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SURVIVAL OF CULEX PIPIENS MOSQUITOES ALONG A LAND-USE GRADIENT

By

CHRISTY JOHNSON

A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2010

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© 2010 Christy Johnson

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To my family and friends

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ACKNOWLEDGMENTS

I thank my supervisory committee chair, Dr. Phil Lounibos; and members Dr.

Marm Kilpatrick and Dr. George O’Meara for their valuable help, guidance and having

unbelievable patience with me throughout this project. Their knowledge and expertise in

statistical analysis and mosquito ecology was a tremendous help to me. I would like to

thank Christopher Uejio for helping me learn R programming. Special thanks are also

due to the neighbors of Takoma Park, MD, the park rangers of Ft. Dupont Park, the

Smithsonian Environmental Research Center, and the city of Baltimore, MD for allowing

me to trap mosquitoes on their properties. I also thank Mary Ashley Laine and Alex

Martin for their assistance with my research. I could not have done the research without

them. I thank all of the interns on the West Nile Virus crew for their support and

friendship. They made the field seasons much less stressful.

I would like to thank my family and friends for their encouragement and

unwavering faith in my ability. I would also like to thank Dan Jones for always being

there for me and for putting up with so much throughout this Master’s project. Finally, I

would like to thank the Winthrop University Biology Department for helping me gain the

skills I needed to follow my dreams and for your continued support even after I

graduated.

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TABLE OF CONTENTS page

ACKNOWLEDGMENTS .................................................................................................. 4

LIST OF TABLES ............................................................................................................ 7

LIST OF FIGURES .......................................................................................................... 8

LIST OF ABBREVIATIONS ............................................................................................. 9

ABSTRACT ................................................................................................................... 10

1 INTRODUCTION .................................................................................................... 12

2 LITERATURE REVIEW .......................................................................................... 15

Culex Mosquito Ecology ......................................................................................... 15 West Nile Virus ....................................................................................................... 17 Urbanization Effects on Vectors and Disease ......................................................... 19 Mark Release Recapture for Survival and Dispersal ............................................. 21

3 SURVIVAL AND DISPERSAL RATES OF CULEX PIPIENS ALONG A LAND-USE GRADIENT ..................................................................................................... 23

Introduction ............................................................................................................. 23 Methods and Materials............................................................................................ 25

Study Sites ....................................................................................................... 25 Baltimore, MD ............................................................................................. 26

Takoma Park, MD ...................................................................................... 26 Fort Dupont, Washington, DC .................................................................... 26 SERC, Edgewater, MD .............................................................................. 26

Collecting Egg Rafts ......................................................................................... 27 Rearing Mosquitoes ......................................................................................... 27 Spermathecal Dissections ................................................................................ 27 Capture Mark Release ..................................................................................... 28 Marking Mosquitoes ......................................................................................... 28 Releasing Mosquitoes ...................................................................................... 28 Recapturing Mosquitoes ................................................................................... 29 Identifying Mosquitoes ...................................................................................... 29 Statistical Analysis ............................................................................................ 29

Results .................................................................................................................... 32 Discussion .............................................................................................................. 34

4 CONCLUSIONS ..................................................................................................... 50

LIST OF REFERENCES ............................................................................................... 52

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BIOGRAPHICAL SKETCH ............................................................................................ 60

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LIST OF TABLES

Table page 3-1 Survival rates (survival ± SE) of Culex pipiens females calculated by

nonlinear least squares at three sites near Washington, DC .............................. 48

3-2 Comparison of survival rates of Culex pipiens females at three sites near Washington, DC ................................................................................................. 49

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LIST OF FIGURES

Figure page 3-1 Study area of Baltimore ...................................................................................... 39

3-2 Study area of Takoma Park 2008 and 2009 ....................................................... 40

3-3 Study area of Fort Dupont .................................................................................. 41

3-4 Study area of SERC ........................................................................................... 42

3-5 Number of recaptured Culex pipiens each day ................................................... 43

3-6 Number of recaptures in light and gravid traps at Takoma Park 2009 ................ 45

3-7 Average dispersal distance each day, corrected for trapping effort .................... 46

3-8 Trap locations and number of recaptured mosquitoes collected in each sixty degree sector of Takoma Park 2009. ................................................................. 47

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LIST OF ABBREVIATIONS

MRR Mark-Release-Recapture

WNV West Nile Virus

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ABSTRACT OF THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE

REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

SURVIVAL OF CULEX PIPIENS MOSQUITOES ALONG A LAND-USE GRADIENT

By

Christy E. Johnson

May 2010

Chair: L. Philip Lounibos Major: Entomology and Nematology

Mosquito survival rates are important in pathogen transmission because they

partly determine the intensity of amplification. For transmission to occur, a vector has to

live long enough to become infected with the pathogen and transmit it to another host. A

mark-release-recapture study was conducted using adult female Culex pipiens

mosquitoes at urban, residential, park, and forested sites to determine survival rates

and dispersal distances. Mosquito survival at one residential site was measured in 2008

and 2009 and survival at 3 additional sites was measured in 2009. At all but one site,

mosquitoes were reared to adults at the same site as their release, and held 3 days to

allow mating. They were then dusted with fluorescent dust and released at dusk.

Twenty CDC light traps and 4-10 gravid traps were used to recapture dusted

mosquitoes over the subsequent 15 nights. Survival was highest at the residential site,

Takoma Park, in 2009 with a daily survival rate (±SE) of 86.0±3.0%, implying an

average longevity of 7.17 days. Daily survival was lowest at Fort Dupont Park, with a

daily survival rate (±SE) of 52.0±13.0%, and this was significantly lower than survival at

Takoma Park in both 2008, and 2009, and in 2009 at the urban site, Baltimore, where

released mosquitoes were adults of unknown age caught the previous night. No

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mosquitoes were recaptured at the forested site. The trapping grid was too small to

measure long distance dispersal rates for Cx. pipiens but the recapture data suggested

that dispersal after the initial release was very low. Future work should include using

larger land areas and more traps for each site in order to better determine dispersal for

each site, which would allow for better comparisons of survival from each site.

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CHAPTER 1 INTRODUCTION

Insects belonging to the family Culicidae have existed for more than 170 million

years and probably evolved during the Jurassic period from a sister group of midges

(Chaoboridae) (Borkent and Grimaldi 2004). Mosquitoes have since evolved into 3,500

species that are found throughout the world and feed from a variety of hosts. Although

there are a great number of mosquito species, only a few species feed on humans.

Several of these species were probably associated with humans since before the Homo

erectus period of human evolution (Desowitz 1991). Mosquitoes have played an

important role in human history. Numerous wars have been fought with solders not only

fighting each other, but the mosquito-borne diseases being transmitted. Napoleon may

have used the presence of malaria in the Dutch Island of Walcheren to defeat the British

force in the early 19th Century (Spielman and D’Antonio 2001). During World War I and

II, mosquito-borne diseases were killing more troops than the opposing army (Spielman

and D’Antonio 2001). Although many of the mosquito-borne diseases have been

eradicated from developing countries, they are beginning to emerge again (Spielman

and D’Antonio 2001).

Female mosquitoes of most species require a blood meal for the production of

eggs, although some species are able to produce one batch of eggs without having a

blood meal. Depending on the species, they feed on mammal, avian, reptile, or

amphibian animals, and in some cases, on all four vertebrate classes. Some mosquito

species that feed on humans and other mammals have evolved to live and breed in

man-made environments (Forattini et al. 1995). The bite of a female mosquito can

cause mild irritation at the site of the bite, although some people have stronger

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reactions than others. These reactions are due to an immune response to the

mosquito’s saliva which she secretes while probing and biting (Ribeiro et al. 1984).

Mosquitoes have been transmitting pathogens for at least 20,000-30,000 years.

Many humans from temperate environments who began to explore foreign lands did not

have the immunity needed to defend themselves against the foreign diseases that they

faced (Spielman and D’Antonio 2001, Desowitz, 1991). These diseases and parasites

include malaria, yellow fever, dengue, and many others. Today we still face the problem

of pathogens and vectors unintentionally being imported into different countries

(Lounibos 2002). Since air travel is so common, it is easy for a mosquito or other vector

to travel thousands of miles in only several hours, infecting numerous people along the

way (Takahashi 1984).

The most recent mosquito-borne disease to emerge in North America is West

Nile Virus (Lanciotti et al. 1999). The virus was discovered in New York in 1999 and has

since rapidly spread across the country (except Hawaii and Alaska) and throughout

Central and South America (Kilpatrick et al. 2007). A novel genotype of WNV that was

first detected in 2001 has since spread across North America (Davis et al. 2005), and is

transmitted more efficiently in Culex mosquitoes than the original virus that was

introduced (Kilpatrick et al. 2008, Moudy et al. 2007). Before 1999, WNV had only been

found in the Old World (Europe, Asia, and Africa) (Spielman and D’Antonio, 2001) and

little research had been done on the potential of it reaching the New World. However,

for the past decade extensive research has been made on the virus and how it is

affecting people and wildlife in North America (Kilpatrick et al. 2007, Marra et al. 2004).

One important gap in our knowledge of WNV transmission ecology is the survival

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of a key vector, Culex pipiens. Variation in mosquito survival can play a critical role in

transmission because a mosquito must survive long enough to feed at least twice (once

to become infected, and once to transmit), and it must survive the full length of the

extrinsic incubation period in order to transmit the virus (Garrett-Jones 1964). Cx.

pipiens is the dominant enzootic (bird-to-bird) vector for WNV in urban areas throughout

the northern half of the USA, and is also a key bridge (bird-to-human) vector (Kilpatrick

et al. 2005, Hamer et al. 2008). Measuring the survival of this mosquito across a range

of habitats will help determine how many times a mosquito could transmit the virus over

its lifetime, and thus how effective control measures must be to reduce or interrupt

transmission.

My research examines the survival rates of Culex pipiens across a land use

gradient. WNV has, so far, predominantly been found in urban environments. It is

unclear if this is due to the amount of vectors that are found in those environments, or to

the differences in survival rates of Culex pipiens found in urban versus rural

environments. While survival of Cx. pipiens has been studied in lab environments (Oda

et al. 1999, Oda et al.2002), survival rates in the field have not been determined.

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CHAPTER 2 LITERATURE REVIEW

Culex Mosquito Ecology

Mosquitoes are members of the Class Insecta, the Order Diptera, and Family

Culicidae. There are over 40 genera of mosquitoes with many species in most genera.

Over 1000 species are assigned to the Culex genus alone. Members of the genus

Culex are found all over the world (except Antarctica). Culex spp. mosquitoes transmit

many pathogens, such as avian malaria, St. Louis Encephalitis Virus, WNV and others

(Ross 1897, Lumsden 1958, Taylor et al. 1953). Diseases caused by these pathogens

are known to affect humans, domestic animals, and wildlife.

Like all holometabolous insects, Culex mosquitoes have 4 main stages in their

life cycle- egg, larva, pupa, and adult. The developmental time (egg to adult) can take

as little as 4 days or as long as one month, depending on the species and temperature

(Mori et al. 1988). Some species of Culex mosquitoes lay their 30-300 eggs in rafts at

night on the surface of highly organic, stagnant water (Horsfall 1955). Larvae usually

eclose from the eggs within 30 hours of being laid (Gerberg et al. 1969).

Mosquito larvae are often called “wrigglers”, based on the way they move in

water. Larvae feed on organic materials and microbes in the water (Beehler and Mulla

1995). Culex larvae breathe by swimming to the surface of the water and obtaining

oxygen by using a breathing tube called a siphon. As larvae grow they molt to a new

instar. Culex larvae go through 4 instars before metamorphosing into pupae. Depending

on temperature and the amount of nutrients in its environment, development from egg

hatch to the pupal stage takes approximately 10 days at 30◦C (Olejnicek and Gelbic

2000).

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Mosquito pupae are often called “tumblers”, based on the way they “tumble”

away from the water surface when disturbed. Since they do not eat; pupae spend most

of their time at the water’s surface. They are lighter than water and easily float at the

surface. They breathe by using two breathing tubes called trumpets. Culex pupae

usually metamorphose into adults within 1-2 days (Gerberg et al. 1969). Adults emerge

to the surface of the water and rest until their bodies dry and harden.

After adults emerge from pupae, it generally takes about 2 days before they are

mature enough to mate. During this time, both sexes of mosquito will feed on flower

nectar or other sources of sugar. For Culex pipiens mating usually takes place around

dusk, with the males forming a swarm and mating with females that enter (Reisen et al.

1985). The male’s sperm is stored in the female’s spermatheca after copulation (Jones

and Wheeler 1965). Digestion of the blood meal usually takes several days, during

which time nutrients are absorbed for egg production (Benach 1970). Females of Cx.

pipiens that emerge during the late summer will overwinter in a sheltered area until the

spring when she can lay her eggs (Main et al. 1968).

Culex pipiens females usually take most of their bloodmeals from avian hosts.

However, they feed on mammals as well, and in some areas take 10-15% of the

bloodmeals from humans (Hamer et al. 2008, Kilpatrick et al. 2006, Savage et al. 2007).

Abundance is often higher in tree canopies than near the ground (Drummond et al.

2006), which may be due to their preference of feeding on birds. This may influence

mosquito surveillance because most mosquito traps are placed at a standard height

(usually 1.5m).

Although survival rates have been conducted on other species of Culex, it is

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unknown how long Culex pipiens adult females live in the wild. In laboratory studies

they can live up to 54 days at 21◦C. Culex quinquefasciatus were found to live up to 64

days at 25◦C (Oda et al. 1999, Oda et al. 2002). The female adult longevity of these

studies would enable them to take multiple blood meals and lay thousands of eggs.

However, survival in the field is hypothesized to be much lower.

West Nile Virus

West Nile Virus is a plus-sense single stranded RNA virus that is a member of

the family Flaviviridae and the genus Flavivirus. It is similar in structure to dengue virus.

It is believed by some to have killed Alexander the Great in 332BC (Marr and Calisher,

2003). However, it was first isolated in 1937 from a woman in the West Nile district of

Uganda (Smithburn et al. 1940). The next isolates of West Nile were not collected until

1950, from three apparently healthy children in Egypt (Melnick et al. 1951). Since then,

hundreds of isolates have been obtained from many places including the Middle East,

Europe, Asia, and most recently in North America.

It is estimated that 4 out of 5 people infected with WNV show no symptoms.

However, about 20% infected develop West Nile Fever, symptoms of which include a

fever, headache, body ache, vomiting, and swollen lymph nodes. Approximately 1 in

150 infected will develop West Nile meningitis, whose symptoms include high fever,

neck stiffness, coma, tremors, muscle weakness and paralysis. People over the age of

50 and those with compromised immune systems are at higher risk of becoming

severely ill when infected with WNV (Centers for Disease Control and Prevention [CDC]

2009). Although vaccines for horses and birds have been developed, vaccines for

humans are not yet available (Kramer et al. 2007).

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WNV is normally transmitted by mosquitoes which obtain the virus by feeding on

an infected bird. However, the virus may also be transmitted to humans through blood

transfusions and organ transplants (Iwamoto et al. 2003, Pealer et al. 2003). The virus’

normal life cycle depends on mosquitoes feeding on birds and transmitting the virus to

them. However, many Culex mosquitoes also feed on other animals such as deer,

humans, reptiles, etc (Miller et al. 2005, Jacobson et al. 2005, Apperson et al. 2002,

Apperson et al. 2004, Savage et al. 2007). The virus rarely reaches high enough titres

to be infectious to a biting mosquito in most of these hosts (Kilpatrick et al. 2007);

however some studies have shown that eastern chipmunks and alligators are capable

of infecting mosquitoes (Platt et al. 2007, Klenk et al. 2004), . Although these dead end

hosts are not infectious to biting mosquitoes, they still may become ill from WNV

infection.

In 1999, WNV was found in New York and in five years had spread throughout

the country. From 1999-2009, the virus has caused 36,236 human illnesses in the

United States and Canada, including 12,088 cases of encephalitis and 1,161 deaths

(Centers for Disease Control and Prevention [CDC] 2009, Health Canada 2009). When

WNV emerged in New York, large numbers of American Crow (Corvus brachyrhynchos)

deaths were observed and were used to follow the spread of the disease (Hayes et al.

2005). A new genotype was discovered in 2001 and is thought to have evolved from

the original 1999 strain (Davis et al. 2005). This new genotype has been shown to be

transmitted more efficiently by Culex mosquitoes, by decreasing the extrinsic incubation

period (the time needed to enter a mosquito and become transmitted to another host)

(Moudy et al. 2007, Kilpatrick et al. 2008, Ebel et al. 2004).

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The North American epidemic over the last ten years is more intense than any

that have occurred in Europe in the last 70 years. There have been many explanations

for this higher intensity. It has been suggested that North American birds, which had no

previous exposure to WNV, lacked acquired and evolved immunity (Spielman et al.

2004). The lack of immunity needed to fight off this new pathogen may have increased

the intensity of the epidemics. It has also been shown that Culex pipiens in North

America are hybrids between two forms of Culex pipiens, one that is “bird biting”

(pipiens) and one that is “human biting” (molestus) (Fonseca et al. 2004). The

higher the ancestry from form molestus, the great the probability that a mosquito will

feed on humans (Kilpatrick et al. 2007). Therefore, the newly evolved virus could not

only infect large numbers of bird species, but because the virus is transmitted by hybrid

Culex pipiens mosquitoes, it would also frequently be transmitted to humans and other

mammals as well. Further, in some locations, a seasonal feeding shift from birds to

mammals (including humans) has been observed which may increase the intensity of

transmission to humans in these areas (Kilpatrick et al. 2006).

Although Culex mosquitoes can transmit WNV to their offspring, the efficiency of

transmission has been shown to be 1.8 out of 1000 for Culex pipiens and 3.0 out of 100

for Cx. quinquefasciatus (Dohm et al. 2002, Goddard et al. 2003). As a result, most are

not infected with West Nile virus until they feed on an infected host. However, WNV has

been detected in overwintering Culex females. Determining mosquito survivorship is

important for understanding the potential of a single mosquito to infect multiple hosts in

her lifetime.

Urbanization Effects on Vectors and Disease

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In recent years, the urbanization of rural areas has increased, replacing forest

with buildings and other structures. Changes in habitat influence mosquito abundance

by changing factors that influence survivorship and reproduction, including temperature,

humidity, and the availability and quality of larval habitat (Yanoviak et al. 2006). In the

USA, certain species are more abundant in urban areas (Culex pipiens and Aedes

albopictus) while others (Culex restuans) are more abundant in rural areas.(Ebel et al.

2005).

Due to the replacement of natural areas with impermeable surfaces, urbanization

not only affects the natural habitat of wildlife, but creates a “heat island” that raises the

temperature of the area 2-3◦C (Thorsteinson 1988). This temperature increase causes a

reduction in the length of time required to digest a blood meal and lay eggs. It also

increases activity and flight speed (Thorsteinson 1988), which may increase the

efficiency of host seeking.

Temperature also affects vector competence, the probability that a mosquito will

transmit a pathogen after feeding on an infected host, and the length of the extrinsic

incubation period. While vector competence varies significantly between and within

species, it increases with extrinsic incubation temperature of up to 32◦C (Kilpatrick et al.

2008, Dohm et al. 2002, Reisen et al. 2006).

Urbanization also affects the avian host population. Differences in the

composition and abundance of bird communities along gradients of urbanization are

well known and may be caused by differences in food availability and predation by

animals associated with humans (cats, rats, etc.) (Marzluff et al. 2001). Bird

communities in intact forests are composed of a diverse range of families and species

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whereas urban communities are mostly composed of a few highly abundant, often

introduced species. Sites between these extremes usually have a mix of both species

(Gavareski 1976).

The changes in avian communities resulting from urbanization thus alter the

reservoir competence of the host community for WNV. Effective reservoir competence

of an avian community is determined by the abundance and reservoir competence (the

infectiousness when infected) of each bird species and the feeding preference of

mosquitoes. Since avian hosts differ significantly in reservoir competence for WNV,

mosquito feeding preference for or against particular avian species will alter

transmission of the virus.

There is growing evidence that urbanization may increase transmission of WNV

and other vector-borne pathogens (Bradley et al. 2008, Savage et al. 2006, Andreadis

et al. 2004). However, it is unclear whether urbanization has an impact on the overall

lifespan of mosquitoes. If mosquitoes live longer in urban environments, they would

have more chances to find and bite hosts, and therefore have a much greater chance at

spreading the virus. They may also be able to produce more offspring that would further

increase pathogen transmission.

Mark-Release-Recapture for Survival and Dispersal

Mark-release-recapture (MRR) studies are the most important method for

studying survival (White and Burnham 1999), and have been conducted on mosquitoes

for almost 100 years (Zetek 1915). This technique is a useful tool in the study of

population ecology and dispersal (Service 1993). This method has been used to study

survival, population size, and dispersal of many different types of animals including fish,

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mammals, reptiles, insects and other invertebrates. The technique includes capturing or

rearing many individuals of a species and “marking” them with a feature that is unique to

that animal or group of animals (e.g., ear tags, paint dots, florescent dust, etc). After the

marking takes place, the animals are then released. Traps are then set up to recapture

the marked specimens. There are four principle assumptions in most MRR methods: 1)

behavior and survival are not affected by capture, mark, or release; 2) survival rate does

not change with age; 3) release does not affect the probability of recapture; and 4)

marked insects do not disperse beyond the sampling area (Southwood 1978).

MRR experiments have been conducted on mosquitoes in order to determine

survival rates, dispersal, and gonotrophic cycle length. Measuring survival rates

requires periodic trapping following marking of animals for a certain period of time.

Accurately measuring dispersal requires an array of traps that encompass an area

larger than that in which mosquitoes move in the interval between release and

recapture.

These three parameters (survival, dispersal rate, and gonotrophic cycle length)

are important to measure because they can be used to predict approximately how long

a mosquito may live to spread a virus, how far she can fly to spread it, and what is the

length of the interval between a blood meal looking for another host.

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CHAPTER 3 SURVIVAL RATES AND DISPERSAL OF CULEX PIPIENS ACROSS A LAND-USE

GRADIENT

Introduction

Longevity of adult female mosquitoes is an important variable for transmitting

pathogens. After ingesting an infectious blood meal, a mosquito must survive the

extrinsic incubation period (EIP) of the pathogen before it can transmit it to a susceptible

host (Davis 1932). Mosquito survival rate is one of several important parameters in

models of vector-borne disease (Tempelis 1989). However, measurements in a

laboratory setting are frequently very different from those obtained in the field. Culex

quinquefasciatus, for example, have been shown to have a life expectancy of 74 days in

the lab at 25-28◦C (Suleman and Reisen 1979), and 6.25 days in residential areas of

California (Reisen et al.1991) Determining the survival rate of different vectors of

diseases can allow a better understanding of disease transmission and help predict the

occurrence of outbreaks.

WNV is a member of the Japanese encephalitis antigenic complex of the genus

Flavivirus, family Flaviviridae. The epidemic in New York City in 1999 quickly spread

across the country and has reached most of Canada, and throughout Central and South

America and the Caribbean (Kilpatrick et al. 2007). The virus is thought to have been

spread by a combination of bird migration and dispersal (Rappole et al. 2006). Since In

2001 a new strain evolved that subsequently displaced the introduced strain, at least

partly through increased replication in mosquitoes (Davis et al. 2005, Kilpatrick et al.

2008, Ebel et al. 2004, Moudy et al. 2007).

Adults of Culex pipiens (L.) are found in urban areas, readily enter houses, and

usually feed during the night (Vinogradova 2000). Host preference has been shown to

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be primarily for birds, but feeding on humans and other mammals increases in the late

summer in some areas (Kilpatrick et al. 2005, Kilpatrick et al. 2006, Tempelis et al.

1967). Culex pipiens (L.) was also determined to be the predominant vector in urban

epidemics of WNV in Romania, southern Russia, and New York City in the 1990s

(Lampman et al. 2006). Culex pipiens have adapted well to urban environments, using

storm drains and other sources of organically rich, stagnant water for eggs and larvae.

Urbanization of areas may increase a mosquito’s ability to transmit the virus,

because of the increased temperatures in urban areas. Increased temperatures of up to

32◦C have also been shown to increase vector competence for pathogens like WNV

(Kilpatrick et al. 2008, Dohm et al. 2002, Reisen et al. 2006). Greater numbers of Culex

pipiens mosquitoes have been collected in the tree canopies than other places

(Anderson et al. 2004), making birds roosting in the area a prime target to be fed on.

Changes in land use are not only associated with the spread of West Nile, but

have also affected the spread of other diseases. Malaria, once considered a rural

disease, has now diversified into various ecotypes due to enormous new vector habitats

created by irrigation systems without proper drainage systems (Sharma, 1996). Also,

houses and cattle sheds made out of organically rich mud are suitable breeding areas

for the sandflies Phlebotomus argentipes, which spreads Kala azar causing major

outbreaks in India, Sudan, and Brazil (Sharma 1996). Although effects of urbanization

on the occurrence of vectors have been studied, the affects of urbanization on survival

rates, especially those of Culex pipiens, have not been studied.

Survival studies have been done in many areas of the world in order to better

understand how vector survival affects disease transmission. In Africa, one study found

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that Anopheles gambiae were estimated to have a survival rate of 80-88% per day and

a flight range of 350-650 meters per day (Costantini et al. 1996). In Australia, Aedes

notosciptus were found to have a survival rate of 77-70% per day and an average flight

distance of 105.2-179.9 meters per day (Watson et al. 2000). In Brazil, Culex

quinquefasciatus had a survival rate of between 60-68% per day (Laporta and Sallum

2008). These studies, however, did not compare survival rates across different land use

types.

The purpose of our study was to determine if the survival rates and dispersal of

Culex pipiens are different among sites along an urbanization land-use gradient. We

attempted to measure survival rates and, to a lesser extent, dispersal, of Culex pipiens

in urban, residential, park, and forested areas. We tested the hypothesis that survival

would increase and dispersal would decrease with urbanization. Urban areas have an

abundance of sources of highly organic water, therefore Cx pipiens do not have to fly

very far to look for a place to lay eggs.

Methods and Materials

We performed mark release recapture studies at 4 sites in one year and in two

years at one site (Table 1).The 2008 study was performed in Takoma Park from August

19- September 2. The study was then repeated at 4 sites from July 25- August 8, 2009.

Study Sites

The study sites were located in Washington, DC and Maryland along a gradient of

anthropogenic land use change. Each site was defined by a 1km diameter circle and

was selected so that land use/land cover was relatively homogenous within the area

and beyond. An urbanization index of UI = (100% – % tree cover + % impervious

surface)/2 was used to determine the classification of each site along an urbanization

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gradient. The sites can also be qualitatively categorized as being a highly urban area

(Baltimore), a residential site (Takoma Park), a suburban park within urban/suburban

areas (Fort Dupont), and a mostly forested site (SERC).

Baltimore, MD. Located within downtown Baltimore, this site was classified as a

high urban area stadium (Figure 3-1). The 1 km diameter site is 82% impermeable

surface and only 1% forest cover, with the rest composed of grass/lawns. It is located

in the Rigley’s Delight neighborhood, which is across from the Oriole’s stadium. This

site mostly consists of row housing, with very few trees.

Takoma Park, MD. Located in a subdivision of Washington, DC; this was

classified as a residential site (Figure 3-2). The 1 km diameter site is 20% impermeable

surface and 35% forest cover, with the rest composed of grass/lawns. This site consists

primarily of houses with yards and overhanging trees, and also includes a grassy park.

Fort Dupont National Park, Washington, DC. Located in the southeast area of

Washington, DC; this was classified as a suburban park within urban/suburban areas

(Figure 3-3). The 1 km diameter site is 19% impermeable surface and 44% forest cover,

with the center of the site composed of a community garden approximately 6 ha in area.

This site consists of a large, roughly circular, forested area of ~800-900m diameter,

surrounded by a residential neighborhood, with the aforementioned community garden

in the middle of the park.

Smithsonian Environmental Research Center (SERC), Edgewater, MD.

Located near the Chesapeake Bay, this was classified as a forested site (Figure 3-4).

The 1 km diameter site is 3% impermeable surface and 90% forest cover. This site

consists of mostly forested area with a few research buildings and some unused

27

pasture land with a large amount of tree cover.

Collecting Egg Rafts

Egg rafts were collected on August 6, 2008 for Takoma Park 2008 and again on

July 12 and 13, 2009 for the 4 sites. Egg rafts were collected by placing approximately

10 ovitraps at each site. Ovitraps were navy blue, 1.5 gallon Rubbermaid® containers.

These containers were filled 1-2 in with highly organic water, and were covered with one

inch chicken-wire screening. Screening was used to attract Cx. pipiens to lay their eggs

and deter other Culex species from laying their eggs in the ovitrap (Walther and Weber

1996). Egg rafts were collected daily and placed in individual container, which were

placed in a 6 ft3 (2’x2’x2’) cage that was located at each site. Two days of egg rafts

were collected, and each day had a designated cage. The cages had screen on all

sides to allow airflow and clear covers were placed over the cages to protect from rain.

Rearing Mosquitoes

After 2-3 days, two larvae were removed from each container for species

determination. Containers that did not contain Cx. pipiens were removed from the cage

before adult emergence. Containers that did contain Cx. pipiens were left in the cage

and allowed to become adults. Larvae were fed 0.5 grams of fish (koi) food which

contains an ample supply of protein needed for larval growth, and adults were fed a

10% sucrose solution needed for adult survival. Poison baits were placed in the cages

after it was discovered that ants were entering the cage and eating both larvae and

adult mosquitoes.

Spermethecal dissections

After the mosquitoes became adults, they were held in the cage for 3 days in

28

order to allow mating. Early on the third day, ten females were removed from cages at

each site, except Baltimore (where all mosquitoes were consumed by ants), and

spermethecal dissections were conducted to determine mating status.

Capture Mark Release (Baltimore, only)

Because ants consumed the reared mosquitoes at Baltimore we used CDC light

traps to collect mosquitoes to be used in a mark-recapture experiment for release on

the following day. Trapped mosquitoes were collected early next morning, mosquitoes

were counted, placed in a cage (1ft3), and given 10% sucrose solution until marking

took place that evening. At this site, previous trapping efforts suggest that 96-99% of

Culex mosquitoes trapped at this site in July were Culex pipiens, with the other Culex

mosquitoes being identified as Culex salinarius. There were also some Aedes

albopictus caught in the traps, but these were easily identifiable from the Culex

mosquitoes and were not used in the experiment.

Marking Mosquitoes

Marking took place approximately an hour before dusk. Mosquitoes were

counted and transported to 1ft3 cages by using Clarke handheld aspirators. These

aspirators were used to collect female mosquitoes from the main cage. Hand held

counters were used to count the mosquitoes collected. Mosquitoes were dusted with

Bioquip fluorescent dusts and 4 oz handheld bulb dusters. Different colors of red, blue,

white, and yellow were used to differentiate the mosquitoes released at different sites

and release dates. Fluorescent dust was deposited into the bulb and applied to the

mosquitoes in the cage.

Releasing Mosquitoes

29

The marked mosquitoes were released at dusk from a location that was

approximately in the center of the study site. Releases were done on one night at

Takoma Park in 2008, at Baltimore in 2009, and took place over 2 days at Takoma Park

and Fort Dupont in 2009. Five hundred marked mosquitoes were released on the first

day at Fort Dupont and Takoma Park, 152 were released at SERC, and 0 at Baltimore.

On the second day 500 were released at Takoma Park, 177 at Fort Dupont, 200 (caught

in CDC light traps, not reared) at Baltimore, and 0 at SERC. Releasing took place on

August 19, 2008 for Takoma Park 2008 and on July 25 and 26 2009 for Takoma Park

2009, Ft. Dupont, Baltimore, and SERC.

Recapturing Mosquitoes

Twenty CDC light traps (baited with CO2) and four gravid traps (baited with hay

and rabbit chow infused water that was incubated in warm (75-90ºC) temperatures for

~2 weeks) were distributed within each site, with the exception of Takoma Park, which

had 20 light traps and 10 gravid traps. Traps were set up in tree canopies the afternoon

of the release. Traps were collected at 2pm every afternoon for 15 days. Rainfall

amounts were recorded daily for the duration of the recapture period using weather

stations (Lacrosse Technology, WS-1516-IT) that were set up close to each site.

Identification of Mosquitoes

Mosquitoes were identified to species with keys to adult females (Darsie and

Ward 2005). The mosquitoes were examined for fluorescent dusts under a dissecting

microscope and under UV light.

Statistical Analysis

30

Non-linear least squares regression was used to determine the survival rates

(Buonaccorsi et al. 2003). We fit the number of marked mosquitoes captured on each

night, Cj, to the following equation:

Cj = Nθ(1-θ)j-1πtj (1)

Here N is the number of mosquitoes of that color released, θ is the probability that

a live marked mosquito is recaptured, π is the daily survival probability, j is the trapping

occasion (in this case daily), and tj is the number of days between release and trapping

occasion j (in our case, tj = j, because each trapping occasion was 1 day and

mosquitoes were trapped every day). We analyzed the influence of rain on recapture

probability and survival using the equation:

Cj = Ni(θ+c5*Rain)(1-(θ+c5*Rain))j-1(π+c6*Rain)tj (2)

Here c5 measured the effect of rain on recapture probability, and c6 measured the effect

of rain on survival. We compared the recapture probability and survival rates between

sites (or years for Takoma Park) using the equation:

Cj = Ni(θ+s*c4)(1-(θ+ s*c4))j-1(π+s*c3)tj (3)

Here s was a dummy variable (0 for one site, and 1 for the other), the coefficient c3

measured the difference in survival between the sites or years, and c4 measured the

difference in recapture probability between the two sites. Comparisons between sites

were then made by determining whether the site coefficients (c3 and c4) were

significantly different from 0. The variance of these coefficients is estimated from a

linearized version of the model (Bates and Watts, 1988). The non-linear regression

models were fit to the data using function nls in R (R foundation for Statistical

Computing 2009). We corrected for multiple pairwise comparisons of survival rates

31

using a Bonferroni correction; we made n = 6 pairwise comparisons, and thus used an

adjusted cutoff for statistical significance (to maintain a Type I error rate of 0.05) of

0.05/6 = 0.0083. Finally, we calculated an estimate of the average longevity by

inverting the daily survival rate (average longevity = 1/ (1-survival rate)).

The average daily dispersal distance was estimated by calculating the effort-

weighted average distance that marked individuals were trapped away from the release

site. We estimated trapping effort by calculating the number of traps and dividing by the

area in a disc with the distance between outer and inner circles of 20m. For example, if

there were three traps that were between 20m and 40m from the release site, the

trapping effort in this disc would be 3/(π(402-202)). We then calculated the distance

dispersed, D, using the equation:

∑ ∑= ==n

i i

m

kkki

j

EdC

D1

1,

(4)

Here Cji,k is the number of mosquitoes trapped on day j, in trap k that is within disc i (i=1

to n, with i=1 being a 20m radius circle, i=2, a disc with outer diameter 40m and inner

diameter 20m, etc., and i=n being the disc containing the traps furthest from the release

site; k=1 to m, where m is the number of traps within disk i), dk is the distance between

trap k and the release site, and Ei is the trapping effort in that disc, as described above.

We estimated the dispersal rate by simply regressing the effort-weighted average

distance that marked mosquitoes were caught each day against the number of days

since the release.

32

The direction traveled by the mosquitoes was determined by averaging the

direction (in degrees) of the release site for each site to a trap that captured a marked

mosquito.

A chi-Square test was used to determine if the mosquitoes dispersed in random

directions or showed evidence of directional movement by separating the site into six

sectors (Figure 3-8), the smallest number that could be used and still satisfy the

assumptions of a chi-squared test, and using the equation:

(5)

Here obs is the observed number of marked mosquitoes trapped in the sector, exp is

the total number of mosquitoes recaptured divided by the number of quadrants

measured; x is the number of traps in a sector, n is the total number of traps at the site,

and q is the number of sectors. Numbers of recaptured mosquitoes were large enough

to permit analysis for the study at Takoma Park 2009, but were too small for other sites

or years.

Results

Spermethecal dissections showed the presence of sperm in 100% of the spermethecae

of reared mosquitoes from Takoma Park 2009, SERC, and Ft Dupont. Recapture rates

varied from 1.3-7.6%, with the highest percentage occurring at the Takoma Park 2008

33

site and the lowest percentage occurring at SERC in 2009 (0%). The highest recaptures

usually occurred on the second day of collecting, except for Fort Dupont which had its

highest recaptures on the first day (Figure 3-5). The majority of the mosquitoes

released at all of the sites were captured by CO2-baited CDC light traps, perhaps

because of the larger number of these traps than the CDC gravid traps (20 vs. 4 or 10).

However, in Takoma Park in 2009 approximately 31% of the marked mosquitoes were

caught in gravid traps perhaps due to the increased number of CDC gravid traps (10 vs.

4). Temporal patterns of recaptures of marked mosquitoes at Takoma Park in 2009,

suggested that the gonotrophic cycle length was approximately 4 days (Figure 3-6).

Survival of Culex pipiens mosquitoes varied significantly from site to site (Table3-

2). Survival of Culex pipiens was highest at Takoma Park in 2009 with a daily survival of

0.860 resulting in an average lifespan of 7.0 days. Survival of Culex pipiens at Fort

Dupont was lowest with a daily survival of 0.517 resulting in an average lifespan of 2.0

days (Table 3-1). At SERC no mosquitoes were recaptured.

Rainfall occurred several times during the study and appeared to have a

significant negative effect on the survival of mosquitoes in Baltimore (coefficient c6

(±SE) in equation 2: 0.53±0.07, p=

34

Dupont 2009 survival rates were significantly different from the Takoma Park 2008

(-0.468±0.08), Takoma Park 2009 (-0.451±0.105), and Baltimore 2009 (0.425±0.06, all

p< 0.001) (Table 3-2).

Dispersal rates (mean distance of marked mosquitoes recaptured each day,

weighted by trapping effort) were estimated at all of the sites (Figure 3-7). For the

Takoma Park 2008 study, there was weak marginal support for Culex pipiens

mosquitoes dispersing at a very low rate (Figure 3-7; linear regression: effort weighted

average distance of recaptured mosquitoes = 68.5 + 8.04m/day; n = 7; p = 0.057).

However, at the other sites, and at Takoma Park in 2009 there was no evidence for the

distance of trapped mosquitoes to increase with days since release (all p>0.22).

There was some evidence that dispersal of mosquitoes at Takoma Park in 2009

was not random (χ2 = 12.8, df= 5, p=0.025). More mosquitoes were caught, after

adjusting for trapping effort, in traps in a NNW direction, and fewer in the NNE direction

(Figure 3-8).

Due to low recapture numbers, we were unable to determine if dispersal was random

for the rest of the sites and Takoma Park 2008.

Discussion

Our mark release recapture study suggests that the survival rates for Cx. pipiens

mosquitoes range from 0.50-0.86 The determination of survival rates among different

sites is important for understanding the epidemiology of WNV transmission by Culex

pipiens. Mosquito female longevity is linked to the WNV extrinsic incubation period

(EIP), i.e., the period that a disease-producing organism requires to develop in a

mosquito to the point where it can be transmitted. High daily survival rates increase the

35

chance that a mosquito will blood feed on an infected bird, become infective, and

transmit the WNV to a bird, human, or horse host. Our results show that survival rates

of Culex pipiens in residential and urban sites were higher than survival at a forested

park surrounded by residential housing, but the number of sites studied is too small to

determine whether differences in survival were due to the effect of differences in land

cover or simply differences between sites due to other factors.

Our research suggests that our sites in residential and urban areas have

appropriate conditions for WNV transmission. High daily survival rates increase the

chance that a mosquito may bloodfeed on an infected bird, become infected, and

transmit the virus to another host. Previous studies have shown that ~15-20% (at 30ºC

and 32ºC) of Culex pipiens are able to transmit WNV 4 days after feeding on WNV

infected blood with a titre of 108 PFU/ml, and therefore if females live this long they will

transmit the virus to its next host (Kilpatrick et al. 2008). From the results of this study,

the average longevity for the residential site was long enough to become infected and

transmit the virus to possibly multiple hosts. The urban site average longevity appeared

to allow the mosquito to transmit WNV to at least one host. The average longevity for

the forested park site does not appear to allow the mosquito to have more than one

blood meal, giving it little time to transmit the virus.

The recapture rates for the study sites ranged from 1.2-7.6%, which are

comparable to the 0.34% for Culex quinquefasciatus using the same trapping technique

(Schreiber et al. 1988). The residential site recapture rates are also comparable to the

6.81% for Aedes aegypti using backpack aspirators, BG-Sentinels adult traps, and

sticky ovitraps (Maciel-De-Freitas et al. 2007). Rearing at the urban site was

36

unsuccessful, due to an ant infestation in the rearing cages; therefore captured

mosquitoes were marked and released.

We found weak support that mosquitoes were dispersing from the release sites,

but the sample size of recaptured mosquitoes was relatively low. All of the traps were

within 500m of the release site, so it is possible that the released mosquitoes had

already dispersed throughout our trapping area within the first night after release.

Therefore, if the study is repeated, traps should be placed at further distances from the

release site to determine dispersal beyond the very local area, and to better understand

dispersal differences among urban, residential, and park areas.

The direction of dispersal calculated for Takoma Park 2009 was 142◦ (SSE) and

movement direction appeared to differ from random. Wind measurements that were

taken during the study showed that the wind was blowing towards the South for the

majority of the study. This may explain the directionality preference of these

mosquitoes. However, since the weather stations were not placed directly on the site,

we are unable to confirm if the wind at the site blew the same way. If this study is

repeated it would be beneficial to have a weather station located as close to the release

site as possible.

For the Takoma Park 2009 site, 31% of the recaptured mosquitoes were caught

in gravid traps from day 5-12. This pattern suggests that a possible gonotrophic cycle

had been observed in the study. Culex quinquefasciatus females lay their eggs

approximately 4 days after mating and 2-3 days after receiving a blood meal (Lowe et

al. 1973), which is similar to was observed in this study.

37

The daily survival rate for Culex pipiens in the residential site (Takoma Park) was

82.5% for 2008 and 90.9% for 2009. The survival rates of this study are similar to the

daily survival rate of 90% for Culex quinquefasciatus studied in a residential area of

Burma (Macdonald et al. 1968).

A daily survival rate for the urban site (Baltimore) was 73% which is similar to the

87-88% daily survival rates of Culex quinquefasciatus in an urban area Northern Mexico

(Elizondo-Quiroga et al. 2006), as well as studies conducted in residential areas

(Macdonald et al. 1968). The lower survival rate between Baltimore and Takoma Park

2008 and 2009 could be due to using captured and not reared mosquitoes like studies

at our other sites. Although the mosquitoes were given time to recover from the stress

of being captured in a light trap, the trap could have affected the mosquitoes’ survival

ability in a way that was not noticeable before the marking and release. Another reason

could be the mosquitoes captured were older and survival may decrease with age.

The daily survival rate Culex pipiens in the park area (Fort Dupont) was 50.3%,

which is similar to the survival rate of 60-68% for Culex quinquefasciatus studied in

Brazil in a suburban park (Laporta and Sallum 2008). We found a significantly lower

survival rate between Ft. Dupont Park and our urban and residential sites.

Mosquitoes were found to disperse throughout the study areas in approximately

2 days after the release and little evidence of dispersal afterward. For Takoma Park

2009 there appears to be a lack of dispersal with mosquitoes continuing to be caught in

traps in close proximity to the release site up to day 14. For the Fort Dupont no

mosquitoes were collected after day 4. Since we cannot differentiate between

mosquitoes dispersing out of the study area and mortality according to the fourth mark-

38

release recapture assumption, we must assume that the reason for this is due to a low

survival rate. Future studies should include more traps and larger study areas, to ensure

that survival rates are based on mortality and not mosquitoes dispersing out of the study

area.

We were unable to recover any recaptures for the forested area (SERC). The

reason for this could be a lower survival rate, a lower recapture probability, a higher

dispersal rate, or a combination of all three factors. A previous study of Culex

quinquefasciatus in a forested area had a recapture rate of 1.8% when over 66,000

mosquitoes were released and over 40 CDC light traps were used (Lapointe 2008).

Since we released many fewer mosquitoes than Lapointe (2008) and didn’t use as

many traps to recapture, it is possible that the lack of any recaptures could be due to a

low release number.

39

Figure 3-1. Study area of Baltimore. Outline of trapping area, trap locations, and release site at Baltimore. The larger icons indicate traps that captured marked mosquitoes.

A

40

Figure 3-2. Study area of Takoma Park 2008 and 2009. A) Outline of trapping area, trap location, and release site of Takoma Park 2008. B) Outline of trapping area, trap locations, and release site of Takoma Park 2009. Circles represent light traps. Squares represent gravid traps. The larger icons show a trap that recaptured a marked mosquito.

A

B

41

Figure 3-3. Study area of Fort Dupont. Outline of trapping area, trap locations, and

release site for Fort Dupont. The larger icons show traps that recaptured a marked mosquito.

42

Figure 3-4. Study area of SERC. Outline of trapping area, trap locations and release site for SERC. No mosquitoes were recaptured at this site.

43

Figure 3.5. Number of recaptured Culex pipiens each day. A)Takoma Park 2008.

B)Takoma Park 2009. C)Fort Dupont. D) Baltimore. SERC was not included because it had zero recaptures for the entire study.

A

B

C

44

Figure 3.5. Continued.

D

45

Figure 3-6. Numbers of recaptures in light and gravid traps at Takoma Park in 2009.

46

Figure 3-7. Average dispersal distance each day, corrected for trapping effort. A)

Takoma Park 2008. B) Takoma Park 2009. C) Baltimore. D) Fort Dupont.

B

Avg

. Dis

pers

al D

ista

nce

A

vg. D

ispe

rsal

Dis

tanc

e A

vg. D

ispe

rsal

Dis

tanc

e

Avg

. Dis

pers

al D

ista

nce

A

C

D

47

Figure 3-8. Trap locations and number of recaptured mosquitoes collected in each sixty degree sector of Takoma Park 2009.

48

Table 3-1. Survival rates (survival ± SE) of Culex pipiens females calculated by nonlinear least squares at three sites near Washington DC Study Sites &YR Released Recap % Recap Survival rate

Takoma Park 2008 223 17 7.6 0.835±0.06 a 6.06 Takoma Park 2009 1000 58 5.8 0.860±0.03 a 7.14 Baltimore 2009 200 6 3.0 0.606±0.05 a 2.54 Fort Dupont 2009 766 9 1.2 0.517±0.13 b 2.07 SERC 2009 152 0 0.0 -- -- Means with same letters do not differ significantly (P=0.008), following Bonferroni’s correction for multiple comparisons.

Avg. Longevity (Days)

49

Table 3-2. Comparison of survival rates of Culex pipiens using nonlinear least squares

at three sites near Washington, DC. Cells below the diagonal give the coefficient and standard error for the difference in survival between the sites (coefficient c3 in equation 3), whereas cells above the diagonal give the p-value for that comparison.

Takoma

Park 2009 Takoma Park 2008

Baltimore 2009 Fort Dupont 2009

Takoma Park 2009

0.976 0.408

50

CHAPTER 4 CONCLUSION

Culex pipiens is commonly found in urban environments and usually feeds on

birds. This species is also an important vector of WNV, and the transmission of this

virus is dependent on the vector surviving long enough for the mosquito to take at least

two blood meals (one to receive the virus and one to transmit). The evolution of NY99

into the WN02 genotype of West Nile Virus has allowed transmission to occur more

efficiently (Kilpatrick et al. 2008). Also, the feeding shift of Culex pipiens from birds to

mammals in the late summer is associated with an increase in WNV transmission to

humans and other mammals (Kilpatrick et al. 2006). Since survivorship is one of the

most important factors in vector-borne disease transmission, it is important to determine

and compare survival rates areas across a gradient of land use.

The survival rate of an organism can change depending on certain aspects of its

habitat. I found that Cx. pipiens had higher survival rates in residential and urban areas

than in a park. In my study, mosquitoes at Fort Dupont had a significantly lower survival

rate than those at Takoma Park and Baltimore.

Dispersal is also important in vector-borne disease transmission, since it would

give an indication of how far a vector could spread the disease they carry. However, it is

important to do the study in a wide enough area, in order to avoid the mosquitoes

dispersing throughout the study area in the first couple of days.

More studies need to be conducted to better explain the pattern of survival and

dispersal rates among urban, residential, park, and forested areas. These need to

include more survival and dispersal studies with possibly more mosquitoes released at

each site and possibly a broader study area. By releasing more mosquitoes we would

51

be able to get a better idea of survival and dispersal in more forested areas like SERC.

A broader area would allow dispersal to be better tested. Further studies also need to

include observations of predators and nutrient resources available at each study site.

These additional surveys would allow us to better understand why survival rates are so

high at residential sites like Takoma Park and so low in areas like Fort Dupont.

52

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BIOGRAPHICAL SKETCH

Christy was born in Hartsville, SC and graduated from Mayo High School for Math,

Science and Technology in Darlington, SC. In 2000 she attended Winthrop University

and graduated with a Bachelor of Science degree with a major in biology and a minor in

philosophy and religion. It was while attending Winthrop that she first became interested

in entomology and conducted undergraduate research with milkweed bugs. However,

she was always interested in mosquitoes and parasites, so after college she got a

summer job identifying mosquitoes for West Nile Virus testing in South Carolina. In

2006, she received an entomology internship in Hawaii studying ants; however she

missed working with mosquitoes. Later that year, she jumped at the chance to study

mosquitoes in Washington, DC for 6 months. After the job ended she was able to get a

permanent job in Baltimore, MD studying different insecticides on different insects to

determine their effectiveness. She worked there for a little over a year before she

received the opportunity to go to graduate school and work on the Washington, DC

project again. Since then she have been traveling between Florida and Washington,

DC pursing my Master of Science degree in entomology.

ACKNOWLEDGMENTSLIST OF TABLESLIST OF FIGURESLIST OF ABBREVIATIONSAbstract of thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of master of scienceIntroductionliterature reviewSURVIVAL RATES and dispersal OF cULEX PIPIENS Across a LAND-use GRADIENTIntroductionStudy Sites

Results

ConclusionLIST OF REFERENCESBIOGRAPHICAL SKETCH