PARASITE SURVEY OF FATHEAD MINNOWS, GOLDEN SHINERS, AND WHITE SUCKERS USED AS
BAIT IN WISCONSIN
By Abby M . Purdy
We recommend acceptance ofthis thesis in partial fulfillment ofthe candidate's requirements for the degree of Master of Science in Biology (Aquatic Science Concentration).
The candidate has completed the oral defense of the thesis.
~ ~~ q-J!-1/ Becky~h.D. Date Thesis Committee Co-Chairperson
Greg~ Date Th~sis Committee Co-Chairperson
cr-~ 1- ll Michael Hoffman, Ph.D. Date Thesis Committee Member
9· -~/·-11 Date Thesis Committee Member
Thesis Accepted
llbo/~11 Ropert Hoar, Ph.D. Date' , Interim Associate Vice Chancellor for Academic Affairs UNIVERSITY OF WISCONSIN-LA CROSSE
Graduate Studies
PARASITE SURVEY OF FATHEAD MINNOWS, GOLDEN SHINERS, AND WHITE SUCKERS
USED AS BAIT IN WISCONSIN
A Manuscript Style Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science in Biology
Abby M. Purdy
College of Science and Health Aquatic Science Concentration
May, 2011 ABSTRACT
Purdy, A.M. Parasite survey of fathead minnows, golden shiners, and white suckers used as bait in Wisconsin. MS in Biology, May 2011, 71 pp. (B. Lasee and G. Sandland)
The purpose of this study was to identify and describe parasites species in three Wisconsin baitfish species from five different baitfish sources. Baitfish examined were fathead minnow (Pimephales pramelas), golden shiner (Notemigonus crysoleucas), and white sucker (Catostomus commersoni) from either wild, hatchery, wholesale, import, or retail sources throughout Wisconsin and the North Central Region (NCR). A total of 350 baitfish were sampled from five sources within two states (W I and MN) and at least 20 fish of each baitfish species were necropsied. There was no statistical difference in mean prevalence amoung baitfish sources for all host species combined and for each of the baitfish species. AN OVA results demonstrated that mean abundance and mean intensities differed significantly among sources for both fathead minnow and white sucker. Two parasite species occurred, Neascus spp. and Ornithodiplostomum ptychocheilus, across all five baitfish sources. Five parasite species were observed within all three baitfish species (Hysteromorpha tri/oba, Myxobolus spp., Neascus spp., Neoechinorhynchus spp. and Spiroxys spp.). From results a list of baitfish parasite species was generated and potential transfer pathways between sources were identified. Natural reso urce management within the NCR can use the baseline knowledge to possibly generate management strategies (e.g., pathogen screening, baitfish health certificates ) aimed at recognizing baitfish/parasite transfer risks or pathogen screening protocol used prior to baitfish transfer.
iii ACKNOWLEDGEMENTS
I would like to thank my co-advisors, Dr. Becky Lasee and Dr. Gregory Sand land for sharing their enthusiasm about biological studies and their extensive knowledge of parasitology. Thank you both for all the hours you spent working with me on my thesis research and manuscript. Thank you both for the continued patience and countless hours you spent sharing your career experiences and helping me through the graduate school process. No matter my future endeavors, I know that I have professionally and personally grown through my graduate student experience.
In pa rticular, I would like to thank Dr. Lasee for providing me with a USFWS - STEP position at the La Crosse Fish Health Center. No substitution can be made for the extensive skill set I have received working for the La Crosse Fish Health Center. I am very grateful to have had this opportunity to expand my technical laboratory and field skills.
Many thanks to my graduate committee members Dr. Roger Haro and Dr. Michael
Hoffman for their time, patience, advice, and knowledge they have given and passed along to me. I have learned a great deal from you both in and out of the classroom and truly enjoyed your enthusiasm as professors. Addit ionally, I would like to thank Dr. Sherwin Toribio for the long hours he put in helping me with and guiding me through statistical analysis.
Thank you to the staff at the La Crosse Fish Health center for their support and fish health expertise. In particular, thank you Corey Puzach, Ken Phillips, and the late John Whitney for sharing with me your fish health expertise and continual their advice and support. Thank
iv you, Chris Olds, Dustin Hart, Ryan Katona, Beka McCann, Emma Waffenschmidt, and Corey
Puzach for your help with processing my samples.
I thank Genoa National Fish Hatchery, Governor Tommy G. Thompson's State Fish
Hatchery, the University of Wisconsin- Stevens Point - Northern Aquaculture Demonstration
Facility, Dave Wedan from La Crosse Fisheries Resource Office, and the Wisconsin DNR for the helping me obtain samples.
I thank my parents Lisa and William Purdy for their continuous love, support, and belief in my abilities to achieve anything I set my mind to. Special thanks to Michael Mataya and to my dear fellow graduate students keeping me grounded throughout the process. Last but not least, thank you to my professors/mentors from the University of Wisconsin- Stevens Point for their knowledge and guidance that gave me a good foundation for graduate school.
I dedicate this thesis in memory of John M. Whitney a trusted friend and co-worker at the La Crosse Fish Health Center.
v TABLE OF CONTENTS
PAGE
LIST OF TABLES ...... vii
LIST OF FIGU RE S...... viii
INTRODUCTION ...... l
Ba itfish Species Traits ...... 7
Parasites and Pathology...... 9
OBJ ECTIVES ...... 16
METHODS ...... 17
Fish Collection ...... 17
Necropsy ...... l9
Statistical Analysis ...... 19
RESULTS ...... 21
DISCUSSION ...... 44
REFERE NCES...... 53
vi LIST OF TABLES
TABLE PAGE
1. List of baitfish parasites of national concern ...... 6
2. Collection categories and collection sites ...... l8
3. Mean infection prevalence(%) within 3 baitfish species and among baitfish sources ...... 22
4. Mean infection prevalence(%) for all parasites within all five baitfish sources from white suckers (WHS), fathead minnows (FHM), and golden shiners (GOS) ...... 23
5. Mean parasite abundance, range, and ±SD for parasites found in 3 fish species (fathead minnow, white sucker, and golden shiner) collected from 5 baitfish sources (wild, import, wholesale, retail, and hatchery) ...... 27
6. Mean parasite intensity(± SD) for parasites found in three baitfish species collected from five baitfish sources ...... 30
7. Mean parasite intensity (±_SO) within ailS baitfish sou rces from white suckers (WHS) and fathead minnows (FHM) ...... 30
8. Parasite overlap in fish hosts collected from five baitfish sources ...... 35
9. Amount of overlap among parasite species within three baitfish species ...... 37
vii LIST OF FIGURES
FIGURE PAGE
1. General baitfish transfer flow diagram ...... 2
2. Mean prevalence(%) for all parasites in all fish species from 5 baitfish sources ...... 23
3. Mean prevalence(%) for all parasites in fathead minnows from five baitfish sources (n=147) ...... 24
4. Mean prevalence(%) for all parasites in white suckers from the five baitfish sources (n=153) ...... 24
5. Mean prevalence(%) of golden shiners among sources ...... 25
6. Mean prevalence(%) for all parasites from white suckers (WHS) and fathead minnows (FHM) within ailS baitfish sources ...... 25
7. Parasite mean intensity(± SO) within all fish species from five baitfish sources ...... 31
8. Parasite mean intensity C±_SO) of fathead minnows sampled from five baitfish sources (n=143) ...... 31
9. Parasite mean intensity C± SO) of w hite sucker samples from five baitfish sources (n=82) ...... 32
10. Parasite mean intensity(± SO) of golden shiner samples from two baitfish sources (n=S0) ...... 32
11. Parasite mean intensity (±_SO) for all parasites from white suckers (WHS) and fathead minnows (FHM) within all five baitfish sources ...... 33
12. Photomicrograph of Microspora spp. wet mount...... 39
13. Photomicrograph of Myxobolus spp. cysts attached to the gill lamellae of a fathead minnow ...... 39
viii LIST OF FIGURES
FIGURE PAGE
14. Wet mount photomicrographs of Myxobolus spp. diversity within white suckers and fathead minnows ...... 40
15. Photomicrograph of Monogenean; Pseudomurraytrema alabarrum stained wet mount...... 40
16. Photomicrograph of immature Digenean; Diplostomum spathaceum within eye lens of fathead minnow ...... 41
17. Photomicrograph of immature encysted Digenean; Ornithodiplostomum ptychocheilus infecting fathead minnow brain tissue ...... 41
18. Photomicrograph of immature Cestoide; Valipora spp from fathead minnow ...... 42
19. Photomicrographs of immature Nematode; Spiroxys spp. from all three baitfish species ...... 42
20. Photomicrograph of Acanthocephalan; Acanthocephalus spp. from fathead minnow and white sucker ...... 43
ix INTRODUCTION
The North American baitfish industry generates approximately 1 billion dollars in annual sa les (Litvak and Mandrak 1993; Gunderson and Tucker 2000). In Wisconsin, the latest report of annual sales was in 2005 when baitfish sa les were 3.8 million dollars.
This was an increase of almost 1.4 million dollars since the prior ce nsus in 1997 (USDA
2000, 2006). High baitfish sales in the state have resulted in high numbers of registered baitfish hatcheries (14 of 257 hatcheries nationwide), wholesale dealer permits (800 per year), and wild baitfish harvest permits (6,000 per year) (WDNR 2006, USDA 2006).
Each year the Wisconsin baitfish industry supplies 105 federal, state, tribal, and cooperative game fish hatcheries and about 1.4 million anglers with baitfish (WDNR
2006; WAA 2009). However, wholesale dealers struggle to meet local baitfish demands of hatcheries and anglers. This has led to wholesale dealers either wild-harvesting baitfish or initiating baitfish culture programs. A recent estimate showed that 39% of the baitfish so ld in the North Central Region (NCR) (WI, Ml, IL, IN, lA, KS, MN, MS, NB,
ND, SD, OH) were cu ltured (USDA 2000). Both baitfish culture and wild-harvest generate concerns because both practices may facilitate the movement of these fish and their parasites (Litvak and Mandrak 1993; Gunderson and Kinnunen 2001; Engle and Stone
2003; Chaudry et al. 2006) into novel habitats which could introduce parasites/pathogens into na·ive wild populations of baitfish or game-fish .
1 Baitfish transport, from initial collection sites to the anglers themselves often follows a common route. First, wholesale dealers transfer wild baitfish (often collected from multiple water bodies and/or watersheds) and/or cultured baitfish (including imported stock) to retail baitfish shops. Anglers then purchase, transfer, and re lease baitfish from retail shops back into the aquatic environment (i.e., bait-bucket transfers)
(Ludwig Jr. and Leitch 1996), usually at sites that are different from the original collection locales (Figure 1).
( Imported Stock)
~ I r-----"-./eofbanfish lor?I Wholesale Dealers ~ansfcr andse.t~tf.shto-•~S(• to--48
M~~l \ I '---.,----'1 I \ I . : :.:::=:1 I j I l Ha~~roe; ) I I ~sed br cuttu~t or { I I I I I I I ! \ i : '"'"'"""''"'"" j I
------releascfishbackioto ---~
Figure 1. General baitfish transfer flow diagram. Main focus is the movement of baitfish through wholesale dealers.
2 These transfer steps are often complicated by the fact that baitfish collections
from the wild often include numerous non-target (incidental catch) species. For
example, a single stream sampling for fathead minnows in Wood County, WI, reported
15 non-target species of baitfish (Becker 1983). This increases the chance that
pathogens (i.e., Plistophora ovariae) are inadvertently transferred from non-target
species to baitfish (Summerfelt and Warner 1970) or that the non-target species themselves could be transferred to new environments. For example, Ludwig and Leitch
(1996) calculated that 28.5% of baitfish sampled from retail holding tanks were non target species and that the odds of these species being transferred to different habitats via a bait-bucket was 100%.
The conditions surrounding baitfish capture and maintenance may facilitate pathogen transmission within baitfish species. First, transportation stressors such as low oxygen, crowding, lack of feeding, excessive temperatures, and the mixing of baitfish with similar non-target species (Engle and Stone 2003) may increase host susceptibility (Noga 1996). Second, temporary holding tanks and/or outdoor ponds may expose baitfish to parasitic infections via naturally infected snails and birds within these areas. Thus problems due to stress and holding facilities likely facilitate increased infection events within baitfish species, which, in turn could increase transfer of baitfish parasites among environments.
There have been numerous parasites species reported from baitfish that could be potentially transferred into novel environments and to non-target fish species
3 including gamefish (Litvak and Mandrak 1993; Gunderson and Kinnunen 2001; Engle and
Stone 2003; DATCP 2008; Whelan 2009). For example, a commonly used baitfish (golden shiners) has been recently identified as the source of a Bothriocephalus achel/ognathi
(Asian Fish Tapeworm) introductions to Peter Lake near Land o' Lakes, Wisconsin in
2001 (Choudhury et al. 2006). This pathogen species is of major concern because of its ability to infect numerous fish species and its potentially pathogenic effects on these host species. Another example, Ovipleistophora ovariaem which was introduced by golden shiners used as bait. This resulted in natural populations of golden shiner being potentially being infected (Summerfelt and Warner 1970).
Risk assessments are used by fisheries managers to assess the potential for parasite and pathogen transfer by baitfish. Factors that are incorporated into these assessments include: the number of fish being transferred, where the fish are being transferred from, what potential pathogens are harbored by the fish being transferred, and the location the fish are being transferred to (USFWS 2004). Factors are scored and then summed up for a final "risk" score. The final score identifies particular mitigation strategies and actions that best reduce the risk of pathogen transfer. High risk scores strongly suggest that fish should not be transferred due to the high potential for virus, bacteria, or parasite transmission to novel baitfish or game fish species. Ultimately, the strength of these risk assessments depends on the empirical data available for baitfish and their associated parasites/parasite communities.
4 Researchers have identified that parasites can be carried by baitfish (Litvak and
Mandrak 1993; Gunderson and Kinnunen 2001; Engle and Stone 2003; Choudry et al.
2006} and subsequently transferred to na'ive fish populations. Therefore, my work focused on regional parasite populations in three baitfish species at a single point in time. There has been little empirical work done to identify the parasites infecting
Wisconsin baitfish. Previous studies on baitfish parasite communities have either focused on a single baitfish species (Table 1) or a single parasite species. Due to this gap in our knowledge, I investigated parasite communities in three baitfish species from five difference baitfish sources. The ultimate goal of this research was to describe the parasite species within these baitfish and identify those of greatest concern. This will provide fish health specialists and fisheries managers w ith the ability to make better assessments of the risks of parasite transfer and the potential impacts of pathogen introductions within the North Central Region of the U.S.
5 Table 1. Baitfish parasites of national concern. Parasite Species
Baitflsh Micros~ora Myxozoa Monogenea Digenea Cestoidea Co pepoda Fathead minnow (Pimephales Myxobolus Doctylogyrus Bothriocephalus Ergosilus promelas) Glugeospp. angusrus bifurcates Balbapharus canfusu" acheilagnathi canfusus Pleistaphara spp. M . aureatus D. bychawskyi B. daminiftcus •• Ligula mtestinalis E. cyprinaceus Heterasparis spp. • M D. pectenatus Dip/ostomulum L. cyrinaceo hendericksanus huronense•• M haffmanus Gyrodactylus D. scheuringi • • hoffmani CTI "vv. hyborhynchus G. lacustris D. spathaceum .. Golden shiner Oviple1staphara M (Natemlgonaus D. aureus D. spathaceum .. B. acheilognathi E. cyprinaceus ova rio~ alganqumensis crysa/eucas) Heterosparis spp • M orgenteus D. parvicirrus D. carti E megaceros M . artus D. bulbus L. cyprinacea M . bilabus G. crysoleucas M . cyprini G. elegans M mortim G. kathorineri M musculi G. rachelae M . notemigoni G. variabilis M . notrop1s G. wellborni M--- >tiOOI White sucker (Castostamus cammersani) Heterosporis spp • M bi bullatum G. cammersoni B. conjusus 3. acheilognathi E caeruleus M e>tsu/arus G spathulatus D. corti 8 bilaculotdes E canjusus G. stunkardi D. fle>tcaudum' • 8 CUSpldOtus E. magaceros Dactylogyrus spp. D. spothaceum Ltgula intestine/is E. nerkoe E. vers1colar L cyrinoceo •experimentallaboratary exposures.- · Metacercanal stages. Ba itfish Species Traits
The goal of my resea rch was to provide initial assessments of the parasite communities found in a number of baitfish species acquired and sold in Wisconsin.
This study focused on three important WI baitfish species: fathead minnows
(Pimephales promelas; FHM) (WI - 11 farms and 1.6 million in total sales), golden shiners (Notemigonus crysoleucas; GOS) (WI - 6 farms and 993 thousand in total sales), and white suckers (Catostomus commersoni; WHS} (WI - 6 farms and 725 thousand in total sales). These three species rank in the top five of the most economically important baitfish within WI and the U.S. (Meronek et al. 1997a,
1997b; USDA 2006) and are the most frequently cultured baitfish species within the
U.S. (Engle and Stone 2003; USDA 2006).
Fathead Minnow
Fathead minnows, Pimephales promelas (Cyprinidae), are common throughout Wisconsin. These minnows are omnivorous opportunistic bottom feeders. Due to their feeding habits, fatheads can be cultured with little or no supplemental feeding. Equally important for rearing is the fact that fathead minnows mature within the first year of life, spawn multiple times between May and
August, and have high fecundity (12 thousand eggs/nest) (Pflienger 1975). Because of these characteristics, fathead minnows play a large economic role in the baitfish industry, not only as bait, but also as forage fish for cultured game fish. Fathead minnows frequently co-exist with other baitfish species in a wide range of habitats from clear lakes to silt-laden vegetated ditches (Becker 1983).
7 Golden Shiner
Golden shi ners, Notemigonus crysoleucas (Cyprinidae), are common throughout most of Wisconsin, with the exception of the lower southwestern portion of the state. Wild populations often occur in slow moving or stag nant waters with high vegetation and patchy sandy bottoms. Golden shiners feed primarily on zooplankton at or near the water's surface. Spawning occurs throughout the summer months (June-August). During this period, 7-10% of a mature female's body weight is made up of eggs (Becker1983). Golden shiners are extensively used as forage fish or baitfish during colder periods of t he year. Restricted use of go lden shiners during the summer is due to temperature hypersensitivity which makes golden shiners prone to injury and mortality during hauling and transport throughout the warmer months. Culturing of golden shiners has occurred in natural ponds for decades, but golden sh in er supplies still fall short of demand. Therefore, cultured and wild-harvested golden shiners are often imported into Wisconsin.
White sucker
The range of white suckers, Castostomus commersoni (Catostomidae), extends throughout lakes and streams of Wisconsin. They are often more abundant than either fathead minnows or golden shiners since they are more tolerant of poor water quality. Because of their tolerance, white suckers have been used as indicator species in many biological assessme nts (Lyons, Wang, and Simonson 1996; La ngdon
1999; Grabarkiewicz and Davis 2008). Spawning occurs during April and May just after ice -off. During these months 16% of a 400 mm female sucker's body weight is comprised of ovaries, which can harbor about 35,600 eggs (Becker 1983). White 8 suckers that are approximately 356 mm (14 inches) in length are commonly harvested as forage fish or baitfish for a variety of predatory fish (i.e., walleye
(Sander vitreus), northern pike (fsox lucius), or muskellunge (Esox masquinongy)).
The feeding strategies (bottom feeders) and spawning success of white suckers make them relatively easy to culture in a wide variety habitats, but they do best in moderately fertile and mud lined ponds (Becker 1983).
Parasites and Pathology Microspora
Although most single celled microsporidian protist infections manifest as asymptomatic lesions, some may result in high host morbidity and/or mortality especially to small hosts (Table 1). Microsporida are obligate intracellular parasites which infect multiple host organs/tissues. These histozoic parasites typically form either white- yellow nodules or large tumors. These abnormal cellular formations are filled with microsporidian spores (1.5 to 20 11m length) which can be etrimental to fish health (Roberts and Janovy 2005). Cell hypertrophy or cell enlargement can cause pressure atrophy that disrupts organ and tissue function and in creases inflammation (Ward 1912; Dykova and Lorn 1980; Ferguson 1989; Noga 1996).
Microspores, such as Ovipleistophora ovariae also infect oocytes reducing host fecundity and body condition. Microspora infections in fish hosts can result in any number of the following broad pathologies: reduced host growth, anorexia, intestinal blockage, decreased fecundity, parasitic castration, and/or muscle degradation. For example, Glugea species interfere with the host's gastro-intestinal tract or visceral connective tissues through the development of xenomas. Xenomas
9 are enlarged host cells containing multiple spores at various life stages (Noga 1996;
Phelps and Goodwin 2008). In contrast, Pleistohora ovariae (recently reclassified as
Ovipleistophora ovariae by Phelps and Goodwin 2008) does not form xenomas but instead infects, reproduces, and matures in oocytes. Portions of the infected ovaries then turn into white opaque masses which may destroy host reproductive tissues.
Typically, 0. ovariae is vertically transmitted in longer lived hosts (Dunn and Smith
2001) since virulence and mortality rates are typically low (AFS 2007). Go lden shiners infected with 0. ovariae had 50% less gonadal mass than uninfected fish after one year (Rheui-Fehlert et al. 2005). Lastly, infection by Heterosporis spp. will result in white opaque lesions that degrade muscle. Fishermen describe infected fillets as having "freezer-burn" (Miller 2009).
Myxozoa
The single-celled protists, Myxozoa, are histozoic and coelozoic parasites producing white-yellowish nodules or pseudocysts within fish hosts. Myxozoans of fish are commonly host and site specific (Ward 1912; Post 1983; Ferguson 1989;
Roberts and Janovy 2005). Worldwide there are about 1,350 myxozoan species.
Myxozoan infections can be asymptomatic and elicit relatively little host immune response (Longshaw et al.2003)or when fish become heavily infected they may suffer from serious local mechanical tissue and organ damage. Tissue pathology is often the result of intracellular spore development causing pressure atrophy, inflammation, and necrosis (Molnar and Kovac's- Gayer 1985; Ferguson 1989;
Longshaw 2003; Roberts and Janovy 2005). In addition, Cone (2005) reports that,
10 cells infected with Myxobolus bibobus may rupture and hemorrhage thus faci litating spore dispersal.
The myxozoan, Myxobolus notemigoni is common in golden shiners. It produces characteristic white spots that develop under or between the scales of infected shiners. Infection is concentrated along the ventral portion of the host and produces little pathological effect but may affect host aesthetic appearance and thus marketability. M. notemigoni has also been reported to infect large portions of pond-raised baitfish. Lewis and Summerfelt (1964) reported that 20% of 30,000 golden shiners were infected with M. notemigoni. Many other myxozoa species have been reported from the three baitfish species (Table 1).
Monogenea
The majority of monogenean flatworms are small (S. 1 mm) multicellular ectoparasites that infest the gill s, skin, and/or fins of fish . Signs of infestations are gross lesions, epidermal hyperplasia, hemorrhaging, necrosis, increased mucus production, erosion of fins, and behavioral responses such as gasping or itching
(Post 1983; Noga 1996; Roberts and Janovy 2005; AFS 2007). In wild populations, monogeneans typically exhibit host specificity, but can potentially infect other species in aquaculture settings due to high fish densities and reduced host immune system. Once transmitted, heavy infestations in fry or small baitfish species frequently result in mortality because of excess mucus production, tissue damage, deformation, and hemorrhaging of gills, skin, or fins. Dactylogyrus spp. infestations have resulted in mortality rates as high as 80-100% in carp fry populations (AFS
2007). 11 The two monogenean genera infecting baitfish are Gyrodactylus spp. and
Dactylogyrus spp., which commonly infest the skin and gills of cultured freshwater
fish (Noga 1996) (Table 1).
Digenea
Digenean trematodes are macro parasites that always possess complex life
cycles consisting of multiple hosts (i.e., invertebrate and vertebrate hosts).
Definitive hosts most often are vertebrates that eat infected fish. Fish can serve as
both intermediate and definitive hosts in the life cycles. In life cycles with fish as an
intermediate host, parasites encyst as metacercariae in external and internal organs
after migration. During migration through host tissues digeneans damage host
tissues and/or disrupt organ function. Diplostomum spathaceum cercariae migrate
to the host's eyes and transform into metacercariae where they can cause blindness
or parasitic cataracts. Heavily infected hosts have reduced feeding ability and
predator detection (Post 1983; Noga 1996). Ornithodiplostomum ptychocheilus
metacercaria encyst in the brain tissue of fathead minnows. Heavy infections of 0.
ptychocheilus have the potential to negatively affect its host and the host's
population by disrupting central nervous system functioning and modifying host
behavior therefore reducing host or host population survival (Sand land, 1999).
Metacercarial stages of invasive Bolbophorus spp. can generate
hemorrhages, necrosis (i.e., kidney), inflammation, and high mortality in channel catfish (Terhume 2002). Pathology varies depending upon host and parasite
12 species, but large mortality events of cultured fish are due to B. confusus
(Overstreet et al. 2004; Mitchell et al. 2006) (Table 1).
Cestoidea
Cestodes (or tapeworms) have complex life cycles in which fish can be either intermediate or definitive hosts. Cestodes typically reside in fish host intestinal tracts as adults and in the peritoneal cavity, liver, gonads, or muscles as larvae
(Ferguson 1989). Most infections are innocuous at low infection levels but damaging at high infections. Although many cestode species tend to be host-specific, many others can infect a wide variety of fish species. For example, Ligula intestinalis specifically infect many cyprinid, catostomid, and other species worldwide (Hoffman
1999). Ligula intestinalis damages hosts by producing peritoneal adhesions, causing castration, or generating liver, gonad, or muscle atrophy. Tissue atrophy is due to the pressure caused by larval (plerocerciod) growth and migration through or out of host tissues (Ferguson 1989; Groves and Shields 2001). Also, high-intensity infections may result in reduced host growth and fecundity (Szalai et al. 1989) or increased susceptibility to secondary infections and anemia (Post 1983; Ferguson
1989; Noga 1996).
A cestode of extreme concern is the Asian fish tapeworm, Botheriocephalus achei/ognathi. Importation of grass carp may have introduced B. acheilognathi into the U.S. (Noga 1996) (Table 1). It is w idely distributed and pathogenic to native fish within the U.S. This cestode infects a wide range of hosts (i.e. multiple minnow, shiner, and carp species) and has the potential to devastate commercial baitfish farm populations (Noga 1996; AFS 2007). Infected fish populations exhibit higher 13 mortality rates when the anterior portion of the intestinal tract is obstructed and
perforated by the enlarged adult cestode parasite. Botheriocephalus acheilognathi
mortality rates have been estimated to reach 90% in non-native grass carp
(Ctenopharyngodon idella).
Nematoda
Nematodes or round worms often live within the hosts intestinal tract or
viscera where they feed off the hosts blood, tissue, and fluid. These parasites have
a non- segmented hard cuticle and can live in a multitude of environments and hosts. Nematodes are capable of parasitizing fish at two points within its complex life cycle either as definitive or intermediate hosts (Roberts and Janovy 2005).
Those species infecting baitfish are Carnal/anus spp., Contracaecum spp.,
Raphidascaris spp., and Philometra spp.
Acanthocephala
Acanthocephala or "spiny headed worms" are distinguished by their anterior proboscis which is armed with a single or multiple rows of hooks. Adult worms infect fish intestinal tracts by attaching to intestinal walls and absorbing nutrients through body surfaces (Roberts and Janovy 2005; Hoffman 1999).
Acanthocephalans infecting baitfish are Neoechinorhynchus spp. and
Pomphorhynchus spp. (Muzzall 1980). Pathologically, acanthocephala rarely cause mortality events in fish but heavy infections may causes inflammation, ulceration, and possibly necrosis {Hoffman 1999).
14 Copepoda
Adult female Copepoda (i.g., Ergasi/us or Lernaea) are ectoparasites which infest cultured or wild fish hosts populations by penetrating and attaching to host gills, skin, fins, or muscles. Localized attachment sites cause damage such as erosion, ulceration, scale lifting, hemorrhaging, inflammation, and/or secondary infections (Post 1989; Noga 1996; Roberts and Janovy 2005; Lester and Hayward
2006; AFS 2007). Vital organs are damaged when t he anterior anchors of Lernaea spp. (the anchor worm) penetrate host body walls (Khalifa and Post 1976). Tissues in/on eyes, nostrils, and/or cranium are often infested with Lernaea spp. In these tissues, the anchors can penetrate deeper into the host and damage vital organs especially within small bodied fish hosts (such as baitfish). Because of this, mortality is often higher due to damage done to vital organs (such as the brain or visceral organs) (Khalifa and Post 1976; AFS 2007). Adult female Ergasilus spp. grasp and/or anchor to host gills, skin, muscle, or between scales. Gill damage can often result in host mortality because of changes in respiratory functions by parasitic attachment and grazing (Lester and Hayward 2006). Copepod grazing also increases t he risk of secondary infections and host mortality (Roberts and Janovy 2005). Overall, cope pod infestations are of concern to fish culturists because of the high rate of fish bodily disfigurement consequently decreasing their economic value (Table 1).
15 OBJECTI VES
My research objectives are to: 1} provide baseline descriptions of parasite communities that occur in three important Wisconsin baitfish species (fathead minnows (Pimephales promelas), go lden shiners (Notemigonus crysoleucas}, and white suckers (Catostomus commersoni}. 2} statistically determine if parasite community prevalence, mean abundance, mean intensity, and range differ within/between baitfish species and sources (wild populations, hatchery populations, retail businesses, wholesale facilities, and imports), and 3) generate additional parasite lists for baitfish which can be used as baselines for risk assessment and fisheries management.
These objectives will allow me to test two hypotheses:
Hypothesis 1: Baitfish from wholesale dealers will have greater parasite diversity than baitfish from other sources. Wholesale dealers will have greater parasite diversity because baitfish are mixed and received from multiple sources
(i.e. wild and imported fish).
Hypothesis 2: Fathead minnows will have higher diversity than golden shiners or white suckers. The abundance of individual parasite species will be higher in fathead minnows due to parasite specificity, source type, and/or minnow life history traits.
16 METHODS Fish Collection
Between 2009 and 2011, approximately twenty individuals of each baitfish species (fathead minnow, golden shiner, and white sucker) were collected from five sources within Wisconsin. The five source categories were: wild populations,
hatcheries, retail businesses (baitfish shops), wholesale facilities, and imported stock. For each source at least two baitfish species were sam pled from different
locations (Table 2). Golden shiners could not be assessed in all source categories since the sampled wholesale facilities obtain golden shiner stock from outside of the
NCR.
After collection, fish were transported to the USFWS- La Crosse Fish Health
Center, Onalaska, WI for necropsy. Euthanasia occurred either on site or at the Fish
Health Center using an overdose of Finquel MS-222 (Tricaine Methanesulfonate).
Total length (mm), standard length (mm), weight (g), and sex was recorded for each fish after euthanization and prior to final necropsy. Fish were frozen using super cooled (-80°C) 100% ethanol and stored in chest freezers ( -20 °C) until necropsy
(Bush and Holmes1986).
17 Table 2. Species collection sites, number sampled, and source category (n=350).
Number Category Species Site Sampled Hatchery FHM Genoa NFH*, WI 20 Hatchery WHS Governor Tommy T. Thompson SFH **, WI 20 Hatchery WHS Northern Aquaculture Demonstration Faci lity, WI 20
Retail FHM Gander Mountain, Onalaska, WI 20 Retail GOS Bob's Bait & Tackle, La Crosse, WI 20 Retail WHS Gander Mountain, Onalaska, WI 20
Wholesale 20 Import FHM Ken's Bait, MN Wholesale 20/20 Import WHS/FHM Rockies Bait, MN Wholesale 20 Import WHS Ben's Bait, MN
Wholesale WHS/FHM Friesse's Minnow Farm Inc., WI 20/20 Wholesale WHS/FHM Hayward Bait & Tackle Inc., WI 20/20
Wi ld FHM Pheasant Branch, WI 20 Wild GOS Black River Falls- Flowage 17, WI 30 Wild WHS Bad Axe River - North Fork, WI 20
18 Necropsy
Internal and external parasite screening were done following protocols outlined in the Laboratory Procedures Manual for the National Fish Health Survey
(4 ed., September 2006, online at http://www.fws.gov/wildfishealthsurvey) and the American Fisheries Society- Fish Health Section blue book (AFS 2007). A stereomicroscope was used to systematically examine all external surfaces (skin, fins, eyes, gills) and internal organs (including muscle tissue) for para sites. All macroparasites were stored and subsequently stained, dehydrated, and mounted on slides prior to identification. After staining, parasites were identified at least to genus using taxonomic keys and parasite species descriptions by Hoffman (1999},
Arai (1989), Beverley-Burton (1984}, Gibson (1996), and Kabata (1988).
Statistical Analyses
Prevalence (number of hosts infected/number hosts examined x 100) of infected hosts, mean parasite abundance (mean number of individual parasite species per all hosts sampled), mean parasite intensity (total number parasites recovered/ number of fish infected), and parasite range (minimum number to max number of parasites) were used to describe and compare parasite communities found within baitfish species. Comparisons were made within and between baitfish species and sources using mean infection prevalence(%), mean abundance, parasite range, and mean parasite intensity. Quantitative statistical procedures were not run for every baitfish sou rce combination due to the fact that some categories lacked replication in time or space. Kruskai -Wallis tests were 19 used to compare prevalence and mean intensities of fathead minnow and w hite sucker between sources {Rozsa 2000) using SPSS. Mann-Whitney U-test was used to test for differences in golden shiner parasite infection prevalence and mean intensity between sources using SPSS. Linear regression was used to determine correlations between parasite load and fish length {Muzzall et. al. 1992).
Significance was set at p-value is~ 0.05. If the P-value falls below this you reject the null hypothesis and accept the alternative that there are differences between/among the groups. For microscopic parasites, primarily the protists,
1000 was used as the upper limit for parasite species intensities and abundances that were too numerous to count reliably {Table 6 and 7).
20 RESULTS
A total of 350 baitfish were sampled from 14 sources in two states (WI and MN) (Table 2). General comparisons among sources were made using
infection metrics such as prevalence, mean abundance, range, and mean
intensity. There was no statistical difference in mean prevalence
(presence/absence of parasites within all hosts sampled) among baitfish sources
(i.e. wild, import, wholesale, retail, or hatchery) for all hosts combined (P > 0.05 of all comparisons) (Table 3 and Fig. 2) . Moreover, there was no significant difference in mean prevalence among baitfish sources for each species (fathead minnow, white sucker, and golden shiner) (P > 0.05 for all comparisons) (Tables 3-
4 and Fig. 2-6). Nearly 100% of fathead minnows were infected with at least one parasite species whereas only about 50% of white suckers were infected (Tab le 4 and Fig. 6).
21 Table 3. Mean infection prevalence(%) within 3 baitfish species and between baitfish sources. Prevalence= (#of hosts infected/# of hosts examined) x 100. FHM =Fathead minnow, WHS =White sucker, and GOS =Golden shiner. Species Source N Prevalence (%) FHM Wild 22 100.00 Import 41 95.00 Wholesale 44 98.00 Retail 20 95.00 Hatchery 20 100.00 WHS Wild 20 70.00 Import 41 76.00 Wholesale 30 60.00 Retail 21 67.00 Hatchery 42 14.00 GOS Wild 30 50.00 Retail 20 40.00 Total Wild 72 71.00 Import 82 85.00 Wholesale 74 82.00 Retail 61 67.00 Hatchery 62 42.00
22 Table 4. Mean infection prevalence(%} for all parasites within all 5 baitfish sources from white suckers (WHS}, fathead minnows (FHM), and golden shiners (GOS}. Species N % Prevalence FHM 147 97.00 WHS 153 54.00 GOS so 46.00
100.00
~ Qj 1.1c: ~ "'> 50.00 ....Qj c. c: ro Qj ~
0.00 Wild Import Wholesale Retail Hatchery Baitfish sources Figure 2. Mean prevalence(%) for all parasites in all fish species (fathead minnow, white sucker, and golden shiner) from 5 baitfish sources. Golden shiners were on ly sampled from wild and retail sources (n = SO).
23 100.00
~ OJ v c OJ iii > 50.00 ...OJ Cl. c nl OJ ~
0.00 Wild Import Wholesa le Retail Hatchery Baitfish sources Figure 3. Mean prevalence(%) for all parasites in fathead minnows from 5 baitfish sources (n=147).
100.00
'*~ c OJ ~ 50.00 ...OJ Cl. c nl OJ ~
0.00 Wild Import Wholesale Retail Hatchery Baitfish sources Figure 4. Mean prevalence(%) for all parasites in white suckers from the five baitfish sources (n=153).
24 100.00
~ C1l u r:: ~ ~ 50.00 ...C1l c. r:: tO C1l ~
Wild Retail Baitfish sources Figure 5. Mean prevalence(%) of golden shiners between sources (n=50 is total for both sources).
100
C1l '*'u r:: ~ tO > C1l ...c. II) so ·;:;C1l C1l c. II) ....tO 0 1- 0 FHM WHS Baitfish species Figure 6. Mean prevalence (%)for all parasites from white suckers (WHS) and fathead minnows (FHM) within all 5 ba itfish sources.
25 A total of 248 parasites recovered from sampled baitfish and these parasites represented thirty-e ight different species from eight taxa (Tab le 5). In most instances mean abundance was less than one (Table 5). However, Hysteromorpha triloba (X= 1.97:!:: 16.73, range: 0-225), Ornithodiplostomum ptychocheilus (X=28.70
:!:: 96.96, range: 0-827), Posthodiplostomum minimum (X=2.76:!:: 14.87, range: 0-
189), Microspora spp. {X=2 .86:!:: 53.45, range: 0-1000) and Myxobolus spp. {X=10.46
:!:: 92.63, range: 0-1000) had mean abundances greater than one {Table 5 and Figures
12-20). Standard deviations were higher in these groups since parasites numbers ranged between 0-1000 per host most (Table 5). Kruskai-Wallis analysis revealed that parasite abundance of both FHM {X=91.48 ±.164.02, P < 0.05) and WHS
{X=11.63 ±31.94, P < 0.05) were significantly different across baitfish sources. Trends show that in FHM the hatchery sources had a higher mean abundance {212.30) and in WHS sources the retail sources (33.62) had a higher mean abundance. Mann
Whitney test of parasite abundance in golden shiners between wild and retail sources showed no significant difference between these sources (X=61.18 :!::239.61, p ;;: 0.98).
26 Table 5. Mean parasite abundance, range, and .±SD for parasites found in 3 fish species (fathead minnow, white sucker, and golden shiner) collected from 5 baitfish sources (wild, import, wholesale, retail, and hatchery). Abundance = the number of parasites (of a given species)/all hosts sampled (infected and uninfected). Table 5 continued on following page.
Host Mean Parasite Range Phylum Genus species Species Abundance Min· Max ±SD Microspora Microspora spp. FHM,WHS 2.86 0-1000* 53.45 Myxozoa FHM, 92.63 Myxobolus spp. WHS,GOS 10.46 0-1000* Monogenea Pseudomurroytrema WHS 0.01 0-2 0.11 alaborrum Digenea Clinostomum marginatum** WHS 0 0-1 0.05 Crassiphiala bulboglossa** FHM 0.04 0·8 0.50 Diplostomum spathaceum** FHM,WHS 0.34 0-18 1.96 Diplostomum spp. ** FHM,WHS 0.14 0·10 0.96 Hysteromorpha triloba** FHM, 1.98 0-225 16.76 WHS,GOS Lissorchis attenuatus WHS 0.65 0-42 3.78 Lissorchis spp. WHS 0.08 0-20 1.12 Neascus spp. ** FHM, 2 0-96 7.51 WHS,GOS Ornithodiplostomum FHM 28.8 0-827 97.08 ptychocheilus * * Ornithodiplostomum spp. ** FHM 0.22 0-66 3.57
27 Table 5. continued Host Mean Parasite Range Phylum Genus species Species Abundance Min · Max ±SO Posthodiplostomum FHM 2.77 0-189 14.89 minimum•• Strigeidae spp. FHM 0.03 0-9 0.49 Uvulifer ambloplitis.,. FHM 0.01 0-2 0.11 Trematode unknown FHM, 0.99 0-68 5.12 WHS,GOS Cestoidea Atractolytocestus spp. WHS 0.01 0-2 0.11 Biacetabulum spp.,.,. FH M 0.18 0-64 3.42 Glaridacris spp. WHS 0 0·1 0.05 lsoglaridacris spp. WHS 0.03 0-7 0.43 Valipora sp. ** FHM 0 0-1 0.05 Caryophyllid spp. WHS 0.01 0-3 0.18 Cestode unknown FHM 0 0·1 0.05 Nematoda Capillaria spp. GOS 0.01 0·2 0.12 Contraceaum spp. •• FHM, WHS 0.02 0-7 0.38 Hunterella nodulosa WHS 0.02 0·6 0.32 Philometra nodulosa WHS 0 0-1 0.05 Philometra spp. WHS 0 0-1 0.05 Raphidascaris spp. FHM, GOS 0.01 0-2 0.13 Rhabdochona spp. FHM 0.12 0-35 1.89 Spiroxys spp. ** FHM, 1.84 WHS,GOS 0.37 0-16 Nematode unknown WHS, GOS 0.02 0-3 0.21 Acanthocephala Acanthocephalus spp. FHM, WHS 0.03 0-5 0.31 Neoechinorhynchus spp. FHM, 0.14 WHS, GOS 0.01 0-2 M ollusca Glochidia spp. *"' WHS 0.02 0-3 0.23 *A value of 1000 was used for those parasites that were too numerous to count due to microscopic sizes. **Immature parasite.
28 There was no apparent difference in mean intensity (total number of
parasites recovered/ number of fish infected) for all hosts (combined) among all
three species and baitfish sources (Table 6). Hatcheries (X = 163.58 ± 128.82) had
the largest total mean intensity and comparatively lowest standard deviation
among baitfish sources (Fig. 7). Comparisons of FHM mean intensities within each
baitfish source varied slightly and had large standard deviations {Fig. 8). Hatchery
sites for FHM were found to have the highest mea n intensity {X=212.3 ± 104.92)
and compared to wholesa le and import sources hatcheries had a relatively low
standard deviation (Fig. 8). Parasite mean intensities among baitfish sources for
WHS showed more variation, but there were large standard deviations (Fig. 9).
Parasite mean intensities for golden shiners were found to be higher from retail
sources and lower from wild sources. Retail sources had a much large r standard
deviation (Fig. 10). Parasite mean intensity per baitfish species within all sites (not
including GOS) was higher in FHM (X=94.04) but had a high standard deviation
(±165.59) (Table 7 and Fig. 11). Kruskai-Wallis analyses of parasite intensities in
both FHM {X=94.04±165.59, P < 0.05) and WHS (X=21.71 ±41.15, P < 0.05) across
baitfish sources showed significant differences. Trends again show FHM hatchery sources {212.30) and WHS retail sources (50.43) to have the highest mean intensities. Mann Whitney U-test for GOSs (X=133.00 ± 343.36, P = 0.05) showed that wild and retail source mean intensity distributions were not significantly difference among so urces.
29 Table 6. Mean parasite intensity(± SO} for parasites found in 3 baitfish species (FHM = Fatheand minnow, WHS= White sucker, GOS=Golden shiner) collected from 5 baitfish sources. Intensity = number of parasites/ infected hosts of baitfish species within each source. Species Source N Mean Intensity ±SO FHM Wild 22 38.95 54.27 Import 39 78.51 151.44 Wholesale 43 79.49 116.03 Retail 19 98.16 69.01 Hatchery 20 212.3 104.92 WHS Wild 14 11.86 22.15 Import 31 16.90 16.99 Wholesale 18 21.89 47.21 Retail 14 50.43 72.55 Hatchery 6 1.17 0.41 GOS Wild 15 2.13 1.64 Retail 8 378.38 514.79 Totals Wild 51 20.69 40.46 Import 69 51.72 117.84 Wholesale 61 62.49 159.15 Retail 41 136.54 294.50 Hatcher~ 26 163.58 128.82
Table 7. Mean parasite intensity (±_SO} within all 5 baitfish sources from white suckers (WHS} and fathead minnows (FHM}. Species N M ea n ±SO FH M 143 94.04 165.59 WHS 82 21.71 41.15 GOS 23 133.00 343.36
30 500
.~ 400 "'c Ql .E 3oo c 10 Ql E 200 ....Ql 'iii ~ 100 10 Q. 0
Baitfish sources Figure 7. Parasite mean intensity(± SD) within all fish species (fathead minnow, white sucker, and golden shiner) from five baitfish sources. Golden shiners were only sampled from wild and retail sources (n = 50).
150
....> 100 'iii c ....Ql ·=c 10 50 Ql E ....Ql 'iii 10... 10 0 Q. Baitfish sources Figure 8. Parasite mean intensity(± SD) of fathead minnows sampled from five baitfish sources (n=143).
31 400 ....> 'iii c ....Cll .:: 300 c ra Cll E 200 Cll ·;;;.... ra ...ra Q. 100
0
Wild Import Wholesale Retail Hatchery
Baitfish sources Figure 9. Parasite mean intensity C± SD) of white sucker samples from five baitfish sources (n=82).
1000
> .t: "'c ....Cll 100 c c ra Cll E Cll ·;;;.... 10 ...ra ra Q.
1 Wild Baitfish sources Figure 10. Parasite mean intensity(±. SD) of golden shiner samples from two baitfish sources (n=SO).
32 500
> 400 'iii... c: ...Cll c: 300 c: ro GJ E Cll 200 'iii...... ro ro a. 100
0
FHM WHS Baitfis h specie s Figure 11. Parasite mean intensity (±_SO) for all parasites from white suckers (WHS) and fathead minnows (FHM) within all five baitfish sources.
In order to assess parasite overlap among baitfish sources, a matrix was developed to compare presence/absence data for parasite species found in this study (Table 8). A total of 15 parasite species were found in fish collected from 2 or more baitfish sources. Most commonly, 2 sources contained the same parasite species. However, in the case of two species, Neoscus spp. and
Ornithodiplostomum ptychocheilus, overlap occurred across all five baitfish sources (Table 8). There were a total of 33 parasite species were recovered from all baitfish sources. An overlap matrix was also created to compare parasite species presence/absence across baitfish species (Table 9). Overlap ana lysis of parasite species within baitfish species found that there were a total of 10 parasite species which occurred in 2 or 3 of the baitfish species (Table 9).
Parasites infecting all three baitfish species were Hysteromorpho trilobo,
Myxobolus spp., Neoscus spp., Neoechinorhynchus spp., and Spiroxys spp.
33 (Table 9). There was no significant correlation between parasite load and fish length for FHM (r = 0.050), WHS (r=0.093), or GOS (r=0.077).
34 Table 8. Parasite overlap in fish hosts collected from five baitfish sources. Colored codes indicate amount of overlap among sources; Blue = 2 sources, Green= 3 sources, Yellow= 4 sources, Red = 5 sources. Source Type Species Wild Import Wholesale Retail Hatchery
Microspora spp. X I X
Myxobolus spp. X X X X
Pseudomurraytrema alabarrum X w V1 Clinostomum marginatum * X Crassiphia/a bu/boglossa* X Diplostomum spathaceum * X X ; --- Diplostomum spp. * I X X Hysteromorpha triloba* X X X X
Lissorchis attenuates X X X Lissorchis spp. -- X Neascus spp. * ilx X X X X Ornithodiplostomum ptychochei/us * l_x -- X X X X Posthodiplostomum minimum* X X X X
Strigeidae spp. X Uvulifer ambloplitis* X
Atractolytocestus spp. X Table 8. continued Biacetabulum spp. * X caryopnyJl/a spp. X Glariaacris spp. X lsoglaridacris spp. I X X ~ Valipora sp. * X Cestode spp. X
Capillaria spp. . X X Contraceaum spp. * X Hunterelia noauiosa X w Pnilometra spp. X 0'1 Pnilometra noaulosa X Raphidascaris spp. X Rhabdochona spp. X X Spiroxys spp . * X X X ' X
Acanthocepha/aus spp. X l X Neoechinorhynchus spp. • X i X
Mollusca* X *Immature parasites. Table 9. Amount of overlap among parasite species within three baitfish species. Colors indicate amount of overlap among species; Blue= 2 sources, Green= 3 sources. Baitfish Species Parasite Species FHM WHS GOS
Microspora spp. X
Myxobolus spp. X X X
Pseudomurraytrema alabarrum X
OJ --.J Clinostomum marginatum * X Crassiphiala bulboglossa * X
Diplostomum spathaceum * X X Diplostomum spp. * X X Hysteromorpha triloba* X X X Llssorchts attenuates X
Lissorchis spp. X
Neascus spp. * X X X Ornithodiplostomum ptychocheilus* X
Posthodiplostomum minimum* X
Strigeidae spp. X Uvulifer ambloplitis* X Table 9. continued. Atracto/'t_tocestus s~~· X Biacetabulum spp. * X Caryophyllid spp. X
Glaridacris spp. X
/soglaridacris spp. X Valipora sp. * X
Cestode spp. X
Contraceaum spp. * X X I ------·- Huntere/la nodulosa X
Philometra spp. X
Philometra nodulosa X Raphidascaris spp. I X I X w Rhabdochona spp. X 00 Spiroxys spp. * X X X
Acanthocephalaus spp. X X I , ·-· - ~------__ -- --- Neoechinorhynchus spp. X X X
Mollusca* X *immature parasites. Figure 13. Photomicrograph of Myxobolus spp. cysts (arrows) attached to the gill lamellae of a fathead minnow.
39 Figure 14. Photomicrographs of Myxobolus spp. wet mounts from Fathead minnow.
/
Figure 15. Photomicrograph of Pseudomurraytrema alabarrum wet mount from white sucker.
40 Figure 16. Photomicrograph of Diplostomum spathaceum (arrow) within lens of fathead minnow.
Figure 17. Photomicrograph of immature encysted Ornithodiplostomum ptychochei/us infecting fathead minnow brain tissue.
41 Figure 18. Photomicrograph of immature Va/ipora sp. from fathead minnow.
Figure 19. Photomicrographs of Spiroxys spp. found in all three baitfish species. A. Anterior end. B. Posterior end.
42 A B Figure 20. Photomicrograph of male Acanthocepha/us spp. from fathead minnow and white sucker. A. Anterior proboscis with spines. B. Posterior end.
43 DISCUSSION
High demands for baitfish in the North Central Region (NCR) increases the transport of baitfish within and between states. Transportation of baitfish enhances the potential for the transfer of parasites associated with these fish species
(Summerfelt and Warner 1970; Litvak and Mandrak 1993; Engle and Stone 2003).
Studies investigating baitfish and their parasites within the NCR have been limited.
Most research has focused on either a single parasite species or a single baitfish species of interest (Hoffman 1999). Recently, with the threat of increasing pathogen transfers to na'lve species in aquatic systems, researchers have identified a need for baseline data on fish parasite communities across a number of potential sources.
Moreover, such broader studies are required to help managers 1) identify and potentially mitigate the risk of baitfish/pathogen transfers and 2) establish pathogen screening procedures to identify infected hosts prior to their use as baitfish in natural systems (Daszak et al. 2000; Kerr and Grant 2000; Gaughan 2002; Poulin 2007;
Okamura and Feist 2011).
Common practice for anglers is to re lease excess baitfish into local freshwater environments (Litvak and Mandrak 1993). These observations suggest that there is high potential for parasitic transfers within aquat ic environments of the NCR. Thus angler-release practices may allow for the spread of parasitic disease outbreaks within
NCR water bodies with high densities of cyprinids (Kerr and Grant 2000). In addition, 44 it is possible that fish movement by baitfish industry could be inadvertently facilitating
regional parasite transfer into novel environments through their importation, rearing,
and transfer practices (Passarel li 2010). Because of this, the management of baitfish
transfers will be critical for minimizing the potential spread of both baitfish and their
parasites throughout the NCR (Kiak 1940; Summerfelt and Warner 1970; Gunderson
and Kinnunen 2001; Engle and Stone).
Ornithodiplostomum ptychocheilus and Neascus spp. were the only two
parasites observed within all five baitfish sources. The result for 0. ptychocheilus
was not surprising given that FHM from all sources are typically wild harvested
from shallow lakes (Gunderson and Tucker 2000) prior to distribution. Within
these habitats, 0. ptychocheilus prevalence often approaches 100% (Sandland
1999). Furthermore, FHMs are often maintained in small outdoor ponds
(Gunderson and Tucker 2000) where intermediate hosts (e.g., snails such as
Physa) are likely present. Berra and Au (1978) also observed in Ohio that parasite
prevalence in multiple host species from stagnant waters was about 89%.
Therefore these artificial conditions and high FHM densities further increase the
potential for 0. ptychochei/us infections.
The ability of Neascus spp. to infect multiple species of intermediate fish hosts may explain the parasite's appearance in al l 5 ba itfish sources (Hoffman 1999). Neoscus spp. is also a parasite that utilizes a complex lifecycle, but unlike 0. ptychocheilus it has been reported from a wide range of baitfish host species, including the three species examined in this study (Hoffman 1999). These fish species can act as compatible links in
45 the parasite's life cycle or may be either accidental or dead-end hosts (Roberts and
Janovy 2005). Again, given t he high frequency of this parasite in natural ba itfish populations and the rearing conditions at different sources, it was not surprising to see this parasite in fish from all sources.
Peeler and Feist (2011), suggest that human movement of baitfish may extend
parasites ranges by intentional or unintentional stocking of baitfish in open water
systems. Both O.ptychocheilus and Neascus spp. encyst within intermediate hosts for
life and as such, they could be carried by baitfish hosts throughout transfer events
until death, consumption by definitive bird host, (Sandland 1999) or, in the case of
angling, consumed by a secondary predatory fish (dead-end host). Immature
metacercarie of 0. tychocheilus and Neascus spp. cou ld be transported among the five
baitfish sources studied.
High numbers of Neascus spp. can cause mortality by increasing physiological
stress on fish hosts, especially for small fish (Kiak 1940). Additionally, as generalists
Neascus spp. infect a wide range of hosts including game fish (e.g., Centrarchidae
(bass), Esocidae (pike), Percidae (perch), and Salmonidae (trout)). Infection by
Neascus spp. often causes hosts to react and deposit black melanin around encysted
metacercarie. These black spots are undesirable to most sportsmen and considered
not suitable for human consumption (Hunter and Hunter 1938; Miller 1940;
Choq uette 1956). As a result, if Neascus spp. are transferred to game fish by the
introduction of infected baitfish then many areas in the NCR may see a decrease in
economic gain from anglers. In Wisconsin decreased angling could cause a loss of
46 economic activity and potential ly affect resource management options because the
majority of funding for the aquatic ecosystem management comes from the sale of
sport fishing goods and services under the Sport Fish Restoration Act (WDNR 2007).
Parasites with indirect lifecycles tend to infect hosts across a broader
taxonomic ra nge than those with simple life cycles, which may al low them to better
infect novel hosts if they are encountered (Sasal et al. 1998). Hysteromorpha triloba,
Myxobolus spp., Neascus spp., Neoechinorhynchus spp., and Spiroxys spp. were
observed within all three baitfish species. These five genera exhibit indirect life cycles
and have been reported to infect multiple fish hosts and/or host organs throughout
North America (Hoffman 1999) suggesting that they have generalist host preferences.
A number of intermediate hosts can be infected with developing parasites that
may cause damage to host tissues/organs and/or use host to transfer to the next
available host in life cycle. Hosts that exhibit similar life history characteristics, occupy similar habitats, and are related phylogenetically; are likely to be more susceptible to the same suite of parasites (Poulin 1992). Given these similar traits of baitfish hosts in the NCR the transmission of similar parasites may be more likely. The generalist
nature of these five parasite species in combination with host handling stress and overcrowding may enhance transmission among taxonomically similar baitfish hosts
which may increase the probability of such species being spread throughout ba itfish sources and introduced into new habitats. This possibility is further supported by the fact that cyprinids are common throughout natural habitats in the NCR which may
47 allow these parasites to spread across a number of species beyond the baitfish studied.
Myxobo/us spp. pathogens of wild and farm raised fish are of significant importance (Kent et.al. 2001). The best known species is Myxobolus cerebra/is which infects salmon ids and causes whirling disease. In addition, Myxobolus spp. have been found to increase mortality, reduce fecundity, and increase predation events due to changes in host behavior (Shaw and Kent 1999; Feist and Longshaw 2008; Longshaw et al. 2010). Within cyprinids such as northern pikeminnow, redside shiner, or largescale sucker, pathogenic effects can range from minor tissue damage to organs to major vertebral deformities (Markle et al. 2002; Kent et al. 2004). Myxobolus spp. were found in all baitfish species and in various organs/tissues. Given host preference and their pathogenic impacts on numerous host species, the presence of this parasite in baitfish in this study is concerning, particularly due to the high potential for baitfish release into natural water bodies. In Montana, researchers have deduced that
Myxobo/us spp. is most likely being transferred w ithin and between states due to angler movements (Gates et al. 2009). Likewise, transfer of baitfish throughout t he
NCR may be facilitating Myxobolus spp. movement among baitfish sources and water bodies. Myxobolus spp. have always been of high importance to resource managers and as such information on infected baitfish movement throughout the NCR would be of value to managers mitigate potential threats on fish populations.
It is difficult to accurately identify Myxobo/us species morphologically because different species have similar spore sizes, shapes, and infection sites (Eszterbauer et
48 al. 2001). Species level identification for Myxobo/us spp. was beyond the scope of this
study since PCR is used for positive confirmation. However, based on previous
research it is likely that there are a number of Myxobolus spp. in baitfish species of
the NCR that have the potential to cause disease. This idea is supported by a
Canadian study that found at least five new species of Myxobolus spp. in golden
shiners alone (Salim and Desser 2000). Identification of Myxobo/us spp. infecting
baitfish species and the determination of possible effects on fish populations are just
beginning throughout North America (Longshaw et al. 2010). In the NCR, pathogenic
consequences of Myxobolus spp. infecting baitfish could lead to the possible
economic loses for hatcheries, wholesalers, and retailers.
The parasites of national concern found infecting multiple baitfish species were Myxobo/us spp. (discussed above) and Dip/ostomum spathaceum.
If baitfish are infected withthese parasites and subsequently transferred and/or introduced into NCR water bodies we may see an expansion of the host range of these parasites. Muzzall et al. (2006) found Diplostomum spathaceum metacercariae infecting the lenses of the eyes of nearly 72% of pond- reared walleye where it was hypothesized that the infection had come from imported baitfish used as feeder fish for the walleyes. Diplostomum spathaceum also has the ability to cause blindness or parasitic cataracts which can reduce baitfish feeding ability and predator detection (Post 1983; Noga
1996). Therefore, if baitfish have the potential to transfer and increase the rate of Diplostomum spathaceum infections within NCR fish populations then
49 cataracts and loss of vision may also become more prevalent and thereby reduce fish hosts ability to detect food, predators, and increase mortality rates.
Valipora sp., is an unusual larval cestode with an armed rost ellum that infects
multiple organs of freshwater f ish. It was identified within a fathead minnow from a
retail source. Interestingly, cyprinid fish are the most frequent hosts for Valipora spp.;
however, w it hin the U.S., specimens have mostly been identified only to genus,
misidentified, or identified within unpublished research (Scholz et al. 2004). There is
no information on Va/ipora spp. distributions or rates of infection within the U.S ..
Baitfish are often intermediate hosts for metacestodes such as Valipora (Scholz et al.
2004). Infected baitfish may be transferring Valipora throughout the NCR. One
Canadian parasitological survey of an aquaculture introduction of tench (cyprinid fish species) reported that 20% of infected specimens were carrying Va/ipora campylancristrota (Marcogliese et al. 2009). The movement of infected ba itfish into t he NCR may potentially introduce Valipora spp. to novel ecosystems that have compatible hosts to complete the life cycle (i.e. heron and copepod) (Becker, 2004) and have the potential to increase rates of infection wit hin fish populations.
Total mean parasite abundance and intensity differed across baitfish sources for both FHM and WHS. One explanation for this difference in FHM, is that hatcheries of fathead minnows typically raise FHMs for multiple years, at high densities, and in outdoor mud-bottom ponds (Engle and Stone; Gunderson and Tucker 2000). These ponds may help to facilitate and maintain optimal conditions for parasite life cycle
50 completion. Continual contact with multiple parasite stages, and increased access to
transfer, develop, and reproduce in intermediate or definitive host allows for multiple
baitfish infection events (Engle and Stone; Dobson and May 1987; Price and Nickum
1995). Furthermore, prolonged elevated temperatures in hatchery ponds, particularly
at certain times of the year, may enhance parasite development and reproduction in
intermediate hosts leading to higher overall parasite intensities (Dobson and Carper
1992). Overcrowding in hatchery ponds may also be responsible for the observed
differences in parasite abundance/intensities as more hosts can lead to increased
parasite contact and higher host numbers can lead to higher stress and reduced
immune responses (Engle and Stone; Rottmann et al. 1992; Noga 1996; Gunderson
and Tucker 2000).
Differences in parasite prevalence and mean intensity among white sucker
sources may be explained by the fact that hatchery-reared WHS were cultured in
indoor re-circulating aquaculture systems as eggs (Gregory Fischer, Northern
Aquaculture Demonstration Faci lity Manager, personal communication). These in door
conditions would remove baitfish contact with parasites released from (or contained
within) intermediate hosts. Although, rearing conditions are likely to be a factor
influencing the parasite communities, a lack of sample replication for some categories
may also have influenced the results.
Information on parasites infecting baitfish within the NCR is limited (Engle and
Stone2003). Baseline information of parasite species infecting major baitfish species within the NCR may support that baitfish could be transferring unknown pathogens
51 among baitfish within sources which may cause disease within baitfish populations from any of the five sou rces. Further studies with more replication in space and t ime are needed to better characterize general baitfish parasite communities and make comparisons among baitfish sources. Parasite pathogen lists can be used to help identify risks of baitfish transfer and associated parasites to ecosystems. Resource managers can then use the information to make transfer guidelines for the NCR that w ill sustain the economic viability of the baitfish industry for baitfish wholesalers, retailers, and hatcheries without sacrificing ecosystem integrity.
Passarelli (2010) reviewed factors for pathogen transfer in the California live bait trade and found that more research is needed to increase our understanding of pathogens, transfer pathways, and parasite-host interactions for managers to make more informed decisions when working to prevent baitfish mediated parasite introductions. My research has contributed to this goal by identifying the parasite communities in three baitfish species collected from five baitfish sources. This has allowed me to generate a list of ba itfish parasite species and identify the potential transfer pathways between sources. This baseline knowledge can be used by natural resource management within the NCR to generate preventative strategies (e.g., pathogen screening, baitfish health certificates) aimed at recognizing baitfish/parasite transfer risks and the possible establishment of pathogen screening prior to baitfish transfer (Fen ichel et al. 2008).
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