PARASITES OF LARGEMOUTH BASS (MICROPTERUS SALMOIDES) IN NORTHERN CALIFORNIA. Daniel J. Troxel. A Thesis Presented to

PARASITES OF LARGEMOUTH BASS (MICROPTERUS SALMOIDES) IN NORTHERN CALIFORNIA By Daniel J. Troxel A Thesis Presented to The Faculty of Humboldt State University In Partial Fulfillment of The Requirements for the Degree Masters of Science Natural Resources, Fisheries November, 2010

ABSTRACT Parasites of largemouth bass (Micropterus salmoides) in northern California Daniel J. Troxel A total of fifty largemouth bass (Micropterus salmoides) were collected from Clear Lake, Lake Berryessa, Lake Sonoma, Trinity Lake and the Sacramento San Joaquin River Delta. All fish were infected with at least one parasite, with the exception of one juvenile fish from Trinity Lake in which no parasites were found. The following parasites were observed in largemouth bass: Actinocleiudus unguis, Clavunculus bursatus, Clinostomum complanatum, Proteocephalus pearsei, Contracaecum sp., Hysterothylacium (?) sp., Spinitectus carolini, Camallanus sp., Neoechinorhynchus cylindratus, Myzobdella lugubris, Batracobdella phalera, Ergasilus centrarchidarum, and Argulus flavescens. In addition to these identified parasites, I also found an unidentified leech and Proteocephalus sp. plerocercoids, which could not be identified to species. All of these parasites have been previously reported infecting largemouth bass. Clinostomum complanatum, Contracaecum sp. and Myzobdella lugubris, have been previously reported in California; but these are the first known reports from largemouth bass in California. Actinocleidus unguis was the only parasite found that has previously been reported to infect largemouth bass in California. iii

ACKNOWLEDGMENTS I would first and foremost like to acknowledge my parents, without their support over the past two years this project would not have been possible. I would like to thank Dr. Gary Hendrickson for his contributions in the laboratory during the long process of identifying parasites. Thank you to my other committee members, Dr. Kristine Brenneman and Dr. Rick Brown, who were very accommodating in making time for me and sharing valuable input during the review process. A thanks goes out to Steve Monk, HSU Boat Safety Officer, for his help in allowing me to use my own boat for this project. Thank you to Kyle Murphy and Monty Currier from California Department of Fish Game, for taking an interest in my study and offering what help they could. I would especially like to thank the many anglers; Paul, Scott, Jen, Derrick, John, Len, Bill and Eric from National Bass West, The Contra Costa Bass Club, and Redwood Empire Bass Club who donated their time and effort to help me get my fish. iv

TABLE OF CONTENTS ACKNOWLEDGEMENTS...iv LIST OF TABLES.......vi LIST OF FIGURES. vii INTRODUCTION.....1 STUDY SITES...... 5 MATERIALS AND METHODS....11 RESULTS.........16 DISCUSSION..... 44 LITERATURE CITED.....55 PERSONAL COMMUNICATIONS...59 v

LIST OF TABLES Table Page 1 Common fish species reported from five collection locations: Trinity Lake, Clear Lake, Lake Sonoma, Lake Berryessa and the Sacramento-San Joaquin River Delta.... 7 2 Abundance of parasite species recovered from largemouth bass at five collection locations in northern California..17 vi

LIST OF FIGURES Figure Page 1 Map of northern California featuring five collection locations: Trinity Lake, Clear Lake, Lake Sonoma, Lake Berryessa and the Sacramento San Joaquin River Delta.......6 2 Haptoral hooks of Actinocleidus unguis from gills of largemouth bass collected from Clear Lake, Lake County, California, March 2009...19 3 Clavunculus bursatus from gills of largemouth bass collected from Lake Berryessa, Napa County, California, November 2009....20 4 Clinostomum complanatum from gills of largemouth bass collected from the Sacramento San Joaquin River Delta San Joaquin County, California, October 2009. 22 5 Scolex of an adult Proteocephalus pearsei from the intestine of a largemouth bass collected from Clear Lake, Lake County, California, March 2009... 23 6 Scolex of an immature adult Proteocephalus pearsei from the intestine of a largemouth bass collected from the Sacramento San Joaquin River Delta, San Joaquin County, California, November 2009..... 24 7 Scolex of a Proteocephalus sp. plerocercoid from the mesenteries of a largemouth bass collected from Clear Lake, Lake County, California, March 2009...25 8 Anterior (featuring intestinal caecum) of juvenile Contracaecum sp. from the mesentaries of a largemouth bass collected from Clear Lake, Lake County, California, September 2009.......27 9 Anterior of Hysterothylacium (?) sp. from the stomach of a largemouth bass collected from the Sacramento San Joaquin River Delta, San Joaquin County, California, October 2009.. 28 10 Anterior of a female Spinitectus carolini from the intestine of a largemouth bass collected from the Sacramento San Joaquin River Delta, San Joaquin County, California, October 2009......29 vii

LIST OF FIGURES (CONTINUED) 11 Posterior of a male Spinitectus carolini from the intestine of a largemouth bass collected from Lake Sonoma, Sonoma County, California, November 2009....30 12 Anterior of a Camallanus sp. from the intestine of a largemouth bass collected from Clear Lake, Lake County, California, September 2009 31 13 Male Neoechinorhynchus cylindratus from the intestine of a largemouth bass collected from the Lake Berryessa, Napa County, California, November 2009.......32 14 Proboscis of a female Neoechinorhynchus cylindratus from the intestine of a largemouth bass collected from the Sacramento San Joaquin River Delta, San Joaquin County, California, October 2009..... 34 15 Anterior of a Myzobdella lugubris (featuring separate eye spots) from the fins of a largemouth bass collected from Lake Berryessa, Napa County, California, November 2009......35 16 Anterior of a Myzobdella lugubris (alternate body form) from the pectoral fin of a largemouth bass collected from Clear Lake, Lake County, California, March 2009.......... 36 17 Batractobdella phalera from the pectoral fin of a largemouth bass collected from the Sacramento San Joaquin River Delta, San Joaquin County, California, October 2009.......37 18 Fused eye spots of a Batractobdella phalera from the pectoral fin of a largemouth bass collected from the Sacramento San Joaquin River Delta, San Joaquin County, California, October 2009.... 38 19 Unidentified leech (Batracobdella phalera(?)) from the gills of a largemouth bass collected from the Sacramento San Joaquin River Delta, San Joaquin County, California, October 2009.....39 20 Ergasilus centrarchidarum from the gills of a largemouth bass collected from Sacramento San Joaquin River Delta, San Joaquin County, California, October 2009..........40 viii

LIST OF FIGURES (CONTINUED) 21 Secondary antenna of an Ergasilus centrarchidarum from the gills of a largemouth bass collected from Sacramento San Joaquin River Delta, San Joaquin County, California, October 2009......42 22 Argulus flavescens from a largemouth bass collected from Clear Lake, Lake County, California, March 2009....43 ix

INTRODUCTION Largemouth bass (Micropterus salmoides) are the most widely pursued game fish in California (Moyle 2002). Historically, largemouth bass ranged north from northeastern Mexico through the Mississippi River drainage into Ontario and Quebec, east into the southeastern US south of South Carolina, and west to the Rocky Mountains. Largemouth bass have been introduced to countries across the globe, including Japan, Guatemala, Italy, Spain, South Africa and Zimbabwe (Moyle 2002). There are two subspecies of largemouth bass: the northern strain (M. s. salmoides) inhabiting most of the native range and the Florida strain (M. s. floridanus) from Florida. Northern strain largemouth bass were introduced into California in 1891 and 1895 in aquarium tanks on rail cars by the Central Pacific Railroad. Initial introductions were done by the U.S. Fish Commission (Smith 1895) but anglers and agency biologist spread them throughout the state. Florida largemouth bass were first introduced into San Diego County in 1959 (Moyle 2002). They were subsequently spread throughout California and have since hybridized with northern largemouth bass. Largemouth bass parasites have been extensively studied in their native range and are known hosts to many types of parasites including fungi, protozoa, Monogenea, Trematoda, Cestoidea, Nematoda, Gordiacea, Acanthocephala, Hirudinea, Mollusca and Crustacea (see Hoffman 1999 for a review). However, relatively few parasite studies have been conducted in places where centrarchid bass have been introduced. Surprisingly, very little is known about parasites of bass in California, given the importance of largemouth bass to sport angling in the state. Mizelle and Crane (1964) looked at relative sizes of 1

2 monogenetic trematodes between those collected in California and those collected at locations within the native range of largemouth bass. Becker (1972) did a similar study on the Columbia River, Washington. He studied monogenean parasites from juvenile largemouth bass. Because monogenea are host specific, it is likely that these parasites came to California with the introduction of largemouth bass. Parasites of freshwater fishes have been studied sporadically in California (see for example Haderlie 1953 and Hensley and Nahhas 1975). However, such studies did not focus on non-native fishes, particularly black basses. Some known parasites of largemouth bass have been previously reported in California. Mizelle and Crane (1964) reported Actinocleidus unguis (Monogenea) from largemouth bass in a pond near Sacramento, California. Haderlie (1953) reported Clinostomum complanatum (Digenea) and Contracaecum sp. (Nematoda). However, Haderlie focused on native fish species. Klemm (1985) reported Myzobdella lugubris (Hirudinea) from California, but not from largemouth bass. Hendrickson (2009, personal communication) reported Neoechinorhynchus cylindratus (Acantocephala) from spotted bass (Micropterus punctulatus) from Lake Oroville, California. It is possible that some of these parasites were introduced with largemouth bass to California, while others may have already been present before the introduction of largemouth bass. Introduced fish often serve as a vector for the introduction of non-native fish parasites. The introduction of Myxobolus cerebralis (Myxozoa), the causative agent of salmonid whirling disease, and its spread throughout North America, is a classic example (Bartholomew and Reno, 2002). The persistence of an introduced parasite in a new

3 geographic area depends on presence and sufficient density of suitable hosts, water quality, habitat and several other factors (Torchin et al. 2003). Poulin and Morand (2000) suggested that the diversity of introduced parasites is dependent on the probability of parasites successfully colonizing the new location and undergoing speciation following the introduction versus the probability of failing to establish and going extinct in the new location. Torchin et al. (2003) noted that the entire parasite fauna from the native location is not transplanted. Thus, the parasite fauna of an introduced species in its new location includes a small portion of the entire parasite fauna from the native location and new parasites acquired after introduction. Sakanari and Moser (1990) suggested that many parasites infecting native fishes could be non-host specific, and therefore would be capable of infecting an introduced fish species. This ability can vary greatly depending upon host specificity of the parasite in question, the drainage area involved, and(or) the abundance of introduced host species. Parasite diversity may increase due to the species richness in the ecosystem (Poulin 1997), particularly as the species richness of potential hosts increases (Esch 1971). This suggests that ecosystems with greater diversity will support more species of parasites. Eutrophic systems tend to exhibit high species richness while oligotrophic systems tend to exhibit lower species richness (Esch 1971). Therefore, collection locations such as the Sacramento San Joaquin River Delta and Clear Lake, should have a greater diversity of bass parasites than locations like Sonoma and Trinity Lake, which have a less varied fauna of potential fish hosts. Fish species richness varies substantially

4 between collection locations, but there are several species which are found at most or all collection locations, including several non-native centrarchid species (Moyle 2002). Largemouth bass collected for this study should have a fairly diverse assortment of parasite species with varying degrees of infection as they are a top predator species and are able to inhabit diverse environments. Parasite diversity and intensity of infection is likely less than that of bass sampled in their native range (Poulin and Morand 2000, Torchin et al. 2003). There are two reasons for this. First, only some parasite species will be able to survive the introduction from their native range. For example, some parasites may not be able to complete their life cycle in the new location. Second, largemouth bass have only been in California a short time. It may take a long time for native parasites to adapt to this host. Some parasites may be difficult to acquire due to their general rarity or seasonal variation in abundance, but California largemouth bass should have most of the commonly known groups of bass parasites. The objective of this study is to identify parasites of largemouth bass in northern California.

STUDY SITES Fish were sampled from five fisheries in Northern California (Figure 1): Clear Lake, the Sacramento/San Joaquin River Delta, Lake Sonoma, Lake Berryessa and Trinity Lake. These locations were chosen because of their broad geographic range in northern California and their large populations of largemouth bass. Trinity Lake is located approximately 16 km northeast of Weaverville in Trinity County California. Trinity Dam was built in 1962 by the U.S. Bureau of Reclamation for the Central Valley Project. The dam was constructed for increased water storage for use in the central valley. It is the third largest body of water in California with over 64 square km, 234 km of shoreline and holds nearly 2.5 million acre feet capacity at full pool (United States Department of the Interior, Bureau of Reclamation 2010b). Trinity Lake is an oligotrophic reservoir characterized by steep rocky banks. Largemouth bass were introduced to Trinity Lake after the completion of the dam in 1962. Trinity Lake supports some species of fish common to California (Table 1). Clear Lake is the largest natural lake contained entirely within the boundaries of California. Clear Lake is situated in the California Coast Range in Lake County. Lakes have existed at the site of Clear Lake for over 2.5 million years, making Clear Lake one of the oldest lakes in North America (Lake County Water Resources 2010). At full pool, Clear Lake has a capacity of over one million acre feet, a surface area of nearly 178 square km and 205 km of shoreline (Lake County Water Resources 2010). Clear Lake is an extremely eutrophic lake supporting a variety of aquatic macrophytes. 5

Figure 1. Map of Northern California featuring collection locations: Trinity Lake, Clear Lake, Lake Sonoma, Lake Berryessa and the Sacramento San Joaquin River Delta. 6

7 Table 1. Common fish species from five collection locations: Trinity Lake, Clear Lake, Lake Sonoma, Lake Berryessa and the Sacramento-San Joaquin River Delta. Species Trinity Lake Clear Lake Lake Sonoma Lake Berryessa Sacramento- San Joaquin River Delta largemouth bass (Micropterus salmoides) X X X X X smallmouth bass (Micropterus dolomieu) X X X X X spotted bass (Micropterus punctulatus) X X X redeye bass (Micropterus coosae) X X X black crappie (Pomoxis nigromaculatus) X X X white crappie (Pomoxis annularis) X X X X bluegill (Lepomis macrochirus) X X X X X green sunfish (Lepomis cyanellus) X X X X X redear sunfish (Lepomis gibbosus) X X X channel catfish (Ictaluris punctatus) X X X X X brown bullhead (Amerius nebulosus) X X X X threadfish shad (Dorosoma petenense) X X X X carp (Cyprinus carpio) X X X X X rainbow trout (Onchorhynchus mykiss) X X X X Chinook salmon (Onchorhynchus tshawytscha) X X kokanee salmon (Onchorhynchus nerka) X X brown trout (Salmo trutta) X X - X denotes recorded presence of species

8 This characteristic lends itself to a highly complex food web, with many prey species available for largemouth bass. Northern largemouth bass were introduced into Clear Lake in 1897. A subsequent introduction of Florida largemouth bass occurred in 1967 (Giusti 2009). Clear Lake hosts a variety of common fish species (Table 1) and some other species of note including hitch (Lavinia exilicauda), and Sacramento blackfish (Orthodon microlepidotus) (Moyle 2002). Lake Sonoma is situated in the fertile California wine country in Sonoma County California. The reservoir is located approximately 8 km West of Geyserville, California and Highway 101. Lake Sonoma was constructed in 1983 with the building of Warm Springs Dam, by the U.S. Army Corps of Engineers. It was built for flood control and irrigation. Below Warm Springs Dam is the Congressman Dan Clausen Fish Hatchery, which was put in place to mitigate the loss of salmon and steelhead spawning grounds (United States Army Corps of Engineers 2010). Lake Sonoma has a capacity of just over 100,000 acre feet. The lake has a surface area of 10 square km and has 80 km of shoreline (United States Army Corps of Engineers 2010). Lake Sonoma is mainly an oligotrophic reservoir characterized by steep rocky banks and flooded timber. Small portions of the lake support limited weed growth, which is primarily during the warm months. Largemouth bass were introduced into Lake Sonoma shortly after completion of Warm Springs Dam in 1983. Lake Sonoma supports a variety of fish species (Table 1). Lake Berryessa is located approximately 32 km West of the city of Davis, California. It is one of California s largest reservoirs. It was formed by the damming of

9 Putah Creek, and is the reservoir for the Solano Project, which provides irrigation and flood control for surrounding communities (United States Department of the Interior, Bureau of Reclamation 2010a). The reservoir has 266 km of shoreline. It stores a maximum capacity of 1.6 million acre feet of water at full pool. Lake Berryessa is a mesotrophic lake with moderate amounts of aquatic vegetation, which is restricted to the littoral zone. Largemouth bass were present in the Cache Creek drainage from the stocking of Clear Lake when the dam was completed in 1963. Lake Berryessa supports several species fish (Table 1). The Sacramento/San Joaquin River Delta was historically a tidal freshwater marshland created by the confluence of the Sacramento and San Joaquin Rivers flowing to San Francisco Bay. Historically this marshland was covered in peat and alluvium from nearby streams and rivers (California Department of Water Resources 1995). The late 1800 s brought large scale agricultural operations to the region. Marshland islands were surrounded by levees to prevent flooding. These islands were then drained, cleared and tilled for agricultural use. By the 1930 s most of the islands had been isolated and established as farmland. Today the Delta is a complex network of over 1,600 km of waterways, with nearly 60 islands which are surrounded by over 1,800 km of natural and man-made levees. The approximate Delta boundary reaches North to Sacramento, South to Tracy, East to Stockton, and West to Pittsburgh (California Department of Water Resources 1995). As a whole, the Sacramento San Joaquin River Delta is a eutrophic ecosystem. It has complex and varied habitats which include highly eutrophic backwater sloughs and flooded islands, to free flowing sections of major rivers. Much of the Delta s

10 banks and in-river islands are covered with, bulrush (Scirpus sp.), cattails (Typha sp.) and other emergent aquatic vegetation. Usually the islands are surrounded by heavy vegetation in the form submergent macrophytes or floating algae. Similar to Clear Lake, the Delta exhibits a highly complex food web. This means more potential prey species for bass and a broad forage base for other predatory species. Northern largemouth bass were introduced into the Sacramento San Joaquin River Delta in 1890 s with the initial introductions done by the United States Fish Commission (Smith 1895). Florida largemouth bass were first introduced to the Sacramento San Joaquin River Delta in the 1960 s following their initial introduction to the state in 1959 (Moyle 2002). The Delta supports an extremely wide variety of fish species (Table 1) and several other species of fish found in California, including Sacramento perch (Archoplites interruptus), striped bass (Morone saxatilis), Sacramento pikeminnow (Ptychocheilus grandis), tule perch (Hysterocarpus traskii), California roach (Lavinia symmetricus), delta smelt (Hypomesus transpacificus), hitch (Lavinia exilicauda), Sacramento blackfish (Orthodon microlepidotus), Sacramento splittail (Pogonichthys macrolepidotus), and white sturgeon (Acipenser transmontanus) (Brown and Moyle 1993, Moyle 2002).

MATERIAL AND METHODS The three individuals from Trinity Lake were collected by Monty Currier, Associate Fishery Biologist with California Department of Fish and Game, using electrofishing techniques. All other sampling was done using hook and line with artificial lures. Hook and line collection was used for two reasons: it allowed for minimal stress to be put on the fish (unlike gill netting or electrofishing) and allowed for quick release of fish that were not retained. This method also lent itself to size selective capture. For this study, I collected fish that were 12 inches in total length or greater, with the exception of the fish from Trinity Lake. Fish over 12 inches were adult fish and therefore were more likely to have parasites because they have had more time to contact parasites and intermediate hosts in their environment (Fischer and Kelso 1990). During sampling, any small fish were immediately released at the location of capture. Fish were euthanized in a solution of 400mg/L of Finquel (MS-222, tricaine methanesulfonate; Argent Chemical Laboratories, Redmond, Washington). Fish remained in Finquel solution for five minutes, which was sufficient time to ensure euthanization. Several fish used in this study were acquired from other anglers. All fish obtained from other anglers were obtained live and euthanized using Finquel or carcasses were transported to me on ice. Following euthanization, fish were placed into insulated coolers with ice for transport to the laboratory. All fish collections and handling were done in accordance with Humboldt State University Institutional Animal Care and Use Committee Protocol number IACUC 08/09.F.82-A. 11

12 Collected fish were taken to the fish pathology laboratory at Humboldt State University. Once at the lab, fish were either left on ice or transferred to a refrigerator for temporary storage. All fish were processed within 48 hours of capture. Location of capture, date of capture, total length, and sex were recorded. Fish were examined externally for any abnormalities or parasites. External parasites were placed directly into lake water. Blood smears were taken directly from the heart to minimize any contamination with parasites from adjacent tissues and organs (Pritchard and Kruse 1982). Blood smears were stained in Wright-Giesma Stain according to Woronzoff- Dashkoff (2002). Slides were placed in stain for 40 minutes. They were then removed from stain and placed directly into Wright-Giemsa buffer for 20 minutes. Slides were then removed from the buffer, gently rinsed with tap water to remove excess stain and buffer and left to air dry. Blood smears were then viewed using a Leica DME microscope using oil immersion 1000x magnification. Each slide was systematically scanned for five mintues, concentrating on the feathered end of the smear (Pritchard and Kruse 1982). Gill arches were removed, placed into a dish of water from the location of collection, and examined under a dissecting microscope. Any parasites collected from the gills were placed into a small dish of cold-blooded Ringers saline solution and then set aside for later fixation and staining (Pritchard and Kruse 1982). Internal organs were thoroughly examined for parasites. Internal organs were completely removed from the fish. First the stomach was cut away from the esophagus of the fish. Then the liver was cut away from pericardial cavity and heart. Lastly, the posterior intestine was cut from the vent and the entire visceral mass was removed from

13 the fish. Internal organs including the liver, spleen, kidney, gonads and gas bladder were separated and examined either by unaided eye or under a dissecting microscope. Eyes were removed and examined under a dissecting microscope (Pritchard and Kruse 1982). Special attention was paid to the digestive tract. First the intestine was separated from the stomach and pyloric caeca. The intestine was separated into upper, middle and lower thirds. Each section was opened and thoroughly scraped into a dish of coldblooded Ringers saline solution. Visible parasites were immediately removed. This mixture was then decanted several times and examined under a dissecting microscope for any remaining parasites (Pritchard and Kruse 1982). The stomach was cut open and examined grossly. It was then scraped, and the gut contents were emptied into a dish of cold-blooded Ringers saline solution. The mixture was decanted several times and examined under a dissecting microscope. The exterior surface of pyloric caeca was thoroughly examined for juvenile nematodes and cestodes. Pyloric caeca were then cut open and the contents were emptied into a dish of coldblooded Ringers saline solution and examined for parasites. The liver, gonads, spleen and gas bladder were all examined for encysted parasites. Musculature was examined primarily for encysted trematodes. Muscle was removed from the skin and viewed by holding it in front of a bright light source to locate any encysted parasites (Pritchard and Kruse 1982). Immediately after collection, acanthocephalans and cestodes were rinsed in coldblooded Ringers saline solution to remove any contaminant material. Prior to fixation, acanthocephalans were placed in distilled water and put under refrigeration for 12 hours

14 to extend the proboscis. Prior to fixation, cestodes were placed in distilled water for approximately 30 minutes (depending on size) to allow for relaxation and expulsion of eggs. Nematodes were placed in cold-blooded Ringers saline solution. Monogeneans, digeneans, cestodes, acanthocephalans, crustaceans, or hirudineans were fixed in hot AFA (alcohol/formalin/acetic acid). Larger parasites were fixed under light coverslip pressure to keep them flat. Cestodes were fixed by extending them on a flat surface and pipetting hot AFA over their length. Specimens were fixed in AFA for 24 hours and then transferred to 70% ethanol (Pritchard and Kruse 1982). All specimens except for nematodes were stained in Semichon s carmine (Cable 1977) for eight to twelve hours. Specimens were then de-stained in acid alcohol (1.5% hydrochloric acid in 70% ethanol). De-staining times varied based on size of the specimen and intensity of staining. De-staining was neutralized using basic alcohol (1.5% potassium acetate in 70% ethanol) for ten minutes. Specimens were then dehydrated in 95% ethanol for ten minutes. They were then counter-stained in fast green diluted in 95% ethanol. After counter-staining, specimens were fully dehydrated in three changes of absolute (100%) ethanol. Once completely dehydrated, they were cleared in methyl salicylate (synthetic oil of wintergreen) and mounted on slides with Permount with a single cover slip (Cable 1977). Some monogeneans collected from Clear Lake were cleared in Grey-Weiss solution. This allowed for clear viewing of the haptoral hooks. Cleared specimens were covered with a single cover slip and sealed with clear nail polish.

15 Nematodes were fixed and stored in hot GL-70 (glycerine in 70% ethanol). The alcohol was allowed to evaporate so that glycerine cleared the specimen. Nematodes were then mounted in glycerine jelly between two coverslips. Coverslips were then mounted on slides with Permount (Cable 1977). Two nematodes were too big to completely clear in GL-70. These specimens were cleared in a saturated solution of phenol crystal in 95% ethanol (Hendrickson 2010, personal communication). Nematodes were fully cleared within seven days and were permanently stored in this solution. Once mounted, specimens were viewed using a Ziess compound microscope, or a Leica DME phase-contrast microscope. Standard light microscopy was used for all stained specimens, while phase-contrast microscopy was used to view all unstained monogeneans and nematodes. Identification of parasites utilized total magnification levels ranging from 40x to 1000x. Magnification depended on size of the specimen and what identifying characteristics were being viewed. Identification of parasites were made using original descriptions, keys, taxonomic summaries and published articles including Cort (1913), Schmidt (1970), Jilek and Crites (1982b), Beverly-Burton (1984, 1986), Klemm (1985), Kabata (1988), Arai (1989), Beverly-Burton and Klassen (1990) and Hoffman (1999). Parasites were photographed using a Nikon Coolpix 8800 digital camera equipped with a microscope adapter (Martin Microscopes, Easley, South Carolina).

RESULTS For this study, I collected a total of 50 largemouth bass. I collected 13 from the Sacramento San Joaquin River Delta, 13 from Clear Lake, ten from Lake Berryessa, 11 from Lake Sonoma and three from Trinity Lake. All fish were at least 12 inches in total length, in accordance with the state mandated minimum size limit for rivers, lakes and reservoirs except the three fish from Trinity Lake (provided to me by Monty Currier, California Department of Fish and Game). All bass collected for this study were infected by at least one parasite, with the exception of one fish from Trinity Lake. Forty-eight of 50 (96%) fish in the study were infected with two or more species of parasite. No one fish was infected with more than six species of parasite. Intensity of infections ranged widely between species. Some parasites were common, while other species were rare (Table 2). I did not find any parasites infecting blood of largemouth bass. Two species of monogenean were found; Actinocleidus unguis (Figure 2) and Clavunculus bursatus (Figure 3). Both species of monogenean were found on the gills in between gill filaments. Actinocleidus unguis was found on three of 13 (23.1%) fish from Clear Lake. Fish were infected with five to 12 individuals (x = 8.6). Only two individuals of Clavunculus bursatus were found in one of ten (10%) fish from Lake Berryessa. 16

17 Table 2. Abundance of parasite species recovered from largemouth bass at five collection locations: Trinity Lake, Clear Lake, Lake Sonoma, Lake Berryessa and the Sacramento San Joaquin River Delta. # of fish infected (% infected) [range: low to high # parasites per infected fish (mean # parasites per infected fish)]. Monogenea Parasite species Clear Lake n=13 Lake Berryessa n=10 Lake Sonoma n=11 Trinity Lake n=3 Actinocleidus unguis 3(23.1%) [5-12(8.6)] - - - - S-SJ Delta n=13 Clavunculus bursatus - 1(10%) [2(2)] - - - Digenea Clinostomum complanatum - - - - 3(23.1%) [1-6(3)] Cestoidea Proteocephalus pearsei (adult) 3(23.1%) [2-6(3.6)] - - - - Proteocephalus pearsei (immature) - 3(30%) [1-6(3)] - - 1(7.7%) [2(2)] Proteocephalus 1(7.7%) [5(5)] 6(60%) [1-47(17.2)] 2(18.1%) [9-16(12.5)] 1(33.3%) [3(3)] 6(46.2%) [7-25(15.3)] (plerocercoid)

18 Table 2. Abundance of parasite species recovered from largemouth bass at five collection locations: Trinity Lake, Clear Lake, Lake Sonoma, Lake Berryessa and the Sacramento San Joaquin River Delta. # of fish infected (% infected) [range: low to high # parasites per infected fish (mean # parasites per infected fish)] (continued). Nematoda Contracaecum sp. 13(100%) [1-10(100%) [2-10(90.9%) [1-58(15.2)] 2(66.6%) [3-13(100%) [1-100 s(*)] 100 s(*)] 61(16.8)] 7(5)] Hysterothylacium (?) sp. - - - - 1(7.7%) [2(2)] Spinitectus carolini - - 3(27.3%) [2-3(2.3)] - 1(7.7%)[2(2)] Camallanus sp. 1(7.7%) [1(1)] - - - - Acanthocephala Neoechinorhynchus cylindratus 4(30.7%) [3-5(50%) [6-28(16.4)] 9(81.8%) [3-12(6.9)] - 7(53.8%) [1-30(11.8)] 68(22.75)] Hirudinea Myzobdella lugubris 1(7.7%) [1(1)] 3(30%) [1-2(1.3)] - - 2(15.4%) [1(1)] Batracobdella phalera - - - - 2(15.4%) [1(1)] Unidentified leech - - - - 1(7.7%) [1(1)] Copepoda Ergasilus centrarchidarum - - - - 4(30.7%)[1-2(1.5)] Branchiura Argulus flavescens 1(7.7%) [1(1)] - - - - *value not calculated

19 50 µm Figure 2. Haptoral hooks of Actinocleidus unguis from gills of largemouth bass collected from Clear Lake, Lake County, California, March 2009. Note the presence of four large anchors, used to attach to the gills of the host. Specimen was cleared using Gray and Weiss solution.

20 200 µm Figure 3. Clavunculus bursatus from gills of largemouth bass collected from Lake Berryessa, Napa County, California, November 2009. Specimen was stained with Semichon s carmine and counterstained with fast green.

21 Clinostomum complanatum (syn. Clinostomum marginatum) (Figure 4) was found in three of 13 (23.1%) fish from the Sacramento San Joaquin River Delta. Intensity of infection varied. One fish had one individual encysted in the body cavity. The second fish had two individuals encysted in the fins and the body cavity. The third fish had six individuals encysted in the gills and fins. Cysts were yellow in color, hence the common name yellow grub. Adult Proteocephalus pearsei (Figure 5) were found in three of 13 (23.1%) fish from Clear Lake. A total of 11 individuals were found. Infected bass had from two to six adult parasites (x =3.6). All Proetecephalus pearsei were found in the intestine. I found several individuals which exceeded 50 cm in length, with one individual measuring 75 cm in length. Immature adults of Proteocephalus pearsei (Figure 6) were found in three of ten (30%) fish from Lake Berryessa and one of 13 (7.7%) fish from the Sacramento San Joaquin River Delta. Intensity of infection varied. The one fish from the Delta had only two individuals, while the fish from Berryessa had one to six individuals (x =3). All immature Proteocephalus pearsei were found in the intestine. Individuals measured approximately 4 to 8 cm in length. Proglottidization was not evident and internal organs were not visible. Proteocephalus sp. plerocercoids (Figure 7) were found in one of 13 (7.7%) fish from Clear Lake, six of ten (60%) fish from Lake Berryessa, two of 11 (18.1%) fish from Lake Sonoma, one of three (33.3%) fish from Trinity Lake and six of 13 (46.2%) fish from the Sacramento San Joaquin River Delta. Plerocercoids were found mainly in the mesentaries of the fish. There appeared to be no site specificity, as they were found

22 550 µm Figure 4. Clinostomum complanatum from gills of largemouth bass collected from the Sacramento San Joaquin River Delta San Joaquin County, California, October 2009. Specimen was stained with Semichon s carmine and counterstained with fast green.

23 200 µm Figure 5. Scolex of Proteocephalus pearsei adult from intestine of largemouth bass collected from Clear Lake, Lake County, California, March 2009. Specimen was stained with Semichon s carmine and counterstained with fast green.

24 130 µm Figure 6. Scolex of Proteocephalus pearsei immature adult from intestine of largemouth bass collected from the Sacramento San Joaquin River Delta, San Joaquin County, California, October 2009. Specimen was stained with Semichon s carmine and counterstained with fast green.

25 140 µm Figure 7. Scolex of Proteocephalus sp. plerocercoid from mesenteries of largemouth bass collected from Clear Lake, Lake County, California, March 2009. Specimen was stained with Semichon s carmine and counterstained with fast green.

26 throughout the body cavity. In one fish from Clear Lake, they were also found in the intestine. Proteocephalus sp. plerocercoids were significantly smaller in size compared to immature Proteocephalus pearsei, with individuals ranging from 5-30 mm. Contracaecum sp. (Figure 8) was found in 47 of the 50 (94%) fish collected. It was collected from fish from all five collection locations. Infection intensity ranged from fish having only a single individual, to a few fish that were infected with over 100 individuals. All individual Contracaecum sp. were found either encysted or free in the mesenteries of the fish. Two juvenile Hysterothylacium (?) sp. (Figure 9) were found on one of 13 (7.7%) fish from the Sacramento San Joaquin River Delta. These were large nematodes. One individual measured 75 mm and the other measured 60 mm. Spinitectus carolini (Figure 10) was found in one of 13 (7.7%) fish from the Sacramento San Joaquin River Delta and in three of 11 (27.3%) fish from Lake Sonoma. A total of nine S. carolini were collected, two were from the Delta and seven were from Lake Sonoma. Infected fish from both locations had an average of 2.2 S. carolini per infected fish. All S. carolini were collected from the anterior intestine of the fish. Both males and gravid females were found. Male and females can be easily differentiated, as males have spirally coiled tails (Figure 11). Only one individual of Camallanus sp. (Figure 12) was found from one of 13 (7.7%) fish from Clear Lake. It was found in the posterior intestine of the fish. Neoechinorhynchus cylindratus (Figure 13) was found in twenty-five of fifty (50%) fish collected in this study. It was found in fish from all locations except

27 250 µm Figure 8. Anterior (featuring intestinal caecum) of juvenile Contracaecum from mesentaries of largemouth bass collected from Clear Lake, Lake County, California, September 2009. Specimen was cleared in glycerine alcohol and mounted in glycerine jelly.

28 250 µm Figure 9. Anterior of Hysterothylacium (?) sp. from stomach of largemouth bass collected from the Sacramento San Joaquin River Delta, San Joaquin County, October 2009. Specimen was cleared in a saturated solution of phenol crystal in 95% ethanol.

29 50 µm Figure 10. Anterior of female Spinitectus carolini from intestine of largemouth bass collected from the Sacramento San Joaquin River Delta, San Joaquin County, California, October 2009. Specimen was cleared in glycerine alcohol and mounted in glycerine jelly.

30 200 µm Figure 11. Posterior of male Spinitectus carolini from intestine of largemouth bass collected from Lake Sonoma, Sonoma County, California, November 2009. Specimen was cleared in glycerine alcohol and mounted in glycerine jelly.

31 BC 150 µm Figure 12. Anterior of Camallanus sp. from intestine of largemouth bass collected from Clear Lake, Lake County, California, September 2009. Note the chitinous buccal capsule (BC). Specimen was cleared in glycerine alcohol and mounted in glycerine jelly.

32 600 µm Figure 13. Male Neoechinorhynchus cylindratus from intestine of largemouth bass collected from the Lake Berryessa, Napa County, California, November 2009. Specimen was stained with Semichon s carmine and counterstained with fast green.

33 Trinity Lake. Intensity of infection across all locations ranged from one to sixty-eight, with an average of 14.5 parasites per infected fish. There was an average of 2.8 females for every 1 male. Counts and measurements of hooks were all within the published measurements for Neoechinorhynchus cylindratus (Figure 14) (Arai 1989). Myzobdella lugubris (Figure 15) was found on a total of six fish collected in this study. It was found on one of 13 (7.7%) fish from Clear Lake, two of 13 (15.4) fish from the Sacramento San Joaquin River Delta and three of ten (30%) fish from Lake Berryessa. Intenstiy of infection was low, with infected fish having two or fewer individuals (x =1.2). Myzobdella lugubris was found attached to fins and (or) skin of the fish, usually on the inside of the pectoral fins or below the lower jaw. Myzobdella lugubris is known to exhibit several body forms. The single individual found at Clear Lake exhibited one of the alternate body forms (a wide body with a trunk-like anterior end) of the species (Figure 16). Two specimens of Batracobdella phalera (Figure 17) were found. They were attached to the fins of two of 13 (15.4%) fish from the Sacramento San Joaquin River Delta. Batracobdella phalera is characterized by fused eye spots on the anterior end and a white or lightly pigmented collar towards the anterior end (Figure 18). One unidentified leech (Figure 19) was found on the gills off a fish from the Sacramento San Joaquin River Delta. It exhibited similar body form and sucker placement as Batractobdella phalera, but a positive identification could not be made. Ergasilus centrarchidarum (Figure 20) was collected from the gills of four of 13 (30.8%) fish from the Sacramento San Joaquin River Delta. Intensity of infection was

34 25 µm Figure 14. Proboscis of female Neoechinorhynchus cylindratus from intestine of largemouth bass collected from the Sacramento San Joaquin River Delta, San Joaquin County, California, October 2009. Note the proboscis has three circle of hooks with six hooks in each circle. Specimen was stained with Semichon s carmine and counterstained with fast green.

35 150 µm Figure 15. Anterior of Myzobdella lugubris (featuring separate eye spots) from fins of largemouth bass collected from Lake Berryessa, Napa County, California, November 2009. Specimen was stained with Semichon s carmine and counterstained with fast green.

36 1 mm Figure 16. Anterior of Myzobdella lugubris (alternate body form) from pectoral fin of largemouth bass collected from Clear Lake, Lake County, California, March 2009. Alternate body form consists of a narrow, trunk-like anterior and a robust body. Specimen was stained with Semichon s carmine and counterstained with fast green.

37 600 µm Figure 17. Batractobdella phalera from fins of largemouth bass collected from the Sacramento San Joaquin River Delta, San Joaquin County, California, October 2009. Specimen was stained with Semichon s carmine and counterstained with fast green.

38 140 µm Figure 18. Fused eye spots of Batractobdella phalera from fins of largemouth bass collected from the Sacramento San Joaquin River Delta, San Joaquin County, California, October 2009. Specimen was stained with Semichon s carmine and counterstained with fast green.

39 200 µm Figure 19. Unidentified leech (B. phalera (?)) from gills of largemouth bass collected from the Sacramento San Joaquin River Delta, San Joaquin County, California, October 2009. Specimen was stained with Semichon s carmine and counterstained with fast green.

40 250 µm Figure 20. Ergasilus centrarchidarum from gills of largemouth bass collected from Sacramento San Joaquin River Delta, San Joaquin County, California, October 2009. Specimen was stained with Semichon s carmine and counterstained with fast green.

41 low with one to two individuals collected per infected fish (x =1.5). Ergalisus centrarchidarum were identified by the broad antennae and the inflation between the first and second segments of the second antenna (Kabata 1988) (Figure 21). Only E. centrarchidarum females are parasitic. All E. centrarchidarum found in this study were gravid females. Only one individual of Argulus flavescens (commonly known as fish louse ) (Figure 22) was found from one of 13 (7.7%) fish from Clear Lake. I do not know where it was attached to the fish, as it was found in the ice and water in which the fish were transported.

42 50 µm Figure 21. Secondary antenna of Ergasilus centrarchidarum from gills of largemouth bass collected from Sacramento San Joaquin River Delta, San Joaquin County, California, October 2009. Note the inflation between the first and second segments of the secondary antenna. Specimen was stained with Semichon s carmine and counterstained with fast green.

43 600 µm Figure 22. Argulus flavescens from largemouth bass collected from Clear Lake, Lake County, California, March 2009. Specimen was stained with Semichon s carmine and counterstained with fast green.

DISCUSSION Largemouth bass are hosts to a variety of parasites (Hoffman 1999). This is probably because bass can survive in many different environments. Some parasites collected in this study (Clinostomum complanatum, Myzobdella lugubris and Contracaecum sp.) show little host specificity. These species commonly infect other species of fish and C. complanatum and M. lugubris have been found infecting amphibians as well (Hoffman 1999). Host-specific parasites such as Actinocleidus unguis and Clavunculus bursatus, are found infecting a single species or group of closely related species (Collins and Janovy 2003). Mizelle and Crane (1964) described Actinocleidus unguis from largemouth bass in California. It seems likely that it was introduced with largemouth bass to California. Actinocleidus unguis is known only to infect fish of the family Centrarchidae (Micropterus spp., Lepomis spp., etc.), but the extent of host specificity within the family is not known. Infection intensity of monogeneans is often at least partly due to seasonal fluctuations in parasite population abundance (Fellis and Esch 2004). I found Actinocleidus unguis only at Clear Lake in spring. Typically, monogenean populations bloom in the spring and summer, and die back in the fall and winter (Fellis and Esch 2004). However, when Clear Lake was sampled again in August, no monogeneans were found. This is probably due to the relatively small sample size. Clavunculus bursatus was found in very low numbers and only at Lake Berryessa. The low numbers of parasites is likely due to the seasonal population fluctuations of monogeneans, where their population decreases in fall. This is a time when monogenean 44

45 populations are usually very low (Fellis and Esch 2004). Clavunculus bursatus is known only to infect centrarchids. It is likely that Clavunculus bursatus was introduced with largemouth bass to California, along with Actinocleidus unguis, as suggested by Mizelle and Crane (1964). Clavunculus bursatus has been previously reported from largemouth bass. To my knowledge, this is the first report of Clavunculus bursatus in California. Clinostomum complanatum shows very little host specificity. It is known to infect many species in many families of fish (Hoffman 1999). The life cycle of Clinostomum complanatum is very complex. Adults release eggs, which are passed out through the feces or saliva of a piscivorous bird. Eggs hatch in water releasing a miracidium, which infects a freshwater snail. The parasite develops into a mother sporocyst, which produces rediae, and subsequently cercariae. Cercariae are released from the snail and penetrate and encyst in fish as metacercariae. Metacercariae in fish are commonly known as yellow grub (Cort 1913). The infected fish is then eaten and the worm infects the mouth cavity of the bird (Hoffman 1999). This complex life cycle, which includes a piscivorous bird as the definitive host, contributes to the fact that it is a broadly distributed and highly cosmopolitan parasite. It also has great potential for dispersal (Hazen and Esch 1978). In the fish second intermediate host seasonal variation in abundance has not been reported (Ingram and Dronen 1982). This is probably due to the fact that metacercariae are fairly long lived (Cort 1913). Clinostomum complanatum has been widely reported in largemouth bass. It has also been previously reported in California (Haderlie 1953). However, it has never been reported from largemouth bass in California (Hoffman 1999).

46 Proteocephalus pearsei has been described from largemouth bass and several other species of fish, including smallmouth bass, yellow perch (Perca flavescens), black crappie (Pomoxis nigromaculatus) and rainbow trout (Oncorhynchus mykiss) (Hoffman 1999). Proteocephalan cestodes utilize copepods as first intermediate hosts, but the species of copepod varies. Largemouth bass are likely infected in one or both of two ways. Either bass actively prey upon copepods, and(or) bass eat smaller fish (which acts as a second intermediate host) and the cestode is transmitted to the bass (Schmidt 1970). It is possible that the life cycle of Proteocephalus pearsei is similar to that of Proteocephalus ambloplitis, in which case, juvenile cestodes migrate into the viscera of the host and reside there as plerocercoids. Plerocercoids then migrate into the intestine. They grow to adults and reproduce, releasing mature proglottids to which are passed out of the fish in the feces (Fischer and Freeman 1969). Proteocephalus pearsei has been reported in largemouth bass. This is the first report of Proteocephalus pearsei in California that I am aware of. Immature adults have been reported from several species of fish, but not from largemouth bass (Hoffman 1999). The immature individuals I found did not exhibit distinct proglottidization. Proteocephalus sp. plerocercoids have been reported in many fish species (Hoffman 1999). In my study, intensity of infection varied greatly. There was a greater average abundance and higher prevalence of infection of plerocercoids in Lake Berryessa and the Sacramento San Joaquin River Delta, than the other three locations. However, Lake Berryessa and the Delta are dissimilar habitats. Habitat characteristics are probably not a direct factor in tapeworm abundance, but moreso a factor in abundance of first

47 intermediate host copepods. Parasite abundance and infection intensity could be attributed to several factors, such as small sample size, seasonal variation in tapeworm abundance and(or) relative abundance of first or second intermediate hosts. It is likely that the variation in infection intensity is attributed to ecological parameters, as tapeworms induce little immunity in the host (Schmidt 1970). Some fish were infected with Proteocephalus pearsei and Proteocephalus sp. plerocercoids. Thus, it is likely that at least some of the plerocercoids were P. pearsei. Proteocephalus sp. plerocercoids have been reported in California (Haderlie 1953) and in largemouth bass, however, they have not been reported from largemouth bass in California. (see Hoffman 1999 for a complete review). Contracaecum sp. was found in forty-seven of fifty fish sampled. High prevalence of infection with Contracaecum is common in many populations of largemouth bass, with infection prevalence often exceeding 90% (Szalai and Dick 1990). Contracaecum sp. has been reported in several species of fish throughout North America (Hoffman 1999), and it is suggested that juveniles do not exhibit fish host specificity. Definitive hosts for Contracaecum sp. are piscivorous birds. This helps explain why Contracaecum sp. is such a highly cosmopolitan parasite. Because of this, it is likely that Contracaecum was already established in California. In my study infection intensity varied greatly from fish to fish (Table 2). This variation could be attributed to several factors such as abundance of first intermediate host, environmental conditions to support juveniles before infecting first intermediate hosts, or the likelihood of bass eating a first intermediate or paratenic host. The first intermediate host for Contacaecum is a