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1988 Ultrastructural studies of two coelozoic myxozoans Tom Alvin Bailey Iowa State University

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Ultrastructural studies of two coelozoic myxozoans

Bailey, Tom Alvin, Ph.D. Iowa State University, 1988

UMI SOON.ZeebRd. Ann Aibor, MI 48106

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Ultrastructural studies of two coelozolc myxozoans by Tom Alvin Bailey

A Dissertation Submitted to the Graduate Faculty in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY

Department: Zoology Major: Zoology (Parasitology)

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Iowa State University Ames, Iowa 1988 11

TABLE OF CONTENTS p^gg

GENERAL INTRODUCTION AND LITERATURE REVIEW 1 SECTION I. ULTRASTRUCTURAL OBSERVATIONS ON 26 SPOROGENESIS OF CHLOROMYXUM TRIJUGUM (MYXOSPOREA: CHLOROMYXIDAE) FROM FOUR CENTRARCHID FISHES INTRODUCTION 27 MATERIALS AND METHODS 29 RESULTS 31 DISCUSSION 49 REFERENCES CITED 53 SECTION II. PATHOLOGY OF THE GALL BLADDER OF 56 FOUR CENTRARCHID FISHES INFECTED WITH THE MYXOZOAN CHLOROMYXUM TRIJUGUM INTRODUCTION 57 MATERIALS AND METHODS 58 RESULTS 60 DISCUSSION 73 REFERENCES CITED 76 SECTION III. LIGHT AND ELECTRON MICROSCOPY OF THE 78 MYXOZOAN MYXOBILATUS NOTURI GUILFORD FROM THE TADPOLE MADTOM fNOTURUS GYRINUS1 INTRODUCTION 79 MATERIALS AND METHODS 80 RESULTS 82 DISCUSSION 92 REFERENCES CITED 97 iii

SUMMARY AND DISCUSSION 99 ADDITIONAL LITERATURE CITED 102 ACKNOWLEDGMENTS 110 APPENDIX 111 1

GENERAL INTRODUCTION AND LITERATURE REVIEW

The Nyxozoa (Grasse 1960) 1s a protlstan phylum In the Subklngdom Protozoa (Levine et a1. 1980). Myxozoans are primarily parasites of lower vertebrates, especially freshwater and marine fishes, and some Invertebrates. The phylum Is characterized by plurlcellularly developed spores containing one to seven nematocyst-llke polar capsules enclosing polar filaments, and one to many amoeboid sporoplasms (Lom and Noble 1984). The spore walls are formed by two to seven valves which join to form a suture. Myxozoans are grouped Into two classes, Myxosporea and Actlnosporea. The smaller class, Actlnosporea, Is characterized by spores with three polar capsules, three valves, several sporoplasms, and a reduced Plasmodium stage. Actlnosporeans parasitize Invertebrates, primarily annelids. The vast majority of myxozoan species are members of the Class Myxosporea. Myxosporeans have multicellular Plasmodia which harbor vegetative nuclei and generative cells producing spores. Plasmodia can be mono- or polysporous, and reproduce sexually by autogamy (Lom and Noble 1984). The Class Myxosporea Is further subdivided into two orders, Bivalvulida and Multivalvulida, three suborders (Sphaeromyxina, Variisporlna, and Platysporina), and 16 families. Shulman (1966) based ordinal differences on number of shell valves, and subordinal differences on the arrangement of polar capsules relative to the suturai plane. In a revised classification system, Lom and Noble (1984) retained the above ordinal characteristics, but added polar filament structure as a 2

subordlnal guideline. Myxozoan parasites are either histozolc (Intercellular or Intracellular) In tissues or coelozolc In hollow organs. Plasmodia of coelozolc species attach to the walls of hollow organs or float free In lumlna. Plasmodia of histozolc species usually form cysts. Those that Inhabit the gills, Integument, or musculature are generally more pathogenic to fishes than coelozolc species (Mitchell 1977). Increased attention has been given to myxosporean fish pathogens Important In Intensive aquaculture (Lom and Noble 1984). Histozolc species responsible for substantial losses In fish stocks Include those In the genera Ceratomvxa (Ching and Munday 1984; Wade 1986), Henneouva (Current and Janovy 1976; Bowser and Conroy 1985), Kudoa (Stehr 1986; Stehr and Whitaker 1986), and Mvxobolus (-Myxosoma) (Hoffman and Putz 1969; Prihoda 1983; Marklw and Wolf 1983; Wolf and Marklw 1984b; Hamilton and Canning 1985, 1987).

Histozolc Myxozoans Species Infecting the Integumentary system and serosal membranes The most common foci of infection by myxozoans in the Integumentary system are the epidermal and subdermal regions of cutaneous tissues. Serosal membranes of Internal organs also serve as sites of infection by myxozoans. Common genera of myxozoans inhabiting cutaneous tissues are Mvxobolus. Sphaerospora. and Thelohanellus. Mvxobolus pharvnoeus and & cvprini form close associations with epithelial tissues in the gills and 3

general body surface of fishes. Spall (1973) reported three Plasmodia!

forms of MJ, oharvnaeus infecting Gambusia affinis. The first type was found in large multilocular cysts in connective tissues underlying the scales and epithelium. The second type was a vesicular cyst that was non-invasive in host tissues, loosely associated with pharyngeal tissues, and found free in the pharyngeal cavity. The third plasmodial type was a diffuse cyst within the subepithelial connective tissues of the pharyngeal bones. Only one plasmodial form of & cvorini has been reported. Spall (1973) found small and spherical Plasmodia of this species forming cysts on the epithelial tissues of gill lamellae in red shiners fNotroois lutrensisl and golden shiners fNotemioonus crvsoleucasK Molnar (1980) found Sohaerospora carassii. thought to be primarily a gill parasite, in the epithelial linings of the gill cavity and body of juvenile carp (Cvorinus caroio). Developmental stages were restricted to the stratum germinativum, but spores were present at all levels of epithelium. Molnar (1980) postulated that the cutaneous and renal forms of Sohaerospora occurring in the same fish are distinct species. Thelohanellus hovorkai forms cysts on the serosal membrane of the swim bladder in amur carp fCvorinus caroio haematooterus) (Molnar and Kovacs-Gayer 1982). Thelohanellus hovorkai is both host- and organ- specific.

Species infecting ihâ musculoskeletal system Myxozoans inhabiting the fins of fishes include Henneauva pinnae whose Plasmodia are enclosed by host derived epithelioid cysts (Schubert 4

1968). Similar cysts were reported for Thelohanellus nikolskii infecting the fins of common carp (Nolnar 1982; Desser et al. 1983a). The cyst walls surrounding young Plasmodia of L. nikolskii contain collagen fibrils which are less distinct in mature Plasmodia (Desser et al. 1983a). Fin damage appears to be significant only in Juvenile fish (Molnar 1982). In studying infections of Henneauva adioosa. which localizes in the connective tissue bands of the adipose fin of host species, Current (1979) found that surrounding collagen fibrils prevented direct contact between the parasite and host fibrocytes. Henneauva Pinnae. L nikolskii. and tL adioosa are all apparently non-pathogenic (Schubert 1968; Nolnar 1982; Desser et al. 1983a; Current 1979). The best known myxozoan parasites that infect muscle tissue are members of the genus Kudoa. Species of this genus may cause spoilage of the muscle tissue of captured fish (Kabata and Whitaker 1981). Kudoa paniformis and {L thvrsites in muscle cells of the Pacific whiting fMerluccius oroductusK for example, produce proteolytic enzymes, and may cause deterioration of muscle tissue after death (Patashnik et al. 1982; Harrell and Scott 1985; Stehr and Whitaker 1986). Whitaker and Kabata (1987) reported three stages of host response to |C thvrsites commencing only four months after the parasite was established in the muscle fiber. Kudoa paniformis and jL thvrsites may often infect the same host. Stehr and Whitaker (1986) compared unencapsulated Plasmodia containing developing and mature spores with encapsulated Plasmodia containing degenerating spores. They found that microvillar borders of 5

unencapsulated Plasmodia of !L oanlformls and thvrsites were closely associated with host muscle. Kudoa thvrsites produced only acidic enzymes and contained multivesicular bodies. Kudoa oanlformls produced both acidic and neutral enzymes, and contained vesicles, microfilaments, and microtubules In Its Plasmodia. The presence of Plasmodia In the muscle cells did not stimulate host response until most of the muscle fiber had been replaced. Subsequently, the Plasmodia were encapsulated by fibroblast-like cells, and phagocytes infiltrated the area. No proteolytic activity was apparent in the encapsulated Plasmodia. In yearling Atlantic salmon fSalmo salarK the spores of |L thvrsites were found only in muscle fibers, where they formed pseudocysts, characterized by assemblages of spores within somatic muscle fiber and surrounding sarcolemma. Infected fish exhibited signs of anemia, inflammation of the kidneys, and muscle deterioration after death. Surviving smolts transferred to salt water showed increased mortality and post-mortem deterioration. Harrell and Scott (1985) postulated that L. thvrsites is a relatively new parasite in Salmo salar host, and as a result, effective defense systems have not evolved in this host. Lom et al. (1983c) described five species of Kudoa in the musculature of seven species of fishes, and found that these myxozoans were either interfibrillar (located between muscle cells) or Intracellular (within muscle fibers). The interfibrillar species formed cysts with connective tissue envelopes. The intracellular species filled the muscle fibers, but did not become surrounded by connective tissue envelopes. The intracellular species were surrounded by their own 6

plasmalemma, and caused minimal damage to the muscle tissue. Lom et al. (1983c) described the host-parasite interface of Kudoa as a zone of narrow channels and vesicles. Host-parasite interaction among species of Kudoa varies, and is species-specific; this may account for the Inconsistency with which proteolysis is observed (Voelker et al. 1978). Lom et al. (1983a) found no proteolysis associated with five species of Kudoa. One of the most pathogenic myxozoans, the cartilagophagous species Mvxobolus f-Mvxosoma) cerebralis. was introduced Into the United States accidentally in the 1950s (Wolf and Markiw 1984b). This species is most harmful to rainbow trout fSalmo aairdneril. probably a new host for this parasite; a Eurasian host, the brown trout fSalmo trutta). is an Important carrier of this parasite throughout the world. The disease in rainbow trout commonly Is referred to as whirling disease, because infected fish display tail-chasing behavior. The pathology of & cerebralis is dependent upon the age of the host when exposed and on the number of infective units present (Halliday 1973). Trout 4-6 months old are generally resistant to infection, because of ossification of the skeleton surrounding the central nervous system. The infection results in deformities such as shortened mandibles and operculae, lordosis, torsion of the caudal region, and dark coloration. Transmission of the parasite requires aging or maturation of the spores (Hoffman and Putz 1969; Prihoda 1983). Wolf and Markiw (1984a) showed that hot (66 C, 40 min) smoking of Infected trout tissue inactivated the spores of (L cerebralis. and prevented transmission. 7

Species infecting ihfi nervous system The nervous system of teleosts commonly Is Infected by species of the Family Nyxobolldae. Recently, Kent and Hoffman (1984) reported Mvxobolus Inaeouus in the brain of Eloenmannla virescens. Mitchell et al. (1985) described & hendrlcksoni from the brain and meninx primltlva of the fathead minnow fPlmeohales oromelas). Sporulated Plasmodia of & hendrlcksoni replaced large areas of the optic lobes and corpus cerebelll. Unencapsulated spores were observed In the brain ventricles. Monocytes, histiocytes, fibrocytes, and rodlet cells were sited In meningeal connective tissues In association with aggregates of spores. Despite extensive Infections, fish survived and exhibited normal feeding and swimming behavior.

Species infecting tM respiratory system A rich supply of nutrients and ready accessibility to the aquatic environment make the gills prime targets for parasitic infections (Roberts 1978). Common myxozoan invaders of gill tissues are species of Mvxobolus (including Mvxosoma) (Daniels et al. 1976; Current et al. 1979; Desser and Paterson 1978; Pulsford and Matthews 1982; Kovacs-Gayer and Molnar 1983), Henneouva (Lom and Vavra 1965; Current and Janovy 1976, 1977, 1978; Bowser and Conroy 1985), Sohaerospora (Lom et al. 1983c, 1985), and Thelohanellus (Dykova and Lom 1987).

Cysts on gills generally are inter- or intralamellar, and Plasmodia are often pinocytotic. Cysts of Myxobolus so. observed on the gills of 8

the common shiner fNotroois cornutus) contained Plasmodia enveloped by syncytial walls bounded by 2 membranes (Desser and Paterson 1978). Pulsford and Matthews (1982) compared the Plasmodia of an Interlamellar and subdermal form of Mvxobolus exiauus In grey mullet fCrenlmuall labrosus). Both Plasmodia! forms showed ptnocytotic activity, and were coated with a filamentous glycocalyx. Host response In juvenile fish Infected with Interlamellar Plasmodia Included hyperplasia of epithelial cells and reduced respiratory surface. Infection with subdermal Plasmodia resulted In distension of the skin surface, slight fibrosis, and the deposition of collagen and melanin. Kovacs-Gayer and Molnar (1983) found that (L basilamellarls formed cysts at the base of gill filaments or gill arches, and caused significant deformities of blood vessels In the gills. Two types (Inter- and intralamellar) of Henneauva exil Is Infections have been described (Current and Janovy 1978). Spore morphology and sporogenesis of the two types are similar, but the interlamellar Plasmodia were surrounded by two outer membranes, and there was a close association with the host cells. By contrast, the intralamellar Plasmodia were surrounded by a single outer membrane, and a granular coating prevented any association with host cell tissue. Bowser and Conroy (1985) observed multifocal interlamellar gill lesions of iL. exil is in channel catfish. Henneauva has been involved in significant losses of channel catfish (NcCraren et al. 1975; Ninchew 1977) by causing proliferative gill lesions. When coupled with environmental stressors, Henneauva infections may lead to death of the host (Bowser and Conroy 9

1985). Lorn et al. {1983c) described Sohaerospora molnari from the gills, skin, and blood of yearling common carp. This myxozoan Infects the stratified epithelium of gill filaments and lamellae. Resulting inflammation reduces surface area of lamellae, which in turn reduces gas exchange. Thelohanellus ovriformls Plasmodia develop In gill filament arteries. Dykova and Lom (1987) found that this myxozoan encloses uninfected hypertrophic host cells. The epithelial cells of the host coat the plasmodial wall.

Coelozoic Myxozoans Coelozoic myxozoans commonly inhabit fluid-filled organs and ducts of the biliary and urinary systems of fishes.

Myxozoans inhabiting the biliary system The key organ of the biliary system commonly infected with myxozoan parasites is the gall bladder. Invasion of the bile ducts and liver may also occur via the gall bladder (Walliker 1968; Lom and Dykova 1981). Although most investigations have been performed with the light microscope, several myxozoan genera that Inhabit the gall bladder have been studied with the electron microscope. Lom and dePuytorac (1965) described the ultrastructure and polar capsule development in Chloromvxum; Listebarger and Mitchell (1980) described the surface morphology of spores of two species of Chloromvxum with scanning electron 10

microscopy. Bajpal and Haldar (1982) described the spores of Mvxidium aoocrvptae from the gall bladder of Aoocrvotes bato. and Delvlnquler (1986) reported scanning electron microscope observations of Mvxidium Immersum from the gall bladder of the cane toad fBufo marlnus). Delvlnquler (1986) postulated that an Intermediate host was required In the life cycle of & Immersum. Bajpal and Haldar (1982) described the spores of Mvxobolus ounctatus from the gall bladder of Oohiceohalus Dunctatus. Lom (1969a) described plasmodlal and spore ultrastructure and development for Sohaeromvxa found In the gall bladder of Lloarls cvcloous and Dasvcottus so.. Grasse and LaVette (1978b) did likewise for a Sohaeromvxa in the gall bladder of Hiopocampus auttulatus. Lom and dePuytorac (1965) described the fine structure of Plasmodia and morphogenesis of sporoblasts and spores of Zschokkella from the gall bladder of Rutilus rutilus. Davies (1985) looked at the prevalence of Zschokkella in the gall bladder of Ciliata mustela. and found that these Infections produced proliferation, enlargement and thickening of hepatic ducts, lowering of the duct epithelium and pericholangitis. Species of Chloromvxum (Mingazzini 1890) commonly Infect bile ducts and gall bladders of teleosts. This genus is characterized by spores with smooth or ridged valves, rarely with caudal extensions. Chloromvxum spores have four polar capsules of equal or nearly equal size, and typically, binucleate sporoplasms. Plasmodia are monosporous or polysporous, and can form pansporoblasts (Lom and Noble 1984). One of the most common species of Chloromvxum. ^ triiuaum. infects 11

the gall bladder of centrarchid fishes in North America (Jameson 1931; Neglitsch 1937; Otto and Jahn 1943; Mitchell 1978a, b), and exhibits marked host specificity; this species was first described by Kudo (1920) from the longear sunfish fLeoomis meqalotis) in Illinois. Plasmodia attach to the mucosa of the gall bladder or float free in the bile. The prevalence of free-floating Plasmodia in bluegill gall bladders is highest during winter and spring, but decreases by half during summer and fall (Mitchell et al. 1980). Mitchell (1978b) in a survey of fishes in the Columbia river drainage (western Montana) found over half of the pumpkinseed fLeoomis oibbosus) examined to be infected with L. tri.iuaum. Mitchell et al. 1980) confirmed the occurrence of plasmotomy in Plasmodia, and offered the first description of sporonts in these species. Listebarger and Mitchell (1980) found that the valves of Lu tri.iuaum have a thick ridge running parallel to the suturai ridge and uncapped polar capsule pores that open into the extrasutural ridges. The only published report on the pathology of Li tri.iuaum infections is that of Mitchell et al. (1980), who described the attachment of Plasmodia to underlying mucosal cells via pseudopodia-like projections. Epithelial cells associated with Plasmodia of L. tri.iuaum showed Indistinct boundaries, loss of nuclear detail, reductions in height, and vacuolation between the infected mucosa and underlying connective tissue. Species of genus Mvxidium occur mainly in fishes, but also have been reported in amphibians. Mvxidium species primarily infect the gall bladder (Jayasri and Hoffman 1982). Delvinquier (1986), using scanning electron microscopy to study the spores of & immersum from the gall 12

bladder of the cane toad found that the longitudinal and transverse valve strlatlons were not as constant as formerly thought. Delvlnquler (1986) also questioned the supposed host specificity of & Immersum. but was unsuccessful in experimentally Infecting hosts. Bajpal and Haldar (1982) described the spores of & aoocrvotae In Apocrvotes bato and Mvxobolus punctatus In Oohlceohalus punctatus. but did not mention pathological effects. Uspenskaya (1966) reported that Mvxidlum lleberkuehni developed special cytoplasmic extensions that penetrate the epithelial cells of the urinary bladder In pike fEsox luclusK She also found no hyaluronic acid or chondroltin sulphate C In the Intercellular spaces and concluded that hyaluronldase did not occur In the Plasmodium. Uspenskaya (1966) further demonstrated acid phosphatase only In the body of the Plasmodium, and postulated that & lleberkuehni absorbed nutrients from the urine. In a later study, Uspenskaya (1969) reported pinocytotic activity by & oasterostel In the gall bladders of Gasterosteus aculeatus and Punaltlus punaitlus. Ceratomvxa shasta. another species Infecting the gall bladder, causes ceratomyxosis, a disease Involving general visceral degeneration, in susceptible salmonids. This myxozoan has caused several epizootics among juvenile and adult salmonid fish in the western United States (Ratiiff 1981). Sequelae include loss of appetite, distended abdomens, hemorrhaging around the vents, darkened coloration, lethargy, and death (Conrad and DeCew 1966). Ceratomvxa shasta Is geographically restricted to northern California, Oregon, Washington, Idaho, and British Columbia (Ching and Munday 1984). The life cycle of Li. shasta is virtually 13

unknown (Wade 1986} and spores of this parasite do not seem capable of directly Infecting other fish (Johnson et al. 1979).

Mvxozoans inhabiting excretory systems Nyxozoans Inhabiting kidney tubules and urinary bladders Include Hoferellus cyorlnl (Lorn and Dykoya 1985), Kudoa sjl. (Paperna 1982), Myxidlum (Dykova et al. 1987), three species of Myxobllatus (Guilford 1965; Booker and Current 1981), and flye species of Sphaerosnora (Hamilton 1980; Dykova and Lom 1982; Lom et al. 1982; Grupcheya et al. 1983; Hermanns and Korting 1985; Arthur and Lom 1985; Desser et al. 1986). Lom and Dykoya (1985) described the fine structure of iL cyorlnl. Sporogeny was absent and Plasmodia underwent a gradual degradation of cell structure, rendering themselves Incapable of further development. Infected epithelial cells formed syncytial masses of parasites, and were subsequently eliminated by host tissue reaction. Because no spore formation was ever apparent during the infection, Lom and Dykova (1985) suggested that the intracellular stages, assumed to be those of iL. cvprini. may actually be abortive developmental stages of another myxozoan, Sohaerosoora renicola. Paperna (1982) reported that 85% of observed glomeruli in Soarus aurata were Infected with Kudoa so. He observed sporonts in mesangial and visceral epithelium. From one to eight sporonts were seen per glomerulus, resulting in swollen Bowman's capsules, obstructed capillary lumina, and encapsulation within visceral epithelium. Changes in hematopoietic tissues or renal tubules were not observed. 14

Plasmodia of Mvxidlum rhodei In the Bowman's capsule were associated with hypertrophy and atrophy In adjacent renal parenchyma (Dykova et al. 1987). Only mature Plasmodia In renal corpuscles were associated with granulomatous reactions, but In the interstitlum, young Plasmodia were found in granulomatous Inflammatory tissues. Dykova et al. (1987) showed that heavy infections of & rhodei in the kidney of Rutilus rutilus could result in a reduced number of glomeruli, alteration of the interstitlum, or atrophy of surrounding tissues. Paperna et al. (1987) studied the attachment of Mvxidlum aiardl to the epithelium of the urinary bladder of juvenile eels (Anguilla mossambica). These researchers suggested that the projections from Plasmodia, which form firm attachments to the host cells, were actually pseudopodia, not microvilli. They also described a network of large tubules and cisternae associated with endoplasmic reticulum in Plasmodia. Plasmodia of (L aiardl contained only vegetative cells. Species of Sohaerospora may infect all types of kidney tubules (Hamilton 1980), and have been reported from the kidneys of goldfish (Hamilton 1980), common carp (Dykova and Lom 1982; Lom et al. 1982), tench. Tinea tinea. (Hermanns and Korting 1985), skates (Arthur and Lom 1985), and bullfrog tadpoles (Desser et al. 1986). Most species of Sohaerospora appear to be highly host specific (Arthur and Lom 1985). Hamilton (1980) isolated Sohaerospora so. from the kidneys of goldfish diagnosed with "goldfish ulcer disease". The degree of infection among fish varied, Sohaerospora so. was present in some healthy fish, and the degree of infection did not correlate with the 15

presence of ulcers on the goldfish. It was concluded that the ulcers were not associated with the myxozoan Infection. Ultrastructural1y, microvilli and cilia In kidney tubules were Interdlgltated with Plasmodia! extensions of Sohaerospora iq. Lorn et al. (1982) described the ultrastructure of Sohaerospora renlcola In carp kidney, and Dykova and Lom (1982) discussed Its pathogenicity. Sohaerospora renlcola usually is attached to the microvilli of renal tubule epithelia rather than floating free in the lumen. Both the host cell microvilli and the plasmalemma of the parasite become electron dense along the host-parasite interface. The membranes form close associations resembling gap- junctions. Further, pseudopodia formed by the Plasmodia were seen extending down between the microvilli (Lom et al. 1982). Sohaerospora renlcola causes swelling, hyperplasia and dystrophy within the renal tubule cells. Luminal infections were reported to cause eventual necrosis of renal epithelium (Dykova and Lom 1982). Sohaerospora tincae is host specific in tench, and associated mortalities may be high (Hermanns and Korting 1985). This myxozoan forms large aggregations in the anterior portion of the body, and may displace the head kidney. Sohaerospora tincae has not been found In the excretory kidney. Arthur and Lom (1985) described Sohaerospora araii from the longnose skate fRala rhina). This myxozoan, the first species of Sohaerospora described from an elasmobranch, forms large thin walled developing spores, perhaps unique among myxozoans. Cesser et al. (1986) provided the first electron microscopic study of 16

it ohlmâcheri. a species Infecting bullfrogs, first described by Kudo (1920). Pseudoplasmodia of L. ohlmacherl from bullfrog tadpoles were observed In the space of Bowman's capsule. The presence of parasites caused degeneration of renal tubule epithelium. Cesser et al. (1986) postulated that degeneration of the epithelial tissues contributed to cell necrosis. Members of the genus Mvxobllatus are coelozolc parasites of the urinary system In freshwater and marine fishes (Davis 1944). Kudo (1920) originally placed these parasites in the Genus Henneauva. but they were removed to the Genus Mvxobi1atus by Davis (1944). The major characteristics distinguishing Mvxobllatus from Henneauva are that spores of Mvxobllatus are ridged and have a suture in frontal view. Mvxobllatus also seems to Infect only the urinary system, whereas species of Henneauva are chiefly histozoic. Little is known about Mvxobllatus species. Guilford (1965) described & noturi from the tadpole madtom fNoturus avrlnus). but to date no other studies have been made on this species. Booker and Current (1981), in the only report to date of an electron microscopical study of a Mvxobllatus species, provided evidence of distinctively different morphological forms of Plasmodia occurring at different times of the year. Sheetlike Plasmodia in ÎL mictospora existed throughout the year, while fingerlike and spherical Plasmodia occurred during the spring and summer in specimens obtained From the urinary bladders of largemouth bass fMicrooterus salmoides). Because a greater number of mature spores were observed in urinary bladder contents when the finger-like or spherical 17

Plasmodia were present, Booker and Current (1981) proposed that the sheetlike structure was an overwintering form. The Plasmodia of (L leaerl In the urinary bladder of Cobltis barbatula (Cepede 1905) were also thought to be overwintering forms, because Plasmodia grew during winter and did not produce spores until spring. Booker and Current (1981) also provided ultrastructural evidence of endocytosis of host cell cytoplasm by & mlctosoora. formerly reported only for histozolc species.

Life Cycle Postulated two-host Ufg cvcle In sharp contrast to previous assumptions that the myxozoan life cycle is direct, Marklw and Wolf (1983) and Wolf and Marklw (1984b) postulated that myxozoans have two hosts. Marklw and Wolf (1983) found that tublflcid ollgochaetes play an essential role in the development of infectivity of Mvxobolus cerebral is spores. They showed that the infectivity of IL cerebralis was not developed endogenously as previously thought, but that the aging process discussed by Hoffman and Putz (1969) and Uspenskaya (1978) actually required the presence of aquatic ollgochaetes. Marklw and Wolf (1983) aged spores of (L cerebralis in inert, sterilized, pasteurized, and natural aquatic substrates. Aquatic soils from hatcheries with fish known to have whirling disease were also tested. Whirling disease developed in young rainbow trout fed ollgochaetes collected from pond soil of hatcheries with whirling disease epizootics. In other experiments, four genera of ollgochaetes, Aeolosoma. Dero. Stvlaria. and Tublfex. obtained from biological supply houses were 18

added to containers of pasteurized soil. Tublflcid worms from whirling disease free hatcheries were also used In these experiments. Four months subsequent to Inoculations with £L cerebral is spores, whirling disease occurred only in trout held with Tubifex or hatchery tubificids. It is unclear whether Tubifex is the only annelid genus involved, because accurate identification of hatchery tubificids was not provided. Nevertheless, it is interesting that fresh spores of IL cerebralls have never been shown to Infect fishes (Wolf and Markiw 1984b). Wolf and Markiw (1984b) postulated that the whirling disease life cycle is blphasic, with two dissimilar spore stages. The & cerebral 1 s spores are lenticular (8.0-10.0 pm), and Infectious to the second host, Tubifex. Spores of (L cerebral is. thought to be liberated when the fish dies or is eaten by a predator, reach the tublflcid per os. Inside the tublflcid, the spores gain in size and number, and change morphologically to a large (170-180 nm) tripartite spore formerly described for the genus "Triactinomvxon". The "Triactinomvxon" is waterborne and present in the tublflcid (Wolf and Markiw 1984b). Susceptible fish may contract whirling disease after ingesting "Triactinomvxon" spores released by the tublflcid. Wolf and Markiw (1984b) named the tublflcid form a new species Triactinomvxon qvrosalmo. However Corliss (1985) pointed out that a single species cannot be known concurrently by two names; this violates the International Code of Zoological Nomenclature (12, Art. 23). Corliss (1985) emphasized that the species name Mvxobolus cerebralis holds precedence over all others for this parasite. Markiw (1986) reported on the dynamics of development and production 19

of Triactlnomvxon spores. In two experiments she found that specific- parasite free tublflcid ollgochaetes (held at 12.5 C) exposed to spores of ÎL cerebral is released 26-50 million Triactinomvxon spores after 113 days. Triactlnomvxon spores were released in trace amounts for up to 9 months. Markiw (1986) reported a 20% prevalence of infection. These results have been recently refuted by Hamilton and Canning (1987). On the basis of spore dimensions (epispore 36 im; 32-50 sporoplasms; style 90 /mi; projecting arms &170 /mi), Hamilton and Canning (1987) considered L. qvrosalmo described by Wolf and Markiw (1984b) to be synonymous with L. dubium. Further, after adding spores of & cerebralis to sterilized medium containing Tubifex tubifex. they observed no significant change in the prevalence of Triactinomvxon dubium (syn. L. qvrosalmo). Tubifex tubifex ingested & cerebralis spores, but hatching of spores or further development was not observed. Hamilton and Canning (1987) found no obvious correlation between the occurrence of L. dubium (syn. L. qvrosalmo) and & cerebralis. They strongly disagree with the two host life cycle proposed by Wolf and Markiw (1984b). They believe that the small number of actinosporeans known to date makes these parasites unlikely as secondary hosts. Clearly, additional studies involving several myxozoan parasites and additional species of ollgochaetes are required to confirm or refute the two host hypothesis. Possibly monoclonal antibody techniques and other immunocytochemical procedures will aid in determining the life cycle of Mvxobolus cerebralis. and possibly other myxozoan species. 20

Development within IM fiih Msi Most researchers concur that when an Infective myxozoan spore is ingested by a suitable fish host, attachment to the intestine is accomplished by extrusion of the polar filament (Mitchell 1977). The mature spore consists of shell valves, polar capsules (each containing a coiled polar filament), and a sporoplasm cell with two nuclei that fuse before or after Infection of the host. Once the spore is ingested, the sporoplasm is released and transferred to the infection site where it is termed an amoebula. The amoebula stage undergoes repeated nuclear divisions and cytoplasmic growth to become large inter- or Intracellular Plasmodia. The Plasmodia continue to Increase in size, and become multicellular coenocytes. The multicellular Plasmodia of histozoic species are generally surrounded by host tissue capsules. Coelozoic Plasmodia attach to host tissues or may float freely in luminal spaces. The cytoplasm of a Plasmodium is sometimes segregated into a clear outer ectoplasm, and an internal granular endoplasm. Sporoaenesis. As myxosporean Plasmodia increase in size, much of their cytoplasm differentiates in the process of sporogenesis. The earliest events of sporogenesis have been largely ignored in ultrastructural studies (Lom et al. 1983b), but two types of nuclei, vegetative and generative, can be distinguished with the light microscope as this process begins. Generative nuclei become surrounded by aggregates of cytoplasm, and eventually become generative cells. Cesser et al. (1983a) described the early generative cells of Thelchanellus nikolskii. Uspenskaya (1976) suggested from cytophotometric studies that 21

vegetative nuclei In Mvxidlum lleberkuehni are polyploid and that melosls occurs during early sporogenesis, producing haplold sporoblast cells and spores. In most species that have been studied, sporogenesis continues as generative cells pair. The membranes of paired cells become closely adherent and may Interdlgltate. Eventually, one cell of each pair engulfs the other. The engulfing cell, sometimes called the pericyte, does not develop further. The engulfed generative cell differentiates Into sporoblasts. These general life cycle patterns have been confirmed In several genera, Including Henneouva (Schubert 1968; Current and Janovy 1977; Current 1979), Mvxobolus (Lom and dePuytorac 1965; Desser and Paterson 1978; Current et a1. 1979; Pulsford and Matthews 1982), SohaerosDora (Lom et al. 1982; Desser et al. 1983b), Sohaeromvxa (Grasse and Lavette 1978b), and Thelohanellus (Desser et al. 1983a). Although pairing and engulfment seem to be typical for myxosporeans, these processes may not occur In Kudoa oanlformls (Stehr 1986), Ceratomvxa shasta (Yamamoto and Sanders 1979), Sohaerospora renlcola (Lom et al. 1982), or In Mvxidlum zealandlcum (Hulbert et al. 1977). Later events In sporogenesis Include repeated amitotic divisions of engulfed or unpaired generative cells. In most myxosporeans that have been studied, an engulfed cell produces 10 cells, which differentiate into 2 units (sporoblasts) of five cells each. Each unit, consisting of two valve cells, two polar capsule cells, and a single, binucleate sporoplasm cell forms a single spore. Except for Sohaerospora renlcola which is polysporous, all species that do not exhibit engulfment have monosporous sporoblasts. 22

Capsuloaenesis Capsulogenesis Is the developmental stage during which polar capsules and associated filaments are produced. During capsulogenesis, both smooth (Stehr 1986) and granular endoplasmic reticulum In capsulogenic cells appear swollen (Desser et al. 1983a; Grasse and Lavette 1978a; Weldner and Overstreet 1979). These swollen cisternae may play a role In polar capsule origination (Pulsford and Matthews 1982; Schubert 1968; Desser and Paterson 1978; Lom 1969a). Other characteristics of capsulogenic cells Include mitochondria with tubular cristae, nuclei with or without heterochromatin, and polar capsules at various stages of development. Subsequent to differentiation and final positioning of the capsulogenic cells, capsular primordia appear with short external tubules. Later in development, the external tubules lengthen, form extensive loops throughout the capsulogenic cells, and terminate at filament discharge canals. The external tubules are surrounded by microtubules, which may be involved in Intracellular movements and contractions (Wessels et al. 1971). These events are common in Chloromvxum (Lom and dePuytorac 1965), Henneauva (Schubert 1968; Current and Janovy 1977; Current 1979; Current et al. 1979), Kudoa (Stehr 1986), Mvxosoma (Current et al. 1979), Mvxobolus (Desser and Paterson 1978; Pulsford and Matthews 1982), Mvxldium (Lom and dePuytorac 1965), Sohaeromvxa (Lom 1969a; Grasse and Lavette 1978b), Sohaerospora (Hamilton 1980; Lom et al. 1982; Desser et al. 1983b), Thelohanellus (Desser et al. 1983a), and Zschokkella (Lom and dePuytorac 1965). All studied 23

myxozoans, except Fabespora vermicola. which occurs in piatyhel(ninths (Weidner and Overstreet 1979), exhibit external tubules. In nearly mature spores, the polar filament develops within the external tubule and cortex of the capsular primordium. Eventually the external tubule shortens, the capsular primordium increases in volume, and the polar filament appears in the cortex of the capsular primordium. The conversion of external tubule to polar filament occurs very rapidly (Desser et al. 1983a). Although the process has never been observed, it is presumed that the external tubule invaginates into the polar capsule, and differentiates into the polar filament (Desser and Paterson 1978; Lom 1969a; Lom and dePuytorac 1965; Schubert 1968).

Valvoaenesis Valvogenesis is the developmental process by which the shell valves enclosing myxozoan spores are formed. Valvogenic cells join to form suturai ridges and polar filament discharge canals. The valvogenic cells are identifiable in immature spores by their positions on either side of the capsulogenic and sporoplasm cells. Valvogenesis in Henneouva. Kudoa. Mvxobolus cvorini. ÊL. pharvnaeus. and Thelohanellus usually begins with the appearance of electron dense inclusions near the suturai ridges and beneath the valvogenic cell plasma membrane (Current 1979; Current and Janovy 1977; Stehr 1986; Spall 1973; Desser et al. 1983a). This electron-dense substance, or valve forming material (Current 1979) occurs much later in the development of Mvxobolus funduli. Sphaerospora. and Sphaeromvxa (Pulsford and Matthews 1982; 24

Desser and Paterson 1978; Current et al. 1979; Lorn et a1. 1982; Lorn 1969a). In Kudoa paniformis (Stehr 1986), Mvxobolus s!^ (Desser and Paterson 1978), and Sohaerornvxa (Lorn 1969a) striated, amorphous material appears at the apical end of the spore. In Immature spores of several tnyxozoans, microtubules occur along the suturai plane (Lom 1969a; Desser et al. 1983a), although Stehr (1986) did not observe these microtubules in L. paniformis. The cytoplasm of Immature valvogenic cells concentrates around the suturai ridges and polar filament discharge canal region (Current 1979). After maturation of the spores, the microtubules disappear and the cytoplasm Is reduced to only a small portion separating the opposing membranes of mature valve cells.

Sporoplasm development The majority of myxozoan species studied to date have exhibited binucleate sporoplasm cells with dense Inclusions in their cytoplasms (Schubert 1968; Lom 1969b; Desser and Paterson 1978; Pulsford and Matthews 1982). It Is assumed that in the mature spore the sporoplasm nuclei fuse to form a single diploid nucleus. However, in Sohaerospora renicola (Lom et al. 1982) and Ceratomvxa shasta (Yamamoto and Sanders 1979), the sporoplasm divided into two separate cells. Stehr (1986) observed two cells of different sizes in the sporoplasm of paniformis. The smaller, more electron dense cell, was encapsulated by the larger cell. Mature sporoplasm cells contain numerous free ribosomes, granular endoplasmic reticulum, and prominent mitochondria. The sporoplasm cell contains j8-glycogen particles, which give it a dense appearance. 25

Explanation of Dissertation Format The outline of this dissertation Includes a general Introduction, an explanation of format, three manuscripts (Sections I-III), a general summary, and bibliography. The format of the three manuscripts conforms to that prescribed by the Iowa State University thesis manual rather than a journal format. Each manuscript represents a publlshable entity, and Includes Introduction, Materials and Methods, Results, Discussion, and References cited. 26

SECTION I.

ULTRASTRUCTURAL OBSERVATIONS ON SPOROGENESIS OF CHLOROMYXUM TRIJUGUM (MYXOSPOREA: CHLOROMYXIDAE) FROM FOUR CENTRARCHID FISHES 27

INTRODUCTION

Chloromvxum triiuaum is a coelozolc myxosporldan commonly Infecting the gall bladder of several centrarchid fishes (Jameson 1931; Neglitsch 1937; Mitchell 1978a, 1978b; Mitchell et al. 1980). This species was first reported by Kudo (1920) in longear sunfish fLeoomis meoalotist in Illinois. Plasmodia of this myxozoan are found floating free in bile and/or attached to the mucosal surface of the gall bladder. The complete life cycle of myxozoans has not yet been determined. Wolf and Markiw (1984) described a developmental scheme involving a vertebrate and invertebrate host for Mvxobolus cerebral is. but any assumptions of this being common among other myxosporean species are speculative. Research on culture techniques and experimental infections are needed to elucidate details of myxozoan life cycles. There is a concensus that ultrastructural and histopathological studies will greatly advance our knowledge of myxozoans (Lom and Noble 1984). While ultrastructural studies have been done on numerous myxosporeans such as Ceratomvxa shasta (Yamamoto and Sanders 1979), Chloromvxum cristatum (Lom and dePuytorac 1965b), Henneauva (Schubert 1968; Current and Janovy 1977; Current 1979), Kudoa (Stehr 1986), Mvxobolus (Desser and Paterson 1978; Current et al. 1979; Pulsford and Matthews 1982), Mvxidium (Hulbert et al. 1977; Dykova et al. 1987), Sohaeromvxa (Lom 1969; Grasse and Lavette 1978), Sohaerospora (Lom et al. 1982; Desser et al. 1983b), and Thelohanellus (Desser et al. 1983a), the fine structure of £s. tri.iugum has largely been ignored. Aside from the 28

examination of spores of two species of Chloromvxum by scanning electron microscopy (Listebarger and Mitchell 1980) and a brief unpublished transmission electron microscope study by Listebarger (1981), studies of Chloromvxum have been limited to the light microscopical level. The purpose of this report Is to describe sporogenesis In trl.luaum. The host-parasite Interface and pathogenesis associated with gall bladder Infections of Chloromvxvm trl.luaum will be described In a subsequent paper. 29

MATERIALS AND METHODS

Four species of fishes, 30 blueglll fLepomls macrochlrus). two white crapple fPomoxIs annularis), two black crapple fPomoxIs nIaromaculatusK and five pumpklnseed (Leoomls qlbbosus) were collected between September 1985 and May 1987 from three artificial Impoundments in Story County, Iowa (Hickory Grove Lake, Horticulture Pond, and Petersen Pits), and from backwaters of Pool 7 of the Upper Mississippi River (LaCrosse County, Wisconsin). Infections of Chloromvxum tri.iuaum were diagnosed by observing characteristic spore stages in fresh bile with the light microscope.

Transmission Electron Microscopy Normal and Infected gall bladders were removed from pithed fish, placed in Karnovsky's fixative (2.0% paraformaldehyde / 2.5% glutaraldehyde) in O.IM cacodylate buffer (0-4 C) for 30 min to 1 h, minced into small pieces (0.5-1.0 mm) and transferred to fresh fixative for 2h to 24h at 0-4 C. Tissue was post-fixed in 1.0% osmium tetroxide in 0.1 M cacodylate buffer for 2h, washed in sterile deionized water, and in some cases stained en bloc with 2.0% uranyl acetate for 2h. Stained tissues were dehydrated in a graded series of acetone, and embedded in Medcast epoxy resin. Thick sections were stained with.either toluidine blue (1.0% in borax) or azure II/methylene blue and observed with the light microscope. Ultrathin sections of grey or silver interference colors were cut with a diamond knife on a Sorvall MT-2 or Relchert Om-U2 30

uUramlcrotome. Sections on copper grids were post-stained with uranyl acetate and lead citrate. In some cases, uranyl magnesium acetate was substituted for uranyl acetate. Grids were examined with a Hitachi HS-8 or HU-llC electron microscope at 50 kV.

SBfifilâl ilâM Ruthenium red stain was used to locate areas with mucopolysaccharides. Reagent grade ruthenium red was added to both buffered Karnovsky's fixative and osmium tetroxide at a concentration of 0.15% (Luft 1971a,b). Ultrathin sections were observed either unstained or subsequent to post-staining with uranyl acetate and lead citrate (Brooks 1969). Lanthanum nitrate was used to examine permeability pathways in host tissue and at the host-parasite interface. Tissue was fixed in Karnovsky's fixative and post-fixed with 1.0% osmium tetroxide plus 3.0% lanthanum nitrate (Revel and Karnovsky 1967). Specimens were dehydrated and embedded normally. Ultrathin sections were post-stained with uranyl acetate and lead citrate. 31

RESULTS

Early Sporogenesis The youngest Plasmodia! stages of L. trliuoum seen In this study lacked sporoblasts and contained numerous spherical electron lucent structures (Fig. 1). Generally the ectoplasmic regions of the cytoplasm were more densely stained. The endoplasmic region of many Plasmodia observed In blueglll contained numerous cells with dividing nuclei and

mitochondria (Fig. 2). Filaments (0.05 fm diameter) were scattered throughout the ectoplasm. Some Plasmodia contained membranous figures bounded by double membranes. These structures appeared to originate as small, electron-dense spheres themselves surrounded by a double membrane (Fig. 3). Most Plasmodia on the gall bladder epithelium of blueglll were elongated and extended over 5-6 mucosal cells. Microvlllosltles were present, but regions of Plasmodium completely devoid of microvlllosltles were common (Fig. 4). Microvlllosltles varied In size (1.0-3.0pm long) and location. Plasmodia contained numerous lipid inclusions, nuclei (generative or vegetative), and finger-like tubules (0.8-1.2 jm long). Some Plasmodia contained distinctive ectoplasmic regions with fibrillar nets, rounded vesicles, small ovoid mitochondria, and multivesicular bodies (Fig. 5). The endoplasm contained developing cells closely associated with mitochondria and numerous vesicles. Several membrane- bounded vesicles (0.1 im), glycogen granules, and golgi apparatuses 1

Figure 1. Electron micrograph of early Plasmodium of Chloromvxum tri.iuaum attached to the mucosa of a bluegill. P, Plasmodium; G, generative cell; H, host cell; V, vesicles. 6,613X Figure 2. Electron micrograph of plasmodial endoplasm. (2a) membranous figures bounded by double membrane; (2b) cell within Plasmodium containing dividing nucleus. F, filaments. 6,048X Figure 3. Ultrastructure of small, electron-dense spheres surrounded by double membrane. P, Plasmodia. 20,790X Figure 4. Electron micrograph of Plasmodium extended over several host cells. Mv, microvillosities; In, lipid inclusions; Nu, nuclei; t, tubules. 8,352X Figure 5. Ultrastructure of ectoplasm in Plasmodium of L. tri.iuaum. fb, fibrillar net; V, vesicles; Mt, mitochondria; Mvb, multivesicluar bodies. 8,352X 33 34

occurred In the endoplasmic zone. The developing cells within Plasmodia had granular cytoplasm and were approximately 10 /un In diameter. They also contained dividing nuclei near the center, and mitochondria at their periphery. Paired generative cells were observed in the final stages of early sporogenesis. The larger of the two generative cells typically enveloped the smaller cell. This stage of development was seen in both free floating and attached Plasmodia (Figs. 6 and 7).

Envelopment Stage Plasmodia in blueain Microvillosities covered portions of the luminal surfaces of Plasmodia containing envelopment stages. Enveloping cells surrounded two developing sporoblasts. The cytoplasm of the enveloping cells contained ellipsoid nuclei, mitochondria, and inclusions (Fig. 8). In some cases the membrane of the enveloping cell appeared to be fused with the plasmalemma of the luminal surface of the Plasmodium. Microvillosities were present, but did not encompass the entire Plasmodium. Mitochondria with numerous vermiculatlng cristae often occurred in the ectoplasmic region of the Plasmodium. The enveloped generative cells had divided and were populated with golgi apparatuses, mitochondria, and spherical structures with dense striae (Figs. 9 and 10). Some of the mitochondria contained dilated cisternae. Divided generative cells contained large nuclei (0.7-1.9 pm), surrounded by a narrow rim of cytoplasm. Free ribosomes were abundant in 35

the cytoplasm of sporoblast cells. The membranes of the sporoblast and enveloping cells were elaborately folded, and In some cases formed

pseudopod-like extensions (0.5 /m) with pouch-like terminal ends. Sporoblast cells In later stages of development possessed 25-30 membrane enclosed spherical structures. These structures ranged In size from 0.1 to 0.4 im In diameter, and had loosely granular electron-lucent centers. Plasmodia In srmis. Plasmodia In Pomoxis annularis and & niaromaculatus were similar to those infecting bluegill. Microvillositles were short (0.2-0.6 im long) and numbered 5-20 per ultrathin section of Plasmodium. The ectoplasmic regions of the Plasmodia were populated with vesicles and mitochondria. The vesicles (0.1-0.8 im in diameter) were rounded or ovoid shaped and contained a loosely granular electron-lucent Interior stippled with electron-dense material. The mitochondria (about 0.6 ^m in diameter) had thin symmetrical cristae (Fig. 11). The cytoplasm of the enveloping cells contained rounded mitochondria, but generally lacked other organelles. Enveloped sporoblast cells were of unequal sizes. The smaller sporoblast cells were surrounded by considerably more mitochondria (8-10 per section) than the intermediate (1-2) or larger (0- 1) sized sporoblast cells. Free nbosomes, golgi apparatuses, and inclusion bodies were seen primarily in the cytoplasm of the larger sporoblast cells. Other structures observed in the cytoplasm included rough endoplasmic reticulum (with swollen cisternae) and numerous Figure 6. Electron micrograph of paired generative cells. Env, enveloping cell; Nu, ndeus; G, generative cell; Mv, microvlHosltles. Arrows point to host tissue. 11,790X Figure 7. Envelopment stage of sporogenesls. Larger of two generative cells envelops the second cell. P;, plasmalemma of Plasmodium; Pe, plasmalemma of enveloping cell; Mt, mitochondria; Sp, sporoblast. Note how enveloping cell plasmalemma forms junction with plasmodlal plasmalemma (arrows). 8,352X Figure 8. Ultrastructure of cytoplasm of enveloping cells containing nuclei (Nu), mitochondria (Mt), and Inclusion bodies (In). 8,352X Figures 9-10. Electron micrograph of divided and differentiated generative cells. Nu, nucleus; In, Inclusions; Go, golgi apparatus; Mt, mitochondria; Env, enveloping cell; EvN, enveloping cell nucleus. Unidentifiable spherical structures (large arrow). Fig. 9, 20,700X; Fig. 10, 6,048X Figure 11. Electron micrograph of enveloped generative cells of L. tri.luaum In Pomoxis. Enveloped sporoblast were of unequal sizes. Nu, nucleus; V, vesicles; Mt, mitochondria; In, Inclusion bodies. 8,3S2X 37 38

pinocytotic vesicles, which were closely associated with the nuclear membrane.

Plasmodia in pumokinseed The majority of Plasmodia were long, narrow, bounded by a single limiting membrane, and polysporous. Anastomosing microvillosities (0.2- 0.8 /un long) covered most of the Plasmodia! surface. The ectoplasm contained isolated regions of golgl apparatus, multivesicular bodies, and mitochondria. The mitochondria were elongated (0.7 to 1.5 im length), round (0.3 to 0.7 im diameter) or oval (0.3 to 0.6 /zm diameter), and more prevalent in regions distal to the developing sporoblasts (Fig. 12). The envelopment of generative cells and their subsequent amitotic divisions were asynchronous within a single Plasmodium. The enveloping cell membrane was surrounded by an electron-dense band (900 A" wide). The only organelles observed in the enveloping cell cytoplasm were free ribosomes. Enveloped generative cells ranged in diameter from 0.7 fim to 2.7 im. These cells were very tightly compacted. Granular, electron- lucent structures were prominent in the sporont cytoplasm, but other cellular organelles were absent. Mitochondria were not seen in the vicinity of developing sporonts (Figs. 13 and 14).

Capsulogenesis Evsnt; in biueoin Plasmodia undergoing capsulogenesis were occupied with one to several developing spores. Microvillosities were present, and ranged in 39

number from 5 to 10 per ultrathin section of Plasmodium. The ectoplasm was reticulated, and contained crescent shaped mitochondria. Fewer vesicles were observed in the ectoplasmic region than In earlier stages. The maturing spores contained units of capsular cell nuclei and sporoplasm cells surrounded by valvogenic cells (Fig. 15). Capsulogenic cells contained endoplasmic reticulum, golgl apparatus, and small mitochondria. The polar capsules were located in the apical region of the capsular cells (Fig. 16). Early polar capsules were sac- like and possessed external tubules. Distal segments of external tubules were observed throughout the capsular cell. Capsular primordia were composed of amorphous electron-dense cores surrounded by trilaminar walls (Fig. 17) of an inner electron-lucent band (0.06 (im wide), a medial electron-dense band (0.1 pm), and an outer electron-lucent band (0.06 /un wide). The outer sheath tapered anteriorly, ending in what appeared to be a discharge tube. The dense inner core did not extend as far anteriorly as the outer sheath. The medially located dense band encircled the capsular primordium, and joined anteriorly to form a dense cap. Microtubules were not seen surrounding capsular primordia or external tubules. The external tubule was absent in mature polar capsules. Approximately half of the central core in mature polar capsules remained electron-dense; however, the remainder became granular. Polar filament material was evident in the lucent regions of the polar capsule core (Fig. 18). Valve formation was clearly evident at this time. Figure 12. Electron micrograph of ectoplasmic region of Plasmodium in L. tri.iuaum from the gall bladder of L. aibbosus. Mv, microvillosities; Go, golgi apparatus; Mvb, multivesicular bodies; Mt, mitochondria. 5,500X Figure 13. Electron micrograph showing asynchronous development of enveloped generative cells, and a developing sporoblast (arrow), rb, ribosomes; Env, enveloping cell; Sp, sporoblast cell; Nu, nucleus. 5,500X Figure 14. Electron micrograph of Plasmodium of tri.iuaum containing developing sporoblast. Mvb, multivesicular bodies; Mt, mitochondria; Cp, capsulogenic cell; Vc, valvular cell material; Et, external tubule; 8,352X Figure 15. Electron micrograph of developing capsulogenic cells, Cp, capsulogenic cell; Nc, capsulogenic cell nucleus; Et, external tubule. Bluegill fL. macrochirus): 20,790X Figure 16. Ultrastructure of polar capsule in L. tri.iuaum from bluegill it. macrochirusl. Dense central core, (d), Pc, polar capsule; Et, external tubule; Mt, mitochondria, Nc, capsulogenic cell nucleus. 8,618X Figure 17. Ultrastructure of nearly mature polar capsule in L. tri.iuaum from bluegill li. macrochirusK Arrows point to trilaminar wall. Df, filament discharge tube; Vv, valvular material (shell); Polar filament (large arrow). 20,790X 41 42

Events 1q cranole The ectoplasmlc regions of Plasmodia observed during capsulogenesis contained golgl and crescent-shaped mitochondria with thickened cristae. Membrane bounded vesicles with granular, electron-lucent centers and pockets of amorphous 1ip1d-11ke material were also dispersed throughout the ectoplasm (Fig. 19). The capsulogenic cell cytoplasm was occupied by endoplasmic reticulum with swollen cisternae, numerous mitochondria, and polar capsules. Developing polar capsules contained an electron-dense core surrounded by a wide (0.2 im) band of electron-lucent material, which was bound by a narrow dense band (Fig. 20). The polar capsules were separated from the capsulogenic cell cytoplasm by a single membrane. External tubules observed were continuous with the polar capsule. Sections of external tubule were seen throughout the capsulogenic cell cytoplasm, and contained cores similar in structure to those seen in polar capsules. Microtubules were not observed around the external tubules.

Events in pumokinseed Plasmodia contained immature spores and early sporoblasts (Fig. 21). Multlveslculated bodies were present in the ectoplasm at this stage of development, as well as several round and elongated mitochondria with electron-dense bodies in the cristae. The earliest evidence of capsulogenic formation was seen at the eleven cell stage of development (Fig. 22a, b). Small polar capsules 43

containing crescent shaped electron-opaque structures were observed. Sections of external tubule were also observed adjacent to the polar capsules. Coincident with spore maturation, the polar capsule wall became progressively thicker and the central core Increased In electron density (Fig. 23). Mitochondria were also observed at this stage In the cytoplasm of capsulogenic cells. At this stage of capsulogenesis, valvogenic cell development was well underway. Suturai depressions were evident at the anterior ends, and contained large mitochondria and membrane fragments.

Valvogenesis Valvogenesis usually accompanied capsulogenesis and was similar In all four hosts. Valvogenic cells were recognizable by their peripheral location, and the dense staining material In the cytoplasm. Ultrastructural details Indicated that as development proceeded, the valvular cells Increased In length and thickness, completely surrounded developing capsulogenic and sporoplasm cells, and joined at either end to form sutures. Three ridges were formed on either side of the suturai lines by infoldings of valvular material (Figs. 24 and 25). Numerous vesicles and a few mitochondria were observed in the endoplasm of the Plasmodium adjacent to the maturing spores. In advanced stages of valvogenesis, the valve cells continued to decrease in width, and became increasingly more compartmentalized. In maturing spores the cytoplasm was limited to the regions of valve process Figure 18. Electron micrograph of Plasmodium of L. tri.iuaum from white Crappie fPomoxis annularis) during capsulogenesis. Mt, mitochondria; V, vesicles. 8,352X Figure 19. Electron micrograph of late stage of capsulogenesis. Polar capsules (Pc) have rounded and reduced electron-dense cores (d). Sp, sporoplasm cell; Nc, capsulogenic cell nucleus; Et, external tubule; Mt, mitochondria. 8,352X Figures 20-21. Electron micrographs of attached and free-floating Plasmodia of L. tri.iuaum from the gall bladder of the pumpkinseed II. oibbosus). Mvb, multivesicular bodies; Mt, mitochondria; Sp, sporoblast; Vg, vegetative nuclei. 5,500X Figure 22a. Ultrastructure of maturing polar capsule showing thickened capsular wall. Cp, polar capsule; Vv, valvular material; tu, tubuli. 11,700X Figure 22b. Electron micrograph of anterior end of nearly mature spore. Suturai depressions contained large mitochondria (Mt) and membrane fragments (arrow). 11,700X Figure 23. Electron micrograph of mature spore from the gall bladder of pumpkinseed (L. aibbosus) in cross section. Large uninucleate sporoplasm cell (Sc) and four polar capsules (Pc) with polar filaments (Fp). Spore is surrounded by a thick valve shell (Vs). In the center is a polar filament discharge tube (Df). 11,700X 45 46

formation. The valve wall of mature spores assumed a laminated structure (Fig. 26). In sections from infected white crappie and from a pumpkinseed, valve material appeared to separate the spore into two equal halves (Fig. - 27). The original site of contact between the valve cells seemed to become the suturai line. The anterior portions of each valve cell involuted toward the center line to form the three ridges characteristic of the valves of L. triiuoum.

Sporoplasm Development Sporoplasm development in tri.iuaum was typical of that reported for other coelozoic myxozoans. The sporoplasm cell was distinguishable by its location and dense cytoplasm (Fig. 28). Sporoplasm cells were binucleate, and contained ribosomes, endoplasmic reticulum, and mitochondria. Sizes of nuclei were not always equal. The sporoplasm cell was usually binucleate in both immature and mature spores. Inclusions such as those described for Kudoa oaniformis (Stehr 1986) and for Mvxobolus hendricksoni (Mitchell et al. 1985) were not observed.

Special stains Ruthenium red and lanthanum nitrate, employed to localize areas of mucopolysaccharides and examine permeability pathways in host tissue and at the host-parasite interface respectively, produced only minimal tissue staining. These results were inconclusive. Figure 24. Ultrastructure of Plasmodium containing sporoblast undergoing valvogenesls. Vc, valvular cells; Cp, capsulogenic cell; Vv, valvular material; Mt, mitochondria; Mvb, multivesicular bodies; EvN, enveloping cell nucleus. C. triiuaum. bluegill fL. macrochirus). 8,352X Figure 25. Electron micrograph of ridge formation in spore of tri.iuQum. Valve suture is indicated by arrow. 20,700X Figure 26. Ultrastructure of a developing spore showing the laminated wall structure (arrows). 20,700X Figure 27. Electron micrograph showing compartmentallzatlon of maturing spore. Valve suture (arrow); Mt, mitochondria; Vv, valvular material. 8,352X Figure 28. Electron micrograph of sporoplasm cell (Sc). Nc, capsulogenic cell; Vc, valvogenic cell; Mt, mitochondria; Of, filament discharge tube. 8,352X 48 49

DISCUSSION

The ultrastructure of Plasmodia prior to sporogenesis has been largely ignored. Because Identification of early structures Is difficult, cytochemlcal analyses could provide Insight Into similarities among varied myxozoan species. This study revealed several types of vesicles In presporoblastic Plasmodia, Although morphologically similar, the cytochemlcal profiles of these vesicles could be quite different. Identification of enzymes or metabolic products within these structures could offer a better understanding of their role in sporogenesis. Several structures have been commonly observed in the presporoblastic Plasmodia of myxozoans. Pinocytotic channels and vesicles, mitochondria with a plethora of cristae forms, and lipid droplets have been reported in the ectoplasmic regions of Plasmodia from Henneouva (Current and Janovy 1977; Lom and dePuytorac 1965b; Current 1979), Kudoa (Stehr and Whitaker 1986), Mvxobolus (Current et al. 1979), Sphaeromvxa (Lom 1969; Grasse and Lavette 1978), and Thelonellus (Desser et al. 1983a). However, a few organelles such as multivesicular bodies (Stehr and Whitaker 1986), ribosomes (Lom 1969), tubular elements (Grasse and Lavette 1978), and vacuoles (Current et al. 1979) do not occur in the ectoplasmic regions of all myxozoans. There is general agreement among reports on myxozoans that the endoplasmic region of Plasmodia contain vegetative nuclei, generative cells, cell aggregates, developing sporoblasts, and mature spores. Chloromvxum tri.luoum generally contained structures similar to those listed above. 50

Sporogenesis in Chloromvxum tri.iuoum infections in the four species of centrarchids was similar to that reported for other species of myxosporeans. The process was initiated by the envelopment of one generative cell by a second. The paired generative cells initiating sporogenesis were of equal size, as is true for Henneouva osorosoermica (Lom and dePuytorac 1965b); iL pinnae (Schubert 1968); Mvxobolus so. (Oesser and Paterson 1978); Chloromvxum cristatum. Mvxidium lieberkuehni. Zschokkela nova (Lom and dePuytorac 1965b); Sohaerornvxa sabrazesi (Grasse and Lavette 1978); and Thelohanellus nikolskii (Desser et al. 1983a). In contrast, paired generative cells observed in Henneouva exil is (Current and Janovy 1977); iL adiposa (Current 1979); Mvxobolus funduli (Current et al. 1979), and Mvxobolus exiauus (Pulsford and Matthews 1982) differed in size and morphology. The cell-adherence and envelopment stages have not been observed in Sohaerospora renicola (Lom et al. 1982), Ceratomvxa shasta (Yamamoto and Sanders 1979), Mvxidium zealandicum (Hulbert et al. 1977), or Kudoa oaniformis (Stehr 1986). Whether this developmental sequence differs with varied physiological conditions of the host or parasite, or is a basic genetic difference among species remains to be determined. As in other myxozoans, in L. tri.iuaum subsequent to the envelopment stage, the enclosed generative cell, through a number of divisions, differentiated into its respective cellular components. Capsulogenic development was similar in all four host species. External tubules accompanied polar capsules prior to the differentiation of polar filament material. Chloromvxum tri.iuoum differed from most 51

myxosporeans In that the external tubule was not ensheathed with microtubules. Current (1979), Lom et al. (1982), Current and Janovy (1977), Lom (1969), Lom and dePuytorac (1965a), Desser et al. (1983a, 1983b), Desser and Paterson (1978), and Grasse and Lavette (1978) all found microtubules associated with external tubules. The conversion of external tubule to polar filament was not observed In LL trl.luQum, Desser et al. (1983a) reported that external tubule In Thelohanellus nikolskll was converted to polar filament rapidly. Desser and Paterson (1978), Lom (1969), Lom and dePuytorac (1965b), and Schubert (1968) proposed that the external tubule Invaginates Into the polar capsule In Mvxobolus Sohaerornvxa. Chloromvxum cristae. Henneauva Dsorospermlca. Mvxidlum lleberkuehnl. Zschokkela nova, and Henneauva Pinnae. The conversion of external tubule to polar filament In L. trl.luQum probably occurs rapidly as In L. nikolskli. Valvogenesis In Ç. trl.luoum varied slightly among the four species of fishes Investigated. Valvular material was observed relatively early In sporogenesis In all four hosts. The granular, lucent material described In this study may be similar to the striated material described In the apical walls of valve cells In Kudoa oanlformls (Stehr 1986), Mvxobolus DL. (Desser and Paterson 1978), Sohaerornvxa (Lom 1969), Chloromvxum cristatum. Henneauva osorospermlca. Mvxobolus Mvxidlum lleberkuehnl. and Zschokkela nova (Lom and dePuytorac 1965b). In most myxozoans this dense material appears to extend around the boundaries of the developing sporogenic cells, eventually widening laterally to form the valve shell. Likewise in L. triiuaum. the apically located dense 52

material Increased In volume and became the characteristic laminated valvular wall. Although microtubules were observed along the valvular suture lines In Henneouva exil is (Current and Janovy 1977), Thelohanellus nikolskll (Desser et al. 1983a), Mvxidlum zealandlcum (Hulbert et al. 1977), Henneouva adioosa (Current 1979), and Mvxobolus fundul1 (Current et al. 1979), microtubules were not observed along the valvular suture lines In L. tri.luaum. Microtubules may be less Important In species of Chloromvxum and other myxozoan genera in which polar capsules occupy only one end of the spore. Perhaps microtubules provide a structural base for aligning polar capsules at opposite poles of the spore and for elaborating extensions of the valve shell. Sporoplasm cell development in C^ tri.luaum was typical of other myxosporeans with binucleate cells. Fusion of the sporoplasm cell nuclei probably occurs sometime after spore maturation. Isolation of the sporoplasm cell and subsequent cytochemical studies should provide better understanding of this development stage. 53

REFERENCES CITED ' Brooks, R. E. 1969. Ruthenium red stalnable surface layer on lung alveolar.cells; electron microscopic interpretation. Stain Tech. 44:173-177. Current, W. L. 1979. Henneauva adioosa Minchew (Myxosporida) in the channel catfish: Ultrastructure of the Plasmodium wall and sporogenesis. Journal of Protozoology 26:209-217. Current, W. L. and J. J. Janovy, Jr. 1977. Sporogenesis in Henneauva exilis infecting the channel catfish: An ultrastructural study. Protistolog. 13:157-167. Current, W. L., J. Janovy, Jr., and S. A. Knight. 1979. Mvxosoma funduli Kudo (Myxosporida) in Fundulus kansae: Ultrastructure of the Plasmodium wall and of sporogenesis. Journal of Protozoology 26:574- 583. Cesser, S. S. and W. B. Paterson. 1978. Ultrastructural and cytochemical observations on sporogenesis of Mvxobolus ag. (Myxosporida: Myxobolidae) from the common shiner Notroois cornutus. Journal of Protozoology 25:314-326. Cesser, S. S., K. Molnar, and I. Weller. 1983a. Ultrastructure of sporogenesis of Thelohanellus nikolskii Akhmerov, 1955 (Myxozoa: Myxosporea) from the common carp, Cvorinus caroio. Journal of Parasitology 69:504-518. Desser, S. S., K. Molnar, and I. Horvath. 1983b. An ultrastructural study of the myxosporeans, Sohaerospora anoulata and Sohaerosoora carassii. in the common carp, Cvorinus caroio L. J. Protozool. 30:415-422. Dykova, I., J. Lorn, and G. Grupcheva. 1987. Pathogenicity and some structural features of Mvxidium rhodei (Myxozoa: Myxosporea) from the kidney of the roach Rutilus rutilus. Dis. Aquat. Org. 2:109-115. Grasse, P. P. and A. Lavette. 1978. La myxosporidie Sphaeromyxa sabrazesi et le nouvel embranchement des myxozoaires (Myxozoa). Recherches sur l'état pluricellulaire primitif et considerations phylogenetiques. Annales des Sciences Naturelles, Zoologie, Paris, 12 Serie 20:193-285. Hulbert, W. C., M. P. Komourdjian, T. W. Moon and J. C. Fenwick. 1977. The fine structure of sporogony in Mvxidium zealandicum (Protozoa: Myxosporida). Can. J. Zool. 55:438-447. Jameson, A. P. 1931. Notes on California Myxosporldia. J. Parasitology 54

18:59-68. Kudo, R. R. 1920. Studies on myxosporldla. A synopsis of genera and species of Myxosporldla. Illinois Biological Monographs 5:1-265. Listebarger, J. K. 1981. Scanning and transmission electron microscopy of two coelozolc myxosporeans: Genus Chloromvxum. Unpublished M.S. Thesis. Iowa State University, Ames, Iowa. Listebarger, J. K. and L. 6. Mitchell. 1980. Scanning electron microscopy of spores of the Myxosporidans Chloromvxum trl.iuaum Kudo and Chloromvxum catostomi Kudo. Journal of Protozoology 27:155-159. Lom, J. 1969. Notes on the ultrastructure and sporoblast development In fish parasitizing myxosporldlan of the genus Sohaeromvxa. Zeitschrlft fur Zel1forschung 97:416-437. Lom, J. and P. dePuytorac. 1965a. Observations sur 1'ultrastructure des trophozoites de myxosporldles. Comptes Rendus de 1' Academie Sciences, Paris 260:2588-2590. Lom, J. and P. dePuytorac. 1965b. Studies In the myxosporldlan ultrastructure and polar capsule development. Protlstologica 1:53-65. Lom, J. and E. R. Noble. 1984. Revised classification of the class myxosporea Butschli, 1881. Folia Parasitology (Praha) 31:193-205. Lom, J., I. Dykova, and S. Lhotakova. 1982. Fine structure of sDhaerosDora renicola Dykova and Lom, 1982 a myxosporean from carp kidney and comments on the origin of pansoroblasts. Protlstologica 18:489-502. Luft, John H. 1971a. Ruthenium red and violet. I. Chemistry, purification, methods of use for electron microscopy and mechanism of action. Anatomical Record 171:347-368. Luft, John H. 1971b. Ruthenium red and violet. II. Fine structural localization in animal tissues. Anatomical Record 171:369-416. Meglltsch, P. A. 1937. On some new and known Myxosporldla of the fishes of Illinois. J. Parasitology 23:467-477. Mitchell, L. G. 1978a. Myxosporidan infections in some fishes of Iowa. J. Protozool. 25:100-105. Mitchell, L. G. 1978b. Coelozolc Myxosporida of fishes of Western Montana: Genus Chloromvxum. Northwest Sci. 52:236-242. Mitchell, L. G., J. K. Listebarger and W. C. Bailey. 1980. 55

Epizootlology and histopathology of Chloromvxum tri.iuaum (Myxospora: Nyxosporlda) in centrarchid fishes from Iowa. Journal of Wildlife Diseases 16:233-236. Mitchell, L. G., C. L. Seymour, and J. M. Gamble. 1985. Light and electron microscopy of Mvxobolus hendricksoni sp. nov. (Myxozoa: Myxobolidae) infecting the brain of the fathead minnow, Pimeohales promelas Rafinesque. Journal of Fish Diseases 8:75-89. Pulsford, A. and R. A. Matthews. 1982. An ultrastructural study of Mvxobolus exiauus Thelohan, 1895 (Myxosporea) from grey mullet, Crenimuail labrosus (Risso). Journal of Fish Diseases 5:509-526. Revel, J. P. and M. J. Karnovsky. 1967. Hexagonal array of subunits in intercellular junctions of mouse heart and liver. Journal of Cell Biology 33:C7. Schubert, G. 1968. Elekrtonenmikroskopische untersuchungen zur sporenentwicklung von Henneauva pinnae Schubert (Sporozoa, Nyxosporidea, Myxobolidae). Zeit. Parasitenk. 30:57-77. Stehr, C. 1986. Sporogenesis of the myxosporean Kudoa oaniformis Kabata and Whitaker, 1981 infecting the muscle of the Pacific whiting, Merluccius oroductus (Ayres). Journal of Fish Diseases 9:493-504. Stehr, C. and D. J. Whitaker. 1986. Host-parasite interaction of the myxosporeans Kudoa oaniformis Kabata and Whitaker, 1981 and Kudoa thvrsites (Gilchrist, 1924) in the muscle of Pacific whiting, Merluccius oroductus (Ayres): An ultrastructural study. J. Fish Dis. 9:505-517. Wolf, K. and M. E. Markiw. 1984. Biology contravenes taxonomy in the Myxozoa: New discoveries show alternation of invertebrate and vertebrate hosts. Science 225:1449-1452. Yamamoto, T. and J. E. Sanders. 1979. Light and electron microscopic observations of sporogenesis in the myxosporida, Ceratomvxa shasta (Noble, 1950). J. Fish Dis. 2:411-428. 56

SECTION II. PATHOLOGY OF THE GALL BLADDER OF FOUR CENTRARCHID FISHES INFECTED WITH THE MYXOZOAN CHLOROMYXUM TRIJUGUM 57

INTRODUCTION

Chloromvxum trl.iuaum. a coelozolc myxozoan Inhabiting ga11 bladders, exhibits remarkable host specificity within the sunfish Family Centrarchidae (Mitchell et al. 1980). Mitchell et al. (1980) reported prevalence of this parasite in bluegill fLeoomis macrochirus) to be 100% during winter and spring, but only 50% during summer and fall. Sporulation and plasmotomy apparently occur throughout the year. Most reports on trl.iuaum have been based on light microscopic observations (Kudo 1920; Mitchell 1978; Mitchell et al. 1980). Listebarger and Mitchell (1980) studied spore surfaces of L. trl.iuaum with the scanning electron microscope. They reported that the valves of Lt. trl.iuaum have a thick ridge that runs parallel to the suturai ridge, and that the uncapped cnidocyst pores open into the extrasutural ridges. L. trl.iuaum does not appear to be associated with any major disease symptoms (Kudo 1920; Mitchell 1978); however, Mitchell et al. (1980) described several localized tissue changes in gall bladders of infected centrarchids. They noted a general breakdown of gall bladder mucosa involving loss of nuclear detail, reduction in cell height, and vacuolation, as well as thickened submucosa, and submucosal aggregation of leucocytes.

The purpose of this paper is to describe the surface topography of

Plasmodia and host cell mucosa, the pathology, and host-parasite interface associated with infections of trl.iuaum in four closely related host species. 58

MATERIALS AND METHODS

Infected ga11 bladders were excised from 30 blueglH fLeoomis macrochlrus). two white crapple fPomoxIs annularis), two black crapple fPomoxIs niaromaculatus). and five pumpklnseed fLeoomIs albbosus) to determine the pathological effects of Chloromvxum tri.luaum. All four species of fishes were collected between September 1985 and May 1987 from artificial Impoundments In Story County, Iowa (Hickory Grove Lake, Horticulture Pond, and Petersen Pits), and from backwaters of Pool 7 of the Upper Mississippi River (LaCrosse County, Wisconsin). Samples of gall bladder containing L. triiuoum were prepared for observation by light and electron microscopy.

Scanning Electron Microscopy Samples prepared for freeze-fracture analysis were fixed in 2.5% glutaraldehyde in O.IM phosphate buffer (pH 7.3) overnight (ca. 8h, 0-4 C) followed by a post-fixation in phosphate buffered (O.IM; pH 7.3) osmium tetroxide for 2h. Specimens were washed in buffer, dehydrated in a standard ethanol gradient, immersed in liquid propane (-196 C), and fractured on a metal stage under liquid nitrogen. Fractured units were rehydrated in 100% ethanol, infiltrated in three steps (3:1, 1:1, 1:3) with freon 113, and critical point dried using COg. Dried tissue blocks were mounted on copper disks using colloidal silver paste, sputter coated with gold-palladium and examined on a Jeol JSM-35 scanning electron microscope at an accelerating voltage of 15 Kv. 59

Transmission Electron Microscopy Normal and infected gall bladders were removed from pithed fish, placed in Karnovsky's fixative (2.0% paraformaldehyde/2.5% glutaraldehyde) in O.IM cacodylate buffer (0-4 C) for 30 min to Ih, 3 minced into small pieces (1.0 mm ) and transferred to fresh fixative for 2h to 24h at 0-4 C. Tissue was post-fixed in 1.0% osmium tetroxide in 0.1 M cacodylate buffer for 2h, washed in sterile deionized water, and in some cases stained en bloc with 2.0% uranyl acetate for 2h. Stained tissues were dehydrated in a graded series of acetone, and embedded in Nedcast epoxy resin. Thick sections were stained with either toluidine blue (1.0% in borax) or azure II/methylene blue and observed with the light microscope. Ultrathin sections of grey or silver interference colors were cut with a diamond knife on a Sorvall MT-2 or Reichert 0m-U2 ultramicrotome. Sections on copper grids were post-stained with uranyl acetate and lead citrate. In some cases, uranyl magnesium acetate was substituted for uranyl acetate. Grids were examined with a Phillips 400, Hitachi HS-8 or HU-llC electron microscope at 50 or 60 kV. 60

RESULTS

Scanning Electron Microscopy The mucosal relief of gall bladder was covered with mucoid material. In uninfected regions the apical surfaces of closely packed, columnar epithelial cells were visible In areas where the mucous layer was absent. The luminal surfaces of uninfected mucosal cells often showed short microvilli and protruding bullae, measuring about 3.5 /m x 3.5 lun. Host cells were tightly adherent In areas devoid of Chloromvxum tri.luaum Plasmodia. Attachment sites of Plasmodia were surrounded by numerous (about 75) cytoplasmic bullae measuring about 1.5 x 0.95 im. These bullae were round to elongate, and extended from short stalks attached to the epithelial cell surfaces. Small Plasmodia (about 5.4 x 5.8 jum) showed several microvlllosltles (about 0.3 im long) and larger protuberances (Figs, la, lb). Smaller Plasmodia exhibited numerous microvlllosltles but no protuberances, and were not associated with cytoplasmic bullae (Fig. 2). In severe Infections, Plasmodia were attached to virtually every mucosal cell. Plasmodia were attached In gaps between epithelial cells of about 0.19 im to 0.6 im (Fig. 3). Figure 1. Scanning electron micrograph of Plasmodia of triiuaum. (a) the surface is covered with several microviHosities (Mv) and protuberances (arrows); (b) some Plasmodia were associated with numerous cytoplasmic bullae (bu). 6,000X Figure 2. Scanning electron micrograph of smaller Plasmodium with numerous microvillosities and no protuberances or bullae. 780X Figure 3. Scanning electron micrograph showing gaps (arrows) between epithelial cells caused by the attachment of L. triiuaum Plasmodia (P). 5,400X Figure 4. Transmission electron micrograph. Plasmodia (P) of L, triiuaum overlay several host (H) cells. 3,845X 62 63

Transmission Electron Microscopy Infected mucosal cells in a11 four hosts Generally contained a variety of vesicles, mitochondria, vacuoles, and elliptical nuclei. Migrating leucocytes were also observed in the extracellular spaces at the bases of infected epithelial cells. No inflammatory or granulomatous reactions were observed, but host cells did show general necrosis, separation of junctional complexes, and in some cases erosion of epithelial cells down to the basal membrane. Cellular destruction was markedly worse in & annularis and & niaromaculatus.

Blueaill Plasmodia often spanned several epithelial cells and were attached to host cells at irregular intervals (Fig. 4). Attachment sites of unsporulated Plasmodia ranged in length from 0.3 nm to 1.8 nm. Plasmodial ectoplasm proximal to the host-parasite interface contained multivesicular bodies and large, elongated, mitochondria (Fig. 5). Sporulating Plasmodia exhibited pseudopodia, which often extended into a depression (up to 1.5 /im deep) in the apex of the host cell, and formed rhizoidal extensions between adjacent epithelial cells. Rhizoidal extensions were firmly attached by junctional complexes resembling Intermediate junctions (zonula adherens) (Fig. 6). Microvilli were absent from most host cells that were attached to Plasmodia. When microvilli were present, they contained vase-like, basal electron-dense bodies (Fig. 7). The apical regions of Infected host cells lacked organelles, except 64

that rlbosomes were generally evenly distributed throughout the cytoplasm. Cytoplasmic bullae on the apical borders of the host cells were associated with lucent, peanut-shaped vesicles (about 0.6 x 0.2 im). The cytoplasm beneath the bullae contained short, swollen cisternae of endoplasmic reticulum and mitochondria with dilated cristae. In several mucosal cells, minute channels (0.5 to 3.0 /un long) were observed In the vicinity of sites of attachment to Plasmodia. However, the presence of pinocytic channels in L. tri.lugum was not established. Generally, the basal regions of host cells contained an elongate nucleus, mitochondria, and numerous vacuoles. Chromatin material appeared throughout the nucleoplasm, but was denser at the periphery. Occasionally, rough endoplasmic reticulum and beta-cytomembranes (Bader 1966) surrounded the nucleus (Fig. 8). Numerous membrane bounded vesicles derived from endoplasmic reticulum were also associated with the host cell nucleus. Several types of spheroid vesicles appeared in the basal cytoplasm of infected mucosal cells. One type was clear and vacuole-!ike; others contained varied amounts of granular or vesicular material (Fig. 9). crsppie Junctional complexes observed between uninfected mucosal cells were normal. Tight (zonula occludens) junctions were 0.5 pan in depth, and intermediate (zonula adherens) junctions covered intercellular spaces of 435 A. Desmosomes (macula adherens) and associated cytoplasmic fibrils Figure 5. Transmission electron micrograph. Plasmodia! organelles bordering the host-parasite Interface Include elongate mitochondria (Mt) and multivesicular bodies (Mvb). 20,790X Figure 6. Ultrastructure of Junctional complexes formed between rhizoldal extensions (large arrow) of the parasite and host mucosa (H). 20,790X Figure 7. Electron micrograph of host-parasite Interface. Microvilli (Ml) are generally absent from Infected host cells, but when present are accompained by vase-like electron dense bodies (small arrows). 8,352X, Figure 8. Ultrastructure of basal region of host cell. Present are nucleus (Nu), mitochondria (Mt), vacuoles (Vo), rough endoplasmic reticulum (rER), and beta-cytomembranes (cy). 11,700X Figure 9. Electron micrographs of the basal region of host cells revealed several types of vesicles (1-5). Vesicles observed were 1) clear and vacuole-like 2) amorphous containing opaque, crescent-shaped structures, 3), spherical with an electron-dense center surrounded by separated concentric tubules 4), spherical with unidentifiable structures at the center, and 5) slightly spherical with electron-dense material covering half of the core. 20,790X 66

m 67

were not observed. Microvilli and cytoplasmic extensions (bullae) were present on the cell surfaces, but lacked filamentous material (antennulae mlcrovlllares) at their tips. The cytoplasm in the apical region of Infected mucosal cells contained only vacuoles and sac-like vesicles. Mitochondria, short cisternae of rough endoplasmic reticulum, and golgl apparatus appeared subaplcally. Adjacent host cells were Interdlgltated, and Intercellular spaces ranged from 0.04 /un to 0.1 /un. Unsporulated Plasmodia were tightly associated with mucosal cell surfaces (Fig. 10). Intercellular spaces at the host-parasite Interface were about 513 A° wide. Pseudopodia either penetrated the host cell cytoplasm or Interdlgltated with the lateral surfaces of the mucosal cell membrane. Microvilli were absent from Infected mucosal cells, and junctional complexes of Infected host cells were disrupted (Fig. 11). In some sections even tight junctions (zonula occludens) were separated. Pinocytic channels were seen in some Plasmodia. In light infections, involving unsporulated Plasmodia, infected mucosal cells contained numerous, swollen apical vesicles. Some host cells also contained large, apical, multivesicular bodies (Fig. 12). Subaplcally In host cells, cisternae of both endoplasmic reticulum and golgi apparatus were swollen. Several paired cisternae of endoplasmic reticulum were seen, and rough endoplasmic reticulum was degranulated. More golgi apparatuses occurred in the apical region of infected cells than in uninfected ones. Mitochondria were more numerous in the central region of mucosal cells infected with unsporulated Plasmodia (Fig. 13). Some infected mucosal cells were severely eroded. Cytoplasm of Figure 10. Transmission electron micrograph of unsporulated Plasmodium (P). Shown are penetrating or Interdlgltating pseudopodia (Ps) and host tissue (H). 2,500X Figure 11. Electron micrograph showing disruption of junctional complexes between Infected host cells (H). In some cases even tight Junctions (zonula occludens) were separated (arrows). 19,500X Figure 12. Ultrastructure of Infected mucosal cells (H) containing swollen apical vesicles (av). Multivesicular bodies (Mvb) were also present. 19,500X Figure 13. Ultrastructure of the subapical region of Infected gall bladder cells (H). Observed were paired cisternae of endoplasmic reticulum (ER) and degranulated rough endoplasmic reticulum (rER). 19,500X Figure 14. Transmission electron micrograph of severely eroded gall bladder epithelial cell (H). Infected host cells were vacuolated and contained swollen nuclei (Nu). 7,000X Figure 15. Ultrastructure of Infected host cell (H). The plasmalemma (Ph) folded into elongate tubular projections. 19,500X 69 70

these cells was highly vacuolated; nuclei appeared swollen and contained clumped chromatin; and plasmalemmae typically were folded into elongate tubular projections (Figs. 14, 15). Pseudopodate leukocytes and rodlet cells were common among the disrupted mucosal cells.

Pumokinseed Plasmodia of tri.iuaum were closely associated with the gall bladder epithelia in L. aibbosus. Mitochondria were abundant in the Plasmodia! cytoplasm contiguous with the host-parasite Interface (Fig. 16). The host-parasite interface was characterized by intermediate-type junctions interrupted frequently by inward reflections of the plasmodial membrane. Some mucosal cells contained pinocytic channels (2.0 to 3.0 pm long) at the host-parasite interface. Infected mucosae in Ls. aibbosus generally were more intact than those in Lt. macrochirus or Pomoxis (Fig. 17). Infected host cell membranes generally lacked microvilli, cytoplasmic bullae, and concentric tubular forms. Irregular vesicles were numerous just beneath the host- parasite interface (Fig. 18). Junctional complexes between host cells remained intact. Host cell nuclei, mitochondria, endoplasmic reticulum, and golgi apparatus appeared normal. In 10 infected cells several large inclusions were observed (Figs. 19a, 19b). Figure 16. Electron micrograph of Infected ga11 bladder mucosae (H) In pumpklnseed (L. gibbosus). The host-parasite Interface was characterized by Intermediate-type junctions (arrows). Pseudopodia, Ps; Mitochondria, Mt. 7,000X Figure 17. Electron micrograph showing mostly Intact mucosae of pumpklnseed (L. gibbosus) gall bladder (H) Infected with L. trljuoum. Note Junction of host cells (large arrow). 11,700X Figure 18. Ultrastructure of apical region of Infected host cells (H). Numerous, Irregular vesicles (V) were observed just beneath the host-parasite Interface (H-P). 25,000X Figure 19. Electron micrograph of Infected gall bladder mucosa (H). Several large Inclusions (In) were apparent throughout the host cell cytoplasm, (a) apical region of host cell; (b) basal region of host cell. 11,700X 72 73

DISCUSSION

Protective mucus, microvilli, and cytoplasmic bullae are common features of normal gall bladder cells In fishes (Viehberger 1983). Cytoplasmic bullae were observed In twelve species of freshwater teleosts by Togarl and Okada (1960), and ultrastructure described in tench (Tinea tinea L.) and rainbow trout fSalmo oairdneri R.) by Viehberger (1983). Viehberger (1982) reported diameters of cytoplasmic bullae up to 5.0 /m, and postulated that the function of these bullae were not secretory, but rather a means of extruding degenerated cytoplasmic structures. In this study, bullae were generally smaller, ranging in diameter from 1.0 to 3.5 im. Cytoplasmic bullae accompanying the attachment of Plasmodia to the mucosa of Lt macrochirus. L. annularis and & niaromaculatus were agranular, and contained no other secretory organelles. Viehberger (1982) also noted that Cytoplasmic bullae are not permanent structures; thus the absence of cytoplasmic bullae from the mucosa of L. aibbosus may Indicate a slower rate of regeneration of cytoplasmic structures. Furthermore, increased cellular destruction due to L. triiuoum infections may explain the numerous cytoplasmic bullae on the mucosae of Ls. macrochirus and Pomoxis. Plasmodia of L. triiuoum were free floating in the gall bladder lumen, or firmly attached to mucosal cells in all four host species. The plasmodial extensions of L. tri.iuqum into the epithelial cells of all four host species were classifiable as pseudopodia as described by Grasse and Lavette 1978; Booker and Current 1981; Lorn et al. 1982); or as 74

rhizolds as described by Paperna et al.(1987). The extension of pseudopodia or rhizolds onto host cell cytoplasms or between adjacent host cells has been described for several myxozoans Including Sohaerornvxa (Grasse and Lavette 1978; Lom 1969), Ceratomvxa. Leptotheca (Desportes and Theodorides 1982), Mvxidlum (Uspenskaya 1969; Paperna et al. 1987), Mvxobilatus (Booker and Current 1981), Zschokkela. and Chloromvxum (Lom and dePuytorac 1965; Mitchell et al. 1980). Lom et al. (1982) reported that pseudopodia of Sohaerospora renicola in the kidney of Cvprinus carpi0 formed junctions with host cell microvilli rather than with the general epithelial cell surfaces. The junctional complexes formed between the parasite and host cells of all four fish species resembled those found in most coelozoic myxozoans. Desportes and Theodorides (1982) and Lom et al. (1982) termed them hemidesmosomal in Ceratomvxa alobuHfera. Leptotheca elonaata. and Sphaerospora renicola. respectively. In contrast, Hoferellus cvorini in the kidney of L. carolo forms loose connections with host cell microvilli. Greater stability may be required of bladder inhabiting myxozoan parasites, because of bulk flushing of bladder contents. The apical regions of host cells exhibited the most severe changes associated with C. tri.luaum infections. Reduction in cell width, loss of cellular organization, reduction or eradication of microvilli, and proliferation of swollen apical vesicles indicated a direct effect of the Plasmodia on the host cells. In the subapical region swollen cisternae of either endoplasmic reticulum or golgi apparatus and mitochondria were abundant in Lt macrochirus and Pomoxis. but not In L. aibbosus. Perhaps 75

the regeneration processes of these organelles In L. olbbosus were Inhibited by the attachment of Lt. triiuaum. Evidence of pinocytic channels in host cell mucosae at the host- parasite interface was apparent only in L. macrochirus and L. aibbosus. Some nutrient transfer along these channels seems possible. Unlike Mvxobilatus mictosoora (Booker and Current 1981), which apparently absorbs nutrients from the host cell, Mvxidium lieberkuehni (Uspenkaya 1966) and genera such as Chloromvxum. Sohaeromvxa. and Zschokkela (Lom 1969) seem to depend on surface absorption. Nevertheless, the presence of golgi apparatus and endoplasmic reticulum in the apical cytoplasm of L. annularis and & niaromaculatus may indicate transport activity. Further, the presence of these organelles coupled with the absence of channels possibly supports transfer of cytoplasmic materials to the host- parasite Interface by methods not visible with TEN (i.e., diffusion and active or passive transport). In summary, Chloromvxum triiuaum infections were associated with a general disruption of host gall bladder mucosae. Gall bladder epithelial cells are both absorptive and secretory (Viehberger 1983); thus compromise of the apical surfaces and disruption of cell to cell contacts by pseudopodia most likely interfere with these normal processes. Severe cell damage was more prevalent in L. aibbosus and Pomoxis than in Lt. mscroqhlrw;. 76

REFERENCES CITED Bader, G. 1966. Die submlkroskoplsche struktur des gallenblasenepithels und seiner regeneration 1. Mittellung: Karpfen fCvorinus caroio. L.) und frosch fRana esculenta. L.) Z. Mikrosk. Anat. Forsch. 74:92-107. Booker, 0. J. and Current W. L. 1981. Mvxobllatus mlctosoora (Kudo 1920) (Myxozoa: Myxosporea) in the largemouth bass (Microoterus salmoides Lacepede): Plasmodium morphology and fine structure. J. Parasitology 67:859-865. Desportes, I. and J. Theodorldes. 1982. Donnees ultrastructurales sur sporogenese de deux myxosporidies rapportées aux genres Leototheca et Ceratomvxa parasites de Merluccius merluccius (L) (Teleosteen, Merluciidae). Protistologica 18:533-557. Grasse, P. P. and A. Lavette. 1978. La myxosporidie Sohaeromvxa sabrazesl et le nouvel embranchement des Myxozoaires (Myxozoa). Recherches sur l'état pluricellulaire primitif et considerations phylogentelques. Annales des Sciences Naurelles Zoologie et Biologie Animale (Paris) 20:193-285. Kudo, R. R. 1920. Studies on mycosporidia. A synopsis of genera and species of Myxosporidla. Illinois Biological Monographs 5:1-265. Listebarger, J. K. and L. G. Mitchell. 1980. Scanning electron microscopy of spores of the Myxosporidans Chi oromvxum tri.iuaum Kudo and Chioromvxum catostomi Kudo. J. Protozool. 27:155-159. Lom, J. 1969. Notes on the ultrastructure and sporoblast developnment in fish parasitizing myxosporidian of the genus Sohaeromvxa. Zeit. Zellforsch. 97:416-437 Lom, J. and P. dePuytorac. 1965. Observations sur 1'ultrastructure des trophozoites de myxosporidies. Comptes Rendus de T Academie Sciences, Paris, 260:2588-2590. Lom, J., I. Dykova, and S. Lhotakova. 1982. Fine structure of SohaerosDora renicola Dykova and Lom, 1982. A myxosporean from carp kidney and comments of the origin of pansporoblasts. Protistologica 18:489-502. Mitchell, L. G. 1978. Coelozoic myxosporida of fishes of Western Montana: genus Chioromvxum. Northwest Science 52:236-242. Mitchell, L. G., J. K. Listebarger, and W. C. Bailey. 1980. Epizootiology and histopathology of Chioromvxum triiuoum (Myxospora: Myxosporida) in centrarchid fishes from Iowa. Journal of Wildlife Diseases 16:233-236. 77

Paperna, I., A. H. Hartley and R. H. M. Cross. 1987. Ultrastructural studies on the Plasmodium of Mvxidlum alardi (Myxosporea) and its attachment to the epithelium of the urinary bladder. International Journal for Parasitoology 17:813-819. Togari, C. and T. Okada. 1960. Cytological studies of the gallbladder epithelium of the fish. Okajimas Folia Anat. Jap. 35:11-25. Uspenskaya, A. V. 1966. On the mode of nutrition of vegetative stages of Mvxidium lieberkuehni (Butchli). Protozoologica 4:81-86. Uspenskaya, A. V. 1969. Ultrastructure of some stages of development of Myxidium gasterostei Noble, 1943. Acta Protozoologica 7:71-79. Viehberger, G. 1982. Apical surface of the epithelial cells in the gallbladder of the rainbow trout and the tench. Cell Tissue Res. 224:449-454. Viehberger, G. 1983. Ultrastructural and histochemical study of the gallbladder epithelia of rainbow trout and tench. Tissue and cell 15:121-135. 78

SECTION III. LIGHT AND ELECTRON MICROSCOPY OF THE MYXOZOAN MYXOBILATUS NOTURI GUILFORD FROM THE TADPOLE MADTOM (NOTURUS GYRINUS) 79

INTRODUCTION

Mvxobilatus noturi was described by Guilford (1965) from urinary bladders in two tadpole madtoms, Noturus ovrinus (Mitchill). The tadpole madtom, the only known host of this myxozoan, is widely distributed in Canada and the United States east of the Rocky Mountains, and has been introduced in the Columbia River drainage. Herein, I augment Guilford's brief description with additional measurements and analyses of intraspecific variability of Plasmodia and spores. I also provide a description of Plasmodia attached to urinary bladder mucosa in tissue sections observed by light and electron microscopy. 80

MATERIALS AND METHODS

Adult and juvenile tadpole madtoms (standard length 2.5-5.4 cm) were collected by seining from a first order prairie stream (herein referred to as DG Creek) draining Dan Greene Slough In Clay County, Iowa. Infections of & noturl were diagnosed in wet mounts of the contents of urinary bladders and by the plankton centrifuge technique of O'GrodnIck (1975). Three to five specimens of each of the following fishes were collected from DG Creek with infected tadpole madtoms and processed with the plankton centrifuge for myxozoan infections: Culaea Inconstans (Kirtland), Esox lucius Linnaeus, Ictalurus nebulosus (Lesueur), Leoomis cvanellus Rafinesque, Notropis dorsal is (Agassiz), and Pimeohales promelas Rafinesque. Urinary bladders and attached portions of the kidneys of five tadpole madtoms were fixed in 10% buffered formalin, paraffin wax embedded, sectioned at 6-8 im, and stained with haematoxylin and eosin. Similar material from six brown bullheads fIctalurus nebulosus Lesueur) collected with the madtoms was also sectioned. For scanning electron microscopy pieces of urinary bladders were fixed in cold (0-5 C) 2.5% O.IM phosphate buffered-glutaraldehyde (pH 7.3) for 5h and post-fixed in 1% buffered osmium tetroxide for 2.5h at room temperature. Subsequent to dehydration in a standard ethanol gradient, and a 3-step (3:1, 1:1, 1:3) replacement of ethanol with freon 113, samples were critical point dried using COg, mounted on copper disks using colloidal silver paste, sputter coated with gold-palladium, and examined in a Jeol JSM-35 scanning electron microscope at an accelerating 81

voltage of 15 Kv and 20 Kv. For transmission electron microscopy urinary bladders were fixed in 2.0% (w/v) paraformaldehyde/ 2.5% (w/v) glutaraldehyde at 4 C in 0.1 N cacodylate buffer (pH 7.0) for 2h, washed 3 times in cold 0.1 M buffer (approx. 15 min each), and post-fixed in 1.0% (w/v) osmium tetroxide in 0.1 M cacodylate buffer for 2h. Some material was further stained en bloc with 2.0% uranyl acetate for 2h. Subsequent to dehydration in a graded series of acetone, samples were embedded in Medcast epoxy resin. Thick sections were stained with either toluidine blue (1.0% in borax) or azure II/methylene blue and observed with the light microscope. Ultrathin sections of grey or silver interference colors were cut with a diamond knife on a Sorvall MT-2 or Reichert 0m-U2 ultramicrotome, placed on formvar-coated copper grids, and post-stained with uranyl acetate and lead citrate. Sections were examined with a Phillips 400 or Hitachi HU- IIC electron microscope at 50 or 60 kV. 82

RESULTS

Epizootiology No Mvxobilatus infections were found In any fishes other than tadpole madtoms collected from DG Creek. Likewise, sectioned urinary bladders of the brown bullhead were not infected with & noturi. Six (60%) of ten tadpole madtoms collected during June and July, 1985 were Infected with fL. noturi; all of five madtoms collected during September, 1987 were Infected.

Plasmodia Unsporulated Plasmodia observed in fresh smears were generally spherical (diameters of five: 14-20 im) with no distinction between ecto- and endoplasm. Sporulated Plasmodia were ovoid or Irregular, often with slow moving lobopodia formed of clear ectoplasm (dimensions of five specimens: 20-25 x 23-30 im). Sporulated Plasmodia contained 1 to 3 (typically 1) mature spore. Spore development appeared to be asynchronous within Plasmodia. Three of the five urinary bladders and attached tissues examined in tissue sections were Infected with & noturi. Histologically, the uninfected bladders (from both madtoms and brown bullheads) were markedly similar to that of the channel catfish Hctalurus punctatus) described by Grizzle and Rogers (1976). Mucosae were highly Irregular, ranging from one to several squamous, cuboidal, or low columnar cells thick, and contained diffuse goblet cells and giant cells similar to alarm substance 83 cells common In the epidermis of catflshes and minnows. Giant cells were surrounded by a single layer of flattened epithelial cells. Hvxobllatus Plasmodia in tissue sections were attached to the mucosa of the urinary bladder and free-floating in the bladder lumen (Fig. 1). The distal opisthonephric ducts were also Infected, but with fewer Plasmodia than any of the infected urinary bladders. Attached Plasmodia were underlain with an eosinophilic zone of cytoplasm or with flattened epithelial cells, and were often lodged in a shallow concavity in the mucosa. In light infections (Plasmodia on less than 10% of the bladder mucosa), attached Plasmodia were generally ovoid or spheroidal; detached Plasmodia were larger and often irregular in outline. Light infections were often associated with loose infiltration of lymphoid cells in the bladder submucosa and mild hyperplasia of mucosa underlying clusters of several attached Plasmodia. In heavy infections (over 90% of the bladder mucosa covered by a single or double layer of Plasmodia), mucosal cells were typically vacuolated, attached Plasmodia were generally larger and more irregular than those in light infections, and most Plasmodia were attached to a single flattened cell layer or to submucosa. Many Plasmodia appeared to be attached to underlying cells by cytoplasmic processes. Giant cells were seen in only one infected bladder in which they were surrounded by a uni layer of epithelial cells covered with attached Plasmodia (Fig. 2). Heavily infected bladders also contained dense clusters of free-floating Plasmodia. 84

Scfirsâ The structure of spores In the tadpole madtoms from DG creek generally conformed to Guilford's original description of & noturl. although valve striae and caudal processes on Individual spores were more varied than those described by Guilford (1965). Striae (8-10 per valve) were typically ridged, and ridges on some spores were nearly as high as the suturai ridges. Most caudal processes were equal, but some differed by approximately 1 /un in length. Spores with processes that were widely divergent in suturai view (as illustrated by Guilford) occurred in approximately equal numbers to those with virtually parallel processes. Several spores had straight caudal processes. The following dimensions of fresh spores from five infected madtoms from Iowa extend the known range of variability for this species--total g length: 17-39, mean = 22.0, n-10, s = 38.2; body length: 8-11, mean » 9.8, n • 10, s 2 =0.84; breadth; 6.5-7.5, mean = 7.0, n= 10, s ? - 0.19; thickness: 6.5-7.0, mean • 6.7, n = 3, s^ - 0.33; cnidocysts--length: g 2.5-4.0, mean = 3.5, n - 10, s = 0.03; breadth: 2.0-2.5, mean = 2.3, n • 10, S2 » 0.07; length: 2.5-4.0, mean = 3.4, n = 10, s2 = 0.49; breadth: 2.0-2.5, mean - 2.3, n - 10, s2 = 0.07; caudal processes: 9.0- 21.0, mean • 13.0, n = 10,s^ = 19.3.

Electron microscopy As seen with the scanning electron microscope, Plasmodia of (L noturl had an irregular surface and protruded from the mucosal surface (Figs. 3, 4). Two plasmodial shapes were evident; Rounded Plasmodia were 85

most common, and measured 5.6 pm x 5.0 (n-5). Some Plasmodia had apical protuberances (1.9 tm x 1.6 /im, n-3), and rested deeply within the mucosal folds of the urinary bladder. The second type of Plasmodia was ovoid, crenated, and covered with microvlllosltles. Ovoid Plasmodia were larger (13.2 im x 4.5 jum) and more loosely attached to the apical

surfaces of host cells. Filament discharge pores 0.5 im In diameter were observed along the suturai ridges of some ovoid Plasmodia. Transmission electron micrographs revealed that Plasmodia were attached by pseudopod-like processes to underlying mucosal cells. Plasmodia contained numerous generative cells, lipid vesicles, vacuoles, spherical mitochondria, and a network of peripheral tubules associated with endoplasmic reticulum. Some of the tubules had electron dense stained regions at their centers. Similar tubules were also noticed adjacent to the generative cells. Sporulated Plasmodia were polysporous and limited by a single membrane. Nuclear material (unbounded), which may have represented developing vegetative cells dispersed throughout the Plasmodium. Free floating Plasmodia had ectoplasmic and endoplasmic regions. The endoplasm contained developing spores associated with multivesicular bodies. Microvlllosltles were generally abundant on attached Plasmodia, and sparse or absent on free-floating Plasmodia (Fig. 5). Sporoaenesis Plasmodia observed during early sporogenesis did not have pinocytotic channels, but contained endoplasmic reticulum, multivesicular bodies, and generative cells. In some cases, as many as four generative cells were seen adjacent to each other (Fig. 6). Figure 1. Light micrograph of Mvxobilatus Plasmodia (P) attached to mucosa of urinary bladder (H) or free-floating in lumen (large arrow). Figure 2. Light micrograph showing giant cells (GC) surrounded by a uni layer of epithelial cells (H). Note cytoplasmic processes (arrows). Figure 3. Scanning electron micrograph of round Mvxobilatus Plasmodia (P) rested within folds of urinary bladder mucosa (H). Arrow points to apical protuberances. Figure 4. Scanning electron micrograph of ovoid, crenated Plasmodia (P) loosely attached to urinary bladder mucosa (H). Of, filament discharge tube. Figure 5. Transmission electron micrograph of Mvxobilatus Plasmodia (P) attached to urinary bladder mucosa via pseudopod-like processes (Ps). Plasmodia contained generative cells (6), lipid vesicles (Lv), vacuoles (Vo), mitochondria (Mt), and peripheral system of tubules associated with endoplasmic reticulum. 10,000X Figure 6. Electron micrograph of Plasmodia during early sporogenesis. Present are endoplasmic reticulum (sER), multivesicular bodies, and generative cells (G). 10,000X 87 88

Mitochondria, with dense staining material in their matrices, occupied the cytoplasm of the generative cells. Ribosomes were scattered throughout the ectoplasm and endoplasm. Evidence of the envelopment process common to most myxozoans was not seen. Capsulogenic cells were situated in a plane perpendicular to the developing suturai ridge (Fig. 7). They contained vesicles, mitochondria, and extensive endoplasmic reticulum that often completely encircled the capsular primordia. Capsular primordia were sac-like and contained rounded, dense cores enclosed by a thick (0.3 im in diameter), lucent wall. Fragments of external tubule were also observed, and were surrounded by microtubules (Fig. 8). Maturing polar capsules contained coarse, granular cores with polar filaments (11-12 coils) and a markedly reduced lucent region (0.1 /un in diameter) surrounded by a narrow electron-opaque wall (0.05 jum diameter). Openings to a filament discharge tube were not observed (Fig. 9). Valvogenesis was coincident with capsulogenesis. Valvogenic cells surrounded capsulogenic and sporoplasm cells and contained dense staining material located intermittently along the wall of the developing spore. Suture lines formed between valve cells were straight and surrounded by multiple rows of approximately 20-25 microtubules. In maturing spores, a discharge canal capped with an electron-dense plug appeared in the valvular material (Fig. 9). Valvular material encasing capsulogenic and sporoplasm cells varied in thickness and ornamentation; valvular cytoplasm occurred at opposite ends of the sporoplasm cell. Sporoplasm cells in immature spores were dense, binucleated, and Figure 7. Electron micrograph of sporoblast cell during capsulogenesls. Capsulogenic cells (Cp) are perpendicular to the suturai ridges (Su), and contained vesicles (V), mitochondria (Mt), and endoplasmic reticulum (sER) surrounding the capsulogenic cells. 10,000X Figure 8. Electron micrograph of sac-like capsular primordia (Pc). The capsular primordia contained rounded, dense cores (d) surrounded by a thick, lucent wall (w). Fragments of external tubules (Et) were surrounded by numerous microtubules (tu). 22,000X Figure 9. Electron micrograph of maturing polar capsules (Pc) containing polar filaments (Fp). Valvogenic cells (Vc) contained intermittent pockets of dense material (arrows) along the spore wall. Valvogenic cells joined to form straight suture lines (Su), which were associated with microtubules (tu). A filament discharge tube (Df) appears to be capped with a dense plug (large arrow). 6,300X Figure 10. Electron micrograph of host-parasite interface. Junctions formed resembled tight junctions (zo) and formed dense plaques (large arrows). Fragments of sloughed epithelial tissues (small arrows) are visible in the spaces along the host-parasite Interface. 22,000X 90 91

contained electron-lucent regions speckled with droplets of electron- dense material. Golgl apparatus and endoplasmic reticulum were present In the cytoplasm. In maturing spores, the sporoplasm cell nuclei were larger, and contained electron-opaque granules. Sporoplasm cells lacked membrane-bounded organelles.

Host-parasite Interface As seen with the transmission electron microscope, & noturl showed numerous pseudopodlum-like projections bounded by a single limiting membrane. The ectoplasmic region of the Plasmodia adjacent to the urinary bladder mucosa contained rounded mitochondria, microtubules, and glycogen granules. Pinocytosis was not apparent. Attachment of Plasmodia to epithelial cells was intimate, but intermittent. The junctions formed by fL noturl resembled tight junctions, and formed dense plaques. Spaces formed from the arch-like arrangement of parasite pseudopodia contained fragments of sloughed epithelial tissue (Fig. 10). Pathosis associated with the attachment of Plasmodia to host cells included reduction in number and size of host cell microvilli, cytoplasmic vacuolatlon, erosion of the cell surfaces, and the formation of giant glycogen globules. The apical regions of Infected host cells lacked vesicles, but contained microtubules and membranous figures. The nuclear region showed extensive swollen cisternae of endoplasmic reticulum. In areas where pseudopodia extended between adjacent host cells, cell junctions were disrupted. Loose Infiltration of histiocytes and other leukocytes was also indicated. 92

DISCUSSION

This is the only report of Mvxobilatus noturi since the original species description by Guilford (1965). Like Guilford's, my study indicates that this species is strongly host specific. In contrast to Guilford's description, I have recorded a greater range in plasmodial shape, pseudopodial activity in Plasmodia, longer caudal processes on spores, and generally shorter cnidocysts. These differences are an indication of the intraspecific variability of this species. Mvxobilatus noturi does not elicit a strong inflammatory response in madtoms. Also, there was no indication in any of the sectioned material that Plasmodia induce hemorrhaging. Plasmodia are, however, associated with local cellular changes in urinary bladder mucosa and may virtually cover the mucosal surface. Whether these changes impair bladder function is not known. The Plasmodia appear to be released from the urinary bladder mucosa, with little trauma to the host, as mucosal cells are regularly sloughed and replaced. The only coelozoic myxozoans studied with scanning electron microscopy are Chloromvxum triiuoum. L. catostomi (Listebarger and Mitchell 1980), and Mvxidium aiardi (Paperna et al. 1987). Two plasmodial forms were observed in Mvxobilatus noturi. but no seasonal relationships applied such as was proposed by Booker and Current (1981) for Mvxobilatus mictospora. The pore observed on ovoid and elliptical Plasmodia, may be an exit pore for spore release. The peripheral tubules observed in tL noturi Plasmodia were similar 93

to those mentioned by Paperna et al. (1987) In Mvxidlum olardi. As discussed by Uspenskaya (1969) for Mvxidlum oasterostel. the tubular network and other peripheral organelles may be Important In nutrient absorption and assimilation. Lom (1969) found that Plasmodia of Sohaeromvxa have a tubule system separate from the endoplasmic reticulum. Endocytosis does not appear to be a universal feature of myxozoans. Booker and Current (1981) reported that Plasmodia of Mvxobllatus mictosDora are phagocytic in largemouth bass fMicrooterus salmoidesK On the other hand, the apparent lack of pinocytosis or phagocytosis in & noturi agrees with the findings of Lom and dePuytorac (1965) for Mvxidlum lieberkuehni and Lom (1969) for Sohaerornvxa. These results indicate that nutritional modes of myxozoans may vary with species; it seems likely that many coelozolc myxozoans may obtain dissolved nutrients by membrane transport mechanisms other than endocytosis. Paperna et al. (1987) suggested that because myxozoan surface microvillosities appear to be retractable, they should be called pseudopodia rather than microvilli. That microvillosities are reduced or absent, as seen in & noturi during spore maturation, may have resulted from their retraction. The taxonomic value of size, shape, or retractability of microvillosities remains unclear. An envelopment process between paired generative cells has been confirmed in several myxozoan genera, including Henneauva (Current 1979), Mvxobolus (Pulsford and Matthews 1982), Sohaerospora (Lom et al. 1982), Sohaeromvxa (Grasse and Lavette 1978), and Thelohanellus (Desser et al. 1983), and seems to be typical of myxosporeans. The apparent lack of 94

that process in (L noturi and several other species, including Kudoa oaniformis (Stehr 1986), Ceratomvxa shasta (Yamamoto and Sanders 1979), SohaerosDora renicola (Loin et al. 1982), and Hvxidium zealandicum (Hulbert et al. 1977), may indicate a fundamental developmental difference between these two groups of myxozoans. Except for Sphaeromvxa. the presence or absence of envelopment seems to separate two generic groups. Capsulogenesis in & noturi was typical of that reported for other myxozoans. The only myxozoan not known to produce external tubules during polar capsule formation is Fabespora vermicola (Weidner and Ovestreet 1979), a hyperparasite of the platyhelminth fCrassicutis archosarai). External tubules surrounded by microtubules as seen in fL noturi. have also been reported for Sohaerospora renicola (Lorn et al. 1982), Sphaeromvxa (Lom 1969), Henneauva (Current 1979), Thelohanellus (Desser et al. 1983), and Kudoa (Stehr 1986). Events in valvogenesis, beginning with the appearance of electron dense inclusions near the suturai ridges and beneath the valvogenic cell plasma membrane in ÎL noturi. were similar to those reported for Henneauva. Kudoa. Mvxobolus cvorini. (L Pharvnaeus. and Thelohanellus. (Current 1979; Stehr 1986; Spall 1973; Desser et al. 1983). The electron-dense substance, termed valve forming material by Current (1979) appears later in the development of Mvxobolus funduli. Sohaerospora. and Sphaeromvxa (Pulsford and Matthews 1982; Desser and Paterson 1978; Current et al. 1979; Lom et al. 1982; Lom 1969). In Kudoa oaniformis (Stehr 1986), Mvxobolus sp. (Desser and Paterson 1978), and Sphaeromvxa 95

(Lorn 1969) striated, amorphous material appears at the apex of the spore. Microtubules, as observed In & noturl. occur along the suturai plane In Immature spores of several myxozoans (Lorn 1969; Cesser et al. 1983), although Stehr (1986) did not observe these microtubules In L. oanlformls. The b1nucleated sporoplasm cells as seen In noturl are typical of myxozoans. However, the sporoplasm of Kudoa oanlformls (Stehr 1986), Sohaerosoora renicola (Lom et al. 1982), and Ceratomvxa shasta (Yamamoto and Sanders 1979) contains two cells. Nuclear division in the sporoplasm cell of fL noturl probably occurs sometime during capsulogenesis, because the sporoplasm cells observed during early capsulogenesis appeared to be uninucleated. The host-parasite interaction between & noturi and urinary bladder mucosa was similar to the Interaction observed for Chloromvxum tri.iuQum on gall bladder mucosa of centrarchids (Mitchell 1978; Mitchell et al. 1980). Both species of myxozoans form pseudopodium-like extensions that attach to the apical surfaces of epithelial cells, and extend down between adjacent host cells. The junctions formed by both LL trl.luaum and IL noturi resemble tight junctions, and form dense plaques. Likewise, the arch-like attachment of L. tri.iuaum to host tissue was similar to that observed in & noturi. One difference, however, was the absence of organelles in the apical region of noturl. whereas the apical region of Cj, tri.iuaum was occupied by vesicles, tubules, golgi apparatus, and in some cases pinocytotic-like channels. This type of attachment seems to elicit only mild host-tissue response, resulting in 96 an apparently harmless host-parasite Interaction. Because the mucosa of urinary and gall bladders are constantly being replaced, parasites living on the cells that do not penetrate below the mucosa are not likely to cause serious disease. In contrast, histozolc myxozoans cause marked host tissue responses and often are associated with significant pathosls. 97

REFERENCES CITED Booker, 0. J.and W. L. Current. 1981. Mvxobilatus mictospora (Kudo 1920) (Myxozoa: Nyxosporea) in the Targemouth bass fmicrooterus salmoides Lacepede): Plasmodium morphology and fine structure. J. Parasitology 67:859-865. Current, W. L. 1979. Henneauva adiposa Minchew (Myxosporida) in the channel catfish: Ultrastructure of the Plasmodium wall and sporogenesis. J. Protozool. 26:209-217. Current, W. L., J. Janovy, Jr., and S. A. Knight. 1979. Mvxosoma funduli Kudo (Myxosporida) in Fundulus kansae; Ultrastructure of the Plasmodium wall and of sporogenesis. J. Protozool. 26:574-583. Desser, S. S. and W. B. Paterson. 1978. Ultrastructural and cytochemical observations on sporogenesis of Mvxobolus sg. (Myxosporida: Myxobolidae) from the common shiner Notropis cornutus. J. Protozool. 25:314-326. Cesser, S. S., K. Molnar, and I. Wei1er. 1983. Ultrastructure of sporogenesis of Thelohanellus nikolskii Akhmerov, 1955 (Myxozoa: Myxosporea) from the common carp Cvorinus carpio. J. Parasitology 69:504-518. Grasse, P. P. and A. Lavette. 1978. Sur la reproduction des Myxosporidies et la valeur des cellules dites germinal es. C. R. Acad. Sci., Paris, 286 (Serie D):757-760. Grizzle, J. M. and W. A. Rogers. 1976. Anatomy and histology of the channel catfish. Auburn University Agricultural Experiment Station, Auburn, Alabama. 94p. Guilford, H. G. 1965. New species of Myxosporida from Green Bay (Lake Michigan). Trans. Amer. Microsc. Soc. 84:566-573. Hulbert, W. C., M. P. Komourdjian, T. W. Moon and J. C. Fenwick. 1977. The fine structure of sporogony in Mvxidium zealandicum (Protozoa: Myxosporida). Can. J. Zool. 55:438-447. Listebarger, J. K. and L. G. Mitchell. 1980. Scanning electron microscopy of spores of the Myxosporidans Chloromxum triiuoum Kudo and Chloromvxum catostomi Kudo. J. Protozool. 27:155-159. Lom, J. 1969. Notes on the ultrastructure and sporoblast development in fish parasitizing myxosporidian of the genus Sohaeromvxa. Zeit. Zellforsch. Mikroskop. Anat. 97:416-437. Lom, J. and P. dePuytorac. 1965. Studies in the myxosporidian 98

ultrastructure and polar capsule development. Protlstologica 1:53-65. Lorn, J., I. Dykova, and S. Lhotakova. 1982. Fine structure of SDhaerosDora renlcola Dykova and Lorn, 1982 A Myxosporean from carp kidney and comments of the origin of pansporoblasts. Protlstologica 18:489-502. Mitchell, L. G. 1978. Coelozolc Myxosporlda of fishes of Western Montana: Genus Chloromvxum. Northwest Scl. 52:236-242. Mitchell, L. G., J. K. Listebarger, and W. C. Bailey. 1980. Epizootlology and histopathology of Chloromvxum tri.iuaum (Myxospora: Myxosporlda) In centrarchid fishes from Iowa. J. Wildlife Dis. 16:233-236. O'GrodnIck, J. J. 1975. Whirling disease Mvxosoma cerebral Is spore concentration using the continuous plankton centrifuge. J. Wildlife Dis. 11:54-57. Paperna, I., A. H. Hartley, and R. H. M. Cross. 1987. Ultrastructural studies on the Plasmodium of Mvxidium olardl (Mysosporea) and Its attachment to the epithelium of the urinary bladder. International Journal for Parasitology 17:813-819. Pulsford, Ann and R. A. Matthews. 1982. An ultrastructural study of Mvxobolus exiouus Thelohan, 1895 (Myxosporea) from grey mullet, Crenlmuoil labrosus (Risso). J. Fish Dis. 5:509-526. Spall, R.D. 1973. Studies on the Ultrastructure, ontogeny and transmission of Mvxosoma oharvnaeus and fL cvorini (Protozoa: Myxosporlda). Ph.D. dissertation. Oklahoma State University, Stillwater, Oklahoma. Microfilm No. 74-8126, 89 pp. Stehr, C. 1986. Sporogenesis of the myxosporean Kudoa oaniformis Kabata and Whitaker, 1981 infecting the muscle of the Pacific whiting, Merluccius oroductus (Ayres). J. Fish Dis. 9:493-504. Uspenskaya, A. V. 1969. Ultrastructure of some stages of Mvxidium aasterostei. Acta Protozoologica 7:71-79. Weidner, E. and R.M. Overstreet. 1979. Sporogenesis of myxosporidan with motile spores. Cell and Tissue Res. 201:331-342. Yamamoto, T. and J.E. Sanders. 1979. Light and electron microscopic observations of sporogenesis in the myxosporlda, Ceratomvxa shasta (Noble, 1950). J. Fish Dis. 2:411-428. 99

SUMMARY AND DISCUSSION

This research constitutes the first ultrastructural studies reported for Chloromvxum trl.iuaum or Mvxobllatus noturi. These are coelozoic myxozoans that commonly Infect the gall bladder and urinary bladder, respectively. Sporogenesis and the pathology of Infections of four centrachid fishes with tri.luqum and the Infection of tadpole madtoms fNoturus ovrinus) by & noturi were described. Sections I and II discussed, respectively, the development and pathology of Chloromvxum trl.iuaum in the gall bladders of Leoomls macrochlrus. L. albbosus. Pomoxis annularis, and L. niaromaculatus. Section III described the development of Mvxobllatus noturi in the urinary bladder of the tadpole madtom. Although the infective sites and hosts differ between the two parasites, the resulting host-parasite interactions are very similar. A concurrent study of Chloromvxum trl.iuaum in the gallbladder of centrarchids and Mvxobllatus noturi in the urinary bladder of tadpole madtoms revealed basic similarities and differences between two host- specific coelozoic myxozoans. The early plasmodial stages of C^ trl.iuaum contained numerous multivesicular bodies, vegetative nuclei, and generative cells. Sporogenesis in £A triluoum was comparable to that seen in other myxosporeans. Spore development was initiated by the encapsulation of a generative cell. Polar capsule cells began as sac-like structures, which extended into an external tubule. Subsequent to the disappearance of the 100

external tubules, polar filament formation was prevalent In the polar capsule core. Valve material appeared early In the development of the sporoblast. No microtubules were associated with the valvular suture. The thick masses of valve material became more narrow as the spore matured, and the valvular wall assumed a laminated appearance. The Indentations and convolutions of valvular material formed the characteristic three ridged structure as well as shaping the various compartments of the host cell. Sporoplasm development In L. tri.luaum was typical of that reported for many myxosporeans. The paired nuclei were usually of equal size. The apical region of the host cells exhibited the most severe changes relative to L. trliuoum Infections. Cellular organization In the gall bladder of all four species of centrarchlds diminished when associated with the Plasmodia of C. triiuaum. Epithelial cells in crappie gallbladders experienced some shrinkage. Other pathological events observed in infected mucosal cells were, Increased vesicle formation, swollen cisternae of endoplasmic reticulum and golgi apparatus, and disruption of host cell gap junctions. The absorptive function of centrarchid gall bladders was most likely compromised.

Mvxobilatus noturi from the urinary bladders of tadpole madtom

fNoturus avrinus) were investigated using light, scanning, and

transmission electron microscopy. Mvxobilatus noturi was observed free

floating in luminal spaces and attached to the mucosa of the urinary bladder. Scanning electron microscopy revealed two morphological forms of Plasmodia. Oval Plasmodia were seen seated in the crevices of the 101

urinary bladder mucosa, and prominent protrusions were present on their

surfaces. Ellipsoidal Plasmodia observed were more loosely attached to

host cells than the oval Plasmodia. A structure presumed to be a spore

filament exit pore was observed along one of the suture lines on the medial surface of the Plasmodium. Ultrathin sections revealed that the peripheral region of Plasmodia contained vesicles, rounded mitochondria, and vegetative cells. A unique feature of & noturi was the secondary system of microtubules closely associated with existing endoplasmic reticulum. Mvxobilatus noturi did not elicit a strong Inflammatory response in the urinary bladder of tadpole madtoms. In most cases Plasmodia were released from mucosal cells with minimal damage to the host. The envelopment of generative cells by pericytes was not observed in & noturi. Otherwise sporogenesis In & noturi resembled that reported for other species of myxozoans. However, in contrast to Cj. triiuoum. external tubules observed in & noturi were associated with microtubules. One point of commonality between Mvxobilatus (Booker and Current 1981) and Chloromvxum (Mitchell et al. 1980) is the formation of pseudopodium-like structures, which extend down between host cells. Both myxozoan species form junctions which resemble tight junctions. 102

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ACKNOWLEDGMENTS

I wish to thank my major professor, Dr. Lawrence G. Mitchell, for his guidance, patience, and encouragement throughout my doctoral program. I thank my committee. Dr. G. J. Atchison, Dr. J. G. Nickum, Dr. E. C. Powell, and Dr. L. H. Tiffany for their guidance, support, and diligent review of this dissertation. I also wish to acknowledge the Region 8 Directors of the U. S. Fish and Wildlife Service, my station Director, Dr. F. P. Meyer, my supervisor, Mr. L. L. Marking, and the entire staff of the National Fisheries Research Center, LaCrosse, Wisconsin, for granting me this unique opportunity. I thank the Iowa Cooperative Fish and Wildlife Research Unit for financial support, and especially Ms. Linda Ritland who willingly did whatever was requested. I also wish to thank Bruce Wagner, Glenn Kietzman, Pat Abel-Aleff, and the EM staff at Mayo Clinic for technical assistance with the scanning electron microscopy, and Dr. Carl Korschegen for his priceless expertise In computer skills, word processing, and printing. I am grateful for the moral support and encouragement from friends and family. It was their faith in me that gave me the will to continue when the going got tough. I especially thank my loving wife, Peggy Sue Nunley-Bailey, who showed much patience, perseverance, and willpower during this ordeal, my mother, Berdie Bailey Haston, who continually encouraged me, and to my father, the late Tom A. Bailey, Sr., to whom this dissertation is dedicated. Ill

APPENDIX

Table 1. Summary notes on the early sporogenic stage of chloromvxum tri.iuaum from three genera of centrarchid fishes

Item Characteristics

1. Youngest plasmodlal stages were devoid of sporoblasts Ectoplasm--densely stained; filaments (0.05 /un) Endoplasm--cells with dividing nuclei; --mitochondria --membranous figures (double membranes) 2. Plasmodia were elongated and extended over 5-6 host cells -microvillosities (1-3 fim long) -lipid inclusions -nuclei (generative and vegetative) -finger-like tubules (0.8-1.2 fm) 3. Ectoplasm -mitochondria -multivesicular bodies -rounded vesicles -small ovoid mitochondria 4. Endoplasm -mitochondria (closely associated vesicles) -membrane bound vesicles (0.1 pm) -glycogen granules -golgi 5. Paired generative cells observed 112

Table 2. Comparison of envelopment stage structures of chloromvxum tri'iuQum from three genera of centrarchid fishes

Structure Blueglll Crapple Pumpkinseed

Microvillosities present present present Env membrane fused yes no (band) no

Env cytoplasm . . • nuclei ellipsoidal Ibid. Ibid. mitochondria yes no yes inclusions yes no no ribosomes no no yes

Ectoplasm - » « mitochondria vermiculated symmetrical vermiculated

Enveloped generative - - - cells - - - golgi yes yes no mitochond. yes no no free ribosomes yes yes no spheres w/dense yes no no striae 113

Table 3. Comparison of capsulogenic stage structures of chloromvxum tri'iuQum from three genera of centrarchid fishes

Structure Bluegill Grapple Pumpkinseed

Capsulogenic cell ER yes yes no mitochondria yes yes yes golgi yes no no

Polar capsules primordia/core yes yes yes tri 1am. wall yes yes yes ext. tubule yes yes yes microtubules no no no 114

Table 4. Summary notes on valvogenesis and sporoplasm development In chloromvxum triiuaum from three genera of centrarchid fishes

Item Characteristics

Valvogenesis (early stage) occurred concurrent with capsulogenesis recognized by peripheral location valvular material (thick) completely surrounded capsular and sporoplasm cells ridges of valve shell formed by infolding of valvular material

Valvogenesis (advanced stage) valve material reduced in width valve wall assumed laminated structure original sites of contact between valve cells formed sutures compartmentalization increased

Sporoplasm development development typical of other coelozoic myxozoans distinguished by location and dense cytoplasm sporoplasm cells were binucleate (ribosomes, mitochondria and ER) inclusions not observed 115

Table 5. General summary of pathosis associated with Infections of the gall bladders of three genera of centrarchid fishes Infected with the myxozoan Chloromvxum trliuoum observed with the scanning and transmission electron microscope

Microscopy Type Summary

Scanning mucosa covered with mucoid material uninfected mucosal cells showed microvilli and bullae (3.5 X 3.5/im) attachment sites of Plasmodia associated w/numerous bullae (1.5 X 0.95pm) smaller Plasmodia not associated with bullae

Transmission mucosal cell organelles: vesicles, mitochondria, vacuoles, elliptical nuclei migrating leucocytes in extracellular spaces no inflammatory or granulomatous reactions general necrosis separation of junctional complexes erosion of epithelial cells cellular destruction markedly worse in Pomoxis 116

Table 6. Detailed comparisons of pathosis associated with Infections of the gall bladder of three genera of centrarchid fishes Infected with the myxozoan Chloromvxum tri.luaum observed with the transmission electron microscope

Feature Bluegill Grapple Pumpkinseed

Attachment Irregular uninterrupted uninterrupted Pseudopodia extensive slight very slight Junction intermed. ibid. ibid. Channels/Intf. 0.5-3/im not seen 2-3ffm Pinocytosis inconclusive Ibid. Ibid. Microvilli absent absent absent Mv w/vesicles yes no no Bullae present present absent Apical org. none golgi none

Sub-apical org. RER SER few to none mitochondria mitochondria ------golgi Basal org. Nucleus Ibid. Ibid. mitochondria Ibid. Ibid. vacuoles Ibid. Ibid. Nu/chromatin clumped Ibid. Ibid. /}-cytomembrane present absent absent '>sicle types 5 11 117

Table 7. Summary of observations using light microscopy of the myxozoan Mvxobllatus noturl Guilford from the tadpole madtom (Noturus avrlnus)

Item Characteristics

Epizootlology Mvxobllatus not found In any other fish species collected from DG urinary bladders of brown bullhead uninfected eleven of fifteen tadpole madtoms infected with & noturl

Plasmodia unsporulated Plasmodia were spherical; indistinct ecto- and endoplasm sporulated Plasmodia were ovoid; lobopodia (clear ectoplasm) uninfected urinary bladders similar to CCF irregular mucosae contained goblet cells and giant cells & noturl in tissue attached to mucosa or luminal distal opisthonephric ducts infected

Spores structure of spores in Iowa generally similar to original description by Guilford (1965). valve striae and caudal processes varied from earlier description 118

Table 8. Summary of observations using electron microscopy of the myxozoan Mvxobilatus noturl Guilford from the tadpole madtom (Noturus avrlnus)

Microscopy Type Characteristics

Scanning Plasmodia! surface of (L noturl Irregular; protrudes two shapes observed: round--5.6 X 5.0/im (closely attached) ovo1d--13.2 X 4.5jum (loosely attached)

Transmission genera: plasmodlal attachment by pseudopod-like processes Plasmodia polysporous and limited by single membrane unbound nuclear material dispersed throughout Plasmodia

sporogenesis: Plasmodia had no pinocytotic channels mitochondria contained dense bodies in matrix envelopment process of generative cells not seen capsulogenic, valvogenic, and sporoplasm development followed generally that described for other myxozoans

host-parasite: junctions formed by & noturl resembled interface tight junctions 119

Table 9. Electron microscopy of the pathosIs caused by the myxozoan Mvxobllatus noturi Guilford from the tadpole madtom fNoturus avrinus)

Item Summary of Events

Pathosis reduction in no. and size of host cell microvilli cytoplasmic vacuolatlon erosion of cell surfaces formation of giant glycogen globules loose Infiltration of histiocytes and other leukocytes