THE EFFECT OF NITROGEN ON THE LARVAL GROWTH OF THE INVASIVE MOSQUITO
AEDES JAPONICUS JAPONICUS (THEOBALD)
by
Matthew J. Lundquist

A THESIS
Submitted
to Michigan State University
in partial fulfillment of the requirements
for the degree of
Entomology - Master of Science
2013

ABSTRACT
THE EFFECT OF NITROGEN ON THE LARVAL GROWTH OF THE INVASIVE MOSQUITO
AEDES JAPONICUS JAPONICUS (THEOBALD)
by
Matthew J. Lundquist
Invasions by non-native disease vectoring mosquito species have become important considerations
in mosquito control and public health. The Asian rock pool mosquito, Aedes japonicus japonicus
(Theobald), which was first found in the US in 1998, has been increasing both its range and
abundance. This invasive mosquito has the potential to displace the native La Crosse vectoring
mosquito, Aedes triseriatus (Say). Several studies have shown that Ae. j. japonicus larvae have
shorter development times to pupation than Ae. triseriatus. The overarching hypothesis for this
thesis was that Ae. j. japonicus is able to emerge from larval habitats faster than Ae. triseriatus by
better utilizing nutrients that accelerate its growth. Single larvae nanocosms were used to monitor
individual larval development in the absence of competition, and multi-larvae microcosms were
used to test effect of additional nitrogen on larval development. In these studies larvae of both
species depressed the total amount of nitrogen in their habitats similarly, but newly emerged
Ae. j. japonicus adults retained less nitrogen in their body tissues. It was also found that Ae.
j. japonicus growth was not limited by nitrogen availability and that Ae. triseriatus may have
been more sensitive to pathogenic microorganisms that were stimulated by the addition of soluble
nitrogen.

Copyright by
MATTHEW J. LUNDQUIST
2013

ACKNOWLEDGEMENTS

I would like to acknowledge the support and guidance of my major advisor Michael G. Kaufman
throughout the course of my Masters program. The time and effort that my guidance committee
has put into reading and critiquing this thesis was also appreciated. Thank you to the graduate
and undergraduate assistants in the Insect Microbiology Lab at Michigan State University for their
part in facilitating a pleasurable and supportive work environment. I appreciate the Department
of Entomology for providing financial support and the Insect Microbiology Lab for providing
supplies and laboratory space. I would also like to thank Stacey VanderWulp at Kellogg Biological
Field Station for her facilitation of prompt C:N analysis.

iv

TABLE OF CONTENTS

LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ix

CHAPTER 1
INTRODUCTION
Mosquito Invasions . . . . . . . . .
Container Habitat Food Web . . . .
Ae. j . japonicus vs. Ae. triseriatus .
Research Objectives and Rationale .

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COMPARING AEDES JAPONICUS JAPONICUS (THEOBALD)
AND AEDES TRISERIATUS (SAY) EFFECTS ON NITROGEN
IN NANOCOSMS . . . . . . . . . . . . . . . . . . . . . . . . . .
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Mosquito Larvae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Experiment 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Experiment 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Carbon and Nitrogen Analysis . . . . . . . . . . . . . . . . . . . . . . . . . .
Statistical Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Experiment 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Experiment 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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CHAPTER 2

CHAPTER 3

THE EFFECT OF NITROGEN ON AEDES JAPONICUS JAPONICUS
(THEOBALD) AND AEDES TRISERIATUS (SAY) GROWTH
IN MICROCOSMS . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Mosquito Larvae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Nitrogen Addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Carbon and Nitrogen Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Statistical Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

CHAPTER 4
GENERAL CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . 44
Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Single vs. multi-larvae rearing . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

v

Larval feeding preferences on microorganisms
Partitioning of nitrogen compounds . . . . . .
Ae. j. japonicus habitat utilization . . . . . .
Ae. j. japonicus larval physiology . . . . . . .
Ae. j. japonicus natural enemies . . . . . . .

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APPENDIX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
LITURATURE CITED . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

vi

LIST OF TABLES

Table 2.1

ANCOVA analysis of E1 nanocosms N changes between larva and no larva
nanocosms. * = significant with Bonferroni adjustment, α=0.05. . . . . . . . . . 13

Table 2.2

ANCOVA analysis of leaf mass and nutrient changes between E1 larva and no
larva nanocosms. * = significant with Bonferroni adjustment, α=0.05. . . . . . . 14

Table 2.3

ANCOVA analysis of larval nutrient changes between species in E1. * =
significant with Bonferroni adjustment, α=0.05. . . . . . . . . . . . . . . . . . . 15

Table 2.4

MANOVA analysis of final N measurements in E2 nanocosms. Variates were
water column N, leaf surface N, and dissolved N. . . . . . . . . . . . . . . . . . 17

Table 2.5

ANOVA analysis of final N in E2 nanocosms. * = significant with Bonferroni
adjustment, α=0.05. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Table 2.6

ANOVA analysis of E2 leaf tissue nitrogen and carbon concentrations. . . . . . . 19

Table 2.7

MANOVA analysis of overall mosquito development in E2. Variates were
development time and weight. . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Table 2.8

ANOVA analysis of overall mosquito growth parameters and nitrogen and
carbon concentrations in E2. * = significant with Bonferroni adjustment, α=0.05. 21

Table 2.9

ANOVA analysis of overall mosquito nitrogen and carbon concentrations in
E2. * = significant with Bonferroni adjustment, α=0.05. . . . . . . . . . . . . . 22

Table 3.1

MANOVA analysis of overall mosquito growth parameters. Variates were
survival, total adult biomass, total number of adults produced, average female
weight, number of females produced, average female development time. . . . . . 35

Table 3.2

ANOVA of total microcosm production. * = significant with Bonferroni adjustment,
α = 0.05. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Table 3.3

ANOVA of female growth production. * = significant with Bonferroni adjustment,
α = 0.05. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

Table 3.4

ANOVA of male growth production. * = significant with Bonferroni adjustment,
α = 0.05. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

Table 3.5

ANOVA of female nitrogen and carbon. * = significant with Bonferroni
adjustment, α = 0.05. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Table 3.6

ANOVA analysis of leaf decay. . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

vii

Table A.1 Catalog of preserved specimens . . . . . . . . . . . . . . . . . . . . . . . . . . 51

viii

LIST OF FIGURES

Figure 2.1

E1 nanocosm nitrogen concentrations over time. Lines are from ANCOVA
analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Figure 2.2

Leaf decay, nitrogen and carbon over time in E1 nanocosms. Lines are from
ANCOVA analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Figure 2.3

Larval growth and nutrient accumulation over time in E1. Lines are from
ANCOVA analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Figure 2.4

E2 nanocosm nitrogen concentrations. Values are means ± SE (n=20, 27, 16, 18). 18

Figure 2.5

E2 nanocosm leaf nutrient concentrations. Values are means ± SE (n=20,
27, 16, 18). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Figure 2.6

Mosquito growth parameters and nitrogen in E2. Values are means ± SE
(n=20, 27, 16, 18). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Figure 2.7

Larval nitrogen and carbon accumulation in E2. Values are means ± SE
(n=20, 27, 16, 18). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Figure 3.1

Total microcosm production. Values are means ± SE (n=8). . . . . . . . . . . . 36

Figure 3.2

Female growth parameters. Values are means ± SE (n=8). . . . . . . . . . . . . 37

Figure 3.3

Male growth parameters. Values are means ± SE (n=8). . . . . . . . . . . . . . 38

Figure 3.4

Female nitrogen and carbon. Values are means ± SE (n=8). . . . . . . . . . . . 39

Figure 3.5

Remaining leaf mass in microcosms. Values are means ± SE (n=8). . . . . . . 40

ix

CHAPTER 1
INTRODUCTION

Mosquito Invasions
High-speed world commerce has increased both the mobility and distribution of non-native organisms
and biological invasions have become commonplace. In many cases, invasive organisms can reduce
or displace local populations and disrupt ecosystems. In the field of medical entomology, the
biological invasions of arthropod born virus (arbovirus) vectoring mosquitoes (Diptera: Culicidae)
have been of increasing importance (Juliano and Lounibos 2005). The introduction of any novel
vector or disease can have a resounding impact on public health. The understanding and tracking
of mosquito invasions are paramount.
The shipment of tires to the United States from Asia in the mid-1990’s has facilitated the
establishment of a number of mosquitoes in the genus Aedes, including the Asian rock pool
mosquito, Ae. japonicus japonicus (Theobald) (Peyton et al. 1999). This species was first detected
in the Eastern US in 1998, and has since been reported in both the Southern and Midwestern
US (Peyton et al. 1999; Morris et al. 2007; Hughes et al. 2008; Neitzel et al. 2009; Gaspar et al.
2012), Pacific Northwest (Irish and Pierce 2008), and Hawaii (Larish and Savage 2005). Aedes j.
japonicus has also expanded its range into Canada (Thielman and Hunter 2006) and throughout
central Europe (Schaffner et al. 2003, 2009; Versteirt et al. 2009). Their larvae can be found in
a diverse array of aquatic container habitats including rock pools, tree holes, and discarded tires
(Scott et al. 2001; Andreadis et al. 2001). Mosquitoes that already inhabit these containers can
interact with and can be affected by Ae. j. japonicus (Hardstone and Andreadis 2012). In discarded
tire habitats in the North Eastern US, larvae of native mosquito Aedes triseriatus (Say) appear to
be in the process of being replaced by Ae. j. japonicus (Andreadis and Wolfe 2010).
The increased presence of Ae. j. japonicus and the reduction of Ae. triseriatus could have
an effect on US arbovirus transmission (Leisnham and Juliano 2012). Aedes j. japonicus has

1

been implicated as an important vector of Japanese encephalitis in Asia (Takashima and Rosen
1989) and is also a highly competent vector of West Nile virus (Sardelis and Turell 2001; Turell
et al. 2000), eastern equine encephalitis virus (Sardelis et al. 2002a), La Crosse virus (Sardelis
et al. 2002b), and St. Louis encephalitis virus (Sardelis et al. 2003) based on laboratory studies.
While not considered an important disease vector in the US, Ae. j. japonicus is known to feed on
both birds and humans (Williges et al. 2008; Molaei et al. 2009) and some field collected females
have been found to be infected with West Nile virus (CDC 2000) and La Crosse encephalitis
virus (Westby et al. 2011). Aedes triseriatus is the primary vector of La Crosse encephalitis virus
(LACV) (Puntuwat et al. 1974; Beaty et al. 2000) and has also been implicated in West Nile virus
transmission (Turell et al. 2005). The effect of the displacement of Ae. triseriatus by Ae. j.
japonicus on disease transmission will depend on the efficiency of Ae. j .japonicus to transmit
disease (Juliano and Lounibos 2005). Indeed, if Ae. j. japonicus is a less efficient it may decrease
disease transmission; however, the opposite may be more likely.
The mechanism by which Ae. j. japonicus can displace Ae. triseriatus has not been fully
elucidated. The most compelling finding has been that Ae. j. japonicus larvae tend to develop
faster than Ae. triseriatus larvae under similar developmental conditions (Alto 2011; Hardstone and
Andreadis 2012). Accelerated development time could allow for a quicker exposure to potential
hosts and potential oviposition sites, as well as escaping from larval habitats that have restricted
resources or may dry out (Juliano and Stoffregen 1994). It is currently not known how Ae. j.
japonicus larvae develop faster, but it may be related to how the larvae interact with their habitat
and compete for limited food resources.

Container Habitat Food Web
Container habitats usually contain a variety of aquatic organisms including fungi, bacteria, algae
and insects (Kitching 2001; Kaufman et al. 2001, 2002; Lorenz et al. 2013). Nutrients in these
containers are provided by the input of allochthonous particulate plant detritus (Fish and Carpenter
1982; Walker et al. 1997), animal matter (Daugherty et al. 2000; Yee and Juliano 2006; Harshaw
2

et al. 2007) and dissolved organic and inorganic nutrients from stemflow and rain (Carpenter
1982b; Kaufman et al. 1999; Kaufman and Walker 2006).
Larval mosquitoes cannot access most detrital nutrients directly. Fungi and bacteria are able
to break down and obtain nutrients from decaying plant and animal material for use in metabolic
processes (Fish and Carpenter 1982; Kaufman et al. 2001, 2008), and mosquito larvae rely heavily
on this microbial biomass for their nutritional requirements (Carpenter 1983). Plant derived detritus
is high in carbon (C) but low in nitrogen (N), and the decay of plant material in larval mosquito
habitats can often be limited by N (Kaufman and Walker 2006); therefore, microbial biomass and
mosquito development is limited by the relative amounts of C and N in detritus (Walker et al. 1997).
The addition of dissolved forms of C and N provided by stemflow and rain can also stimulate
microbial production and can alleviate larval nutritional stress (Kaufman et al. 2002; Kaufman and
Walker 2006).

Ae. j . japonicus vs. Ae. triseriatus
Containers with decaying leaf detritus are attractive to ovipositing females and can sometimes
support large larval populations (Beehler et al. 1992; Bartlett-Healy et al. 2012). High larval
densities can cause both a reduction in larval survival and adult size (Alto 2011; Hardstone and
Andreadis 2012); therefore, the fitness of a species is greatly influenced by competitive interactions
in the larval stage.
Direct competition between larval Ae. j. japonicus and Ae. triseriatus has been examined only
in a few laboratory studies (Ingrassia 2006; Alto 2011; Hardstone and Andreadis 2012). In most
cases, competitive pressure was related to overall larval density and not interspecific competition;
however, Ae. j. japonicus has consistently been shown to develop faster (Alto 2011; Hardstone
and Andreadis 2012), including under very low food rations (Ingrassia 2006). This suggests that
Ae. j. japonicus may have an intrinsic advantage in growth through physiological mechanisms
independent of its interaction with Ae. triseriatus.
Stressful conditions have been shown to shorten larval development and this results in a decrease
3

in adult size in mosquitoes (Juliano and Stoffregen 1994). Even with a shorter development time,
Ae. j. japonicus adults have not been found to differ greatly in size than those of Ae. triseriatus
(Alto 2011; Hardstone and Andreadis 2012; Lorenz 2012). Factors that can determine body size
in insects are food quality (Davidowitz et al. 2003), rearing temperature (Dodson et al. 2012), and
genetics (Nijhout 2003).
Larvae of Ae. j. japonicus and Ae. triseriatus have been described as active grazers and filter
feeders (Walker and Merritt 1991; Ingrassia 2006; O’Donnell and Armbruster 2007), though it
has been reported that Ae. triseriatus tends to filter feed near the surface of the water column
and Ae. j. japonicus larvae tend to graze near the bottom and on the container walls (Ingrassia
2006). Regardless, it is likely that both species can access similar food resources in containers;
however, analysis of C and N in field-collected, newly emerged female Ae. j. japonicus and Ae.
triseriatus collected from the same larval habitats showed that while C concentration was similar,
Ae. j. japonicus had a lower N concentration (M. G. Kaufman unpublished data). Differences
in N concentration has been shown between different mosquito species (Winters and Yee 2012).
This may indicate that Ae. j . japonicus requires less N than Ae. triseriatus and can produce a
similar amount of body mass with less food consumption (Fagan et al. 2002). This would allow
for a competitive edge, especially when resources are limited.

Research Objectives and Rationale
With the arrival of Ae. j. japonicus and the displacement of Ae. triseriatus (Andreadis and Wolfe
2010), the ecology of disease transmission may change (Leisnham and Juliano 2012). Observations
suggest that Ae. j. japonicus accumulates nutrients differently and this may point to an explanation
as to how it is able to out-compete Ae. triseriatus (M. G. Kaufman unpublished data). This thesis
aimed to examine the accumulation and effect of N on Ae. j. japonicus and Ae. triseriatus larval
growth in the laboratory. The two main objectives were to:
1. Contrast the effects of individual Ae. j. japonicus and Ae. triseriatus larva on the levels

4

of particulate and dissolved N in laboratory nanocosms throughout development against
nanocosms without larvae, and to measure and compare the accumulation of N in larval
tissues throughout development
2. Compare the effect of additional nitrogen on the growth and N accumulation of Ae. j.
japonicus and Ae. triseriatus larvae under intraspecific competition in laboratory microcosms

5

CHAPTER 2
COMPARING AEDES JAPONICUS JAPONICUS (THEOBALD) AND AEDES
TRISERIATUS (SAY) EFFECTS ON NITROGEN IN NANOCOSMS

Introduction
Larvae of the invasive mosquito Aedes japonicus japonicus (Theobald) appear to be displacing
Aedes triseriatus (Say) in discarded tire habitats (Joy and Sullivan 2005; Andreadis and Wolfe
2010). Studies have shown that Ae. j. japonicus generally develop faster than Ae. triseriatus
females when under intraspecific and interspecific competition (Ingrassia 2006; Alto 2011; Hardstone
and Andreadis 2012; Lorenz 2012). The underlying mechanism of this acceleration in growth is
unknown, though it may be related to nutrient acquisition or assimilation.
Natural and artificial containers that act as mosquito larval habitats rely heavily on allochthonous
inputs of particulate plant and animal detritus and dissolved nutrients (Carpenter 1982b; Fish
and Carpenter 1982; Walker et al. 1997; Daugherty et al. 2000; Kaufman and Walker 2006).
A common source of organic detritus in container habitats are senescent plant leaves (Fish and
Carpenter 1982). These leaves initially leach out the more labile nutrients into the water column
(Pelz-Stelinski et al. 2010). The majority of leaf particulates remain and are colonized by fungi and
bacteria and is further decomposed (Fish and Carpenter 1982; Ponnusamy et al. 2008; Kaufman
et al. 2010). The microbial biomass produced during leaf decay is utilized by mosquito larvae as
their primary source of energy and nutrients for growth (Fish and Carpenter 1982; Walker et al.
1991; Kaufman et al. 2001, 2002; Kaufman and Walker 2006). Larval competition is common in
these types of habitats and larval survival and production can be limited by the quantity and quality
of detritus and associated microorganisms (Fish and Carpenter 1982; Walker et al. 1997).
Decomposition of carbon (C) polymers is generally limited by nitrogen (N), and phosphorus
(P) (Howarth and Fisher 1976; Güsewell and Gessner 2009). Leaf detritus generally has a high C:N
ratio; therefore, the decomposition of leaf material is limited by the availability of N (Kaufman and

6

Walker 2006; Güsewell and Gessner 2009). Fungi that colonize leaf surfaces also tend to have a
relatively high C:N ratio, while bacteria tend to have a comparatively low C:N ratio (Güsewell
and Gessner 2009). Larval feeding has been shown to depress bacterial productivity in the water
column (Kaufman et al. 2001), and larvae actively feed on leaf surface bacteria and fungi, causing
reductions and community shifts (Kaufman et al. 1999, 2008). Larval feeding can also affect the
accumulation of inorganic N compounds in water columns (Kaufman et al. 1999).
Larval feeding modes have been shown to be similar between Ae. j. japonicus and Ae.
triseriatus (Ingrassia 2006); however, newly emerged Ae. j. japonicus females from discarded
tires were found to have a significantly lower N concentrations than Ae. triseriatus females (M.
G. Kaufman unpublished data). This indicated that Ae. j. japonicus may have different nutrient
requirements and it may interact differently with microbial resources to accelerate growth. This
finding has not been corroborated in the laboratory, nor has it been investigated in larvae. The
purpose of this study was twofold:
1. Investigate N accumulation in individual Ae. j. japonicus and Ae. triseriatus during the
larval stage and at adult emergence
2. Determine if Ae. j. japonicus and Ae. triseriatus larvae alter available N in their habitats
differently

7

Methods
All experimental units were housed in nanocosms consisting of plastic 50 ml conical tubes with 20
ml of deionized water, 50 mg of oven dried white oak (Quercus alba; ± .5 mg) leaves (collected
at Kellogg Forest, Augusta, MI), and 100 µl of microbial inoculum collected from tree holes in
Toumey woodlot (MSU, East Lansing, MI). Each nanocosm was incubated for 24 h at 24 ◦ C to
facilitate microbial colonization and leaf decay before larval addition.

Mosquito Larvae
New Jersey strain Ae. j. japonicus larvae (Rutgers University, New Brunswick, NJ) and MSU
strain Ae. triseriatus newly hatched larvae were used for these experiments. Hatching was induced
by flooding the eggs in a nutrient-rich broth. The eggs of Ae. triseriatus required an additional
24 h soaking period in deionized water prior to flooding. After approximately 9 h, newly hatched
larvae of each species were removed from the broth using a glass Pasteur pipette and transferred
to a plastic Petri dish for counting. To remove the confounding factors of larval competition and
mortality, each nanocosm received only one Ae. j. japonicus larva or one Ae. triseriatus. An
additional 50 newly hatched larvae of each species were collected on glass cover slides and oven
dried for 48 h for initial first instar weight and nitrogen content estimation. Nanocosms were stored
at 24 ◦ C with a 12:12 light cycle. Each larval nanocosm was checked daily for newly shed cuticles
to determine instars.

Experiment 1
To track the effect of larvae on N through time, three experimental groups were created consisting
of (1) Ae. j. japonicus, (2) Ae. triseriatus, and (3) a no larvae control, with 50 replicates in each
group. To insure adequate collection of second, third, and fourth instars, five randomly selected
nanocosms from each group were destructively sampled on d 2, 4, 6, 9, 11, and 13 after hatching.
Additional destructive sampling occurred at adult emergence or at larval death. Prior to processing,

8

the gender was determined for all adults and pupae. Sampling was ended after no pupae were
produced ten days after the final pupa was observed (d=40). Mortality was not calculated for these
nanocosms.

Experiment 2
To measure the concentration of N at pupation or adult emergence for both species, nanocosms
were divided into a Ae. j. japonicus group, and a Ae. triseriatus group, with 60 replicates in
each group. Larvae were allowed to develop to adulthood and were sampled at adult emergence
or pupal death. Any larvae that died or did not make it to pupation were not sampled and were
only included in the calculation of overall survival. An additional 5 nanocosms of each group
were run and destructively sampled on days 4 and 7 to compare with E1 nanocosms. Data from
these cursory observations did not differ from E1 observations and are not presented in this thesis.
Four nanocosms were discovered to have received a larva of both species and were excluded from
calculations.

Sampling
Larvae and pupae were removed from nanocosms using a glass Pasteur pipette and transferred to
a glass cover slide and dried at 50 ◦ C for 48 h. Adults were knocked down in nanocosms at -20 ◦ C
for 30 min and transferred to 1.5 ml plastic centrifuge tubes using forceps and dried at 50 ◦ C for
48 h. All dried samples were weighed to the nearest .001 mg and were saved for C:N analysis.
Leaves were placed in plastic scintillation vials with 15 ml of deionized water and sonicated for
12 min to remove any particulates from the leaf surface. The leaves were then transferred to glass
scintillation vials and dried at 50 ◦ C for at least 48 h and weighed. Dissolved N was partitioned
from total N by filtering 5 ml of nanocosm water through 1 µm glass filters. The remaining
nanocosm water, dissolved N samples and leaf sonicate was stored at -20 ◦ C for N analysis.

9

Carbon and Nitrogen Analysis
The concentrations of N in nanocosms were measured in 4 ml samples from the total and dissolved
water column and the leaf sonicate. Total water column samples were initially diluted 1:1 with
deionized water due to high nutrient concentration. Each sample received 600 µl of oxidizing
reagent solution (1:1 NaOH and K2 SO8 ), was vortexed, and then autoclaved for 30 min at 121 ◦ C.
After cooling, 8 µl of concentrated H2 SO4 was added lower the pH. Samples were individually
run through UV N analysis (Lambda 25, PerkinElmer, Inc.). Spectrometry curves were collected
using UV WinLab software (PerkinElmer, Inc.) and total N concentration in each sample was
determined using second derivative analysis (Crumpton et al. 1992). The total concentration of N
in each sample was computed using the linear equation of a NaNO3 standard curve.
The C:N ratios of larvae, pupae, adults, and leaves were determined using elemental combustion
analysis (Costech Inc., Valencia, CA ) at Kellog Biological Station (Hickory Corners, MI). Whole
mosquito samples were prepared in 5x9 mm tin cups (Costech Inc.) for analysis. For processing,
leaf samples were transferred to flat-bottomed centrifuge tubes with metal shot and shaken at 1,800
rpm for 10 min to produce a fine powder. Two 3 mg samples from each leaf was then prepared in
5x9 mm tim cups (Costech, Inc.) for analysis.

Statistical Analysis
Analysis of covariance (ANCOVA) was performed to analyze nanocosm N measurements, leaf
decay and leaf tissue C:N, and mosquito C, N and C:N over time separately between larvae and
no larvae treatments and between mosquito species from E1 nanocosms. Any significant larval
effects were further analyzed for species effects. Multivariate analysis of variance (MANOVA)
was performed to analyze mosquito growth parameters from E2. Growth parameters included
adult development time, weight, and the total C and N accumulation on variation between species
and genders. Comparisons of E2 nanocosm nitrogen concentrations between species and genders
were also performed using MANOVA analysis. Univariate analysis of variance (ANOVA) was
also performed. All related post hoc p-values were Bonferroni adjusted to control for Type 1 error.
10

When necessary, data were transformed to meet assumptions of normality and homoscedasticity
and all proportional data were arcsine square-root transformed. All reported data are untransformed.

11

Results
Experiment 1
In the absence of larvae, total water column N decreased (Fig 2.1), and leaf surface N (Fig 2.1)
and dissolved N (Fig 2.1) increased. In the presence of larvae, total water column N increased (Fig
2.1), dissolved N decreased (Fig 2.1), and leaf surface N remained at a constant level (Fig 2.1). The
decrease of total water column N was accelerated by larval presence (Fig 2.1, Table 2.1). Larvae
also significantly reduced N accumulation on leaf surfaces (Fig 2.1, Table 2.1). The amount of
dissolved N present in nanocosms was not significantly affected by larval presence (Fig 2.1, Table
2.1), and these three measured N concentrations did not differ between larval species.
The loss of leaf mass increased significantly over time (Fig 2.2, Table 2.2) but was not affected
by larval presence (Table 2.2). Leaf N and C concentration did not differ significantly through time
(Table 2.2).
Larvae of both species gained weight similarly through time (Fig 2.3 A, Table 2.3). Throughout
larval development, %N increased significantly in the larval body (Fig 2.3, Table 2.3) and %C
significantly decreased (Fig 2.3, Table 2.3). The ratio of C:N in larval tissues also decreased
significantly over time (Fig 2.3, Table 2.3).

12

Table 2.1: ANCOVA analysis of E1 nanocosms N changes between larva and no larva
nanocosms. * = significant with Bonferroni adjustment, α=0.05.

Source
Days
Larval Presence
Days X Presence

Total Water Column
df
F
P
1 51.8
<0.0001*
1 11.8
<0.0001*
1 3.3
0.04

Leaf Surface
F
P
11.0
0.001*
21.1 <0.0001*
2.4
0.09

Dissolved
F
P
38.2 <0.0001*
0.34
0.72
0.63
0.53

200
100

qq
qq
qqq
qq
qqq
q
q
qqq
qqq
qqq
q
qq
q

q
q
q
qq
qqq q
qq
q q q
q
q
q
qq
qqqqq
q
qqq
qqqq
qqqqq
qq qq
q
q
q

q

Ae. j. japonicus
Ae. triseriatus
No Larvae

q

q
q
q
q
q
q
q q
q
q
q
q q
q
q q

q
q

0

ug/ nanocosm

Total Water Column Nitrogen

0

10

20

30

40

Days

0

10

q
q
q
q
q
q
q

q qq
q q
q q
q
q
q
q q

20

100
qq
q q
q
qq
q
q
qq
qq
qq
qqq
qq
q
qqq
q
q q
q

50

qq
qqq q
q q q
q
qq qqq
q
qqqqq
q q q
qqqq q
qq q
qqq
qqq qq
q
q q
q
q

0

q
qqq
q
qq
qq
qqq
qqq
q
qqq
q
qqq
qqq
q q

ug/ nanocosm

20 40 60 80

150

Dissolved Nitrogen

q

q
q

0

ug/ nanocosm

Leaf Surface Nitrogen

30

40

0

Days

q
q
qqq q
q
qq qq
qqqq
q
q
q
qqqqq
qqqq
qq qq
qq

q
q
q
q
q qq
q
q
q
q q
q
q q

q
q
q
q
q

q
q
q
q
q

10

20

30

40

Days

Figure 2.1: E1 nanocosm nitrogen concentrations over time. Lines are from ANCOVA analysis.

13

Table 2.2: ANCOVA analysis of leaf mass and nutrient changes between E1 larva and no larva
nanocosms. * = significant with Bonferroni adjustment, α=0.05.
Remaining leaf
df
F
P
1 37.1 <0.0001*
1 1.2
0.30
1 0.58
0.56

Source
Days
Larval Presence
Days X Presence

%N
%C
C:N
F
P
F
P
F
P
0.64 0.43 4.8 0.03 1.27 0.27
1.86 0.17 2.2 0.11 1.65 0.26
0.64 0.53 1.0 0.39 0.58 0.56

Percent Nitrogen
4

30 40 50 60 70

q
q q
q
qqq
qq
q q
qq
q
q q

qqq
qqq q
qqqqq
qqqqqq
q q qq
q
qqqqq
q
qqq q

q
q q
q q
q
q qq
q
q q
q
q
q
q

q
q
q
q
q

0

q
q

Ae. j. japonicus
Ae. triseriatus
No Larvae

q
q
q
qqq qq
q
q
qq
qqq qq
qq

q
q
q q
q
q

10

20

30

40

0

10

20

30

q

Ae. j. japonicus
Ae. triseriatus
No Larvae

q
q
q
qqq
q
q q
q
q q
q
q
q
q q
qqq q
q
q

10

q
q
q
q
q
q

20

q
q
q

q
q
q
q

q
q

60 100

q

q

30

40

0

Days

Ae. j. japonicus
Ae. triseriatus
No Larvae

qq
q
qq
q q
q
q
qqq qq
q
q q
q
q
q

20

q

160

Carbon/Nitrogen Ratio

C:N/ leaf

40 50 60 70 80

40

Days

Percent Carbon

%C/ leaf

Days

0

q
q

0

q

3

q

2

q

Ae. j. japonicus
Ae. triseriatus
No Larvae

1

q

%N/ leaf

mg/ nanocosm

Remianing Leaf Mass

10

q
q q
q
q
q q
q

20

30

Days

Figure 2.2: Leaf decay, nitrogen and carbon over time in E1 nanocosms. Lines are from
ANCOVA analysis.

14

q
q

40

Table 2.3: ANCOVA analysis of larval nutrient changes between species in E1. * = significant
with Bonferroni adjustment, α=0.05.
Source
df
Days
1
Species
1
Days X Species 1

Weight
F
P
11.23 0.0011*
4.79
0.03
0.126
0.73

%N
F
P
14.6 0.0003*
1.64
0.21
0.30
0.59

%C
F
P
7.93 0.007*
0.08 0.77
2.99 0.09

q

0

q q
qqq
q
qqq qq
qqqqq
qqqqq
q q
q qq
q
q q
q q
q
q

q
q q
q q
q q
q
q

q
q
q
q

10 20 30 40

q
q
q
q q
q
qq
q
qqq
q
qqq
qq

Ae. j. japonicus
Ae. triseriatus

q

0

1.0

q

10

20

30

P
0.0008*
0.28
0.49

Percent Nitrogen

%N/ larva

q

0.5
0.0

mg/ larva

1.5

Larval Weight

C:N
F
12.67
1.21
4.05

40

q
q

Ae. j. japonicus
Ae. triseriatus

q q
q q
qqqqq
q
qqqq qqq qq
q q qqq
qq
q
q
q

0

10

q
q
q q
q q

20

q

30

25

Ae. j. japonicus
Ae. triseriatus

q
q
q
qqqq
qq q
q q q qqqqq
q
qqq q
q q
q
q
q
q
q
q
q
q
q
q

q
q q
q q
q

0

10

20

q

30

40

q

15

q

0 5

q

C:N

80
40

Carbon:Nitrogen Ratio

0

%C/ larva

Days

Percent Carbon
120

Days

40

q
q
qq
q
qqqq qqq q
qq qq
q q q qqqq
q
q
qq q
q
q
q
q
q

0

Days

10

q q
q
q q

20

q

30

40

Days

Figure 2.3: Larval growth and nutrient accumulation over time in E1. Lines are from ANCOVA
analysis.

15

Experiment 2
The final amount of all total, leaf surface, and dissolved N in nanocosms were nearing significance
between mosquito species and was significant between genders in the MANOVA analysis (Table
2.4). Total water column and dissolved N were significantly higher in Ae. j. japonicus nanocosms
and in nanocosms that produced females in post hoc ANOVA analysis (Fig 2.4, Table 2.5). The
final leaf nutrient concentrations were not significantly different between either species or gender
in the MANOVA analysis (Table 2.6), nor were they significant different in post hoc ANOVA
analysis (not reported); however, %C in appears to be lower in nanocosms with Ae. j. japonicus
that produced females (Fig 2.5), even though the mean was not statistically significant.
Larval growth parameters and nutrient accumulation in nanocosms were significantly different
between species and genders in the MANOVA analysis (Table 2.7). The adults of Ae. j. japonicus
were slightly smaller than Ae. triseriatus but statistical significance was not found after Bonferroni
adjustment (Fig 2.6, Table 2.8). There was no difference in size between males or females within
either species (Fig 2.6, Table 2.8). Total development time was not significantly different between
species, but males emerged sooner than females (Fig. 2.6, Table 2.8). Survivorship to pupation
from nanocosms was 72% in Ae. j. japonicus nanocosms and 93% in Ae. triseriatus nanocosms.
The %N in emerged adults was not significantly different between species or genders, but there
was a significant interaction between these two main effects (Fig 2.7, Table 2.9). Female Ae. j.
japonicus had a lower %N than either sex of Ae. triseriatus (F=19.45, P=0.003; Fig 2.7) or male
Ae. j. japonicus (F=15.88, P=0.0004; Fig 2.6). Adult %C was significantly different between
species and genders (Fig 2.7, Table 2.9) and C tended to be higher in Ae. triseriatus, as well as
higher in adult males (Fig 2.7). The ratio of C:N in adults was significantly different between
genders (Table 2.9). Females had a lower C:N ratio than males (Fig 2.7).

16

Table 2.4: MANOVA analysis of final N measurements in E2 nanocosms. Variates were water
column N, leaf surface N, and dissolved N.
MANOVA
Source
Species
Gender
Species X Gender
Error

Roy’s max root
0.105
0.375
0.0514

17

df
1
1
1
76

P
0.06
<0.0001
0.292

Table 2.5: ANOVA analysis of final N in E2 nanocosms. * = significant with Bonferroni
adjustment, α=0.05.
Source
Species
Gender
Species x Gender

df
1
1
1

Total Water Column
F
P
7.20
0.009*
6.83
0.011*
3.443
0.067

Leaf Surface
F
P
1.05 0.309
3.65 0.06
0.074 0.786

Dissolved
F
P
7.61
0.007*
19.5 <0.0001*
2.74
0.102

50 100

Male
Female

0

ug/ nanocosm

200

Total Water Column Nitrogen

Ae. triseriatus Ae. j. japonicus

80
0

40

ug/ nanocosm

30
0 10

ug/ nanocosm

120

Dissolved Nitrogen

50

Leaf Surface Nitrogen

Ae. triseriatus Ae. j. japonicus

Ae. triseriatus Ae. j. japonicus

Figure 2.4: E2 nanocosm nitrogen concentrations. Values are means ± SE (n=20, 27, 16, 18).

18

Table 2.6: ANOVA analysis of E2 leaf tissue nitrogen and carbon concentrations.
%N
Source
Species
Gender
Species x Gender

df
F
1 0.008
1 0.98
1 1.41

%C

C:N

P
F
P
0.93 0.77 0.40
0.34 1.89 0.19
0.26 4.29 0.06

F
P
0.30 0.59
0.013 0.91
0.0102 0.97

2.0
1.0

Male
Female

0.0

%N/ nanocosm

3.0

Percent Nitrogen

Ae. triseriatus Ae. j. japonicus

Carbon:Nitrogen Ratio

Ae. triseriatus Ae. j. japonicus

60
0 20

C:N/ nanocosm

60
40
20
0

%C/ nanocosms

80

100

Percent Carbon

Ae. triseriatus Ae. j. japonicus

Figure 2.5: E2 nanocosm leaf nutrient concentrations. Values are means ± SE (n=20, 27, 16, 18).

19

Table 2.7: MANOVA analysis of overall mosquito development in E2. Variates were development
time and weight.
MANOVA
Source
Species
Gender
SXG
Error

Roy’s max root
0.10
1.16
0.05

20

df
1
1
1
77

P
0.027
<0.0001
0.17

Table 2.8: ANOVA analysis of overall mosquito growth parameters and nitrogen and carbon
concentrations in E2. * = significant with Bonferroni adjustment, α=0.05.
Source
Species
Gender
Species x Gender

df
1
1
1

Weight
F
P
5.9 0.02
3.3 0.07
0.02 0.9

Development Time
30

0.5

Adult Weight

Devel Time
F
P
2.3
0.1
55.2 <0.0001*
0.4
0.08

Male
Female

0

0.0

5

0.1

10

15

days/ individual

0.3
0.2

mg/ individual

20

0.4

25

Male
Female

Ae. triseriatus Ae. j. japonicus

Ae. triseriatus Ae. j. japonicus

Figure 2.6: Mosquito growth parameters and nitrogen in E2. Values are means ± SE (n=20, 27,
16, 18).

21

Table 2.9: ANOVA analysis of overall mosquito nitrogen and carbon concentrations in E2. * =
significant with Bonferroni adjustment, α=0.05.
%N
Source
Species
Gender
Species x Gender

df
1
1
1

F
5.3
5.7
10.5

%C

P
0.03
0.02
0.002*

F
15.5
29.6
0.4

P
0.0002*
<0.0001*
0.5

C:N
F
4.4
11.7
2.5

P
0.04
0.001*
0.12

Figure 2.7: Larval nitrogen and carbon accumulation in E2. Values are means ± SE (n=20, 27,
16, 18).

22

Discussion
This study shows that larval mosquitoes directly affect the nutrient dynamics of their aquatic
habitats. The depression of N in the water column and on leaf surfaces is likely due to microbial
biomass reduction caused by larval feeding (Kaufman et al. 2001). Bacteria, which tend to be
C limited, and fungi, which tend to be N limited, readily take up N compounds for metabolic
processes (Kaufman and Walker 2006). It is likely that the decrease in water column N and
the increase in leaf surface N in larvae-free nanocosms can be attributed to the accumulation
of N by fungi and bacteria on the leaf surfaces. The presence of larvae has been shown to
decrease populations of bacteria and fungi on leaf surfaces (Fish and Carpenter 1982; Kaufman
et al. 1999, 2001, 2002; Kaufman and Walker 2006) and larvae are known to feed directly on
bacteria and particles in the water column (Walker and Merritt 1991; O’Donnell and Armbruster
2007). Dissolved N on the other hand was unaffected by larval presence. When in water, leaf
detritus readily leaches out its soluble nutrients (Carpenter 1982a) and these nutrients can be
incorporated by free floating bacteria and attached microorganisms which can then be utilized by
larvae (Pelz-Stelinski et al. 2010). An initial increase in dissolved N would be expected because of
leaching. The incremental increase in dissolved N throughout the experiment was likely related to
larval excretion and the slow release of N from decaying leaf material (Pelz-Stelinski et al. 2010).
Differences between Ae. j. japonicus and Ae. triseriatus N utilization were seen in the percent
of N present in newly emerged females. Both C and N percentages were higher in Ae. triseriatus
adults at the end of the experiment. These results confirm earlier observations that Ae. j. japonicus
females emerge with lower N, but did not reveal any evidence that N is obtained or processed
differently by either species. Lower %N in insects is usually indicative of a lower N requirement
for development (Fagan et al. 2002) and a higher concentration of N is correlated with a larger body
size (Hood-Nowotny et al. 2012), though this is not always the case (Winters and Yee 2012). High
%N can also associated with high %C (Hood-Nowotny et al. 2012) which translates to higher lipid
and carbohydrate stores and a larger body size (Fagan et al. 2002; Winters and Yee 2012) unless
a species is particularly N efficient (Winters and Yee 2012). In this study, adult body size did not
23

vary between species even though Ae. triseriatus had a higher %C and %N. While carbon can be
found in both lipid and carbohydrate stores that make up much of the mosquito biomass, it can
also be respired as CO2 . It is possible that differences in respiration rates between Ae. j. japonicus
and Ae. triseriatus may account for some of the differences in %C even though adult body mass
was similar between species.
Contrary to expectations, the development of both Ae. j. japonicus and Ae. triseriatus
in this study followed similar trajectories. In previous multi-larvae studies, Ae. j. japonicus
larvae generally developed faster than Ae. triseriatus (Alto 2011; Hardstone and Andreadis 2012).
Indeed, the development time of Ae. triseriatus females was accelerated to what was seen by Alto
(2011) at a much higher leaf ration. It is unclear why only Ae. triseriatus development time was
accelerated and Ae. j. japonicus development was not. Low larval densities tend to decrease
the development time of both Ae. j. japonicus and Ae. triseriatus (see Alto 2011; Hardstone
and Andreadis 2012) and density effects are related to leaf ration (Leonard and Juliano 1995) .
Hardstone and Andreadis (2012) postulated that larval density may affect Ae. triseriatus growth to
a greater extent than Ae. j. japonicus, but this was using highly nutritious brewers yeast. This may
not be the case when rearing with purely leaf detritus (Alto 2011). It is possible that in this study
Ae. triseriatus larvae were able to accelerate their development time to that of the natural rate for
Ae. j. japonicus due to the leaf ration and lack of interspecific competition. This does not explain
why Ae. j. japonicus larvae did not also develop faster, even though faster larval development
times for Ae. j. japonicus have been previously reported by Hardstone and Andreadis (2012).
Larval developmental acceleration can be caused by both nutritional abundance and nutritional
stress at particular temperatures. Reiskind and Zarrabi (2012) found that female Aedes albopictus
were smaller and developed faster when raised at a comparable temperature to this study (24 ◦ C)
and at a lower food ration. The food source used by Reiskind and Zarrabi (2012), even at lower
levels, most likely had a higher nutrient content than leaf detritus. It is possible that larvae in
this study were nutritionally stressed, resulting in a smaller body size and a faster development
time. On the other hand, the growth of mosquitoes has been postulated to be associated with

24

an optimal weight at pupation, or pupation window, that is related to available leaf ration and
temperature through time (Gilpin and McClelland 1979; Carpenter 1984; Walker et al. 1997).
Generally, an increase in leaf ration results in a decrease in development time and a higher optimal
pupation weight (Carpenter 1984; Walker et al. 1997). Walker et al. (1997) postulated that higher
temperatures could increase development time and lower the optimal pupation weight, decreasing
the effect of a high leaf ration. The per larva leaf ration in this study was about 70% more than the
what was used by Walker et al. (1997) but females of both species emerged at a smaller size and Ae.
triseriatus development was accelerated. In fact, Ae. triseriatus was growing as fast as previously
reported with higher leaf rations and similar rearing temperature (Alto 2011). It is unlikely that
leaf ration and rearing temperature alone can explain the accelerated growth observed in this study
and other factors will have to be explored.
It was clear through N analysis that larvae were feeding on leaf surface microbes; however,
leaf decay rates were not increased in larval nanocosms when compared to those without larvae.
Increases in leaf decay rates have been previously associated with multiple larvae grazing on leaf
surface microbial biomass (Fish and Carpenter 1982; Kaufman and Walker 2006); however, a
single larva appears not to have the ability to affect overall leaf decay. Larval effects on leaf
surface N was similar between species, indicating that overall larval feeding on microbial biomass
may have been similar. There were also no differences in total or dissolved N through time in
E1 larval nanocosms; however, higher water column N was observed in nanocosms that produced
female mosquitoes. This may have been caused by gender differences in nutrient accumulation
(Chambers and Klowden 1990). This gender effect could not be established in E1 larvae because
mosquitoes can only be definitively sexed at the pupal or adult stage. The higher total and dissolved
N in female Ae. j. japonicus nanocosms also suggests that, overall, Ae. j. japonicus females were
not feeding on water column microbes as much as Ae. triseriatus females (Kaufman et al. 2001),
even though both emerged at the same time and at a similar size. The efficient utilization of N can
be associated with weight gain in certain mosquitoes (Winters and Yee 2012) and although females
appeared to have a lower N requirement in this study, the lack of size dimorphism between males

25

and females suggests that there was some other factor keeping females small.
The similar size between males and females produced from the nanocosms was surprising.
Male Ae. j. japonicus and Ae. triseriatus weights were similar to previous multi-larvae studies,
but female weight was considerably lower (Walker et al. 1997; Lorenz 2012). The availability
of nutrients in microbial biomass for mosquito larvae limits the development time and body size
of emerging adults (Kaufman et al. 2002). The concentration of N increased in the larval tissues
of both species through time, eventually reaching levels found in adults, indicating a constant
feeding on microbial biomass. The ratio of C:N in larval tissues decreased through time, perhaps
indicating a differential metabolism of proteins and lipids as larvae neared pupation. Chambers
and Klowden (1990) showed that mosquito larvae require a critical level of carbohydrates and
lipids before pupation is initiated and variations in this critical level can depend on gender and
rearing temperature. Furthermore, Klowden and Chambers (1992) found that these reserves can
vary between mosquito species. It is also possible that the availability of quality C compounds
in larval food resources was diminishing as larvae harvested microbial biomass (Kaufman et al.
2001). A declining C:N ratio would be apparent, for example, if accumulation of lipids did not
match the accumulation of proteins in larval tissues. The accumulation of C was also different
between species even though body size was similar. Hood-Nowotny et al. (2012) found that the
concentration of N is more related to body size than concentration of C in the malaria mosquito
Anopheles arabiensis and that fatty acid profiles can be different between males and females. The
fatty acid reserves in Ae. j. japonicus and Ae. triseriatus females may be different, accounting for
different developmental strategies for nutrient accumulation and weight gain.
It is also possible that female larvae require fatty acids and other compounds released by
recently deceased larvae in container habitats. The survival rates of larvae in nanocosms, taken as
a whole, were higher for Ae. triseriatus, but survivorship for both species were at levels previously
seen in microcosms with low larval density and high nutrient sources (Alto 2011; Hardstone and
Andreadis 2012). Indeed, the survival of Ae. triseriatus was higher than in studies using highly
nutritious brewer’s yeast (Hardstone and Andreadis 2012). This measurement of mortality only

26

reveals individual larval success within each nanocosm because larvae cannot interact with each
other. In studies with multiple larvae per container, mortality can decrease the competition for
food resources and the decaying carcasses of deceased larvae could act as an additional nutrient
resource for microorganisms and larvae (Yee et al. 2007b). This lack of differentiation of size has
been previously reported in both Ae. albopictus and Culex restuans reared in low larval density
microcosms on a similar leaf per larva ration as in these nanocosms, (Winters and Yee 2012).
Winters and Yee (2012) also showed that the addition of an invertebrate carcass increased both
adult weight the dimorphism between males and females within each species. Decaying larvae
may be important to Ae. j. japonicus and Ae. triseriatus female weight gain.
Adult Ae. triseriatus were slightly larger than Ae. j. japonicus in this study even though
statistical significance was not established. Taken with the similar development time for both
species, this may indicate that under these conditions Ae. triseriatus larvae may have been slightly
more efficient at converting food into body weight. The correlation between larval efficiency and
leaf decay has been postulated by Carpenter (1984) but the relationship between microbial quality
and larval food assimilation will have to be studied further.
The results of this study indicated that there is a differential utilization of N in the larval
environment between Ae. j. japonicus and Ae. triseriatus but the faster development of Ae. j.
japonicus previously reported was not replicated in this study and may or may not be related to
N accumulation; therefore, the utilization of N by Ae. j. japonicus may not explain much of its
observed displacement of Ae. triseriatus in some larval habitats. Further multi-larval studies will
need to be performed to replicate the findings in nanocosms and elucidate any factors specific to
nanocosm rearing that may have affected larval development. If larvae of Ae. j. japonicus are
replacing those of Ae. triseriatus in tire habitats (Andreadis and Wolfe 2010) and the utilization of
available nutrients by Ae. j. japonicus is not a factor in this phenomenon, then other characteristics
of Ae. j. japonicus such as early season egg hatch rates and oviposition behavior should be
examined. Additionally, research will be needed to explore the consequences of adults emerging
with lower N than conspecifics. This should include studies on the physiology and behavior of

27

specific larval nutrient utilization such as protein accumulation and body amino acid composition.
The establishment of Ae. j. japonicus throughout the US can be considered permanent and it
will likely change the ecology of existing disease vectors. A better understanding of the larval
physiology of this invasive species will be important in future studies of mosquito borne disease
dynamics in many locales.

28

CHAPTER 3
THE EFFECT OF NITROGEN ON AEDES JAPONICUS JAPONICUS (THEOBALD)
AND AEDES TRISERIATUS (SAY) GROWTH IN MICROCOSMS

Introduction
The invasion of non-native mosquitoes into the US has both ecological and public health implications
(Juliano and Lounibos 2005). Successful biological invaders must be able to utilize the resources
available to them in their non-native range to integrate with the local inhabitants. Invasive mosquitoes
can utilize common habitats with both native and other invasive species. Competition between
the larvae of these mosquitoes for limited nutritional resources can impact both development and
survival (Carpenter 1983; Alto 2011; Hardstone and Andreadis 2012).
The invasive species Aedes japonicus japonicus (Theobald) has been expanding its range in the
US since its introduction in the mid-1990’s (Peyton et al. 1999; Morris et al. 2007; Hughes et al.
2008; Neitzel et al. 2009; Gaspar et al. 2012) and appears to be displacing the native mosquito
Aedes triseriatus (Say) in discarded tire habitats (Andreadis and Wolfe 2010). The success of Ae.
j. japonicus can be attributed to a faster development time to a comparable sized female than Ae.
triseriatus (Alto 2011). The growth rate of Ae. j. japonicus females is not significantly affected by
direct competition with Ae. triseriatus (Alto 2011; Hardstone and Andreadis 2012). This may be
related to nutrient utilization, which was shown to be different between Ae. j. japonicus and Ae.
triseriatus (Ch. 2); however, single larva rearing of both species did not produce the differences in
development time, and females were much smaller than expected (Ch. 2).
The nutrients in mosquito larval habitats are provided by allochthonous particulate detrital
inputs including leaves and invertebrate carcasses (Fish and Carpenter 1982; Daugherty et al.
2000) and soluble nutrient inputs from stemflow and rain (Carpenter 1982b; Kaufman et al. 2002;
Kaufman and Walker 2006). In most situations, larvae coexist with many conspecifics in container
habitats and mortality is common, reducing resource competition between survivors. Decaying

29

larval carcasses can also provide another source of nutrients in containers. All of these inputs
can be broken down by fungi and other microbes and made available to mosquitoes through their
consumption of microbial biomass (Fish and Carpenter 1982). The growth of these microorganisms
is generally limited by the availability of N (Kaufman and Walker 2006). Mosquito growth can
be limited by the availability of this high quality microbial biomass; therefore, larvae tend to be
indirectly limited by the availability of N.
The addition of soluble nitrate has also been shown to increase both decay and microbial
productivity in microcosms (Kaufman et al. 2002; Kaufman and Walker 2006). This increase in
microbial activity also been shown to increase Ae. triseriatus larval growth (Kaufman and Walker
2006). A survey of newly emerged Ae. triseriatus and Ae. j. japonicus adults from discarded tires
on the campus of Michigan State University (MSU) found that body %N content of Ae. j. japonicus
females was lower than that of Ae. triseriatus females (Kaufman, unpublished data). This has also
been corroborated in laboratory nanocosms (Ch. 2). This suggests that Ae. j. japonicus may be
less limited by N availability than Ae. triseriatus.
Single and multi-larval rearing may have some fundamental differences that can affect larval
growth. The lack of developmental differences between Ae. j. japonicus and Ae. triseriatus in
single larval nanocosms in the previous chapter may have been caused by the lack of interaction
with conspecifics or N compounds provided by deceased larvae. It is possible that in a multi-larvae
situation, the previously seen developmental differences (Alto 2011; Hardstone and Andreadis
2012) could be replicated.
The purpose of this study was to attempt to corroborate the reported developmental differences
between Ae. j. japonicus and Ae. triseriatus in experimental microcosms and elucidate abnormalities
seen in nanocosm rearing and to test the effect of additional N on the growth and survival of Ae.
j. japonicus and Ae. triseriatus in microcosms. It was expected that the interaction and mortality
of conspecifics in microcosms would cause the accelerated Ae. j. japonicus growth and reduce
the development time of Ae. triseriatus seen in nanocosms and increase female weight above that
of the males. Furthermore, if the development time was different between species in microcosms,

30

then the addition of N was expected to increase microbial activity and because it is less limited
by the availability of N, Ae. j. japonicus development would respond less to it than that of Ae.
triseriatus.

31

Methods
Microcosms were used as experimental units to test the effect of multi-larval rearing and additional
N on Ae. j. japonicus and Ae. triseriatus larval growth. Microcosms were housed in plastic 500
ml cups with aerated tops and filled with 300 ml of deionized water, 1 g of oven dried white oak
(Quercus alba) leaves (Kellogg Forest, Augusta, MI) and 1000 µl of microbial inoculum collected
from tree holes in the Toumey woodlot (MSU, East Lansing, MI). Each microcosm was incubated
for 24 h at 24 ◦ C to facilitate microbial colonization of the leaves before larval addition.

Mosquito Larvae
New Jersey strain Ae. j. japonicus (Rutgers University, New Brunswick, NJ) and MSU strain
Ae. triseriatus eggs were hatched by flooding in a nutrient broth. The eggs of Ae. triseriatus
required an additional 24 h soaking period in deionized water prior to flooding with nutrient broth.
After approx. 9 h, newly hatched larvae of each species were removed from the broth using a
glass Pasteur pipette and transferred to a plastic Petri dish for counting. Each microcosm (N=32)
received 30 newly emerged larvae of either Ae. j. japonicus or Ae. triseriatus.

Nitrogen Addition
To test the effect of N to larval growth, the equivalent of 5 ppm KNO3 -N (the amount of N
provided by a stem flow event, see Kaufman and Walker (2006)) was added on d 4, 10, and 15
to experimental microcosms (N=8:8). An equivalent amount of deionized water was added to
control microcosms (N=8:8) to account for dilution by N addition. Microcosms were incubated at
24 ◦ C with a 12:12 light cycle and checked daily for adults.

Sampling
Adults were collected from microcosms using an aspirator and separated by gender and species.
Individual adults were transferred to plastic tubes and oven dried at 50 ◦ C for at least 48 h. Dried

32

samples were weighed to the nearest .001 mg. The experiment was ended after <3 adults were
emerging per day from all microcosms (d=33). Leaf samples were oven dried at 50 ◦ C and weighed
to the nearest .001 g. Any surviving larvae or pupae were collected, counted, and dried at 50 ◦ C.

Carbon and Nitrogen Analysis
Randomly selected adult females from each group were added to individual tin cups for C/N
elemental combustion analysis using an elemental analyzer (Costech Inc., Valencia, CA) at Kellogg
Biological Field Station (Hickory Corners, MI) (n=50 for all groups except Ae. triseriatus with
additional N, n=37 due to low female emergence).

Statistical Analysis
Multivariate analysis of variance (MANOVA) was used to analyze mosquito growth parameters
between species and N addition. Individual mosquito growth parameters were individually analyzed
using univariate analysis of variance (ANOVA). Overall growth parameters were total survival
(adults produced plus larvae remaining), total adult weight and total number of adults produced.
Average male and female weight, adults produced, and development time were also analyzed.
All related p-values were Bonferroni adjusted to control for Type 1 error. One-way ANOVA was
performed to analyze total leaf mass loss with and without N addition. Two-way ANOVA was
used to analyze body N concentration between adult female Ae. j. japonicus and Ae. triseriatus
and between the control and N addition treatments. If necessary, data was transformed to meet
parametric assumptions of normality and homoscedasticity and all proportional measurements
were arcsine square-root transformed. All reported data are untransformed.

33

Results
Overall mosquito parameters were significantly different between species and were significantly
different between control and nitrogen treatments in MANOVA analysis (Table 3.1). Total mosquito
survival was significantly different between N and control treatments (Table 3.2), with a lower
overall survival in N added microcosms (Fig 3.1) with no significant difference between species
(Table 3.2). The total number of adults produced was significantly different between species (Table
3.2) with Ae. j. japonicus microcosms producing more adults (Figure 3.1), and fewer adults were
produced by both species overall in N treated microcosms (Fig 3.1). The total amount of adult
biomass produced was also significantly lower in microcosms with added N (Fig 3.1, Table 3.1).
Fewer females were produced from microcosms with additional N (Fig 3.2 , Table 3.3). There
was also an indication that fewer Ae. triseriatus females were produced than Ae. j. japonicus
females in microcosms with additional N though significance was not upheld with Bonferroni
adjustment (Fig 3.2). Neither average female weight nor average female development time was
affected by N addition and did not vary significantly between species (Fig 3.2, Table 3.3). Male
development was not significantly affected by the addition of N (Fig 3.3, Table 3.4), but male Ae.
triseriatus were significantly larger than Ae. j. japonicus males (Fig 3.3, Table 3.4).
The %N in adult female Ae. j. japonicus was lower than in Ae. triseriatus females but was
not affected by N addition (Fig 3.4, Table 3.5). Overall female %C and C:N ratios were not
significantly different between species or treatments (Fig 3.4, Table 3.5).
There was no difference between decay of leaf matter between mosquito species (Fig 3.5, Table
3.6), but leaf decay was increased in microcosms with additional N (Fig 3.5, Table 3.6).

34

Table 3.1: MANOVA analysis of overall mosquito growth parameters. Variates were survival,
total adult biomass, total number of adults produced, average female weight, number of females
produced, average female development time.
MANOVA
Source
Species
Nitrogen
Species X Nitrogen
Error

Roy’s max root
2.94
1.24
0.28

35

df
1
1
1
28

P
<0.0001
0.003
0.404

Table 3.2: ANOVA of total microcosm production. * = significant with Bonferroni adjustment, α
= 0.05.
No. of
adults

Survival
Source
Species
Nitrogen
Species X Nitrogen

df
F
1 0.008
1 11.55
1 2.16

P
0.93
0.0002*
0.15

F
9.87
21.94
1.03

P
0.004*
<0.0001*
0.32

Tot adult
bimass
F
P
0.19
0.67
12.86 0.001*
2.49
0.13

30
20
10

Ae. j. japonicus
Ae. triseriatus

0

individuals/ microcosm

Survival

Control

N

4
2
0

5

15

mg/ microcosm

6

25

Total Adult Biomass

0

number/ microcosm

Total Number of Adults

Control

N

Control

N

Figure 3.1: Total microcosm production. Values are means ± SE (n=8).

36

Table 3.3: ANOVA of female growth production. * = significant with Bonferroni adjustment, α =
0.05.

Source
Species
Nitrogen
Species X Nitrogen

No. of
females
df
F
P
1 5.28
0.029
1 19.54 0.0001*
1 2.92
0.098

Avg. female Avg. female
wt
devel. time
F
P
F
P
7.86 0.009 6.15 0.019
0.524 0.48 0.61 0.44
0.20 0.66 0.56 0.46

12
4

8

Ae. j. japonicus
Ae. triseriatus

0

number/ microcosm

Females Produced

Control

N

Female Development Time
30
20
0

10

days

0.4
0.2
0.0

mg/ individual

0.6

Female Weight

Control

N

Control

N

Figure 3.2: Female growth parameters. Values are means ± SE (n=8).

37

Table 3.4: ANOVA of male growth production. * = significant with Bonferroni adjustment, α =
0.05.
No. of
males
Source
Species
Nitrogen
Species X Nitrogen

df
F
1
7.07
1 <0.0001
1
0.04

P
0.013
0.99
0.82

Avg. male
wt
F
P
11.81 0.002*
0.12
0.73
1.61
0.23

Avg. male
devel. time
F
P
0.19 0.65
0.18 0.67
1.35 0.26

12
4

8

Ae. j. japonicus
Ae. triseriatus

0

individuals/ mirocosm

Males Produced

Control

N

Male Development Time

20
0

10

days

0.4
0.2
0.0

mg/ individual

30

Male Weight

Control

N

Control

Figure 3.3: Male growth parameters. Values are means ± SE (n=8).

38

N

Table 3.5: ANOVA of female nitrogen and carbon. * = significant with Bonferroni adjustment, α
= 0.05.
%N
Source
Species
Nitrogen
Species X Nitrogen

df
F
1 13.33
1 2.70
1 2.14

P
0.001*
0.11
0.15

%C

C:N

F
P
F
P
3.53 0.07 0.21 0.65
0.05 0.82 0.89 0.35
2.14 0.16 0.77 0.39

Figure 3.4: Female nitrogen and carbon. Values are means ± SE (n=8).

39

Table 3.6: ANOVA analysis of leaf decay.
Source
Species
Nitrogen
Species X Nitrogen

df
1
1
1

F
0.007
13.78
0.013

P
0.935
0.0009
0.909

Figure 3.5: Remaining leaf mass in microcosms. Values are means ± SE (n=8).

40

Discussion
The developmental differences previously seen between Ae. j. japonicus and Ae. triseriatus
(Ingrassia 2006; Alto 2011; Hardstone and Andreadis 2012; Lorenz 2012) were not replicated
in microcosms as expected. In control and treatment microcosms there was no difference in
development time between Ae. j. japonicus and Ae. triseriatus and development times were
similar to what was found in nanocosms (Ch. 2); however, female weights were higher than what
was seen in nanocosms and females were larger than males. There were very few differences
between the results in this study and those from nanocosms, even though microcosms did have a
lower leaf ration per larva and had more larvae present. Survival was relatively high in microcosms
and did not differ between species; however, it is possible that the competitive interactions between
larvae and the availability of particular nutrients provided by any deceased conspecifics increased
female biomass production (Yee et al. 2007b).
Surprisingly, and in contrast to previous studies (Kaufman and Walker 2006, M. G. Kaufman
unpublished data) the addition of nitrate had an overall negative effect on Ae. j. japonicus
and Ae. triseriatus growth. Fewer adults were produced from N added microcosms than in
the control microcosms and the adults produced also had longer development times and were
generally smaller. Control microcosms in this study were also relatively more productive than
in similar studies with additional N (Kaufman et al. 2002; Kaufman and Walker 2006), which
was highly unexpected. Leaf decay was enhanced by the addition of nitrate, as seen in previous
studies (Kaufman et al. 2002; Kaufman and Walker 2006), and it was apparent that leaf-associated
decay microorganisms were stimulated as predicted. In this study microbial stimulation may have
elevated compounds or microorganisms that were inhibitory to growth. Although unexpected,
the inhibition of mosquito larvae by the growth of microorganisms, particularly fungi, is not
unprecedented (Washburn et al. 1988; Yee et al. 2007a; R. Morningstar unpublished data).
Observations of the surfaces of leaves used for microcosms and nanocosms revealed numerous
unidentified fungal colonies. These fungi were present on leaf surfaces prior to their addition into
microcosms and could represent leaf pathogens that were present in high abundance in the field.
41

Fungal biomass, as measured by the concentration of the compound ergosterol has been shown
to increase with dissolved N addition (Kaufman and Walker 2006). An exploratory analysis of
ergosterol in additional samples of these fungal colonized leaves without larvae showed that initial
ergosterol levels were at least double what has been previously seen on leaves from the same source
(M. Lundquist, unpublished data; M. G. Kaufman personal correspondence). Fungi are readily fed
on (Fish and Carpenter 1982) and the presence of this additional fungal source may have increased
total amount of available nutrients for larvae in both microcosms and nanocosms. It is possible
that access to this additional fungal food source allowed the growth acceleration of otherwise more
nutrient limited Ae. triseriatus larvae.
Counter-intuitively, this fungus found on the leaf surfaces may have also been an inhibitory
factor on mosquito production. The spores of some species of fungi, including members of the
genus Aspergillus, have been associated with a dose dependent decrease in the survival of early
instar Ae. triseriatus and Ae. j. japonicus larvae (R. Morningstar and M. G. Kaufman unpublished
data). Other genera of fungi have been utilized as commercial larvicides of Anopheles sp. for
malaria control (Scholte et al. 2005). Overall larval survival was lower in microcosms with
additional N; however, the production of adult males was not affected by N addition. Males
emerged between the addition of N on d 10 and d 15; therefore, male larvae would only have
been affected by the first N addition. The later N additions may have stimulated the proliferation
of fungal spores, moving the fungus from a beneficial state to a pathogenic state, which is not
unprecedented (see Washburn et al. 1988; R. Morningstar, personal correspondence). This would
have increased larval exposure and negatively impacted the slower developing female larvae.
Experiments with Aspergillus niger have also found that pathogenetic effects tended to differ
between species, with Ae. j. japonicus larvae tending to be more resilient to pathogenesis (R.
Morningstar and M. G. Kaufman unpublished data). It is likely that the fungus found on the leaf
surfaces was not Aspergillus niger (R. Morningstar, personal correspondence); however, Ae. j.
japonicus female production was slightly higher than that of Ae. triseriatus in this study and this
may indicate that Ae. j. japonicus is more resilient to the pathogenic effects of this fungus.

42

Although the experimental manipulation of N effects were obfuscated by the presumed fungal
related inhibition of growth, adult Ae. j. japonicus females did emerge with a lower concentration
of N in their tissues compared to Ae. triseriatus females in both control microcosms and microcosms
with added N. This follows what has been found in the field (M. G. Kaufman unpublished data)
and in the laboratory without N addition in nanocosms (Ch. 2). The concentration of C, which was
lower in Ae. j. japonicus than Ae. triseriatus in nanocosms (Ch. 2), was the same between species
in both control and treatment microcosms. This more closely resembles what is expected if Ae. j.
japonicus is more N efficient, which is that Ae. j. japonicus requires less N to produce the same
amount of biomass as Ae. triseriatus (Fagan et al. 2002). The difference in %C seen in nanocosms
(Ch. 2) may have been caused by lack of nutrients provided by decaying larvae or as previously
postulated, that Ae. triseriatus respiration is different than that of Ae. j. japonicus.
The lack of growth differences between species in both this study and in nanocosms (Ch.
2) may have been due to the lack of nutritional stress caused by the presence of the unknown
leaf fungus. The difference in %N between species indicates that Ae. j. japonicus has a lower
N requirement for development (Fagan et al. 2002) but differences in development between the
species in this study were ameliorated by a higher quality food source present in the leaf material.
The addition of soluble nutrients has been shown to increase both microbial abundance and mosquito
development (Carpenter 1982b; Kaufman et al. 2002; Kaufman and Walker 2006; Pelz-Stelinski
et al. 2010); however, the effect that these nutrients have on inhibitory and pathogenetic organisms
is relatively unknown but has potentially been demonstrated in this study.
This experiment suggests that there are fundamental processes that are influential in female
larval development in multi-larval situations that are absent when larvae are reared individually. It
is also apparent that Ae. j. japonicus does have some tolerance for both N limitation and perhaps
microbial pathogenesis but these need to be explored more extensively in future research. The
expansion of Ae. j. japonicus is likely caused by a number of factors that allow it be successful.
A greater understanding of the ecology of its invasion will be important in the future dynamics of
arbovirus transmission in the US.

43

CHAPTER 4
GENERAL CONCLUSIONS

Through these studies, it is still unclear how Aedes japonicus japonicus (Theobald) is potentially
reducing Aedes triseriatus (Say) populations in tire habitats in the US; however, what is clear
is that Ae. j. japonicus does process nitrogen differently. Female larvae of Ae. j. japonicus
tended to reduce non-particulate nitrogen compounds to a lesser extent than Ae. triseriatus in
their habitats. When they reach adulthood, Ae. j. japonicus females had a lower %N in their
bodies. This phenomenon has now been observed in the field and in both laboratory nanocosms
and microcosms.
Through these experiments, the lower N accumulation by Ae. j. japonicus could not be
adequately coupled with the expected faster larval development of that species over Ae. triseriatus.
Both the nanocosm and microcosm experiments were initially designed to nutritionally stress
larvae to the point that developmental differences would be more pronounced. Indeed, in both
habitats, adults of both species emerged at the same time and in nanocosms, males and females
were similarly sized at emergence. This lack of larval developmental stress may have been caused
by the inadvertent introduction of a nutritious fungus on the leaf surfaces. This additional food
source may have allowed Ae. triseriatus to develop just as fast as Ae. j. japonicus, which
apparently could not take full advantage of this resource. This fungus may also have acted as a
larval pathogen in microcosms where additional N was added, and may have affected Ae. triseriatus
to a greater extent. These types of organisms may play an important role in the dynamics of larval
populations in natural containers. If Ae. j. japonicus is more pathogen resistant than the native
species, it may increase larvae survival in containers.
The differences in adult female sizes between nanocosm and microcosm experiments were
also unexpected. A possible reason for the smaller sized females produced from nanocosms
may be the lack of certain factors only present in multi-larvae rearing situations. These may
include interactions with conspecifics or the availability of certain essential nutrients released from

44

decaying larval carcasses that are ubiquitous in containers due to larval mortality.
The results from this thesis have revealed a possible interaction between fungal availability and
larval development rates that was unexpected, and the initial question of how Ae. j. japonicus
develops faster than Ae. triseriatus still remains to be addressed. In future studies, more controlled
larval assays with leaves clear of confounding fungi or other organisms, or the addition of these
organisms, could be performed to more closely examine the differences in nutritional reserves in
connection to previously reported differential development times between Ae. j. japonicus and Ae.
triseriatus.

Future Directions
There are a number of potential avenues for further study of the interactions between Ae. j.
japonicus and Ae. triseriatus and the effects of fungi and other nutrient sources on larval development.
There are also many other possible explanations for the expansion of Ae. j. japonicus throughout
the eastern US. While not an exhaustive list, some possible directions for further study into this
particular system and mosquito ecology in general are provided below.

Single vs. multi-larvae rearing
The development of mosquito larvae, especially female larvae, was different when reared individually
in nanocosms than when reared in groups within microcosms. The major differences between
nanocosm and microcosm rearing are that in nanocosms, there is no resource competition between
larvae and there is no additional microbial decay of deceased larvae. The impacts of decaying
carcasses have been previously studied in mosquito microcosms and have been found to generally
enhance larval growth (Daugherty et al. 2000; Harshaw et al. 2007; Yee et al. 2007a,b); therefore,
a test of the effect of larval carcasses directly on individual larvae in nanocosms may reveal that
compounds released during larval decay are important for proper larval growth, at least for females,
which take longer to develop than males.

45

Larval feeding preferences on microorganisms
The faster development time of reported Ae. j. japonicus females was originally postulated in
this thesis as the mechanism for the displacement of Ae. triseriatus in tire habitats; however, the
development times of these species were similar in both nanocosm and microcosm experiments.
This departure from observations from previous studies may have been caused by the introduction
of a leaf-associated fungus into both nanocosms and microcosms. The presence of this fungus
appeared to increase the growth rate of Ae. triseriatus relative to Ae. j. japonicus, but this
observation still remains to be investigated in the laboratory. Both bacteria and fungi are important
for mosquito growth and recent gnotobiotic work indicates that fungal availability may be essential
to mosquito late instar development (M. G. Kaufman personal correspondence). The feeding of
larvae on fungal biomass can be visualized on leaf surfaces (Fish and Carpenter 1982), but the
florescent marking and quantification of specific fungal and bacterial feeding by larvae may be
more revealing (see Geavgaard et al. 2010). Carbon and nitrogen stable isotope analysis of fungal
and bacterial biomass and mosquito tissues could also be used to track the utilization of particular
microbial nutrient bases by the two species (Kaufman et al. 2010; Winters and Yee 2012). This
approach may be difficult because the marking of specific bacteria and fungi in a heterogeneous
microbial community could be problematic. This could be done by marking specific bacteria and
fungi initially and feeding these microorganisms directly to larvae. It also may be reasonable to
use these methods to track the ingestion and assimilation of these food sources into larval biomass
and determine the growth efficiency of both Ae. j. japonicus and Ae. triseriatus and how this
relates to possible developmental differences between each species.

Partitioning of nitrogen compounds
Female Ae. j. japonicus did accumulate less nitrogen in their tissues in both nanocosm and
microcosm experiments. While this finding cannot be adequately associated with development
time, when paired with the higher water column nitrogen in nanocosms with female Ae. j. japonicus
adults, it indicates some differences in nutritional needs. The measurements of N in both water
46

columns and adults was relatively broad, taking into account every form of nitrogen in a single
measurement. The nitrogen compounds available to larvae can be in the form of microbial biomass
consisting of various nucleic acids, proteins, and amino acids. The availability and quality of
these compounds could be important to larval development and the accumulation of high quality
nitrogen compounds by Ae. j. japonicus may account for a lower N requirement. Additional
research, including the partitioning and tracking of these different nitrogen compounds may help
elucidate the differential accumulation of these compounds by Ae. j. japonicus and Ae. triseriatus.
A potential way to do this is stable isotope analysis of labeled particulate organic matter and
microorganisms (Kaufman et al. 2010; Winters and Yee 2012). Protein analysis could also be
used to determine any differences in amino acid compositions of either species and any potential
differences in amino acid requirements.

Ae. j. japonicus habitat utilization
If the displacement of Ae. triseriatus by Ae. j. japonicus in tire habits can not be explained solely
by faster emergence times caused by differential larval nutrient accumulation, other factors will
need to be explored. It may therefore be important to explore the ability of adult Ae. j. japonicus
to disperse and colonize tires and other containers. For example, if Ae. j. japonicus is able to get
out faster, it may have the ability to access habitats sooner or get out of habitats that are drying
out or becoming unsuitable. This may also be important because the eggs of Ae. j. japonicus have
been observed to be prone to dessication in the laboratory (Williges et al. 2008). Female Ae. j.
japonicus oviposition site choices may therefore be broad to insure that more eggs will survive and
more larvae will develop successfully and larval success may be higher in containers not prone to
dessication (i.e. tires or large buckets; Bartlett-Healy et al. 2012). It is known that Ae. j. japonicus
can be found in multiple types of container habitats (Bartlett-Healy et al. 2012) but only seem to
be displacing native mosquitoes in tires (Andreadis and Wolfe 2010) and in rockpools (Armistead
et al. 2008). It is possible that these artificial habitats provide something that enhances either Ae. j.
japonicus oviposition, egg hatch success, or larval developmental success and these factors should

47

be explored.

Ae. j. japonicus larval physiology
The physiology of Ae. j. japonicus development should also be explored in future work. The
phases of larval and adult development may be timed differently between Ae. j. japonicus and
Ae. triseriatus, perhaps accounting for the difference in nitrogen accumulation. Vitellogensis, for
example, may be delayed in Ae. j. japonicus females until after acquiring additional nitrogen
from their first blood meal. The fecundity has been shown to be similar in Ae. j. japonicus and
Ae. triseriatus in the field (Oliver and Howard 2005). This may suggest that fecundity of Ae. j.
japonicus females is affected by nutrient accumulation during the adult stage. The comparison of
Ae. j. japonicus nutrient reserves and fecundity to Ae. triseriatus as well as autogenous and other
anautogenous mosquito species would be highly useful as both nitrogen and carbon stores have
been shown to differ between both types (see Telang et al. 2006). The difficulty of rearing Ae. j.
japonicus in laboratory colonies may render direct comparisons infeasible; however, the collection
and nutritional analysis of newly emerged Ae. j. japonicus and other mosquitoes from the same
containers in the field, coupled with live blood feeding and fecundity analysis could be used to
elucidate any differences between species.

Ae. j. japonicus natural enemies
The invasion of any animal can be attributed to many factors. It is common for scientists to search
for predators or other natural enemies for the control of invasive insects. The natural enemies
of mosquitoes can include predatory larva (i.e. Toxorhynchites sp.; Kesavaraju et al. 2011) and
intracellular parasites like gregarines (reviwed by Juliano and Lounibos 2005 in Ae. albopictus). A
recent study of the gregarine Ascogregarina taiwanensis which is a natural enemy of Ae. albopictus
has been shown to be infective to Ae. j. japonicus but the effects on larval survival were not
explored (Erthal et al. 2012). It is possible that there are natural enemies of Ae. j. japonicus or
closely related mosquito species that could be used to control larval populations. A further study
48

into the effects of gegarines and other parasites in Ae. j. japonicus may be a reasonable starting
point to see if there are mosquito pathogens that will negatively affect the success of the species.

49

APPENDIX

50

RECORD OF DEPOSITION OF VOUCHER SPECIMENS

The specimens listed below have been deposited in the named museum as samples of those species
or other taxa, which were used in this research. Voucher recognition labels bearing the voucher
number have been attached or included in fluid preserved specimens.

Voucher Number: 2013-05

Author and Title of thesis: Matthew J. Lundquist

THE EFFECT OF NITROGEN ON THE LARVAL GROWTH OF THE INVASIVE MOSQUITO
AEDES JAPONICUS JAPONICUS (THEOBALD)

Museum(s) where deposited:
Albert J. Cook Arthropod Research Collection, Michigan State University (MSU) Specimens:

Table A.1: Catalog of preserved specimens
Family
Culicidae
Culicidae
Culicidae
Culicidae
Culicidae
Culicidae

Genus-Species
Aedes japonicus japonicus ♂
Aedes japonicus japonicus ♀
Aedes japonicus japonicus
Aedes triseriatus ♂
Aedes triseriatus ♀
Aedes triseriatus

Life Stage
adult
adult
larva
adult
adult
larva

51

Quantity
10
10
10
10
10
10

Preservation
pinned
pinned
alcohol
pinned
pinned
alcohol

LITERATURE CITED

52

LITERATURE CITED
Alto, B. W. (2011). Interspecific larval competition between invasive Aedes japonicus and native
Aedes triseriatus (Diptera: Culicidae) and adult longevity. Journal of Medical Entomology,
48(2):232–242.
Andreadis, T. G., Anderson, J. F., Munstermann, L. E., Wolfe, R. J., and Florin, D. A.
(2001). Discovery, distribution, and abundance of the newly introduced mosquito Ochlerotatus
japonicus (Diptera: Culicidae) in Connecticut, USA. Journal of Medical Entomology,
38(6):774–779.
Andreadis, T. G. and Wolfe, B. J. (2010). Evidence for reduction of native mosquitoes with
increased expansion of invasive Ochlerotatus japonicus japonicus (Diptera: Culicidae) in the
Northeastern United States. Journal of Medical Entomology, 47(1):43–52.
Armistead, J. S., Nishimura, N., Escher, R. L., and Lounibos, L. P. (2008). Larval competition
between Aedes japonicus and Aedes atropalpus (Diptera: Culicidae) in simulated rock pools.
Journal of Vector Ecology, 33(2):238–246.
Bartlett-Healy, K., Unlu, I., Obenauer, P., Hughes, T., Healy, S., Crepeau, T., Farajollahi, A.,
Kesavaraju, B., Fonseca, D., Schoeler, G., Gaugler, R., and Strickman, D. (2012). Larval
mosquito habitat utilization and community dynamics of Aedes albopictus and Aedes japonicus
(Diptera: Culicidae). Journal of Medical Entomology, 49(4):813–824.
Beaty, B., Rayms-Keller, A., Borucki, M., and Blair, C. (2000). LaCrosse encephalitis virus and
mosquitoes: a remarkable relationship. ASM News, 66(6):349–357.
Beehler, J., Lohr, S., and Defoliart, G. (1992). Factors influencing oviposition in Aedes triseraitus
(Diptera, Culicidae). Great Lakes Entomologist, 25(4):259–264.
Carpenter, S. R. (1982a). Comparisons of equations for decay of leaf litter in tree-hole ecosystems.
Oikos, pages 17–22.
Carpenter, S. R. (1982b). Stemflow chemistry: Effects on population dynamics of detritivorous
mosquitoes in tree-hole ecosystems. Oecologia, 53(1):1–6.
Carpenter, S. R. (1983). Resource limitation of larval treehole mosquitoes subsisting on beech
detritus. Ecology, 64(2):219–223.
Carpenter, S. R. (1984). Experimental test of the pupation window model for development of
detritivorous insects. Ecological Modelling, 23(3):257–264.
(CDC) Centers for Disease Control and Prevention (2000). Update: West Nile virus activity —
Eastern United States, 2000. MMWR, 49:1044–1047.
Chambers, G. and Klowden, M. (1990). Correlation of nutritional reserves with a critical weight
for pupation in larval Aedes aegypti mosquitoes. Journal of the American Mosquito Control
Association, 6(3):394–399.

53

Crumpton, W. G., Isenhart, T. M., and Mitchell, P. D. (1992). Nitrate and organic N analyses with
second-derivative spectroscopy. Limnology and Oceanography, 37(4):907–913.
Daugherty, M., Alto, B., and Juliano, S. (2000). Invertebrate carcasses as a resource for competing
Aedes albopictus and Aedes aegypti (Diptera : Culicidae). Journal of Medical Entomology,
37(3):364–372.
Davidowitz, G., D’Amico, L. J., and Nijhout, H. F. (2003). Critical weight in the development of
insect body size. Evolution & Development, 5(2):188–197.
Dodson, B. L., Kramer, L. D., Rasgon, J. L., et al. (2012). Effects of larval rearing temperature on
immature development and West Nile virus vector competence of Culex tarsalis. Parasites &
Vectors, 5(1):1–6.
Erthal, J. A., Soghigian, J., and Livdahl, T. (2012). Life cycle completion of parasite Ascogregarina
taiwanensis (Apicomplexa: Lecudinidae) in non-native host Ochlerotatus japonicus (Diptera:
Culicidae). Journal of Medical Entomology, 49(5):1109–1117.
Fagan, W. F., Siemann, E., Mitter, C., Denno, R. F., Huberty, A. F., Woods, H. A., and Elser, J. J.
(2002). Nitrogen in insects: implications for trophic complexity and species diversification. The
American Naturalist, 160(6):784–802.
Fish, D. and Carpenter, S. R. (1982). Leaf litter and larval mosquito dynamics in tree-hole
ecosystems. Ecology, pages 283–288.
Gaspar, J. P., Mckay, T., and Huss, M. J. (2012). First report of Aedes japonicus japonicus in
natural and artificial habitats in Northeastern Arkansas. Journal of the American Mosquito
Control Association, 28(1):38–42.
Geavgaard, J. C., Turnbull, M. W., and McNealy, T. L. (2010). A novel technique for feeding and
confirming uptake of bacteria in larvae of the southern house mosquito Culex qunquefasciatus
(Diptera: Culicidae). Florida Entomologist, 93(4):577–583.
Gilpin, M. E. and McClelland, G. (1979). Systems analysis of the yellow fever mosquito Aedes
aegypti. Fortschritte der Zoologie, 25.
Güsewell, S. and Gessner, M. O. (2009). N: P ratios influence litter decomposition and colonization
by fungi and bacteria in microcosms. Functional Ecology, 23(1):211–219.
Hardstone, M. C. and Andreadis, T. G. (2012). Weak larval competition between the invasive
mosquito Aedes japonicus japonicus (Diptera: Culicidae) and three resident container-inhabiting
mosquitoes in the laboratory. Journal of Medical Entomology, 49(2):277–285.
Harshaw, L., Chrisawn, C., Kittinger, B., Carlson, J., Metz, G., Smith, L., and Paradise,
C. J. (2007). Decaying invertebrate carcasses increase growth of Aedes triseriatus (Diptera
: Culicidae) when leaf litter resources are limiting. Journal of Medical Entomology,
44(4):589–596.

54

Hood-Nowotny, R., Schwarzinger, B., Schwarzinger, C., Soliban, S., Madakacherry, O., Aigner,
M., Watzka, M., and Gilles, J. (2012). An analysis of diet quality, how it controls fatty acid
profiles, isotope signatures and stoichiometry in the malaria mosquito Anopheles arabiensis.
PloS One, 7(10):e45222.
Howarth, R. W. and Fisher, S. G. (1976). Carbon, nitrogen, and phosphorus dynamics during leaf
decay in nutrient-enriched stream microecosystems. Freshwater Biology, 6(3):221–228.
Hughes, T. H., Irwin, P. M., Kaufman, A., Sage, H., Pagac, Jr., B. B., and Paskewitz, S. M. (2008).
First records of Aedes japonicus japonicus in Wisconsin. Journal of the American Mosquito
Control Association, 24(4):583–584.
Ingrassia, A. (2006). Larval competition between the native treehole mosquito, Ochleotatus
triseriatus, and the invasive mosquito, Ochlerotatus japonicus, using natural diets. Master’s
thesis, Binghamton University.
Irish, S. R. and Pierce, C. S. (2008). Update on the distribution of Ochlerotatus japonicus in Oregon
and Washington. Journal of the American Mosquito Control Association, 24(1):110–111.
Joy, J. E. and Sullivan, S. N. (2005). Occurrence of tire inhabiting mosquito larvae in different
geographic regions of West Virginia. Journal of the American Mosquito Control Association,
21(4):380–386.
Juliano, S. and Lounibos, L. (2005). Ecology of invasive mosquitoes: Effects on resident species
and on human health. Ecology Letters, 8(5):558–574.
Juliano, S. and Stoffregen, T. (1994). Effects of habitat drying on size at and time to metamorphosis
in the tree hole mosquito Aedes triseriatus. Oecologia, 97(3):369–376.
Kaufman, M., Bland, S., Worthen, M., Walker, E., and Klug, M. (2001). Bacterial and fungal
biomass responses to feeding by larval Aedes triseriatus (Diptera : Culicidae). Journal of
Medical Entomology, 38(5):711–719.
Kaufman, M., Goodfriend, W., Kohler-Garrigan, A., Walker, E., and Klug, M. (2002). Soluble
nutrient effects on microbial communities and mosquito production in Ochlerotatus triseriatus
habitats. Aquatic Microbial Biology, 29(1):73–88.
Kaufman, M., Walker, E., Smith, T., Merritt, R., and Klug, M. (1999). Effects of larval mosquitoes
(Aedes triseriatus) and stemflow on microbial community dynamics in container habitats?
Applied and Environmental Microbiology, 65(6):2661–2673.
Kaufman, M. G., Chen, S., and Walker, E. D. (2008). Leaf-associated bacterial and fungal taxa
shifts in response to larvae of the tree hole mosquito, Ochlerotatus triseriatus. Microbial
Ecology, 55(4):673–684.
Kaufman, M. G., Pelz-Stelinski, K. S., Yee, D. A., Juliano, S. A., Ostrom, P. H., and Walker, E. D.
(2010). Stable isotope analysis reveals detrital resource base sources of the tree hole mosquito,
Aedes triseriatus. Ecological Entomology, 35(5):586–593.

55

Kaufman, M. G. and Walker, E. D. (2006). Indirect effects of soluble nitrogen on growth
of Ochlerotatus triseriatus larvae in container habitats. Journal of Medical Entomology,
43(4):677–688.
Kesavaraju, B., Khan, D. F., and Gaugler, R. (2011).
container-dwelling mosquitoes to a native predator.
48(3):526–532.

Behavioral differences of invasive
Journal of Medical Entomology,

Kitching, R. (2001). Food webs in phytotelmata: “Bottom-up” and “top-down” explanations for
community structure. Annual Review of Entomology, 46:729–760.
Klowden, M. J. and Chambers, G. M. (1992). Reproductive and metabolic differences between
Aedes aegypti and Ae. albopictus (Diptera: Culicidae). Journal of Medical Entomology,
29(3):467–471.
Larish, L. B. and Savage, H. M. (2005). Introduction and establishment of Aedes (Finlaya)
japonicus japonicus (Theobald) on the island of Hawaii: implications for arbovirus transmission.
Journal of the American Mosquito Control Association, 21(3):318–321.
Leisnham, P. T. and Juliano, S. A. (2012). Impacts of climate, land use, and biological invasion on
the ecology of immature Aedes mosquitoes: implications for La Crosse emergence. EcoHealth,
9(2):217–228.
Leonard, P. M. and Juliano, S. A. (1995). Effect of leaf litter and density on fitness and population
performance of the hole mosquito Aedes triseriatus. Ecological Entomology, 20(2):125–136.
Lorenz, A. R. (2012). The role of algae in the invasion ecology of the mosquito species Aedes
japonicus japonicus (Diptera: Culicidae). Master’s thesis, Michigan State University.
Lorenz, A. R., Walker, E. D., and Kaufman, M. G. (2013). Does autocthonous primary production
influence oviposition by Aedes japonicus japonicus (Diptera: Culicidae) in container habitats?
Journal of Medical Entomology, 50(1):69–78.
Molaei, G., Farajollahi, A., Scott, J. J., Gaugler, R., and Andreadis, T. G. (2009). Human
bloodfeeding by the recently introduced mosquito, Aedes japonicus japonicus, and public health
implications. Journal of the American Mosquito Control Association, 25(2):210–214.
Morris, J. A., Lampman, R. L., Ballmes, G., Funes, J., Halvorsen, J., and Novak, R. J. (2007). First
record of Aedes japonicus japonicus in Illinois: Defining its spatial distribution and associated
mosquito species. Journal of the American Mosquito Control Association, 23(3):243–251.
Neitzel, D. F., Johnson, K. A., Brogren, S., and Kemperman, M. M. (2009). First collection records
of Aedes japonicus in Minnesota. Journal of the American Mosquito Control Association,
25(3):367–369.
Nijhout, H. (2003). The control of body size in insects. Developmental biology, 261(1):1–9.
O’Donnell, D. and Armbruster, P. (2007). Comparison of larval foraging behavior of Aedes
albopictus and Aedes japonicus (Diptera: Culicidae). Journal of Medical Entomology,
44(6):984–989.
56

Oliver, J. and Howard, J. J. (2005). Fecundity of naturally blood-fed Ochlerotatus japonicus.
Journal of Medical Entomology, 42(3):254–259.
Pelz-Stelinski, K. S., Walker, E. D., and Kaufman, M. G. (2010). Senescent leaf exudate increases
mosquito survival and microbial activity. Ecological Entomology, 35(3):329–340.
Peyton, E., Campbell, S., Candeletti, T., Romanowski, M., and Crans, W. (1999). Aedes(Finlaya)
japonicus japonicus (Theobald), a new introduction into the united states. Journal of the
American Mosquito Control Association, 15(2):238–241.
Ponnusamy, L., Xu, N., Stav, G., Wesson, D. M., Schal, C., and Apperson, C. S. (2008).
Diversity of bacterial communities in container habitats of mosquitoes. Microbial Ecology,
56(4):593–603.
Puntuwat, S., Thomson, W., Watts, D., Yuill, T., and Hanson, R. (1974). Isolation of La Crosse
virus from field collected Aedes triseriatus larvae. American Journal of Tropical Medicine and
Hygiene, 23(2):246–250.
Reiskind, M. H. and Zarrabi, A. A. (2012). Is bigger really bigger? differential responses to
temperature in measures of body size of the mosquito, Aedes albopictus. Journal of Insect
Physiology.
Sardelis, M., Dohm, D., Pagac, B., Andre, R., and Turell, M. (2002a). Experimental transmission
of eastern equine encephalitis virus by Ochlerotatus j. japonicus (Diptera : Culicidae). Journal
of Medical Entomology, 39(3):480–484.
Sardelis, M. and Turell, M. (2001). Ochlerotatus j. japonicus in Frederick County, Maryland:
Discovery, distribution, and vector competence for West Nile virus. Journal of the American
Mosquito Control Association, 17(2):137–141.
Sardelis, M., Turell, M., and Andre, A. (2002b). Laboratory transmission of La Crosse virus by
Ochlerotatus j. japonicus (Diptera : Culicidae). Journal of Medical Entomology, 39(4):635–639.
Sardelis, M. R., Turell, M. J., and Andre, R. G. (2003). Experimental transmission of St. Louis
encephalitis virus by Ochlerotatus j. japonicus. Journal of the American Mosquito Control
Association, 19(2):159–162.
Schaffner, F., Chouin, S., and Guilloteau, J. (2003). First record of Ochlerotatus (Finlaya)
japonicus japonicus (Theobald, 1901) in metropolitan France. Journal of the American
Mosquito Control Association, 19(1):1–5.
Schaffner, F., Kaufmann, C., Hegglin, D., and Mathis, A. (2009). The invasive mosquito Aedes
japonicus in Central Europe. Medical and Veterinary Entomology, 23(4):448–451.
Scholte, E.-J., Ng’habi, K., Kihonda, J., Takken, W., Paaijmans, K., Abdulla, S., Killeen, G. F.,
and Knols, B. G. J. (2005). An entomopathogenic fungus for control of adult African malaria
mosquitoes. Science, 308(5728):1641–1642.
Scott, J. J., Carle, F. L., and Crans, W. J. (2001). Ochlerotatus japonicus collected from natural
rockpools in New Jersey. Journal of the American Mosquito Control Association, 17(2):91–92.
57

Takashima, I. and Rosen, L. (1989). Horizontal and vertical transmission of Japanese encephalitis
virus by Aedes japonicus (Diptera, culicidae). Journal of Medical Entomology, 26(5):454–458.
Telang, A., Li, Y., Noriega, F., and Brown, M. (2006).
Effects of larval nutrition
on the endocrinology of mosquito egg development. Journal of Experimental Biology,
209(4):645–655.
Thielman, A. and Hunter, F. F. (2006). Establishment of Ochlerotatus japonicus (Diptera:
Culicidae) in Ontario, Canada. Journal of Medical Entomology, 43(2):138–142.
Turell, M. J., Dohm, D. J., Sardelis, M. R., O’guinn, M. L., Andreadis, T. G., and Blow, J. A.
(2005). An update on the potential of North American mosquitoes (Diptera: Culicidae) to
transmit West Nile virus. Journal of Medical Entomology, 42(1):57–62.
Turell, M. J., Sardelis, M. R., Dohm, D. J., and O’Guinn, M. L. (2000). Potential for North
American mosquitoes to transmit West Nile Virus. American Journal of Tropical Medicine and
Hygiene, 62:413–414.
Versteirt, V., Schaffner, F., Garros, C., Dekoninck, W., Coosemans, M., and Van Bortel, W. (2009).
Introduction and establishment of the exotic mosquito species Aedes japonicus japonicus
(Diptera: Culicidae) in Belgium. Journal of Medical Entomology, 46(6):1464–1467.
Walker, E., , D., Merritt, R., Morgan, W., and Klug, M. (1991). Nutrient dynamics, bacterial
populations, and mosquito productivity in tree hole ecosystems and microcosms. Ecology,
72(5):1529–1546.
Walker, E., Kaufman, M., Ayres, M., Riedel, M., and Merritt, R. (1997). Effects of variation in
quality of leaf detritus on growth of the eastern tree-hole mosquito, Aedes triseriatus (Diptera:
Culicidae). Canadian Journal of Zoology, 75(5):706–718.
Walker, E. and Merritt, R. (1991). Behavior of larval Aedes triseriatus (Diptera; Culicidae).
Journal of Medical Entomology, 28(5):581–589.
Washburn, J. O., Egerter, D. E., Anderson, J. R., and Saunders, G. A. (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(5):307–314.
Westby, K., Fritzen, C., Huang, J., Jaske, E., Paulsen, D., Jones, C., and Moncayo, A. C. (2011).
La Crosse encephalitis in Eastern Tennessee: Evidence of invasive mosquito (Aedes albopicus
and Ochleotatus japonicus) involvement in the transmission of an in digenous disease. American
Journal of Tropical Medicine and Hygiene, 85(supplement):374.
Williges, E., Farajollahi, A., Scott, J. J., McCuiston, L. J., Crans, W. J., and Gaugler, R. (2008).
Laboratory colonization of Aedes japonicus japonicus. Journal of the American Mosquito
Control Association, 24(4):591–593.
Winters, A. E. and Yee, D. A. (2012). Variation in performance of two co-occurring mosquito
species across diverse resource environments: Insights from nutrient and stable isotope analyses.
Ecological Entomology, 37(1):56–64.
58

Yee, D. and Juliano, S. (2006). Consequences of detritus type in an aquatic microsystem: Effects
on water quality, micro-organisms and performance of the dominant consumer. Freshwater
Biology, 51(3):448–459.
Yee, D. A., Kaufman, M. G., and Juliano, S. A. (2007a). The significance of ratios of detritus types
and micro-organism productivity to competitive interactions between aquatic insect detritivores.
Journal of Animal Ecology, 76(6):1105–1115.
Yee, D. A., Kesavaraju, B., and Juliano, S. A. (2007b). Direct and indirect effects of animal
detritus on growth, survival, and mass of invasive container mosquito Aedes albopictus (Diptera:
Culicidae). Journal of Medical Entomology, 44(4):580.

59