Group Title: Life cycle and epidemiology of Amblyospora sp. (Microspora: Thelohaniidae) in the mosquito Culex salinarius Coquillett /
Title: Life cycle and epidemiology of Amblyospora sp. (Microspora: Thelohaniidae) in the mosquito Culex salinarius Coquillett
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Title: Life cycle and epidemiology of Amblyospora sp. (Microspora: Thelohaniidae) in the mosquito Culex salinarius Coquillett
Physical Description: x, 59 leaves : ill. ; 28 cm.
Language: English
Creator: Andreadis, Theodore George, 1950-
Publication Date: 1978
Copyright Date: 1978
 Subjects
Subject: Microsporidia   ( lcsh )
Mosquitoes -- Biological control   ( lcsh )
Entomology and Nematology thesis Ph. D
Dissertations, Academic -- Entomology and Nematology -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Thesis: Thesis--University of Florida.
Bibliography: Bibliography: leaves 52-57.
Additional Physical Form: Also available on World Wide Web
General Note: Typescript.
General Note: Vita.
Statement of Responsibility: by Theodore George Andreadis.
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Bibliographic ID: UF00097465
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000013236
oclc - 04803275
notis - AAB6257

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










By

Tm Iqi.L,,,, / i, ,t 'u'C ,-.T I.," n lI


, L I'. I T j I ll f-11 F .- i ;r Tif_ T.i TH E I;i [I Ul 'ITL f11f.i, .i: I If
TH1 1.111 ii i T i l. Fi .i> II.1
'1 J 'I Ti l, .IL L -LI:I IT ii: TIl I" nI .nliFl rll-i :rfi I' : !H
LE III, r i_ f l. .:1. 11i 1f 1 .III I Il H









lir i F IT I : Fi I" l m

I.,













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 ... .

LIST OF FIGURES . . . . . . . . . vi

ABSTRACT ............ .. . . . ...... i

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 Amblyospora Hazard and Oldacre . . . . . 9

DEVELOPMENT, ULTRASTRUCTURE AND MODE OF TRANSMISSION OF
Amblyospora sp. (MICROSPORA:THELOHANIIDAE) IN THE
MOSQUITO Culex salinarius COQUILLETT . . . . .... 11

Abstract . . . . . . ......... ... 11

Introduction . . . . . . . ... . .. . 11

Materials and Methods ................. 13

Results . . . . . . . . . .... . 14

Discussion . . . . . . . . . . . . 17






iii








Page

SIGNIFICANCE OF TRANSOVARIAL INFECTIONS OF Amblyospora sp.
(MICROSPORA:THELOHANIIDAE) 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. salinarius . . . . . . . . 45

2 Egg production and hatch during each gonotrophic cycle
for healthy and Amblyospora sp.-infected C. salinarius .46

3 Developmental periods for healthy and Amblyospora sp.-
infected C. salinarius . . . . . . .. .. 47

4 Survival rates for healthy and Amblyospora sp.-
infected C. salinarius ................ .48

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













LIST OF FIGURES


Figure Page

1 Life cycle of Amblyospora 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 ............... ...... 224

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. salinarius infected with
Amblyospora sp. .................... 26

23 Amblyospora sp.-infected oenocyte containing
diplokarya lying next to the ovaries of a
newly emerged adult female C. salinarius ...... 26

24 Amblyospora sp.-infected oenocyte containing
mature spores in close association with the
developing oocytes of an adult C. salinarius
female 48 hr after a blood meal . . . . ... 26

25 Electron micrograph of a binucleate 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 . . . . . . . . . 36

42 Electron micrograph of a mature sporoblast
from the male host . . . . . . . . .. 36

43 Electron micrograph of a mature spore from
the male host . . . . . . . . . . 36

44 Electron micrograph of a mature macrospore
from the male host . . . . . . . . 36

45 Prevalence rate of infection for successive
generations of a theoretical population of
C. salinarius infected with Amblyospora sp.
where the parasite is maintained by trans-
ovarial transmission alone . . . . .. . 51


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
Amblyospora sp. (MICROSPORA:THELOHANIIDAE)
IN THE MOSQUITO Culex salinarius COQUILLETT

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 salinarius Coquillett.




2



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 Microsporidia

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 unspecialized. With the exception of the absence of mito-

chondria, their structural organization is typical of other eucaryotic

cells (Vdvra, 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 (Vdvra,

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; Lom 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 metabolically 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 (Lom 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 (Lom 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). Lom 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 (Vavra, 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 (Vavra, 1976b).

This entire developmental sequence occurs intracellularly 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 (Loubas et al., 1976; V5vra, 1976a; Hazard et al.,

1978) suggests a sexual phase.


Transmission

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 susceptability, 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 Amblyospora 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. Reduced 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. Reduced 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. Impairment 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 Amblyospora 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








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 Amblyospora 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 perorally 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 Ambyospora sp. (MICROSPORA:THELOHANIIDAE) IN THE
MOSQUITO Culex salinarius COQUILLETT


Abstract

Amblyospora sp. in Culex salinarius 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








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

1977).

Most, if not all, species of Anblyospora 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 Coquillett (Chapman et al., 1966). This study was under-

taken to describe the complete development and ultrastructure 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 californica 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.








Materials and Methods

Experimental animals

The healthy and Amblyospora sp.-infected colonies of C. salinarius

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







fixed in Carnoy's solution, embedded in paraffin, sectioned at 6 pm and

stained with Heidenhains hematoxylin and Eosin y.

For ultrastructural 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 ultrastructural 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. salinarius, 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. Ib-d, 2-4).








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.








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. Ib-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.

ln-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 ultrastructural 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.








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 ultrastructural

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 Amblyospora 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.







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.








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 ultrastructural 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 Amblyospora 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 binucleate

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







ultrastructural evidence for meiosis in Gurleya chironomi and Tuzetia

debaisieuxi, respectively. Verification of these ultrastructural find-

ings in the same species of Amblyospora 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. salinarius. (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.













merogony t
"LV



/





i( .,,


sporogony


sporogony


,'at ll


*

*- --

q P


.Q c\


b





e


ST
f


.. l
-

' *


//merogony
2

g








Figs. 2-21. Photomicrographs of giemsa stained and living material of
Amblyospora 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.















Iir
4
jk


4 5






81 9






12 13


U.-


.in ^ ^21








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


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


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



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












-4







Fb
22











ovQo
1 :



'/ ii

Ov,
' S
" ,.00








Figs. 25-44. Electron micrographs of the developmental stages of
Amblyospora sp. in C. salinarius; (25-31 stages in the female host;
T32--41 stages in the iale host.


Abbreviations:


CM, cytoplasmic membrane; D, diplokaryon; EN, endospore
wall; EX, exospore wall; MG, metabolic granules; N, N1-
N = microsporidium nucleus(i); Nfb, host fatbody nu-
cfeus; 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 (N1 and N2). 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.









A~ ifl -'.


:ii







I do
Slit








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.











/ **
{ a .


. * '",:1

. 31 :


.I
p


--D D







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 division of the nuclei (N1 and N2). X 5,700.

Fig. 35. Secondary diplokaryon showing the distinct separation of the
two nuclei (N1 and N2). 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.








n,': ''' 'I

. 't

-*.











34


S 36








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 (N1 -
N3). X 7,400.










































.*~ y..-r,"


' * t
.- < ; -. ,J '
- *..* /', .pi .'
^....,,,' ; W

ir'7./ '.W5 v;.-',.''
* i*4B *ri' ; -* Ir*.-'..


.38
<* 38


39


i.4.

i~i ?t


. ._ 40








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

Fig. 42. Mature sporoblast or imnature 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.









Fl


b. *


i-


C r.1


CM'-













SIGNIFICANCE OF TRANSOVARIAL INFECTIONS OF
Amblyospora sp. (MICROSPORA:THELOHANIIDAE)
IN RELATION TO PARASITE MAINTENANCE IN THE
MOSQUITO Culex salinarius COQUILLETT


Abstract

Adult females of Culex salinarius, 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 congenitally 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).








The expression of this host-parasite relationship is typically

exemplified by an undescribed Amblyospora sp. and its natural mosquito

host,Culex salinarius 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








anopheline and culicine mosquitoes perorally infected with Nosema

algerae and Pleistophora culicis, 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.

salinarius 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. salinarius

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








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.









Results and Discussion

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 preoviposition 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).








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 mosquitoes.

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:


S[B, e (1 B + Baa) + Bav (1 B + Baa Baar)]
a [Ba ar (1 B + + Baa) + Bav (1 Ba + Ba0 Bacr)] +
(1 Ba + a Bar) (1 Ba + Bao Baav)

where:


B, = prevalence rate of infection among adult progeny

Ba = initial prevalence rate

r = maternal vertical transmission rate, the prevalence rate of

infection among progeny of infected females when mated with

uninfected males








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

B = 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 BS for Ba, 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.) 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

congenitally infected young to reproductive age were identical to

healthy controls, relative survival potential (8) 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. salinarius where infections are maintained by trans-

ovarial transmission alone (Fig. 45). These calculations show a








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. salinarius. 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.













TABLE 1


Physiological longevity of healthy and Amblyospora sp.-
infected C. salinarius. 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 3.16 0.16 3.22 + 0.19b


aAverage/female + S.E.

bNot statistically different from healthy mean.





46






,: O l 1
C=$~m


o- ) C) U)

N- 0-. (0 LO C3)


= - C C




47






TABLE 3


Developmental periods for healthy and Amblyospora sp.-
infected C. salinarius (average days/female S.E.)


Juvenile
No. of development Preoviposition
observations period period


Healthy 158 9.45 0.10 5.05 0.06

Infected 161 9.51 0.09a 5.10 t 0.05a


aNot statistically different from healthy mean.













TABLE 4


Survival rates for healthy and Amblyospora sp.-infected
juvenile female C. salinarius expressed as the % of the
total larval hatch surviving to reproductive age
(females only)


Total no. females
Total larval surviving to
hatcha reproductive age %


Healthy 12,472 4,348 34.9

Infected 5,937 1,952 32.9b


alncludes males and females and assumes 1:1 sex ratio.

Not statistically different from healthy mean.













TABLE 5


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



Total no. of adult female progeny
from infected females
Gonotrophic cycle Healthy Infected % infected


I 0 646 100

II 97 890 90.2

III 65 146 69.2

IV 27 60 69.0

V 9 12 57.1


overall average 198 1754 90.0




































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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.




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.




Donald W. Hall, Chairman
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.




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. Walke'
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.




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




Dean, Colege o Agrical e




Dean, Graduate School




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