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UNIVERSITY OF WISCONSIN-LA CROSSE Graduate Studies FISH HEALTH ASSESMENTS OF GREAT LAKES COREGONIDS A Manuscript Style Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science in Biology Christopher M. Olds College of Science and Health Aquatic Science Concentration May, 2012 FISH HEALTH ASSESSMENTS OF GREAT LAKES COREGONIDS By Christopher M. Olds We recommend acceptance of this thesis in partial fulfillment of the candidate's requirements for the degree of Master of Science in Biology (Aquatic Sciences Concentration) The candidate has completed the oral defense of the thesis. A- r-1;)_ Date Gregory Sanclt n , Ph.D. Thesis Committee Co-Chairperson Roger H ro, P .D. Thesis Committee Member ( - f--ZVIZ... Date .x IJ(..;{. 6 / 2 I ate 'd.--+ -J.o 12 Date Thesis Committee Member Thesis accepted 2 Robert H. Hoar, Ph.D. Associate Vice Chancellor for Academic Affairs -"1~ -.?a/~ Date ABSTRACT Olds, C. M. Fish health assessments of Great Lakes Coregonids. MS in Biology, May 2012, 70pp. (B. Lasee and G. Sandland) Health of Great Lakes coregonids is continuously monitored for the spread of pathogens because of their economic importance. An infectious disease and parasite survey was conducted to build on previous data collected in the past but to also examine other factors that may affect distribution of pathogens in Great Lakes lake whitefish, round whitefish, · and bloater populations. Coregonids (n=394) were collected from 3-4 regions in Lake Michigan, Lake Huron and Lake Superior and screened for target pathogens according to the procedures outlined in the AFS Blue Book Fish Health Section Inspection Manual. Parasite and age data were collected from a sub-sample (n = 2-30) of each fish species from each region sampled. No target viral pathogens were detected and one bacterial pathogen was detected in a lake whitefish from Western Lake Superior. A total of7,404 parasites were recovered representing 12 genera. Prevalence of infection ranged from 50% to 90% in each host species. Results indicate that host age and condition factor do not appear to influence the presence or absence of parasite communities of coregonids in the Great Lakes. iii ACKNOWLEDMENTS I would like to thank everyone at the United States Fish and Wildlife Service, La Crosse Fish Health Center for their patience and time as I learned a new part of the Region 3 Fisheries Program and for funding my research. Thank you Becky Lasee for bringing me into your facility and sharing your knowledge and passion for health of the Great Lakes and its fish species. I have learned so much from my experience with you and your office. I would like to thank all of my committee me.mbers: Dr. Becky Lasee, Dr. Gregory Sandland, Dr. Roger Haro, and Dr. Michael Hoffman for their time and patience as we worked through this process. Your encouragement and guidance is greatly appreciated. Thank you for sharing the passion you have in your subject areas and sharing it with me. I would like to thank Dr. Gregory Sandland and Dr. Sherwin Toribio for their patience and time explaining new statistical concepts to me. I would like to thank all the collecting agencies that took the time to individually package each fish and get the samples to me quickly. These agencies include: Alpena FWCO, Michigan Department ofNatural Resources (Marquette and Charlevoix Research Stations), Ontario Department ofNatural Resources, Chippewa Ottawa Resource Authority, Mackinaw Fish Company, Wilcox Fish Company, Wisconsin Department of Natural Resources, United States Geological Survey (Ashland Office). I would like to thank my friends and family for their guidance, support, and patience throughout my college career. I want to thank God for giving me the guidance and direction that I needed as I made big decisions for school and family. Lastly, I would lV like to thank my loving wife Jade for all her support and patience throughout my college career. v TABLE OF CONTENTS PAGE viii .............. . .. LIST OF TABLES ................. ............ . ...... ...... ........... . .......... LIST OF FIGURES ... ......... .. . ................ .......... ......... .. .... .. ....... . ... . ......... .. .x LIST OF APPENDICES .... ..... .................... ... .................................. ..... .... xiii INTRODUCTION ............... ...... .. . .. .............. .... ..... .................................. . 1 Morphology and Habitat Preferences ........ . ..... ... .. . ..... . .. .. .. . .......... .. .... .... 2 Great Lakes Whitefish Status ................. ................... ... .... ................... 3 Great Lakes Bloater Status ............... .......................... ........................ .4 Coregonid Population Management Objectives ....... .... ........... . ................... 4 Great Lakes Viral Pathogens .......... . ..... ...................... ... .. .. . ...... .. ......... 5 Great Lakes Bacterial Pathogens .... ........... : ............. .... ... .. .................... 7 Great Lakes Parasite Fauna .............. . .......... ........... ............................ 8 Large-Scale Fish Parasite Sampling .... . ...... ... ... . ......................... .... :.. ... .. 9 Study Objectives ................ : ............. ..... ..... .............. . .. .. .. ... ... ......... 10 METHODS ......... . ........ .. ............... .... .. .. .... .... ............................ . .......... 11 Site Selection ............ .......... .................... ....... . . ... ..... ............ .... ... .11 Fish Collection .. ............ .............. ............... .......... .. ... ........... ....... 12 Virology ........... ...... ... ............. ...................... ........ ..... .......... ...... 14 Bacteriology .... .......... . .......... ..... ... .... .. .... ........ .. . ........................... 14 Parasitology . .. .... ................ . ..... . .. .. ........ .. ... ...... ... ....... ..... ........... . 15 Statistical Analysis .................. ...................... ...................... . .. ....... 15 Vl PAGE RESULTS ......... .. ......................................... . ........................ .. ............. 17 Bacteriology and Virology ...... . .. .. .......................... ........... .. ...... . .... ... 17 Parasitology ........................... ..... . ..... ........ .... .. ............ .... ...... .... ... 17 Lake Whitefish ......................................... . ........ . ............. . ... :22 · Round Whitefish ......... ................ ... ... ..... ....... .. .... . ................. 26 Bloater. ..... .... ..... .. ..... .............. .......... . .... .. . ... .... . .. ... . ........... 31 Parasite abundances between host species .......... ... .............. . .. ...... .36 DISCUSSION .. ............... .. ...... ............................ . . ....... ........................ . 39 REFERENCES .... ......................................... .. ........................ ............... 45 APPENDICES .................... . ............ .... . ...... . .... .. ............... ................ ..... 52 Vll LIST OF TABLES PAGE TABLE 1. Lake, sampling region, and collection dates of lake whitefish, round whitefish, and bloater collected from the Upper Great Lakes. Plus, the number of fish examined for target pathogens and the number of individuals examined for parasites with the mean length, weight, and depth of fish sampled from each region ...... ..... .... .. .............. ..... .13 2. Infection prevalence, mean intensity(± S.D.) and the range of intensities for parasites found in lake whitefish (Coregonus clupeaformis) from sampling sites in Lake Superior. ................................... ................ .. 20 3. Infection prevalence, mean intensity (±S.D.) and range of intensities for parasites found in lake whitefish from sampling sites in Lake Michigan and Lake Huron ................ ...... .................. .... .... .. ... ........ ... ...... ..... .. .21 4. Gender-based estimates of parasites and their abundances of lake whitefish using the Mann-Whitney U Test for significance ................................. ..... 26 5. Infection prevalence, mean intensity (± S.D.) and range of intensities found in round whitefish (Prosopium cylindraceum) from sampling sites in Lake Superior, Lake Michigan, and Lake Huron ...... ...................... ..28 6. Gender-based estimates of parasites and their abundances of round whitefish using the Mann-Whitney U Test for significance ................................. . ....31 7. Infection prevalence, mean intensity(± S.D.) and range of intensities found in bloater (Coregonus hoyi) from sampling sites in Lake Michigan and Lake Superior. ................................. ................................. .................... 33 Vlll PAGE TABLE 8. Gender-based estimates of parasites and their abundances of bloater using the Mann-Whitney U Test for significance ................. ..... ........... .. .......... 36 lX LIST OF FIGURES PAGE FIGURE 1. Sampling regions of A. Lake Michigan, B. Lake Huron, and C. Lake Superior using the Whitefish Management Units established by the U.S. vs. Michigan Consent Decree. Yellow rectangles indicating sampling regions within each of the three Great Lakes sampled ........... .. ............................. ............... .. .. .. . . .... . . ............ . .. 11 2. Photomicrograph of the posterior end of the nematode Cystidicolafarionis at 40x magnification. Nematode preserved in glycerin alcohol. . ... . . .. . ..... . ... ..... . ... .. .... .......... .. .. .... .. ..... ... .. .. . ... ... .... 18 3. Photomicrograph of the acanthocephalan Acanthocephalus dirus with a protracted proboscis (Semi chon's acetic-carmine stain) at 1Ox magnification .. . .. .. .. ... ....... .. . . ....... ..... .. .. .. . ..... ... ... .. .. ........ . .. .. ... ..... . 18 4. Photomicrograph ofthe cestoda Cyathocephalus truncatus scolex. (Semichon' s acetic-carmine stain) at 1Ox magnification .... .................. .. ..... . 19 5. Distribution of the number of parasite taxa infecting lake whitefish in the Great Lakes ... ... ... .......... .... . . ... .. ....... .. ...... .. . . .. . .. . .. . .. . .... ....... .. ... 23 6. Mean parasite abundance(± 1 standard error) infecting lake whitefish in Lake Michigan (LM), Lake Huron (LH), Lake Superior East (LS-E), West (LS-W), Middle (LS-M), and North (LS-N) sampling regions. A. All parasite species; B. Cystidicolafarionis; C. Cyathocephalus truncatus; D. Acanthocephalus dirus ................ .. . .. .. .. .... 24 X PAGE FIGURE 7. Fulton's K (condition factor) of lake whitefish examined for parasites from all sampling regions in the Great Lakes .................... .. . ....24 8. Age distribution of lake whitefish sampled from Lakes Michigan, Huron, and Superior. ............ .. ........... ... .. ............ .. ....... .. . ... .. ..... .. ..... 25 9. Distribution of the number of parasite taxa infecting round whitefish in the Great Lakes ............ . ... ... ..... ........ .. ................ .. .......... .......... .. 27 10. Parasite abundance(± standard error) infecting round whitefish from Lake Huron (LH), Lake Michigan (LM), and Lake Superior West (LS-W). A. All parasite species; B. Acanthocephalus dirus; C. Salmincola thymalli ............. ..... .. .... .. . ..... .. . .. ...... .... ... ... .. ....... ..... .... ... .... ...... .. 29 11. Mean Fulton's K (condition factor) of round whitefish examined for parasites from all sampling locations in the Great Lakes .......... .. ............. 30 12. Age distribution of round whitefish sampled from Lakes Michigan, Huron and Superior................. .. . .... . .. .. ................................. .......... 30 13. Distribution of the number of parasite taxa infecting bloater from Lake Michigan and Lake Superior ..... . .... ...... .............. .. ... ... .... ........... .. 32 14. Parasite abundance(± 1 standard error) infecting bloater from Lake Michigan (LM) and Lake Superior North (LS-N) and West (LS-W) sampling regions. A. All parasite species; B. Cystidicola farionis; C. Acanthocephalus dirus ...... .. 34 15. Mean Faulton's K (condition factor) of bloater examined for parasites from Lake Michigan and Lake Superior North and West sampling regions ....... 35 16. Age distribution of bloater in Lake Superior and Lake Michigan ................... 36 XI PAGE FIGURE 17. Parasite abundance(± 1 standard error) infecting lake whitefish and bloater of Lake Michigan (LM) and Lake Superior North (LS-N) and West (LS-W) sampling regions. A. All parasite species; B. Cystidicolafarionis; C. Acanthocephalus dirus ....................... . ... .. . ....... 37 18. Parasite abundance (± 1 standard error) infecting lake whitefish and round whitefish from Lake Huron (LH), Lake Michigan (LM) and Lake Superior West (LS-W) sampling regions. A. All parasite species; B. Acanthocephalus dirus ... ... .......... .. ........ . ....... ... ... .. .. . ... . ...... ..... .. ... 38 Xll LIST OF APPENDICES PAGE APPENDIX A. List of Target pathogens screened for by fish health methods ............ ....... ... .. 52 B. Species affected by viral hemoragic septicemia (VHS) federal order ... ... .......... 54 C. Inner ear bone (Otolith) for ageing before and after being cracked and burned ...... .. ..... .... .. ... .... ...... ...... ... .... ..... ......... .... .. ...... .. ........ ... 56 Xlll Introduction Great Lakes commercial fishing is a multi-million dollar industry, with whitefish being the primary source of revenue (DeBmyne et al. 2008; Pothoven and Medenjian 2008). Between the 1930s and 1950s, thousands of commercial operations targeted whitefish and lake trout (Salvelinus namaycush) in Lake Superior (Ebener 2007). Subsequent reductions in stocks led to fewer fishing operations in the region. Now, there are fewer than 200 commercial operators remaining (Ebener 2007). Due to reductions in fishing pressure, whitefish abundance in Lake Superior has increased dramatically over the past 40 years, but populations are still threatened by new diseases, invasive species, and environmental change (Ebener 2007; Wagner et al. 201 0). Fish management agencies in states and provinces surrmmding the Great Lakes are encouraged to manage each fishery to promote abundant healthy populations (Pothoven et al. 2006; Wagner et al. 2009). Due to recent changes in fish populations, feeding· grounds, and spawning habitat, fishery managers have been required to manage each species differently based on their specific biologies. This has not been an issue for managing many fish species because much is known about their morphologies, behaviors, and genetics. However, less is known about whitefish and bloaters (Coregonidae: Salmonifmmes). One of the few studies to investigate coregonid stocks found that individual lake whitefish tend to stay within 16 kilometers of their spawning reef (Smith and Oosten 1940; Dryer 1963). Movement of whitefish within these areas can be food related as they are generalist benthivores that feed on energy-rich invertebrates such as Diporeia sp. (Ihssen et al. 1981). Unfortunately, native prey species like Diporeia sp. have declined in abundance which has con-elated with a decline in the condition and growth of Lake Huron and Lake Michigan whitefish (Pothoven et al. 2001 ). Because of a decline in native prey, whitefish health in the Great Lakes is continuously assessed with the goal of monitoring responses to perturbations from pathogens, parasites, malnutrition, management actions, and environmental change (Wagner et al. 2009). The deepwater chub or bloater (Coregonus hoyi) is also a coregonid of concern in the Great Lakes. Bloaters are the smallest of the coregonids and inhabit deep pelagic waters (Brown et al. 1985). They are an impmiant ecological link in the Great Lakes food web where they primarily forage on invetiebrates such as Mysis relicta and Diporeia sp. (Moeffet 1957). Bloaters also have experienced population reductions in the Great Lakes. From the late 1800s until the 1950s, the commercial fishing industry overharvested top predators. Reductions of top predators and increased abundance of bloaters led the Great Lakes commercial fishetmen to target bloaters to boost their commercial harvest (Brown et al. 1985; Ebener et al. 2005). Commercial fishing of bloater was shmi-lived but had a negative impact on the populations. Morphology and Habitat Preferences Lake whitefish (Coregonu_s clupeiformis) and round whitefish (Prosopium cylindraceum) are found near the bottom of lakes at a depth of 15-50 meters. Lake whitefish are silver in color with a black edging around their scales, whereas the round whitefish are smaller and have different scale color patterns. 2 Bloaters are a much deeper dwelling fish species that tend to inhabit depths of 60160 meters. Unlike whitefish, bloaters are pelagic and differ morphologically. They have a terminal mouth, larger eyes, and longer fins proportional to their body size. Great Lakes Whitefish Status In lakes Michigan and Huron, harvest limitations facilitated whitefish population peaks in the early 1990s. Unfortunately, this timing corresponded with the introduction of invasive mussel species (Pothoven et al. 2001; Bence et al. 2004; Schneeberger et al. 2005). In 1989, zebra mussels (Dreissena polymorpha) were introduced and quickly established throughout the Great Lakes. A second invasive mollusk, the quagga mussel (Dreissena bugensis), followed shortly after and established in the deeper waters of the Great Lakes (Nalepa et al. 2001). After establishment of dreissenid mussels, a decline in Diporeia sp., the primary nutrient-rich food source for whitefish, was observed (Nalepa et al. 1998; DeBrunye et al. 2008). Currently Lake Superior has the greatest abundance of native prey and the lowest abundance of dreissenid mussels. This has maintained healthy coregonid populations across Lake Superior (Ebener et al. 2004). The decline in growth and condition of whitefish in lakes Michigan and Huron may have been due in part to nutritional stress (Ebener et al. 2004). With the absence of an important food source, whitefish switched prey sources from Diporeia sp. to dreissenid mussels (Pothoven et al. 2008; Tillit et al. 2009). Current research has shown that dreissenid mussels contain high levels of thiaminase which, when ingested by fish in large numbers, can lead to thiamine deficiency and affect future offspring. Thiamine deficiency is a lack of vitamin B 1 in fish eggs and fry and is called early mortality syndrome (EMS). This leads to poor development and high fry mortality in a number offish species. This condition has been 3 observed in whitefish from both Lakes Michigan and Huron (Pothoven and Nalepa 2006; Pothoven and Medenjian 2008; Tillitt et al. 2009). Great Lakes Bloater Status Commercial fishing has played an important role in supporting the economy of local ports around the Great Lakes basin, but has led to overfishing of many important species (Lawrie and Rahrer 1973; Clapp and Horns 2008). Bloater fishing began when lake trout populations declined during the early 1950s, and commercial fishing shifted focus to the increasingly abundant bloater populations. The decline oflake trout allowed bloater growth rates and habitat ranges to increase throughout the region (Smith 1964; Dryer and Beil1968). Overfishing ofbloaters in the late 1950s and 60s led to the species' decline. As a result, fisheries agencies in the 1960s had to change their management strategy from unrestricted commercial fishing to limited harvest for commercial and recreational fishing (Brown et al. 1999). These actions were designed to protect small populations of bloaters (and whitefish) throughout the Great Lakes. EMS has not been identified in Great Lakes bloater populations. Coregonid Population Management Objectives The Great Lakes Fishery Commission is an agency formed by members of academic, state, federal, and tribal fishery agencies of the United States and Canada. It is designed to protect the Great Lakes and eradicate the sea lamprey. The commission has distinct lake committees for each ofthe Great Lakes (e.g., Lake Michigan Committee) that create management objectives for the fish communities within that lake. Management objectives addressing bloater and whitefish species have been created for Lake Michigan, Lake Huron, and Lake Superior. 4 Management objectives for all coregonids include maintaining present coregonid populations while restoring certain threatened or endangered species. Maintaining selfsustaining stocks of lake whitefish, round whitefish, and bloater for commercial harvest is also goal of the commission (Busiahn 1990; DesJardine et al. 1995; Eshenroder et al. 1995). Managing important fish species is a difficult task when current populations face many challenges. Rehabilitation of native planktivores through stocking by fish hatcheries is one example of how to increase the biological integrity and diversity of planktivore populations (Busiahn 1990; DesJardine et al. 1995; Eshenroder et al. 1995). This approach has recently been combined with modeling to enhance restocking success. For example, Zimmerman and Krueger (2009) created a conceptual model on how to reestablish native deepwater fish communities. The model uses an ecosystem approach that incorporated exotic species, parasites, environmental changes and challenges, to reestablishing extirpated or depleted stocks of deepwater fish species. The model examined the individual life cycle within the context of the population, metapopulation, community, and ecosystem level processes of coregonids. In addition to overfishing and diet-related factors, micro- and macroparasites may also influence coregonid populations in the Great Lakes. Parasites can influence the foraging ability, spawning success, and predator avoidance oftheir host. Factors influencing parasite communities that establish within these species are fish habitat, range, food preference, gender, size, and age (Wagner et al. 2010). Great Lakes Viral Pathogens Viral pathogens have been shown to cause large die offs of fish in water bodies around the world. In the Great Lakes, the loss of important commercial fish species to a 5 viral pathogen could be devastating to local economies. Viral pathogens of recent concern to Great Lakes biologist include Viral Hemorrhagic Septicemia (VHSv), Spring Viremia of Carp Virus (SVCV), and Largemouth Bass Virus (LMBV). These viruses have been detected within the Great Lakes basin and have been associated with fish kills (G-rizzle and Brunner 2003; Elsayed et al. 2006; Goodwin 2011). SVCV and LMBV both tend to infect a narrow range of hosts (Grizzle and Brunner 2003; Goodwin 2011) whereas VHSV has broad host specificity (Grizzle and Brunner 2003). Viral Hemorrhagic Septicemia (VHS) was originally reported to cause high mortality rates in rainbow trout (Oncorhyncus mykiss) and other salmonids in Europe (Elsayed et al. 2006; Bergmann and Fichtner 2008). The first outbreak in the Great Lakes was recorded in muskellunge (Esox musquinongy) in southern Lake St. Clair in 2003, but was not identified as VHS until2005. Since then, it has been identified in 28 different fish species and has caused large fish kills around the Great Lakes region (Appendix B). It is unknown how VHS was introduced into the Great Lakes or how long it has been present. The genotype of VHS found in the Great Lakes is VHS IVb has been shown to be a different genotype than those from other parts of the country, such as the West Coast strain of VHS IVa that infects marine fish species (Hope et al. 201 0). Fish infected with VHS may appear to be normal or may exhibit some external clinical signs of disease such as bulging eyes, bloated abdomens, inactive or over active behavior, and hemorrhages in the eyes, skin, gills, and at the base of the fins (http://www.aphis.usda.gov/animal_healthl emergingissues/downloads/vhsgreatlakes.pdf). Some fish will have lesions that can resemble other viral pathogens, which makes it important to test the fish to confirm the presence ofVHS. Recently, VHS has been confirmed in lake whitefish in the Great 6 Lakes (http://www.aphis.usda.gov/animal_healthlemergingissues/ downloads/vhsgreatlakes.pdf). Due to the importance oflake whitefish for commercial fishing it is necessary to monitor the spread ofVHSV to other species and regions. Great Lakes Bacterial Pathogens Bacterial Kidney Disease (BKD) is a serious, and often fatal, disease among wild and hatchery reared salmonids worldwide (Fryer and Sanders 1981 ). BKD is caused by infections of Renibacterium salmoninarum, which appear to become virulent when fish are stressed or experience a drastic environmental change. Renibacterium salmoninarum has been found in whitefish and bloaters from Lakes Michigan and Huron by serological and molecular assays (Jonas et al. 2002; Faisal et al. 2010a). Feeding and swimming behaviors of infected juveniles may become erratic, making them more susceptible to predation (Mesa et al. 1998). Infected adults may lack clinical signs ofthe disease and may feed and behave normally. Over time, destruction of kidney tissue by the bacteria leads to mortality in these adults. Recent research has shown a higher prevalence of BKD in coregonids sampled from the Great Lakes in 2009 compared to previous sampling (Faisal et al. 2010a). Faisal et al. (2010a) sampled 1,284lake whitefish in northern Lakes Huron and Michigan over a three-year period; 62.31% of the whitefish tested positive for R. salmoninarum. The high prevalence of BKD may indicate that the fish are stressed which could result in lower reproductive success (Ebener et al. 2004). BKD is less prevalent in Lake Superior compared to Lakes Michigan and Huron (Faisal et al 201 Oa). Another group of bacterial pathogens that causes fish disease are the motile Aeromonas species. Motile aeromonad species cause Motile Aeromonad Septicemia 7 (MAS). MAS can cause an aiTay of disease signs in fish such as lesions, gastroenteritis, and extra-intestinal infections. MAS often is associated with stressors such as spawning, handling, temperature fluctuations, and poor nutrition (Austin and Adams 1996; Aoki 1999). MAS could pose problems to a fish population that is already experiencing a decline in growth and condition (Pothoven et al. 2001). Loch and Faisal (2010) sampled 443 lake whitefish from Lakes Huron and Michigan over a 11 month span and 63 tested positive for Aeromonas species. MAS is significant because of the economic losses it has caused in salmonids worldwide (Austin and Austin 2007). Many of the salmonids introduced into the Great Lakes are known to be infected with A. salmonicida (Cipriano and Bullock 2001) and they may serve as a source of infection for lake whitefish. In addition, Wagner et al. (2009) reported that bacterial infections might be a secondary infection caused by the swim bladder nematode Cystidicolafarionis (Wagner et al. 2009; Faisal et al2010a). Great Lakes Parasite Fauna Coregonids are distributed throughout North America with species overlap among lakes (Hanzelova and Scholz 1999; Baldwin and Goater 2003). In inter-connected lakes of Alberta, Canada, lake whitefish were shown to have higher parasite diversity than any other fish species sampled (Baldwin and Goater 2003). Karvonen and Valtonen (2004) examined parasite similarities in whitefish of interconnected lakes in Finland and found whitefish to have similiar parasite species across lakes. However, in the Great Lakes little is known about the parasite communities of coregonids. Understanding parasite species overlap among these hosts could allow researchers to also understand the spread of secondary bacterial and/or viral pathogens. Cystidicola farionis is one parasite species 8 that has been documented to overlap in many coregonids found in Lakes Michigan, Huron, and Superior (Sutherland et al. 1985; Faisal et al. 2010b). Distribution of parasites across fish species and bodies of water can be altered with the introduction of exotic species (Matitsky et al. 201 0). An invasive parasite species introduced into the United States is the Asian fish tapeworm (Bothriocephalus acheilognathi). It was most likely brought into North America in 1975 with the importation of Asian carp species (Hoffman 1999). To date, it has been reported in the Great Lakes region but not in any coregonid species in the Great Lakes. Large-Scale Fish Parasite Sampling Historically, fish parasite surveys have only examined one host species from one sampling location or a single body of water. Few studies have assessed multiple species from different sites (Muzzall1995). In rare instances where multiple sites and lakes were taken into account, sampling was not consistent across lakes or season, and focused on specific parasite taxa (Bangham 1955, Muzzall et al. 1995). For example, Karvonen and Valtonen (2004) examined the influence of geographical separation on helminth infections of whitefish in Finland, but only examined one parasite. Cone et al. (2004) assessed spatial dynamics of parasites in spottail shiner (Notropis hudsonius) from the Great Lakes, but studied the myxozoan fauna. There is a need for large multi-lake, site, and host species assessments to fill in the information gap for parasite distributions across multiple species within the Great Lakes (Brenden et al. 2010). This information would not only be useful for monitoring parasite distribution over time, but also to monitor parasite community changes across species of fish. 9 Study Objectives Research examining and comparing the parasite community structure of multiple fish species across multiple areas of each upper Great Lake (Lake Michigan, Lake Huron, Lake Superior) are lacking. Specifically, there has been little or no comparative work on the parasite-communit{' structure of whitefish and bloater populations. The four main objectives of this study are to: 1) screen for viral or bacterial pathogens that may be present in coregonids (lake whitefish, round whitefish and bloaters); 2) examine and compare the parasite communities from coregonids in Lake Michigan, Lake Huron, and Lake Superior; 3) determine differences in parasite community structure among fish species, sampling sites and lakes; and 4) investigate the influence of age and gender on parasite abundance and composition. Lake whitefish from Lake Superior would probably have the greatest diversity of parasites because their native prey is more abundant than Lakes Michigan and Huron. Furthermore, I predicted that fish from Lake Michigan and Lake Huron would have similar parasite communities due to declining prey abundance and close proximity of sampled spawning reefs. My research findings can be used as a tool for future comparisons of coregonid parasite communities that aid in identifying spatial and temporal differences. 10 Methods Site Selection Collection sites were selected using the Whitefish Management Units (Figure 1) from the U.S. vs. Michigan Consent Decree of 1836. Based on this document, Lake Michigan was separated into northern, middle and southern sampling regions (Figure 1a). Lake Huron was divided into a northern and middle sampling region (Figure 1b), and Lake Superior divided into four sampling regions (western, middle, eastern, and northern zones) (Figure 1c). +_.. ..._ . .... _..-=i _ _ _ ....... _.__..,_ ...., FIGURE 1. Sampling regions of A. Lake Michigan, B. Lake Huron, and C. Lake Superior. Regions were determined using the Whitefish Management Units established by the U.S. vs. Michigan Consent Decree. Yellow rectangles indicate sampling regions within each of the three Great Lakes sampled. 11 Fish Collection Up to 60 whitefish and bloaters were collected from each of the 9 sites by trawling, gill netting, and/or trap netting from late July to late November 2009 (Table 1). Collections were made with assistance from the United States Fish and Wildlife Service, United States Geological Service, Michigan Department ofNatural Resources, Wisconsin Department ofNatural Resources, Chippewa Ottawa Resource Authority, and Ontario Ministry ofNatural Resources. All fish were held on ice until processing, and when possible, fi~h were examined within 48 h of collection to prevent breakdown of parasites and reductions of viral titers. In the laboratory, each fish was measured for total length (rnrn), weighed (g), and then opened with a longitudinal incision along the abdomen to note gender and maturity. After the initial incision, the viral and bacterial samples were acquired as noted below. 12 TABLE 1. Lake, sampling region, and collection dates of lake whitefish, round whitefish, and bloater collected from the Upper Great Lakes. Plus, the number offish examined for target pathogens and the number of individuals examined for parasites with the mean length, weight, and depth of fish sampled from each region. Depth(m) Mean Weight (g .±SD) Mean Length (mm .±SD) No. of Fish Examined for Date Collected Location Target Pathogens Lake Whitefish Lake Michigan North Middle Lake Huron North Lake Superior North West Middle East Parasites - October 2009 November 2009 20 31 0 31 479.4 ±36.2 514.9 ±35.5 1463.1 ±317.5 60-100 50-100 October 2009 40 30 487.3 ±18.5 1015.4 ±134.3 80-100 November 2009 July 2009 August 2009 October 2009 9 35 32 36 9 21 32 30 388.8 ±74.3 385.1 ±123.2 381.5 ±184.7 520.8 ±44.7 558.9 ±329.4 677.5 ±482.9 516.3 ±330.8 1319.3 ±368.3 50-100 50-100 105-200 60-100 August 2009 23 23 434.0 ±20.5 905.0 ±176.4 60-100 August 2009 32 32 387.8 ±34.7 483.3 ±144.7 100 July 2009 August 2009 17 2 17 2 246.7 ±150.3 354.5 ±17.7 263.2 ±280.7 342.5 ±88.4 20-50 115-200 November 2009 60 30 249.1 ±23.0 164.3 ±41.1 288-318· November 2009 July 2009 Totals 30 27 30 22 212.3 ±16.7 228.0 ±45.5 96.2 .±22.5 80.8 ±48.4 250-300 100-200 394 309 ~ w Round Whitefish Lake Michigan Middle Lake Huron Middle Lake Superior West Middle Bloater Lake Michigan South Lake Superior North West Virology Virology assays were conducted on individual fish or pooled samples of up to five fish. Approximately 0.2 g ofkidney and spleen tissues was removed and diluted 1:10 in Hank's Balanced Salt Solution (HBSS). Samples were homogenized with a stomacher (Stomacher 80 Biomaster-Seward) and centrifuged at 2,800 rpm for 5 minutes at a temperature of 4°C. Once centrifuged, samples were diluted 1:1 (1ml of sample to 1ml HBSS) and incubated at either 4°C overnight or at 15°C for 2 hours, and centrifuged at 2,800 rpm for 5 minutes to pellet any large material left in the sample. Duplicate wells of Bluegill Fry Cells (BF -2), Epithelioma Papulosum Cyprini (EPC), and Chinook Salmon Embryo cells (CHSE) in 24-well cell culture plates were inoculated with 0.2 ml of sample per well. The EPC and BF -2 are acceptable cell lines that facilitate growth of such vimses such as VHSV and SVCV (USFWS and AFS-FHS 2010; Hope et al. 2011). The CHSE cell line facilitates growth of vimses such as Infectious Pancreatic Necrosis (IPNV) and VHSV. The inoculated EPC and CHSE cell lines were incubated at 15°C and BF-2 cells at 30°C for 14 days. Culture plates were examined twice per week for cytopathic effect (CPE) caused by target pathogens (Appendix A) (USFWS and AFSFHS 2010). Bacteriology The kidney of each whitefish and bloater was stabbed with a 1 ~1 sterile disposable loop and inoculated onto a brain-herui infusion agar (BHIA) slant tube. The samples were then incubated for 5 to 7 days at 20°C. After incubation, bacteria was removed from the original tube and streaked onto a BHIA plate to isolate individual bacterial colonies. Isolated colonies were then identified following the procedures 14 outlined in the USFWS and AFS-FHS (2010). Kidney tissue samples were also collected for Enzyme-linked Immunosorbent Assay (ELISA) analysis for the presence of Renibacterium salmoninarum following the sampling protocol outlined in the National Wild Fish Health Survey Laboratory Procedures Manual (http://www.fws.gov/wildfishsurvey/manuall NWFHS _Lab_ Manual_5.0_ Editiqn.pdf). Parasitology When possible, fish were examined immediately after collection. However, when this was not possible, fish were flash frozen by pouring super cooled ethanol (-80°C) over the whole fish and stored in a freezer (-20°C) for later necropsy. Whitefish and bloaters were examined for external and internal parasites using a stereomicroscope. Parasites were removed from the fins, gills, stomach, gastro-intestinal tract, pyloric caecae, eyes, and swim bladder. Nematodes and copepods were preserved in glycerin alcohol for later identification. All other parasites were stored in Alcohol-fonnol-acetic Fixative (AF A). Staining and identification of parasites was completed by following the steps outlined by Daily (1996), Hoffman (1999) and Beverly-Burton (1984). While performing the necropsies, the otoliths were removed and stored in a coin envelope. Otoliths were aged using the crack and bum method (Claramunt 2005). Using the otolith for aging has been proven to be more accurate than using other hard structures such as fin rays and scales (Isermann et al. 2003). Statistical Analysis For all three fish species, parasite prevalence (number of infected fish/ number examined x 100), mean intensity (average number of parasites/ number of infected fish), range (minimum-maximum), and mean abundance (average number of parasites/ all hosts 15 examined) were calculated across all three lakes (Margolis et al. 1982). SPSS (Statistical Package for Social Sciences) software was used for statistical analysis of parasite data using non-parametric tests. The Mann-Whitney U test was used to test gender differences and parasite abundance difference between two sites. Pearsons Correlation Coefficient was used to examine the relationship between age and parasite abundance across fish hosts. Lastly, the Kruskal-Wallis Tests were used to compare fish from multiple sites and to compare multiple fish and parasite species at a single time point. All tests were run using an alpha of 0.05 and results were considered significant at P < 0.05. 16 Results Bacteriology and Virology No target viral pathogens were detected in the 394 whitefish and bloaters examined from Lake Michigan, Lake Huron, and Lake Superior. Only one sample tested positive for Renibacterium salmoninarum in western Lake Superior. Target bacterial pathogens were not detected in any other samples from any of the lakes sampled. Parasitology Of the 309 fish examined for parasites, 7,404 parasites were recovered, representing 11 genera. Parasites collected represented the following taxa: Crustacea (Sa/mineola sp.), Monogenea (Discocotyle sagittata), Digenea (Diplostomum spathaceum), Nematoda (Cystidicolafarionis (Figure 2) Acanthocephala (Metechinorhynchus sp., Neochinorhynchus sp., Acanthocephalus dirus (Fig. 3), Cestoda (Cyathocephalus truncatus (Figure 4), larval and adult Eubothrium salvelini, and Proteocephalus exiguus), Myxozoa (Henneguya zschokkei) (Tables 2,3,5,7). None ofthe parasites identified were considered invasive species. 17 FIGURE 2. Photomicrograph ofthe posterior end of the nematode Cystidicolafarionis. Nematode preserved in glycerin alcohol. FIGURE 3. Photomicrograph ofthe acanthocephalanAcanthocephalus dirus with a protracted proboscis (Semichon's acetic-carmine stain). 18 FIGURE 4. Photomicrograph of the cestode Cyathocephalus truncatus scolex. (Semichon's acetic-carmine stain). 19 TABLE 2. Infection prevalence(%), mean intensity(± S.D.) and the range of intensities for parasites found in lake whitefish (Coregon.us clupeaformis) from sampling sites in Lake Supe1ior. N 0 Parasite Protozoa Henneguya sp. Mongenea Disc:ocotyle sagittata Digenea Diplostomum spathacewn Cestoda Cyathocephalus truncatus Eubothrium salvelini Proteocephalus exiguus Proteocephalus sp." Acanthocephala Acanthocephalus dirus Metechinorhynchus salmonis Neochinorhynchus tumidus salmon is rutlii Nematoda Cystidicola farionis North (n=9) West (n=21) Middle (n=32) East (n=30) 31.3,3 ±.2.1 (1-9) 30.0,2.7 ±.1.4 (1-7) 3.1,4 ±.0.7 (1-4) 30.0,2.3 ±.1.2 (1-6) 62.5, - b(3-tntc) 3.1,8 ±._undc (8) 56.3,14.1 ±.)2.5(1-50) 10.0,9.5 ±.4.9 (1-29) 4. 76,8±. undc (8) 11.1, 1 ±.undc (1) 9.5,4 ±.undc (4) 44.4,120.3 ±134.0 (4-408) 14.3,6.7 ±2.8 (3-12) 9.5,2 ±. undc (2) 55.6,7.6 ±.8.2 (6-25) 100,12.2 ±.11.2 (2-35) 44.4,21.8 ±16.8 (4-41) 33.3,10 ±.6.1 (4-18) 4.8,2 ±.0.4 (1-2) 4.8,1 ±.und' (1) 9 .5,4 ±.1.5 ( 1-7) 4.8,6 ±. undc (6) 4.8,3 8 ±. undc (3 8) " Larval parasite n Could not be determined because parasites were too numerous to count (tntc ). 37.5,18.3 ±.21.0 (2-115) 25.0,5.25 ±.3.0 (3-13) 22 .2,36.3 ±.6.6 (1-27) 8.3, - b(l-tntc) 44.4,23.6±.19.0 (1-80) 15.6,7.6 ±.3.4 (2-13) 3.0,3 ±.0.6 (2-3) 3 .1, 118 ±. undc ( 118) 33.3,2.5 ±.1.6 (1-6) ~ Mathematical operation cannot be defined. (und =undefined) TABLE 3. Infection prevalence(%), mean intensity (±S.D.) and range of intensities for parasites found in lake whitefish from sampling sites in Lake Michigan and Lake Huron. Parasite Digenea Diplostomum spathaceum Lake Michigan (n=31) Lake Huron (n=30) 22.6,1.4 ±0.7 (1-3) 10.0,2.5 ±0.7 (1-4) 9.7,10 ±3.7 (5-20) 29.0,- "(1-tntc) 12.5,111.2 .±57.6 (39-306) 85.0,13.6 .±14.8 (1-70) 5.0,70 ±11.1 (2-70) 25.8,1.9 +1.0 (1-4) 70.0,18.1_±19 (1-95) b 5.0,1 + und (1) b 15.0,2 ±und (2) Cestoda N Cyathocephalus truncatus Proteocephalus exiguus Proteocephalus .sp. Acanthocephala Acanthocepha/us dirus Metechinorhynchus salmonis Neoch inorhynchus tumidus Nematoda 22.6,2 +1.2 (1-6) "Could not be determined because parasites were too numerous to count (tntc) b Mathematical operation cannot be defined. (und =undefined) Cystidicolafarionis 40.0,7.2 ±6.7 (1-39) Lake Whitefish.- Lake whitefish were infected with 0-6 parasite species, with 72% offish being infected with at least one parasite species across all lakes (Fig. 5). Cystidicolafarionis, A. dirus, C. truncatus, and P. exiguus were the more common parasite species recovered. Lake Superior had >75% offish infected with one or more parasite species. Whereas, Lake Michigan and Lake Huron had < 70% of fish infected with one or more parasite species. Common parasite species such as C. farionis had a mean prevalence of 22%, with a mean intensity of 8.5 parasites/fish and a range of 0-118 parasites across Lake Superior (Fig. 2). Lake Superior-East had the highest prevalence of C. farionis (33%), whereas Lake Superior-Middle had the lowest prevalence (3.1 %) (Table 2). Lake Huron C. farionis prevalence ( 40%) was much greater than Lake Michigan C. farionis (22.6%) (Table 3). A second common parasite, Acanthocephalus dirus had a mean prevalence of36%, with a mean intensity of 16.7 parasites per fish and a range of 1-115. In Lake SuperiorNorth A. dirus had a prevalence of 100%, which is more than double the second highest prevalence found in Lake Superior-East (44.4%) (Table 2). Prevalence ofLake Huron A. dirus was 70.0%, which is greater than Lake Michigan at 25.8% (Table 3). Cyathocepalus truncatus had the highest prevalence (62.5%) in Lake Superior-Middle (Tables 2). Lake Michigan had the lowest prevalence of C. truncatus (9.7%). 22 18 ISJLake Huron 16 D Lake Michigan 12 Lake Superior-North 14 12 .....= ~ ~ II-< 10 Q 8 z 6 4 2 0 . • Lake Superior-East ~ ~ ~~ 0 raLake Superior-Middle ~ ~ rl} DLake Superior-West § ~ ~ ~ ~ ~ ~~ ~ ~ ~ ~ ~ ~ § ~ ~ ~ ~ 0 ~ n ~ 2 l ~ ~~ 3 ~ ~ = ~ ~ ~ ~ ~~ ~ ~~ == ~ 4 § ~~ 5 ~~ 6 No. of Parasite Species FIGURE 5. Distribution ofthe number of parasite species infecting lake whitefish in the Great Lakes. Total parasite abundance in lake whitefish was significantly different in Lake Superior, Lake Michigan and Lake Huron (Kruskal-Wallis Test, df=2, p = 0.001) (Fig. 6a). Of the parasites recovered from lake whitefish, only P. exiguus had the same distribution across the three lakes sampled. The dominant parasite species C. farionis, C. truncatus, and A. dirus differed significantly in abundance among lakes (All KruskalWallis Test, df= 2, p < 0.001) (Fig. 6b,c,d). Cystidicolafarionis had the lowest mean abundance in Lake Michigan fish and Lake Huron had the highest overall abundance (Fig. 6b). Cyathocephalus truncatus had the greatest abundance in Lake Superior, while Lake Michigan had the lowest abundance (Fig. 6c ). Lake Superior and Lake Huron had similar abundances of A. dirus while Lake Michigan had the lowest abundance (Fig. 6d). The condition factor of lake whitefish, which could be influenced by parasite infection 23 was also compared across the three lakes and was not significantly different (KruskalWallis Test, df= 2, p > 0.05) (Fig. 7). 50 45 40 ·~ "'a 4., '0 ci z .,"'c ::E 4 B 3.5 3 35 30 25 20 2.5 2 1.5 IS 10 5 0.5 0 0 70 12 c D 60 10 50 8 40 6 JO 4 20 2 10 0 0 LM LII LII LS LM LS FIGURE 6. Mean parasite abundance(± 1 standard error) infecting lake whitefish in Lake Huron (LH), Lake Michigan (LM), and Lake Superior (LS) sampling regions. A. All parasite species; B. Cystidicolafarionis; C. Cyathocephalus truncatus; D. Acanthocephalus dirus. L2 ::..:: "" ·~:: 0.8 Q ;:: :I 011 '"1:011"' .. 0.6 0.4 :E 0.2 0 Lll LM LS FIGURE 7. Fulton's K (condition factor) oflake whitefish examined for parasites from all Lakes. 24 Whitefish movement has been documented to be within 40 miles of spawning reefs (Dryer 1963). The two sampling regions, Lake Superior-East and Lake Huron are within that range. Total parasite abundance of lake whitefish between the sampling regions was not significantly different (Mann-Whitney U Test, df= 1, p = 0.37). However, individual parasite abundance of C. farionis between Lake Huron and Lake Superior-East was significantly different (Mann-Whitney U Test, df= 1, p = 0.048). Lake whitefish ranged in age from 2 to 31 years across lakes (Fig. 8). Lake whitefish parasite abundance had no correlation with age fo~ any parasite species identified (Pearson Correlation Coefficient, r < 0.25). Parasite abundance was examined in relation to host age at different sampling points in Lake Superior because a sufficient range offish sizes were collected (and they represented the necessary year classes). There was no correlation between host age and parasite abundance for the North, Middle and East sampling regions of Lake Superior (Pearson Correlation Coefficient, r < 0.30). There was a weak negative relationship for parasite abundance and host age from Lake Superior-West (Pearson Correlation Coefficient, r = -0.603). 10 9 ....-= <ll ~ ._ 8 7 - 6 5 = 0 4 z 3 2 I 0 - I' I II -.- I 0 1 2 3 4 56 7 8 9101112131415161718192 0212223242526272829303 1 Age (years) FIGURE 8. Age distribution of lake whitefish sampled from Lakes Michigan, Huron, and Superior. 25 Lake whitefish pooled across all lakes showed no significant difference in overall parasite abundance between host sexes (Table 4). Abundance of C. farionis was significantly lower in male than in female lake whitefish. Abundance of C. truncatus was also significantly different between sexes of lake whitefish. Males had a higher abundance of C. truncatus than females. Acanthocephalus dirus and P. exiguus had similar parasite abundances between sexes of lake whitefish pooled across all lakes. Lake Huron samples only included females, therefore a comparison between male and female fish was not possible. TABLE 4. Gender-based estimates of parasites and their abundances oflake whitefish using Lhe _Mann-Whitney U Test for significance. Abundance Sample Size Parasite Species Male/Female Male (±SD) Female (±SD) p-value 0.396 All Parasites 68/89 31.5 (±3.2) 21.8 (+80.5) Cystidicola farionis 68/89 0.2 (±0.8) 1.6 (±4.7) Cyathocephalus truncatus 68/89 40.3 (±29.3) Acanthocephauls dirus 68/89 7.6 (±1.5) 9.4 (±0.3) 0.979 Proteocephauls exiguus 68/89 4.5 (±7.9) 4.1 (±22.7) 0.258 10.8 (±57.1) <0.001 0.003 Round Whitefish.- The number of parasite species infecting round whitefish from Lakes Michigan, Huron and Superior ranged from 0 to 3, with >50% of the hosts infected with at least one parasite species (Fig. 9). The most common parasite species was the acanthocephalan, A. dirus. Round whitefish from Lake Superior had an 11% infection rate of at least one parasite species, whereas 60% of round whitefish from Lake Michigan 26 and 59% of Lake Huron round whitefish were infected with at least one parasite (Table 5). 20 •Lake Huron DLake Michigan u Lake Superior-West IR 16 14 -= .~ ""'~ z0 12 10 8 6 4 2 0 2 0 3 No. of Parasite Species FIGURE 9. Distribution of the number of parasite species infecting round whitefish in the Great Lakes. 27 TABLE 5. Infection prevalence(%), mean intensity(± S.D.) and range of intensities found in round whitefish (Prosopium cylindraceum) from sampling sites in Lake Superior, Lake Michigan, and Lake Huron. N 00 Lake Michigan (n=23) Parasite Protozoa Henneguya sp. Digenea 13 .0,5.3 ±2.1 (2-9) Diplostomum spathaceum Cestoda 34.8,3.8 ±2.5 (1-10) Eubothrium salvelini Proteocephalus exiguus Acanthocephala 52.2,1.3 ±0.8 (1-3) Acanthocephalus dints Crustacea Sa/mineola thymalli " Mathematical operation cannot be defined. (und =undefined) Lake Huron (n=32) Lake Superior West (n=17) Middle (n=2) 5.9,8.0±. und"(8) 18.8,0.3 ±4.3 (10-18) 3.1,0.1 ±. und" (3) 3.1,0.01 ±und" (1) 11.8,2.5 ± und" (4) 50.0,5.0 ± unda (5) 34.4,0.2 ±4.2 (1-22) 5.9,2.0 ± unda (2) 50.0,13 ±und" (13) Overall parasite abundance was not significantly different among round whitefish from Lake Michigan, Lake Huron and Lake Superior (Kruskal-Wallis Test, df= 2, p = 0.055) (Fig. lOa). However, the abundances of more common parasite species including A. dirus and S. thymalli were significantly different between lakes (All Kruskal-Wallis Test, df= 2, p < 0.004) (Fig. lOb,c). Lake Michigan round whitefish had the highest abundance of A. dirus, whereas Lake Huron had the lowest value (Fig. 1Ob). However, the greatest abundance of S. thymalli was found in fish in Lake Huron whereas fish from Lake Michigan where not infected by this parasite (Fig. 1Oc). The condition factor of round whitefish sampled was not significantly different between lakes (Kruskal-Wallis Test, df= 2, p = 0.354) (Fig. 11) 5 4.5 4 3.5 3 2.5 2 1.5 ·~., a c.. '0 0 ;z_ c: "' ~ 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0. 1 0 I 0.5 0 LH LM LS-W 2.5 B LH LM LS-W ~c-----------., 2 1.5 0.5 0 LH LM LS-W FIGURE 10. Parasite abundance (± standard error) infecting round whitefish from Lake Huron (LH), Lake Michigan (LM), and Lake Superior West (LS-W). A. All parasite species; B. Acanthocephalus dirus; C. Sa/mineola thymalli. 29 1.4 l.2 ~ ~"' .s=0.8 '; ~ =0.6 ~ ~ ~ 0.4 0.2 0 LS-W LM LH FIGURE 11. Mean Fulton's K (condition factor) (±1 standard error) of round whitefish examined for parasites from all sampling locations in the Great Lakes. Round whitefish ranged in age from 1 to 19 years (Fig. 12). Round whitefish parasite abundance was not correlated with host age using total parasite abundance, or correlated with any parasite species (all Pearson Correlation Coefficients, r < 0.20). 16 14 12 ..r:l "' ii: .... 10 0 8 c z; 6 4 ..II I 2 0 I 0 2 3 4 5 6 7 8 9 I0 II 12 13 14 15 F I 16 *"i 17 18 19 20 Age (years) FIGURE 12. Age distribution of round whitefish sampled from Lakes Michigan, Huron and Superior. Total parasite abundance was not significantly different between sexes of round whitefish across all lakes (Table 6). There was no significant difference in total parasite abundance between male and female round whitefish from either Lake Huron or Lake 30 Superior (Table 6). However, male and female fish from Lake Michigan did show a significant difference in total parasite abundance. However, female round whitefish had a greater parasite abundance than male round whitefish from Lake Michigan. No significant difference was observed in abundance ofthe most common parasite species between male and female round whitefish across all lakes. TABLE 6. Gender-based estimates of parasites and their abundances of round whitefish using the Mann-Whitney U Test for significance. Abundance Parasite Species/ Sample Size Location Male/Female Male (±SD) Female (±SD) 23/44 3.2 (±4.8) 2.7 (±4.8) 0.798 Lake Michigan 5/15 0.4(±0.5) 3.4 (±4.4) 0.39 Lake Huron 14/17 4.1 (±5.5) 3.1 (±5.8) 0.325 3.07 (±52.2) 31.5 (±77.9) All Parasites Lake Superior 4/9 p-value <0.001 Diplostomum spathaceum 23/44 1.8 (±4.7) 0.7 (±2.2) 0.340 Acanthocephalus dirus 23/44 0.3 (±0.9) 0.4 (±0.7) 0.139 Eubothrium salvellini 23/44 0 (±0) 0.7 (±1.9) 0.790 Salmincola thymalli 23/44 0.6 (±1.5) 0.8 (±3.5) 0.500 Bloater.- The number of parasite taxa in bloaters collected from Lake Michigan and Lake Superior ranged from 0-5, with 90% of fish with at least one parasite species (Table 7). Bloaters were not collected from Lake Huron because their population is considered to be extirpated in most areas. The infection rate in Lake Superior-North (81%) was lower than the Lake Superior-West (91% ). All of the bloaters from Lake Michigan were infected. The most common parasite species in Lake Michigan were C. farionis and A. dirus. Prevalence of C. farionis was 90% in Lake Michigan with a mean 31 intensity of 27.1 parasites and a range of 1-112. Lake Michigan had a lower prevalence and mean intensity (24.8 with range 0-76) of C. farionis (9%) compared to Lake Superior. Prevalence of E. salmonis was 45%, with a mean intensity of 11.6 parasites (range of 1-76) (Fig. 13). 16 • Lake Superior-North 14 DLake Superior-West 12 • Lake Michigan ~10 ~ ~ Q z 8 6 4 2 0 0 3 2 4 5 No. of Parasite Species FIGURE 13. Distribution of the number of parasite taxa infecting bloater from Lake Michigan and Lake Superior. 32 TABLE 7. Infection prevalence(%), mean intensity(± S.D.) and range of intensities found in bloater (Coregonus hoyi) from sampling sites in Lake Michigan and Lake Superior. Lake Superior w w Lake Michigan (n=30) Parasite Protozoa Henneguya sp. Mongenea Discocotyle sagittata Digenea 6.7,1 ±. und" (1) Diplostornurn spathaceum Cestoda 33 .3,2.7 ±1.8 (1-8) Cyathocephalus truncatus 13 .3,6 ±.2.9 (1-15) Eubotltrium salvelini Proteocephalus exiguus Proteocephalus sp. Acanthocephala 83.3,13.8 ±16.1 (1-76) Acanthocephalus dints Metechinorhynchus salmonis 3.3,1 ±und"(l) Neochinorhynchus tumidus Nematoda 90.0,27.1 ±.30.9 (2-112) Cystidicolafarionis Crustacea Salmineola thymali 16.7,1.2 ±0.5 (1-2) corpulentus • Mathematical operation cannot be defined. (und =undefined) North (n=30) 19.4,7.3 ±.3.5 (2-16) West (n=22) 22.7,3 ±(1-10) 6.5,1.5 ±0.4 (1-2) 19 .4,3 ±.1.7 ( 1-8) 9.1,7.5 ±2.8 (2-13) 35.5,10.5 ±6.5 (3-25) 40.9,19.9 ±.3.5 (1-11) 22.7,46 ±36.3 (3-169) 19.4,5 ±.2.3 (1 -9) 45.1,5.8 ±.3.8 (2-12) 9.7,3.3 ±.1.3 (1 -7) 27.3,8.8 ±.8.1 (1-36) 38.7,24.8 ±.21.8 (2-95) 40.9,21.3 ±.19.5 ( 10-78) 9.7,1 ± und" (1) 12.9,1 ±und" (1) 4.6, 1 :!:. und' (I) Overall parasite abundance was significantly different between Lake Superior and Lake Michigan bloaters (Mann-Whitney U Test, df= 1, p = 0.011) (Fig. 14a). Lake Michigan had a greater abundance of parasites than Lake Superior. In terms of the most common parasite, abundance of C. farionis was significantly different between lakes (Mann-Whitney U Test, df= 1, p < 0.05) (Fig. 14b). The less abundant parasite C. truncatus did not significantly differ between the two lakes (Mann-Whitney U Test, df = 1, p > 0.05). Bloater parasite abundance was also compared between two regions of Lake Superior that were close in distance. Cystidicola farionis abundance was not significantly different between the North and West sampling regions (Mann-Whitney U Test, df= 1, p = 0.935) (Fig. 14b). Moreover, the mean abundance ofCyathocephalus truncatus, P. exiguus, and A. dirus were not significantly different between sites (MannWhitney U Test, df= 1, p > 0.229). The condition factor ofbloaters between the two regions was significantly different (Mann-Whitney U Test, df= 1, p < 0.001) (Fig. 15). 50 45 35 A ....~"' 40 35 ~ j, 25 ~ 30 ..... Q 20 25 ~ 20 15 ; 15 10 ~ ~ B 30 10 5 5 0 0 LM LM LS LS FIGURE 14. Parasite abundance (± 1 standard error) infecting bloater from Lake Michigan (LM) and Lake Superior (LS). A. All parasite species; B. Cystidicolafarionis. 34 1_-1 I__:> ~ Jl -= = 0 ().X ('l ;.,.., = ('l ~ ;:: (l_h fl_~ (} __2 n LS L:'vl FIGURE 15. Mean Faulton's K (condition factor)(± 1 standard error) of bloater examined for parasites from Lake Michigan (LM) and Lake Superior (LS). There was no correlation with host age and total parasite abundance or with that of individual parasite species (Pearson Correlation Coefficient, r < 0.25). Bloater age ranged from 8 to 28 years (Fig. 16). 14 12 ..c 10 "' ~ 8 = 0 6 ...... z 4 2 II 0 I I 0 1 2 3 4 56 7 8 9101112131415161718192021222324252627282930 Age (years) FIGURE 16. Age distribution of bloater in Lake Superior and Lake Michigan. There was a significant difference in total parasite abundance between sexes in bloaters sampled in Lakes Michigan and Superior (Table 8). Female bloaters had a higher abundance of parasites than the male bloaters sampled from all lakes. Bloaters sampled in Lake Superior-North did not have significantly different parasite abundance 35 between males and females (Mann-Whitney U Test, df=l, p=0.068). In Lake SuperiorWest, parasite abundance between the two sexes was not significantly different. Lake Michigan-South bloater parasite abundance did significantly differ between sexes. Female bloater had a much greater parasite abundance compared to males. Abundance of C. farionis was significantly different between sexes in bloaters sampled from all lakes (Table 8). Conversely, C. truncatus, A. dirus, and P. exiguus had similar parasite abundances between sexes from all lakes. TABLER . Gender-based estimates of parasites and their abundance of bloater using the MannWhitney U Test for significance. Abundance Parasite Species/ Sample Size Location Male/Female Male (±SD) Female (±SO) p-value All Parasites 24/40 18.7 (±38.3) 31.5 (±32.2) 0.004 Lake Superior-W 10/12 23.8 (±51.5) 26.75 (±29.7) 0.275 Lake Superior-N 11/20 I 4.5 (±30.5) 21.H (±20.3) 0.06H Lake Michigan-S 3/27 13.0 (+4.4) 40.7 (+38.3) 0.25 Cystidicola farionis 24/40 5.7 (±19.5) 11.6 (±27.2) 0.042 Cyathocephalus truncates 24/40 0.1 (±1.1) 0.9 (±2.3) 0.328 Acathocephalus dirus 24/40 0 0 0.201 Proteocephauls exiguus 24/40 3.5 (±5.8) 2.8 (±4.0) 0.594 Parasite abundances between host species.- Bloater and lake whitefish had significantly different parasite abundances when data from Lake Michigan and Lake Superior were combined (Mann-Whitney U Test, df = 1, p = 0.002) (Fig. 17a). Bloaters had significantly higher abundances of C. farionis compared to lake whitefish (MannWhitney U Test, df= 1, p < 0.001) (Fig. 17b). 36 Parasite abundance of round whitefish, lake whitefish and bloater were compared across regions in Lake Superior because it was the only lake to have all three species present in samples. Parasite abundance was significantly different between the three species (Kruskal-Wallis Test, df= 2, p < 0.001). Lake whitefish had the greatest parasite abundance whereas round whitefish had the lowest. The abundance of dominant parasite species, such as C. farionis was significantly different between fish species (KruskalWallis Test, df= 2, p < 0.001). Bloater was found to have the greatest abundance of C. farionis and round whitefish had the lowest abundance. However, less abundant parasites such as N tumidus and N rutlii did not differ between fish species (Kruskal-Wallis Test, df= 2, p > 0.328). ....."' 2 3.5 ~ A ·;;.; ~ !. 1.5 3 ~ 2.5 0 1.5 c.. .....Q z = •LWF B BLO 2 0.5 ~ ~ ~ T 0.5 0 0 LM LS-W LS-N FIGURE 17. Parasite abundance (± 1 standard error) infecting lake whitefish (L WF) and bloater (BLO) ofLake Michigan (LM), Lake Superior West (LS-W) and North (LS-N) sampling regions. A. All parasite species; B. Cystidicolafarionis. Parasite abundance was significantly different between round whitefish and lake whitefish species across all sites (Mann-Whitney U Test, df= 1, p = 0.033) (Fig. 18a). Lake whitefish sampled had a significantly higher abundance of C. farionis, A. dirus, and P. exiguus compared to round whitefish (all Mann-Whitney U Tests, df= 1, p < 0.001). Lake Huron lake whitefish had significantly higher total parasite abundance, and a higher 37 abundance of A. dirus compared to round whitefish (Mann-Whitney U Test, df= 1, p < 0.033) (Fig. 18a,b). In Lake Michigan overall parasite abundance did not differ between the two whitefish species (Mann-Whitney U Test, df = 1, p = 0.836) (Fig. 18a). Abundance of more common parasite species found in Lake Michigan was only significantly different for C. farionis (Mann-Whitney U Test, df= 1, p = 0.016). For A. dirus, abundance was similar between species (Mann-Whitney U Test, df= 1, p = 0.101) (Fig. 18b). Overall parasite abundance did not differ between the two whitefish species in Lake Superior (Mann-Whitney U Test, df= 1, p= 0. 352) (Fig. 18a). More common parasite species were not significantly different between round whitefish and lake whitefish (Mann-Whitney U Test, df= 1, p > 0.091) (Fig. 18b). 12 A !JO ~ LWI' I ·~ ; . RWF 8 p.. '1:; 6 Q z4 = _l ~ <l.l ~ 2 0 -~ LH LM 45 40 35 30 25 20 B l 15 10 5 0 . LS-W LH LM LS-W FIGURE 18. Parasite abundance (± 1 standard error) infecting lake whitefish (L WF) and round whitefish (RWF) from Lake Huron (LH), Lake Michigan (LM) and Lake Superior West (LS-W) sampling regions. A. All parasite species; B. Acanthocephalus dirus. 38 Discussion Coregonid fish populations have been negatively impacted as the Great Lakes undergo environmental changes. Despite fewer numbers, whitefish are the most important commercially harvested fish species and a key biological indicator of health for the Great Lakes. Because of the current decline in condition factor of coregonids due to the switching from native prey to exotic prey species, I predicted to find a higher incidence of microbial pathogens than previously reported, and an altered parasite community than previously documented. No target bacterial pathogens other than Renibacterium salmoninarum were detected from the coregonids sampled and even Renibacterium salmoninarum was found at a very low incidence (one positive fish) in lake whitefish from western Lake Superior using ELISA. Moreover, the single infected whitefish did not exhibit any visible signs of bacterial kidney disease such as white granulomatous lesions in the kidney. Conversely, Faisal et al. (2010a) detected R. salmoninarum in 305 of371 (82%) lake whitefish sampled from northern Lakes Michigan and Huron between the years 2003 and 2006. One explanation for this large discrepancy in R. salmoninarum incidence between studies may lie in the different techniques employed to detect these pathogens. In the work by Faisal et al. (2010a) bacteria were cultured on Modified Kidney Disease Medium (MKDM), which is highly selective for R. salmoninarum from the kidney (White et al. 1995). Furthermore, Faisal et al. (2010a) also used q-ELISA which detects a protein secreted by the bacteria. The standard ELISA used in my work was not as sensitive as 39 those of the above study (White et al. 1995) suggesting that infected fish may have not have been detected as readily in my study. Unlike this study, Loch and Faisal (2010) foundAeromonas salmonicida in four lake whitefish from northern Lake Huron (n= 1,286 fish) during the course of three years (fall2003-summer 2006). This was a very low infection incidence (0.3%) given the number of hosts examined. The sample size (n=20) used in my work was much less than that of Loch and Faisal (2010). Because of this, I may not have detected this pathogen given its potentially low incidence in the fish population in combination with my small sample size. Even though no viral target pathogens were detected in this study, they have been reported in previous work. For example, VHS has been documented in Great Lakes lake whitefish and in lake herring (Coregonus artedi) from the Apostle Islands of Lake Superior (www.wisconsin.dnr.gov/fish/vhs). Interestingly the infected lake herring were collected in close proximity to where my Lake Superior-West samples were taken, but at different time points. Seasonal variability in the transmission of this virus may help to explain why previous work detected VHS, yet it was absent in my study. Although viral pathogens were not detected in fish from my collections, reduced condition factor and increased consumption of exotic prey species in coregonids may eventually lead to increases in the incidence of viral pathogens, such as VHS, in these Great Lakes fish (Wagner et al. 2010) Recent switches in diet could have altered the parasite species present in the three coregonid species in Lakes Michigan, Huron, and Superior. Lake Superior has a stable forage base and was found to have a higher abundance of parasites and a greater diversity 40 of parasites species. However, in Lakes Michigan and Huron, where the forage base has declined, parasite abundance and parasite species diversity is lower. The native forage base ofmacroinvertebrates (Diporeia sp.) has been reduced in Lakes Michigan and Huron, causing bottom-up limitations in the food web (Wells and Beeton 1963; Pothoven and Madenjin 2008; Strayer 2009). When native prey abundance declines, predators switch to different prey sources. Recently, coregonids have switched to consuming the exotic dreissenid mussels and spiny water fleas could explain the differences in parasite abundances between Lake Superior and Lakes Michigan and Huron (Pothoven and Madenjin 2008; Strayer 2009). This idea is also supported by patterns seen in C. farionis which was at its highest prevalence and abundance in Lake Superior. The intermediate hosts of C. farionis are the amphipods Gammarus sp. and Diporeia sp. Declines in amphipods in Lakes Huron and Michigan versus stable populations in Lake Superior (Scharold et al. 2004) could account for the differences in C. farionis infections from hosts collected from these different waterbodies. Overall prevalence of C. farionis in lake whitefish in this study varied compared to past work. For example, previous investigations of lake whitefish from Lake Superior (Sutherland et al1985; Nepszy 1988) reported relatively high prevalence values (2087%) for C. farionis relative to this study (14%). Conversely, Faisal et al. (2009; 2010b) found lower prevalence values of C. farionis in whitefish from Lakes Michigan (3 .310%) and Huron (12-33%) compared to this work (Lake Michigan- 22.6%; Lake Huron - 40%). Seasonal variation in fish sampling could account for differences in C.farionis prevalence (Faisal et al. 2010a). The relatively short sampling window employed in this study likely provided a snapshot in the overall seasonal and annual patterns exhibited by 41 this parasite species. Collecting over a longer time scale would capture more of the temporal variation in C. farionis infection dynamics potentially leading to a broader estimate of nematode infections. Thus the longer collection periods employed in previous studies (Sutherland et al. 1985) may help to explain differences between this work and previous studies. In past studies Cystidicola farionis has been identified in round whitefish; however none were present in this study. Nepszy (1988) and Sutherland et al. (1985) reported prevalences of C.farionis ranging from 7-59%. The absence of C. farionis may be due to a loss of prey species that are intermediate hosts for C. farionis or an increase in the consumption of exotic prey (Pothoven et al. 2006; Carlsson et al. 2009; Faisal et al. 2010b). Also in many sampling locations few round whitefish were collected in this study providing limited representation of parasites present. Thus larger sample sizes of round whitefish like in previous studies (Nepzy 1988; Sutherland et al. 1985) could enhance the probability of capturing this nematode. Prevalence of C. farionis in bloaters was also observed to differ between this work and previous research. For instance, C. farionis prevalence from Lake Michigan was 90% in this study compared to work by Sutherland et al. (1985) who reported a value of6.7%. In Lake Superior, Nepszy (1988) reported C.farionis prevalence at 75% compared to 40% in this study. As alluded to above, seasonal variation in sampling could account for differences in prevalence of C. farionis because coregonids have been shown to have a lower prevalence in the fall compared to other seasons (Faisal et al. 201 Oa). The lower prevalence of C. farionis in Lake Superior bloaters could be due to bloaters inhabiting deeper waters. The deep waters of the Great Lakes are limited by 42 light and food therefore reducing the number of intermediate hosts that could survive reducing the parasite distribution and exchange between deeper dwelling hosts (Wells and Beeton 1963; Scharold et al. 2004; Pothoven and Madenjin 2008). No relationship between mean parasite intensity and host age was identified for any of the host species examined in this study. This was somewhat unexpected, as correlations between parasite intensity and host age have been documented in other studies (Hanek and Fernando 1978; Watson and Dick 1979). Hanek and Fernando (1978) examined rock bass infected with three species of monogeneans and found rock bass older than four years of age have a greater parasite load than younger fish (Hanek and Fernando 1978). Coregonids are a long lived fish species which could allow for parasite load to increase with age (Dryer and Beil 1968; DeBryune 2008). However, a lack of individuals making up each age ~lass may have provided a very limited representation of parasite intensity in young versus old fish. There was little overlap of parasite species between lake and round whitefish in this study. This was surprising, given that the species overlap in feeding habits, movement, and spawning areas. Diplostomum spathaceum was one species where there was overlap, but it exhibits broad host specificity and is ubiquitous in Great Lakes fishes (Nepszy 1988). Similarly, lake whitefish and bloater shared few parasite species. Two uncommon parasites, D. sagittata and E. salmonis had similar parasite abundances in these hosts. Nepszy (1988) also reported overlap ofthese species in coregonids from the Great Lakes. Climate change and alterations in food web dynamics may create competition for habitat and prey among these species, and in turn, may disrupt parasite life cycles leading to variation in parasite distributions. 43 Prior to European settlement, the Great Lakes were maintained by a balanced diverse fish assemblage. Now, stocking of native and exotic fish species such as pacific salmon and rainbow trout.( Onchorhynchus mykiss) has resulted in predatory fish species becoming dominant in the Great Lakes. Prior to the 1930s, the planktivore: piscivore fish ratio was 16:2 (lake trout, burbot). Today the ratio is 1.3:1 (Eby et al. 2006). This trophic cascade over time could have influenced parasite dispersal and intermediate hosts across the Great Lakes due to changing behavioral patterns of native fish species in competition with exotic species (Eby et al. 2006). Parasite communities of lake whitefish, round whitefish, and bloater were different between water bodies and sampling regions in Lakes Michigan, Huron, and Superior. Hudson et al. (2006) described an ecosystem rich in parasite species to generally be a healthy community. Based on parasite analysis, the three coregonid species in Lake Superior from this study reside in what could be considered a healthy fish community and harbored a diverse fauna of parasites. As the sampling gradient went from North to South (northern Lake Superior to southern Lake Michigan), parasite diversity decreased. This could be due to habitat and food web degradation found in Lakes Michigan and Huron and stable in Lake Superior. This was the first study to examine parasite communities of coregonid species from multiple lakes and regions within the Great Lakes. Previous work has documented parasites recorded in these species; however, parasite data did not include a viral and bacterial component. 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North American Journal of Fisheries Management 29:1352-1371. 51 APPENDIX A LIST OF TARGET PATHOGENS SCREENED FOR BY FISH HEALTH METHODS Viral Target Pathogens Viral Hemorrhagic Septicemia Infectious Pancreatic Necrosis Infectious Hematopoietic Necrosis Spring Viremia of Carp Virus Largemouth Bass Virus Bacterial Target Pathogens Aeromonas spp. Edwardsiella ictaluri Edwardsiella tarda Yersinia ruckeri Renebacterium salmoninarum 53 APPENDIXB SPECIES AFFECTED BY VIRAL HEMORAGIC SEPTICEMIA (VHS) FEDERAL ORDER (http://www.aphis. usda. gov/animal_health/emergingissues/downloads/vhsgreatlakes. pdf) Black crappie Bluegill Bluntnose minnow Brown bullhead Brown trout Burbot Channel catfish Chinook salmon Emerald shiner Freshwater drum Gizzard shad Lake whitefish Largemouth bass Muskellunge Shorthead redhorse Northern pike Pumpkinseed Rainbow trout Rock bass Round goby Silver redhorse Smallmouth bass Spottail shiner Trout-Perch Walleye White bass White perch Yell ow perch Poxomis nigromaculatus Lepomis macrochirus Pimphales notatus Ictalurus nebulosus Salmo trutta Lota Iota Ictalurus punctatus Oncorhynchus tshawytscha Notropis atherinoides Aplodinotus grunniens Dorosoma cepedianum Coregonus clupeaformis Micropterus salmoides Esox musquinongy Moxostoma macrolepidotum Exos lucius Lepomis gibbosus Onchorhynchus mykiss Ambloplites rupestris Meogobius melanstomus Moxostona anisurum Micropterus dolomieu Notropis hudsonius Percopsis omiscomaycus Sander vitreus Marone chrysops Marone americana P erca jlavescens 55 APPENDIXC INNER EAR BONE (OTOLITH) FOR AGEING (BEFORE AND AFTER BEING CRAC;KED AND BURNED) BEFORE AFTER 57