MORTALITY AND DEVELOPMENT OF AEDES LARVAE
EXPOSED TO POTENTIAL NATURAL PATHOGENS
ASPERGILLUS NIGER, FUSARIUM OXYSPORUM, AND PYTHIUM ULTIMUM

By
Rebecca Jean Morningstar

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Comparative Medicine and Integrative Biology - Master of Science

2013

ABSTRACT
MORTALITY AND DEVELOPMENT OF AEDES LARVAE
EXPOSED TO POTENTIAL NATURAL PATHOGENS
ASPERGILLUS NIGER, FUSARIUM OXYSPORUM, AND PYTHIUM ULTIMUM
By
Rebecca Jean Morningstar
Survival and development of Aedes triseriatus, the mosquito vector of La Crosse
Encephalitis, were found to be inhibited in the presence of dried leaves of certain
populations of the sugar maple Acer saccharum. It is suspected that these effects are
due to fungal contaminants carried on the leaf surface. Pure cultures of Aspergillus
niger and Fusarium oxysporum were isolated from experimental containers in which
these effects were observed. Pure cultures were used to challenge larvae of Ae.
triseriatus and the invasive species Aedes japonicus japonicus. Results indicate that
Aspergillus spores have a significant and repeatable lethal effect on larvae of the tested
species, particularly those at the first and second instar. Results also indicate a
significant inhibition of development in some experiments. Also implied is a possible
competitive advantage of Ae. j. japonicus over Ae. triseriatus when exposed to spores
of the fungus Aspergillus, as Ae. j. japonicus rates of survival were significantly higher
than those of Ae. triseriatus when run concurrently.

ACKNOWLEDGEMENTS

This work was funded by NIH grant # AI21884. I would like to thank Michigan State
University and in particular the departments of Comparative Medicine and Integrative
Biology, Entomology and Microbiology and Molecular Biology for support. This thesis
would not have been possible without the constant support and guidance of my major
advisor, Dr. Edward D. Walker and the members of my guidance committee: Dr.
Michael Kaufman, Dr. Linda Mansfield, Dr. Terence Marsh, and Dr. A. Leonel Mendoza.
I would also like to thank Drs. Vilma Yuzbasiyan-Gurkan and Victoria Hoelzer-Maddox
for personal and program support, and of course, my friends, family, and coworkers for
all of their advice and support throughout my entire academic career, and my husband
Eric for everything he does every day.

iii

TABLE OF CONTENTS

LIST OF TABLES ........................................................................................................... vii
LIST OF FIGURES ........................................................................................................ viii
CHAPTER 1
INTRODUCTION AND BACKGROUND INFORMATION................................................ 1
Treehole environments ........................................................................................ 2
Aedes triseriatus as a vector of disease ............................................................... 3
Aedes japonicus as a vector of disease ............................................................... 7
Vector development.............................................................................................. 7
Pathogenic and mosquitocidal effects of fungi and oomycetes on mosquito
larvae. ................................................................................................................... 8
Objectives and Hypotheses ................................................................................ 12
LITERATURE CITED ......................................................................................... 14
CHAPTER 2
OBSERVATION OF INCREASED MORTALITY OF AEDES TRISERIATUS LARVAE
EXPOSED TO POTENTIAL ENVIRONMENTAL CONTAMINANTS AND
CHARACTERIZATION OF POTENTIAL ETIOLOGIC AGENTS ASPERGILLUS
NIGER AND FUSARIUM OXYSPORUM ...................................................................... 20
Introduction ......................................................................................................... 21
Materials and Methods ....................................................................................... 22
Mosquito Culture ...................................................................................... 22
Initial Observations and Analysis ............................................................. 22
Ergosterol Analysis .................................................................................. 23
Confirmation of Effect .............................................................................. 24
Oomycete Analysis .................................................................................. 26
Natural Microcosm Survey ....................................................................... 26
Maple Survey ........................................................................................... 27
Fungal Isolations ...................................................................................... 28
Identification............................................................................................. 29
Results ............................................................................................................... 30
Initial Observations and Analysis ............................................................. 30
Ergosterol Analysis .................................................................................. 30
Confirmation ............................................................................................ 30
Oomycete Analysis .................................................................................. 32
Surveys .................................................................................................... 32
Fungal Isolation and Identification ........................................................... 34
Discussion .......................................................................................................... 34
LITERATURE CITED ......................................................................................... 37
iv

CHAPTER 3
QUANTIFICATION OF MORTALITY AND INHIBITION OF DEVELOPMENT OF
MOSQUITO LARVAE (CULICIDAE) FOLLOWING EXPOSURE TO PURIFIED SPORE
SUSPENSIONS OF ASPERGILLUS NIGER, FUSARIUM OXYSPORUM,
AND PYTHIUM ULTIMUM ........................................................................................... 40
Introduction ......................................................................................................... 41
Objectives and Hypotheses ..................................................................... 43
Materials and Methods ....................................................................................... 44
Media Preparation ................................................................................... 44
Fungal Spore Suspension Preparation .................................................... 46
Single Species Laboratory Microcosms ................................................... 47
Mixed Species Laboratory Microcosms ................................................... 48
Mixed Species Simulated Field Microcosms ............................................ 49
Single Species Laboratory Nanocosms ................................................... 51
Multi Species Laboratory Nanocosms...................................................... 52
Adult Emergence Nanocosms ................................................................. 54
Anopheles nanocosms............................................................................. 54
Results ............................................................................................................... 55
Single Species Laboratory Microcosms ................................................... 55
Mixed Species Laboratory Microcosms ................................................... 57
Mixed Species Simulated Field Microcosms ............................................ 58
Single Species Laboratory Nanocosms ................................................... 59
Multi Species Laboratory Nanocosms...................................................... 61
Adult Emergence Nanocosms ................................................................. 61
Anopheles nanocosms............................................................................. 63
Discussion .......................................................................................................... 63
Single Species Laboratory Microcosms ................................................... 64
Mixed Species Laboratory Microcosms ................................................... 65
Mixed Species Simulated Field Microcosms ............................................ 65
Single Species Laboratory Nanocosms ................................................... 65
Multi Species Laboratory Nanocosms...................................................... 66
Adult Emergence Nanocosms ................................................................. 66
Anopheles nanocosms............................................................................. 67
LITERATURE CITED ......................................................................................... 68
CHAPTER 4
EXAMINATION OF POTENTIAL MECHANISMS OF ACTION
OF ASPERGILLUS NIGER, FUSARIUM OXYSPORUM, AND PYTHIUM ULTIMUM
AGAINST LARVAE OF THE MOSQUITO SPECIES AEDES TRISERIATUS
........................................................................................................................... 71
Introduction ......................................................................................................... 72
Materials and Methods ....................................................................................... 73
Mycotoxin Assay ...................................................................................... 73
Microscopic Analysis................................................................................ 74
Results ............................................................................................................... 74
Mycotoxin Assay ...................................................................................... 74
v

Microscopic Analysis................................................................................ 75
Discussion .......................................................................................................... 77
LITERATURE CITED ......................................................................................... 79
CHAPTER 5
CONCLUSIONS AND DISCUSSION ............................................................................ 81
LITERATURE CITED ........................................................................................ 87

vi

LIST OF TABLES

Table 1.1. Fungal/oomycete genera known to be pathogenic to Aedes triseriatus.
................................................................................................................... 11
Table 2.1. Thermocycler program for fungal 18s PCR. ............................................... 23
Table 2.2. Thermocycler program for oomycete ITS1/5.8S PCR. ................................. 26
Table 3.1. Distribution of treatment groups for Single Species Laboratory Microcosms
experiment. ................................................................................................ 53

vii

LIST OF FIGURES

Figure 2.1 Experimental Microcosms. (For interpretation of the references to color in
this and all other figures, the reader is referred to the electronic version of
this thesis.) ................................................................................................. 24
Figure 2.2. Larval survival after 15 days growth on oak or maple leaves.
Note: OL= Oak microcosm, ML=maple microcosm. .................................. 31
Figure 2.3. Development of larvae after 15 days growth on oak or maple leaves. Note
the pronounced difference in general developmental progress between oak
and maple microcosms. Light grey indicates early (first and second) instars,
dark grey indicates late (third and fourth) instars, black indicates pupae.
.................................................................................................................. 32
Figure 2.4. Average percent survival of larvae in microcosms constructed with the
indicated source of leaves. Error bars indicate standard error. n=12.
................................................................................................................... 33
Figure 2.5: Average number of larvae per microcosm in the designated developmental
stage. Light grey indicates early (first and second) instars, dark grey
indicates late (third and fourth) instars, black indicates pupae. Error bars
indicate standard error. n=12. ................................................................... 34
Figure 3.1. Average survival of Ae. triseriatus larvae after exposure to various doses of
spores of the fungus Aspergillus niger. Error bars indicate standard error.
n=6. ........................................................................................................... 55
Figure 3.2. Average survival of Ae. j. japonicus larvae after exposure to various doses
of spores of the fungus Aspergillus niger. Error bars indicate standard error.
n= 6. ......................................................................................................... 56
Figure 3.3. Average survival of Ae. triseriatus larvae after exposure to various doses of
spores of the fungus Fusarium oxysporum. Error bars indicate standard
error. n= 6. ............................................................................................... 57
Figure 3.4. Average survival of Aedes larvae after challenge with mixed species
(Aspergillus and Fusarium) fungal inoculum in laboratory microcosms. Error
bars indicate standard error. a) Aedes triseriatus survival b) Ae. triseriatus

and Ae. j. japonicus survival. n=6. *p<0.005. ............................................ 58

viii

Figure 3.5. Average survival of Aedes larvae after challenge with mixed species
(Aspergillus and Fusarium) fungal inoculum in simulated field microcosms.
Error bars indicate standard error. a) Trial 1 b) Trial 2. Trial 1 n= 8, Trial 2
n=9. ........................................................................................................... 59
Figure 3.6. Ae. triseriatus survival by treatment group. Note the correlation between
survival and age of the larva at the beginning of the experiment in
Aspergillus groups. (Tris= Aedes triseriatus, Asp= Aspergillus, Fus=
Fusarium, Ctrl= Untreated Control.) n=24. ................................................ 60
Figure 3.7. Ae. j. japonicus survival by treatment group. Note the low survival of first
instar larvae challenged with Aspergillus. (Jap= Aedes japonicus japonicus,
Asp= Aspergillus, Fus= Fusarium, Ctrl= Untreated Control.) n=24
................................................................................................................. 61
Figure 3.8. Survival of Ae. triseriatus larvae when challenged with single- and mixedspecies fungal inocula. Ctrl: Control, Asp: Aspergillus, Fus: Fusarium, Pyth:
Pythium, A.F: Aspergillus/Fusarium, A.P: Aspergillus/Pythium, F.P:
Fusarium/Pythium, All: All three fungal species. n=24. ............................... 62
Figure 3.9. Cumulative adult emergence of Ae. triseriatus challenged with Aspergillus
or Fusarium spores as larvae. n=24. ......................................................... 62
Figure 3.10. Survival of Anopheles gambiae larvae challenged with spores of the
fungus Fusarium oxysporum. n=48. .......................................................... 63
Figure 4.1. Survival of Aedes triseriatus larvae exposed to fungal extracellular filtrate.
No significant differences are present between groups. n=5. .................... 75
Figure 4.2. Photographs taken under a standard dissecting microscope of visible
filamentous growth around the anal papillae of moribund Ae. triseriatus
larvae after growth in maple-based microcosms. Unusual growth marked
with arrows. ................................................................................................ 76

ix

CHAPTER 1: INTRODUCTION
AND BACKGROUND INFORMATION

1

Mosquitoes vector a broad range of diseases of both human and veterinary
medical importance. Among these are the arboviruses, such as the La Crosse
Encephalitis Virus and West Nile Virus, which can cause severe neuroinvasive disease
resulting in lasting neurological effects or death (Rust et al. 1999, Turell et al. 2001,
Whitley and Gnann 2002, CDC 2009). As a result of their role in the transmission of
these potentially devastating diseases, native and invasive mosquito species such as
Aedes triseriatus and Aedes japonicus japonicus are of particular interest in current
entomological research. It has been shown that various factors that affect the
development of these mosquitoes during their immature stages can have a significant
effect on the ability of the adults to successfully transmit disease (Grimstad and
Haramis 1984, Grimstad and Walker 1991, Paulson and Hawley 1991), and that
exposure to certain fungal species may have detrimental and/or fatal effects on
developing larvae (Nnakumusana 1985, Moraes et al. 2001, Seye et al. 2009). Thus it
is that the current research focuses on the effects of challenge with native
fungal/oomycete genera on the larvae of treehole/container breeding mosquito species
Aedes triseriatus and Aedes japonicus japonicus.

Treehole environments. As indicated by its common name, the Eastern Tree
Hole Mosquito, Aedes triseriatus is a tree hole-dwelling species. Tree holes are, as the
name implies, holes that form in the body of a tree and collect water. These can form in
a variety of ways. Two of the main varieties are pans and rot holes. Pans, which tend to
be shallow and have a solid bark base, form when parts of the tree, such as aboveground root sections, grow together and form depressions or pockets. These pans

2

collect water and insects, such as mosquitoes, subsequently utilize them for oviposition
and larval development. Pans tend to be more ephemeral than deeper treeholes, such
as rot holes. Rot holes form when a wound in the tree leads to the rotting away of
tissue and eventual formation of a hole (Kitching 1971). These holes can be very deep
or quite shallow, and the size of the opening may vary greatly, depending on the origin
and development of the hole. Both pans and rot holes can collect a wide range of inputs
such as leaves, soil, flowers, and decaying insect matter (Unpublished data). These
inputs serve to increase the microbial community and dissolved nutrient content of the
treehole, which in turn aids in and affects the development of larval insects such as
mosquitoes (Fish and Carpenter 1982, Walker et al. 1991, Kaufman et al. 2010).
Relatively few studies have been conducted examining the fungal community
typically found in tree holes. Several of those that have were conducted in beech-maple
forests in the area near the campus of Michigan State University in East Lansing, MI
(Kaufman et al. 2001, Yee et al. 2007, Kaufman et al. 2008). Interestingly, neither
Aspergillus nor Fusarium species, which are the focus of this study, were among the 32
fungal species identified in the 2008 study by Kaufman, et al, which looked at bacterial
and fungal species associated with leaf surfaces in naturally occurring tree holes
(Kaufman et al. 2008) or the 2003 paper by Gönczöl and Révay which looked at
hyphomycetous fungi in natural treehole microcosms in Hungary (Gonczol and Revay
2003). However, species within both genera have been isolated from tree holes
containing larval mosquito habitats in Brazil (Luz et al. 2007).
Aedes triseriatus as a vector of disease. Aedes triseriatus is most well-known
as the primary vector of La Crosse Encephalitis (Sudia et al. 1971, Watts et al. 1972),

3

which is a viral infection caused by a virus within the family Bunyaviridae. It is a member
of the California encephalitis group and, prior to the outbreak of West Nile Virus in 1999,
was the causative agent behind the majority of identified cases of arboviral encephalitis
in the United States (Rust et al. 1999, Whitley and Gnann 2002). It is still the cause of
most cases of California serogroup neuroinvasive disease (CDC 2009), and has been
shown to infect both humans and occasionally dogs, who can then transfer the disease
back to owners/caretakers (Tatum et al. 1999).
La Crosse virus is transmitted by mosquitoes in the genus Aedes, specifically Ae.
triseriatus and the invasive species Ae. albopictus in the United States (Watts et al.
1972, Gerhardt et al. 2001). The virus amplifies in small mammal hosts such as
chipmunks, squirrels, and rabbits and is picked up by female mosquitoes when they
take a blood meal (Gauld et al. 1974, Yuill 1984). After establishing an infection in the
mosquito, the virus replicates and disseminates throughout the body of the mosquito.
One to two weeks post-infected blood meal, the virus infects the salivary glands and
amplifies. From here, it is passed to small mammal hosts during subsequent blood
meals. In addition to this cross-species transmission, the virus can be passed directly to
offspring via viral infection of the mosquito ovaries that then passes to the eggs (Borucki
et al. 2002). This transovarial transmission allows the virus to survive the winter season
in the protected environment of the mosquito egg (Watts et al. 1974). The La Crosse
virus can also be passed from mosquito to mosquito via venereal transmission
(Thompson and Beaty 1977).
La Crosse Encephalitis is rarely fatal. Rather, it usually manifests as a mild viral
infection in humans, frequently without noticeable symptoms. When symptoms do

4

occur, they are typically mild and include fever, headache, fatigue, and nausea. The fact
that the symptoms are so minor and generic leads to significant underreporting and/or
misdiagnosing of La Crosse Encephalitis (McJunkin et al. 2001, CDC 2009). However,
in the United States, approximately 80-100 cases each year develop into a severe,
potentially fatal neuroinvasive disease characterized by seizures (CDC 2009) that
closely resembles the disease caused by herpes simplex viral encephalitis (McJunkin et
al. 1997, Sokol et al. 2001). These neuroinvasive infections occur when the virus,
which replicates in muscle tissue, is able to reach a high enough viremia to penetrate
the blood brain barrier and cause apoptosis in neural cells of the brain (Hollidge et al.
2010). Approximately 3,600 cases of neuroinvasive encephalitis caused by a member
of the California encephalitis group (mostly La Crosse Encephalitis) were reported in the
United States from 1964 to 2010. This severe presentation is most prevalent in children
in the Eastern half of the U.S. and occurs between five and fifteen days after being
bitten by an infected mosquito. Most cases resolve without lasting effects, but
occasionally the disease does prove fatal (CDC 2009). In a small number of cases,
lasting neurological effects such as cognitive impairment, attention deficit, and impaired
voluntary movement may also develop. These can be transient and disappear after
only a short time, or they may be permanent (McJunkin et al. 2001, Utz et al. 2005,
CDC 2009, Hollidge et al. 2010). These neurologic after-effects can lead to severe
impairment, changes in behavior and personality, disabilities, and social impairment.
They may also cause significant hardship and emotional distress for the families of the
affected children (Utz et al. 2005).
Recently, the range of La Crosse Encephalitis has been increasing, and more

5

cases are being reported in the Southeastern United States than have been historically
(Jones et al. 1999, Nasci et al. 2000, CDC 2009). Unfortunately, awareness of this
disease has not been increasing. There is a surprising lack of public awareness of this
and related diseases (Soldan and Gonzalez-Scarano 2005), and Utz, et al (2005) found
when studying this disease in North Carolina, that “LAC encephalitis cases have been
reported from the mountains of NC since the mid 1960s […], yet the majority (80%) of
families who participated in our study were not aware of the disease even though they
resided in endemic areas.”
In addition to its role in the transmission of the La Crosse Encephalitis Virus, Ae.
triseriatus is a competent vector for several other diseases as well. This means that it is
able to become infected by a virus or other pathogen, maintain and amplify the
infection, and transmit said infection to naïve hosts. Of note, it has been shown that Ae.
triseriatus could serve as an effective bridge vector for West Nile Virus based on its
ability to become infected by feeding on infected chickens and its subsequent ability to
transmit that infection to uninfected birds (Turell et al. 2005). However, given that the
primary hosts of this mosquito species are small mammals such as chipmunks and
squirrels, rather than the avian hosts which are vital to the West Nile Virus transmission
cycle, its role in transmission of West Nile to humans is minimal at present. Ae.
triseriatus is also a competent vector for Eastern Equine Encephalitis (Chamberlain et
al. 1954) and the canine heartworm Dirofilaria immitus (Intermill 1973, Debboun et al.
2005), which has been shown to affect humans as both a pulmonary infection (Dashiell
1961, Merrill et al. 1980) and occasionally as an ocular infection (Moorhouse 1978,
Avellis et al. 2011).

6

Aedes japonicus as a vector of disease. In recent years, the mosquito Aedes
japonicus has spread from its natural range in Asia to regions of North America and
Europe (Peyton et al. 1999, Schaffner et al. 2009, Werner and Kampen 2013). Ae. j.
japonicus has been shown to be a highly competent vector for West Nile Virus in the
laboratory (Turell et al. 2001), even more so than Ae. triseriatus, as it was rated more
highly as both an enzootic vector and a bridge vector (Turell et al. 2005). West Nile
Virus has also been detected in wild populations of Ae. j. japonicus (Molaei et al. 2009,
CDC 2012). Fortunately, while it may serve as an occasional bridge vector, its feeding
habits are such that it is not likely to cause an immediate and dramatic increase in
human cases of this disease, as it feeds primarily on mammals, rather than the bird
species that are the amplifying vectors for West Nile Virus (Molaei et al. 2009). Ae. j.
japonicus was also shown to develop disseminated infections of La Crosse Encephalitis
Virus, which it could transmit, causing new infections in naïve mammal hosts (Sardelis
et al. 2002b, Sardelis et al. 2002a). Ae. j. japonicus has also demonstrated an ability to
transmit Eastern Equine Encephalitis to chickens in the laboratory (Sardelis et al.
2002b). However, the ability of a vector to transmit disease in the laboratory is not
always indicative of its role in disease cycles in nature, as there are many more factors
in play in the natural world than in most laboratory experiments.
Vector development. It is well established that factors affecting larval
development have a subsequent effect on the physiology of the adult mosquito and the
ability of adult mosquitoes to transmit arboviral diseases such as La Crosse
Encephalitis. Larval nutrition plays a critical role in establishing the fitness and vectorial
capacity of adult Aedes mosquitoes (Grimstad and Haramis 1984, Grimstad and Walker

7

1991). Additionally, competition is also a significant component to the development of
mosquitoes that can act as competent vectors of disease as adults (Grimstad and
Haramis 1984, Grimstad and Walker 1991, Paulson and Hawley 1991, Alto et al. 2005).
Generally, mosquitoes that have experienced malnutrition or other stressors as larvae
develop into smaller adults (Grimstad and Haramis 1984, Briegel 1990). These smaller
adults have an increased capacity to transmit the La Crosse Encephalitis Virus. Early
theories were that this increase in infection might be a result of the higher dose relative
to body size when small adults were given a dose equivalent to that given to larger
adults. However, subsequent research has shown that it is more likely due to enhanced
dissemination of infection through the barrier of the midgut. In fact, the basement
membrane of the midgut, which is one of several layers separating the contents of the
midgut from the rest of the body, was found to be nearly twice as thick in smaller
nutritionally deprived mosquitoes as it was in larger mosquitoes (McLintock 1978,
Grimstad and Haramis 1984, Grimstad and Walker 1991, Paulson and Hawley 1991).
Pathogenic and mosquitocidal effects of fungi and oomycetes on mosquito
larvae. There are many documented cases of fungi and oomycetes demonstrating
pathogenic effects against mosquito larvae. For example, several members of the
genus Aspergillus have been shown to be pathogenic to Aedes, Anopheles, and Culex
mosquitoes (Nnakumusana 1985, Moraes et al. 2001, Seye et al. 2009). Aspergillus
clavatus was shown to be effective at killing larvae of all three genera, though it was
more effective against mosquitoes of the genus Aedes than against mosquitoes of the
genus Culex (Seye et al. 2009). In another study, Moraes, et al (2001) tested eleven
different strains of Aspergillus fungi against Aedes and Culex mosquitoes and found up

8

to 100% mortality, in some cases within 24 hours. The Moraes, et al study looked
specifically at Aspergillus flavus, Aspergillus kanagawaensis, Aspergillus ochraceus,
Aspergillus sclerotiorum, and Aspergillus sulphureus. These strains were tested against
Culex nigripalpus and Aedes fluviatilis mosquitoes. In three tests, two with Aedes
mosquitoes and one with Culex, mortality rates above 65% were observed within 24
hours.
Another group of fungi known to be pathogenic to immature mosquitoes is the
collection of filamentous fungi within the genus Fusarium (Hasan and Vago 1972,
Teetor-Barsch and Roberts 1983, Prakash et al. 2010). A classic study by Hasan and
Vago (1972) showed that larvae exposed to conidia of the fungus Fusarium oxysporum
reached between 51% and 83% mortality, with higher levels being noted in populations
that had been injured prior to exposure. This pattern of spore invasion at mosquito
wound sites has been found in other studies. In a study by Clark, et al (1966), for
instance, an oomycete in the genus Pythium was found to invade Aedes triseriatus
larvae preferentially at wound sites.
Studies indicate that both Aspergillus and Fusarium species can cause negative
effects in mosquito larvae via multiple pathways. Species in both genera are known to
produce mycotoxins (Govindarajan et al. 2005). Some species of Aspergillus produce
aflatoxin, a highly potent

ycotoxins that has been shown to cause carcinogenic effects

in vertebrates, particular in the liver (Alpert et al. 1971, Eaton and Gallagher 1994,
Wang et al. 1996). Species in the genus Fusarium produce several types of
mycotoxins, including the trichothecenes, the fumonisins, and zaeralenone (D’Mello
et al. 1999, Placinta et al. 1999, Eriksen and Pettersson 2004). Extracellular filtrates of

9

cultures of both Aspergillus and Fusarium have been found to cause high mortality in
mosquito bioassays utilizing the species Culex quinquefasciatus (Govindarajan et al.
2005). In addition, both Aspergillus and Fusarium can infect mosquito larvae directly.
Invasion of the mosquito body cavity can occur through ingestion of spores, invasion at
wound sites, or direct penetration of the larval cuticle (Hasan and Vago 1972, TeetorBarsch and Roberts 1983, Moraes et al. 2001, Seye et al. 2009).
Despite the abundance of literature on interactions between mosquitoes and their
fungal pathogens, very little information exists regarding what fungi have pathogenic
effects on larvae of the treehole mosquito Aedes triseriatus. However, Ae. triseriatus
larvae have been shown to be susceptible to several different genera of fungi and
oomycetes. One such genus is Coelomomyces, a generalist pathogen that infects many
different insects, but is found most often in mosquitoes (Anthony et al. 1971, Whisler et
al. 1974, Couch and Bland 1985). Another important genus that has been shown to
have significant pathogenic effect against Ae. triseriatus is Lagenidium. It is important to
note that Lagenidium is an oomycete, not a true fungus (McCray Jr et al. 1973). In fact,
Lagenidium was found to be so effective at killing mosquito larvae that it was approved
in three formulations under the name Laginex for use as a larvicidal agent by the United
States Environmental Protection Agency (EPA) in 1996. It was used in an aquatic
suspension that was sprayed into natural larval habitats such as drainage sewers, tires,
streams, and rice paddies (Kerwin and Washino 1988, USEPA 2001). In August of
2011, the EPA published a request from Agraquest to voluntarily cancel production and
sales of Laginex (USEPA 2011).

10

Table 1.1. Fungal/oomycete genera known to be pathogenic to Aedes triseriatus.
Genus
Coelomomyces
Funicularius
Lagenidium
Leptolegnia
Metarhizium
Pleistophora
Pythium
Smittium
Tolypocladium
Verticillium

Reference
Couch and Bland, 1985
Zaim et al, 1979
Couch, 1935, McCray, 1973
Seymour, 1984
Andrade, 1993
Chapman, 1974
Clark et al, 1966, Nnakumusana 1985
Williams and Lichtwardt, 1972
Nadeau and Boisvert, 1994
Ballard and Knapp, 1984

A list of fungal/oomycete genera reported to be pathogenic to Ae. triseriatus can
be found in Table 1. Notably, neither Aspergillus nor Fusarium appear to have been
evaluated for activity against Ae. triseriatus larvae. As these are both fairly ubiquitous
fungi, they are species that Ae. triseriatus could be reasonably expected to encounter in
their natural habitat, so understanding more about the interactions between these
species is desirable for expanding our base of knowledge regarding mosquito
development in the wild.
The third potentially pathogenic genus utilized in this study, Pythium, is an
oomycete genus typically associated with blights and wilts of plants (Martin and Loper
1999). However, some species are also pathogens of both vertebrate and invertebrate
hosts. Most famous among the animal pathogens is the species Pythium insidiosum,
which causes very serious, and often fatal, disease in vertebrates such as horses, dogs,
cats, and humans, among others (Gaastra et al.). P. insidiosum has also recently been
isolated from a mosquito in India (Schurko et al. 2003). Other species of Pythium have
been reported as pathogens of mosquitoes as well. In fact, both Clark et al (1966) and
Nnakumusana (1985) have shown Pythium spp. To be pathogenic to Ae. triseriatus,
11

though in both cases, identification is restricted to “Pythium sp.” Pythium guiyangense
has also been identified as a mosquito-pathogenic species (Su 2008).
Objectives and Hypotheses. Based on the above information, the following
studies aim to further explore the relationship between Aedes larvae and potentially
entomopathic fungal/oomycete genera Aspergillus, Fusarium, and Pythium. The
rationale behind this set of studies was twofold: both to look at the basic science of the
interactions between these naturally occurring species and to examine potential natural
pathogens of disease-vectoring mosquito species in an attempt to identify fungi that
may utilize chemicals or processes that could be exploited in future mosquito control
agents. The objectives and major hypotheses are as follows:

Objective 1: Assess the presence and magnitude of mosquitocidal or inhibitory
effects of isolated fungal/oomycete species against Ae. triseriatus larvae.
Hypothesis 1: Aspergillus, Fusarium, and Pythium isolates will all
demonstrate significant mosquitocidal effects when used individually to
challenge Ae. triseriatus larvae.
Hypothesis 2: Aspergillus, Fusarium, and Pythium isolates will all
demonstrate significant inhibition of growth and development when used
individually to challenge Ae. triseriatus larvae.
Hypothesis 3: Mosquitocidal effects will increase with mixed-species
inoculum as compared with single-species inoculum.

12

Objective 2: Characterize the mode of action of mosquitocidal fungal/oomycete
isolates against Ae. triseriatus larvae.
Hypothesis 1: Mortality and inhibition of development of Ae. triseriatus
larvae challenged with Aspergillus and Fusarium isolates is due to a
combination of secondary metabolites and direct infection of larvae.
Hypothesis 2: Pythium isolate effects are due to direct infection of
mosquito larvae by motile zoospores, not secondary metabolites.

13

LITERATURE CITED

14

LITERATURE CITED

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19

CHAPTER 2: OBSERVATION OF INCREASED MORTALITY OF
AEDES TRISERIATUS LARVAE EXPOSED TO POTENTIAL
ENVIRONMENTAL CONTAMINANTSAND CHARACTERIZATION
OF POTENTIAL ETIOLOGIC AGENTS
ASPERGILLUS NIGER AND FUSARIUM OXYSPORUM

20

Introduction
Aedes triseriatus, the Eastern Treehole Mosquito, is the primary vector for
LaCrosse Encephalitis and competent to vector several other diseases, including the
canine heartworm Dirofilaria immitus (Intermill 1973) and West Nile Virus (Turell et al.
2005). Due to its potential for disease transmission, and its prevalence in Southern
Michigan, as well as its ability to survive and even thrive in established laboratory
conditions, it is an ideal organism for laboratory testing and evaluation of larval
development and factors affecting development.
In the laboratory, mosquito colonies are generally raised on a standardized diet.
Often the nutrients necessary for growth are supplied via dried and powdered
compounds such as liver powder (Arrivillaga and Barrera 2004), yeast (Rahuman et al.
2008), or ground pet foods, such as dog chow or fish food (Naksathit et al. 1999, Onken
et al. 2004). However, during experimental procedures with treehole mosquitoes, it is
common to see dried leaves used to encourage microbial growth as a nutrient source,
to more closely mimic the conditions that would be seen in the mosquito’s natural
habitat (Fish and Carpenter 1982). This does add a level of complexity to an
experimental container, as it is difficult to anticipate what microbes, fungal spores, etc,
may be carried into the container on the surface of the leaves. In some cases, these
contaminants can be incredibly detrimental to the development of the mosquitoes being
reared, and may cloud experimental results. Such was the case with a recent
experiment, during which dried leaves of the sugar maple Acer saccharum were used
as a microbial substrate and a subsequent growth of filamentous material in the water

21

column and on the leaf surface seemed to have a dramatic inhibitory and lethal effect
on developing larvae of the mosquito Aedes triseriatus.
The sudden and significant die-off of mosquitoes in the laboratory is a surprising
phenomenon, and one that with further exploration might yield information that could be
used to develop improved methods of control for these disease-carrying organisms. As
such, this observational study aimed to identify and characterize potential causative
agents of this effect.

Materials and Methods
Mosquito Culture. The mosquitoes used for this work are Aedes triseriatus,
Walton strain, and Aedes japonicus japonicus. Larvae are maintained in laboratory
culture and provided ground Tetramin brand fish food (Tetra Werke, Melle, Germany) as
a nutrient source. Laboratory colonies are regularly fed bovine blood (Hemostat
Laboratories, Dixon, CA) via an artificial feeder made of blown glass. Ae. j. japonicus
eggs were obtained from Dr. Linda McCuiston at Rutgers University in New Brunswick,
New Jersey. Unless otherwise stated, larvae used for all experiments were less than 24
hours old and hatched in the laboratory.
Initial Observations and Analysis. It was observed during an unrelated
experiment designed to evaluate the toxicity of ionic copper to larval Aedes triseriatus
that first instar larvae of this mosquito species unexpectedly demonstrated dramatically
increased levels of mortality regardless of treatment group (unpublished data). This
increased mortality appeared to be associated with the use of dried leaves of the sugar
22

maple Acer saccharum as a source of nutrients in the experimental microcosms.
Additionally, there was a distinct inhibition of development observed in those larvae that
did survive to the end of the experimental timecourse. Surviving larvae appeared
significantly smaller and underdeveloped when compared to larvae of the same age in
previous experiments and in the laboratory colony. Microcosms showing this increase in
mortality all had visible filamentous growth associated with the leaf surface and water
column. To facilitate further analysis of the suspected contaminating factor, two 50 mL
water samples were taken from each experimental microcosm. DNA was extracted from
one sample per microcosm using the MoBio Ultra Clean Soil DNA Isolation Kit (MoBio
Laboratories, Carlsbad, CA). DNA was then amplified via PCR using primers targeting
the 18S region of the fungal genome (Forward: 5’- TTA GCA TGG AAT AAT RRA ATA
GGA -3’, Reverse: 5’- ATT GCA ATG CYC TAT CCC CA -3’) (Borneman and Hartin
2000). Thermocycler program was as indicated in Table 2.1. This gene fragment was
cloned into E. coli using the pGEM T-easy Vector system (Promega; Madison, WI). The
cloned region was then sequenced and used for BLAST searches of the GenBank
database.
Table 2.1. Thermocycler program for fungal 18s PCR.
Stage
Cycles
Temp (°C)
Time

Stage 1
x1
94.0
2:00

94.0
0:45

Stage 2
x30
58.0
0:30

72.0
1:00

Stage 3
x1
72.0
4.0
7:00
infinity

Ergosterol Analysis. Ergosterol is one of the major components of the fungal
cell membrane and is a well-established biomarker for evaluating the amount of live
fungal biomass in a sample (Seitz et al. 1979). Ergosterol analysis was conducted as in
23

previous studies (Kaufman et al. 2001, Kaufman et al. 2002). Briefly, ergosterol was
extracted from 50 mL water samples as follows: Samples were centrifuged to
concentrate fungal biomass and pellets were resuspended in 5 mL of HPLC grade
methanol. To this suspension, 2 mL of a 4% potassium hydroxide in 95% ethanol
solution were added and samples were heated in an 80° C water bath for 30 minutes.
Samples were cooled and deionized water (2 mL) was added. Hexanes (4 mL) were
added to this solution and the sample was mixed thoroughly, then allowed to settle.
After a clear bilayer formed, the upper layer, containing hexanes and extracted
ergosterol, was removed and transferred to a new vial. To this vial, 3 mL hexanes were
added and another bilayer was allowed to form. Again, the upper layer was removed
and transferred to the same vial as the previous hexanes solution. This
hexane/ergosterol solution was dried under nitrogen in a 40° C sand bath and the dried
ergosterol was resuspended in methanol. This ergosterol solution was run through a
Shimadzu Prominence UFLC (Shimadzu Scientific Instruments, Columbia, MD) unit and
analyzed using the LCSolution software (Shimadzu Scientific Instruments, Columbia,
MD) and Microsoft Excel (Microsoft Corporation, Redmond, WA).
Confirmation of Effect. To confirm that the observed phenomenon was not an
isolated incident, a limited bioassay was conducted. For this bioassay, White Oak
(Quercus alba) leaves were used as a control. These leaves had been used
extensively in previous experiments and shown to promote healthy growth and
development of mosquito larvae. There was no documented evidence of increased
mortality associated with these leaves. Experimental microcosms were constructed as
follows: 345 mL milli-Q filtered water, 1 g dried maple or oak leaf (depending on
24

treatment group), and 5 mL treehole inoculum (water/debris collected from a natural
treehole and homogenized in a lab blender) were combined in a 16 oz clear plastic food
storage container (Figure 2.1). Four microcosms each were set up for treatment and
control groups. Microcosms were allowed to sit one week at room temperature, covered
with vented lids, to develop a robust microbial community prior to larval addition. After
one week, 20 first instar Ae. triseriatus larvae were added to each microcosm. Larvae
were maintained in the microcosms for 15 days, at which point the experiment was
terminated due to larval mortality. For the confirmation of effect, a t-test with Welch’s
correction for unequal variances between groups was run. To examine the distribution
of developmental stages between the two groups, a Wilcoxon Rank Sum test was run.
All statistics were completed in Program R (R Foundation for Statistical Computing,
Wien, Austria).

Figure 2.1 Experimental
Microcosms. (For interpretation
of the references to color in this
and all other figures, the reader
is referred to the electronic
version of this thesis.)
25

Oomycete analysis. Due to the conflict between the copious amounts of
filamentous growth in experimental microcosms and the low ergosterol levels returned
on analysis, it was determined that analyses should be run to survey for the presence of
oomycetes in the experimental containers. Oomycetes could potentially explain these
conflicting data, as they look and behave like fungus, but do not produce ergosterol,
instead relying on exogenous cholesterol in the construction of cell membranes
(McCorkindale et al. 1969, Gaulin et al. 2010) DNA was extracted using the MoBio
Ultra Clean Soil DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA) and amplified at
the ITS1/5.8s region using Pythium specific primers (Forward: 5’-GAA GGA TCA TTA
CCA CAC-3’, Reverse: 5’-TAC GGA CAC TGA TAC AG-3’, (Geraats et al. 2002))and
thermocycler conditions as outlined in Table 2.2. This gene fragment was cloned into
E. coli using the pGEM T-easy Vector system (Promega; Madison, WI). The cloned
region was then sequenced and used for BLAST searches of the GenBank database.

Table 2.2. Thermocycler program for oomycete ITS1/5.8S PCR.
Stage
Cycles
Temp (°C)
Time

Stage 1
x1
94.0
1:00

94.0
0:30

Stage 2
x30
52.0
0:30

72.0
1:00

Stage 3
x1
72.0
4.0
7:00
infinity

Natural Microcosm Survey. In order to confirm that the isolated fungi are
present in naturally occurring larval habitats, a survey of habitats on and around the
campus of Michigan State University was conducted. Survey sites included treeholes
and ponds on the main campus of Michigan State University, as well as in two woodlots
26

south of campus, Toumey and Hudson Woodlots. Water samples of 50 mL each were
collected from 24 known or potential larval mosquito habitats, including natural
treeholes, tires, and ornamental ponds. Polymerase chain reactions and gel
electrophoresis were conducted with fungal and oomycete primers as described above.
Maple Survey. In order to determine whether the observed effect was generally
associated with leaves of the sugar maple Acer saccharum or specific to leaves found in
the same location as the original collection, a survey of maple leaves from select
locations was conducted. Maple leaves were collected at the same location on the
Michigan State University campus as the original contaminated stock, from both the
ground and directly from the tree. This dual collection was to determine whether
contamination from the soil or grass might be responsible for the observed effect.
Leaves were also collected from a tree and the surrounding ground in one location in
Hudson Woodlot south of campus. White Oak (Quercus alba) and Beech (Fagus sp.)
leaves were used as controls, as both have been shown to consistently facilitate rapid,
healthy growth of Ae. triseriatus larvae. These leaves were used in experimental
microcosms set up as previously, with 345 mL milli-Q filtered water, 1 g dried leaf, and 5
mL treehole inoculum. Twelve microcosms were set up for each experimental group
(Campus Ground, Campus Tree, Woodlot Ground, Woodlot Tree, Beech Control, Oak
Control). After one week, the experiment was ended. Larvae were preserved in 70%
ethanol. Mortality and developmental stage of preserved larvae were recorded and 50
mL water samples were taken for further analysis. Non-parametric statistical analyses
were run to analyze these data. These tests included a Wilcoxon Rank Sum test

27

comparing mean mortality values, and Kruskal-Wallis and Mann-Whitney tests
comparing the distribution of developmental stage.
Fungal Isolations. Aspergillus cultures were isolated as follows. Samples taken
from suspect maple leaves were plated on Czapac Yeast Agar (CYA) to look for and
isolate both epiphytic and endophytic fungi. For epiphytic fungi, intact leaf surfaces
approximately equivalent in area to a 13 mm diameter leaf disc were swabbed with a
sterile cotton-tipped applicator dipped in milli-Q filtered water. The cotton tip was then
placed in 1 mL milli-Q water, swirled gently and allowed to soak for one minute. After
one minute, the cotton was removed and 200 μL water was spread on CYA plates.
Plates were incubated at room temperature. For endophytic fungi, two 13 mm leaf discs
were cut using a standard cork borer and surface sterilized as follows; Leaf discs were
soaked for 60 seconds in 70% ethanol, transferred to 75% commercial bleach for three
minutes, transferred to a new beaker of 70% ethanol and rinsed thoroughly in milli-Q
water (Lodge et al. 1996). Discs were then transferred to CYA plates and incubated at
room temperature. After eight days, selected colonies were passed to new CYA plates
containing chloramphenicol and incubation was continued at room temperature. After
14 additional days, spores of selected cultures were picked from individual fruiting
bodies under a dissecting scope using a sterile probe and transferred to new CYA
plates containing chloramphenicol and kanamycin to yield pure colonies of sampled
fungi.
Fusarium cultures were isolated via a modification of the protocol by Ho and Ko
(Ho and Ko 1997). Briefly, three pieces were cut from an actively growing culture,
approximately 5 mm x 5 mm in size. These culture pieces were transferred to 20 mL of
28

sterile water in a standard 100 mm petri plate and allowed to sit for 48 hours at room
temperature (24° C). After 48 hours, water cultures were transferred to a 4° C
refrigerator and allowed to sit for 15 minutes. After 15 minutes, the chilled liquid culture
was transferred to a 50 mL polypropylene centrifuge tube (Corning Incorporated,
Tewksbury, MA) and vortexed for one minute. Plates of 2% water agar had been
previously divided into 60 equally-sized squares, marked on the bottom of the plate. A
predetermined amount of the chilled and agitated spore solution was added to each
square. One full plate was inoculated with 0.25 μL per square. A second full plate was
inoculated with 0.5 μL per square. Inoculated plates were incubated for 24 hours at
room temperature. After 24 hours, each square of the grid was examined to evaluate
spore presence and/or germination. Eight sections containing a single germinated
spore were selected, carefully cut from the original culture plate, and transferred to new
2% water agar plates. These plates were monitored for growth and healthy colonies
from seven of eight plates were passed to new plates after two weeks. One successful
culture was selected for future use and continually passed on water agar and CYA
media. Media recipes are given in Chapter 3.
Identification. Isolated colonies were identified using both morphological
characterization and molecular analysis. Morphological observations were made using
microscopic analysis of plated culture material, slide culture techniques, and digital
imaging/measurement of characters such as hyphal width, sexual structures, and
septation, as well as measurements of average growth rate on selected media.
Molecular analysis was via PCR and sequencing of either the 18S or ITS regions, as
described previously, followed by a BLAST search of resulting sequences.
29

Results
Initial Observations and Analysis. Analysis of 280 cloned samples containing
DNA extracted from initially contaminated water samples yielded mostly sequences
from the genus Aspergillus. The most common results were A. niger and A. awamori.
Subsequent analysis of purified spore solutions confirmed the identification as
Aspergillus niger.
Ergosterol Analysis. Ergosterol samples from tested containers showed very
low values (Averages: 0.035008 μg/leaf disc, standard deviation 0.03516, and 0.006479
μg/mL water, standard deviation 0.003531, n=94 in both cases), despite the copious
visible growth in the containers.
Confirmation. After one week of growth, there was no noticeable difference
between the appearance and activity level of larvae grown in maple and oak
microcosms. However, by Day 12 of larval growth, there was a marked difference
between the two treatment groups. There was a massive die-off of larvae in maple
containers, and the few surviving larvae appeared small and sluggish, whereas the
larvae in oak containers were large and very active. One oak container appeared to
have more filamentous growth and smaller larvae than the others in its treatment group.
On Day 13 of larval growth, deceased specimens were examined under a dissecting
scope. A few were found to have extensive microbial growth on the anal papillae. By
Day 14, only one of 80 larvae grown in maple microcosms was still alive. The
experiment was ended on Day 15 and water samples were taken for ergosterol, DNA,
and culture. Larvae were counted, sorted by instar, and preserved in 70% Ethanol.

30

Larval counts and distribution are given in Figures 2.2 and 2.3. The average survival in
untreated controls was 79%, and in treatment groups was 1.25%. Statistical analysis
showed that survival was significantly different between the two treatment groups (t-test,
p=0.001563). A Wilcoxon rank sum test also showed that the distribution of larvae
among developmental stages was significantly different between the two groups (p <
2.2 x 10

-16
), with the maple-based microcosms demonstrating a much higher proportion

of early instars, implying that there is a significant inhibition of development when

Percentage of surviving larvae

contaminated maple leaves are used in laboratory microcosms.

100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%

OL1

OL2

OL3
OL4
ML1
ML2
Container Identification

ML3

Figure 2.2. Larval survival after 15 days growth on oak or maple leaves.
Note: OL= Oak microcosm, ML=maple microcosm.

31

ML4

Oomycete analysis. Analysis of extracted DNA from initial microcosms showed
a strong presence of sequences within the genus Pythium. The most frequent results
were P. sterilum, P. litorale, and P. delawarii.

Number of larvae at
developmental stage

16
14
12
10

1st

8

2nd

6

3rd

4

4th
Pupae

2
0

OL1

OL2

OL3
OL4
ML1
ML2
Container Identification

ML3

ML4

Figure 2.3. Development of larvae after 15 days growth on oak or maple leaves. Note
the pronounced difference in general developmental progress between oak and maple
microcosms. Light grey indicates early (first and second) instars, dark grey indicates
late (third and fourth) instars, black indicates pupae.

Surveys. All natural samples produced positive amplicons for both Pythium and
fungal DNA in PCR analysis. The maple survey showed a significant difference in effect
based on location. Wilcoxon rank sum tests showed that larvae grown with leaves
collected from the ground around the campus-located tree showed a much higher level
of mortality at the end of two weeks than those grown with leaves either directly from
32

the tree (p < 0.001) or from Hudson Woodlot (p < 0.001). Kruskal-Wallis and MannWhitney tests on developmental stage showed a significant effect of leaf source on
larval development, with “Campus Ground” leaves producing a much higher proportion
of larvae at early developmental stages (first and second instars) than other maple
sources, which produced larvae that had nearly all reached the third and fourth instars,
and a small number of pupae (p < 0.001). The other maple collections did not differ
significantly from the beech or oak control microcosms in mortality (Kruskal-Wallis, p >
0.5). Developmental stages were only compared between maple groups. Beech and
Oak controls were not included in development data. These data are summarized in

Average Percent Survival

Figures 2.4 and 2.5.
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%

*
Woodlot
Tree

Woodlot
Ground

Campus
Campus
Tree
Ground
Leaf Source

Oak
Control

Beech
Control

Figure 2.4. Average percent survival of larvae in microcosms constructed with the
indicated source of leaves. Error bars indicate standard error. n=12.

33

Fungal Isolation and Identification. Two species of fungus were isolated from
contaminated leaves for further experimentation and characterization. Species isolated
were Aspergillus niger and Fusarium oxysporum. Both were confirmed by 18s
sequencing as described above.

Average Number of Larvae in
Developmental Stage

14
12
10
8

1st Instar

6

2nd Instar
3rd Instar

4

4th Instar

2
0

Pupae
Woodlot Tree

Woodlot
Campus Tree
Ground
Leaf Source

Campus
Ground

Figure 2.5: Average number of larvae per microcosm in the designated developmental
stage. Light grey indicates early (first and second) instars, dark grey indicates late (third
and fourth) instars, black indicates pupae. Error bars indicate standard error. n=12.

Discussion
Initial observations and confirmation tests demonstrate a definite and significant
effect when first instar larvae of the mosquito species Aedes triseriatus are grown in the
presence of certain populations of leaves from the Sugar Maple, Acer saccharum. Most
dramatically, the pronounced die-offs of larvae would suggest that there is a component
34

or combination of components present in the natural mosquito habitat that could be of
great interest to both microbiology and entomology as a system that can be explored for
potential mosquito control measures or products in the future. In fact, the presence of a
copious amount of apparent fungal growth, which, if it were truly of fungal origin would
be expected to produce a high level of ergosterol, demonstrating uncommonly low
levels of associated ergosterol in the water column lends another interesting aspect to
this exploration. The implication that perhaps it is an oomycete, and not a true fungus,
that is responsible for the mortality effect is, in itself, interesting given the history of
oomycetes in mosquito control in the past. In 1996, the oomycete Lagenidium
giganteum was approved for use as a larvicide under the name Laginex in three
different formulations, by the Environmental Protection Agency (USEPA 2001).It was
considered established that this product was not harmful to mammals (Kerwin et al.
1990) or other off target organisms. In fact, the EPA Fact Sheet from 2001 states that
“Lagenidium giganteum has no observable effects on any organisms except
susceptible mosquitoes. Because the fungus is specifically used in aquatic
environments, its potential harmful effects on aquatic organisms were thoroughly
studied. No harmful effects were found in any aquatic or terrestrial species
tested, including birds, beneficial insects, aquatic fish and invertebrates,
mammals, and other animals.”
However, there was some evidence to the contrary, showing that the organism
Lagenidium giganteum may be harmful to several species of aquatic organisms,
including both insects and small crustaceans (Couch 1935, Nestrud and Anderson
1994). It now appears that L. giganteum may also be pathogenic to mammals,
35

specifically, to canines (Grooters et al. 2003). Additionally, it was voluntarily removed
from the market in the fall of 2011 (USEPA 2011). Given this recent development, it is
of interest to look more closely at fungus and oomycetes that are pathogenic to
mosquitoes, as they may provide insights into potentially mammal-pathogenic species
or strains.
The isolation of Aspergillus niger and Fusarium oxysporum from the experimental
microcosm is not surprising, as they are both very common species. In fact, according
to the Centers for Disease Control, “Most people breathe in Aspergillus spores every
day”. However, previous research has indicated that related species can infect or kill
mosquitoes of multiple genera (Hasan and Vago 1972, Teetor-Barsch and Roberts
1983, Moraes et al. 2001, Seye et al. 2009, Prakash et al. 2010), so it is of interest to
see whether they are similarly effective against Ae. triseriatus.

36

LITERATURE CITED

37

LITERATURE CITED

Arrivillaga, J., and R. Barrera. 2004. Food as a limiting factor for Aedes aegypti in
water-storage containers. Journal of Vector Ecology 29: 11-20.
Borneman, J., and R. J. Hartin. 2000. PCR primers that amplify fungal rRNA genes
from environmental samples. Applied and environmental microbiology 66: 4356-4360.
Couch, J. N. 1935. A New Saprophytic Species of Lagenidium, with Notes on Other
Forms. Mycologia 27: 376-387.
Fish, D., and S. R. Carpenter. 1982. Leaf Litter and Larval Mosquito Dynamics in TreeHole Ecosystems. Ecology 63: 283-288.
Gaulin, E., A. Bottin, and B. Dumas. 2010. Sterol biosynthesis in oomycete
pathogens. Plant signaling & behavior 5: 258-260.
Geraats, B. P. J., P. A. H. M. Bakker, and L. Van Loon. 2002. Ethylene insensitivity
impairs resistance to soilborne pathogens in tobacco and Arabidopsis thaliana.
Molecular plant-microbe interactions 15: 1078-1085.
Grooters, A. M., E. C. Hodgin, R. W. Bauer, C. J. Detrisac, N. R. Znajda, and R. C.
Thomas. 2003. Clinicopathologic findings associated with Lagenidium sp. infection in 6
dogs: initial description of an emerging oomycosis. Journal of veterinary internal
medicine 17: 637-646.
Hasan, S., and C. Vago. 1972. The pathogenicity of Fusarium oxysporum to mosquito
larvae. Journal of Invertebrate Pathology 20: 268-271.
Ho, W. C., and W. H. Ko. 1997. A simple method for obtaining single-spore isolates of
fungi. Botanical Bulletin of Academia Sinica 38.
Intermill, R. 1973. Development of Dirofilaria immitis in Aedes triseriatus Say. Mosquito
News 33: 176-181.
Kerwin, J. L., D. A. Dritz, and R. K. Washino. 1990. Confirmation of the safety of
Lagenidium giganteum (Oomycetes: Lagenidiales) to mammals. Journal of economic
entomology 83: 374-376.
Lodge, D. J., P. J. Fisher, and B. C. Sutton. 1996. Endophytic Fungi of Manilkara
bidentata Leaves in Puerto Rico. Mycologia 88: 733-738.
McCorkindale, N. J., S. A. Hutchinson, B. A. Pursey, W. T. Scott, and R. Wheeler.
1969. A comparison of the types of sterol found in species of the saprolegniales and
leptomitales with those found in some other phycomycetes. Phytochemistry 8: 861-867.
Moraes, A. M. L. d., G. L. d. Costa, M. Z. d. C. Barcellos, R. L. d. Oliveira, and P. C.
d. Oliveira. 2001. The entomopathogenic potential of Aspergillus spp. in mosquitoes
vectors of tropical diseases. Journal of Basic Microbiology 41: 45-49.
38

Naksathit, A. T., J. D. Edman, and T. W. Scott. 1999. Amounts of Glycogen, Lipid,
and Sugar in Adult Female Aedes aegypti (Diptera: Culicidae) Fed Sucrose. Journal of
Medical Entomology 36: 8-12.
Nestrud, L. B., and R. L. Anderson. 1994. Aquatic safety of Lagenidium giganteum:
Effects on freshwater fish and invertebrates. Journal of Invertebrate Pathology 64: 228233.
Nnakumusana, E. 1985. Laboratory infection of mosquito larvae by entomopathogenic
fungi with particular reference to Aspergillus parasiticus and its effects on fecundity and
longevity of mosquitoes exposed to sporal infections in larval stages. Current Science
54: 1221.
Onken, H., S. B. Moffett, and D. F. Moffett. 2004. The anterior stomach of larval
mosquitoes (Aedes aegypti): effects of neuropeptides on transepithelial ion transport
and muscular motility. Journal of Experimental Biology 207: 3731-3739.
Prakash, S., G. Singh, N. Soni, and S. Sharma. 2010. Pathogenicity of Fusarium
oxysporum against the larvae of Culex quinquefasciatus (Say) and Anopheles stephensi
(Liston) in laboratory. Parasitology research 107: 651-655.
Rahuman, A. A., G. Gopalakrishnan, P. Venkatesan, and K. Geetha. 2008.
Larvicidal activity of some Euphorbiaceae plant extracts against Aedes aegypti and
Culex quinquefasciatus (Diptera: Culicidae). Parasitology research 102: 867-873.
Seitz, L. M., D. B. Sauer, R. Burroughs, H. E. Mohr, and J. D. Hubbard. 1979.
Ergosterol as a measure of fungal growth. Phytopathology 69: 1202-1203.
Seye, F., O. Faye, M. Ndiaye, E. Njie, and J. M. Afoutou. 2009. Pathogenicity of the
fungus, Aspergillus clavatus, isolated from the locust, Oedaleus senegalensis, against
larvae of the mosquitoes Aedes aegypti, Anopheles gambiae and Culex
quinquefasciatus. Journal of Insect Science 9.
Teetor-Barsch, G. H., and D. W. Roberts. 1983. Entomogenous Fusarium species.
Mycopathologia 84: 3-16.
Turell, M. J., D. J. Dohm, M. R. Sardelis, M. L. O'guinn, T. G. Andreadis, and J. A.
Blow. 2005. An update on the potential of North American mosquitoes (Diptera:
Culicidae) to transmit West Nile virus. Journal of Medical Entomology 42: 57-62.
USEPA. 2001. Lagenidium giganteum (129084) Fact Sheet. In U. S. E. P. Agency [ed.].

39

CHAPTER 3: QUANTIFICATION OF MORTALITY AND INHIBITION
OF DEVELOPMENT OF MOSQUITO LARVAE (CULICIDAE) FOLLOWING
EXPOSURE TO PURIFIED SPORE SUSPENSIONS OF
ASPERGILLUS NIGER, FUSARIUM OXYSPORUM,
AND PYTHIUM ULTIMUM

40

Introduction
Development of larval mosquitoes has a pronounced effect on their vectorial
capacity as adults. Mosquitoes that are stunted as larvae, either by starvation or
competition, have been shown to be more effective at transmitting diseases such as La
Crosse Encephalitis (Grimstad and Haramis 1984, Grimstad and Walker 1991). By the
same token, mosquitoes that do not survive to eclosion, and thereby never develop into
adults, cannot transmit disease at all. It is therefore of interest to delve further into the
relationships between the larval development of disease-transmitting mosquito species
and their potential pathogens.
One such disease-transmitting mosquito species is Aedes triseriatus, the primary
vector for La Crosse Encephalitis (Sudia et al. 1971, Watts et al. 1972). This tree holedwelling mosquito is a daytime biter and has been known to feed on humans (Walker
1992). Another closely related species that also occasionally feeds on humans is Aedes
japonicus japonicus, an invasive species which established populations in the United
States in the late 1990’s (Molaei et al. 2009). As an invasive species which shares a
very similar habitat preference to that of Ae. triseriatus, it has been speculated that Ae.
j. japonicus may be in competition with Ae. triseriatus in its natural habitat(Alto 2011). If
this is the case, investigation of potential pathogens which may yield a competitive
advantage to either speices are also of interest in current studies.
Following the observation of lethal and inhibitory effects when contaminated
maple leaves were used to encourage microbial growth in experimental microcosms
and the subsequent isolation and characterization of potential etiologic agents, it was

41

determined that bioassays should be run utilizing the isolated fungi and oomycetes.
These tests are of value to see which, if any, of the isolated fungi are contributing most
to the observed effect. Determination of the primary causative agents will allow for
more in-depth examination of how they cause the observed effects. While unlikely that
any of the isolated agents would be a candidate for direct use as a larvicidal agent,
based on the unexpected off-target results of Lagenidium giganteum use (Couch 1935,
Nestrud and Anderson 1994, Grooters et al. 2003), this examination may shed light on a
compound or process that could be of use in the development of future mosquitocidal
products.
Of additional interest is whether or not these agents act in a synergistic manner
such that the combination of two or more of the isolated species provide a more robust
effect than would be observed normally. There is evidence to support the idea that fungi
can work in a synergistic manner when infecting host tissue. In fact, it has been shown
that “parasitizing Fusarium sp. may weaken the insect to the extent that the latter will be
predisposed to pathologies of other causes.” (Teetor-Barsch and Roberts 1983). It
seems logical that this would be the case with nearly any combination of pathogenic
influences on an organism, as the host system would be taxed further by each new
challenge and thus unable to react as strongly to any one pathogen as it might have
otherwise been able to do. In order to determine whether the isolated fungi and
oomycetes produced the previously observed effect either individually or in combination,
a series of bioassays were run, exposing larvae of the mosquito species Aedes
triseriatus to the isolated cultures and observing subsequent mortality and/or inhibition
of development.
42

Another point of interest in determining the efficacy of these agents in causing
mortality in mosquito larvae is investigating potential differences in efficacy based on
the developmental stages of the larvae at the time of exposure. It was expected that
the first instars of both Ae. triseriatus and Ae. japonicus larvae would be more
susceptible to fungal pathogens, based on the susceptibility of early instars of both
mosquitoes and other insects to infection by microbes and parasites (Bukhari et al. ,
Stairs 1965, Magnoler 1975). Based on these studies, first instars were used for the
following studies unless otherwise indicated.
An additional aspect of the effects of fungal challenge on mosquito larvae that
was evaluated in this study was whether there was a decrease in adult emergence after
challenge. It was thought that, even if high levels of mortality were not seen when larvae
were challenged with some of these fungi, there may be decreased adult emergence
due to inhibited development. This would be directly relevant to mosquitoes as vectors
of disease, as it is the adult stage of the mosquito that is responsible for transmission of
disease in most, if not all, cases.
Objectives and Hypotheses. Bioassays evaluating the mortality and
development of mosquito larvae exposed to fungal or oomycete pathogens were used
to evaluate the following hypotheses:
Hypothesis 1: Aspergillus, Fusarium, and Pythium isolates will all demonstrate
significant mosquitocidal effects when used individually to challenge Ae.
triseriatus larvae.

43

Hypothesis 2: Aspergillus, Fusarium, and Pythium isolates will all demonstrate
significant inhibition of growth and development when used individually to
challenge Ae. triseriatus larvae.
Hypothesis 3: Mosquitocidal effects will increase with mixed-species inoculum
as compared with single-species inoculum.

Materials and Methods
Media Preparation. Mung bean broth was prepared according to a modification
of the protocol from the University of Kentucky Department of Plant and Soil Sciences
website (2006). Briefly, 1 L of milli-Q filtered water was brought to a boil in a covered 2 L
Erlenmeyer flask. Once boiling, the water was removed from the heat source and
allowed to sit for 30 seconds. At this point, 40 g of dried mung beans were added and
the flask was swirled gently to disperse them. The beans were allowed to steep for ten
minutes undisturbed. After ten minutes, the broth was poured into a clean flask or
flasks, depending on the volume needed, being sure that no beans were transferred in
the process. Flasks were covered with clean aluminum foil and autoclaved for 20
minutes at 123°.
3% Water Agar: Thirty grams of Bacto-agar (Becton Dickinson and Company,
Franklin Lakes, NJ) were added to 1 L milli-Q filtered water and dissolved. The solution
was autoclaved for 20 minutes at 123°. Plates were poured on a benchtop, using
standard aseptic technique.

44

Czapek Yeast Agar: For Czapek Yeast Agar, a concentrate first had to be
made using the following ingredients and amounts: sodium nitrate (30.0 g), potassium
chloride (5.0 g), magnesium sulfate heptahydrate (5.0 g), ferrous sulfate heptahydrate
(0.1 g), zinc sulfate heptahydrate (0.1 g), cupric sulfate pentahydrate (0.05 g), Milli-Q
filtered water (100 mL). Components were combined and dissolved with stirring and
low heat. Once Czapek concentrate was finished, Czapek Yeast Agar plates were made
as follows: potassium phosphate (dibasic- 1.0 g), Czapek concentrate (10 mL),
powdered yeast extract (5.0 g), sucrose (15.0 g), and agarose (15.0 g) were combined
and dissolved in 1.0 L Milli-Q filtered water with stirring. Media was autoclaved for 20
minutes at 123° C. After autoclaving, media was cooled in a water bath to approximately
55° C, then Chloramphenicol and Kanamycin were added in a sterile biological safety
cabinet to a final concentration of 0.05 g/L. Plates were poured on a benchtop using
standard aseptic technique (Klich and voor Schimmelcultures 2002).
CMA-PARP: For Corn Meal Agar (CMA) plates, 17 g of Corn Meal Agar was
dissolved in Milli-Q filtered water in an Erlenmeyer flask and autoclaved for 20 minutes
at 123° C. The media was allowed to cool to approximately 55° C, then the following
antibiotics were added with gentle swirling to distribute: Ampicillin (0.25 g dissolved in
50% ethanol), Rifampicin (0.01 g dissolved in DMSO), Pimaricin (25 mg dissolved in
Milli-Q water), Penta-Chloro-Nitro-Benzene (PCNB, 0.025 g dissolved in warn 95%
ethanol). Plates were poured on a benchtop using standard aseptic technique (Vitoreli).
cV8A: Clarified v8 juice was made as follows: Calcium carbonate was added to
room temperature V8 juice (CSC Brands LP, 2 g per 200 mL). The mixture was
centrifuged for 20 minutes at 4000 rpm to clarify. To make 3% clarified V8 Agar (cV8A),

45

30 mL of clarified V8 juice and 15 g of Bacto-agar were added to 1 L of Milli-Q filtered
water and autoclaved for 20 minutes at 123° C. The media was cooled to
approximately 55° C and plates were poured on the benchtop using standard aseptic
technique (Jeffers 2006).
Fungal Spore Suspension Preparation. Fungal spore suspensions were
produced as follows: Isolated Fusarium cultures were grown on 3% water agar for five
to seven days at room temperature, until plates reached nearly full coverage. At this
point, plates were divided into approximately sixteen pieces each and transferred into
prepared mung bean broth- one 100 mm plate per liter of broth. Inoculated broth was
allowed to sit on a benchtop at room temperature for approximately two weeks, with
occasional swirling of the flask. After two weeks of growth, the broth was filtered
through sterile gauze to remove hyphae and transferred to 50 mL centrifuge tubes.
Spore solutions were centrifuged for 15 minutes at 4000 rpm and the pellets were
collected and combined into one vessel. The concentration of spores in this solution
was determined using an improved Neubauer phase contrast hemocytometer (Hausser
Scientific, Horsham, PA). Concentrations were calculated as the mean of ten to twelve
counts. Spore suspensions were then diluted to a standard concentration using mung
bean broth.
Aspergillus cultures were grown on CYA slants in sterile 50 mL centrifuge tubes
with polypropylene screw-on caps at room temperature for approximately one week.
After sufficient growth was observed, 15 mL sterile Milli-Q filtered water was added to
each tube and tubes were capped tightly and shaken vigorously and/or vortexed to
suspend the hydrophobic spores in the water (Brown and Salvo 1992). Spore

46

concentrations were determined using an improved Neubauer phase contrast
hemocytometer (Hausser Scientific, Horsham, PA). Spore suspensions were then
diluted to a standard concentration using sterile water.
Pythium cultures were grown continually on CMA-PARP. A 5 mm x 5 mm piece
of actively growing culture was transferred to the center of a 3% cV8A plate and allowed
to grow for one week in the dark at room temperature. After one week, this plate was
flooded with 15 mL sterile water and allowed to sit for one to two hours (Pettitt et al.
2002). The water was then pipetted off the surface of the plate and collected in a
centrifuge tube. Spore concentrations were not able to be accurately measured by the
researcher, so a standardized amount of inoculum was used in laboratory microcosms,
rather than a final spore concentration. It was estimated that spore concentrations
would be relatively high (similar to those seen in Aspergillus solutions), but this could
not be confirmed.
Single Species Laboratory Microcosms. Laboratory microcosm bioassays
were run with single fungal species to establish the efficacy of each species in killing
first instar larvae of the mosquito species being studied, with the emphasis on Ae.
triseriatus, though Ae. japonicus was also challenged with Aspergillus. Different
dosages of each fungal species were also evaluated.
Aspergillus/Ae. triseriatus: Aspergillus spores were prepared as discussed
above. Microcosms were constructed in 16 ounce food storage containers, as
discussed in the previous chapter and contained: 200 mL Milli-Q filtered water, 2 mL
treehole inoculum, 1 g dried ground-collected beech leaves, 20 first instar Ae. triseriatus
larvae. Aspergillus spores were added to reach the following final concentrations:
47

5
6
Control= no inoculum, Low Dose= 1.45 x 10 spores/mL, Medium Dose= 1.45 x 10
6
spores/mL, High Dose= 2.9 x 10 spores/mL. Microcosms were run for one week at

room temperature. After one week, larvae were checked for survival, counted, and
preserved in 70% ethanol.
Aspergillus/Ae. japonicus. Bioassays evaluating the survival of Ae. japonicus
exposed to Aspergillus spores were run as above, with all concentrations the same.
These experiments were not, however, run concurrently, so data from the two trials
could not be compared directly during statistical analysis.
Fusarium/Ae. triseriatus. Bioassays evaluating the survival of Ae. triseriatus
exposed to Fusarium spores were run as above, with final concentrations as follows:
3
Control= no inoculum, Low Dose= 2.5 x 10 spores/mL, Medium Dose= 2.5 x
4
5
10 spores/mL, High Dose= 2.5 x 10 spores/mL.

Mixed Species Laboratory Microcosms. Two laboratory microcosm trials were
run in preparation for simulated field microcosms. Initial mixed species microcosms
were run using a modification of the 16 ounce microcosm system described in the
previous chapter. Microcosms contained 200 mL Milli-Q filtered water, 1 g dried
ground-collected beech leaves, 2 mL treehole inoculum, and spore solutions to reach
6
the following final concentrations: Aspergillus- 1.45 x 10 spores/mL, Fusarium- 25,000

spores/mL. Microcosms were set up and twenty first instar larvae were added
immediately. For Trial 1, all larvae were Ae. triseriatus. In Trial 2, ten Ae. triseriatus
48

larvae and ten Ae. japonicus larvae were added to each microcosm. Microcosms were
run for one week in an incubator set to 28° C, then larvae were removed, counted, and
preserved in 70% ethanol for further analysis. Water samples (50 mL each) were taken
for DNA extraction. Statistical analysis was via two-factor analysis of variance (two-way
ANOVA) and Tukey’s Honest Significant Difference.
Mixed Species Simulated Field Microcosms. Due to the fact that laboratory
conditions are typically severely limited when compared with the immense complexity of
natural systems, simulated field microcosms were created using standard used
automotive tires. These microcosms were designed to more closely represent the
conditions found in the natural habitats of these tire-dwelling mosquitoes. Elements that
contribute to the natural simulation of these microcosms include mixed species locallycollected leaf inputs, tire-based systems, and placement in a beech-maple woodlot in
which many native populations of Aedes triseriatus are found.
Tire preparation was as follows: tires were screened off using 1 x 1.5 mm
aluminum window screening and silicone caulk to create an enclosed space in the base
of the tire. This enclosure was intended to exclude wild mosquitoes and other insects
that might otherwise oviposit in the experimental area and to prevent the escape of any
experimental mosquitoes that might eclose prior to the end of the trial.
Trial 1: Microcosms contained: 1 L Milli-Q filtered water, 5 g dried leaf litter
(mixed species collected from the floor of the woodlot in which tires were stationed).
After one week, on Day 1, 100 first instar Ae. triseriatus larvae and 100 first instar Ae.
japonicus larvae were added for a total of 200 larvae per tire. Larvae were allowed to

49

acclimate in the tire microcosms for 24 hours prior to the addition of fungal inocula.
Fungal inocula were added to experimental containers on Day 2. Fungal inocula were
6
added to the following final concentrations: Aspergillus: 1.45 x 10 spores/mL,

Fusarium: 25,000 spores/mL. In Trial 1, treatment tires received fungal spores and
controls were untreated Microcosms were allowed to run for one week. After one week,
the entire contents of the tire microcosms were removed and transferred to the
laboratory where they were processed.
Trial 1 Processing: Tire contents were transferred to ceramic-coated larval
rearing trays. Leaves were removed and stored at -80° C. Water samples (50 mL each)
were taken for DNA extraction. Larvae were checked for survival, counted, and
incubated at 28° C for 48 hours, then recounted and preserved in 70% ethanol.
Trial 2: Microcosms contained: 1 L Milli-Q filtered water, 5 g dried leaf litter
(mixed species collected from the floor of the woodlot in which tires were stationed). For
this trial, poor larval hatch led to a necessary staggering of the addition of larvae to
experimental containers. After one week, on Day 1, 50 first instar Ae. triseriatus larvae
and 50 first instar Ae. japonicus larvae were added for a total of 100 larvae per tire. The
remaining 100 larvae, 50 Ae. triseriatus and Ae. japonicus larvae, were added the
following day for a final total of 200 larvae per tire. Larvae were allowed to acclimate in
the tire microcosms for 24 hours after the second set of larvae were added, prior to the
addition of fungal inocula. Fungal inocula were added on Day 3. Fungal inocula were
6
added to the following final concentrations: Aspergillus: 1.45 x 10 spores/mL,

Fusarium: 25,000 spores/mL. In Trial 2, control tires received autoclaved fungal inocula,
50

to provide a more appropriate negative control. Autoclaved fungal spores were also
tested for viability by plating on appropriate media, as discussed in the fungal growth
section. After one week, the entire contents of the tire microcosms were removed and
transferred to the laboratory where they were processed.
Trial 2 Processing: Tire contents were transferred to cold storage (4° C)
overnight. Then leaves were removed and stored at -80° C, water samples were taken
for DNA extraction, and larvae were counted and preserved in 70% ethanol.
Preserved larvae from both trials were examined under a dissecting microscope
to determine developmental stage and look for any visible signs of infection. Statistical
analysis was via two-factor analysis of variance (two-way ANOVA) and Tukey’s Honest
Significant Difference.
Single Species Laboratory Nanocosms. Single species laboratory nanocosms
were run with single larvae in the wells of a standard 24 well tissue culture plate
(Costar, Corning Incorporated, Tewksbury, MA). Plates were set up as follows: 0.2 mg
Tetramin brand fish food (Tetra Werke, Melle, Germany) and approximately 2.5 mL of
water were added to each well (water volumes were adjusted to account for the volume
of the fungal inoculum, for a final volume of 2.5 mL). Once plates were set up, larvae
were added. For this experiment, larvae at four different developmental stages were
used. Larval instars were estimated based on developmental time (ie- Larvae less than
24 hours old were considered first instars, larvae approximately three days old were
considered second instars, and larvae five days old were considered third instars).
Populations were checked to ensure that the majority of the larvae were of appropriate

51

size and development to meet the indicated developmental stage, but each individual
larva was not checked prior to use. Larvae were divided into 32 treatment groups, as
outlined in Table 3.1, with 24 larvae per group. After all larvae were in plates, fungal
inoculum was added to appropriate wells as follows: Aspergillus/Fusarium- Fungal
6
inoculum was added to reach a final concentration of 3.0 x 10 spores/mL, Pythium-

0.095 mL were added to each well. Nanocosms were allowed to run for one week with
regular feeding (approximately 0.1 mg /larva/day). At the end of one week, larvae were
checked for survival, counted, and preserved in 70% ethanol.
Multi Species Laboratory Nanocosms. Multi-species laboratory microcosms
were set up as with the single species nanocosms described above, but only Ae.
triseriatus larvae were used. Treatment groups for this experiment were: Untreated
controls, Aspergillus, Fusarium, Pythium, Aspergillus/Fusarium, Aspergillus/Pythium,
Fusarium/Pythium, Aspergillus/Fusarium/Pythium, where names in the format X/Y
indicate that multiple fungal species were used in combination in those wells. There
were 24 mosquitoes per treatment group. Final fungal concentrations were 1.45 x
107 spores/mL where accurate counts could be obtained. For Aspergillus/Fusarium
treatment group wells, inocula were standardized to an equal concentration and the
same amount was added of both, to reach the desired final total concentration. For
Pythium-treated wells, consistent amounts of Aspergillus and Fusarium were used and
a volume of Pythium consistent with that added for the other two species was used. The
experiment was allowed to run for one week, then mortality was recorded and larvae
were preserved in 10% neutral buffered formalin.

52

Table 3.1. Distribution of treatment groups for Single Species
Laboratory Microcosms experiment.
Group Mosquito Species

Fungal Inoculum

Instar

n

1

Ae. triseriatus

Aspergillus

1

24

2

Ae. triseriatus

Aspergillus

2

24

3

Ae. triseriatus

Aspergillus

3

24

4

Ae. triseriatus

Aspergillus

4

24

5

Ae. triseriatus

Fusarium

1

24

6

Ae. triseriatus

Fusarium

2

24

7

Ae. triseriatus

Fusarium

3

24

8

Ae. triseriatus

Fusarium

4

24

9

Ae. triseriatus

Pythium

1

24

10

Ae. triseriatus

Pythium

2

24

11

Ae. triseriatus

Pythium

3

24

12

Ae. triseriatus

Pythium

4

24

13

Ae. triseriatus

Control

1

24

14

Ae. triseriatus

Control

2

24

15

Ae. triseriatus

Control

3

24

16

Ae. triseriatus

Control

4

24

17

Ae. japonicus

Aspergillus

1

24

18

Ae. japonicus

Aspergillus

2

24

19

Ae. japonicus

Aspergillus

3

24

20

Ae. japonicus

Aspergillus

4

24

21

Ae. japonicus

Fusarium

1

24

22

Ae. japonicus

Fusarium

2

24

23

Ae. japonicus

Fusarium

3

24

24

Ae. japonicus

Fusarium

4

24

25

Ae. japonicus

Pythium

1

24

26

Ae. japonicus

Pythium

2

24

27

Ae. japonicus

Pythium

3

24

28

Ae. japonicus

Pythium

4

24

29

Ae. japonicus

Control

1

24

30

Ae. japonicus

Control

2

24

31

Ae. japonicus

Control

3

24

32

Ae. japonicus

Control

4

24

53

Adult Emergence Nanocosms. Single larva nanocosms were also utilized to
analyze adult emergence after exposure to fungal challenge. Fungal treatments were
with Aspergillus and Fusarium spores. There were 24 larvae per treatment group.
Nanocosms were set up in 24 well tissue culture plates (Costar, Corning Incorporated,
Tewksbury, MA) as follows: 2.325 mL Milli-Q filtered water, 0.075 mL fungal inoculum to
6
reach a final concentration of 1.45 x 10 spores/mL, and 0.2 mg Tetramin brand fish

food (Tetra Werke, Melle, Germany). One first instar larva was added to each well.
Plates were checked every day and pupae were removed to eclosion containers
consisting of a 1 ounce cup of Milli-Q filtered water inside a ventilated 16 ounce food
storage container, where they remained until they eclosed. Time to pupation and time to
eclosion were both recorded.
Anopheles nanocosms. Based on findings in a multi-species pathology assay
(unpublished results), it was determined that an addition bioassay should be run in
which larvae of the mosquito species Anopheles gambiae (Kisumu strain), one of the
primary vectors for human malaria in Africa, were challenged with Fusarium spores.
Nanocosms were set up as above. Briefly, 2.325 mL Milli-Q filtered water, 0.075 mL
6
fungal inoculum to reach a final concentration of 1.45 x 10 spores/mL, and 100 μL of a

10 mg/mL solution of active dry yeast in water were combined in each well of 24 well
tissue culture plates (Costar, Corning Incorporated, Tewksbury, MA). One larva
(approximately 48 hours old) was added to each well, with a total of 48 larvae per
treatment group (Fusarium-treated and untreated control). Larvae were fed another 100
μL of active dry yeast solution on day 4 of the experiment. On day 7, mortality was
54

recorded and larvae were preserved in 10% neutral buffered formalin. Body length of
preserved larvae was recorded as an indicator of development.

Results
Single Species Laboratory Microcosms. Aspergillus/Ae. triseriatus: In the
Aspergillus/Ae. triseriatus microcosm bioassay, a dose-response pattern was evident
based on ANOVA (p< 0.001)and pairwise t-tests with Bonferroni’s adjustment. Only the
high dose treatment group showed increased mortality when compared with the
untreated control (p< 0.001). The mortality in the high dose group was also significantly
higher than in the low and medium dose treatment groups (p< 0.001). The average
survival was 77% for controls, 68% for the low dose treatment group, 69% for the

Average Survival of
Ae. triseriaus

medium dose treatment group, and 20% for the high dose treatment group (Figure 3.1).
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%

a

a

a

b

No spores 1.45 x 10⁵ 1.45 x 10⁶

2.9 x 10⁶

Dosage of Aspergillus niger spores (spores/mL)

Figure 3.1. Average survival of Ae. triseriatus larvae after exposure to various doses of
spores of the fungus Aspergillus niger. Error bars indicate standard error. n=6.
55

Aspergillus/Ae. j. japonicus. In the Aspergillus/Ae. j japonicus microcosm
bioassay, a robust dose-response pattern was observed (Figure 3.2). The nonparametric Kruskal-Wallis test was used for initial analysis of the data to accommodate
the zero variance present in the high dose group. This test indicated that there was at
2
least one significant difference present (X =20.5, df=3, p<0.005). Further testing using

Bonferroni-corrected t-tests indicated that there were significant differences between all
groups except the control and low doses (p<0.005 in all cases). That is, the medium and
high dose treatment groups both showed increased mortality when compared to the
control and low dose groups, and the high dose treatment group showed increased
mortality when compared to the medium dose group. The average survival was 99% for
both control and low dose treatment groups, 33% for the medium dose treatment group,
and 0% for the high dose treatment group.

Average survival of
Ae. j. japonicus

100%

a

a

80%
60%

b

40%
20%

c

0%

No spores 1.45 x 10⁵ 1.45 x 10⁶ 2.9 x 10⁶
Dosage of Aspergillus niger spores (spores/mL)

Figure 3.2. Average survival of Ae. j. japonicus larvae after exposure to various doses
of spores of the fungus Aspergillus niger. Error bars indicate standard error. n= 6.

56

Fusarium/Ae. triseriatus. In the bioassay examining the effect of challenge with
Fusarium spores on Ae. triseriatus, Kruskal-Wallis’ test showed no significant
2
relationship between dosage and mortality (X =6.85, df=3, p=0.0767, Figure 3.3).

Mixed Species Laboratory Microcosms. In Trial 1, survival was 83% in
controls and 8.3% in treatment containers (Figure 3.4a, t-test, df=8.45, p< 0.001). In
Trial 2, a two-way ANOVA showed that significant results were obtained based on
mosquito species (p<0.005), treatment group (p<0.005), and the interaction between
those two factors (p<0.05). Ae. triseriatus survival was 82% in controls and 13% in
treatment containers (Figure 3.4b. Tukeys Honest Significant Difference, p<0.005). Ae.
japonicus survival was 87% in controls and 63% in treatment containers, which was not
significantly different (Figure 3.4b. Tukey’s Honest Significant Difference, p=0.217). In
both treatments (p<0.005) and controls (p<0.005), Ae. j. japonicus survival was

Average survival of Ae.
triseriatus

significantly higher than that of Ae. triseriatus.
100%

a

95%
90%
85%

a
a

a

80%
75%
70%

No spores

2.5 x 10³

2.5 x 10⁴

2.5 x 10⁵

Dosage of Fusarium oxysporum spores
Figure 3.3. Average survival of Ae. triseriatus larvae after exposure to various doses of
spores of the fungus Fusarium oxysporum. Error bars indicate standard error. n= 6.
57

120%

*

Percent Survival

100%

Percent Survival

100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%

b

a

80%

Ae. japonicus

60%
40%
20%
0%

Control Treatment
Treatment Group

Ae. triseriatus

Control
Treatment
Treatment Group

Figure 3.4. Average survival of Aedes larvae after challenge with mixed species
(Aspergillus and Fusarium) fungal inoculum in laboratory microcosms. Error bars
indicate standard error. a) Aedes triseriatus survival b) Ae. triseriatus and Ae. j.
japonicus survival. n=6. *p<0.005

Mixed Species Simulated Field Microcosms. In Trial 1, two-way ANOVA
showed that there was a significant effect based on mosquito species (p<0.005), but not
based on treatment group (p=0.572) or interaction of these two factors (p=0.858).
Based on the results of a Tukey’s Honest Significant Difference analysis, Ae. japonicus
demonstrated significantly higher survival in treatment containers (p<0.05). Survival was
not significantly different between treatment and control microcosms for either species
(p> 0.05). In Trial 2, there was no significance found between any of the experimental
groups in a two-way ANOVA (p>0.05). Survival was not significantly different between
treatment and control microcosms for either species (p> 0.05, Figure 3.5b). No visible
signs of infection were noted in larvae from either Trial.
58

90%

a

Percent Survival

80%
70%
60%
50%
40%
30%
20%
10%
0%

Control Treatment
Treatment Group

100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%

Ae triseriatus

b

Ae japonicus

Control
Treatment
Treatment Group

Figure 3.5. Average survival of Aedes larvae after challenge with mixed species
(Aspergillus and Fusarium) fungal inoculum in simulated field microcosms. Error bars
indicate standard error. a) Trial 1 b) Trial 2. Trial 1 n= 8, Trial 2 n=9.

Single Species Laboratory Nanocosms. When Ae. triseriatus larvae were
challenged with Aspergillus spores, a clear correlation was evident between
age/development of the larvae and survival. In nanocosms containing first instar larvae,
there were no survivors. Survival was 8%, 33%, and 75% for second, third, and fourth
instars, respectively. As a result of the binomial nature of the data (alive vs dead),
pairwise tests of proportion were run and showed significant differences between
treatment groups of different developmental stages within the Ae. triseriatus/Aspergillus
family, as indicated in Figure 3.6. No other significant differences were seen within the
Ae. triseriatus treatment groups. In the Ae. japonicus/Aspergillus treatment groups, the
first instar larvae showed significantly lower survival than any of the other groups (p<
0.01), and the second instar survival was significantly lower than that of third instars
59

(p= 0.042). No other significant differences were seen within Ae. japonicus treatment
groups (Figure 3.7). Due to the fact that not all larvae were examined prior to use, It is
possible that not all larvae were within the indicated developmental stage at the time the
experiment was run, however, this does not seem likely to have caused a significant
change in the outcome of the experiment, and cursory examination showed that the
vast majority of the larvae were of the appropriate developmental stage.

Percent Survival

120%
100%
80%
Tris-Asp

60%

Tris-Fus

40%

Tris-Pyth
Tris-Ctrl

20%
0%

1

2
3
Developmental Stage (instar)

4

Figure 3.6. Ae. triseriatus survival by treatment group. Note the correlation between
survival and age of the larva at the beginning of the experiment in Aspergillus groups.
(Tris= Aedes triseriatus, Asp= Aspergillus, Fus= Fusarium, Ctrl= Untreated Control.)
n=24.

60

Percent Survival

120%
100%
80%
Jap-Asp

60%

Jap-Fus

40%

Jap-Pyth
Jap-Ctrl

20%
0%

1

2
3
Developmental Stage (instar)

4

Figure 3.7. Ae. j. japonicus survival by treatment group. Note the low survival of first
instar larvae challenged with Aspergillus. (Jap= Aedes japonicus japonicus, Asp=
Aspergillus, Fus= Fusarium, Ctrl= Untreated Control.) n=24.

Multi Species Laboratory Nanocosms. Aspergillus-treated nanocosms
appeared to show decreased survival when compared with other treatment groups,
although in a pairwise test of proportion, this relationship did not reach statistical
significance. Survival is shown in Figure 3.8.
Adult Emergence Nanocosms. Aspergillus-treated larvae showed significantly
decreased total emergence when compared to Fusarium-treated and untreated control
larvae in pairwise tests of proportion (p < 0.05). Fusarium-treated larvae did not show a
difference in survival when compared with untreated controls. Cumulative emergence
patterns are shown in Figure 3.9.

61

Total Percent Survival

100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%

Ctrl

Asp

Fus

Pyth
A.F
Treatment Group

A.P

F.P

All

Figure 3.8. Survival of Ae. triseriatus larvae when challenged with single- and mixedspecies fungal inocula. Ctrl: Control, Asp: Aspergillus, Fus: Fusarium, Pyth: Pythium,
A.F: Aspergillus/Fusarium, A.P: Aspergillus/Pythium, F.P: Fusarium/Pythium, All: All
three fungal species. n=24.

Total Emergence

25
20
15

Control

10

Aspergillus
Fusarium

5
0

14

15

16

17 18 19 20 21
Day of Development

22

23

24

Figure 3.9. Cumulative adult emergence of Ae. triseriatus challenged with Aspergillus
or Fusarium spores as larvae. n=24.

62

Anopheles nanocosms. There was a clear and significant difference in survival
between the untreated control Anopheles gambiae larvae and the Fusarium-treated
larvae (pairwise test of proportion, p<0.001). Survival was 65% for control larvae and
12.5% for treated larvae (Figure 3.10). In addition to showing much higher mortality, the
treatment group larvae were considerably smaller than their untreated counterparts
when body lengths were measured.
70%
Total Percent Survival

60%
50%
40%
30%
20%
10%
0%

Control

Fusarium
Treatment Group

Figure 3.10. Survival of Anopheles gambiae larvae challenged with spores of the
fungus Fusarium oxysporum. n=48.

Discussion
Overall, the bulk of the hypotheses for these studies were unsupported by the
findings of the experiments. Aspergillus was the only tested isolate that showed
repeated significant lethal or developmental effects against larvae of Aedes mosquitoes.
In addition, despite the lack of a significant effect, the lowest survival in the mixed
63

species microcosm assay was observed in the groups exposed to strictly Aspergillus
spores, rather than a combination of species, as predicted.
Single Species Laboratory Microcosms. There were significant dose-response
effects apparent for both Ae. triseriatus and Ae. japonicus after exposure to Aspergillus
spores. Interestingly, the relationship between the survival rates of the two mosquito
species are inverted from what would be expected based on the results of the mixed
species microcosms, both in the laboratory and in the field, where Ae. japonicus shows
increased survival when compared with Ae. triseriatus. However, the larvae in the
mixed species microcosms were in the same experimental containers, and thus
conditions were all identical. This was not the case with the single species microcosms,
which were run at two separate times, rather than concurrently. As such, it is possible
that, despite efforts to control all variables, something was different between the two
setups that would account for the different results, and no direct comparison between
the two experiments can reasonably be made. For example, there may have been
something that caused a decrease in viability of the Aspergillus spores used for the Ae.
triseriatus bioassay, which could easily have been missed, as there were no checks for
viability during all experiments. This is something that should be added in future
experiments of this variety to avoid such possible interfering factors.
The lack of a significant mortality effect in the Fusarium bioassay is not terribly
surprising, as relatively low spore concentrations were used. These numbers were
originally based on the amounts of spores that could be generated using the prepared
materials and were maintained after preliminary trials (unpublished data) with mixed
Aspergillus/Fusarium inoculum showed a robust, observable mortality effect.
64

Mixed Species Laboratory Microcosms. The results of Trial 2 show a very
clear difference in survival between the two species in a competitive situation, with Ae.
japonicus showing significantly higher survival. This is very interesting, as little is
currently known about what competitive advantages Ae. japonicus might have that
would allow it to out-compete Ae. triseriatus in the natural treehole environment. (Bevins
2007, Andreadis and Wolfe 2010). This provides a potential starting point for future
research into the competitive qualities of these two species as they reflect the ability of
the mosquitoes to survive fungal and bacterial challenges in these microbe-rich
environments.
Mixed Species Simulated Field Microcosms. Again in this set of trials, Ae.
japonicus demonstrates an apparent increased ability to survive a challenge with
common fungal spores when compared with Ae. triseriatus. This effect was not as
universal in the second trial as it appears to have been in the first trial or the laboratory
microcosms. However, this repeated observation of differential survival does contribute
to the potential for interesting future work investigating the competitive difference of
these two species relating to microbial challenge.
Single Species Laboratory Nanocosms. Based on the known susceptibility of
early instars of other insect species to infectious agents (Stairs 1965, Magnoler 1975),
as well as prior studies that had shown increased susceptibility of early instars to fungal
infection (Hasan and Vago 1972), it was expected that first instar larvae would show the
highest levels of mortality of the tested developmental stages. This was the case in
those Ae. triseriatus groups exposed to Aspergillus spores. A similar survival pattern
was suggested in the other Ae. triseriatus groups, but was not robust enough to reach
65

statistical significance (Figure 3.6). This hypothesis was also supported with Ae.
japonicus larvae exposed to Aspergillus spores. However, contrary to expectations, the
overall survival of third and fourth instar larvae of Ae. japonicus was lower than that of
second and, in most cases, first instar larvae, though this relationship did not reach
statistical significance. Of the different fungi species tested, only Aspergillus showed
significant levels of mortality in any of the treatment groups. The lack of effect with
Pythium treatment groups may be due, in part, to the fact that zoospores were not able
to be quantified, and as a result, may have been present only in low quantities or absent
entirely. In future work, protocols should be improved to ensure the presence and
viability of accurately quantifiable Pythium zoospores in the experimental microcosms.
Multi Species Laboratory Nanocosms. No significant results were evident in
this experiment, though it was suggested that Aspergillus showed decreased survival
and, in fact, if statistical analysis is run for individual groups, rather than pairwise
comparisons across the board, there does appear to be a significant difference between
the Aspergillus-treated group and untreated controls. The lack of significant results in
this experiment is likely due to insufficient sample size given the number of treatment
groups or decreased efficacy of spore solutions as compared to previous experiments.
Adult Emergence Nanocosms. Aspergillus spores showed a significant
decrease in adult emergence when compared with untreated controls and Fusariumtreated microcosms. This is directly relevant to both increasing the knowledge base
regarding mosquito development in nature, where they may be encountering these
fungi, and to the capacity of these species for disease transmission. As it is the adult

66

stage of the Aedes mosquitoes that transmits disease, if fewer mosquitoes are reaching
adulthood, this has a direct effect on potential disease transmission.
Anopheles nanocosms. The ability of any agent to cause mortality or inhibition
of development in the malaria vector Anopheles gambiae is of significant interest to the
field of malariology. To date, it appears that little to no research has been done on the
effects of Fusarium oxysporum infection on Anopheles gambiae. Thus, while the
current study evaluated this only at a very preliminary level, it provides an intriguing
avenue for further research into the relationship and interactions existing between these
two species. For instance, it was observed during the course of the experiment that
some of the larvae appeared tangled in fungal growth, but as observations were not
made of each larva each day, it is impossible to know whether this entanglement was
the cause of death or occurred posthumously. Regarding the difference in body length,
all measurements were made after the experiment was concluded and mortality was not
recorded prior to day 7, so it is probable that some or all of the difference in body length
is due to the treatment group larvae dying toward the beginning of the experiment,
before they were able to reach the later developmental stages that the surviving larvae
did. Future experiments should involve a more direct comparison of daily growth,
perhaps via destructive sampling to check for average daily growth in both treatment
groups, along with daily monitoring and recording of mortality.

67

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68

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Nestrud, L. B., and R. L. Anderson. 1994. Aquatic safety of Lagenidium giganteum:
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Stairs, G. R. 1965. Quantitative differences in susceptibility to nuclear-polyhedrosis
virus among larval Instars of the forest tent caterpillar, Malacosoma disstria (Hübner).
Journal of Invertebrate Pathology 7: 427-429.
Teetor-Barsch, G. H., and D. W. Roberts. 1983. Entomogenous Fusarium species.
Mycopathologia 84: 3-16.
Vitoreli, A. PARP, University of Florida Plant Disease Clinic-GNV mediabook.

70

CHAPTER 4: EXAMINATION OF POTENTIAL MECHANISMS OF ACTION
OF ASPERGILLUS NIGER, FUSARIUM OXYSPORUM, AND PYTHIUM ULTIMUM
AGAINST LARVAE OF THE MOSQUITO SPECIES AEDES TRISERIATUS

71

Introduction
It has been shown that many different fungal and oomycete species act as
pathogens against mosquitoes. Among those genera that have demonstrated
significant pathogenic effects are Aspergillus (Moraes et al. 2001, Seye et al. 2009),
Fusarium (Hasan and Vago 1972), Coelomomyces (Couch and Bland 1985),
Lagenidium (Couch 1935, McCray Jr et al. 1973, Kerwin and Washino 1988), and
Pythium (Clark et al. 1966, Nnakumusana 1985). These fungi achieve larvicidal or
inhibitory effects through a variety of methods, such as mycotoxins production and
physical disruption of tissues (Bennett and Klich 2003, Seye et al. 2009).
With any significant effect such as mortality, there is a drive to know not just what
the effect is, but how and why it is occurring. With the aforementioned experiments,
there was significant mortality observed when Aedes larvae were challenged with
spores of the fungus Aspergillus. There are several different reasons that this could be
occurring. First, Aspergillus species are known to produce mycotoxins. Having been
identified as Aspergillus niger, the species of Aspergillus used in this study does not
produce the potent aflatoxin that some Aspergillus species produce, but it does produce
ochratoxin A and oxalic acid, both of which can have significant toxic effects in animals
(Abarca et al. 1994, Schuster et al. 2002, Bennett and Klich 2003). Alternatively, the
Aspergillus fungus could be causing mortality in these mosquitoes by sheer physical
disruption of host tissue. Fungi in the genus Aspergillus have been shown to possess
the capability to penetrate the cuticle of larval mosquitoes and thereby cause internal
proliferation of hyphae (Seye et al. 2009). The following experiments take a preliminary

72

approach to determining the mode of action of this fungus against mosquito species.
The hypotheses tested were as follows:
Hypothesis 1: Mortality and inhibition of development of Ae. triseriatus larvae
challenged with Aspergillus and Fusarium isolates is due to a combination of
secondary metabolites and direct infection of larvae.
Hypothesis 2: Pythium isolate effects are due to direct infection of mosquito
larvae by motile zoospores, not secondary metabolites.

Materials and Methods
Mycotoxin Assay. Fungal extracellular filtrates were prepared using sterile
mung bean broth as a liquid growth medium. Briefly, fungal tissues were added to 250
mL mung bean broth in Erlenmeyer flasks as follows: Untreated controls- no addition,
Aspergillus- suspended spores from one 50 mL slant of actively growing culture was
added, Fusarium- one plate of actively growing culture on CYA was cut into
approximately 2 cm x 2cm blocks and four blocks were added, Pythium- one plate of
actively growing culture on 3% cV8A was cut into approximately 2 cm x 2 cm blocks and
four blocks were added. Cultures were gently swirled and left to grow on a benchtop at
room temperature for two weeks. After two weeks, cultures were filtered through glass
fiber filters (Pall Corp, Type A/E, pore size 1.0 μm) to remove hyphal fragments and
spores.

73

Microcosms were set up as follows: 9 mL Milli-Q water, 1 mL fungal extracellular
filtrate, and 2 mg Tetramin fish food (Tetra Werke, Melle, Germany) suspended in water
were combined in each well of a six-well plate (Costar, Corning Incorporated,
Tewksbury, MA). Ten Ae. triseriatus larvae were added to each well. There were five
wells of ten larvae in each treatment group. Treatment groups were: Untreated control,
Aspergillus, Fusarium, and Pythium, with treatment groups differing only in which of the
previously described extracellular filtrates was added. The experiment was allowed to
run on the benchtop for one week. Larvae were fed 0.2 mg/larva on days 2, 5, and 6.
After one week, mortality was evaluated and recorded and larvae were preserved in 3%
formalin.
Microscopic analysis. Larvae from all previously described experiments were
preserved for microscopic analysis. Analysis was conducted using a standard laboratory
dissecting microscope. Larvae were examined for obvious injury, external fungal
growth, visible internal fungal proliferation, or other unusual features.

Results
Mycotoxin Assay. Survival in the mycotoxin assay was very high overall, at
98%, 88%, 94%, and 94% for Aspergillus, Fusarium, Pythium, and untreated controls,
respectively (Figure 4.1). There was no significant difference between these levels.

74

Average Percent Survival

105%
100%
95%
90%
85%
80%
75%

Aspergillus

Fusarium
Pythium
Treatment Group

Control

Figure 4.1. Survival of Aedes triseriatus larvae exposed to fungal extracellular filtrate.
No significant differences are present between groups. n=5.

Microscopic Analysis. General observations were as follows: in most cases,
there was no obvious external fungal growth or sign of infection prior to death.
Occasionally, copious filamentous growth could be seen around the anal papillae of
moribund larvae (Figure 4.2), although this was not common and tended to occur in
those experimental containers where maple leaves were used in place of fungal spore
suspensions. Post-mortem analysis revealed very little external growth on cadavers.
Growth was observed on a small portion of the sampled larvae, but due to the nature of
the experiments it could not be determined based on examination of the intact larvae
whether this growth was the cause of death or occurred posthumously. Larvae from an
unreported experiment were found to have dark spots on the cuticle, particularly on and
around the thorax and between abdominal segments, but these occurred in all
treatment groups, and so did not appear to be associated with treatment.
75

Figure 4.2. Photographs taken under a standard dissecting microscope of visible
filamentous growth around the anal papillae of moribund Ae. triseriatus larvae after
growth in maple-based microcosms. Unusual growth marked with arrows.
76

Discussion
As a result of the preliminary nature of these studies, a clear conclusion could not
be drawn regarding the acceptance or rejection of the hypotheses. It is also not possible
to draw any decisive conclusions on whether there are mycotoxins involved in the
pathology seen when these Aedes mosquitoes are challenged with Aspergillus, as there
was no significant difference detected between treatment groups. Unfortunately, due to
poor larval hatch, this study was restricted to five wells of ten larvae in each treatment
group, and treatments at only one dosage per fungal species. In future tests, it would be
beneficial to run chemical assays to quantify the amount of known mycotoxins in the
media so that dosage can be more accurately judged, and to use a range of dosages.
The microscopic analysis conducted was also of a very preliminary nature.
Samples are, at the time of this writing, being processed for histological analysis and
analyzed for internal hyphal proliferation and spore germination. These data will
hopefully help lend insight into the mode of action of these fungal pathogens. Based on
external analysis, it is evident that occasional fungal growth prior to death does occur on
the tested mosquito larvae. However, due to the fact that this was generally seen in
containers where maple leaves had been used in place of fungal spore solutions, it is
possible that the observed growth was due to a fungal species that was not isolated for
this work and remains as yet unidentified. Another possibility is that the growth
observed on the anal papillae of infected mosquitoes may have been due to the
Pythium oomycete, which has been shown to infect the anal papillae and wound sites
(Clark et al. 1966, Washburn et al. 1988), but the concentrations used in this study were
not great enough or the correct infectious stage was not present at the time of
77

challenge. Also, the species of Pythium used in this study was Pythium ultimum, which
has not previously been shown to have pathogenic effects on invertebrates, but is a
well-established plant pathogen which can be isolated from natural samples in Michigan
(Martin Chilvers -Personal Communication).

78

LITERATURE CITED

79

LITERATURE CITED

Abarca, M., M. Bragulat, G. Castella, and F. Cabanes. 1994. Ochratoxin A production
by strains of Aspergillus niger var. niger. Applied and environmental microbiology 60:
2650-2652.
Bennett, J. W., and M. A. Klich. 2003. Mycotoxins. Clinical Microbiology Reviews: 497516.
Clark, T. B., W. R. Kellen, J. E. Lindegren, and R. D. Sanders. 1966. Pythium sp.
(Phycomycetes:Pythiales) pathogenic to mosquito larvae. Journal of Invertebrate
Pathology 8: 351-354.
Schuster, E., N. Dunn-Coleman, J. Frisvad, and P. Van Dijck. 2002. On the safety of
Aspergillus niger: a review. Applied microbiology and biotechnology 59: 426-435.
Seye, F., O. Faye, M. Ndiaye, E. Njie, and J. M. Afoutou. 2009. Pathogenicity of the
fungus, Aspergillus clavatus, isolated from the locust, Oedaleus senegalensis, against
larvae of the mosquitoes Aedes aegypti, Anopheles gambiae and Culex
quinquefasciatus. Journal of Insect Science 9.
Washburn, J. A. N. O., D. E. Egerter, J. R. Anderson, and G. A. Saunders. 1988.
Density reduction in larval mosquito (Diptera: Culicidae) populations by interactions
between a parasitic ciliate (Ciliophora: Tetrahymenidae) and an opportunistic fungal
(Oomycetes: Pythiaceae) parasite. Journal of Medical Entomology 25: 307-314.

80

CHAPTER 5: CONCLUSIONS AND DISCUSSION

81

The overarching goals of this study were to examine an unexpected
phenomenon causing increased mortality of larval mosquitoes in the laboratory, to
attempt to identify and characterize the etiologic agent or agents for the phenomenon,
test the identified and isolated candidate agents against known mosquito species, and
to attempt to determine by what method they are achieving the described effect. More
specifically, the objectives and hypotheses for these studies were as follows:
Objective 1: Assess the presence and magnitude of mosquitocidal or inhibitory
effects of isolated fungal/oomycete species against Ae. triseriatus larvae.
Hypothesis 1: Aspergillus, Fusarium, and Pythium isolates will all
demonstrate significant mosquitocidal effects when used individually to
challenge Ae. triseriatus larvae.
Hypothesis 2: Aspergillus, Fusarium, and Pythium isolates will all
demonstrate significant inhibition of growth and development when used
individually to challenge Ae. triseriatus larvae.
Hypothesis 3: Mosquitocidal effects will increase with mixed-species
inoculum as compared with single-species inoculum.
Objective 2: Characterize the mode of action of mosquitocidal fungal/oomycete
isolates against Ae. triseriatus larvae.
Hypothesis 1: Mortality and inhibition of development of Ae. triseriatus
larvae challenged with Aspergillus and Fusarium isolates is due to a
combination of secondary metabolites and direct infection of larvae.
82

Hypothesis 2: Pythium isolate effects are due to direct infection of
mosquito larvae by motile zoospores, not secondary metabolites.

Of the isolated fungal species, only Aspergillus showed consistent pathogenic
effects against either Aedes triseriatus or Aedes japonicus japonicus larvae. This is
contrary to that the hypothesis that all three species would show significant
mosquitocidal and developmental effects. Moreover, given its relatively ubiquitous
distribution in nature (Schuster et al. 2002), it was expected that mosquitoes of both
species would have been exposed to Aspergillus more frequently than any of the other
species tested and would therefore be less likely to be severely affected by it. It was, in
fact, expected that Pythium would generate the highest levels of larval mortality, based
on the highly pathogenic nature of several species of Pythium against both vertebrate
and invertebrate animals (Clark et al. 1966, De Cock et al. 1987) and the fact that the
closely-related organism Lagenidium giganteum has been licensed by the
Environmental Protection Agency for use as a larvicidal agent against mosquitoes
(USEPA 2001).
It was expected that Fusarium would be moderately effective against both
species of Aedes larvae. This was not the case, as Fusarium did not show significant
lethal effects in any of the experiments with these larval species. It did, however,
produce significant levels of mortality in larvae of the African malaria vector Anopheles
gambiae (Kisumu strain). This is surprising, as larvae of Anopheles gambiae have been
shown to be highly competent at mobilizing an immune response called melanization to

83

protect against these fungal pathogens and thereby surviving infection (Golkar et al.
1993). It is of definite interest that Fusarium does show pathogenic/lethal effects against
Anopheles gambiae, and more studies should be conducted to further explore this
relationship.
Unfortunately, very little was able to be concluded regarding the second objective
of this study, which was to characterize the mode of action of these fungi against their
mosquito hosts based on the mycotoxin assay or external microscopy. Data is currently
being collected/analyzed for a more in-depth look at potential physical invasion of the
mosquito by the tested fungi, in which samples preserved in 10% neutral buffered
formalin were submitted to the Investigative Histopathology Laboratory on the campus
of Michigan State University for histological sectioning and staining with hematoxylin
and eosin. The slides prepared by the lab are being analyzed for penetration of the
cuticle, spore germination within the digestive tract, hyphal penetration and proliferation
within the larval body, and fungal growth on the external surface of the cuticle. Larvae
from several different treatment groups will be evaluated and compared.
Assays conducted in the field did not show marked mortality of either Ae.
triseriatus or Ae. j. japonicus upon exposure to mixed Aspergillus and Fusarium spores.
This is in direct contrast to the preliminary laboratory bioassays, which were essentially
equivalent in their setup, with consistent final concentrations of fungus. The larval
density was approximately quadrupled, with 200 larvae per liter in the field microcosm
where there had been 10 larvae per 200 mL (50 larvae per liter) in the laboratory
microcosms to more closely mimic larval densities observed in the field. It is possible
that this contributed to the difference in effect, as the concentration of fungal spores per
84

larva would then be reduced to one quarter its original amount. Also, there were many
environmental factors which could not be controlled in the field, such as temperature,
light, rainfall, etc. Any of these could potentially have affected the outcome of these
experiments, and it is likely that it was a combination of factors, rather than one in
isolation, that ultimately were responsible for the lack of a significant lethal effect in the
field that was equivalent to that observed in the laboratory.
Another interesting result of this work is the observation that Aedes japonicus
japonicus appears to be less susceptible to the tested strain of Aspergillus than Aedes
triseriatus¸which could contribute to a possible competitive advantage in the wild. To
date, little has been published regarding competition between these two species, but it
has been suggested that Ae. j. japonicus may be able to outcompete Ae. triseriatus in
the wild, leading to decreased occurrence of the latter species in their former habitats
(Bevins 2007, Andreadis and Wolfe 2010). In addition to the intrinsic ecological
importance of invasive species biology, this relationship may be of additional public
health importance, as larval competition has been shown to affect the competence of
adult mosquitoes as vectors of disease and longevity of the adult mosquito (Alto et al.
2005, Alto 2011).
There are many directions that can be taken as future explorations of these
complex relationships are planned. For example, it would be of value to more
specifically determine the dosages at which these fungi have demonstrable action
against the development and survival of Aedes triseriatus larvae. Perhaps more
interesting would be to look at the potential competitive advantage of Aedes japonicus
japonicus when compared with Aedes triseriatus after exposure to these pathogens.
85

There is little known about the competitive interactions of these two species. The
potential for Ae. j. japonicus to spread as an invasive species and develop as a disease
vector makes this an intriguing area of research for future studies. Finally, it would be of
great value to further examine the mode of action of these fungal species, particularly
Aspergillus niger, which was the most effective at killing Aedes mosquitoes.

86

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