THE BIOLOGY AND HISTOPATHOLOGY OF Proteocephalus ambloplitis LEIDY, 1887 (CESTODA; PROTEOCEPHALIDAE) INFECTING WALLEYE ( Stizostedion v i tr eu^n vi tr eum ) AND YELLOW PERCH ( Perea flavescens ) IN LAKE OF THE WOODS, ONTARIO A Thesis presented to The Faculty of Graduate Studies o f Lakehead University by KIMBERLY BLYTHE ARMSTRONG In partial fulfilment of the requirements for the degree of Master of Science March 1985 ProQuest Number: 10611713 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Pro ProQuest 10611713 Published by ProQuest LLC (2017). Copyright of the Dissertation is held by the Author. All rights reserved. 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ISBN 0-315-31681-0 ABSTRACT Walleye ( S tizos tedion vitreum vitr eum ) and yellow perch ( Perea flavescens ) from Lake of the Woods were examined for parenteral Proteocephalus ambloplitis from May to November, 1982 and 1983. One hundred percent of the age 1 and older walleye and 747o of the age 1 and older yellow perch harboured p1erocercoids. In corresponding age classes, walleye were generally 10 times more heavily infected than yellow perch. Mean intensity of live p1erocercoids increased with age of walleye until age class 5 then declined significantly in older fish. Intensity increased from a mean of 23 (in age class 0) to a maximum of 171 in age class 5 and was only 58 in the 7+ age class. Mean intensity of plerocercoids increased continuously with the age of yellow perch from 2 (age class 0) to a maximum of 20 in the 5+ age class. All age classes of walleye (0 to 7+) became infected by preying on yellow perch, particularly the young-of-the-year (YOY). Small yellow perch became infected by eating copepods but older perch obtained plerocercoids by cannibalism. The transmission of plerocercoids to walleye and yellow perch was greatest during late summer. Young-of-the-year walleye and YOY yellow perch first harboured plerocercoids in early Augus t. The liver was the first organ of walleye and yellow perch to be invaded by migrating plerocercoids. However, in walleye, the mesenteries ultimately contained the greatest proportion of p1erocercoids in age 1 and older fish. The liver remained a relatively Important site for p1erocercoids in all age classes of yellow perch. Relatively few p1erocercoids were found in the gonads of walleye or yellow perch. Walleye fecundity was not correlated with plerocercoid intensity. Ninespine sticklebacks ( Fungitiu s pungitiu s ) and logperch ( Percina caprodes ) were found to harbour P. ambloplitis p1erocercoids . These are new host records. Migrating p1erocercoids caused the greatest pathological change in the liver of walleye and yellow perch. Zones of compressed and necrotic hepatocytes were evident adjacent to live, unencapsulated pierocerco ids . The mesenteries of walleye were often fibrosed in response to large numbers of invading plerocercoids. The gastro-intestina1 tract, posterior gonads and associated mesenteries were often compacted with fibrous tissue. Obstruction of the passage of gametes is possible. Constriction of the oviduct may result from the fibrous reaction. Also, encapsulated plerocercoids were found in the lumen of the oviduct creating a physical barrier. The wall of capsules encompassing plerocercoids in walleye was relatively thin (maximum of 90 microns) while in yellow perch, it was up to 290 microns thick. ACKNOWLEDGEMENTS I would like to extend my gratitude to my supervisor, Dr. M. W. Lankester, for his contributions to, and criticisms of this thesis. As well, I would like to thank the members of my committee. Dr. W. T. Momot, Dr, P. J. Colby and Mr. V. Macins for their time and advice during the past 2-plus years. The Ministry of Natural Resources, Kenora District, Fish and Wildlife Division also contributed significantly to this project. The field work could not have been accomplished without the kind support of Val Macins and the rest of the Lake of the Woods F. A. U. The F. A. U. kindly lent equipment, donated boat time, collected additional specimens from various parts of the lake and provided accomodation at French Portage Narrows. The F. A. U. also aged the fish caught during 1982. Beverlee Ritchie found some time during her own thesis work to age the fish caught during 1983, for which I am very grateful. The time and effort volunteered by Jann Atkinson and Scott Lockhart during the field seasons 1982-83 is recognized and greatly appreciated. Without their help much of this work would not have been completed. Ms. L. Hauta kindly advised on statistical methods and computer workings and Dr. A. MacDonald helped enormously with histological procedures, their contributions are greatly appreciated. Many thanks are extended to Carol Martin for typing and sometimes re-typing the enclosed - i - tables . Finally I would like to thank my parents and my friends in Thunder Bay, and elsewhere, for their support and encouragement during all phases of this thesis. TABLE OF CONTENTS PAGE ACKNOWLEDGEMENTS i LIST OF FIGURES v LIST OF TABLES vii INTRODUCTION 1 MATERIAL AND METHODS 5 S tudy Area 5 Examination of Fish for Parenteral P, amb loplitis 5 Age 1 and older walleye and yellow perch 5 Young-of-the-year walleye and yellow perch 9 Forage fish 10 Feeding Analysis 11 Examination of Fish for Enteral Cestodes 11 Fecund ity 13 Histopatho1ogy 14 RESULTS 15 Parenteral P. ambloplitis 15 Walleye 15 Yellow perch 30 Young-of-the-year walleye and yellow perch ....... 43 Forage fish 51 Feeding Analysis 53 Enteral Cestodes 63 Fecund ity 6 6 - i i i - TABLE OF CONTENTS (Continued) Histopatho1ogy 66 Gross description 66 Light microscopy 71 Liver 71 Mesenteries 73 Gastro-intestinal tract 73 Spleen and pancreas 75 Gonads 75 DISCUSSION 78 Biology of P. amb loplitis 78 Histopathology 103 LITERATURE CITED 109 iv LIST OF FIGURES Figure Page 1. Map of Lake of the Woods showing the borders of the s tudy area 6 2. Mean intensity of parenteral P. ambloplitis infecting age 1 and older walleye 17 3. Mean intensity of parenteral P. ambloplitis infecting age 1 and older walleye in relation to month of sampl ing 19 4. Percent of total parenteral P. ambloplitis invading each visceral organ of walleye 20 5. Percent of total parenteral P. amb1o p1itis invading each visceral organ of walleye in relation to month of samp 1 ing 2 2 6. Frequency of occurrence of parenteral P. ambloplitis intensities in age 1 and older walleye 29 7. Mean intensity of parenteral P. ambloplitis infecting age 1 and older yellow perch 32 8. Mean intensity of parenteral P. ambloplitis infecting age 1 and older yellow perch in relation to month of samp 1 ing 34 9. Percent of total parenteral ambloplitis invading each visceral organ of yellow perch 35 10. Percent of total parenteral P. ambloplitis invading each organ of yellow perch in relation to month of s amp 1 ing 3 6 11. Frequency of occurrence of parenteral P. ambloplitis intensities in age 1 and older yellow perch, 44 12. Fibrosis of the stomach wall and connecting mesenteries with adhesions to pyloric ceacae and liver 70 13. Fibrosis of mesenteries including the lower intestine and posterior gonads 70 14. A light plerocercoid infection in a walleye 70 15. Encapsulated p1erocercoids in the mesenteries of a yellow perch 70 - V - LIST OF FIGURES (Continued) 16. Enlarged view of figure 15 70 17. Liver necrosis adjacent to a live plerocercoid in a wa II eye 72 18. Extensive hepatic necrosis in a walleye 72 19. Plerocercoid in the mesenteries of a YOY walleye 72 20. Concentration of inflammatory cells adjacent to dead and encapsulated p 1 e r o c er c o i d s 72 21. Live and dead p 1 erocercoids in the mesenteries of an older walleye 74 22. Accumulation of dead p1erocercoids in the mesenteries of a wa Ileye 74 23. Thick capsule wall surrounding a plerocercoid in the mesenteries of a yellow perch 74 24. Encapsulated p1erocercoids in the stomach wall of a wa Ileye 74 25. A migrating plerocercoid in the spleen of a walleye. .. 76 26. Ruptured pancreatic cells adjacent to a migrating plerocercoid in the liver of a walleye 76 27. Oocytes caugt in the fibrous tissue encompassing some dead p1erocercoids in the ovary (walleye) 76 28. Encapsulated plerocercoid in the anterior oviduct of a wa Ileye 76 - vi - LIST OF TABLES Table Page 1. Prevalence and mean intensity of parenteral P. ambloplitis pIerocercoids in age one and older wa Ileye 16 2. Mean number of parenteral P. amb1op1itis p1erocercoids in visceral organs of age one and older walleye 23 3. Mean number of parenteral P. ambloplitis p1erocercoids in visceral organs of age one and older walleye in relation to month of sampling 25 4. Percent occurrence of parenteral P. ambloplitis p1erocercoids in visceral organs of age one and older wa Ileye 26 5. Percent occurrence of parenteral P. amb1op1itis p1erocercoids in visceral organs of age one and older walleye in relation to month of sampling 28 6. Prevalence and intensity of parenteral P. ambloplitis plerocercoids in age one and older yellow perch 31 7. Mean number of parenteral P. amblop litis plerocercoids in visceral organs of infected age one and older yellow perch 38 8. Mean number of parenteral P_^ amblop litis plerocercoids in visceral organs of infected age one and older yellow perch in relation to month of sampling. .. 40 9. Percent occurrence of parenteral P^_ ambloplitis in visceral organs of infected age one and older yellow perch 41 10. Percent occurrence of parenteral P. ambloplitis plerocercoids in visceral organs of infected age one and older yellow perch in relation to month of s amp 1 ing 4 2 11. Prevalence and intensity of parenteral P_^ ambloplitis p 1 er o c er CO i d s in YOY walleye 45 12. Percent of total parenteral P. ambloplitis p1erocercoids in visceral organs of infected YOY walleye 46 - vl i - LIST OF TABLES (Continued) 13. Mean number of parenteral P . amblo plitis p1erocerocids in visceral organs of infected YOY wal leye 47 14. Percent occurrence of parenteral P_^ ambloplitis p1erocercoids in visceral organs of infected YOY walleye 49 15. Prevalence and intensity of parenteral P. ambloplitis p 1 er oc e r c o i d s in YOY yellow perch 50 16. Prevalence and intensity of parenteral P. ambloplitis recovered from forage fish 52 17. Percent occurrence of prey items in stomachs of age one and older walleye 55 18. Percent total volume of prey items in stomachs of age one and older walleye 56 19. Percent occurrence of prey items in stomachs of age one and older yellow perch 58 20. Percent total volume of prey items in stomachs of age one and older yellow perch 60 21. Percent occurrence of prey items in stomachs of YOY wa 11 eye 62 22. Percent occurrence of prey items in stomachs of YOY yellow perch 64 23. Prevalence and intensity of enteral cestodes from three fish species 65 24. Mean fecundity of walleye 67 25. Linear regression formulae for relationships between fecundity and length, weight and age of walleye 68 26. Bibliographic summary of parenteral P. ambloplitis in North American fish 79 27. Bibliographic summary of enteral P. ambloplitis in North American fish 100 -viii- INTRODUCTION Smallmouth bass ( Micropterus dolomieui ), and presumably Proteocephalus amb1o p1itis Leidy, 1887 , were not known in Lake of the Woods prior to 1920. An initial survey of fishes from Lake of the Woods did not find smallmouth bass in these waters (Evermann and Latimer 1910). Smallmouth bass were first intoduced into Lake of the Woods during the early 1920's and they were probably infected with P. ambloplitis . Since the initial introductions, the fish and parasite both, have flourished. In fact, many non-bass species in Lake of the Woods now harbour the larvae of P. ambloplitis (Dechtiar 1972). The presence of P. ambloplitis larvae in the body cavity of walleye ( Stizostedion vitreum vitreum ), the most economically important species in Lake of the Woods, has become a concern in recent years (V. Macins pers. comm.). The accepted life history of P. ambloplitis , which includes an obligate parenteral stage in the viscera of its definitive host, was determined by Fischer (1972). Eggs shed into the water column are consumed by copepods (Hunter 1928; Hunter and Hunter 1929; Fischer 1972). In the haemocoel of copepods the oncosphere develops to a plerocercoid I (terminology of Freeman 1964). Infected copepods are then eaten by many species of fish, including young bass, where the plerocercoid penetrates into the body cavity, is encapsulated and grows to a plerocercoid II. The life cycle -1- -2- is completed when a large bass consumes an infected small fish. If a large bass eats an infected small bass the plerocercoid II may mature directly in the intestine or it may enter the viscera of the large bass. But, if a large bass eats an infected non-bass fish then the plerocercoid II must enter the viscera of the definitive host. It can not mature directly in the intestine. Fischer and Freeman (1973) determined that P. ambloplitis p1erocercoids have an obligate parenteral development in bass before they can ma ture. The p lerocercoids in the viscera of large bass are not at a dead end as was originally believed by Cooper (1915) and Hunter (1928). Parenteral plerocercoids can be stimulated to leave their parenteral sites and migrate back to the intestine. Fischer and Freeman (1969) determined that an increase in water temperature from 4 to 7^C was the required stimulus in the laboratory as well as in smallmouth bass from Lake Opeongo. However, the migration of parenteral plerocercoids into the intestine was noted to occur only in mature bass suggesting the hormonal state of the host may enhance the effect of temperature (Fischer and Freeman 1969). Esch et al. (1975) also found that plerocercoid migration only occurred in mature bass from Michigan, however, the migration was stimulated at higher temperatures (14°C or greater) than reported by Fischer and Freeman (1969). Esch et al. (1975) then stressed the possibility that the migratory stimulus was hormonal, and -3- not temperature dependent. At present, the controversy remains unresolved. Apparently, P. amb1op litis can survive only where smallmouth and/or largemouth bass ( Micropterus salmoides ) reside (Fisher and Freeman 1973; Eure 1976; Freeman pers. comm.). Therefore, smallmouth and largemouth bass which are probably the only definitve hosts, will be referred to as 'natural' hosts. Other fish species in which there is no development further than the plerocercoid II stage and in which, the parasite is unlikely to be transferred to a suitable host are herein termed 'accidental' hosts. In this sense walleye and large yellow perch ( Perea flavescens ) are referred to as accidental hosts. The terms plerocercoid I and plerocercoid II were proposed by Freeman (1964) to describe the life stages of P. parallacticus . Apparently, the p1erocercoids in copepods and fish differed only in size with the plerocercoid II being 10 times as large as the plerocercoid I. Fischer and Freeman (1969) found the developmental stages of P. ambloplitis to be similar to P. para 1lacticus and proposed the same terminology for the former species. Throughout this study, 'plerocercoid' refers to the parenteral stage in fish, unless stated otherwise. When p1erocercoids invade the body cavity of fish pathological change may be Incurred by the host, especially in heavy infections. Pathological damage to fish, particularly smallmouth bass, as a result of heavy -4- plerocercoid infections has been well documented (Moore 1925; Bangham 1927a; Bangham 1927b; Langlois 1936; Esch and Huffines 1973; Hoffman 1975). It is not known if heavy plerocercoid infections will effect pathological change in accidental hosts, such as walleye and large yellow perch. The biology of P. amb loplitis has been studied intensively, however, the fate of p1erocercoids infecting accidental hosts has not. The present study was initiated to examine the biology and histo patho1ogy of P. amb1o p 1 itis plerocercoids in two percid accidental hosts, the walleye and yellow perch. The main objectives of this study were to: 1) determine the prevalence and intensity of plerocercoid infections in walleye and yellow perch with respect to age, size and sex; 2) determine which visceral areas harboured plerocercoids ; 3) identify the source of plerocercoids infecting walleye and yellow perch; 4) describe the pathological changes associated with a plerocercoid infection in walleye and yellow perch and 5) assess the affect of a plerocercoid infection on walleye fecundity. MATERIALS AND METHODS S tudy area Lake of the Woods (49*^20^ N; 94°40’ W) is located approximately 500 km west of Thunder Bay, Ontario. The lake has a total area of 387,151 ha, of which 647<> (246,718 ha) is in Ontario (Ontario Ministry of Natural Resources, Kenora; unpubl.), 337o in Minnesota, U.S.A. (Carlander 1949), and the remainder in Manitoba, Canada. The portion in Ontario has a mean and maximum depth of 7.9 m and 68.8 m respectively. Lake of the Woods is part of the Hudson Bay drainage area (Carlander 1949) with Rainy River as its major tributary and the Winnipeg River the outlet. This study was restricted to the southwest section of the lake within Ontario. This portion of the lake is bordered by French Portage Narrows, the International border. Rainy River and Sabaskong Bay to the north, west, south and east, respectively (Fig. 1). Examination of fish for parenteral P. ambloplitis Age one and older walleye and yellow perch Age 1 and older walleye and yellow perch were sampled monthly from May to September 1983 and examined for P. ambloplitis p1erocercoids . Walleye data were supplemented with preliminary collections made from June to August and November 1982. Walleye and yellow perch were collected using gill nets -5- - 6 - Fig. 1. Map of Lake of the Woods showing the borders of the study area. Inset shows geographical location of Lake of the Woods. -7- (1.9 to 12.7 cm, in 1.3 cm increments, stretched mesh) set overnight. Total length (nearest 1 mm), weight (nearest 10 g if greater than 100 g and nearest 1 g if less than 100 g) and sex were recorded for each fish. The left opercular bone, second dorsal spine and scales were removed for age determination. Walleye and yellow perch were selected according to predetermined length classes to ensure equal sampling over most of the available size range. Walleye were separated into 7 length classes from 10 to 40+ cm in 5 cm increments. Yellow perch were separated into 6 length classes from 5 to 20+ cm in 3 cm increments. Thirty-five walleye and 30 yellow perch (5 from each length class) were sampled each month. Walleye age classes 7 and older and yellow perch age classes 5 and older were grouped for statistical analyses. The grouped walleye and yellow perch age classes are referred to as 7+ and 5+, respectively. Fish were examined by carefully removing the lateral musculature to expose the intact viscera. The viscera were washed with fresh water to collect p1erocercoids free in the peritoneal cavity. The wash was poured into a Baermann funnel and left to settle for a minimum of three hours. Contents were drained from the bottom and examined for P. ambloplitis p1erocercoids. The gastro-intestina1 tract was severed at the esophagus and rectum and the viscera were removed from the peritoneal cavity. The gonads, spleen, gallbladder, liver -8- atid kidney were removed and examined individually. Each organ was pressed between two glass plates and scanned for p lerocercoids using a Bausch & Lomb dissecting microscope at 20-30X. If the organ was large, it was sectioned into manageable pieces prior to pressing. Stomach contents were removed (see feeding analyses) and the gastro-intestina1 tract and mesenteries placed in a plastic bag with approximately 250 mL of pepsin digest solution (Meyer and Olsen 1971). When the mesenteries were digested the solution was poured into a Baermann funnel, diluted with fresh water and left to settle for a minimum of three hours. Contents were drained from the bottom and examined for P . ambloplitis plerocercoids. The entire viscera of small fish, less than 15 cm, were pressed between glass plates instead of being digested. Proteocephalus ambloplitis was recognized by the presence of four distinct suckers, a prominent end organ, many calcium corpuscles and no acetabular glands (Befus and Freeman 1973; R. Freeman pers. comm.). These criteria apply only to live p1erocercoids . Preliminary analyses of walleye in 1982 indicated that although some infections appeared severe with large numbers of cysts visible in the viscera, few plerocercoids were obtained on digestion of the tissues. In these fish, many p1erocercoids may have been dead. Only live plerocercoids were recovered using the digest technique. For this reason, walleye sampled in 1983 were assigned an index relative -9- intensity upon visual examination. This assessment was based on the apparent number of live and dead p1erocercoids visible in the viscera and peritoneal cavity. Each fish was assigned an intensity index of light (number of cysts visible estimated to be from 1 to 50), medium (51 to 200) or heavy (201+). The number of live plerocercoids from indexed fish was later determined by pressing and digesting the tissues. Also, in this manner, an index of intensity was recorded for 280 walleye sampled during July and August, 1982 to determine if apparent plerocercoid intensity was related to the size of fish. Statistical analyses followed Daniel (1978) and Sokal and Rohlf (1981). The criterion for significance of all analyses was at the 957o level. Differences between sexes were tested with a Mann-Whitney U analysis. Age and season variations in plerocercoid intensity were examined using the Kruska 1-Wa11is ANOVA with multiple comparisons analysis. Tests of association were accomplished using Kendall's Tau rank correlation unless otherwise specified. Statistics were performed with the aid of a Vax 11/780 mainframe computer using the Statistical Package for the Social Sciences (SPSS) (Nie et al. 1975). Young-of-the-year walleye and yellow perch Young-of-the-year (YOY) walleye and yellow perch were captured from June to September, 1983 using a 30 m bag seine and a small (1.9 cm stretched mesh) monofilament gill net. -10- YOY walleye data were supplemented with preliminary collections made during July and August, 1982. YOY walleye were differentiated from YOY sauger ( S. canadense ) according to the criteria of Nelson (1968). Total length (nearest 1 mm) was recorded for each fish, the peritoneal cavity opened and the viscera extracted. The organs were separated from the mesenteries where possible and examined for parenteral p1erocercoids by pressing each viscus between two glass plates or two microscope slides. The viscera of YOY yellow perch less than 40 mm long were examined with the aid of a Leitz/Wetzlar compound microscope at lOOX. The viscera of YOY fish greater than 40 mm were examined using a Bausch & Lomb dissecting microscope at 20-30X. Forage fish Fishes considered to be potential prey items of walleye and yellow perch were examined for parenteral P. ambloplitis by inspecting the peritoneal cavity and pressing the viscera between glass plates. Fish were collected using seine, trawl or gill nets during July and August, 1982 and 1983 and identified to species according to Scott and Crossman (1973). All fish examined during this study were identified according to Scott and Crossman (1973) but scientific and common names used herein follow Robins et al. (1980). -11- Feeding analyses The stomach contents of walleye and yellow perch were collected and preserved in 107o formalin. The buccal cavity was examined for regurgitated food items and the intestine flushed with water with the aid of a 50 mL syringe and a 5 mm diameter plastic tube. All items recovered were included as stomach contents. Invertebrate food items were identified according to Pennak (1978) and Merritt and Cummins (1978). Amphipods were identified to genus where possible; all other invertebrates were classified to order. Fish prey items were identified to species, where possible, according to Scott and Crossman (1973). Body shape, colouration, bone structure, number of pyloric caecae and scales were criteria used to identify partially digested fish. Volume (nearest 0.1 mL) and number of each prey item were determined for each stomach sample. Individual samples were combined to obtain a monthly total. The relative importance of each prey item was determined by comparing percentage frequency of occurrence and percentage total volume. Only the frequency of occurrence of each prey item was calculated for YOY walleye and YOY yellow perch. Enteral cestodes Walleye, yellow perch, smallmouth bass, black crappies ( Pomoxis nigromaculatus ), rock bass ( Ambloplites rupes tris ) and pumpkinseeds ( Lepomis gibbosus ) were collected by gillnetting, trapnetting and angling from May -12- to September, 1982 and 1983 and examined for adult P. ambloplitis . As it had been noted previously that some enteral cestodes of walleye were evacuated after the death of the host, only those fish that were alive when removed from the nets were examined for enteral P, amblopltis . Fish were sacrificed by cervical dislocation and the peritoneal cavity opened. The esophagus and rectum were severed and the gastro-intestina1 tract removed. The stomach was opened and the gastro-intestina1 tract placed in a plastic bag with fresh water for a minimum of two hours which facilitated the release of cestodes. When removed from the plastic bag, more water was flushed through the intestine with the use of a 50 mL syringe and a 5 mm plastic tube to dislodge any cestodes still attached. After a number of such washings, the intestine and pyloric caecae were opened and examined visually for any remaining tapeworms. All tapeworms were saved and placed in a 12 cm diameter Petri plate with fresh water. To enhance the relaxation of the strobilae, the tapeworms were refrigerated in a fresh-water bath for a minimum of 12 hours. When the strobilae were relaxed, the scolex of each tapeworm was examined using a Bausch & Lomb dissecting microscope at 20-30X. The number of individuals of each genus was recorded. Representative specimens were preserved in hot 107o AFA. Cestodes greater than 10 cm in length were stretched on dry paper towelling and the fixative poured over them. Smaller tapeworms were placed directly in the fixative (R. -13- Appy pers. comm.). For long term storage of the cestodes the 107o AFA was replaced with 707. EtOH. All cestodes were stained with Semichon’s aceto-carmine (Meyer and Olsen 1971), cleared in Oil of Cedarwood and mounted in Permount. Fee undity Twenty mature female walleye were collected in early November, 1982 for fecundity analyses. Fish were obtained from commercial fishermen at Windy Point (on Lake of the Woods), Ontario. The ovaries were separated from the remaining viscera, patted dry, weighed to the nearest 0.0001 g with a Sauter balance and preserved in 107. buffered formalin. The remaining viscera were examined for parenteral P. ambloplitis in the manner previously described for age 1 and older walleye and yellow perch. The 20 walleye examined here comprise the total number of fish sampled for plerocercoids during this month and alone, represent the November sample. Fecundity was estimated by the gravimetric method (Bagenal and Braum 1978; Serns 1982). Preserved ovaries were weighed to the nearest 0.0001 g with a Mettler AC 100 balance. The tunica albuginea was removed so that the weight of the eggs could be determined. A minimum 57. subsample of eggs was excised from the mid-section of the left ovary and counted. The total number of eggs was obtained by direct proper tion. -14- Histopathology Fifty walleye and 20 yellow perch were collected for histopathologica1 examination from June to September, 1982 and 1983, from Lake of the Woods. Fish were collected by gill netting, trap netting and seining. Only the fish that were alive when removed from the nets were kept for this study. Fish were sacrificed by cervical dislocation and then the peritoneal cavity was opened. Selected sections of the liver, spleen, gonads, gastro-intestina1 tract and mesenteries were excised and preserved in 107o phosphate buffered formalin. Tissues were embedded in paraffin, sectioned at 6 to 10 microns, stained in Lillie's A & B (Lillie 1954) or Gomori's triple stain (Humason 1979). Photomicrographs were produced using a Zeiss C 35 camera mounted on a Zeiss compound microscope. RESULTS Parenteral P. ambloplitis Walleye - One hundred percent of the age 1 and older walleye sampled were infected with parenteral P. ambloplitis (Table 1). There was no difference in intensity of infection between sexes (U = 3915; 79,95 df.)* However, male and female walleye did differ with respect to gonadal infections. Only 46.87<> (37 of 79) of the males examined had p1erocerCOids in the testes with a mean of 3.3 per fish, whereas 84.27o (80 of 95) of the ovaries contained p1erocerCOids (mean = 7.5). However, gonadal infections constituted a relatively small proportion of the total number of p1erocercoids recovered and therefore, males and females were combined for statistical analyses. Mean intensity of parenteral P. ambloplitis in walleye increased significantly from 41.5 at age 1 to a maximum of 170.9 by age 5 (Table 1; Fig. 2). Mean intensity was significantly lower in older fish (36.8 and 57.6 in age 2 classes 6 and 7+ respectively) (K-W ANOVA; X = 28.8; 6 df.). The intensity of parenteral P. ambloplitis infection was positively correlated with length (Tau = 0.26), weight (Tau = 0.27) and age (Tau = 0.21) of walleye in age classes 1 to 5. Since length and weight were not correlated over the entire size range of walleye, further statistical analyses were performed only with respect to age. The mean number of parenteral P. amb1op1itis -15- -16- >> LCDM o o o CM +J CO I— CO CM 00 CO CO CM CM CO I— CM LO CT> CO icn CM O 00 CM 00 CD (D CO CO 00 CT> CO LO OJ -o ue - OJ U o o O o o O o fO ^ CU o o O o o o o tct- > (U S- Q. o o o o o o LO Ch o I— ^ 1— o o LO CM CO CM LO I— CM CM CM CO CO 'st- LO +1 I +1 +I -H +1 I +I +1 • CM o CroO CM o o o CM o LO 00 LO LO LO LO CO CM CO CO CO 00 o LO JZ -l-> cr> o CcMn CM o 'd" 0) >5l‘ o o LT> CM ro 00 CM O CO cr» CM CO cr> CO CM CO CM CO CM CO CO CO CO CO CM LO o CO CM CM * CO 00 CM CM CO CM CM CO CO >> OC7J> ■k < ■k -k TABLE 1. Prevalence and mean intensity of parenteral ambloplitis plerocercoids in age one and older walleye from Lake of the Woods, Ontario, May to November, 1982 and 1983 Excludes one walleye with 971 plerocercoids. Age class 7+ includes 13, 6, 1 and 1 fish, 7, 8, 9 and 10 yr old respectively. Values are means ± S.E. subtended by range. -17 - Fig. 2. Mean intensity (+/- S.E.) of parenteral P, ambloplitis infecting age 1 and older walleye sampled May to November, 1982 and 1983. Sample sizes are indicated above each point. 220 15 200 180 160 140 120 100 80 60 40 20 T 1 1 r "T" 1 2 3 4 5 7+ AGE CLASS -18- recovered from all age classes of walleye remained relatively constant from May to November (mean = 60), except for the month of September (mean = 154) (Fig. 3). The importance of some visceral organs as sites for harbouring parenteral P. ambloplitis varied with age (Fig. 4). The mesenteries harboured the largest percentage (42.57o) of all parenteral P. ambloplitis present in age 1 walleye. The proportion of p1erocercoids recovered from the mesenteries increased to 807. by age 5 and remained at approximately 807o in age classes 6 and 7 + . As walleye aged, the percentage of all parenteral plerocercoids found in the liver and free in the peritoneal cavity varied inversely with that found in the mesenteries. In YOY walleye, 477o of all parenteral P. ambloplitis occurred in the liver. By age 3, and continuing to age 7+, less than 107o of the total infection was found in the liver. Similarly, 537. of all plerocercoid in YOY walleye were free in the peritoneal cavity during August, but one month later only 357o of all plerocercoids were free and by age 4 just 127o of all parenteral P. ambloplitis were free. Approximately 107o of all plercercoids were free in the peritoneal cavity of walleye in age classes 5 to 7+. The proportion of parenteral P. ambloplitis infecting the gonads and spleen remained constant from September of the walleye's first year of life to age 7+ (Fig. 4). The gonads contained approximately 57. of the total plerocercoid burden and the spleen approximately 2.07o. The -19- Fig. 3. Mean intensity (+/- S.E.) of parenteral P. ambloplitis infecting age 1 and older walleye in relation to month of sampling. Sample sizes are indicated above each point. 180 27 160 140 120 100 80 60 40 20 —I” —1 1— —I— MAY JUN JUL AUG SEP NOV MONTH -20- Fig. 4. Percent of total parenteral P. ambloplitis invading each visceral organ of walleye sampled May to November, 1982 and 1983. Young-of-the-year data is for August and September. Mesenteries (■) Free (A); Liver (•) ; Gonads (O); Spleen (□). 100 90 80 70 60 50 40 30 20 10 AGE CLASS -21- kidney and gallbladder of age one and older walleye each harboured less than 1% of the total parenteral P. atnbloplitis infection. Little monthly variation occurred with respect to the proportion of p 1 erocerco i ds found in each visceral organ (Fig. 5). The percentage recovered from the mesenteries remained relatively constant at about 607o from May to September but increased to 897o by November. The percentage of all p 1 erocercoids recovered from the liver declined from 197o in May to 57o by November. However, the November sample was comprised exclusively of age 5 to 7+ walleye that were obtained for the fecundity estimate. The percentages of tota1p lerocercoids found in the gonads and spleen changed little from May to November. The percentage free in the peritoneal cavity increased from 57» in May to 187o by June and remained at approximately 187o until September. However, the percentage free in walleye was only 57o by November. The mean number of parenteral P_^ ambloplitis recovered from some visceral organs varied with age (Table 2). The mean number of plerocercoids infecting the liver was significantly higher in age 1 and 2 walleye (11.6 and 12.7) 2 than in fish older than age 5 (means less than 5) (X = 55.5; 6 df.). The mean number of p1erocercoids free in the viscera of walleye ages 1 to 5 was significantly greater than in age 6, but not age 7+ fish (X^ = 25.2; 6 df.). The mean number of parenteral plerocercoids in the mesenteries demonstrated the greatest magnitude of change with age. The -22- Fig. 5, Percent of total parenteral P. ambloplitis invading each visceral organ of walleye in relation to month of sampling. Mesenteries (■); Free (^); Liver (•) ; Gonads (O) ; Spleen (□). 100 90 80 70 60 50 40 30 20 10 MONTH -23- «>o 00 CO CO m (3^ LO LO CO CM CM CM ro LO o CVJ CO o o 00 CO CT> CM ccu CM CM LO XJ O O o CD Ln LO CM CO CO LO i- XJ Xrt>J CM oo CO o o o o fO C3 c (U (U (T» cr> CO CO Q. o I— O o CO (/) * ■•KK XfO3 c CM m ■K ■}c o CM o CM CO CO CM CO o CO CM LO LO CO o CM CM CO CO II CM CO CO OJ M ■K •K > CO CO CO CO CO o to CO CO CO CO 00 CM CO O o cr> CO LO CM (>U> -cO CO ir> o o o V LO CO o « CM CO 00 CO CM i<-u T•oD fd 1— CM CO CM CD O O o O fO O c ((UU CO CO CO 00 • • (Q/> . o I— o CM o •(o/) c ro to CM CO o LD CM CO CO CD o r>* cr> CO CO CO CM S- O) S- +J JD JcOu +c-> Eto (U E o >> O) fd c13z =3 cor> Q. > •-0 «a: (U o 00 TABLE 3. Mean number of parenteral P. ambloplitis plerocercoids in visceral organs of age one and older walleye in relation to month of sampling (1982 and 1983) -26- to (U •r“ s- 03 to CM cu •cfj to to o o LO 00 03 C33 03 o o <73 O) to - CO 00 o lO LO u_ cr> 03 00 o CO >» O) to to C73 00 CO o cr ■a CM LO CO LO •r~ CO 03 03 to to to LO C33 C33 00 00 to LO i- *OoJ -a 03 o to C33 LO LO to to o CM CM CM o03 c d to o>j o LO o o o 00 Q. CO to to to LO CO CO -tao CO CO CO LO 03 E Od CM CM cOs 00 00 to <73 to LO CO CM r>. LO LO O CO f— "!d- CM CM CM CO LO to (U c TABLE 4. Percent occurrence of parenteral amblopiItis plerocercoids in visceral organs of age one and older walleye sampled May to November, 1982 and 1983 -27 - than 807o of the walleye sampled. Free plerocercoids were recovered from at least 15% of the walleye sampled from all age classes. The percent occurrence of plerocercoids found in the liver appeared to decrease, with increasing age of fish. Approximately 987o of the age 1 walleye had plerocercoids in the liver whereas only 51% of the age 7+ fish had a liver infection. The percent occurrence of plerocercoids in the gonads, spleen, gallbladder and kidney varied little with age. The monthly percentage occurrence of plerocercoids in the visceral organs examined was constant from May to November, except for September which consistently provided values greater than the other months (Table 5). By the indexing method, it was apparent that some of the older and larger walleye in the population had infections that visually appeared heavy. Of the 193 walleye for which plerocercoid counts were done, 4 (2.17o) were judged to have heavy infections. These fish were a mean of 368 mm in length, weighed a mean of 465 g and were 4.3 years old. However, actual counts revealed that 13 (6.77o) had heavy infections of live plerocercoids, also 104 (53.97o) and 76 (39.47o) had light (1 - 50 plerocercoids) and medium (51 - 200) Infections, respectively (Fig. 6). Thirteen (4.67o) of the additional 280 walleye indexed during 1982 were judged to harbour a heavy infection. These fish were a mean of 417 mm in length, weighed a mean of 911 g and were an average of 6.3 years old. While the indexing method underestimated the -28- «/i QJ •r— s- 4O->) cn C CO o O o O) 00 cn o o lO O) cn o >> Q; ■c CO OJ o CO a CCJ CO o LO LO CO CO CO 00 C\J 00 o CO LO 00 00 cr> 00 00 00 CD -ao» "O fO CO cn cn LO 00 00 00 CO 00 lO CsJ to CD 00 to 00 r>«. OsJ o CL cn CO cn <=3- CO in TD LO cn (O c cn cn 00 o cn co CO LO 00 CD o cn CO CO CO CCJ i. > cn o. > fO =3 Z5 CU o •~o < LO TABLE 5. Percent occurrence of parenteral K ambloplitis plerocercoids in visceral organs of age one and older walleye in relation to month of sampling Q982 and 1983) -29- Fig, 6. Frequency of occurrence of parenteral P, ambloplitis intensities in age 1 and older walleye sampled May to November, 1982 and 1983. 60 50 40 30 20 10 T "1 T 1- 26- al- 76- 201 + 25 so 75 100 125 150 175 200 INTENSITY -30- number of walleye harbouring a heavy infection it did suggest that walleye appear to acquire p 1 erocercoids continuously with increasing age. The distribution of parenteral ambloplitis in the walleye sampled did not fit a negative binomial distribution although the variance to mean ratio was much greater than 1 (6461/73 = 88.5). Such a large ratio indicates a very overdispersed population of live plerocercoids. Yellow perch - Seventy-four percent (143/193) of the age 1 and older yellow perch sampled were infected with parenteral P. ambloplitis (Table 6). Prevalence increased from 59.47o at age 1 to almost 1007o by age 5+. There was no difference in intensity between sexes (U = 2201; 54,89 df.). Only 8 of 54 (14.87,) infected male yellow perch had pleroceroicds in the testes with a mean of 2.9 per fish while 7 of 89 (7.97.) infected females had plerocercoids in the ovaries (mean = 1.3). Since plerocercoid infections in the gonads of male and female yellow perch were relatively rare and essentially the same, the sexes were combined for statistical analyses. Mean intensity of parenteral P . amb1o p1itis increased significantly with age of yellow perch (K-W ANOVA; = 35.3: 4 df.) (Table 6; Fig. 7). Mean intensity increased from 3.2 at age 1 to 19.6 by age 5+. The intensity of parenteral P. ambloplitis infection was positively correlated with length (Tau = 0.37), weight (Tau = 0.29) and -31 - +>->> CD CM 00 CD n CO CM CO CM CM LO (/) c +1 +1 +1 +1 +1 CD to Lf) CM +-> o> Lf) 00 00 CD CD o s_ 0) Du Qto. CU s- T3 O) CM 4->hM un CO CM U O) S- (A 50 -a 't— oo CM O CM CM ■o CO CO CD to to CM CO CD sz VO CM cn^ +1 I +1 +1 +1 +1 CD dj CO CM O Lf) O VO CO Lf) CO CD VO O CO CM Jd • s to d) ■I- CD i- «»- E CU ta jz r— S_ 4-> ■ O 4-> o >> CD O Lf) O Lf) C ^ 0) c: +- CD CO to o fO -E O) CO 1— CO I— CM Lf) CM CD CO T3 -P CM CU +1 I +1 +1 +1 +1 I . -a " I— c CO (O • CD Lf) CD r— CM CD CO O CD -p O CM I— Lf) ^ Jd 3 -P to •(— LO ^ S -o •»« U) LU CU LiJ TJ S- + CM Lf) O)' CD < TABLE 6. Prevalence and mean intensity of parenteral ambloplitis plerocercoids in age one and older yellow perch from Lake of the Woods, Ontario, May to September, 1983 -32- Fig. 7. Mean intensity ( + /- S.E.) of parenteral P_^ ambloplitis infecting age 1 and older yellow perch sampled May to September, 1983. Sample sizes are indicated above each point. T T 1 1 r 1 2 3 4 5+ AGE CLASS -33- age (Tau = 0.37) of yellow perch. To be consistent with the analyses for the walleye, all statistics were performed only with respect to age. The mean number of parenteral P. ambloplitis recovered from age 1 and older yellow perch varied only slightly with month of sampling (Fig, 8). The proportion of all plerocercoids infecting the mesenteries and liver of yellow perch exhibited the greatest change with age (Fig. 9). Only 87o of all parenteral p1erocercoids were found in the mesenteries of YOY yellow perch in September but increased to 487o by age 2. By age 5+, the mesenteries accounted for 697o of all plerocercoids found in the viscera of yellow perch. Conversely, the liver of YOY yellow perch harboured 667. of all plerocercoids by September but dropped to only 367o by age 2 and contained no more than 207o by age 5+. The percentage of all plerocercoids free in the viscera of yellow perch did not exhibit the same trend as in the walleye. Although the percentage free was greatest in younger fish it never exceeded 127o of total worm burden. The percentage of all plerocercoids recovered from the gonads and spleen remained relatively constant and low in all age classes of yellow perch. The gonads harboured from less than 17o to approximately 1,5% of all plerocercoids, and the spleen from 1% to 4.57.. From all organs, the percentage of parenteral P. amb1op litis that were free in the viscera, in the mesenteries or in the liver varied the most from May to September (Fig. 10). The percentage of all plerocercoids -34- Fig. 8. Mean intensity (+/- S.E.) of parenteral P. ambloplitis infecting age 1 and older yellow perch in relation to month of sampling. Sample sizes are indicated above each point. MONTH -35- Fig, 9. Percent of total parenteral P, ambloplitis invading each visceral organ of yellow perch sampled May to September, 1983. Young-of-the-year data is for September. Mesenteries (■); Free (^); Liver (•); Gonads (O); Spleen (□). 80 70 60 50 40' 30' 20 10' -36- Fig. 10. Percent of total parenteral P. ambloplitis invading each organ of yellow perch in relation to month of sampling. Mesenteries (■) ; Free (-^) ; Liver (•) ; Gonads (O) ; Spleen (□). 80 60 50 40 30 20' 10- MONTH -37- recovered from the mesenteries varied inversely with the percentage of all p1erocercoids recovered from the liver. The percentage recovered from the mesenteries declined from 527o in May to 327o by July but was again 527o during September. Conversely, the percentage recovered from the liver increased from 427o in May to 567o in July but then was lower (327o) in August and September. Unlike in walleye, the percentage of all p1erocercoids that were free in the viscera of yellow perch increased gradually from 17o in May to a high of 12.57o by August. The percentage free was somewhat lower (7 7o) in September. The proportion of p1erocercoids harboured in the spleen and the gonads was relatively constant throughout the sampling period. The mean number of parenteral P. amb1op1itis in the visceral organs of yellow perch did not increase greatly with age until the 5+ age class (Table 7). The mean number of p1erocercoids in the gonads and spleen was less than 1 for all age classes. The gallbladder and kidney of yellow perch were rarely infected with p1erocercoids. There was no significant difference in the mean number of free p1erocercoids in the viscera, with increasing age of yellow 2 perch (X = 5.0; 4 df.). The mean number of p1erocercoids in the liver of age 5+ fish was greater than that in age 2 fish. The mean number of p1erocercoids found in the mesenteries increased significantly from 0.5 in age 1 fish to 13.5 in age 5+ fish (X^ = 44.6; 4 df.). There were no differences in the mean number of -38- CO fO + o CM CO ir> S- O) 00 CM CO +-> £Z in o CO 00 O) in CO 00 00 Ca>O s: CO in CO CM 00 CO CO Oc) C33 ■O CO in 00 in 0CO3 00 in CO o CO o S- (U X3 T3 fO CT3 CO in 03 CD C TABLE 9. Percent occurrence of parenteral P. ambloplitis in visceral organs of infected age one and olB’er yellow perch sampled May to September, 1983 42 o CD CO -O *r- LO oo LO LO •r— O SZ o u S- i- O) (U O CL o CD C30 oo o i- 5 > S- (U o (U C x» ■O O oo o o Qj O o -O c <0 fO cn oo o o . a» n LO CD CO o CO tX C 00 00 00 CD o cr> fO T3 S- 4-> rtJ oo rO O CL cu oo oo 4- 4- C O - r- fO O OJ 4- U O E c CO 0) (U "O o o ra i- CO c (U -r- o Q- > C3 o CO c 4-> C o -43- from 6.97o in May to 46.77o in August. A slight decline to 36.07o was apparent by September. There were 10 or fewer parenteral p1erocercoids in 89.57o (128 of 143) of the infected yellow perch (Fig. 11). Only 2 of 143 infected age 1 and older yellow perch harboured more than 50 p lerocercoids. As in walleye, the distribution of parenteral P. amb1op1itis in the yellow perch did not follow a negative binomial or a Poisson distribution. However, the population was determined to be over-dispersed as indicated by a variance to mean ratio of 13.5 (81/6). Young-of-the-Year Walleye and Yellow Perch Young-of-the-Year (YOY) walleye were first vulnerable to seining in July of both 1982 and 1983. However, YOY walleye were not infected with parenteral P. amb1 op litis until August (Table 11). Prevalence and mean intensity were greater in August 1983 (93.27>; 5.6) than at the same time in 1982 (57.17c.; 1.5). Virtually all (97.17o) of the YOY walleye sampled in September 1983 were infected with parenteral P. ambloplitis . The YOY walleye sampled in 1983 were generally larger than those sampled in 1982. The majority of plerocercoids in newly infected walleye were free in the viscera (527o) or in the liver (47.7 7o) (Table 12). Mean numbers of plerocercoids recovered from each organ increased from August to September (Table 13). However, mean numbers of plerocercoids recovered from the -44- Fig, 11. Frequency of occurrence of parenteral ambloplitis intensities in age 1 and older yellow perch sampled May to September, 1983 INTENSITY -45- TABLE n. Prevalence and intensity of parenteral P. ambloplitis plerocercoids in young-of-the-year walleye sampled July to September, 1982 and 1983, from Lake of the Woods, Ontario Fish total length(mm) Prevalence Intensity Year Month N Mean Range (%) Mean Range 1982 July 52 43-76 0 0 0 Aug. 14 60-95 57.1 1.5 1-3 1983 July 51 64.8 45-94 Aug. 44 117.3 81-141 93.2 5.6 1-19 Sept. 34 144.3 111-197 97.1 22.8 2-85 46 CM o ■o OJ to ca ~o to CM CU O Q> cu CM o i- Ln ro i- O) Li- > ■5^- (U CU r— i- £I o O. fO ■O O) •I— to >> CD CM Q. O o> fO S- -r- I— CD <0 cm- « o ^ I— cu c o fO to ^ cu 4-» C E cu o CM o o> +J cn+-> CL S- Q. oo tf- o Oi O C/) 4- to J fC O C 4-> "O to > £= IX) 5- to r— o ■"3 ■a CM cu • +J *vj- o u IX) ro s: 4-3 (C C7) £3. o 3 3 CU ■3 C o o cvj 03 to 03 > CT\ 03 o 0> O r— o OsJ t. r- Q) ns O S O S- S- >> (U 03 03 •— (U C Q. >> XJ o o o I (/) (U r>. 00 Q. 0 O 1 o OJ o C7> XS C E 3 CO 03 O 00 >>cr> s~ Q_ 1X3 03 03 "O I— 4-> S- -o 03 O 03 03 U 03 jQ 03 M- E o o C 03 C -I- +J 03 O- 03 i- M- 03 O 03 O to a. SZ ■ c 03 03 LO cn >» 03 S- S- f— o o o 03 o 3 Q. V X3 o to E 3 03 -O c 03 03 I— to c O Q. X3 OJ 03 CO E 03 03 •I— 03 C o o > to o CD CO *o 03 UJ • -4-> O U LT> ro QQ z: 03 C M- o CO £= CO >> 4-> C3> Q. Z5 03 to -48- visceral organs were still relatively low. Pleroceroids free in the viscera occurred in almost 887o of the YOY walleye examined in August (Table 14). Liver infections were not as frequent at that time. However, by September 947o of the YOY walleye harboured a liver infection whereas 797o had p 1 er o c er o i cd s free in the viscera. Also, by September all the visceral organs, except the gallbladder, harboured at least one P. ambloplitis plerocercoid. YOY yellow perch were first vulnerable to seining in June. YOY yellow perch did not harbour parenteral P. ambloplitis until August (Table 15). Prevalence of parenteral P. amb loplitis was 36.07o in August but doubled to 72.0 7o by September. However, mean intensity did not change from August to September. The location of parenteral P. ambloplitis in the viscera of YOY yellow perch was recorded only in September. The liver harboured 82.17o (92 of 112) (mean intensity = 2.6) of all parenteral plerocercoids, 8.07> (9 of 112) (mean intensity = 0.3) were free in the peritoneal cavity and 9.87o (11 of 112) (mean intensity = 0.3) were already encapsulated. No plerocercoids were found in the other organs. Unidentifiable plerocercoids were found in both YOY walleye and yellow perch during June and July 1983, but not later in the season. Two species or two developmental forms were seen. The first type which had four distinct suckers but no discernable end organ was found in 13.7 7o (mean -49- O o CNJ c/> O ■a +J o >> o I— s- 3 > i/) CU E o O CD >> OL (1) O to CD (KS S- o Lf) C>0 • fO CD Q- (U I— I ns I C M- XJ CU O fO 5- I fO cn o o o OL C rj o fO o >> CD CU XJ o E o o o S- CD CL o CO OJ S- CU CD Q_ O CO XI CU • 4-J O CJ CO CO ZC CU M- o CO CO E CO o: • 4-> CD Q. Z3 ZJ CU rD c. ea cd S'-/i . J‘^]prj i mm .S‘^ vV:: 4//i vfvfSih wyc. i