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1979 Glugea Hertwigi (Protozoa: Microsporida) in Osmerus Mordax (Teleosteii: Osmeridae): Field Ecology, Laboratory Transmission and Ultrastructural Study of Parasite Development and the Host Cell-Parasite Interface. Ann L. Scarborough Louisiana State University and Agricultural & Mechanical College

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Recommended Citation Scarborough, Ann L., "Glugea Hertwigi (Protozoa: Microsporida) in Osmerus Mordax (Teleosteii: Osmeridae): Field Ecology, Laboratory Transmission and Ultrastructural Study of Parasite Development and the Host Cell-Parasite Interface." (1979). LSU Historical Dissertations and Theses. 3463. https://digitalcommons.lsu.edu/gradschool_disstheses/3463

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SCARBOROUGH, ANN L.

GLUGEA HERTIVIGI (PROTOZOA:MICROSPORIDA) IN OSMERUS MORDAX (TELEOSTEII:OSMERIDAE): FIELD ECOLOGY, LABORATORY TRANSMISSION AND ULTRASTRUCTURAL STUDY OF PARASITE DEVELOPMENT AND THE HOST CELL-PARASITE INTERFACE

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300 Z = E= RD.. ANN A R 30P Ml J8106 '3131 761-4700 Gluqea hextwigi (ProtozoarMicrosporida) in Osmerus mordax (Xeleosteii:Osmeridae): Field Ecology, Laboratory Transmission and Ultrastructural Study of Parasite Development and the Host Cell-Parasite Interface

A Dissertation

Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy

in

The Department of Zoology and Physiology

by Ann L. Scarborough B.A., University of California, San Diego, 1972 M.Sc., Louisiana State University, Baton Rouge, 1977 December 1979 EXAMINATION AND THESIS REPORT

Candidate: Ann L. Scarborough

Major Field: Zoology

Title of Thesis: Glugea hertwigi (Protozoa: Microsporida) in Osmerus mordax (Teleosteii: Osmeridae): Field Ecology, Laboratory Transmission and Ultrastructural Study of Parasite Development and the Host Cell-Parasite Interface

Approved:

Major Professor and Chairman

Dean of the Graduate

EXAMINING COMMITTEE:

Date of Examination:

3 December, 1979 ACKNOWLEDGEMENTS

I wish to thank my major professor Dr. Earl Weidner for guidance and encouragement during all phases of this investigation. Additional appreciation to my committee members; Drs. Kenneth C. Corkum, William B. Stickle,

J. Michael Fitzsimmons and Albert H. Meier of the

Department of Zoology and Physiology and Dr. James A. Avault of the Department of Wildlife and Fisheries, for unlimited academic interchange.

Thanks are recorded to Mr. Charlie Wheeler of the

National Marine Fisheries Service Aquarium, Woods Hole,

Massachusetts; to Mr. Stephen Nepszy of the Canadian

Ministry of Natural Resources, Lake Erie Fisheries

Research Station, Wheatley, Ontario, and special appreciation to Mr. James Kennedy of the Massachusetts

Fish and Wildlife Service, Buzzards Bay, Massachusetts.

Electron Microscopy facilities were supplied by

Dr. Mort Maser of the Marine Biological Laboratory,

Woods Hole, Massachusetts.

I dedicate this work to my father William E. Scarborough xvho started it all, and to my husband David L. Bull, who lived through it all.

This research was supported by NSF Doctoral Research Grant #DEB 77-08413. ii TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ...... ii

LIST OF TABLES ...... iv

LIST OF FIGURES...... v

ABSTRACT ...... vii

INTRODUCTION...... 1

MATERIALS AND METHODS ...... 7

Adult Smelt ...... 7

Transmission Experiments ...... 7

Development of Xenomas ...... 9

RESULTS ...... 10

Adult Smelt ...... 10

Parasite Transmission ...... 10

Parasite Development ...... 16

Light Microscopic Observations...... 16

Electron Microscopic Observations ...... 16

DISCUSSION...... 54

LITERATURE CITED ...... 77

VITA ...... 89 LIST OF TABLES

Table Page

1. Experimental infection of laboratory-reared and collected smelt with Glugea hertwigi s p o r e s ...... 11

iv LIST OF FIGURES

Young specimens of the smelt, Osmerus mordax, experimentally infected with Glugea hertwigi ...... 12

Young smelt, 12 days after feeding on spore-carrying plankton ...... 14

Young smelt infected by both spore suspension and spore-carrying plankton 15

Whole intestine 1 week after exposure to spore-carrying plankton ...... 17

Cross section of intestine 1 week after exposure to spore-carrying plankton . . 18

Cross section of intestine from lightly infected smelt 2 weeks after feeding on a spore suspension ...... 19

Xenoma, 5 days after parasite infection 21

Xenoma, 1 week after exposure to spore- carrying plankton ...... 22

Host-parasite interface, schizont, endo­ plasmic reticulum and mitochondria . . 23

Host-parasite interface, schizont plasmodium and endoplasmic reticulum 25

Host response to xenoma 14 days after parasite infection ...... 26

Host-parasite interface, schizonts and endoplasmic reticulum ...... 28

Cross section of schizonts ...... 29

Schizont plasmodium ...... 30

v LIST OF FIGURES (concluded)

Figure Page

15. Asexual multiplication of schizont .... 32

16. Flurinucleate plasmodium ...... 33

17. Onset of sporogony ...... 34

18. Division of plasmodium into primordial s p o r o n t s ...... 35

19. Early sporonts ...... 37

20. Sporonts during karyokenesis ...... 39

21. Early sporogonial plasmodium ...... 40

22. Sporogonial plasmodium ...... 41

23. Cross section of nuclei within sporogonial p l asmod ium...... 43

24. Division of sporogonial plasmodium .... 44

25. Cytokenesis of sporogonial plasmodium . . 45

26. Stages of G. hertwigi development .... 47

27. Young sporoblasts ...... 48

28. Sporogenesis...... 50

29. Sporogenesis...... 51

30. G. hertwigi spores ...... 52 ABSTRACT

Glugea hertwigi-induced microsporidosis is a disease of the smelt Osmerus mordax. The yearly incidence of infection reaches over 50% in adult smelt and as high as

90% in juveniles. Primary infections localize as large intracellular colonies in submucosal cells of the digestive tract. Field observations indicate the ovaries of spawning females are a secondary site of infection. G. hertwigi was successfully transmitted to both laboratory reared and collected young smelt at 20° C by small filter- feeding vectors and by direct ingestion of spores.

Infections transmitted by spore-carrying vectors numbered hundreds per animal, and were visible along the intestine one week after feeding. Large parasitized host-cells

(xenomas) extended from the intestinal serosa and were easily recovered. G. hertwigi infections acquired by direct spore feeding numbered one or two per animal; these fish had the capacity to develop many new infections by feeding on spore-carrying vectors. This microscopic study revealed that G. hertwigi-induced xenomas are products of infected host cells which undergo extensive hypertrophy. After 1 week of growth, 20 to 50 pm xenomas

vii contained a few enlarged host nuclei and vegetative G. hertwigi; after 2 weeks, the xenomas measured 100 to 125

Vim, exhibited multiple host nuclei and numerous G. hertwigi sporoblasts and spores. At 2 weeks, the host elaborated a capsule of collagen in opposing layers around the xenoma.

An extensive trabecular network (TBN) of membrane lamellae established continuity between Gluqea and host cell components. The cisternae of the TBN contained condensed material in direct contact with parasite schizonts. Pine structure corresponds with the basic pattern revealed for other Glugea. Features of schizogony included a multilayered outer coat, multinucleate plasmodia and karyokinesis by an intranuclear centriolar plaque. Sporonts formed plasmodia within parasitophorous vacuoles. Sporoblasts were ovoid, contained a classical

Golgi, endoplasmic reticulum and remained connected by a cytoplasmic bridge until sporogenesis. Sporogenesis organized the polar tube, polaroplast, endospore and exospore into a typical uninucleate microsporidan. Neither diplokaryon nor mitochondria were observed in any of the parasite developmental stages. INTRODUCTION

Glugea hertwigi-induced microsporidosis is a disease of the smelt, Osmerus mordax. mordax is an anadromous species that has been successfully introduced to temperate freshwater areas. Haley (1957) has provided some evidence which indicates G. hertwigi was in part responsible for the decline of the smelt fishery in the Atlantic. The incidence of Glugea infection reaches a seasonal peak of nearly 90% in juvenile Lake Erie smelt each summer and fall; Nepszy, Budd, and Dechtiar (1978) estimate vast economic losses in the smelt fishery in the Great Lakes each year due to mortality of infected juveniles. G. hertwigi cysts range from 0.4 to 5.0 mm in diameter; heavy smelt infections number over 200 xenomas per host (Legault and Delisle, 1967; Nepszy and Dechtiar, 1972).

G. hertwigi infections typically localize as parasite colonies in the submucosal layer of the intestine

(Schrader, 1921). Mortality of the smelt host is believed to occur by starvation; in addition, infected fish have navigation problems, are more susceptible to predation, and less able to recover from environmental stress (Legault and Delisle, 1967; Nepszy and Dechtiar, 1972). Spawning 2 female smelt in Lake Erie characteristically have numerous

G. hertwigi cysts in the ovaries as well as along the

intestine. Chen and Power (1972) reported a 42% decrease

in fecundity of Glugea infected females. The microsporidan

Pleistophora ovariae infecting the golden shiner

Notemigoneous crysoleucas is an example of transovarian

parasite transmission (Summerfelt and Warner, 1970).

McVicar (1975), Olson (1976) and Weissenberg (1968)

transmitted Glugea species to fish held in the laboratory.

Weidner (1973) and Stunkard and Lux (1965) suggested that

invertebrate filter-feeders may serve as natural vectors

or transport hosts for fish microsporidans. Olson (1976)

determined a low level of Glugea stephani infection

occurred by ingestion of spores directly. McVicar (1975)

transmitted C3. stephani through spore-carrying vectors

and by injection of spores into the peritoneal cavity

of adult fish. Weissenberg (1968) did not determine whether Glugea anomala was initiated by a spore-carrying

vector or by direct ingestion of the spores.

G_. hertwigi is an obligate intracellular parasite

completing its life cycle within a single host cell

(Debaisieux, 1920; Honiberg, 1964; Kudo, 1966; Moniez,

1920; Theolohan, 1S91). After ingestion by a smelt, the

spore stage is activated to extrude the polar filament 3

apparatus and discharges the infective sporoplasm stage

intracellularXy into the submucosal environs of the

intestine (Dissanaike and Canning, 1957; Ishihara, 1968;

Kramer, 1960; Lom and Vavra, 1963a; Vavra, 1976a; Weidner,

1972, 1976c). There are essential differences in the effect of microsporidan invasion on the host cell (Sprague,

1969; Sprague and Vernick, 1974; Weissenberg, 1976). One

such effect is exemplified by the cytopathology of Glugea

infections. Initially the parasite does not cause host

cell degeneration but stimulates hypertrophy and abnormal

development (Weissenberg, 1968, 1976; Weidner, 1976b).

Hypertrophic growth is a common response to intracellular parasite invasion; however, the enlarged host-cell nucleus usually remains undivided (Jirovec, 1930; Moulder, 1979;

Mrazek, 1910, Petri, 1976; Sprague, 1969; Weissenberg,

1949, 1965, 1976). On the other hand, development of

Glugea is unique since it stimulates amitotic multiplica­

tion of the host-cell nucleus (Loubes et al 1976; Sprague

and Vernick, 1968; Weissenberg, 1976). In his initial

descriptions of G. anomala, Weissenberg (1921, 1949)

followed by light microscopy transformation of the host

cell. He referred to the association between an

hypertrophied host cell and developing intracellular

parasites as a "xenoma" (Weissenberg 1922, 1968, 1976). 4

The host cell component is induced to undergo extensive growth during vegetative development of the parasite.

Eventually the parasite differentiates into mature spores which fill the central region of the xenoma. By this stage, a com’oination of host animal response and parasite growth transform the "xenoma" into a thick-walled "Glugea- cystM filled with innumerable spores (Sprague and Vernick,

1968; Weidner, 1974, 1976b, Weissenberg, 1968, 1976).

The "Glugea-cyst" or cell-hypertrophy tumor represents the most complex formation of an host cell and microsporidan parasite (Nigrelli, 1953; Sprague, 1969;

Sprague and Vernick, 1974; Weissenberg, 1976).

The microsporidan life cycle has two distinct phases:

(1) schizogony, during which the number of parasites rapidly increases by asexual multiplication and the infection is spread through the host cells and (2) sporogony, during which additional multiplication, involving autogametic recombination occurs and resistant spores are formed (Kudo, 1966, Vavra, 1976b; Sprague,

1977; Loubes, 1979). Among the microsporida life cycle stages of schizogony and sporogony differ in their structure and relationship to the host cell; however, many species are similar in the site of infection, number of parasite generations and the duration of the two 5

developmental phases (Cali, A. 1971; Gray, Cali & Briggs,

1969; Hazard and Anthony, 1974; Orimeres and Sprague, 1973;

Sprague, 1977; Vavra, 1976b). Similarity of features does not imply similarity of biological characteristics (Loubes,

1979). Biological and biochemical characteristics are known for only a small number of microsporida and as yet are not used in their taxonomy (Fowler & Reeves, 1974a,

1974b; Sprague, 1977; Sprague and Vernick, 1971).

Morphological features such as spore size, number of coils in the intrasporal polar filament, and length of extruded filament, and life cycle characteristics such as presence of a single nucleus or a diplokaryon in any life stage, formation of a pansporoblast or parasitophorous vacuole, and number of spores from a sporont are used as taxonomic determinants (Burges, Canning and Hulls, 1974; Canning,

1970; Fowler, 1974b; Hazard and Anthony, 1974; Loubes et al, 1976, 1979; Loubes, 1979; Milner and Beaton, 1977;

Ormieres and Sprague, 1973; Sprague, 1977; Sprague and

Vernick, 1971; Weiser, 1976).

The minutia of the microsporida urges morphological investigation at the ultrastructural level. Although the genus Glugea was established in 1891 by Thelohan, much of our present knowledge remains based on light micro­ scopic examination of mature cyst infections (Debaisieux, 6

1920; Kudo, 1924, 1966; Moniez, 1920; Weissenberg, 1949,

1968, 1976). The few electron microscopic studies of

Glugea have been concerned with stages of spore formation and spore structure (Loubes et al, 1976; McLaughlin, 1969;

Sprague and Vernick, 1969; Vernick, Tousimis and Sprague,

1969). Lorn and Vavra (1963a, 1963b) were first to publish electron microscopic studies on several genera of microsporidan spores, including Glugea, but gave details concerning only Pleistophora. Sprague and Vernick (1969) published a limited study of the fine structure of Glugea weissenbergi development. The authors admit the poor condition of their specimens and their descriptions have raised considerable controversy (Vavra, 1976a). Weidner

(1976b) was first to publish a fine structure and cytochemical characterization on the periphery of the

Glugea stephani-induced xenoma.

This study is the first ultrastructural investigation of Glugea"induced xenoma development. Observations made during this study produced data on: (a) the determination of the ecology of parasite transmission; (b) the establish­ ment of a reliable laboratory method of inducing parasite infection; and (c) an understanding of the general life cycle, parasite-host cell relationship and ultrastructure of Glugea hertwigi development within host cells. MATERIALS AND METHODS

Adult smelt

Spawning female smelt were collected with a cast-net

from Wheatley, Ontario, on Lake Erie and from the Jones

River, near Plymouth, Cape Cod, Massachusetts. A total

of 150 fish from each location were examined internally for the presence of Glugea hertwigi cysts. The intestine

and ovaries from infected and non-infected fish were

excised, cut into small pieces and fixed in pH 7.4 phosphate buffered guitaraldehyde overnight at 4° C.

After several buffer rinses, the material was post-fixed

in phosphate buffered 2% osmium tetroxide for 2 hrs at

4° C, dehydrated in ethanol and embedded in Epon. One micron sections were cut on a Dupont-Sorvail MT-2B microtome and stained with 1% toluidine blue. Parasite cysts were removed from adult smelt, homogenized, pelleted

in pH 7.4 phosphate buffer, stored at 4° C and used for the transmission experiments.

Transmission experiments

Eggs and milt were stripped from spawning smelt, mixed 1:5 respectively and kept in a well-aerated nylon

7 8 mesh cone. Naturally fertilized egqs were also collected from the riverbed. Anticipating large mortalities, a non­ infected smelt population was located in Long Pond, Cape

Cod, Massachusetts. Several years* examination of smelt from this pond had indicated they were completely free of C3. hertwigi infection. Young smelt (20-25 mm) in Lonq

Pond were attracted at night to a strong light at the surface and collected by hand-net. All fish were maintained at the National Marine Fisheries Service Aquarium, Woods

Hole, in 20° C filtered fresh water taken from the Jones

River well above the spawning sites. All fish were maintained on a diet of phyto- and zooplankton seined from Long Pond. The followinq methods of parasite transmission were attempted.

Experiment 1 . Laboratory reared and collected smelt were exposed to a suspension of hertwigi spores placed in the tanks. The water was well aerated but not filtered for the following 48 hrs.

Experiment 2 . Laboratory reared smelt (10 mm, 6 weeks post spawning) were fed spore-carrying zooplankton

(cladocerans and copepods) on 2 consecutive days. The plankton was first exposed to a spore suspension for 30 min, washed once with water, examined to ensure the 9

presence of spores in their digestive tracts and then fed to the smelt.

Experiment 3 . Collected smelt (15 mm, approximately

8 weeks post spawninq) were fed spore-carrying plankton

as above.

Experiment 4 . Smelt from Experiment 1, 4 weeks after

exposure to a spore suspension, were fed spore-carrying

plankton as above. As a control, a number of laboratory

reared and collected smelt were maintained unexposed to

spores.

Development of Xenomas

Young smelt from all tanks were observed and

photographed with a Wild M4 Makroskope 5 days, 1, 2 and

3 weeks after spore feeding. Intestinal tissue from

experimental and control smelt were prepared for light

and electron microscopy as outlined above. Serial, 2

micron thick sections were cut from 10 day old xenomas

and stained for periodic-acid-Schiff’s (PAS) positive

material according to Huger, (1969). After a 1 hr

incubation in IN HC1 at 55° C, the sections were thoroughly

rinsed and stained in Schiff’s reagent for 3 hr, rinsed

briefly in 1% sodium metabisulfite, and counter stained

with 1% toluidine blue. RESULTS

Adult smelt

In spawning females, Glugea hertwigi cysts were

found in the ovaries of more than 50% of the infected

fish in Lake Erie and in 25% of infected fish in the

Jones River during 1977, 1978 and 1979. The ovaries of

Lake Erie fish were heavily infected, whereas those of

the Jones River were lightly infected. Light microscopy

and ultrastructural examinations of infected ovaries

showed that Glugea parasites were not within egg cells.

Parasite transmission

The results of the experiments are summarized in

Table I. Infections were obtained from both methods of

spore transmission; however, the intensity of infection

differed {Fig. 1).

Experiment 1. Laboratory reared and field collected fish were exposed to suspensions of G. hertwigi spores.

Four weeks after exposure, 40% of the smelt exhibited one

or two Glugea cysts in the posterior region of the

intestine. These cysts were uniformly dense and protruded from the submucosa distending the peritoneal cavity. The

10 11

TABLE 1

Number of smelt infected Experiment Source of Sample Method of Period of light < 5 heavy > 50 number smelt size exposure incubation (per host) (per host) mixture of spore 1 lob reored 20 28 days 8 0 suspension and collected

spore - corrying 2 iab reared 5 7 days 0 5 plankton

II II 3 collected 14 0 14

II H 4 * mixture 8 0 8

* Smelt from Experiment 1, carrying light infections 28 days after exposure to a spore suspension. Figure 1. Young specimens of the smelt, Osmerus mordax, experimentally infected with Glugea hertwigi. The yearly incidence of natural infection nears 90% in juveniles with consequent vast mortalities. Seven days post infection, xenomas (white arrows) develop proximate to larger 2-week- old "Glugea cysts" (black arrows). Bar represents 2.5 mm. 13 fish fed continually throughout the experimental period.

Several fish died during the period, xvere examined and

one was found infected with a single Glugea cyst.

Experiment 2 . Laboratory reared fish were exposed

to spore-carrying plankton. One week after exposure all fish exhibited numerous small xenomas beginning just

behind the stomach and continuing along the entire length

of the intestine to the vent (Fig. 2). Gradually the

fish stopped feeding, had difficulty swimming and all

died by 16 days after exposure to the spores.

Experiment 3. Collected smelt were exposed to spore-

carrying plankton. The results were similar to those in

Experiment 2; however, these fish stopped feeding and

died at about 25 days after exposure to the parasite.

Experiment 4 . Smelt from Experiment 1, 4 weeks

after being exposed to a spore suspension, were fed spore-

carrying plankton. One week after feeding, all fish

exhibited numerous small xenomas along the intestine as

well as the large posterior xenomas (Fig. 3). All fish

died within 2 weeks after exposure to the spore-carrying

vectors. Controls were examined periodically and found

free of infection. Figure 2. Young smelt, 12 days after feeding on spore- carrying plankton. Numerous xenomas (arrows) begin just be­ hind the stomach and continue along the gut to the vent.

S, stomach; sb, swimbladder. Bar represents 180 jim. Figure 3. Young smelt previously infected after exposure to a spore suspension developed numerous new xenomas (arrows) along the intestine when fed spore-carrying plankton. X, xenomas from exposure to spores directly. Sb, swimbladder.

Bar represents 750 urn. 16

Parasite development

Light Microscopic Observations: Parasite growth was rapid at 20° C. Examination of the intestine from a heavy infection (Experiments 2, 3, and 4) showed the extensive tissue involvement. Infections protruded to the serosa and were easily dissected away intact (Fig. 4). Extensive host cell hypertrophy was the obvious feature of one micron sectioned material (Fig. 5). Xenomas ranged from

20 to 50 pm in diameter. Smaller xenomas contained one or two greatly enlarged host cell nuclei; whereas the larger xenomas were multinucleate with many nuclei lobed or branched. At 1 week the parasite formed vegetative colonies peripheral to the host cell nuclei. After 2 weeks of growth there was considerable increase in host cell hypertrophy and Glugea maturation to spores (Fig. 6).

Xenomas ranged from 100 to 125 pm in diameter with host cell nuclei, cytoplasmic components and vegetate G. hertwigi particularly obvious in the peripheral region of the xenomas; spore stages of the parasite were common in the central region.

Electron Microscopic Observations: Glugea parasite invasion into host cells occurs without apparent effect on the integrity of the host cell. Host cell responded 1?

Figure 4. Whole intestine 1 week after exposure to spore- carrying plankton. Xenomas (arrows) protrude to the serose and are easily dissected away intact. Bar represents 80 pm. 18

Figure 5. Cross section of intestine similar to that shown in Figure 4. At least 40 xenomas (arrows) 20 to 50 pm in diameter develop within the submucosa and protrude from the mucosa. Infected host cells hypertrophy and contain early . schizont stages of the parasite. £, epithelium. One micron

Epon section; 1% toluidine blue stain. Bar represents 50 Mmt 19

Figure 6. Cross section of intestine from lightly infected smelt 2 weeks after feeding on a spore suspension. Progres­ sive hypertrophy increased the xenomas to 100 to 125 ym in diameter. Xenomas develop in the submucosal layer causing mechanical distension of the epithelium fE). Sporogony stages and free denr?e spores fill the central region of the xenoma. One micron Epon section; 1% toluidine blue stain.

Bar represents 50 ym. 20

to invasion with general hypertrophy of the cytoplasm.

Host cells maintained a normal complement of cytoplasmic organelles such as mitochondria, ribosomes, endoplasmic reticulum and scattered vesicles (Fig. 7). Host-cell nuclei characteristically transformed from spherical to branching shapes with one or more diffuse nucleoli (Figs.

7 and 8). Condensed chromatin, karyokinesis or spindle microtubules were not observed in the host-cell nuclei during xenoma development (Figs. 8 and 9). Host-cell nuclei and mitochondria had close proximity to the vegetative population of parasites throughout xenoma development (Figs. 8 and 9). An extensive increase of host endoplasmic reticulum (ER) occurred and Figure 10 shows that layered arrangements of the ER paralleled the surface of schizonts.

The formation of the capsule was a distinguishing feature in the development of 10 to 14 day old xenomas.

Hypertrophied host cells appear to elicit a cellular, inflammatory response from the host. Seven days after parasite invasion, lymphocytes and fibrocytes border the xenoma surface (Fig. 7). Light microscopy showed that

7 day old xenomas had shape plasticity (Fig. 5). As the xenomas matured from day 10 to day 14, numerous host fibroblasts were observed to interdigitate with one 21

Figure 7. Xenoma, 5 days after parasite infection. The hypertrophied host cell-parasite complex consists of host

nuclei (N), early schizont stages of the parasite (Sc), numerous mitochrondria (M) and an extensive network formed by the endoplasmic reticulum (arrows). Host nuclei are

lobed and undergo amitotic blebbing (bold arrow). Host

lymphocytes (L) and fibrocytes (F) infiltrate the site of in­

fection. Nu, host nucleoli. Bar represents .74 ym. 22

-i • V i ' / . tV'rLrJj 'iV’-’

Figure 8. Xenoma from intestine shown in Figure 4. Host nuclei

Figure 9. Schizont (Sc) surrounded by lamellar profiles of host endoplasmic reticulum (arrow) and mitochondria (M).

Note narrow isthmus between lobes of host nucleus (double arrows). Bar represents .38 ym. 24

another. Layers of collagen fibers covered the xenoma

surface with both circular and longitudinal orientation

(Fig. 11).

The youngest parasite stages observed by electron

microscopy were amoeboid, uninucleate schizonts measuring

3 x 5 urn (Figs. 7, 8 and 9). This stage had centrally

located nuclei with dispersed chromatin and a homogeneous nucleoplasm surrounded by a typical double envelope. The

cytoplasm was limited by a unit membrane and contained

areas of unequal electron density. Several schizonts were seen in each xenoma; these stages were situated within the host cell cytoplasm (Figs. 7 and 8). The transformation of uninucleate schizonts into schizont plasmodia was gradual. Schizonts became elongate and

irregular in dimensions. The nucleus enlarged and diversification of the cytoplasm and cell limiting membrane occurred (Fig. 10). Figure 12, a cross-section of schizont plasmodia, shows these proliferative forms roughly ovoid

and bounded by 3 distinct layers. The outer most was a thin single unit membrane which covered an intermediate lucent layer over a thick inner envelope. This trilaminar boundary was unique to this stage. The cytoplasm attained homogeneity, contained few inclusions, scattered vestiges of endoplasmic reticulum and numerous ribosomes. Golgi, 25

Figure 10. Early schizont plasmodium (PSc) surrounded by

overcoats of host endoplasmic reticulum (arrows). Bar repre­ sents .28 urn. 26

■ v#sc-v ytifs •$&? ^=£S»,,?£

Figure 11. Host response to xenoma 14 days after parasite infection. Longitudinal and circular layers of collagen fibers (arrows) secreted by host fibroblasts (F) encapsu­ late the xenoma. Bar represents .18 ym. 27

smooth-surfaced vesicles and mitochondria were not

observed at this stage. As in all other microsporidans,

nucleoli were not observed and condensed chromatin was

uniformly distributed throughout a double membrane

bound nucleus (Fig. 12).

The outer most membrane of schizont plasmodia formed

occasional tubular elaborations which gave the appearance

of a trabecular network (TBN) extending throughout the

host cytoplasm, but particularly along the confines of

the cisternae of the endoplasmic reticulum (Figs. 13, 14

and inserts). In cross section, the TBN exhibited a

single, double-layered annulus that had a denser outer

layer (Fig. 13, insert). Condensed material within the

TBN was in perpetuity with the middle layer of the parasite boundary (Figs. 13 and 14, inserts).

Cytokinesis was evident in elogate schizont

plasmodia (Fig. 14). The TBN junctions were found at

various sites along plasmodia including the point of

cytoplasmic constriction (Figs. 13 and 14). Karyokinesis

proceeded as acentriolar pleuromitosis maintaining a

nuclear envelope throughout the division cycle (Fig. 15).

Microtubules approximately 0 . 0 2 5 p in diameter attached

to chromosomes and formed an intranuclear spindle that

terminated at the centriolar plaque on the nuclear membrane. 28

i * * * & * ? ■ *?&• :.*«£■& v.^a t - ;■ ssftfi

P%lg»

Figure 12. Schizonts (Sc) surrounded by host endoplasmic reticulum. These stages are not observed within parasito- phorous vacuoles. Parasite boundary delimited by 3 layers; a standard outer membrane, intermediate .lucent layer and a thick inner envelope (arrows) N, nucleus. Bar represents

,27 ym. 2$

Figure 13. Cross section of schizonts (Sc). These stages characteristically display a trabecular network extending in to the host cytoplasm from the surface of the developing parasites (arrow). Condensed material within the cisternae of these trabeculae appear to have continuity with the middle layer of the parasite boundary (insert). Bar repre­ sents .24 ym (a), .14 ym (b). Figure 14. Schizont plasmodia tPSc). Trabecular junction is at the point of schizont cytokinesis (asterisk^) (insert)...

Bar represents .34 urn (a), .24 ym (b). 31

A cross-sectional view of this structure displayed a hollow, barrel-like form (Fig. 15, insert). During division, the spindle plaque, and adjacent nuclear membrane appeared to invaginate into the nucleus (Fig. 15).

Elongate schizont plasmodia, with a single row of nuclei, became the dominant feature of the xenoma shortly before sporogony (Fig. 16). These plasmodia had an outer envelope similar to mature schizont; however, the glycocalyx appeared thinner. Cytoplasmic organelles were not discernible but condensed chromatin and the presence of centriolar plaques indicated recent karyokinesis.

Multinucleate schizonts appeared to be the last stage in schizogony prior to sporogony.

Parasitophorous vacuoles (PV) formed in the cytoplasm of the host cell component when xenomas reached a diameter of about 90 p (Fig. 6). The end of schizogony and the onset of sporogony was marked by several morphological features (Figs. 17 and 18). Chromatin dispersed within the nucleoplasm and appeared as in interphase. The cytoplasm lost density and contained small vesicles, tubular structures, Golgi, flattened profiles of endoplasmic reticulum and numerous free ribosomes (Figs. 17 and 18).

An irregular PV vacuole formed around schizont plasmodia as cytokinesis divided each nuclei and a portion of 32

p P q

Figure 15. Asexual multiplication of schizont nucleus with­ in schizont plasmodium. Microtubules (MT) form spindle to a centriolar plaque (CP) on the surface of the nuclear"membrane.

Hollow barrel-like appearance suggests true centriole (insert).

M, mitrochondria; ER, endoplasmic reticulum of host cell.

Bar represents .20 ym (a), .15 ym (b). 33

Figure 16. Plurinucleate plasmodium (PSc). N, nucleus;

S, spore. Bar represents .53 ym. 3k

ii-.. ■

Figure 17. Onset of sporogony. Cytoplasmic constrictric- tions (arrows) of plurinucleate plasmodium (PSc) forms pri­ mordial sporonts (Sp). Note pores along host nuclear membrane

(bold arrows). Bar represents .44 ym. 35

§ P * ' ^ a ^ p r \

Figure 18. Division of plasmodium (PSc) into primordial sporonts (Sp). Note scalloped surface of sporont (double arrows). Cytoplasm contains ribosomes and loose membrane lamellae (arrows). Chromatin (Ch) condensed within nucleus (N). Bar represents .38 pm. surrounding cytoplasm into primordial sporonts (Figs. 17

and 18). The boundary of the PV was apparently absent

or poorly delineated. There was an increase in the number

of pores between the host-cell nuclei and host-cell

endoplasmic reticulum adjoining FVTs.

Structural changes of the sporont boundary and

cytoplasmic components were rapid. Irregular patches of

glycocalyx organized onto the plasma membrane of sporonts

(Fig. 19). The initial manifestation of these patches

took place during formation of primordial sporonts (Figs.

17 and 18). After division of the schizont plasmodia,

the sporonts remained proximate to the periphery of the

PV boundary; these stages did not affix to the boundary

or develop tight junctions to the vacuolar envelope (Fig.

19). Gylcocalyx deposition was preceded by the

differentiation of the sporont Golgi apparatus and

endoplasmic reticulum. The Golgi was composed of small

vesicles loaded with electron dense material. Nuclei had

dispersed chromatin and were centrally located within

sporonts (Fig. 19). As sporogony continued, chromosomes

condensed and a series of nuclear divisions took place

(Fig. 19). Karyokinesis was again observed within membrane bound nuclei (Fig. 20). Chromosomes attached to microtubules

terminating on the nuclear membrane at the centriolar 37

Figure 19. Early sporonts (Sp) adjacent to parasitophorous vacuole membrane. Outer surface forms patches of unequal density. Bar represents .32 ym. 38 plaque (Fig. 20). Small perinuclear vesicles were often observed proximate to the centriolar plaque; however, no continuity was observed between the vesicles and the spindle apparatus. Extensive layers of rough endoplasmic reticulum developed within the differentiating sporont cytoplasm. The glycocalyx became organized as a continuous coat measuring 20-30 pm on the sporont surface (Fig. 20).

Cytokinesis did not directly follow karyokinesis in the sporont; rather, the sporont became a multinucleated plasmodium (Fig. 21). Early sporogonial plasmodia had eccentric nuclei, rough endoplasmic reticulum (RER) with long flattened lamellae and a Golgi complex with extensive membrane cisternae adjacent to the RER (Fig. 21). A conspicuous feature within parasitophorous vacuoles was the presence of amorphous material (Figs. 21, 22 and 23).

This material was observed in the vacuolar cavity throughout sporogony. Shape of the vacuolar material varied from tubules to granules of different size and density with a distinct tendency to assemble itself into tubules (Figs.

17, 21 and 22). Sporogonial plasmodia became very irregular in shape. Nuclei of these forms displayed chromatin condensed into a unique ring configuration (Fig.

22). The chromatin ring was observed within a single membrane bound nucleus and consisted of granules and a 39

v* ■

Figure 20. Sporont during karyokenesis. Condensed chroma­

tin (Ch) attach to intranuclear spindle microtubules (MT)

that terminate at centriolar plaque (CP) on nuclear membrane.

Note extensive endoplasmic reticulum (ER) and sporont surface

(double arrows). Bar represents .23 ym. k0

Figure 21. Early sporogonial plasmodia (PSp). Tubules and amorphous material accumulate within parasitophorous vacuole

(arrows). Outer surface of plasmodia thicken (double arrows) and Golgi (g ) form membrane lamellae adjacent to endoplastic reticulum (ER). Bar represents .42 ym. s^PpN^ai

Figure 22. Sporogonial plasmodium (PSp) with condensed chromatin in unique configuration within nucleii (N). Note tubules in pansporoblastic cavity (arrows). G, Golgi. Bar represents .40 ym. 42

few dense strands (Figs. 22 and 23). The nuclear membrane

remained intact during this period. Membrane cisternae

of the Golgi and endoplasmic reticulum expanded into

lamellar profiles (Fig. 23).

Nuclear activity decreased concurrent with cytokinesis

of the sporogonial plasmodia. Cytokinesis was incomplete

dividing the plasmodia into sporoblast buds which remained

connected by a short cytoplasmic bridge at the center.

Sporoblast buds were seen in groups of two, three and most

often four (Fig. 24). The sporoblast cytoplasm was similar

to the sporont stage but the membrane lamellae were tightly coiled and interpreted as primordia of the polar filament (Fig. 24), The loss of ground substance during fixation seen in Figure 25 afforded an opportunity to observe the Golgi and endoplasmic reticulum during sporoblast differentiation. Dense aggregates of vacuolar and vesicular Golgi lay interspersed within an expanded system of endoplasmic reticulum. Several small uniform tubes formed inside this complex and appeared hollow in cross-section (Fig. 25). Sporoblast maturation occurred within an ever-increasing parasitophorous vacuole.

Typically the cell wall of the sporoblast had the same structure as the sporogonial plasmodium, but measured

50 to 60 pm. b3

sfpwi

Figure 23. Cross section of nuclei within sporogonial plas- modium. Chromatin condenses to form unique circular array.

Golgi (G) membranes are continuous with endoplasmic reticu­ lum (ER) (arrow). Bar represents .34 ym. 4 sporoblasts (Spb). Chromatin disperses within nucleus (N) . Arrows indicate cleavage furrows. Bar represents .30 ym. Figure 25. Cytokinesis of sporogonial plasmodium. . Narrow isthmus (asterisk) connects primordial sporoblasts. Outer surface becomes uniformly dense (bold arrow). Vesicular

Golgi (G), distended endoplasmic reticulum (ER) and vesicles

(arrows) form in a cytoplasm devoid of other organelles.

Bar represents .29 ym. 46

Sporogony and consequent FV expansion was initiated in the central zone of the xenoma; after sporogenesis, fusion of vacuoles constructed a central storage space of large dimension (Fig. 6), The mass of stored spores restricted the growth area to the xenoma periphery. In such a stage, the central area of sporogenesis was surrounded by schizonts, plasmodia and a margin of host cell components (Fig. 26). Individual parasites moved from the FV boundary as cytokinesis separated the sporoblast buds. Host-cell nuclei exhibited numerous pores to the host endoplasmic reticulum which constituted a thin stratum between the PV and host cell components.

A large number of host cell mitochondria and nuclei surrounded the PV (Fig. 26).

A loss of cytoplasmic density was characteristic of sporoblasts in early sporogenesis (Fig. 27). Golgi vesicles, vacuoles and cisternae were observed in close association with extensive rough endoplasmic reticulum

(RER). The primordial polar tube appeared to form from

Golgi and RER (Fig. 27, insert). Late sporoblast stages exhibited increased cytoplasmic density, general cellular compaction and elongation with the nucleus toward one end (Fig. 28). A unique feature of elongated sporoblasts was a crenulated surface topography (Fig. 28). &'V

Figure 26. Several stages during Glugea hertwig1 develop­ ment. Sc, schizont; PSc, schizont plasmodipm; Sp, sporont;

Spb, sporoblast; PV, parasitophorous vacuole; N, host nuc­ leus; Nu, nucleolus; arrows indicate nuclear pores. Bar represents .44 ym. Figure 27. Young sporoblasts. Extensive rough endoplasmic reticulum fills the cytoplasm (arrows). Golgi (G) and associated particulate material have close association with endoplasmic reticulum. N, nucleus; CP, centriolar plaque.

Bar represents .56 ym (a), .36 (b). 49

The polar cap structure originated prior to polar filament formation and appeared as an irregular vacuole limited by a unit membrane (Fig. 28). The vacuole was partially filled by vesicles of the Golgi apparatus and was situated in close proximity to the sporoblast nucleus

(Fig. 28). The filament proper originated within the polar sac whose perinuclear position moved toward the anterior pole of the spore as the coils of the polar tube were formed (Fig. 29). Membrane sheaths from the endoplasmic reticulum surrounded the filament proper as coils spiraled within the spore posterior (Fig. 29).

The spore wall was a trilamellar structure consisting of an outer dense exospore, an electron transparent middle endospore and an inner plasma membrane (Fig. 29). No surface ornamentation, appendages or substructure were observed; however, the exospore remained crenulated until final spore maturation. The endospore varied from 50-200 pm in thickness, being thinner at the apical pole of the spore (Fig. 30).

Figure 30 demonstrates the structure of mature G. hertwigi spores. Several planes of section were required to visualize all mature spore structures. Spores were oval and had average dimensions of 3.9 x 2.3 pm. The extrusion apparatus was composed of the polaroplast, 5 0

Figure 28. Sporogenesis. Vacuole (V) is closely associated with nucleus (N). Polar filament (PF) (arrow) is anchored to the polar sac by Golgi . PV, parasitophorous vacuole. Cren ulation (assumed artifact) is common appearance during sporogenesis. Bar represents .40 pm. 51

Figure 29. Spores. Surface wall has outer dense envelope and an inner lucent layer (double arrow). Membrane sheaths surround the polar filament (arrows). G, Golgi; PS, polar sac. Bar represents .22 ym. 52

Figure 3,0. Glugea her twig i sppres. W, wall; PF, polar filament; Pol, polarplast; Pov, vesicular polarplast; N, nucleus; PC, polar cap. Bar represents .34 ym; 53

polar tube, and posterior vacuole. The polaroplast

appeared as tightly-packed arrangements of membrane pleats.

These pleats extended from the anterior end of the spore

as folds around the descending polar tube. The polar tube

anchored to the inner spore wall, descended through the polaroplast and turned to form 10-12 coils arranged in rows around the posterior end of the spore. In cross section, the polar tube consisted of several concentric

layers. The infective sporoplasm with a single nucleus filled the middle third of the spore interior. DISCUSSTON

Transovarian parasite transmission is known from a

number of microsporidan species (Kudo, 1966). Recently,

Summerfelt and Warner (1970) demonstrated a Pleistophora

ovariae infection in viable eggs of the golden shiner,

Notemigoneous crysoleucas. Although the ovaries of

spawning female smelt were often loaded with Glugea

hertwig i par as it es . thorough examinations indicate G.

hertwigi are not present in germinative or egg cells.

Previously, there has been limited success in experimental

peroral transmission of fish microsporidans (Delisle, 1969;

McVicar, 1975; Summerfelt and Warner, 1970, Stunkard and

Lux, 1965). However, Olson (1976) successfully transmitted

Glugea stephani to the english sole, Paraphyrys vetulus in

- water temperatures above 15° C. Several authors are

convinced that transport vectors are necessary to

concentrate Glugea spores for natural transmission of

certain fish microsporidosis (Haley, 1957; Putz and

McLaughlin, 1970; Stunkard and Lux, 1965). Small filter-

feeding animals may serve as transport hosts and in

addition, may stimulate the spores to hatch and infect

the fish. Weidner (unpublished observations) has observed

54 55

such a phenomenon with C3. stephani in the winter flounder,

Pseudopleurenectes americanus. In this study, transmission of G. hertwiqi to both laboratory-reared and collected smelt was successful at 20° C either by direct spore consumption or ingestion of spore-carrying vectors; however, a major magnitude of difference exists in the intensity between direct spore and vector transmitted infections. Vector transmission produced massive infections along the entire intestine in all test subjects.

Smelt are selective predators, taking cyclopoid and calanoid copepods and several species of cladocerans as their first food (Reif and Tappa, 1966; Siefert, 1972).

Presumably, the natural G. hertwigi infection occurs through the ingestion of spore-carrying filter feeders by very young smelt. Release of spores from infected adult smelt occurs via two routes. Scarborough (unpublished observations) has observed the expulsion of parasite xenomas from ovaries during spawning. In this manner, female adult smelt may unwittingly concentrate G. hertwigi spores in the immediate vicinity of developing young. Further, infected smelt carrion were seen being preyed upon by small crustaceans in the nursery areas after spawning. Nepszy and Dechtiar (1972) found that 56 heavily infected adult smelt were unable to recover from spawning stress; mass mortalities in the spawning grounds consisted of infected adults.

Massive infections of £. hertwiqi consequent to

ingestion of spore vectors are fatal to both collected

and laboratory reared smelt. Mechanical distention of the

intestinal tissue and starvation are thought to be the cause of death. Osmerus mordax is a difficult species to raise in the laboratory and the minimal condition of the reared fish probably precluded a greater susceptibility to the effects of multiple infections. Young smelt tolerate light infections for at least several weeks

and likely carry them into adulthood. Nepszy and Dechtiar

(1972) found that hertwigi colonies can remain in smelt for much of the host’s life, and stress will significantly

increase the mortality rate in these fish over uninfected smelt.

It is well documented that the life cycle of microsporidans begins with injection of the sporoplasm through a spore tube into a host cell (Lorn and Vavra,

1963a; Vavra, 1976, Weidner, 1972). It is assumed that the discharging tube of G. hertwigi spores penetrates through the gut basal lamina delivering the parasite into submucosa cells. Although Weissenberg (1968, 1976) 57 believed the host cell to be a presumptive macrophage, the cell types which can support CL hertwigi development remain undetermined. The present observations indicate that massive infections will produce xenomas easily separated from the submucosal layer of the intestinal wall. Xenomas were observed associated with various visceral organs, including cells below the peritoneal lining. It is not known how G. hertwigi enters ovarian tissue; presumably, initially infected host cells enter the blood stream and are delivered to favorable environs for growth, such as the highly vascularized ovaries.

Nosema michaelis, a microsporidan infecting the blue crab,

Callenectes sapidus, undergoes vegetative growth in the gut wall and these cells subsequently circulate to muscle tissue for continued development (Weidner, 1972).

Moulder (1979) believes that the host-parasite interface is the juxtaposition in space and time of the regulatory mechanisms of the host and parasite. Although they have separate origins, the host-cell and Glugea spore become structurally and physiologically integrated.

Once inside the host cell, parasite adaptions are concerned with avoidance of destruction and the maintenance and control of host-cell function (Moulder, 1974, 1979).

Some intracellular parasites, e.g. Leishmania donovani, 58

are resistant to lysosomal enzymes; while others, e.g.

Toxaplasma gondii and Encephelatozoon cuniculi appear to block lysosomal fusion with the phagocytized parasite

vacuole (Jones and Hirsh, 1972; Jones et al, 1972;

Moulder, 1979; Shadduck and Pakes, 1971; Trager, 1970;

Weidner, 1975, 1976a). G. hertwigi multiplies while in

direct contact with the host’s cytoplasm. This is a

common survival mechanism among microsporidans and a

distinct feature of Glugea (Canning and Nicholas, 1974;

Loubes, 1979, Loubes and Akbarieh, 1977; Loubes et al,

1976; McLaughlin, 1969; Sprague and Vernick, 1969;

Weissenberg, 1976; Youssef and Hammond, 1971).

The interfacing association observed between G. hertwigi and host cell mitochondria and endoplasmic reticulum is seen in other obligate intracellular parasite

infections. The regular overcoating of vacuoles containing

X* and _E. cuniculi and the apposition of vegetative

G* stephani with host-cell mitochondria and endoplasmic reticulum is thought to play a significant role in providing nutrients to the parasites (Gelderman et al,

1968; Hirsch et al, 1974; Jones and Hirsch, 1972; Jones et al, 1972; Pakes et al, 1975; Weidner, person, comm.).

The trabecular network of vegetative G. hertwigi may be involved in molecular transport at the host-parasite 59

interface. The observation of continuous karyokinesis of the schizont, formation of multinucleate schizont plasmodia, absence of parasite mitochondria and parasite cytoplasmic simplicity supports the conclusion that the parasite relies exclusively on host-cell function for metabolites during schizogony.

Initially the host cell is not destroyed but stimulated to an abnormal hyper-growth. Cell hypertrophy is a frequent response to intracellular parasitism; however, amitotic proliferation of the host-cell nuclei is a feature unique

Glugea. In contrast, the host cell remains uninucleate in the hypertrophy of glomerular cells in Esox lucius invaded by Pleistophora oolytica, in the hypertrophy of ganglion cells of Lophius lophius invaded by Spr ague a lophii, and in the hypertrophy of fibroblasts of

Pleuronectes sp. invaded by lymphocytis virus (Canning,

1976; Loubes et al, 1976; Nigrelli, 1953; Sprague, 1969;

Weissenberg, 1949, 1968). Giant nuclei are present in neoplastic tissues infected with Stempellia simulli,

Pleistophora debaisieux, Thelohania chaetogastiis and

Thelohania solenopsae (Knell et al, 1977; Loubes and

Maurand, 1976; Weiser, 1976).

The mechanism of G. hertwigi influence and stimulation of the host cell to amitosis and hypertrophy is unknown. 60

Pavan et al (1969), (1971) and Roberts et al (1967) have described microsporidan invasions of Sciaridae and the effect on polytene chromosomes. Characteristically the size of the nucleus and chromosomes increased under parasite influence. The total amount of DNA and the amount of RNA synthesis was greatly enhanced. There was an increase in the number of mitochondria and in the activity of the host endoplasmic reticulum. Although infected cells behaved abnormally, they did not degenerate and successfully survived insect metamorphosis to form giant juvenile cells. Fisher and Sandborn (1964) isolated a type of j'uvenile hormone that caused giant cell stages in infected insects.

The enlargement of the nucleus in cells infected with microsporidans is thought to be a consequence of polyteny and generalized hyperfunction of the entire genome leading to an overall increase in DNA-dependent RNA synthesis

(Diaz and Pavan, 1966; Roberts et al, 1967). Petri and

SchieSdt (1966) and Petri (1966, 1976) report the nuclei of Yosida rat sarcoma cells infected with E. cuniculi are characterized by an extraordinary number of large nuclear pores. According to current views, large nuclear pores provide a direct channel for RNA transit to the endoplasmic reticulum (DuPraw, 1970). Preliminary studies 61 by de la Torre and Gemenez-Martin (1975) on induced

amitosis in onion root-tip provides evidence that

transcription of ribosomal cistrons is necessary for the

normal progression of mitosis and that any changes in the

rate of RNA transcription at a particular point of the

cycle can be a mechanism leading to amitosis. Band and

Mohrlok (1974) found that Ac an t h amoeb a sp. grown in

deficient media exhibited an increase in nuclear number

and size and that cytokinesis following karyokinesis was

delayed. The induced amitosis was due to the metabolism

of nucleotides as an energy source and consequent

"unbalanced RNA synthesis:" Gross (person, comm.) has

determined that the time and plane of cell division

during sea urchin embryogenesis is controlled by levels

of mRNA coding for histones. The present morphological

evidence of nuclear edema, diffuse nucleoli, organelle proliferation, and the formation of nuclear pores in host-cell nuclei infers that during schizogony, G. hertwigi may in some way influence host RNA synthesis

and by this mechanism induce cellular hypertrophy and

nuclear amitosis.

In the present study, the interpretation of host- parasite interface is broadened to include the interaction

of the xenoma and/or its products and the host's defense mechanisms. The portal of cell entry for the microsporidan excludes the elaboration of specific antibodies to the sporoplasm; however, hypertrophic growth of the xenoma and consequent mechanical distention of host tissue provokes-an inflammatory response from the host. This cyst-forming response benefits the host in localized containment of the parasite. The capsule formation seen in Glugea infections parallel that observed in many helminth diseases (Lumsden, 1979). There is local accumulation of lymphocytes, eosinophils and fibrocytes.

The combined influence of metabolic by-products of the parasite and eosinophils induces fibrocytes to fibroblast differentiation and collagen deposition (Jackson, 1968;

Warren, 1974). The chronic phase persists with prolifera­ tive fibrosis (Thomas, 1964). As seen in the mechanical distention of the intestine by G. hertwigi xenomas, fibrosis itself is potentially pathogenic where tissues of a given organ are displaced by collagenous connective tissue.

Abundant blood vessels within the capsule is a common feature of Glugea-cysts (Sprague and Vernick, 1968;

Weidner, 1976b; Weissenberg, 1968, 1976). In an examination of cysts produced by G. stephani, Weidner

(1976b) found the xenoma glycocalyx acted as a sieve allowing passage of metabolites between the xenoma and host. 63

The xenoparasitic complex is a special category of host-parasite interactions distinctly different from simple infections (Canning, 1976; Weiser, 1976a). The term

"tissue parasite" has been suggested as a substitute for the term xenoma (Loubes, 1979). The new terminology would encompass several types of cell-hypertrophy tumors

(Sprague, 1969). Xenoparasitic complexes with microsporida are represented by 3 types: (1) syncytial xenomas with host cells confluent in one common syncytium; (2) Gluqea- cysts with a unicellular origin, and (3) neoplastic xenomas in which the parasite provokes an increase in the number of host cells inside the lobe of infected tissue.

The syncytial xenoma produced by Icthyosporidium sp. in the spot, Leiostomus xanthurus, has been studied by

Schwartz (1963), Sprague (1969) and Sprague and Vernick

(1974). The tumors result from fusion of neighboring cysts to produce a compound lesion of spectacular dimensions.

After fusion of the cysts a massive invasion of host connective tissue divides the parasitic mass.

The transition between a syncytical and neoplastic xenoma is the cel1-hypertrophy tumor (Weiser, 1976).

This type of microsporidan infection is often seen in fish and occurs in several microsporidan genera as well as in

Glugea. Kabata (1959) recognized the swelling of gill filaments in Gadus aeglefinus to be a result of Nosema branchiale. Canning (1976) and Putz et al (1965) described the xenoma as a product of a single cell invasion. £i. lophii parasitizes the ganglion cells of Lophius sp. and the formation of a cyst has been described by a number of authors (Canning, 1976; Loubes et al, 1979; Weidner, 1974).

Pleistophora cepedianae (Putz et al, 1965) in Dorsoma cepedianum and Nosema pimephales (Fantham et al, 1941) in Fimephalus promelas produce large cysts in the body cavity. Pleistophora hyphessobryconis causes enlargement of skeletal muscle bundles to many times the normal diameter (Lorn and Corliss, 1967). Nosema invadens elicits an inflammatory response and cyst formation in Lepidoptera sp. (Kellen and Lindgren, 1973).

Neoplastic xenomas usually occur without an enveloping cyst wall and are common to members of the genus Pleistophora

(Canning, 1976; Sprague, 1969). Pleistophora salmonae in Salmo gairdneri occur as diffuse infiltrations in the connective tissue of the gill lamellae (Putz et al, 1965).

The infections exhibit rather extensive epithelial proliferation (Canning, 1976). j?. ovariae causes the same type of host neoplasm in ovaries of N. crysoleucas

(Summerfelt and Warner, 1970). Pleistophora typicalis causes extensive damage in skeletal muscle of Cottus sp. 65

(Canning, 1976). Pleistophora macrozoarcidis produces large neoplastic masses in deep musculature near the vertebral column of Macrozoarces americanus (Nigrelli,

1953).

Some aspects of development are common to all microsporidan infections (Vavra, 1976a, 1976b).

Microsporidan schizonts are initially simple cells limited by a unit type membrane with one rather compact nuclear component (Canning and Nicholas, 1974; Lom and

Corliss, 1967; Vavra, 1976b; Weidner, 1970). The presence of two schizont generations in G. hertwigi is similar to that reported for a number of microsporida

(Gray et al, 1969; Vavra, 1976b; Youssef and Hammond,

1971). The first generation is represented by small cells and small, rounded plasmodia. In the second generation the parasite grows into long plasmodial cylinders with several large nuclei. Occasionally an infected host-cell is filled with sprouting multinucleated schizont-cylinders

(Weissenberg, 1976). Unlike other microsporidan develop­ ment, the present observations indicate a considerable simplicity of the cytoplasm during schizogony of G. hertwigi. The development of an endoplasmic reticulum and/or primitive Golgi apparatus is reported in schizonts of Nosema sp., and Pleistophora sp. (Loin and Corliss, 1967

Milner, 1972; Weidner, 1972; Youssef and Hammond, 1971).

The limiting membrane of many microsporidan schizonts exhibits structural evidence of an active interaction with host cell cytoplasm. The surface area of the schizont membrane can be substantially increased by vesicular or tubular projections interdigitated with the host cell cytoplasm (Codreanu and Vavra, 1970; Vavra, 1976b).

Sprague and Vernick (1968) reported the presence of an

"annulate lamellae" in Glugea weissenberqi xenomas. They observed the lamellae continuous with all host cell and parasite components via the host endoplasmic reticulum.

The trabecular network (TBN) observed in xenomas of G. hertwigi is analogous to the "annulate lamellae" reported by Sprague and Vernick (1969). Weidner (person, comm.) has observed such a trabeculae in GS. stephani xenomas.

Sprague and Vernick (1969) observed a very small granule or "gemmule" continuous with the annulate lamellae and suggested that this represented the earliest stage of parasite development. Electron microscopic examination in the present study failed to reveal any such stage.

It is likely that the condensed material within the TBN is the granular material observed by Sprague and Vernick

(1969); however, present evidence cannot support their 67 interpretation. When G. hertwigi was made to discharge

into unfertilized oocytes of Stronglyocentrotus purpuratus an extensive array of lamellae containing dense material formed within the egg cell in the region of the parasite

(Scarborough and Byrd, unpublished). Elaboration of the

TBN between host-cell components and the parasite may be for the purpose of nutrient exchange.

The trilaminar boundary observed in hertwigi schizont plasmodia has not been observed in the development of other microsporidans. Usually the schizont boundary remains as a simple unit membrane (Vavra, 1976a, 1976b).

The continuity of the trabecular network with the outer envelope of G. hertwigi schizonts suggests that the outer layer may be a closely applied envelope during growth but not a true limiting membrane. Furthermore, the TBN appears to be continuous with the endoplasmic reticulum of the host cell.

Typically, centrioles are not observed in microsporidan parasites (Vavra, 1976a). A specialized area on the nuclear membrane serves as an attachment point for the intranuclear spindle apparatus. In the present study, microtubular spindles occurring in the dividing nuclei of the schizont and sporoblast are similar to those reported for G. weissenbergi by Sprague and Vernick (1968), 68 for Nosema nelsoni by Sprague and Vernick (1969) for

Nosema algerae by Canning and Sinden (1973) and Vavra and

and Undeen (1970), and for N. apis by Youssef and Hammond

(1971). The structure has morphological similarity with

the kinetic center of yeasts (Sprague and Vernick, 1971;

Moens and Rapport, 1971). The persistence of the centriolar plaque without attached microtubules in G. hertwigi

sporoblasts was also observed in Nosema sp. by Walker and

Hinsch (1972). Perinuclear vesicles are commonly observed

adjacent to the spindle plaque (Vavra, 1976a, Vavra and

Undeen, 1970; Youssef and Hammond, 1971). Their significance

is unknown.

There is some evidence from microsporidans grown in

tissue culture and from insect hosts subject to various conditions that the passage from schizogony into sporogony

is due to the influence of environmental conditions

(Fowler and Reeves, 1974b; Hazard and Anthony, 1974;

Ishihara, 1969; Sprague and Vernick, 1971; Vavra, 1976b).

It is not known if the environmental condition of the host or host-cell influences G^. hertwigi sporogony. It is observed that sporogony is initiated after the majority of schizonts are in the multinucleate plasmodial stage

and fill the central region of the xenoma. The occurrence of irregularly spaced patches on the outside of the parasite limiting membrane and the appearance of a clear space between the parasite and the adjacent host-cell cytoplasm are used to mark the onset of sporogony and to identify the sporont (Cali, 1971; Vavra, 1976b).

These outer patches have been described as "ramified cisternae" by Codreanu and Vavra (1970) as a "ragged outer coat" by Krinsky and Hayes (1978), as "incomplete amorphous material" by Canning and Nicholas (1974), as "tubular projections" by Colley et al (1975), as "secretion products" by Loubes and Maurand (1976), and as "wall material" by

Sanders and Poinar (1975). According to Canning and

Nicholas (1974) and Sprague and Vernick (1969) the coat material is produced by the Golgi vesicles and endoplasmic reticulum of the sporont. The appearance of a Golgi apparatus and endoplasmic reticulum in G. hertwigi sporonts is concurrent with the disposition of the spore coat. Glycoprotein is known to be synthesized in endoplasmic reticulum and conjugated with carbohydrate in the Golgi complex (Peterson and Leblond, 1964).

Therefore, it is likely that substances needed for the elaboration of the spore coat are manufactured by the parasite. In some species the thickening of the sporont membrane is due to the appearance of an additional unit 70

membrane (Overstreet and Weidner, 1974). During sporoblast differentiation the extramembranous material or membrane sheaths are disseminated more uniformly until a continuous thick coat is formed (Vavra, 1976a). In some microsporidan species this exospore becomes decorated with appendages or tubular structures. In Pleistophora sp. numerous flexible tubules radiate in all directions (Szollosi,

1971). Smaller tubules described as bristles (Sprague et al, 1968) occur at the surface of sporoblasts and spores of FJ. michaelis (Weidner, 1970) and of Qrmiersia carcini

(Vivares et al, 1977). Dwyer and Weidner (1973) demonstrated that these bristles have some of the character­ istics of microtubules. These microtubules may participate in the flow of material to and from the parasite (Lorn and

Corliss, 1967; Overstreet and Weidner, 1974). The sporoblasts and spores of G. hertwigi are not decorated and have a smooth surface. The Golgi complex and endoplasmic reticulum continue to develop and become the dominant cytoplasmic feature of sporonts and sporoblasts of G_. hertwigi. The increased activity of these organelles provides the substance for exospore formation during schizogony. The mode of transport of the exospore material to the parasite surface is unknown. There is no evidence in the present study of surface pores described by Canning and Nicholas (1964). 71

£* wig1 sporonts transform into multinucleate plasmodia from which individual sporoblasts are cleaved by multiple fission. This development has been reported for a number of microsporidan species (Codreanu and Vavra,

1970; Cossins and Bowler, 1973; Gassouma and Ellis, 1972;

Sprague et al, 1968). The clear space that separates

G^ hertwigi sporonts from the host cytoplasm increases to form a vacuolar space during the formation of sporcgonial plasmodia. The vacuole is likely not bounded by a membrane of parasite origin; rather, it appears to be delimited by membranes of the host endoplasmic reticulum and is described as a parasitophorous vacuole (PV). A pansporoblast, being the complex of a sporogonial plasmodium, vacuolar cavity, and limiting membrane of parasite origin is reported for numerous microsporidans (Sprague, 1977;

Vavra, 1976a). Overstreet and Weidner (1974) hypothesize that a vascuolar cavity acts as a "growth chamber" and regulates the flow of material from host to parasite.

A common feature shared by both pansporoblast and parasitophorous vacuoles is the presence of some electron dense material within the vacuole cavity (Canning and

Sinden, 1974; Knell et al, 1976; Liu, 1973; Loubes and

Arbariek, 1977; Pakes et al, 1975; Weidner, 1970, 1975,

1976a). This material apparently is of parasite origin 72 and is termed "secretion" by Vavra (1976). The appearance of this material varies and is observed as granules and tubules in the present study. Tubular formation within the FV is also seen in N. michaelis (Sprague and Vernick,

1979; Weidner, 1972) in Pleistophora debaisieux (Vavra,

1976) in Encephelatozoon cuniculi (Weidner, 1976a) and in

Thelohania sp. and Pleistophora sp. (Gassouma and Ellis,

1972). What function the secretion material has remains unknown. Since sporoblast appendages function to transport material to and from the parasite (Dwyer and Weidner,

1974), these tubules may transport material within the

PV cavity.

In some microsporidans, cells with 2 closely adjacent nuclei (the diplokaryon) appear at the onset of sporogony

(Canning and Sinden, 1973; Canning and Madhavi, 1977;

Cossins and Bowler, 1974; Vavra, 1976a, 1976b; Weidner,

1970). The presence or absence of the diplokaryon during certain stages of microsporidan development has recently been suggested as a taxonomic character (Loubes, 1979;

Weiser, 1976b). The diplokaryon was not observed during any stage of G. hertwigi development.

Whether unikaryon or diplokaryon, the behavior of the sporont nucleus is the most controversial part of the microsporidan life cycle (Vavra, 1976b). There is no 73 useful evidence that karyogamy occurs in microsporidans.

Sprague and Vernick (1968) report what appears to be nuclear fusion in G. weissenbergi; however, this interpre­ tation is admittedly tentative. Loubes (1979) observed lengthy nuclear condensation in sporogonial plasmodia of several species of microsporidans. The nuclear structures observed by Vavra (1976a) in Gurleya chironomi are thought to represent synaptonemal complexes. These structures resemble chromatin strands in the ring-nuclei seen in

G. hertwigi during early sporogony. Similar ring-nuclei were observed by Kellen and Lindgren (1973) in Nosema invadens and by Sprague (1940) in Collosporidium periplanetae; however, these authors made no comment as to their implication- The occurrence of synaptonemal complexes which in turn indicate the presence of meiosis, would establish sexuality in the microsporidans. Since there is no information about the DNA content of the nucleus of G. hertwigi or of any other microsporidan during any life stage, this question remains unanswered.

The single cells resulting from the division of the sporogonial plasmodia are sporoblasts. Multiple fusion and the formation of sporoblast buds that remain connected by a cytoplasmic bridge until sporogenesis is common in microsporidans that form a plasmodia during sporogony 74

(Codreanu and Vavra, 1970; Cossins and Bowler, 1973;

Gassouma and Ellis, 1972; Vavra, 1976b). It is in the

sporoblast that spore organelles begin to form. As seen

in the present study the sporoblast cytoplasm contains

extensive endoplasmic reticulum and a Golgi apparatus.

All 3 elements of a "classical" Golgi occur in G. hertwigi

(Dalton, 1961; Sprague and Vernick, 1974; Vernick et al,

1977 ) .

The final event of the microsporidan life cycle is

gradual transf armation of the sporoblast to the spore.

The present electron microscope study demonstrates that

the polar filament and polar sac are the spore organelles

differentiated first. The last event is formation of the

thick endospore layer of the spore wall. Sporogenesis

is recognized to be a complicated process (Vavra, 1976b).

It is a rather rapid process; generally not many

transforming sporoblasts are observed in ultrathin sections.

Although the density of the spore and problems encountered

during EPON embedding render electron microscopic studies

difficult, there have been a number of exhaustive ultrastructural studies on sporogenesis and the structure

of the mature spore (Burges et al, 1974; Colley et al,

1975; Erickson et al, 1968; Gassouma and Ellis, 1973;

Jensen and Welling, 1972; Liu 1973; Liu and Davies, 75

1972; Sprague et al, 1978; Vavra, 1976a, 1976b; Weidner,

1970).

Although there is no unanimous agreement concerning

the origin of the polar filament, recent data and the

present observations support the idea that it originates

from the Golgi zone. The filament is built from the basal

end. The bulbous part representing the future polar sac

appears as a vacuole in the vicinity of the nucleus. The

coils are synthesized and their formation progresses from

the basal part to the terminal end. Membrane sheaths from

the endoplasmic reticulum are added after coil formation.

This mode of filament development is in agreement with the

observations of Jenson and Wellings (1972), Loubes and

Maurand (1976), Walker and Hinsch (1972) and Weidner,

(1970). During G. hertwigi sporogenesis the polar sac

vacuole is surrounded by vesicular Golgi and continuous with elements of the endoplastic reticulum; however, it

could not be determined if the spore polaroplast was a

derivative of the Golgi, the endoplasmic reticulum or

both.

The taxonomic character of disporous development has

been applied to the genus Glugea (Canning and Nicholas,

1974; Kudo, 1924, 1966; Sprague and Vernick, 1972). This

condition was not commonly observed in this study of G. 76

hertwigi. Loubes (1979) provided evidence from his ultrastructural survey of several microsporidan genera

that the number of spores from a sporont division can be highly variable. Loubes et al (1976) found that G. habrodesmi was at least tetrasporous and occasionally octosporous. Loubes (1979) points out that nuclear configuration, presence or absence of sporogonial plasmodia, the presence of a pansporoblast or parasitophorous vacuole, type .of cyst formation and host specificity are constant characters. The high temperature used in this study likely influenced the rate of G. hertwigi maturation and may well have influenced the spore number. LITERATURE CITED

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Ann L. Scarborough was born August 2, 1949 in

San Diego, California and graduated from the Academy of

Our Lady of Peace High School in 1967. She moved to

San Francisco and attended the University of San Francisco in 1967 and 1968. She returned to San Diego, attended the University of California (UCSD), and graduated with a

Bachelor of Arts degree in Biology. After 3 1/2 years as a Research Associate at UCSD Medical School Department of

Pathology, she moved to Baton Rouge, Louisiana. In 1975 she entered Louisiana State Oliversity Department of

Zoology and Physiology as a graduate student and received a Master of Science degree in 1977. She was awarded a

Doctoral Dissertation Grant from the National Science

Foundation in 1977 and continued in the Department of

Zoology. She is currently a candidate for a Doctor of

Philosophy degree.

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