Title: Susceptibility of non-target organisms to Nosema algerae Vavra and Undeen, a microsporidian parasite of mosquitoes /
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Title: Susceptibility of non-target organisms to Nosema algerae Vavra and Undeen, a microsporidian parasite of mosquitoes /
Physical Description: v, 82 leaves : ill. ; 28 cm.
Language: English
Creator: Van Essen, Frank William, 1947-
Publication Date: 1975
Copyright Date: 1975
 Subjects
Subject: Antimalarials   ( lcsh )
Nosema -- Disease and pest resistance   ( lcsh )
Entomology and Nematology thesis Ph. D
Dissertations, Academic -- Entomology and Nematology -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Thesis: Thesis (Ph. D.)--University of Florida, 1975.
Bibliography: Includes bibliographical references (leaves 71-81).
Statement of Responsibility: by Frank William Van Essen.
General Note: Typescript.
General Note: Vita.
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Bibliographic ID: UF00098163
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 - 000431396
oclc - 38046190
notis - ACJ0820

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SUSCEPTIBILITY OF NON-TARGET ORGANISMS
TO Nosema algerae VAVRA AND UNDEEN,
A MICROSPORIDIAN PARASITE OF MOSQUITOES







By

FRANK WILLIAM VAN ESSEN


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


1975














ACKNOWLEDGEMENTS


I would like to express my sincere appreciation to

D.W. Anthony of the Insects Affecting Man Research Labora-

tory (USDA) for his helpful suggestions and constant

interest in my work. Ken Savage, Sherlee Oldacre and

Genie Crosby of the same laboratory were also helpful in

acquainting the author with some of the laboratory pro-

cedures employed.

Dr. Jerry Butler was always a great source of encour-

agement and a substantial help in the preparation of the

manuscript.

I would like to thank Dr. Reese Sailer for his prompt

identification of the hemipterans tested and Edwin Hazard

for his identification of the megalopteran.

John Knell was very helpful in the histological work

and Durland Fish aided in the photography.

Thanks are also due to Dr. Donald Weidhaas for allow-

ing me to use the facilities of the USDA laboratory.

Finally, I would like to thank my wife, Barbara, for

moral support during the past 3 years and for typing this

dissertation.


















TABLE OF CONTENTS


ACKNOWLEDGEMENTS . . . . .. . .

ABSTRACT . . . . . . . . .

INTRODUCTION AND LITERATURE REVIEW . . .

Potential for Control . . .....

Nomenclature and Life History . . .

Effect of Microsporidian Parasites on
Vectored Disease ....

Host Specificity of Other Microsporida

The Predators Tested . . .

Purpose of the Present Study . . .

MATERIALS AND METHODS . . .

Maintenance of Infected Mosquitoes .

Collection and Maintenance of Predators

Standard Feeding Procedure . .

Tests for Presence of Nosema . .

RESULTS . . . . .

DISCUSSION . . . . . . . . .

LITERATURE CITED.... . . . .

BIOGRAPHICAL SKETCH . . . . . .


. . ii

. . iv

. . 1

. 1

. 3

8


. 9

. 15

. 21

. 22

. . 22

. . 24

. . 26

. . 28

. . 31

. . 62

. . 71

. . 82










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


SUSCEPTIBILITY OF NON-TARGET ORGANISMS
TO Nosema algerae VAVRA AND UNDEEN,
A MICROSPORIDIAN PARASITE OF MOSQUITOES

By

Frank William Van Essen

March, 1975

Chairman: Jerry F. Butler
Co-Chairman: Donald E. Weidhaas
Major Department: Entomology and Nematology

Nosema algerae Vavra and Undeen, a microsporidian

parasite of mosquitoes, significantly reduces the longe-

vity and egg-laying capacity of the adult female and

adversely effects the developmental cycle of malarial

parasites in Anopheles albimanus Wiedemann, a vector of

the disease in Mexico and Central America.

If an organism such as N. algerae is to be released

into the environment, in an effort to control malaria,

information should be available as to the effect it may

have on organisms other than mosquitoes. Therefore,

laboratory studies were initiated to determine if various

predators of mosquito larvae would become infected with

N. algerae after having fed upon diseased larvae.

The 9 predators studied were: Anax junius (Drury)

(Odonata); Hydrophilus sp., Coptotomus interrogatus










(Fabricius) (Coleoptera); Notonecta undulata Say, Belo-

stoma fluminea Say, Ranatra australis Hungerford (Hemi-

ptera); Chauliodes rastricornis Rambur (Megaloptera);

Procambarus sp. (Decapoda); and Gambusia affinis (Baird

and Girard) (Pisces).

Test animals were continuously fed infected mosqui-

toes containing 1 x 10 to 3.8 x 10 spores per larva

for 14-24 days. Since some predators consumed up to 240

larvae, they were exposed to as many as 2.3 x 108 spores

during the testing period.

Examination of the predators at an average of 10-14

days after feeding on infected larvae was discontinued

revealed Notonecta undulata as the only susceptible host,

with infection rates averaging 47.9%.

The following tissues were found to be infected in

the adult notonectids: gut, muscle, fat body, Malpighian

tubules, tracheal epithelium, testes, brain, hypodermis

and ommatidia.















INTRODUCTION AND LITERATURE REVIEW


Potential for Control


In recent years, the problems involved with chemi-

cal control of insects, such as resistance of the

target organism, persistence of some of the chemicals

employed and the harmful effects on non-target organisms,

have created considerable interest in the use of preda-

tors, parasites and pathogens as biological control

agents. The reviews by Tanada (1959), Cameron (1963),

Jenkins (1964), Gerberich and Laird (1968), Laird

(1970), Weiser (1961, 1963, 1970), Burges and Hussey

(1971), and Chapman (1974) amply illustrate the large

amount of research that has been done in this area.

These also point out that while considerable progress

has been made, much of the work is still in the pre-

liminary stages.

The impetus for the present study arose primarily

from the work of Anthony et al. (1972). These inves-

tigators examined in the laboratory the effects of

infection by Nosema algerae Vavra and Undeen on the










fecundity and longevity of Anopheles albirnanus Weide-

mann, a vector of malaria in Mexico and Central Ameri-

ca. Infection produced by continuous exposure of lar-

val stages to a dosage of 5 x 104 spores/ml of rearing

water reduced the longevity of female mosquitoes from

32 days to 16 days at the LT-90 level. Since the ma-

laria parasite requires a minimum of 9 days to de-

velop to the infective stage, the reduced longevity

could have an adverse effect upon malaria transmission.

Their data also indicated that even at low dosages,

egg production, viability and, therefore, production

of FI generation larvae were significantly reduced.

With the aid of a population model designed to relate

the effect of the nosematosis on malaria transmission,

it was proposed that an infection which reduces the

female LT-90 by one-half would cause a reduction of

85-97% in the number of infective females produced

in a hypothetical population. Modeling was based on

previous work with malaria by MacDonald (1952) and on

various assumptions related to adult and immature sur-

vival values and rates of increase of natural popu-

lations of mosquitoes, as discussed more fully by

Weidhaas (1974).

Although Reynolds (1972) reported that his experi-

mental release of Plistophora culicis Weiser on the










Pacific island of Nauru did not adversely affect the

natural population of Culex pipiens fatigans Widerma-n,

it is felt that a similar release of N. algerae to

control A. albimanus will be much more successful.

Preliminary field tests in Panama during 1974 gave

very promising results, where, in one plot 86% of A.

albimanus present were found to be infected (Anthony,

1974).


Nomenclature and Life History


Some confusion exists in the literature regarding

Nosema stegomyiae Marchoux, Salimbeni and Simond 1903

and N. algerae Vavra and Undeen 1970.

N. stegomyiae was first described from Aedes aegypti

(Linn.) (Marchoux et al., 1903). Fox and Weiser (1959)

reported N. stegomyiae from adults of Anopheles gambiae

Giles and A. melas (Theobald) even though minor differ-

ences were observed between their material and that of

Marchoux et al. N. lutzi Kudo (Kudo, 1929) is identical

with N. stegomyiae Lutz and Splendore (Lutz and Splendore,

1908) which is also considered to be the same as N.

stegomyiae of Marchoux et al. (Fox and Weiser, 1959).

N. anophelis Kudo (Kudo, 1925a) which had also been

synonymized with N. stegomyiae (Vavra and Undeen, 1970)

was found in A. quadrimaculatus Say. This species is










now known as Parathelohania anophelis (Kudo) (Hazard

and Anthony, 1974). Canning and Hulls (1970) reported

a Nosema from a laboratory colony of A. gambiae from

Tanzania. Although these authors report the Nosema as

similar to N. stegomyiae as described by Fox and Weiser

(1959), they preferred to call it N. algerae Vavra and

Undeen. N. algerae was first reported from a laboratory

colony of A. stephensi Liston reared at the Department

of Zoology, University of Illinois but obtained from the

National Communicable Disease Center, Savannah, Georgia

(Vavra and Undeen, 1970). Hazard (1970) reported Nosema

infections in two other laboratory colonies of mosqui-

toes. Nosema was found in A. albimanus, A. gambiae,

A. quadrimaculatus and A. stephensi colonies at the

University of Maryland and in A. quadrimaculatus, A.

stephensi and A. balabacensis Baisas in colonies at the

Walter Reed Army Institute of Research, Washington, D.C.

The Nosema described by Vavra and Undeen (1970) was

found in mosquitoes obtained from the NCDC but this

colony was originally obtained from the London School

of Hygiene and Tropical Medicine, as was the colony

maintained at WRAIR. The staff at WRAIR and at the

University of Maryland often exchanged mosquitoes when

one of the insectories was not producing sufficient

numbers of mosquitoes for malaria studies. With all










this in mind, Hazard (1970) considered the Nosema in

the WRAIR and University of Maryland colonies to be

identical to the Nosema reported by Fox and Weiser (1959)

Vavra and Undeen (1970), Canning and Hulls (1970) and

Hazard and Lofgren (1971).

The material for the present study was originally

obtained from WRAIR and is considered to be N. algerae.

The following description of the life cycle of N.

algerae is taken from Vavra and Undeen (1970) and their

studies with A. stephensi. At 260 C the first spores

were usually found in the third instar about 4 days

after spores had been fed to newly hatched larvae.

Infection could be produced by feeding spores to any

larval instar. In larvae the following tissues were

infected: gut, Malpighian tubules, hypodermal cells,

muscle, fat body, tracheal epithelium, pericardial cells

and testes. Heavily infected larvae usually died before,

during or after pupation. Lightly infected larvae pupa-

ted normally. In the adult mosquito, the infection

spreads further, with the gut being the most heavily

infected. The Malpighian tubules, intersegmental

muscles, thoracic muscles, gonads, hypodermal cells,

fat bodies, tracheal epithelium and pericardial cells

were also invaded. The first detected stages of the

parasite are round or ovoid cells with 2 nucleii.










As the organism grows, its nucleii divide, resulting in

a 4-nucleate plasmodium. Usually the whole cell then

divides, giving rise to 2 daughter cells, each containing

2 nucleii. These apparently repeat the cycle. Some-

times the cell division is delayed and the 4 nuclei

divide again producing an 8-nucleate plasmodium, al-

though this stage is rare. The binucleate cells even-

tually produced from the 4 or 8-nucleate stages either

repeat the whole cycle or change into sporoblasts. Each

sporoblast then develops into a single spore, which

always has 2 nucleii. The mature spores are ovoid, with

one pole more pointed than the other. The extruded

polar filaments were about 70p long, although complete

extrusion was not confirmed. The spores average 4.34p

x 2.71P in larvae and 4.41p x 2.80p in adults.

In laboratory studies by Hazard and Lofgren (1971),

the following tissues of A. quadrimaculatus were found

to be infected with N. algerae: accessory glands, brain,

fat body, gut, Malpighian tubules, muscle, nerve ganglia,

rectum, ventral diverticula, and ventriculus. In Culex

pipiens quinquefaciatus Say, fat body, gut and Malpighian

tubules were infected. In Culex salinarius Coquillett,

fat body, gut, Malpighian tubules and muscle were infect-

ed. In Aedes aegypti, only the brain and nerve ganglia

were invaded. While A. quadrimaculatus was heavily










infected, the degree of infection in the non-anopheline

mosquitoes was markedly less.

Savage and Lowe (1970) also reported extensive

infections in A. quadrimaculatus in laboratory studies.

In some cases the infection was so intense that there

were no recognizable organs in the mosquito.

Nosema stegomyiae has also been reported in nature

from Aedes detritus (Haliday) and Anopheles maculipennis

Meigen (Tour et al., 1971). In laboratory studies,

Reynolds (1971) was able to infect Culex pipiens fatigans

by hatching mosquito eggs in water to which a known con-

centration of spores of N. stegomyiae had been added.

He concluded that N. stegomyiae would be of little value

as a biological control agent for C. p. fatigans because

of the limited pathological effects produced. In labora-

tory studies, Savage (1975) was able to produce infections

per os in the following species of mosquitoes: Anopheles

crucians Wiedemann, A. triannulatus (Neiva and Pinto),

Aedes triseriatus Say, A. taeniorhynchus Wiedemann, Culex

restuans Theobald, C. tarsalis Coquillett, C. nigripalpus

Theobald, C. territans Walker, C. tritaeniorhynchus Giles

and Wyeomyia medioalbipes Lutz. Although these mosqui-

toes were infected, only C. tarsalis and C. nigripalpus

were highly susceptible. Little pathology was noted in

the other species tested. Attempts to infect Psorophora










ciliata Fabricius and Toxorhynchites rutilus sepLc'nrio-

nalis Dyar and Knab were unsuccessful.


Effect of Microsporidian Parasites on Vectored Disease


Several workers have observed that the developmental

cycle of the malaria parasite in adult mosquitoes is

impaired by simultaneous microsporidian infection.

Garnham (1956) reported that Plistophora culicis

influenced the normal development of the oocysts and

sporozoites in laboratory colonies of Anopheles gambiae

and A. stephensi. Bano (1958) observed an inhibitory

effect on the development of Plasmodium cynomolgi Mayer

in A. stephensi, reflected primarily in low oocyst counts

and a retardation in the growth of the oocysts. Simi-

larly, Fox and Weiser (1959) fed A. gambiae on patients

with gametocytes of P. falciparum (Welch) and found that

only about 4% of mosquitoes infected with Nosema stego-

myiae developed oocysts, while 46% of the Nosema-free

mosquitoes developed oocysts. In a study of A. stephensi

infected with N. algerae, it was discovered that control

mosquitoes produced 10,000 sporozoites of P. berghei

Vincke and Lips per female salivary gland, while those

infected with the microsporidan contained only 1621 sporo-

zoites per female (Hulls, 1971). Studies by Savage et al.

(1971) indicated that when A. quadrimaculatus was infected










with Nosema algerae, fewer of the adults subsequently

developed sporozoites of P. gallinaceum Brumpt. Con-

current infection of A. stephensi with N. algerae and

P. cynomolgi produced a reduction in the mean number of

oocysts which developed in the adult female mosquito

midgut possibly because the midgut wall was so disin-

tegrated that suitable sites were not available (Ward

and Savage, 1972).


Host Specificity of Other Microsporida


Although Microsporida as a group are sometimes

considered to be rather host-specific, often this is

not the case. As pointed out by Weiser (1961), the

basic principle of old taxonomy that a new host species

automatically means a new parasite species can no longer

be considered valid.

Stempellia magna (Kudo) has been recorded in nature

from a number of mosquitoes, including Culex pipiens

Linn., C. territans Walker (Kudo, 1925b),C. restuans

Theobald (Kudo, 1962; Bailey et al., 1967; Simmers,

1974), Anopheles punctipennis (Say) (Simmers, 1974),

Aedes detritus (Tour et al., 1971), A. sierrensis

Ludlow (Clark and Fukuda, 1967) and Culiseta inornata

Williston (Simmers, 1974). Plistophora collessi Laird

was reported from Culex tritaeniorhynchus and C. gelidus










Theobald in Singapore (Laird, 1959). Species of Thelo-

hania have been reported from at least 37 species of

mosquitoes in 5 genera: Aedes, Culex, Anopheles, Culi-

seta and Psorophora (Kellen et al., 1965; Wills and

Beaudoin, 1965; Chapman et al., 1966; Anderson, 1968;

Chapman et al., 1973). In Europe, Weiser (1963) report-

ed Thelohania opacita Kudo from Aedes vexans Meigen, A.

sticticus Meigen, A. annulipes (Meigen) and A. communis

DeGeer and found T. legeri Hesse in Anopheles maculi-

pennis and A. bifurcatus Linn.

This non-host-specificity is not restricted to

mosquitoes by any means. Octosporea muscaedomesticae

Flu has been reported in nature from 2 species of

Drosophila, 2 species of Musca and 4 species of other

muscoid flies (Kramer, 1964a). Nosema king Kramer, a

parasite of Drosophila willistoni Sturtevant was found

to be infective to Phormia regina (Meigen), Phaenicia

cuprina (Weidemann) and Musca domestic Linn., although

attempts to infect the lepidopterous larvae Bombyx mori

(Linn.), Galleria mellonella (Linn.), Pseudaletia uni-

puncta (Haworth) and a beetle,Tribolium confusum Duval,

were unsuccessful (Kramer, 1964b).

Thelohania pristiphorae Smirnoff, originally des-

cribed from Pristiphora erichsonii (Hartig) was shown










to infect 10 other species of sawflies (Hymenoptern),

including 6 genera and 2 families, in laboratory feedinrT

tests performed by Smirnoff (1974). This same species

also infected 2 species of tent caterpillars (Smirnoff,

1968). Fantham and Porter (1913) reported Nosema apis

Zander as infective to a wide variety of insects, inclu-

ding bumble bees, mason bees, wasps, 4 lepidopterans,

and 3 dipterans. A species of Nosema, originally isolated

from Tribolium, infected Tenebrio molitor Linn., Galleria

mellonella and Bombyx mori per os (Fisher and Sanborn,

1962). These authors also found that 3 species of cock-

roaches, 3 species of Lepidoptera and Tribolium were in-

fected when the microsporidan was introduced surgically.

Lipa (1968) found Nosema coccinellae Lipa in 3

of 16 species of coccinellid beetles examined and Milner

(1972) found Nosema white Weiser infective to 3 species

of Tribolium and to Oryzaephilus surinamensis Linn.

Henry (1967) observed Nosema acridophagus Henry

infecting 4 species of grasshoppers in nature and reported

Nosema cuneatum Henry as experimentally transmissible to

6 species of grasshoppers (Henry, 1971a). In studies

with Nosema locustae Canning to control grasshoppers, the

same author found that 17 species had become infected

in the field in tests conducted in 1969 (Henry, 1971b)










and 20 species became infected in later tests (Henry

et al., 1973). This microsporidan is known to infect

a total of 59 species of Orthoptera including 3 families

(Henry, 1969).

In a study of Nosema oryzaephili Burges, Canning

and Hurst, Burges et al. (1971) found 8 species of

granivorous insects, including 5 beetles and 3 moths, to

be susceptible to the infection.

Perhaps the microsporida of Lepidoptera are the

best known as having wide host ranges. Nosema infesta

Hall occurs naturally in 3 species of Lepidoptera and

experimentally infected per os 11 other species, all in

different genera (Hall, 1952, 1954). Brooks (1970)

reported Nosema sphingidis Brooks ofManduca sexta (Linn.)

to be experimentally transmissible to 5 species of

sphingids and 3 noctuids. Thirteen lepidopteran species

in 5 families were susceptible to N. trichoplusiae Tanabe

and Tamashiro but 4 species of Diptera tested were not

susceptible (Tanabe and Tamashiro, 1967). Finlayson and

Walters (1957) were successful in infecting 7 lepidop-

terans and 1 dipteran with a Nosema species found in

Hyalophora cecropia (Linn.). N. bombycis Nageli is known

to infect experimentally at least 5 species of lepidop-

terans (Kudo and DeCoursey, 1940). N. destructor










Steinhaus and Hughes is experimentally infective to 9

species of moths, and Plistophora californica is infec-

tive to 9 species of Lepidoptera, 2 species of hymenop-

terous parasites and Chrysopa californica Coquillett

(Steinhaus and Hughes, 1949). Of 5 moths tested for

susceptibility to N. phryganidiae Lipa and Martignoni,

only 2 became infected in tests conducted by Lipa and

Martignoni (1960). Kellen and Lindegren (1969) were able

to infect the naval orangeworm and the greater waxmoth

with 2 species of Microsporida found in the Indian-meal

moth, but could not infect 3 other moths and 2 beetles

tested. These same authors, working with N. invadans

Kellen and Lindegren, were able to infect 6 lepidopterous

hosts (Kellen and Lindegren, 1973a). Pleistophora schubergi

Zwolfer, a microsporidan of the orange-striped oakworm,

also infected 10 other forest lepidopterous insects in

laboratory studies in Connecticut (Kava, 1973).

Several cases have been reported of a microsporidian

infection in an insect also occurring in its hymenopter-

ous parasite. Allen (1954) found a Nosema of the potato

tuberworm also present in one of its parasites, Macro-

centrus ancylivorus Rohwer. Tanada (1955) reported

Perezia mesnili Paillot as infecting both Pieris rapae

(Linn.) and its parasite Apanteles glomeratus Linn.

York (1961) reported Perezia pyraustae Paillot as










infecting 3 parasites of the European corn borer.

Smirnoff (1971) found Thelohania pristiphorae parasi-

tizing a eulophid parasite of 4 species of sawflies in

Canada.

Collections by Cort et al. (1960) revealed a Nosema

sp. infecting 12 species of strigeoid trematodes of

snails. This species was subsequently named Nosema

strigeoideae by Hussey (1971). Lie and Nasemary (1973)

found that Nosema eurytremae Canning could easily be

transmitted from one trematode species to another by

feeding spores to snails. They were also able to infect

Aedes aegypti, A. albopictus (Skuse) and A. togoi

(Theobald) by feeding them spores. Canning et al. (1974)

also found that microsporidian parasites of trematodes

were not host-specific. Dissanaike (1958) experimentally

infected 2 species of tapeworms and 2 oribatid mites

with Nosema helminthorum Moniez, a parasite of Moniezia

cestodes.

Wide host ranges are also known from other insect

parasites. For example, Helicosporidium parasiticum

Keilin was infective to 15 species of insects in 3

orders, including Culex pipiens, as well as 3 species

of mites (Kellen and Lindegren, 1973b). Mattesia is

infective to 12 species of Lepidoptera, 3 Coleoptera, 1










Siphonaptera and 1 Hymenoptera. The fungus Metarrhiziurn

anisopliae (Metschnikoff) is known to attack over 50

species of insects in 7 orders and Beauvaria bassiana

(Balsamo) may have an even wider host range (Baile,, 1971).


The Predators Tested


The following predators were used as test animals

in this study: Gambusia affinis (Baird and Girard),

Anax junius (Drury), Hydrophilus sp., Coptotomus interro-

gatus (Fabricius), Notonecta undulata Say, Belostoma

fluminea Say, Ranatra australis Hungerford, Chauliodes

rastricornis Rambur, and Procambarus sp.

Gambusia affinis is a prolific live-bearer known

as the mosquito fish. Because of its widespread use in

mosquito control programs (Bay, 1967; Gerberich and Laird,

1968; Hoy et al., 1971), it is now considered to be the

most widely distributed fresh-water fish in the world

(Bay, 1967). Females can begin to produce young at 6

weeks of age, although more often they overwinter before

beginning to breed at 8-10 months. In their lifetime,

3-4 broods are produced, each containing up to 300 young.

Given favorable conditions, several thousand specimens

can be obtained by September or October from as few as

50-100 stocked in a pond in June (Bay, 1967).










Anax junius is a large species of dragonfly in the

family Aeshnidae. The nymph passes through 13 instars.

In laboratory rearing studies conducted by Calvert (1934),

the minimum time from egg to adult was 177 days. A

reported development time of 4 months in nature has not

been verified. Calvert (1934) states that the age of

a given nymph can be determined only within an approxi-

mation of 1 or 2 instars because different individuals

and different parts of the same individual grow at dif-

ferent rates. Odonata are often reported as predators

of mosquitoes. Sailer and Lienk (1954) reported Odonata

as consuming Culiseta, Anopheles, and Culex larvae in

Alaska. Pritchard (1964) reported Aeshna, Leucorrhinia

and Sympetrum as feeding upon Culex territans and Ano-

pheles earlei Vargas. Brooke and Proske (1946) reported

Anax junius as a predator of A. quadrimaculatus using

radioactive tracers. In laboratory studies, Lee (1967)

found Anax junius to feed readily upon Culiseta incident

Thomson.

Arnett (1960) reported 206 species of hydrophilids

occurring in the United States. Eggs are laid in silken

cases, all larvae pass through 3 instars and all except

Enochrus pupate out of water in earthen cells. The larval

stage of Hydrophilus triangularis (Say) is said to be 15










days while H. obtusatus Say became full grown in ;ibouit

30 days, depending on temperature. A species of Enochris

was reported to have a larval period of 2 months (Bnlduf,

1935). H. obtusatus was an important predator of Aedes

stimulans Walker and A. trichurus Dyar in radioactive

tracer studies conducted by Baldwin et al. (1955) and

James (1961). James (1965) reported Enochrus and Berosus

as important predators of Aedes atropalpus (Coquillett).

Two species of Tropisternis were also found to be mos-

quito predators in Canada (James, 1967). In laboratory

studies, Clarke (1938) claimed that 3 larvae of H. trian-

gularis consumed 1000 mosquitoes each in one day.

Arnett (1960) reported 329 species of Dytiscidae

from the United States. All have 3 larval instars. Eggs

of Coptotomus are inserted in submerged plants and pupa-

tion takes place out of water in a mud cell. The larval

stage of Dytiscus semisulcatus Muller varied from 16 days

at 260 C to 15 weeks at 60 C. In Cybister lateralimar-

ginalis DeGeer the total larval stage was passed in 42-

47 days in the summer in Germany. Hydroporus depressus

Fabricius was reported to have a 21-day larval stage

(Balduf, 1935). Service (1973) reported Agabus and

Dytiscus as the most important predators of Aedes cantans

(Meigen) using the precipitin test. A large number of










dytiscids, including Coptotomus interrogatus, have been

reported as important predators of mosquitoes, including

Aedes atropalpus, A. trichurus, A. stimulans and A.

communis in radioactive tracer studies by James (1961,

1965, 1966, 1967). Two species of Laccophilus readily

fed on Culex pipiens and a chironomid in laboratory

studies (Roberts et al., 1967).

Notonectids, or back swimmers, have long been known

as predators of mosquito larvae (Hungerford, 1917; Clark,

1928; Toth and Chew, 1972a) and recent laboratory studies

indicated a preference of Notonecta undulata for mosquito

larvae when other food was available (Ellis and Borden,

1970). Twinn (1931) reported a Notonecta sp. as feeding

on Culex larvae. Lee (1967) reported N. undulata as an

important predator of mosquito larvae. Hinman (1934)

referred to N. undulata as the most voracious of all

hemipterans known to him. Clark (1928) found notonectids

to feed on a wide variety of organisms including Copto-

tomus interrogatus. In laboratory rearing studies, Toth

and Chew (1972a) reported 57.3 days as the minimum genera-

tion time for N. undulata at 230 C. Eggs hatched in 14

days, nymphal stages I-IV required 22.1 days, stage V

required 12.7 days,and 8.5 days was necessary from adult

to the appearance of the first eggs. Nymphs and adults

were cannibalistic.










Belostomatids are known as effective predators of

mosquitoes (Jenkins, 1964). Severin and Severin (1911)

reported Belostoma fluminea as feeding on many insects

including N. undulata. Blatchley (1926) refers to this

species as a predacious carnivore. Young (1921) reported

a Belostoma sp. as a natural enemy of Aedes aegypti and

Culex pipiens fatigans. In radioactive tracer studies,

Baldwin et al. (1955) found B. fluminea preying upon A.

stimulans and A. trichurus. Eggs of Belostoma are glued

to the backs of the males and are carried there until

they hatch. In Canada, Brooks and Kelton (1967) report

that the 5 nymphal instars require about 7 weeks to

develop. In laboratory studies by Torre-Bueno (1906a),

3 specimens of B. fluminea were reared from eggs. The

time required from the date of oviposition to mature

adults was 43, 53 and 54 days, respectively.

Ranatra australis was reported by Blatchley (1926)

to be the most common Ranatra in Florida. Ranatra fusca

Beauvois, a close relative, was reported to feed on

Anopheles and Culex larvae by Jenkins (1964), A. quadri-

maculatus by Brooks and Proske (1946) and on Aedes spp.

by Baldwin et al. (1955). Several other genera of

Nepidae are also reported as predators of mosquito larvae

(Jenkins, 1964). Torre-Bueno (1906b) presented the










life cycle of R. quadridentata Stal. The nymph passed

through 5 instars and matured in 61 days in the labora-

tory. In nature it is known to overwinter in the boLtom

mud.

Cuyler (1958) reported only 2 species of Chauliodes

in North America. Usinger (1971) reported that eggs are

deposited on stones, branches or bridges that overhang

the water. The length of the larval period is not known

but may be 3 years. In discussing Megaloptera in general,

Pennak (1953) reported the larvae as passing through 10

instars in 2-3 years. They pupate on shore. Chauliodes

sp. was reported as a predator of Aedes stimulans and A.

trichurus by James (1961). The hellgrammite, Corydalus

cornutus (Linn.) is a close relative.

Procambarus is a large genus of common crayfish, with

many species occurring in Florida (Hobbs, 1942). Cray-

fish do not leave the parent until the third instar and

individuals hatching in the spring have 6-10 molts by

autumn. Copulation usually occurs in the first autumn

of life although some females are not sexually mature

until early the following spring. Subsequent to the

first mating season, most crayfish have only 2-4 molts

before death (Pennak, 1953). Barnes (1963) reports most

decapods as predators or predators and scavengers. Pennak










(1953) refers to crayfish as mainly scavengers, but

they will eat animal food. In the present study, nos-

quitoe larvae were fed upon quite readily.


Purpose of the Present Study


If an organism such as Nosema algerae is going to

be released into the environment in an effort to control

anopheline mosquitoes and hopefully malaria, information

about its effects on organisms other than mosquitoes

should be obtained. Therefore, studies were initiated

at the USDA, ARS,Insects Affecting Man Research Labora-

tory, Gainesville, Florida, to see if various predator's

of mosquito larvae would become infected with Nosema

after having fed upon diseased larvae.















MATERIALS AND METHODS


Maintenance of Infected Mosquitoes


A supply of Anopheles quadrimaculatus larvae infected

with a strain of Nosema algerae originally obtained from

the Walter Reed Army Institute of Research, Washington,

D.C. (Hazard, 1970) was maintained continuously in the

laboratory. First instar larvae obtained from the labora-

tory stock colony were exposed to a concentrated aqueous

spore suspension of approximately 5 x 106 spores/ml in a

small petri dish for 1-2 hours and then transferred to

white enamel rearing pans (30 x 19 x 5 cm) containing

about 700 ml of water. One hundred fifty to 200 larvae

were reared in each pan on a diet consisting of a 1:1:1

mixture of liver powder, dried brewer's yeast and finely

ground hog supplement (Ralston Purina Company). A slurry

was made with 2 gm of this mixture per 100 ml of water and

then 15 ml of the slurry was added to each pan. No addi-

tional food was added until day 4 when a small amount of

hog supplement was dusted on the water surface. This was

continued daily until day 12 or 13. Pupation began on

day 9 or 10. Pupae were then collected with a pipette and










transferred to cages (28 x 22 x 22 cm) to await ;idult

emergence.

Adults of the stock laboratory colony were maintained

at 290C and 70% R.H. under natural light conditions

(through windows in the room) during the day and were

provided with only a small light in one corner of the

room during the night. Cages measured 76 x 76 x 64 cm.

Rabbits with closely shaved backs were provided for 2 hours

each day for the females to feed on and cotton wads soaked

in 20% sugar water were provided as a supplemental food

source. The cage was supplied with a white enameled pan

(25 cm diam. x 7.6 cm deep) containing about 2.5 cm of

distilled water for oviposition. All viable eggs float.

For hatching, eggs were placed in a pan (30 cm diam. x 5 cm

deep) containing distilled water mixed with 200 mg of

brewer's yeast. Newly hatched larvae were transferred to

the 30 x 19 x 5 cm pans and reared as mentioned above at

290C and 70% R.H. under conditions of constant light.

All rearing of infected larvae, as well as maintenance

of all predators tested and exposure of the larvae to

Nosema, was carried out in one room under the following

conditions: 300C, 70% R.H. and a 16 hr photoperiod.

Infected adult mosquitoes were maintained in a separate

room at 26.50C, 70% R.H. and a 12 hr photoperiod. Cotton










balls soaked in 20% sugar water were provided is a food

source for the adults.

Adult mosquitoes that were about 1 week old were

crushed in a Ten Broeck glass tissue grinder to produce

the spore suspension that was immediately used to infect

the next set of first instar larvae. The spore suspension

was filtered through KimwipesTM to remove large debris

and was then centrifuged at 3000 rpm (1700G) for 5 minutes

to help eliminate bacteria and other extraneous material

before being fed to the larvae. In this manner, a con-

stant supply of infected larvae was available to be fed

to the test organisms.


Collection and Maintenance of Predators


Gambusia affinis and Chauliodes rastricornis were

collected from Hatchet Creek near Gainesville, Florida.

The crayfish, Procambarus sp., was collected from Lake

Alice on the University of Florida campus, Gainesville.

All other predators studied were collected in the Gaines-

ville area from a small temporary pond near Castle Gate

Mobile Home Park.

Pregnant females of Gambusia were held in 20 t

aquaria and fed on uninfected mosquito larvae (any species

that was available) until the young were born. These were










then fed on progressively larger larvae until fourth

instar A. quadrimaculatus could be taken (at about 1

month), whereupon they were separated and kept indivi-

dually in pint Mason jars for the subsequent feeding

tests using infected larvae. Aeration was unnecessary

when the water was changed about once a week, and mor-

tality was negligible using this simple system.

Adults of Notonecta undulata were held in 20

aquaria until egg-laying took place. As suggested by

Ellis and Borden (1969) and Toth and Chew (1972b), rubber

sink matting suspended in the water provided a substrate

for the eggs as well as a resting place for adults (Figs.

1-3). Mating presumably had already taken place in the

field since eggs were usually seen only 2 or 3 days after

the adults had been brought into the laboratory. Since

the young are cannibalistic, they were separated into

small glass rearing dishes of about 300 ml capacity (11.5

cm diam. x 5 cm deep) as soon as the eggs hatched. As

with young Gambusia, progressively larger mosquito larvae

(uninfected) were used as food until fourth instars could

be taken (about 1 week), at which time infected larvae

were used for the duration of the tests. Again, aeration

was unnecessary when the water was changed once a week.

Mortality was negligible after an initial die off due to










improper hatching or molting, cannibalism before sepa;ra-

tion, etc.

Crayfish were collected in 3 size ranges. Small

and medium size specimens were maintained separately in

the small glass rearing dishes. Larger individuals were

kept in enamel mosquito rearing pans when the bowls ap-

peared to be too small and confining.

All other predators studied were held separately

in the small glass rearing bowls.

In all cases, except the 7 large specimens of Procam-

barus, it was the young, immature stages of the predators

that were exposed to the Nosema.

These organisms were chosen because they are known

mosquito predators, they were readily collected in nature,

they were rather easy to maintain in the laboratory and

they represent a fairly wide range of animals taxonomically

speaking.


Standard Feeding Procedure


Each individual predator was held in a separate con-

tainer, as described above, to make sure that each had

eatenaknown number of mosquitoes. Infected fourth instar

A. quadrimaculatus that were 10 days or older were used

as food and were counted out for each predator. The










number fed per ind ividual varied according t to hle :;ize

of the test animal, from small notoncctids eit ing, only

2 or 3 a day to large crayfish consuming 20-25 a day

(Tables 1-9). Each container was checked the following

day to determine how many larvae had been ingested during

the previous 24 hr period.

Each day 10 larvae were chosen at random, macerated

in a tissue grinder, and spore counts were taken with the

aid of a hemacytometer. Knowing the number of larvae and

the amount of water they were crushed in allowed a calcu-

lation of the average number of spores of N. algerae per

mosquito. Thoughout the testing, the number of spores

per larva was within the range of 1.0 x 105 to 3.8 x 106.

By keeping a record of the number of mosquitoes fed to

each predator and of the number of spores per mosquito,

it was possible to calculate the approximate number of

spores ingested by each test animal.

In most cases they were fed infected larvae daily

for about 3 weeks and then examined 7-10 days later

(Tables 1-9) to determine if they had become infected

with N. algerae.










Tests for Presence of Nosema


Several standard laboratory techniques were utilized

in searching for the presence of Nosema in the predators

tested. Spores of most Microsporida are highly refractive

and can be easily observed using phase microscopy (Fig. 4),

The usual procedure was to cut the specimen in two loca-

tions, smear some body fluid and tissue on a slide, add

a cover slip and immediately look for spores. On the

larger species (i.e.,Anax, Gambusia), organs were dissected

out and smeared, while on very small ones (i.e., Coptotomus)

the whole body was smeared. Giemsa smears were also pre-

pared in order to identify the presence of vegetative

stages of the pathogen (Figs. 5-8). The specimen or tissue

was crushed on a slide, allowed to air dry, fixed in metha-

nol for 5 minutes, stained in a 9:1 mixture of pH 7.41

buffer and Giemsa for 10 minutes, washed gently with tap

water and allowed to air dry (Humason, 1967).

If spores were discovered in the initial body fluid

smears, then permanent wet mounts were prepared. Tissue

was smeared on a cover slip and dropped immediately into

aqueous Bouin's fixitive for at least 2 hrs. It was then

soaked in 70% ethanol overnight, treated with iron alum

mordant for 5 hours and stained overnight in Heidenhain's

hematoxylin diluted 1:1 with distilled water. The pre-










paration was then destined in iron alum, dehydrated in

graded ethanols, cleared in xylene and mounted (Humason,

1967).

As additional proof that the infection was indeed

Nosema algerae, some infected material was retained for

electron microscope preparation, using 4% gluturaldehyde

and 1% osmium tetroxide as fixatives, propylene oxide

as the transitional solvent and Epon-Araldite as the

embedding medium (Pease, 1964; Kay, 1965) (Figs. 15-16).

Also, some material was saved to be fed back to mosquitoes.

In this case, infected predators were crushed in a tissue

grinder and a concentrated spore suspension was produced.

First instar Anopheles albimanus and A. quadrimaculatus

were then exposed to the resulting spore suspension, as

previously discussed, under the standard laboratory con-

ditions and then transferred to the rearing pans until

old enough to check for infection.

In order to determine what tissues became infected,

paraffin sections were prepared. Infected predators were

fixed in Carnoy's solution for 2-4 hrs, rinsed in 70%

ethanol for 1 hour and then soaked in 70% ethanol over-

night. The tissue was dehydrated in graded ethanols,

rinsed in a 1:1 mixture of absolute ethanol and butanol,

soaked in 3 changes of 100% butanol, transferred to a 1:1

mixture of butanol and paraffin, infiltrated with paraffin










under vacuum and finally embedded in fresh paraffin

Tissue thus embedded was sectioned and allowed to air

dry on slides before being stained. Paraffin was re-

moved in 3 changes of xylene, tissue was hydrated through

graded ethanols to distilled water, soaked in iron alum

for 5 hrs and stained overnight in Heidenhain's hematoxy-

lin. The preparation was then destined in iron alum,

counterstained in Eosin, dehydrated through graded ethan-

ols back to xylene and mounted in HistocladTM (Humason,

1967).













RESULTS


The results of the tests are presented in Tables

1-9. In most cases, more than one attempt was made

to infect the test animals. This is indicated by

test no. 1, 2, etc. The number of animals fed may

include ones that had died only 2 or 3 days after the

feeding of infected larvae had ceased. But the ma-

jority were examined on the day indicated in the last

column or, where large numbers were involved, on the

following day as well. As mentioned previously, the

predators were exposed continuously to Nosema-infected

larvae and the extent of the exposure is recorded in

column 3. The maximum time available for development of

the pathogen, therefore, is the sum of columns 3 and 7.

Careful records were also kept of the number of larvae

fed to each predator and the number of spores contained

within each larva. This data is presented in columns 4 and

5. Many of the predators consumed a large number of infec-

ted mosquitoes and consequently were exposed to a high do-

sage of spores. For example, in 1 test with Anax, 222 lar-

vae were consumed by each nymph over a 24-day period (Table

1). During this time they ingested over 190 million spores

each. In a test using Gambusia 125 larvae were consumed,










representing about 230 million spores (Table 9). In 2

instances (Procambarus and Hydrophilus), size variation

in the collected material was obvious and so they were

divided into 2 or 3 groups. This is indicated in paren-

theses as small, medium and large under column 2.

Table 10 summarizes the data for all tests conduc-

ted, indicating the number of tests, the number of pre-

dators tested and the percentage of those which subse-

quently became infected. Only 1 of 9 species tested was

susceptible to Nosema algerae after having fed upon

diseased larvae under laboratory conditions.

Nosema infection was found during all 4 tests con-

ducted with Notonecta. The percentage did not vary appre-

ciably from test to test and averaged 47.9%. All adults

used during these tests were collected from the same tem-

porary pond. Eggs were laid by only 2 or 3 of the females

collected for each test and it was felt that transovarial

transmission from the female to the egg of a natural micro-

sporidian infection resembling N. algerae could have ac-

counted for the infections observed in the nymphs. There-

fore, during the last test (#4), 20 controls, which were

fed uninfected larvae, were set up along with the test

animals. None of the control nymphs were found to be

infected, while 56.2% of the test insects were. Also,

many more adults than were necessary to recover enough










eggs for testing were collected. After enough eggs were

laid, all 28 specimens collected were sacrificed and

examined for the presence of a natural infection. All

were negative.

As further proof that N. algerae was the pathogen

involved, infected notonectids were ground up to produce

a spore suspension to feed back to mosquitoes. Four

notonectids, containing an average of 5.5 x 107 spores

per individual, were used for this purpose. Both Ano-

pheles quadrimaculatus and A. albimanus became infected

after an exposure of 1 hr to spore suspension of 5.5 x

106 spores/ml. This procedure was performed twice, once

for test 3 nymphs and once for test 4 nymphs. On both

occasions, 90% of A. quadrimaculatus and 100% of A. albi-

manus were found to be infected after 8 days.

Some material was prepared for electron microscope

observations in order to compare the appearance of N.

algerae in N. undulata and in A. quadrimaculatus. Fixa-

tion of the microsporidan in the notonectid was not as

good as in the mosquito. Nevertheless, comparison of

spore size, spore shape, spore wall thickness, number of

turns of the polar filament and the binucleate nature of

the spore indicate the parasite is identical in the 2

hosts (Figs. 15-16).










Microscopic examination ofiparaffin sections revealed

the following tissues to be infected in the adult nolo-

nectids: gut, muscle, fat body, Malpighian tubules, tra-

cheal epithelium, testes, brain, ommatidia and hypodermis

(Figs. 9-14). The most heavily infected tissues were fat

and thoracic musculature. The Malpighian tubules, testes

and gut tissue were moderately infected. Tracheal epi-

thelium was usually lightly infected. At times, infection

was so heavy that identification of the tissue involved

was quite difficult. In some specimens, the fat body was

practically non-existent. The thoracic musculature was

always much more heavily infected than the abdominal mus-

culature. Spores were generally evenly dispersed through-

out the tissue, although they did occur in batches in the

testes. This is not surprising, as the testes are com-

posed of small sacs or follicles.

Examination of infected A. quadrimaculatus adults re-

vealed the most heavily infected tissues to be gut and

Malpighian tubules. The ommatidia and fat body were some-

what less heavily infected. Infection of the tracheal

epithelium was much heavier than that of N. undulata,

while the thoracic musculature and fat body usually were

not as heavily infected as in the notonectids.

Mortality records were only kept for tests 3 and 4.

In test 3, 12 of 52 backswimmers tested died by the end










of the examination period (23.1%) and all 12 were infect .'

In test 4, 5 of 16 died (31.2%) and all 5 were infected

This compares with a mortality of 20% for the controls.

It is assumed that had the tests been continued for a

longer period of time, a higher rate of mortality would

have been recorded for the test animals since 10 of those

still alive in test 3, and 4 still alive in test 4 were

subsequently found to be infected and probably would have

died.

The tissue specificity of the microsporidan in N.

undulata and mosquitoes, the comparison of spores of the

pathogen in N. undulata and A. quadrimaculatus using

electron microscopy, the positive results obtained in

feeding spores from infected notonecti back to mosqui-

toes, and the results of the fourth test with notonectids

in which controls were not infected while 56.2% of the

test insects were, all demonstrate transmission of N.

algerae to N. undulata.

Thus, of 9 mosquito predators tested for per os sus-

ceptibility to Nosema algerae, only Notonecta undulata

became infected. This result was obtained in all 4 tests

in which Notonecta was used.














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Table 10. Summary of per os susceptibility tests for
Nosema algerae to 9 predators of mclsquitop-.


Total
No.
No. of animals "
Host Tests fed infected



Odonata

Anax junius 4 25 0

Coleoptera

Hydrophilus sp. 2 21 0

Coptotomus 1 6 0
interrogatus

Pnmiptera

Notonecta undulata 4 127 47.9

Belostoma fluminea 3 22 0

Ranatra australis 3 17 0

Megaloptera

Chauliodes 1 5 0
rastricornis

Decapoda

Procambarus sp. 1 25 0

Chordata

Gambusia affinis 4 45 0





















Fig. 1 Set up for rearing Notonecta undulata,
showing 20t aquarium and sinkmatting
suspended in the water. Note several
adults on the water surface and Mn the
matting.


















Fig. 2 Close up of Fig. 1, showing an adult
N. undulata, eggs, and one newly-
ha tcher-yiymph.























Fig. 3 Close up of N. undulata egg on a por-
tion of sink matting.


Fig. 4 Spores
instar
an oil
scopy.


of Nosema algerae from fourth
A. uarimacuatus as seen in
suspension using phase micro-
(1000 X)






49





















Fig. 5 Spores of N. algerae from adult N.
undulata fixed Vinmethanol and
stained in Giensa. (1000 X)


















Fig. 6 Binucleate vegetative stages of N.
algerae from adult N, undulata frxed
in methanol and staineT ITri'Gemsa.
(1000 X)









*t, I'

* C


a L


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e '


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a


0


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/


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Fig. 7 Binucleate stages of N. algerae from
fourth instar A. quadrimaculatus with
several spores noticeable in top Let t
portion of the photograph, Giemsa
stain. (1000 X)


















Fig. 8 Later binucleate stage cf N. algerae
from adult N. undulata, Gi,.msa stain.
(1000 X)





















Fig. 9 Spores of N. algerae (s) in muscle
tissue of adutI N. urdulata. (250 Y)




















Fig. 10 Heavily infected portion o4 muscle
of adult N. undulata. (250 X)










i'I
I.
i.


F'
I





















Fig. 11 Infection of gut (g), Malpigbian
tubule (m) and testes (t) of adult
N. undulata. (250 X)


















Fig. 12 Spores of N. algerae (s) in testes
of adult N. undulata. (250 X)




























,A t -




















Fig. 13 Section through brain of adult N.
undulata showing spores of N.
algerae (s). (250 X)




















Fig. 14 Spores of N. algerae (s) in hypodermal
cells of adult N. undulata. (250 X)




















Fig. 15 Electron micrograph of spore of N.
algerae from adult A. quadrimacu-
atus, showing the T n'jeieii (n
and coils c the polar fiiamcnt (1).
(20,000 Y)















Fig. 16 Electron micrograph of spore of N.
algerae from adult N. undulata.
(20,000 X)






























t --- --I


0



Si-


I A


~,

,sg~Q~*,~;;~IILP~aZ


"tr
`4














DISCUSSION


As previously indicated, although wide host ranges

are known among various terrestrial insects, this pheno-

menon is not as widespread in aquatic species. It is

therefore somewhat surprising that in the present study

Nosema algerae was infective to Notonecta undulata. A

species of Notonecta is also susceptible to Coelomomycec

(Ignoffo and Hink, 1971) and Laird (1971) thinks it con-

ceivable that C. notonectae Bogoyavlensky represents an

infection derived from larval mosquito prey. This is the

only report of fungi of the genus Coelomomyces outside of

the Diptera (Roberts, 1970).

Having a wide host range is not necessarily detri-

mental. In the case of Nosema oryzaephili, which can

infect 8 species of granivorous insects, including beetles

and moths, its non-host-specificity is a distinct advan-

tage in a situation where many pests often occur together.

Although it has not been demonstrated in the field, the

infectivity of N. algerae to a large number of mosquito

species may also be an advantage. And, as McLaughlin

(1971) points out, the larger the host range, the more

persistent the pathogen is likely to be in nature.

62










It should be emphasized that the infection of I7.

undulata was produced in the laboratory ;ind not in lie

field. Infectivity in the field is undoubtedly a nulLi-

factorial quality and changes of various factors in nature

that would allow infection to occur in a new host are

perhaps not probable. However, there is no way of knowing

whether the changes that are necessary will or will not

occur (Bailey, 1971). As pointed out by Laird (1973),

the fungus, Beauvaria bassiana, has been shown to repro--

duce in bees under laboratory conditions, but no inifec-

tion of bees has ever been shown in nature, despite the

world-wide distribution of the fungus and the fact that

bees are among the best known of insects.

Another point is that the infection was produced by

continuous exposure of the animal to a large dosage cf

spores a dosage that is not likely to occur in nature.

And, since the tests were conducted in the laboratory,

the animals involved may have been stressed, although all

other animals tested would also have been stressed and

yet were not susceptible.

The results also indicate that those which do be-

co"e infected eventually die of the disease.

In studies with N. algerae, Undeen and Maddox (1973)

were able to infect Heliothis zea (Boddie) per cs but were

unable to infect Anopheles atroparvus Van Thiel, a Chiro-











nomus species, Corydalus cornutus (Linu.), Blaberus dis-

coidalis Serville and 3 other species of lepidopterans.

Although not a natural infection route, injecting animals

with spores produced infections in 11 species of insects

and a crayfish. They were unsuccessful when injecting a

tri Lad platyhelminth, an annelid, a frog and a mc.se.

Savage (1975) was also able to infect H. zea, Musca

domistica Linn. and Stomoxys calcitrans (Linn.) with N.

algerae. In fact, mass production of spores has been

undertaken using H. zea, although 24 hr food deprivation

is required to produce a large number of spores. These

hosts are not likely to come in contact with spores re-

leased for control of mosquito larvae.

Although Notonecta undulata became infected in the

laboratory, another aspect to be considered is that infec-

ted mosquitoes served as the entire diet, while in nature,

other prey may be available. Quantitative information as

to what percentage of the natural diet is composed of mos-

quitoes is not known. In most cases this would vary from

place to place depending on the variety of other food pre-

sent. In Frink Spring, California, mosquitoes were the

most available natural food for backswimmers (Toth and Chew,

1972i) and, as already mentioned, these authors reported

N. undriliata as preferring mosquitoes to other prey that

wa; available to them in the laboratory. Ellis and Borden










(1970) considered N. undulata well adapted as a rosqu to

predator. But, in a locality where a wide variety of

aquatic life is present, mosquitoes would certainly not

be the sole source of food, as notonectids are known to

feed on a large number of prey species (Hungerford, 1917;

Clark, 1928).

The tissue specificity of N. algerae in N. undulata

was very similar to that of N. algerae in A. stephensi

(Vavra and Undeen, 1970). Both hosts showed infections

in gut, muscle, fat body, Malpighian tubules, tracheal

epithelium, testes and hypodermis. Vavra and Undeen

(1970) reported the gut as the most heavily infected

tissues in adult mosquitoes. Fat and muscle were the

most heavily invaded tissues in N. undulata in the present

study. Hazard and Lo.gren (1971) reported essentially tie

same results with Nosema infections in A. quadrimaculatus

They found the fat body, gut, muscle and Malpighian tu-

bules were the tissues most heavily attacked. Although

these authors found Culex pipiens quinquefasciatus and

Culex salinarius to be less susceptible to Nosema algerae,

the fat body, gut and Malpighian tubules were the most

commonly invaded tissues in both species. They found the

muscles rarely infected in C. p. quinquefasciatus and

never found the testes or ovaries to be invaded in any of










the 3 species of mosquitoes mentioned above. In Aedes

aegypti only nerve tissue was parasitized. Fox and Weiser

(1959) also found gut, Malpighian tubules and fat to be

the most heavily infected tissues in their studies of Ano-

pheles gambiae. Although the trachea were not invaded,

the ovaries were. Esophageal and salivary gland tissues

were the first to be invaded, the infection spread along

the alimentary tract, including the Malpighian tubules,

and then to fatty tissues, muscle, nerve and finally to

gonadal tissue. In heavy infections the parasites were so

numerous that none of the midgut tissue could be seen.

Similarly, Savage and Lowe (1970) found Nosema infections

of A. quadrimaculatus to be so intense at times that in-

vaded tissues could not be recognized. This was also the

case with N. undulata in the present study.

Savage (1975) found a wide range of susceptibility

of various mosquitoes to N. algerae. Anopheline mosqui-

toes, such as A. albimanus and A. stephensi were highly

susceptible while Culex nigripalpus and Culex tarsalis

appeared to be of intermediate susceptibility. In Aedes

taeniorhynchus and A. aegypti, the infection was very

limited, usually involving only nerve tissue. Psorophora

ciliata and Toxorhynchites rutilus septentrionalis, 2

predacious species of mosqitoes, were not susceptible.










Vavra and Undeen (1970) found that Anopheles atropar-

vus was not susceptible to N. algerae, altb- ..'i Aedes

aegypti was. They reported the same tissues infecLtd in

A. aegypti and Anopheles stephensi.

As mentioned previously, Savage (1975) was also able

to infect Musca domestic, Stomoxys calcitrans and a

chironomid. All 3 were lightly infected. Gut and fat

tissue were invaded in the housefly and chironomid, just

as they were in all previously reported susceptible hosts

except A. aegypti tested by Hazard and Lofgren (1971).

Muscle tissue was the most heavily invaded tissue in the

housefly, as was the case with N. undulata in the preseitt

study.

It appears that the primary sites of infection of

N. algerae are fat, muscle, gut and Malpighian tubules.

These tissues were reported to be infected in nearly all

species of insects that have been tested. While the gut

was most heavily invaded in the majority of mosquitoes

studied, the thoracic musculature was the most heavily

attacked in at least 2 non-mosquito hosts examined M.

domestic and N. undulata.

Anopheline mosquitoes appear to be the primary hosts

of N. algerae. But, Vavra and Undeen (1970) could not

infect Anopheles atroparvus even though the name tissue










which were infected in Anopheles stephensi were also

invaded in Aedes aegypti. Histological studies, as well

as laboratory transmission experiments, indicate that N.

undulata, a hemipteran, may be more susceptible to N.

algerae than some species of mosquitoes. Obviously, more

host susceptibility studies should be undertaken, not

only with N. algerae, but with other pathogens as well.

Further studies with notonectids could involve the

determinati of minimum dosage levels required to produce

infection, elucidation of the sequence of tissues invaded

as the infection proceeds, discovery of any effects th

parasite may have on adult longevity and fecundity, ard

determination of how the pathogen might be transmitted.

Although transovarial transmission has not been reported

for N. algerae, eggs could possibly be mechanically con-

taminated in N. undulata since the testes are ln,'wr to

become infected.

Why N. undulata became infected and the other predators

did not, is not readily apparent, especially when one con-

siders that other hemipterans were tested, as well as a

megalopteran and several coleopterans, which are inter-

mediate to the Diptera and Hemiptera phylogenetically

speaking. Smith (1973) points out that conditions in which

the microsporidan extrudes its polar filament are the mirin










factors in determining its host-specificity. Similarly,

Fisher and Sanborn (1962) believe specificity is deter-

mined by properties of the host's gut, observing that

insects are more susceptible during or immnldiitely f ,lw-

ing a molt when the cuticular lining of the foregut and

midgut is shed. In the present study, the predators we're

fed infected larvae for a prolonged period of time so

that molting would occur during the time of exposure.

Age is also a factor. At least in some species,

younger individuals are more susceptible than older ones

(C-nning, 1970). Therefore, as indicated previously, yuong

immature stages of each predator were chosen for study.

As indicated by Baily (1971), tests with beneficial

insects are too few in number to give a clear picture of

the potential host ranges of most insect pathogens.

Marshall Laird (1970, p. 578) put it this way:


Clearly, a great deal more work lies ahead
of us before the integrated control of
mosquitoes becomes more than a fond hope...
And, by tempering with a degree of realism
our enthusiasm for the course that we all
desire, let us ensure that our anxieties
about preservation of the ecosystem will
become the more credible to those charged
with the responsibility for executive deci-
sions.


In the final analysis, the true susceptibility of

non-target organisms to various insect pathogens will











only come with detailed studies of field tests. Never-

theless, laboratory studies should be undertaken as

the first line of investigation. As an extension of

the present study, tests on scavenger-type organisms,

such as blackflies, psychodids, annelids an( other i -

vertebrates, would also be beneficial.















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BIOGRAPHICAL SKETCH


Frank William Van Essen was born on February 6, 1947

in Pittsburgh, Pennsylvania. He attended high school in

Bethel Park, a suburb of Pittsburgh, and graduated in

1965. He then attended Allegheny College in Meadville,

Pennsylvania and received a B.S. in biology in 1969.

During this time, one summer was spent at the Theodore

Stone Laboratory of Ohio State University at Put-in-Bay,

Ohio.

He was awarded a graduate assistantship in Entomology

at the University of Delaware to work on mosquito biology

and received his M.S. in June, 1971. He married the

former Barbara Jean Ross of Norfolk, Massachusetts on

August 29, 1970.

Following a summer of work on insect identification

for the Department of Entomology at Delaware, hf began

work toward the PhD degree in September, 1971.

During his stay in Florida, a strong interest in

karate was fostered and he was promoted to black belt in

January, 1974 in Cuong Nhu, a Vietnamese style. He has

also had an avid interest in motorcycles.










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 Phi losr-phv





/Jerr 4 Butler, Chairm in
Associate Professor of EntomoLngy

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.




Dcnald E. Weidhaas, Co-haii-man
Professor of Entomology

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of s-holarly
presentation and is fully adequate, in scope and quality,
as a dissertation for'the degree of Doctor of Philosophy.




Dale H. Habeck
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.




Minter J. WestfTll s
Professor of Zoology










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 dissertatiop for the degree of Doctor of Philo.sophy.



,> i
Darrell W. Anthony
Assistant Prolessor o\ Eiif oolopv


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 Philrsophy.

Tirch, 1?75



an ollegef Agr cilt e


Dean, Galuate SK-FT.




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