FIELD AND LABORATORY STUDIES OF THE MOSQUITO
FUNGAL PATHOGEN COELOMOMYCES STEGOMYIAE
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
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
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
TABLE OF CONTENTS
ABSTRACT..................... .............................. vi
I LITERATURE REVIEW...................................4
II A FIELD EVALUATION OF THE FUNGAL
PATHOGEN COELOMOMYCES STEGOMYIAE
AGAINST AEDES AEGYPTI............................... 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
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
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
Alan D. Gettman
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.
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
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
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
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.
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.
Corethrella appendiculata Grabham
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
Simulium metallicum Bellardi
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)
Heterocypris incongruens Sars
Potamocypris smaragdina (Vavra)
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
(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.
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
Q./~ \V96/^ )
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.
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
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
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
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
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
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
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
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
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.
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
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.
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
A FIELD EVALUATION OF THE FUNGAL PATHOGEN
AGAINST AEDES AEGYPTI
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
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
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
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)
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.
Figure 2-3. One of the 5 field sites used for the 1986
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.
The five field sites were sampled twice weekly from
July 1 to August 25. Sampling was continued at site A until
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)
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
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
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
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 #
of sampling days.
at P < .05 (t-test).
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
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
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)
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
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
I II III
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.
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
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.
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
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
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.
WW~r- u, ".
Figure 2-8. A modified tire in the field. The lid is opened
to reveal the water pool.
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
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
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.
DATE I II III
J1 27 T2
Jl 31 T2
Au 3 T2
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
I I I
0o (0 qt
i i i I I
CO Q n
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
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 )
T8 ( 3)
T8 ( 5)
T7 ( 9)
T9 ( 7)
Table 2-5. Percent infected larvae from treated tires
in row II in 1987.
TIRE I.D. + ( % INFECTION )
Aug. 31 T3 ( 8)
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 )
T4 ( 5)
T4 ( 3)
T4 ( 9)
T4 ( 4)
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
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
I I I I I I I
I I -- T
tires. This was true even for tires that were supporting
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
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.
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
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.
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
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
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
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
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
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
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
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
* = 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
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
O 00 (D N 0 O '- NO 0
SI I I I
D m C
i I I I i I o
L O 0 O 0 LO 0
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.
I II III
T1 T2 T3
T1 T2 T3
T2 T3 T5 T9
T2 T3 T10
T2 T3 T5 T10
T1 T2 T3 T6
T1 T2 T3 T6
T2 T6 T7 T8
T1 T2 T6 T7
T1 T2 T6
T2 T6 T7
T2 T6 T7 T8
T11 T2 T6 T7 T8
T2 T3 T6 T8
T2 T3 T6
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
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.
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
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