform this function. However, there is little doubt that the negative effects on host

251 populations can be much more devastating for endangered populations (McCallum and Dobson, 1995).

My study and previous investigations by Ebersole et al. (2006) indicate that only a few parr from the lower mainstem of WFSR survive to the smolt stage. Hence, fish from this location contribute very little to the genetic structure of this coho salmon population. For most Pacific salmon population, it is assumed that there is little genetic separation within a river, which appears to be the case for coho salmon in the

WFSR (Rodnick, K., Idaho State University, pers. comm.). This heterogeneity would reflect that there is interbreeding of the sub-populations, presumably from fish competing for quality spawning habitats. Therefore, this amount of gene flow would negate selective pressure of the evolution of resistant fish. In other words, the parents of lower mainstem parr almost always are derived from regions of the river with little genetic pressure to develop resistance to parasitism, and thus resulting in a constant pool of susceptible fish in the lower mainstem. This could affect the recovery efforts for this population because such naïve fish would always be subjected to parasite associated mortality.

Negative effects on the coho salmon population from WFSR due to limiting factors are of serious concern to management programs, as they are attempting to aid in the recovery of this population. Certainly, based on the results from my studies, parasitism should be included as such a presupposing factor. Furthermore, negative impacts of parasitism may be acting on other populations or sub-populations within this threatened ESU. This would be especially true if there has been some disturbance

252 to the host-parasite dynamics, possibly mediated through environmental changes.

Perhaps the negative effects of parasitism on these fish may be contributing to the poor recovery of conservation programs. Although, the level of reduced recovery associated with these infections are often difficult to discern because many variables, such as habitat, may be interacting. This stresses the complexity involved with attempting to assign proportions of limiting factors to recovery and the relative definition of “a recovery”. Lastly, when studying threatened populations, I suggest collecting as much data as possible from these valuable samples. For example, physiological studies could also incorporate parasite data, as was performed by

Rodnick et al. (2008). I used multiple approaches to examine parasite-associated morality in my field study and these same fish were also used in my laboratory studies

(which I discuss later).

In conclusion, evaluation of field data has shown that parasites in my studies, especially Apophallus sp., are associated with parasite-induced mortality in the juvenile freshwater phase of coho salmon. The threatened disposition of these fish emphasizes that these impacts may be substantially worse, as recovery from such effects may be difficult for small, fragmented, populations. Moreover, these effects may be masked by many interactive variables, such as habitat loss, which in concert may have cumulative effects on fish fitness. Such complexities should be considered by management programs and evaluated for each system.

253 LABORATORY STUDY

My laboratory studies corroborated my findings of parasite-associated mortality, based on field studies, by evaluating potential mechanisms. These experiments also examined the relative strength of associations for individual parasite species. Here, Apophallus sp. was once again shown to be the most associated with host impacts. The other two most associated species were N. salmincola and M. insidiosus, but there were no associations for neascus. This suggests that not all parasites impact the host the same. I did not detect any interactions that were both significant and interpretable among the multiple parasite species on fish performance.

However, the positive correlation between some parasites, particularly Apophallus sp. and N. salmincola infecting muscle, suggests a synergistic relationship between these two parasites. All of the fish performances used in my study (i.e., size, growth, swimming, and ATPase activity) are related to overall fitness. Although I have already discussed the importance of these chosen performances in Chapter 7, a more in depth discussion highlighting the implications of my findings is warranted.

Size

Size, mediated by growth, is an integral component of salmon survival, which may be directly or indirectly linked (Figure 8.1). Smaller fish have reduced survival due to increased predation risk and reduced physiological fitness (Beamish and

Mahnken, 2001). Additionally, fish size influences freshwater juvenile overwinter survival (Quinn and Peterson, 1996), smolt success (Holtby et al., 1990) and ocean

254 migration (Beckman et al., 1998) of salmonids. For juvenile salmon, size can be a very important predictor of overwinter survival in years with severe winter conditions, as smaller fish may struggle more with swimming in higher river flow rates (Quinn and

Peterson, 1996). I found that Apophallus sp. and N. salmincola were both negatively correlated with fish size, which was extraordinarily consistent between both study years, even with different study designs. Consistent with my findings, Johnson and

Dick (2001) found that yellow perch (Perca flavescens) heavily infected with

Apophallus brevis have significantly less total length and somatic mass than lightly infected fish.

Figure 8.1. Simplified diagram of some complex interactions of parasites, fish performance, and environment, leading to mortality.

255 Reduced growth and size are frequently associated with chronic parasite infections in fishes (reviewed in Barber and Wright, 2005). For example, sockeye salmon (Oncorhynchus nerka) experimentally infected with Eubothrium salvelini

(Cestoda) have reduced growth compared to uninfected control fish (Boyce, 1979).

The main mechanism of such effects is thought to be a loss of vitality (i.e., energy).

Energy loss is associated with an excessive energy demand of infection, which may have many sources. For example, parasites divert energy from their hosts to undergo growth and development, and even seemingly quiescent parasites utilize host energy for basal maintenance (Barber et al., 2000). Additionally, the accompanying immune response to infection requires additional energy expenditure (Barber et al., 2000). The net result is that there is a depletion of available energy, which may lead to either compensation or exhaustion (i.e., death) of the fish. Compensation to chronic stressors may result in fish returning to a new homeostatic level; often this is referred to as an allostatic load (see Schreck, 2000). Parasitism would fall under this category, thus the cost of infection results in a decreased homeostatic level of fish performance. The physiology of bioenergetics in most animals, including fishes, has been well studied and a ‘bioenergetic equation’ has been proposed to describe this process (Warren and

Davis, 1967). Clearly parasite infections would affect the balance of this equation.

An example of compensation for the increased energetic cost of parasitism is a change in fish behavior, such as devoting increased time to foraging. Changes in host foraging time have been well studied in three-spined sticklebacks (Gasterosteus aculeatus) infected with plerocercoids of Schistocephalus solidus. Exhaustion due to

256 parasitism has also been shown. For example, Lemly and Esch (1984) confirmed that bluegill sunfish heavily infected with Uvulifer ambloplitis (neascus) did not survive the winter because of depleted fat reserves, which was directly linked to oxygen consumption of encapsulating parasites. In our study, we fed fish to satiation, so the effects of these parasites under natural conditions would likely be even more dramatic, as fish would need to balance foraging for limited resources with exposure to predation. Therefore, coho salmon in my study may be subjected to mortality directly

(exhaustion) or indirectly from exposure to predators in attempts for compensation.

Swimming performance

Swimming performance is also affected by fish size (e.g., Taylor and McPhail,

1985; Ojanguren and Brana, 2003), and thus most studies investigating swimming performance with salmonids account for size. However, the effect of parasitism on swimming could be indirect; therefore I did not account for size in my analyses. I did, however, use structural equation modeling and found that the strongest correlation with parasitism and swimming was indirect via effects on size (data not shown).

Furthermore, parasites and size were both strongly correlated with each other (r > 0.5), meaning these were confounding variables and thus compensating for size would negate the effects of parasitism. This indirect effect, which was confirmed by my growth study, still represents an important finding because reduced swimming performance, regardless of the mechanism, can influence fish survival.

257 Salmon live in fast moving streams and rivers during their freshwater phase, and thus their swimming ability is implicitly tied to their survival. Reduced swimming performance could lead to mortality either directly by smaller fish being purged from systems during heavy winter water flows (Pearsons et al., 1992) or indirectly by decreasing predator avoidance (Taylor and McPhail, 1985). Other parasites have been shown to be associated with reduced swimming performance. For example, Boyce

(1979) demonstrated that sockeye salmon experimentally infected with E. salvelini had significantly reduced critical swimming speeds (Ucrit) compared to controls. Butler and Millemann (1971) examined both burst and sustained swimming abilities of juvenile salmonids experimentally infected with N. salmincola and reported the biggest impairment to swimming occurred shortly after initial infection. They concluded this was due to the migration of cercariae destroying tissue and that encysted metacercariae may impact swimming only when occurring in very high numbers. Likewise, myxozoans have also been associated with decreased swimming performance (e.g., Moles and Heifetz, 1998; Wagner et al., 2005).

Reduced swimming performance in the coho salmon in my study may represent a significant source of mortality, as Ebersole et al. (2006) suggested that reduced overwinter survival in lower mainstem fish in the WFSR may be associated with fish being flushed out of the system during heavy overwinter flows. These authors concluded that this was due to the simplified substrate at this location, which was caused by logging practices. Parasites could further exacerbate this situation by reducing swimming ability. This exemplifies the complexity of infectious agents,

258 environment, and disease that was proposed by Sniezko (1974), which is illustrated simplistically in Figure 8.1.

Many studies have illustrated the importance of predation on salmon survival

(e.g., Ward and Larkin, 1964; Parker, 1968, 1971; Larsson, 1985), and both size

(Bams, 1967; Parker, 1971; Pattern, 1977; Beamish and Mahnken, 2001) and swimming performance (Bams, 1967; Taylor and McPhail, 1985) are principle factors influencing predation. Therefore, reduction of either fish size or swimming performance would be advantageous to parasites with complex life cycles involving piscivorous definitive hosts. There are many examples host manipulation by parasites with complex life cycles (referred to as Parasite Induced Trophic Transmission; PITT) involving fish (reviewed in Barber et al., 2000), which may be either direct or indirect.

For example, Lafferty and Morris (1996) showed that the digenean Euhaplorchis californiensis infecting brains of killifish (Fundulus parvipinnis) directly alters swimming behavior, presumably involving neurochemicals, to increase predation susceptibility. Although the digenean species infecting fish in my study indirectly affected swimming, it is possible that very heavy infections could also have direct effects by applying pressure to muscles involved in swimming. The digeneans in my study also complete their life cycle in piscivorous avian and/or mammalian hosts, so reduced swimming stamina and growth of fish associated with these infections is consistent with the strategy of PITT.

259 Gill ATPase activity

Smoltification involves changes in energy storage and is thought to be a sensitive life stage for salmon (Hoar, 1988). Osmoregulation is crucial for the physiological preparedness of migrating smolts, and hence is an important factor for survival (Moser et al., 1991). Therefore, I evaluated gill Na+,K+-ATPase activity, which is a major component of osmoregulation (reviewed in McCormick, 2001), as a proxy for evaluating the degree of smoltification.

Aside from the direct mortality associated with poor osmoregulation, poor enzyme activity has also been linked to increased avian predation risk (Kennedy et al.,

2007). There have been previous reports that parasites are associated with decreased osmoregulation of fishes. For example, Boyce and Clarke (1983) showed that sockeye salmon infected with E. salvelini had significantly higher mortality and plasma sodium levels when challenged to saltwater than controls. Interestingly, these investigators did not find fish size to be significantly correlated with parasites, suggesting that the parasite effects on osmoregulation were direct. In our study, a decrease in ATPase activity was associated with all parasites, except for neascus. Therefore, when these fish migrate with the rest of the population in the system, they are likely to either directly die due to poor saltwater adaptation or are subjected to greater predation when lingering in the estuary awaiting maturation (Schreck et al., 2006).

Given that the metamorphic state of smolting salmon is stressful, the effects of infectious agents could be augmented during this sensitive stage. For example, Price and Schreck (2003) showed that Chinook salmon (Oncorhynchus tshawytscha)

260 infected with Renibacterium salmoninarum, a chronic bacterial infection that causes bacterial kidney disease, had less preference for seawater than control fish. Therefore, the effects of parasites could be worse for coho salmon from WFSR than resident, non-metamorphosizing, fish species.

Non-anadromous fish species, such as Umpqua dace, can become infected with these same parasites in WFSR (pers. obs.), which could represent a piscine reservoir for increasing infections in this system. Alternative host species, which exhibit lesser signs of clinical disease, may be a source for infection spill over into susceptible populations (see Gog et al., 2002). This may represent an important source of mortality for endangered populations, especially if involving conspecifics that compete for resources. This would be interesting to evaluate with Oncorhynchus mykiss, as migrating steelhead may be more susceptible to the effects of infection than resident rainbow trout. Indeed, reservoir hosts represent another layer of complexity that may be added to disease ecology models.

It is possible that parasite species may compete within the same host. My study indicated that not all species affect the host the same, as Apophallus sp. was always highly associated with host impacts, but this never occurred with neascus. Examining the effects of mixed infections is a relatively nascent field. This is likely due to the difficulty in analysis, which is multidemensional. However, multivariate ecological models, such as Principle Component Analysis, might resolve this question better.

However, my analysis revealed that parasites, such as Apophallus sp. and N. salmincola infecting muscle were positively correlated and both of these species were

261 negatively associated with fish fitness after accounting for mixed infections, suggesting a synergistic relationship between these two parasites. More importantly, I showed that the effects of each parasite species in my study were multiplicative, which was evidenced by the need to log transform data on both axes in the analysis. A multiplicative effect reflects the nature of parasitism being density dependent, where very high infection levels are substantially more detrimental than light infections.

In conclusion through my lab studies I consistently showed digenean and myxozoan parasites were negatively associated with size, growth, ATPase activity, and swimming stamina of coho salmon. Besides the importance in their own right, these performance traits are all associated with the parr-smolt transformation process.

The most dramatically negative association with parasitism was size involving fish from the lower mainstem of WFSR, which are most heavily parasitized. This was incredibly consistent between studies. The impacts these infections have on coho salmon in my study are mostly indirect and complex. Given the importance of the measured fish performances, these findings have merit for including parasites as an influence on fish fitness.

GENERAL CONCLUSION

Results from all my studies, taken together, provide strong evidence of parasites impacting juvenile coho salmon in Oregon. Following a survey of fish from

10 rivers, I chose to focus my research on one, the WFSR. Here, impacts of parasites were best illustrated by my studies involving fish from the lower mainstem of this

262 river, which were heavily infected by Apophallus sp. and other important parasites.

The results from my field studies indicated Apophallus sp. as the most associated with parasite-induced mortality. Thus, in this system, parasites were indicated to have population level effects, as 95% of parr from the epizootic zone were above the estimated threshold for associated mortality, and these heavily infected fish were never seen in the smolt samples. The detrimental effects of parasites were then further investigated in the laboratory. The most profound finding was that parasites were consistently associated with negative impacts on size and growth, which influenced other parameters, such as swimming ability. Furthermore, it was remarkable how similar results were between both study years, even with different year classes of fish and different laboratory regimens. This indicates that the magnitude of these effects is rather stable. These impacts on individual fish all translated into potentially important factors affecting this population.

The interplay of parasites, fish performances, and the abiotic and biotic environment were all involved. The complexity of multiple parasites and multiple host species reflects the ecological aspect of disease. Fishery management programs should be concerned with these seemingly subtle effects. The difficulty in documenting the impacts of parasites often precludes this aspect to management. However, as seen here, parasite burden may have potentially profound impacts at many host levels, especially for endangered stocks.

My dissertation research has clearly demonstrated that these parasites can impact coho salmon. My parasitological survey of fish from 10 rivers in Oregon

263 showed that some watersheds had fish much more heavily infected than others, which may indicate that this phenomenon is more widespread. Thus, the next step would be to determine how landscape and land use practices influence these parasite infections.

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