Field and laboratory studies of the mosquito fungal pathogen Coelomomyces stegomyiae

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Field and laboratory studies of the mosquito fungal pathogen Coelomomyces stegomyiae
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Thesis (Ph. D.)--University of Florida, 1989.
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Includes bibliographical references (leaves 138-145).
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by Alan D. Gettman.
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FIELD AND LABORATORY STUDIES OF THE MOSQUITO
FUNGAL PATHOGEN COELOMOMYCES STEGOMYIAE




















By

ALAN D. GETTMAN


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

UNIVERSITY OF FLORIDA


1989















ACKNOWLEDGMENTS


This study could not have been conducted without the

support of many others. Dr. Donald W. Hall's wisdom, gentle

guidance, and patience kept me on course. His generous

support was invaluable in many ways. I was indeed fortunate

to have him as a committee chairman and my fortune continues

in our friendship. The remaining committee members also

deserve recognition for their thoughtful guidance and

generous support. To Drs. Jerry F. Butler, J. Howard Frank,

and James W. Kimbrough I am most grateful. Thank you,

gentlemen. I've always felt lucky to have such a fine

committee.

Dr. Howard C. Whisler provided necessary materials and

technical advice. The project could not have begun without

his ambition and for his continual encouragement I am most

grateful. Dr. Gregory S. Wheeler generously assisted with

statistical analyses. We are highly significant friends.

The Entomology Department at the University of Florida is

comprised of an impressive group of faculty and staff

members. I've learned a great deal from them and enjoyed

their company as well. A number of employees at the USDA

IAMARL deserve mention. Dr. Jeffrey C. Lord, Dr. Albert H.








Undeen, Dr. James J. Becnel, Tokuo Fukuda, and S. W. Avery

were sources of both information and humor. My tenure at U.

F. was shared with many wonderful colleagues. I retain many

fond memories. Thank you for your company, fellow students.

I could never have made the journey without support

from some special persons close to home. My parents, John

and Janet Gettman, have been a continual source of love and

support. For them, I am eternally grateful. Finally, I am

indebted to my wife, Eileen. She perservered with the

greatest love, patience, and understanding. I am a most

fortunate husband.


iii















TABLE OF CONTENTS


page

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

ABSTRACT..................... .............................. vi

INTRODUCTION............................................... 1

CHAPTERS

I LITERATURE REVIEW...................................4

II A FIELD EVALUATION OF THE FUNGAL
PATHOGEN COELOMOMYCES STEGOMYIAE
AGAINST AEDES AEGYPTI............................... 24

Introduction....................................24
Materials and Methods (1986 Study)..............27
Results and Discussion (1986 Study).............36

Materials and Methods (1987 Study)..............44
Results and Discussion (1987 Study).............55

Materials and Methods (1988 Study)..............81
Results and Discussion (1988 Study).............83

Summary (1986 1988) ...........................95

III HOST SUSCEPTIBILITIES TO
COELOMOMYCES STEGOMYIAE............................ 97

Introduction....................................97
Materials and Methods (4 Aedine Species).......100
Results and Discussion (4 Aedine Species)......103

Materials and Methods
(Larval Predators Study).....................107
Results and Discussion
(Larval Predators Study).....................112

SEM Studies of Resting Sporangia...............115









IV COELOMOMYCES STEGOMYIAE DIPLOID
SPORE YIELD FROM 3 AGE CLASSES OF
PHYLLOGNATHOPUS VIGUIERI..........................123

SUMMARY.................................................... 130

REFERENCES..................................................... 138

BIOGRAPHICAL SKETCH... ..................................146














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

FIELD AND LABORATORY STUDIES OF THE MOSQUITO
FUNGAL PATHOGEN COELOMOMYCES STEGOMYIAE

By

Alan D. Gettman

December, 1989

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


The mosquito fungal pathogen Coelomomyces stegomyiae

was evaluated during 3 seasons as a microbial control agent

against Aedes aegypti. In 1986, 5 open-field sites were

established, each consisting of 16 automobile tires as

larval habitats and 1 caged chicken for adult mosquito

bloodmeals. Pupal production and larval infection data

acquired twice weekly from the 8 treatment and 8 control

tires at each site indicated that pathogen activity was at a

very low level. Phyllognathopus viquieri, the obligate

intermediate copepod host, failed to survive during the

summer when water temperatures were as high as 36*C but

survived under cooler temperatures later in the season.

During 1987 and 1988, similar records were acquired

from 72 tires located at 1 site supplied with 4 chickens.

Black shadecloth supported on a frame above the plot








provided shade, consequently lowering water temperatures.

During 1987, pupal production was ca. 12% lower from

treatment tires during the latter part of the season.

Infected larvae were noted at various times in 15 of the 36

treatment tires. Infections ranged from 0% to 100% with an

average of 37.8% when calculated from the 125 positive

observations only. Copepods maintained sizeable populations

in all tires for the entire season.

During the latter part of the 1988 season, pupal

production was ca. 43% lower from treatment tires. Infected

larvae were recorded from 30 of the 36 treatment tires at

various times. Again, copepod populations were maintained

in all tires and were not significantly different between

treatment and control tires.

In a replicated comparative susceptibility study,

mortality rates for Ae. aegypti, Aedes albopictus, Aedes

triseriatus, and Aedes bahamensis larvae averaged 82%, 36%,

98%, and 3%, respectively. The pathogen failed to complete

its development in the vast majority of Ae. triseriatus

larvae that died. In a separate experiment, larvae of

Toxorhynchites splendens and Corethrella appendiculata were

found refractory to the pathogen. Only 2 of 150

Toxorhynchites rutilus rutilus larvae tested became

infected. When 3 age groups of P. viQuieri were exposed to

the pathogen, early stages produced more spores than adults.




vii


L














INTRODUCTION


Aedes aegypti is established in all of the major

tropical and subtropical regions of the world. It is of

particular importance as a vector of dengue and yellow fever

in lesser developed nations. While neither of those

diseases is currently established in the United States, the

possibility of introduction is real. Introduction would

most likely occur in a southern city where the vector is

established. Cities such as Miami and New Orleans are

particularly at risk because many people from tropical

endemic regions arrive there daily.

A subsequent outbreak of dengue could become

established in a community if 1 or more infected persons

were bitten by the vector. This vector's close association

to human habitation, particularly in lesser developed

neighborhoods, makes it an ideal vector of human disease.

Several mosquito abatement programs in Florida target Ae.

aegypti in order to lessen the probability of a disease

outbreak and to reduce its status as a nuisance pest.

Programs rely on insecticides and source reduction as the

main tools. Several cities in Florida are currently

experimenting with mass production and release of the










predatory mosquitoes Toxorhynchites rutilus rutilus and

Toxorhynchites splendens.

The current field study was designed to assess the

potential of reducing Ae. aegypti populations by using the

fungal pathogen Coelomomyces stegomyiae. It has been

demonstrated in numerous laboratory studies to be of

potential because it can produce very high mortality rates

in the larval stage.

Automobile tires were used as natural larval habitats

because they are regarded as the most prolific sites for

larval development for Ae. aegypti and several other species

in Florida and in other parts of the world. Aedes

albopictus was recently introduced into the western

hemisphere in shipments of used tires from Asia. This

species also commonly develops in tires and that fact has

prompted many states to control used tire shipments and

disposal via legislation. A renewed interest in tire

recycling research has also emerged, in part due to the

introduction of Ae. albopictus.

Tires were observed for the presence of infected

larvae, which, during the latter stages, are readily

apparent without the aid of a microscope. Pupae were

counted and regarded as a gauge of fungal activity, since

pathogen induced mortality usually occurs in the larval

stages.










This study also considered the copepod Phyllognathopus

viquieri because it is an obligate intermediate host for the

pathogen. It is a frequent inhabitant of treeholes and

plant leaf axils throughout the world. Populations of the

copepod were monitored in both treatment and control tires

in order to assess any relationship between population size

and fungal activity.

Two laboratory studies were designed to address the

impact that release of Coelomomyces stegomyiae into the

Florida environment might have on 7 additional dipterans.

One study was conducted to determine the relative

susceptibilities of 4 aedine mosquitoes found in treeholes

and artificial containers in Florida. Another study was

designed to test for pathogen susceptibility in 3 beneficial

dipteran predatory species (2 native and 1 exotic species)

that also inhabit treeholes and artificial containers in

Florida.

Finally, C. stegomyiae haploid spore production was

compared among groups of copepods that had been infected at

different ages. This experiment was designed to consider

the influence that copepod population age structure has on

mosquito infection levels.















CHAPTER I
LITERATURE REVIEW


Introduction

The genus Coelomomyces was originally described from

the sporangia found in a single Aedes aeqypti larva (Keilin

1921). (For convenience, the full scientific names of all

organisms discussed in the text are presented in Table 1-

1). Following that description, Coelomomyces species have

been studied as potential biological control agents for

mosquitoes. At least 64 species and variants have been

described from infections in 1 or more of some 140 mosquito

species in 21 genera (Couch and Bland 1985). Additionally,

four species (C. chironomi, C. beirnei, C. canadensis, and

C. tuzetae) can infect several species in the family

Chironomidae (Rasin 1928, Weiser and Vavra 1964, Manier et

al. 1970, McCauley 1976, and Nolan 1978). An account of a

Coelomomyces species in S. metallicum (Garnham and Lewis

1959) is reported but considered questionable. Excepting

these accounts, infections in insects are apparently limited

to the family Culicidae (mosquitoes).

General bibliographies of Coelomomyces species are

reported by Strand and Chin (1977), McNitt and Couch

(1978),Castillo and Roberts (1980), and Roberts et al.










Table 1-1. List of organisms discussed in the text.


Diptera:
Chaoboridae:
Corethrella appendiculata Grabham

Culicidae:
Aedes aeqypti (Linnaeus)
Aedes africanus (Theobald)
Aedes albopictus (Skuse)
Aedes australis (Erichson)
Aedes bahamensis Berlin
Aedes cantator (Coquillett)
Aedes melanimon (Meigen)
Aedes polynesiensis Marks
Aedes simpsoni (Theobald)
Aedes sollicitans (Walker)
Aedes taeniorhynchus (Wiedemann)
Aedes triseriatus (Say)
Aedes trivittatus (Coquillett)
Aedes vexans (Meigen)
Anopheles costalis Loew
Anopheles crucians Weidemann
Anopheles freeborni Aitken
Anopheles funestus Giles
Anopheles gambiae Giles
Anopheles pharoensis Theobald
Anopheles quadrimaculatus Say
Anopheles tesselatus Theobald
Armigeres obturbans (Walker)
Armigeres subalbatus (Coquillett)
Culex annulirostris (Skuse)
Culex antennatus Becker
Culex duttoni Theobald
Culex modestus Fica
Culex pipiens Linnaeus
Culex quinquefasciatus Say
Culex restuans Theobald
Culex tigripes Grandpre & Charmoy
Culiseta inornata (Williston)
Opifex fuscus Hutton
Stegomyia scuttelaris Walker
Topomyia yanbarensis Mayagi
Toxorhynchites brevipalpus Theobald
Toxorhvnchites gravelyi (Edwards)
Toxorhynchites kaimosi (van Someren)
Toxorhynchites rutilus rutilus (Coquillett)
Toxorhynchites rutilus septentrionalis (Dyar & Knab)
Toxorhynchites splendens (Wiedemann)
Tripteroides bambusa (Yamada)
Wyeomyia vanduzeei Dyar & Knab










Table 1-1 continued


Simuliidae:
Simulium metallicum Bellardi

Copepoda:
Bryocyclops fidjiensis Lindberg
Cyclops navus Herrick
Cyclops (Acanthocyclops) vernalis Fischer
Cyclops (Acanthocyclops) viridis (Jurine)
Elaphoidella taroi Chappuis
Phyllognathopus viquieri (Maupus)
Tigriopus angulatus (Lang)
Tigriopus fulvus (Fischer)

Ostracoda:
Heterocypris incongruens Sars
Potamocypris smaragdina (Vavra)

Amphibia:
Hyla squirella Sonnini & Latreille

Fungi (Blastocladiales: Coelomomycetaceae):
Coelomomyces africanus Walker ex Couch & Bland
Coelomomyces beirnei Weiser & McCauley
Coelomomyces canadensis (Weiser & McCauley) Nolan
Coelomomyces chironomi Rasin
Coelomomyces dodgei Couch & Dodge ex Couch
Coelomomyces iliensis Dubitskij
Coelomomyces indicus Iyenger ex Iyenger
Coelomomyces macleayae Laird ex Laird
Coelomomyces musprattii Couch
Coelomomyces opifexi Pillai & Smith
Coelomomyces psorophorae Couch ex Couch
Coelomomyces punctatus Couch & Dodge ex Couch
Coelomomyces stegomyiae Keilin
Coelomomyces tuzetae Manier et al.
Coelomomyces utahensis Romney, Couch & Nielsen
Coelomomyces walker van Thiel ex van Thiel








7

(1983). The latter two include brief abstracts. Additional

general works include those by Roberts (1970), Chapman

(1974), Federici (1981), Lucarotti et al. (1985), and Couch

and Bland (1985). While most of the ca. 150 publications

are either laboratory oriented or concern host records

and/or observations of field infections, numerous works also

deal with ecological studies and, to a lesser extent, the

role of Coelomomyces spp. as biological control agents.


Host Range Data

Host ranges vary considerably, although pathogen and

host species tend to be habitat-associated, indicating

coevolution. However, that does not imply that all mosquito

species that naturally share a particular habitat are

susceptible to the same species of the pathogen. Some

Coelomomyces species have been recorded from only 1 mosquito

species while others infect numerous species from different

genera. Likewise, some mosquito species are infected by

only 1 pathogen species while others have had more than 1

Coelomomyces species reported from them. Most of the

publications concerning host ranges are records of natural

occurrences. Very few Coelomomyces species have been

extensively tested against mosquitoes in controlled studies.

Therefore, much host range data remain to be established.

Current knowledge on host ranges and the taxonomy of the

pathogen are thoroughly covered by Couch and Bland (1985).










Because natural epizootics have been reported on

numerous occasions, the genus has received considerable

interest as a potential microbial control agent.

Unfortunately, early investigators were frustrated in their

attempts to transmit the infection from mosquito to

mosquito. While that problem was eventually solved, the

lack of in vitro culture methods remains a significant

impediment to the development of Coelomomyces species as

practical biological control agents (Bland 1985).

The reason for the former problem was discovered by

Whisler et al. (1974). In the laboratory, they investigated

water from a pond that was producing C. psorophorae infected

Cs. inornata larvae. Their discovery that copepods (Cy.

vernalis in this case) are obligate intermediate hosts

marked an historic transition in Coelomomyces research

efforts. This represented the first discovery of a

heteroecious aquatic fungus and the first discovery of a

heteroecious fungal pathogen of insects. Based on their

finding, other workers subsequently noted the same

phenomenon with all Coelomomyces species investigated. Two

exceptions have been noted. The ostracods H. incongruens

and Po. smaraqdina are alternate hosts, respectively, for C.

chironomi and C. utahensis (Weiser 1976 and Whisler pers.

commun.). Apparently all other species of Coelomomyces must

undergo a stage of development in copepods.










Life Cycle

The unusual life cycle is graphically displayed in

Figure 1-1. The mosquito is considered the primary host

because the diploid stage occurs in it. The copepod is

defined as the alternate host because it contains the

haploid stage of the pathogen. This definition of host

status was established for other heteroecious fungi. The

yellow-orange resting sporangia are evident in mosquito

larvae in the latter stages of infection. Following host

death and decomposition, they are liberated into the aquatic

environment. Numerous studies have shown the sporangia to

be particularly resistant to desiccation, undoubtedly an

adaptation to ephemeral aquatic environments. Depending on

a drop in oxygen content and possibly other environmental

factors, they undergo meiotic division and subsequently

dehisce, releasing several hundred haploid motile spores.

These uniflagellate zoospores (+ and mating types) encyst

on the cuticle of copepods, penetrate, and give rise to

abbreviated hyphae that eventually occupy most of the

coelom. [The only reported exception concerns C. chironomi

infection in the ostracod H. incongruens (Weiser 1977).

Apparently, diploid spores are ingested and gain access to

the coelom by crossing the gut wall.].

Subsequently, mitotic divisions produce numerous

gametes, evident as a swarming mass of zoospores upon

copepod death. The gametes mate either within the dead

































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copepod or after their escape from the decomposed body,

forming motile diploid biflagellate spores (zygotes). The

development described above has been worked out by Whisler

et al. 1975 and 1983, Federici 1975, Zebold et al. 1979,

Travland 1979a, Wong and Pillai 1980, and others. It is

summarized by Couch and Bland (1985).

The motile zygotes encyst on the intersegmental

membranes of mosquito larvae. Each subsequently produces an

appresorium and a tube that penetrates the cuticle.

Cytoplasm is injected through this tube into a host

epidermal cell or into the hemocoel, giving rise to ovoid

hyphal bodies (hyphagens). These either adhere to various

tissues or float freely in the hemocoel. Apparently,

hyphagens can proliferate by simple division. Eventually

these bodies grow into branched mycelia that ramify

throughout the hemocoel. Mycelia are usually recognized as

units with only a few branches. Depending on the number of

parent hyphal bodies (i.e., the severity of infection),

mycelia may completely fill the mosquito hemocoel or be

rare. Resting sporangia are produced at the tips of the

branches and they eventually absorb mycelial cytoplasm,

becoming the only stage present. An infected larva may

remain alive, exhibiting sluggish behavior, for more than 1

week before succumbing to the infection. The life cycle is

completed when the sporangia are liberated from the

decomposing larva. Developmental periods in the copepod and










in the mosquito require ca. 1 week and 2 weeks,

respectively. The sequence of events that occurs in the

mosquito has been studied in detail by Martin (1969),

Whisler et al. (1983), Zebold et al. (1979), Travland

(1979a,b), Wong and Pillai (1980), and others. The subject

is adequately summarized in Couch and Bland (1985).

Three additional points deserve mention. According to

Whisler (pers. commun.) strong evidence suggests that the

pathogen can remain viable within copepods that have

encysted to survive drought conditions. If so, that

represents another resting stage, which is a remarkable

adaptation. Another fascinating adaptation concerns

dispersal. Mosquito larvae infected lightly and/or as later

instars can survive to become infected pupae. Depending on

the severity of infection and possibly the stage of the

infection, pupae may either die or produce infected adults.

Adult males and females may then contain sporangia in the

hemocoel, which presumably can get dispersed, by an infected

adult which dies and decomposes at another breeding site.

This phenomenon has been reported for several species of

mosquito and several species of the pathogen. Walker (1938)

reported C. africanus in An. costalis male and female adults

and in An. funestus adult females. Garland and Pillai

(1979) noted C. opifexi infections in pupae and both sexes

of adult Ae. australis. Taylor et al. (1980) noted

sporangia of an unidentified species of Coelomomyces in the










hemocoel of Ae. trivittatus females. Andreadis and

Magnarelli (1984) found 1 female Ae. sollicitans with

sporangia of C. psorophorae in the hemocoel. Lucarotti

(1987) reports C. stegomyiae infections in the hemocoels of

Ae. aegypti adult females.

Finally, infected females are capable of "mock-

ovipositing" resting sporangia. Van Thiel (1954) reported

C. walker resting sporangia in the ovaries of An.

tesselatus. Lum (1963) found C. psorophorae sporangia in

the ovaries of Ae. taeniorhynchus. Kellen et al. (1963)

noted C. psorophorae sporangia in the ovaries of Ae.

melanimon. Nnakamusana (1986) reported C. stegomyiae

sporangia in the ovaries of Ae. aegypti. Finally, Lucarotti

(1987, 1988) conducted a particularly detailed study of the

phenomenon utilizing transmission electron microscopy. He

reports that C. stegomyiae hyphae can be present between the

germarium and the follicle of each ovariole in Ae. aegypti

females that have not received bloodmeals. The hyphae did

not differentiate into resting sporangia if a bloodmeal was

not taken, even after 7 days. However, differentiation was

complete 72 hours following a bloodmeal. Oviposited

sporangia were subsequently found to be viable. Gettman

(unpublished) also found "mock-oviposited" C. stegomyiae

sporangia to be viable.










Field Studies

The emphasis of most field oriented publications has

been on reporting natural occurrences, usually including

only limited quantitative and ecological detail. The

majority of these were conducted prior to the discovery that

copepods serve as obligate intermediate hosts.

Nevertheless, several studies yielded pertinent ecological

information.

Walker (1938) collected and examined thousands of An.

gambiae and An. funestus immatures and adults in Sierra

Leone in search of C. africanus. From collections made over

several years, he found infections in 544 larvae, 2 pupae,

and 56 adults (primarily in An. gambiae). That was a very

low overall infection rate, but one pond collection yielded

ca. 35% infected larvae and a subsequent experiment in a

pond yielded a 66% larval An. gambiae infection rate. He

subsequently transferred that pond's water and some sediment

into an experimental cement tank. Larvae were added on two

occasions and infection levels of 7% and 0% resulted. A

final experiment in the tank used brook water and yielded no

infected larvae.

Muspratt (1946) reported C. indicus infection levels as

high as 95% in An. gambiae breeding in temporary sunlit

pools in Rhodesia. His attempts to induce infection in

experimental pot-holes dug near an infectious pool were

apparently successful without adding inoculum. Soil










retrieved from infected areas failed to induce infection

during laboratory experiments. Muspratt (1963) returned to

the same area for further research after 15 years to find

continued C. indicus activity. He determined that the

pathogen can withstand extended dry periods and conjectured

that the sporangia are the resistant stage. Water

temperature and pH readings were not different between pools

where infections occurred and pools where they did not.

Again, he claimed infection levels of nearly 100%, even in

newly created ditches, especially those near older

infectious pools. After observing a noninfectious pool for

3 weeks, he successfully inoculated it with the resting

sporangia from 4 larvae. Laboratory experiments utilizing

soil as inoculum were principally unsuccessful.

Madelin (1968) studied the same system in the

laboratory in England. He imported soil from Muspratt's

Rhodesian location and used dried resting sporangia that

Muspratt had saved for several years. Infection trials in

plastic pans were principally negative, but it is noteworthy

that water fleas (an undetermined species not native to

England) were present in all pans that yielded infected

larvae.

In Sri Lanka, Rajapaksa (1964) organized the collection

and examination of ca. 20,000 larvae from 1,256 breeding

sites within a 1,620 km2 area. The ambitious project was

directed at documenting Coelomomyces spp. infections. Three










species of Coelomomyces were found, each infecting one of

three mosquito species. When the 3 mosquito species were

totalled, 4.4% were infected. Of these, 15.4% of Ae.

albopictus larvae were infected with C. stegomyiae.

Infected larvae were found in a total of 16 different

habitats from the 24 habitats where Ae. albopictus was

recovered. Infection levels ranged from 0% to 50% in these

habitats, including 21.5% of 396 larvae collected from

discarded tires.

Gad et al. (1967) conducted a survey of natural enemies

of the malaria vector An. pharoensis in Egypt. From a total

of the four habitats found infectious, 27.4% of Cx.

antennatus larvae were infected with C. musprattii and 4.1%

of An. pharoensis were infected with C. indicus. A variety

of habitats was sampled, but infected larvae were recovered

only from rice fields.

Pillai (1969) recorded C. opifexi infecting 55% of Ae.

australis larvae breeding in one supralittoral rock pool in

New Zealand. Subsequently Pillai (1971) found 2 foci of the

infection where a total of 21 of 400 pools were infectious.

Measurements of temperature, pH, pool size, and bottom

sedimentation revealed no meaningful differences between

infectious and noninfectious pools. However, pools with

lower salinity (those furthest from the ocean's splash zone)

were more likely to be infectious. Most importantly, he

recorded the copepod T. fulvus, among other invertebrates,










from the more sheltered pools. A Tigriopus species near T.

angulatus, also an inhabitant of these rock pools, was

subsequently shown to be one alternate copepod host for C.

opifexi (Pillai 1976).

In Russia, Deshevykh (1973) studied ecological aspects

of C. iliensis infections in Cx. modestus breeding in the

floodplain of the Ili River. He found more infected larvae

in pools with water temperatures ranging between 30*C and

39*C than at lower temperatures. During a 2-year study,

infected larvae were found from May through September, with

peak infection levels reaching 100% during July and August.

Chapman and Woodard (1966) reported natural C.

psorophorae var. psorophorae infections in Cx. restuans at

levels of 10% and 15% during December, 1964, and January,

1965, collections (respectively) from one pond in Louisiana.

They also reported the same pathogen consistently infecting

20-40% of Cs. inornata in a large saltmarsh during a

December to April collection period. Chapman and Glenn

(1972) studied C. punctatus and C. dodgei infections in An.

crucians during 4 and 2 year periods, respectively. In the

pond harboring C. punctatus, infections ranged from 12% to

67% with an average of 33.2% for the 4-year period. The

highest infection levels occurred in January, but rates

varied remarkably little throughout the year. In the other

pond, C. dodgei infections ranged between 24% and 59%, with

an average of 47.6% for the 2-year period. The highest








19

infection levels occurred in September, and rates were much

lower during midsummer than at other periods during the

year. The low summer rates were probably due to several

periods when the pond dried, according to the authors. Of

particular interest was the natural reestablishment of both

pathogens following dry periods in both ponds.

Umphlett (1968) recorded C. punctatus infections and

ecological parameters during 1 season in a lake in North

Carolina. Infection levels in An. quadrimaculatus ranged

from 6% to 80% with the highest levels occurring in August.

This followed a period of mosquito population increase in

July. High dissolved oxygen contents and high pH were noted

in a "control" area of the lake where infected larvae were

not found. Lower infection rates were noted following

periods of highest air temperatures. Based on laboratory

evidence that the higher temperatures reduce the release of

zoospores from resting sporangia, it was speculated that

high water temperatures had suppressed C. punctatus activity

in the lake. During two subsequent seasons, Umphlett (1969)

noted similar seasonal infection patterns but an overall

decline in infections over the 3 year study. A severe

drought during the final season certainly contributed to the

trend.

Couch (1972) used C. punctatus resting sporangia from

the same lake for numerous laboratory experiments and one

field experiment in North Carolina. He seeded several










ditches with resting sporangia and, over a 4-season study

period, noted infection levels ranging from 0% to near 100%,

with an average of 66%.

A particularly ambitious study was conducted by Laird

(1967 and 1985) on the Tokelau Islands in the southern

Pacific Ocean. In 1958 he located and seeded 761 Ae.

polynesiensis larval habitats with C. stegomyiae resting

sporangia. One year later 118 of these were relocated, 14

of which contained infected larvae or viable sporangia in

the sediment. Three additional previously unseeded habitats

were also found infectious, indicating some dispersal. A

return trip to the islands in 1963 yielded infected larvae

from 37% of the habitats sampled. Collections conducted by

others in 1967 and 1968 yielded very few infected larvae.

In 1980 Laird found only 7.5% of the 40 habitats examined to

be infectious. By that time the role of copepods in the

life cycle had been elucidated. Laird sampled for copepods

and found 2 harpacticoids which later proved to be

intermediate hosts of C. stegomviae. These copepods had

fortunately been established on the islands prior to the

introduction of the pathogen in 1958. That good fortune

surely enabled the establishment of the pathogen at a time

when the necessary role of copepods was unknown.

Unlike the previous studies discussed, several field-

oriented studies were instituted after the role of copepods

as intermediate hosts was discovered. From extensive









21

collections from South Dakota and Nebraska, Mitchell (1976)

recovered Ae. vexans larvae infected with C. psorophorae

from only 11 (3.1%) of the 360 collections. Only 5.7% of

the larvae from these 11 collections were infected and

overall, less than 1% of the 9,016 Ae. vexans larvae

examined were infected. The copepod Cy. navus, a known host

of C. psorophorae, was also present in some water samples.

In an extensive study of mortality factors affecting

larval An. qambiae, Service (1977) recorded C. indicus

infections in 15.9% of larvae collected from rice fields in

Kenya. Only 2.1% of larvae from pools and small ponds were

infected. He noted a patchy distribution of infected larvae

in some rice fields where certain parts contained much

higher levels (46.6%). Cyclops spp. and other copepods were

noted in higher numbers in rice fields containing infected

larvae.

Otieno et al (1985) conducted a survey of natural

enemies of An. gambiae in Kenya for a 1-year period. They

reported C. indicus infection levels of 48% to 82% in a

small, shallow, sunlit, temporary pool. Levels gradually

increased during the course of the 2 rainy seasons sampled.

A copepod host was not mentioned.

Finally, Andreadis and Magnarelli (1984) reported C.

psorophorae infection levels from 5,832 Ae. cantator larvae

examined. Infection levels ranged from 0% to 6.7% from 13

collections and infected larvae were recorded only from










spring and fall broods. They speculated that higher

salinities during the summer months may be detrimental to

the pathogen. The copepod A. vernalis was recovered from

the study site but its role was unproven.


Copepod Data

It bears mention that information concerning many

aspects of the copepod's role in the life cycle of

Coelomomyces species is frequently lacking. While host

records and pertinent ecological data concerning copepod

population dynamics continue to accrue, copepods are perhaps

the least understood component of the system. A better

understanding of the copepods' role is necessary if the

pathogen is to be manipulated as a biological control agent

of mosquitoes.

The taxonomy of copepods is covered by Gurney 1932,

Wilson and Yeatman 1959, Kaestner 1970, and Pennak 1978.

While these works are principally taxonomic, some collection

and ecological data are recorded. The majority of

ecological and/or population studies concern marine or

lacustrine species and do not concern Coelomomyces.

Nevertheless, many reports provide methodology and life

history details of the group (Coker 1934, Carter and

Bradford 1972, Nilssen 1982, and Bergmans 1984).

Toohey et al. (1982) conducted replicated infection

trials with an unknown species of Coelomomyces and five

different copepod species collected from container habitats.








23

Additional species identifications and additional collection

data are given in Yeatman 1983. Elaphoidella taroi was the

only species that became infected. Studies by Federici

(1977a and 1980) and by Federici and Roberts (1976) present

details concerning the rearing and infection of Cy.

vernalis, the alternate host for C. dodgei and C. punctatus.

Federici (1980) reports that copepod overcrowding reduces

the number of infected copepods and slows the development of

the gametophytic stage in those that are infected. He also

noted that younger copepod stages are significantly more

susceptible to infection than older stages. It is clear

from his laboratory studies that the size, nutritional

status, and age structure of a copepod population can have

profound effects on the pathogen's level of activity in the

field. These are probably some reasons why researchers have

noted gross irregularities in fungal activity in field

studies.















CHAPTER II
A FIELD EVALUATION OF THE FUNGAL PATHOGEN
COELOMOMYCES STEGOMYIAE
AGAINST AEDES AEGYPTI


Introduction

The methods and results reported from numerous field

studies conducted with several species of Coelomomyces have

been highly variable. The following is a brief summary of

the pertinent findings from most of those studies. Walker

(1938), working with C. africanus in An. gambiae, noted

natural larval infection levels up to 35% and 66% in two

ponds in Sierra Leone. Muspratt (1946) reported C. indicus

infection levels as high as 95% in An. gambiae larvae

occupying sunlit pools in Rhodesia.

Rajapaksa (1964) organized the examination of ca.

20,000 larvae collected from 1,256 breeding sites in Sri

Lanka. Three species of Coelomomyces were found, each

infecting one mosquito species. Overall, Ae. albopictus

larvae were infected with C. stegomviae at a level of 15.4%,

the rate varying considerably among habitats. Gad et al.

(1967) noted C. musprattii infection levels averaging 27.4%

in Cx. antennatus larvae occupying four rice fields in

Egypt. Pillai (1969, 1971, and 1976) recorded C. opifexi

infecting 55% of Ae. australis larvae occupying one










supralittoral rock pool in New Zealand. He associated

infections with pools that had low salinities and recorded

the copepod T. fulvus as one alternate host occurring in the

pools.

In Russia, Deshevykh (1973) noted C. iliensis

infections in Cx. modestus reaching 100% in floodplain pools

along the Ili River. Chapman and Woodard (1966) studied C.

psorophorae var. psorophorae infections in ponds in

Louisiana. Culex restuans infection levels reached 15% and

Cs. inornata levels were consistently 20%-40%. Also in

Louisiana, Chapman and Glenn (1972) noted C. dodgei and C.

punctatus infections in An. crucians averaging 33.2% and

47.6% respectively. Umphlett (1968) also studied C.

punctatus infections in An. crucians. From a lake in North

Carolina, he recorded infection levels of 6%-80% and

associated seasonal declines with higher water temperatures.

Also in North Carolina, Couch (1972) seeded ditches with C.

punctatus sporangia and recorded a variable infection rate

that averaged 66% in An. quadrimaculatus larvae over four

seasons.

Laird (1967 and 1985) seeded 761 Ae. polynesiensis

larval habitats on the Tokelau Islands with C. stegomviae.

Subsequent surveys taken over a span of 22 years indicated a

decline in the number of infective habitats although several

unseeded habitats had become infectious, indicating some

natural dispersal. Service (1977) recorded C. indicus









26

infections in 15.9% of An. gambiae larvae sampled from rice

fields in Kenya. He noted larger populations of Cyclops

spp. copepods in fields that produced infected larvae.

Finally, Andreadis and Magnarelli (1984) reported C.

psorophorae infection levels in Ae. cantator ranging between

0% and 6.7% during one seasonal study in a saltmarsh in

Connecticut. They also noted the presence of the copepod C.

vernalis at the study site.

The current field study was designed to evaluate C.

steqomviae as a biological control agent of Ae. aegypti.

Automobile tires were chosen as larval development sites

because they represent a major source of Ae. aeqypti

production throughout the world. The field sites were

provided with chickens as blood sources in order to maintain

self-perpetuating mosquito populations. Natural oviposition

and rainfall produced the immatures, which were the primary

focus of attention. The copepods and the pathogen, once

seeded, were not under any control by the researcher.

Extensive sampling of the main elements copepodss and

immature mosquitoes) was conducted in order to quantify the

effects caused by the pathogen.










Materials and Methods (1986 Study)


Field Design

The pilot field study was conducted during the 1986

summer. Eighty automobile tires served as mosquito breeding

sites. In order to enhance sampling, tires were modified as

shown in Figure 2-1. Tires were positioned at an angle of

250 30 as shown. Each tire was rotated to a position at

which water in excess of 2-2.5 liters drained through an

overflow hole cut into the lower sidewall. Thus, the

maximum water holding capacity was standardized for all

tires. The lid, when hinged open, provided unimpeded and

well illuminated access to the crescent shaped water pool.

Sixteen tires were distributed to each of five sites

(Figure 2-2). The sites consisted of weed-infested fields

adjacent to deciduous forest (Figure 2-3). Tires were

positioned in the open fields in order to preclude predation

by larvae of the mosquito Tx. rutilus rutilus. Invasion of

the plot would be prevented, it was hoped, because females

oviposit exclusively in phytotelmata and artificial

containers in shaded habitats.

At each site tires were arranged in a 4 X 4 grid

pattern as defined in Figure 2-4. Treatment and control

tires were alternately positioned in the grid in order to

address the question of migration of the copepod P. viquieri

from treatment tires into control tires. The 8 control

tires at each site (which were not inoculated with copepods)















































Figure 2-1. Automobile tire modified to enhance data
collection of immature mosquitoes.























































Figure 2-2. Locations of the 5 field sites used during 1986.















































J: .
*ies' 7"C'"-~~:i~


Figure 2-3. One of the 5 field sites used for the 1986
season.


























CHICKEN
CAGE

FOREST FOREST







Cl T3 C5 T7



T1 C3 T5 C7



C2 T4 C6 T8



T2 C4 T6 C8


Figure 2-4. The relative positions of control and treatment
tires at each of the 5 field sites used in 1986.










were each adjacent to a minimum of 2 treatment tires.

Specifically, 2 control tires were adjacent to 2 treatment

tires, 4 control tires were adjacent to 3 treatment tires,

and 2 control tires were adjacent to 4 treatment tires.

This standardized arrangement provided the potential for

quantifying migration should it occur. At each site a caged

chicken was positioned in the forest ca. 5 meters from the

nearest tire. The chicken was provided as a source of

mosquito bloodmeals in order to reduce mosquito emigration

from the study area.

On June 18 each tire was filled to its maximum water

holding capacity with 2-2.5 liters of tap water. Four grams

of ground oak leaves were provided to each tire as a natural

food source for the mosquito larvae. Twenty second instar

and 20 fourth instar larvae from a laboratory colony of Ae.

aevypti were also added to each tire. The colony was

derived from a colony that has been maintained at the USDA

IAMARL in Gainesville, Fla. for ca. 20 years. It was

originally developed from collections made in the

Gainesville area. Adults were maintained on a regular diet

of 10% sucrose and mice were provided as the bloodsource.

Eggs were collected on moist filter paper in oviposition

cups. Larvae were reared on a diet of finely ground alfalfa

and fish food (1:1), a modification from Gerberg (1970).

On June 20, ca. 2,000 adult Ae. aevypti were released

at each plot. Females had bloodfed ca. 48 hours previously.










Also, on June 20 ca. 1,600 P. viquieri (mixed ages) were

released into each treatment tire. Copepods were derived

from a laboratory colony which originated from collections

made by Dr. H. C. Whisler from bromeliad leaf axils from a

greenhouse at the University of Washington, Seattle.

Colonies were maintained in tap water in 3 liter plastic

vessels at ca. 25" C under a light:dark regime of ca. 12 hr:

12 hr. Finely ground alfalfa leaves were provided as the

food source as needed. One day prior to field release each

of the 40 subcolonies was inoculated with C. stegomviae

resting sporangia liberated from 2 heavily infected fourth

instar Ae. aeqypti larvae.

Coelomomyces stegomyiae was cultured and stored in the

laboratory as follows. Approximately 300 P. viquieri were

removed with a pipette from stock laboratory colonies and

subcolonized in a 9 cm diameter petri dish in ca. 100 ml of

tap water. Copepods were provided with ca. 20 mg of finely

ground alfalfa and held for 8-10 days at room temperature.

Following this period, the population contained all stages

and was inoculated. Inoculum consisted of the resting

sporangia liberated from 1 heavily infected fourth instar

Ae. aegypti larva that had been stored in a sealed container

under refrigeration (ca. 8 C). Following an exposure

period of 7 days, the infected copepods were transferred to

an enamel pan containing 100 first instar Ae. aegypti

larvae. Larvae were maintained on a diet of finely ground










alfalfa leaves and fish food (1:1). After ca. 2 weeks,

fourth instar larvae were harvested, rinsed, and stored

under refrigeration for future use as inoculum.

Based on poor initial results, two additional releases

were made into treatment tires. On July 17, C. stegomyiae

resting sporangia liberated from one heavily infected Ae.

aevgyti larva were released into each of the 40 treatment

tires. On August 12, 40 subcolonies of infected copepods

were released into the 40 treatment tires. Each subcolony

consisted of ca. 2,000 P. viquieri of mixed ages. The

infection sequence preceding the latter release was as

follows. Coelomomyces stegomyiae resting sporangia

liberated from one heavily infected Ae. aegvpti larva were

added to each of 48 copepod subcolonies. At seven days

post-exposure, 40 of the subcolonies (randomly chosen) were

released into the 40 treatment tires. The remaining 8

subcolonies were retained in the laboratory, observed for

the presence of infected copepods, and transferred to eight

18 X 30 cm enamel baking pans. One hundred Ae. aegvpti

first instar larvae were added to each pan on the day of

field release. All of the 8 pans yielded a mosquito

infection level in excess of 90%, indicating that the

copepod subcolonies released into the field were infective.


Sampling

The five field sites were sampled twice weekly from

July 1 to August 25. Sampling was continued at site A until








35

November 24. Mosquito sampling was as follows. Pupae were

counted in each tire twice weekly. Because C. steqomyiae

principally causes mortality in the larval stage, a tire

exhibiting infection should exhibit reduced pupal

production. Pupal production was regarded as the most

readily attainable correlation to a tire's production of

adult mosquitoes the ultimate gauge of this pathogen's

efficacy. Up to 10 pupae were retrieved from each tire and

returned to the laboratory for species identification. The

sample size never exceeded ca. 50% of the pupae counted in a

tire on that day. Pupae were held in the laboratory until

eclosion and identified based on adult characters. Adults

were identified to the species level for the genus Aedes.

Other genera were determined to the generic level only. The

resultant ratio of Ae. aegypti/other genera was used to

adjust the field pupal counts to reflect the number of Ae.

aevypti pupae produced per tire per sampling period.

Additionally, up to ten late instar larvae were

retrieved from each tire and returned to the laboratory. To

prevent oversampling, the sample size never exceeded ca. 10%

of a tire's larval population. Samples were transferred to

petri dishes in the laboratory and larvae were provided with

50-100 mg of finely-ground alfalfa and fish food (1:1)/dish.

Larvae were observed under a dissecting microscope at 8X

magnification to assess infection 5-7 days after sampling.

Approximately 50 ml of water were retrieved from each










tire twice weekly and returned to the laboratory to

determine copepod presence/absence. Samples were suctioned

from an area central to the water pool and immediately above

the oak leaf substratum upon which the copepods browse.

Samples were returned to the laboratory where each vial was

provided with ca. 2 mg of finely ground alfalfa as a food

source for the copepods. Sample assessment was conducted

within one week using a dissecting microscope at 8X

magnification. Meat basters were used to sample both the

mosquito immatures and the copepods. In order to prevent

contamination of samples and tires, the basters were rinsed

between samplings from each tire. Further, one baster was

reserved for control tires and the other for treatment

tires. Rainfall was recorded from a rain gauge at each site

for most sampling dates. Finally, water temperatures were

recorded from one tire at each site for many sampling dates.


Results and Discussion (1986 Study)


Field Design

The analysis of the results indicated the attributes

and flaws of this pilot study. Tires proved to be effective

units for mosquito breeding and for monitoring the system.

Tires readily collected rainwater and lost water to

evaporation at a surprisingly slow rate. Apparently,

evaporation is not substantial due to a tire's unique shape.

Water levels in several tires dropped to ca. 0.5 liters on








37

only one occasion following a prolonged dry period. At this

time (July 15) all tires were refilled to capacity to

preclude any tires becoming dry. This was the only occasion

when tires were artificially replenished with water. The

lid, when hinged back to reveal the water pool, proved to be

an indispensable attribute of the design. This arrangement

provided for rapid pupal counting and sampling of both

immature mosquitoes and copepods. Placing tires in an open

field apparently precluded invasion by Tx. rutilus rutilus.

This species was noted in only two tires at site A on Nov. 3

and Nov. 24. At that time the sun's lower angle shaded most

of site A for most of the day providing a suitable habitat

for oviposition.


Sampling

A summary of mosquito identification follows. A total

of 1,616 observations (one observation is the record from

one tire on one day) was made in the field. Of these, only

238 observations yielded no pupae. Therefore,samples were

not obtained for species identification from those

observations. An additional 77 observations yielded such

low pupal numbers that samples were not acquired. Of the

remaining 1,301 observations, only 81 contained one or more

Culex pupae. For each of the 81 observations, the % of Ae.

aegypti pupae was calculated and used to adjust the pupal

counts acquired from the field for the corresponding dates.

The overwhelming majority of pupae collected for species










identification was Ae. aeqypti, indicating that the field

sites were appropriate breeding habitats. However, pupal

production was lower than hoped for, probably because Ae.

aegypti females prefer to oviposit in shaded or partly

shaded containers.

For purposes of analysis and discussion, the sampling

season was divided into two periods. The summer period

(July 1 Aug. 25) comprised 17 observations/tire at all

sites. Sampling was continued through the fall (Aug. 29 -

Nov. 24) at site A only. Sixteen observations/tire were

conducted during this latter period.

Summer period. T-tests were conducted to test for

differences in pupal production between treated and control

tires at each of the five sites. Analyses of production

totalled for the full 17 date summer sampling period

revealed that significantly fewer pupae were produced at

sites C, D, and E. This positive result is misleading

because it includes data from the artificially produced

epizootics at these 3 sites. That is, the inoculation was

highly successful at these 3 sites, producing epizootics

affecting the high population of larvae originally

introduced into the tires. This activity subsided within

two weeks. Separate analyses considering only the latter 13

dates of the summer period yielded different results (Table

2-1). Significantly fewer pupae were produced from

treatment tires only at site C.
























Table 2-1. Within site comparisons of pupal
production between treated and control tires
at each of the five 1986 study sites.

PUPAE / TIRE
PERIOD SITE TREATMENT N(a) MEAN + S.E.

summer A control 104 11.3 0.9
A treated 14.1 1.6

B control 7.5 1.0
B treated 6.3 0.7

C control 4.1 0.5
C treated 2.3(b) 0.4

D control "3.3 0.4
D treated 2.8 0.4

E control "2.7 0.4
"E treated "2.3 0.5

fall A control 128 4.4 0.6
A treated 5.3 0.5


8 tires X #
significant


of sampling days.
at P < .05 (t-test).


(a) =
(b) =










The seemingly encouraging data from site C do not

correlate with other evidence that indicates a lack of

sustained fungal activity at all sites. The results from

site C must be discounted as having occurred for an

alternate reason. The highly variable pupal counts (a

partial result of variable oviposition) acquired from only 8

replications per site apparently produced the results by

chance. Following the initial fungal activity, no infected

larvae were noted either in the field or from field samples

observed in the laboratory until late August. Following the

Aug. 12 release of infected copepods, 13 of the final 160

larval samples (for the summer period) yielded one or more

infected larvae. Twelve of these were from site A. Thus,

larval infection data do not support the pupal data which

suggests that C. stegomyiae was influential at site C.

Phyllognathopus viguieri was never retrieved from the

680 observations from control tires, indicating that

migration did not occur. Populations were not sustained in

most treatment tires during the summer. Of the 520 water

samples taken from treated tires up until Aug. 11, only 59

contained copepods. Following the Aug. 12 reintroduction of

copepods into all treated tires, copepods were retrieved

from 75 of the final 160 treated tire observations made

through Aug. 25. The number of copepod-positive tires

declined as the summer progressed and declined following the

Aug. 12 release, indicating that the environment was not










conducive to P. viquieri survival. Water temperatures

recorded from the field averaged 31*C with a range of 25*C -

360C. Because temperatures were always recorded prior to

midday, it is reasonable to presume that the highest daily

temperatures occurred in the afternoons and averaged above

31*C. A subsequent laboratory experiment indicated that

water temperatures above 30C limit P. viguieri survival.

The high water temperatures documented during the summer

period were probably the limiting factor.

Fall period. No significant difference in pupal

production between treated and control tires was noted at

site A for the fall period (Table 2-1). Treated tires

actually produced more pupae than did control tires.

Likewise, no significant differences emerged when each of

the 16 sampling dates was analyzed with a t-test.

Coelomomyces stegomyiae -infected larvae were noted in the

field from 6 observations (from a total of 3 tires) during

the fall. These observations were confirmed with the

laboratory bioassays for the corresponding days. Eleven

additional laboratory observations (from the same 3 tires)

yielded infected larvae. One additional tire yielded

infected larvae from one lab bioassay observation. Thus, 4

of the 8 treated tires exhibited infection.

The tires at site A became increasingly shaded as the

fall period (Aug. 29 Nov. 24) progressed due to increasing

shade from the adjacent trees as the sun's angle receded.










In addition, air temperature decreased during this period.

These two factors led to a decrease in tire water

temperature. Maximum water temperatures (taken at 11:00 AM)

averaged 25.8C with a range of 16C to 34C. Apparently,

the cooler water was conducive to copepod survival. Of the

128 water samples taken from the 8 treated tires, 110

contained copepods. The 18 samples containing no copepods

were acquired early in the fall period (Aug. 29 Oct. 6)

when water temperatures averaged 28.0C with a range of 26C

to 34C. During the latter part of the fall period (Oct.13-

Nov.24), when the water temperature averaged 21.6*C with a

range of 16C to 25C, all samples contained copepods.

Evidently copepods were present in all treatment tires

during the entire fall period. Their absence in 18 samples

from the early fall is probably because populations remained

low due to the higher water temperatures.

Collectively, the evidence from the entire season

supports the notion that high water temperature was a factor

limiting copepod survival and abundance. Further, the

absence of this obligatory host from the system would

obviously preclude C. stegomyiae from cycling, hence the

paucity of infected mosquitoes. This field study did not

address the effect of temperature on survival of the

pathogen. In a study comparing the effects of different

temperatures on susceptibility of second instar Ae. aegypti

larvae to C. stegomyiae, Nnakamusana (1987) reported









43

infection levels of 99.8%, 92.4%, 20%, and 0% at 23"C, 28C,

320C, and 350C respectively. The copepod host was not

identified in this study. Because the experiment was

initiated with patently infected copepods, the system

apparently failed at the higher temperatures because C.

stegomyiae is heat-sensitive. Nolan (1985) reviewed

laboratory and field studies concerning the relationship of

temperature and the storage, germination, growth, and

cycling of several Coelomomyces species. The general

findings are that other species of Coelomomyces operate

optimally at temperatures in the 20C to 30C range. Nolan

concludes "a much better understanding of the role of

temperature in all aspects of the biology of Coelomomyces is

required." In a review concerning the stability of other

entomopathogenic fungi, Roberts and Campbell (1977) conclude

"the optimum temperature for most entomogenous fungi for

spore germination, growth, and sporulation lies between 20

and 300C."

The overall results of the 1986 pilot study were

principally discouraging. However, the study was extremely

valuable in identifying the major research flaws. Four of

the five field sites were only marginally conducive to the

establishment of a self-perpetuating Ae. aegvDti population.

Although the five study sites were chosen on the basis of

ecological similarity, pupal production (both overall and

per site) was unacceptably variable. Finally, operating the










system in open fields clearly produced tire water

temperatures high enough to be detrimental to P. viguieri

populations and probably detrimental to C. stegomyiae.

These principal flaws were addressed during the design of

the 1987 field study.


Materials and Methods (1987 Study)


Field Design

The 1987 field study consisted of seventy two tires

located at site A only. They were arranged in 3 rows

(blocks) of 24 tires per row. Within each row, 12 treated

and 12 control tires were randomly assigned positions

(Figure 2-5). This arrangement constituted a randomized

complete block design. The distance between the bloodmeal

sources and the tires was considered the gradient because

females tend to oviposit in the nearest site encountered.

It was reasoned that the tires closest to the woods would

acquire more eggs than more distant tires. Therefore,

blocks were arranged perpendicular to the oviposition

gradient.

The effect of shading tires (to reduce the maximum

daily water temperatures) was experimentally established

during the previous autumn. On Oct. 3, 1986 the following

arrangement was constructed in an open field. Twelve tires

were positioned on the ground in a 4 x 3 grid pattern.

Tires were positioned at a 25" 30 angle and filled with 2









ROWS
I II III


CHICKEN
CAGE











CHICKEN
CAGE


C12

T12

T11

T10

T9

Cl1

T8

C10

C9

C8

C7

T7

T6

C6

C5

T5

T4

T3

T2

C4

C3

C2

C1

T1


C12

T12

Cl1

T11

C10

T10

C9

T9

T8

C8

C7

C6

T7

T6

T5

T4

T3

T2

C5

T1

C4

C3

C2

C1


T12

C12

T11

C11

C10

C9

T10

C8

T9

C7

C6

T8

T7

T6

C5

C4

T5

T4

C3

C2

T3

T2

T1

C1


CHICKEN
CAGE


CHICKEN
CAGE


Figure 2-5. Relative positions of the 72 tires and 4 chicken
cages for the 1987 and 1988 study seasons.










- 2.5 liters of tap water. A 4 x 4 meter frame was

constructed to support the shadecloth ca. .75 meters above 9

tires. The 3 tires comprising row # 1 were covered with 5

layers of shadecloth. Row # 2 was covered with 1 layer and

row # 3 with 2 layers. The final 3 tires comprising row # 4

were exposed to direct sunlight. Water temperatures were

recorded from all tires at midday on Oct. 6 (a cloudless

day) and averaged for the 3 tires in each row. Average

maximum water temperatures of 32*C, 29"C, 28C, and 27C

were recorded for 0, 1, 2, and 5 layers respectively. Based

on these results, it was concluded that one layer would

probably provide an appropriate degree of shade. While the

difference between one and two layers was minimal, (1C), it

was reasoned that the second layer would provide insurance

that temperatures would remain below 30C. As mentioned,

this temperature approximates the upper limits for survival

of several species of Coelomomyces species.

The 1987 plot was covered with two layers of black

polyethylene shadecloth (rated as 80 % shade value per

layer) supported by a 20 x 4 meters wooden frame ca. 0.75

meters above the ground (Figure 2-6). A light meter was

used to quantify the amount of shade produced midday during

a cloudless day in June. The meter (International Light,

Inc., Newburyport, Mass., 01950, model # IL710A, sensor #

SEE015) registered values of 8.98 x 103 footcandles in the

open sunshine and 1.30 x 103 under two layers of shadecloth.

































































































Figure 2-6. The field design used for the 1987 88 seasons.


. -










On June 30, all tires were thoroughly scrubbed and

rinsed in order to remove mosquito eggs and/or C. stegomyiae

resting sporangia remaining from the 1986 season. All tires

were filled with 2-2.5 liters of well water. Four grams

(dry weight) of crushed oak leaves were added to each tire

as the initial nutrient source. An additional nine grams was

added to each tire on Aug.18 in order to boost the nutrition

level. Leaves were pulverized in a blender and screened

with 1/4 inch hardware cloth. Leaf pieces retained above

the screen were discarded because it was learned that pupae

conceal themselves under whole leaves or large leaf pieces,

confounding field pupal counts.

On July 1, ca. 1,465 P. viguieri (mixed ages) were

added to each of the 72 tires. Colonies originated from

bromeliads in a greenhouse in Seattle and were maintained in

the laboratory as described previously in this chapter.

Thus, unlike the 1986 study, copepod migration into control

tires was not a secondary research question. For the 1987

study, fungal dissemination (via infected adult females)

into control tires was a secondary research question.

Successful dissemination of C. stegomyiae into control tires

would be exhibited as infected larvae noted during field

observations. The copepods were acquired from a laboratory

colony, randomly divided into 80 subcolonies with the aid of

a water-splitting device (Figure 2-7). Seventy-two of these

noninfected colonies were transferred to the field site.





















'N


in


Figure 2-7. The water splitting device, screening vial, and
plate used for partitioning, concentrating, and
counting copepod samples.










Three were sampled for a quantitative assessment of the

population, and the remaining five were retained as lab

stock.

Aedes aegyvti were released at the field site on July 1

also. Mosquitoes were F1 offspring from a wild population

collected in the Gainesville area as larvae earlier in the

season. Colony maintenance was as previously described in

this chapter. The original release consisted of 24 4th

instars, 24 1st-2nd instars (mixed) and 2 pupae per tire.

In addition, 350 1-3 day old (non-bloodfed) adults were

released at each chicken cage. Finally, on July 1, C.

stegomyiae resting sporangia from 6 heavily infected Ae.

aevypti 4th instar larvae were added to each of the 36

treated tires as the inoculum. The infected larvae had been

produced in the laboratory in April 1987 by the methods

previously described and had been refrigerated until the

time of release. Prior to release the 6 larvae in each vial

were shaken in order to fully liberate the sporangia from

the partly decomposed larvae.

Based on poor results, two additional releases of

copepods and C. stegomyiae were made. On July 9, ca. 613

healthy copepods were released into each of the 72 tires and

the resting sporangia liberated from 6 larvae were released

into each of the 36 treated tires. The final release was

made on July 28. Approximately 832 copepods (mixed ages)

were released into each of the 72 tires. Each of the 36










subcolonies released into the 36 treatment tires had been

exposed to the resting sporangia liberated from one larva.

Exposure began 7 days prior to release.

In response to decreasing oviposition, two releases of

mosquito adults were made during the course of the 1987

season. Adults were produced from the laboratory colony

that had originated from the colony maintained at the USDA

IAMARL. Adults were allowed to emerge from cups containing

pupae that were placed near each of the 4 chicken cages. On

July 17, 49 pupae were released at each cage. On Aug. 22,

ca. 500 pupae were set at each cage. The nutrient level in

all tires was supplemented with 9 grams (dry wt.) of crushed

oak leaves on Aug. 18. Finally, due to low rainfall, all

tires were filled to capacity with well water on Aug. 26,

Sept. 10 and 29, and Oct. 14. No tire was permitted to dry

out during the sampling season.


Sampling

As with the 1986 study, pupal production was considered

the principle gauge of fungal activity. Pupae were counted

in each tire twice weekly from July 20 until Oct. 29. A

battery-powered headlamp was used because the shadecloth

markedly reduced the amount of sunlight available for

observation. With experience, it became possible to discern

Aedes spp. pupae from Culex spp. pupae in the field.

Therefore, the need to return samples of pupae to the

laboratory for identification to genus was eliminated. Only










62 Culex pupae were noted in the field during the entire

season. These were discarded during sampling. A

potentially confounding problem in this regard was the

possibility that Aedes triseriatus would invade the plot.

Pupal appearance and behavior are similar to Ae. aegypti.

The most conspicuous character notable in the field is the

larger size of the former. Suspect pupae were returned to

the laboratory for emergence and subsequent identification.

All such pupae were identified as Ae. aegypti. Also, during

field observations, adult mosquitoes were noted hovering

near or resting in tires. Ae. triseriatus adults were never

noted. Thus, field counts of Ae. aeQyvti pupae were not

confounded by the presence of other mosquitoes.

Larval infection was recorded in two ways. During

field observations the numbers of infected and healthy

larvae were recorded from each tire containing obviously

infected larvae. Only fourth instars were so recorded

because younger instars were difficult to count and

infections are not obvious in young larvae. In addition, up

to 10 late instar larvae were retrieved from each tire and

returned to the laboratory to observe for the development of

infection. The protocol for this latter routine is the same

as that used in the 1986 study.

Copepod sampling was considerably modified from the

1986 methodology. On a weekly basis, 100-120 ml of water

were suctioned from each tire and returned to the laboratory








53

for copepod quantification. A separate meat baster was used

at each tire to preclude contamination. A baster's capacity

was ca. 30 ml. Therefore, each sample was produced by

suctioning four times. The tip of the baster was moved

along the leaf debris/tire wall interface as the sample was

taken. Thus, the entire perimeter of the debris was

suctioned to produce each sample (Figure 2-8). The tip was

held slightly above the debris (ca. 0.5 mm) to preclude

clogging.

In the laboratory the copepod samples were processed in

the following manner. Samples were randomly divided into

two 50-60 ml units using the water splitting device pictured

in Figure 2-7. One unit was chosen to process and the other

was discarded. The field sample was split because it was

determined early in the sampling program that it often

contained an excessive number of copepods to count within a

reasonable time period. It was presumed that this protocol

was less variable than if a 50% smaller field sample was

retrieved and not split. The sample was then poured through

the screening device (Figure 2-7). The vial's bottom was

removed and replaced with nylon mesh with 55 micron openings

(Tetko, Inc. 420 Saw Mill River Rd., Elmsford, N.Y., 10523).

The mesh retained the copepods and some debris. The vial

was then immersed in near-boiling water for several minutes.

This process killed the copepods and imparted an opaque

white coloration to the naturally translucent bodies.












































-w..'p cZ
WW~r- u, ".


Figure 2-8. A modified tire in the field. The lid is opened
to reveal the water pool.


k'










Finally, the dead copepods were rinsed into the flat-black

rectangular counting chamber (Figure 2-7). The immobile

copepods were readily counted at 12X magnification.

Maximum water temperatures were recorded with maximum-

registering thermometers (Fisher Scientific Corp.,

Pittsburgh, Pa.). One thermometer was submersed permanently

in each of three representative tires. A raingauge provided

rainfall data at the field site.


Results and Discussion (1987 Study)


The shadecloth notably reduced the maximum water

temperatures compared to the temperatures attained during

the 1986 study when tires were exposed to direct sunlight.

From July 20 until Oct. 29, a total of 124 water

temperatures was recorded. While the measurements were

taken prior to the hottest part of the day, the thermometers

clearly registered and maintained the maximum temperatures

attained between sampling periods. Temperatures ranged from

22"C to 30*C with an average of 27.1*C. The lowest values

were recorded in October and the highest value was recorded

only 3 times during the summer. Because the plot was

distanced from the forest by ca. 3 meters, a "curtain" of

sunlight was maintained in that zone during most of the

season. Apparently this barrier of sunlight prevented adult

Tx. rutilus rutilus from invading the plot. The zone became

increasingly shaded as the season progressed through the










fall. Toxorhynchites rutilus rutilus larvae were entirely

absent from all tires until Oct. 1, when 2 were captured

from 1 tire. Five were captured from 1 tire on Oct. 26 and

1 on Oct. 29. This pattern was also noted at the same field

site during the 1986 season. To prevent further predation,

captured larvae were discarded. As was noted during the

1986 season, the Ae. aevgyti population was relatively self-

sustaining at this site. The two releases of pupae appeared

to augment the population, consequently increasing

oviposition and the population of immature stages in the

tires.

An ANOVA was conducted comparing pupal production from

treated and control tires totalled for the 29 sampling dates

comprising the entire season. The treatment alone did not

produce significantly fewer pupae (P=0.24). However, both

the row (block) and the row X treatment interaction were

significant (at P<0.01 and P=0.01 respectively). Because

the interaction significance can "mask" a difference due to

the treatment, three t-tests were performed to test the

effect of the treatment within each row (Table 2-2). This

analysis revealed that significantly fewer pupae were

produced only from treated tires in row II over the entire

season. However, treated tires in row II did not produce

noticeably more infected larvae. Also, row II did not

contain a particularly high number of infectious tires.

Therefore, it appears unlikely that fungal infections were




























Table 2-2. Within row comparisons of pupal production
between treated and control tires for the 1987 study.

PUPAE / TIRE
PERIOD ROW TREATMENT N(a) MEAN + S.E.

season I control 336 4.7 0.2
I treated 4.3 0.3

II control 328 4.6 0.2
II treated 329 3.7(b) 0.2

III control 316 3.1 0.2
III treated 316 3.6 0.3

fall only I control 168 3.2 0.2
I treated 2.8 0.2

II control 3.5 0.3
II treated 2.7(b) 0.2

III control 162 2.5 0.2
III treated 163 2.5 0.3


(a) = 12 tires X # of sampling
(b) = significant at P < 0 .05


days.
(t-test).








58
responsible for the significant decrease in pupal production

noticed in row II.

Over the entire season, larval infections were noted

during 125 (12.7%) of the 981 field observations from

treated tires. These infection records were acquired from a

total of 15 (41.7%) of the 36 tires. Clearly, the same

tires were repeatedly responsible for infected larvae (Table

2-3). It appears that once established, the pathogen tended

to perpetuate. The reason C. stegomyiae was highly

successful in some tires and totally absent from others

remains a mystery. The majority (96) of positive recordings

occurred after Sept. 10 (Figure 2-9). On Sept. 14, nine of

the 36 treated tires were noted to contain infected larvae,

a marked increase over the summer months. Fungal activity

continued through the duration of the sampling period (-

Oct. 29) at an average of 7.1 infectious tires per sampling

day. During the summer period (July 20 Sept. 10) the

average was 2.0 infectious tires per sampling day. Thus,

Sept. 14 designated a transition into a period of increased

activity. Maximum water temperatures are also charted on

Figure 2-9. The lower temperatures beginning in late

September and continuing until the experiment was terminated

may be partially responsible for the continued fungal

activity observed. This seems unlikely because Nnakamusana

(1987), in a study comparing C. stegomyiae infection of Ae.

aevypti at different temperatures, attained the highest





















Table 2-3. Treated tires that produced Aedes aegypti
larvae infected with Coelomomyces stegomyiae during 1987.


ROW
DATE I II III

J1 27 T2
Jl 31 T2
Au 3 T2
Au 7
Au 10 T3
Au 14 T2
Au 17 T9 T2
Au 21 T8 T9 T2
Au 24 T8 T9 T2
Au 28 T9
Au 31 T9 T3 T2
Sp 3 T9 T2
Sp 7 T8 T9 T2
Sp 10 T8 T9 T3 T2
Sp 14 T1 T7 T8 T9 T3 T7 T2 T4 T7
Sp 17 T1 T7 T8 T3 T7 T2 T4 T7
Sp 21 T1 T7 T8 T9 T3 T2
Sp 24 T9 T3
Sp 28 T7 T8 T9 T3 T7 T2 T4
Oc 1 T1 T7 T8 T9 T3 T7 T2 T4
Oc 5 T7 T9 T7 T2 T4
Oc 8 T7 T7 T2 T4
Oc 12 T8 T9 T3 T2 T4 T8
Oc 15 T8 T9 T3 T2 T4 T8 T9 T10
Oc 20 T1 T8 T9 T3 T5 T2 T4 T8 T9 T10
Oc 23 T1 T8 T9 T3 T5 T1 T4 T8 T9
Oc 26 T8 T9 T3 T5 T2 T4 T8 T9 T10
Oc 29 T8 T9 T3 T5 T2 T8 T9 T10















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infection levels at 28 C, which closely approximates the

average highest temperatures recorded in this field study.

Tables 2-4, 2-5, and 2-6 list the percent infected

larvae observed in the field in rows I, II, and III

respectively and Table 2-7 is a summary thereof. From the

125 observations from which infected larvae were noted,

37.8% were infected. That is an encouraging figure but it

does not imply a 37.8% reduction in pupal production from

those tires. It must be acknowledged that most healthy

fourth instar larvae were probably counted only once but

most infected fourth instars were probably counted more than

once. That is, healthy fourth instars remain in that stage

for only several days (if nutrition is adequate). Most would

probably get counted only once since observations were made

at 3 or 4 day intervals. Infected fourth instars, on the

other hand, can remain alive in that stage for up to 2 weeks

(in the laboratory). If the situation in the field is

similar, then surely many were counted more than once.

Thus, the percent larval infection must be somewhat higher

than the resultant percent pupal reduction from the 125

tires.

An ANOVA conducted for the fall period indicated a

significant reduction in pupal production from treated tires

(P=0.04). From calculations of the mean number of

pupae/treatment/row it was determined that 12.3% fewer pupae

were produced in treated tires throughout the fall period.



















Table 2-4. Percent infected larvae from treated
tires in row I in 1987.


TIRE I.D. + ( % INFECTION )


DATE


T8 ( 3)
T8 ( 5)


T1 (25)
T1 (20)
T1 (11)


T1 (10)


T7 (28)
T7 (17)
T7 ( 9)


(31)
(54)
(44)
(47)


T1 (100)
T1 (50)


( 1)
(17)
(83)
(100)
(50)


T8 (38)
T8 (100)


(88)
(82)
(67)
(43)
(27)
(29)


T9 ( 7)
T9 (10)
T9 (44)
T9 (90)
T9 (100)
T9 (80)
T9 (20)
T9 (88)
T9 (100)


(17)
(67)
(83)
(71)
(67)

(50)
(25)
(40)
(67)
(50)
(50)


J1 27
Jl 31
Au 3
Au 7


Au 10
Au 14
Au 17
Au 21
Au 24
Au 28
Au 31
Sp 3
Sp 7
Sp 10
Sp 14
Sp 17
Sp 21
Sp 24
Sp 28
Oc 1
Oc 5
Oc 8
Oc 12
Oc 15
Oc 20
Oc 23
Oc 26
Oc 29



















Table 2-5. Percent infected larvae from treated tires
in row II in 1987.

TIRE I.D. + ( % INFECTION )

DATE


July 27
July 31
Aug. 3
Aug. 7
Aug. 10
Aug. 14
Aug. 17
Aug. 21
Aug. 24
Aug. 28
Aug. 31 T3 ( 8)
Sep. 3
Sep. 7
Sep. 10 T3 (67)
Sep. 14 T3 (100) T7 (35)
Sep. 17 T3 (100) T7 (19)
Sep. 21 T3 (88)
Sep. 24 T3 (60)
Sep. 28 T3 (67) T7 (13)
Oct. 1 T3 (100) T7 ( 9)
Oct. 5 T7 ( 7)
Oct. 8 T7 ( 7)
Oct. 12 T3 (50)
Oct. 15 T3 (100)
Oct. 20 T3 (71) T5 (20)
Oct. 23 T3 (50) T5 (15)
Oct. 26 T3 (60) T5 ( 8)
Oct. 29 T3 (17) T5 ( 4)




















Table 2-6. Percent infected larvae from treated
tires in row III in 1987.


TIRE I.D. + ( % INFECTION )


DATE


T2 (13)
T2 (33)
T2 (25)


(31)
(63)
(50)
( 9)


T2 (93)
T2 (67)
T2 (100)
T2 (100)
T2 (56)
T2 (100)
T2 (67)

T2 (100)
T2 (100)
T2 (100)
T2 (100)
T2 (84)
T2 (90)
T2 (43)
T1 (20)
T2 (25)
T2 (43)


T3 (44)


(28)
(11)


T4 ( 5)
T4 (45)
T4 (94)
T4 (94)
T4 (56)
T4 (50)
T4 ( 3)
T4 ( 9)
T4 ( 4)


T7 (75)
T7 (13)


Jl 27
Jl 31
Au 3
Au 7
Au 10
Au 14
Au 17
Au 21
Au 24
Au 28
Au 31
Sp 3
Sp 7
Sp 10
Sp 14
Sp 17
Sp 21
Sp 24
Sp 28
Oc 1
Oc 5
Oc 8
Oc 12
Oc 15
Oc 20
Oc 23
Oc 26
Oc 29


T9 (10)
T9 (25)
T9 (43)
T9 (50)
T9 (50)


T10 (6)
T10 (6)

T10 (3)
T10 (3)


T8 (37)
T8 (82)
T8 (68)
T8 (27)
T8 (22)
T8 (25)











Table 2-7. A summary of the 125 field observations
containing infected lar i 1987


ROW TOTAL # LARVAE # INFECTED % INFECTED
I 565 233 41.2
II 282 86 30.5
III 753 285 37.8
TOTALS 1600 604 37.8



Thus, the increase in infectious tires supports the pupal

data. When rows were individually analyzed with t-tests

only row II was significant (Table 2-2). Thus, it appears

that row II was of primary importance in producing the

significant reduction observed for the entire plot for the

fall period. However, the larval data are not supportive

because, as mentioned, row II contained the lowest number of

tires exhibiting infected larvae.

Each of the 14 fall sampling dates was individually

analyzed with an ANOVA. On only one date (Sept. 17) were

significantly fewer pupae produced from treatment tires.

While 4 treated tires were harboring larval infections one

week prior to this date (a reasonable time lag to notice a

pupal reduction), the pupal reduction was not necessarily

due to fungal activity. When individually analyzed, the

evidence from the other 13 fall sampling dates might suggest

that C. stegomyiae was not significantly reducing pupal

production. In all likelihood, that is because pupal

production was too variable when analyzed by date. The fall

analysis, on the other hand, included many more










observations. Consequently, the variability was

considerably reduced and a significant difference became

apparent. Of course, it is the seasonal data that deserve

emphasis in this study.

Because larval populations were frequently considered

too low for sampling, only 24 of the 126 positive field

observations have associated laboratory data. Of these 24,

only 10 yielded infected larvae and 14 produced only healthy

larvae. Infected larvae were noted in an additional 4

laboratory observations which corresponded to field

observations containing only healthy larvae. Three of these

4 tires eventually produced infected larvae evident during

field observations. Thus, only one tire (IIT9) produced a

(one) positive laboratory result without a corresponding

field result. The process of sampling larvae from the field

for laboratory observations provided only minimal

confirmations of the infections evident during field

observations. In fact, more laboratory observations failed

(14) to confirm field infections than succeeded (10) in

doing so. Because infected larvae were never observed in

control tires, apparently C. stegomyiae was not disseminated

into them by infected adult mosquitoes.

Copepods were sampled quantitatively on 15 dates. The

results of an ANOVA test indicated that the treatment

significantly (P=0.01) reduced the number of copepods when

the entire season was considered. T-tests conducted for










each row alone indicated that treated tires produced

significantly fewer copepods only from row II, although all

rows exhibited a reduction from treated tires (Table 2-8).

That is, row II was singularly responsible for the overall

significance. ANOVA and t-test analyses of copepod

populations for the fall period yielded similar results.

Population samples were significantly smaller from treated

tires (P<0.01) and row II alone was responsible (Table 2-

8). While this is an interesting finding, it is incorrect

to ascribe it to elevated cycling of the pathogen. Three

facts demand caution in this regard. (1) Larval infection

data (from both the field and laboratory) are not

supportive. That is, row II had only 3 treated tires which

produced infected larvae, fewer than either of the other 2

rows. (2) The copepod populations were not noticeably lower

in those 3 tires in comparison to other treated tires in row

II. (3) A subsequent calibration experiment indicated that

the copepod sampling technique often provided inaccurate

values. In other words, the quantitative copepod population

data must be considered only in the broadest terms.

The sampling calibration was conducted as follows.

Four tires were arranged in a greenhouse under partial

shade. Each was filled to capacity with tap water and ca.

9.5 gm of crushed oak leaves. All tires were stocked with

known numbers of adult copepods and sampled repeatedly

within a two day period for each of the 4 population levels.
























Table 2-8. Within block comparisons of copepod samples
between treated and control tires for the 1987 study.

COPEPODS / TIRE
PERIOD ROW TREATMENT N(a) MEAN + S.E.

season I control 168 65.9 7.4
I treated 168 52.3 4.7

II control 164 84.9 7.4
II treated 164 63.2(b) 5.2

III control 161 54.9 4.8
III treated 160 52.5 5.5

fall only I control 84 77.9 9.0
I treated 61.5 6.5

II control 121.0 11.8
II treated 75.8(b) 7.4

II control 77.8 7.6
II treated 61.7 7.2


(a) = 12 tires # of sampling days.
(b) = significant at P < 0.05 (t-test).










Following the counting of each sample, the tire of origin

was restocked with that same number of copepods prior to the

next sampling. At least two hours elapsed before subsequent

samples were taken in order to allow copepods to "settle"

onto the substratum. In all, each tire was sampled 5 times

at each of 4 different population levels (1,000, 2,000,

3,000, and 5,000 copepods per tire). A regression analysis

indicated a rather weak relationship (R2=0.51) between the

true population sizes and the corresponding sample sizes.

When samples are plotted against populations (Figure 2-10),

it is evident that sampling provided little more than

trends. Most importantly, sample ranges overlap

considerably among the 4 population categories. When

"significantly different" means from the field data are

plotted on this graph, it becomes apparent that statements

concerning the true population size differences between

treatment and control tires cannot be made with appreciable

confidence.

ANOVA analyses of copepod samples by date for the 7

sampling dates of the fall period revealed significant

reductions due to the treatment on 2 dates Oct. 12 (P<

0.00) and Oct. 20 (P=0.01). While this coincides generally

with elevated larval infections in the fall, these dates do

not correlate with reduced pupal production. Perhaps the

most important and encouraging finding regarding copepods is

simply that populations were never extinguished in treated












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tires. This was true even for tires that were supporting

larval epizootics.

The reason for increased fungal activity beginning

Sept. 14 is not clear. Because the maximum water

temperatures remained remarkably constant throughout the

entire season, that is not likely to be a factor. As

mentioned, C. stegomyiae was released on 3 occasions July

1, July 9, and July 28. This final release (of infected

copepods) preceded Sept. 14 by 7 weeks a period much too

long to account for larval infection noticed on the latter

date.

A more likely reason involves copepod age structure in

the tires. While the age structure was not quantified, it

was clearly evident during laboratory counts that the

populations were comprised principally of adults and older

copepodids (juveniles). Although nauplii were retained by

the 55 micron nylon screen, they were rarely observed. While

the former 2 stages are indistinguishable unless viewed

under high magnification, laboratory observations strongly

suggest that the field populations were comprised

principally of adults. Adults are apparently poor hosts for

C. stegomyiae (Whisler, pers. comm. and Gettman,

unpublished). It would appear that the populations

"stagnated" at a plateau comprised principally of adults.

This phenomenon clearly occurs with neglected

laboratory colonies. If a colony is strained with a screen










and provided with fresh food (ground alfalfa) and water, a

flush of eggs and subsequent immatures results. It is

reasonable to assume that a similar situation was occurring

in the field. The shadecloth covering the plot prevented

falling leaves and other falling organic debris from

reaching the tires. Thus, the nutritional status was

principally regulated by the researcher. Ground oak leaves

were provided to all tires on June 30 and Aug. 18.

Coincidentally, tires received significant amounts of fresh

water (10.16 cm of rainfall) on Aug. 14. Also, all tires

were filled to capacity with well water on Aug. 26. The

high rainfall caused all tires to overflow and the process

of adding well water agitated the debris, perhaps making

nutrients more available. While the importance of the oak

leaves and water additions is unknown, it is reasonable to

expect that their combined effect would induce copepods to

reproduce as "stagnant" laboratory colonies do.

Because the latter addition of oak leaves preceded

Sept. 14 by 4 weeks, a reasonable timetable of events can be

constructed to account for the increase in the number of

infectious tires noted on the latter date. A cohort of

immature copepods would reasonably be available for

infection as early as one week after the nutrient level was

augmented. Immatures infected would begin to die (as

adults) and release zoospores in another week. Thus,

mosquito larvae could become infected as early as Sept. 1.








75

Another week would elapse before symptoms were recognized in

the field. Theoretically then, infected larvae could be

recognized as early as Sept. 7. Allowing for lags in the

idealized scenario presented, it is not unreasonable to

expect a delay of one week. Thus, the sequence of events

leading up to infected larvae could have been set into

motion by copepod populations revitalized four weeks

earlier.

The above hypothesis is put forward only tentatively

because it relies on scant evidence (few tires) and unknown

factors may have been involved. Also, the hypothesis fails

to explain why most treated tires did not become infectious

at or near the same time. Nevertheless, some factors)

obviously induced an increase in fungal activity late in the

season, and the evidence presented supports the notion that

a rise in nutritional status for the copepod could have been

the critical factor.

Copepod population size varied considerably among tires

within a treatment. However, tires tended to maintain

rather consistent sample sizes over time. That is, tires

supporting low or high populations usually did so

consistently. Also, population size was evidently not

correlated with either larval infections or pupal

production. As an attempt to ascertain why tires supported

radically different copepod populations, several water

quality parameters were recorded and compared to copepod










populations by regression analyses. On Aug. 31, a water

sample was acquired from each of the 72 tires and the pH was

recorded in the laboratory. Readings were remarkably

consistent and, not surprisingly, regression analysis

indicated no relationship (R2 =0.03). The average pH value

was 6.80 quite close to the pH 6.6 value Laird (1959a)

used for successful infection of Ae. aeyvpti with C.

stegomyiae.

The dissolved oxygen content in each of the 72 tires

was also regressed with copepod populations. On Sept. 15,

measurements were taken in the field with a portable meter

(YSI). The average of the 72 values was 3.60 ppm with a

range of 2.6 to 4.8. The relationship proved insignificant

(R2 = 0.16), as might be expected within such a narrow range

of dissolved oxygen values. The concentration of dissolved

oxygen is recognized as a critical factor for crustaceans

(Cameron and Mangum 1983) because oxygen is much less

abundant in aquatic systems than in air. While the

relationship of dissolved oxygen and P. viquieri has not

been reported, numerous studies of marine and lacustrine

copepods have indicated that dissolved oxygen levels limit

the abundance of copepods to defined strata in these

systems. Dadswell (1974) quantified the stratification of

two species of calanoid copepods in numerous lakes in

eastern Canada. He found both copepods absent at depths

where the dissolved oxygen level was less than 0.6 ppm a










value much lower than the low of 2.6 ppm noted in the

present study. While P. viquieri survival is undoubtedly

limited by the availability of oxygen, the present results

suggest that oxygen was sufficiently abundant in all of the

72 tires.

Finally, water samples from 24 tires were chemically

analyzed. On Sept. 9, samples were retrieved from the 12

tires supporting the lowest copepod populations and the 12

tires supporting the highest populations. Values (in ppm)

for 9 soluble elements and compounds, in addition to total

soluble salts, were regressed against copepod sample counts

(Table 2-9). The soluble salts value represents electrical

conductivity. Copper, iron, and manganese were omitted from

the analysis because most values were at or near zero. The

10 independent factors and copepod density (the dependent

variable) were also entered into a stepwise multiple

regression model. Although 7 of the 10 factors were

significant when regressed individually, the model rejected

9 of the 10 as insignificant contributors to the overall

model. Calcium alone was retained as the factor which best

explained copepod density.

It is interesting to note that Whisler (pers commun.)

recommends the addition of several of the significant

elements in media used for rearing P. viquieri in the

laboratory. His medium is as follows: 50 mg NaHCO3, 25 mg

MgSO4 x 7H20, 12.5 mg KC1, and 50 mg Ca(NO3)2 x 4H20 in 1










Table 2-9. Relationships of water chemistry to
copepod density.

FACTOR R2 FACTOR R2

ammonium-nitrogen .01 phosphorus .20.
calcium .41. potassium .26.
chlorine .22. sodium .31.
magnesium .29. soluble salts .35.
nitrate-nitrogen .09 zinc .06

* = significant at P < 0.05 (regression analysis).


liter of distilled water. (However, this author has

repeatedly used only tap water from several different

sources for maintaining P. viguieri in colony with no

apparent ill effects.) Henning (1975) and Bliss (1968) note

that crabs and crayfish may consume their exuviae apparently

as a source of calcium. Graf (1978) notes that several

crustaceans store calcium salts in the hemolymph and midgut

glands prior to molting and subsequently export them to the

new shell for hardening. Thus, it appears that calcium is

an essential and possibly limiting element for some

crustaceans.

While the current field data indicate that calcium may

be a significant contributor to the welfare of P. viquieri

populations, the results must be viewed with caution, given

the many unknowable factors that frequently influence field

studies. The results are possibly meaningful, but the issue

must be addressed in the laboratory, where the control of

extraneous factors and sampling are not problems, before

firm conclusions can be reached. The reason for differing









79
calcium levels in the tires is unknown. Three explanations

are proposed. 1) Perhaps varying amounts of calcium were

introduced into the tires by calcareous sand and/or by

isopods. Most tires contained only trace amounts of sand

but drowned isopods commonly littered the surface of the oak

substratum. Conceivably, the numbers of isopods were

sufficiently variable to account for the variation of

calcium quantities. 2) While rainfall was essentially

equally distributed to all 72 tires, the amount of flushing

was not because tires were not all shaped equally. Thus, it

is conceivable that the tires with greater exchange of water

could be those which exhibited lower calcium levels. 3)

Finally, the notion that the relationship between copepod

populations and calcium levels may be reversed cannot be

ignored. Perhaps copepods process resident complex salts

and one product is calcium which consequently leaches from

exuviae. In this (speculative) scenario, higher copepod

populations might lead to elevated calcium levels.

In the spring of 1988, data concerning the survival of

copepods and fungus through the previous winter were

collected. Water samples taken on April 28 revealed that 28

of the 72 tires retained copepods. Unfortunately, the water

conditions had not been monitored up until April 28. It was

not determined if water in any tires had frozen during the

winter. It is presumed that the 28 tires never completely

dried out during the winter. That presumption is supported










by data acquired from April 28 to May 27 during which time

water levels and copepod populations were monitored. On May

27 only 22 (of the same 28) tires contained copepods. The 6

tires devoid of copepods had become completely dry (the

debris measured 7% moisture by weight).

The survival of C. stegomyiae through the winter was

determined in the laboratory. The complete contents of each

of the 36 treatment tires were retrieved and transferred

into 36 enamel baking pans on June 2, 1988. During a five

week period, the pans were monitored for fungal activity as

follows. Several hundred healthy copepods were added to

each pan on June 4. First instar Ae. aeqypti larvae were

added to the pans on five occasions. Twenty were added to

each pan on June 4, 10, 12, and 16. Fifty larvae were added

to each pan on June 20. Pans were observed ca. twice weekly

for the presence of visibly infected larvae. In addition,

older larvae were observed regularly under a dissecting

microscope to detect nonpatent infections. When this

experiment was terminated on July 6, 22 pans had produced

infected larvae. Of these 22 pans, 15 were derived from

tires that had produced field infections the previous

summer. Thus, 7 tires had harbored viable resting sporangia

since July 28, when tires were inoculated the final time.

Also, resting sporangia survived in 11 tires in which debris

had completely dried during the April 28 May 27 period.










In summary, the 1987 season represented a marked

improvement over the 1986 season. Mosquito populations were

more persistent, copepod survival was greatly enhanced,

fungal activity (while not dramatic) was increased, and

resting sporangia survived seemingly rigorous conditions.



Materials and Methods (1988 Study)


On June 6, all tires and meat basters were thoroughly

cleansed and rinsed to remove residual debris, copepods, and

resting sporangia from the previous season. All tires were

refilled with 2 2.5 liters of well water and 26 grams of

pulverized oak leaves on June 8. On June 9, ca. 740

copepods (mixed ages) were added to each tire.

Additionally, the 36 treatment tires received infected

copepod colonies on June 10. Five infected colonies were

retained in the laboratory to ascertain the viability of the

field released colonies. One was observed regularly for

dead copepods containing swarming zoospores. Many were

noted in that condition on June 21. The remaining 4

infected colonies were released into 4 automobile tires

maintained in the laboratory for detailed monitoring of

events. These 4 tires were stocked with the same well water

and oak leaves as the tires in the field. On June 10, the

72 field tires and the 4 laboratory tires received 50 first

instar (ca. 36 hrs. post-hatch) Ae. aegypti larvae. Larvae

were derived from a laboratory colony that was derived from








82

a colony maintained at the USDA IAMARL. Colony maintenance

was as described in this chapter.

Coelomomyces stegomyiae resting sporangia liberated

from 2 large larvae were added to all treatment tires on

June 10. The inoculum was produced as previously described

in this chapter. The 4 laboratory tires subsequently

produced larval epizootics, indicating that the field

releases were probably successful. Four cups, each

containing ca. 1,000 Ae. aegypti pupae were set near the

chicken cages at the field site on June 10 in order to re-

establish a mosquito population.

Based on poor initial results, treatment tires were

reinoculated with infected copepod colonies on July 8. Also

on July 8, 50 first instar (ca. 24 hrs. post-hatch) Ae.

aevypti larvae (laboratory colony) were added to each tire.

Other inputs included filling all tires with well water on

July 12 and adding 9.5 grams (dry wt.) of crushed oak leaves

to each tire on July 25 and again on Sept. 12. Finally, the

mosquito population was augmented with ca. 2,000 pupae on

July 26, ca. 2,000 on Aug. 26, and ca. 1,000 on Sept. 16.

Several adjustments were made to the 1987 sampling

protocol. Larval infections were only recorded in the

field. Larvae were not sampled for laboratory assessment of

infection because that activity did not provide substantial

information during the previous season. Copepods were

sampled ca. twice monthly because the weekly samplings taken










in 1987 proved particularly time-consuming and population

trends could be readily assessed, it was reasoned.


Results and Discussion (1988 Study)


Maximum water temperatures were similar to those

recorded in 1987. From 84 recordings, temperatures ranged

from 26*C to 30*C with an average of 28.30C. Toxorhynchites

rutilus rutilus activity was greater than during the

previous 2 years. Larvae were captured from a total of 26

observations between Sept. 9 and Sept. 30. While the impact

of Tx. rutilus rutilus predation on the pupal production was

not addressed, it is presumed to be minimal because the vast

majority (478) of tire observations during that period

yielded no Tx. rutilus rutilus larvae. Any impact on the

effect of the treatment was further diminished because

larvae were relatively evenly distributed among control (16)

and treatment (10) tire observations. As in 1987, the Ae.

aegypti population seemed relatively self-sustaining and the

additional pupal releases apparently augmented the

population.

An ANOVA was conducted comparing pupal production from

treated and control tires totalled for the 28 sampling dates

comprising the entire season (June 17 Sept. 30). The

treatment produced significantly fewer pupae (P=0.01) (Table

2-10). Based on larval infection data, it was determined

that fungal activity increased in midsummer. Separate









84

Table 2-10. A summary of mean pupal production for 3 periods
during the 1988 season.
PUPAE / TIRE
PERIOD TREATMENT N(a) MEAN + S.E.
Jun 17 Sep 30 control 1008 6.0 0.2
treated 1002 5.2(b) 0.2

Jun 17 Jul 29 control 468 8.1 0.3
treated 468 8.4 0.4

Aug 1 Sep 30 control 540 4.2 0.2
treated 534 2.4(b) 0.1

(a) = 36 tires # of sampling days.
(b) = significant at P < 0.01 (ANOVA).


ANOVAs conducted for the periods preceding and following

Aug. 1 indicated that, as expected, pupae were not

significantly reduced (P=0.59) prior to Aug. 1, but were

significantly reduced (P<0.01) after that date (Table 2-

10). For this latter period, 42.9% fewer pupae were

produced from treated tires than from control tires. This

corresponds well with the fact that larval infections first

became apparent on July 15 and continued until sampling

terminated on Sept. 30.

Table 2-11 indicates that fewer pupae were produced

from treated tires on each of the 15 fall dates when means

were calculated for each date. ANOVA tests by date revealed

that the reduction was significant on 8 of these dates.

Finally, pupal production from 8 tires which regularly

produced infected larvae over a 6 week period (Aug. 8 -

Sept. 26) were compared (t-tests) to pupal production from

the 36 control tires during the same period. Production was

decreased by 55.2% a significant (P<0.01) reduction. This












Table 2-11. Comparisons of pupal production between treated
and control tires b date for the lat 8


PUPAE / TIRE
DATE TREATMENT N MEAN + S.E.
Aug. 1 control 36 7.4 0.6


It

Aug.


Aug.
Aug.
Au

Aug.


Aug.
Sept.

Aug.
Sept.

Aug.



Sept.
iS

Sept.


Sept.

oI
Sept.


Sept.


Sept.
I'

Sept.
I'

Sept.


Sept.
11


treated

control
treated

control
treated

control
treated

control
treated

control
treated

control
treated

control
treated

control
treated

control
treated

control
treated

control
treated

control
treated

control
treated

control
treated


4.9


5.6
3.6*

6.8
3.6*

6.3
3.0*

4.3
2.1*


3.7
2.3


3.4
1.5*

5.0
2.1*


2.8
1.9

2.4
2.2

4.5
2.7


3.3
1.6*


3.3
1.9

2.8
1.7

1.9
1.0


0.9

0.8
0.6

0.9
0.7

0.8
0.6

0.6
0.4

0.6
0.5

0.6
0.3

0.7
0.4

0.5
0.4

0.4
0.6

0.7
0.8

0.5
0.4

0.6
0.5

0.6
0.5

0.5
0.3


* = significant at P < 0.05 (ANOVA).


* = significant


at P < 0.05 (ANOVA).










final analysis lends credence to the presumption that

infections were causing the reduction in pupal production.

Over the entire season, larval infections were noted

during 270 (26.8%) of the 1,002 field observations from

treated tires. These positive recordings were acquired from

a total of 33 (91.7%) of the 36 treated tires. By both

measures, fungal activity was more than twice that noted in

1987. All of the positive recordings occurred after July 11

(Figure 2-11). On July 15, 30 treated tires suddenly

exhibited infected larvae. While some pathogen cycling

activity persisted through the duration of the sampling

period (-Sept. 30), the majority of treated tires failed to

support fungal activity following the initial infection

sequence. The average highest water temperature data

recorded on Figure 2-11 indicate an insignificant

contribution by that factor. However, a brief drop in

temperature occurring on September 9 correlates roughly with

increased fungal activity over a subsequent 2 week period.

Quantifying larval infection levels in individual tires was

found to be prohibitively time consuming. Unlike the

conditions in 1987, many more tires initially exhibited

infections and generally higher larval populations made

counting difficult.

Table 2-12 lists the identities of the tires which

exhibited larval infections during August and September in

1988. As was the case in 1987, several tires were












4)


0
:3




x


(0






tic





0)
t)) -4.
r.



.r-4 :j-
-1-4 l
x

r'd

p4 0
4J ()


(ro




4JU
Ui)
$4-


114
0
























14J
W


























U)

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O 00 (D N 0 O '- NO 0

SI I I I
D m C

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00
w- c'J




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cc 0)








CCM

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i I I I i I o
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Y)-


H- 4- w (-







Co co CM CM ( Nr


























Table 2-12. Treated tires that produced Aedes aevypti
larvae infected with Coelomomyces stegomyiae during the
latter 2 months of the 1988 sampling season.


ROW


I II III


T1 T12
T1
T1 T2 T3
T1 T2 T3
T1
T1 T2
T1 T2
T1 T2
T1 T2
T1 T2
T1 T2
T1 T2
T1 T2
T1
T1


T8 T9
T8 T9
T8 T9
T8
T8 T9
T8 T9
T8
T8 T9
T8
T8
T8
T8
T8
T8


T2 T3 T5 T9
T2 T3 T10
T3 T10
T2 T3 T5 T10
T3 T5
T3 T5
T3 T5
T3 T5


T1 T2 T3 T6
T1 T2 T3 T6
T2 T6 T7 T8
T1 T2 T6 T7
T1 T2 T6
T2 T6
T2 T6
T2 T6
T2 T6 T7
T2 T6 T7 T8
T11 T2 T6 T7 T8
T2 T3 T6 T8
T2 T3 T6
T6
T6


DATE

Au 1
Au 5
Au 8
Aul2
Au22
Au26
Au29
Sp 1
Sp 9
Spl2
Spl6
Spl9
Sp23
Sp26
Sp30








90

repeatedly responsible for producing infected larvae. When

the tires' identification numbers are compared to those that

persistently produced infected larvae in 1987 (Table 2-13),

it is evident that both lists are strikingly similar. This

is surprising because all tires were thoroughly scrubbed and

rinsed and restocked with inoculum between the two seasons.

Table 2-13 contains only tires with 4 or more entries on

each of the 2 seasonal tables.

The similarities appear to be due to something more

than chance alone. The factors) that caused these

particular tires to harbor infections regularly is(are)

unknown. One hypothesis considers that these tires were

manufactured differently, thereby affecting the chemical

environment in the water. All tires' brands were recorded

and no relationship could be made in that regard. Tires

were retained in the same locations within the plot both

years but no relationships to oviposition or other factors

possibly affected by location are evident. No relationship

to water chemistry can be made because samples were only

taken in 1987 and were not retrieved from many of the tires

in question.

It is possible that copepod population structure in

these tires was more appropriate for the pathogen's welfare

but that contention cannot be supported for lack of age

structure data. It is conceivable that those tires received

a greater rainwater flux than the others, which might




































Table 2-13. Tires that persistently produced infected
larvae during 1987 and 1988.
ROW
YEAR I II III
87 T1 T7 T8 T9 T3 T5 T7 T2 T4 T8 T9 T10
88 T1 T2 T7 T8 T9 T3 T5 T1 T2 T3 T6 T7 T8 T9 T10








92

facilitate more vibrant copepod colonies. That could occur

because tires are not all shaped identically and rainfall

was not entirely equally distributed due to the nature of

the shadecloth covering. It was noticed, particularly

during light rains, that, to a limited extent, water

collected in and dripped from areas where the shadecloth had

sagged. To reiterate, factors that may have favored certain

tires' fungal activity both seasons are not at all evident.

The dramatic event recorded on July 15 is directly

linked to the second (and final) inoculation of treated

tires with infected copepods on July 8. The inoculum was

highly infectious, owing to a successful process of

producing many infected copepods in the laboratory. One day

prior to field release, substantial numbers of dead copepods

containing swarming zoospores were evident in all of the

infection dishes observed. The appearance of visibly

infected larvae on July 15 is exactly as expected because

spores were available to infect them as early instars 7 days

earlier. Obviously, the initial infections in the 30 tires

was artificially induced. Laboratory-produced diploid

zoospores were sufficiently abundant to cause patent

infections in many mosquito larvae. Therefore, the sudden

reduction in infectious tires is most likely due to

insufficient diploid spore production, which is probably due

to a low copepod infection level. This can be postulated

because the inoculation procedure proved that when diploid










zoospores are available, they infect mosquito larvae

successfully in the tire environment. Finally, infected

larvae were never found in control tires, indicating that C.

steqomyiae was not disseminated into them by infected adult

mosquitoes nor by other modes.

Copepods were sampled quantitatively on 7 dates. The

results of ANOVAs comparing the numbers of copepods in

samples from treated and control tires are presented in

Table 2-14. Separate analyses for the entire season, for

the periods preceding and following Aug. 1, and for each of

the 7 sampling dates indicated no significant differences.

As the data from 1987 suggest, the most important and

encouraging finding regarding copepods is simply that

populations were never extinguished in treated tires.

In the spring of 1989, data concerning the survival of

copepods and fungus through the previous winter were

collected. On April 24 the complete contents of each of the

36 treated tires were returned to the laboratory in 1 liter

jars for assessment. No copepods were found when each jar

was observed at 8X magnification. Apparently the debris in

all 36 tires became completely dried and/or freezing

temperatures recorded during the first week of March killed

the copepods. Tire contents were transferred subsequently

to 36 enamel pans and assayed for the presence of viable C.

stegomyiae resting sporangia. The protocol used for this

assay in 1988 was repeated for 1989. The assay was