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Full text of "Life cycle and epidemiology of Amblyospora sp. (Microspora: Thelohaniidae) in the mosquito Culex salinarius Coquillett"

LIFE CYCLE AND EPIDEMIOLOGY OF 
Aniblyospora sp. (MICROSPORA:THELOHANIIDAE) 
IN THE "MOSQUITO Culex s alinarius COQUILLETT 



By 

THEODORE GEORGE ANDREADIS 



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 
1978 



ACKNOWLEDGMENTS 

I would like to express my sincere appreciation to Dr. D. W. Hall 
for his continual advice, encouragement and friendship throughout the 
course of this study. 

I would like to thank Mr. E. I. Hazard of the Insects Affecting 
Man and Animals Research Laboratory, USDA, Gainesville, Florida, for 
his assistance in interpreting many aspects of the life cycle. 

Special thanks are extended to Mrs. S. W. Avery and Miss E. A. 
Ellis, also of the Gainesville lab, for their assistance in the ultra- 
structural studies. 

I would also like to acknowledge Dr. T. J. Walker and Dr. J. L. 
Nation for their critical appraisal of the dissertation. 

Finally, I would like to thank the entire staff of the Insects 
Affecting Man and Animals Research Laboratory who graciously provided 
research space and facilities without which this study would not have 
been possible. 



TABLE OF CONTENTS 

Page 

ACKNOWLEDGMENTS ii 

LIST OF TABLES v 

LIST OF FIGURES vi 

ABSTRACT i x 

INTRODUCTION 1 

LITERATURE REVIEW 3 

The Microsporidia 3 

Definition and taxonomic status 3 

Structure 3 

General scheme of the life cycle 5 

Transmission 6 

Effects of microsporidian infections on their 7 

insect hosts 

Genus Ambly ospora Hazard and Oldacre 9 

DEVELOPMENT, ULTRASTRUCTURE AND MODE OF TRANSMISSION OF 

Amblyospora sp. (MICROSPORA:THELOHANI IDAE) IN THE 

MOSQUITO Culex salinarius COQUILLETT 11 

Abstract 11 

Introduction II 

Materials and Methods 13 

Results 14 

Discussion 17 



Page 

SIGNIFICANCE OF TRANSOVARIAL INFECTIONS OF Atnblyospora sp. 
(MICROSPORArTHELOHANIIDAE) IN RELATION TO PARASITE MAIN- 
TENANCE IN THE MOSQUITO Culex salinarius COQUILLETT 37 

Abstract 37 

Introduction 37 

Materials and Methods 39 

Results and Discussion 41 

REFERENCES 52 

BIOGRAPHICAL SKETCH 58 



LIST OF TABLES 



Table Page 

1 Physiological longevity of healthy and Amblyospora sp.- 
infected C. sal inarius 45 

2 Egg production and hatch during each gonotrophic cycle 

for healthy and Amblyospora s p. -infected C_. sal inarius . . 46 

3 Developmental periods for healthy and A mblyospora sp.- 
infected C. sal inarius 47 

4 Survival rates for healthy and Amblyospora sp.- 

infected C. sal inarius 48 

5 Prevalence rate of infection among adult female 
progeny produced by infected females with each 
gonotrophic cycle 49 



LIST OF FIGURES 

Figure Pa 9e 

1 Life cycle of Amblyospor a sp. in C. salinarius .... 22 

2 Photomicrograph of a primary diplokaryon 24 

3 Photomicrograph of a dividing diplokaryon 24 

4 Photomicrograph of a dividing diplokaryon 24 

5 Photomicrograph of an intermediate diplokaryon .... 24 

6 Photomicrograph of a secondary diplokaryon 

from the female host 24 

7 Photomicrograph of a dividing diplokaryon 

from the female host 24 

8 Photomicrograph of divided diplokarya from 

the female host 24 

9 Photomicrograph of a sporoblast from the 

female host 24 

10 Photomicrograph of a mature spore from 

the female host 24 

11 Photomicrograph of a secondary diplokaryon from 

the male host 24 

12 Photomicrograph of a dividing diplokaryon from 

the male host 24 

13 Photomicrograph of a dividing diplokaryon 

from the male host 24 

14 Photomicrograph of a dividing diplokaryon 

from the male host 24 

15 Photomicrograph of a binucleate sporont 

from the male host 24 

16 Photomicrograph of a quadrinucleate 

sporont from the male host 24 



Figure Page 

17 Photomicrograph of an octonucleate sporont 

from the male host 24 

18 Photomicrograph of a pansporoblast with 

eight sporoblasts from the male host 24 

19 Photomicrograph of spores in a pansporo- 
blast membrane from the male host 24 

20 Photomicrograph of mature spores from 

the male host 24 

21 Photomicrograph of macrospores from 

the male host 24 

22 Sagittal section of a fourth instar male 
larva of C. sal inarius infected with 

Amblyospor a sp 26 

23 Amblyospora sp. -infected oenocyte containing 
diplokarya lying next to the ovaries of a 

newly emerged adult female C. sal i narius 26 

24 Amblyospora sp. -infected oenocyte containing 
mature spores in close association with the 
developing oocytes of an adult C. sal inari us 

female 48 hr after a blood meal 26 

25 Electron micrograph of a bi nucleate sporo- 

plasm from the hoemocoel of an adult female 28 

26 Electron micrograph of an infected oenocyte 
containing secondary diplokarya from a re- 
cently emerged adult female 28 

27 Electron micrograph of a secondary diplo- 

karyon from the female host 28 

28 Electron micrograph of a young sporoblast 
from an adult female 42 hr following a 

blood meal 28 

29 Electron micrograph of a mature sporoblast 
from an adult female 44 hr following a 

blood meal 30 

30 Electron micrograph of a mature spore from 

an adult female 48 hr following a blood meal 30 

31 Electron micrograph of a mature spore dis- 
charging its sporoplasm in an adult female 

60 hr following a blood meal 30 



Figure Page 

32 Electron micrograph of an oenocyte from a 

first instar male larva containing diplokarya 30 

33 Electron micrograph of a diplokaryon from 

the fatbody of a first instar male larva 32 

34 Electron micrograph of a diplokaryon in 
the state of mitotic division from the 

male host 32 

35 Electron micrograph of a secondary diplo- 
karyon from the male host 32 

36 Electron micrograph of a diplokaryon at 

the onset of sporogony from the male host 32 

37 Electron micrograph of an early binucleate 

sporont from the male host 34 

38 Electron micrograph of a binucleate sporont 

from the male host 34 

39 Electron micrograph of a meiotically dividing 

sporont from the male host 34 

40 Electron micrograph of a quadrinucleate 

sporont from the male host 34 

41 Electron micrograph of young sporoblasts 
contained within a pansporoblast membrane 

from the male host 35 

42 Electron micrograph of a mature sporoblast 

from the male host 35 

43 Electron micrograph of a mature spore from 

the male host 35 

44 Electron micrograph of a mature macrospore 

from the male host 35 

45 Prevalence rate of infection for successive 
generations of a theoretical population of 
C. sal inarius infected with Amblyospora sp. 
where the parasite is maintained by trans- 

ovarial transmission alone 5] 



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 



LIFE CYCLE AND EPIDEMIOLOGY OF 
Ampjyospora sp. (MICROSPORA:THELOHANIIDAE) 
IN THE MOSQUITO Culex salinarius C0QU1LLETT 

By 

Theodore George Andreadis 

December 1978 

Chairman: Donald W. Hall 

Major Department: Entomology and Nematology 

Amblyospora sp. in Culex salinarius is transovarially transmitted 
and exhibits two developmental sequences, one in each host sex. In fe- 
males, the entire life cycle is restricted to host oenocytes which be- 
come greatly hypertrophied due to the multiplication of diplokarya 
during merogony and come to lie next to the host ovaries. Sporogony 
occurs only after a blood meal is taken and is shortly followed by in- 
fection of the developing oocytes and subsequent transmission to the 
next host generation. In the male host, infections spread from oeno- 
cytes to fatbody tissue where diplokarya undergo a second merogony. 
During this merogonic cycle, the number of diplokarya greatly increase 
and the infection is spread throughout the body of the larval host. 
Sporogony is initiated with the physical separation of the diplokaryo- 
tic nuclei and the simultaneous secretion of a pansporoblastic membrane. 
Subsequent meiotic division and morphogenesis result in the formation 



of eight haploid spores enclosed within a pansporoblastic membrane. 
Buildup of spores and subsequent destruction of host fatbody tissue 
prove fatal to the male during the fourth larval stadium. 

Adult females infected with the microsporidium showed no signifi- 
cant differences in overall fecundity, physiological longevity and 
preoviposition periods when compared to healthy adults under laboratory 
conditions. Development times and survival rates for congenitally in- 
fected young to reproductive age were also indistinguishable from those 
of healthy controls. A significant reduction of 52% in egg hatch was 
observed for infected eggs when compared to healthy eggs. Prevalence 
rates of infection for progeny produced by infected females declined 
with each successive gonotrophic cycle and averaged 90%. Transovarial 
transmission is not sufficient for the maintenance of the microsporidium 
in a population of mosquitoes. An alternate host is suggested as a 
mechanism whereby the microsporidium can re-enter a healthy mosquito 
population. 



INTRODUCTION 

The microsporidia are a large group of obligate, intracellular 
parasites which occur worldwide and exhibit a broad host range, in- 
fecting all phyla of the animal kingdom from protozoa to man (Bulnheim, 
1975). However, they are most frequently encountered as parasites of 
arthropods and are important pathogens of many insect groups. 

They are among the most common and widely distributed pathogens 
found infecting natural populations of mosquitoes, often causing severe 
diseases (Chapman, 1974). Hazard and Chapman (1977) list more than 100 
mosquito species worldwide as known hosts of these parasites and it is 
likely that all mosquito species somewhere, sometime are hosts (Chap- 
man, 1974) . 

Because of their wide distribution, common occurrence and ap- 
parent pathogenicity, microsporidia appear to be promising candidates 
for the biological control of many mosquito species. However, the life 
cycles and mode of transmission of many of these parasites are poorly 
understood. An elucidation of these mechanisms is vital to future re- 
search programs which seek to utilize these parasites for mosquito 
control . 

This study was undertaken to determine the relationship between 
a microsporidian parasite, Amblyospora sp., and its natural mosquito 
host, Culex sal inar ius Coquillett. 



This dissertation consists of a literature review which provides 
information on the biology of mosquito microsporidia and two chapters 
containing all experimental work, written in manuscript form, which 
are currently being submitted for publication. 



LITERATURE REVIEW 

The Microspor idia 
Definition and taxonomic status 

Microsporidia are obligatory intracellular parasites, incapable 
of development and multiplication outside of the host cell. They are 
characterized by the formation of unicellular spores equipped with an 
extrusible polar filament and the absence of mitochondria. Formerly 
considered a distinct class of protozoans they have most recently been 
elevated in rank to an independent phylum, the Microspora (Sprague, 
1977). 

Structure 

All microsporidian cells, other than spores, are structurally 
simple and unspecial ized. With the exception of the absence of mito- 
chondria, their structural organization is typical of other eucaryotic 
cells (Va^vra, 1976a). 

The microsporidian nucleus is typically round or oval and en- 
closed in a unit membrane, perforated with pores (Vavra, 1965). The 
outer membrane is studded with ribosomes and has continuities with 
the endoplasmic reticulum (Maurand, 1966). Many times the microsporid- 
ian nuclear component exists as two structurally identical nuclei in 
intimate association which behave in synchrony - the diplokaryon (V^vra, 
1968). 

The microsporidian cytoplasm has the usual organelles including 
both rough and smooth endoplasmic reticulum, free and bound ribosomes, 



golgi apparatus, and vesicles of endocytosis all enclosed in a cell 
limiting membrane (Vavra, 1965; Lorn and Corliss, 1967; Sprague and 
Vernick, 1968, 1969; Vernick et al., 1977). 

There are no reserve substances in microsporidian cells (Maurand 
and Loubes, 1973). Their absence, according to Vavra (1976a) is in 
keeping with their obligatory, intracellular existence which excludes 
any metabol ical ly active stage outside the host cell. 

In contrast to its developmental stages, the microsporidian 
spore is a highly differentiated cell, specifically adapted to its 
function of transmitting infectious material to a new host. 

It is endowed with a protective trilaminar envelope or wall con- 
sisting of a thin proteinaceous outer layer (exospore), a thick middle 
layer composed of chitin (endospore), and a thin cytoplasmic limiting 
membrane (Vavra, 1964, 1967, 1968). 

Within this protective wall exist one or more nuclei and cyto- 
plasm which constitute the sporoplasm or infective germ (Weidner, 1972) 
and a highly specialized extrusion apparatus consisting of three dis- 
tinct parts (Lorn and Vavra, 1963): (1) polar filament (tube) - a solid, 
tubular, threadlike structure of great elasticity attached to the inner 
surface of the spore and coiled within, which when extruded provides 
the vehicle through which the infective sporoplasm is ejected, (2) 
polaroplast - a laminar or vesicular complex which swells to provide 
the initial intrasporal pressure necessary for polar filament extru- 
sion, and (3) posterior vacuole - a membrane limited vacuole capable 
of expansion providing the additional pressure necessary for further 
polar filament extrusion and ejection of the infective sporoplasm. 



General scheme of the life cycle 

The typical microsporidian life cycle is initiated with the 
liberation from the spore of the sporoplasm which passes through an 
evaginated polar filament and enters a host cell (Lorn and Vavra, 1963; 
Ishihara, 1968; Weidner, 1972). 

The stimulus for spore filament extrusion and release of the in- 
fective sporoplasm in the host is complex and varied but most evidence 
indicates this process to be affected by pH and the presence of cer- 
tain cations which cause an osmotic shift within the spore (Ohshima, 
1964; Ishihara, 1967). Presumably, this results in an influx of water, 
producing a sudden buildup in hydrostatic pressure necessary for forci- 
ble extrusion of the filament (Weidner, 1976). Lorn and Vavra (1963) 
have shown polar filament discharge intensity to be directly propor- 
tional to the osmotic condition or the viscosity of the medium exterior 
to the spore. 

Within the host cell, the microsporidium undergoes an initial 
multiplicative phase (merogony) during wiich the number of parasites 
rapidly increases and the infection is spread throughout the body of 
the host (VSvra, 1976b). 

After merogony the parasites enter a second phase of development 
called sporulation. During sporulation additional multiplication by 
binary or multiple fission (sporogony) occurs followed by morphogenesis 
culminating in the formation of new spores capable of infecting new 
susceptible hosts (Vcivra, 1976b). 

This entire developmental sequence occurs intracel lularly in a 
variety of tissue types dependent upon the host and the species of 
microsporidia. Insect tissues infected include fatbody, gut, muscle, 



nerve, gonads, salivary gland, malpighian tubules and oenocytes (Weiser, 
1976). 

To date all reproduction in microsporidia has been reported to 
be asexual. However, recent cytological evidence of meiotic divisions 
in certain species (Loubes et al., 1976; Vavra, 1976a; Hazard et al., 
1978) suggests a sexual phase. 

Transmissi on 

Microsporidia are normally transmitted when spores from feces or 
cadavers of infected hosts are ingested by new hosts (Canning, 1971). 

Transmission may also take place between hymenopterous parasites 
and their insect hosts, the spores being carried on the contaminated 
ovipositor (Brooks, 1973). In many cases the hymenopterous vector is 
infected by the same microsporidia as its host (Tanada, 1955; Lipa, 
1957), although pure mechanical transmission, with no development of 
the microsporidia in the wasp, may also occur (Lipa, 1963; Laigo and 
Tamashiro, 1967). 

The major route of transmission of microsporidian parasites in 
insects is through the mouth, but transmission by way of the ovary 
(transovarial ) or surface of the egg (transovum) has also been shown 
to commonly occur in many insect groups (Kellen and Wills, 1962a; Kellen 
et al., 1965, 1966; Chapman et al., 1966; Brooks, 1968; Hazard and 
Weiser, 1968; Chapman, 1974; Nordin, 1975). In mosquitoes, the former 
appears to be the principal, if not only, means of transmission of 
certain microsporidian genera (Kellen et al., 1965; Chapman et al . , 
1966) and in some cases has been considered sufficient to account for 
the low levels of infection observed in the field (Kellen et al., 1965, 
1966; Chapman et al., 1967) . 



When entry occurs via the egg, infective stages are incorporated 
into the developing ova or embryos within the female reproductive tract 
and subsequently passed on to her progeny (Hazard and Weiser, 1968; 
Nordin, 1975). 

Successful transmission of microsporidia to new hosts is depen- 
dent upon three primary factors (Weiser, 1969): (1) successful spore 
germination, (2) host tissue susceptabil i ty, and (3) active host re- 
sistance. 

Effects of microsporidian infections on their insect hosts 

The main characteristic of microsporidian infections is the 
chronic and debilitating effect they produce in their insect hosts. 
Often these effects are expressed by a general loss of vigor and dis- 
ruption of vital physiological functions. However, infections may be 
expressed in a wide variety of ways ranging from complete host destruc- 
tion to latency. Those effects, reported to date, can be categorized 
as follows. 

1. No perceivable effect - Kellen et al. (1965, 1966) report 
that in certain genera of mosquitoes infected with Ambl yosp ora spp., 
infected female larvae pupate normally and emerge as apparently healthy 
adults which transmit the parasite transovarially. 

2. Retarded development - Lengthening of the larval period is 

a common effect of microsporidian infections (Thompson, 1958; Gaugler 
and Brooks, 1975). In some cases microsporidia have been shown to pro- 
duce a hormonal substance that results in supernumerary molts, slowing 
down or preventing pupation (Fisher and Sanborn, 1964). 

3. Re duced longevity - Numerous studies have shown adult longev- 
ity to be significantly reduced for infected anopheline mosquitoes 



(Anthony et al., 1972, 1978; Undeen and Alger, 1975) and many lepidop- 
terous pests (Zimmack et al., 1954; Zimmack and Brindley, 1957; Kramer, 
1959; Gaugler and Brooks, 1975; Windels et al . , 1976). 

4. Redu c ed reproductive potential - The principal effect of 
microsporidian infections is a reduction in the reproductive potential 
of the host. This is expressed by a reduction in overall fecundity 
(Veber and Jasic, 1961; Reynolds, 1971; Anthony et al., 1972, 1978; 
Gaugler and Brooks, 1975; Windels et al . , 1976), egg hatch (Reynolds, 
1971; Windels et al., 1976; Anthony et al., 1978), oviposition cycles 
(Anthony et al . , 1972, 1978) or mating success (Gaugler and Brooks, 1975) 
Such effects, as suggested by Veber and Jasic (1961) and Thompson (1958), 
are probably the result of depleted nutritional reserves and reduced 
ability to assimilate food efficiently. 

5. Impai r ment of diapause development - The initiation, main- 
tenance, and termination of diapause in some lepidopterous pupae is af- 
fected by microsporidian infections (Issi and Maslennikova, 1964; 
Gaugler and Brooks, 1975). These effects may be due to a hormonal im- 
balance or nutritient reserve deficiency caused by microsporidian de- 
velopment (Gaugler and Brooks, 1975). 

6. Death - Progressive multiplication of the parasite and sub- 
sequent destruction of host tissue may cause death (Weiser, 1976). 

In general, the virulence and pathogenicity of microsporidian 
infections are dependent upon three primary factors (Weiser, 1963): 
(1) infective dose, (2) age of the host, and (3) tissues infected. 
Where peroral transmission occurs, the higher the dosage, the more acute 
and lethal is the infectious process. Young individuals are generally 
more susceptible to and succumb faster from infections than do older 



individuals. Microsporidia which infect the gut and musculature and 
those which are systemic produce more acute infections than those con- 
fined to fatbody tissue which are generally more chronic. 

Genus Amblyospora Hazard and Oldacre 

Of the eleven genera of microsporidia known to infect mosquitoes, 
members belonging to the genus Amblyospor a are by far the most common 
and widespread. To date, they have been reported from 47 species of 
mosquitoes representing 8 genera (Hazard and Chapman, 1977). 

Most, if not all, species of Amblyospora are transovarially trans- 
mitted and exhibit two developmental sequences in their mosquito hosts: 
one in male or both male and female larvae, and another in adult fe- 
males. Parasite development in larvae characteristically results in 
the production of eight thick-walled, oval, octospores enclosed in a 
pansporoblast membrane while that in adult females produces a variable 
number of thin-walled, cylindrical, free spores (Hazard and Oldacre, 
1975). 

The relationship between many of these microsporidian parasites 
and their mosquito hosts has been categorized into four types based on 
the sex of the larva in which sporogony occurs and the tissues attacked 
(fatbody or oenocytes) (Kellen et al . , 1965; Chapman et al . , 1966). In 
types I and II sporogony occurs in male larvae only producing massive 
infections which invariably prove fatal to the host during the fourth 
larval stadium. In females sporogony is delayed or suppressed. Fe- 
male larvae pupate normally and emerge as apparently healthy adults 
which transmit the parasite transovarially when mated with healthy males, 
In types III and IV sporogony occurs in larvae of both sexes but is 



10 



progressive and usually fatal in type III only, those infections in 
type IV being relatively benign. 

In types I and II, transovarial transmission is continuous for 
successive host generations and in some instances has been reported to 
be sufficient to account for the levels of infection observed in the 
field (Kellen et al., 1965, 1966; Chapman et al., 1967). Transovarial 
transmission may or may not be continuous in types III and IV. In 
some mosquitoes which develop infections characteristic of type III, 
transovarial transmission is limited to one generation (Kellen et al . , 
1966). In these mosquitoes, peroral transmission probably is the more 
common mode of transmission and would be expected for survival of the 
microsporidia (Kellen et al., 1966; Chapman et al . , 1967). 

While transovarial transmission of A mblyospo ra has been clearly 
demonstrated, peroral transmission has always been an enigma. Unlike 
many other microsporidia, spores produced in larvae do not appear to be 
infectious when fed directly back to their mosquito hosts. Successful 
transmission in- the laboratory has been claimed only once (Kellen and 
Lipa, 1960). However, a succeeding report indicated that peroral trans- 
mission for the same microsporidia and mosquito host was not attainable 
(Kellen and Wills, 1962a). Subsequent attempts to transmit these micro- 
sporidia by rearing larvae in water contaminated with spores have also 
been unsuccessful (Kellen et al., 1965; Chapman, 1974). 

Similar tests conducted under field conditions, by exposing lar- 
vae in screened containers in ponds with a past history of Amblyospora 
occurrence, have met with limited success, indicating that transmission 
may occur peroral ly when certain conditions are met (Kellen et al . , 
1966; Chapman et al., 1970). To date, what these conditions are has 
not been determined. 



DEVELOPMENT, ULTRASTRUCTURE AND MODE OF TRANSMISSION 

OF AmpVypsppra sp. (MICROSPORArTHELOHANI IDAE) IN THE 

MOSQUITO Culex salinarius COQUILLETT 



Abstract 
Amblyospora sp. in Culex sal inarius is transovarially trans- 
mitted and exhibits two developmental sequences, one in each host sex. 
In females, the entire life cycle is restricted to host oenocytes which 
become greatly hypertrophied due to the multiplication of diplokarya 
during merogony and come to lie next to the host ovaries. Sporogony 
occurs only after a blood meal is taken and is shortly followed by in- 
fection of the developing oocytes and subsequent transmission to the 
next host generation. In the male host, infections spread from oeno- 
cytes to fatbody tissue where diplokarya undergo a second merogony. 
During this merogonic cycle, the number of diplokarya greatly increase 
and the infection is spread throughout the body of the larval host. 
Sporogony is initiated with the physical separation at the diplokaryo- 
tic nuclei and the simultaneous secretion of a pansporoblastic membrane. 
Subsequent meiotic division and morphogenesis result in the formation 
of eight haploid spores enclosed within a pansporoblastic membrane. 
Buildup of spores and subsequent destruction of host fatbody tissue 
prove fatal to the male host during the fourth larval stadium. 

Introduction 

Microsporidia of the genus Amblyospora Hazard and Oldacre are 

among the most common and widely distributed pathogens found infecting 

natural populations of mosquitoes. To date, they have been reported 

11 



12 



from 47 mosquito species representing 8 genera (Hazard and Chapman, 
1977). 

Most, if not all, species of Amblyospor a are transovarially 
transmitted by their mosquito hosts and typically exhibit two develop- 
mental sequences, one usually, but not exclusively, in each host sex. 
In males, parasite development is rapid. Sporogony (formation of 
spores) occurs in larvae, producing massive infections which prove 
fatal to the host during the fourth larval stadium. In females, how- 
ever, sporogony is delayed and limited to the oenocytes. Female lar- 
vae pupate normally and emerge as apparently healthy adults which trans- 
mit the parasite transovarially to their progeny when mated with healthy 
males (Kellen et al., 1965; Hazard and Oldacre, 1975). 

Because of their wide distribution, common occurrence and apparent 
pathogenicity, these microsporidia appear to be promising candidates for 
the biological control of certain mosquito species. However, the man- 
ner in which these parasites develop in and are transmitted to their 
mosquito hosts has never been completely understood thus restricting 
their use. 

A similar host-parasite relationship has been reported for an un- 
described species of Amblyospora and its natural mosquito host, Culex 
salinarius Coguillett (Chapman et al., 1966). This study was under- 
taken to describe the complete development and ul trastructure of this 
microsporidium and to elucidate the mechanisms involved in transovarial 
transmission of the parasite to its mosquito host. 

Because of the morphological similarities of this Amblyospora sp. 
from C. salinarius to that of Amblyospora c alifornica Kellen and Wills, 
taxonomic description of the former species was delayed until a complete 
life cycle study of the latter species could also be made. 



13 



Materials and Methods 
Exper i mental animals 

The healthy and A mblyospora sp. -infected colonies of C. sal inarius 
used in this study were originally obtained from Dr. Harold Chapman, 
Gulf Coast Mosquito Research Laboratory, Lake Charles, Louisiana. Adults 
were maintained in 38x46x38 cm cages at 24 C under natural photoperiod 
and were constantly supplied with a 5% sucrose solution in distilled 
water. Since almost all males from the infected colony died during the 
fourth larval stadium, males from the healthy colony were used to in- 
seminate infected females. 

Females were fed on guinea pigs placed directly into the cage. 
Egg rafts infected with the microsporidia were deposited into half-pint 
containers and individually transferred into white enamel pans (18x29x 
4.5 cm) containing 500 ml of well water for larval rearing. Rearing 
was at 25 C. The water was infused with 10 ml of an aqueous suspension 
containing 1.5% of a 3:2 mixture of dried liver powder and brewers 
yeast. Larvae were fed on alternate days until all had pupated or died. 
Simultaneous maintenance of the healthy colony was performed in a simi- 
lar manner. 

Life cycle studies 

To determine the developmental sequences and complete life cycle 
of the parasite, all stages of both sexes of the mosquito host were 
chronologically examined for the microsporidia. 

General characterization of the microsporidian stages at the 
light microscope level was made from giemsa-stained smears of infected 
host tissues as described by Hazard and Oldacre (1975). Sites of in- 
fection within the mosquito host were determined from whole mosquitoes 



14 



fixed in Carnoy's solution, embedded in paraffin, sectioned at 6 wm and 
stained with Heidenhains hematoxylin and Eosin y. 

For ul trastructural studies, infected specimens were dissected 
in 2.5% gluteraldehyde buffered with 0.1 M sodium cacodylate (pH 7.5) 
and fixed for 2 hr at room temperature, in the dark in 2.5% gluteralde- 
hyde, 0.1% peroxide in 0.1 M cacodylate buffer, pH 7.5 (Peracchia and 
Mittler, 1972). After several buffer washes, specimens were post fixed 
in 1% osmium tetroxide, dehydrated in an ethanol series, en bloc stained 
with 0.5% uranyl acetate in 70% ethanol and embedded in either Spurrs 
(Spurrs, 1969), a Spurr-Epon mixture (Ellis and Avery, 1978), or Epon- 
Araldite. Sections were poststained with 5% methanolic uranyl acetate, 
followed by lead citrate (Reynolds, 1963) and viewed with a Hitachi 
HU-125 E electron microscope at an acceleration voltage of 75 kV. 

In addition to eggs, larvae and pupae, adult females of different 
physiological age, ranging from recently emerged to fully gravid, were 
examined at the light microsope and ul trastructural level to determine 
the mechanism by which the microsporidia infected the ovaries and were 
subsequently transmitted to the next generation. 

Results 

The complete developmental sequence of Amblyospora sp. in both 
sexes of the mosquito host, C. sal inarius , is shown in Figure 1. 

Infections are initiated when small binucleate sporoplasms (Figs, 
la, 25) infect the developing eggs within the female host and are sub- 
sequently transferred to the next generation when the eggs are laid. 
Within embryonated eggs and newly hatched larvae of both sexes small, 
oval, diplokaryotic stages (Figs, lb, 2) invade host oenocytes and under- 
go an initial multiplicative phase (merogony) where they divide mitoti- 
cally to produce more diplokarya (Figs, lb-d, 2-4). 



15 



In the female host, initial parasite development within these 
oenocytes is slow and infections relatively benign. Infected larvae 
develop normally, pupate and emerge as apparently healthy adults. How- 
ever, at adult emergence the parasites enlarge, become fusiform in shape 
(Figs. 5, 6, 27) and enter a second merogonial phase of development 
where they now begin to rapidly divide within the same oenocytes which 
become greatly hypertrophied due to the multiplication of the parasite 
(Figs, le-h, 5-8, 26-27). 

Infected oenocytes circulate or actively migrate through the 
hemocoel of the female host until they come to reside in very close 
proximity and in many cases lie next to the host ovaries (Fig. 23). 
Within these oenocytes, the microsporidia remain in the diplokaryotic 
stage until a blood meal is taken by the female host at which time they 
begin sporogony (Figs. li-j). During this final phase of development, 
which requires 40-48 hrs to complete, the diplokarya undergo a tremen- 
dous amount of internal reorganization and develop directly into binu- 
cleate spores (Figs. 28-29). Mature spores are elongate and character- 
ized by having a relatively thin wall and a large conspicuous posterior 
vacuole (Figs. 10, 30). 

Spores, still contained within host oenocytes (Fig. 24), are 
short lived and within a 12-24 hr period begin to evert their polar 
filaments and forcibly discharge their amoeboid-shaped sporoplasms into 
the surrounding hemocoel (Fig. 31). These binucleate sporoplasms (Fig. 
25) subsequently infect the developing oocytes and thus complete the 
cycle when the eggs are laid. Sporulation in other oenocytes and 
ovarian infection are repeated during each successive gonotrophic 
cycle of the host. 



16 



In the male host, the developmental sequence of the parasite re- 
sembles that in the female during the initial stages of development but 
then quickly diverges. Infections begin in the embryonated egg where 
the microsporidium initially invades host oenocytes and rapidly multi- 
plies to produce numerous diplokarya during merogony (Figs, lb-d, 2-4, 
32). However, unlike female infections which are confined to the host 
oenocytes, the diplokarya in the male host break out of the oenocytes 
and invade thoracic and abdominal adipose tissue of late first and early 
second instar larvae (Fig. 33). Here they enter a second multiplicative 
phase (merogony 2) during which the number of diplokarya greatly in- 
crease and the infection is spread throughout the body of the larval 
host (Figs. If-h, 11-14, 34). 

After this merogonic multiplication, the parasite enters its last 
phase of development, sporogony, where additional division and morpho- 
genesis result in the formation of spores in groups of eight (Figs. 
In-r). The process begins with separation of the diplokaryotic nuclei 
and the simultaneous secretion of a pansporoblastic membrane (Figs. 
35-37) to form a binucleate sporont (Figs. 15, 38). The two nuclei of 
the sporont divide synchronously (Fig. 39) to produce a quadrinucleate 
stage (Figs. 16, 40) which divides again (Fig. 17) before undergoing 
cytokinesis to form eight sporoblasts, all still contained within the 
pansporoblastic membrane (Figs. 18-19, 41-42). Also within this mem- 
brane are numerous metabolic products (granules) secreted by the develop- 
ing sporonts and readily observed at the ul trastructural level (Figs. 
37-41). The repeated occurrence of synaptonemal complexes in binu- 
cleate sporonts (Fig. 38) indicates this division process to be meiotic 
and the resulting sporoblasts haploid. 



17 



Each of the eight sporoblasts develops directly into a uninu- 
cleate spore characterized by the possession of a thick exospore wall, 
a conspicuously lamellated polaroplast, and a long polar filament 
abruptly constricted near its middle (Figs. 20, 43). Occasionally spores 
almost double in size (macrospores) are observed, which with the excep- 
tion of their size appear structurally identical at the ul trastructural 
level to normal sized spores (Fig. 21, 44). Presumably, they arise 
from the incomplete division of one or more of the nuclei contained in 
quadrinucleate sporonts. They are however, still haploid, having com- 
pleted the first meiotic division. 

The massive buildup of spores and subsequent destruction of host 
fatbody tissue (Fig. 22) usually prove fatal to the male host during 
the fourth larval stadium. Occasionally a few individuals pupate and 
emerge as adults but always die within a 24-hr period. Dissections of 
these adults has always revealed mature spores but in relatively low 
numbers. 

Discussion 

The development of this species of Amblyo spora in the mosquito 
C_. salinarius is clearly dimorphic, exhibiting two complete develop- 
mental sequences: one fatal in males, producing spores in groups of 
eight enclosed by a pansporoblastic membrane, and another in females, 
producing a variable number of free spores which infect the ovaries and 
ensure passage to the next host generation. 

Similarities between the two sequences are exhibited only during 
the initial phase of development where diplokarya invade oenocytes and 
undergo merogony within the embryonated eggs and newly hatched larvae. 



18 



As early as late first instar larvae, however, differences in parasite 
development can be observed. 

In the female host, the entire life cycle is restricted to host 
oenocytes which become greatly hypertrophied due to the multiplication 
of diplokarya by the time of adult emergence. Infected oenocytes, 
readily observed throughout the thorax and abdomen at this time, sub- 
sequently circulate or actively migrate through the hemocoel of the 
female host until they come to lie next to the developing eggs. Sporog- 
ony is initiated only after a blood meal is taken by the female host 
and requires 40-48 hr to complete. The repeated observation that spore 
maturation will not occur until a blood meal is taken by the female 
host regardless of her age, clearly demonstrates the close relationship 
between the biology of the microsporidian parasite and the physiology 
of the mosquito host. While the physiological mechanism for sporulation 
is not known at this time, the involvement of host hormones, shown to 
be released with a blood meal (Hagedorn, 1974; Hagedorn et al., 1975) 
should be investigated. 

Mature spores are short lived and within a 12-24 hr period begin 
to extrude their sporoplasms within the oenocytes. The exact mechanism 
by which these sporoplasms infect the ovaries is not understood. Sporo- 
plasms may actively invade the ovaries or they may be nonselectively 
taken up by the developing oocytes which are sequestering vitellogenins 
by pinocytosis (Roth and Porter, 1964). 

Sporulation in other oenocytes and subsequent ovarian infection 
are repeated during each successive gonotrophic cycle. Thus by this 
unique method, the microsporidium appears to ensure its survival in a 
continually breeding population of host mosquitoes. 



19 



In the male host, initial parasite development is also restricted 
to oenocytes but is much more rapid and prolific than that occurring in 
the female host. Diplokarya subsequently break out of oenocytes and 
invade host fatbody cells where they multiply repeatedly. This second 
merogony sequence provides the primary instrument by which the parasites 
increase in number. It appears these diplokarya are capable of invading 
additional fatbody cells because it is during this phase of development 
that infections are spread throughout the body of the larval host. 

The end of merogony and the onset of sporogony are characterized 
by the physical separation of the diplokaryotic nuclei and the simulta- 
neous secretion of a pansporoblastic membrane. The observation that 
the nuclei of the diplokaryon simply separate during this phase of de- 
velopment is unquestionably confirmed at the ul trastructural level and 
provides further evidence that no karyogamy of diplokaryotic nuclei 
exists as has been suggested by previous investigators (Mercier, 1909; 
Kudo, 1924; Debaisieux, 1928; Weiser, 1977). Furthermore, no evidence 
exists for the presence of either uninucleate sporonts or meronts as 
have been described for other species of Amb lyospora with similar de- 
velopmental sequences (Kellen and Lipa, 1960; Kellen and Wills, 1962b; 
Anderson, 1968). Based on these findings, I now suspect that uninu- 
cleate meronts described by these investigators for Amblyospora spp. 
at the light microscope level are in fact diplokaryotic and can only 
be resolved at the ultrastructural level. 

The repeated observation of synaptonemal complexes in bi nucleate 
sporonts indicates the division process during sporogony to be meiotic 
and the resulting spores to be haploid. These findings support the 
work of Loubes et al . (1976) and Vavra (1976a) who observed similar 



20 



ul trastructural evidence for meiosis in Gurleya chironomi and Tuzetia 
debaisieuxi , respectively. Verification of these ul trastructural find- 
ings in the same species of Ambly ospora has recently come from Hazard 
et al . (1978) who documented meiosis through the examination of chrom- 
osome squashes. 

The significance of these events in the life cycle of this micro- 
sporidium are unclear. All attempts to transmit this parasite by the 
direct feeding of haploid spores to healthy mosquito larvae have been 
unsuccessful both in our lab and by previous investigators (Kellen and 
Wills, 1962a; Kellen et al., 1966). However, from an evolutionary stand- 
point it is hard to rationalize an organism putting that much energy into 
the production of spores that have no function and are produced by the 
millions within male larvae of each host generation. Therefore, our ob- 
servations confirm the contention of Hazard et al. (1978) that a sexual 
process involving these haploid spores may be completed in an alternate 
host. 



Fig. 1. Life cycle of Amblyospora sp. in C. sal inari us. (a) Binucleate 
sporoplasm, (b-d) primary diplokaryon and stages of the 1st merogony 
in oenocytes of embryonated eggs and young larvae, (e) transitional 
diplokaryon, (f-h) secondary diplokaryon and stages of the 2nd mero- 
gony in oenocytes of newly emerged adult females, (i) sporoblast, 
(j) mature spore, (k-m) secondary diplokaryon and stages of the 2nd 
merogony in adipose tissue of young male larvae, (n) binucleate 
sporont, (o) quadrinucleate sporont, (p) octonucleate sporont, (q) 
pansporoblast containing eight sporoblasts, (r) mature haploid spore. 



22 



sporogony 



merogor 
l 



>ny | 



Qm 



/ 



merogony . 
2 I 



merogony 
2 



sporogony 



\ 




. mmm 



«*> • 




Figs. 2-21. Photomicrographs of giemsa stained and living material of 
Amblyospor a sp.; (6-10), stages in the female host; (11-12), stages 
"in the male host. X 1 ,000. 



Fig. 2. Primary diplokaryon. 

Fig. 3. Dividing diplokaryon. 

Fig. 4. Dividing diplokaryon. 

Fig. 5. Intermediate diplokaryon. 

Fig. 6. Secondary diplokaryon. 

Fig. 7. Dividing diplokaryon. 

Fig. 8. Divided diplokarya. 

Fig. 9. Sporoblast. 

Fig. 10. Mature spore, Nomarski phase. 

Fig. 11. Secondary diplokaryon. 

Fig. 12. Dividing diplokaryon. 

Fig. 13. Dividing diplokaryon. 

Fig. 14. Dividing diplokaryon. 

Fig. 15. Binucleate sporont. 

Fig. 16. Quadinucleate sporont. 

Fig. 17. Octonucleate sporont. 

Fig. 18. Pansporoblast with eight sporoblasts. 

Fig. 19. Spores in pansporoblast membrane. 

Fig. 20. Mature spores, Nomarski phase. 

Fig. 21. Macrospores, Nomarski phase. 



24 



* >Ji 




^ • 



14 



11 IP 12 



# 
J 



16 " 17 



18^ 19^^ / *° V V V 21 



Fig. 22. Sagittal section through the thorax and first few abdominal 
segments of a fourth instar male larva of C. sal inarius infected with 
Amblyospora sp. X 60. 



Fig. 23. Amblyos pora sp. -infected oenocyte containing numerous diplo- 
karya lying next to the ovaries of a newly emerged adult female C. 
sal inarius. X 390. 



Fig. 24. A mblyospora sp. -infected oenocyte containing mature spores in 
close association with the developing oocytes of an adult C. sal ina riu 
female 48 hr after a blood meal. X 340. 



Abbreviations: Fb, uninfected fatbody; I, infected fatbody; Oe, infected 
host oenocyte; 0o, host oocyte; Ov, host ovary; S, mature 
spore. 



26 











0o ^ 
3 




Figs. 25-44. Electron micrographs of the developmental stages of 
Amblyosp ora sp. in C. salinarius; (25-31 stages in the female host; 
(32-457 stages in the male host? 



Abbreviations: CM, cytoplasmic membrane; D, diplokaryon; EN, endospore 
wall; EX, exospore wall; MG, metabolic granules; N, N-<- 
Nc = microsporidium nucleus(i); Nfb, host fatbody nu- 
cleus; NM, nuclear membrane; No, host oenocyte nucleus; 
P, polaroplast; PC, polar cap; PF, polar filament; PM, 
pansporoblastic membrane; PV posterior vacuole; RER, 
rough endoplasmic reticulum; Sb, sporoblast; SC, synapto- 
nemal complex; SW, spore wall. 



Fig. 25. Recently extruded binucleate sporoplasm observed in the hemo- 
coel of an adult female 60 hr following a blood meal. X 10,700. 

Fig. 26. Heavily infected oenocyte from a recently emerged adult fe- 
male containing numerous secondary diplokarya (D) in cross and longi- 
tudinal section. X 4,100. 

Fig. 27. Enlarged, fusiform, secondary diplokaryon. Note the arrange- 
ment of the two nuclei (N ] and N 2 ) . X 6,900. 

Fig. 28. Young sporoblast with developing polaroplast (P) and polar 
filament (PF) from an adult female 42 hr following a blood meal. 
Note the arrangement of the nuclei which remain in the diplokaryotic 
state. X 8,800. 



28 




&*• I 



*> 



*7 



28 



Fig. 29. Mature sporoblast from an adult female 44 hr following a blood 
meal. Note the large conspicuous posterior vacuole (PV), relatively 
thin spore wall (SW) and the uniform thickness of the polar filament 
(PF). X 14,900. 

Fig. 30. Fully mature spore from an adult female 48 hr following a 
blood meal. Note the attachment of the polar filament (PF) to the 
inner surface of the spore wall (SW) in the polar cap (PC) region. 
X 17,100. 

Fig. 31. Mature spore in the process of forcibly discharging its sporo- 
plasm through the everted polar filament (PF). From an adult female 
60 hr following a blood meal. X 10,000. 

Fig. 32. Heavily infected oenocyte from a first instar male larva con- 
taining numerous primary diplokarya (D). X 4,000. 



33 





- 

...No. 


• 
v. 








grf 








&Jtf 


IS-T^D 




■J&*~ 




\ 








,i >, 


••* 






.JK'irf- . 








'**T/ r v 








V,ft 








• 








31 



32 



Fig. 33. Isolated diplokaryon (D) within the cytoplasm of an individual 
fatbody cell of a late first instar male larva. X 4,800. 

Fig. 34. Diplokaryon in the state of mitotic division. Note the simul- 
taneous dividion of the nuclei (N-j and N 2 ) . X b,700. 

Fig. 35. Secondary diplokaryon showing the distinct separation of the 
two nuclei (N-| and N 2 ) . X 8,100. 

Fig. 36. Diplokaryon at the onset of sporogony. Note the physical 
separation of the nuclear membranes (NM) (arrow and insert) and the 
beginnings of the panosporoblastic membrane (PM). X 7,400. Insert 
X 18,000. 



32 







WF V- » * : 



• • • jr 




Fig. 37. Early binucleate sporont. Note the metabolic granules (MG) 
released by the microsporidium which collect within the pansporo- 
blastic membrane ( PM) . X 7,000. 

Fig. 38. Binucleate sporont. Note synaptonemal complex (SC) within 
the nucleus and the concentric stacks of rough endoplasmic reticulum 
(RER). X 4,500. 

Fig. 39. Meiotically dividing sporont. X 7,200. 

Fig. 40. Quadrinucleate sporont showing three of the four nuclei (N-j - 
N 3 ). X 7,400. 



34 




Fig. 41. Young sporoblasts (Sb) contained within a pansporoblastic 
membrane (PM) . X 4,400. 

Fig. 42. Mature sporoblast or immature spore showing the developing 
polar filament OPF) and spore wall (SW). X 14,500. 

Fig. 43. Fully mature spore. Note the thick exospore wall (EX), con- 
spicuously lamellated polaroplast (P), single nucleus (N) and abruptly 
constricted polar filament (PF). X 14,800. 

Fig. 44. Mature macrospore. Note its similarities to the normal-sized 
spore (Fig. 43) as well as the additional coils in the polar filament 
(PF) indicative of its increased size. Because of its U-shaped na- 
ture, the nucleus appears double in this section, but is, in fact, 
single. X 11,300. 



36 




SIGNIFICANCE OF TRANSOVARIAL INFECTIONS OF 

Amblyospo ra sp. (MICROSPORArTHELOHANIIDAE) 

IN RELATION TO PARASITE MAINTENANCE IN THE 

MOSQUITO Culex salinarius COQUILLETT 



Abstract 



Adult females of Culex sal i narius , transovarially infected with 
the microsporidian Amblyospora sp. showed no significant differences 
in overall fecundity, physiological longevity and preoviposition periods 
when compared to healthy adults under laboratory conditions. Develop- 
ment times and survival rates for congenital ly infected young to repro- 
ductive age were also indistinguishable from those of healthy controls. 
A significant reduction of 52% in egg hatch was observed for infected 
eggs when compared to healthy eggs. Prevalence rates of infection for 
progeny produced by infected females declined with each successive gono- 
trophic cycle and averaged 90%. Transovarial transmission is not suffi- 
cient for the maintenance of the microsporidium in a population of 
mosquitoes. An alternate host is suggested as a mechanism whereby the 
microsporidium can re-enter a healthy mosquito population. 

Introduction 



Transovarial transmission of microsporidian parasites in mosqui- 
toes is a well-known and widespread phenomenon (Kellen and Wills, 1962a; 
Kellen et al., 1965, 1966; Chapman et al., 1966; Hazard and Weiser, 
1968; Chapman, 1974). In certain microsporidian genera it appears to 
be the principal, if not only,means of transmission (Kellen et al., 1965; 
Chapman et al . , 1966) . 

37 



38 



The expression of this host-parasite relationship is typically 
exemplified by an undescribed Ambl yospora sp. and its natural mosquito 
host,Culex s alinari us Coquillett. These microsporidians exhibit two 
developmental sequences, one in each host sex. In males, parasite de- 
velopment occurs in larvae producing massive infections which usually 
prove fatal to the host during the fourth larval stadium. In females, 
parasite development is suppressed or delayed and restricted to the 
oenocytes. Female larvae pupate normally and emerge as apparently 
healthy adults which transmit the parasite transovarially to their prog- 
eny when mated with healthy males (Kellen et al., 1965; Chapman et al., 
1966). 

Kellen et al. (1965, 1966) reported that in host-parasite relation- 
ships of this type, female fecundity was normal and concluded that trans- 
ovarial transmission was sufficient to account for the levels of infec- 
tion observed in the field. However, they presented no quantitative 
data comparing the reproductive potential of infected to healthy females. 

Surprisingly, very few studies have been conducted to demonstrate 
the sublethal effects of transovarially transmitted infections on their 
hosts. Gaubler and Brooks (1975) showed that in the corn earworm 
Heliothis zea, transovarial infections with Nosema heliothidis result 
in significant reductions in adult longevity and mating success thus 
reducing overall reproductive potential. On the other hand, Krinsky 
(1977) stated that transovarial infections of Nosema parkeri in the 
tick Ornithodoros parkeri do not appear to adversely affect host develop- 
ment or reproduction. Although several quantitative reports (Reynolds, 
1970, 1971; Anthony et al., 1972, 1978; Undeen and Alger, 1975) have 
shown reduced physiological longevity, fecundity and egg hatch for 



39 



anopheline and culicine mosquitoes perorally infected with Nosema 
algerae and P leistophora cul ici s, no such reports exist for trans- 
ovarially induced microsporidian infections in mosquitoes. 

This study was undertaken to determine the sublethal effects of 
Amblyospora sp. on the reproductive potential and physiology of C. 
sal inarius and to assess quantitatively the contribution of transovarial 
transmission to the maintenance of the infectious agent in a continuously 
breeding population of mosquitoes. 

Materials and Methods 

The healthy and Amblyospora sp. -infected colonies of C. sal inarius 
used in this study were originally obtained from Dr. Harold Chapman, 
Gulf Coast Mosquito Research Laboratory, Lake Charles, Louisiana. Adults 
were initially maintained for mating and blood feeding in separate cages 
38x46x38 cm at 24 C under natural photoperiod. Since almost all males 
from the infected colony died as fourth instar larvae, males from an 
additional healthy colony were used to inseminate females. 

A blood meal was provided when adults were four days old by placing 
guinea pigs directly into the cage. Engorged females were then removed 
and placed individually into half-pint screened paper containers con- 
taining a 60x15 mm petri dish filled with water for oviposition. Fe- 
males were maintained at a temperature of 24 C and relative humidity of 
75-80% and constantly supplied with a 5% sucrose solution as a source 
of carbohydrates. Blood meals were offered after each oviposition. 
The number of gonotrophic cycles completed and the number of eggs and 
percent hatch per female per gonotrophic cycle were recorded until ovi- 
position ceased or until the female died. Records were kept on the 



40 



time required for oviposition following each blood meal and compared 
with those for healthy control females. 

To more fully assess the contribution of transovarial transmission 
to the maintenance of the microsporidium within a mosquito population, 
it was also necessary to determine the relative survival potential to 
reproductive age of congenitally infected young when compared with 
healthy controls. This was done by recording female juvenile mortality 
and development times for individually reared egg rafts collected from 
these isolated females throughout their life time. Larval rearing was 
conducted in individual white enamel pans (18x29x4.5 cm) containing 
500 ml of well water at 25 C. The water was infused with 10 ml of an 
aqueous suspension containing 1.5% of a 3:2 mixture of dried liver pow- 
der and brewers yeast. Larvae were fed on alternate days until all had 
pupated. Pupae were isolated and the number of adult females success- 
fully emerging was tabulated. Since transmission of the parasite oc- 
curs entirely through the female line (males die as larvae), mortality 
rates and development times for males were disregarded. 

Only a proportion of the progeny produced by an infected female 
during her life time receive the infection from the maternal parent. 
Therefore, it was necessary to determine the prevalence rate of infection 
among the progeny of infected females. Since the presence of infection 
was more easily recognizable in males and previous records for a two- 
year period indicated an equal infection rate for both sexes, the 
prevalence rate of infection for female progeny was determined from 
the infection rate for sibling males produced from the same egg raft. 

In all experiments, five replicates of ten females each (healthy 
and infected) were conducted and the data were combined for statistical 
analysis. 



41 



Results and Discussioii 

The effect of the microsporidian on the physiological longevity 
of the adult female host is presented in Table 1. A similar decline 
in the number of infected and healthy females completing each successive 
gonotrophic cycle was observed. The average number of gonotrophic 
cycles completed by healthy (3.16 ± 0.16) and infected (3.22 ± 0.19) 
females did not differ significantly. 

Overall egg production (Table 2), expressed as the average num- 
ber of eggs produced by a female during her lifetime, was also statisti- 
cally indistinguishable for healthy (327.7 ± 13.0) and infected (324.3 
± 15.7) females. A significant reduction in the average number of eggs 
laid by infected females when compared to healthy controls was observed 
during the first gonotrophic cycle. However, this reduction was offset 
by an equally significant increase in egg production by infected females 
during the second gonotrophic cycle. 

While no detrimental effects could be observed for physiological 
longevity and overall fecundity, a great effect was observed in the 
viability of eggs produced by infected females. When compared to 
healthy controls, infected eggs showed a 52% reduction in overall hatch 
(Table 2). This difference was found to be highly significant (p < 0.01) 
The reduction in hatch was manifest during the first three gonotrophic 
cycles only and the degree of hatch reduction actually attributed to 
the infection (% healthy hatch minus % infected hatch) was reduced with 
each successive gonotrophic cycle. 

The preoviposi tion period or time required to develop and lay eggs 
after a blood meal was not significantly different for healthy and in- 
fected females (Table 3). 



42 



Once hatched, juvenile females showed little or no effect from 
the infection. Development times (Table 3) and survival rates (Table 4) 
of congenitally infected young to reproductive age were indistinguish- 
able from those of healthy controls. 

The prevalence rate of infection among adult female progeny pro- 
duced by infected females was 90% (Table 5). The percentage of adult 
females acquiring the infection from the maternal parent declined with 
each successive gonotrophic cycle. 

Utilizing data collected on the reproductive potential of infected 
females, prevalence rate of infection among progeny and survival poten- 
tial to reproductive age of congenitally infected young, I determined 
quantitatively the contribution of transovarial transmission to the 
maintenance of the microsporidium in a continually breeding population 
of mosqui toes. 

Using the above parameters, Fine (1975) proposed a model which 
could be used to determine the prevalence rate of infection among adults 
of the progeny generation where transovarial transmission was the sole 
mechanism for transmission of the parasite. The model is defined below: 

B [B a «r (1 - B fl + B^) + B Q aV ( 1 - B Q + B a a - B a ar»)] 

a 6 [B a ar (1 - B a + B a a) + B a av ( 1 - B a + B^ - B a ar)}~ + 
(1 - B a + B a a - B a ar) ( 1 - B a + B a a - B a <xv) 

where: 

B' a = prevalence rate of infection among adult progeny 

B a = initial prevalence rate 

v = maternal vertical transmission rate, the prevalence rate of 

infection among progeny of infected females when mated with 

uninfected males 



43 



v = paternal vertical transmission rate, the prevalence rate 

of infection among progeny of infected males when mated with 

uninfected females 
a = relative fertility (number of progeny) of infected adults 

when compared with their uninfected peers 
3 = relative survival potential (to reproductive age) of con- 

genitally infected young when compared with uninfected young 

This model assesses the contribution of transovarial transmission 
to the prevalence rate of infection in subsequent generations. By re- 
peatedly substituting the solution B' a for B a , one can determine the 
prevalence rates of infection that would be found in successive genera- 
tions of hosts . 

In this model I began with an initial prevalence rate {B a ) of 
50% which was arbitrarily chosen. The maternal vertical transmission 
rate (r) was 0.9 as 90% of the progeny produced by an infected female 
during her lifetime were themselves infected. Since transmission of 
the microsporidium occurs entirely through the female line, v, the pa- 
ternal vertical transmission rate was 0. Infected eggs showed a 52% 
reduction in hatch when compared to healthy controls, therefore relative 
fertility of infected adults (a) was 0.48. And since survival rates of 
congenital ly infected young to reproductive age were identical to 
healthy controls, relative survival potential (3) was 1. 

Applying these values to the equation I generated a curve de- 
scribing the levels of infection with Amblyospora sp. for a theoretical 
population of C. sal inarius where infections are maintained by trans- 
ovarial transmission alone (Fig. 45). These calculations show a 



44 



dramatic decline in infection rates of adult progeny from 50% to less 
than 1% within six host generations. 

The 52% reduction in hatch of egg produced by infected females 
relative to healthy controls and the escape of 10% of the adult female 
progeny from infection significantly reduce the number of females capa- 
ble of transmitting the parasite to subsequent generations. With 
each generation proportionally more healthy females are produced thus 
reducing the prevalence rate of infection over a period of time. 

Based on these results, and assuming that values derived from 
laboratory studies are similar to those existing under field conditions, 
I can conclude that transovarial transmission, by itself, is not capable 
of maintaining the microsporidium in a continually breeding population 
of C. sal inarius . Some other mechanism must exist whereby the parasite 
can re-enter a healthy population. Otherwise, infections would rapidly 
disappear. 

These findings are most interesting since in host-parasite re- 
lationships of this type, transovarial transmission has been reported 
to be the sole mechanism by which transmission of the parasite occurs 
(Kellen et al . , 1965; Chapman et al . , 1966) and all attempts to trans- 
mit the microsporidium horizontally by feeding spores produced in male 
larvae back to larval hosts have been unsuccessful (Kellen and Wills, 
1962a; Chapman, 1974). At the same time, infection rates, determined 
from field collected eggs for natural mosquito populations, have re- 
vealed values ranging from 6-17% (Kellen and Wills, 1962a; Kellen et 
al., 1966; Chapman et al., 1967). 

Based on these reports and the data presented herein, I believe 
an alternate host may be infected by the larval spores and eventually 
bring the microsporidium back to the mosquito host. 



45 



TABLE 1 



Physiological longevity of healthy and Ambly ospora sp.- 

infected C. sal inarius . Number of females completing 

each gonotrophic cycle. 



Gonotrophic cycle Healthy Infected 

I 50 50 

II 45 43 

III 38 36 

IV 19 22 

V 6 10 

Overall average 3 3.16 ± 0.16 3.22 ± 0.19 1 



a Average/ female ± S.E. 
Not statistically different from healthy mean. 



46 



c o 

>> II 



cr 


1/1 




=1 


C 




U 


s_ 


on 




<D 


c 






o. 




C 


to 


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47 



TABLE 3 



Developmental periods for healthy and Ambl yospora sp. 
infected C_. sal in arius (average days/female ± S.E.) 





No. of 
observations 


Juvenile 

development 

period 


Preovi position 

period 


Healthy 
Infected 


158 
161 


9.45 ± 0.10 
9.51 + 0.09 a 


5.05 ± 0.06 
5.10 ± 0.05 a 



a Not statistically different from healthy mean. 



48 



TABLE 4 



Survival rates for healthy and Amblyospora sp. -infected 

juvenile female C. sal inarius expressed as the % of the 

total larval hatch surviving to reproductive age 

(females only) 





Total larval 
hatch 3 


Total no. females 

surviving to 
reproductive age 


% 


Healthy 
Infected 


12,472 
5,937 


4,348 
1,952 


34.9 
32. 9 L 



a Includes males and females and assumes 1:1 sex ratio. 

b 
Not statistically different from healthy mean. 



49 



TABLE 5 



Prevalence rate of infection among adult female progeny 
produced by infected females with each gonotrophic cycle 



Gonotrophic cycle 



Total 


no. c 
from 


if adult female 
infected femal 


progi 
es 


sny 


Healthy 




Infected 


% 


infected 







646 




100 


97 




890 




90.2 


65 




146 




69.2 


27 




60 




69.0 


9 




12 




57.1 



I 

II 
III 

IV 
V 



overall average 198 1754 90.0 



CD -t-> i— 



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i — n3 


<D 


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U O 


SZ 


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o. 


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BIOGRAPHICAL SKETCH 

Theodore G. Andreadis was born on March 22, 1950, in Chelsea, 
Massachusetts. He attended secondary school in Hanover, Massachusetts, 
and graduated in 1968. In September of the same year, he entered the 
University of Massachusetts at Amherst. He received the Bachelor of 
Science degree in Fisheries Biology in 1972. 

In January of 1973 he began his graduate studies in Entomology 
at the University of Massachusetts under the direction of Dr. Donald W. 
Hall. Here, he served as a graduate research assistant studying the 
defense reactions of mosquitoes to nematode parasites and received the 
Master of Science degree in January 1975. 

Following the completion of his studies at the university, he 
joined the staff of the Cape Cod Museum of Natural History in Brewster, 
Massachusetts, where he served as a field naturalist and education in- 
structor. 

In September 1975 he entered the Department of Entomology and 
Nematology at the University of Florida and began his doctoral studies. 
During this time he served as a graduate research and teaching assis- 
tant and was the recipient of the Entomological Society of America's 
Southeastern Branch Student Award. 

He has recently accepted a position at the Connecticut Agricul- 
tural Experiment Station in New Haven, Connecticut, and plans to con- 
tinue research in the field of insect pathology. 



58 



59 



He holds membership in the Society of Sigma Xi, the American Assoc- 
iation for the Advancement of Science and the Entomological Society of 
America. 



I certify that I have read this study and that in my opinion it 

conforms to acceptable standards of scholarly presentation and is fully 

adequate, in scope and quality, as a dissertation for the degree of 
Doctor of Philosophy. 



Do nald W. Hall , Chairma n 
Assistant Professor of Entomology 



I certify that I have read this study and that in my opinion it 

conforms to acceptable standards of scholarly presentation and is fully 

adequate, in scope and quality, as a dissertation for the degree of 
Doctor of Philosophy. 



/ 



)a 



James L. Nation 
Professor of Entomology 



' ( 



I certify that I have read this study and that in my opinion it 

conforms to acceptable standards of scholarly presentation and is fully 

adequate, in scope and quality, as a dissertation for the degree of 
Doctor of Philosophy. 



) 




Thomas J. Walker' 
Professor of Entomology 



I certify that I have read this study and that in my opinion it 

conforms to acceptable standards of scholarly presentation and is fully 

adequate, in scope and quality, as a dissertation for the degree of 
Doctor of Philosophy. 



*3u^J^} 



Jonathan Reiskind 

Associate Professor of Zoology 



This dissertation was submitted to the Graduate Faculty of the College 
of Agriculture and to the Graduate Council, and was accepted as partial 
fulfillment of the requirements for the degree of Doctor of Philosophy. 

December 1978 



a iv College of Agricultu 



Dean, Graduate School 



fl