Pathobiology of Burenella dimorpha Jouvenaz and Hazard (Microspora: Microsporida)

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Title:
Pathobiology of Burenella dimorpha Jouvenaz and Hazard (Microspora: Microsporida)
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x, 95 leaves : ill. ; 28 cm.
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English
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Jouvenaz, Donald P., 1936-
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Subjects

Subjects / Keywords:
Burenella dimorpha   ( lcsh )
Microsporidia   ( lcsh )
Ants -- Control -- Biological control   ( lcsh )
Fire ants -- Biological control   ( lcsh )
Solenopsis invicta -- Biological control   ( lcsh )
Solenopsis richteri -- Biological control   ( lcsh )
Genre:
bibliography   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1982.
Bibliography:
Includes bibliographical references (leaves 89-93).
Statement of Responsibility:
by Donald P. Jouvenaz.
General Note:
Typescript.
General Note:
Vita.

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University of Florida
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All applicable rights reserved by the source institution and holding location.
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aleph - 000319143
notis - ABU5994
oclc - 09311614
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AA00003858:00001

Full Text








PATHOBIOLOGY OF BURENELLA DIMORPHA JOUVENAZ
AND HAZARD (MICROSPORA: MICROSPORIDA)














By

DONALD P. JOUVENAZ


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY




UNIVERSITY OF FLORIDA


1982













gC ACKNOWLEDGMENTS
00%

00 Many people have assisted, advised, and encouraged me

Sin my graduate studies. I am deeply grateful to the members

of my graduate committee, Drs. Donald W. Hall, Clifford S.

Lofgren, Edward M. Hoffman, and Robert C. Wilkinson, for

their patience and guidance, and for the time and effort

they have spent on my behalf. I also wish to thank Dr.

George E. Allen, who served as chairman of the graduate

committee before his departure from the University for

another position.

In addition to the members of my graduate committee,

several people have given me invaluable assistance. Fore-

most among these is Ms. E. Ann Ellis, Electron Microscopist

at the Insects Affecting Man and Animals Research Laboratory,

USDA, Gainesville. Mr. John D. Atwood, Mrs. Anita Lemire,

Mrs. Susan W. Avery, and Dr. Albert Undeen have also helped

me in various ways. Dr. William F. Buren of the University

of Florida encouraged me over a long period of time.

I cannot adequately express my gratitude to my wife,

Judy, for her unfailing encouragement and patience throughout

my graduate studies.












TABLE OF CONTENTS


Page

ACKNOWLEDGMENTS........................................... ii

LIST OF TABLES.................................... V

LIST OF FIGURES.................................... i

ABSTRACT.......................................... ix

CHAPTER

ONE INTRODUCTION............................ 1

TWO GENERAL LITERATURE REVIEW............. 4
Synopsis of the Microsporidia......... 4
Microsporidia Parasitic in Ants....... 6
Solenopsis geminata................... 9

THREE GENERAL MATERIALS AND METHODS......... 11
Collection and Maintenance of Colonies 11
Temperature and Humidity.............. 15
Diet.................................. 15
Laboratory Propagation of B. dimorpha 16
Harvest of Spores...................... 16

FOUR TRANSMISSION AND INFECTIVITY OF SPORES
OF BURENELLA DIMORPHA................. 18
Materials and Methods ................ 19
Results................................ 22
Discussion.............................. 25

FIVE HOST SPECIFICITY OF BURENELLA DIMORPHA 27
Materials and Methods ................. 29
Results............................... 30
Discussion............................. 32

SIX ABUNDANCE OF BURENELLA DIMORPHA IN
NATURE................................. 37
Materials and Methods.................. 37
Results and Discussion................ 37


iii








CHAPTER


SEVEN PATHOLOGY OF BURENELLA DIMORPHA
INFECTION..............................
Materials and Methods ................
Results...............................
Discussion.............................

EIGHT TEMPERATURE-DEPENDENT SPORE DIMORPHISM
IN BURENELLA DIMORPHA................
Materials and Methods.................
Results...............................
Discussion..............................

NINE AMENDMENTS TO THE DESCRIPTION OF
BURENELLA DIMORPHA....................
Materials and Methods.................
Results...............................
Discussion..............................

TEN SUMMARY AND CONCLUSIONS...............

GLOSSARY..........................................

LITERATURE CITED...................................

BIOGRAPHICAL SKETCH...............................












LIST OF TABLES


Table Pa&g

1. B. dimorpha infection rates in colonies
of S. invicta and S. richteri. 31

2. Susceptibility of selected species of ants
and a moth to infection by B. dimorpha. 33

3. Relative abundance of B. dimorpha MB spores
in pupae of S. geminata reared from larvae
at various temperatures. 69

4. B. dimorpha spore yield of pupae of
S. geminata reared from larvae at 20,
28, or 32 C. 69

5. Relative abundance of B. dimorpha MB spores
in pupae of S. geminata placed at 20 or 32 C
at different ages. 71











LIST OF FIGURES


Figure Page

1. A six-cell colony of S. geminata. 14

2. A miniature nest cell. 14

3. Infrabuccal cavity. 24

4. Enlarged view of infrabuccal cavity and
pellet. 24

5. Diseased S. richteri pupa compared to
healthy and diseased S. geminata pupae. 36

6. Diseased (left) and healthy male pupae
of S. geminata. 44

7. Diseased (left) and healthy female pupa
of S. geminata. 44

8. Healthy (left) and diseased worker pupae
of S. geminata. 44

9. The development of pathognomonic signs
in pupae of S. geminata infected by
B. dimorpha .47

10. Frontal section through the head of a
young (ca 10 days postpupation) pupa
infected by B. dimorpha. 51

11. Eye, optic nerve, and optic stalk of a
young (ca 10 days postpupation) pupa
infected by B. dimorpha. 51

12. Frontal section through the head of an
older (ca 16 days postpupation) pupa
infected by B. dimorpha. 51

13. Cuticle of a healthy pupa. 54

14. Body surface of a pupa infected by
B. dimorpha. 54








Figure Pagi

15. Detail of the cuticle in a healthy
pupa. 54

16. Detail of microvilli extending into
the fluid space of a pupa infected by
B. dimorpha. 54

17. Sagittal section through the developing
eye of a healthy pupa. 56

18. Sagittal section through the eye of a
pupa infected by B. dimorpha. 56

19. Sagittal section through the eye of a
healthy pupa. 56

20. Sagittal section through the eye of a
diseased pupa. 56

21. Sagittal section through the developing
eye of a healthy pupa showing detail of
optic nerve and base of the developing
ommatidium. 58

22. Sagittal section through the eye of a
pupa infected by B. dimorpha. 58

23. Fat body of a healthy pupa. 58

24. Fat body of a pupa infected by B. dimorpha. 58

25. Tissue specificities of MB and NMB spores
of B. dimorpha. 60

26. Tissue specificities of MB and NMB spores
of B. dimorpha. 60

27. Tissue specificities of MB and NMB spores
of B. dimorpha seen at higher magnification. 60

28. Transmission electronmicrograph of a
mature NMB spore of B. dimorpha. 80

29. Transmission electronmicrograph of a
mature MB spore of B. dimorpha. 80

30. Scanning electronmicrograph of a mature
NMB spore of B. dimorpha. 80


vii








Figure Page

31. Scanning electronmicrograph of a
mature MB spore of B. dimorpha. 80

32. Transmission electronmicrograph of an
immature NMB spore. 83

33. Transmission electronmicrograph of two
immature MB spores within a delicate
pansporoblast membrane. 83


viii












Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

PATHOBIOLOGY OF BURENELLA DIMORPHA JOUVENAZ
AND HAZARD (MICROSPORA: MICROSPORIDA)


By

Donald P. Jouvenaz

December, 1982

Chairman: Donald W. Hall
Major Department: Entomology and Nematology

Burenella dimorpha Jouvenaz and Hazard is a host-specific,

dimorphic microsporidium that parasitizes the tropical fire

ant, Solenopsis geminata (F.). The red and black imported fire

ants, Solenopsis invicta Buren and Solenopsis richteri Forel,

can be infected perorally by B. dimorpha, but the parasite

survives only a few generations in colonies of these factitious

hosts. Nine species of ants other than fire ants (representing

eight genera) were refractory to infection.

Infected pupae develop pathognomonic signs (eye teratology

and blister-like clearings in the occiput and petiole) that are

due to destruction of the cuticle. In an advanced stage of
disease, the pupa ruptures and is cannibalized by worker ants.

The spores and other particulate matter are not ingested, but

are diverted to the infrabuccal cavity, formed into a pellet,







expelled, and fed to fourth-instar larvae only. The intra-

colonial cycle of infection is thus from ruptured, diseased

pupae to fourth-instar larvae via the adults, who are mechan-

ical vectors. The binucleate, nonpansporoblast membrane-

bounded (NMB) spores are infective perorally for larvae; the

uninucleate, pansporoblast membrane-bounded (MB) spores are

not infective, and their function is unknown. These spore

types are tissue specific: NMB spores develop from disporous

sporonts in the hypodermis; MB spores develop in octets from

multinucleate sporonts in the fat body.

The development of MB spores is temperature-dependent.

The lower thermal threshold for MB spore development is

between 20 and 22.5 C; the upper thermal threshold is below

32 C. A hypothesis is advanced that these limits are set by

the stability of an enzyme(s), and that the physiological

function blocked in MB sporulation is meiosis.

Burenella dimorpha has two cycles of merogony (vegetative

multiplication). The meronts of the first cycle were originally

described as uninucleate cells that become binucleate and divide.

However, these nuclei are actually pairs of nuclei in a diplo-

caryotic arrangement.

Surface structure has been seen for the first time in MB

spores. The surface of NMB spores is smooth.












CHAPTER ONE

INTRODUCTION


The red and black imported fire ants, Solenopsis

invicta Buren and Solenopsis richteri Forel, are medical

and agricultural pests which infest ca 9.3 x 107 hectares

(2.3 x 10 acres) in the Southeastern United States. Both

species were apparently introduced into the United States

with products shipped from South America to Mobile, Alabama,

about 1918 and 1940, respectively. The tropical fire ant,

Solenopsis geminata (Fabricius), may also be an introduced

species; however, it has been a resident of this country

for so long it is generally regarded as native. S. geminata

is not important as a pest, except in Hawaii where it has

been introduced.

Efforts to control the imported fire ants by chemical

means have been the subject of serious controversy since

before 1960. Consequently, the discovery of microsporidian

infections in these ants in their native lands by Drs. G. E.

Allen, W. F. Buren, and A. Silviera-Guido greatly stimulated

interest in research on their possible use for biological

control. Earlier, surveys by several investigators had

failed to detect specific pathogens of fire ants in the

United States.







In response to the reports of Allen, Buren, and Silviera-

Guido, the Agricultural Research Service of the United States

Department of Agriculture funded several trips to South

America by federal and university scientists to search for

potential biological control agents for fire ants. I was

priviledged to participate in the first trip to Mato Grosso

and Mato Grosso do Sul, the homeland of S. invicta, as a

member of a team of five investigators. For five weeks

we collected fire ants and laboriously examined them indi-

vidually for evidence of infection. Despite the handicap

of not knowing how to efficiently screen ants for disease,

we collected spores of two species of microsporidia. Unfor-

tunately, all of our attempts to infect healthy laboratory

colonies of S. invicta with these spores failed.

Since we were unable to propagate and study the exotic

microsporidia, a survey of pathogens of S. geminata in Florida

was initiated to find a locally available host-pathogen

model for studies of basic pathobiology, and to develop

techniques for the detection and study of disease in ants.

The pathogen selected for study in detail was Burenella

dimorpha Jouvenaz and Hazard, a microsporidium that is trans-

missible per os and which has the advantage of producing

pathognomonic signs of infection in pupae of S. geminata.

I have attempted to trace the history of this microsporidiosis

form the transmission of spores to a healthy host, through

the life cycle of the parasite and pathogenesis in the host,

to the production of spores of the succeeding generation.




3


An understanding of the biology of the natural enemies

of insects is fundamental to the development of strategies

for their employment in pest management. The information

gained in this study will contribute to our knowledge of

the microsporidia in general, and, hopefully, in some small

way to the development of effective biological control of

the imported fire ants.












CHAPTER TWO

GENERAL LITERATURE REVIEW


As a general reference to the microsporidia, the reader

is referred to the two volume monograph by Bulla and Cheng

(1976, 1977). These two volumes constitute the only recent

monograph on these protozoa. Earlier monographs on the

microsporidia were published by Kudo (1924) and Weiser (1961);

however, the development and recent extensive application

of electron microscopy to the study of microsporidia has

rendered these works largely obsolete. Since microsporidia

are little known to most entomologists and even protozoolo-

gists, a brief synopsis of their systematics and biology is

presented. A glossary of terms specifically used in the

study of microsporidia and abbreviations used in this disser-

tation has been included.


Synopsis of the Microsporidia

Microsporidia are extremely small protozoa which are

obligate (lacking mitochondria), intracellular parasites of

invertebrate (primarily arthropod) and, less commonly, verte-

brate animals. Their life cycles include the production of

spores which contain a single sporoplasm and a long, coiled,




5




tubular organelle, the polar filament (or a rudiment thereof).

Upon ingestion by a suitable host, the polar filament uncoils

and extrudes with extreme rapidity from the spore, remaining

attached anteriorly. The sporoplasm is expelled through

the polar filament, which apparently evaginates as it ex-

trudes from the spore. If the spore is near the gut wall

and is properly oriented, the wall is penetrated by the

violently extruding filament and the sporoplasm is injected

into a host cell as though by a hypodermic syringe. There

it multiplies and develops in direct contact with the host

cytoplasm, there being no parasitophorous vesicle. After a

large number of vegetative parasites have been produced by

cycles of multiplicative development (merogony), they trans-

form into sporonts, or cells which give rise to spores after

a set number of divisions characteristic of the species

(sporogony).

Sprague (1977), considering the protozoa a polyphyletic

group, assigned the microsporidia to a new phylum, Microspora,

consisting of two classes, Rudimicrosporea and Microsporea.

The typical microsporidia, including the subject of this

dissertation, are assigned to the order Microsporida, one

of the two orders of Microsporea (Levine et al., 1980).








Microsporidia Parasitic in Ants

Only two species of microsporidia, Thelohania solenopsae

Knell et al. (Thelohaniidae), and the subject of this disserta-

tion, Burenella dimorpha Jouvenaz and Hazard (Burenellidae),

have been described from ants.


Thelohania Solenopsae

T. solenopsae, the first specific pathogen known from

fire ants and the first protozoan known from Formicidae,

was discovered by Dr. W. F. Buren during a taxonomic study

of S. invicta (Allen and Buren, 1974). While examining alcohol-

preserved specimens from the state of Mato Grosso, Brazil,

Buren observed subspherical, cyst-like bodies in the partially

cleared gasters of workers. These cysts contained spores

of the microsporidium, which was subsequently described from

fresh material by Knell et al. (1977). Very soon after Buren's

observation, Allen and Silviera-Guido (1974) reported similar

microsporidia from S. richteri in Uruguay and Argentina,

and from an unidentified Solenopsis species in Uruguay.

T. solenopsae (or T. solenopsae and sibling species which

cannot be differentiated) has since been detected in ca 22

described and undescribed species of fire ants in South

America (Jouvenaz et al., 1977).

T. solenopsae infects fat body cells of workers and

sexual, and the ovaries of females. Infected cells hyper-

trophy, forming the cysts observed by Buren. Within the








cysts, the spores occur in octets bounded by a membrane

(pansporoblast membrane). The infection is not rapidly

fatal, but destruction of the fat body occurs which results

in premature death of adult ants. Consequently, colonies

are debilitated (Knell et al., 1977). Attempts to transmit

T. sdlenopsae to healthy colonies in the laboratory have

failed, and the mode of transmission of this parasite is

unknown (Jouvenaz et al., 1981).


Burenella Dimorpha

Burenella dimorpha was described by Jouvenaz and Hazard

(1978) as the type species of a new genus which represents

a new family, Burenellidae. This family includes those

species of microsporidia having two sporogonic sequences,

one producing non-pansporoblast membrane-bounded (NMB)

spores from disporous sporonts, and the other producing

octets of spores bounded by a pansporoblast membrane (MB).

Hazard et al. (1981) listed three additional genera as

members of Burenellidae: Culicosporella Weiser and Hazardia

Weiser (monotypic genera parasitic in mosquitoes) and

Vairimorpha Pilley (parasitic in a variety of Lepidoptera).

Certain genera of the family Thelohaniidae Hazard and Oldacre

also have dimorphic (producing two morphologically distinct

types of spores) species; however, their NMB spores arise

from plasmodia rather than disporous sporonts.

Little is known of the pathobiology of B. dimorpha beyond

those minimal aspects of spore morphology and life cycle







necessary for classification. This information will be

reviewed in conjunction with the specific studies reported

in this dissertation.


Other Microsporidia

In addition to B. dimorpha, at least three undescribed

species of microsporidia infect S. geminata (Jouvenaz et

al., 1977), and at least one undescribed species infects

Solenopsis spp. in South America (Jouvenaz et al., 1980).


Other Pathogens of Ants

Because of the paucity of information on the diseases

of ants, it is feasible to summarize briefly the literature

on pathogens of ants other than microsporidia. The only

remaining pathogen of ants which has been described is

Mattesia geminata Jouvenaz and Anthony (Neogregarinida:

Ophrocystidae), which also infects S. geminata in Florida.

This protozoan develops in the oenocytes of the hypodermis,

causing destruction of the hypodermis, melanization, and

eye malformation in pupae. The infection appears to be

invariably fatal in the pupal stage of development. Attempts

to transmit the infection per os (using fresh, aged, and

variously treated spores) and by placing infected pupae

in healthy colonies (conspecific pupae are adopted) have

failed (Jouvenaz and Anthony, 1979). A similar or identical

neogregarine occurs in fire ants in Brazil (Jouvenaz et

al., 1980).








Virus-like particles have been detected in an undescribed

Solenopsis species from Brazil and in S. geminata from

Florida (Avery et al., 1977). These particles are morpho-

logically similar, being non-occluded, rod-shaped, and

bound by double membranes. They occur in hypertrophied

nuclei of fat body cells, but their pathogenicity is as

yet undetermined. Virus-like particles have also been

reported from apparently healthy wood ants, Formica lugubris

Zett. (Steiger et al., 1969). These particles occurred

in the cytoplasm of nerve cells, and were morphologically

quite different from those found in fire ants.

Only one species of fungus, which remains unidentified,

is known to be specifically associated with fire ants (Jouvenaz

et al., 1977). Other fungi which have been reported as

causing infections in various species of ants include

Metarrhizium anisopliae (Metschnikoff) Sorokin, Beauveria

bassiana (Balsamo) Vuillemin, and several species of Cordyceps.

Allen and Buren (1974) summarized and discussed these reports.

Broome et al. (1976) discussed the mechanism by which B.

bassiana infection is initiated in S. richteri.


Solenopsis geminata

The literature on the tropical fire ant is very sparse;

most studies of fire ants have been concerned with the

imported species, which are pests. For information on

the biology and behavior of S.geminata and fire ants in general,

the reader is referred to the excellent review by Lofgren et al.,

(1975).







An outline of the life cycle of Solenopsis species

is as follows: mating takes place during nuptial flights,

after which the females (queens) return to earth, dealate,

and secrete themselves in a closed, subterrainian chamber.

During this claustrall" period, the queens, subsisting

on their histolyzing flight muscles and food stored in

their fat body and crop, lay eggs and rear their first

offspring. As the number of workers increases, the queen

stops caring for the immatures, but continues to lay eggs

throughout her lifespan. The workers forage for food,

extend and maintain the nest structure, defend the nest

and territory, and care for the queen and brood. A mature

colony may contain as many as 200,000 or more individuals.

The developmental stages of fire ants are egg, four

larval instars, pupa, and adult. The development period

is temperature dependent, but averages about three to four

weeks.












CHAPTER THREE

GENERAL MATERIALS AND METHODS


Collection and Maintenance of Colonies

Laboratory colonies of fire ants were obtained by

collecting queens and contingents of their workers from es-

tablished field colonies, or (during the spring of the year)

by capturing newly mated queens from under debris or as

they wandered over the surface of the ground. Queens of

other species of ants (used for host range studies) were

collected exclusively by the latter method.

Ants were collected from field colonies by excavating

the mounds with shovels and transporting the soil containing

ants to the laboratory in plastic buckets, the inner walls

of which were coated with Fluon GP-1 (ICI United States,

Wilmington, Delaware 19897) to prevent escape of the ants.

The soil in the buckets was slightly moistened (if necessary)

and left undisturbed overnight to allow the ants to establish

tunnels and to collect buried immatures. Water was then

slowly dripped from medical intravenous fluids tubes into

the buckets, forcing the ants to the surface of the soil.

When the soil was completely submerged, masses of adult

and immature ants floated or clung to the sides of the bucket,

and were easily transferred with a ladle to Fluon-coated








trays (ca 52 x 40 x 7 cm) containing several layers of paper

towels on their bottoms. After the wet masses of ants had

dried and dispersed, the collections of ants were searched

for queens. Those containing queens were transferred to

laboratory nests in Fluon-coated trays held in metal racks

(Fig 1).

Newly mated queens were placed in miniature nest cells

or in glass culture tubes (150 x 17 mm) containing a mass

of wet cotton and held in a Fluon-coated tray.

Nest cells for large colonies were constructed by pouring

liquid Castone (Ransom and Randolph Co., Toledo, Ohio) to

a depth of 10-12 mm in 25 x 150 mm disposable plastic petri

dishes, allowing the Castone to solidify, and melting four

exit ports in the sides of the bottom dish above the surface

of the Castone (ports were unnecessary in the loose-fitting

tops). Solidified Castone is too hard for the ants to tunnel

through, yet remains slightly moist, providing adequate

(but not excessive) humidity and substrate moisture within

the cells for two to three months.

When the nest cells became dry and soiled, their tops

were removed, and fresh cells were placed in the tray. The

ants quickly moved into the new cells, and the abandoned

old cells were discarded. Up to four cells were used simul-

taneously in each tray, and were replaced on a rotating

basis.

Miniature nests for newly mated queens, very small

colonies, or groups of experimental subjects held in isolation























Fig 1. A six-cell colony of S. geminata. Two glass culture
tubes plugged with cotton and containing water or
dilute honey, a dish of insect pupae, and part of a
boiled egg are on the left. Alate sexual are con-
centrated in the reat top cell.








Fig 2. A miniature nest cell. The nest cell has a layer
of Castone (dental casting compound) in the bottom
and an entry port in the side. A wad of wet cotton
helps maintain high humidity. The wall of the large
petri dish is coated with Fluon to prevent escape
of the ants. The large petri dish top is normally
taped securely to the bottom and labeled.





















" 9 '
.1




_Asd
4-

**-* ''*, ., S


(


I'i!








from their parent colonies were constructed from 15 x 60 mm

and 25 x 150 mm disposable plastic petri dishes. Liquid

Castone was poured to a depth of ca 5 mm in the smaller

dish, allowed to solidify and two or three exit ports were

melted into the wall. This small nest, with lid in place,

was placed inside the larger dish, the inner walls of which

were coated with Fluon. A wad of wet cotton was also placed

in the larger dish to help maintain high humidity (the much

smaller mass of Castone in these cells dried within a few

days) and the lid was taped to the petri dish bottom to

provide additional security. Thus, a nest and a secure

foraging area were combined in a single, compact unit (Fig 2).

Nest cells of both sizes were prepared in advance and

stored in a refrigerator until needed. Prior to use, they

were soaked in distilled water to saturation, then blotted

with paper towels to remove excess water.


Temperature and Humidity

The colonies were maintained at a ca 280 C in a labora-

tory room having no humidity control; however, the Castone

nest substrate and wet cotton balls (described above) main-

tained humidity within tolerable limits.


Diet

Laboratory colonies were fed live or dead (frozen)

insectary-reared insects (cockroaches, mealworms, cabbage

looper pupae), supplemented with boiled eggs, fruit, and 50%







aqueous honey solution. A diet composed of housefly or stable-

fly pupae (1 liter) pureed with cooked ground beef (ca 250 g)

in a blender and thoroughly mixed with whole eggs (1 dozen)

and vitamins (5.0 ml Poly Vi-Sol, Mead Johnson & Co., Evansville,

Indiana 47721) in hot liquid agar solution (50 g agar dissolved

in 1.5 L distilled water) was also used as a supplement

when insects were in short supply. The warm liquid diet

was poured to a depth of ca 1.5 cm in shallow enamel pans

and allowed to gel. It was then cut into cubes for dispens-

ing to the ants. The excess diet was covered with plastic

film and frozen or stored in the refrigerator.


Laboratory Propagation of Burenella dimorpha

Colonies of ants were infected with B. dimorpha by

feeding them boiled egg yolk wetted with a suspension of

spores to a consistency of paste. These colonies were then

maintained and diseased pupae were harvested periodically

as spores were needed. Prior to infection, colonies were

carefully examined for other diseases using the methods

described by Jouvenaz et al. (1977).


Harvest of Spores

Suspensions of spores free of cellular debris were

obtained by density gradient centrifugation using Percoll

(Pharmacia Fine Chemicals, 800 Centennial Ave., Piscataway,

N.J. 08854). Pupae exhibiting signs of advanced infection








were homogenized in an equal volume of distilled water using

a glass tissue grinder, and the homogenate was centrifuged

at 10,000 g for ca 20 minutes through a discontinuous gradient

consisting of layers of 25, 50, 75, and 100% Percoll. Over

97% of B. dimorpha NMB spores purified in this manner ex-

truded their polar filaments when ingested by ant larvae

(determined by phase microscopy of meconia), indicating

that most immature spores had been removed. Slightly less

highly purified suspensions suitable for use in routine

propagation of the parasite were obtained by centrifuging

the crude homogenate at 750 g for 20 minutes in a 50% solution

of Percoll. After all centrifugations with Percoll, the

spores were washed three times with distilled water, and

the volume was adjusted to ca 107 spores/ml. Spore suspen-

sions were stored at 40 C.












CHAPTER FOUR

TRANSMISSION AND INFECTIVITY OF SPORES OF
BURENELLA DIMORPHA


Burenella dimorpha produces two morphologically distinct

types of spores. Binucleate NMB spores develop from disporous

sporonts in the hypodermis; uninucleate, MB spores develop

in octets from multinucleate sporonts in the fat body. The

infection is characterized by the development of clear,

blister-like areas in the occiput and petiole which are

due to destruction of the cuticle. As the infection pro-

gresses, the clear areas increase in size, the cuticle becomes

extremely fragile, and eventually it ruptures. The pupa

is then cannibalized by workers (I have observed this in

laboratory colonies).

Suspensions of spores of B. dimorpha containing both

types of spores are infective perorally for S. geminata

(Jouvenaz and Hazard, 1978). However, attempts to separate

the spore types by density gradient centrifugation were

unsuccessful (the pansporoblast membrane is very delicate,

rupturing on dissection of the host, and the free MB spores

are nearly identical in bouyant density to NMB spores).

Therefore, Jouvenaz and Hazard were unable to determine

which or if both types are infective. They described this








parasite as a dimorphic species on the basis of light micro-

scopy studies of the life cycle, and the statistical improb-

ability of a dual infection occurring at high frequency,

but they were not able to experimentally demonstrate by

feeding tests that both spore types were produced by the

same microsporidium.

Adult fire ant workers feed liquid food to larvae in

all instars, but solid foods to fourth-instar larvae only

(Petralia and Vinson, 1978). A pellet of solid food is

expelled from the infrabuccal cavity and placed on the prae-

saepium of a larva by a worker, and the pellet is consumed

by the pupa. This method of feeding larvae suggested a

cycle of infection from ruptured pupae to fourth-instar

larvae vectored mechanically by adults via the infrabuccal

pellet. This study was conducted to 1) determine the mode

of intracolonial transmission of infection, 2) to determine

whether one or both spore types are infective, and 3) to

verify that B. dimorpha is a dimorphic species.


Materials and Methods

Boiled egg yolk wetted to a paste consistency with

a suspension of spores was offered to a small, healthy colony

of S. geminata, which was allowed to feed for 24 hr. A

sample of workers trapped immediately after feeding was

held one hour, killed by freezing, sectioned into the three

body parts, and examined by phase-contrast microscopy to








determine whether spores were ingested or diverted to the

infrabuccal cavity microsporidiann spores are visible in

slightly compressed, intact fire ant body segments at a

magnification of 300X). Heads of these workers were fixed,

stained with heavy metals, embedded in Spurr-Quetol resin,

sectioned, and examined by phase-contrast microscopy. The

details of these procedures are given in the section on

host pathology. Eight fresh infrabuccal pellets were removed

from the praesaepia of larvae and examined by phase micro-

scopy for spores.

After 24 hours, 109 fourth-instar larvae were removed

from the nest and held in a miniature nest cell with conspe-

cific workers that had not been exposed to B. dimorpha.

These workers functioned as nurses (immatures held in isola-

tion from adults are quickly attacked by fungi). After

21 days these immatures (now pupae) were examined for infec-

tion. A group of 79 prefourth (primarily third) instar

larvae were also removed from the same nest and held in

a similar manner until they closed as adults.

A suspension of mature spores of both types was prepared

by homogenizing diseased pupae in distilled water with a

glass tissue grinder and centrifuging the extract in a dis-

continuous Percoll gradient (100%, 75%, 50%, and 25%) for

20 minutes at ca 10,000 g (Jouvenaz, 1981). This procedure

produced a clean suspension of spores of both types, almost

all of which appeared to be mature. These spores were fed







to fourth-instar larvae in a paste of finely powdered (mortar

and pestle) dry baby cereal and spore suspension. Infrabuccal

pellet-sized quantities of this preparation were placed

on the praesaepia of fourth-instar larvae with a flattened

insect pin, and the larvae were held in a miniature nest

cell until pupation. Adult workers were provided to care

for these larvae ca 4 hours after feeding. When the larvae

pupated, 20 meconia were recovered and examined by phase-

contrast microscopy to determine whether spores had extruded

their polar filaments.

A suspension of NMB spores only was obtained by selecting

diseased pupae which, on the basis of pathognomonic signs,

were estimated to harbor some mature NMB spores but no mature

MB spores (NMB spore development precedes MB spore development).

Wet squashes of these pupae were examined individually by

phase-contrast microscopy, and those that were free of mature

MB spores were washed from the slide, pooled, and cleaned

and concentrated by centrifugation. An examination of 10,000

spores individually in a diluted aliquot (0-5 spores/field)

and careful scanning of the concentrated suspension confirmed

the absence of MB spores.

The suspension of NMB spores was mixed with boiled

egg yolk and fed to a small healthy colony of S. geminata.

After 20 days, 25 pupae in advanced infection were individually

homogenized in ca 0.5 ml water and examined by phase-contrast

microscopy (0-6 spores/field) to determine the ratio of the








spore types in the first 200 spores seen. Twenty-five infected

pupae from a laboratory colony that had been infected with a

crude suspension of mixed spores for routine propagation

of the parasite were also examined.

Results

Solenopsis geminata adults did not ingest spores into

the crop; instead, they diverted them to the infrabuccal

cavity (Figs 3 and 4). One hour after exposure to food

containing spores, 100 of the 160 workers that were examined

contained spores in their infrabuccal cavities. Not one

contained even a single spore in the digestive tract. All

eight infrabuccal pellets removed from fourth-instar larvae

shortly after deposition by workers contained numerous spores.

Only fourth-instar larvae became infected with B. dimorpha.

Seventy-one of the 109 that were exposed as fourth-instar

larvae became infected; none of the 79 immatures that were

exposed to spores as prefourth-instar larvae became infected.

Nonmembrane bound spores were infective perorally for

S. geminata and produced infections with pathognomonic
signs and both spore types. Furthermore, the spore types

were produced in normal ratios. Pupae that had been fed

NMB spores only contained 28.6% 10.2 (range 16-48%) MB

spores; those that had been fed both spore types contained

30.8% 7.8 (range 16-43%) MB spores.

In the meconia of larvae fed the suspension of mature

spores of both types, 1,723 (97.1%) of the 1,774 spores found
























Fig 3. Infrabuccal cavity. This medial section of an adult
fire ant worker head shows an infrabuccal pellet in
the infrabuccal cavity. P = pharynx; IBC = infra-
buccal cavity; IBP = infrabuccal pellet.















Fig 4. Enlarged view of infrabuccal cavity and pellet.
Numerous MB and NMB spores may be seen in the pellet.
Arrows point to valves of the orifice of the infra-
buccal cavity.




24












pp









F, A 4 r f
Ph



r j .y ,



ieillli








had extruded their polar filaments. The 51 (2.9%) NMB spores

that had not extruded their polar filaments were probably not

quite mature, as they were very slightly less refractile

or less deep amber internally than mature spores.

Membrane bound spores did not appear to be infective

per os for S. geminata. Examination of meconia showed that

few if any MB spores had extruded their polar filaments

while in the larval gut. A total of 274 mature, nonextrud-

ed MB spores were seen in the 20 meconia, but only one body

was seen which strongly appeared to be an extruded MB spore.

Five additional bodies were seen that resembled extruded

MB spores but almost certainly were not. The meconia con-

tained occasional empty walls of ingested or gut unicellular

fungi, some of which superficially resembeled extruded MB

spores; extruded NMB spores were distinct.


Discussion

The cycle of B. dimorpha infection within an ant

colony may be summarized as follows: NMB spores develop

in the hypodermis, producing clear areas in the heads,

petioles, and gasters of pupae. As the infection

progresses, the cuticle becomes very fragile and eventually

ruptures. The adult ants cannibalize these ruptured pupae

but do not ingest the spores. Instead, the spores, together

with other particulate matter, are diverted to the infra-

buccal cavity and formed into an infrabuccal pellet. This








pellet is expelled and placed on a specialized anteroventral

area, the praesaepium, of fourth-instar larvae. The prae-

saepium, which bears spines specialized for holding solid

food while the larva feeds, is absent from earlier instars,

which are fed only liquid. Because of this method of feeding,

the fourth-instar larva is the only stage which is vulnerable

to infection. Both spore types are ingested, but only the

NMB spore is infective. The MB spores are expelled unextrud-

ed in the meconium upon pupation.

It is evident that B. dimorpha is indeed a dimorphic

microsporidium, since ingestion of NMB spores only resulted

in typical infection in which both spore types were produced

in normal ratios.

The function of the MB spore remains unknown. A most

attractive hypothesis is that it either infects an alternate

host or is primed in the gut of a mechanical vector for

extrusion upon subsequent ingestion by ant larvae. Either

would explain the mode of intercolonial transmission of

the infection (fire ants are territorial and aggressive

towards conspecific ants). Many candidate species exist

for the role of vector; a large and varied arthropod fauna

is associated with fire ants. Collins and Markin (1971)

listed 52 species of insects that have been collected from

fire ant nests; other invertebrates also occur. At least

some of these organisms have symbiotic relationships with fire

ants and are known to travel between fire ant nests (Wojcik,

1975).












CHAPTER FIVE

HOST SPECIFICITY OF BURENELLA DIMORPHA


Until recently, microsporidia were generally assumed

to be highly host specific, and the discovery of an infection

in a new host frequently led to the description of a new

species. Now microsporidia are known to vary greatly in

host specificity. Some species, particularly the parasites

of lepidoptera, are able to infect numerous hosts in different

genera, families, and even orders. Other species appear

to be highly host specific; however, in some cases the diffi-

culty of transmitting infection in the laboratory makes

it impossible to determine host range. The host ranges

of only a few species have been extensively investigated

(Brooks, 1974).

The successful per os transmission of B. dimorpha infec-

tion to healthy colonies of S. invicta, S. richteri, and

the southern fire ant, Solenopsis xyloni McCook, as well

as the natural host, S. geminata, was reported by Jouvenaz

and Hazard (1978). High rates of infection were obtained

in colonies of these species by feeding them spores mixed

with boiled egg yolk. The infected pupae exhibited typical

pathognomonic signs, and Jouvenaz and Hazard concluded that

"the course of the disease is identical in all four species."
(p. 27)








After these initial transmission tests had been published,

I attempted to propagate the parasite S. invicta. This

species is not only the pest we ultimately hope to control,

but I have found it easier to obtain and culture than S.

geminata. Several colonies were fed spores and, as expected,

successfully infected; however, after two or three months,

these colonies were free of the parasite. It appeared then,

that B. dimorpha might not be able to maintain itself in

factitious fire ant hosts, even though these hosts were

readily infected. The studies reported here were conducted

to determine the degree of host specificity of B. dimorpha.

In addition to fire ants, several species of ants of other

genera were tested for susceptibility to infection by B.

dimorpha.

The corn earworm, Heliothis zea (Boddie) (Lepidoptera:

Noctuiidae) is susceptible to infection by Nosema algerae Vavra

and Undeen, a microsporidium which is highly pathogenic

to anophiline mosquitoes. Heliothis zea was being used

for mass production of N. alterae spores at the Insects

Affecting Man and Animals Research Laboratory, USDA, Gaines-

ville, during part of the period in which I conducted my

research. I therefore took advantage of the availability

of this moth and tested it for susceptibility to infection

by B. dimorpha.








Materials and Methods

Three small (ca 500 workers) colonies of S. invicta and

one larger colony (ca 10,000 workers) of S. richteri (the

only colony of this species that was available) were infected

by allowing them to consume diseased pupae of S. geminata.

The colonies of S. invicta were fed 15 selected pupae in

the advanced stages of infection on each of the following

days: Colony #1, days 1 and 3; colony #2, days 1, 2, and

3; colony #3, days 1, 3, and 5. The colony of S. richteri was

fed 50 selected pupae on one occasion.

The colonies were examined 3 weeks after the final

feeding to determine the initial rate of infection, and

at monthly intervals thereafter. Pupae old enough (judged

by eye development) to exhibit pathognomonic signs if infec-

ted were examined individually against a black background

with a stereomicroscope illuminated by an annular fiber

optic. All, or a maximum of 1,000, of these pupae were

examined from each colony.

Ants of other species were gifts from colleagues or

were collected after mating flights and reared as described

in General Materials and Methods. Identifications of species

were made or confirmed by Dr. Daniel P. Wojcik, Agricultural

Research Service, USDA, Gainesville, or by Mr. James Trager,

Department of Entomology and and Nematology, University

of Florida. These ants were also fed selected diseased








pupae of S. geminata (ingestion of the pupae was confirmed

by observation), and spores mixed with their regular diet.

In addition to examining pupae for pathognomonic signs,

aqueous extracts of pupae were examined by phase-contrast

microscopy for spores in case external signs were not evident.

Larvae of H. zea and their rearing media were obtained

from Mr. D. W. Anthony, Agricultural Research Service, USDA,

Gainesville. The protocols for conducting infectivity tests

with B. dimorpha included those used by Anthony et al. (1978)

to infect H.zea with H. algerae for mass production of spores.

Twenty larvae 4-5 days old were held in isolation without

food or water for 24 hr, fed 0.1 ml of a suspension containing

ca 106 spores per ml, and then placed on fresh media. Twenty

larvae 8-10 days old were allowed to consume one diseased

S. geminata pupa each, and an additional 20 larvae 8-10

days old were injected with 2-10 ul of spore suspension.

The adult moths were examined by phase-contrast microscopy

ca one month after they had ingested or been injected with

spores.


Results

The B. dimorpha did not persist in S. invicta or S.

richteri for more than a few generations (Table 1). Three

weeks after the final feeding of B. dimorpha spores, the

S. invicta colonies were infected at the rates of 6% (colony

#1), 44% (colony #2), and 4% (colony #3). The colony of










Table 1.


B. dimorpha infection rates in colonies of S. invicta
and S. richteri.


Number of days after
ingestion of spores


S. richteri


S. invicta


colony #1


#2 #3


44%

8.5%

< 1%a


110

140


aOne pupa in the nest population of 786 was infected.







S. richteri had an infection rate of 5%. One month later

(day 49), however, infection could be detected only in S.

invicta colony #2, and the infection rate in this colony

had declined from 44% to 8.5%. One month later (day 80),

only one infected pupa (out of 786 pupae in the age group

that exhibits pathognomonic signs) was found in the colony.

By day 110, all of the 1,000 pupae that were examined were

free of disease. All four colonies remained free of B.

dimorpha on all subsequent examinations. In contrast, the

colony of S. geminata from which the diseased pupae (those

fed to the test colonies) were taken maintained an infection

rate of ca 50% for 14 months.

None of the other species of ants tested were susceptible

to infection by B. dimorpha (Table 2). The H. zea was

not susceptible to infection either per os or by injection.


Discussion

The B. dimorpha is host specific for S. geminata. Although

S. invicta and S. richteri may be readily infected, the

parasite does not persist in these factitious hosts beyond

a few generations. The 9 species (representing 8 genera)

of ants other than Solenopsis spp. proved refractory to

infection. While this is a very small sample of the living

species of ants, it is highly improbable that a parasite

that cannot maintain itself in factitious species of the

genus of its host would be able to infect species of other

genera.









Table 2. Susceptibility of selected species of ants and a
moth to infection by B. dimorpha.


Susceptibilitya
HYMENOPTERA: FORMICIDAE

Subfamily Myrmicinae
Solenopsis geminata (Fabricius) +
S. xyloni McCook ?
S. invicta Buren
S. richteri Forel
Pheidole morrisi Forel
Monomorium minimum (Buckley)
M. floricola (Jerdon)
Crematogaster clara Mayr
Aphenogaster ashmeadi (Emery)
Ochetomyrmex auropunctatus (Roger)
(= Wasmannia auropunctata)

Subfamily Pseudomyrmecinae
Pseudomyrmex elongata Mayr

Subfamily Formicinae
Paratrechina longicornis (Latreille)
Camponotus floridana (Buckley)


LEPIDOPTERA: NOCTUIDAE

Heliothis zea (Boddie)
per os
injection

a+ = susceptible to infection
= susceptible, but infection does not persist
- = not susceptible to infection








Following infection with spores of B. dimorpha from

S. geminata, signs typical of infection in S. geminata

develop in pupae of S. invicta and S. richteri. However,

pupae that are infected subsequently in these colonies (by

spores produced in siblings) develop atypical signs. The

blister-like clearings in the head and gaster are not apparent,

although faint clearing in the petiole may occur. Abnormal

eye development typical of the disease was noted, however

(Fig 5). Jouvenaz and Hazard were premature in their state-

ment that the course of the disease is identical in all

four species; they had examined only pupae infected with

spores produced in S. geminata.

The reasons) for the failure of B. dimorpha to cycle

indefinitely in S. invicta and S. richteri is unknown. Ob-

viously, they are "not well adapted" to these species, but

this merely restates our observations. Perhaps the cuticle

is less likely to rupture and cannibalization is less likely

in these species due to dehydration or putrification in

the intact state. The cadavers may then simply be discarded.

































Fig 5. Diseased S. richteri pupa compared to healthy and
diseased S. geminata pupae. The pupa in 5A is
healthy. The signs exhibited by the S. richteri
pupa (5B) are as pronounced as they will become.
Note the faint clearing in the petiole and the
slight blurriness and irregularity of outline of
the eye. The diseased S. geminata pupa (5C)
exhibits typical pathognomonic signs at maximal
expression. X30.





36




























4P













CHAPTER SIX

ABUNDANCE OF BURENELLA DIMORPHA IN NATURE


As part of a survey for pathogens of native and imported

fire ants in the United States, Jouvenaz et al. (1977) examined

307 colonies of S. geminata from74 collection sites in Florida

and Georgia. Twelve (3.9%) of these colonies from seven

collection sites in Florida were infected with B. dimorpha.

The infection rate at the type locality of B. dimorpha (which

was not included in the survey) was obviously much higher

than 3.9%. Therefore, I decided to investigate the infec-

tion rate at this specific site.


Materials and Methods

Sixty-nine colonies of S. geminata from the type locality

of B. dimorpha (State Road #26, ca 1-3 km east of Interstate

Highway 75, Alachua County, Florida) were screened for disease

as described in General Materials and Methods. Forty of

these colonies were examined in August, 1975, and the remain-

ing 29 were examined in September, 1977.


Results and Discussion

The infection rate at the type locality of B. dimorpha was

much higher than that of the 307 colonies examined by Jouvenaz

et al. Seventeen (42.5%) of the 40 colonies in the first







group, and 4 (13.8%) of the 29 colonies in the second group

(collected two years later) were infected. These limited

data suggest that B. dimorpha may be quite common in specific

localities, but not homogeneously distributed in nature.

Unfortunately, surveys for disease are very expensive, time-

consuming, and laborious, and the prospects of amassing

data sufficient to draw conclusions about the distribution

and abundance of B. dimorpha in nature appear remote. The

S. geminata has now been almost completely displaced from

the type locality of B. dimorpha by S. invicta.













CHAPTER SEVEN

PATHOLOGY OF BURENELLA DIMORPHA INFECTION


Very few protozoan diseases of insects may be diagnosed

by simple observation of pathognomonic signs. Pebrine (infec-

tion of the silkworm, Bombyx mori L., by the microsporidium

Nosema bombycis Naegeli) provides a classic example of patho-

gnomonic signs in insects, being characterized by the appear-

ance of dark, pepper-like spots on the integument. Infection

of the tropical fire ant, Solenopsis geminata (Fabricius),

by the neogregarine Mattesia geminata Jouvenaz and Anthony

(1979), may be readily diagnosed by eye teratology and a

pattern of abnormal melanization in pupae beginning with

a "sooty" black discoloration of the legs. In the same

host, infection by Burenella dimorpha Jouvenaz and Hazard

is manifested by signs of a type that appears to be unique

in insects. In general, however, specific manifestations

of protozoan infections, if present at all, are related

to particular tissues and may be detected only by histolog-

ical examination (Brooks, 1974).

Insects infected by protozoa may exhibit nonspecific

signs such as loss of appetite, diarrhea, sluggishness,

irregular growth, stunted or malformed adults, reduced fecun-

dity, and premature death of immatures or adults. Often,







however, there are no indications of disease other than

premature death (Brooks, 1974). Individual fire ant workers

infected with Thelohania solenopsae (Knell et al., 1977)

or any of four undescribed species of microsporidia, for

example, cannot be differentiated from healthy specimens

by either appearance or behavior. Diagnosis of these infec-

tions must be made by microscopic examination or, in the

case of T. solenopsae, by observing cysts in dissected gas-

ters. Similarly, bees, Apis spp., infected by Nosema apis

Zander are completely devoid of outward signs of disease,

and even histological diagnosis is difficult (Bailey, 1981).

Pupae of S. geminata infected by B. dimorpha exhibit

pathognomonic signs that are obvious, and, to the best of

my knowledge, unique. Jouvenaz and Hazard (1978) attributed

these manifestations to destruction of the hypodermis and

described them as follows:

A clear area in the occipital region of the head,
which appears about the time the developing eyes
become prominent, is usually the first noticeable
change. Later, similar clear areas appear in
the petiole and gaster, and the eyes become irreg-
ular in outline and appear sunken. Pupae having
such changes do not mature or even melanize. In-
stead, the clear areas increase in size, and the
cuticle eventually ruptures.(p. 27)

Jouvenaz and Hazard also noted that NMB spores develop in the

hypodermis, and MB spores develop in the fat body.

This brief description of the appearance of diseased

worker pupae and notation of tissue specificity of the spore

types is all that has been published on the pathology of







B. dimorpha infection. The signs of infection in sexual

pupae have not been described. The development and histo-

pathological basis of the unique manifestations of this

disease are described here.


Materials and Methods

The progressive development of pathognomonic signs

was recorded by periodically photographing healthy and dis-

eased pupae. Diseased pupae exhibiting the minimal degree

of eye development necessary for diagnosis and healthy pupae

of corresponding age and size were paired and held in minia-

ture nest cells. The specimens were positioned on a plate

of nonglare glass above a black parabolic background, illum-

inated by an annular fiber optic illuminator, and photographed

through a stereomicroscope fitted with a polarizing filter.

Photographs were made ca every 48 hours until the healthy

specimen had closed and the diseased specimens had reached

maximum expression of pathognomonic signs.

Specimens for both light and transmission electron

microscopy were fixed and embedded in epoxy resin. Tissue

specimens (heads, gasters, whole larvae, etc.) were prefixed

in buffered 1% osmium tetroxide (0.1 M sodium cacodylate

buffer, pH 7.5) for 30-60 minutes at room temperature, rinsed

in the same buffer, and partially hardened in buffered 2.5%

gluteraldehyde-l% acrolein (same buffer). Specimens were

washed in buffer and usually stored in Histocon (polyvinyl-

pyrrolidone, Tris-HCl, 2% chlorhexidine, distilled water;








Polysciences, Inc.) in the refrigerator overnight or for

several days prior to postfixation. Specimens were washed

in buffer, postfixed in buffered 1% osmium tetroxide for

2 hours at room temperature, washed in deionized water,

and en bloc stained in 0.5% aqueous uranyl acetate overnight.

Specimens were dehydrated with acidified 2,2-dimethoxypropane

(Lin et al., 1977) and infiltrated and embedded in a Spurr-

Quetol 651 resin (Ringo et al., 1979).

Blocks were sectioned with a LKB Huxley ultramicrotome.

For light microscopy, 2-4 ur sections were cut on dry glass

knives, spread in a drop of 10% acetone, mounted in immersion

oil, and studied by phase-contrast microscopy. Thinner

sections, 0.5-1.0 um, were stained with 1% aqueous toluidine

blue in 1% aqueous borax, rinsed in deionized water, mounted

in immersion oil, and examined by either bright-field or

phase-contrast microscopy. For transmission electron micros-

copy, gold sections were poststained with 2% aqueous uranyl

acetate followed by lead citrate (Reynolds, 1963). Grids

were examined and photographed at an accelerating voltage

of 75 kV in a Hitachi H-600 electron microscope.


Results

Portraits of healthy and diseased male, female, and

worker pupae are presented in Figs 6-8. The clearing in

the dorsal thorax of the sexual pupae does not occur in

worker pupae, and occasionally is reduced or absent in













Fig 6. Diseased (left) and healthy male pupae of S. geminata.
Note the clearing of the occiput and dorsal thorax.
The light color of the eye is due to destruction of
the lens and a fluid space over the eye. X 10.


Fig 7. Diseased (left) and healthy female pupa of S. geminata.
The apparent faint clearing in the head of the healthy
specimen is a photographic artifact. X10.






















Fig 8. Healthy (left) and diseased worker pupae of S. geminata.
Note eye pathology and clearing in occiput and petiole
of the diseased pupa. X 40.




44







I


I







dlik







sexual pupae. The eyes of the sexual pupae appear to be

less affected than the eye of the worker pupa; this may

be an artifact due to the larger (ca 16-20X in area) eyes

of sexual pupae. The diseased worker pupa is in an advanced

stage of infection, and exhibits typical pathognomonic signs.

Note the irregular outline of the eye, the derangement of

the facets, and its fainter color.

The development of pathognomonic signs is recorded

in Fig 9. In the first pair of photographs, the healthy

and diseased pupae (1H and lD, respectively) are almost

indistinguishable. The diseased pupa exhibits a faint clearing

of the petiole, and in life--but not in the photograph--

the earliest faint signs of eye teratology could be detected.

In the photograph taken 48 hours later (3D), the clearing

in the petiole is not evident due to a slight change in

lighting. Subsequent photographs show the development of

clearing in the head and petiole. The eye of the diseased

pupa is rather blurry in these photographs. This is not

due to focus, but to a fluid space over the eye.

The healthy pupa closed on day 18. The last photo-

graph of the diseased pupa (3D) shows an indentation in

the gaster, indicating dehydration. In a colony, rupture

would probably have occurred by this time as a result of

manipulation (being moved, groomed, etc.) by the workers.

Frontal sections of whole heads of younger and older

diseased pupae are presented, respectively, in Figs 10 and 12.



























Fig 9. The development of pathognomonic signs in pupae of
S. geminata infected by B. dimorpha. The numerals
indicate the number of days after the series began
that the photographs were taken. The letters H and
D indicate, respectively, the healthy and diseased
specimens. X 30.




47









I I I i 3

*





I


U SI






























Fig 9 (cont.) The development of pathognomonic signs in pupae
of S. geminata infected by B. dimorpha. The
healthy pupa closed on day 18 (18H). X 30.





















' I


r./%k


'


')1 I4
r-^
^'















Fig 10. Frontal section through the head of a young (ca 10
days postpupation) pupa infected by B. dimorpha.
Note the fluid space between the head and pupal
sheath. B = brain; IBC = infrabuccal cavity; arrows
point to the eyes. X 90.








Fig 11. Eye, optic nerve, and optic stalk of a young (ca 10
days postpupation) pupa infected by B. dimorpha.
OS = optic stalk; ON = optic nerve; arrow points to
eye. X 600.







Fig 12. Frontal section through the head of an older (ca 16
days postpupation) pupa infected by B. dimorpha.
Note the shrunken, fiberous appearance of the brain
and sunken, amorphous eyes. B = brain; IBC =
infrabuccal cavity; arrows point to eyes. X 90.





51













.. -








Note the fluid spaces in both specimens, and the shrunken,

fiberous appearance of the brain and the amorphous, sunken

eyes of the older specimen. The optic stalk, optic nerve, and

flattened, unstructured mass of developing eye tissue of a

younger pupa (corresponding in age to the specimen in Fig

10) are shown in Fig 11.

The destruction or inhibition of development of the

cuticle in infected pupae is shown in Figs 13-16. The healthy,

developing cuticle with its trophic microvilli and two

pores (Fig 13) is completely absent in the diseased specimen

(Fig 14). The microvilli of the latter extend randomly

into the fluid space. Details of the microvilli are pre-

sented at higher magnification in Figs 15 and 16. The tis-

sues shown in these electronmicrographs are from the head

where pathognomonic clearing develops. In tissues from

the gaster, where little or no clearing occurs, some cuticle

is present (Fig 26).

The structures of a healthy, developing pupa eye and

a diseased eye of a similar age are compared at increasing

magnifications in Figs 17-22. The lenses, composed of cu-

ticle, are absent in the diseased eye, and the rhabdoms

and associated cells are twisted, tangled masses. Also,

the basement membrane is absent, and only remnants of the

optic nerve may be seen. Pigment granules are present in

the pigment cells of the diseased eye.


















Fig 13. Cuticle of a healthy pupa. Note the trophic
microvilli and two pores. PS = pupal sheath;
MV = microvilli; Cu = cuticle. X 1,500.

Fig 14. Body surface of a pupa infected by B. dimorpha.
There is no cuticle, and the microvilli that
normally penetrate the cuticle extend into the
fluid space. PS = pupal sheath; MV = microvilli.
X 1,500.















Fig 15. Detail of the cuticle in a healthy pupa. Note
the microvilli penetrating the cuticle. MV =
microvilli. X 12,500.

Fig 16. Detail of microvilli extending into the fluid space
of a pupa infected by B. dimorpha. X 12,500.
























I'l.
,b ,
4
Ji. i


MV
/

























Fig 17. Sagittal section through the developing eye of
a healthy pupa. L = lens; Rh = rhabdom and
developing associated cells; ON = optic nerve.
X 450.

Fig 18. Sagittal section through the eye of a pupa
infected by B. dimorpha. There is no lens,
and the rhabdoms and the associated cells are
twisted and tangled in an amorphous mass.
Rh = rhabdom and associated cells; ON = optic
nerve. X 450.









Fig 19. Sagittal section through the eye of a healthy
pupa. X 1,500.

Fig 20. Sagittal section through the eye of a diseased
pupa. Rh = rhabdom and associated cells.
X 1,500.





























p.


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4 W


Ic *




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iu


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9 5


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: a ,i




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*- v "' '""




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Fig 21. Sagittal section through the developing eye of a
healthy pupa showing detail of optic nerve and
base of the developing ommatidium. Rh = rhabdom
and associated cells; BM = basement membrane;
PG = pigment granules; ON = optic nerve; FB =
fat body. X 3,000.

Fig 22. Sagittal section through the eye of a pupa infected
by B. dimorpha. Only remnants of the optic nerve
may be seen. The basement membrane area is destroy-
ed. ON = optic nerve; FB = fat body; PG = pigment
granules; Rh = rhabdom and associated cells.
X 3,000.


Fig 23. Fat body of a healthy pupa.


X 7,500.


Fig 24. Fat body of a pupa infected by B. dimorpha. Note
the depleted appearance of fat body and the presence
of several MB spores. X 7,500.











/Tt-iM,
yw
d ,
A 't'c
a.. *


w r


s


. *
*
V .
S.",ed t.Po

S %


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4G ,,,'
S **4

a ** *
, *


*
%0


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# t.' .


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i 9


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4 41
'r


rj 7


a


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a 4
"*.9^.


4.


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--1^


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Fig 25. Tissue specificities of MB and NMB spores of
B. dimorpha. The MB spores in the darkly stained
fat body are seen in groups of less than eight
because of the thinness of this section (o.5 mu).
X 150.


Fig 26.


Tissue specificities of MB and NMB spores of
B. dimorpha. Nonmembrane bound spores are con-
fined to the hypodermis; MB spores are confined
to the fat body. The MB spores are seen in
octets in this section because of its thickness
(6-8 mu). Some cuticle is seen in this section
from the gaster. X 240.


Fig 27. Tissue specificities of MB and NMB spores of
B. dimorpha seen at higher magnification. X 375.




60








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k, ,~~ S.


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JI^ r


-^syAI


Fi-I s5a
I`. ^~
^Ti~- r *I








The fat body is diminished in infected pupae, as is

evident in Figs 23 and 24. Several MB spores may be seen

in the latter electronmicrograph.

The tissue specificities of the two spore types are

shown at increasing magnifications in Figs 25-27. The MB

spores are confined to the fat body; NMB spores are confined

to hypodermal and connective tissues, including that in

the deeper parts of the body such as the tissue encasing

the brain. The tissue section in Fig 25 is only 0.5 mu

thick; therefore, MB spores are seen in groups of 2-4 rather

than octets. The tissue sections in Figs 26 and 27 are

ca 6-8 mu thick, and thus show the octosporous pansporoblasts

better.


Discussion

The pathognomonic signs of B. dimorpha infection in

S. geminata reflect damage or destruction of the developing

adult cuticle. In the pupal stage of development, the integu-

ment of the adult ant forms under the integument of the

fourth-instar larva. During this period, the larval integument

is transformed into a protective sheath that adheres closely

to the developing adult integument and is moulted when the

development of the latter is complete (eclosion). Over

most of the body, the adult cuticle of pupae infected by

B. dimorpha appears to be damaged or inhibited only to the

extent that tanning is inhibited (Fig 26; also, the adult








morphology is essentially developed). In parts of the head

and in the petiole, however, the cuticle is completely destroy-

ed (Figs 13-16). In these areas, tissue fluids seep into

the space between the denuded hypodermis and the pupal sheath.

As the infection progresses, the fat body is diminished and

the brain shrinks (due to loss of lipids?). The mass of

these tissues, covered by hypodermis, decreases in size and

recedes from the pupal sheath, which is extended by fluid.

Jouvenaz and Hazard (1978) attributed the development of

clear areas in the head and petiole to destruction of the

hypodermis. More correctly, infection of the hypodermis

results in destruction or inhibition of formation of the

cuticle.

The malformation of the eyes is also due to destruction

of the cuticle. The lens are part of the cuticle, and the

remaining components of the ommatidium (rhabdom and pigment,

retinular, and semper cells) extend between the lens and

a basement membrane. The lens are nonexistent in eyes in-

fected by B. dimorpha, and the rhabdoms and associated cells

become twisted masses. Perhaps the surrounding connective

tissue matrix is also destroyed. The eyes of pupae infected

by B. dimorpha develop after cuticle destruction has begun,

and are abnormal from the time they first become visible.

Thus, the derangement of the eyes is teratologic in nature.












CHAPTER EIGHT

TEMPERATURE-DEPENDENT SPORE DIMORPHISM
IN BURENELLA DIMORPHA


The effect of temperature on the relative abundance

of MB and NMB spores was first studied by Maddox (1966)

in the dimorphic microsporidium Vairimorpha necatrix (Kramer).

This parasite was originally thought to be a mixed infection

involving two species of microsporidia, Nosema necatrix Kramer

and Thelohania diazoma Kramer, that produced NMB and MB

spores, respectively, in lepidopteran hosts (Kramer, 1965).

Maddox demonstrated that only NMB spores are produced if

infected armyworm larvae, Pseudaletia unipuncta (Haworth),

are reared at temperatures of 320 C or above. At lower

temperatures (160 C) 40% or more of the spores are the MB

type. Maddox infected armyworm larvae with an apparently

pure suspension of NMB spores obtained from larvae reared

at elevated temperature, and found that both types of spores

were produced in larvae held at temperatures below 320 C.

This led Maddox to suggest (as the more radical of two pos-

sibilities) that N. nectarix and T. diazoma were not

two species at all, but rather one species with two distinct

developmental cycles and resulting spore forms, the ratio

of which is influenced by temperature" (p. 112).







The dimorphic nature of this microsporidium was experi-

mentally demonstrated by Fowler and Reeves (1974) through

the use of mechanical as well as thermal and temporal methods

to separate the two spore types for transmission studies.

They retained the name N. necatrix, suppressing the name

T. diazoma. Pilley (1976), recognizing that N. necatrix does

not conform to the definitive characteristics of the genus

Nosema, reassigned it to a new genus, Vairimorpha. Jouvenaz

and Hazard (1978) observed that V. necatrix and B. dimorpha

are related and suggested that the genus Vairimorpha properly

belongs in the family Burenellidae.

The temperature dependence of spore dimorphism in V.

necatrix was also demonstrated by Maddox and Sprenkel (1975),

who serially passed V. necatrix spores through eight genera-

tions of P. unipuncta held at 21.10 and 320 C. Maddox and

Sprenkel also demonstrated temperature-dependence of spore

dimorphism of "Nosema plodiae -Thelohania nana" Kellen and

Lindegren, and in an undescribed microsporidium. Maddox

(1966) described the V. necatrix infections of P. unipuncta

reared at elevated temperature as "light."

The primary purpose of the experiments reported here

was to determine whether spore dimorphism in B. dimorpha

is temperature dependent. The effects of temperature on

spore yield ("light infections", Maddox) and size were also

measured. In addition, since the MB spores of B. dimorpha

develop in the fat body while NMB spores have been found








only in the hypodermis (Jouvenaz and Hazard, 1978), I attempted

to answer the following questions: 1) If MB spores do

not develop at all temperatures at which NMB spores develop,

is it because sporulation is inhibited in the fat body?

Or, 2) do NMB spores develop in lieu of MB spores in the

fat body? And 3) are the effects of heat and cold the same

in this respect? Resolution of these questions was attempted

by histological examination of diseased pupae reared at

high or low temperatures.

Materials and Methods


Ratios of Spore Types

Fourth-instar larvae were removed from a heavily infected

laboratory colony and held in miniature nest cells at the

desired temperature (20, 22.5, 28, 32, and 35 C) overnight.

The temperatures in the incubators were monitored by hygro-

thermographs and mercury thermometers. Humidity was main-

tained close to 100% by the design of the cells (see General

Materials and Methods). Larvae that pupated during the

first 16 hr were discarded, as were those that had not pupated

during the next 24 hr. A contingent of nurses (young workers

primarily care for the immatures) captured from the brood

piles was introduced to care for the immatures. These were

removed shortly before the end of pupal life (at that time

they were no longer needed to groom the pupae) to facilitate

the examination and harvest of specimens in advanced disease.








The uninfected pupae served as controls to monitor the rate

of maturation at the different temperatures. Infected pupae

were harvested only after eclosion of the controls, when

the disease had progressed to near the point of cuticle

rupture and spore production was essentially complete. The

completeness of spore production was judged by the percentage

of immature spores seen in phase-contrast microscopic examina-

tion. Spore ratios were determined only for those pupae

in which > 98% of the NMB spores appeared to be mature.

The initial rearing study indicated that the develop-

ment of MB and NMB spores of B. dimorpha is inhibited by

both high and low temperatures. Therefore, an additional

study was conducted to determine the maximum age of the

host at which MB spore inhibition can be effected by raising

or lowering the temperature. Immature workers were divided

into five age groups and half of each group were incubated

at 20 C and the remaining half at 32 C. The age groups

were 1) fourth-instar larvae; 2) larvaform pupae; 3) early

pupaform pupae (eye development not yet evident); 4) pupae

whose eyes had developed sufficiently to permit diagnosis

of infection; 5) pupae slightly older, having first evidences

of clearing in the occiput. These groups were reared and

examined as described above.

Spore ratios were determined by homogenizing individual

pupae in ca 1 ml distilled water in a 3 ml glass tissue

grinder, and examining this extract by phase-contrast








microscopy at a magnification of 600X. The concentration

of spores in these extracts (0-6 spores/field) was low enough

to facilitate counting and individual study of spores. The

percentage of mature and immature MB spores was based on

the first 200 spores observed.


Spore Measurements

Spore measurements were made using an A. E. I. Cook

Image-Splitting micrometer calibrated for the microscope

used. The spores were immobilized for measurement by trap-

ping them between a layer of agar and the coverslip. This

was accomplished by pipetting a small pool of warm, liquid

Noble Agar (Difco Laboratories, Detroit, Mich.) (1.5%) on

the slide with a dropping pipette and bulb, and withdrawing

a drop of the agar just before gelling occurred. This pro-

duced an agar bed which was rather flattened on top. A

very small drop of spore suspension was then placed on a

coverslip, inverted and placed on the agar.


Spore Yield

Spore yield was determined by weighing groups of 25

pupae in advanced disease on an electric balance, homogeniz-

ing these in distilled water, adjusting the volume appropri-

ately, and counting spores with a hemacytometer (phase-

contrast microscopy, 300X).







Histology

Tissue specimens from gasters of pupae held at 20 or 32 C

until infection was very advanced were fixed, stained with

heavy metals embedded in Spurr-Quetol resin, sectioned, and

examined by phase-contrast microscopy. The details of these

procedures are given in the section on host pathology.


Results

The development of MB spores was inhibited in pupae

held at high (32 or 35 C) or low (20 C) temperature. At

28 C, the temperature at which S. geminata colonies seem

to live best in my insectary, an average of 35.9% 2.6

of the spores in 25 pupae were of the MB type (Table 3).

Jouvenaz and Hazard (1978) stated that MB spores typically

constitute ca 25-40% of the spores in diseased pupae collected

in the field.

Only 10 of the pupae held at 35 C survived to an advanc-

ed stage of infection. These pupae were devoid of MB spores,

and a small number (ca 2%) of the NMB spores from these

pupae were morphologically aberrant (pairs of spores fused

laterally or in tandem, Y-shaped or triangular spores, giant

spores, etc.). Thirty-five degrees appears to be very near

the upper thermal limit of survival for host and parasite.

Twenty-two of the 25 pupae held at 32 C were negative

for MB spores. Only four immature MB spores were among

the 600 spores from the remaining three pupae (200 spores

from each specimen were examined).









Table 3. Relative abundance of B. dimorpha MB spores in
pupae of S. geminata reared from larvae at various
temperatures.


Temperature
(degrees C)


Number of
pupae


No. (%) positive
for MB spores


% MB spores in
positive specimens


5 (100)

25 (100)

3 (12)


3 (1-5)a

35.9 2.6 (31-39)

1 (0-1)


aMean and (range)
bMean and standard deviation (range)




Table 4. B. dimorpha spore yield of pupae of S. geminata
reared from larvae at 20, 28, or 32 C.


Temperature NMB Spores MB Spores
(degrees C) Spores/pupa Spores/mg Spores/pupa Spores/mg

20 6.7 x 105 3.9 x 105 0 0

28a 6.3 x 105 4.4 x 105 2.7 x 105 2.0 x 105

32 4.8 x 105 2.7 x 104 0 0

aTotal spore production at 28 C = 8.9 x 105/pupa or 3.9 x 105/mg.


22.5







The development of MB spores was completely inhibited

in 25 pupae held at 20 C. A few immature MB spores were

found in pupae held at 22.5 C, indicating that the lower

thermal threshold of MB sporulation is between 20 and 22.5 C.

Pupae reared from larvae at 20 and 28 C produced similar

numbers of NMB spores (Table 4). Pupae reared at 32 C produced

ca 75% as many spores per pupa and ca 60% as many spores per mg

bodyweight as did those reared at 28 C.

The inhibitory effect of temperature on MB sporulation

in pupae placed at high or low temperature at comparable

ages was essentially the same (Table 5). Specimens placed

at 20 or 32 C as larvae or larvaform pupae (a total of 40

specimens) were negative for MB spores. Of the 10 specimens

placed at 20 C as early pupaform (lacking visible eye develop-

ment) pupae, five had no MB spores, two had one MB spore

each, two had two MB spores each, and the remaining pupa

had seven MB spores among the 200 spores that were examined.

Of the 10 specimens placed at 32 C as early pupaform pupae,

nine had no MB spores and the remaining pupa had only one

MB spore among the 200 spores. Thus, mean spore production

by this age group was less than one percent at either tempera-

ture.

The production of MB spores in pupae that had developed

eyes to the extent that diagnosis of disease was possible

at the time they were placed at 20 or 32 C was lower than

in those reared at 28 C for their entire life. Those held










Table 5. Relative abundance of B. dimorpha MB spores in pupae
of S. geminata placed at 20 or 32 C at different ages.

% MB Spores
Age Group 20 C 32 C


larvae

larvaform pupae

lacking eye development

minimum eye development

slight clearing, occiput


<1

10.37.6

18.714.0


<1

16.86.8

24.43.7


aMean and standard deviation







at 20 C produced an average of 10.3% 7.6 (range 2-25%)

MB spores; those held at 32 C produced an average of 16.8%

6.8 (range 6-23%). Pupae exhibiting minimal clearing

of the occiput produced averages of 18.7% 14.0 (range

1.5-40%) and 24.4% 3.7 (range 18.5-33.5%) at 20 and 32 C

respectively. Approximately 90% of the spores in these

latter two age groups appeared to be mature.

The measurements of NMB spores from pupae reared from

larvae at 20, 28 or 32 C were, respectively, 3.0 0.2 X

6.8 0.3 mu, 3.0 0.1 X 6.8 0.3, and 3.0 0.0 X 7.0

0.4 mu. Obviously, there were no differences in NMB spore

sizes (MB spores were not produced at 20 or 32 C).

Neither MB or NMB spores were seen in tissue sections

of fat body of pupae reared at 20 or 32 C. Very few MB

spores were seen in sections of fat body of pupae reared

at 22.5 C.


Discussion

The development of NMB spores precedes the development

of MB spores, and NMB spores predominate in number. In

field colonies, MB spores typically constitute ca 25-40%

(occasionally fewer) of the spores from pupae in advanced

stages of infection (Jouvenaz and Hazard, 1978). The present

study has demonstrated that MB sporulation also occurs in

a more restricted range of temperature than NMB sporulation.

The lower thermal limit of MB spore development appears

to be between 20 and 22.5 C. At 28 C production ofMB spores








was consistently near maximum. The upper thermal limit

of MB spore development is below 32 C. Thus, optimal MB

spore production occurred near the upper thermal limit of

spore production. This is reminiscent of the activity curve

of heat-liabile enzymes and the growth curves of poikilo-

therms over a range of temperatures. Very possibly the

upper thermal limit of MB spore development is the tempera-

ture of inactivation of an enzyme(s).

The B. dimorpha MB spore production is also inhibited

by low temperature (20-22.5 C). In contrast, V. necatrix MB

spores are produced at temperatures at least as low as 16 C.

This difference in cold sensitivity may reflect different

requirements for enzyme stability due to host behavior.

Loss of quaternary enzyme structure at low temperature is

relatively common in homeotherms and their microbial symbiotes.

The homologous enzymes of poikilotherms do not, at least

in some cases, lose their quaternary structure at low temper-

ature (Hochachka and Somero, 1973). The hosts of both V.

necatrix and B. dimorpha are, of course, poikilotherms;

however, the host of the latter microsporidium is a subtrop-

ical, social insect that actively tends its brood, moving

them in a subterranian environment to regulate temperature

and humidity. The tumulus of the nest is a solar heating

device, and there is evidence that metabolic heat production

is significant (Seeley and Heinrich, 1981). The various

lepidopteran hosts of V. necatrix, however, are exposed







to ambient temperatures as are most poikilotherms. Thus,

the stability of quaternary enzyme structure may be more

critical in this microsporidium.

We may only speculate as to the physiological functions)

that may be inhibited by extremes of temperature. However,

the critical event in MB sporulation may well be meiosis.

If the sister nuclei of the diplocaryon found in NMB spores

and the vegetative stages of B. dimorpha are each diploid,

meiosis would produce eight uninucleate haploidd) spores

(MB spores occur in octets bound by a membrane). Evidence

that meiosis does indeed occur in microsporidia has been

published by Loubes et al. (1976) and Hazard et al. (1979).

The hypothesis that meiosis may be the critical event

in MB sporulation inhibition by extremes of temperature

is at least circumstantially supported by the essentially

complete absence of MB spores in specimens placed at 20

or 320 C at an age before eye development becomes visible,

and the partial and highly variable degree of inhibition

in specimens exhibiting early eye development. The appear-

ance of MB sporoblasts approximately coincides with the

earliest visible eye development. At this stage, second

cycle meronts enlarge to the full size of mature pansporo-

blasts, and the diplocaryotic nuclei become very diffuse.

Successive nuclear divisions then produce plasmodia containing

two, four, and finally eight nuclei. Endogenous cytoplasmic

budding around these nuclei produces an octet of uninucleate




75



spores bound by a membrane. Thus, meiosis occurs at the

age limit for thermal inhibition of MB spore development.

The high variability of inhibition among pupae in this age

group simply reflects the slight variability in age of indi-

viduals and the degree to which MB sporulation had advanced

at the time of temperature change. Once sporogony is under

way (meiosis has occurred?), it apparently cannot be stopped

by temperatures at least as low as 20 C or as high as 32 C.













CHAPTER NINE

AMENDMENTS TO THE DESCRIPTION OF BURENELLA DIMORPHA


The vegetative stages of B. dimorpha were described

by Jouvenaz and Hazard (1978) from light microscope studies

as follows:

Burenella dimorpha appears to have two sequences
of merogony. The first involves uninucleate
cells with deeply staining cytoplasm (Giemsa)
and compact nuclei that become binucleate and
divide. The second sequence involves binucleate
cells with moderately staining cytoplasm and
less dense nuclei that become tetranucleate and
divide to produce two binucleate cells.(p. 27)

The accuracy of this description is challenged by the follow-

ing considerations.

Studies on the transmission of B. dimorpha have demon-

strated that only the NMB spores are infective for fire

ant larvae (see Transmission and Infectivity of Spores).

The NMB spores are diplocaryotic (Fig 32); therefore, the

infecting sporoplasm is (presumably) also diplocaryotic.

The formation of uninucleate meronts would require either

fusion of the sister nuclei or separation of the nuclei

followed by cell division. Studies of Nosema spp. (Sprague

et al., 1968; Vavra and Undeen, 1970; Cali, 1971) and an

Amblyospora sp. (Andreadis and Hall, 1979) indicate that in

these species, the diplocaryotic condition persists throughout

the life cycle.








Recently I collected an undescribed microsporidium

(a parasite of S. invicta in Brazil) that appears to be

closely related to B. dimorpha. This dimorphic species

also has two sequences of merogony; however, its meronts

are diplocaryotic. These observations and reports prompted

me to reexamine the life cycle of B. dimorpha, with particular

attention to the nuclear condition of early meronts. Addi-

tional studies of ultrastructure, including scanning electron

microscopy, were also conducted.

The appearance of sexual castes in colonies of S. geminata

is seasonal (unpublished), and Jouvenaz and Hazard were

unable to examine diseased sexual pupae. Spores from sexual

pupae have now been examined, and found to differ in one

respect from spores from worker pupae.


Materials and Methods

Light Microscopy

Specimens were smeared on acid-alcohol cleaned, galss

microscope slides, air-dried, and fixed in acetone-free

methanol for 5 minutes. Slides were stained with 10% Giemsa

stain in phosphate buffer, pH 7.41 (Fisher Gram Pac Buffer)

for 12 minutes, rinsed in tap water or acidified deionized

water deionizedd water adjusted to pH 6.8 with acetic acid),

blotted with filter paper, and examined under oil.








Scanning Electron Microscopy

Spores from a suspension in deionized water were attached

to carbon-stabilized, formvar coated grids with polylysine

(Mazia et al., 1975), fixed overnight in buffered 1% osmium

tetroxide, and dehydrated in 2,2-dimethoxypropane. Specimens

were critical point dried, sputter coated with palladium-

gold, and examined in a Hitachi H-6010A high resolution

scanning electron microscope at an accelerating voltage

of 50 kV.


Results

Jouvenaz and Hazard (1978) were correct in their state-

ment that B. dimorpha has two sequences of merogony; however,

I am now convinced that these cells are diplocaryotic and

bidiplocaryotic, rather than uninucleate and binucleate.

The cells of the first merogonic sequence are very small,

and their compact nuclei are very closely appressed; the

cells of the second merogonic sequence are larger, and their

nuclei are rather diffuse. The diplocaryotic condition

of these nuclei is not readily apparent, and the published

diagrammatic life cycle adequately reflects their appearance

in stained smears.

Jouvenaz and Hazard also stated that the surface of

both spores is smooth. This is correct only with respect

to the NMB spore (Figs 28 and 30). The surface of the MB

spore has a reticular pattern of ridges that are evident















Fig 28. Transmission electronmicrograph of a mature NMB
spore of B. dimorpha. Note smooth spore surface.
N=nucleus; Pf=polar filament. X 17,100. Inset:
NMB spores as they appear in phase-contrast micros-
copy. X 2,000.


Fig 29. Transmission electronmicrograph of a mature MB
spore of B. dimorpha. Note the surface sculpture
(arrow) and the polar cap (upper end of spore).
N=nucleus; Pf=polar filament. X 18,000. Inset:
MB spore as it appears in phase-contrast micros-
copy. X 2,000.






Fig 30. Scanning electronmicrograph of a mature NMB spore
of B. dimorpha. Note the smooth surface and lack
of a morphologically differentiated polar cap area.
X 16,500.


Fig 31. Scanning electronmicrograph of a mature MB spore
of B. dimorpha. Note the surface sculpture and
morphologically differentiated polar cap area
(bottom of spore). X 16,500.





00 i


A








in both the transmission (Fig 29) and scanning (Fig 31)

electronmicrographs. The ridges may also be seen in the

immature spores in Fig 33.

The pansporoblast membrane of B. dimorpha in worker

pupae was described by Jouvenaz and Hazard (1978) as sub-

persistent and delicate, and ruptured during dissection

of the host. Mature spores are never, and immature spores

are rarely, seen in octets in smears. This is also true of

B. dimorpha from male pupae; however, the pansporoblast mem-

brane is quite persistent in specimens from female sexual

pupae. In the latter, mature spores are commonly seen in

octets in smears or aqueous extracts.


Discussion

The diplocaryotic condition of meronts of B. dimorpha is

most difficult to observe. I have been unable to obtain

convincing photomicrographs of diplocaryia in these cells,

despite the use of high contrast technical pan film and

manipulation of staining protocols. After studying a very

large number of these cells with a microscope of excellent

quality, focusing up and down to distinguish depth of stain-

ing and faint curvatures, I have become convinced that these

cells are indeed diplocaryotic. Interestingly, I have con-

sistently obtained much superior definition in stains of

an undescribed, closely related microsporidium from S. invicta.



















Fig 32. Transmission electronmicrograph of an immature NMB
spore. The spore wall has not yet developed.
N=nuclei; Pf=polar filament. X 12,000.


Fig 33. Transmission electronmicrograph of two immature
MB spores within a delicate pansporoblast membrane.
The surface sculpture is already prominent, although
the endospore is not yet fully developed. PM=pan-
sporoblast membrane. X 9,000.















PM
I


E~~d
'~













CHAPTER TEN

SUMMARY AND CONCLUSIONS


Burenella dimorpha was described by Jouvenaz and Hazard

(1978) as the type species of a new genus which represents

a new family, Burenellidae. This family includes those

species of microsporidia having two sporogonic sequences,

one producing nonpansporoblast membrane-bounded (NMB) spores

and the other producing octets of spores bounded by a pansporo-

blast membrane (MB). Jouvenaz and Hazard described this

microsporidium as a dimorphic species on the basis of light

microscope studies of the life cycle. They were unable

to separate the two spore types, and therefore could not

confirm by feeding tests that B. dimorpha is a single species.

Neither, of course, could they determine which or if both

spore types are infective. Also, the degree of host-speci-

ficity of the parasite was unknown. Three species of Solenopsis

other than S. geminata were shown to be susceptible to infection,

but later observations cast doubt on the ability of the

parasite to persist in populations of these species.

Fire ant pupae infected by B. dimorpha develop pathogno-

monic signs--malformation of the eyes and blister-like clear

areas in the occiput and petiole. These signs increase

in severity as the infection becomes more advanced, and








eventually the pupa ruptures and is cannibalized by workers.

The histopathological basis of these signs was not understood,

nor was the exact mode of transmission of the parasite.

An effort was made to extend our limited knowledge

of B. dimorpha in the areas outlined above. In addition,

the effect of temperature on spore dimorphism was investi-

gated, and certain aspects of the life cycle and morphology

of the parasite were reexamined. The conclusions reached

in this study are summarized below.

1. It was experimentally confirmed that B. dimorpha is

a dimorphic species, producing two morphologically distinct

types of spores, and not a dual infection of two species

of microsporidia.

2. Only NMB spores are infective; unextruded MB spores

are expelled in the meconium upon pupation.

3. The intracolonial cycle of transmission of B. dimorpha

infection is from ruptured, diseased pupae to fourth-instar

larvae via the adult workers, who act as mechanical vectors.

4. Adult workers do not ingest spores into the crop,

but divert them to the infrabuccal cavity where they are

formed into infrabuccal pellets with particulate food material.

5. Only fourth-instar larvae are vulnerable to infection.

6. Burenella dimorpha is host-specific for S. geminata.

Infections in S. invicta and S. richteri do not persist

in the colony.








7. Burenella dimorpha may be locally abundant, even

though it is not common in the S. geminata population as

a whole.

8. Spore dimorphism is temperature dependent. Mem-

brane bound sporulation is inhibited by both low (20-22.5 C)

and high (32 C) temperatures; NMB sporulation is affected

little, if at all.

9. Nonmembrane bound spores do not develop in lieu

of MB spores in the fat body at low or high temperatures.

10. The pathognomonic signs of B. dimorpha infection

are due to destruction or inhibition of formation of the

adult cuticle.

11. Meronts are diplocaryotic and bidiplocaryotic,

not uninucleate and binucleate as originally described.

12. The NMB spore surface is smooth, but the MB spore

surface has a complex pattern of ridges and a well defined

polar cap area.

13. The pansporoblast membrane is extremely delicate

and subpersistent in workers (as originally described),

and in male pupae; in female pupae it is persistent.













GLOSSARY


Diplocaryon. Two nuclei in intimate contact, their membranes

adhering to each other over a large area. These nuclei

divide in a plane perpendicular to the plane of their

physical contact, in synchrony.

Disporous. Producing two sporoblasts.

Endogenous sporogony. Sporogony within the limiting membrane

of the sporogonial plasmodium.

Endospore. The chitinous inner spore wall.

Exospore. The proteinaceous outer spore covering or envelope.

MB. As used in this dissertation--bounded by a pansporoblast

membrane.

Merogony. Vegetative multiplication. Synonym: schizont.

Meront. A cell that undergoes binary or multiple fission in

the vegetative phase of the life cycle. Synonym: schizont.

NMB. As used in this dissertation--not bounded by a pansporo-

blast membrane.

Pansporoblastic membrane. A somewhat modified membrane that

encloses a group of spores or sporoblasts.

Polar cap. An internal, chromophilic area at the anterior end

of the spore.

Sporogenesis. The transformation of the sporoblast into a

spore.








Sporogenesis. The transformation of the sporoblast into a

spore.

Sporogony. The production of sporoblasts.

Sporont. A cell that gives rise to a sporoblast.

Sporoplasm. The nucleus (nuclei) and cytoplasm contained

within the spore that is injected into a host cell.

Sporulation. Spore production; sporogony plus sporulation.

Vegetative stage. That phase of the life cycle when the

parasite is actively feeding and multiplying prior to

sporulation.












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