<%BANNER%>

Larval ecology and adult vector competence of invasive mosquitoes Aedes albopictus and Aedes aegypti for chikungunya virus

Permanent Link: http://ufdc.ufl.edu/UFE0041830/00001

Material Information

Title: Larval ecology and adult vector competence of invasive mosquitoes Aedes albopictus and Aedes aegypti for chikungunya virus
Physical Description: 1 online resource (137 p.)
Language: english
Creator: Westbrook, Catherine
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: climate, dengue, density, development, diet, disease, immature, mosquito, vector, virus
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: LARVAL ECOLOGY AND ADULT VECTOR COMPETENCE OF INVASIVE MOSQUITOES Aedes albopictus AND Aedes aegypti FOR CHIKUNGUNYA VIRUS Abiotic and biotic features of the mosquito larval environment shape life history traits important in arbovirus dynamics (e.g., fecundity, life span, biting rate) and can directly affect characteristics that influence adult arbovirus susceptibility. Chikungunya virus (CHIKV) has recently emerged as an important agent of human arboviral disease, and the invasive container mosquitoes Aedes albopictus and Ae. aegypti are the epidemic vectors. When the effect of larval rearing temperature on Ae. albopictus growth and susceptibility to CHIKV was investigated, results showed that temperature had a significant effect on size, development time, and CHIKV infection and dissemination rates. Adult females produced from the coolest temperature, had the largest mean body size, took the longest to mature, and were 6 times more likely to be infected with CHIKV than females reared at the highest temperature. In a separate experiment, Ae. aegypti larvae were reared at different temperatures and food levels to explore relationships among attributes of the larval habitat, body size, and CHIKV susceptibility. Larval temperature and food availability had significant effects on mean adult body size, and female size and quantity of CHIKV ingested were positively correlated. Larval temperature, but not food quantity nor the temperature x food level interaction, had a significant effect on CHIKV infection, but temperature, food level, and their interaction had a significant effect on dissemination. Significant wing length - infection correlations disappeared after the extrinsic incubation period, suggesting that mosquito size alone may not be a good predictor of viral susceptibility. Larval competition between Ae. aegypti and Ae. albopictus within the container habitat is modulated by temperature, and these factors may interactively influence adult susceptibility to CHIKV. The outcome of competition between the two species did not change with temperature, and Ae. aegypti was found to be the superior competitor under these experimental conditions. Temperature and larval competition did not affect the likelihood of infection or disseminated infection with CHIKV. However, mean body titer of CHIKV-infected Ae. albopictus females was significantly affected by larval rearing temperature, with females reared at lower temperatures having higher mean CHIKV body titers than counterparts from the highest temperature.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Catherine Westbrook.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Lounibos, Leon P.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041830:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041830/00001

Material Information

Title: Larval ecology and adult vector competence of invasive mosquitoes Aedes albopictus and Aedes aegypti for chikungunya virus
Physical Description: 1 online resource (137 p.)
Language: english
Creator: Westbrook, Catherine
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: climate, dengue, density, development, diet, disease, immature, mosquito, vector, virus
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: LARVAL ECOLOGY AND ADULT VECTOR COMPETENCE OF INVASIVE MOSQUITOES Aedes albopictus AND Aedes aegypti FOR CHIKUNGUNYA VIRUS Abiotic and biotic features of the mosquito larval environment shape life history traits important in arbovirus dynamics (e.g., fecundity, life span, biting rate) and can directly affect characteristics that influence adult arbovirus susceptibility. Chikungunya virus (CHIKV) has recently emerged as an important agent of human arboviral disease, and the invasive container mosquitoes Aedes albopictus and Ae. aegypti are the epidemic vectors. When the effect of larval rearing temperature on Ae. albopictus growth and susceptibility to CHIKV was investigated, results showed that temperature had a significant effect on size, development time, and CHIKV infection and dissemination rates. Adult females produced from the coolest temperature, had the largest mean body size, took the longest to mature, and were 6 times more likely to be infected with CHIKV than females reared at the highest temperature. In a separate experiment, Ae. aegypti larvae were reared at different temperatures and food levels to explore relationships among attributes of the larval habitat, body size, and CHIKV susceptibility. Larval temperature and food availability had significant effects on mean adult body size, and female size and quantity of CHIKV ingested were positively correlated. Larval temperature, but not food quantity nor the temperature x food level interaction, had a significant effect on CHIKV infection, but temperature, food level, and their interaction had a significant effect on dissemination. Significant wing length - infection correlations disappeared after the extrinsic incubation period, suggesting that mosquito size alone may not be a good predictor of viral susceptibility. Larval competition between Ae. aegypti and Ae. albopictus within the container habitat is modulated by temperature, and these factors may interactively influence adult susceptibility to CHIKV. The outcome of competition between the two species did not change with temperature, and Ae. aegypti was found to be the superior competitor under these experimental conditions. Temperature and larval competition did not affect the likelihood of infection or disseminated infection with CHIKV. However, mean body titer of CHIKV-infected Ae. albopictus females was significantly affected by larval rearing temperature, with females reared at lower temperatures having higher mean CHIKV body titers than counterparts from the highest temperature.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Catherine Westbrook.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Lounibos, Leon P.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041830:00001


This item has the following downloads:


Full Text





LARVAL ECOLOGY AND ADULT VECTOR COMPETENCE OF INVASIVE
MOSQUITOES Aedes albopictus AND Aedes aegypti FOR CHIKUNGUNYA VIRUS
















By

CATHERINE JANE WESTBROOK


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

UNIVERSITY OF FLORIDA

2010

































2010 Catherine Jane Westbrook


























To my husband and daughter, Bill and Seneca Turechek









ACKNOWLEDGMENTS

I would like to acknowledge my dissertation committee, L.P. Lounibos, C. Smartt,

W. Tabachnick and P. Gibbs for their constructive comments and guidance. I feel very

fortunate that I had such a supportive, motivating, and intellectually stimulating advisor

in L.P. Lounibos and I am grateful that he was able to support my work through his NIH

grant (R01 Al-044793).

I am grateful to B. Alto for allowing me to build on much of the work he started in

the Lounibos lab at FMEL. I am also very thankful that M. Reiskind and K. Pesko were

such great teachers, collaborators and friends. I would like to thank K. Greene and N.

Nishimura for all their hard work on all of the experiments. I would like to thank E.

Blosser and S. Anderson for making themselves available at critical times in my final

experiment. I want to thank S. Yost for providing me with cultured cells. I appreciate C.

Lord for permitting me to monopolize her incubators for nearly three years. I would like

to thank Drs. Stephanie Richards and George O'Meara for their critical reading of the

manuscript resulting from Chapter 2. I also need to thank a long list of people at FMEL

that helped me pick and identify pupae. The list includes, but is probably not limited to

T. Stenn, S. O'Connell, S. Lynn, R. Ortiz, and H. Robinson.









TABLE OF CONTENTS

paqe

A C KNOW LEDG M ENTS ............................ ................... ......................................... 4

LIST O F TABLES .............. ............................................................................. 7

LIST O F F IG U R E S .................................................... 8

A BST RA C T ............... ... ..... ......................................................... ...... 10

CHAPTER

1 INTRODUCTION AND REVIEW OF THE LITERATURE ................................. 12

Intro d u cto ry S ta te m e nt .................................. ........... ................... ............... 12
Mosquitoes: Aedes aegypti and Aedes albopictus .......... ........... .............. 14
Invasion Biology of Disease Vectors ......................... ...... ........... ....... 15
Host Preference and Feeding..................................... ............................ 17
Short-Range Dispersal and Longevity.............. ......... .. .. .............. 19
Oviposition....................... ........ 21
Thermal Tolerance ............... .. ......... ................. 22
Eradication and Control ........... .... ..... .. .................. .. ...... ............ 24
Competition Between Aedes Albopictus and Ae. Aegypti.............................. 24
C hikungunya V irus.................. ........................... ........ .............. 26
Discovery of Virus and Vectors ........... ..... ........ ... ....... ............... 26
Viral Characterization and Phylogenetics............. .. ......... .. ........... .... 28
Historical and Current Epidem ics .............. .................. ............. .... .......... 29
CHIKV Interactions in Epidemic Vectors: Aedes aegypti and Ae. albopictus... 30
V sector C om petence ........................ ................. .................... .............. 33
The Environment and Vector Competence.................... ................... 34
The Larval Environment, Vector Competence, and Mosquito Size .................. 35

2 LARVAL ENVIRONMENTAL TEMPERATURE AND THE SUSCEPTIBILITY OF
Aedes albopictus SKUSE (DIPTERA: CULICIDAE) TO CHIKUNGUNYA VIRUS .. 38

Introduction ............... ......... ....... .................. .... .... ......... 38
Materials and Methods................................ ............... 41
Mosquitoes and Viruses ......................................... .... .............. 41
Vector Competence.............................. ............... 42
S tatistica l A na lysis ........................................................................ ......... 4 4
Results ......... ............ .................... ................. ......... 45
Growth and Mortality ......... ......... ......... ........ ....... 45
Chikungunya Infection and Dissemination....................... ................... 45
D is c u s s io n ......................... ................................................... 4 6









3 LARVAL TEMPERATURE AND NUTRITION ALTER THE SUSCEPTIBILITY
OF Aedes aegypti L. (DIPTERA: CULICIDAE) MOSQUITOES TO
CHIKUNGUNYA VIRUS ......... .. ....... ..................................... 54

Introduction ............... ..... .. ...... ......... .......... .... .... ......... 54
Materials and Methods........................................ .......... 57
Mosquitoes and Viruses .... ... ................................. .... .............. 57
Mosquito Infection ........ .......... ......................... ............... 59
S statistical A nalysis............................................ ............ ....... 62
Results ..... ......................... ... ...... ... ...... ..... ...................... 64
Chikungunya Titer of Freshly Engorged Mosquitoes............... ............... 64
Growth and Mortality ............. .............. ........ ............... 64
Chikungunya Infection and Dissemination.......................................... 65
D is c u s s io n ......................... ................................................... 6 6

4 LARVAL TEMPERATURE, COMPETITION, AND THE VECTOR
COMPETENCE OF Aedes aegypti AND Aedes albopictus FOR
CHIKUNGUNYA VIRUS ........ ......... ........................ ................ 81

Introduction ............... ......... ....... .......... ........ .... .... ......... 81
M materials and M ethods.................................................................... 84
Mosquitoes, Temperature, and Competition............... ..................... 84
Virus and Mosquito Infection ............... .......... .... .. ................. 86
S tatistica l A na lysis ........................................................................ ......... 8 9
Results .......................................... ............................ 92
Mosquitoes, Temperature, and Competition............... ..................... 92
Virus and Mosquito Infection ............... .......... .... .. ................. 94
D iscussio n ................... ................. ...... .................................... 96

5 GENERAL CONCLUSIONS AND FUTURE DIRECTIONS .............................. 113

LIST OF REFERENCES .................. ........... ....................... ..... .......... 119

B IO G RA PH IC A L S K ETC H ............. ................. ................. .................. ............... 137









LIST OF TABLES
Table page

2-1. Aedes albopictus treatment medians and interquartile ranges (IQR= 25th
percentile -75th percentile) for development ................................................ 52

3-1. Aedes aegypti LS means and standard error for development time to
adulthood and female wing length. ................ ....... .. ....... .. ............. 73

3-2. Maximum likelihood (ML) contrasts for comparisons of mortality rates for
340C-high food treatment ........... ......... ........................ 74

4-1. MANOVA for temperature and competitive treatment effects and their
interaction on population growth parameters............................. 103

4-2. Multivariate pairwise contrasts of temperature treatment effects on female
Aedes albopictus and Aedes aegypti for growth measurementsh................. 104

4-3. Multivariate pairwise contrasts of competitive treatment effects on female
Aedes albopictus and Aedes aegypti for growth measurements ................. 105









LIST OF FIGURES


Figure page

2-1. Bivariate plots of mean wing lengths (SE) and CHIKV susceptibility ............... 53

3-1. Correlation between log transformed whole mosquito body titers of CHIKV
and wing lengths for engorged Aedes aegypti............... ............................ 75

3-2. Bivariate plot of LS means (SE) for wing lengths and log transformed
CHIKV body titers for engorged Aedes aegypti females ................ ............... 76

3-3. Juvenile mortality rates at the low and high food levels within the three
temperature treatments for Aedes aegypti ........... .... .... .... ............... 77

3-4. Proportion of Aedes aegypti females (SE) in each temperature treatment
infected w ith C H IKV .................................... .......... ............... .............. 78

3-5. Proportions of infected Aedes aegypti females (SE) from temperature and
food level treatments with disseminated CHIKV infections.............................. 79

3-6. Least squared means (SE) for sizes of adult female Ae. aegypti mosquitoes
in CHIKV infection status categories. ............................... .............. ........... 80

4-1. Proportion of female Ae. albopictus (SEM) surviving to adult emergence...... 106

4-2. Female Ae. albopictus mean (SEM) days to pupation........................... 106

4-3. Female Ae. albopictus mean (SEM) wing length. ................. ............ ..... 107

4-4. Proportion of female Ae. aegypti (SEM) surviving to adult emergence ......... 108

4-5. Female Ae. aegypti mean (SEM) days to pupation. ................................... 108

4-6. Bivariate means (SEM) of replicates for proportions of Ae. aegypti and Ae.
albopictus with infections and disseminated infections...... ........................ 109

4-7. Bivariate plot of least squares means for proportion of Ae. albopictus with
infections and disseminated infections .......... ........... ...... ........... .... 109

4-8. LS means (SEM) of Aedes albopictus CHIKV body titer for temperature
tre a tm e n ts .................................................. ................ 1 1 0

4-9. Correlation analysis of log transformed of CHIKV whole mosquito body titer
and wing length. ............ .............................. ............... 111

4-10. Correlation analysis of log transformed of CHIKV whole mosquito body titers
and wing lengths for Aedes aegypti.............................. .............. 111









4-11. Least squared means (SE) for sizes of adult female Ae. albopictus
mosquitoes in CHIKV infection status categories. ................. .......... ......... 112

4-12. Least squared means (SE) for sizes of adult female Ae. aegypti mosquitoes
in CHIKV infection status categories. .............. ..................... .................. 112









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

LARVAL ECOLOGY AND ADULT VECTOR COMPETENCE OF INVASIVE
MOSQUITOES Aedes albopictus AND Aedes aegypti FOR CHIKUNGUNYA VIRUS

By

Catherine Jane Westbrook

August 2010

Chair: L. Philip Lounibos
Major: Entomology and Nematology

Abiotic and biotic features of the mosquito larval environment shape life history

traits important in arbovirus dynamics (e.g., fecundity, life span, biting rate) and can

directly affect characteristics that influence adult arbovirus susceptibility. Chikungunya

virus (CHIKV) has recently emerged as an important agent of human arboviral disease,

and the invasive container mosquitoes Aedes albopictus and Ae. aegypti are the

epidemic vectors.

When the effect of larval rearing temperature on Ae. albopictus growth and

susceptibility to CHIKV was investigated, results showed that temperature had a

significant effect on size, development time, and CHIKV infection and dissemination

rates. Adult females produced from the coolest temperature, had the largest mean body

size, took the longest to mature, and were 6 times more likely to be infected with CHIKV

than females reared at the highest temperature.

In a separate experiment, Ae. aegypti larvae were reared at different temperatures

and food levels to explore relationships among attributes of the larval habitat, body size,

and CHIKV susceptibility. Larval temperature and food availability had significant effects

on mean adult body size, and female size and quantity of CHIKV ingested were









positively correlated. Larval temperature, but not food quantity nor the temperature x

food level interaction, had a significant effect on CHIKV infection, but temperature, food

level, and their interaction had a significant effect on dissemination. Significant wing

length infection correlations disappeared after the extrinsic incubation period,

suggesting that mosquito size alone may not be a good predictor of viral susceptibility.

Larval competition between Ae. aegypti and Ae. albopictus within the container

habitat is modulated by temperature, and these factors may interactively influence adult

susceptibility to CHIKV. The outcome of competition between the two species did not

change with temperature, and Ae. aegypti was found to be the superior competitor

under these experimental conditions. Temperature and larval competition did not affect

the likelihood of infection or disseminated infection with CHIKV. However, mean body

titer of CHIKV-infected Ae. albopictus females was significantly affected by larval

rearing temperature, with females reared at lower temperatures having higher mean

CHIKV body titers than counterparts from the highest temperature.









CHAPTER 1
INTRODUCTION AND REVIEW OF THE LITERATURE

Introductory Statement

Fluctuating temperatures, limited food, and high inter- and intraspecific competition

are common features in container habitats occupied by the aquatic larvae of many

holometabolous insects, including the Asian tiger mosquito, Aedes albopictus and the

yellow fever mosquito, Aedes aegypti (Juliano 2009). The effect of the mosquito larval

environment on adult life history traits and other characteristics such as growth rate, age

at maturity, biting rates, gonotrophic cycle lengths, vector size, fecundity, and life span

are well documented (Madder et al. 1983, Briegel 1990, Rueda et al. 1990, Scott et al.

1993a). However, the larval environment may also influence adult vector-pathogen

interactions, by altering a vector's competence for a virus resulting in changes in the

distribution and transmission intensity of an arbovirus. Vector competence is the

capacity of an arthropod to acquire a pathogen and transmit it to a subsequent host. For

successful transmission to occur arbovirus taken in from an infectious blood meal must

overcome internal barriers in the mosquito midgut, disseminate into other organs, and

then pass through an additional barrier into the salivary glands in order to infect a new

host during the next blood feeding (Hardy et al. 1983). Vector competence can vary

greatly among individuals and between mosquito populations (Lorenz et al. 1984,

Tabachnick et al. 1985, Turell et al. 1992, Paupy et al. 2001) and has been shown to be

influenced by both the genetic background of a vector and environmental conditions

(Davis 1932, Hurlbut 1973, Hardy et al. 1990, Kilpatrick et al. 2008).

Most research has focused on the adult environment with fewer studies exploring

the influence of larval ecology on mosquito-arbovirus interactions. There is evidence









that larval habitat variables, such as temperature, food resources, density and

competition can affect vector competence in certain mosquito-arboviral systems (Baqar

et al. 1980, Grimstad and Walker 1991, Turell 1993, Sumanochitrapon et al. 1998, Alto

et al. 2005, Alto et al. 2008a, Bevins 2008). However, the strength of the effect and the

direction seems to vary with different species of mosquitoes and different viruses.

Furthermore, previous research has primarily focused on the effect of a single factor in

the larval environment without consideration for the potential effects of interactions

between variables.

The research presented in this dissertation explores the interactions of larval

ecological factors and their impact on the transmission of chikungunya virus (CHIKV) by

invasive container mosquitoes Aedes albopictus and Ae. aegypti. The hypothesis tested

here is that variation in environmental factors during larval development affects the

physical and physiological characteristics of adults to alter their ability to become

infected and/or transmit an arbovirus. Experiments were conducted manipulating: (1)

larval rearing temperature, (2) larval rearing temperature and larval food levels, and (3)

larval rearing temperature and intra- and inter-specific larval competition in Ae.

albopictus and/or Ae. aegypti from South Florida. To determine the effect of larval

habitat quality on life-history characteristics of these mosquitoes, juvenile mortality,

development time, and adult body size were measured. Adult females were given a

blood meal containing CHIKV, and susceptibility to the virus was assessed by

measuring infection and dissemination rates and whole mosquito body viral titer. This

research is novel for exploring the interactions among multiple factors in the larval

environment and their impact on transmission potential of an arbovirus by adult









mosquitoes. Furthermore, this work is particularly relevant in light of predicted

alterations in temperature due to global climate change and the ongoing ecological and

public health problems caused by the continued intercontinental dispersal of invasive

vectors and pathogens.

The following literature review provides information on the mosquito vectors,

CHIKV, and the influence of the environment on vector competence. Chapters two,

three, and four contain original research on the influence of Ae. albopictus and Ae.

aegypti larval ecological factors on adult CHIKV susceptibility, followed by chapter five

which provides conclusions and recommendations for future research directions.

Mosquitoes: Aedes aegypti and Aedes albopictus

As the volume of global trade and travel has greatly increased in the past fifty years,

so have the number of accidental introductions of exotic organisms to new geographic

areas (Mooney and Hobbs 2000). Invading organisms can have grave detrimental

effects on indigenous communities, but when the invader is also a potential vector of an

exotic human pathogen then the introduction and spread of the organism can also have

a major public health consequence (Juliano and Lounibos 2005). The container

mosquito species, Ae. aegypti and Ae. albopictus have globally invasive ranges. Aedes

aegypti is believed to have traveled to the New World from Africa in water storage jars

aboard slave ships (Christophers 1960), while the spread of Ae. albopictus from its

native Asian range has been a more recent phenomenon (Hawley et al. 1987).

Arboviruses that are transmitted by Ae. aegypti and Ae. albopictus threaten the health

of millions of people worldwide, and the continued range expansion of these species will

add to the populations at risk.









Invasion Biology of Disease Vectors

Aedes (Stegomyia) aegypti (L.), the yellow fever mosquito, is the primary epidemic

vector of yellow fever virus (YFV), dengue virus (DENV) CHIKV. The preferred habitat

of Ae. aegypti is the urban environment and although it is limited to tropical and

subtropical regions, within these limits it has a very cosmopolitan distribution. In East

Africa Ae. aegypti exists in two forms: (1) Aedes aegypti formosus, a sylvan and likely

ancestral form with darker scales which oviposits primarily in treeholes and is often

found away from human habitats and (2) Ae. aegypti aegypti, a domestic form, which

exhibits a preference for human habitats (Tabachnick and Powell 1979). In coastal

Kenya, the sylvan and domestic Ae. aegypti represent two distinct gene pools,

maintained through habitat selection, where sympatry exists gene flow between the two

subspecies is limited (Tabachnick and Powell 1979).

Though Ae. aegypti formosus has been found in other regions of subSaharan

Africa, it is the domestic form, Ae. aegypti aegypti, through its ability to exploit human

water storage containers and human habitats, that has spread from Africa to tropical

and subtropical regions across the globe (Christophers 1960, Tabachnick 1991). Key

behavioral characters of Ae. aegypti aegypti are: oviposition in human water storage

containers, feeding on human blood, and entering into homes in search of hosts, mates

and oviposition and resting sites (Trpis and Hausermann 1975). Oviposition containers,

such as water cisterns, aboard New World bound slave ships during the 15th to the 19th

centuries are believed to be a major mode of introduction for this mosquito (Tabachnick

1991). The spread of Ae. aegypti across the globe likely involved the movement of

multiple life stages of the mosquito: aquatic larvae, adults, and eggs (Christophers









1960). For the remainder of this dissertation Ae. aegypti will be used as a synonym for

Ae. aegypti aegypti.

Aedes (Stegomyia) albopictus (Skuse), the Asian tiger mosquito, is also an

efficient and important vector of DEN and CHIKV and, like Ae. aegypti, Ae. albopictus

has successfully spread across the globe through the exploitation of man-made

environments. Although common in suburban and rural settings, this mosquito

originated in the forest edges of Southeast Asia, but human migration towards the

Malay peninsula and the Indian Ocean islands, including Madagascar, may have led to

an early movement of Ae. albopictus out of its native Asian range (Paupy et al. 2009).

In the late nineteenth century Ae. albopictus began spreading onto the Pacific

islands, such as Hawaii and Guam (Lounibos 2002). In 1985 it was discovered in the

United States, where it was identified as the most abundant artificial container-inhabiting

mosquito in Houston, Texas (Sprenger and Wuithiranyagool 1986). Since its discovery,

Ae. albopictus has become established in most states in the eastern part of the USA,

extending as far north as Illinois, Indiana, Ohio, Pennsylvania and New Jersey (Moore

1999). Between 1985 and 1998, Ae. albopictus was recovered from many countries in

the Americas and the Caribbean (Benedict et al. 2007). In Europe, it was first recorded

in Albania in 1979, Italy in 1990, then France in 1999, and currently Ae. albopictus is

present in at least 12 European countries (Knudsen et al. 1996, Vazeille et al. 2008).

Established populations were recorded in Nigeria in 1991 (Savage et al. 1992) and,

since, Ae. albopictus has spread to other nations in West and Central Africa. The

establishment of this mosquito in numerous countries, in Africa, the Middle East, Europe









and the Americas is primarily due to the trade and movement of used tires that contain

Ae. albopictus eggs (Hawley et al. 1987, Benedict et al. 2007).

Host Preference and Feeding

Humans are the most common bloodmeal source of Ae. aegypti (Scott et al.

1993a, Ponlawat and Harrington 2005), and much work has been done to determine

how the consumption of human blood compares to blood from other vertebrate hosts in

terms of fecundity and longevity in this mosquito (Briegel 1985, Harrington et al. 2001).

Females of most mosquito species require both blood and sugar (Foster 1995), and

both male and female Ae. aegypti have been observed feeding on plant nectars

(Christophers 1960). However, certain populations of Ae. aegypti in Thailand have

adapted to an environment low in sugar, but where human blood is readily available.

These females seldom feed on plant sugars and take more frequent blood meals

(Edman et al. 1992). Laboratory studies on Ae. aegypti suggest that a diet of human

blood without sugar increases the reproductive success of this mosquito through a

greater age-specific survival (Ix), estimated reproductive output per day (mx) and the net

replacement rate (Ro), although total egg production was greater in mosquitoes fed

blood and sugar (Harrington et al. 2001).

Aedes albopictus is considered an opportunistic feeder, with a preference for

mammals (Savage et al. 1993). In suburban North Carolina humans (24%), cats (21%)

and dogs (14%) were the primary hosts (Richards et al. 2006). However, in villages in

Thailand and Singapore humans were the primary sources of blood (Colless 1959,

Ponlawat and Harrington 2005). This species is thought to have progressively adapted

to anthropogenic changes in the environment, moving from a zoophilic host feeding

regime to a greater dependence on blood meals from humans and domesticated









animals (Paupy et al. 2009). Laboratory studies on Ae albopictus also suggest that a

diet of human blood without sugar increases the reproductive success of this species,

and thus the benefit of feeding exclusively on blood is not limited to the highly

anthropophilic Ae. aegypti (Braks et al. 2006).

Aedes aegypti and Ae. albopictus females often take multiple blood meals during a

single gonotrophic cycle (MacDonald 1956, Scott et al. 1993b, Scott et al. 2000, Delatte

et al. 2009). This behavior, known as multiple feeding, can lead to an exponential

increase in the vectorial capacities of these two mosquitoes because the daily

probability of being fed upon is a squared function in the vectorial capacity equation

resulting from the product of host preference index and the frequency of feeding (Black

and Moore 1996). Furthermore, multiple feeding can increase the probability of

concurrent infection and viral genetic mixing (Kuno and Chang 2005).

Aedes aegypti preferentially feeds in the daytime, but individuals will on occasion

feed at night if a suitable host is present (Christophers 1960). Ae albopictus is also a

diurnal feeder with peak feeding times occurring at daybreak and two hours before

sunset (Delatte et al 2009). Nocturnal feeding sub-populations of Ae. aegypti in the

Ivory Coast (Diarrassouba and Dossou-Yovo 1997) and in Trinidad (Chadee and

Martinez 2000) are documented. In Trinidad nocturnal feeding was recorded at the

urban site and not at the rural site and accounted for approximately 10% of urban indoor

feeding totals and 9.4% of outdoor urban feeding totals. It was suggested that the

nocturnal extension of Ae. aegypti feeding may be an adaptation to increased electrical

lighting in and around houses, and also explains the absence of nocturnal feeding in the

poorly lit rural site (Chadee and Martinez 2000).









Short-Range Dispersal and Longevity

The movement of mosquitoes has been studied because of the important role of

dispersal and flight range in vector-borne disease. Many studies support the theory that

Ae. aegypti takes fairly short flights and does not disperse over far distances. Muir and

Kay (1998), in a mark-release-recapture study in northern Australia found the mean

distance Ae. aegypti traveled was 56 meters (m) for females and 35 m for males.

Harrington et al. (2005), in an 11 year study, involving 21 mark-release-recapture

experiments in Puerto Rico and Thailand, concluded that the mean dispersal distance

for Ae. aegypti ranged from 28 to 199 m and Maciel-de-Freitas et al. (2007) found a

similar pattern in Rio de Janeiro, Brazil where the average distance travelled ranged

between 81 to 86 m. In contrast a few studies have reported, through assaying rubidium

labeled eggs, longer distance dispersal of up to 800 m in Rio de Janeiro (Honorio et al.

2003) and means ranging between 221-279 m in Puerto Rico (Reiter et al. 1995). A

major difference in the studies assaying labeled eggs is that they measured Ae. aegypti

dispersal during an oviposition cycle and this may account for some of the differences

between the two types of studies.

Dispersal has been investigated in Ae albopictus and compared to Ae. aegypti,

Aedes albopictus generally takes longer flights and disperses over farther distances. In

a mark-release-recapture study in Missouri the maximum distances Ae albopictus

traveled were 525 m for females and 225 m for males (Niebylski and Craig 1994).

Through ovitrapping of rubidium labeled eggs, dispersal by Ae albopictus of up to 800 m

was found in Rio de Janeiro and did not differ in distance from Ae. aegypti in this study

(Honorio et al. 2003). In Singapore Ae albopictus rubidium labeled eggs were found at

distances as far as 640 m from the release site (Liew and Curtis 2004).









The life span of adult mosquitoes in the field is determined by factors such as

quality of the environment, climate, predation, and genetic background. Laboratory

studies in which Ae. aegypti and Ae. albopictus are given ample sugar and blood meals

while being held at a constant and suitable temperature with a high level of humidity,

record maximum longevity for females at over 100 days and the mean life spans

between 4-6 weeks (Christophers 1960, Hawley 1988). Daily survival probabilities of

adult females are an important element of vectorial capacity, and, to transmit an

arbovirus, mosquitoes must survive longer than the time prior to taking an infectious

blood meal combined with the extrinsic incubation period (EIP) of the pathogen, which is

the time interval between ingestion of an infective blood meal and oral transmission of a

virus (Davis 1932). The EIP for CHIKV in Ae. aegypti and Ae. albopictus can be as short

as two days (Dubrelle et al. 2009).

The assumption can be made that wild mosquito individuals at the mercy of biotic

and abiotic factors in the environment experience greatly reduced longevity; the

difficulty is measuring it. Mark-release-recapture experiments have been the primary

method of assessing probability of daily survival (PDS), and in Rio de Janeiro, Brazil

female Ae. aegypti PDS varied from 0.71 to 0.87 depending on the site and season

(Maciel-de-Freitas et al. 2007) and in Kenyan field studies PDS was 0.77 for male Ae.

aegypti which corresponds to a 4.4 day life span, and 0.89 (9.2 days) for females

(McDonald 1977). For Ae. albopictus in a scrap tire yard in Potosi, Missouri PDS was

0.89 (8.2 days) for females and 0.77 (3.9 days) for males (Niebylski and Craig 1994).

Very low recapture rates in survival studies make the reliability of PDS estimates









questionable, and predictions of mosquito age in future studies may make use of newer

technologies such as gene transcription profiling (Cook et al. 2006).

Oviposition

Gravid female Ae. aegypti and Ae. albopictus oviposit desiccation resistant eggs

that can survive in a dried state for several months or longer until submersion in water

triggers hatching. A common feature of oviposition containers selected by both female

Ae. aegypti and Ae. albopictus is clean water with a high organic content (Clements

1992, Delatte et al. 2008). A gravid Ae. aegypti female will select both indoor, because

of its close association with man, and outdoor containers for oviposition (Christophers

1960) and for Ae. albopictus peridomestic and rural oviposition sites are frequently used

(Hawley 1988).

Egg-laying in both these mosquitoes is diurnal with the majority of eggs laid two

hours after sunrise and two hours before sunset (Corbet and Chadee 1990, Delatte et al

2009). Based on the results of field studies on Ae. aegypti in Puerto Rico and Trinidad

and on Ae. albopictus in Honolulu, Hawaii, it was determined that females deposit their

eggs from the same batch at several oviposition sites, a behavior known as "skip

oviposition" (Rozeboom et al. 1973, Chadee and Corbet 1987, Apostol et al. 1994,

Reiter et al. 1995). Skip oviposition may benefit the species by decreasing sibling

competition and spreading risk of mortality over multiple sites.

Aedes albopictus is remarkable for the wide range of natural and artificial

containers in which it is found, ranging from tree holes, bamboo stumps, rock holes, leaf

axils, flower plates, pots, catch basins, and discarded tires (Hawley 1988, Sota et al.

1992, Delatte et al. 2008). On the island of Reunion, where it is the most common

Aedes species, Ae. albopictus showed a strong ecological plasticity. In the wet season









this mosquito occurred most frequently in small artificial disposable containers and in the

dry winter season, natural containers (bamboo stumps and rock holes) were clearly

important (Delatte et al. 2008). In North America, the most common microhabitat for Ae.

albopictus has been discarded tires (Sprenger and Wuithiranyagool 1986). However in

Florida Ae. albopictus immatures are found in the water-holding tanks and axils of

ornamental bromeliads, although in southern Florida their use of this phytotelmata is

kept in check by two endemic Wyeomyia spp. of mosquitoes (Lounibos et al. 2003). In

Florida Ae. albopictus is also found sharing tree holes with the native inhabitant Ae.

triseriatus (Lounibos et al. 2001).

Thermal Tolerance

No life stage of Ae. aegypti undergoes diapause, and 160C seems to be close to

the lower limit for adult activity of this species (Christophers 1960). Larvae continue to

develop and pupate at temperatures as low as 150C, but the duration of the larval stage

is approximately 31-32 days, and at temperatures between 8.2-10.6 C development

completely ceases (Kamimura et al. 2002). In terms of upper thermal limits, Ae. aegypti

larvae do not thrive in water temperature much above 340C and adults begin to die if air

temperatures exceed 40 C. Desiccation resistant eggs are also susceptible to

temperature extremes. Prolonged exposure to a low temperature of 100C and a high

temperature of 400C resulted in 100% egg mortality (Christophers 1960).

In its native Asian range, Ae. albopictus is abundant in both tropical and temperate

regions and, as a result, this mosquito species can survive over a broad spectrum of

temperatures and relative humidity. At 110C Udaka (1959) found that larval

development ceased for a Japanese strain of Ae. albopictus. A similar result was

recorded with a strain of Ae. albopictus from Reunion island in the Indian Ocean, where









no development beyond first instar took place at 50C or 100C (Delatte et al. 2009). At

temperatures as low as 120C Ae. albopictus larvae developed and pupated with an 28

day larval duration, but two-thirds of adult females did not mature eggs or died (Briegel

and Timmermann 2001). Adults from a Reunion strain fared worse with no egg laying

observed among females at 150C (Delatte et al. 2009). The limiting upper temperature

of Ae. albopictus larval development is 350C (Monteiro et al. 2007, Delatte et al. 2009),

and at 43.30C 100% adult mortality was observed after approximately 30 minutes (Smith

et al. 1988). Analysis of life tables, combining developmental rates, reproduction and

mortality, suggest maximum population growth (r) between 25 and 300C for Ae.

albopictus tested at eight constant temperatures (5, 10, 15, 20, 25, 30, 35 and 400 C)

(Delatte et al. 2009).

Strains of Ae. albopictus found in temperate regions are sensitive to short day-

lengths during the pupal and adult stage leading to the production of diapause eggs

(Hawley et al. 1987). Not long after Ae. albopictus was found in the United States, this

species was also identified in Brazil (Forattini 1986), but the lack of a photoperiodic egg

diapause in Brazilian populations suggested distinct origins for the two populations

(Hawley et al. 1987). Sequence data from the mitochondrial ND5 gene confirmed that

the North American populations of Ae. albopictus originated from Asian temperate

regions, while Brazilian populations are tropical in origin (Birungi and Munstermann

2002). This was in contrast to an earlier allozyme study inferring a common Northern

Asian (Japan) origin for both populations (Kambhampati et al. 1991). Future use of

recently identified polymorphic microsatellite loci (Porretta et al. 2006) in population









genetic studies may help to further elucidate the structure and relationships among

invasive Ae. albopictus populations.

Eradication and Control

In 1947 the countries comprising the Pan American Health Organization (PAHO)

proposed a resolution to eradicate Ae. aegypti, primarily to control yellow fever. At the

time of the resolution Ae. aegypti was known from all of the western hemisphere except

Canada and Bolivia. Between 1958 and 1965 eradication was accomplished in 17 of 23

targeted countries, primarily through perifocal application of DDT insecticide to infested

containers (Schliessmann and Calheiros 1974). However, countries and territories in the

Caribbean had a more difficult time achieving eradication and, by the 1970s with

depleting financial resources, and subsequent social upheaval in countries such as El

Salvador, re-infestations became widespread.

Over the past 50 years, Aedes control methods, often for dengue control, have not

greatly changed. Larval source reduction, remains the primary method of Ae. aegypti

and Ae. albopictus control and is accomplished through the removal of disposable

containers, or the treatment of water storage containers with one of three common

larvicides: (1) temephos (an organophosphate), (2) methoprene (insect growth

regulator), or (3) BTI (Bacillus thuringiensis var.israelensis). For adult control, which

becomes a focus when an outbreak of Aedes-vectored disease is already underway,

ultra-low volume aerosols of insecticide, such as malathion, are applied with either truck

mounted units or airplanes (World Health Organization 1997)

Competition Between Aedes Albopictus and Ae. Aegypti

Aedes albopictus and Ae. aegypti currently have sympatric distributions in many

parts of the world, and the co-occurrence of larvae and pupae of both species within the









same container is common (MacDonald 1956, Fontenille and Rodhain 1989, O'Meara et

al. 1992, Braks et al. 2003). In larval competition experiments on North American and

Brazilian populations of the two species, Ae. albopictus exhibited a competitive

advantage over Ae. aegypti under field conditions (Juliano 1998, Braks et al. 2004).

These results support a role for interspecific competition in the observed decline or local

extinction of Ae. aegypti in a large portion of the United States now inhabited by Ae.

albopictus (O'Meara et al 1995). Nevertheless, there are regions in Asia, North and

South America, and Africa where these two Aedes species successfully coexist and

much effort has gone into elucidating the mechanisms behind their sustained co-

occurrence (Sota and Mogi 1992, Juliano et al. 2002, Costanzo et al. 2005, Lounibos et

al. 2002, Leisnham and Juliano 2009, Leisnham et al. 2009).

A probable process at work is condition-specific competition, where seasonal and

spatial variation in environmental conditions cause a change in which competitor is

favored (Costanzo et al. 2005, Leisnham and Juliano 2009). Although Ae. albopictus is

a better larval competitor, superior desiccation resistance in the egg stage by Ae.

aegypti allows greater numbers of this mosquito to survive during the dry season (Sota

and Mogi 1992, Juliano et al. 2002). Therefore, early in the wet season as eggs that

persisted during the drying period hatch, Ae. albopictus populations are at a low due to

greater egg mortality (Leisnham and Juliano 2009). Condition-specific competition can

also explain the spatial partitioning of the two mosquitoes in the environment, the

distribution of Ae. aegypti populations is associated with lower humidity and higher

temperatures common in more urbanized settings. In contrast, Ae. albopictus, is

negatively associated with hot, dry climate and is more common in suburban, rural, and









forest edge areas where vegetation is more abundant and there is an excess of humid

and shaded resting and oviposition sites (Hawley 1988, Sota and Mogi 1992, Juliano et

al. 2002, Braks et al. 2003, Rey et al. 2006, Reiskind and Lounibos 2009).

Chikungunya Virus

In parts of their geographic ranges both Ae. aegypti and Ae. albopictus are

important epidemic vectors of CHIKV, a single stranded enveloped, positive sense RNA

virus. In its native African range, CHIKV is a zoonosis, with a transmission cycle

involving wild primates and sylvatic Aedes species. However, in the invasive range of

CHIKV, the viral transmission cycle is urban or suburban, and the primary vectors are

Ae. aegypti and Ae. albopictus with humans as the host. Since the first isolation and

identification of the virus in Africa in the 1950s, CHIKV has spread into new geographic

areas with human epidemics documented on multiple continents.

Discovery of Virus and Vectors

Chikungunya is an alphavirus in the Family Togaviridae and, based on serological

cross-reactivity, CHIKV is grouped more specifically into the Semliki Forest virus (SFV)

antigenic serocomplex (Powers and Logue 2007). The prototype virus was isolated by

Ross during the 1953 dengue epidemic in the Newala district of Tanzania (formerly

Tanganyika) from the blood of a febrile patient (Ross 1956). The name chikungunya is

derived from the Makonde word meaning "that which bends up" or "walking bent over" in

reference to the stooped posture developed as a result of the incapacitating arthralgia

that can last for months (Sourisseau et al. 2007). Although CHIKV is rarely fatal,

symptoms of the disease include high fevers, rashes, headache, photophobia, vomiting,

and excruciating joint and muscle pain. Clinical symptoms follow an incubation period of









2 to 7 days, and acute illness is short in duration, lasting 3 to 5 days with recovery in 5

to 7 days (Ligon 2006, Robinson 1955).

The vectors of the virus are Aedes mosquitoes in the subgenera Diceromyia,

Stegomyia and Aedimorphus. Sylvan transmission cycles have been documented in

tropical Africa in moist forest and semiarid savannah-woodland involving sylvatic Aedes

species, such as Ae. africanus, Ae. furcifer, Ae. luteocephalus, Ae. neoafricanus and

Ae. taylori and wild primates, such as vervet monkeys (Cercopithecus aethiops) and

baboons (Papio ursinus) (Jupp and Mclntosh 1988, Jupp and Mclntosh 1990, Diallo et

al. 1999). In monkeys and baboons infection is characterized by a short incubation

period and a subsequent viremia lasting approximately five days. There is no mortality

and resulting immunity is likely life-long (de Moor and Steffens 1970). The average life

expectancy of C. aethiops is between three and four years and it is possible that CHIKV

sylvan epidemics follow a 3-4 year cycle that correspond with the renewal of non-

immune wild primate populations (de Moor and Steffens 1970; Powers and Logue

2007). Studies in the Zika forest of Uganda have also provided data suggesting that a 5

to 7 year cycle in CHIKV activity may correspond with the replacement of non-

susceptible red-tailed monkey (C. ascanius schmidti) with susceptible individuals

(Macrae et al. 1971).

How the virus is maintained long-term in the wild is unknown. No field or laboratory

data can confirm that the virus is maintained transovarially in mosquito eggs (Vazeille et

al. 2009). Computer simulated epidemiological models generated by de Moor and

Steffens (1970) suggest that CHIKV could exist endemically by continuous transmission

in the vertebrate (C. aethiops) population. Urban epidemic transmission of CHIKV is









sustained by the mosquitoes Ae. aegypti and/or Ae. albopictus in a human-mosquito-

human cycle (Jupp and Mclntosh 1988).

Viral Characterization and Phylogenetics

Chikungunya virus, like all known alphaviruses, is arthropod-borne with

mosquitoes being the predominant vector. Alphaviruses are enveloped particles, and

their genome consists of a single stranded, positive sense RNA molecule of ~12,000

nucleotides (nt). The 5 prime end is capped with a 7-methylguanosine while the 3 prime

end is polyadenylated. The CHIKV genome is approximately 11.8 kb and is divided into

two major regions: the first two-thirds of the genome which forms the 5 prime end

encodes the four non-structural proteins (nsP 1-4) and a structural domain encoding the

three structural proteins of the virus capsidd (C), E2 and El). The non-structural proteins

are translated directly from the genomic RNA into a polyprotein that through proteolytic

cleavage produces nsP1, nsP2, nsP3, and nsP4 in addition to important cleavage

intermediates (Khan et al. 2002). The structural proteins are translated through a

subgenomic mRNA intermediate called the 26S RNA into a single polyprotein that is

also cleaved to produce the three structural proteins as well as two small polypeptides

E3 and 6K (Strauss and Strauss 1994). Thus the genome of CHIKV is 5 prime cap-

nsP1-nsP2-nsP3-nsP4-(junction region)-C-E3-E2-6K-E1-poly(A) 3 prime.

CHIKV appears to have originated in Central/West Africa and spread to other parts

of the world based on chronology of outbreaks, their infrequency and high morbidity in

Asia (Carey 1971), the absence of a vertebrate reservoir and a sylvan transmission

cycle outside of tropical Africa (Jupp and Mclntosh 1988), and phylogenetic data

(Powers et al. 2000). Phylogenetic analysis based on El sequences groups CHIKV into









three district genotypes: (1) Asian, (2) East/Central/South African, and (3) West African

(Parola et al. 2006; Schuffenecker et al. 2006).

Historical and Current Epidemics

Since its initial discovery in 1953, epidemics of CHIKV have occurred on multiple

continents. In the 1960s and 1970s, outbreaks were recorded in Thailand, India,

Vietnam, Cambodia, Myanmar and Sri Lanka (Jupp and Mclntosh 1988; Rao 1971). In

the 1962 to1964 outbreak in Bangkok, Thailand, seroprevalence rates were between

70-85% in 20 to 70 year olds (Halstead et al. 1969). Outbreaks from 1963 to 1973 in

India sickened hundreds of thousands of people (Rao 1971). From the late 1950s

through the 1990s CHIKV was isolated from multiple countries in central and southern

Africa as well as from West Africa (Powers et al. 2000). More recent outbreaks include

the 1999 to 2000 epidemic in the Democratic Republic of Congo (Pastorino et al. 2004)

and the 2001-2003 outbreak in Indonesia and Malaysia (Kit 2002; Laras et al. 2005).

A large-scale epidemic of CHIKV began in 2004 on Lamu Island, Kenya and then

spread to Mombasa (Chretien et al. 2007; Sergon et al. 2008). In 2005 and 2006 CHIKV

moved onto the Indian Ocean island nations starting with the Comoros, then Reunion,

and on to the Seychelles, Mauritius, and Madagascar. Extrapolation from

seroprevalence data suggests that on Grande Comore island (population 341,000)

nearly 215,000 people (63% of the population) may have been infected during the

outbreak (Sergon et al. 2007) and on the island of Reunion (population 770,000) alone

approximately 241,000 clinical cases (31% of the population) were reported (Paquet et

al. 2006). As the epidemic continued, 1.39 million suspected cases were reported in

India in 2006 and tens of thousands of additional suspected cases were identified in

2007 (Arankalle et al. 2007; NVBDCP 2007). Local transmission was reported in two









small towns in the Italian province of Ravenna in the summer of 2007 resulting in 200

human cases (Rezza et al. 2007; Watson 2007). The epidemic continues with additional

cases reported throughout South East Asia, India, and Sri Lanka where cases were

reported through 2009 and into 2010 (International Society for Infectious Diseases

2009-2010). Furthermore, multiple cases have been imported into other areas of

Europe, the United States, Canada and many other countries through the movement of

infected travelers (Lanciotti et al. 2007, International Society for Infectious Diseases

2009-2010)

Isolates from the current outbreaks of CHIKV are most closely related to strains in

the East/Central/South African genotype (Parola et al. 2006, Schuffenecker et al. 2006,

Arankalle et al. 2007, Njenga et al. 2008). Since the outbreak, full genome sequences

have been published for multiple isolates from Kenya, Indian Ocean islands, India, and

South East Asia and are available in GenBank.



CHIKV Interactions in Epidemic Vectors: Aedes aegypti and Ae. albopictus

In past CHIKV outbreaks, Ae. aegypti has been implicated as the dominant vector,

with virtually all Asian mosquito isolates coming from this species (Powers and Logue

2007). Early on in the recent outbreaks (2004-2005), both in Kenya and on the Comoros

islands, Ae. aegypti was the vector responsible for CHIKV transmission (Sang et al.

2008, Njenga et al. 2008). However, as the epidemic moved onto Reunion Island Ae.

albopictus was the only vector present (Delatte et al. 2008). Aedes albopictus was also

the sole vector on the Lakshadweep islands in the Indian Ocean (Samuel et al. 2009),

and in Ravenna, Italy (Rezza et al. 2007), which all experienced CHIKV outbreaks

between 2005 and 2008. Similar patterns are documented in DENV epidemics, in that in









the absence of Ae. aegypti, Ae. albopictus acts as the primary vector of DENV (Ali et al.

2003; Effler et al. 2005; Xu et al. 2007). There were also some outbreak localities where

although Ae. aegypti is common, Ae. albopictus was still the primary vector of CHIK as

was the case in the West African country of Gabon (Pages et al. 2009).

In other recent CHIKV outbreak regions where Ae. aegypti and Ae. albopictus

distributions overlap there is evidence that both mosquitoes are transmitting the virus. In

Singapore, in 2008, larval surveys identified Ae. albopictus as the predominant species

in certain locations, with Ae. aegypti also present and adult mosquito surveillance

yielded both Ae. albopictus and Ae. aegypti adult females positive for CHIKV (Ng et al.

2009). Similar patterns were found in Thailand, where wild-caught adults of both

species were positive for CHIKV (Thavara et al. 2009). Entomological surveys done

during the 2006 CHIKV outbreak in the southern Indian state of Kerala found high

densities of Ae. albopictus, and in 2007 in some of the worst CHIKV-affected districts

(Alappuzha, Kottayam and Pathanamthitta) of Kerala state, Ae. albopictus constituted

85-92% of the total mosquito juveniles collected and in a follow-up survey this mosquito

made up 58-76% of the totals (Kumar et al. 2008). In the north eastern Indian state of

Orissa, entomological surveys revealed the presence of both Aedes species with Ae.

albopictus having a slightly higher density than Ae. aegypti (Dwibedi et al. 2009).

The increased global presence of Ae. albopictus, along with a largely non-immune

population, likely fueled the magnitude and speed of the CHIKV outbreaks. However,

there is evidence that the acquisition of a mutation in the viral gene coding the El

envelope protein, during the outbreak may have also played a role in acceleration of the

spread of CHIKV (Schuffenecker et al. 2006). In the laboratory this mutation, which









results in a substitution of valine for alanine at the 226 amino acid position (A226V) of

El, significantly increases the susceptibility of Ae. albopictus to the virus. The

mechanism causing increased viral fitness in Ae. albopictus is unknown, but there may

be some association with cholesterol dependence (Tsetsarkin et al. 2007). Aedes

albopictus became infected with, disseminated, and transmitted a Reunion island isolate

with the E1-A226V isolate at much higher rates at all blood meal titers, while there was

no change in susceptibility in Ae. aegypti when compared with a back mutated isolate

with alanine at the E1-226 position (Tsetsarkin et al. 2007). Tsetsarkin et al. (2007) then

took a historic West African CHIKV isolate, mutated it to contain the same valine El -226

substitution, and found when compared to the original West African isolate there was

increased susceptibility in Ae. albopictus, but not Ae. aegypti.

Previous laboratory studies with East African and Thai isolates of CHIKV had

already indicated that in the laboratory Ae. albopictus was a significantly more

competent vector than Ae. aegypti (Mangiafico 1971; Turell et al. 1992). Jupp and

Mclntosh (1988) suggest that since historic human viremias do not circulate much

above 7.0 logloTCIDso/ml, some populations of Ae. aegypti would be inefficient vectors

in human-to -human transmission. The superior laboratory competence of Ae.

albopictus over Ae. aegypti is also evident in the Tsetsarkin et al. (2007) study with the

emergent E1-A226V CHIKV isolate. This pattern was again confirmed in another

laboratory study in which first generation Ae. albopictus and Ae. albopictus from South

Florida were given one of four blood meals each with a 10-fold reduced titer of the

emergent E1-A226V CHIKV. Aedes aegypti showed an overall susceptibility









significantly lower than Ae. albopictus, and only Ae. albopictus individuals were infected

at the two lowest viral doses (Pesko et al. 2009).

Early on in the epidemic in 2004 and 2005 the A226V mutation was absent from

Kenyan and Comoros isolates, but the mutation then appeared in CHIKV isolates from

Reunion island in 2005 and 2006 (Njenga et al. 2008, Gould and Higgs 2009). Isolates

from the 2006 epidemic in India did not have the mutation, but isolates sequenced from

2007 Indian outbreaks did have the A226V (Yergolkar et al. 2006, Arankalle et al. 2007,

Santhosh et al. 2008). Chikungunya viral samples from the 2007 Ravenna, Italy

outbreak did have the A226V mutation where, as mentioned earlier, Ae. albopictus was

the sole vector, and the virus was introduced to Italy by an Indian national visiting

Ravenna (Rezza et al. 2007). In Singapore, CHIKV strains from the 2008 epidemic were

mixed, isolates from Ae. aegypti abundant areas did not have the A226V mutation, but

the mutation was present in virus identified from Ae. albopictus adults or in human blood

samples from Ae. albopictus dominated neighborhoods. According to Gould and Higgs

(2009), during the 2004-2009 epidemics the only mosquitoes that tested positive for

CHIKV with the El -A226V mutation were Ae. albopictus, and all positive Ae. albopictus

tested were infected with this mutant. How the continuing geographic spread of Ae.

albopictus combined with the E1-A226V mutation in CHIKV contributed to the

magnitude of the epidemic of the past five years is currently unknown.

Vector Competence

After taking a viremic blood meal a mosquito will only be capable of orally

transmitting the virus after a series of barriers to infection are overcome (Hardy et al.

1983). Assuming there is ingestion of a sufficient quantity of virions to exceed the

infectious dose threshold (Chamberlain and Sudia 1961), the virions that enter the









midget lumen generally bind to the membrane of the midgut epithelial cells, enter the

cell cytoplasm and replicate. Infectious virions must then disseminate from the midgut

epithelial cells to the haemocoel and infect other tissues. Finally, for transmission to

occur, virions must infect and replicate in salivary gland tissue and then be secreted in

saliva during feeding on a subsequent host. The relationship between mosquito species

and virus is often specific and the presence of one or more of the barriers in the

described processes results in a mosquito that is an incompetent vector for a given

virus.

However, even when a mosquito is determined to be a 'competent vector', both

intrinsic and extrinsic factors can affect the susceptibility of a vector for a pathogen

(Hardy et al. 1983). A vector's susceptibility to and ability to transmit an arbovirus can

vary both inter- and intra-specifically, as is the case with Ae. albopictus and Ae. aegypti

susceptibility for certain strains of CHIKV (Turell et al. 1992). Intrinsic (genetic) factors

of the mosquito that influence vector competence include: behavior, physiology, and

metabolism and thus, vector competence is thought to be a complex phenotypic trait

under the control of multiple genetic loci (Bosio et al. 1998).

The Environment and Vector Competence

Extrinsic (environmental) factors can also exert a strong influence on mosquito

vector competence, with temperature studies accounting for the majority of research on

the topic. Within limits, increased temperature during the adult life stage has a positive

influence on vector competence. In studies with West Nile Virus (WNV) (Dohm et al.

2002, Reisen et al. 2006, Richards et al. 2007), Western equine encephalomyelitis

(Reisen et al. 1993) and multiple other viruses, temperature is shown to increase the

number of individuals infected with or transmitting the virus. In addition, temperature has









an inverse relationship with an arthropod vector's EIP (Chamberlain and Sudia 1955),

shortening the time between infection and the subsequent ability to transmit the virus.

The Larval Environment, Vector Competence, and Mosquito Size

Arboviral diseases are ecologically complex, and interactions between larval

mosquitoes and their aquatic environment can influence adult transmission dynamics.

Biotic and abiotic variables such as temperature, larval nutrition, and inter- and intra-

specific competition can alter the life history and the intrinsic physiology of mosquitoes

in ways that affect the ease with which individuals become infected and transmit a virus.

Previous studies have shown that larval rearing temperature can affect mosquito

competence for several arboviruses, including Ae. taeniorhynchus for Rift Valley fever

(RVFV) and Venezuelan equine encephalitis (VEEV) (Turell 1993), Culex annulirostris

for Murray Valley encephalitis (MVEV) (Kay et al. 1989a), Cx. tritaeniorhynchus for

Japanese encephalitis (JEV) (Takahashi 1976), and Cx. tarsalis for western equine

encephalitis (WEEV) (Hardy et al. 1990) viruses. Unfortunately, none of these studies

reared mosquitoes individually to separate temperature and density effects. In contrast,

no rearing temperature effect was found in Cx. tritaeniorhynchus for WNV (Baqar et al.

1980) and Aedes vigilax for Ross River virus (RRV) (Kay and Jennings 2002). With the

exception of Cx. tritaeniorhynchus for JEV (Takahashi 1976), adult females showed

reduced vector competence with increased rearing temperature.

Larval nutrition can also influence the ability of adult mosquitoes to become

infected with and transmit virus. Cx. tritaeniorhynchus and Ae. triseriatus nutrient-

deprived larvae were more susceptible than their well-fed counterparts, for WNV (Baqar

et al. 1980) and better transmitters of La Crosse virus (LACV) to suckling mice

(Grimstad and Haramis 1984; Grimstad and Walker 1991). However, in other studies,









nutritional deprivation of larvae had no effect on vector competence of Cx. annulirostris

for MVEV (Kay et al. 1989b) or Ae. vigilax for RRV (Jennings and Kay 1999). When an

effect was detected, low nutritional resources produced adult mosquitoes more

susceptible to virus.

Aedes triseriatus adults produced in low intra- and interspecific competition

treatments with Ae. albopictus had higher infection and dissemination rates for LACV

(Bevins 2008). The opposite was found with Ae. albopictus reared in highly competitive

larval environments, wherein adults were smaller and had higher rates of infection and

dissemination for Sindbis virus and DENV, while within the same studies a competitive

larval environment showed the same trends but did not have a significant effect on

vector competence in Ae. aegypti for the two viruses (Alto et al. 2005; Alto et al. 2008a).

In a number of the studies investigating the influence of larval environmental

factors on vector susceptibility to arboviruses, mosquito body size is measured in

addition to infection outcomes. Mosquito body size is an easily measurable physical

manifestation of larval habitat quality, and larvae reared at low temperatures are

generally larger as adults, while juveniles reared with low food availability and/or in a

competitive environment will be smaller as adults. In a few studies larval factors were

varied just as a means to produce mosquitoes of different size classes.

Sumanochitrapon et al. (1998) produced three size classes of adult Ae. aegypti, by

varying both the quantity of food and density of larvae, and found that large Ae. aegypti

females showed higher rates of oral infection with dengue virus (DENV) compared to

small and medium mosquitoes. Similar findings were reported when two size classes of

Ae. aegypti were generated through variation in larval diet, and larger individuals were









more susceptible to RRV (Nasci and Mitchell 1994). In contrast, smaller Ae. triseriatus

adults generated from field collected pupae were more likely to transmit LACV to

suckling mice (Paulson and Hawley 1991), and when Alto et al. (2008b) carried out

additional analyses in which size was examined independent of rearing conditions,

small Ae. aegypti females were more susceptible to DENV; however it should be noted

that the size range of individuals measured in this study was extremely narrow.

However, body size may not be a good predictor of how an individual mosquito

may respond to viral challenge in the form of an infectious blood meal. There may be

more critical, but not as easily measurable physiological and physical features of adult

mosquitoes that vary with larval conditions and are more correlated than size with viral

susceptibility. Furthermore, the type of larval condition that is being manipulated may

influence the strength and even possibly the direction of the response. For example,

variations in larval temperature or food level may produce mosquitoes of a similar size

range, but their responses to infection may be very different.









CHAPTER 2
LARVAL ENVIRONMENTAL TEMPERATURE AND THE SUSCEPTIBILITY OF Aedes
albopictus SKUSE (DIPTERA: CULICIDAE) TO CHIKUNGUNYA VIRUS

Introduction

Climate is one of the principal determinants of the distribution of vector-borne

diseases (van Lieshout et al. 2004). In particular, both vector development and survival

and arbovirus replication are greatly influenced by temperature. In mosquito vectors,

temperature can influence larval development time, larval and adult survival, biting

rates, gonotrophic cycle lengths, and vector size (Madder et al. 1983, Rueda et al.

1990, Scott et al. 1993). Ambient temperature can affect arboviral dynamics within the

mosquito vector by altering the length of the extrinsic incubation period (EIP), which is

the time between ingestion of an infectious blood meal to when transmission to a

subsequent host is possible (Chamberlain and Sudia 1955, Reeves et al. 1994, Patz et

al. 1996). Furthermore, temperature often defines the latitudinal and altitudinal ranges of

a vector. Species range may limit the distribution of disease when pathogens are

species specific. Temperature may also limit viral transmission in areas where the

vector is present but the temperature precludes efficient transmission (Purse et al.

2005).

Vector competence, which is the capacity of an arthropod to acquire an infection

and transmit it to a subsequent host, can greatly vary among individuals and between

populations (Lorenz et al. 1984, Turell et al. 1992) and is influenced by genetics

(Mercado-Curiel et al. 2008) as well as by climate variables, such as temperature (Davis

1932, Turell 1993, Dohm et al. 2002). An increase in environmental temperature for

adult mosquitoes reduces the EIP (Davis 1932, Chamberlain and Sudia 1955), most









likely due to an increase in the metabolism of the adult mosquito and the replication

speed of the virus.

Although the majority of research on mosquito-virus interactions has focused on

adult mosquitoes, temperature changes experienced in the immature stages of

holometabolous vectors prior to infection, may affect vector-virus interactions by

changing physical and physiological characteristics of midgut and salivary gland

barriers, which could have direct consequences on viral infection, replication, and

transmission. Previous studies have shown that larval rearing temperature can affect

mosquito competence for several arboviruses, including Murray Valley encephalitis

(MVEV) (Kay et al. 1989a), Japanese encephalitis (Takahashi 1976), and western

equine encephalitis (Hardy et al. 1990) viruses. In a specific study with Aedes

taeniorhynchus, mosquitoes reared at 19C had higher infection rates for Rift Valley

fever and Venezuelan equine encephalitis viruses than counterparts reared at 26C

(Turell 1993). In view of global climate change models, which predict changes in

temperature that will directly impact larval mosquito habitats, this study, which

investigates a previously unexplored relationship between Aedes albopictus larval

environmental temperature and chikungunya virus (CHIKV) susceptibility, could help

increase the predictability of disease transmission patterns and future outbreaks.

The intercontinental dispersal of invasive arbovirus vectors, such as the Asian

tiger mosquito Ae. albopictus, is accompanied by an increase in human vulnerability to

the exotic diseases vectored by these invaders (Juliano and Lounibos 2005). In 2005-

2006 CHIKV, a single stranded positive sense RNA enveloped Alphavirus in the family

Togaviridae, emerged as an important pathogen in the Indian Ocean Basin. On the









island of Reunion alone, 241,000 clinical cases of chikungunya fever, representing 31%

of the population, were reported (Paquet et al. 2006). A sylvan transmission cycle of

CHIKV involving mosquitoes, such as Ae. furcifer and Ae. luteocephalus, and wild

primates is limited to tropical Africa, and epidemic transmission of the virus is sustained

though infection of the mosquitoes Ae. aegypti and Ae. albopictus in urban and

peridomestic environments (Jupp and Mclntosh 1990, Diallo et al. 1999).

An unusual feature of the South West Indian Ocean CHIKV outbreak was the

increased importance of Ae. albopictus as a vector. The enhanced role of Ae. albopictus

as a CHIKV vector on Reunion island was in part due to the rarity of the primary vector

Ae. aegypti (Delatte et al. 2008), however, an amino acid substitution in the Reunion

CHIKV isolates, from alanine to valine at the 226 position of the El envelope structural

protein, has been shown to increase Ae. albopictus, but not Ae. aegypti, susceptibility to

the virus in the laboratory (Tsetsarkin et al. 2007). Following the Indian Ocean Basin

epidemic, a strain of CHIKV nearly identical (99.9% nucleotide identity) to isolates from

La Reunion emerged in India where 1.3 million human cases were reported in 13 states

in 2005-2006 (Arankalle et al. 2007). This widespread epidemic was not restricted to the

tropics, with autochthonous transmission reported in northern Italy in 2007 (Rezza et al.

2007). The epidemic continued to spread to Indonesia, Sri Lanka, and Singapore where

cases were reported through 2008 and into 2009 (Seneviratne et al. 2007, International

Society for Infectious Diseases 2008-2009).

In this study we explore how variation in temperature during larval development

affects the susceptibility of Ae. albopictus for CHIKV by measuring infection and

dissemination rates, and viral titer. We also assess how larval temperature affects









growth and survivorship by measuring larval mortality, development time to adulthood,

and adult body size.

Materials and Methods

Mosquitoes and Viruses

Aedes albopictus used in this experiment were generated from field collections of

approximately 4000 larvae and/or eggs made from June to August 2007 in Palm Beach

County, Florida. This population of Ae. albopictus was previously shown to be highly

susceptible to the Reunion (LR2006-OPY1) CHIKV strain (Reiskind et al. 2008).

Females reared from field-collected immatures were given 20% sucrose ad libitum,

blood fed weekly on live chickens, and kept in cages at 14 L:10 D, 260C (+/- 1 C SD)

and >80% rh. Chicken care followed federally mandated animal use and care policies

(University of Florida, IACUC Protocol VB-17). Eggs (F1) were hatched in tap water and,

within 24 hours after hatching, individual larvae were placed in 50 ml Falcon conical

tubes with 35 ml of tap water and 0.0105g 1:1 yeast:albumin food. Based on preliminary

studies, 0.0105g of food at the beginning of the experiment was adequate for the

completion of individual development but did not increase mortality at the higher

temperature. Larvae were reared at 180C, 240C and 320C with a 14L:10D cycle in a

Percival (Percival Corporation, Perry, IA) incubator. The experimental treatment units in

this study were different incubators which were identical in all respects except for

rearing temperature and, thus, it was assumed that differences in treatments were

caused by rearing temperature. These temperatures are within the range encountered

in the treehole environment occupied by Ae. albopictus in Florida (Lounibos 1983).

Larvae in each temperature treatment were from the same cohort of eggs whose hatch

was staggered to synchronize adult emergence among all three temperature









treatments. After the final larval instar, pupae were removed from rearing tubes, sexed

and stored in groups of 10 in water-filled vials to record adult emergences. After

emergence, all adults were held at 240C, 95-99% rh with a 14L: 10D cycle in a biosafety

level-3 facility and given 20% sucrose ad libitum.

The LR2006-OPY1 CHIKV strain, (GenBank accession number DQ443544) was

isolated in France from a febrile patient who had been infected on the island of Reunion

(Parola et al. 2006). This recently emergent strain of CHIKV contains the alanine to

valine substitution at the 226 position of the El envelope structural protein that has

been identified as a feature in many current CHIKV epidemics (Rezza et al. 2007) and

has been shown to increase Ae. albopictus susceptibility (Tsetsarkin et al. 2007). To

produce virus for infectious blood meals, a T-75 cm2 flask with a confluent monolayer of

Vero cells in 10 mL of cell culture media (M199 media supplemented with fetal bovine

serum, antibiotics and antimycotics (Invitrogen, Carlsbad, CA) was inoculated with 150

pL of previously frozen stock virus, and allowed to incubate in a 5% CO2 and 35C

atmosphere for 24 hours.

Vector Competence

Groups of 50 five to seven day-old mosquitoes were placed in 1-L cylindrical,

waxed cardboard containers (Dade Paper Co., Miami, FL) with mesh screening.

Mosquitoes were starved for 24 hours prior to being offered an infectious blood meal of

1:1000 dilution of freshly propagated CHIKV in citrated bovine blood (Hemostat

Laboratories, Dixon, CA) supplemented with ATP [5mM] as a phagostimulant. Water-

jacketed glass membrane feeders (Rutledge et al. 1964) covered with Edicoll collagen

film (Devro, Sandy Run, SC) and connected to a Haake Series F water circulator

(Thermo Haake, Paramus, NJ) were used to maintain the blood meal at 370C.









Mosquitoes were given 30 minutes to feed. Low feeding success of Ae. albopictus

made it necessary to conduct three consecutive days of feeding for individuals from all

three temperature treatments. The blood meals from the three consecutive feeding days

were assayed using qRT-PCR, with copy number standardized to plaque forming unit

(pfu) by plaque-assay performed on 10-fold serial dilutions of virus stock (Bustin 2000).

Virus titers in blood meals were loglo 4.7, 4.5, and 3.4 pfu/mL for the three feeding days,

respectively. After feeding, mosquitoes were cold anesthetized, and fully engorged

mosquitoes were removed, placed in new cages, and given 20% sucrose ad libitum.

After a 10 day EIP at 24C, surviving Ae. albopictus females were killed by

freezing. Females were stored at -80C and, after thawing, wings were removed for

measurements, bodies were assayed to determine infection status and titer, and legs

were tested to check for a disseminated infection (Turell et al. 1984). Wing length was

measured as an indicator of body size (Blackmore and Lord 2000), in millimeters, from

alula to wing tip, using digital images and a computer imaging and measurement

program (i-Solution lite, AIC Inc., Princeton, NJ).

Bodies and legs were triturated separately in 2 mL microcentrifuge tubes

containing 900pL of BA-1 media (Lanciotti et al. 2000) and two zinc-plated beads.

Samples were homogenized at 25 Hz for 3 min using a Tissuelyzer tissue

homogenizer (Qiagen Inc., Valencia, CA) and then clarified by centrifugation (3,000 x g

for 4 min). Viral RNA was extracted from 250 pL of the sample with Trizol-LS

(Invitrogen, Carlsbad, CA) following the manufacturer's instructions and using a final

elution volume of 50 pl in DEPC treated water.









One-step qRT-PCR was used to determine infection and dissemination status and

body titer by previously established protocols (Reiskind et al. 2008). Primers from the

CHIKV El gene were designed with the following sequence: forward: 5'-ACC CGG TAA

GAG CGA TGA ACT-3'; reverse: 5'-AGG CCG CAT CCG GTA TGT-3'. The probe

sequence was: 5'-/5Cy5/CCG TAG GGA ACA TGC CCA TCT CCA /3BHQ_2/-3' (IDT

DNA, Coralville, IA).

Statistical Analysis

Kruskal-Wallis tests followed by Dunn's pairwise multiple comparisons (family a =

0.05) were used to determine differences among treatments in distributions of wing

lengths and development times to adulthood (SAS 9.1, SAS Institute, Inc., Cary, NC).

Males and females were analyzed separately because of gender-specific sizes and

developmental times in this species. Chi-squared tests of independence were used to

determine the effect of temperature on survivorship to adulthood. If significant effects

were detected, post-hoc pairwise comparisons were made with an alpha adjusted

(Bonferroni) correction to account for multiple comparisons (Minitab 15, Minitab Inc.,

State College, PA).

A generalized linear mixed model (PROC GLMMIX) was used to describe

relationships among temperature (fixed effect), feeding day (random effect), infection

rate (# with virus/# fed), dissemination rate (# with virus in their legs/# with virus), and

population dissemination rate (# with virus in their legs/# fed) specifying a logistic link

function and a binomial error distribution. Odds ratios (OR) and 95 % confidence

intervals for infection at each temperature treatment were also calculated (SAS 9.1). A

linear mixed model (PROC MIXED) was used to test for effects of temperature

treatments (fixed) and feeding day (random), which were both class variables on body









titer, a continuous variable. Titer data did not fit the model assumptions of normality, but

approximate normality was achieved through a log transformation of titer values (SAS

9.1). Wing length comparisons, pooled across all temperature treatments, between

binomial variables: infected versus uninfected and disseminated versus non-

disseminated were analyzed with t-tests (Minitab 15).

Results

Growth and Mortality

Wing lengths and development times to adulthood were significantly affected by

temperature for both females (wing length: H= 417.5; df = 2; P<0.0001, development

time: H= 1279.6; df = 2; P <.0001) and males (wing length: H= 2127.4; df = 2; P

<0.0001 and development time: H= 1697.3; df = 2; P <0.0001) (Table 2-1). There was

an inverse relationship between wing length and temperature with the largest adult

mosquitoes produced at 180C and the smallest mosquitoes produced at 320C.

Mosquitoes reared at the lowest temperature (180C) took over two times longer to

develop than those at 24C and 32C. Juvenile mortality rates at 180C, 24C, and 32C

were 9.16% (n = 1454), 7.34% (n = 1520), and 16.90% (n = 1473), respectively. There

was a significant relationship between survivorship to adulthood and temperature (X2 =

54.123, df = 2, P<0.0001). Aedes albopictus reared at 32C were significantly more

likely to die as larvae when compared with individuals reared at 180C and 24C, with no

differences between the two lower temperatures.

Chikungunya Infection and Dissemination

There was a significant temperature treatment effect on the percentage of females

that developed CHIKV infections (F=16.92, df = 2, P<0.0001) (Figure 2-1). Infection was

6 times more likely in adult females reared at 180C than at 32C (Odds Ratio (OR) =









6.052; 95% CI 3.22-11.373), and females reared at 24C were 2.7 times more likely to

be infected than those reared at 32C (OR = 2.722; 95% CI 1.385-5.351). Females

reared at 180C were 2.2 times more likely to be infected than those reared at 240C (OR

= 2.223; 95% CI 1.328-3.722). Among the infected individuals the proportion of females

that developed disseminated infections did not vary significantly among larval rearing

temperatures (F = 0.85, df = 2, P > 0.4293), however there was a significant

temperature effect on population dissemination rate (F=6.20, df = 2, P<0.0022) (Figure

2-1). Population dissemination rate was approximately 5 times higher in adult females

reared at 180C than at 320C (OR = 4.905; 95% CI 1.814-13.271) and 2.3 times higher in

females reared at 180C than at 240C (OR = 2.291; 95% CI 1.088-4.827). There was no

significant difference in population dissemination rates between the 240C and 32C

treatments.

After the 10-day EIP, no significant variation in body titer of virus positive

mosquitoes was observed among the temperature treatment groups (F = 1.14, df = 2,

P>0.4300). Aedes albopictus females that were positive for CHIKV infection, in all three

temperature treatments were significantly larger than uninfected females (t = -3.59, df =

327, P = 0.0004) as measured by mean wing length. There was no significant difference

in mean wing lengths between females with disseminated and non-disseminated

infections (t = -0.41, df = 94, P = 0.6830).

Discussion

Due to the impact of climate on vector ecology, mosquito-borne diseases will be

sensitive to projected changes in global temperatures. In this laboratory study we

demonstrated that larval rearing temperature can influence survival, development time,

and wing length, and may directly impact disease transmission by influencing the









likelihood of infection with CHIKV. Although the proportion of infected females that

developed disseminated infections did not differ significantly among the three larval

rearing temperatures, the population dissemination rates were significantly higher at

18C, when compared to the two higher temperatures of 24C and 32C. Disseminated

infection is generally accepted as a measure of a mosquito's ability to transmit a virus

through biting (Turell et al. 1984). The rate of dissemination, when expressed as a

percentage of the number of mosquitoes infected, may provide information about the

effect of a midgutt escape barrier" moderating whether gut infections are able to

disseminate into the hemolymph. In this study, individuals reared at 32C had

significantly lower infection rates, but no significant difference was found in

dissemination rate. Thus, it can be speculated that there may be a reduced midgutt

escape barrier" in mosquitoes derived from the higher rearing temperatures. On the

other hand, the population dissemination rate, expressed as a percentage of the total

number of mosquitoes tested, was greater for mosquitoes from 180C, and is

epidemiologically more important in that it gives an estimate of the vector competence

or the transmission potential of a population. Body titers did not differ among

temperature treatments for mosquitoes with disseminated infections. Although this

result was somewhat surprising, it is possible that after the 10-day EIP virus titers

stabilized to the extent that treatment effects on titer were diminished. Additionally, only

a limited number of mosquitoes developed disseminated infections and, although the

mean titer of disseminated mosquitoes from 180C was higher, it was not significantly

different from the other treatments.









The higher temperature of 32C decreased survivorship, when compared with

180C and 24C. This was unexpected because results from preliminary experiments

showed no difference in survivorship between the three temperature treatments. The

increased mortality could have been due to the stress of high temperature or the

interaction of high temperature and excess nutritional resources resulting in the

proliferation of detrimental micro-organisms in the larval environment. Larvae took

longer to develop at cooler temperatures which produced larger adults. Mean wing

length differences between successive temperature treatments were approximately 0.2

mm, confirming for Ae. albopictus that adult body size, within limits, exhibits an inverse

relationship with larval rearing temperature (Briegel and Timmermann 2001). Upper and

lower thermal limits for the growth of Ae. albopictus larvae are approximately 11 C and

35C, at which temperatures larval development is inhibited, eventually resulting in

mortality (Hawley 1988, Monteiro et al. 2007).

Our data indicate that at lower larval rearing temperatures there is an increased

likelihood of an adult female becoming infected with CHIKV virus. This may have had a

positive effect on CHIKV infection rates in locations such as highlands of Reunion

island, where entomological surveys recovered Ae. albopictus at elevations of 1200

meters and temperatures as low as 12.6C (Delatte et al. 2008). However, 12.6C is a

lower larval environmental temperature than what was investigated in this study and

therefore it is uncertain whether the relationship between reduced temperature and

higher infection rates would hold true at this temperature. The ability of Ae. albopictus to

tolerate low temperatures and adapt to diverse ecological environments combined with

their vector competence for currently circulating CHIKV isolates may help to explain the









2007 northern Italy CHIKV outbreak and increases the potential for future epidemics in

other temperate areas where Ae. albopictus is abundant.

This study focused only on the influence of larval temperature, and adults were

maintained at a common temperature of 24C. How combinations of different adult and

larval temperatures may affect vector competence was not addressed. It is likely that

adults maintained at the lower temperature will have decreased virogenesis and a

longer EIP resulting in a decreased probability of transmission. Therefore, the

maintenance of low adult temperatures may result in a reduction or elimination of any

benefit low rearing temperature may have on increasing vector competence.

Results from this study are consistent with other systems where arboviral vector

competence was reduced in female mosquitoes that were reared at higher compared to

lower temperatures (Kay et al. 1989a, Hardy et al. 1990, Turell 1993). Unfortunately,

none of the previous studies that explored larval temperature effects on adult arboviral

susceptibility, reared mosquitoes individually to separate temperature and density

effects, nor did they measure variables such as wing or body size and survivorship, as

we have done in this study.

In our work, lower rearing temperature not only produced mosquitoes that were

more susceptible to viral infection but were also significantly larger. Blood consumption

by females is a function of size and large females are known to imbibe more than twice

as much blood as smaller females (Briegel 1990). Thus, in smaller mosquitoes reared at

the higher temperature, the imbibing of a lower blood volume would decrease the initial

viral dose and, in combination with a low CHIKV titer in the blood meal, limit the

establishment of infection. Low initial exposure to virus may not affect dissemination,









which requires post-infection virus replication. If this explains why large mosquitoes

derived from cooler temperatures have higher infection rates than smaller mosquitoes

then we would expect that titers of freshly engorged mosquitoes will increase with body

size, which will be tested in future studies. We also predict that different sized

mosquitoes derived from different temperatures and fed an infectious blood meal with a

high virus titer would overcome a threshold infectious dose and may result in similar

infection rates.

Because the variation in size was achieved through different temperature

treatments it is difficult to separate the effect of temperature from the response variable

body size. There may be other temperature dependent phenotypic traits that vary in a

way so as to cause an increase in infection when adults are subjected to a lower

temperature larval environment that we did not measure. Previous studies show a lack

of consistency in relationships between vector size and pathogen transmission. Large

adult Ae. aegypti females from low density larval conditions showed higher rates of oral

infection with dengue virus (DENV) compared to two other size classes from higher

density larval conditions (Sumanochitrapon et al. 1998), and similar findings were

reported for Ae. aegypti and Ross River virus (Nasci and Mitchell 1994). In contrast, Ae.

albopictus adults reared in competitive larval environments were smaller and had higher

rates of infection and dissemination for Sindbis virus and DENV, while within the same

studies a competitive larval environment did not have a significant effect on vector

competence in Ae. aegypti for the two viruses (Alto et al. 2005; Alto et al. 2008a). In Ae.

aegypti, when size was examined independent of rearing conditions small adults were

more susceptible to DENV, however the size range of individuals measured was









extremely narrow (Alto et al. 2008b). In contrast, larger Ae. triseriatus adults produced

through variation in competitive treatments had higher infection and dissemination rates

for La Crosse virus (LACV) (Bevins 2008). In nutritional deprivation studies with Cx.

tritaeniorhynchus and Ae. triseriatus, smaller mosquitoes derived from nutrient-deprived

larvae were more susceptible than their well-fed, larger counterparts, for West Nile Virus

(Baqar et al. 1980) and better transmitters of LACV to suckling mice (Grimstad and

Haramis 1984; Grimstad and Walker 1991). Smaller Ae. triseriatus adults generated

from field collected pupae were more likely to transmit LACV to suckling mice (Paulson

and Hawley 1991). However, nutritional deprivation which led to small mosquitoes had

no effect on vector competence in Cx. annulirostris for MVEV (Kay et al. 1989b) and Ae.

vigilax for Ross River virus (Jennings and Kay 1999). These inconsistencies in the

effect of size on vector competence could be based on the intrinsic differences between

vector-viral systems, however it is also possible that larval conditions, such as high

temperature and low nutrients or competition produce small mosquitoes by different

mechanisms that differently effect their competence as vectors.

In summary, cooler rearing temperatures produced mosquitoes that were larger,

had higher survival, and were more likely to become infected with CHIKV, emphasizing

the importance of the mosquito larval environment in determining adult vector-virus

interactions. Future studies should explore the connection between larval rearing

temperature-infection patterns observed in the laboratory to patterns in the field and

how climate and climate change may continue to impact the mosquito larval

environment and the epidemiology of CHIKV.









Table 2-1. Aedes albopictus treatment medians and interquartile ranges (IQR= 25th percentile -75th percentile) for
development time to adulthood and wing length. Medians with different letters in the same row are significantly
different from one another.


Female time to adulthood (days)
Interquartile range
(n)
Male time to adulthood (days)
Interquartile range
(n)
Female wing length (mm)
Interquartile range
(n)
Male wing length (mm)
Interquartile range
(n)


180C
24.5a
23.0-26.0
(515)
22.5a
21.5-24.0
(817)
3.40a
3.29-3.51
(347)
2.83a
2.77-2.90
(745)


240C
11.0b
10.0-11.5
(556)
10.0b
9.5-10.5
(860)
3.14b
3.00-3.26
(150)
2.61b
2.55-2.66
(810)


320C
9.0c
8.5-9.5
(445)
8.0c
7.5-8.5
(791)
2.91c
2.80-3.01
(207)
2.44c
2.37-2.51
(740)
















18*C (190)
50 24*C
32C
C
.o 40 (110)
o

30


(93)
20 -


A
10
2.6 2.8 3.0 3.2 3.4
55

50

45
c
0
0 40 (100)

E 35

S 30 (18) (40)

25

20

15
2.6 2.8 3.0 3.2 3.4
22

20 -(190)
o
1 18

E 16

.M 14

O 12
0
", (110)
10

o 8

6 (93)
SC

2.6 2.8 3.0 3.2 3.4

Wing length (mm)


Figure 2-1. Bivariate plots of mean wing lengths (SE) and CHIKV susceptibility A)
percent infection, B) percent dissemination, and C) percent population
dissemination grouped by treatment. Numbers in parentheses above symbols
represent number of mosquitoes tested.









CHAPTER 3
LARVAL TEMPERATURE AND NUTRITION ALTER THE SUSCEPTIBILITY OF Aedes
aegypti L. (DIPTERA: CULICIDAE) MOSQUITOES TO CHIKUNGUNYA VIRUS

Introduction

In recent years chikungunya virus (CHIKV) has emerged as an important agent of

human arboviral epidemics sickening millions of people worldwide (National Vector

Borne Disease Control Programme (NVBDCP) 2007, International Society for Infectious

Disease 2005-2010). Chikungunya, a single stranded enveloped, positive sense RNA

alphavirus (Family Togaviridae), was first isolated by Ross in 1953 from the blood of a

febrile patient in Tanzania (Ross 1956) and, although endemic CHIKV was known to be

rarely fatal, symptoms of the disease include high fevers, rashes and severe and

debilitating arthralgia (Robinson 1955, Ligon 2006). In its native African range, CHIKV is

a zoonosis, with wild primates serving as hosts and sylvatic Aedes spp. as vectors.

However, in the invasive range of CHIKV, humans are the main host and Ae. aegypti

and Ae. albopictus are the vectors (Jupp and Mclntosh 1988). Aedes aegypti, which

through the exploitation of man-made habitats spread from Africa to tropical and

subtropical regions across the globe, is the primary epidemic vector of dengue, yellow

fever, and, historically, CHIKV (Tabachnick 1991, Powers and Logue 2007). During

previously documented Asian CHIKV epidemics all mosquito isolates were solely from

Ae. aegypti (Powers and Logue 2007) and, although Ae. albopictus has recently risen in

importance as a CHIKV vector, Ae. aegypti has continued to play an important role in

recent CHIKV outbreaks.

An epidemic of CHIKV began in Kenya in 2004 (Chretien et al. 2007) and spread

in 2005 and 2006 to the African island nations of the Comoros, Reunion, Seychelles,

Mauritius, and Madagascar in the Indian Ocean (Sergon et al. 2007). Chikungunya then









moved into India, where 1.39 million suspected cases were reported in 2006 and tens of

thousands of additional cases were identified in 2007 (Arankalle et al. 2007; NVBDCP

2007). Local transmission of CHIKV was reported in 2007 in the northern Italian

province of Ravenna (Rezza et al. 2007), and the epidemic continues with additional

infections confirmed in 2010 in the Maldives, Madagascar, Sri Lanka, and many South

East Asia countries, such as Indonesia, Malaysia, Thailand, and Myanmar (International

Society for Infectious Diseases 2009-2010). Furthermore, multiple cases have been

imported into other areas of Europe, the United States, Canada and many other

countries through the movement of infected travelers (Lanciotti et al. 2007, International

Society for Infectious Diseases 2009-2010).

Arboviral diseases, such as CHIKV, are ecologically complex, and the interaction

between immature mosquitoes and factors in their aquatic environment can influence

the ability of adult mosquitoes to transmit an arbovirus. Aedes aegypti larvae feed on

microorganisms and organic detritus available in their container habitats. Containers

hold all the nutrients needed by developing larvae, whose resources are often limited

leading to nutritionally stressed adult populations of Ae. aegypti (Barrera et al. 2006).

Abiotic factors such as temperature also influence such factors as larval development

and adult body size, and many variables interact with food availability to alter mosquito

life history traits (Padmanabha unpublished data).

Previous studies have shown that temperature and food availability during the

immature stages can exert a strong influence on adult mosquito vector competence,

which is the capacity of an arthropod to acquire a pathogen and transmit it to a

subsequent host (Hardy et al. 1990). Larval rearing temperature has been shown to









affect mosquito competence for viruses of Rift Valley fever (RVFV), Venezuelan equine

encephalitis (VEEV) (Turell 1993), Murray Valley encephalitis (MVEV) (Kay et al.

1989a), Japanese encephalitis (JEV) (Takahashi 1976), and western equine

encephalitis (WEEV) (Hardy et al. 1990), while nutritional deprivation has been shown

to affect vector competence for West Nile Virus (WNV) (Baqar et al. 1980) and La

Crosse virus (LACV) (Grimstad and Haramis 1984, Grimstad and Walker 1991).

It is well established that a larval environment with high temperatures and/or low

food availability will produce smaller adult mosquitoes (Keirans and Fay 1968, Briegel

1990, Rueda et al. 1990). Thus, mosquito body size is an easily measurable physical

manifestation of larval habitat quality, which has been documented in many studies

investigating larval environmental factors and arboviral susceptibility. In a few studies

larval factors were varied specifically to produce mosquitoes of different size classes to

test the effect of adult body size on arboviral susceptibility to viruses of dengue (DENV)

(Sumanochitrapon et al. 1998) and Ross River (RRV) (Nasci and Mitchell 1994).

Overall, the relationships among larval habitat quality, body size, and vector

competence are not well worked out, and results from different experiments are

conflicting. More controlled and well-designed investigations into unexplored vector-viral

systems and diverse combinations of larval ecological factors will add to a growing

understanding of this subject.

This study explores how features of larval habitat shape Ae. aegypti competence

for CHIKV, an important emerging arbovirus causing human disease. Specifically

investigated are the relationships among larval rearing temperature, food availability,

adult body size, and Ae. aegypti susceptibility to CHIKV. In Chapter Two research was









done investigating the influence of temperature at a few discrete levels in Ae.

albopictus, while in this experiment two factors are crossed to express more realistically

the variation experienced in the field by developing Ae. aegypti, another key vector of

CHIKV. A further objective of this work was to establish the effect of rearing temperature

and food availability on larval mortality and development time to adulthood. Since the

first isolation and identification of CHIKV in Africa in the 1950s, this virus has spread to

new geographic areas with human epidemics documented on multiple continents.

Understanding how larval ecological factors can affect the interaction of adult Ae.

aegypti with CHIKV may help in making predictions as to the direction and magnitude of

future outbreaks.

Materials and Methods

Mosquitoes and Viruses

Aedes aegypti used in this study were first generation progeny of approximately

3000 field collected eggs and larvae, which were collected from April to July 2008 in

Palm Beach County, Florida. Field collected females were given 20% sucrose ad

libitum, blood fed weekly on live chickens, and kept in cages under constant

environmental conditions (26 1C, 14:10 L:D photoperiod, >80% rh). Chicken care

followed federally mandated animal use and care policies (University of Florida, IACUC

Protocol VB-17). First generation eggs were hatched in tap water and, within 24 hours

(h) after hatching, individual larvae were placed in 50 ml FalconTM (BD Biosciences,

Franklin Lakes, NJ) conical tubes with 35 ml of tap water and 10.5 mg or 3.0 mg 1:1

yeast:albumin food. Based on preliminary studies, 3.0 mg given to a larva at the

beginning of the experiment was the lowest level of food that allowed for the completion

of development to adulthood without significant reduction in mortality, and 10.5 mg was









the highest level of food that could be given to an individual without a marked increase

in mortality due to fouling of the aquatic environment.

Larvae were individually reared at 20, 27, and 34C with a 14L:10D cycle. Thus,

the experiment was a 3x2 factorial design with temperature as one factor at three levels

and food as the second factor at two levels. Larvae in each temperature and food

treatment were from the same cohort of eggs whose hatch was staggered to

synchronize adult emergence among all treatments. After the final larval instar, pupae

were removed from rearing tubes, sexed and stored in groups of 20 in water-filled vials

to record adult emergences. After emergence, all adults were held at 27C, 95-99%

relative humidity (rh) with a 14 L:10 D cycle in a Percival (Percival Corporation, Perry,

IA) incubator in a biosafety level-3 facility and given 20% sucrose ad libitum.

The LR2006-OPY1 CHIKV strain, (GenBank accession number DQ443544) was

isolated in France from a febrile patient who had been infected on the island of Reunion

in 2006 (Parola et al. 2006). Previously tested Ae. aegypti individuals from Palm Beach

County were shown to be highly susceptible to the Reunion (LR2006-OPY1) CHIKV

strain (Reiskind et al. 2008), which contains the alanine to valine substitution at the 226

position of the El envelope structural protein (El A226V) that has been identified as a

dominant genotype in many current CHIKV epidemics (Rezza et al. 2007). Virus for

infectious blood meals was produced by inoculating a confluent monolayer of Vero cells

in a T-75 cm2 flask with 250 pL of previously frozen stock virus, and incubating them in

a 5% CO2 atmosphere (atm) at 350C for 48 h. After 48 h, infectious blood meals were

made by combining freshly recovered media-viral suspension with citrated bovine blood

(Hemostat Laboratories, Dixon, CA) in a 1 to 20 ratio. Concentration of virus in fresh









blood meals was 6.3 Loglo plaque-forming units (PFU)/mL, which was measured by

plaque assay performed in duplicate 6-well plates of confluent Vero cells. Ten-fold serial

dilutions (to the 10-9 dilution) of infectious blood meal samples were prepared by

combining 0.1 ml of the CHIKV infected blood meal with 0.9 ml BA-1 media (Lanciotti et

al. 2000) and repeating the process. Each cell well was inoculated with 0.1 ml of a

dilution, plates were incubated for 1 h at 5% C02 atm at 35C, before a first overlay of

agarose was applied to the cell monolayer. The second overlay was applied two days

later, the plate was read the following day, plaques were counted, and final viral

concentrations were expressed in PFUs per ml of blood meal.

Mosquito Infection

Groups of 100 five to seven day-old Ae. aegypti mosquitoes were placed in 1 -L

cylindrical, waxed cardboard containers (Dade Paper Co., Miami, FL) with mesh

screening. Mosquitoes were starved for 24 hours and then offered an infectious blood

meal using a water-jacketed glass membrane feeder (Rutledge et al. 1964) covered

with Edicoll collagen film (Devro, Sandy Run, SC) and connected to a Haake Series F

water circulator (Thermo Haake, Paramus, NJ) used to maintain the blood meal at

37C. Mosquitoes were given 30 minutes to feed. Immediately after feeding, mosquitoes

were cold anesthetized, and 10 fully engorged mosquitoes were removed, from each

temperature-food treatment, frozen in individual microcentrifuge tubes at -80C for

subsequent wing removal and measurement, trituration, viral RNA extraction, and

quantitative RT-PCR. Wings were removed from each mosquito using forceps that were

sterilized with 100% ethanol followed by intense flaming using a portable one-touch

burner (Daigger). Wing length was measured in millimeters as an indicator of body

size (Blackmore and Lord 2000) from the alula to wing tip, excluding wing fringe. Digital









images of the wing were captured and measured using a computer imaging and

measurement program (i-Solution lite, AIC Inc., Princeton, NJ). These individuals were

used to determine the relationship between mosquito size and quantity of virus initially

ingested by freshly feed females.

The remainder of the engorged mosquitoes were held for a 10 day EIP at 27C

and provided with 20% sucrose ad libitum, after which surviving Ae. aegypti females

were killed by freezing. Females were stored in individual microcentrifuge tubes at -

800C and, after thawing, wings were removed for measurements, bodies were assayed

to determine infection status and titer, and legs were tested to check for a disseminated

infection. An assayed mosquito could be, (1) uninfected, have an (2) isolated infection,

which specified a CHIKV positive body, but legs negative for the presence of the virus

or have a (3) disseminated infection, which meant virus was found in the legs signifying

the infection had spread beyond the midgut and on to secondary organs (Turell et al.

1984). Both wings and legs of individual mosquitoes were removed using the sterilized

force technique described previously. Samples were homogenized at 25 Hz for 3 min

using a Tissuelyzer tissue homogenizer (Qiagen Inc., Valencia, CA). RNA was

extracted separately from bodies and legs. Mosquito bodies were homogenized in TRI

Reagent (Molecular Research Center, Inc., Cincinatti, OH) and then RNA was

extracted according to the manufacturer's protocol using 50 pl of DEPC treated water as

the final elution volume. Mosquito legs were homogenized in 250 pl BA-1 media

(Lanciotti et al. 2000) with two zinc-plated BBs (Daisy), which was then added to 750

pl of TRI Reagent-LS (Molecular Research Center, Inc., Cincinatti, OH) for RNA









extraction, following the manufacturer's protocol and also with a final elution volume of

50 pl in DEPC treated water.

One-step quantitative RT-PCR was used to determine infection status and body

titer of samples. Primers were designed from the El gene and had the following

sequences: forward: 5'-ACC CGG TAA GAG CGA TGA ACT-3'; reverse: 5'AGG CCG

CAT CCG GTA TGT-3'; and probe: 5'-/5cy5/CCG TAG GGA ACA TGC CCA TCT CCA

/3BHQ_2/-3' (IDT DNA, Coralville, IA). Reactions were performed in a 96-well reaction

plate, with each reaction containing: 0.4pl SuperScript III RT/Platinum Taq mix

(Invitrogen, Carlsbad, CA), 10pl 2x reaction mix (a buffer system, MgSO4, dNTPs and

stabilizers), 1 l forward primer (10 pmol/L), 1 l reverse primer (10 pmol/L), 0.4pl

fluorogenic probe (10 pmol/L), 2.2pl DEPC-treated H20, and 5pl of the test sample

RNA. Viral RNA was quantified using a Roche LC480 light-cycler (Roche Applied

Sciences, Indianapolis, IN) with the following thermal conditions: 20 minutes at 480 C

and 2 minutes at 950 C, followed by 40 cycles of PCR, 10 seconds at 950 C and 15

seconds at 600 C followed by a cool down for 30 seconds at 500 C. A negative control

(DEPC-treated water in place of sample) and a positive control (CHIKV stock virus, 10-2

dilution) were included in each reaction run.

A standard curve was generated by assaying a full range of ten-fold serial dilutions

of CHIKV virus stock (7.8 Loglo PFU/ml) by plaque assay which determined PFUs per

dilution. Viral RNA was then isolated from three replicates of each dilution using TRI

Reagent-LS (as previously described for leg aliquots), and all dilutions were assayed

using qRT-PCR. Viral concentrations and crossing point (Cp) values determined from

qRT-PCR from dilutions 10-2 through 10-6 constituted the six values used to establish a









linear regression (Cp = -3.455*Log10(PFU) + 32.2, n=6, p<0.0001, r2 =0.9985 ).

Mosquito body titers in each test sample were then calculated by comparing the test

sample with standard curve values that had been transformed into plaque-forming unit

(Cp) equivalents.

Statistical Analysis

All statistical analysis was performed using SAS software, version 9.2 (SAS

Institute, Carey NC). CHIKV mosquito titer data for the engorged female mosquitoes

killed directly after blood feeding did not fit assumptions, of normality and approximate

normality was achieved through a log transformation of titer values. Product moment

correlation analysis was then carried out between the log transformed CHIKV mosquito

body titer and the wing lengths of the engorged females. Two-way analysis of variance

(ANOVA) (PROC GLM) was used to compare the effects of temperature and food level

and their interaction on CHIKV titer in engorged females. Main effects means

(temperature and food) were compared by Tukey's studentized range tests. To

determine differences among the mean wing lengths of the engorged females from the

six larval treatment groups (temperature-food combinations) a two-way ANOVA (PROC

GLM) was followed by Tukey's studentized range tests. Temperature and food were

categorical variables in the two-way ANOVA analysis.

Two-way ANOVA (PROC GLM) followed by Tukey's studentized range tests was

used to determine differences among the temperature and food treatments in

distributions of the wing lengths of blood-fed females that were killed by freezing after

the 10 day EIP. Two-way ANOVA (PROC GLM) followed by Tukey's studentized range

tests was also used to detect development time differences among the temperature-

food treatments. Mean development times were determined from all mosquitoes reared,









not just blood fed females and males and females were analyzed separately because of

gender-specific developmental times in this species. The proportion of mosquitoes that

died as larvae during rearing was analyzed for significant effects of the six treatments

by maximum likelihood categorical analyses of contingency tables (PROC CATMOD).

Comparisons of mortality rates between treatments were performed with maximum

likelihood contrasts using a Bonferroni adjustment to maintain an experiment-wise a =

0.05.

Logistic regression (PROC LOGISTIC) were used to model mosquito CHIKV body

infection (# with virus/# fed) and disseminated infection (# with virus in their legs/# with

virus) by temperature, food level, and temperature x food interaction, specifying a

logistic link function and a binomial error distribution. Odds ratios (OR) and 95 %

confidence intervals for infection and disseminated infection by treatment were also

calculated.

Two-way ANOVA was used to test for effects of temperature, food level, and a

temperature x food interaction on mosquito body titer following the 10 day EIP.

Differences between main effect means (temperature and food) were further analyzed

by Tukey's studentized range tests. Titer data did not fit the model assumptions of

normality, but approximate normality was achieved through a log transformation. To

determine if there was a difference in size among uninfected, infected (non-

disseminated), and disseminated females a one-way ANOVA (PROC GLM) was used.

The one-way ANOVA was used to compare differences in mean wing length among the

females of the three infection status categories. These were females that blood fed,









completed the 10 day EIP and were then pooled across all larval treatments for the size

analysis.

Results

Chikungunya Titer of Freshly Engorged Mosquitoes

Correlation analysis showed a significant positive correlation between mosquito

wing length and log transformed-CHIKV body titer (r = 0.5787, P<0.0001, df = 56)

(Figure 3-1). When tested by two-way ANOVA, the log-transformed mean titers of

CHIKV in freshly engorged females were significantly affected by larval rearing

temperature (F= 20.97, df = 2, P<0.0001), but not larval food level (F= 1.25, df = 1,

P<0.2683), nor the temperature x food level interaction (F= 1.59, df = 2, P<0.2140)

(Figure 3-2). Females reared at two lower temperatures of 20 and 27C had significantly

higher CHIKV titer in mosquito bodies than those reared at 34C (Tukey's studentized

range tests, P < 0.05). Wing lengths of freshly engorged females were significantly

affected by larval rearing temperature (F= 103.88, df = 2, P<0.0001), larval food level

(F= 33.69, df = 1, P<0.0001), but not the temperature x food level interaction (F= 1.50,

df = 2, P<0.2316). When followed up with pairwise comparisons using Tukey's

studentized range tests (P < 0.05) all temperature levels and food levels were

significantly different from eachother (Figure 3-2).

Growth and Mortality

Wing lengths of blood-fed females that were held through the 10 day EIP varied

significantly due to temperature (F = 577.76; df = 2; P<0.0001), food (F = 235.66; df = 1;

P<0.0001), and the temperature x food interaction (F= 7.58, df = 2, P= 0.0006).

Development time to adulthood in females was also significantly affected by larval

rearing temperature (F = 16187.6; df = 2; P<0.0001), food (F = 100.48; df = 1;









P<0.0001), and the temperature x food interaction (F= 46.34, df = 2, P<0.0001). There

were also significant effects in male development due larval rearing temperature (F =

16614.9; df = 2; P<0.0001), food (F = 130.55; df = 1; P<0.0001), and the temperature x

food interaction (F= 51.97, df = 2, P<0.0001) (Table 3-1). Larger mosquitoes were

generated from the lower temperatures and higher food treatments.

Juvenile mortality rates at the low and high food levels within the three

temperature treatments, 20C, 27C, and 34C, respectively were 2.37% (n = 969),

2.05% (n = 975), 2.90% (n = 966), 2.53% (n = 1029), 2.89% (n = 1073), and 6.42% (n =

1028) (Figure 3-3). There was a significant difference in mortality among treatments (X2

= 39.67, df = 5, P<0.0001). The maximum likelihood contrasts showed that the 340C-

high food level treatment at 6.42%, had a significantly higher juvenile mortality rate than

all other treatments (Table 3-2).

Chikungunya Infection and Dissemination

There was a significant temperature (X2=26.0248, df = 2, P<0.0001) effect on the

likelihood of females developing CHIKV infections, but food level (X2=1.1108, df = 1, P =

0.2919) and the interaction between temperature and food level (X2=4.5452, df = 2, P =

0.1030) were not significant (Figure 3-4). Infection was 5.4 times more likely in adult

females reared at 27C than at 20C (Odds Ratio (OR) = 5.428; 95% Confidence

Interval (CI): 2.798-10.532) and females reared at 270C were 4.7 times more likely to be

infected than those reared at 340C (OR = 4.768; 95% CI: 1.980-11.485). There was no

significant difference in CHIKV infection between females reared at 200C and at 34C

(OR = 1.138; CI: 0.525-2.468).

Among the infected individuals the proportion of females that developed

disseminated infections was significantly affected by larval rearing temperature









(X2=8.7265, df = 2, P = 0.0127), food level (X2=5.0123, df = 1, P = 0.0252), and their

interaction (X2= 6.9914, df = 1, P = 0.0303) (Figure 3-5). Dissemination was 5.4 times

more likely at 27C compared to 34C (OR = 5.466; 95% CI: 1.4515-20.585) and 2.1

times more likely at 27C compared to 20C (OR = 2.109; CI: 1.0380-4.2832). There

was no significant difference in disseminated infections between 20 and 340C.

Dissemination was also 2.7 times more likely at the higher food level (OR = 2.7000; CI:

1.1317-6.4417).

After the 10-day EIP, when tested by a two-way ANOVA, log titer of CHIKV in

infected females was not significantly affected by larval rearing temperature (F= 2.01; df

= 2; P = 0.1353), food level (F= 0.45; df = 1; P = 0.5037), nor the temperature x food

level interaction (F= 0.56; df = 2; P= 0.5706). A one-way ANOVA showed that there was

no significant variation in wing lengths among CHIKV infection status categories (F =

1.66, df = 2, P = 0.192) (Figure 3-6).

Discussion

Ecological factors in the larval environment influence mosquito life history traits

that are important in infectious disease dynamics (i.e. growth rate, life span, biting rate)

and can directly affect traits that affect arbovirus susceptibility. Specific results from this

experiment demonstrate that temperature and food availability influence body size,

development time and CHIKV infection status, although the nature of the relationship

between body size and viral susceptibility is not clear.

Among engorged Ae. aegypti females, assayed immediately after taking a blood

meal, there was significant correlation between body size, as measured by wing length,

and body titer (Figure 3-1). Prior to the experiment it was hypothesized that larger

females would take in a greater volume of blood and, thus have a higher initial titer of









virus when assayed immediately after feeding and this hypothesis was partially

supported by the correlation analysis results. However, when the effect of larval

temperature and food quantity on freshly engorged female mosquito body titer was

analyzed via two-way ANOVA it was found that only temperature and not food level had

a significant effect on body titer, yet food level definitely had a significant effect on size

(Figure3-2). Mosquitoes reared at 34C, but given two different quantities of food were

significantly different in size, but not in body titer, while the mean titer of mosquitoes

reared at 34C was significantly lower than the mean titers of mosquitoes from 27 and

20C treatments. Thus how the body size was achieved, either by temperature

differences or food differences, was an important factor in determining the amount of

blood and virus ingested and that size alone was not the best predictor of ingested

blood volume and initial viral dose.

Larval habitat features are important in regulating the growth of individuals and

populations of mosquitoes (Rueda et al. 1990; Scott et al. 1993; Juliano 2009). In this

experiment, temperature and food availability had measurable effects on mosquito

development rate, size, and mortality. As expected, males had shorter development

times than their female counterparts due to developmental dimorphism between the

sexes. Growth rate was phenotypically plastic with respect to temperature and food

level and at the lowest temperature of 20C mosquitoes took the longest to develop with

median development time to adulthood for the low level food females of 12 days and for

the high level food treatment of 13 days. As treatments increased in temperature the

differences between development time decreased so at 27C mosquitoes only took

approximately one day longer than individuals reared at 34C to reach adulthood.









Surprisingly, mosquitoes (both male and female) reared at 20C and at 34C took

longer to develop if they were given more food as larvae. Most studies show a decline in

development time when food quantity per larvae is increased (Wada 1965, Black et al.

1989, Teng and Apperson 2000). However, Ae. aegypti reared at 60oF (15.6C) fed a

finely ground laboratory chow took 33 days to pupate at the full food treatment and 28

days to pupate at the half-food treatment (Keirans and Fay 1968), and among Ae.

aegypti larvae given different daily amounts of Brewer's yeast larvae from the lowest

food treatment did not differ from the other treatments in mean rate of pupation (Peters

et al. 1969). It is possible that at 34C the interaction between temperature and the

higher food quantity produced a polluted environment leading to a longer development

time, while the low temperature of 20C is close enough to the lower thermal

development limit of Ae. aegypti that resource utilization was unpredictable. At 27C

there was no significant difference in median development-time to adulthood between

the food levels, but the interquartile range for the low food level is wider and skewed to

include a longer development period. This temperature is probably close to the optimal

physiological temperature for Ae. aegypti (Christophers 1960) and is commonly used in

experiments, which may explain the more expected relationship of increased food

availability and decreased development time.

The three temperature and two food level combinations produced six significantly

different wing length classes of mosquitoes with differences between adjacent groups

ranging from 0.06 mm to 0.33 mm (Table 3-1). At 27C the two food levels, produced

females with a wing length difference of approximately 0.25 mm, but no difference in

development time to adulthood. The same difference in wing length, achieved through









different rearing temperatures also produced difference in development time to

adulthood. The low temperature of 20C had a more dramatic effect on size and

development time, that the two higher temperatures.

The experiment was designed to maximize adult production at temperatures and

food levels that would produce markedly different outcomes in size, development times

and responses to virus. To that effect there was an attempt to keep larval mortality at a

minimum and equal among all six temperature-food combinations. However, there was

a significant difference in mortality between the highest temperature-food combination

and all the other treatments (Figure 3-3). Although results from preliminary experiments

showed no difference in survivorship between the larval treatments the very large

sample size probably increased the ability to see even a small effect. The increased

mortality could have been due to the interaction of high temperature and excess

nutritional resources resulting in the proliferation of detrimental micro-organisms in the

larval environment.

Only temperature had an effect on CHIKV infection status, with a significantly

greater proportion of individuals infected when reared at the middle temperature of 27C

and no difference between the lower and higher temperature of 20 and 34C. In similar

studies with Aedes vigilax, larvae reared at 32C and held at 25C had lower RRV

infection rates than counterparts reared at 18 and 25C and held at 25C (Kay and

Jennings 2002), and in Ae. albopictus there was a reduction in CHIKV infection with

increasing temperature (Westbrook et al. 2009). Non-linear responses to temperature

are common in biological systems (Zhou et al. 2008) and, based on the pattern of the

results in Figure 3-4, the Ae. aegypti response to the effect of larval rearing temperature









on CHIKV infection may not be linear. Sub-optimal temperature conditions, represented

by the 20 and 34C may lessen the susceptibility of Ae. aegypti to CHIKV. This study

was designed to explore the general question of whether rearing temperature and food

availability effect CHIKV infection status in Ae. aegypti and the use of a generalized

linear model with temperature as a classed predictor variable was appropriate

considering the limited number of temperatures used. However, now that an effect has

been established future experiments will be designed with the intent of predictability.

Increasing the number of temperature levels, reducing the difference between levels

and treating temperature as a continuous variable would provide data to model the

response pattern more thoroughly.

Temperature, food availability, and their interaction had an effect on the probability

of having a disseminated infection. Significance in the interaction terms specified that

temperature had a different effect on the probability of having a CHIKV disseminated

infection dependent on food level. At 20 and 34C mosquitoes generated from high food

treatments were more likely to have disseminated infections, while the opposite pattern

was found at 27C. In the overall model disseminated infections were 2.7 times more

likely in the high food treatments. This result is in contrast with other studies on larval

nutrition and vector competence of Cx. tritaeniorhynchus for WNV and Ae. triseriatus for

LACV which showed reduced susceptibility of adults generated from nutrient-deprived

larvae (Baqar et al. 1980, Grimstad and Haramis 1984; Grimstad and Walker 1991).

Although wing length was positively correlated with the initial quantity of virus

ingested, significant wing length-infection correlations disappeared after the extrinsic

incubation period, suggesting that mosquito size alone in this vector-viral system is not









a good predictor of viral infection, dissemination or body titer (Figure 3-6). It was

originally hypothesized that a larger initial viral dose, even after the ten day EIP, may

lead to a higher proportion of larger mosquitoes with isolated infections and

disseminated infections. There was support for this hypothesis in results from other

studies. For example, large Ae. aegypti females, produced by varying food and density,

showed higher rates of oral infection with DENV compared to small and medium sized

individuals (Sumanochitrapon et al. 1998), larger Ae. aegypti generated through

variation in larval diet which were more susceptible to RRV (Nasci and Mitchell 1994),

and large Ae. triseriatus adults from competition treatments had lower infection and

dissemination rates for LACV (Bevins 2008). In contrast, smaller Ae. albopictus

generated from high competition larval environments had higher rates of infection and

dissemination for Sindbis (SINV) and dengue (DENV) viruses (Alto et al. 2005; Alto et

al. 2008a).

In this study rearing temperature and food level affected the ease with which Ae.

aegypti became infected with and disseminated CHIKV which may impact the

epidemiology of this disease. Failure to consider the importance of the larval

environment may lead to incorrect estimates of vector susceptibility. Variations in

different ecological factors in the larval habitat larval may produce mosquitoes of a

similar size range, but with very different responses to infection. Thus, in this

experiment body size was not a very good predictor of how a mosquito will respond to

arboviral infection and there may be more critical, but not as easily measurable

physiological and anatomical features of adult mosquitoes that vary with larval

conditions and are more substantially correlated with viral susceptibility. This









experiment demonstrates the significant role of larval ecology in adult vector-viral

interactions, but additional well designed experiments with predictability in mind are

required to determine more quantitatively the effects of factors such as food,

temperature, and interactions with other individuals or organisms during juvenile

development on adult vector-viral interactions.









Table 3-1. Aedes aegypti LS means and standard error for development time to adulthood and female wing length.
Temperature 200C 270C 340C
Food Level Low High Low High Low High
Male time to adulthood (days) 11.470.04a 12.180.04b 6.530.04c 6.480.04c 5.170.04d 5.550.04e
(n) (459) (488) (547) (526) (550) (513)

Female time to adulthood (days) 11.970.04a 12.680.04b 6.650.04c 6.550.04c 5.360.04d 5.750.04e
(n) (487) (467) (391) (477) (492) (449)
Female wing length (Y) (mm) 3.340.02a 3.670.02b 3.030.02c 3.280.01d 2.700.02e 2.870.02f
(n) (116) (60) (90) (91) (33) (58)


* Means from PROC GLM analysis with different letters in the


same row are significantly different from one another









Table 3-2. Maximum likelihood (ML) contrasts for comparisons of mortality rates for
340C-high food treatment with all other temperature and food level treatment
groups
ML Contrast df Chi square P
340C-high food vs.
200C-high food 1 17.72 <.0001
270C-low food 1 20.94 <.0001
270C-high food 1 13.07 0.0003
340C-Low food 1 17.02 <.0001
340C-high food 1 14.13 0.0002













4.4




E o O o
4. -4
00.4.0 -0


S3.8 -
.

3.6 -
I

3.4 -
0)




3.0
3.0 4--------
2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0

Wing length (mm)
Figure 3-1. Correlation between log transformed whole mosquito body titers of CHIKV
and wing lengths for engorged Aedes aegypti females killed immediately after
feeding (r= 0.5787, P<0.0001).
















9.6 -






0
-j-
9.4 -


9.2- b b



O

8.8


8.6 +


8.4
2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8

wing length (mm)


Figure 3-2. Bivariate plot of LS means (SE) for wing lengths and log transformed
CHIKV body titers for engorged Aedes aegypti females killed immediately
after feeding. Filled symbols represent the low food treatment and open
symbols the high food treatment, squares are 34C, triangles 27C, and
circles 20C. Different letters indicate significant differences among means of
log transformed CHIKV body titers. Symbols within dashed ellipses do not
have significantly different wing lengths.










0.08 *
(1028)


? 0.06-



4- 0.04
o (966) (1073)
c- (1029)
.o (969)
t (975)
2 0.02



0.00
Low High Low High Low High
20C 20C 27C 27C 34C 34C

Food Level
Temperature

Figure 3-3. Juvenile mortality rates at the low and high food levels within the three
temperature treatments for Aedes aegypti. *Significantly different from other
five treatments based on results from maximum likelihood contrasts.










1.0
(91)
0.8 (92)
(58)
(60) (33)
4- 0.6 -
S0.6 (116)











200C 200C 27C 27C 34C 34C

Food Level
Temperature

Figure 3-4. Proportion of Aedes aegypti females (SE) in each temperature treatment
infected with CHIKV. Numbers in parentheses are the number of blood fed
females in that treatment group.
females in that treatment group.










0.8

(35)
(76)
.6 (67) (36)


((56)
C


S0.4
C
I (17)
0
2 0.2



0.0
Low High Low High Low High
20C 20C 27C 27C 34C 34C

Food Level
Temperature

Figure 3-5. Proportions of infected Aedes aegypti females (SE) from temperature and
food level treatments with disseminated CHIKV infections. Numbers in
parentheses are the number of blood fed females in that treatment group.













3.35
I1I Uninfected
eZZ] Infected (non-disseminated)
3.30 Disseminated Infection


3.25 (160) (155)
E
S3.20 (133)


3.15


3.10


3.05


3.00

Figure 3-6. Least squared means (SE) for sizes of adult female Ae. aegypti
mosquitoes in CHIKV infection status categories.





































80









CHAPTER 4
LARVAL TEMPERATURE, COMPETITION, AND THE VECTOR COMPETENCE OF
Aedes aegypti AND Aedes albopictus FOR CHIKUNGUNYA VIRUS

Introduction

Competition is often an important biotic mechanism in the shaping of insect

distributions and abundances (Craig et al. 1990, Settle and Wilson 1990, Human and

Gordon 1996, Kaplan and Denno 2007). In nature, resources may be scarce in a limited

area, and competing organisms often have a choice as to whether to stay and compete

or disperse to another resource patch. However, in artificial and natural containers that

house developing Ae. aegypti and Ae. albopictus, the immature competitors cannot

leave the container environment, and competition can have considerable effects on

population growth components such as, development time, size, fecundity, and survival

to adulthood (Teng and Apperson 2000, Armistead et al. 2008, Reiskind and Lounibos

2009). Furthermore, larval competition can affect adult mosquito susceptibility to an

arbovirus, which can result in changes in the distribution and transmission intensity of

an arbovirus (Baqar el al 1980, Alto et al. 2005, Alto et al. 2008, Bevins 2008).

Aedes albopictus and Ae. aegypti are invasive vectors with geographic ranges that

span large portions of the globe. Aedes aegypti is believed to have traveled to the New

World from its native Africa in water storage jars aboard slave ships (Christophers

1960), while the spread of Ae. albopictus from its native Asian range has mostly been a

more recent event in part due to the trade in used tires (Hawley et al. 1987). In many

parts of the world larvae and pupae of both Aedes species may be found developing

and feeding in the same container (MacDonald 1956, Fontenille and Rodhain 1989,

O'Meara et al. 1992, Braks et al. 2003), and resource competition within and between

these species is well documented (Juliano 1998, Braks et al. 2004).









In larval competition experiments using leaf detritus as a basal resource, Ae.

albopictus exhibited a competitive advantage over Ae. aegypti (Juliano 1998, Braks et

al. 2004), which might account for the observed decline of Ae. aegypti in areas of the

United States now inhabited by Ae. albopictus (O'Meara et al. 1995, Lounibos 2007).

Nevertheless, there are many regions of sympatry of these two species, including

southern Florida (Rey et al. 2006), and condition-specific competition has been

proposed as the process behind their sustained coexistence (Costanzo et al. 2005,

Leisnham and Juliano 2009). Aedes albopictus is a better larval competitor, but superior

desiccation resistance of the egg stage of Ae. aegypti allows greater numbers of this

species to survive during the dry season (Sota and Mogi 1992, Juliano et al. 2002).

Furthermore, Ae. aegypti presence or abundance is positively associated with lower

humidity, higher temperature, and urbanization while Ae. albopictus is negatively

associated with hot, dry climates and is more common in sites with shade and

vegetation (Hawley 1988, Braks et al. 2003, Rey et al. 2006, Reiskind and Lounibos

2009). Thus, in nature the outcome of competitive interactions between the two

mosquito species changes temporally with the dry and wet seasons and spatially with

environmental features associated with humidity and temperature.

Both Ae. aegypti and Ae. albopictus are important epidemic vectors of

chikungunya virus (CHIKV), a single stranded, positive sense enveloped RNA

alphavirus. Chikungunya virus was first isolated in 1953 from a febrile patient in

Tanzania (Ross 1956), and sporadic epidemics were recognized subsequently from

Africa, Asia, and India, but a particularly explosive epidemic of CHIKV began in 2004 in

coastal Kenya and spread throughout African nations in the Indian Ocean, infecting high









proportions of island inhabitants, and subsequently spreading to India, Southeast Asia,

Italy and other countries (Powers and Logue 2007, Gould and Higgs 2009). In previous

epidemics. Ae. aegypti was implicated as more important, with virtually all Asian vector

isolates coming from this mosquito species (Powers and Logue 2007), but in recent

CHIKV outbreaks in regions where Ae. aegypti and Ae. albopictus distributions overlap,

both species have tested positive for the virus. In Singapore, in 2008, larval surveys

identified Ae. albopictus as more common than Ae. aegypti, testing of wild-caught

mosquitoes yielded both Ae. albopictus and Ae. aegypti adult females positive for

CHIKV (Ng et al. 2009). Similar patterns were found in Thailand, where wild-caught

adults of both species were positive for CHIKV (Thavara et al. 2009). Entomological

surveys done during the 2006 CHIKV outbreak in the north eastern Indian state of

Orissa, revealed the presence of both Aedes species with Ae. albopictus having a

slightly higher abundance than Ae. aegypti (Dwibedi et al. 2009).

This study addresses whether larval rearing temperature modulates the

competitive larval interactions between Ae. albopictus and Ae. aegypti and how that

may in turn influence adult susceptibility to CHIKV. Most laboratory larval competition

studies on Ae. albopictus and Ae. aegypti have been carried out at temperatures

between 250 and 27C (Black et al.1989, Barrera 1996, Daugherty et al. 2000, Alto et al.

2005, Alto et al. 2008), with the exception of Lounibos et al. (2002) which compared

competition between these two species at 240 and 30C. The container habitat of Ae.

albopictus and Ae. aegypti larvae may be subjected to temperature lows of

approximately 120 to 14C and highs ranging from 300 to 35C or above (Lounibos

1992, Reiskind unpublished data). Physical features of the environment, such as









temperature may act in concert with, or in opposition to, biotic factors like competition to

cause changes in physical or physiological traits in mosquito vectors that alter their

susceptibility to arbovirus. Previous studies in other insect systems have shown that a

change in temperature can reverse the outcome of interspecific competition between

insects that occupy the same environment (Birch 1953, Park 1954, Ayala 1970, Russell

1986). It is also possible that an increase or a decrease in temperature may increase

the magnitude of competitive effects without changing the direction of the interspecific

competitive outcomes between Ae. aegypt and Ae. albopictus.

Materials and Methods

Mosquitoes, Temperature, and Competition

Aedes aegypti and Ae. albopictus used in this experiment were second generation

(F2) progeny of individuals collected as eggs, larvae, and pupae in St. Lucie and Palm

Beach counties in southern Florida. Adults maintained in cages under constant

environmental conditions (26 1C, 14:10 L:D photoperiod, >80% relative humidity)

were provided with 20% sucrose ad libitum, and blood meals from restrained chickens

(housed and maintained in accordance with federally mandated animal use and care

policies as part of the University of Florida, IACUC Protocol VB-17). Oviposited F2 eggs

were collected on seed germination paper. Eggs from both species were simultaneously

hatched in separate Erlenmeyer flasks with tap water under a vacuum for 30 minutes to

approximately synchronize hatching time. Newly hatched (<16 hours after hatching)

larvae were counted and added to respective competition and temperature treatments.

Competition treatments consisted of Ae. aegypti:Ae. albopictus species abundance

ratios of 200:0, 100:0, 100:100, 0:200, 0:100, with five replicates per competitive

treatment. All five competitive treatments, replicated five times, were run in incubators at









temperatures of 22, 27 and 32C, each with a 14:10 L:D photoperiod. Larvae in each

temperature treatment were from the same cohort of eggs whose hatch was staggered

to synchronize adult emergence among all treatments, which ensured that individuals of

the same species were approximately the same adult age when blood fed.

Five-liter white plastic buckets partially filled with 2500ml of tap water, 500ml of

oak leaf infusion water (O'Meara et al, 1989), and 0.15 grams (g) particulate food (1:1

lactoalbumin: Brewer's yeast) were used as larval rearing containers. Oak leaf infusion

was made by collecting fallen oak (Quercus virginiana) leaves, oven drying them for 48

hours at 80C, combining 35.5 g of leaves per liter of tap water and letting it incubate at

27C for ten days. A total of 50 liter of infusion was prepared and then frozen so that all

buckets, some of which were set up on different days because of staggered

temperature treatments, would receive infusion that was derived from the same starting

material and treated identically. Enough oak leaf infusion to set up all the competitive

treatments and replicates was thawed for 12 hours before adding it to each 5 L bucket.

The 0.15 g of particulate food allowed for competitive interactions between Ae. aegypti

and Ae. albopictus in preliminary experiments with limited mortality so that enough

adult females were produced for the CHIKV infection portion of the study, as

accomplished in similar experiments using other arboviruses (Alto et al. 2005, 2008a).

Pupae were collected daily, sexed and identified to species in the mixed

treatments, and the pupation date in days since hatching was recorded. Pupae were

collected until all individuals emerged or died. All female pupae from a given replicate

were placed in a water-filled 10 ml cup inside a 1-L cylindrical, waxed cardboard

container (Dade Paper Co., Miami, FL) with fine mesh screening. Enough adult males









were retained so that there was approximately a 1:5 ratio of males to females for mating

to take place in the cages; the remaining male pupae were discarded. All pupae inside

adult 1-L cylindrical cages were maintained at 270C and as adults emerged humidity

was maintained at >90% rh and adults were given 20% sucrose ad libitum. The idea

was that larvae completed development in one of the three different temperature

treatments, but once pupation occurred and feeding in the aquatic stage ceased, pupae

were moved to 270C and as adults maintenance continued at 270C though the blood

feeding and the EIP.

Virus and Mosquito Infection

The LR2006-OPY1 CHIKV strain, (GenBank accession number DQ443544) was

isolated in France from a febrile patient who had been infected on the island of Reunion

in 2006 (Parola et al. 2006). Aedes aegypti and Ae albopictus females from Palm Beach

County were shown previously to be highly susceptible to this recently emergent CHIKV

strain (Reiskind et al. 2008), which contains the alanine to valine substitution at the 226

position of the El envelope structural protein (El A226V) that has been identified as a

feature in many recent CHIKV epidemics (Rezza et al. 2007). Virus for infectious blood

meals was produced by inoculating a confluent monolayer of Vero cells in a T-75 cm2

flask with 250 pL of previously frozen stock virus, and incubating them in a 5% CO2

atmosphere (atm) and 350C for 36 h. After 36 h, infectious blood was prepared by

combining freshly recovered media-viral suspension with defibrinated bovine blood

(Hemostat Laboratories, Dixon, CA) in a 1 to 10 ratio. Concentration of virus in blood

meals offered to mosquitoes was measured at 7.4 Loglo plaque-forming units (PFU)/mL

by one-step quantitative RT-PCR.









Each 1-L cylindrical, waxed cardboard containers contained all the emerged

females of the same species from each replicate. Aedes albopictus females from all

treatments were approximately 6-10 days old and Ae. aegypti were approximately 10-14

days old at blood feeding. Because intense larval competition increased development

time, very young mosquitoes that had recently emerged from highly competitive

treatments were not offered a blood meal, and only those mosquitoes within the

previously stated age range were brought into the biosafety level-3 laboratory for blood

feeding. Mosquitoes were sucrose-starved for 24 hours, but water was available to

them, before blood was offered. Adult mosquitoes were initially offered an infectious

blood meal using water-jacketed glass membrane feeders (Rutledge et al. 1964)

covered with 38-42 mm hog casings (SausageMaker, Buffalo, NY) with an additional

layer of Parafilm MTM (American National Can, Chicago, IL) as a barrier between the

infectious blood and hog casing. Membrane feeders were attached to each other and to

a Haake Series F water circulator (Thermo Haake, Paramus, NJ) which maintained the

blood meal at 370C. No mosquitoes fed from the Parafilm M plus hog casing

membranes, and 24 hours later all cages of adult females were offered an infectious

blood meal from cotton pledgets, each containing 3 ml of infectious blood pre-heated to

350C for 20 min.

Mosquitoes were given 30 minutes to feed. Immediately afterwards, mosquitoes

were cold anesthetized, and engorged mosquitoes were separated from unfed, which

were removed from the experiment. Engorged mosquitoes were held for a 10 day

extrinsic incubation period (EIP) at 270C and provide with 20% sucrose ad libitum, after

which surviving mosquitoes were killed by freezing. Mosquitoes were stored individually









in 1.5 ml microcentrifuge tubes at -800C and, after thawing, wings were removed for

measurements, bodies were assayed to determine infection status and titer, and legs

were tested to check for disseminated infections. An assayed mosquito could be, (1)

uninfected, have an (2) isolated infection, which specified a CHIKV positive body, but

legs negative for the presence of the virus or have a (3) disseminated infection, which

meant virus was found in the legs, indicating that the infection had spread beyond the

midgut and on to other organs. A disseminated infection signifies that a mosquito is

capable of transmitting the virus (Turell et al. 1984). Wings and legs were removed from

each mosquito using forceps that were sterilized with 100% ethanol followed by intense

flaming with a portable one-touch burner (Daigger). Wing length was measured in

millimeters as an indicator of body size (Blackmore and Lord 2000) from the alula to

wing tip, excluding wing fringe. Photographic images of the wing were captured with a

digital camera mounted on a dissecting microscope and measured with a computer

imaging and measurement program (i-Solution lite, AIC Inc., Princeton, NJ).

Mosquito bodies were homogenized at 25 Hz for 3 min using a Tissuelyzer

tissue homogenizer (Qiagen Inc., Valencia, CA). RNA was extracted separately from

bodies and legs. Mosquito bodies were homogenized in TRI Reagent (Molecular

Research Center, Inc., Cincinatti, OH), and then RNA was extracted according to the

manufacturer's protocol using 50 pl of DEPC treated water as the final elution volume.

Mosquito legs were homogenized in 250 pl BA-1 media (Lanciotti et al. 2000) with two

zinc-plated BBs (Daisy), whereafter the homogenate was added to 750 pl of TRI

Reagent-LS (Molecular Research Center, Inc., Cincinatti, OH) for RNA extraction, also

with a final elution volume of 50 pl in DEPC treated water.









One-step quantitative RT-PCR was used to determine infection status and body

titer of samples. Primers were designed from the El gene and had the following

sequences: forward: 5'-ACC CGG TAA GAG CGA TGA ACT-3'; reverse: 5'AGG CCG

CAT CCG GTA TGT-3'; and probe: 5'-/5cy5/CCG TAG GGA ACA TGC CCA TCT CCA

/3BHQ_2/-3' (IDT DNA, Coralville, IA). Reactions were performed in a 96-well reaction

plate, with each reaction containing: 0.4pl SuperScript III RT/Platinum Taq mix

(Invitrogen, Carlsbad, CA), 10pl 2x reaction mix (a buffer system, MgSO4, dNTPs and

stabilizers), 1 l forward primer (10 pmol/L), 1 l reverse primer (10 pmol/L), 0.4pl

fluorogenic probe (10 pmol/L), 2.2pl DEPC-treated H20, and 5pl of the test sample

RNA. Viral RNA was quantified using a Roche LC480 light-cycler (Roche Applied

Sciences, Indianapolis, IN) with the following thermal conditions: 20 minutes at 480 C

and 2 minutes at 950 C, followed by 40 cycles of PCR, 10 seconds at 950 C and 15

seconds at 600 C followed by a cool down for 30 seconds at 500 C. A negative control

(DEPC-treated water in place of sample) and a positive control (CHIKV stock virus, 10-2

dilution) were included in each reaction run. A standard curve was generated by

assaying a full range of ten-fold serial dilutions of CHIKV virus stock (7.8 Loglo PFU/ml)

as previously described in Chapter 3.

Statistical Analysis

All statistical analyses were conducted using SAS 9.2 (SAS Institute, Cary, NC).

Only data from female mosquitoes were included in the analysis. Each 5 L bucket

containing a developing cohort of larvae was considered a replicate, and survivorship to

adulthood per replicate was calculated as the proportion of adults that emerged from the

initial cohort of first-instar larvae. Female survivorship was estimated by assuming each

original cohort contained 50% of each sex. Using individual time to pupation, the mean









female time to pupation was calculated for each replicate. Female wing-length was only

measured for females assayed for CHIKV infection. Two-way multivariate analysis of

Variance (MANOVA) (PROC GLM) was used to analyze the effects of temperature and

competitive treatment on Ae. albopictus female time to pupation, wing length, and

survival to emergence simultaneously. Proportional data for survival to emergence were

transformed using an arcsine transformation, which is recommended when percentages

are outside the range 30% to 70% (Sokal and Rohlf 1995). Because of poor blood

feeding by female Ae. aegypti very few wing-length measurements were taken of this

species, therefore, this variable was removed from the female Ae. aegypti MANOVA

analysis. MANOVAs were done separately for each mosquito species.

MANOVA creates a composite index of all measured response variables, which

provides a distinct advantage over separate ANOVAs because the correlations among

the variables are a factor in the model (Bray and Maxwell 1985). It is valuable to have

several measures of group differences, and using multivariate methods to assess the

influence of treatment on groups provide a more valid assessment of effects. All

dependent variables had multivariate normal distributions within each group

(temperature and competitive treatments). Pillai's trace was used to assess significance

because this test statistic is robust to violations of assumptions concerning homogeneity

of the covariance matrix and provides maximum protection against finding a statistical

significance when there is none, with small samples (Bray and Maxwell 1985).

Significant temperature and competitive treatment effects were further analyzed by

multivariate pairwise contrasts of main effect multivariate means with a Bonferroni

correction for experiment wise a = 0.05 (a=0.05/3 = 0.017). Standardized canonical









coefficients were used to determine the relative contribution of each response variable

to significant multivariate effects as well as their relationship to each other (e.g., positive

or negative).

Differences in CHIKV susceptibility in Ae. albopictus were evaluated by two-way

MANOVA and standardized canonical coefficients on the response variables proportion

infected (# with virus/# fed) and proportion with disseminated infection (# with virus in

their legs/# with virus). Proportional data for infection and disseminated infection were

transformed using an arcsine transformation. Differences in CHIKV infection and

disseminated infection were also analyzed using a generalized linear mixed model

(PROC GLMMIX), with temperature and competition as fixed effects and replicate as a

random effect, specifying a logistic link function and a binomial error distribution. A

multiplicative overdispersion component was added to the generalized linear mixed

models using a simple R-side residual effect because of a higher than expected

variance in the distribution of the data (Schabenberger 2007). Few numbers of blood

fed Ae. aegypti precluded analyses of infection and disseminated infection data with

MANOVA or a generalized linear mixed model (PROC GLMMIX). Overall, Ae. aegypti

and Ae. albopictus mean infection and disseminated proportions and standard errors of

the mean (SEM) were calculated from replicate means from 40 Ae. albopictus and 20

Ae. aegypti replicates pooled across all temperature and competitive treatments.

A generalized linear mixed model (PROC GLMMIX) was used to test for effects of

temperature and competition (fixed) and replicate (random) on Ae. albopictus body titer,

after the 10 day EIP. Body titer was a continuous variable and the model specified an

identity link function and a gaussian error distribution. Titer data did not fit the model









assumptions of normality, but approximate normality was achieved through a log

transformation of titer values. Differences between main effect means (temperature and

competition) were further analyzed by pairwise comparisons using the LS means

statement in PROC GLIMMIX.

To investigate the relationship between size and titer correlation analysis was

performed on the mean size of Ae. albopictus females per replicate, pooled across all

competitive and temperature treatments, and titer values. This analysis was also

performed on the mean size of Ae. aegypti individuals, pooled across all competitive

and temperature treatments, and titer values. In addition, to determine if there was a

difference in size among uninfected, infected (non-disseminated), and disseminated

females a one-way ANOVA (PROC GLM) was used. The one-way ANOVA was used to

compare differences in mean wing length among the females of the three infection

status categories, which were .categorical variables in the model. The females in the

analysis were those that blood fed, completed the 10 day EIP, and were pooled across

all larval treatments. Each species was analysed separately.

Results

Mosquitoes, Temperature, and Competition

Results from the MANOVAs showed that in both Ae. aegypti and Ae. albopictus

female growth and development varied significantly as a result of temperature and

competition, with no significant interaction between these variables in Ae. albopictus

and a significant interaction term in Ae. aegypti (Table 4-1). An examination of the

standardized canonical coefficients for temperature effects on growth parameters,

shows that temperature had the greatest effect on time to pupation in both Ae.

albopictus and Ae. aegypti (Table 4-1). For Ae. albopictus time to pupation varied in the









same direction as wing length (Table 4-1), so lower temperature produced larger

mosquitoes that took longer to pupate. Also in Ae. albopictus survival to emergence was

negatively correlated with time to pupation and wing length, but the SCC value was

small, so differences in survivorship contributed very little to the overall significance of

temperature. For Ae. aegypti survival to emergence varied positively with time to

pupation (Table 4-1), but similar to Ae. albopictus the small SCC value indicated that

differences in survivorship contributed only slightly to differences among temperatures.

Significant temperature effects of treatments on growth parameters of both Ae.

albopictus and Ae. aegypti were further investigated in pairwise contrasts (Table 4-2).

All pairwise temperature contrast for both Ae. albopictus and Ae. aegypti were

significant. Associated SCC indicated that time to pupation was the primary source of

differences between the pairs. Wing length contributed secondarily to pairwise

differences in Ae. albopictus (Table 4-2). In examination of the SCC values for

survivorship to adulthood in Ae. albopictus, survival was the highest at the middle

temperature of 27C with little difference between survival at 22C and 32C. In Ae.

aegypti survivorship to adulthood was the lowest at 32C with little difference between

22C and 27C (Table 4-2).

Significant competitive effects of treatments on growth parameters of both Ae.

albopictus and Ae. aegypti were further investigated in pairwise contrasts (Table 4-3).

All pairwise competitive contrast for both Ae. albopictus and Ae. aegypti were

significant. As was found in examination of the SCC form temperature, SCC for

competitive pairwise contrasts indicated that time to pupation was the primary source of

differences between the pairs. Aedes albopictus from the 0:100 treatment replicates









were larger, took less time to emerge, and suffered less larval mortality than the two

high competition treatments of 0:200 and 100:100 (Figures 4-1, 4-2, 4-3). There were

also significant differences between the 0:200 and 100:100 competitive treatments for

Ae. albopictus. The interspecific larval competition treatment produced Ae. albopictus

that took longer to develop, had lower survivorship to emergence, and were smaller

than Ae. albopictus produced under intraspecific competitive conditions of the same

density (Table 4-3). In contrast, Ae. aegypti generally did better under the interspecific

than intraspecific competitive conditions (Table 4-3). However, at 27C Ae. aegypti

survivorship to adulthood was higher in the intraspecific than the interspecific treatment

(Figures 4-4, 4-5). This pattern accounts for the significant interaction between

temperature and competition in this species (Table 4-1). The pairwise contrast between

Ae. aegypti 100:100 and 0:100 was only marginally significant (P= 0.0487). Thus, the

100 Ae. aegypti larvae housed with 100 heterospecific Ae. albopictus larvae only slightly

differed in survival to emergence or time to pupation from the 100:0 Ae. aegypti

treatment.

Virus and Mosquito Infection

A total of 317 blood fed Ae. albopictus survived the 10 day EIP and were

processed and assayed for CHIKV susceptibility. The 57 Ae. aegypti that survived

through the 10 EIP were also processed and assayed for CHIKV, but replicate size and

number were not large enough to include Ae. aegypti in any kind of treatment effect

analysis. When pooled across treatments, the mean ( SEM) replicate infection and

disseminated infection rates for Ae. aegypti were 0.690.085 and 0.720.093 and for

Ae. albopictus 0.950.017 and 0.700.037 (Figure 4-6).









Results from the MANOVA show that temperature did not have a significant effect

on the proportion of infected and the proportion with disseminated infections for Aedes

albopictus (Pillai's trace = 0.112, F4,62 = 0.92, P = 0.4578) nor did competition (Pillai's

trace = 0.009, F4,62 = 0.07, P = 0.9906) or their interaction (Pillai's trace = 0.107, F8,62=

0.44, P = 0.3736) (Figure 4-7). Similarly, when Ae. albopictus infection data were

analyzed with generalized linear mixed models (PROC GLMMIX) there were no

significant effects from temperature (F=0.01, df = 2, P = 0.9940), competition (F=0.01,

df = 2, P = 0.9912) or the interaction of these factors (F=0.58, df = 4, P = 0.6786) on the

likelihood of infection, and on the probability of disseminated infection there were also

no significant effects from temperature (F=0.67, df = 2, P = 0.5106), competition

(F=2.05, df = 2, P = 0.1307) or their interaction (F=0.82, df = 4, P = 0.5150).

After the 10-day EIP, results from the generalized linear mixed model (PROC

GLMMIX) showed titer of CHIKV in infected Ae. albopictus females was significantly

affected by larval rearing temperature (F= 8.36; df = 2; P = 0.0003), but not by

competition (F= 1.62; df = 2; P= 0.1999), nor the temperature x competition interaction

(F= 1.33; df = 2; P<0.2580). There was no significant difference in body titer between

the 22 and 270C treatments, but both were signicantly different than the body titer of Ae.

albopictus reared at 320C (Figure 4-8). Correlation analysis showed no relationship for

Ae. albopictus (r = 0.255; P= 0.1385, df=34) or Ae. aegypti r = 0.311; P= 0.1480, df=21)

between wing length and log transformed-CHIKV body titer of infected mosquitoes

(Figures 4-9,4-10). A one-way ANOVA showed that there were no significant wing

length differences among CHIKV infection status categories for Ae. albopictus (F = 0.97,

df = 2, P = 0.3795) or Ae. aegypti (F = 0.61, df = 2, P = 0.5457) (Figures 4-11, 4-12).









Discussion

In this experiment Ae. albopictus was the inferior larval competitor. An interspecific

competive environment (100:100) produced the smallest sized Ae. albopictus females,

with the lowest survival, and the longest pupation times. Intraspecific competition

(0:200) also had an effect on Ae. albopictus, but the effect was not as severe on the

three growth measurements as the interspecific competitive environment. In contrast,

Ae. aegypti from the interspecific competitive environment were only slightly affected in

survivorship to emergence and days to pupation (P = 0.0487), compared to conspecifics

reared in the 100:0 treatment. Essentially, the presence of 100 Ae. albopictus had very

little effect on the 100 Ae. aegypti developing in the same 5L bucket. Intraspecific

competition affected Ae. aegypti and the 200:0 treatment was the poorest performer.

This confirmed similar findings by Lounibos et al. (2002) in which Ae. aegypti female

growth was uniquely retarded by a high density of its own species.

In the water-filled containers that serve as habitat for Ae. aegypti and Ae.

albopictus in Florida the primary food source is microorganisms such as bacteria and

fungi that grow on decaying oak and other leaf detritus and parts of dead invertebrates

(Fish and Carpenter 1982, Lounibos et al. 1992, Lounibos et al. 1993). In this

environment, where the dominant resource is leaf litter, Ae albopictus is the superior

competitor (Barrera 1996, Juliano 1998). To successfully execute an experiment on

CHIKV vector competence it was essential to generate enough adult mosquitoes that

also emerged within a reasonable time span, and leaf litter as the sole food source in

preliminary studies did not produce enough adults in an acceptable age range. A

combination of oak leaf infusion and an artificial food source of a one-to-one mixture of

brewer's yeasts and lactoalbumin, was used as a compromise between the need for a









reliable number of mosquitoes within a narrow age range and the maintenance of

ecological relevance for the experiment. Because competitive interactions

betweenthese two mosquitoes are context dependent, and the relative effects of

competition may change under different ecological conditions (Juliano 2009) it was not

surprising that Ae. aegypti was the superior larval competitor. In previous studies the

type of food resource affected the outcome of competition between these two Aedes

species, with the addition of a high protein food (i.e. yeast, lactoalbumin, liver powder)

favoring Ae. aegypti (Black et al. 1989, Barrera 1996, Daugherty et al. 2000).

Differences in temperature did not change the outcome of competition, which

confirmed previous results of Lounibos et al. (2002), when competition between these

two species was investigated at 240 and 30C. As expected, based on results in

Chapters 2 and 3, temperature did have an effect on pupation time and size in both

Aedes species, with low temperature increasing development time and leading to larger

sized adults. Temperature also had a small effect on survivorship to adulthood in Ae.

albopictus, survival was the highest at the middle temperature of 27C and in Ae.

aegypti survivorship to adulthood was high at both 22C and 27C. Therefore, the low

temperature only seemed to have a negative effect on survival in Ae. albopictus. For

Ae. aegypti there was a significant interaction between temperature and competition

and how temperature modified the effect of competition is seen in Figure 4-4, where the

200:0 treatment had greater survival than the 100:100 at 27C, while at the other

temperatures and for the other growth measurement, time to pupation, the 100:100

treatment out performed the 200:0 for Ae. aegypti.









Neither competitive interactions nor temperature treatment had an effect on

CHIKV infection or disseminated infection for Ae. albopictus. However, temperature did

have a significant effect on CHIKV body titer in Ae. albopictus. The lack of a

temperature effect on infection and disseminated infection in Ae. albopictus was

unexpected because Chapters Two and Three demonstrated that aquatic larval

temperature had an effect on CHIKV infection in both Ae. aegypti and Ae. albopictus

and larval food level and temperature had an effect on disseminated infections in Ae.

aegypti. In addition, larval rearing temperature has been shown to affect mosquito

competence for viruses of Rift Valley fever (RVFV), Venezuelan equine encephalitis

(VEEV) (Turell 1993), Murray Valley encephalitis (MVEV) (Kay et al. 1989a), Japanese

encephalitis (JEV) (Takahashi 1976), and western equine encephalitis (WEEV) (Hardy

et al. 1990). Larval competition has been shown to affect Ae. triseriatus vector

competence for LaCrosse virus (LACV) (Bevins 2008) and Ae. albopictus vector

competence for viruses of Sindbis (SINV) (Alto et al. 2005) and dengue (Alto et al.

2008a).

Body titer of CHIKV infected Ae. albopictus females was significantly affected by

larval rearing temperature, with females reared at the two lower temperatures of 220

and 270C having higher meanCHIKV body titers than counterparts from 320C. This

effect is in agreement with results from Chapter Two, in which CHIKV susceptibility was

greater in Ae. albopictus adults reared at lower temperatures compared to individuals

from higher rearing temperature treatments.

The lack of a significant effect due to larval competition and temperature on

CHIKV infection and disseminated infection in this study is most likely due to low









sample size and the general logistical difficulties of bringing an ecological experiment

into a laboratory setting. The mosquitoes used were first generation progeny of field

collected individuals. It was very difficult to get recently derived field colonies of Ae.

albopictus and Ae. egypti to feed on an artificial blood sources. The original

experimental design used water-jacketed glass feeders with hog casing membranes,

with parafilm added to prevent the membranes from leaking. In preliminary experiments

sufficient feeding took place on the hog casing leading to the belief that is would be a

good feeding method. However, the parafilm was a last minute required addition in

order to comply with BL-3 protocols established at FMEL, and the added barrier seems

to have inhibited feeding on the parafilm-hog casing combination. Subsequently, an

alternative protocol using blood-soaked, heated cotton pledgets was attempted.

Because previous studies showed that only when Ae. aegypti fed on pledgets soaked in

a very high titer of CHIKV infectious blood, is this species able to establish a midgut

infection (Pesko et al. 2009), it was decided that a high ratio of 1:10 virus supernantant

to defibrinated bovine blood would be used. This produced a blood meal titer of 7.4

Loglo plaque-forming units (PFU)/mL, which was markedly higher than blood meal titers

used in prior experiments and historic human viremias, which have generally not

circulated above 7.0 logloTCIDso/ml (Jupp and Mclntosh 1988).

Unfortunately, a combination of low feeding on the pledgets, combined with a large

amount of mortality during the 10 day EIP led to entire missing replicates from the

different temperature and competitive treatments for Ae. aegypti, and it was necessary

to remove this species from the infection and dissemination analyses. Aedes aegypti

developed faster than Ae. albopictus maintained in an identical growth environment, but









both species in interspecific larval treatments needed to be reared simultaneously

leading to adult Ae. aegypti that were older than Ae. albopictus. The older age of Ae.

aegypti combined with the somewhat stressful rearing conditions may have caused the

higher adult mortality that was noted for this species over the 10 day EIP.

For Ae. albopictus the high blood meal titer led to a mean infection rate of

approximately 95% among replicates pooled across temperature and competitive

treatments, which likely prevented any observed treatment effect on infection rates.

Previous laboratory studies with historic epidemic CHIKV isolates have indicated that, in

the laboratory Ae. albopictus is a significantly more competent vector than Ae. aegypti

(Mangiafico 1971; Turell et al. 1992) and this superior laboratory competence of Ae.

albopictus is even more exaggerated with the emergent E1-A226V CHIKV isolate

(Tsetsarkin et al. 2007, Pesko et al. 2009). Titers of between 4.0 and 5.0 logloTCIDso/ml

resulted in about a 90% infection rate in Ae. albopictus and a <10% infection rate in Ae.

aegypti and at titers above 6.0 logloTCIDso/ml infection approached 100% in Ae.

albopictus and 30% in Ae. aegypti (Tsetsarkin et al. 2007). In this study, replicate

means and SEMs for the overall proportion with infections and with disseminated

infections pooled across treatments. suggest lower infection rates in Ae. aegypti

compared with Ae. albopictus for the emergent El -A226V CHIKV isolate (Figure 4-6).

With such high infection rates in Ae. albopictus absence of a treatment effect on

infection was partly expected. Among all nine temperature-competitive treatments

average infection rates were between 90 and 100% (Figure 4-7). The lowest proportions

of infected individuals were among the three competitive treatments run at 22C. The

overall disseminated infection rate among infected individuals for Ae. albopictus was


100









70% (Figure 4-6). Among the nine temperature-competitive treatments proportions of

disseminated infections varied between approximately 50 and 80% (Figure 4-7),

however low sample sizes and large standard errors decreased the ability to find

statistically significant differences between treatments.

Body size for neither Ae. albopictus nor Ae. aegypti was correlated with CHIKV

body titer, and body size had no significant effect on whether either species established

an isolated or disseminated infection. In prior experiments, size was shown to influence

susceptibility of Ae. aegypti for DENV (Sumanochitrapon et al. 1998, Alto et al. 2008b)

and Ross River virus (RRV) (Nasci and Mitchell 1994). In this experiment the absence

of significant correlations was somewhat expected because of the mostly non-significant

temperature and competitive treatment effects on CHIKV susceptibility, since both

temperature and competition have significant influence on size. However, temperature

did have an effect on CHIKV body titer in Ae. albopictus, but size was not found to be

significantly correlated with body viral titer in either species of mosquitoes. This may be

because CHIKV body titers were very high overall, which possibly resulted from the high

CHIKV titers in the blood meal these mosquitoes were fed. In both figures 4-9 and 4-10

the majority of the CHIKV body titer values are clustered in a fairly narrow range of titers

for Ae. albopictus between 6.0-6.5 Loglo pfu/0.1ml and Ae. aegypti between 6.3-7.0

Loglo pfu/0.1ml. These high ranges of titers may represent biological maximum titers for

these species, which may have led to the lack of significant correlations.

In summary, inter- and intra-specific competitive interactions between larval Ae.

aegypti and Ae. albopictus influenced immature survival, the length of the larval period,

and body size. Under these specific experimental conditions, in which a protein-rich


101









artificial food source was provided to developing larvae, Ae. aegypti was the superior

competitor across all three temperature treatments. Larval temperature and competition

did not influence the likelihood of CHIKV infection or disseminated infection, but CHIKV

body titers were significantly greater in female Ae. albopictus from the lower larval

temperatures. The larval environment strongly influences adult size, but in this study

there was no significant relationship between mosquito size and measures of CHIKV

susceptibility. Future studies will be aimed at exploring what other physical or

physiological traits may play roles in predicting vector susceptibility.


102











Table 4-1. MANOVA for temperature and competitive treatment effects and their interaction on population growth
parameters of female Aedes albopictus and Aedes aegypti: time to pupation, juvenile survivorship, and wing length
Pillai's trace Standardized canonical coefficients
Comparison P
(num df, den df) Time to Survivorship Wing
Pupation
Aedes albopictus

Temperature 1.12 (6, 58) <0.0001 3.398 -0.153 0.978

Competitive treatment 0.96 (6, 58) <0.0001 -2.633 1.049 0.821

Temperature x competitive treatment 0.50 (12, 90) 0.1351

Error d.f. 30


Aedes aegypti

Temperature 1.30 (4, 72) <0.0001 4.854 0.245

Competitive treatment 0.88 (4, 72) <0.0001 4.671 -0.222

Temperature x competitive treatment 0.52 (8, 72) 0.0040 2.821 1.231

Error d.f. 36


103









Table 4-2. Multivariate pairwise contrasts of temperature treatment effects on female Aedes albopictus and Aedes
aegypti for growth measurements time to pupation, juvenile survivorship, and wing length.
Pillai's trace Standardized canonical coefficients
Competitive treatment P
pairwise comparisons (num df, den df) Time to Survivorship Wing
Pupation
Aedes albopictus

22C vs. 27C 0.86 (3, 28) <0.0001 3.411 -0.435 0.954

22C vs. 32C 0.91 (3, 28) <0.0001 3.374 -0.029 0.983

27C vs. 32C 0.47 (3, 28) 0.0004 2.740 0.985 0.897


Aedes aegypti

22C vs. 27C 0.94 (2, 35) <0.0001 4.849 0.072

22C vs. 32C 0.96 (2, 35) <0.0001 4.834 0.313

27C vs. 32C 0.59 (2, 35) <0.0001 3.757 1.013










Table 4-3. Multivariate pairwise contrasts of competitive treatment effects on female Aedes albopictus and Aedes aegypti
for growth measurements time to pupation, juvenile survivorship, and wing length.
Pillai's trace Standardized canonical coefficients
Competitive treatment P
pairwise comparisons (num df, den df) Time to Survivorship Wing
Pupation
Aedes albopictus

0:100 vs. 0:200 0.83 (3, 28) <0.0001 -2.409 1.281 0.691

0:100 vs. 100:100 0.92 (3, 28) <0.0001 -2.676 0.998 0.848

0:200 vs. 100:100 0.54 (3, 28) <0.0001 -3.046 0.406 1.101


Aedes aegypti

100:0 vs. 200:0 0.72 (2, 35) <0.0001 4.518 -0.356

100:0 vs. 100:100 0.16 (2, 35) 0.0487 -0.883 1.337

200:0 vs. 100:100 0.67(2, 35) <0.0001 4.688 -0.243


105













0:100
S0:200
100:100
I


22C 27C 32C

Larval temperature treatments

Figure 4-1. Proportion of female Ae. albopictus (SEM) surviving to adult emergence


0:100
0:200
S100:100


22C 27C 32C

Larval temperature treatments

Figure 4-2. Female Ae. albopictus mean (SEM) days to pupation.


106



















3.2

E

E 3.0


a,
--



2.8



2.6


0:100
0:200
S100:100


2.4 1 n
22C 27C 32C

Larval temperature treatments


Figure 4-3. Female Ae. albopictus mean (SEM) wing length.


107




















0 0.9
'--

(/)
cjn
D 0.8

E


0.7
0
t-
Q-
0
L 0.6
-


100:0
S200:0
S100:100


0.5 '
22C 27C 32C

Larval temperature treatments


Figure 4-4. Proportion of female Ae. aegypti (SEM) surviving to adult emergence.


100:0
S200:0
S100:100


22C 27C 32C


Larval temperature treatments


Figure 4-5. Female Ae. aegypti mean (SEM) days to pupation.









108













0.85



0.80



0.75



0.70



0.65



0.60


O Ae. albopictus
A Ae. aegypti


Proportion infected
Figure 4-6. Bivariate means (SEM) of replicates for proportions of Ae. aegypti and Ae.
albopictus with infections and disseminated infections.



0.9
C

1 O
I 0.8 -

SA
C D
E 0.7
a)

A A D

i 0.6 -
o
0
a- 0
0. O
2 0.5


0.88 0.90 0.92 0.94 0.96 0.98 1.00
Proportion infected
Figure 4-7. Bivariate plot of least squares means for proportion of Ae. albopictus with
infections and disseminated infections. Open symbols specify 22C, grey
symbols 27C, and black symbols 32C. Triangles represent 0:100, circles
0:200, and squares 100:100 treatment.


109
















6.4 (P')
(1 13)
E 6.2 -

6.0

C,
5.8
-- B

I 5.6 (58)

o 5.4

5.2

5.0
220C 270C 320C

Larval rearing temperature treatments
Figure 4-8. LS means (SEM) of Aedes albopictus CHIKV body titer for temperature
treatments, LS means with different letters are significant at a per comparison
P-level of 0.017.


110
















6 *
S* *


*.
*





*





2.4 2.6 2.8 3.0 3.2 3.4


Wing length (mm)
Figure 4-9. Correlation analysis of log transformed of CHIKV whole mosquito body titer
and wing length replicate means for Aedes albopictus pooled across
temperature and competitive treatments (r= 0.255; P = 0.1385).


4.5 I I I I I I
2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8

Wing length (mm)
Figure 4-10. Correlation analysis of log transformed of CHIKV whole mosquito body
titers and wing lengths for Aedes aegypti infected individuals pooled across
temperature and competitive treatments (r= 0.311; P = 0.1480).





111


S S
*e *g
SC


S S













3.15


1 Uninfected
77ZZ Infected (non-disseminated)


3.10 Disseminated Infection

(15)
3.05
E
E
S3.00-
t--


2.95
(89) (211)

2.90


2.85


2.80

Figure 4-11. Least squared means (SE) for sizes of adult female Ae. albopictus
mosquitoes in CHIKV infection status categories.




3.30 -

3.25 I Uninfected
77ZZ Infected (non-disseminated)
3.20 Disseminated Infection

3.15 (22)
E (26)
E 3.10

3.05 -

0 3.00

2.95 -

2.90

2.85

2.80

Figure 4-12. Least squared means (SE) for sizes of adult female Ae. aegypti
mosquitoes in CHIKV infection status categories.


112









CHAPTER 5
GENERAL CONCLUSIONS AND FUTURE DIRECTIONS

The primary focus of this study was to explore the extent to which vector

susceptibility is influenced by the larval environment within the Ae. aegypti, Ae.

albopictus and chikungunya virus system. Previous studies with Ae. aegypti and DENV

serotype-2 estimated that environmental variance accounted for up to 78-91% of the

total phenotypic variance in vector competence (Bosio et al. 1998). Clearly,

environmental effects are an important component of vector susceptibility and overall

disease epidemiology (Tabachnick 2009). Many studies have looked at the effect of

temperature, humidity, and other environmental effects on adult mosquito competence,

but little attention has been paid as to how the larval environment may shape traits

involved in adult vector competence.

Varying temperature was a common treatment in the three experiments described

in this dissertation. Larval rearing temperature influenced Ae. albopictus and Ae. aegypti

growth and susceptibility to CHIKV. In Ae. albopictus, CHIKV infection rates decreased

with increasing larval rearing temperature and decreasing adult size. Also in Ae.

albopictus, decreased larval temperature was associated with higher CHIKV body titers

among infected individuals. In Ae. aegypti, larval temperature also influenced the

likelihood of infection and disseminated infection, with the intermediate temperature of

27C (22-32 degree range) resulting in the highest rates of infection and dissemination.

The larval habitat in nature is composed of multiple biotic and abiotic features that

interact. To simulate some of the complexity that exists in the natural larval

environment, differing levels of nutritional resources and intra- and interspecific

competitive treatments were added to temperature treatments in experiments described


113









in Chapters 3 and 4, respectively. For Ae. aegypti higher levels of nutritional resources

significantly increased the likelihood of having a disseminated infection. On the other

hand, larval competition had no effect on susceptibility to CHIKV infection in either

species of mosquito, but it did have a significant effect on growth parameters in both Ae.

aegypti and Ae. albopictus.

It is unclear why Ae. aegypti and Ae. albopictus reared at different temperatures

and different food levels were more susceptible to CHIKV. When the mesenteronal

tissues from large and small Ae. triseriatus females reared at differing food levels, were

examined with electron microscopy physical differences in the basement membrane

(basal laminae) were found. Small Ae. triseriatus exhibited higher rates of transmission

and disseminated infections for LACV and their basement membranes had fewer

laminae resulting in a reduced mean thickness when compared with larger individuals

(Grimstad and Walker 1991). It was suggested that the thinner basement membrane

might allow for the more rapid release of LACV in to the hemocoel (Grimstad and

Walker 1991). With the use of microscopy, a rapid release of EEEV into hemocoel due

to disruptions in the posterior midgut was observed in Culiseta melanura immediately

following an infectious blood meal (Weaver et al. 1991) and the concept of a "leaky

midgut" has been supported by the rapid appearance of arbovirus in the hemocoel in

other vector-viral systems (Boorman 1960, Miles et al. 1973, Hardy et al. 1983, Weaver

1986).

In Chapter 3, Ae. aegypti reared at two different food levels crossed with three

different temperatures had significantly different dissemination rates, with the proportion

of Ae. aegypti from the higher food level having a greater odds of developing a


114









disseminated infection. This contrasts with results for small Ae. triseriatus from the low

food level (Grimstad and Walker 1991), although there was no significant relationship

between size and susceptibility in the experiment with Ae. aegypti described in Chapter

3. It is possible that the mechanism responsible for differences in CHIKV susceptibility

in Ae. aegypti from different food levels also involves anatomical features of the midgut

cells and electron microscopy studies could help elucidate a mechanism.

Large Ae. albopictus generated, in Chapter 2, through low temperatures were

more susceptible to CHIKV, which is in agreement with increased infection in Ae.

taeniorhynchus for RVF and VEEV (Turell 1993), but contradicts previously described

patterns between small Ae. triseriatus and LACV (Grimstad and Walker 1991). In other

studies, large Ae. aegypti mosquitoes generated by increasing larval food quantities

were significantly more susceptible to RRV (Nasci and Mitchell 1994) and DENV

(Sumanochitrapon et al. 1989) than small mosquitoes. It is likely that virus enters midgut

cells through receptor mediated endocytosis (Hardy el al. 1983) and viral determinants

for mosquito midgut infection have been studies for a number of arboviruses (Ludwig et

al 1989, Mourya et al. 1989, Houk et al. 1990, Mertens et al. 1996, Xu et al. 1997,

Pletnev et al. 2001, Molina-Cruz et al. 2005, Smith et al. 2008). It is possible that the

expression of viral determinants or receptors in the midgut is influenced by rearing

temperature or larger mosquitoes generated through different larval conditions have a

greater number of receptors or enhanced binding (Turell 1993). This is certainly a

mechanism worth exploring and identification of viral binding sites or receptors would

greatlt increase our understanding of vector-viral interactions.


115









Because all growth of mosquitoes is accomplished during the aquatic larval period,

which may be long at colder temperatures and under stressful conditions of limited food

and high competition, it seems likely that these environmental features are shaping

insect immune pathways, and other physiological and anatomical features of the adult

mosquito that may be more strongly correlated than body size to measures of arbovirus

susceptibility. It is also possible that how body size is achieved can completely change

the susceptibility of a mosquito for an arbovirus. Future experiments should investigate

the underlying physiological and/or molecular mechanisms that are influenced by the

larval environment and lead to differential vector competence.

Because laboratory colonization can cause significant changes in phenotype and

genotype of organisms, first or second generation Aedes, the progeny of field collected

parents or grandparents, were used in this study to more realistically represent the

types of interaction that would take place in natural populations. Colonization often

leads to a decrease in heterozygosity and a shift in allele frequencies due to selection,

drift, non-random mating, and founder effect (Munstermann 1980, Lorenz et al. 1984,

Mason et al. 1987). Frequencies of genes involved in vector competence may be

influenced by evolutionary forces that accompany colonization, and the use of colonies

freshly derived from field collected mosquitoes may limit the effect of selection.

However, it was a challenge to get newly established colonies of Ae. aegypti and Ae.

albopictus to feed on an artificial blood source. In the first two experiments Edicoll

collagen film was used as membrane for the mosquitoes to feed through in combination

with water jacket glass feeders connected to a water circulator. Edicoll collagen film is

a manufactured product used as a casing for sausages and hotdogs. Low to moderate


116









feeding was accomplished with the film, and for the third experiment hog casing was

used after a preliminary experiment established that its use increased blood feeding

success. As described in Chapter 4, the hog casing and water jacketed feeder system

was replaced by blood-soaked pledgets because the addition of parafilm as a

secondary membrane over the hog casing inhibited feeding. The unanticipated switch to

a different feeding method is one example of difficulties encountered while trying to get

a substantial number of F1 Ae. aegypti and Ae. albopictus to feed in the laboratory

during this study.

This dissertation is a general exploration as to whether variations in temperature,

food, and competition in the larval environment affect adult CHIKV susceptibility. An

effect from temperature and food was established, and now the more difficult task of

modeling mosquito viral susceptibility response patterns (infection, disseminated

infection, and viral titer) to changes in individual and combined larval environmental

factors may be a future goal. Some of the other factors in the study that most likely

influenced mosquito susceptibility to CHIKV, but were held constant among treatments,

were blood meal titer, adult holding temperature and the length of EIP. These factors

are known to influence absolute values of infection, disseminated infection, and body

titer, and variation in response to changing values of titer, adult temperature, and EIP

would need to be included in future work in which predictability is a goal.

If relationships between viral susceptibility and larval environment can be

elucidated and, in addition, a mosquito trait or traits are identified that are a product of

the environment and are correlated to viral susceptibility, this could contribute to the

predictability and risk assessment of epidemics. Pupal productivity surveys are common


117









in areas with container inhabiting mosquitoes and endemic disease such as dengue,

yellow fever, chikungunya and filariasis. Surveys of immature mosquitoes are normally

used as a method to assess vector population densities in a given area. More

specifically, estimates of pupal abundance of Aedes vectors of DENV, have been

promoted as a more accurate index of potential female vectors than traditional larval

surveillance (Strickman and Kittayapong 2003). If pupal surveys could incorporate

measurements of habitat quality and pupal or adult mosquito physical attributes related

to susceptibility, the additional information obtained could result in a much more

powerful and directed approach of vector and disease control.

Lastly, a clear understanding of how ecological factors in the larval environment

influence vector competence, will be an important element in the use of genetically

modified mosquitoes to control vector-borne disease. Genetically modified mosquitoes

are not completely refractory to the pathogens they transmit. Vector competence may

be significantly limited, as is the case with Anopheles stephensi that express the bee

venom phospholipase A2 (PLA2) gene leading to a reduction in Plasmodium berghei

oocyst formation by 87% (Moreira et al. 2002) or that express the C-type lectin CEL-III

from the sea cucumber, Cucumaria echinata, resulting in only a moderate inhibition

against P. falciparum (Yoshida et al. 2007). Because vector competence is heavily

influenced by the environment it seems likely that the expression of the inserted genes

may also be influenced by how the mosquito responds and develops to a changing

environment.


118











LIST OF REFERENCES


Ali M, Wagatsuma Y, Emch M, Breiman RF. Use of a geographic information system for
defining spatial risk for dengue transmission in Bangladesh: Role for Aedes
albopictus in an urban outbreak. Am J Trop Med Hyg 2003; 69:634-640.

Alto, BW, Lounibos, LP, Higgs, S, Juliano, SA. Larval competition differentially affects
arbovirus infection in Aedes mosquitoes. Ecol 2005; 86:3279-3288.

Alto, BW, Lounibos, LP, Mores, CN, Reiskind, MH. Larval competition alters
susceptibility of adult Aedes mosquitoes to dengue infection. Proc R Soc Lond B
Biol Sci 2008a; 275:463-471.

Alto, BW, Reiskind, MH, Lounibos, LP. Size alters susceptibility of vectors to dengue
virus infection and dissemination. Am J Trop Med Hyg 2008b; 79:688-695.

Apostol BL, Black WC, Reiter P, Miller BR. Use of randomly amplified polymorphic DNA
amplified by polymerase chain-reaction markers to estimate the number of Aedes
aegypti families at oviposition sites in San Juan, Puerto Rico. Am J Trop Med
Hyg 1994; 5:89-97.

Arankalle, VA, Shrivastava, S, Cherian, S, Gunjikar, RS, et al. Genetic divergence of
chikungunya viruses in India (1963-2006) with special reference to the 2005-
2006 explosive epidemic. J Gen Virol 2007; 88:1967-1976.

Armistead JS, Arias JR, Nishimura N, Lounibos LP. Interspecific larval competition
between Aedes albopictus and Aedesjaponicus (Diptera : Culicidae) in northern
Virginia. J Med Entomol 2008; 45:629-637.

Baqar, S, Hayes, CG, Ahmed, T. The effect of larval rearing conditions and adult age on
the susceptibility of Culex tritaeniorhynchus to infection with West Nile virus.
Mosquito News 1980; 40:165-171.

Barrera R. Competition and resistance to starvation in larvae of container-inhabiting
Aedes mosquitoes. Ecol Entomol 1996; 21:117-127.

Barrera R, Amador M, Clark GG. Ecological factors influencing Aedes aegypti (Diptera:
Culicidae) productivity in artificial containers in Salinas, Puerto Rico. J. Med
Entomol 2006; 43:484-492.

Benedict MQ, Levine RS, Hawley WA, Lounibos LP. Spread of the tiger: Global risk of
invasion by the mosquito Aedes albopictus. Vector-Borne Zoonot Dis 2007; 7:76-
85.


119









Bevins, SN. Invasive mosquitoes, larval competition, and indirect effects on the vector
competence of native mosquito species (Diptera: Culicidae). Biological Invasions
2008; 10:1109-1117.

Birch LC. Experimental background to the study of the distribution and abundance of
insects .3. The relation between innate capacity for increase and survival of
different species of beetles living together on the same food. Evolution 1953;
7:136-144.

Birungi J, Munstermann LE. Genetic structure of Aedes albopictus (Diptera: Culicidae)
populations based on mitochondrial ND5 sequences: Evidence for an
independent invasion into Brazil and United States. Annals of the Entomol
Society of America 2002; 95:125-132.

Black, WC IV, Moore CG. Population biology as a tool for studying vector-borne
diseases. In BJ Beaty, WC Marquardt, eds. The Biology of Disease Vectors.
Niwot, CO: University Press of Colorado 1996; 393-416

Black WC, Rai KS, Turco BJ, Arroyo DC. Laboratory study of competition between
United States strains of Aedes albopictus and Aedes aegypti (Diptera: Culicidae).
J Med Entomol 1989; 26:260-271.

Blackmore, MS, Lord, CC. The relationship between size and fecundity in Aedes
albopictus. J Vect Ecol 2000; 25:212-217.

Boorman J. Observations on the amount of virus present in the haemolymph of Aedes
aegypti infected with Uganda S, yellow fever and Semliki Forest viruses, Trans R
Soc Trop Med Hyg 1960; 54:362-365.

Bosio CF, Beaty BJ, Black WC. Quantitative genetics of vector competence for dengue-
2 virus in Aedes aegypti. Am J Trop Med Hyg 1998; 59:965-970.

Braks MAH, Honorio NA, Lounibos LP, Lourenco-De-Oliveira R, Juliano SA.
Interspecific competition between two invasive species of container mosquitoes,
Aedes aegypti and Aedes albopictus (Diptera: Culicidae), in Brazil. Annals of the
Entomol Society of America 2004; 97:130-139.

Braks M, Honorio N, Lourenco-de-Oliveira R, Juliano SA, Lounibos LP. Convergent
habitat segregation of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in
southeastern Brazil and Florida. J Med Entomol 2003; 40:785-794.

Braks MAH, Juliano SA, Lounibos LP. Superior reproductive success on human blood
without sugar is not limited to highly anthropophilic mosquito species. Med Vet
Entomol 2006; 20:53-59.

Briegel H. Mosquito reproduction: Incomplete utilization of the blood meal protein for
oogenesis. J of Insect Physiol 1985; 31:15-21.


120









Briegel, H. Fecundity, metabolism, and body size in Anopheles (Diptera: Culicidae),
vectors of malaria. J Med Entomol 1990; 27:839-850.

Briegel, H, Timmermann, SE. Aedes albopictus (Diptera: Culicidae): Physiological
aspects of development and reproduction. J Med Entomol 2001; 38:566-571.

Bustin, SA. Absolute quantification of mRNA using real-time reverse transcription
polymerase chain reaction assays. J Mol Endocrinol 2000; 25:169-193.

Carey DE. Chikungunya and dengue: a case of mistaken identity? J Hist Med Allied Sci
1971; 26:243-262.

Chadee DD, Corbet PS. Seasonal incidence and diel patterns of oviposition in the field
of the mosquito, Aedes aegypti (L) (Diptera, Culicidae) in Trinidad, West Indies a
preliminary study. Annals of Trop Med and Parasitology 1987; 81:151-161.

Chadee DD, Martinez R. Landing periodicity of Aedes aegypti with implications for
dengue transmission in Trinidad, West Indies. J Vector Ecol 2000; 25:158-163.

Chamberlain, RW, Sudia, WD. The effects of temperature upon the extrinsic incubation
of eastern equine encephalitis in mosquitoes. Am J Hyg 1955; 62:295-305.

Chamberlain RW, Sudia WD. Mechanism of transmission of viruses by mosquitoes.
Annu Rev Entomol 1961; 6:371-390.

Chretien JP, Anyamba A, Bedno SA, Breiman RF, et al. Drought associated
chikungunya emergence along coastal East Africa. Am J Trop Med Hyg 2007;
76:405-407.

Christophers SR. Aedes aegypti (L.), the yellow fever mosquito; its life history,
bionomics, and structure. Cambridge, England: Cambridge University Press,
1960.

Clements AN. The Biology of Mosquitoes, Vol. 1-11. New York, New York, Chapman and
Hall, 1992.

Colless DH. Notes on the Culicine mosquitoes of Singapore. VII. Host preferences in
relation to the transmission of disease. Ann Trop Med Parasitol 1959; 53:259-
267.

Cook PE, Hugo LE, Iturbe-Ormaetxe I, Williams CR, et al. The use of transcriptional
profiles to predict adult mosquito age under field conditions. PNAS 2006;
103:18060-18065.

Corbet PS, Chadee DD. Incidence and diel pattern of oviposition outdoors of the
mosquito, Aedes aegypti (L) (Diptera, Culicidae) in Trinidad, WI in relation to
solar aspect. Annals of Trop Med and Parasitology 1990; 84:63-78.


121









Costanzo KS, Kesavaraju B, Juliano SA. Condition-specific competition in container
mosquitoes: The role of noncompeting life-history stages. Ecology 2005; 86:
3289-3295.
Craig TP, Itami JK, Price PW. Intraspecific competition and facilitation by a shoot-galling
sawfly. Journal of Animal Ecology 1990; 59:147-159.

Daugherty MP, Alto BW, Juliano SA. Invertebrate carcasses as a resource for
competing Aedes albopictus and Aedes aegypti (Diptera : Culicidae). J Med
Entomol 2000; 37:364-372.

Davis, NC. The effect of various temperatures in modifying the extrinsic incubation
period of the yellow fever virus in Aedes aegypti. Am J Hyg 1932; 16:163-176.

de Moor PP, Steffens FE. Computer-simulated model of an arthropod-borne virus
transmission cycle, with special reference to chikungunya virus. Trans Royal
Society of Trop Med Hyg 1970; 64:927-934.

Delatte, H, Dehecq, JS, Thiria, J, Domerg, C, et al. Geographic distribution and
developmental sites of Aedes albopictus (Diptera: Culicidae) during a
chikungunya epidemic event. Vector Borne Zoonot Dis 2008; 8:25-34.

Delatte H, Gimonneau G, Triboire A, Fontenille D. Influence of temperature on immature
development, survival, longevity, fecundity, and gonotrophic cycles of Aedes
albopictus, vector of chikungunya and dengue in the Indian Ocean. J Med
Entomol 2009; 46:33-41.

Diallo, M, Thonnon, J, Traore-Lamizana, M, Fontenille, D. Vectors of chikungunya virus
in Senegal: current data and transmission cycles. Am J Trop Med Hyg 1999; 60:
281-286.

Diarrassouba S, Dossou-Yovo J. Atypical activity rhythm in Aedes aegypti in a sub-
sudanian savannah zone of Cote d'lvoire. Bull Soc Pathol Exot 1997; 90:361-
363.

Dohm, DJ, O'Guinn, ML, Turell, MJ. Effect of environmental temperature on the ability of
Culex pipiens (Diptera: Culicidae) to transmit West Nile virus. J Med Entomol
2002; 39:221-225.

Dubrulle M, Mousson L, Moutailler S, Vazeille M, Failloux AB. Chikungunya virus and
Aedes mosquitoes: saliva is infectious as soon as two days after oral infection.
PLoS One 2009; 4:e5895.

Dwibedi B, Mohapatra N, Beuria MK, Kerketta AS, et al. Emergence of chikungunya
virus infection in Orissa, India. Vector Borne Zoonot Dis 2009; Online ahead of
print http://www.liebertonline.com/doi/pdfplus/10.1089/vbz.2008.0190.

Edman JD, Strickman D, Kittayapong P, Scott TW. Female Aedes aegypti (Diptera,
Culicidae) in Thailand rarely feed on sugar. J Med Entomol 1992; 29:1033-1038.


122









Effler PV, Pang L, Kitsutani P, Vorndam V, et al. Dengue fever, Hawaii, 2001-2002.
Emerging Infectious Diseases 2005; 11:742-449.

Fish D, Carpenter SR. Leaf litter and larval mosquito dynamics in tree-hole ecosystems.
Ecology 1982; 63:283-288.

Fontenille D, Rodhain F. Biology and distribution of Aedes albopictus and Aedes aegypti
in Madagascar. J Am Mosq Control Assoc 1989; 5:219-225.

Forattini OP. Aedes (Stegomyia) albopictus (Skuse) Identification in Brazil. Revista De
Saude Publica 1986; 20:244-245.

Foster WA. Mosquito sugar feeding and reproductive energetic. Annual Review of
Entomology 1995; 40:443-474.

Gould EA, Higgs S. Impact of climate change and other factors on emerging arbovirus
diseases. Trans R Soc Trop Med Hyg 2009; 103:109-121.

Grimstad, PR, Haramis, LD. Aedes triseriatus (Diptera: Culicidae) and La Crosse virus.
III. Enhanced oral transmission by nutrition-deprived mosquitoes. J Med Entomol
1984; 21: 249-256.

Grimstad, PR, Walker, ED. Aedes triseriatus (Diptera: Culicidae) and La Crosse virus.
IV. Nutritional deprivation of larvae affects the adult barriers to infection and
transmission. J Med Entomol 1991; 28:378-386.

Halstead SB, Scanlon JE, Umpaivit P, Udomsakdi S. Dengue and chikungunya virus
infection in man in Thailand, 1962-1964. IV. Epidemiologic studies in the
Bangkok metropolitan area. Am J Trop Med Hyg 1969; 18:997-1021.

Hardy JL, Houk EJ, Kramer LD, Reeves WC. Intrinsic factors affecting vector
competence of mosquitoes for arboviruses. Annu Rev Entomol 1983; 28:229-
262.

Hardy, JL, Meyer, RP, Presser, SB, Milby, MM. Temporal variations in the susceptibility
of a semi-isolated population of Culex tarsalis to peroral infection with western
equine encephalomyelitis and St. Louis encephalitis viruses. Am J Trop Med Hyg
1990; 42:500-511.

Harrington LC, Edman JD, Scott TW. Why do female Aedes aegypti (Diptera :
Culicidae) feed preferentially and frequently on human blood? J Med Entomol
2001; 38:411-422.

Harrington LC, Scott TW, Lerdthusnee K, Coleman RC, et al. Dispersal of the dengue
vector Aedes aegypti within and between rural communities. Am J Trop Med Hyg
2005; 72:209-220.


123









Hawley, WA. Biology of Aedes albopictus. J Am Mosq Control Assoc 1988;
4(Supplement#1 ): 1-39.

Hawley WA, Reiter P, Copeland RS, Pumpuni CB, et al. Aedes albopictus in North
America Probable introduction in used tires from northern Asia. Science 1987;
236:1114-1116.

Holt RA, Subramanian GM, Halpern A, Sutton GG, et al. The genome sequence of the
malaria mosquito Anopheles gambiae. Science 2002; 298:129-149.

Honorio NA, Silva Wda C, Leite PJ, Goncalves JM, et al. Dispersal of Aedes aegypti
and Aedes albopictus (Diptera: Culicidae) in an urban endemic dengue area in
the State of Rio de Janeiro, Brazil. Mem Inst Oswaldo Cruz 2003; 98:191-198.

Houk EJ, Arcus YM, Hardy JL, Kramer LD. Binding of western equine encephalomyelitis
virus to brush border fragments isolated from mesenteronal epithelial cells of
mosquitoes. Virus Res 1990; 17:105-118.

Human KG, Gordon DM. Exploitation and interference competition between the invasive
Argentine ant, Linepithema humile, and native ant species. Oecologia 1996;
105:405-412.

Hurlbut HS. Effect of environmental temperature upon transmission of St. Louis
Encephalitis virus by Culex Pipiens Quinquefasciatus. J Med Entomol 1973;
10:1-12.

International Society for Infectious Diseases. ProMED mail archive numbers
20081217.3963, 20081211.3895, 20090302.9854, 20100224.0617,
20100323.0918; Accessed March 1, 2010.

Jennings, CD, Kay, BH. Dissemination barriers to Ross River virus in Aedes vigilax and
the effects of larval nutrition on their expression. Med Vet Entomol 1999;
13:43108.

Juliano SA. Species introduction and replacement among mosquitoes: Interspecific
resource competition or apparent competition? Ecology 1998; 79:255-268.

Juliano SA. Species interactions among larval mosquitoes: Context dependence across
habitat gradients. Annual Review of Entomology 2009; 54:37-56.

Juliano SA, Lounibos LP. Ecology of invasive mosquitoes: effects on resident species
and on human health. Ecology Letters 2005; 8:558-574.

Juliano SA, O'Meara GF, Morrill JR, Cutwa MM. Desiccation and thermal tolerance of
eggs and the coexistence of competing mosquitoes. Oecologia (Berlin) 2002;
130:458-469.


124









Jupp PG, Mclntosh BM. Chikungunya disease. In: Monath T, ed. The arboviruses:
Epidemiology and ecology. Boca Raton, FL: CRC Press; 1988:137-157.

Jupp, PG, Mclntosh, BM. Aedes furcifer and other mosquitoes as vectors of
chikungunya virus at Mica, northeastern Transvaal, South Africa. J Am Mosq
Control Assoc 1990; 6:415-420.

Kambhampati S, Black WCt, Rai KS. Geographic origin of the US and Brazilian Aedes
albopictus inferred from allozyme analysis. Heredity 1991; 67:85-93.

Kamimura, K, Matsuse, IT, Takahashi, H, Komukai, J, et al. Effect of temperature on the
development of Aedes aegypti and Aedes albopictus. Med Entomol Zool 2002;
53:53-58.

Kaplan I, Denno RF. Interspecific interactions in phytophagous insects revisited: a
quantitative assessment of competition theory. Ecology Letters 2007; 10:977-
994.

Kay, BH, Edman, JD, Fanning, ID, Mottram, P. Larval diet and the vector competence of
Culex annulirostris (Diptera: Culicidae) for Murray Valley encephalitis virus. J
Med Entomol 1989a; 26:487-488.

Kay, BH, Fanning, ID, Mottram, P. The vector competence of Culex annulirostris, Aedes
sagax and Aedes alboannulatus for Murray Valley encephalitis virus at different
temperatures. Med Vet Entomol 1989b; 3:107-112.

Kay BH, Jennings CD. Enhancement or modulation of the vector competence of
ochlerotatus vigilax (Diptera : Culicidae) for Ross River virus by temperature.
Journal of Medical Entomology 2002; 39:99-105.

Keirans JE, Fay RW. Effect of food and temperature on Aedes aegypti (L) and Aedes
triseriatus (Say) larval development. Mosquito News 1968; 8:338-342.

Khan AH, Morita K, Parquet Md Mdel C, Hasebe F, et al. Complete nucleotide
sequence of chikungunya virus and evidence for an internal polyadenylation site.
J Gen Virol 2002; 83:3075-3084.

Kilpatrick AM, Meola MA, Moudy RM, Kramer LD. Temperature, viral genetics, and the
transmission of West Nile virus by Culex pipiens mosquitoes. PLoS Pathogens
2008, 4(6) e1000092.

Kit LS. Emerging and re-emerging diseases in Malaysia. Asia Pac J Public Health 2002;
14:6-8.

Knudsen AB, Romi R, Majori G. Occurrence and spread in Italy of Aedes albopictus,
with implications for its introduction into other parts of Europe. J Am Mosq
Control Assoc 1996; 12:177-183.


125









Kumar NP, Joseph R, Kamaraj T, Jambulingam P. A226V mutation in virus during the
2007 chikungunya outbreak in Kerala, India. Journal of General Virology 2008;
89:1945-1948.

Kuno G, Chang GJ. Biological transmission of arboviruses: reexamination of and new
insights into components, mechanisms, and unique traits as well as their
evolutionary trends. Clin Microbiol Rev 2005; 18:608-637.

Lanciotti, RS, Kerst, AJ, Nasci, RS, Godsey, MS, et al. Rapid detection of West Nile
virus from human clinical specimens, field-collected mosquitoes, and avian
samples by a TaqMan reverse transcriptase-PCR assay. J Clin Microbiol 2000;
38:4066-4071.

Lanciotti RS, Kosoy OL, Laven JJ, Panella AJ, et al. Chikungunya virus in US travelers
returning from India, 2006. Emerging infectious diseases 2007; 13:764-767.

Laras K, Sukri NC, Larasati RP, Bangs MJ, et al. Tracking the re-emergence of
epidemic chikungunya virus in Indonesia. Trans R Soc Trop Med Hyg 2005;
99:128-141.

Leisnham PT, Juliano SA. Spatial and temporal patterns of coexistence between
competing Aedes mosquitoes in urban Florida. Oecologia 2009; 160:343-352.

Leisnham PT, Lounibos LP, O'Meara GF, Juliano SA. Interpopulation divergence in
competitive interactions of the mosquito Aedes albopictus. Ecology 2009;
90:2405-2413.

Liew C, Curtis CF. Horizontal and vertical dispersal of dengue vector mosquitoes,
Aedes aegypti and Aedes albopictus, in Singapore. Med Vet Entomol 2004;
18:351-360.

Ligon BL. Reemergence of an unusual disease: The chikungunya epidemic. Semin
Pediatr Infect Dis 2006; 17:99-104.

Lorenz, L, Beaty, BJ, Aitken, TH, Wallis, GP, et al. The effect of colonization upon
Aedes aegypti susceptibility to oral infection with yellow fever virus. Am J Trop
Med Hyg 1984; 33:690-694.

Lounibos, LP. The mosquito community of treeholes in subtropical Florida. In
Phytotelemata: Terrestrial Plants as Hosts forAquatic Insect Communities (eds.
J. H. Frank and L. P. Lounibos), pp. 223-246. Medford, NJ. Plexus Publishing
Inc., 1983.

Lounibos LP. Invasions by insect vectors of human disease. Annual Review of
Entomology 2002; 47:233-266.


126









Lounibos LP. Competitive displacement and reduction. In Biorational Control of
Mosquitoes (eds.TE Floore and J Becnel), pp. 276-282 American Mosquito
Control Association Bulletin No.7 2007; 23 (Suppl. No 2).

Lounibos LP, Nishimura N, Escher RL. Seasonality and components of oak leaf litterfall
in Southeastern Florida. Florida Scientist 1992; 55:92-98.

Lounibos LP, Nishimura N, Escher RL. Fitness of a treehole mosquito: influences of
food type and predation. Oikos 1993; 66:114-118.

Lounibos LP, O'Meara GF, Escher RL, Nishimura N, et al. Testing predictions of
displacement of native Aedes by the invasive Asian tiger mosquito Aedes
albopictus in Florida, USA. Biological Invasions 2001; 3:151-166.

Lounibos LP, O'Meara GF, Nishimura N, Escher RL. Interactions with native mosquito
larvae regulate the production of Aedes albopictus from bromeliads in Florida.
Ecological Entomology 2003; 28:551-558.

Ludwig GV, Christensen BM, Yuill TM, Schultz KT. Enzyme processing of La
Crosse virus glycoprotein G1: A bunyavirus-vector infection model. Virology 1989;
171:108-113.

Macdonald WW. Aedes aegypti in Malaya. II. Larval and adult biology. Annals of tropical
medicine and parasitology 1956; 50:399-414.

McCrae AWR, Henderson BE, Kirya BG, Sempala SDK. Chikungunya virus in the
Entebbe area of Uganda: isolations and epidemiology. Trans R Soc Trop Med
Hyg 1971; 65:152-168.

Maciel-de-Freitas R, Codeco CT, Lourenco-de-Oliveira R. 2007. Daily survival rates and
dispersal of Aedes aegypti females in Rio de Janeiro, Brazil. Am J Trop Med Hyg
2007; 76:659-665.

Madder, DJ, Surgeoner, GA, Helson, BV. Number of generations, egg production, and
developmental time of Culex pipiens and Culex restuans (Diptera: Culicidae) in
southern Ontario. J Med Entomol 1983; 20:275-287.

Mangiafico J. Chikungunya virus infection and transmission in five species of mosquito.
Am J Trop Med Hyg 1971; 20:642-645.

Mason LJ, Pashley DP, Johnson SJ. The laboratory as an altered habitat: Phenotypic
and genetic consequences of colonization. Florida Entomologist 1987; 70:49-58.

McDonald PT. Population characteristics of domestic Aedes aegypti (Diptera: culicidae)
in villages on the Kenya Coast I. Adult survivorship and population size. J Med
Entomol 1977; 14:42-48.


127









Mercado-Curiel, RF, Black, WC, Munoz, Mde.L. A dengue receptor as possible genetic
marker of vector competence in Aedes aegypti. BMC Microbiol 2008, 15:118-
133.

Mertens PP, Burroughs JN, Walton A, Wellby MP, et al. Enhanced infectivity of modified
bluetongue virus particles for two insect cell lines and for two Culicoides vector
species. Virology 1996; 217:582-593.

Miles JA, Pillai JS, Maguire T, Multiplication of Whataroa virus in mosquitoes. J Med
Entomol 1973; 10: 176-185.

Minitab. Minitab 15.1 for Windows. Minitab Inc. State College, PA, 2006.

Molina-Cruz A, Gupta L, Richardson J, Bennett K, et al. Effect of mosquito midgut
trypsin activity on dengue-2 virus infection and dissemination in Aedes aegypti,
Am J Trop Med Hyg 2005; 72:631-637.

Monteiro, LC, de Souza, JR, de Albuquerque, CM. Eclosion rate, development and
survivorship of Aedes albopictus (Skuse)(Diptera: Culicidae) under different
water temperatures. Neotropical Entomol 2007; 36:966-971.

Mooney, HA, Hobbs RJ (eds.). 2000. Invasive species in a changing world. Island
Press, Washington DC.

Moore CG. Aedes albopictus in the United States: Current status and prospects for
further spread. J Am Mosq Control Assoc 1999; 15:221-227.

Moreira LA, Ito J, Ghosh A, Devenport M, et al. Bee venom phospholipase inhibits
malaria parasite development in transgenic mosquitoes. J Biol Chem 2002;
277:40839-40843.

Mourya DT, Ranadive SN, Gokhale MD, Barde PV, et al. Putative chikungunya virus-
specific receptor proteins on the midgut brush border membrane of Aedes
aegypti mosquito, Indian J Med Res 1998; 107:10-14.

Muir LE, Kay BH. Aedes aegypti survival and dispersal estimated by mark-release-
recapture in northern Australia. Am J Trop Med Hyg 1998; 58:277-282.

Munstermann LE. Distinguishing geographic strains of the Aedes atropalpus group
(Diptera, Culicidae) by analysis of enzyme variation. Ann Entomol Soc Am 1980;
73:699 1980.

National Vector Borne Disease Control Programme (NVBDCP) Chikungunya situation in
India. 2006-2009. http://www.nvbdcp.gov.in/Chikun-cases.html,
http://www.nvbdcp.gov.in/Doc/chikun-update07.pdf. Accessed March 1, 2010


128









Nasci, RS, Mitchell, CJ. Larval diet, adult size, and susceptibility of Aedes aegypti
(Diptera, Culicidae) to infection with Ross River virus. J Med Entomol 1994;
31:123-126.

Nene V, Wortman JR, Lawson D, Haas B, et al. Genome sequence of Aedes aegypti, a
major arbovirus vector. Science 2007; 316:1718-1723.

Ng LC, Tan LK, Tan CH, Tan SS, et al. Entomologic and virologic investigation of
chikungunya, Singapore. Emerging Infectious Diseases 2009; 15:1243-1249.

Niebylski ML, Craig GB. Dispersal and survival of Aedes Albopictus at a scrap tire yard
in Missouri. J Am Mosq Control Assoc 1994; 10:339-343.

Njenga MK, Nderitu L, Ledermann JP, Ndirangu A, et al.Tracking epidemic chikungunya
virus into the Indian Ocean from East Africa. Journal of General Virology 2008;
89:2754-2760.

O'Meara GF, Evans LF, Jr., Gettman AD, Cuda JP. Spread of Aedes albopictus and
decline of Ae. aegypti (Diptera: Culicidae) in Florida. J Med Entomol 1995;
32:554-562.

O'Meara, GF, Gettman, AD, Evans, LF Jr. et. al. Invasion of cemeteries in Florida by
Aedes albopictus. J Am Mosq Control Assoc 1992; 8: 1-10.

O'Meara, GF, Vose, FE, Carlson, DB. Environmental factors influencing oviposition by
Culex (Culex) (Diptera:Culicidae) in two types of traps. J Med Entomol 1989;
26:528-534.

Pages F, Peyrefitte CN, Mve MT, Jarjaval F, et al. Aedes albopictus mosquito: The main
vector of the 2007 chikungunya outbreak in Gabon. PLoS One 2009;4:e4691.

Paquet, C, Quatresous, I, Solet, JL, Sissoko, D, et al. Chikungunya outbreak in
Reunion: Epidemiology and surveillance, 2005 to early January 2006. Euro
Surveill 2006; 11:E0602023.

Park T. Experimental studies of interspecies competition .11. Temperature, humidity, and
competition in two species of Tribolium. Physiol Zool 1954. 27:177-238.

Parola, P, de Lamballerie, X, Jourdan, J, Rovery, C, et al. Novel chikungunya virus
variant in travelers returning from Indian Ocean islands. Emerg Infect Dis 2006;
12:1493-1499.

Pastorino B, Muyembe-Tamfum JJ, Bessaud M, Tock F, et al. Epidemic resurgence of
chikungunya virus in democratic Republic of the Congo: Identification of a new
central African strain. J Med Virol 2004; 74:277-282.

Patz, JA, Epstein, PR, Burke, TA, Balbus, JM. Global climate change and emerging
infectious diseases. J Am Mosq Control Assoc 1996; 275:217-223.


129









Paulson, SL, Hawley, WA. Effect of body size on the vector competence of field and
laboratory populations of Aedes triseriatus for La Crosse virus. J Am Mosq
Control Assoc 1991; 7: 170-175.

Paupy C, Delatte H, Bagny L, Corbel V, et al. Aedes albopictus, an arbovirus vector:
From the darkness to the light. Microbes Infect 2009; 1:1177-1185.

Paupy C, Girod R, Salvan M, Rodhain F, et al. Population structure of Aedes albopictus
from La Reunion Island (Indian Ocean) with respect to susceptibility to a dengue
virus. Heredity 2001; 87:273-283.

Pesko K, Westbrook CJ, Mores CN, Lounibos LP, et al. Effects of infectious virus dose
and bloodmeal delivery method on susceptibility of Aedes aegypti and Aedes
albopictus to chikungunya virus. J Med Entomol 2009; 46:395-369.

Pletnev SV, Zhang W, Mukhopadhyay S, Fisher BR, et al. Locations of carbohydrate
sites on alphavirus glycoproteins show that El forms an icosahedral scaffold.
Cell 2001; 105:127-136.

Ponlawat A, Harrington LC. Blood feeding patterns of Aedes aegypti and Aedes
albopictus in Thailand. J Med Entomol 2005; 42:844-849.

Porretta D, Gargani M, Bellini R, Calvitti M, et al. Isolation of microsatellite markers in
the tiger mosquito Aedes albopictus (Skuse). Molecular Ecology Notes 2006;
6:880-881.

Powers AM, Brault AC, Tesh RB, Weaver SC. Re-emergence of chikungunya and
O'nyong-nyong viruses: Evidence for distinct geographical lineages and distant
evolutionary relationships. J Gen Virol 2000; 81:471-479.

Powers AM, Logue CH. Changing patterns of chikungunya virus: Re-emergence of a
zoonotic arbovirus. J Gen Virol 2007; 88:2363-2377.

Purse, BV, Mellor, PS, Rogers, DJ, Samuel, et al. Climate change and the recent
emergence of bluetongue in Europe. Nature reviews 2005; 3:171-181.

Rao TR. Immunological surveys of arbovirus infections in South-East Asia, with special
reference to dengue, chikungunya, and Kyasanur Forest disease. Bull World
Health Organ 1971; 44:585-591.

Ravi V. Re-emergence of chikungunya virus in India. Indian J Med Microbiol 2006;
24:83-84.

Reeves, WC, Hardy, JL, Reisen, WK, Milby, MM. Potential effect of global warming on
mosquito-borne arboviruses. J Med Entomol 1994; 31:323-332.


130









Reisen WK, Meyer RP, Presser SB, Hardy JL. Effect of temperature on the
transmission of western equine encephalomyelitis and St. Louis encephalitis
viruses by Culex tarsalis (Diptera: Culicidae). J Med Entomol 1993; 30:151-160.

Reisen WK, Fang Y, Martinez VM. Effects of temperature on the transmission of West
Nile virus by Culex tarsalis (Diptera: Culicidae). J Med Entomol 2006; 43:309-
317.

Reiskind, MH, Pesko, K, Westbrook, CJ, Mores, CN. Susceptibility of Florida
mosquitoes to infection with chikungunya virus. Am J Trop Med Hyg 2008;
78:422-425.

Reiskind MH, Lounibos LP. Effects of intraspecific larval competition on adult longevity
in the mosquitoes Aedes aegypti and Aedes albopictus. Med Vet Entomol 2009;
23:62-68.

Reiter P, Amador MA, Anderson RA, Clark GG. Short report: dispersal of Aedes aegypti
in an urban area after blood feeding as demonstrated by rubidium-marked eggs.
Am J Trop Med Hyg 1995; 52:177-179.

Rey JR, Nishimura N, Wagner B, Braks MAH, et al. Habitat segregation of mosquito
arbovirus vectors in south Florida. J Med Entomol 2006; 43:1134-1141.

Rezza, G, Nicoletti, L, Angelini, R, Romi, R, et al. Infection with chikungunya virus in
Italy: An outbreak in a temperate region. Lancet 2007; 370:1840-1846.

Richards SL, Apperson CS, Ghosh SK, Cheshire HM, et al. Spatial analysis of Aedes
albopictus (Diptera : Culicidae) oviposition in suburban neighborhoods of a
piedmont community in North Carolina. J Med Entomol 2006; 43:976-989.

Richards SL, Mores CN, Lord CC, Tabachnick WJ. Impact of extrinsic incubation
temperature and virus exposure on vector competence of Culex pipiens
quinquefasciatus Say (Diptera: Culicidae) for West Nile virus. Vector Borne
Zoonot Dis 2007; 7:629-636.

Robinson MC. An epidemic of virus disease in Southern Province, Tanganyika Territory,
in 1952-53. I. Clinical features. Trans R Soc Trop Med Hyg 1955; 49:28-32.

Ross RW. The Newala epidemic. Ill. The virus: Isolation, pathogenic properties and
relationship to the epidemic. J Hyg (Lond) 1956; 54:177-191.

Rozeboom LE, Rosen L, Ikeda J. Observations on oviposition by Aedes-(S)-albopictus
Skuse and A-(S)-polynesiensis Marks in nature. J Med Entomol 1973; 10:397-
399.

Rueda, LM, Patel, KJ, Axtell, RC, Stinner, RE. Temperature-dependent development
and survival rates of Culex quinquefasciatus and Aedes aegypti (Diptera:
Culicidae). J Med Entomol 1990; 27:892-898.


131









Russell RC. larval competition between the introduced vector of dengue fever in
Australia, Aedes aegypti (L), and a native container-breeding mosquito, Aedes
notoscriptus (Skuse) (Diptera, Culicidae). Aust J of Zool 1986; 34:527-534.

Rutledge, LC, Ward, RA, Gould, DJ. Studies on the feeding response of mosquitoes to
nutritive solutions in a new membrane feeder. Mosq News 1964; 24:407-419.
SAS.

Samuel PP, Krishnamoorthi R, Hamzakoya KK, Aggarwal CS. Entomo-epidemiological
investigations on chikungunya outbreak in the Lakshadweep islands, Indian
Ocean. The Indian Journal of Medical Research 2009; 129:442-445.

Sang RC, Ahmed O, Faye O, Kelly CL, et al. Entomologic investigations of a
chikungunya virus epidemic in the Union of the Comoros, 2005. Am J Trop Med
Hyg 2008; 78:77-82.

Santhosh SR, Dash PK, Parida MM, Khan M, et al. Comparative full genome analysis
revealed El: A226V shift in 2007 Indian chikungunya virus isolates. Virus
research 2008; 135:36-41.

SAS 9.1 for Windows. SAS Institute, Inc., Cary, NC, 2003.

Savage HM, Ezike VI, Nwankwo AC, Spiegel R, et al. First record of breeding
populations of Aedes albopictus in continental Africa: Implications for arboviral
transmission. J Am Mosq Control Assoc 1992; 8:101-103.

Savage HM, Niebylski ML, Smith GC, Mitchell CJ, et al. Host-feeding patterns of Aedes
albopictus (Diptera: Culicidae) at a temperate North American site. J Med
Entomol 1993; 30:27-34.

Schabenberger O. Introducing the GLIMMIX procedure for generalized linear models.
SUGI 30. Cary, NC: SAS Institute. 2007.

Schliessmann.DJ, Calheiro.LB. Review of status of yellow fever and Aedes aegypti
eradication programs in Americas. Mosquito News 1974; 34:1-9.

Schuffenecker I, Iteman I, Michault A, Murri S, et al. Genome microevolution of
chikungunya viruses causing the Indian Ocean outbreak. PLoS Med 2006;
3:e263.

Scott TW, Amerasinghe PH, Morrison AC, Lorenz LH, et al. Longitudinal studies of
Aedes aegypti (Diptera : Culicidae) in Thailand and Puerto Rico: Blood feeding
frequency. J Med Entomol 2000; 37:89-101.

Scott, TW, Chow, E, Strickman, D, Kittayapong, P, et al. Blood-feeding patterns of
Aedes aegypti (Diptera: Culicidae) collected in a rural Thai village. J Med
Entomol 1993a; 30:922-927.


132









Scott TW, Clark GG, Lorenz LH, Amerasinghe PH, et al. Detection of multiple blood
feeding in Aedes aegypti (Diptera, Culicidae) during a single gonotrophic cycle
using a histologic technique. J Med Entomol 1993b; 30:94-99.

Seneviratne, SL, Gurugama, P, Perera, J. Chikungunya viral infections: an emerging
problem. J Travel Med 2007; 14:320-325.

Sergon K, Yahaya AA, Brown J, Bedja SA, et al. Seroprevalence of chikungunya virus
infection on Grande Comore Island, union of the Comoros, 2005. Am J Trop Med
Hyg 2007; 76:1189-1193.

Sergon K, Njuguna C, Kalani R, Ofula V, et al. Seroprevalence of chikungunya virus
(CHIKV) infection on Lamu Island, Kenya, October 2004. Am J Trop Med Hyg
2008; 78:333-337.

Settle WH, Wilson LT. Invasion by the variegated leafhopper and biotic interactions -
Parasitism, competition, and apparent competition. Ecology 1990; 71:1461-1470.

Smith DR, Adams AP, Kenney JL, Wang, E. Venezuelan equine encephalitis virus in
the mosquito vector Aedes taeniorhynchus: Infection initiated by a small number
of susceptible epithelial cells and a population bottleneck. Virology 2008;
372:176-186.

Smith GC, Eliason DA, Moore CG, Ihenacho EN. Use of elevated temperatures to kill
Aedes albopictus and Ae. aegypti. J Am Mosq Control Assoc 1988; 4:557-558.

Sota T, Mogi, M. Interspecific variation in desiccation survival time of Aedes
(Stegomyia) mosquito eggs is correlated with habitat and egg size. Oecologica
1992; 90:353-358.

Sota T, Mogi M, Hayamizu E. Seasonal distribution and habitat selection by Aedes
albopictus and Ae rivers (Diptera, Culicidae) in Northern Kyushu, Japan. J Med
Entomolo 1992; 29:296-304.

Sourisseau M, Schilte C, Casartelli N, Trouillet C, et al. Characterization of reemerging
chikungunya virus. Plos Pathogens 2007; 3:804-817.

Sprenger D, Wuithiranyagool T. The discovery and distribution of Aedes albopictus in
Harris County, Texas. J Am Mosq Control Assoc 1986; 2: 217-219.

Strauss JH, Strauss EG. The alphaviruses: gene expression, replication, and evolution.
Microbiol Rev 1994; 58:491-562.

Strickman D, Kittayapong P. Dengue and its vectors in Thailand: calculated
transmission risk from total pupal counts of Aedes aegypti and association of
wing-length measurements with aspects of the larval habitat. Am J Trop Med Hyg
2003; 68:209-217.


133









Sumanochitrapon, W, Strickman, D, Sithiprasasna, R, Kittayapong, P, et al. Effect of
size and geographic origin of Aedes aegypti on oral infection with dengue-2 virus.
Am J Trop Med Hyg 1998; 58:283-286.

Tabachnick WJ. Evolutionary genetics and arthropod-borne disease: The yellow fever
mosquito. American Entomologist 1991; 37:14-24.

Tabachnick WJ. Challenges in predicting climate and environmental effects on vector-
borne disease episystems in a changing world. J Exp Biol 2010;213:946-54.

Tabachnick WJ, Powell JR. A world-wide survey of genetic variation in the yellow fever
mosquito, Aedes aegypti. Genet Res 1979; 34:215-229.

Tabachnick WJ, Wallis GP, Aitken THG, Miller BR, et al. Oral infection of Aedes aegypti
with yellow fever virus geographic variation and genetic considerations. American
J Trop Med Hyg 1985; 34:1219-1224.

Takahashi M. The effects of environmental and physiological conditions of Culex
tritaeniorhynchus on the pattern of transmission of Japanese encephalitis virus. J
Med Entomol 1976; 13:275-284.

Teng HJ, Apperson CS. Development and survival of immature Aedes albopictus and
Aedes triseriatus (Diptera: Culicidae) in the laboratory: Effects of density, food,
and competition on response to temperature. J Med Entomol 2000; 37:40-52.

Thavara U, Tawatsin A, Pengsakul T, Bhakdeenuan P, et al. Outbreak of chikungunya
fever in Thailand and virus detection in field population of vector mosquitoes,
Aedes aegypti (L.) and Aedes albopictus Skuse (Diptera: Culicidae). The
Southeast Asian Journal of Tropical Medicine and Public Health 2009; 40:951-
962.

Trpis M, Hausermann W. Demonstration of differential domesticity of Aedes aegypti (L)
(Diptera, Culicidae) in Africa by mark release recapture. Bulletin of Entomol Res
1975; 65:199-208.

Tsetsarkin, KA, Vanlandingham, DL, McGee, CE, Higgs, S. A single mutation in
chikungunya virus affects vector specificity and epidemic potential. PLoS Pathog
2007; 3:e201.

Turell, MJ, Gargan, TP, Bailey, CL. Replication and dissemination of Rift Valley fever
virus in Culex pipiens. Am J Trop Med Hyg 1984; 33:176-181.

Turell, MJ, Beaman, JR, Tammariello, RF. Susceptibility of selected strains of Aedes
aegypti and Aedes albopictus (Diptera, Culicidae) to chikungunya virus. J Med
Entomol 1992; 29:49-53.


134









Turell, MJ. Effect of environmental temperature on the vector competence of Aedes
taeniorhynchus for Rift Valley fever and Venezuelan equine encephalitis viruses.
Am J Trop Med Hyg 1993; 49:672-676.

Udaka, M. Some ecological notes on Aedes albopictus in Shikoku, Japan. Kontyu 1959;
27: 202-208.

van Lieshout, M, Kovats, RS, Livermore, MTJ, Martens, P. Climate change and malaria:
analysis of the SRES climate and socio-economic scenarios. Global Environ
Chang 2004; 14:87-99.

Vazeille M, Jeannin C, Martin E, Schaffnerbl F, et al. Chikungunya: A risk for
Mediterranean countries? Acta Tropica 2008; 105:200-202.

Vazeille M, Mousson L, Failloux AB. Failure to demonstrate experimental vertical
transmission of the epidemic strain of Chikungunya virus in Aedes albopictus
from La Reunion Island, Indian Ocean. Memorias do Instituto Oswaldo Cruz
2009; 104:632-635.

Watson R. Europe witnesses first local transmission of chikungunya fever in Italy. BMJ
2007; 335:532-533.

Wada, Y. Effect of larval density on the development of Aedes aegypti (L.) and the size
of adults. Quaest Entomol 1965; 1:223-249.

Weaver SC. Electron-microscopic analysis of infection patterns for venezuelan equine
encephalomyelitis virus in the vector mosquito, Culex (Melanoconion) taeniopus.
Am J Trop Med Hyg 1986; 35:624-631.

Weaver SC. Detection of eastern equine encephalomyelitis virus deposition in Culiseta
melanura following ingestion of radiolabeled virus in blood meals. Am J Trop Med
Hyg 1991; 44:250-259.

Westbrook CJ, Reiskind MH, Pesko KN, Greene KE, et al. Larval environmental
temperature and the susceptibility of Aedes albopictus Skuse (Diptera: Culicidae)
to chikungunya virus. Vector Borne Zoonot Dis 2009; Online ahead of print
http://www.liebertonline.com/doi/pdfplus/10.1089/vbz.2009.0035.

World Health Organization. Chapter 5. Vector surveillance and control in dengue
haemorrhagic fever: diagnosis, treatment, prevention and control. 1997; 2nd
edition. Geneva, Switzerland:

Wu, HH., Chang N.T. Influence of temperature, water quality and pH value on ingestion
and development of Aedes aegypti and Aedes albopictus (Diptera: Culicidae)
larvae. Chin J Entomol 1993; 13:33-44.


135









Xu G, Wilson W, Mecham J, Murphy K, et al. VP7: An attachment protein of bluetongue
virus for cellular receptors in Culicoides variipennis. J Gen Virol 1997; 78: 1617-
1623.

Xu GZ, Dong HJ, Shi NF, Liu SA, et al. An outbreak of dengue virus serotype 1 infection
in Cixi, Ningbo, People's Republic of China, 2004, associated with a traveler from
Thailand and high density of Aedes albopictus. Am J Trop Med Hyg 2007;
76:1182-1188.

Yergolkar PN, Tandale BV, Arankalle VA, Sathe PS, et al. Chikungunya outbreaks
caused by African genotype, India. Emerg Infect Dis 2006; 12:1580-1583.

Yoshida S, Shimada Y, Kondoh D, Kouzuma Y,et al. Hemolytic C-type lectin CEL-III
from sea cucumber expressed in transgenic mosquitoes impairs malaria parasite
development. PLoS pathogens 2007; 312:e192.

Zhou XH, Weng ES, Luo YQ. Modeling patterns of nonlinearity in ecosystem responses
to temperature, C02, and precipitation changes. Ecological Applications 2008;
18:453-466.


136









BIOGRAPHICAL SKETCH

Catherine Jane Westbrook was born in Washington, D.C. in 1972. She graduated

from the University of California, Berkeley in 1996 with a Bachelor of Arts degree in

integrative biology. In 2003, she received her Master of Science degree from Cornell

University in entomology. In 2007, she began her doctoral work at the University of

Florida under the guidance of Dr. L. Philip Lounibos at the Florida Medical Entomology

laboratory in Vero Beach, FL.





PAGE 1

1 LARVAL ECOLOGY AND ADULT VECTOR COMPETENCE OF INVASIVE MOSQUITOES A edes albopictus AND Aedes aegypti FOR CHIKUNGUNYA VIRUS By CATHERINE JANE WESTBROOK A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF F LORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010

PAGE 2

2 2010 Catherine Jane Westbrook

PAGE 3

3 To my husband and daughter, Bill and Seneca Turechek

PAGE 4

4 ACKNOWLEDGMENTS I would like to ackno wledge my dissertation committee, L.P. Lounibos, C. Smartt, W. Tabachnick and P. Gibbs for their constructive comments and guidance. I feel very fortunate that I had such a supportive, motivating, and intellectually stimulating advisor in L.P. Lounibos and I am grateful that he was able to support my work through his NIH grant ( R01 AI 044793) I am grateful to B. Alto for allowing me to build on much of the work he started in the Lounibos lab at FMEL. I am also very thankful that M. Reiskind and K. Pesko we re such great teachers, collaborators and friends. I would like to thank K. Greene and N. Nishimura for all their hard work on all of the experiments. I would like to thank E. Blosser and S. Anderson for making themselves available at critical times in my final experiment. I want to thank S. Yost for providing me with cultured cells. I appreciate C. Lord for permitting me to monopolize her incubators for nearly three years. I would like to thank for their critical reading of the manuscript resulting from Chapter 2 I also need to thank a long list of people at FMEL that helped me pick and identify pupae. The list includes, but is probably not limited to

PAGE 5

5 T ABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 L IST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 ABSTRACT ................................ ................................ ................................ ................... 10 CHAPTER 1 INTRODUCTION AND REVIEW OF THE LITERATURE ................................ ....... 12 Introductory Statement ................................ ................................ ............................ 12 Mosquitoes: Aedes aegypti and Aedes albopictus ................................ .................. 14 Invasion Biology of Disease Vectors ................................ ................................ 15 Host Preference a nd Feeding ................................ ................................ ........... 17 Short Range Dispersal and Longevity ................................ .............................. 19 Oviposition ................................ ................................ ................................ ........ 21 Thermal Tolerance ................................ ................................ ........................... 22 Eradication and Control ................................ ................................ .................... 24 C ompetition Between Aedes Albopictus and Ae. Aegypti ................................ 24 Chikungunya Virus ................................ ................................ ................................ .. 26 Discovery of Virus and Vectors ................................ ................................ ........ 26 Viral Characterization and Phylogenetics ................................ ......................... 28 Historical an d Current Epidemics ................................ ................................ ..... 29 CHIKV Interactions in Epidemic Vectors: Aedes aegypti and Ae. albopictus ... 30 Vector Competence ................................ ................................ ................................ 33 The Environment and Vector Competence ................................ ....................... 34 The Larval Envi ronment, Vector Competence, and Mosquito Size .................. 35 2 LARVAL ENVIRONMENTAL TEMPERATURE AND THE SUSCEPTIBILITY OF Aedes albopictus SKUSE (DIPTERA: CULICIDAE) TO CHIKUNGUNYA VIRUS .. 38 Introduction ................................ ................................ ................................ ............. 38 Materials and Methods ................................ ................................ ............................ 41 Mosquitoes and Viruses ................................ ................................ ................... 41 Vector Competence ................................ ................................ .......................... 42 Statistical Analysis ................................ ................................ ............................ 44 Results ................................ ................................ ................................ .................... 45 Growth and Mortality ................................ ................................ ........................ 45 Chikungunya Infection and Dissemi nation ................................ ........................ 45 Discussion ................................ ................................ ................................ .............. 46

PAGE 6

6 3 LARVAL TEMPERATURE AND NUTRITION ALTER THE SUSCEPTIBILITY OF Aedes aegypti L. (DIPTERA: CULICIDAE) MOSQUITOES TO CHIKUNGUNYA VIRUS ................................ ................................ ......................... 54 Introduction ................................ ................................ ................................ ............. 54 Materials and Methods ................................ ................................ ............................ 57 Mosquitoes and Viruses ................................ ................................ ................... 57 Mosquito Infection ................................ ................................ ............................ 59 Statistical Analysis ................................ ................................ ............................ 62 Results ................................ ................................ ................................ .................... 64 Chikungunya Titer of Freshly Engorged Mosquit oes ................................ ........ 64 Growth and Mortality ................................ ................................ ........................ 64 Chikungunya Infection and Dissemination ................................ ........................ 65 Discussion ................................ ................................ ................................ .............. 66 4 LARVAL TEMPERATURE, COMPETITION, AND THE VE CTOR COMPETENCE OF Aedes aegypti AND Aedes albopictus FOR CHIKUNGUNYA VIRUS ................................ ................................ ......................... 81 Introduction ................................ ................................ ................................ ............. 81 Materials and Methods ................................ ................................ ............................ 84 Mosquitoes, Temperature, and Competition ................................ ..................... 84 Virus and Mos quito Infection ................................ ................................ ............ 86 Statistical Analysis ................................ ................................ ............................ 89 Results ................................ ................................ ................................ .................... 92 Mosquitoes, Temperature, and Competition ................................ ..................... 92 Virus and Mosquito Infection ................................ ................................ ............ 94 Discussion ................................ ................................ ................................ .............. 96 5 GENERAL CONCLUSIONS AND FUTURE DIRECTIONS ................................ .. 113 LIST OF REFERENCES ................................ ................................ ............................. 119 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 137

PAGE 7

7 LIST OF TABLES Table page 2 1. Aedes albopictus treatment medians and interquartile ranges (IQR= 25th percentile 75th percentile) for development ................................ ...................... 52 3 1. Aedes aegypti LS means and standard error for development time to adulthood and female wing length. ................................ ................................ ..... 73 3 2. Maximum likelihood (ML) contrasts for comparisons of mortality rates for 34C high food treatment ................................ ................................ .................... 74 4 1. MANOVA fo r temperature and competitive treatment effects and their interaction on population growth parameters ................................ .................... 103 4 2. Multivariate pairwise contrasts of temperature treatment effects on female Aedes albopictus and Aedes aegypti for growth measurements h. ................... 104 4 3. Multivariate pairwise contrasts of competitive treatment effects on female Aedes albopictus and Aedes aegypti for growth measurements ..................... 105

PAGE 8

8 LIST OF FIGURES Figure page 2 1. Bivariate plots of mean wing lengths (SE) and CHIKV susceptibil ity ................ 53 3 1. Correlation between log transformed whole mosquito body titers of CHIKV and wing lengths for engorged Aedes aegypti ................................ ................... 75 3 2. Bivariate plot of LS means (SE) for wing lengths and log transformed CHIKV body titers for engorged Aedes aegyp ti females ................................ ..... 76 3 3. Juvenile mortality rates at the low and high food levels within the three temperature treatments for Aedes aegypti ................................ ......................... 77 3 4. Proportion of Aedes aegypti females (SE) in each temperature tr eatment infected with CHIKV ................................ ................................ ........................... 78 3 5. Proportions of infected Aedes aegypti females (SE) from temperature and food level treatments with disseminated CHIKV infections ................................ 79 3 6. Least squared means (SE) for sizes of adult female Ae. aegypti mosquitoes in CHIKV infection status categories. ................................ ................................ 80 4 1. Proportion of female Ae. albopictus (SEM) surviving to adult emergence. ..... 106 4 2. Female Ae. albopictus mean (SEM) days to pupation. ................................ ... 106 4 3. Female Ae. albopictus mean (SEM) wing length. ................................ ........... 107 4 4. Proportion of female Ae. aegypti (SEM) surviving to adult emergence. .......... 108 4 5. Female Ae. aegypti mean (SEM) days to pupation. ................................ ....... 108 4 6. Bivariate means ( SEM) of replicates for proportions of Ae. aegypti and Ae. albopictus w ith infections and disseminated infections. ................................ .... 109 4 7. Bivariate plot of least squares means for proportion of Ae. albopictus with infecti ons and disseminated infections ................................ ............................. 109 4 8. LS means ( SEM) of Aedes albopictus CHIKV body titer for temperature treatments ................................ ................................ ................................ ......... 110 4 9. Correlation analysis of log transformed of CHIKV whole mosquito body titer and wing length ................................ ................................ ............................... 111 4 10. Correlation analysis of log transformed of CHIKV whole mosquito body titers and wing lengths for Aedes aegypti ................................ ................................ .. 111

PAGE 9

9 4 11. Least squared means (SE) for sizes of adult female Ae. albopictus mosq uitoes in CHIKV infection status categories. ................................ ............ 112 4 12. Least squared means (SE) for sizes of adult female Ae. aegypti mos quitoes in CHIKV infection status categories. ................................ ............................... 112

PAGE 10

10 Abstract of Dissertation Presented to the Graduate School of the University of Flor ida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy LARVAL ECOLOGY AND ADULT VECTOR COMPETENCE OF INVASIVE MOSQUITOES Aedes albopictus AND Aedes aegypti FOR CHIKUNGUNYA VIRUS By Catherine Jane Westbrook A ugust 2010 Chair: L. Philip L ounibos Major: Entomology and Nematology Abiotic and biotic features of the mosquito larval environment shape life history traits important in arbovirus dynamics ( e g fecundity, life span, biting rate) and can directly affe ct characteristics that influence adult arbovirus susceptibility. C hikungunya virus (CHIKV) has recently emerged as an important agent of human arboviral disease and the invasive container mosquitoes Aedes albopictus and Ae. aegypti are the epidemic vecto rs. When the effect of larval rearing temperature on Ae. albopictus growth and susceptibility to CHIKV was investigated results showed that temperature had a significant effect on size, development time, and CHIKV infection and dissemination rates. Adult females produced from the coolest temperature, had the largest mean body size, took the longest to mature, and were 6 times more likely to be infected with CHIKV than females reared at the highest temperature In a s eparate experiment Ae. aegypti larvae w ere reared at different temperatures and food levels to explore relationships among attributes of the larval habitat, body size, and CHIKV susceptibility. Larval temperature and food availability had significant effects on m ean adult body size and female size and quantity of CHIKV ingested w ere

PAGE 11

11 positively correlated. Larval temperature, but not food quantity nor the temper ature x food level interaction, had a significant effect on CHIKV infection, but temperature, food level, and the ir interaction had a si gnificant effect on dissemination. Significant wing length infection correlations disappeared after the extrinsic incubation period suggesting that mosquito size alone may n ot be a good predictor of viral susceptibility. Larval competition between Ae. a egypti and Ae. albopictus within the container habitat is modulated by temperature and these factors may interactively influen ce adult susceptibility to CHIKV T he outcome of competiti on between the two species did not change with temperature and Ae. aeg ypti was found to be the superior competitor under these experimental conditions. Temperature and larval competition did not affect the likelihood of infection or disseminated infection with CHIKV However, mean body titer of CHIKV infected Ae. albopictus females was significantly affected by larval rearing temperature, with females reared at lower temperatures having higher mean CHIKV body titers than counterparts from the highest temperature

PAGE 12

12 CHAPTER 1 INTRODUCTION AND REV IEW OF THE LITERATUR E Introductor y Statement Fluctuating temperatures, limited food, and high inter and intraspecific competition are common features in container habitats occupied by the aquatic larvae of many holometabolous insects, including the Asian tiger mosquito, Aedes albopictus and the yellow fever mosquito, Aedes aegypti (Juliano 2009). The effect of the mosquito larval environment on adult life history traits and other characteristics such as growth rate, age at maturity, biting rates, gonotrophic cycle lengths, vector size, fe cundity, and life span are well documented (Madder et al. 1983, Briegel 1990, Rueda et al. 1990, Scott et al. 1993 a ). However, the larval environment may also influence adult vector pathogen ting in changes in the distribution and transmission intensity of a n arbovirus Vector competence is the capacity of an arthropod to acquire a pathogen and transmit it to a subsequent host. For successful transmission to occur arbovirus taken in from an in fectious blood meal must overcome internal barriers in the mosquito midgut, disseminate into other organs, and then pass through an additional barrier into the salivary glands in order to infect a new host during the next blood feeding (Hardy et al. 1983) Vector competence can vary greatly among individuals and between mosquito populations (Lorenz et al. 1984, Tabachnick et al. 1985, Turell et al. 1992 Paupy et al. 2001 ) and has been shown to be influenced by both the genetic background of a vector and en vironmental conditions (Davis 1932 Hurlbut 1973, Hardy et al. 1990 Kilpatrick et al. 2008 ). Most research has focused on the adult environment with fewer studies exploring the influence of larval ecology on mosquito arbovirus interactions. There is evide nce

PAGE 13

13 that larval habitat variables, such as temperature, food resources, density and competition can affect vector competence in certain mosquito arbov iral systems (Baqar et al. 1980, Grimstad and Walker 1991, Turell 1993, Sumanochitrapon et al. 1998, Alto et al. 2005, Alto et al. 2008 a Bevins 2008 ). However, the strength of the effect and the direction seems to vary with different species of mosquitoes and different viruses. Furthermore, previous research has primarily focused on the effect of a single fac tor in the larval environment without consideration for the potential effects of interactions between variables. The research presented in this dissertation e xplores the interactions of larval ecological factors and their impact on the transmission of chik ungunya virus (CHIKV ) by invasive container mosquitoes Aedes albopictus and Ae. aegypti The hypothesi s tested here is that variation in environmental factors during larval development affects the physical and physiological characteristics of adults to alt er their ability to become infected and/or transmit an arbovirus. Experiments were conducted manipulating: (1) larval rearing temperature, (2) larval rearing temperature and larval food level s and (3) larval rearing temperature and intra and inter specif ic larval competition in Ae. albopictus and/or Ae. aegypti from South Florida. To determine the effect of larval habitat quality on life history characteristics of these mosquitoes, juvenile mortality, development time, and adult body size were m easured. A dult females were given a blood meal containing CHIKV and susceptibility to the virus was assessed by measuring infection and dissemination rates and whole mosquito body viral titer. This research is novel for ex ploring the interactions among multiple fac tors in the larval environment and their impact on transmission potential of an ar bovirus by adult

PAGE 14

14 mosquitoes. Furthermore, this work is particularly relevant in light of predicted alteration s in temperature due to global climate change and the ongoing eco logical and public health problems caused by the continued intercontinental dispersal of invasive vectors and pathogens. The following literature review provides information on the mosquito vectors, CHIKV and the influence of the environment on vector com petence Chapters two, three, and four contain original research on the influence of Ae albopictus and Ae. aegypti larval ecological factors on adult CHIKV susceptibility, followed by chapter five which provides conclusion s and recommendation s for future research directions. Mosquitoes : Aedes aegypti and Aedes albopictu s As the volume of global trade and travel has greatly increased in the past fifty years, so have the number of accidental introductions of exotic organisms to new geographic areas (Mooney a nd Hobbs 2000). Invading organisms can have grave detrimental effects on indigenous communities, but when the invader is also a potential vector of an exotic human pathogen then the introduction and spread of the organism can also have a major public healt h consequence (Juliano and Lounibos 2005). The container mosquito species, Ae aegypti and Ae. albopictus have globally invasive ranges. Aedes aegypti is believed to have traveled to the New World from Africa in water storage jars aboard slave ships (Chris tophers 1960), while the spread of Ae. albopictus from its native Asian range has been a more recent phenomenon (Hawley et al. 1987). Arboviruses that are transmitted by Ae. aegypti and Ae. albopictus threaten the health of millions of peop le worldwide an d the continued range expansion of these species will add to the population s at risk.

PAGE 15

15 Invasion Biology of Disease Vectors Aedes ( Stegomyia ) aegypti (L.) the yellow fever mosquito is the primary epidemic vector of yellow fever virus ( YFV ) dengue virus ( D ENV ) CHIKV The preferred habitat of Ae. aegypti is the urban environment and a lthough it is limited to tropical and subtropical regions, within these limits it has a very cosmopolitan distribution. In East Africa Ae. aegypti exists in two forms: (1) Aedes aegypti formosus a sylvan and likely ancestral form with darker scales which oviposits primarily in treeholes a nd is often found away from human habitats and (2) Ae. aegypti aegypti a domestic form, which exhibits a preference for human habitats (Tabach nick and Powell 1979) In coastal Kenya, the sylvan and domestic Ae. aegypti represent two distinct gene pools, maintained through habitat selection, where sympatry exists gene flow between the two subspecies is limited (Tabachnick and Powell 1979) Though Ae. aegypti formosus has been found in other regions of subSaharan Africa, it is the domestic form, Ae. aegypti aegypti through its ability to exploit human water storage containers and human habitats, that has spread from Africa to tropical and subtropi cal regions across the globe (Christophers 1960, Tabachnick 1991). Key behavioral characters of Ae. aegypti aegypti are: oviposition in human water storage containers, feeding on human blood, and entering into homes in search of hosts, mates and ovipositio n and resting sites (Trpis and Hausermann 1975). O viposition containers such as water cisterns aboard New World bound slave ships during the 15 th to the 19th centuries are believed to be a major mode of introduction for this mosquito (Tabachnick 1991). T he spread of Ae. aegypti across the globe likely involved the movement of multiple life stages of the mosquito: aquatic larvae, adults, and eggs (Christophers

PAGE 16

16 1960). For the remainder of this dissertati on Ae. aegypti will be used as a synonym for Ae. aegyp ti aegypti Aedes ( Stegomyia ) albopictus (Skuse), the Asian tiger mosquito, is also an efficient and important vector of DEN and CHIKV and like Ae. aegypti Ae. albopictus has successfully spread across the globe through the exploitation of man made envir onments. Although common in suburban and rural setting s t his mo squito originated in the forest edges of Southeast Asia, but human migration towards the Malay peninsula and the Indian Ocean islands, including Madagascar, may have led to an early movement o f Ae. albopictus out of its native Asian range (Paupy et al. 2009). In the late nineteenth century Ae. albopictus began spread ing onto the Pacific islands, such as H awaii and Guam (Lounibos 2002). In 1985 it was discovered in the United States, where it w as identified as the most abundant artificial container inhabiting mosqui to in Houston, Texas (Sprenger and Wuithiranyagool 1986). Since its discovery, Ae albopictus has be come established in most states in the eastern part of the USA extending as far no rth as Illinois, Indiana, Ohio, Pennsylvania and New Jersey (Moore 1999). Between 19 8 5 and 1998, Ae albopictus was recover ed from many countries in the Americas and the Caribbean (Benedict et al. 2007). In Europe, it was first recorded in Albania in 1979, Italy in 1990, then France in 1999 and currently Ae albopictus is present in at least 12 European countries (Knudsen et al 1996, Vazeille et al. 2008). Established populations were recorded in Nigeria in 1991 (Savage et al. 1992) and since Ae albopi ctus has spread to other nations in West and Central Africa The establis hment of this mosquito in numerous countries in Africa, the Middle East, Europe

PAGE 17

17 and the Americas is primarily due to the trade and movement of used tires that contain Ae. albopictus eggs (Hawley et al. 1987, Benedict et al. 2007). Host Preference and Feeding Hum ans are the most common bloodmeal source of Ae. aegypti (Scott et al. 1993a Ponlawat and Harrington 2005 ), and much work has been done to determine how the consumption of huma n blood compares to blood from other vertebrate hosts in terms of fecundity and longevity in this mosquito (Briegel 1985, Harrington et al. 2001) Females of most mosquito species require both blood and sugar (Foster 1995) and both male and female Ae. aeg ypti have been observed feeding on plant nectars (Christophers 1960). H owever certain populations of Ae. aegypti in Thailand have adapted to an environment low in sugar, but where human blood is readily available. T hese females seldom feed on plant sugars and take more frequent blood meals (Edman et al. 1992). Laboratory studies on Ae. aegypti suggest that a diet of human blood without sugar increase s the reproductive success of this mosquito through a greater age specific survival (l x ), estimated reproduc tive output per day (m x ) and the net replacement rate ( R o ), although total egg production was greater in mosquitoes fed blood and sugar (Harrington et al. 2001). Aedes albopictus is considered a n opportunistic feeder, with a preference for mammals (Savage et al. 1993 ). In suburban North Carolina humans (24%), cats (21%) and dogs (14%) were the primary hosts (Richards et al. 2006 ). However, in villages in Thailand and Singapore humans were the primary sources of blood ( Colless 1959, Ponlawat and Harrington 2005). This species is thought to have progressively adapted to anthropogenic changes in the environment, moving from a zoophilic host feeding regime to a greater dependence on blood meals from humans and domesticated

PAGE 18

18 animals (Paupy et al. 2009). Laborator y studies on Ae albopictus also suggest that a diet of human blood without sugar increases the reproductive success of this species and thus the benefit of feeding exclusively on blood is not limited to the highly anthropophilic Ae. aegypti (Braks et al. 2006). Aedes aegypti and Ae. albopictus females often take multiple blood meals during a single gonotrophic cycle ( MacD onald 1956, Scott et al. 1993b Scott et al. 2000 Delatte et al 2009 ). This behavior, known as multiple feeding, can lead to an exponen tial increa se in the vectorial capacities of these two mosquitoes because the daily probabilit y of being fed upon is a squared function in the vectorial capacity equation resulting from the product of host preference index and the frequency of feeding (Bla ck and Moore 1996). Furthermore, multiple feeding can increase the probability of concurrent infection and viral genetic mixing (Kuno and Chang 2005). Aedes aegypti pref erentially feeds in the daytime, but individuals will on occasion feed at night if a su itable host is present (Christophers 1960) Ae albopictus is also a diurnal feeder with peak feeding times occurring at daybreak and two hours before sunset (Delatte et al 2009). N octurnal feeding sub populations of Ae. aegypti in the Ivory Coast (Diarrass ouba and Dossou Yovo 1997) and in Trinidad ( Chadee and Martinez 2000) are documented In Trinidad noct urnal feeding was recorded at the urban site and not at the rural site and accounted for approximately 10% of urban indoor feeding totals and 9.4% of outd oor urban feeding totals. It was suggested that the nocturnal extension of Ae. aegypti feeding may be an adaptation to increased electrical lighting in and around houses, and also explains the absence of nocturnal feeding in the poorly lit rural site (Chad ee and Martinez 2000).

PAGE 19

19 Short Range Dispersal and Longevity The movement of mosquitoes has been studied because of the important role of dispersal and flight range in vector borne disease. Many studies support the theory that Ae. aegypti takes fairly short flights and does not disperse over far distances Muir and Kay (1998), in a mark release recapture study in northern Australia found the mean distance Ae. aegypti traveled was 56 meters (m) for females and 35 m for males. Harrington et al (2005), in an 11 year study, involving 21 mark release recapture experiments in Puerto Rico and Thailand, concluded that the mean dispersal distance for Ae. aegypti ranged from 28 to 199 m and Maciel de Freitas et al. (2007) found a similar pattern in Rio de Janeiro, Braz il where the average distance travelled ranged between 81 to 86 m. In contrast a few studies have reported, through assaying rubidium label ed eggs, longer distance dispersal of up to 800 m in Rio de Janeiro (Honorio et al 2003) and means ranging between 2 21 279 m in Puerto Rico (Reiter et al. 1995) A major difference in the studies assaying labeled eggs is that they measured Ae. aegypti dispersal during an oviposition cycle and this may account for some of the differences between the two types of studies. Dispersal has been investigated in Ae albopictus and compared to Ae. aegypt i Ae des albopictus generally takes longe r flights and disperses over farther distances. In a mark release recapture study in Missouri the maximum distances Ae albopictus traveled w ere 525 m for females and 225 m for males (N i e bylski and Craig 1994). Through ovitrapping of rubidium labeled eggs, dispersal by Ae albopictus of up to 800 m was found in Rio de Janeiro and did not differ in distance from Ae. aegypti in this study (Honori o et al 2003) I n Singapore Ae albopictus rubidium labeled eggs were found at distances as far as 640 m from the release site (Li ew and Curtis 2004)

PAGE 20

20 The life span of adult mosquitoes in the field is determined by factors such as quality of the environmen t, climate, predation, and genetic background. Laboratory studies in which Ae. aegypti and Ae. a lbopictus are given ample sugar and blood meals while being held at a constant and suitable temperature with a high level of humidity, record maximum longevity for females at over 100 days and the mean life spans between 4 6 weeks (Christophers 1960 Hawley 1988 ). Daily survival probabilities of adult females are an important element of vectorial capacity and to transmit an arbovirus, mosquitoes must survive lo nger than the time prior to taking an infectious blood meal combined with the extrinsic incubation period (EIP) of the pathogen which is the time interval between ingestion of an infective blood meal and oral transmission of a virus (Davis 1932) The EIP for CHIKV in Ae. aegypti and Ae. a lbopictus can be as short as two days (Dubrelle et al. 2009). The assumption can be made that wild mosquito individuals at the mercy of biotic and abiotic factors in the environment experience greatly reduced longevity; th e difficulty is measuring it Mark release recapture experiments have been the primary method of assessing probability of daily survival (PDS) and in Rio de Janeiro, Brazil female Ae. aegypti PDS varied from 0.71 to 0.87 depending on the site and season ( Maciel de Freitas et al. 2007) and in Kenyan field studies PDS was 0.77 for male Ae. aegypti which corresponds to a 4.4 day life span and 0.89 ( 9.2 days ) for females ( McDonald 1977). For Ae. albopictus in a scrap tire yard in Potosi, Missouri PDS was 0.89 (8.2 days) for females a nd 0.77 (3.9 days) for males (N i e bylski and Craig 1994). Very low recapture rates in survival studies make the reliability of PDS estimates

PAGE 21

21 questionable and predictions of mosquito age in future studies may make use of newer techn ologies such as gene transcription profiling (Cook et al. 2006). Oviposition Gravid female Ae. aegypti and Ae. albopictus oviposit desiccation resistant eggs that can survive in a dried state for several months or longer until submersion in water triggers hatching. A common feature of oviposition containers selected by both f emale Ae. aegypti and Ae. albopictus is clean water with a high organic content ( Clements 1992, Delatte et al. 2008). A gravid Ae. aegypti female will select both indoor because of its close association with man, and outdoor containers for oviposition (Christophers 1960) and f or Ae. albopictus peridomestic and ru ral oviposition sites are frequently used ( Hawley 1988). Egg laying in both these mosquitoes is diurnal with the majority of e ggs lai d two hours after sunrise and two hours before sunset (Corbet and Chadee 1990 Delatte et al 2009 ) Based on the results of field studies on Ae. aegypti in Puerto Rico and Trinidad and on Ae. albopictus in Honolulu, Hawaii it was determined that fe males deposit their eggs from the same batch at several ( Rozeboom et al. 1973, Chadee and Corbet 1987 Apostol et al. 1994, Reiter et al. 1995 ) Skip oviposition may benefit the species by decreasin g sibling compet ition and spreading risk of mortality over multiple sites Ae des albopictus is remarkable for the wide range of natural and artificial container s in which it is found ranging from tree holes, bamboo stumps, rock holes, leaf axils, flower p lates, pots, catch basins, and discarded tires (Hawley 1988, Sota et al. 1992, Delatte et al 2008). On the island of Reunion, where it is the most common Aedes species, Ae. albopictus showed a strong ecological plasticity. I n the wet season

PAGE 22

22 this mosquito occured most frequently in small artificial disposable containers and in the dry winter season, natural containers (bamboo stumps and rock holes) were clearly important (Delatte et al. 2008). In North America, the most common microhabitat for Ae. albopictu s ha s been discarded tires (Sprenger and Wuithiranyagool 1986) However in Florida Ae. albopictus immatures are found in the water holding tanks and axils of ornamental bromeliads, although in southern Florida their use of t his phytotelm ata is kept in chec k by two endemic Wyeomyia spp. of mosquitoes (Lounibos et al 2003). In Florida Ae. albopictus is also found sharing tree holes with the native inhabitant Ae triseriatus (Lounibos et al 2001). Thermal T olerance No life stage of Ae. aegypti undergoes diap ause and 16 C seems to be close to the lower limit for adult activity of this species (Christophers 1960). Larvae continue to develop and pupate at temperatures as low as 15 C but the duration of the larval stage is approximately 31 32 days and at tempe ratures between 8.2 10.6 C development completely ceases (Kamimura et al. 2002). In terms of upper thermal limits Ae. aegypti larvae do not thrive in water temperature much above 34 C and adults begin to die if air temperatures exceed 40 C Desi c cation resistant e ggs are also susceptib le to temperature extremes. Prolonged exposure to a low temperature of 10 C and a high temperature of 40 C resulted in 100% egg mortality (Christophers 1960) In its native Asian range Ae. albopictus is abundant in both tr opical and temperate regions and as a result this mosquito species can survive over a broad spectrum of temperatures and relative humidity. At 11C Udaka (1959) found that larval development ceased for a Japanese strain of Ae. albopictus A similar resu lt was recorded with a strain of Ae. albopictus from Reunion island in the Indian Ocean, where

PAGE 23

23 no development beyond first instar took place at 5 C or 10 C (Delatte et al. 2009). At temperatures as low as 12C Ae. albopictus larvae develop ed and pupate d wi th an 28 day larval duration but two thirds of adult females did not mature eggs or died (Briegel and Timmermann 2001). Adults from a Reunion strain fared worse with no egg laying observed among females at 15 C (Delatte et al. 2009). The l imiting upper te mperature of Ae. albopictus larval development is 35C (Monteiro et al. 2007 Delatte et al. 2009 ) and at 43.3C 100% adult mortality was observed after approximately 30 minutes (Smith et al. 1988). Analysis of life tables, combining developmental rates, reproduction and mortality, suggest maximum population growth ( r ) between 25 and 30 C for Ae. albopictus tested at eight constant temperatures (5, 10, 15, 20, 25, 30, 35 and 40 C) (Delatte et al. 2009). S trains of Ae. albopictus found in temperate regions are sensitive to short day lengths during the pupal and adult stage leading to the production of diapause eggs (Hawley et al. 1987). Not long after Ae. albopictus was found in the United S t ates, this species was also identified in Brazil (Forattini 1986) but the lack of a photoperiodic egg diapause in Brazilian populations suggested distinct origins for the two populations (Hawley et al. 1987) Sequence data from the mitochondrial ND5 gene confirmed that the North American populations of Ae. albopictus o riginated from Asian temperate regions, while Brazilian populations are tropical in ori gin (Birungi and Munstermann 2002) This was in contrast to a n earlier allozyme study inferring a common Northern Asian (Japan) origin for both populati ons (Kambhampati et al. 1991). Future use of recently identified p olymorphic microsatellite loci (Porretta et al. 2006) in population

PAGE 24

24 genetic studies may help to further elucidate the structure and relationships among invasive Ae. albopictus populations. Eradication and Co ntrol I n 1947 the countries comprising the Pan American Health O rganization (PAHO) proposed a resolution to eradicate Ae. aegypti primarily to control yellow fever. At the time of the resolution Ae. aegypti was known from al l of the western hemisphere exc ept Canada and Bolivia. Between 1958 and 1965 eradication was accomplish ed in 17 of 2 3 targeted countries primarily through perifocal application of DDT insecticide to infested containers (Schliessmann and Calheiros 1974) H owever countries and territor ies in the Caribbean had a more difficult time achieving eradication and by the 1970s with depleting financial resources and subsequent social uphea val in countries such as El Salvador re infestation s became widespr ead. Over the past 50 years Ae des con trol methods often for d engue control, have not greatly changed. L arval source reduction, remains the primar y method of Ae. aegypti and Ae. a lbopictus control and is accomplished through the removal of disposable containers or the treatment of water stor age containers with one of three common larvicide s : (1) temephos (an organophosphate), (2) metho prene (insect growth regulator), or (3) BTI ( Bacillus thuringiensis var. israelensis ) For adult control, which becomes a focus when an outbreak of Ae des vectore d disease is already und erway, ultra low volume aerosols of insecticide, such as malathion, are applied with either truck mounted units or airplanes (World Health Organization 1997) Competition Between Aedes Albopictus and Ae Aegypti Aedes albopictus and Ae. aegypti currently have sympatric distributions in many parts of the w orld and the c o occurrence of larvae and pupae of both species within t he

PAGE 25

25 al. 1992, Braks et al. 200 3). In larval competition experiments on North American and Brazilian populations of the two species, Ae. albopictus exhibited a competitive advantage over Ae. aegypti under field conditions (Juliano 1998, Braks et al. 2004). These results support a role f or interspecific competition in the observed decline or local extinction of Ae. aegypti in a large portion of the United States now inhabited by Ae. albopictus South America, and Afri ca where these two Aedes species successfully coexist and much effort has gone into elucidating the mechanisms behind their sustained co occurrence (Sota and Mogi 1992, Juliano et al 2002, Costanzo et al 2005, Lounibos et al. 2002, Leisnham and Juliano 2 009, Leisnham et al. 2009). A probable process at work is condition specific competition, where seasonal and spatial variation in environmental conditions cause a change in which competitor is favored (Costanzo et al. 2005, Leisnham and Juliano 2009). Alth ough Ae. albopictus is a better larval competitor, superior desi c cation resistance in the egg stage by Ae. aegypti allows greater numbers of this mosquito to survive during the dry season (Sota and Mogi 1992, Juliano et al. 2002). Therefore, early in the w et season as eggs that persisted during the drying period hatch Ae. albopictus populations are at a low due to greater egg mortality (Leisnham and Juliano 2009). Condition specific competition can also explain the spatial partitioning of the two mosquitoe s in the environment, the distribution of Ae. aegypti populations is associated with lower humidity and higher temperatures common in more urbanized settings. I n contra st Ae albopictus is negatively associated with hot, dry climate and is more common in suburban, rural, and

PAGE 26

26 forest edge areas where vegetation is more abundant and there is an excess of humid and shaded resting and oviposition sites (Hawley 1988 Sota and Mogi 1992, Juliano et al. 2002, Braks et al. 2003, Rey et al. 2006, Reiskind and Louni bos 2009). Chikungunya Virus In parts of their geographic ranges both Ae. aegypti and Ae. albopictus are important epidemic vectors of CHIKV a single stranded enveloped, positive sense RNA virus. In its native African range, CHIKV is a zoonosis, with a tr ansmission cycle involving wild primates and sylvatic Aedes species H owever, in the invasive range of CHIKV, the viral transmission cycle is urban or suburban, and the primary vectors are A e aegypti and A e albopictus with humans as the host. Since the f irst isolation and identification of the virus in Africa in the 1950s, CHIKV has spread into new geographic areas with human epidemics documented on multiple continents. Discovery of Virus and V ectors Chikungunya is an alphavirus in the Family Togaviridae and based on serological cross reactivity, CHIKV is grouped more specifically into the Semliki Forest virus (SFV) antigenic serocomplex (Powers and Logue 2007). The prototype virus was isolated by Ross during the 1953 dengue epidemic in the Newala distict of Tanzania (formerly Tanganyika) from the blood of a febrile patient (Ross 1956). The name chikungunya is reference to the stooped posture developed as a result of the incapacitating arthralgia that can last for months (Sourisseau et al. 2007). Although CHIKV is rarely fatal, symptoms of the disease include high fevers, rashes, headache, photophobia, vomiting, and excruciating joint and muscle pain. Clinical symptoms fol low an incubation period of

PAGE 27

27 2 to 7 days, and acute illness is short in duration, lasting 3 to 5 days with reco very in 5 to 7 days (Ligon 2006, Robinson 1955). The vectors of the virus are Aedes mosquitoes in the subgenera Diceromyia Stegomyia and Aedimorp hus Sylvan transmission cycles have been documented in tropical Africa in moist forest and semiarid savannah woodland involving sylvatic Aedes species, such as Ae. africanus Ae. furcifer Ae. luteocephalus Ae. neoafricanus and Ae. taylori and wild prima tes, such as vervet monkeys ( Cercopithecus aethiop s) and baboons ( Papio ursinus ) ( Jupp and McIntosh 1988, Jupp and McIntosh 1990 Diallo et al. 1999 ). In monkeys and baboons infection is characterized by a short incubation period and a subsequent viremia l asting approximately five days. There is no mortality and resulting immunity is likely life long (de Moor and Steffens 1970). The average life expectancy of C aethiop s i s between three and four years and it is possible that CHIKV sylvan epidemics follow a 3 4 year cycle that correspond with the renewal of non immune wild primate populations ( de Moor and Steffens 1970 ; Powers and Logue 2007). Studies in the Zika forest of Uganda have also provided data suggesting that a 5 to 7 year cycle in CHIKV activity m ay correspond with the replacement of non susceptible red tailed monkey ( C. ascanius schmidti ) with susceptible individuals (Macrae et al. 1971) How the virus is maintained long term in the wild is unknown. No field or laboratory data can confirm that the virus is maintained transovarially in mosquito eggs (Vazeille et al. 2009) Computer simulated e pidemiological models generated by de Moor and Steffens ( 1970) suggest that CHIKV could exist endemically by continuous transmission in the vertebrate ( C aeth iop s) population. U rban epidemic transmission of CHIKV is

PAGE 28

28 sustained by the mosquitoes Ae. aegypti and/or Ae. albopictus in a hu man mosquito human cycle (Jupp and McIntosh 1988). Viral Characterization and P hylogenetics Chikungunya virus, like all known alp haviruses is arthropod borne with mosquitoes being the predominant vector. Alphaviruses are enveloped particles and their genome consists of a single stranded, positive sense RNA molecule of ~12,000 nucleotides (nt). The 5 prime end is capped with a 7 me thylguanosine while the 3 prime end is polyadenylated. The CHIKV genome is approximately 11.8 kb and is divided into two major regions: the first two thirds of the genome which forms the 5 prime end encodes the four non structural proteins (nsP 1 4) and a structural domain encoding the three structural proteins of the virus (capsid (C), E2 and E1). The non structural proteins are translated directly from the genomic RNA into a polyprotein that through proteolytic cleavage produces nsP1, nsP2, nsP3, and nsP4 in addition to important cleavage intermediates (Khan et al. 2002). The structural proteins are translated through a subgenomic mRNA intermediate called the 26S RNA into a single polyprotein that is also cleaved to produce the three structural proteins as well as two small polypeptides E3 and 6K (Strauss and Strauss 1994) Thus the genome of CHIKV is 5 prime cap nsP1 nsP2 nsP3 nsP4 (junction region) C E3 E2 6K E1 poly(A) 3 prime CHIKV appears to have originated in Central/West Africa and spread to other parts of the world based on chronology of outbreaks, their infrequency and high morbidity in Asia (Carey 1971), the absence of a vertebrate reservoir and a sylvan transmission cycle outside of tropical Africa (Jupp and McIntosh 1988), and phylogenetic data (Powers et al. 2000). Phylogenetic analysis based on E1 sequences groups CHIKV into

PAGE 29

29 three distict genotypes: (1) Asian, (2) E ast/Central/South African and (3) West African (Parola et al. 2006; Schuffenecker et al. 2006). Historical and Current E pidemics Since its initial discovery in 1953, epidemics of CHIKV have occurred on multiple continents In the 1960s and 1970s, outbreaks were recorded in Thailand, India, Vietnam, Cambodia, Myanmar and Sri Lanka (Jupp and McIntosh 1988; Rao 1971). In the 1962 to196 4 outbreak in Bangkok, Thailand, seroprevalence rates were between 70 85% in 20 to 70 year olds (Halstead et al. 1969). Outbreaks from 1963 to 1973 in India sickened hundreds of thousands of people (Rao 1971). From the late 1950s through the 1990s CHIKV wa s isolated from multiple countries in central and southern Africa as well as from W est Africa (Powers et al. 2000). More re cent outbreaks include the 1999 to 2000 epidemic in the Democratic Republic of Congo (Pastorino et al. 2004) and the 2001 2003 outbre ak in Indonesia and Malaysia (Kit 2002; Laras et al. 2005). A large scale epidemic of CHIKV began in 2004 o n Lamu I sland Kenya and then spread to M o mbasa (Chretien et al. 2007; Sergon et al. 2008). In 2005 and 2006 CHIKV moved onto the Indian Ocean island nations starting with the Comoros, then Reunion, and on to the Seychelles, Maur itius, and Madagascar. Extrapolation from seroprevalence data suggests that on Grande Comore island (population 341,000) nearly 215,000 people (63% of the population) may have been infected during the outbreak (Sergon et al. 2007) and o n the island of Reunion (population 770,000) alone approximately 241,000 clinical cases (31% of the population) were reported (Paquet et al. 200 6). As t he epidemic continued 1.39 million suspecte d cases were reported in India in 2006 and tens of thousands of additional suspected cases were identified in 2007 (Arankalle et al. 2007; NVBDCP 2007 ). Local transmission was reported in two

PAGE 30

30 small towns in the Italian province of Ravenna in the summer of 2007 resulting in 200 human cases (Rezza et al. 2007; Watson 2007). The epidemic continues with additional cases reported thro ughout South East Asia, India, and Sri Lanka where cases were reported through 2009 and into 2010 (International Soci ety for Infec tious Diseases 2009 2010 ). Furthermore, multiple cases have been imported into other areas of Europe, the United States, Canada and many other countries through the movement of infected travelers (Lanciotti et al. 2007, International Society for Infectious Diseases 2009 2010) Isolates from the current outbreaks of CHIKV are most closely related to strains in the East/Central/South African genotype (Parola et al. 2006, Schuffenecker et al. 2006, Arankalle et al. 2007, Njenga et al. 2008). Since the outbreak, full genome sequences have been published for multiple isolates from Kenya, Indian Ocean islands, India and South East Asia and are available i n GenBank. CHIKV I nteractions in Epidemic V ectors: Aedes aegypti and Ae albopictus In past CHIKV outbreaks A e. aegypti has been implicated as the dominant vector, with virtually all Asian mosquito isolates coming from this species (Powers and Logue 2007) E arly on in th e recent outbreak s (2004 2005) both in Kenya and on the Comoros islands, Ae. aegypti was the vector responsible for CHIKV transmission (Sang et al 2008, Njenga et al. 2008) However, as the epidemic moved onto Reunion I sland Ae. albopictus was the only vector present (Delatte et al. 2008) Aedes albopictus was also the sole vect or on the Lakshadw eep islands in the Indian Ocean (Samuel et al. 2009), and in Ravenna, Italy (Rezza et al. 2007) which all experienced CHIKV outbreaks between 2005 and 2008 Similar patterns are documented in DENV epidemics in that in

PAGE 31

31 th e absence of Ae. aegypti Ae. albo pictus acts as the primary ve ctor of DENV (Ali et al. 2003; Effler et al. 2005; Xu et al. 2007). There were also some outbreak localities where although Ae. aegypti is common, Ae. albopictus was still the primary vector of CHIK as was the case in the West African country of Gabon (Pages et al. 2009). I n other recent CHIKV outbreak regions where Ae. aegypti and Ae. albopictus distributions overlap there is evidence that both mosquitoes are transmitting the virus. In Singapore in 2008, larval surveys identif ied Ae. albopictus as the predominant species in certain locations, with Ae. aegypti also present and adult mosquito surveillance yielded both Ae. albopictus and Ae. aegypti ad ult females positive for CHIKV (Ng et al. 2009) Similar patterns were found in Thailand, where wild caught adults of both species were positive for CHIKV (Thavara et al. 2009). Entomological surveys done during the 2006 CHIKV outbreak in the southern India n state of Kerala found high densities of Ae. albopictus and in 2007 in some o f the worst CHIKV affected district s ( Alappuzha, Kottayam and Pathanamthitta ) of Kerala state, Ae. albopictus constituted 85 92% of the total mosquito juveniles collected and in a follow up survey this mosquito made up 58 76% of the totals (Kumar et al. 2 008). In the n orth eastern Indian state of Orissa, entomolog ical surveys revealed the presence of both Aedes species with Ae. albopictus having a slightly higher density than Ae. aegypti (Dwibedi et al. 2009). T he increased global presence of Ae. albopictu s along with a largely non immune population likely fueled the magnitude an d speed of the CHIKV outbreaks. However, there is evidence that the acquisition of a mutation in the viral gene coding the E1 envelope protein during the outbreak may have also p layed a role in acceleration of the spread of CHIKV (Schuffenecker et al. 2006). In the laboratory th is mutation, which

PAGE 32

32 results in a substitution of valine for alanine at the 226 amino acid position (A226V) of E1, significantly increase s the susceptibility of Ae. albopictus to the virus The mechanism causing increased viral fitness in Ae. albopictus is unknown, but there may be some association with cholesterol dependence (Tsetsarkin et al. 2007). Aedes albopictus became infected with, disseminated, and tr ansmitted a Reunion island isolate with the E1 A226V isolate at much higher rates at all blood meal titers, while there was no change in susceptibility in Ae. aegypti when compared with a back mutated isolate with alanine at the E1 226 position (Tsetsarkin et al. 2007). Tsetsarkin et al. (2007) then took a historic West African CHIKV isolate mutated it to contain the same valine E1 226 substitution and found when compared to the original West African isolate there was increased susceptibility in Ae. albop ictus but not Ae. aegypti Previous laboratory studies with East African and Thai isolates of CHIKV had already indicated that in the laboratory Ae. albopictus was a significantly more competent vector than Ae. aegypti (Mangiafico 1971; Turell et al. 1992 ). Jupp and McIntosh (1988) suggest that since historic human viremias do not circulate much above 7.0 log 10 TCID 50 /ml some populations of Ae. aegypti would be inefficient vector s in hu man to hu man transmission. T he superior laboratory competence of Ae. a lbopictus over Ae. aegypti is also evident in the Tsetsarkin et al. ( 2007) study with the emergent E1 A226V CHIKV isolate This pattern was again confirmed in a nother laboratory study in which first generation Ae. albopictus and Ae. albopictus from South F lorida were given one of four blood meals each with a 10 fold reduced titer of the emergent E1 A226V CHIKV Aedes aegypti showed an overall susceptibility

PAGE 33

33 significantly lower than Ae. a lbopictus and only Ae. albopictus individuals were infected at the two lowest viral doses (Pesko et al. 2009). Early on in the epidemic in 2004 and 2005 the A226V mutation was absent from Kenyan and Comoros isolates, but the mutation then appeared in CHIKV isolates from Reunion island in 2005 and 2006 (Njenga et al 2008, Go uld and Higgs 2009 ). Isolates from the 2006 epidemic in India did not have the mutation, but isolates sequenced from 2007 Indian outbreaks did have the A226V ( Yergolkar et al. 2006, Arankalle et al. 2007, Santhosh et al. 2008 ) Chikungunya viral samples fr om the 2007 Ravenna, Italy outbreak did have the A226V mutation where, as mentioned earlier Ae. albopictus was the sole vector, and the virus was introduced to Italy by an Indian national visiting Ravenna (R ezza et al 2007). In Singapore, CHIKV strains f rom the 2008 epidemic were mixed, isolates from Ae. aegypti abundant areas did not have the A226V mutation, but the mutation was present in virus identified from Ae. albopictus adults or in human blood samples from Ae. albopictus dominated neighborhoods. A ccording to Gould and Higgs (2009), during the 2004 2009 epidemic s the only mosquitoes that tested positive for CHIKV with the E1 A226V mutation were Ae. a lbopictus and all positive Ae. albopictus tested were infected with this mutant How the continuing geographic spread of Ae. albopictus combined with the E1 A226V mutation in CHIKV contributed to the magnitude of the epidemic of the past five years is currently unknown Vector Competence After taking a viremic blood meal a mosquito will only be capable o f orally transmitting the virus after a series of barriers to infec tion are overcome (Hardy et al. 1983). Assuming there is ingestion of a sufficient quantity of virions to exceed the infectious dose threshold (Chamberlain and Sudia 1961) the virions that enter the

PAGE 34

34 midget lumen generally bind to the membrane of the midgut epithelial cells, enter the cell cytoplasm and replicate. Infectious virions must then disseminate from the midgut epithelial cells to the haemocoel and infect other tissues. Finally, for transmission to occur, virions must infect and replicate in salivary gland tissue and then be secreted in saliva during feeding on a subsequent host. The relationship between mosquito species and virus is often specific and the presence of one or more of the barriers in the described processes results in a mosquito that is an incompetent vector for a given virus intrinsic and extrinsic factors can affect the susceptibility of a v ector for a pathogen vary both inter and intra specifically, as is the case with Ae. albopictus and Ae. aegypti susceptibility for certain strains of CHIKV (Turell et al. 1992). Intrinsic (genetic) factors of the mosquito that influence vector competence include: behavio r, physiology, and metabolism and thus, v ector competence is thought to be a complex phenotypic trait under the control of multiple genetic loci (Bos io et al. 1998). The Environment and Vector Competence Extrinsic (e nvironmental ) factors can also exert a strong i nfluence on mosquito vector competence, with temperature studies account ing for the majority of research on the topic. Within limits, increase d temperature during the adult life stage has a positive influence on vector competence. In studies with West Nile Virus (WNV) (Dohm et al. 2002 Reisen et al. 2006, Richards et al. 2007 ), Western equine encephalomyelitis (Reisen et al. 1993) and multiple other viruse s, temperature is shown to increase the number of individuals infected wit h or transmitting the virus In addition, temperature has

PAGE 35

35 an inverse relationship with an arthropod vector EIP ( Chamberlain and Sudia 1955 ) shortening the time between infection and the subse quent ability to transmit the virus The Larval Environment, V ector Competence a nd Mosquito Size Arboviral diseases are ecologically complex and interactions between l arval mosquitoes and their aquatic environment can influence a dult transmission dynamics. Biotic and abiotic v ariables such as temperature, larval nutrition, and inter and intra specific competition can alter the life history and the intrinsic physiology of mosquitoes in ways that affect the ease with which individu als become infected and transmit a virus Previous studies have shown that larval rearing temperature can affect mosquito competence for several arboviruses, including Ae taeniorhynchus for Rift Valley fever (RVFV) and Venezuelan equine encephalitis (VEEV ) (Turell 1993), Cule x annulirostris for Murray Valley encep halitis (MVEV) (Kay et al. 1989a ), C x tritaeniorhynchus for Japanese encephalitis (JEV) (Takahashi 1976), and Cx. t arsalis for western equine encephalitis (WEEV) (Hardy et al. 1990) viruses Unfo rtunately, none of these studies reared mosquitoes individually to separate temperature and density effects. In contrast, no rearing temperature effect was found in C x tritaeniorhynchus for WNV ( Baqar et al. 1980) and Aede s vigilax for Ross River virus (R RV) (Kay and Jennings 2002). With the exception of C x tritaeniorhynchus for JEV (Takahashi 1976), adult females showed reduced vector competence with increased rearing temperature. Larval nutrition can also influence the ability of adult mosquitoes to bec ome infected with and transmit virus. C x tritaeniorhynchus and Ae. triseriatus nutrient deprived larvae were more susceptible than their well fed counterparts, for WNV (Baqar et al. 1980) and better transmitters of La Crosse virus (LACV) to suckling mice (Grimstad and Hara mis 1984; Grimstad and Walker 1991). However, i n other studies,

PAGE 36

36 nutritional deprivation of larvae had no effect on vector competence of C x annulirostris for MVEV (Kay et al. 1989 b ) or Ae. vigilax for RRV (Jennings and Kay 1999). When an effect was detect ed, low nutritional resources produced adult mosquitoes more susceptible to virus Aedes triseriatus adults produced in low intra and interspecific competition treatments with Ae. albopictus had higher infection and dissemination rates fo r LACV (Bevins 2008). The opposite was found with A e. albopictus reared in highly competitive larval environments, wherein adults were smaller and had higher rates of infection and dissemination for Sindbis virus and DENV, while within the same studies a c ompetitive larval environment showed the same trends but did not have a significant effect on vector competence in Ae. aegypti for the two viruses (Alto et al. 2005; Alto et al. 2008a ). In a number of the studies investigating the influence of larval envi ronmental factors on vector susceptibility to arbovirus es, mosquito body size is measured in addition to infection outcomes. Mosquito body size is an easily measurable physical manifestat ion of larval habitat quality and larvae reared a t low temperatures are generally larger as adults, while juveniles reared with l ow f ood availability and/or in a competitive environment will be smaller as adults. In a few studies larval factors were varied just as a means to produce mosquitoes of different size classes. Su manochitrapon et al. (1998) produced three size classes of adult Ae. a egypti by varying both the quantity of food and density of larvae and found that large Ae. aegypti females showed higher rates of oral infection with dengue virus (DENV) compared to sm all and medium mosquitoes. Similar findings were reported when two size classes of Ae. aegypti were generated through variation in larval diet and larger individuals were

PAGE 37

37 more susceptible to RRV (Nasci and Mitchell 1994). In contrast, smaller Ae. triseria tus adults generated from field collected pupae were more likely to transmit LACV to suckling mice (Paulson and Hawley 1991) and when Alto et al. (2008b) carried out additional analyses in which size was examined independent of rearing conditions small Ae. aegypti females were more susceptible to DENV ; however it should be noted that the size range of individuals measured in this study was extremely narrow. However, body size may not be a good predictor of how an individual mosquito may respond to viral challenge in the form of an infectious blood meal. There may be more critical, but not as easily measurable physiological and physical features of adult mosquitoes that vary with larval conditions and are more correlated than size with viral susceptibility Furthermore, the type of larval condition that is being manipulated may influence the strength and even possibly the direction of the response. For example, variations in larval temperature or food level may produce mosquitoes of a similar size range, bu t their response s to infection may be very different.

PAGE 38

38 CHAPTER 2 LARVAL ENV IRONMENTAL TEMPERATU RE AND THE S USCEPTIBILITY OF Aedes a lbopictus SKUSE (DIPTERA: CULI CIDAE) TO CHIKUNGU NYA VIRUS I ntroduction Climate is one of the principal determinants of the di stribution of vector borne diseases (van Lieshout et al. 2004). In part icular, both vector development and survival and arbovirus replication are greatly influenced by temperature. In mosquito vectors, temperature can influence larval development time, lar val and adult survival, biting rates, gonotrophic cycle lengths, and vector size (Madder et al. 1983, Rueda et al. 1990, Scott et al. 1993). Ambient temperature can affect arboviral dynamics within the mosquito vector by altering the length of the extrinsi c incubation period (EIP), which is the time between ingestion of an infectious blood meal to when transmission to a subsequent host is possible (Chamberlain and Sudia 1955, Reeves et al. 1994, Patz et al. 1996). Furthermore, temperature often defines the latitudinal and altitudinal ranges of a vector. Species range may limit the distribution of disease when pathogens are species specific. Temperature may also limit viral transmission in areas where the vector is present but the temperature precludes effici ent transmission (Purse et al. 2005). Vector competence, which is the capacity of an arthropod to acquire an infection and transmit it to a subsequent host, can greatly vary among individuals and between populations (Lorenz et al. 1984, Turell et al. 1992) and is influenced by genetics (Mercado Curiel et al. 2008) as well as by climate variables, such as temperature (Davis 1932, Turell 1993, Dohm et al. 2002). An increase in environmental temperature for adult mosquitoes reduces the EIP (Davis 1932, Chamber lain and Sudia 1955), most

PAGE 39

39 likely due to an increase in the metabolism of the adult mosquito and the replication speed of the virus. Although the majority of research on mosquito virus interactions has focused on adult mosquitoes, temperature changes exper ienced in the immature stages of holometabolous vectors prior to infection, may affect vector virus interactions by changing physical and physiological characteristics of midgut and salivary gland barriers, which could have direct consequences on viral inf ection, replication, and transmission. Previous studies have shown that larval rearing temperature can a ffect mosquito competence for several arboviruses, including Murray Valley encephalitis (MVEV) (K ay et al. 1989a ), Japanese encephalitis (Takahashi 1976 ), and western equine encephalitis (Hardy et al. 1990) viruses. In a specific study with Aedes taeniorhynchus mosquitoes reared at 19 C had higher infection rates for Rift Valley fever and Venezuelan equine encephalitis viruses than counterparts reared at 26 C (Turell 1993). In view of global climate change models, which predict changes in temperature that will directly impact larval mosquito habitats, this study, which investigates a previously unexplored relationship between Aedes albopictus larval envir onmental temperature and chikungunya virus (CHIKV) susceptibility, could help increase the predictability of disease transmission patterns and future outbreaks. The intercontinental dispersal of invasive arbovirus vectors, such as the Asian tiger mosquito Ae. albopictus is accompanied by an increase in human vulnerability to the exotic diseases vectored by these invaders (Juliano and Lou nibos 2005). In 2005 2006 CHIKV a single stranded positive sense RNA enveloped Alphavirus in the family Togaviridae, eme rged as an important pathogen in the Indian Ocean Basin. On the

PAGE 40

40 island of R e union alone, 241,000 clinical cases of chikungunya fever, representing 31% of the population, were reported (Paquet et al. 2006). A sylvan transmission cycle of CHIKV involving mos quitoes, such as Ae. furcifer and Ae luteocephalus and wild primates is limited to tropical Africa, and epidemic transmission of the virus is sustained though infection of the mosquitoes Ae. aegypti and Ae. albopictus in urban and peridomestic environmen ts (Jupp and McIntosh 1990, Diallo et al. 1999). A n un usual feature of the South West Indian Ocean CHIKV outbreak was the increased importance of Ae. albopictus as a vector. The enhanced role of Ae. albopictus as a CHIKV vector on Re union island was in par t due to the rarity of the primary vector Ae. aegypti (Delatte et al. 2008), however, a n amino acid substitution in the Re union CHIKV isolates from alanine to valine at the 226 position of the E1 envelope structural protein has been shown to increase Ae. albopictus but not Ae. aegypti susceptibility to the virus in the laboratory (Tsetsarkin et al. 2007). Following the Indian Ocean Basin epidemic, a strain of CHIKV nearly identical (99.9% nucleotide identity) to isolates from La R e uni n emerged in India where 1.3 million human cases were reported in 13 states in 2005 2006 (Arankalle et al. 2007). This widespread epidemic was not restricted to the tropics, with autochthonous transmission reported in northern Italy in 2007 (Rezza et al. 2007) The epidemic continued to spread to Indonesia, Sri Lanka, and Singapore where cases were reported through 2008 and into 2009 (Seneviratne et al. 2007, International Society for Infectious Diseases 2008 2009). In this study we explore how variation in temperature durin g larval development affects the susceptibility of Ae. albopictus for CHIKV by measuring infection and dissemination rates, and viral titer. We also assess how larval temperature affects

PAGE 41

41 growth and survivorship by measuring larval mortality, development ti me to adulthood, and adult body size. Materials and Methods Mosquitoes and V iruses Aedes albopictus used in this experiment were generated from field collections of approximately 4000 larvae and/or eggs made from June to August 2007 in Palm Beach County, Florida. This population of Ae. albopictus was previously shown to be highly susceptible to t he Re union ( L R2006 OPY1 ) CHIKV strain (Reiskind et al. 2008). Females reared from field collected immatures were given 20% sucrose ad libitum blood fed weekly on live chickens, and kept in cages at 14 L:10 D, 26 C (+/ and >80% rh. Chicken care followed federally mandated animal use and care policies (University of Florida, IACUC Protocol VB 17). Eggs (F 1 ) were hatched in tap water and, within 24 hours aft er hatching, individual larvae were placed in 50 ml Falcon conical tubes with 35 ml of tap water and 0.0105g 1:1 yeast:albumin food. Based on preliminary studies, 0.0105g of food at the beginning of the experiment was adequate for the completion of indivi dual development but did not increase mortality at the higher temperature. Larvae were reared at 18 C, 24 C and 32 C with a 14L:10D cycle in a Percival (Percival Corporation, Perry, IA) incubator. The experimental treatment units in this study were differe nt incubators which were identical in all respects except for rearing temperature and, thus, it was assumed that differences in treatments were caused by rearing temperature. These temperatures are within the range encountered in the treehole environment o ccupied by Ae. albopictus in Florida (Lounibos 1983). Larvae in each temperature treatment were from the same cohort of eggs whose hatch was staggered to synchronize adult emergence among all three temperature

PAGE 42

42 treatments After the final larval instar, pup ae were removed from rearing tubes, sexed and stored in groups of 10 in water filled vials to record adult emergences. After emergence, all adults were held at 24 C, 95 99% rh with a 14L:10D cycle in a biosafety level 3 facility and given 20% sucrose ad li bitum The LR2006 OPY1 CHIKV strain (GenBank accession number DQ443544) was isolated in France from a febrile patient who had been infected o n the island of Re union (Parola et al. 2006). This recently emergent strain of CHIKV contains the alanine to valin e substitution at the 226 position of the E1 envelope structural protein that has been identified as a feature in many current CHIKV epidemics (Rezza et al. 2007) and has been shown to increase A e albopictus susceptibility (Tsetsarkin et al. 2007) To pro duce virus for infectious blood meals, a T 75 cm 2 flask with a confluent monolayer of Vero cells in 10 m L of cell culture media (M199 media supplemented with fetal bovine serum, antibiotics and antimycotics (Invitrogen Carlsbad, CA ) was ino culated with 1 5 0 L of previously frozen stock virus, and allowed to incubate in a 5% CO 2 an d 35C atmosphere for 24 hours. Vector C ompetence Groups of 50 five to seven day old mosquitoes were placed in 1 L cylindrical, waxed cardboard containers (Dade Paper Co., Miami, FL) with mesh screening. Mosquitoes were starved for 24 hours prior to being offered an infectious blood meal of 1:1000 dilution of freshly propagated CHIKV in citrated bovine blood (Hemostat Laboratories, Dixon, CA) supplemented with ATP [5mM] as a phago stimulant. Water jacketed glass membrane feeders (Rutledge et al. 1964) covered with Edicoll collagen film (Devro, Sandy Run, SC) and connected to a Haake Series F water circulator (Thermo Haake, Paramus, NJ) were used to maintain the blood meal at 37 C.

PAGE 43

43 Mosquitoes were given 30 minutes to feed. Low feeding success of A e albopictus made it necessary to conduct three consecutive days of feeding for individuals from all three temperature treatments. The blood meals from the three consecutive feeding days we re assayed using qRT PCR, with copy number standardized to plaque forming unit (pfu) by plaque assay performed on 10 fold serial dilutions of virus stock (Bustin 2000). Virus titers in blood meals were log 10 4.7, 4.5, and 3.4 pfu/mL for the three feeding d ays, respectively. After feeding, mosquitoes were cold anesthetized, and fully engorged mosquitoes were removed, placed in new cages, and given 20% sucrose ad libitum After a 10 day EIP at 24C, surviving Ae. a lbopictus females were killed by freezing. Fe males were stored at 80C and, after thawing, wings were removed for measurements, bodies were assayed to determine infection status and titer, and legs were tested to check for a disseminated infection (Turell et al. 1984). Wing length was measured as an indicator of body size (Blackmore and Lord 2000) in millimeters from alula to wing tip using digital images and a computer imaging and measurement program (i Solution lite AIC Inc., Princeton, NJ). Bodies and legs were triturated separately in 2 mL micro centrifuge tubes 1 media (Lanciotti et al. 2000) and two zinc plated beads. Samples were homogenized at 25 Hz for 3 min using a Tissuelyzer tissue homogenizer (Qiagen Inc., Valencia, CA) and then clarified by centrifugation (3,000 x g for 4 min). Viral RNA was extracted from 250 LS ( Invitrogen Carlsbad, CA elution volume of 50

PAGE 44

44 One step qRT PCR was used to determine in fection and dissemination status and body titer by previously established protocols (Reiskind et al. 2008). Primers from the ACC CGG TAA GAG CGA TGA ACT AGG CCG CAT CCG GT A TGT /5Cy5/CCG TAG GGA ACA TGC CCA TCT CCA /3BHQ_2/ DNA, Coralville, IA). Statistical Analysis Kruskal Wallis tests followed by s (fam 0.05) were used to determine differences among treatment s in distributions of w ing lengths and development times to adulthood (SAS 9.1, SAS Institute, Inc., Cary, NC) Males and females were analyzed separately because of gender specific sizes and developmental times in this species. Chi squared tests of independence were used to determine the effect of temperature on survivorship to adulthood. If significant effects were detected, post hoc pairwise comparisons were made with an alpha adjusted (Bonf erroni) correction to account for multiple comparisons ( Minitab 15, Minit ab Inc., State College, PA ) A generalized linear mixed model (PROC GLMMIX) was used to describe relationships among temperature (fixed effect), feeding day (random effect), infection rate (# with virus/# fed), dissemination rate (# with virus in their legs/# with virus), and population dissemination rate (# with virus in their legs/# fed) specifying a logistic link function and a binomial error distribution. Odds ratios (OR) and 95 % confidence intervals for infection at each temperature treatment were also calculated (SAS 9.1). A linear mixed model (PROC MIXED) was used to test for effects of temperature treatments (fixed) and feeding day (random), which were both class variables on b ody

PAGE 45

45 titer, a continuous variable. Titer data did not fit the model assumptions of normality, but approximate normality was achieved through a log transformation of titer values (SAS 9.1). Wing length comparisons, pooled across all temperature treatments, b etween binomial variables: infected versus uninfected and disseminated versus non disseminated were analyzed with t tests (Minitab 15). R esults Growth and M ortality W ing length s and development time s to adulthood were significantly affect ed by temperature for both females ( wing length: H = 417.5; df = 2; P<0.0001, development time: H= 1279.6 ; df = 2; P <.0001 ) and males ( wing length: H= 2127.4 ; df = 2; P < 0 .0001 and development time: H = 1697.3 ; df = 2; P < 0 .0001 ) ( Table 2 1). There was an inverse relationshi p between wing length and temperature with the largest adult mosquitoes produced at 18 C and the smallest mosquitoes produced at 32 C Mosquitoes reared at the lowest temperature (18 C) took over two times longer to develop than those at 24C and 32 C Juv enile mortality rates at 18 C 24 C and 32 C were 9.16% (n = 1454), 7.34% (n = 1520), and 16.90% (n = 1473), respectively. There was a significant relationship between survivorship to adulthood and temperature ( 2 = 54.123, df = 2, P < 0 .000 1). Aedes albopi ctus reared at 32 C were significantly more likely to die as larvae when compared with individuals reared at 18 C and 24 C with no differences between the two lower temperatures Chikungunya Infection and D issemination There was a significant temperature treatment effect on the percentage of females that developed CHIKV infections (F=16.92, df = 2, P < 0 .000 1) (Figure 2 1 ). Infection was 6 times more likely in adult females reared at 18 C than at 32 C (Odds Ratio (OR) =

PAGE 46

46 6.052; 95% CI 3.22 11.373) and female s reared at 24C were 2.7 times more likely to be infected than those reared at 32C (OR = 2.722; 95% CI 1.385 5.351). Females reared at 18 C were 2.2 times more likely to be infected than those reared at 24C (OR = 2.223; 95% CI 1.328 3.722). Among the in fected individuals t he proportion of females that developed disseminated infections did not vary significantly among larval rearing temperatures (F = 0.85, df = 2, P > 0.4293), however there was a significant temperature effect on population dissemination rate (F=6.20, df = 2, P < 0 .00 22) (Figure 2 1 ). Population dissemination rate was approximately 5 times higher in adult females reared at 18 C than at 32C (OR = 4.905; 95% CI 1.814 13.271) and 2.3 times higher in females reared at 18 C than at 24C (OR = 2. 291; 95% CI 1.088 4.827). There was no significant difference in population dissemination rates between the 24C and 32C treatments. After the 10 day EIP, no significant variation in body titer of virus positive mosquitoes was observed among the temperatu re treatment groups (F = 1.14, df = 2, P>0.4300). Aedes albopictus females that were positive for CHIKV infection, in all three temperature treatments were significantly larger than uninfected females (t = 3.59, df = 327, P = 0.0004) as measured by mean w ing length. There was no significant difference in mean wing lengths between females with disseminated and non disseminated infections (t = 0.41, df = 94, P = 0.6830). D iscussion Due to the impact of climate on vector ecology, mosquito borne diseases will be sensitive to projected changes in global temperatures. In this laboratory study we demonstrated that larval rearing temperature can influence survival, development time, and wing length, and may directly impact disease transmission by influencing the

PAGE 47

47 l ikelihood of infection with CHIKV. Although the proportion of infected females that developed disseminated infections did not differ significantly among the three larval rearing temperatures, the population dissemination rates were significantly higher at 18 C, when compared to the two higher temperatures of 24 C and 32 C. Disseminated through biting (Turell et al. 1984). The rate of dissemination, when expressed as a p ercentage of the number of mosquitoes infected, may provide information about the disseminate into the hemolymph. In this study, individuals reared at 32 C had significantly lower infection rates, but no significant difference was found in other hand, the populati on dissemination rate, expressed as a percentage of the total number of mosquitoes tested, was greater for mosquitoes from 18 C, and is epidemiologically more important in that it gives an estimate of the vector competence or the transmission potential of a population. Body titers did not differ among temperature treatments for mosquitoes with disseminated infections. Although this result was somewhat surprising, it is possible that after the 10 day EIP virus titers stabilized to the extent that treatment e ffects on titer were diminished. Additionally, only a limited number of mosquitoes developed disseminated infections and, although the mean titer of disseminated mosquitoes from 18 C was higher, it was not significantly different from the other treatments.

PAGE 48

48 The higher temperature of 32 C decreased survivorship, when compared with 18 C and 24 C. This was unexpected because results from preliminary experiments showed no difference in survivorship between the three temperature treatments. The increased mortalit y could have been due to the stress of high temperature or the interaction of high temperature and excess nutritional resources resulting in the proliferation of detrimental micro organisms in the larval environment. Larvae took longer to develop at cooler temperatures which produced larger adults. Mean wing length differences between successive temperature treatments were approximately 0.2 mm, confirming for A e albopictus that adult body size, within limits, exhibits an inverse relationship with larval re aring temperature (Briegel and Timmermann 2001) Upper and lower thermal limits for the growth of A e albopictus larvae are approximately 11 C and 35 C, at which temperature s l arval development is inhibited, eventually resulting in mortality (Hawley 1988, Monteiro et al. 2007). Our data indicate that at lower larval rearing temperatures there is an increased likelihood of an adult female becoming infected with CHIKV virus. This may have had a positive effect on CHIKV infection rates in locations such as hig hlands of Re union island where entomological surveys recovered A e albopictus at elevations of 1200 meters and temperatures as low as 12.6 C (Delatte et al. 2008). However, 12.6 C is a lower larval environmental temperature than what was investigated in t his study and therefore it is uncertain whether the relationship between reduced temperature and higher infection rates would hold true at this temperature. The ability of A e albopictus to tolerate low temperatures and adapt to diverse ecological environm ents combined with their vector competence for currently circulating CHIKV isolates may help to explain the

PAGE 49

49 2007 northern Italy CHIKV outbreak and increases the potential for future epidemics in other temperate areas where A e albopictus is abundant. This study focused only on the influence of larval temperature and adults were maintained at a common temperature of 24 C. How combinations of different adult and larval temperatures may affect vector competence was not addressed. It is likely that adults mai ntained at the lower temperature will have decreased virogenesis and a longer EIP resulting in a decreased probability of transmission. Therefore the maintenance of low adult temperatures may result in a reduction or elimination of any benefit low rearing temperature may have on increasing vector competence. Results from this study are consistent with other systems where arboviral vector competence was reduced in female mosquitoes that were reared at higher compared to lower temperatures (Kay et al. 1989a, Hardy et al. 1990, Turell 1993). Unfortunately, none of the previous studies that explored larval temperature effects on adult arboviral susceptibility, reared mosquitoes individually to separate temperature and density effects, nor did they measure varia bles such as wing or body size and survivorship, as we have done in this study. In our work lower rearing temperature not only produced mosquitoes that were more susceptible to viral infection but were also significantly larger. Blood consumption by femal es is a function of size and large females are known to imbibe more than twice as much blood as smaller females (Briegel 1990). Thus, in smaller mosquitoes reared at the higher temperature, the imbibing of a lower blood volume would decrease the initial vi ral dose and, in combination with a low CHIKV titer in the blood meal, limit the establishment of infection. Low initial exposure to virus may not affect dissemination,

PAGE 50

50 which requires post infection virus replication. If this explains why large mosquitoes derived from cooler temperatures have higher infection rates than smaller mosquitoes then we would expect that titers of freshly engorged mosquitoes will increase with body size, which will be tested in future studies. We also predict that different sized mosquitoes derived from different temperatures and fed an infectious blood meal with a high virus titer would overcome a threshold infectious dose and may result in similar infection rates. Because the variation in size was achieved through different temp erature treatments it is difficult to separate the effect of temperature from the response variable body size. There may be other temperature dependent phenotypic traits that vary in a way so as to cause an increase in infection when adults are subjected t o a lower temperature larval environment that we did not measure. Previous studies show a lack of consistency in relationships between vector size and pathogen transmission. Large adult Ae. aegypti females from low density larval conditions showed higher r ates of oral infection with dengue virus (DENV) compared to two other size classes from higher density larval conditions (Sumanochitrapon et al. 1998) and similar findings were reported for Ae. aegypti and Ross River virus (Nasci and Mitchell 1994). In co ntrast, A e albopictus adults reared in competitive larval environments were smaller and had higher rates of infection and dissemination for Sindbis virus and DENV, while within the same studies a competitive larval environment did not have a significant e ffect on vector competence in Ae. aegypti for the two viruses (Alto et al. 2005; Alto et al. 2008a ). In Ae. aegypti when size was examined independent of rearing conditions small adults were more susceptible to DENV, however the size range of individuals measure d was

PAGE 51

51 extremely narrow (Alto et al. 2008b). In contrast, larger Ae triseriatus adults produced through variation in competitive treatments had higher infection and dissemination rates for La Crosse virus (LACV) (Bevins 2008). In nutritional depriva tion studies with Cx tritaeniorhynchus and Ae. triseriatus smaller mosquitoes derived from nutrient deprived larvae were more susceptible than their well fed larger counterparts, for West Nile Virus (Baqar et al. 1980) and better transmitters of LACV to suckling mice (Grimstad and Haramis 1984; Grimstad and Walker 1991). Smaller Ae. triseriatus adults generated from field collected pupae were more likely to transmit LACV to suckling mice (Paulson and Hawley 1991). However, nutritional deprivation which l ed to small mosquitoes had no effect on vector competence in Cx. annulirostris for MVEV (Kay et al. 1989b ) and Ae. vigilax for Ross River virus (Jennings and Kay 1999). These inconsistencie s in the effect of size on vector competence could be based on the intrinsic differences between vector viral systems however it is also possible that larval conditions, such as high temperature and low nutrients or competition produce small mosquitoes by different mechanisms that differently effect their competence as v ectors. In summary, cooler rearing temperatures produced mosquitoes that were larger, had higher survival, and were more likely to become infected with CHIKV, emphasizing the importance of the mosquito larval environment in determining adult vector virus i nteractions. Future studies should explore the connection between larval rearing temperature infection patterns observed in the laboratory to patterns in the field and how climate and climate change may continue to impact the mosquito larval environment an d the epidemiology of CHIKV.

PAGE 52

52 Table 2 1. Aedes albopictus t reatment medians and interquartile ranges (IQR= 25th percentile 75th percentile) for development time to adulthood and wing length. Medians with different letters in the same row are significant ly different from one another 18C 24C 32C Female time to adulthood ( days ) Interquartile range (n) 24.5 a 23.0 26.0 (515) 11.0 b 10.0 11.5 (556) 9.0 c 8.5 9.5 (445) Male time to adulthood ( days ) Interquartile range (n) 22.5 a 21.5 24.0 (817) 10.0 b 9 .5 10.5 (860) 8.0 c 7.5 8.5 (791) Female wing length ( mm ) Interquartile range (n) 3.40 a 3.29 3.51 (347) 3.14 b 3.00 3.26 (150) 2.91 c 2.80 3.01 (207) Male wing length ( mm ) Interquartile range (n) 2.83 a 2.77 2.90 (745) 2.61 b 2.55 2.66 (810) 2.4 4 c 2.37 2.51 ( 740)

PAGE 53

53 Figure 2 1. Bivariate plots of mean wing lengths (SE) and CHIKV susceptibility A) percent infection, B) percent dissemination, and C) percent population dissemination grouped by treatment. Numbers in parenth eses above symbols represent number of mosquitoes tested.

PAGE 54

54 CHAPTER 3 LARVAL TEMPERATURE AND NUTRITION ALTER THE SUSCEPTIBILITY O F A edes aegypti L. (DIPTERA: CULICIDAE) MOSQUITOES TO CHIKUN GUNYA VIRUS Introduction In recent years chikungunya virus (CHIKV) has emerged as an important agent of human arboviral epidemics sickening million s of people worldwide ( National Vector Borne Disease Control Programme (NVBDCP) 2007, International Soc iety for Infectious Disease 2005 2010 ) Chikungunya, a single stranded en veloped, positive sense RNA alphavirus (Family Togaviridae), was first isolated by Ross in 1953 from the blood of a febrile pa tient in Tanzania (Ross 1956) and a lthough endemic CHIKV was known to be rarely fatal, symptoms of the disea se include high feve rs, rashes and severe and debilitating ar thr algia (Robinson 1955, Ligon 2006 ). In its native African range, CHIKV is a zoonosis, with wild primates serving as hosts and sylvatic Aedes spp. as vectors. However, in the invasive range of CHIKV, humans are the main host and Ae. aegypti and Ae. albopictus are the ve ctors (Jupp and McIntosh 1988). Aedes aegypti which t hrough the exploitation of man made habitats spread from Africa to tropical and subtropical regions across the globe is the primary epidemic vect or of dengue, yellow fever and historically CHIKV ( Tabachnick 1991, Powers and Logue 2007 ) During previously documented Asian CHIKV epidemics all mosquito isolates were solely from Ae. aegypti (Powers and Logue 2007) and although Ae. albopictus has re cently risen in importance as a CHIKV vector, Ae. aegypti has continued to play an important role in recent CHIKV outbreaks. An epidemic of CHIKV began in Kenya in 2004 (Chretien et al. 2007) and spread i n 2005 and 2006 to the African island nations of th e Comoros, Reunion, Seychelles, Mauritius, and Madagascar in the Indian Ocean (Sergon et al. 2007). Chikungunya then

PAGE 55

55 moved i nto India, where 1.39 million s uspected cases were reported in 2006 and tens of thousands of additional cases were identified in 200 7 (Arankalle et al. 2007; NVBDCP 2007). L ocal transmission of CHIKV was reported in 2007 in the northern Italian province of Ravenna ( Rezza et al. 2007 ) and t he epidemic continues with additional infections confirmed in 2010 in the Maldives, Madagascar, Sri Lanka, and many South East Asia countries, such as Indonesia, Malaysia, Thailand, and My a nmar (International Society for Infectious Diseases 2009 2010). Furthermore, multiple cases have been imported into other areas of Europe, the United States, Canad a and many other countries through the movement of infected travelers (Lanciotti et al. 2007, International Society for Infectious Diseases 2009 2010) Arboviral diseases, such as CHIKV are ecologically complex and the interaction between immature mosqui toes and factors in their aquatic environment can influence the ability of adult mosquitoes to transmi t a n arbo virus Aedes aegypti larvae feed on microorganisms and organic detritus available in the ir container habitat s C ontainers hold all the nutrients needed by developing larvae whose resources are often limited leading to nutritionally stressed adult populations of Ae. aegypti (Barrera et al. 2006) Abiotic factors such as temperature also influence such factors as larval development and adult body si ze and many variables interact with food availability to alter mosquito life history traits ( Padmanabha unpublished data ). P revious studies have shown that temperature and food availability during the immature stages can exert a strong i nfluence on adult mosquito vector competence which is the capacity of an a rthropod to acquire a pathogen and tr ansmit it to a subsequent host ( Hardy et al. 1990) L arval rearing temperature has been shown to

PAGE 56

56 a ffect mosquito competence for viruses of Rift Valley fever (RVFV ), Venezuelan equine encephalitis (VEEV) (Turell 1993), Murray Valley encephalitis (MVEV) (Kay et al. 1989a ), Japanese encephalitis (JEV) (Takahashi 1976), and western equine encephalitis (WEEV) (Hardy et al. 1990) while nutritional deprivation has been s hown to a ffect vector competence for West Nile Virus (WNV) (Baqar et al. 1980) and La Crosse virus (LACV) (Grimstad and Haramis 1984, Grimstad and Walker 1991). I t is well established that a larval environment with high temperatures and/or l ow f ood availab ility will produce smaller adult mosquitoes (Keirans and Fay 1968, Briegel 1990, Rueda et al. 1990). Thus, mosquito body size is an easily measurable physical manifestat ion of larval habitat quality which has been documented in many studies investigating larval environmental factors and ar boviral susceptibility In a few studies larval factors were varied specifically to produce mosqu itoes of different size classes to test the effect of adult body size on arboviral susceptibility to viruses of dengue (DENV ) (Sumanochitrapon et al. 1998) and R oss R iver (RRV) (Nasci and Mitchell 1994). Overall, the relationships among larval habitat quality, body size, and vector competence are not well worked out and results from different experiments are conflicting. M ore controlled and well designed investigations into unexplored vector viral systems and diverse combination s of larval ecological factors will add to a growing understanding of this subject. T his study explores how fea tures of larval habitat shape Ae. aegypti competence for CHIKV an important emerging arbovirus causing human disease S pecific ally investigated are the relationships among larval rearing temperature, food availability, adult body size and Ae. aegypti susceptibility to CHIKV. In Chapter Two r ese arch was

PAGE 57

57 done investigating the influence of temperature at a few disc rete levels in Ae. albopictus while in this experiment two factors are crossed to exp ress more realistically the variation experienced in the field b y developing Ae aegyp t i another ke y vector of CHIKV A further objective of this work was to establish the effect of rearing temperature and food availability on larval mortality and development time to adulthood Since the first isolation and identification of CHIKV in Africa in the 1950s this virus has spread to new geographic areas with human epidemics documented on multiple continents. Understanding how larval ecological factors can affect the interaction of adult Ae. aegypti with CHIKV may help in making predictions as to the directio n and magnitude of future outbreaks. Materials and Methods Mosquitoes and V iruses Aedes aegypt i used in this study were first generation progeny of approximately 3 000 field collected eggs and larvae, which were collected from April to July 2008 in Palm Be ach County, Florida. Field collected females were given 20% sucrose ad libitum, blood fed weekly on live chick ens, and kept in cages under constant followed federally mandated animal use and care policies (University of Florida, IACUC Protocol VB 17). First generation eggs were hatche d in tap water and, within 24 hours (h) Franklin Lakes, NJ) conical tubes with 35 ml of tap water and 10.5 mg or 3.0 mg 1:1 yeast:albumin food. Based on preliminary studies, 3. 0 mg given to a larva at the beginning of the experiment was the lowest level of food that allowed for the completion of development to adulthood without significant reduction in mortality and 1 0.5 mg was

PAGE 58

58 the highest level of food that could be given to a n individual without a marked increase in mortality due to fouling of the aquatic environment. Thus, the experiment was a 3x2 factorial design with temperature as one factor at three levels and food as the second factor at two levels Larvae in each temperature and food treatm ent were from the same cohort of eggs whose hatch was staggered to synchronize adult emergence among all treatments. After the final larval instar, pupae were removed from rearing tubes, sexed and stored in groups of 20 in water filled vials to record adul t emergences. 99% relative humidity (rh) with a 14 L:10 D cycle in a Percival (Percival Corporation, Perry, IA) incubator in a biosafety level 3 facility and given 20% sucrose ad libitum The LR2006 OPY1 CHIKV strain, (GenBank accession number DQ443544) was isolated in France from a febrile patient who had been infected on the island of Re union in 2006 (Parola et al. 2006). Previously tested Ae. aegypti individuals from Palm Beach County were shown to be highly suscepti ble to the Re union (LR2006 OPY1) CHIKV strain (Reiskind et al. 2008) which contains the alanine to valine substitution at the 226 position of the E1 envelope structural protein (E1 A226V) that has been identified as a dominant genotype in many current CHI KV epidemics (Rezza et al. 2007). Virus for infectious blood meals was produced by inoculating a confluent monolayer of Vero cells in a T 75 cm 2 flask with 25 L of previously frozen stock virus, and incubating them in a 5% CO 2 atmosphere (atm) a t 35C fo r 48 h After 48 h infectious blood meal s were made by combining freshly recovered media viral suspension with citrated bovine blood (Hemostat Laboratories, Dixon, CA) in a 1 to 20 ratio Concentration of virus in fresh

PAGE 59

59 blood meal s was 6.3 Log 10 plaque fo rming units (PFU)/mL, which was measured by plaque assay performed in duplicate 6 well plates of confluent Vero cells. T en fold serial dilutions (to the 10 9 dilution) of infectious blood meal samples were prepared by combining 0.1 ml of the CHIKV infected blood meal with 0.9 ml BA 1 media (Lanciotti et al. 2000 ) and repeating the proce ss Each cell well was inoculated with 0.1 ml of a dilution, plates were incubated for 1 h at 5% CO 2 atm a t 35C, before a first overlay of agarose was applied to the cell m onolayer. The second overlay was applied two days later the plate was read the following day plaques were counted and final viral concentration s were exp ressed in PFUs per ml of blood meal. Mosquito Infection Groups of 100 five to seven day old Ae. aegy pti mosquitoes were placed in 1 L cylindrical, waxed cardboard containers (Dade Paper Co., Miami, FL) with mesh screening. Mosquitoes were starved for 24 hours and then offered an infectious blood meal using a w ater jacketed glass membrane feeder (Rutledge et al. 1964) covered with Edicoll collagen film (Devro, Sandy Run, SC) and connected to a Haake Series F water circulator ( Thermo Haake, Paramus, NJ) used to maintain the blood meal at 37C. Mosquitoes were given 30 minutes to feed. Immediately a fter fee ding, mosquitoes were cold anesthetized, and 10 fully engorged mosquitoes were removed, from each temperature food treatment, frozen in individual microcentrifuge tubes at 80C for subsequent wing removal and measurement, trituration, viral RNA extraction and quantitative RT PCR. Wings were removed from each mosquito using forceps that were sterilized with 100% ethanol followed by intense flaming using a portable one touch burner (Daigger). Wing length was measured in millimeters as an indicator of body size (Blackmore and Lord 2000) from the alula to wing tip excluding wing fringe. D igital

PAGE 60

60 images of the wing w ere captured and measured using a computer imaging and measurement program (i Solution lite AIC Inc., Princeton, NJ). These individuals were use d to determine the relationship between mosquito size and quantity of virus initially ingested by freshly fee d females The remainder of the engorged mosquitoes were held for a 10 day EIP at 27C and provide d with 20% sucrose ad libitum after which surviv ing Ae. aegypti females were killed by freezing. Females were stored in individual microcentrifuge tubes at 80C and, after thawing, wings were removed for measurements, bodies were assayed to determine infection status and titer, and legs were tested to che ck for a disseminated infection. An assayed mosquito could be, (1) uninfected, have an (2) isolated infection, which specified a CHIKV positive body, but legs negative for the presence of the virus or have a (3) disseminated infection, which meant virus was found in the legs signifying the infection had spread beyond the midgut and on to secondary organs (Turell et al. 1984). Both wings and legs of individual mosquitoes were removed using the sterilized forcep technique described previously. Samples were homogenized at 25 Hz for 3 min using a Tissuelyzer tissue homogenizer (Qiagen Inc., Valencia, CA). RNA was extracted separa tely from bodies and legs. Mosquito bodies were homogenized in TRI Reagent (Molecular Research Center, Inc., Cincinatti, OH) and t hen RNA was extracted the final elution volume Mosquito legs were homogenized in 1 media (Lanciotti et al. 2000) with two zinc plated BBs (Daisy) which was then adde d to 750 LS (Molecular Research Center, Inc., Cincinatt i, OH) for RNA

PAGE 61

61 with a final elution volume of One st ep quantitative RT PCR was used to determi ne infection status and body titer of samples. Primers were designed from the E1 gene and had the following ACC CGG TAA GAG CGA TGA ACT CAT CCG GTA TGT /5cy5/CCG TAG GGA ACA TGC CCA TCT CCA / 3BHQ_2/ well reaction stabil of the test sample RNA Viral RNA was quantified using a Roche LC480 light cycler (Roche Applied Sciences, Indianap olis, IN) with the following thermal conditions: 20 minutes at 48 C and 2 minutes at 95 C, followed by 40 cycles of PCR, 10 seconds at 95 C and 15 seconds at 60 C followed by a cool down for 30 seconds at 50 C. A negative control (DEPC treated water i n place of sample) and a positive control (CHIKV stock virus, 10 2 dilution) were included in each reaction run. A standard curve was generated by assaying a full range of ten fold serial dilutions of CHIKV virus stock ( 7.8 Log 10 PFU/ml) by plaque assay w hich determine d PFUs per dilution. Viral RNA was then isolated from thr ee replicates of each dilution using TRI Reagent LS ( as previously described for leg aliquots ), and all dilutions were assayed using qRT PCR Viral c oncentrations and crossing point (C p) values determined from qRT PCR from dilutions 10 2 through 10 6 constituted the six values used to establish a

PAGE 62

62 linear regression (Cp = 3.455*Log10(PFU) + 32.2, n=6, p<0.0001, r 2 =0.9985 ). Mosquito body titers in each test sample were then calculated by comparing the test sample with standard curve values that had been transformed into plaque forming unit (Cp) equivalents. Statistical Analysis All statistical analysis was p e r formed using SAS software, versi on 9.2 (SAS Institute, Carey NC ). CHIKV mosqui to titer data for the engorged female mosquitoes killed directly after blood feeding did not fit assumptions of normality and approximate normality was achieved through a log transformation of titer values. Product moment c orrelation analysis was then car ried out between the log transformed CHIKV mosquito body titer and the wing length s of the engorged females T wo way analysis of variance (ANOVA) (PROC GLM) was used to compare the effects of temperature and food level and their interaction on CHIKV titer in engorged females. M ain effect s means (temperature and food) were compared To determine differences among the mean wing lengths of the engorged females from the six la rval treatment groups (temperature food combination s) a two way ANOVA (PROC GLM) was tudentized range tests T emperature and food were categorical variable s in the two way ANOVA analysis. Two way ANOVA (PROC GLM) as used to determine differe nces among the temperature and food treatments in distributions of the wing lengths of blood fed females that were killed by freezing after the 10 day EIP. Two tests was also used to detect develop ment time differences among the temperature food treatments Mean development times were determined from all mosquitoes reared,

PAGE 63

63 not just blood fed females and m ales and females were analyzed separately because of gender specific developmental times in this species. The proportion of mosquitoes that died as larvae during rearing was analyzed for significant effects of the six treatments by maximum likelihood categorical analy ses of contingency tables ( PROC CATMOD) Comparisons of mortality rates between trea tments were performed with maximum likelihood contrasts using a Bonferroni adjustment to maintain an experiment 0.05. Logistic regression (PROC LOGISTIC) were used to model mosquito CHIKV body infection (# with virus/# fed) and disseminated infect ion (# with vi rus in their legs/# with virus) by temperature, food level, and temperature x food interaction, specifying a logistic link function and a binomial error distribution. Odds ratios (OR) and 95 % conf idence intervals for infection and disseminat ed infection by treatment were also calculated. Two way ANOVA was used to test for effects of temperature, food level, and a temperature x food interaction on mosquito body titer following the 10 day EIP. Differences between main effect means (temperature and food) were further analyzed by Titer data did not fit the model assumptions of normality, but approximate norm ality was achieved through a log transformation. To determine if there was a difference in size among uninfec ted, infected (non disseminated), and disseminated females a one way ANOVA (PROC GLM) was used. The one way ANOVA was used to compare differences in mean wing length among the females of the three infection status categories These were females that blood fed

PAGE 64

64 completed the 10 day EIP and were th en pooled across all larval tre atments for the size analysis. R esults Chikungunya Titer of Freshly Engorged M osquitoes Correlation analysis showed a significant positive correlation between mosquito wing length and log transformed CHIKV body titer ( r = 0.5787 P<0.0001 df = 56 ) (Figure 3 1 ). When tested by two way ANOVA, the log transformed mean titers of CHIKV in freshly engorged females w ere significantly affected by larval rearing temperature (F= 20.97 df = 2 P<0.0001), but not larval food level (F= 1.25 df = 1 P<0.2683), nor the temperature x food level interaction (F= 1.59 df = 2 P<0.2140) (Figure 3 2) higher CHIKV titer in mosquito bodies t r ange tests, P < 0.05) Wing lengths of freshly engorged females were significantly affected by larval rearing temperature (F= 103.88, df = 2, P<0.0001), larval food level (F= 33.69, df = 1, P<0.0001), but not the temperature x food level interaction (F= 1.50, df = 2, P<0.2316). W hen followed up with pairwise comparisons studentized range tests (P < 0.05) all temperature levels and food levels were significantly different from eachother (Figure 3 2 ) Growth and M ortality Wing lengths of blood fed females that were held through the 10 day EIP varied significantly due to temperature (F = 577.76; df = 2 ; P< 0.0001), food (F = 235.66; df = 1; P<0.0001), and the temperature x food interaction (F= 7.58, df = 2, P= 0.0006). D evelopment time to adulthood in females was also significantly affected by larval rearing temperature (F = 16187.6; df = 2; P<0.0001), food (F = 100.48; df = 1;

PAGE 65

65 P<0.0001), and the temperature x food interaction (F= 46.34, df = 2, P<0.000 1). There were also significant effects in male development due larval rearing temperature (F = 1 6614.9 ; df = 2; P<0.0001), food (F = 1 30 55 ; df = 1; P<0.0001), and the temperature x food interaction (F= 51.97 df = 2, P<0.0001) (Table 3 1). Larger mosquit oes were generated from the lower temperatures and higher food treatments. Juvenile mortality rates at the low and high food levels within the three temperature treatments, 20C, 27C, and 34C, respectively were 2.37% (n = 969), 2.05% (n = 975), 2.90% (n = 966), 2.53% (n = 1029), 2.89% (n = 1073), and 6.42% ( n = 1028) (F igure 3 3 ). There was a significant difference in mortality among treatments ( X 2 = 39.67, df = 5, P<0.0001). The maximum l ikelihood contrasts showed that the 34C high food level treatment at 6.42 %, had a significantly higher juvenile mortality rate than all other treatments ( Table 3 2 ). Chikungunya Infection and D issemination There was a significant temperature ( X 2 =26.0248, df = 2, P<0.0001) effect on the likelihood of females developing CH IKV infections, but food level ( X 2 =1.1108, df = 1, P = 0.2919) and the interaction between temperature and food level ( X 2 =4.5452, df = 2, P = 0.1030) w ere not significant (Figure 3 4 ). Infection was 5.4 times more likely in adult females reared at 27C tha n at 20C (Odds Ratio (OR) = 5.428; 95% C onfidence I nterval (CI): 2.798 10.532) and females reared at 27C were 4.7 times more likely to be infected than those rea red at 34C (OR = 4.768; 95% CI: 1.980 11.485). There was no significant difference in CHIKV infection between females reared at 2 0C and at 34C (OR = 1.138; CI: 0.525 2.468). Among the infected individuals the proportion of females that developed disseminated infections was significantly affected by larval rearing te mperature

PAGE 66

66 ( 2 =8.7265, df = 2, P = 0.0127), f ood level ( 2 =5.0123, df = 1, P = 0.0252), and their 914, df = 1, P = 0.0303) (Figure 3 5 ). Dissemination was 5.4 times more likely at 27C compa red to 34C (OR = 5.466; 95% CI: 1.4515 20.585) and 2.1 ti mes more likely at 27C c ompared to 20 C (OR = 2.109; CI: 1.0380 4.2832). There was no significant difference in disseminated infections between 20 and 34C. Dissemination was also 2.7 times more likely at the higher food level (OR = 2.7000; CI: 1.1317 6.4 417 ). After the 10 day EIP, when tested by a two way ANOVA, log titer of CHIKV in infected females was not significantly a ffected by larval rearing temperature (F= 2.01; df = 2; P = 0.1353), food level (F= 0.4 5; df = 1; P = 0.5037), nor the temperature x f ood level interaction (F= 0.56; df = 2; P= 0.5706). A one way ANOVA showed that there w as no significant variation in wing length s among CHIKV infection status categories (F = 1. 66, df = 2, P = 0.192) (Figure 3 6 ). Discussion Ecological factors in the larv al environment influence mosquito life history traits that are important in infectious disease dynamics (i.e. growth rate, life span, biting rate) and can directly affect traits that affect arbovirus susceptibility. Specific results from this experiment de monstrate that temperature and food availability influence body size, development time and CHIKV infection status, although the nature of the relationship between body size and viral susceptibility is not clear. Among engorged Ae. aegypti females, assayed immediately after taking a blood meal, there was significant correlation between body size, as measured by wing length, and body titer (Figure 3 1). Prior to the experiment it was hypothesized that larger females would take in a greater volume of blood and thus have a higher initial titer of

PAGE 67

67 virus when as sayed immediately after feeding and this hypothesis was partially supported by the correlation analysis results. However, when the effect of larval temperature and food quantity on freshly engorged female mosqui to body titer was analyzed via two way ANOVA it was found that only temperature and not food level had a significant effect on body titer yet food level definitely had a significant effect on size (Figure 3 2). Mosquitoes reared at 34 C, but given tw o different quantities of food were significantly different in size, but not in body titer while the mean titer of mosquitoes reared at 34 C was significantly lower than the mean titers of mosquitoes from 27 and 20 C treatments. Th us how the body size was achieved either by temperature differences or food differences, was an important factor in determining the amount of blood and virus ingested and that size alone was not the best predictor of ingested blood volume and initial viral dose. L arval habitat f eatures are important in regulating the growth of individuals and populations of mosquitoes (Rueda et al. 1990; Scott et al. 1993; Juliano 2009). In this experiment, temperature and food availability had measurable effects on mosquito dev elopment rate, siz e, and mortality. As expected, males had shorter develop ment times than their female counterparts due to developmental dimorphism between the sexes. Growth rate was phenotypically plastic with respect to temperature and food level and at the lowest tempera ture of 20 C mosquitoes took the longest to develop with median development time to adulthood for the low level food females of 12 days and for the high level food treatment of 13 days. As treatments increased in temperature the differences between develop ment time decreased so at 27 C mosquitoes only took approximately one day longer than individuals reared at 34 C to reach adulthood.

PAGE 68

68 Surprisingly, mosquitoes (both male and female) reared at 20 C and at 34 C took longer to develop if they were given more f ood as larvae. Most studies show a decline in development time when food quantity per larvae is increased (Wada 1965, Black et al. 1989, Teng and Apperson 2000). However, Ae. aegypti reared at 60 F (15.6 C) fed a finely ground laboratory chow took 33 days to pupa t e at the full food treatment and 28 days to pupate at the half food treatment (Keirans and Fay 1968) and among Ae. aegypti larvae given different daily amounts of B food treatment did not differ from the other t reatments in mean rate of pupation (Peters et al. 1969). It is possible that at 34 C the interaction between temperature and the higher food quantity produced a polluted environment leading to a longer development time, while the low temperature of 20 C is close enough to the lower thermal development limit of Ae. aegypti that resource utilization was unpredictable. At 27 C there was no significant difference in median development time to adulthood between the food levels, but the interquartile range for th e low food level is wider and skewed to incl ude a longer development period This temperature is probably close to the optimal physiological temperature for Ae. aegypti (Christophers 1960) and is commonly used in experiments, which may explain the more exp ected relationship of increased food availability and decreased development time. The three temperature and two food level combination s produced six significantly different wing length classes of mosquitoes with differences between adjacent groups ranging from 0.06 mm to 0.33 mm (Table 3 1). At 27 C t he two food level s produced females with a wing length difference of approximately 0.25 mm, but no difference in development time to ad ulthood The same difference in wing length, achieved through

PAGE 69

69 different re aring temperature s also produced difference in development time to adulthood The l ow temperature of 20 C had a more dramatic effect on size and development time, that the two higher temperatures. The experiment was designed to maximize adult production at temperatures and food levels that would produce markedly different outcomes in size, development times and responses to virus. To that effect there was an attempt to keep larval mortality at a minimum and equal among all six temperature food combinations. However, there was a significant difference in mortality between the highest temperature food combination and all the other treatments (Figure 3 3). Although results from preliminary experiments showed no difference in survivorship between the larval trea tments the very large sample size probably increased the ability to see even a small effect The increased mortality could have been due to the interaction of high temperature and excess nutritional resources resulting in the proliferation of detrimental m icro organisms in the larval environment. Only temperature had an effect on CHIKV infection status, with a significantly greater proportion of individuals infected when reared at the middle temperature of 27 C and no difference between the low er and high er temperature of 20 and 34 C In similar studies with Aede s vigilax larvae reared at 32 C and held at 25 C had lower RRV infection rates than co unterpa rts reared at 18 and 25 C and held at 25 C (Kay and Jennings 2002) and in Ae. albopictus there was a red uction in CHIKV infection with increasing temperature (Westbrook et al. 2009). Non linear responses to temperature are common in biological systems (Zhou et al. 2008) and based on the pattern of the results in Figure 3 4 the Ae. aegypti response to the e ffect of larval rearing temperature

PAGE 70

70 on CHIKV infection may not be linear Sub optimal temperature conditions, represented by the 20 and 34 C may lessen the susceptibility of Ae. aegypti to CHIKV. T his study was designed to explore the general question of w hether rearing temperature and food availability effect CHIKV infection status in Ae. aegypti and the use of a generalized linear model with temperature as a classed predictor variable was appropriate considering the limited number of temperatures used. Ho wever, now that an effect has been established future experiments will be designed with the intent of predictability. Increasing the number of temperature levels, reducing the d ifference between levels and treating temperature as a continuous variable w oul d provide data to model the response pattern more thorough ly Temperature, food availability, and their interaction had an effect on the probability of having a disseminated infection. S ignificance in the interaction terms specified that temperature had a different effect on the probability of having a CHIKV disseminated infection depend ent on food level At 20 and 34 C mosquitoes generated from high food treatments were more likely to have disseminated infections, while the opposite pattern was found at 27 C In the overall model disseminated infections were 2.7 times more likely in the high food treatments. This result is in contrast with other studies on larval nutri tion and vector competence o f C x tritaeniorhynchus for WNV and Ae. triseriatus for LACV w hich showed reduced susceptibility of adults generated from nutrient deprived larvae (Baqar et al. 1980 Grimstad and Haramis 1984; Grimstad and Walker 1991) Although w ing length was positively correlated with the initial quantity of virus ingested, signi ficant wing length infection correlations disappeared after the extrinsic incubation period, suggesting that mosquito size alone in this vector viral system is not

PAGE 71

71 a good predictor of viral infection, dissemination or body titer (Figure 3 6) It was origin ally hypothesized that a larger initial viral dose, even after the ten day EIP, may lead to a higher proportion of larger mosquitoes with isolated infecti ons and disseminated infections. There was support for this hypothesis in results from other studies F or example, large Ae. aegypti females, produced by varying food and density showed higher rates of oral infection with DENV compared to small and medium sized individuals (Sumanochitrapon et al. 1998) larger Ae. aegypti generated through variation in la rval diet which were more susceptible to RRV (Nasci and Mitchell 1994) and large Ae. triseriatus adults from competition treatments had lower infection and dissemination rates for LACV (Bevins 2008). In contrast, smaller A e. albopictus generated from high competition larval environments had higher rates of infection and dissemination for Sindbis (SINV) and dengue (DENV) viruses (Alto et al. 2005; Alto et al. 2008a ). In this study rearing temperature and food level a ffect ed the ease with which Ae. aegypti became infected with and disseminated CHIKV which may impact the epidemiology of this disease Failure to consider the importance of the larval environment may lead to incorrect estimates of vector susceptibility. Variations in different ecological factors in the larval habitat larval may produce mosqui toes of a similar size range, but with very different responses to infection. Thus, in this experiment body size was not a very good predictor of how a mosquito will respond to arboviral infection and t here m ay be more critical, but not as easily measurable phy siological and anatomical feature s of adult mosquitoes that vary with larval conditions and are more substantially correlated with viral susceptibility. This

PAGE 72

72 experiment demonstrates the significant role of larval ecology in adult vector viral interactions, but a dditional well designed experiments with predictability in mind are required to determine more quantitatively the effects of factors such as food temperature, and interactions with other individua ls or organisms during juvenile development on adult vector viral interactions.

PAGE 73

73 Table 3 1. Aedes aegypti LS means and standard error for development time to adulthood and female wing length Temperature 20C 27C 34C Food Level Low High Low High Low H igh Male time to adulthood (days) 11.470.04 a 12 .180.04 b 6.5 30.04 c 6.480.04 c 5 .17 0.04 d 5.5 5 0.04 e (n) (459) (488) (547) (526) (550) (513) Female time to adulthood (days) 11.970.04 a 12 .680.04 b 6.650.04 c 6.550.04 c 5 .360.04 d 5.750.04 e (n ) (487) (467) (391) (477) (492) (449) 3.34 0.02a 3.67 0.02b 3.03 0.02c 3.28 0.01d 2.70 0.02e 2.87 0.02f (n) (116) (60) (90) (91) (33) (58) M eans from PROC GLM analysis with different letters in the same row are sign ificantly different from one another

PAGE 74

74 Table 3 2. Maximum likelihood (ML) contrasts for comparisons of mortality rates for 34C high food treatment with all other temperature and food level treatment groups ML Contrast df Chi square P 34 C high food vs. 20C high food 1 17.72 <.0001 27 C low food 1 20.94 <.0001 27 C high food 1 13.07 0.0003 34 C Low food 1 17.02 <.0001 34 C high food 1 14.13 0.0002

PAGE 75

75 Figure 3 1 Correlation between log transformed whole m osquito body titers of CHIKV and wing lengt hs for engorged Aedes aegypti females killed immediately after feeding ( r = 0.5787 P<0.0001)

PAGE 76

76 Figure 3 2 Bivariate plot of LS means (SE) for wing lengths and log tr ansformed CHIKV body titers for engorged Aedes aegypti females killed immediately after feeding Filled symbols represent the low food tr eat ment and open symbols the hig h food treatment, squares are 3 4 C, triangles 27C, and circles 2 0 C Different letters indicate significant differences among means of log transformed CHIKV body titers. Symbols within dashed ellipses do not have significantly different wing lengths.

PAGE 77

77 Figure 3 3 Juvenile mortality rates at the low and high food levels within the three temperature treatments for Aedes aegypti *S ignificantly different from other five trea tments based on results from maximum likelihood contrasts.

PAGE 78

78 Figure 3 4 Proportion of Aedes aeg ypti females (SE) in each temperature treatment infected with CHIKV Numbers in parentheses are the number of blood fed females in that treatment group.

PAGE 79

79 Figure 3 5 Proportion s of infected Aedes aegypti females (SE ) from temperature and food level treatment s with disseminated CHIKV infections. Numbers in parentheses are the number of blood fed females in that treatment group.

PAGE 80

80 Figure 3 6 Least squared means (SE) for sizes o f adult female Ae. aegypti mosquitoes in CHIKV infection status categories.

PAGE 81

81 C HAPTER 4 LARVAL TEMPERATURE, COMPETITION, AND THE VECTOR COMPETENCE OF A edes aegypti AND A edes albopictus FOR CHIKUNGUNYA VIRUS Introduction Competition is often an important bi otic mech anism in the shaping of insect distributions and abundances (Craig et al. 1990, Settle and Wilson 1990, Human and Gordon 1996, Kaplan and Denno 2007). In nature, resources may be scarce in a limited area and competing organisms often have a choic e as to whether to stay and compete or disperse to another resource patch. However, in artificial and natural containers that house developing Ae. aegypti and Ae. albopictus the immature competitors cannot leave the container environment and competition can have considerable effects on population growth component s such as, development time, size, fecundity, and survival to adulthood (Teng and Appe rson 2000, Armistead et al. 2008 Reiskind and Lounibos 2009). Furthermore, larval competition can affect adul t mosquito susceptibility to an arbovirus which can result in changes in the distribution and transmission intensity of an arbovirus (Baqar el al 1980, Alto et al 2005, Alto et al 2 008, Bevin s 2008 ). Aedes albopictus and Ae. aegypti are invasive vectors with geographic ranges that span large portions of the globe. Aedes aegypti is believed to have traveled to the New World from its native Africa in water storage jars aboard slave ships (Christophers 1960), while the spread of Ae. albopictus from its nati ve Asian range has mostly been a more recent event in part due to the trade in used tires (Hawley et al. 1987). In many parts of the world larvae and pupae of both Aedes species may be found developing and feeding in the same container (MacDonald 1956, Fon tenille and Rodhain 1989, 3) and resource competition within and between these species is well documented (Juliano 1998, Braks et al. 2004).

PAGE 82

82 In larval competition experiments using leaf detritus as a basal resource, Ae albopictus exhibited a competitive advantage over Ae. aegypti (Juliano 1998, Braks et al. 2004), which might account for the observed decline of Ae. aegypti in areas of the United States now inhabited by Ae. albopictus 1995 Lounibos 2007 ). Nevertheless, there are many regions of sympatry of these two species including s outh ern Florida (Rey et al 2006) and condition specific competition has been proposed as the process behind their sustained coexistence (Costanzo et al. 2005, Leisnham a nd Juliano 2009). Aedes albopictus is a better larval competitor, but superior desi c cation resistance of the egg stage of Ae. aegypti allows greater numbers of this species to survive during the dry season (Sota and Mogi 1992, Juliano et al. 2002). Further more, Ae. aegypti presence or abundance is positively associated with lower humidity higher temperature and urbaniz ation while Ae. albopictus is negatively associated with hot, dry climate s and is more common in site s w ith shade and vegetation (Hawley 19 88, Braks et al. 2003, Rey et al. 2006, Reiskind and Lounibos 2009). Thus, in nature the outcome of competitive interactions between the two mosquito species changes temporally with the dry and wet seasons and spatially with environmental features associat ed with humidity and temperature. Both Ae. aegypti and Ae. albopictus are important epidemic vectors of chikungunya virus (CHIKV), a single stranded, positive sense enveloped RNA alphavirus. Chikungunya virus was first isolated in 1953 from a febrile patie nt in Tanzania (Ross 1956) and sporadi c epidemics were recognized subsequently from Africa, Asia, and India, but a particularly explosive epidemic of CHIKV began in 2004 in coastal Kenya and spread throughout African nations in the Indian Ocean, infecting high

PAGE 83

83 proportions of island inhabitant s, and subsequently spreading to India, South e ast Asia, Italy and other countries (Powers and Logue 2007, Gould and Higgs 2009 ). In previous epidemics. Ae. aegypti was implicated as more important with virtually all A sian vector isolates coming from this mosquito species (Powers and Logue 2007), but in recent CHIKV outbreaks in regions where Ae. aegypti and Ae. albopictus distributions overlap both specie s have t ested positive for the virus. In Singapore, in 2008, lar val surveys identified Ae. albopictus as more common than Ae. a egypti testing of wild caught mosquitoes yielded both Ae. albopictus and Ae. aegypti adult females positive for CHIKV (Ng et al. 2009). Similar patterns were found in Thailand, where wild caug ht adults of both species were positive for CHIKV (Thavara et al. 2009). Entomological surveys done during the 2006 CHIKV outbreak in the north eastern Indian state of Orissa, revealed the presence of both Aedes species with Ae. albopictus having a slightl y higher abundance than Ae. aegypti (Dwibedi et al. 2009). This study addresses whether larval rearing temperature mod ulates the competitive larval interactions between Ae. albopictus and Ae. aegypti and how that may in turn influence adult susceptibility to CHIKV. Most laboratory larval competition studies on Ae. albopictus and Ae. aegypti have been carried out at temperatures between 25 and 27 C (Black et al.1989, Barrera 1996, Daugherty et al. 2000 Alto et al. 2005, Alto et al. 2008 ) with the exceptio n of Lounibos et al. (2002) which compar ed competition between these two species at 24 and 30 C. The container habitat of Ae. albopictus and Ae. aegypti larvae may be subjected to temperature low s of approximately 12 to 14 C and highs ranging from 30 to 35 C or above ( Lounibos 1992 Reiskind unpublished data) Physical features of the environment, such as

PAGE 84

84 temperature may act in concert with or in opposition to biotic factors like competition to cause changes in physical or physiological traits in mosqu ito vectors that alter their susceptibility to arbovirus P revious studies in other insect systems have shown that a change in temperature can reverse the outcome of interspecific competition between insects that occupy the same environment (Birch 1953 Pa rk 1954, Ayala 1970, Russell 1986 ) It is also possible that an increase or a decrease in temperature may increase the magnitude of competitive effects without changing the direction of the interspecific competitive outcomes between Ae. aegypt and Ae. albo pictus Materials and Methods Mosquitoes, T emperature, and C ompetition Aedes aegypti and Ae. albopictus used in this experiment were second generation (F2) progeny of individuals collected as eggs, larvae, and pupae in St. Lucie and Palm Beach counties in s outh ern Florida. Adults maintained in cages under constant were provided with 20% sucrose ad libitum and blood meals from restrained chickens (housed and maintained in acco rdance with federally mandated animal use and care policies as part of the University of Florida, IACUC Protocol VB 17). Oviposited F 2 eggs were collected on seed germination paper. Eggs from both species were simultaneously hatched in separate Erlenmeyer flasks with tap water under a vacuum for 30 minutes to approximately synchronize hatching time. Newly hatched (<16 hours after hatching) larvae were counted and added to respective competition and temperature treatments. Competition treatments consisted of Ae. aegypti : Ae. albopictus species abundance ratios of 200:0, 100:0, 100:100, 0:200, 0:100, with five replicates per competitive treatment. All five competitive treatments, replicated five times, were run in incubators at

PAGE 85

85 temperatures of 22, 27 and 32C each with a 14:10 L:D photoperiod. Larvae in each temperature treatment were from the same cohort of eggs whose hatch was staggered to synchronize adult emergence among all treatments, which ensured that individuals of the same species were approximately the same adult age when blood fed. Five liter white plastic buckets partially filled with 2500ml of tap water, 500ml of particulate food (1:1 lactoalbumin: B l rearing containers. Oak leaf infusion was made by collecting fallen oak ( Quercus virginiana ) leaves, oven drying them for 48 hours at 80C, combining 35.5 g of leaves per liter of tap water and letting it incubate at 27C for ten days. A total of 50 lite r of infusion was prepared and then frozen so that all buckets, some of which were set up on different days because of staggered temperature treatments, would receive infusion that was deriv ed from the same starting material and treated identically. Enough oak leaf infusion to set up all the competitive treatments and replicates was thawed for 12 hours before adding it to each 5 L bucket. The 0.15 g of particulate food allow ed for competitive interactions between Ae. aegypti and Ae. a lbopictus in preliminar y experiments with limited mortality so that enough adult females were produce d for the CHIKV infection portion of the study as accomplished in similar experiments using other arboviruses (Alto et al. 2005, 2008a). Pupae were collected daily sexed and i dentified to species in the mixed treatments, and the pupation date in days since hatching was recorde d Pupae were collect ed until all individuals emerged or died. All female pupae from a given replicate were placed in a water filled 10 ml cup inside a 1 L cylindrical, waxed cardboard container (Dade Paper Co., Miami, FL) with fine mesh screening. Enough adult males

PAGE 86

86 were retained so that there was approximately a 1:5 ratio of males to females for mating to take place in the cages ; the remain ing male pupae were discarded. All pupae inside adult 1 L cylindrical cages were maintained at 27C and as adults emerged humidity was maintained a t >90% rh and adults were given 20% sucrose ad libitum The idea was that larvae completed development in one of the three d ifferent temperature treatments, but once pupation occurred and feeding in the aquatic stage ceased, pupae were moved to 27C and as adults maintenance continued at 27C though the blood feeding and the EIP. Virus and Mosquito I nfection The LR2006 OPY1 CHI KV strain, (GenBank accession number DQ443544) was isolated in France from a febrile patient who had been infected on the island of Reunion in 2006 (Parola et al. 2006). Ae des aegypti and Ae albopictus female s from Palm Beach County were shown previously t o be highly susceptible to this recently emergent CHIKV strain (Reiskind et al. 2008) which contains the alanine to valine substitution at the 226 position of the E1 envelope structural protein (E1 A226V) that has been identified as a feature in many rece n t CHIKV epidemics (Rezza et al. 2007). Virus for infectious blood meals was produced by inoculating a confluent monolayer of Vero cells in a T 75 cm 2 2 atmosphere (atm) and 35C for 36 h. After 36 h infectious blood w as prepared by combining freshly recovered media viral suspension with defibrinated bovine blood (Hemostat Laboratories, Dixon, CA) in a 1 to 10 ratio. Concentration of virus in blood meals offered to mosquitoes was measured at 7.4 Log 10 plaque forming units (PFU)/mL by one step quantitative RT PCR.

PAGE 87

87 Each 1 L cylindrical, waxed cardboard containers contained al l the emerged females of the same species from each replicate. Aedes albopictus females from all treatments were approximately 6 10 days old and Ae. aegypti were approximately 10 14 days old at blood feeding. Because intense larval competition increased de velopment time very young mosquitoes that had recently emerged from highly competitive treatment s were not offered a blood meal and only those mosquitoes within the previously stated age range were brought into the biosafety level 3 laboratory for blood feeding. Mosquitoes were sucrose starved for 24 hours, but water was available to them before blood was offered Adult mosquitoes were initially offered an infectious blood meal using water jacketed glass membrane feeders (Rutledge et al. 1964) covered wi th 38 42 mm hog casings (SausageMaker, Buffalo, NY) with an additional layer of P arafilm M (American National Can, Chicago, IL) as a barrier between the infectious blood and hog casing. Membrane feeders were attached to each other and to a Haake Series F water circulator (Thermo Haake, Paramus, NJ) which maintain ed the blood meal at 37C. N o mosquitoes fed from the Parafilm M plus hog casing membranes and 24 hours later a ll cages of adult females were offered an infectious blood meal from cotton pledgets each containing 3 ml of infectious blood pre heated to 35C for 20 min. Mosquitoes were given 30 minutes to feed. Immediately after wards mosquitoes were cold anesthetized, and engorged mosquitoes were separated from unfed, which were removed from the ex periment. Engorged mosquitoes were held for a 10 day extrinsic incubation period (EIP) at 27C and provide with 20% sucrose ad libitum after which surviving mosquitoes were killed by freezing. M osquito es w ere stored individual ly

PAGE 88

88 in 1.5 ml microcentrifuge tube s at 80C and, after thawing, wings were removed for measurements, bodies were assayed to determine infection status and titer, and legs were tested to check for disseminated infections. An assayed mosquito could be, (1) uninfected, have an (2) isolat ed infection, which specified a CHIKV positive body, but legs negative for the presence of the virus or have a (3) disseminated infection, which meant virus was found in the legs indicating that the infection had spread beyond the midgut and on to other o rgans. A disseminated infection signifies that a mosquito is capable of transmitting the virus (Turell et al. 1984). Wings and legs were removed from each mosquito using forceps that were sterilized with 100% ethanol followed by intense flaming with a port able one touch burner (Daigger). Wing length was measured in millimeters as an indicator of body size (Blackmore and Lord 2000) from the alula to wing tip, excluding wing fringe. Photographic images of the wing w ere captured with a digital camera mounted on a dissecting microscope and measured with a computer imaging and measurement program (i Solution lite, AIC Inc., Princeton, NJ). Mosquito bodie s were homogenized at 25 Hz for 3 min using a Tissuelyzer tissue homogenizer (Qiagen Inc., Valencia, CA). R NA was extracted separately from bodies and legs. Mosquito bodies were homogenized in TRI Reagent (Molecular Research Center, Inc., Cincinatti, OH) and then RNA was extracted according to the he final elution volume. 1 media (Lanciotti et al. 2000) with two zinc plated BBs (Daisy), wh ereafter the homogenate Reagent LS (Molecular Research Center, Inc., Cincinatti, OH) for R NA extraction, also

PAGE 89

89 One step quantitative RT PCR was used to determine infection status and body titer of samples. Primers were designed from the E1 gene and had the following sequences: forward: ACC CGG TAA GAG CGA TGA ACT CAT CCG GTA TGT /5cy5/CCG TAG GGA ACA TGC CCA TCT CCA /3BHQ_2/ well reaction SuperScript III RT/Platinum Taq mix 4 dNTPs and treated H 2 RNA. Viral RNA was quantified using a Roche LC480 light cycler (Roche Applied Sciences, Indianapolis, IN) with the following thermal conditions: 20 minutes at 48 C and 2 minutes at 95 C, followed by 40 cycles of PCR, 10 seco nds at 95 C and 15 seconds at 60 C followed by a cool down for 30 seconds at 50 C. A negative control (DEPC treated water in place of sample) and a positive control (CHIKV stock virus, 10 2 dilution) were included in each reaction run. A standard curve was generated by assaying a full range of ten fold serial dilutions of CHIKV virus stock (7.8 Log 10 PFU/ml) as previously described in Chapter 3 Statistical A nalysis All statistical analyses were conducted using SAS 9. 2 (SAS Institute, Cary, NC). Only dat a from female mosquitoes were included in the analysis. Each 5 L bucket containing a developing cohort of larvae was considered a replicate and survivorship to adulthood per replicate was calculated as the proportion of adults that emerged from the initia l cohort of first instar larvae. Female survivorship was estimated by assuming each original cohort contained 50% of each sex. Using individual time to pupation the mean

PAGE 90

90 female time to pupation was calculated for each replicate. Female wing length was onl y measured for females assayed for CHIKV infection Two way multivariate analys i s o f Variance (MANOVA) (PROC GLM) was used to analyze the effects of temperature a nd competitive treatment on Ae. albopictus female time to pupation, wing length, and survival to emergence simultaneously. P roportion al data for survival to emergence w ere transformed using an arcsine transformation, which is recommended when percentages are outside the range 30% to 70% (Sokal and Rohlf 1995) Because of poor blood feeding by femal e Ae. aegypti very few wing length measurements were taken of this species, therefore, this variable was removed from the female Ae. aegypti MANOVA analysis MANOVAs were done separately for each mosquito species. MANOVA creates a composite index of all me asured response variables which provides a distinct advantage over separate ANOVAs because the correlations among the variables are a factor in the model (Bray and Maxwell 1985). It is valuable to have several measures of group differences and using mult ivariate methods to assess the influence of treatment on groups provide a more valid assessment of effects. All dependent variables had multivariate normal distributions within each group assess s ignificance because this test statistic is robust to violations of assumptions concerning homogenei ty of the covariance matrix and provides maximum protection against finding a statistical significance when there is none, with small samples (Bray and Maxwell 1985) Significant temperature and competitive treatment effects were further analyzed by multivariate pairwise contrasts of main effect multivariate means with a Bonferroni correction for experiment wise 3 = 0.01 7). Standardi z ed canonical

PAGE 91

91 coefficients were used to determine the relative contribution of each response variable to si gnificant multivariate effects as well as their relationship to each other (e.g positive or negative). Differences in CHIKV susceptibility in Ae al bopictus were evaluated by two way MANOVA and standardized canonical coefficients on the response variables proportion infected (# with virus/# fed) and proportion with disseminated infection (# with virus in their legs/# with virus) Proportion al data for infection and disseminated infection w ere transformed using an arcsine transformation Differences in CHIKV infection and disseminated infection were also analyzed using a generalized linear mixed model (PROC GLMMIX) with temperature and competition as f ixed effects and replicate as a random effect specifying a logistic link function and a binomial error distribution. A multiplicative overdispersion component was added to the generalized linear mixed model s using a simple R side residual effect because o f a higher than expected variance in the distribution of the data ( Schabenberger 2007) Few number s of blood fed Ae. aegypti precluded analyses of infection and disseminated infection data with MANOVA or a generalized linear mixed model (PROC GLMMIX) Over all Ae. aegypti and Ae albopictus mean infection and disseminated proportions and standard errors of the mean (SEM) were calculated from replicate means from 40 Ae albopictus and 20 Ae. aegypti replicates pooled across all temperature and competitive tr eatments A generalized linear mixed model ( PROC GLMMIX ) was used to test for effects of temperature and competition (fixed) and replicate (random) on Ae. albopictus body titer after the 10 day EIP. Body titer was a continuous variable and the model speci fied a n identity link function and a gaussian error distribution Titer data did not fit the model

PAGE 92

92 assumptions of normality, but approximate normality was achieved through a log transformation of titer values. Differences between main effect means (tempera ture and competition) were further analyzed by pairwise comparisons using the LS means statement in PROC GLIMMIX To investigate the relationship between size and titer c orrelation analysis was performed on t he mean size of Ae. albopictus females per repli cate pooled across all competitive and temperature treatments, and titer values. This analysis was also performed on t he mean size of Ae. a egypti individuals pooled across all competitive and temperature treatments, and titer values. In addition, t o dete rmine if there was a difference in size among uninfected, infected (non disseminated), and disseminated females a one way ANOVA (PROC GLM) was used. The one way ANOVA was used to compare differences in mean wing length among the females of the three infect ion status categories which were categorical variables in the model. The f emales in the analysis were those that blood fed, completed the 10 day EIP and were pooled across all larval tre atments Each species was analysed separately. Results Mosquitoes, T emperature and C ompetition Results from the MANOVAs showed that in both Ae aegypti and Ae albopictus female growth and development varied significantly as a result of temperature and competition w ith no significant interaction between these variables in Ae albopictus and a significant interaction term in Ae aegypti (Table 4 1 ) An examination of the standardized canonical coefficients for temperature effect s on growth parameters shows that temperature had the greatest effect on time to pupation in b oth Ae albopictus and Ae aegypti (Table 4 1 ) For Ae albopictus time to pupation varied in the

PAGE 93

93 same direction as wing length (Table 4 1 ) so lower temperature produced larger mosquitoes that took longer to pupate. Also in Ae albopictus s urvival to em ergence was negative ly correlated with time to pupation and wing length but th e SCC value was small so differences in survivorship contributed very little to the overall significance of temperature For Ae aegypti survival to emergence varied positively with time to pupation (Table 4 1) but similar to Ae albopictus the smal SCC value indicated that differences in survivorship contributed only slightly to differences among temperatures Significant temperature effects of treatments on growth parameters of both Ae. albopictus and Ae. aegypti were further investigated in pairwise contrasts (Table 4 2). All pairwise temperature contrast for both Ae. albopictus and Ae. aegypti were significant. Associated SCC indicated that time to pupation was the primary s ource of differences between the pairs. Wing length contributed secondarily to pairwise differences in Ae. albopictus (Table 4 2). In examination of the SCC values for survivorship to adulthood in Ae. albopictus, survival was the highest at the middle temp erature of 27 C with little difference between survival at 22 C and 32 C. In Ae. aegypti survivorship to adulthood was the lowest at 32 C with little difference between 22 C and 27 C (Table 4 2). Significant competitive effects of treatments on growth para meters of both Ae albopictus and Ae aegypti were further investigate d in pairwise contrasts (Table 4 3 ). All pairwise competitive contrast for both Ae. albopictus and Ae. aegypti were significant. A s was found in examination of the SCC form temperature, SCC for competitive pairwise contrasts indicated that time to pupation was the primary source of differences between the pairs. Aedes albopictu s from the 0:100 treatment replicates

PAGE 94

94 were larger, took less time to emerge, and suffer ed less larval mortality t han the two high c ompetition treatments of 0:200 and 100:100 (Figures 4 1, 4 2, 4 3) There were also significant differences between the 0:200 and 100:100 competitive treatments for Ae albopictus The i nterspecific larval competition treatment produced A e albopictus that took longer to develop, had lower survivorship to emergence, and were smaller than Ae albopictus produced under intra specific competitive conditions of the same density (Table 4 3) In contrast, Ae aegypti generally d id better under th e inter specific than intra specific competitive conditions (Table 4 3). However, at 27 C Ae aegypti survivorship to adulthood was higher in the intraspecific than the interspecific treatment (Figures 4 4, 4 5) This pattern accounts for the significant int eraction between temperature and competition in this species (Table 4 1). T he pairwise contrast between Ae aegypti 100:100 and 0:100 was only marginally significant (P= 0.0487) Thus, the 100 Ae aegypti larvae housed with 100 heterospecific Ae albopictu s larvae only slightly differ ed in survival to emergence or time to pupation from the 100:0 Ae aegypti treatment. Virus and Mosquito I nfection A total of 317 blood fed Ae. albopictus survived the 10 day EIP and were processed and assayed for CHIKV suscept ibility. The 57 Ae. aegypti that survived through the 10 EIP were also processed and assayed for CHIKV, but replicate size and number were not large enough to include Ae. aegypti in any kind of treatment effect analysis. When pooled across treatments the mean ( SEM) replicate infection and disseminated infection rates for Ae. aegypti were 0.69 0.085 and 0.72 0.093 and for Ae. a lbopictus 0.95 0.017 and 0.70 0.037 (Figure 4 6)

PAGE 95

95 Results from the MANOVA show that temperature did not have a significant effect on the proportion of infected and the proportion with disseminated infections for Aedes albopictus 12 F 4,62 = 0.92 P = 0. 4578 ) nor did competition trace = 0.009, F 4,62 = 0.07, P = 0.9906) or the ir ce = 0.107, F 8 ,62 = 0.44, P = 0.3736) (Figure 4 7 ) Similarly, when Ae. albopictus infection data w ere analyzed with generalized linear mixed model s (PROC GLMMIX) there were no significant effects from temperature (F=0.01 df = 2, P = 0. 9940 ), competition ( F=0.01 df = 2, P = 0. 9912) or the interaction of these factors ( F=0. 58 df = 4, P = 0.6786) on the likelihood of infection and on the probability of disseminated infection there were also no significant effects from temperature (F=0.67, df = 2, P = 0.51 06), competition ( F=2.05, df = 2, P = 0.1307 ) or the ir interaction ( F=0.82, df = 4, P = 0.5150). After the 10 day EIP, r esults from the generalized linear mixed model (PROC GLMMIX) showed titer of CHIKV in infected Ae. albopictus females was significantly affected by larval rearing temperature (F= 8.36 ; df = 2 ; P = 0. 000 3 ), but not by competition (F= 1.62 ; df = 2; P= 0. 1999 ), nor the temperature x competition interaction (F= 1.33 ; df = 2; P<0. 2580 ). There was no significant difference in body titer between the 22 and 27C treatments, but both were signicantly different than the body titer of Ae. albopictus reared at 32C (Figure 4 8) Correlation analysis showed no relationship for Ae. albopictus ( r = 0. 255; P= 0. 1 385 df= 34 ) or Ae. aegypti r = 0. 311 ; P= 0. 1 480 df= 21 ) between wing length and log transformed CHIKV body titer of infected mosquitoes (Figure s 4 9 4 10 ). A one way ANOVA showed that there were no significant wing length differences among CHIKV infec tion status categories for Ae. albopictus (F = 0. 97 df = 2, P = 0.3795 ) or Ae. aegypti (F = 0.61, df = 2, P = 0.5457 ) (Figure s 4 11, 4 12 ).

PAGE 96

96 Discussion In this experiment Ae. a lbopictus was the inferior larval competitor. An interspecific competive environment (100:100) produced the smallest sized Ae. a l bopictus females, with the lowest survival, and the longest pupation times. Intraspecific competition (0:200) also had an effect on Ae. a lbopictus but the effect was not as severe on the three growth measurements as the interspecific competitive environme nt. In contrast, Ae. aegypti from the interspecific c ompetitive environment were only slightly affected in survivorship to emergence and days to pupation (P = 0. 0487) compared to conspecifics reared in the 100:0 treatment. Essentially, the presence of 100 Ae. a lbopictus had very little effect on the 100 Ae. aegypti developing in the same 5L bucket. Intraspecific competition affected Ae. aegypti and the 200:0 treatement was the poorest performer. This confirmed similar finding s by Lounibos et al. ( 2002 ) in which Ae. aegypti female growth was uniquely retarded by a high density of its own species. In the water filled containers that serve as habitat for Ae. aegypti and Ae. a lbopictus in Florida the primary food source is microorganisms such as bacteria and fu ngi that grow on decaying oak and other leaf detritus and parts of dead invertebrates (Fish and Carpenter 1982, Lounibos et al. 1992 Lounibos et al. 1993 ). In this environ ment where the dominant resource is leaf litter, Ae albopictus is the superior comp etitor ( Barrera 1996, Juliano 1998 ) To successfully execute a n experiment on CHIKV vector competence it was essential to generate enough adult mosquito es that also emerged within a reasonable time span and leaf litter as the sole food source in prelimina ry studies did not produce enough adults in an acceptable age range. A combination of oak leaf infusion and an artificial food source of a o ne to one mixture of was used as a compromise between the need for a

PAGE 97

97 reliable numb er of mosquitoes within a narrow age range and the maintenance of ecological relevance for the experiment. Because competitive interactions betweenthese two mosquitoes are context dependent, and the relative effects of competition may change under differen t ecological conditions (Juliano 2009) it was not surprising that Ae. aegypti was the superion larval competitor. In previous studies the type of food resource affected the outcome of competiton between these two Aedes species, with the addition of a high protein food (i.e. yeast, lactoalbumin, liver powder) favoring Ae. aegypti ( Black et al. 1989, Barrera 1996, Daugherty et al. 2000 ). Differences in temperature did not change the outcome of competition which confirmed previous results of Lounibos et al. ( 2002) when competition between these two speci es was investigated at 24 and 30 C As ex pected, based on results in C hapters 2 and 3 t emperature did have an effect on pupation time and size in both Aedes species with low temperature increasing develop m e n t time and lead ing to larger sized adults. Temperature also had a small effect on survivorship to adulthood in Ae. albopictus, survival was the highest at the middle temperature of 27 C and i n Ae. aegypti survivorship to adulthood was high at both 22 C an d 27 C Therefore, the low temperature only seemed to have a negative effect on survival in Ae. albopictus For Ae. aegypti there was a significant interaction between temperature and competition and how temperature modified the effect of competition is se en in Figure 4 4 where the 200:0 trea tment had greater survival than the 100:100 at 27 C while at the other temperatures and for the other growth measurement, time to pupation, the 100:100 treatment out performed the 200:0 for Ae. aegypti

PAGE 98

98 Neither compet itive interactions nor temperature treatmen t had an effect on CHIKV infection or dis seminated infection for Ae. albopictus However, temperature did have a significant effect on CHIKV body titer in Ae. albopictus The lack of a temperature effect on infect ion and disseminated infection in Ae. albopictus was unexpected because Chapters T wo and T hree demonstrated that aquatic larval temperature had an effect on CHIKV infection in both Ae. aegypti and Ae. albopictus and larval food level and temperature had an effect on disseminated infections in Ae. aegypti In addition, l arval rearing temperature has been shown to a ffect mosquito competence for viruses of Rift Valley fever (RVFV), Venezuelan equine encephalitis (VEEV) (Turell 1993), Murray Valley encephalitis (MVEV) (Kay et al. 1989a ), Japanese encephalitis (JEV) (Takahashi 1976), and western equine encephalitis (WEEV) (Hardy et al. 1990) Larval c ompetition has been shown to a ffect Ae triseriatus vector competence for LaCrosse virus (LACV) (Bevins 2008) and Ae. albopictus vector competence for viruses of S indbis (S INV) (Alto et al. 2005) and dengue ( Alto et al. 2008a ). Body titer of CHIKV infected Ae. albopictus females was significantly affected by larval rearing temper ature with females reared at the two lower temperatures of 22 and 27 C having higher mean CHIKV body titers than counterparts from 32 C. This effect is in agreement with results from Chapter T wo, in which CHIKV susceptibility was greater in Ae. albopictus adults reared at lower temperatures c ompared to individuals from higher rearing temperature treatments. The lack of a significant effect due to lar val competition and temperature on CHIKV infection and disseminated infection in this study is most likely due to low

PAGE 99

99 sample size and the general logistical difficulties of bringing an ecological experiment into a laboratory setting. The mosquitoes used were first generation progeny of fie ld collected individuals. It was very dif ficult to get recently derived field colonies of Ae. albopictus and Ae. egypti to feed on an artificial blood sources The original experimental design used water jacketed glass feeders with hog casing membrane s with parafilm added to prevent the membranes f r o m leaking. In preliminary experiments sufficient feeding took plac e on t he hog casing leading to the belief that is would be a good feeding method However, the parafilm was a last minute required addition in order to comply with BL 3 protocols established at FMEL, and the added barrier seems to have inhibited feeding on the parafilm hog casing combination. Subsequently, an alternative protocol using blood soaked heated cotton pledgets was attempted. Because pre vious studies showed that only when Ae. aegypti fed on pledgets soaked in a very high titer of CHIKV infectious blood is this species able to establish a midgut infection (Pesko et al. 2009) it was decided that a high ratio of 1:10 virus supernantant to defibrinated bovine blood w ould be used. This produced a blood meal titer of 7.4 Log 10 plaque forming units (PF U)/mL which was marked ly higher than blood meal titers used in prior experiments and historic human vi remias which have generally not circulated above 7.0 log 10 TCID 50 /ml (Jupp and McIntosh 1988). Unfortunate ly, a combination of low feeding on the pledget s, combined with a large amount of mortality during the 10 day EIP led to entire missing replicates from the different temperat ur e and competitive treatments for Ae. aegypti and it was necessary to remove th is species from the infection and dissemination analyse s. Aedes aegypti develop ed faster than Ae. a lbopictus maintained in an identical growth environment, but

PAGE 100

100 both species in interspecific larval treatements needed to be reared simultaneously leading to adult Ae. aegypti that were older than Ae. a lbo pictus The older age of Ae. aegypti combined with the somewhat stressful rearing conditions may have caused the higher adult mortality that was noted for this species over the 10 day EIP. For Ae. albopictus t he high blood meal titer led to a mean infectio n rate of approximately 95% among replicates pooled across temperature and competitive treatme nts which likely prevented any observed treatment effect on infection rates. Previous laboratory studies with historic epidemic CHIKV isolates have indicated tha t in the laboratory Ae. albopictus is a significantly more competent vector than Ae. aegypti ( Mangiafico 1971; Turell et al. 1992 ) and this superior laboratory competence of Ae. albopictus is even more exaggerated with the emergent E1 A226V CHIKV isolate (Tsetsarkin et al. 2007 Pesko et al. 2009 ). Titers of between 4.0 and 5.0 log 10 TCID 50 /ml resulted in about a 90% infection rate in Ae. albopictus and a <10% infection rate in Ae. aegypti and at titers above 6.0 log 10 TCID 50 /ml infection approached 100% in Ae. albopictus and 30% in Ae. aegypti (Tsetsarkin et al. 2007). In this study, replicate means and SEMs for the overall proportion with infections and with disseminated infections pooled across treatments. su ggest lower infection rates in Ae. aegypti compa red with Ae. albopictus for the emergent E1 A226 V CHIKV isolate (Figure 4 6) With such high infection rates in Ae. albopictus absence of a treatement effect on infection was partly expected. Among all nine te mperature competitive treatments average infect ion rates were between 90 and 100% ( F igure 4 7). The lowest proportions of infected individuals were among the three competitive treatements run at 22 C The overall disseminated infection rate among infected individuals for Ae. albopictus was

PAGE 101

101 70% (Figure 4 6). Among the nine temperature competitive treatments proportions of disseminated infections varied between approximately 50 and 80% (Figure 4 7) however low sample size s and large standard errors decreased the ability to find statistically significant differences between treatements Body size for neither Ae. albopictus nor Ae. aegypti was correlated with CHIKV body titer and body size had no significant effect on whether either species established an isolated or disseminated infection. In prior experi ments, size was shown to influence susceptibility of Ae. aegypti for DENV (Sumanochitrapon et al. 1998, Alto et al. 2008b) and Ross River virus (RRV) (Nasci and Mitchell 1994). In this experiment the absence of significant correlations was somewhat expecte d because of the mostly non significant temperature and competitive treatment effects on CHIKV susceptibility, since both temperature and competition have asignificant influence on size. However, temperature did have an effect on CHIKV body titer in Ae. al bopictus but size was not found to be significantly correlated with body viral titer in either species of mosquitoes This may be because CHIKV body titers were very high overall, which possibly result ed from the high CHIKV titer s in the blood meal these mosquitoes were fed In both figures 4 9 and 4 10 the majority of the CHIKV body titer values are clustered in a fairly narrow range of titers for Ae. albopictus between 6.0 6.5 Log 10 pfu/0.1ml and Ae. aegypti between 6.3 7.0 Log 10 pfu/0.1ml. These high ra nges of titers may represent biological maximum titer s for these species which may have led to the lack of significant correlations In summary, inter and intra specific competitive interaction s between larval Ae. aegypti and Ae. albopictus influence d im mature survival the length of the larval period, and body size. Under these speci fic experimental conditions, in which a protein rich

PAGE 102

102 artificial food source was provided to developing larvae Ae. aegypti was the superior competitor across all three temper ature treatments. L arval temperature and competition did not influence the likelihood of CHIKV infection or disseminated infection, but CHIKV body titers were significantly greater in female Ae. albopictus from the lower larval temperatures. The larval env ironment strongly influences adult size, but in this study there was no significant relationship between mosquito size and measures of CHIKV susceptibility. Future studies will be aimed at exploring what other physical or physiological traits may play role s in predicting vector susceptibility.

PAGE 103

103 Tabl e 4 1. MANOVA for temperature and competitive treatment effects and their interaction on population growth parameters of female Aedes albopictus and Aedes a egypti : time to pupation, juvenile survivorship, and wing length Comparison (num df, den df) P Standardized canonical coefficients Time to Pupation Survivorship Wing Aedes albopictus Temperature 1.12 (6, 5 8 ) <0.0001 3. 398 0. 153 0.9 78 Competitive treatment 0. 9 6 (6, 5 8 ) < 0.0001 2. 633 1. 049 0. 821 Temperature x competitive treatment 0 50 (12, 90 ) 0.1 351 Error d.f. 30 Aedes aegypti Temperature 1.30 (4, 72 ) <0.0001 4.8 54 0.2 45 Competitive treatment 0.88 ( 4, 72 ) <0.0001 4 671 0. 222 Temperature x competitive treatment 0.52 (8, 72 ) 0.0040 2. 821 1. 231 Error d.f. 36

PAGE 104

104 Table 4 2 M ultivariate pairwise contrasts of temperature treatment effects on female Aedes albopictus and Aedes a egypti for growth measurements time to pupation juvenile survivorship, and wing length Competitive treatment pairwise comparison s (num df, den df) P Standardized canonical coefficients Time to Pupation Survivorship Wing Aedes albopictus 22C vs. 27C 0 86 (3, 28) <0.0001 3 .4 11 0 435 0. 954 22C vs. 32C 0.91 (3, 28) <0.0001 3.374 0. 029 0. 983 27C vs. 32C 0. 4 7 (3, 28) 0.000 4 2 7 4 0 0. 985 0 89 7 Aedes aegypti 22C vs. 27C 0.94 (2, 35) <0.0001 4. 849 0. 072 22C vs. 32C 0.96 (2, 3 5) <0.0001 4. 834 0.313 27C vs. 32C 0 59 (2, 35) <0.0001 3 757 1.013

PAGE 105

105 Table 4 3 M ultivariate pairwise contrasts of competitive treatment effects on female Aedes albopictus and Aedes a egypti for growth measurements time to pupation, juvenile su rvivorship, and wing length Competitive treatment pairwise comparison s (num df, den df) P Standardized canonical coefficients Time to Pupation Survivorship Wing Aedes albopictus 0:100 vs. 0:200 0 83 (3, 28) <0.0001 2.4 09 1. 281 0. 691 0:100 vs. 100:100 0.92 (3, 28) <0.0001 2.6 76 0.998 0. 848 0:200 vs. 100:100 0.54 (3, 28) <0.0001 3 046 0. 406 1 101 Aedes aegypti 100:0 vs. 200:0 0.72 (2, 35) <0.0001 4. 518 0.356 100:0 vs. 100:100 0.1 6 (2, 35) 0. 0487 0.883 1.337 200:0 vs. 100:100 0 67 (2, 35) <0.0001 4.688 0.243

PAGE 106

106 Figure 4 1. Proportion of f emale Ae. albopictus (SEM) surviving to adult emergence Figure 4 2 F emale Ae. albopictus mean (SEM) days to pupation.

PAGE 107

107 Figure 4 3 F emale Ae. albopictus m ean (SEM) wing length.

PAGE 108

108 Figure 4 4. Proportion of f emale Ae. aegypti (SEM) surviving to adult emergence. Figure 4 5. F emale Ae. aegypti mean (SEM) days to pupation.

PAGE 109

109 Figure 4 6. Bivariate m eans ( SEM) of replicates for proportion s of Ae. a egypti and Ae. a lbopictus with infections and disseminated infections. Figure 4 7 Bivariate plot of least squares means for proportion of Ae. a lbopictus with infections and disseminated infections. Open symbo ls specify 22 C, grey symbols 27 C, and black symbols 32 C. Triang l es represent 0:100, circles 0:200, and squares 100:100 treatment.

PAGE 110

110 Figure 4 8. LS means ( SEM) of Aedes albopictus CHIKV body titer for temperature t reatments LS means with different letters are significant at a per comparison P level of 0.017

PAGE 111

111 Figure 4 9. Correlation analysis of log transformed of CHIKV whole mosquito body tit er and wing lengt h replicate mean s for Aedes a lbopictus pooled across temperature and competitive treatments ( r = 0. 255; P = 0.1385 ) Figure 4 10. Correlation analysis of log transformed of CHIKV whole mosquito body titers and wing lengt hs for Aedes a egypti infected individuals pooled across temperature and competitive treatments ( r = 0. 311 ; P = 0.1 480 )

PAGE 112

112 Figure 4 11. Least squared means (SE) for sizes of adult female Ae. a lbopictus mosquitoes in CHIKV infectio n status categories. Figure 4 12 Least squared means (SE) for sizes of adult female Ae. a egypti mosquitoes in CHIKV infection status categories.

PAGE 113

113 C HAPTER 5 G ENERAL CONCLUSIONS AND FUTURE DIRECTIONS The primary focu s of this study was to explore the extent to which vector susceptibility is influenced by the larval environment with in the Ae. aegypti Ae. albopictus and chikungunya virus system. P revious studies with Ae. aegypti and DENV serotype 2 estimated that envir onmental variance accounted for up to 78 91% of the total phenotypic variance in vector competence (Bosio et al. 1998) Clearly, environmental effects are an important component of vector susceptibility and overall disease epidemiology (Tabachnick 2009 ) M any studies have looked at the effect of temperature, humidity and other environmental effects on adult mosquito competence but little attention has been paid as to how the larval environment may shape traits involved in adult vector competence. Varying t emperature was a common treatment in the three experiment s described in this dissertation L arval rearing temperature influenced Ae. albopictus and Ae. aegypti growth and susceptibility to CHIKV. In Ae a lbopictus CHIKV infection rates decreased with inc reasing larval rearing temperature and decreasing adult size. Also in Ae albopictus decreased larval temperature was associated with higher CHIKV body titers among infected individuals. In Ae. a egypti larval temperature also influenced the likelihood of infection and disseminated infection with the intermediate temperature of 27 C (22 32 degree range) resulting in the highest rate s of infection and dissemination T he larval habitat in nature is composed of multiple biotic and abiotic features that inte ract To simulate some of the complexity that exists in the natural larval environment differing levels of nutritional resources and intra and inter specific competitive treatments were added to temperature treatments in experiments described

PAGE 114

114 in Chapters 3 and 4 respectively For Ae. aegypti higher levels of nutritional resources significantly in creased the likelihood of havi n g a disseminated infection. On the other hand, larval competition had no effect on susceptibility to CHIKV in fection in either spec ies of mosquito but it did have a significant effect on growth parameters in both Ae. aegypti and Ae. albopictus It is unclear why Ae. aegypti and Ae. albopictus reared at different temperatures and different food levels were more susceptible to CHIKV. W hen the mesenteronal tissues from large and small Ae. triseriatus females reared at differing food levels were examined with electron microscopy physical differences in the basement membrane (basal laminae) were fou nd. S mall Ae. triseriatus exhibited high er rates of transmission and disseminated infections for LACV and their basement membranes had fewer laminae resulting in a reduced mean thickness when compared with larger individuals (Grimstad and Walker 1991). It was suggested that the thinner basement membrane might allow for the more rapid release of LACV in to the hemocoel (Grimstad and Walker 1991). With the use of microscopy, a rapid release of EEEV into hemocoel due to disruptions in the posterior midgut was observed in Culiseta melanura immediatel y following an infectious blood meal (Weaver et al. 1991) other vector viral systems (Boorman 1960, Miles et al. 1973, Hardy et al. 1983, Weaver 1986). I n Chapter 3, Ae. aegypti reared at two different food levels crossed with three different temperatures had significantly different dissemination rates, with the proportion of Ae. aegypti from the higher food level having a greater odds of developin g a

PAGE 115

115 disseminated infection This contrasts with results for small Ae. triseriatus from the low food level (Grimstad and Walker 1 991), a lthough there was no significant relationship between size and susceptibility in th e experiment with Ae. aegypti describe d in Chapter 3. It is possible that the mechanism responsible for differences in CHIKV susceptibility in Ae. aegypti from different food levels also involves anatomical features of the midgut cells and electron microscopy studies could help elucidate a mec hanism. Large Ae. albopictus gen erated, in Chapter 2, through low temperatures were more susceptible to CHIKV which is in agreement with increased infection in Ae. taeniorhynchus for RVF and VEEV (Turell 1993), but contradicts previously described pattern s between small Ae. triseriatus and LACV (Grimstad and Walker 1991). In other studies, large Ae. aegypti mosquitoes generated by increasing larval food quantities were significantly more susceptible to RRV (Nasci and Mitchell 1994) and DENV (Sumanochitrapo n et al. 1989) than small mosquitoes It is likely that virus enters midgut cells through r eceptor mediated endocytosis (Hardy el al. 1983) and viral determinants for mosquito midgut infection have been studies for a number of arboviruses (Ludwig et al 198 9, Mourya et al. 1989, Houk et al. 1990, Mertens et al. 1996, Xu et al. 1997, Pletnev et al. 2001, Molina Cruz et al. 2005, Smith et al. 2008). It is possible that the expression of viral determinants or receptors in the midgut is influenced by rearing tem perature or larger mosquitoes generated through different larval conditions have a greater number of receptors or enhanced binding (Turell 1993). This is certainly a mechanism worth exploring and identification of viral binding sites or receptors would gre atlt increase our understanding of vector viral interactions.

PAGE 116

116 Because all growth of mosquitoes is accomplished during the aquatic larval period, which may be long at colder temperatures and under stressful conditions of limited food and high competition, i t seems likely that these environmental features are shaping insect immune pathways, and other physiological and anatomical features of the adult mosquito that may be more strongly correlated than body size to measures of arbovirus susceptibility. It is al so possible that how body size is achieved can completely change the susceptibility of a mosquito for an arbovirus. Future experiments should investigate the underlying physiological and/or molecular mechanisms that are influenced by the larval environment and lead to differential vector competence. Because l aboratory colonization can cause significant changes in phen otype and genotype of organisms, first or second generation Aedes the progeny of field collected parents or grandparents were used in this s tudy to more realistically represent the types of interaction that would take place in natural populations Colonization often leads to a decrease in heterozygosity and a shift in allele frequencies due to selection drift, non random mating, and founder e ffect ( Munstermann 1980, Lorenz et al. 1984, Mason et al. 1987) Frequencies of genes involved in vector competence may be influenced by evolutionary forces that accompany colonization, and the use of colonies freshly derived from field collected mosquitoe s may limit the effect of selection. However, it was a challenge to get newly established colonies of Ae. aegypti and Ae. albopictus to feed on an artificial blood source. In the first two experiments Edicoll collagen film was used as membrane for the mos quitoes to feed through in combination with water jacket glass feeders connected to a water circulator Edicoll collagen film is a manufactured product used as a casing for sausages and hotdogs. Low to moderate

PAGE 117

117 feeding was accomplished with the film and for the third experiment hog casing was used after a preliminary experiment established that it s use increased blood feeding success. As described in Chapter 4 the hog casing and water jacketed feeder system was replaced by blood soaked pledgets because t he addition of parafilm as a seconda ry membrane over the hog casing inhibited feeding The unanticipated switch to a different feeding method is one example of difficulties encountered while trying to get a substantial number of F 1 Ae. aegypti and Ae. albo pictus to feed in the laboratory during this study This dissertation i s a general exploration as to whether variations in temperature, food, and competition in the larval environment affect adult CHIKV susceptibility. An effect from temperature and food w as established and now the more difficult task of modeling mosquito viral susceptibility response patterns (infection, disseminated infection and viral titer) to changes in individual and combined larval environmental factors may be a future goal Some o f the o ther factors in the study that most likely influenced mosquito susceptibility to CHIKV, but were held constant among treatment s were blood meal titer, adult holding temperature and the length of EIP. These factors are known to influence absolute va lues of infection, disseminated infection, and body titer and variation in response to changing values of titer, adult temperature, and EIP would need to be included in future work in which predictability i s a goal. If relationships between viral suscepti bility and larval environment can be elucidated and in addition a mosquito trait or traits are identified that are a product of the environment and are correlated to viral susceptibility this could contribute to the predictability and risk assessment of epidemics. Pupal productivity surveys are common

PAGE 118

118 in areas with container inhabit ing mosquitoes and endemic disease such as dengue, yellow fever, chikungunya and filariasis. S urveys of immature mosquitoes are normally used as a method to assess vector popu lation densities in a given area. More specifically, estimates of pupa l abundance of Aedes vectors of DENV ha ve been promoted as a more accurate index of potential female vector s than traditional larval surveillance (Strickman and Kittayapong 2003). If p u pal surveys could incorporate measurements of h abitat quality and pupal or adult mosquito physical attributes related to susceptibility the additional information obtained could result in a much more powerful and directed approach of vector and disease co ntrol Lastly, a clear understanding of how ecological factors in the larval environment influence vector competence will be an important element in t he use of genetically modified mosquitoes to control vector borne disease Genetically modified mosquitoe s are not completely r efractory to the pathogen s they transmit. Vector competence may be signifi c an t l y limited as is the case with Anopheles stephensi that express the bee venom phospholipase A2 (PLA2) gene leading to a reduc tion in Plasmodium berghei ooc yst formation by 87% (Moreira et al. 2002) or that express the C type lectin CEL III from the sea cucumber, Cucumaria echinata resulting in only a moderate inhibition against P falciparum (Yoshida et al 2007). Because vector competence is heavily influe nced by the environment it seems likely that the expression of the inserted genes may also be influenced by how the mosquito responds and develops to a changing environment.

PAGE 119

119 LIST OF REFERENCES Ali M, Wagatsuma Y, Emch M, Breiman RF. Use of a geographic information system for defining spatial risk for dengue transmission in Bangladesh: Role for Aedes albopictus in an urban outbrea k. Am J Trop Med Hyg 2003; 69 :634 640. Alto, BW, Lounibos, LP, Higgs, S, Juliano, SA. Larval competition differentially affects arbovirus infection in Aedes mosquitoes. Ecol 2005; 86:3279 32 88. Alto, BW, Lounibos, LP, Mores, CN, Reiskind, MH. Larval competition alters susceptibility of adult Aedes mosquitoes to dengue infection. Proc R Soc Lond B Biol Sci 2008a; 275:463 4 71. Alto, BW, Reiskind, MH, Lounibos, LP. Size alters susceptibility of vectors to dengue virus infection and dissemination. Am J Trop Med Hyg 2008b; 79:688 6 95. Apostol BL, Black WC, Reiter P, Miller BR. Use of randomly amplified polymorphic DNA amplified by polym erase chain reaction markers to estimate the number of Aedes aegypti families at oviposition sites in San Juan, Puerto Ric o. Am J Trop Med Hyg 1994; 5 :89 97 Arankalle, VA, Shrivastava, S, Cherian, S, Gunjikar, RS, et al. Genetic divergence of chikungunya viruses in India (1963 2006) with special reference to the 2005 2006 explosive epid emic. J Gen Virol 2007; 88:1967 19 76. Armistead JS, Arias JR, Nishimura N, Lounibos LP. Interspecific larval competition between Aedes albopictus and Aedes japonicus (Dipter a : Culicidae) in northern Virginia. J Med Entomol 2008; 45:629 637. Baqar, S, Hayes, CG, Ahmed, T. The effect of larval rearing conditions and adult age on the susceptibility of Culex tritaeniorhynchus to infection with West Nile virus. Mosquito News 1980 ; 40:165 171. Barrera R. Competition and resistance to starvation in larvae of container inhabiting Aedes m osquitoes. Ecol Entomol 1996; 21:117 127. Barrera R, Amador M, Clark GG. Ecological factors influencing Aedes aegypti (Diptera: Culicidae) productivi ty in artificial containers in Salinas, Puerto Rico. J. Med Entomol 2006; 43:484 492. Benedict MQ, Levine RS, Hawley WA, Lounibos LP. Spread of the tiger: Global risk of invasion by the mosquito Aedes albopictus Vector Borne Zoonot Dis 2007; 7 :76 85.

PAGE 120

120 Bevi ns, SN. Invasive mosquitoes, larval competition, and indirect effects on the vector competence of native mosquito species (Diptera: Culicidae). Biological Invasions 2008; 10:1109 1117. Birch LC. Experimental background to the study of the distribution and abundance of insects .3. The relation between innate capacity for increase and survival of different species of beetles living together on the same food. Evolution 1953; 7:136 144. Birungi J, Munstermann LE. Genetic structure of Aedes albopictus (Diptera: Culicidae) populations based o n mitochondrial ND5 sequences: E vidence for an independent invasion into Brazil and United St ates. Annals of the Entomol Society of America 2002; 95:125 132. Black, WC IV, Moore CG. Population biology as a tool for studying ve ctor borne diseases. In BJ Beaty, WC Marquardt eds. The Biology of Disease Vectors Niwot, CO : University Press of Colorado 1996 ; 393 416 Black WC, Rai KS, Turco BJ, Arroyo DC. Laboratory study of competition between United States strains of Aedes albopi ctus and Aedes aegypti (Diptera: Culicidae). J Med Entomol 1989; 26:260 271. Blackmore, MS, Lord, CC. The relationship between size and fecundity in Aedes albopictus J Vect Ecol 2000; 25:212 21 7. Boorman J. Observations on the amount of virus present in t he haemolymph of Aedes aegypti infected with Uganda S, yellow fever an d Semliki Forest viruses, Trans R Soc Trop Med Hyg 1960; 54:362 365. Bosio CF, Beaty BJ, Black WC Quantitative genetics of vector competence for dengue 2 virus in Aedes aegypti Am J Tr op Med Hyg 1998; 59:965 9 70. Braks MAH, Honorio NA, Lounibos LP, Lourenco De Oliveira R, Juliano SA. Interspecific competition between two invasive species of container mosquitoes, Aedes aegypti and Aedes albopictus (Diptera : Culicidae), in Bra zil. Annals of the Entomol Society of America 2004; 97:130 139. Braks M, Honorio N, Lourenco de Oliveira R, Juliano SA, Lounibos LP. Convergent habitat segregation of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in southeastern Brazil and Florida. J Med Ent omol 2003; 40:785 794. Braks MAH, Juliano SA, Lounibos LP. Superior reproductive success on human blood without sugar is not limited to highly anthropop hilic mosquito species. Med Vet Entomol 2006; 20:53 59. Brie gel H. Mosquito reproduction: I ncomplete uti lization of the blood meal protein for oogenesis. J of Insect Physiol 1985; 31:15 21.

PAGE 121

121 Briegel, H. Fecundity, metabolism, and body size in Anopheles (Diptera: Culicidae), vectors of malaria. J Med Entomol 1990; 27:839 8 50. Briegel, H, Timmermann, SE. Aedes albopictus (Diptera: Culicidae): P hysiological aspects of development and reproduction. J Med Entomol 2001; 38:566 5 71. Bustin, SA. Absolute quantification of mRNA using real time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 200 0; 25:169 1 93. Carey DE. Chikungunya and dengue: a case of mistaken ident ity? J Hist Med Allied Sci 1971; 26:243 2 62. Chadee DD, Corbet PS. Seasonal incidence and diel patterns of o viposition in th e field of the m osquito, Aedes aegypti (L) (Dipter a, Culici dae) in Trinidad, West Indies a preliminary s tudy. Annals of Trop Med and Parasitology 1987; 81:151 1 61. Chadee DD, Martinez R. Landing periodicity of Aedes aegypti with implications for dengue transmission in Trinidad, West I ndies. J Vector Ecol 2000; 25 : 158 163. Chamberlain, RW, Sudia, WD. The effects of temperature upon the extrinsic incubation of eastern equine encephalitis in mosquitoes. Am J Hyg 1955; 62:295 305. Chamberlain RW, Sudia WD. Mechanism of transmission of viruses by mosquitoes. Annu Rev En tomol 1961; 6:371 3 90. Chretien JP, Anyamba A, Bedno SA, Breiman RF, et al. Drought associated chikungunya emergence along coastal East Africa. Am J Trop Med Hyg 2007; 76:405 40 7. Christophers SR. Aedes aegypti (L.), the yellow fever mosquito; its life his tory, bionomics, and structure Cambridge, England: Cambridge University Press, 1960. Clements AN. The Biology of Mosquitoes, Vol. I II New York New York, Chapman and Hall, 1992. Colless DH. Notes on the Culicine mosquitoes of Singapore. VII. Host prefer ences in relation to the transmission of disease. Ann Trop Med Parasitol 1959; 53:259 2 67. Cook PE, Hugo LE, Iturbe Ormaetxe I, Williams CR, et al The use of transcriptional profiles to predict adult mosquito age under field conditions. PNAS 2006; 103 :180 60 180 65. Corbet PS, Chadee DD. Incidence and diel pattern of oviposition outdoors of the mosquito, Aedes aegypti (L) (Diptera, Culicidae) in Trinidad, WI in relat ion to solar aspect. Annals of T rop M ed and P arasitology 1990; 84 :63 78.

PAGE 122

122 Costanzo KS, Kesavar aju B, Juliano SA. Condition specific competition in container mosquitoes: The role of noncompeting life hist ory stages. Ecology 2005; 86 : 3289 3295. Craig TP, Itami JK, Price PW. Intraspecific c ompetition and f acilitation by a s hoot g alling s awfly. Journ al of Animal Ecology 1990; 59:147 159. Daugherty MP, Alto BW, Juliano SA. Invertebrate carcasses as a resource for competing Aedes albopictus and Aedes aegypti (Diptera : Culicidae). J Med Entomol 2000; 37:364 372. Davis, NC. The effect of various temperat ures in modifying the extrinsic incubation period of the yellow fever virus in Aedes aegypti Am J Hyg 1932; 16:163 1 76. de Moor PP, Steffens FE. Computer simulated model of an arthropod borne virus transmission cycle, with special r eference t o chikungunya v irus. Trans Royal Society of Trop Med Hyg 1970; 64:927 934. Delatte, H, Dehecq, JS, Thiria, J, Domerg, C, et al. Geographic distribution and developmental sites of Aedes albopictus (Diptera: Culicidae) during a chi kungunya epidemic event. Vector Borne Zo onot Dis 2008; 8:25 34. Delatte H, Gimonneau G, Triboire A, Fontenille D. Influence of temperature on immature development, survival, longevity, fecundity, and gonotrophic cycles of Aedes albopictus vector of chikungunya and d engue in the Indian Ocean. J Med Entomol 2009; 46:33 41. Diallo, M, Thonnon, J, Traore Lamizana, M, Fontenille, D. Vectors of c hikungunya virus in Senegal: current data and transmission cycles. Am J Trop Med Hyg 1999; 60: 281 28 6. Diarrassouba S, Dossou Yovo J. Atypical activity rhyth m in Aedes aegypti in a sub sudanian savannah zone of Cote d'Ivoire. Bull Soc Pathol Exot 1997; 90 :361 363. Dohm, DJ, O'Guinn, ML, Turell, MJ. Effect of environmental temperature on the ability of Culex pipiens (Diptera: Culicidae) to transmit West Nile vi rus. J Med Entomol 2002; 39:221 22 5. Dubrulle M, Mousson L, Moutailler S, Vazeille M, Failloux AB. Chikungunya virus and Aedes mosquitoes: saliva is infectious as soon as two days after oral infection. PLoS One 2009; 4:e5895. Dwibedi B, Mohapatra N, Beuria MK, Kerketta AS, et al. Emergence of chikungunya virus infection in Orissa, India. Vector Borne Zoonot Dis 2009; Online ahead of print http://www.liebertonline.com/doi/pdfplus/10.1089/v bz.2008.0190 Edman JD, Strickman D, Kittayapong P, Scott TW. Female A edes aegypti (Diptera, Culicidae) in Thailand rarely feed o n sugar. J Med Entomol 1992; 29 :1033 10 38.

PAGE 123

123 Effler PV, Pang L, Kitsutani P, Vorndam V, et al. Dengue fever, Hawaii, 2001 2002. Emerging Infectious Diseases 2005; 11:742 4 49. Fish D, Carpenter SR. L e af litter and larval mosquito dynamics in tree hole ecosystems Ecology 1982; 63 :283 2 88. Fontenille D, Rodhain F. Biology and distribution of Aedes albopictus and Aedes aegypti in Madagascar. J Am Mosq Control Assoc 1989; 5:219 2 25. Forattini OP. Aedes (S tegomyia) albopictus (Skuse) Identification in Brazil. Revista De Saude Publica 1986; 20:244 2 45. Foster WA. Mosquito s ugar f eeding and reproductive energetics. Annual Rev iew of Entomology 1995; 40:443 4 74. Gould EA, Higgs S. Impact of climate change and o ther factors on emerging arbovirus diseases. Trans R Soc Trop Med Hyg 2009; 103:109 1 21. Grimstad, PR, Haramis, LD. Aedes triseriatus (Diptera: Culicidae) and La Crosse virus. III. Enhanced oral transmission by nutrition deprived mosquitoes. J Med Entomol 1984; 21: 249 2 56. Grimstad, PR, Walker, ED. Aedes triseriatus (Diptera: Culicidae) and La Crosse virus. IV. Nutritional deprivation of larvae affects the adult barriers to infection and transmission. J Med Entomol 1991; 28:378 3 86. Halstead SB, Scanlon JE Umpaivit P, Udomsakdi S. Dengue and c hikungunya virus infection in man in Thailand, 1962 1964. IV. Epidemiologic studies in the Bangkok metropolitan area. Am J Trop Med Hyg 1969; 18:997 1021. Hardy JL, Houk EJ, Kramer LD, Reeves WC. Intrinsic factors aff ecting vector competence of mosquitoes for arboviruses. Annu Rev Entomol 1983; 28:229 2 62. Hardy, JL, Meyer, RP, Presser, SB, Milby, MM. Temporal variations in the susceptibility of a semi isolated population of Culex tarsalis to peroral infection with wes tern equine encephalomyelitis and St. Louis encephalitis viruse s. Am J Trop Med Hyg 1990; 42 :500 5 11. Harrington LC, Edman JD, Scott TW. Why do female Aedes aegypti (Diptera : Culicidae) feed preferentially and frequently on human bl ood? J Med Entomol 2001 ; 38 :411 4 22. Harrington LC, Scott TW, Lerdthusnee K, Coleman RC, et al Dispersal of the dengue vector Aedes aegypti within and between rural communitie s. Am J Trop Med Hyg 2005; 72 :209 2 20.

PAGE 124

124 Hawley, WA. Biology of Aedes albopictus J Am Mosq Control Assoc 1988; 4(Supplement#1):1 39. Hawley WA, Reiter P, Copeland RS, Pumpuni CB, et al Aedes albopictus in North America Probable introduction in used tires from northern Asia. Science 1987; 236:1114 11 16. Holt RA, Subramanian GM, Halpern A, Sutton GG, et al The genome sequence of the malaria mosquito Anopheles gambiae Science 2002; 298 :129 149. Honorio NA, Silva Wda C, Leite PJ, Goncalves JM, et al Dispersal of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in an urban endemic dengue area in the State of Rio de Janeiro, Brazil. Mem Inst Oswaldo Cruz 2003; 98:191 19 8. Houk EJ, Arcus YM Hardy JL, Kramer LD. Binding of western equine encephalomyelitis virus to brush border fragments isolated from mesenteronal epithelial cells of mosquitoes. Virus Re s 1990; 17 : 105 118. Human KG, Gordon DM. Exploitation and interference competition between the invasive Argentine ant, Linepithema humile, and native ant species. Oecologia 1996; 105:405 4 12. Hurlbut HS. Effect of e nvironmental t emperature u pon transmissio n of St. Louis Encephalitis v irus by Culex Pipiens Quinquefasciatus J Med Entomol 1973; 10:1 12. International Society for Infectious Diseases. ProMED mail archive numbers 20081217.3963, 20081211.3895, 200903 02.9854, 20100224.0617, 20100323.0918 ; Accessed March 1, 2010 Jennings, CD, Kay, BH. Dissemination barriers to Ross River virus in Aedes vigilax and the effects of larval nutrition on their expression. Med Vet Entomol 1999; 13:43 10 8. Juliano SA. Species introduction and replacement among mosquitoes: I nterspecific resource competition or apparent competition? Ecology 1998; 79:255 268. Juliano SA. Species i nteractions a mong l arval m osquitoes: Context d ependence a cross h abitat g radients. Annual Review of Entomology 2009; 54:37 56. Juliano SA, Lounibos LP. Ecology of invasive mosquitoes: effects on resident species and on human health. Ecology Letters 2005; 8:558 574. Juliano SA, O'Meara GF, Morrill JR, Cutwa MM. Desiccation and thermal tolerance of eggs and the coexistence of competing mosquitoes. Oecologi a (Berlin) 2002; 130:458 469.

PAGE 125

125 Jupp PG, McIntosh B M Chikungunya disease. In: Monath T, ed. The a rboviruses: Epidemiology and ecology. Boca Raton, FL: CRC Press; 1988:137 157. Jupp, PG, McIntosh, BM. Aedes furcifer and other mosquitoes as vectors of chikung unya virus at Mica, northeastern Transvaal, South Africa. J Am Mosq Control Assoc 1990; 6: 415 4 20. Kambhampati S, Black WCt, Rai KS. Geographic origin of the US and Brazilian Aedes albopictus inferred from allozyme analysis. Heredity 1991; 67 :85 93. Kamimu ra, K, Matsuse, IT, Takahashi, H, Komukai, J, et al Effect of temperature on the development of Aedes aegypti and Aedes albopictus Med Entomol Zool 2002; 53 :53 58. Kaplan I, Denno RF. Interspecific interactions in phytophagous insects revisited: a quant itative assessment of competition theory. Ecology Letters 2007; 10:977 994. Kay, BH, Edman, JD, Fanning, ID, Mottram, P. Larval diet and the vector competence of Culex annulirostris (Diptera: Culicidae) for Murray Valley encepha litis virus. J Med Entomol 1 989a ; 26:487 488 Kay, BH, Fanning, ID, Mottram, P. The vector competence of Culex annulirostris Aedes sagax and Aedes alboannulatus for Murray Valley encephalitis virus at different tem peratures. Med Vet Entomol 1989b ; 3:107 1 12. Kay BH, Jennings CD. Enh ancement or modulation of the vector competence of ochlerotatus vigilax (Diptera : Culicidae) for Ross River virus by temperature. Journal of Medical Entomology 2002; 39:99 105. Keirans JE, Fay RW. Effect of food and temperature on Aedes a egypti (L) and Ae des t riseriatus (Say) larval development. Mosquito News 1968; 8 :338 342 Khan AH, Morita K, Parquet Md Mdel C, Hasebe F, et al Complete nucleotide sequence of chikungunya virus and evidence for an internal polyadenylation site. J Gen Virol 2002; 83:3075 3 0 84. Kilpatrick AM, Meola MA, Moudy RM, Kramer LD. Temperature, viral genetics, and the transmission of West Nile virus by Culex pipiens mosquitoes. PLoS Pathogens 2008, 4(6) e1000092. Kit LS. Emerging and re emerging diseases in Malaysia. Asia Pac J Publi c Health 2002; 14:6 8. Knudsen AB, Romi R, Majori G. Occurrence and spread in Italy of Aedes albopictus with implications for its introduction into other parts of Europe. J Am Mosq Control Assoc 1996; 12:177 1 83.

PAGE 126

126 Kumar NP, Joseph R, Kamaraj T, Jambulingam P. A226V mutation in virus during the 2007 chikungunya outbreak in Kerala, India. Journal of G eneral Virology 2008; 89:1945 19 48. Kuno G, Chang GJ. Biological transmission of arboviruses: reexamination of and new insights into components, mechanisms, and unique traits as well as their evolutionary trends Clin Microbiol Rev 2005; 18:608 6 37. Lanciotti, RS, Kerst, AJ, Nasci, RS, Godsey, MS, et al. Rapid detection of West Nile virus from human clinical specimens, field collected mosquitoes, and avian samples by a TaqMan reverse transcriptase PCR assay. J Clin Microbiol 2000; 38:4066 40 71. Lanciotti RS, Kosoy OL, Laven JJ, Panella AJ, et al. Chikungunya virus in US travelers returning from India, 2006. Emerging i nfectious diseases 2007; 13 :764 7 67. Laras K, Su kri NC, Larasati RP, Bangs MJ, et al. Tracking the re emergence of epidemic chikungunya virus in Indonesia. Trans R Soc Trop Med Hyg 2005; 99:128 1 41. Leisnham PT, Juliano SA. Spatial and temporal patterns of coexistence between competing Aedes mosquitoes in urban Florida. Oecologia 2009; 160:343 352. Leisnham PT, Lounibos LP, O'Meara GF, Juliano SA. Interpopulation divergence in competitive interactions of the mosquito Aedes albopictus Ecology 2009; 90:2405 24 13. Liew C, Curtis CF. Horizontal and vertical dispersal of dengue vector mosquitoes, Aedes aegypti and Aedes albopictus in Singapore. Med Vet Entomol 2004; 18:351 3 60. Ligon BL. Reemergence of an unusual disease: The chikungunya epidemic. Semin Pediatr Infect Dis 2006; 17:99 104. Lorenz, L, Beaty, B J, Aitken, TH, Wallis, GP, et al The effect of colonization upon Aedes aegypti susceptibility to oral infection with yellow fever virus. Am J Trop Med Hyg 1984; 33:690 69 4. Lounibos, LP. The mosquito commuity of treeholes in subtropical Florida. In Phytot elemata: Terrestrial Plants as Hosts for Aquatic Insect Communities (eds. J. H. Frank and L. P. Lounibos), pp. 223 246. Medford, NJ. Plexus Publishing Inc., 1983. Lounibos LP. Invasions by insect vectors of human disease. Annual Review of Entomology 2002; 47:233 2 66.

PAGE 127

127 Lounibos LP. Competi tive displacement and reduction In Biorational Control of Mosquitoes (eds.TE Floore and J Becnel), pp. 276 282 American Mosquito Control Association Bulletin No.7 2007; 23 (Suppl. No 2). Lounibos LP, Nishimura N, Escher RL Seasonality and components of oak leaf litterfall in Southeastern Florida. Florida Scientist 1992; 55:92 9 8 Lounibos LP, Nishimura N, Escher RL Fitness of a treehole mosquito: influences of food type and predation Oikos 1993; 66:114 1 18. Lounibos LP, O 'Meara GF, Escher RL, Nishimura N, et al Testing predictions of displacement of native Aedes by the invasive Asian tiger mosquito Aedes albopictus in Florida, USA. Biological Invasions 2001; 3:151 1 66. Lounibos LP, O'Meara GF, Nishimura N, Escher RL. Inte ractions with native mosquito larvae regulate the production of Aedes albopictus from bromeliads in Florida. Ecological Entomology 2003; 28:551 5 58. Ludwig GV, Christensen BM, Yuill TM, Schultz KT. Enzyme processing of La Crosse virus glycoprotein G1: A bu nyavirus vector infection model. Virology 1989; 171:108 113. Macdonald WW. Aedes aegypti in Malaya. II. Larval and adult biology. Annals of tropical medic ine and parasitology 1956; 50 :399 414. McCrae AW R Henderson BE, Kirya BG, Sempala SD K. C hikunguny a vi rus in the Entebbe area of U gand a: isolations and epidemiology. Trans R Soc Trop Med Hyg 1971; 65: 152 1 68. Maciel de Freitas R, Codeco CT, Lourenco de Oliveira R. 2007. Daily survival rates and dispersal of Aedes aegypti females in Rio de Janeiro, Brazil. Am J Trop Med Hyg 2007; 76:659 6 65. Madder, DJ, Surgeoner, GA, Helson, BV. Number of generations, egg production, and developmental time of Culex pipiens and Culex restuans (Diptera: Culicidae) in southern Ontario. J Med Entomol 1983; 20:275 2 87. Mangiafic o J. Chikungunya virus infection and transmission in five species of mosquito. Am J Trop Med Hyg 1971; 20:642 6 45. M ason LJ, Pashley DP, Johnson SJ T he lab oratory as an altered habitat : P henotypic and genetic consequences of colonization Florida Entomolo gist 1987 ; 70 :49 58. McDonald PT. Population characteristics of domestic Aedes aegypti (Diptera: culicidae) in villages on the Kenya Coast I. Adult survivorship and population size. J Med Entomol 1977; 14 :42 48.

PAGE 128

128 Mercado Curiel, RF, Black, WC, Munoz, Mde.L. A dengue receptor as possible genetic marker of vector competence in Aedes aegypti BMC Microbiol 2008, 15:118 1 33. Mertens PP, Burroughs JN, Walton A Wellby MP, et al. Enhanced infectivity of modified bluetongue virus particles for two insect cell lines and for two Culicoides vector s pecies. Virology 1996; 217: 582 593. Miles JA, Pillai JS, Maguire T, Multiplication of Whataroa virus in mosquitoes. J Med Entomol 1973; 10: 176 185. Minitab. Minitab 15.1 for Windows. Minitab Inc. State College, PA, 2006. M olina Cruz A, Gupta L, Richardson J, Bennett K, et al. Effect of mosquito midgut trypsin activity on dengue 2 virus infection and dissemination in Aedes aegypti Am J Trop Med Hyg 2005; 72:631 637. Monteiro, LC, de Souza, JR, de Albuquerque, CM. Eclosion r ate, development and survivorship of Aedes albopictus (Skuse)(Diptera: Culicidae) under different water temperatures. Neotropical Entomol 2007; 36:966 9 71. Mooney, HA, Hobbs RJ (eds.). 2000. Invasive s pecies in a c hanging w orld. Island Press, Washington DC Moore CG. Aedes albopictus in the United States: Current status and prospects for further spread. J Am Mosq Control Assoc 1999; 15:221 2 27. Moreira LA, Ito J, Ghosh A, Devenport M, et al Bee venom phospholipase inhibits malaria parasite development i n t ransgenic mosquitoes. J Biol Chem 2002; 277 :40839 40843. Mourya DT, Ranadive SN, Gokhale MD, Barde PV, et al. Putative chikungunya virus specific receptor proteins on the midgut brush border membrane of Aedes aegypti mosquito, Indian J Med Res 1998; 107:10 14. Muir LE, Kay BH. Aedes aegypti survival and dispersal estimated by mark release recapture in northern Australia. Am J Trop Med Hy g 1998; 58 :277 282. Munstermann LE. D istinguis hing geographic strains of the Aedes atropalpus group (Diptera, C ulicidae) b y analysis of enzyme variation Ann Entomol Soc Am 1980; 73: 699 1980 National Vector Borne Disease Control Programme (NVBDCP) Chikungunya situation in India. 2006 2009 http://www.nvbdcp.gov.in/Chikun cases.html http://www.nvbdcp.gov.in/Doc/chikun update 07.pdf. Accessed March 1, 2010

PAGE 129

129 Nasci, RS, Mitchell, CJ. Larval diet, adult size, and susceptibility of Aedes aegypti (Diptera, Culicidae) to infection with Ross River virus. J Med Entomol 1994; 31:123 1 26. Nene V, Wortman JR, Lawson D, Haas B, et al. Genom e sequence of Aedes aegypti a major arbovirus vector. Science 2007; 316 :1718 17 23. Ng LC, Tan LK, Tan CH, Tan SS, et al. Entomologic and virologic investigation of c hikungunya, Singapore. Emerging I nfectious D iseases 2009; 15 :1243 12 49. Niebylski ML, Crai g GB. Dispersal and s urvival of Aedes Albopictus at a s crap t ire y ard in Missouri. J Am Mosq Control Assoc 1994; 10:339 343. Njenga MK, Nderitu L, Ledermann JP, Ndirangu A, et al .Tracking epidemic c hikungunya virus into the Indian Ocean from East Africa. J ournal of General Virology 2008; 89:2754 2760. O'Meara GF, Evans LF, Jr., Gettman AD, Cuda JP. Spread of Aedes albopictus and decline of Ae. aegypti (Diptera: Culicidae) in Florida. J Med Entomol 1995; 32:554 5 62. al. Invasion of cemeteries in Florida by Aedes albopictus J Am Mosq Control Assoc 1992; 8 : 1 10. Culex (Culex) (Diptera:Culicidae) in two types of traps. J Med Entomol 1989; 26:528 534. Pages F, Peyrefitte CN, Mve MT, Jarjaval F, et al. Aedes albopictus mosquito: The main vector of the 2007 c hikungunya outbreak in Gabon. PLoS One 2009;4:e4691. Paquet, C, Quatresous, I, Solet, JL, Sissoko, D, et al. Chikungunya outbreak i n Reunion: E pidemiology and surveillance, 2005 to early January 2006. Euro Surveill 2006; 11:E0602023. Park T. Experimental studies of interspecies competition .II. Temperature, humidity, and competition in two species of Tribolium Physiol Zool 1954. 27:1 77 238. Parola, P, de Lamballerie, X, Jourdan, J, Rovery, C, et al. Novel chikungunya virus variant in travelers returning from Indian Ocean islands. Emerg Infect Dis 2006; 12:1493 149 9. Pastorino B, Muyembe Tamfum JJ, Bessaud M, Tock F, et al Epidemic re surgence of c hikungunya virus in democratic Republic of the Congo: Identification of a new central African strain. J Med Virol 2004; 74:277 2 82. Patz, JA, Epstein, PR, Burke, TA, Balbus, JM. Global climate change and emerging infectious diseases. J Am Mosq Control Assoc 1996; 275:217 2 23.

PAGE 130

130 Paulson, SL, Hawley, WA. Effect of body size on the vector competence of field and laboratory populations of Aedes triseriatus for La Crosse virus. J Am Mosq Control Assoc 1991; 7: 170 175. Paupy C, Delatte H, Bagny L, Cor bel V, et al Aedes albopictus an arbovirus vector: From the darkness to the light. Microbes Infect 2009; 1:1177 1185. Paupy C, Girod R, Salvan M, Rodhain F, et al Population structure of Aedes albopictus from La Reunion Island (Indian Ocean) with respec t to susceptibility to a dengue virus. Heredity 2001; 87:273 283. Pesko K, Westbrook CJ, Mores CN, Lounibos LP, et al Effects of infectious virus dose and bloodmeal delivery method on susceptibility of Aedes aegypti and Aedes albopictus to chikungunya vir us. J Med Entomol 2009; 46:395 36 9. Pletnev SV, Zhang W, Mukhopadhyay S, Fisher BR et al. Locations of carbohydrate sites on alphavirus glycoproteins show that E 1 forms an icosahedral scaffold. Cell 2001; 105: 127 136. Ponlawat A, Harrington LC. Blood fee ding patterns of Aedes aegypti and Aedes albopictus in Thailand. J Med Entomol 2005; 42 : 844 849. Porretta D, Gargani M, Bellini R, Calvitti M, et al. Isolation of microsatellite markers in the tiger mosquito Aedes albopictus (Skuse). Molecular Ecology Note s 2006; 6:880 881. Powers AM, Brault AC, Tesh RB, Weaver SC. Re emergence of c hikungunya and O'nyong nyong viruses: Evidence for distinct geographical lineages and distant evolutionary relationships. J Gen Virol 2000; 81:471 47 9. Powers AM, Logue CH. Chang ing patterns of chikungunya virus: Re emergence of a zoonotic arbovirus. J Gen Virol 2007; 88:2363 23 77. Purse, BV, Mellor, PS, Rogers, DJ, Samuel, et al. Climate change and the recent emergence of bluetongue in Europe. Nature reviews 2005; 3:171 1 81. Rao TR. Immunological surveys of arbovirus infections in South East Asia, with special reference to dengue, chikungunya, and Kyasanur Forest disease. Bull World Health Organ 1971; 44:585 5 91. Ravi V. Re emergence of chikungunya virus in India. Indian J Med Mic robiol 2006; 24:83 8 4. Reeves, WC, Hardy, JL, Reisen, WK, Milby, MM. Potential effect of global warming on mosquito borne arboviruses. J Med Entomol 1994; 31:323 3 32.

PAGE 131

131 Reisen WK, Meyer RP, Presser SB, Hardy JL. Effect of temperature on the transmission of w estern equine encephalomyelitis and St. Louis encephalitis viruses by Culex tarsalis (Diptera: Culicidae). J Med Entomol 1993; 30:151 1 60. Reisen WK, Fang Y, Martinez VM. Effects of temperature on the transmission of W est N ile virus by Culex tarsalis (Dipt era: Culicidae). J Med Entomol 2006; 43:309 3 17. Reiskind, MH, Pesko, K, Westbrook, CJ, Mores, CN. Susceptibility of Florida mosquitoes to infection with chikungunya virus. Am J Trop Med Hyg 2008; 78:422 42 5. Reiskind MH, Lounibos LP. Effects of intraspeci fic larval competition on adult longevity in the mosquitoes Aedes aegypti and Aedes albopictus Med Vet Entomol 2009; 23:62 68. Reiter P, Amador MA, Anderson RA, Clark GG. Short report: dispersal of Aedes aegypti in an urban area after blood feeding as dem onstrated by rubidium marked eggs. Am J Trop Med Hyg 1995; 52 :177 179. Rey JR, Nishimura N, Wagner B, Braks MAH, et al. Habitat segregation of mosquito arbovirus vectors in south Florida. J Med Entomol 2006; 43:1134 1141. Rezza, G, Nicoletti, L, Angelini, R, Romi, R, et al. Infection with chikungunya virus in Italy: An outbreak in a temperate region. Lancet 2007; 370:1840 184 6. Richards SL, Apperson CS, Ghosh SK, Cheshire HM, et al. Spatial analysis of Aedes albopictus (Diptera : Culicidae) oviposition in s uburban neighborhoods of a piedmont community in Nort h Carolina. J Med Entomol 2006; 43:976 989. Richards SL, Mores CN, Lord CC, Tabachnick WJ. Impact of extrinsic incubation temperature and virus exposure on vector competence of Culex pipiens quinquefasci atus Say (Diptera: Culicidae) for West Ni le virus. Vector Borne Zoonot Dis 2007; 7:629 6 36. Robinson MC. An epidemic of virus disease in Southern Province, Tanganyika Territory, in 1952 53. I. Clinical features. Trans R Soc Trop Med Hyg 1955; 49:28 32. Ros s RW. The Newala epidemic. III. The virus: Isolation, pathogenic properties and relationship to the epidemic. J Hyg (Lond) 1956; 54:177 1 91. Rozeboom LE, Rosen L, Ikeda J. Observations on o viposition by Aedes (S) albopictus Skuse and A (S) p olynesiensis M a rks in n ature. J Med Entomol 1973; 10:397 399. Rueda, LM, Patel, KJ, Axtell, RC, Stinner, RE. Temperature dependent development and survival rates of Culex quinquefasciatus and Aedes aegypti (Diptera: Culicidae). J Med Entomol 1990; 27:892 89 8.

PAGE 132

132 Russell RC. larval competition between the introduced vector of dengue fever in Australia, Aedes aegypti (L), and a native container breeding mosquito, Aedes notoscriptus (Skuse) (Diptera, Culicidae). Aust J of Zool 1986; 34:527 534. Rutledge, LC Ward, RA, Gould, DJ. Studies on the feeding response of mosquitoes to nutritive solutions in a new membrane feeder. Mosq News 1964; 24:407 4 19. SAS. Samuel PP, Krishnamoorthi R, H amzakoya KK, Aggarwal CS. Entomo epidemiological investigations on chikung unya outbreak in the Lakshadweep islands, Indian Ocean. The Indian Journal of Medical R esearch 2009; 129 :442 445. Sang RC, Ahmed O, Faye O, Kelly CL, et al. Entomologic investigations of a chikungunya virus epidemic in the Union of the Comoros, 20 05. Am J Trop Med Hyg 2008; 78 :77 82. Santhosh SR, Dash PK, Parida MM, Khan M, et al. Comparative full genome analysis revealed E1: A226V shift in 2007 Indian c hikungunya virus isolates. Virus research 2008; 135 :36 41. SAS 9.1 for Windows. SAS Institute, Inc., Car y, NC, 2003. Savage HM, Ezike VI, Nwankwo AC, Spiegel R, et al. First record of breeding populations of Aedes albopictus in continental Africa: Implications for arboviral transmission. J Am Mosq Control Assoc 1992; 8:101 10 3. Savage HM, Niebylski ML, Smith GC, Mitchell CJ, et al Host feeding patterns of Aedes albopictus (Diptera: Culicidae) at a temperate North American site. J Med Entomol 1993; 30:27 34. Schabenber ger O. Introducing the GLIMMIX procedure for generalized linear m odels. SUGI 30. Cary, NC: S AS Institute. 2007. Schliessmann.DJ, Calheiro.LB. Review of status of yellow fever and Aedes aegypti eradication programs in Ame ricas. Mosquito News 1974; 34 :1 9. Schuffenecker I, Iteman I, Michault A, Murri S, et al Genome microevolution of chikungunya v iruses causing the Indian Ocean outbreak. PLoS Med 2006; 3:e263. Scott TW, Amerasinghe PH, Morrison AC, Lorenz LH, et al Longitudinal studies of Aedes aegypti (Diptera : Culicidae) in Thailand and Puerto Rico: Blood feeding freq uency. J Med Entomol 2000; 37 :89 101. Scott, TW, Chow, E, Strickman, D, Kittayapong, P, et al. Blood feeding patterns of Aedes aegypti (Diptera: Culicidae) collected in a rural Thai village. J Med Entomol 1993 a ; 30:922 92 7.

PAGE 133

133 Scott TW, Clark GG, Lorenz LH, Amerasinghe PH, et al Detection of multiple blood feeding in Aedes aegypti (Diptera, Culicidae) during a single gonotrophic cycle using a histologic technique. J Med Entomol 1993 b ; 30:94 99. Seneviratne, SL, Gurugama, P, Perera, J. Chikungunya viral infections: an emerging pr oblem. J Travel Med 2007; 14: 320 32 5. Sergon K, Yahaya AA, Brown J, Bedja SA, et al. Seroprevalence of c hikungunya virus infection on Grande Comore Island, union of the Comoros, 2005. Am J Trop Med Hyg 2007; 76:1189 11 93. Sergon K, Njuguna C, Kalani R, Ofu la V, et al. Seroprevalence of c hikungunya virus (CHIKV) infection on Lamu Island, Kenya, October 2004. Am J Trop Med Hyg 2008; 78 :333 337. Settle WH, Wilson LT. Invasion by the variegated leafhopper and biotic interactions Parasitism, competition, and a pparent competition. Ecology 1990; 71:1461 14 70. Smith DR, Adams AP, Kenney JL, Wang, E. Venezuelan equine encephalitis virus in the mosquito vector Aedes taeniorhynchus : Infection initiated by a small number of susceptible epithelial cells and a populatio n bottleneck. Virology 2008; 372:176 186. Smith GC, Eliason DA, Moore CG, Ihenacho EN. Use of elevated temperatures to kill Aedes albopictus and Ae. aegypti J Am Mosq Control Assoc 1988; 4:557 55 8. Sota T, Mogi, M. Interspecific variation in desiccation s urvival time of Aedes (Stegomyia) mosquito eggs is correlated with habitat and egg size. Oecologica 1992; 90: 353 358. Sota T, Mogi M, Hayamizu E. Seasonal d istribution and h abitat s election by Aedes albopictus and Ae riversi (Diptera, Culicidae) in Norther n Kyushu, Japan. J Med Entomolo 1992; 29:296 304. Sourisseau M, Schilte C, Casartelli N, Trouillet C, et al. Characterization of reemerging chikungunya virus. Plos Pathogens 2007; 3:804 817. Sprenger D, Wuithiranyagool T. The d iscovery and d istribution of Aedes albopictus in Harris County, Texas. J Am Mosq Control Assoc 1986; 2: 217 219. Strauss JH, Strauss EG. The alphaviruses: gene expression, replication, and evolution. Microbiol Rev 1994; 58:491 562. Strickman D, Kittayapong P Dengu e and its vectors in Thailand: c alculated transmission risk from total pupal counts of Aedes aegypti and association of wing length measurements with aspects of the larval habitat. Am J Trop Med Hyg 2003; 68 :209 217.

PAGE 134

134 Sumanochitrapon, W, Strickman, D, Sithiprasasna, R, Kittaya pong, P, et al. Effect of size and geographic origin of Aedes aegypti on oral infection with dengue 2 virus. Am J Trop Med Hyg 1998; 58:283 28 6. Tabachnick WJ. Evolutionary genetics and arthropod borne disease: The yellow fever mosquito. American Entomolog ist 1991; 37:14 24. Tabachnick WJ. Challenges in predicting climate and environmental effects on vector borne disease episystems in a changing world. J Exp B iol 2010;213:946 54. Tabachnick WJ, Powell JR. A world wide survey of genetic variation in the yell ow fever mosquito, Aedes aegypti Genet Res 1979; 34:215 2 29. Tabachnick WJ, Wallis GP, Aitken THG, Miller BR, et al. Oral infection of Aedes aegypti with yellow f ever virus g eographic v ariation and g enetic c onsiderations. American J Trop Med Hyg 1985; 34: 1219 1224. Takahashi M. The effects of environmental and physiological conditions of Culex tritaeniorhynchus on the pattern of transmission of Japanese encephalitis virus. J Med Entomol 1976; 13:275 2 84. Teng HJ, Apperson CS. Development and survival of im mature Aedes albopictus and Aedes triseriatus (Diptera: Culicidae) in the laboratory: Effects of density, food, and competition on response to temper ature. J Med Entomol 2000; 37 :40 52. Thavara U, Tawatsin A, Pengsakul T, Bhakdeenuan P, et al Outbreak of chikungunya fever in Thailand and virus detection in field population of vector mosquitoes, Aedes aegypti (L.) and Aedes albopictus Skuse (Diptera: Culicidae). The Southeast Asian J ournal of T ropical M edicine and P ublic H ealth 2009; 40 :951 962. Trpis M, Ha usermann W. Demonstration of d ifferential d omesticity of Aedes aegypti (L) (Diptera, Culicidae) in Africa by m ark r elease r ecapture. Bulletin of Entomol Res 1975; 65:199 208. Tsetsarkin, KA, Vanlandingham, DL, McGee, CE, Higgs, S. A single mutation in chik ungunya virus affects vector specificity and epidemic potential. PLoS Pathog 2007; 3:e201. Turell, MJ, Gargan, TP, Bailey, CL. Replication and dissemination of Rift Valley fever virus in Culex pipiens Am J Trop Med Hyg 1984; 33:176 1 81. Turell, MJ, Beaman JR, Tammariello, RF. Susceptibility of selected strains of Aedes aegypti and Aedes albopictus (Diptera, Culicidae) to chikungunya virus. J Med Entomol 1992; 29:49 53.

PAGE 135

135 Turell, MJ. Effect of environmental temperature on the vector competence of Aedes taeni orhynchus for Rift Valley fever and Venezuelan equine encephalitis viruses. Am J Trop Med Hyg 1993; 49:672 67 6. Udaka, M. Some ecological notes on Aedes albopictus in Shikoku, Japan. Kontyu 1959; 27: 202 208. van Lieshout, M, Kovats, RS, Livermore, MTJ, Ma rtens, P. Climate change and malaria: analysis of the SRES climate and socio economic scenarios. Global Environ Chang 2004; 14:87 99. Vazeille M, Jeannin C, Martin E, Schaffnerbl F, et al. Chikungunya: A risk for Mediterranean countries? Acta Tropica 2008; 105:200 202. Vazeille M Mousson L, Failloux AB. Failure to demonstrate experimental vertical transmission of the epidemic strain of Chikungunya virus in Aedes albopictus from La Reunion Island, Indian Ocean. Memorias do Instituto Oswaldo Cruz 2009; 104:6 32 6 35. Watson R. Europe witnesses first local transmission of chikungunya fever in Italy. BMJ 2007; 335:532 53 3. Wada, Y Effect of larval density on the development of Aedes aegypti (L.) and the size of adults. Quaest Entomol 1965; 1:223 249. W eaver SC. E lectron microscopic analysis of infection patterns for venezuelan equine encephalomyelitis virus in the vector mosquito, C ulex ( M elanoconion ) taeniopus Am J Trop Med Hyg 1986; 35:624 631. Weaver SC. D etection of eastern equine encepha lomyelitis virus dep osition in Culiseta melanura following ingestion of radiolabeled virus in blood meals Am J Trop Med Hyg 1991; 44:250 259. Westbrook CJ, Reiskind MH, Pesk o KN, Greene KE, et al. Larval environmental temperature and the s usceptibility of Aedes albopictus S kuse (Diptera: Culicidae) to chikungunya v irus. Vector B orne Zoonot Dis 2009; Online ahead of print http://www.liebertonline.com/doi/pdfplus/10.1089/vbz.2009.0035. World Health Organization. Chapter 5. Vect or surveillance and control in d engue haemorrhagic fever: diagnosis, treatment, prevention and control. 1997; 2nd edition. Geneva, Switzerland: Wu, HH., Chang N.T. Influence of temperature, water quality and pH value on ingestion and development of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) l arv ae. Chin J Entomol 1993; 13: 33 44.

PAGE 136

136 Xu G, Wilson W Mecham J Murphy K et al. VP7: A n attachment protein of bluetongue virus for cellular receptors in Culicoides variipennis J Gen Virol 1997; 78: 1617 1623. Xu GZ, Dong HJ, Shi NF, Liu SA, et al. An ou tbreak of dengue virus serotype 1 infection in Cixi, Ningbo, People's Republic of China, 2004, associated with a traveler from Thailand and high density of Aedes albopictus Am J Trop Med Hyg 2007; 76:1182 1188 Yergolkar PN, Tandale BV, Arankalle VA, Sath e PS, et al. Chikungunya outbreaks caused by African genotype, India. Emerg Infect Dis 2006; 12:1580 158 3. Yoshida S, Shimada Y, Kondoh D, Kouzuma Y,et al. Hemolytic C type lectin CEL III from sea cucumber expressed in transgenic mosquitoes impairs malaria parasite development. PLoS pathogens 2007 ; 312 :e192. Zhou XH, Weng ES, Luo YQ. Modeling patterns of nonlinearity in ecosystem responses to temperature, CO2, and precipitation changes. Eco logical Applications 2008; 18:453 4 66.

PAGE 137

BIOGRAPHICAL SKETCH Catheri ne Jane Westbrook was born in Washington, D.C in 1972. She graduated from the University of California, Berkeley in 1996 with a Bachelor of Arts degree in integrative biology. In 2003 she received her Master of Science degree from Cornell University in e ntomology. In 2007 she began her doctoral work at the University of Florida under the guidance of Dr. L. Philip Lounibos at the Florida Medical Entomology laboratory in Vero Beach FL