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1 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
2 2010 Christy Johnson
3 To my family and friends
4 ACKNOWLEDGMENTS I thank my supervisory committee chair, Dr. Phil Lounibos; and members Dr. Marm Kilpatrick and Dr. George OMeara 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 programmi ng. 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 Mar y 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 Masters 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.
5 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 ....................................................................................................... 2 5 Baltimore, MD ............................................................................................. 26 Takoma Park, MD ...................................................................................... 26 Fort Dupont, Washington, DC .................................................................... 26 SERC, Edgewater, MD .............................................................................. 2 6 Collecting Egg Rafts ......................................................................................... 27 Rearing Mosquitoes ......................................................................................... 27 Spermathecal Dissections ................................................................................ 2 7 Capture Mark Release ..................................................................................... 28 Marking Mosquitoes ......................................................................................... 28 Releasing Mosquitoes ...................................................................................... 2 8 Recapturing Mosquitoes ................................................................................... 29 Identifying Mosquitoes ...................................................................................... 29 Statistical Analysis ............................................................................................ 29 Results .................................................................................................................... 32 Discussion .............................................................................................................. 34 4 CONCLUSIONS ..................................................................................................... 50 LIST OF REFERENCES ............................................................................................... 5 2
6 BIOGRAPHICAL SKETCH ............................................................................................ 6 0
7 LIST OF TABLES Table page 3 1 Survival rates (survival SE) of Culex pipiens females calculated by nonlinear least squares at thr ee sites near Washington, DC .............................. 48 3 2 Comparison of survival rates of Culex pipiens females at three sites near Washington, DC ................................................................................................. 49
8 LIST OF FIGURES Figure page 3 1 Study area of Baltimore ...................................................................................... 3 9 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
9 LIST OF ABBREVIATION S MRR Mark ReleaseRecapture WNV West Nile Virus
10 ABSTRACT OF THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLME NT 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 releaserecapture 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, m osquitoes 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 410 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 F ort Dupont Park, with a daily survival rate ( SE) of 52.0 3 .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
11 mosquitoes were recaptured at the forested site. The trapping grid was too small to measure long distance dispersal rates for Cx. p ipiens 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.
12 CHAPTER 1 INTRODUCTION Insects belonging to the family Culic idae 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 mosquitoborne 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 DAntonio 2001). During World War I and II, mosquito borne diseases were killing more troops than the opposi ng army (Spielman and DAntonio 2001). Although many of the mosquitoborne diseases have been eradicated from developing countries, they are beginning to emerg e again (Spielman and DAntonio 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 manmade 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
13 reactions than others. These reactions are due to an immune response t o the mosquitos saliva which she secretes while probing and biting (Ribeiro et al. 1984). Mosquitoes have been transmitting pathogens for at least 20,00030,000 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 DAntonio 2001, Desowitz, 1991). These diseases and parasites include malaria, yellow fever, dengue, and many others. Today we still face the problem of pa thogens 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 wa y ( Takahashi 1984) The most recent mosquitoborne 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 v irus 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 DAntonio, 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
14 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 survi ve the full length of the extrinsic incubation period in order to tr ansmit the virus (Garrett Jones 1964). Cx. pipiens is the dominant enzootic (birdto 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 v ectors 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 r ates in the field have not been determined.
15 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 30300 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 metamor phosing 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 30C (Olejnicek and Gelbic 2000).
16 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 waters 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 12 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 males sperm is stored in the females 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 usuall y 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
17 unknown how long Culex pipiens adult females live in the wild. In laboratory studies they can live up to 54 days at 21C. Culex quinquefasciatus were found to live up to 64 days at 25C (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 s tranded 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 i nclude 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).
18 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 t o 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 transmi tted to another host) (Moudy et al. 2007, Kilpatrick et al. 2008, Ebel et al. 2004).
19 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 ancestr y 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 pipie ns 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 t o 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
20 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 wildli fe, but creates a heat island that raises the temperature of the area 23C ( 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. Te mperature 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 32C (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
21 whereas urban communities are mostly c omposed 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 comm unity 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 significa ntly 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 bit e 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 ReleaseRecapture for Survival and Dispersal Mark releaserecapture (MRR) st udies 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,
22 mammals, reptiles, insects and other invertebrates. The technique includes capturing or rearing many individuals of a species an d 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. T here 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 r ate, 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 look ing for another host.
23 CHAPTER 3 SURVIVAL RATES AND D ISPERSAL 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 m osquito 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 28C (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
24 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 als o 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 mosquitos ability to transmit the virus, because of the increased temperatures in urban areas. Increased temperatures of up to 32C 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 div ersified 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 Ph lebotomus 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 thos e 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
25 that Anopheles gambiae were estimated to hav e a survival rate of 80 88% per day and a flight range of 350650 meters per day (Costantini et al. 1996). In Australia, Aedes notosciptus were found to have a survival rate of 7770% per day and an average flight distance of 105.2179.9 meters per day ( Watson et al. 2000). In Brazil, Culex quinquefasciatus had a survival rate of between 6068% 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 determ ine if the survival rates and dispersal of Culex pipiens are different among sites along an urbanization landuse gradient. We attempted to measure survival rates and, to a lesser extent, dispersal, of Culex pipiens in urban, residential, park, and forest ed 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 eg gs. 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 19September 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
26 gradient. The sites can also be qualitatively categorized as being a hi ghly 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 st adium ( 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 Rigleys Delight neighborhood, which is across from the Or i oles 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 ~800900m diameter, surrounded by a residential neighborhood, with the aforementioned community garden in the middle of the park. Smithsonian Environmental Research Center (SER C), 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 12 in with highly organic water, and were covered with one inch chicken wire screening. Screening was used to attract Cx. pip iens 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 (2x2x2) cage that was located at each si te. 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 f rom 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 gra ms 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 Bal timore (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 9699% 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 th e 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 col ors 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 mark ed 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 (7590 C) 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 aft ernoon 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 1516IT) that were set up close to each site. Ide ntification 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 ( B uonaccorsi et al. 2003). We fit the number of marked mosquitoes captured on each night, Cj, to the following equation: Cj = N (1 )j 1tj (1) Here N is the number of mosquitoes of that color released, is the probability that a live marked mosquito is recaptured, 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( +c5* Rain)(1 ( +c5*Rain))j 1( +c6*Rain)tj (2) Here c5 measured the e ffect 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( + s*c4)(1 ( + s *c4))j 1( + 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 probabil ity 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 nonlinear 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 long evity = 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/( (402202)). We then calculated the distance dispersed, D, using the equation: n i i m k k k i jE d C D1 1 (4 ) Here Cj i,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 det ermine 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 usi ng 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 sector s. 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 1.37.6%, 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 ( Fi gure 3 5 ). The majority of the mosquitoes released at all of the sites were captured by CO2baited 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 approximat ely 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 ap proximately 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 reca ptured. Rainfall occurred several times during the study and appeared to have a significant negative effect on the survival of mosq uitoes in Baltimore ( coefficient c6 ( SE) in equation 2: 0.53 0.07, p=<0.001). Rainfall did not have a significant effect on survival in Takoma Park 2008 ( 2.26 1.077, p=1.00), Takoma Park 2009 ( 0.268 0.45, p=0.13), or Fort Dupont ( 2.8485.82, p=0.63). Survival was not significantly different between the two years, 2008 and 2009, at Takoma Park ( coefficient c3 ( SE) in equati on 3: 0.002 0.05, p=0.976). Baltimore 2009 survival rates were not significantly different between the Takoma Park 2008 ( 0.120.07, p=0.113) or Takoma Park 2009 ( 0.100.12, p=0.408). However, the Fort
34 Dupont 2009 survival rates were significantly different from the Takoma Park 2008 ( 0.4680.08) Takoma Park 2009 ( 0.4510.105) and Baltimore 2009 (0.4250.06, 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 ( Fi gure 3 7 ; linear regression: effort weighted average distance of recaptured mosquitoes = 68.5 + 8.04 m/ 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 ( 2 = 12.8, df= 5, p=0.025). More mosquitoes were caught, after adjusting for trapping effort, in traps in a NNW direction, a nd 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 0.500. 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 W NV extrinsic incubation period (EIP), i.e., the period that a diseaseproducing 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 infecte d 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 num ber 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 ~1520% (at 30C and 32C) 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 t his 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 averag e 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 1.27.6%, 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 D e 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. The refore, 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 hav e 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 512. 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 23 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 reside ntial 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 rat e for the urban site (Baltimore) was 73% which is similar to the 8788% daily survival rates of Culex quinquefasciatus in an urban area Northern Mexico (ElizondoQuiroga 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 l ight 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 6068% for Culex quinquefasciatus studied in Brazil in a suburban park (Laporta and Sallum 2008). We found a significantly lower survival rate between Ft. D upont 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 t hree 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 didnt 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 31. Study area of Baltimore. Outline of trapping area, trap locations and r elease site at Baltimore. The larger icons indicate traps that captured marked mosquitoes. A
40 Figure 32. Study area of Takoma Park 2008 and 2009. A) Outline of trapping area, t rap location, and release site of Takoma Park 2008. B ) Outline of trapping area, t rap 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 33. Study area of Fort Dupont O utline of trapping area, trap locati ons, and release site for Fort Dupont. The larger icons show traps that recaptured a marked mosquito.
42 Fi gure 34. 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 36 Numbers of recaptures in light and gravid traps at Takoma Park in 2009.
46 Figure 37. Average dispersal distance each day corrected for trapping effort. A) Takoma Park 2008. B) Takoma Par k 2009. C) Baltimore. D) Fort Dupont. B Avg. Dispersal Distance Avg. Dispersal Distance Avg. Dispersal Distance Avg. Dispersal Distance A C D
47 Figure 38 Trap locations and number of recaptured mosquitoes collected in each sixty degree sector of Takoma Park 2009
48 Table 31 Survival rates (survival SE) of Culex pipiens females calculate d 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 0.8350.06 a 6.06 Takoma Park 2009 1000 58 5.8 0.8600.03 a 7.14 Baltimore 2009 200 6 3.0 0.60 60.05 a 2.54 Fort Dupont 2009 766 9 1.2 0.5170.13 b 2.07 SERC 2009 152 0 0.0 --Means with same letters do not differ significantly (P=0.008), following Bonferronis correction for multiple comparisons Avg. Longevity (Days)
49 Table 32. 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 pvalue for that comparison. Takoma Park 20 09 Takoma Park 2008 Baltimore 2009 Fort Dupont 2009 Takoma Park 2009 0.976 0.408 <0.001 Takoma Park 2008 0.002 0.05 0.113 <0.001 Baltimore 2009 0.10 .12 0.12 .07 <0.001 Fort Dupont 2009 0.45 0.105 0.468 0.08 0.425 0.06 Corrections for mul tiple comparisons were done using Bonferronis correction. (P=0.008).
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 transmis sion 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 t o occ ur 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 vec tor 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 i n areas like Fort Dupont.
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60 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 atte nded Winthrop University and graduated with a Bachelor of Science degree with a major in biology and a minor in philosophy and r eligion. It was while attending Winthrop that she first became interested in e ntomology and conducted undergraduate researc h wit h 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 wit h 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 Wa sh ington, DC pursing my Master of Science d egree in e ntomology.