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Development of a Practical Technique for Sampling the Afrotropical Malaria Vectors Anopheles gambiae S.L. and An. funestus

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PAGE 1

DEVELOPMENT OF A PRACTICAL TECHNIQUE FOR SAMPLING THE AFROTROPICAL MALARIA VECTORS Anopheles gambiae S.L. AND An. funestus By JUSTIN ERIC HARBISON A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2005

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This document is dedicated to my parents, Kent and Judy Harbison.

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ACKNOWLEDGMENTS I would like to thank my parents, Kent and Judy Harbison, for their unwavering support. I thank Dr. Jonathan Day, Dr. Dan Kline, Dr. Roxanne Rutledge-Connelly, and Dr. Sandra Allan for their help in Florida. I thank Dr. Richard Mukabana, Dr. Evan Mathenge, Dr. Bart Knols, Dr. Gerry Killeen, and Dr. Ulrike Fillinger for their willingness to help out an American student. I thank everyone else back in Kenya not only for helping me complete this project, but for their willingness to teach me about their great country. I thank Debbie Hall for all the administrative help. I thank Marinela Capanu for statistical assistance and Scott Weihman for the use of his photos. Finally, special thanks go to Camille Francisco for her undying patience while I was in Africa. iii

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TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................iii LIST OF TABLES .............................................................................................................vi LIST OF FIGURES ..........................................................................................................vii ABSTRACT .....................................................................................................................viii 1 INTRODUCTION........................................................................................................1 Approaches to Malaria Control. ...................................................................................1 History of Malaria Control ...........................................................................................2 Future of Malaria Control .............................................................................................2 Role of Rural Housing in Malaria Transmission ..........................................................3 Sampling Methods for Malaria Vectors Indoors ..........................................................4 Indoor Sampling of Resting Mosquitoes ......................................................................5 2 LITERATURE REVIEW.............................................................................................7 Historical Overview ......................................................................................................7 Importance of the Disease Worldwide .........................................................................8 Description of Disease Transmission ...........................................................................8 Importance of Malaria in Africa .................................................................................10 Important Vector Species in Africa ............................................................................11 Summary .....................................................................................................................12 3 MATERIALS AND METHODS...............................................................................14 Site Descriptions .........................................................................................................14 Survey of Indoor Resting Habitats of Mosquitoes .....................................................14 Representative Accuracy of Hand Collection .............................................................17 Development of Cloth Indoor Resting Box in Semi-Field Conditions .......................18 Comparison of Accuracy of Resting box/Resting Net to Hand Collection ................19 Development of an Alternative to Cloth Box in Semi-Field Conditions ....................22 Field Trials ...........................................................................................................22 Semi-Field ...........................................................................................................23 iv

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4 RESULTS AND DISCUSSION.................................................................................24 Results .........................................................................................................................24 Survey of Indoor Resting Habitats of Mosquitoes ..............................................24 Representative Accuracy of Hand Collection .....................................................25 Development of Cloth Indoor Resting Box in Semi-Field Conditions ...............25 Comparison of Representative Accuracy of Resting Box/Resting Net to Hand Collection .........................................................................................................26 Development of an Alternative to Cloth Box in the Semi-Field .........................28 Discussion ...................................................................................................................32 Indoor Resting Places of Mosquitoes ..................................................................32 Comparison of Sampling Techniques .................................................................33 Summary .....................................................................................................................35 5 NOTES ON THE DEVELOPMENT OF A PRACTICAL Anopheles GRAVID TRAP..........................................................................................................................37 Introduction .................................................................................................................37 Preliminary Semi-Field Trials in Florida ....................................................................38 Semi-Field Trials in Kenya .........................................................................................40 Field Trials in Kenya ..................................................................................................41 Results .........................................................................................................................42 Florida Trials .......................................................................................................42 Kenya Trials ........................................................................................................42 Discussion ...................................................................................................................42 LIST OF REFERENCES ...................................................................................................45 BIOGRAPHICAL SKETCH .............................................................................................50 v

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LIST OF TABLES Table page 4.1. Mean area and height of dwellings in study area .......................................................25 4.2 Percent of total actual number of mosquitoes (437) collected from each location. .....26 4.3 Total actual number of mosquitoes collected at each height. ......................................26 4.4 The mean percent of mosquitoes recaptured in semi-field conditions using a resting box either treated or untreated with deltamethrin. ...................................................26 4.5 Comparison of actual number of mosquitoes collected by the three methods tested. .27 4.6 Comparison of the accuracy (percent of the overall number of mosquitoes found in each dwelling) over the 27 test dates for the three methods tested. .........................29 4.7 Comparison of time taken to complete sampling using a resting box (cloth or cardboard) and resting net to hand collection. .........................................................29 4.8 Actual number of An. gambiae mosquitoes caught by method, including sex and gonotrophic status, during the 27 test days of the Latin Square. .............................30 4.9 Actual number of An. funestus mosquitoes caught by method, including sex and gonotrophic status, during the 27 test days of the Latin Square ..............................30 4.10 Actual number of Culicine mosquitoes caught by method, including sex and gonotrophic status, during the 27 test days of the Latin Square. .............................31 4.11 Actual number of unidentified anopheline mosquitoes caught by method,including sex and gonotrophic status, during the 27 test days of the Latin Square. ................31 4.12 Mean number of mosquitoes caught daily by a resting basket placed in a house in the village of Lwanda. ..............................................................................................31 4.13 Comparison of the resting basket to the cloth resting box of the total percentage of mosquitoes recaptured in semi-field conditions. ......................................................31 4.14 Mean percent of mosquitoes recaptured by each density used for both the resting box and resting basket. .............................................................................................32 vi

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LIST OF FIGURES Figure page 3.1 Map of Kenya showing the study sites. .......................................................................15 3.2 Dwellings in Lwanda. A) Single family structure B) Multiple family structure. ........16 3.3 Objects in dwellings on which mosquitoes can be found resting. ...............................17 3.4 Components of the experimental tests A) Resting box hung in experimental hut. B) Experimental hut in modified greenhouse. C) Modified greenhouse. .....................20 3.5 Treatments used in the Latin Square. A) Cloth resting box hanging in dwelling B) Cardboard box hanging in dwelling C)Diagram of a resting net spread out in a dwelling D) Expert collecting mosquitoes by aspiration. ........................................22 3.6 Alternative to the cloth resting box. A) Cloth resting box and resting basket B) Resting basket hanging in dwelling. ........................................................................23 4.1 Layout of dwellings. A) Single Family B) Multiple Family .......................................25 5.1 Design of oviposition trap. A) Full view. B) Collecting cartridge ..............................39 5.2 Modifications to design. A) with clothes hangers B) with black mesh C) with vial with sugar solution. ..................................................................................................40 5.3 Trap designs in Kenya. A) Mbita (two variations) B) Emergence C) Florida D) Cone (two variations). .......................................................................................................43 vii

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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 DEVELOPMENT OF A PRACTICAL TECHNIQUE FOR SAMPLING THE AFROTROPICAL MALARIA VECTORS Anopheles gambiae S.L. AND An. funestus By Justin Eric Harbison May 2005 Chair: Jonathan Day Major Department: Entomology and Nematology Malaria, transmitted by Anopheline mosquitoes, is not only the most important insect-borne disease but one of the top three infectious diseases in the world. Resting boxes are a well-established method for sampling Anopheline mosquitoes. Few resting box designs, however, are used indoors. A practical method, utilizing cloth resting boxes and resting nets for sampling indoor malaria vectors of Africa, was developed in semi-field (a modified greenhouse) and field conditions. In semi-field conditions, a cloth resting box recaptured 36.1 9.9% (mean SE) of three different densities of An. gambiae s.s. (Giles) females of varying gonotrophic status. The accuracy method then was compared to the collection of mosquitoes using an oral aspirator in a rural Kenyan village. The developed method caught a significantly higher percentage (P=0.05) of resting mosquitoes than hand catches with an oral aspirator and required slightly less time to complete (8.0 3.9 minutes versus 15.0 0.0 minutes). A resting basket was also developed as an even more practical alternative to the cloth resting box. The resting viii

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basket was tested under the same semi-field conditions as the resting box. No significant difference (P=0.47) was found in recapture rates between the resting basket and box, suggesting that it could be used as an alternative to the cloth resting box. The materials used in the method, which requires little training to implement, can be easily obtained in rural settings where malaria is of great concern. ix

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CHAPTER 1 INTRODUCTION Approaches to Malaria Control. Historically, there are two general approaches (vertical and horizontal) to control of diseases such as malaria. Vertical approaches call for centralized national programs that act as an independent entity in national health care systems. Horizontal approaches involve broad based local programs emphasizing basic needs such as health education, safe water, and adequate food supply (Tan et al. 2003). Positive aspects to vertical approaches are a government commitment to standardized health care and attention given to the control of all aspects of the disease (such as prevention and treatment). Also, due to clear hierarchical structures typically found in such programs, there are fewer organizational complications compared to those observed in decentralized systems (Kroeger et al. 2002). Vertical programs, however, are criticized for focusing solely on one health problem and failing to build local capacities with wider health benefits (Tan et al. 2003). In some cases vertical programs must be initiated by larger international organizations when national governments do not have the needed resources. Horizontal control programs are strongly based in decentralized health systems and in community mobilization. They are geared to become more focused to the needs of each specific region. Problems that arise from horizontal approaches are that often decentralized health systems do not have the resources and support held in vertical programs, and in many rural regions people cannot access basic health facilities (Deressa et al. 2003, Garfield 1999, Killeen et al. 2002). 1

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2 History of Malaria Control In the 1950s, the World Health Organization (WHO) realized the severity of malaria in terms of human morbidity and mortality and the resulting reduction of agriculture and industry on a global scale. In 1956, the Global Malaria Eradication campaign was launched. The campaign was a relatively worldwide vertical effort based on interrupting transmission through indoor residual spraying (and other mosquito control measures), management of marshes, creation of agricultural land, and modification of human lifestyle (i.e., window screens, air conditioning, and television). The success of the National Malaria Eradication Program in the United States was, in part, due to vertical disease programs from the Tennessee Valley Authority and the Communicable Disease Center (previously the Office of Malaria Control in War Areas) (CDC 2004a, CDC 2004b). Malaria eradication was realized in much of the worlds temperate areas, but failed or was not attempted in many tropical areas. In Africa, eradication was only attempted in Zimbabwe, South Africa, and Ethiopia since malaria was considered too great a problem in the other countries (Kager 2002). In response to the anticipation that national primary health care systems would take over comprehensive malaria information systems, aiding in management of malaria, the vertical approach programs of the WHO were dismantled in the 1980s. International interest and funding in malaria research waned during this time. Since then malaria transmission in many of the affected regions has increased dramatically (Gilles 2002). Future of Malaria Control In the past 15 years, global efforts have been made to combat the increasing morbidity and mortality associated with the disease. Initiatives such as Roll Back Malaria (RBM), Multilateral Initiatives on Malaria (MIM), and the African Initiative on

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3 Malaria (AIM) have been created to reestablish malaria as a global priority, to increase support from wealthier nations, and to develop sector-wide approaches to malaria control. This global push to create regional programs involves not only peripheral health care systems, but educating and empowering community members of the affected areas (Conteh et al. 2004, Deressa et al. 2003, Magnussen et al. 2001). Because such horizontal approaches still have shortcomings, it is likely that large scale malaria control will only be achieved through the integration of aspects from both horizontal and vertical programs (Kroeger et al. 2002). Role of Rural Housing in Malaria Transmission The physical condition of rural houses plays an important part in the epidemiology of malaria, especially when transmitted by endophagic and endophilic vectors like Anopheles gambiae (Giles). High levels of malaria transmission are usually associated with vectors that prefer to feed on humans indoors (Harwood and James 1979). Successful eradication of malaria in many regions of the world is due in part to mosquito-proofing houses (i.e., screened windows and air conditioning). Many people living in regions where malaria causes the highest morbidity and mortality do not have access to such luxuries. Rural houses and villages (especially those of poor construction) are considered the main foci for malaria transmission in these regions (Muirhead-Thomson 1982, Konradsen et al. 2003, Fullerton and Bishop 1933, Gamage-Mendis et al. 1991). In Kenya, Githeko et al. (1994) and Bogh et al. (1998) found that 74% and 99%, respectively, of An. gambiae sensu lato (Giles) collected resting indoors had fed on human blood. Open eaves provide a ready entry point into rural houses and increase the chance of finding higher densities of mosquitoes resting indoors (Lindsay et al. 2003, Schofield and White 1983).

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4 Houses built without ceilings also have been shown to increase human exposure to malaria vectors. (Lindsay et al. 1995, Schofield and White 1983). Houses also provide suitable habitats for vectors of human filariasis (Culex spp.), yellow fever, and dengue (Aedes aegypti L.). Sampling Methods for Malaria Vectors Indoors Although there are various methods for sampling the eggs and larvae of mosquitoes, the most commonly used approach is to sample adults (Service 1993). There exist numerous methods to sample adults, especially host-seeking females. Many of the techniques used to sample mosquitoes seeking human blood are conducted indoors or in artificial shelters. Such methods include bednet traps, hand net collections, drop net collections, and aspirating mosquitoes attempting to bite a sleeping human subject (Service 1993). However, ethical issues arise when studies attempt to quantify the numbers of mosquitoes actively trying to bite humans. Studies involving methods such as counting the number of mosquitoes landing on a human (human landing catches) and human-baited traps can potentially expose human subjects to malaria through a bite from an infected mosquito. These studies are often conducted during the time of day when many female mosquitoes are actively seeking blood. Ways to circumvent using human subjects include the use of carbon dioxide and light traps, as well as animal baited traps. The accuracy of these methods varies, but their cost and practicality are often beyond the means of community level control programs in developing countries. Also, mosquitoes may be attracted to mechanical devices for reasons other than host-seeking and mosquitoes attempting to feed on animals may not behave in the same manner as anthropophagic species.

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5 Collecting adult resting mosquitoes, however, is a method that is considered to be more representative of mosquito populations than collections made by trapping adults in flight. This is because a wider range of mosquitoes, in terms of bloodmeal status, age, sex, and gonotrophic cycle, are taken in resting collection (Service 1993). Resting collections are often made from outdoor natural shelters, such as vegetation and tree-holes, but a few important vector species can be found in man-made structures. Because of this, sampling a population of vectors resting indoors can be valuable to surveillance and control programs (WHO 1992, Lindblade et al. 2000). Indoor Sampling of Resting Mosquitoes Hand catches with a plastic tube or mechanical aspirator and Pyrethrum Spray Catches (PSC) are two commonly used methods for collecting mosquitoes resting indoors (WHO 1992). However, depending on the ability and willingness of the collector, human error can reduce the accuracy of counts made by hand catches. Pyrethrum spray catches are not only expensive but can cause unnecessary exposure to chemicals. A wide variety of artificial resting places have been tested to sample outdoor resting mosquitoes, but relatively few artificial resting places have been used indoors. Those tested indoors have seen limited success (Service 1993, Komar et al.1995, Smith 1942). Yasuno et al. (1977) tested the use of plywood boxes as a method to sample indoor mosquitoes, but found the boxes worked only in high densities of mosquitoes and low humidities. Resting boxes made of cardboard and black muslin cloth caught 30-60% of all Aedes aegypti collected indoors by hand catches (Edman et al. 1997). Das et al. (1997) developed an insecticide-impregnated fabric (IIF) trap for use indoors, but this is an impractical tool in much of the rural settings in Africa where many of the materials needed for construction are not readily available. In Kenya, Sexton et al. (1990) used a 5 x 6 ft reed ceiling mat from

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6 which to make weekly hand catches of indoor resting densities of Anophelines but did not test this method against other kinds of indoor resting collections

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CHAPTER 2 LITERATURE REVIEW Historical Overview Malaria has long been a plague of mankind. Evidence of this is seen in the high host specificity of the four malaria species that infect humans, suggesting a long association between humans and the parasites (Gilles 2002). Archeological evidence suggests that human malaria existed in the eastern Mediterranean region as early as the beginning of the Neolithic Period (9,500 BC) (Harwood and James 1979). In the fifth century, Hippocrates was the first to describe the clinical picture and some complications of malaria. The English word malaria is derived from the Italian mal aria meaning bad air. The French word paludisme and the Spanish paludismo also refer to malaria and come from the Latin palus meaing swamp. Malaria, paludisme, and paludismo have all originated from the idea that malaria was the result of inhaling bad air from swamps (Foster and Walker 2002, Gilles 2002, Harwood and James 1979). The first description of the malaria parasites in human red blood cells was from the French army surgeon Laveran in 1880. Seventeen years later Ronald Ross, a physician in India, discovered a developing form of malaria parasite in the body of an infected mosquito, greatly aiding the understanding of malaria transmission. In the late 1950s the WHO launched a global campaign that successfully eradicated malaria in much of the worlds temperate zones including parts of North America, Europe, and Australia. Control of malaria was not as successful in many tropical areas. In fact, in the past twenty-five years there has been a significant increase of malaria incidence in tropical 7

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8 areas such as southeastern Asia and tropical Africa. In 1998, the WHO stated malaria to be returned to its list of top priorities and introduced a new initiative called, Roll Back Malaria. The success of this program has yet to be evaluated and reported. Importance of the Disease Worldwide Today, malaria is the most important insect-borne disease in public health (Durden and Mullen 2002, Foster and Walker 2002). Malaria, along with AIDS and tuberculosis, are the three most important infectious diseases in the world. Malaria affects around 40% of the worlds population, much of which live some of the poorest countries in the world (WHO 2004, WHO 1995). In 1995, countries with intense malaria transmission had income levels equal to roughly a third of those in countries without malaria (Gallup and Sachs 2001). In Africa alone, it is estimated that the annual costs of malaria exceed US $2 billion (WHO 2000). Malaria not only affects poor people, but it keeps them poor. The disease is found mainly in subtropical and tropical climates. In the past, malaria was more ubiquitous and was found in temperate regions including areas in the United States (Gilles 2002, Honigsbaum 2001). In the early 1900s, 6 to 7 million cases of malaria were reported in the continental United States annually (Harwood and James 1979). Now malaria is responsible for more than 300-500 million illnesses and a least one million deaths each year (WHO 2004, WHO 1995). Description of Disease Transmission Human malaria is caused by one of four species of parasite in the genus Plasmodium, family Plasmodiidae, suborder Haemosporiidae, order Coccidae (Sinden and Gilles 2002, Marquadt et al 2000). The life cycle of Plasmodium species is complex, including an exogenous phase (a cycle in mosquitoes) and an endogenous phase (a cycle in humans) (Foster and Walker 2002). The four species of Plasmodium infecting humans

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9 are P. falciparum, P. vivax, P. ovale, and P. malariae. These parasites are transmitted to humans by mosquitoes of the genus Anopheles. The first sexual developmental stage of the parasites, the gametocytes, are ingested along with asexual stages by the mosquito in the bloodmeal of an infected human. After fertilization occurs a diploid zygote is formed. The zygote (or ookinete) moves through the peritrophic membrane of the mosquito gut. Depending on the species of parasite, 8 to 16 days later the ookinete ruptures, releasing sporozoites that make their way to the mosquitos salivary glands where they can be passed on to another human upon a subsequent bloodmeal (Sinden and Gilles 2002, Foster and Walker 2002). The severity of disease is dependent on the species of parasite and the general health and immune status of the infected person (WHO 1995). Cases of mixed infections of two or more Plasmodium species are not uncommon (Sinden and Gilles 2002). Nonfatal infections that are left untreated can last more than five months depending on the immune system of the individual (Foster and Walker 2002). Common symptoms are febrile paroxysm (violent attack of fever), myalgia (muscle pain), headache, nausea, diarrhea, and vomiting. In areas of stable malaria transmission (little variation in transmission over several years) some degree of acquired immunity is common in adults who survived bouts of malaria as children. This means that infected adults may be asymptomatic or exhibit only slight clinical symptoms. Effects of immunity to malaria on individual are; 1) prevention of infection with the same species of parasite, 2) reduction in parasite multiplication, 3) destruction of parasite, and 4) aid in tissue repair (Marquadt et al. 2000). This immunity is no doubt beneficial to the individual, but typically in such cases proper treatment is not

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10 sought and the infected immune person can serve as a suitable host for the disease. In areas of unstable malaria transmission (much variation in transmission over several years), epidemics become a problem since people in these regions arent exposed to the disease long enough to develop immunity. Children under the age of five and pregnant women are the most vulnerable to malaria infection. Mortality in children results from three main presentations of malaria. One being an acute infection (often presented as seizures or coma) killing the infected child quickly. The second is the development of severe anemia from repeated infections of malaria parasites. The final is a low birth weight often the consequence of the mother being infected with malaria during her pregnancy. This last presentation is a major risk factor during the childs first month of life. Malaria can also cause children to become more susceptible to other infections including respiratory illnesses, diarrhea, and other common childhood illnesses. In regions of unstable malaria transmission, pregnant women are at extremely high risk of maternal and perinatal death. In regions of stable malaria transmission, infection is usually asymptomatic due to some acquired immunity, but commonly causes severe maternal anemia and babies with a low birth weight. A curious occurrence that results from malaria during pregnancy is that there is often a heavy infection of parasites concentrated in the placenta, impairing fetal nutrition. The reason for this is poorly understood (Shulman and Dorman 2002). Importance of Malaria in Africa Of at least one million annual deaths attributed to malaria, 90% occur in sub-Saharan Africa with the majority (90%) of these deaths occurring in children (WHO 2003). For many of the sub-Saharan countries more than a quarter of hospital admissions are due to malaria. Data suggests that the number of malaria cases have increased over

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11 the last decade (WHO 2004). Plasmodium falciparum is most common in Africa and is responsible for the majority of malaria deaths worldwide (WHO 2003). The appearance of a P. falciparum infection can range from almost asymptomatic, to an acute febrile, illness, to severe life-threatening cerebral malaria sometimes resulting in coma. Cerebral malaria can also cause anemia (lower than normal amount of hemoglobin or red blood cells), hemoglobinuria (presence of red blood cells in the urine), jaundice, hypoglycemia (low blood sugar), renal dysfunction (improperly functioning kidneys), psychosis, shock, and pulmonary edema (fluid in the lungs) (Warrell 2002). Important Vector Species in Africa The most important and well-studied vectors of malaria are found in the An. gambiae complex. The complex consists of seven described species: An. gambiae sensu strictu (s.s.), An. arabiensis, An. merus, An. melas, An. quadriannulatus, An. quadriannulatus species B, and An. bwambae (Service 2002). Their behavior, vector status, and distribution differ in various aspects. Anopheles quadriannulatus and An. quadriannulatus species B feed mainly on cattle and are not vectors of malaria. Anopheles bwambae is a rare mosquito found only to breed in the Semiliki forest in Uganda and is not considered an important malaria vector. Anopheles merus is a malaria vector found in lagoons and mangrove swamps along the coast of West Africa. This species breeds only in salt-water. Anopheles melas is considered to be the East African equivalent to An. merus breeding in salt-water lagoons and swamps. The two most important species in the complex are An. arabiensis and An. gambiae s.s. The first is found in regions of dry savannah. The second is found in more humid climates and readily becomes anthropophilic and endophilic (preferring to feed on humans both outdoors and indoors and to rest indoors). Because most of its time is spent

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12 indoors close to people, it is considered a highly efficient vector for malaria. Anopheles arabiensis also exhibits some endophily but will also rest and feed outdoors, sometimes feeding on domestic animals. Often An. gambiae s.s. will feed at least two times before beginning its gonotrophic cyle (Foster and Walker 2002). The extra blood meals act as a substitute for sugar (Foster and Walker 2002). Anopheles gambiae s.s. is found in almost all sub-Saharan countries. Collectively all the species in the An. gambiae complex are referred to as An. gambiae sensu lato (s.l). The most important vector in Africa after An. gambiae s.s. and An. arabiensis is An. funestus. It has a widespread distribution south of the Sahara and feeds predominately indoors. The larvae are found in more permanent waters associated with vegetation such as swamps and marshes. Anopheles funestus feeds both indoors and outdoors and prefers to rest indoors after feeding. Summary Malaria has been, and continues to be, one of the worlds most important diseases. Despite extensive attempts at eradication and control, the malaria situation has worsened over the past two decades and parasites are re-emerging in areas where they had been successfully or nearly eradicated, such as the Republic of Korea, Iraq, and Turkey (WHO 2000). Because malaria affects many of the worlds poorest nations, control of the disease has been difficult due to a lack of a solid health infrastructure. Sub-Saharan Africa is most affected by disease and is home to An. gambiae, one of the worlds most efficient malaria vectors. It is considered a highly efficient vector because it prefers to feed on humans and will take multiple blood meals (Foster and Walker 2002, Service and Townson 2002).

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13 African malaria vector control and research programs often focus on sampling adult populations of An. gambiae s.l. (Giles), An. funestus (Giles), and other important vectors. A major constraint in many developing countries where malaria is rampant is the lack of funds available to such programs. Commonly used methods for sampling adult mosquitoes include hand catches with oral or mechanical aspirators, Pyrethrum Spray Catch, and CDC light traps. These methods can become costly and labor-intensive, especially for community-based malaria control programs. The goal of this study was to develop a technique for sampling adult malaria mosquitoes for use in research and community control programs. To accomplish this, the following five objectives were delineated: 1. Test the efficacy of simple resting boxes for sampling An. gambiae s.s. mosquitoes resting indoors in semi-field conditions (screen-walled greenhouse). The design of the semi-field test structure is described by Mathenge et al. (2002). 2. Identify the preferred natural indoor resting sites for common malaria vectors, An. gambiae s.l. and An. funestus. 3. Assess the representative accuracy of the sampling of mosquito specimens through hand catches by an experienced collector using an oral aspirator. 4. Compare the accuracy of sampling common malaria vectors using a technique utilizing indoor resting boxes and bednets to 15 minutes of hand catches by an experienced collector using an oral aspirator. 5. Test the efficacy of resting baskets for sampling An. gambiae mosquitoes indoors in semi-field conditions (screen-walled greenhouse).

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CHAPTER 3 MATERIALS AND METHODS Site Descriptions All study sites were located on the shores of Lake Victoria in the Suba District, within Nyanza Province, Western Kenya (altitude 1100-1300m)(Fig. 3.1). All semi-field trials were conducted at the International Centre for Insect Physiology and Ecologys (ICIPE) Biological Station in the town of Mbita Point. Fields trials were conducted in the nearby village of Lwanda Nyamasare. The two sites are within approximately 9 km of each other. The area has two rainy seasons from March to June and October to December with an average annual rainfall of 700-1800mm. Average temperatures range from 16 to 34 Celsius. Malaria is considered holoendemic in the region. Suba District is home to around 156,000 people with the majority of these people belonging to the patrilineal Luo ethnic group. Luos make a living primarily through fishing and subsistence agriculture. Description of the area is given in Mathenge et al. (2002), Geissler et al. (2000), Okech et al. (2003), and Knols et al. (2002). Survey of Indoor Resting Habitats of Mosquitoes From March 27 th to April 18 th 2004 at varying times of the day (8:25 to 16:45 hr.), thirty-two dwellings were visited for inspection by a local expert mosquito collector with eight years experience in collecting resting mosquitoes with a plastic tube oral aspirator. This method for sampling adult mosquitoes resting indoors, often referred to as hand collection or hand catches, is a widely used method and provides a suitable technique to quantify the resting site of each collected mosquito (Service 1993, WHO 1992). 14

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15 Figure 3-1. Map of Kenya showing the study sites. The technician thoroughly searched each dwelling (Fig. 3.2) collecting mosquitoes and aspirating them into paper cups covered with netting. Searching involved not only walking around the dwelling and collecting visible mosquitoes, but also looking under and behind furniture, behind curtains, around and under pots and pans, etc. Cups containing live collected specimens were placed in a sealable container with cotton balls soaked with ethyl acetate to prepare the specimens for processing. Nine dwellings were revisited five to ten days after the first collection. Two separate rooms, each of similar size to a single dwelling, were checked in the same large house. The technician was given

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16 as much time needed to collect all mosquitoes seen. The time spent collecting at each house was noted. Dwellings were made of wood and mud with a thatch or a corrugated iron sheet roofs or made completely out of wood and corrugated iron sheets. All dwellings were built without ceilings with the underside of the roof exposed. Dwellings were all rectangular except for one circular house. The lengths of two adjacent walls inside all rectangular dwellings were measured to find the total area inside. For the circular dwelling, the diameter was measured to find the area. The height from the floor to the highest point of all dwellings was also measured from the inside. A) B) Figure 3-2. Dwellings in Lwanda. A) Single family structure B) Multiple family structure. The height where each mosquito was collected was recorded as high, medium, or low. A mark of high required the technician to lift his arm above his shoulder (roughly 161 to 240 cm from the ground). Medium was designated as any collection made from the technicians shoulders to his waist (approximately 81 to 160 cm from the ground). Mosquitoes caught below the waist of the technician, causing him to bend over or kneel, were designated as low (0 to about 80 cm from the ground). The object (wall, jug, pots, hanging shirt, etc.) the mosquito was collected from was also recorded. The objects

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17 on which the mosquitoes were collected were later divided into 5 different categories (Fig. 3.3): 1. wall/floor/door 2. plastic (plastic items such as bottles and basins) 3. cloth (items such as clothes and bed-nets) 4. furniture (more permanent items less likely to be moved around such as a bed, a cabinet, chairs, etc.) 5. temporary (found on items likely to be disturbed or moved such as lanterns or bicycles). Finally, the number of all mosquitoes that were collected in an area deemed hidden or protected was noted (Fig. 3.3). These categories are similar to the studies completed by Pal et al. (1960) and Wattal and Kalra (1960). Figure 3-3. Objects in dwellings on which mosquitoes can be found resting. Representative Accuracy of Hand Collection Twenty-one dwellings of similar size and make were visited by the expert collector and the investigator from April 1 st to April 16 th 2004. The walls were constructed of either wood and mud or wood and corrugated iron sheets. The roofs were made from either corrugated iron sheets or thatch. All dwellings were without ceilings. Fourteen were in multiple family units and seven were single family units. Mosquitoes were collected by hand using a plastic tube oral aspirator by the collector until an entire search of the house was completed (no more mosquitoes found). The time needed to hand collect all mosquitoes was noted. The time of day the collection took place ranged from

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18 8:25 to 16:30 hr. Immediately after finishing a search of the dwelling, a Pyrethrum Spray Catch (PSC) was performed. A commercial-grade pyrethroid insecticide (Doom Fast Kill, manufactured by Mortein, Australia) in 300g/494 mls containers were substituted for pyrethrum because it could be easily purchased and was safer (higher dilution of chemicals than the industrial grade) to use for the investigator and families living in the dwellings. The active ingredients of the insecticide were d-phenothrin (Pyrethroid) 1.0 g/kg and imiprothrin (Pyrethroid) 0.4 g/kg. For each dwelling the combined number of mosquitoes collected from the PSC and hand collection was found giving a total complete catch for each dwelling. From this combined number, the percentage collected by hand was found. Development of Cloth Indoor Resting Box in Semi-Field Conditions Semi-field trials were conducted from April 3 rd to June 15 th 2004 at the ICIPE, Mbita Point Biological Station at Mbita Point, western Kenya (00 25S, 34 13E). A 30cm 30cm 30cm cloth resting box, similar to the design described by Crans (1989) was tested for its ability to sample indoor resting mosquitoes. The box was made of a plain 2 cm thick galvanized wire frame with blue cotton cloth. Blue cloth was sewn to cover the outside of the box with black cotton cloth sewn to cover the inside. The attractiveness of dark colors to mosquitoes is discussed in Bidlingmayer (1994). A flap made of mosquito netting (mesh size 196) with a sleeve was sewn to the top of the box. This was closed to facilitate capture when the number of mosquitoes in the box was relatively high. The box was hung approximately 50 cm from the ground in the opposite right corner from the door of an experimental hut made of plywood (3.2m x 2.7m x 1.7m) inside a modified screen-walled 11.4m x 7.1 m x 4.2m greenhouse (Cambridge Glass

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19 House Co. Ltd., U.K.) (Fig. 3.4). Both hut and greenhouse are described by Mathenge (2002). The box was hung from the far right corner because it was the opposite corner from the door, a large source of light in the hut. Each night one of three different densities of female An. gambiae s.s. mosquitoes from Mbita strain colony at ICIPE (low = 50, medium = 100, and high = 200, as in Mathenge et al. 2002) was released into the greenhouse at approximately 21:00 hr. For each density, mosquitoes were allowed to feed on 10% sugar solution for at least 24 hours after emergence. Half of the females were not allowed a bloodmeal and half were allowed to feed on blood for ten minutes daily for three nights prior to release. This adult rearing protocol is approved by the Kenya Medical Research Institute and the Kenyan National Ethical Review Committee (2001). The resting box was checked hourly from 7:00 to 12:00 hr and again at 15:00 hr. All mosquitoes caught in the box were counted and removed each time. The number of mosquitoes collected each hour was noted. After the final collection of the day, two technicians searched and collected the remaining females in the greenhouse and then disposed of them. One similarly designed resting box treated with deltamethrin (the cloth soaked in 2 liters of water with a 25% m/m concentration) was also tested in preliminary tests but was discarded when almost 15% fewer mosquitoes were caught. The deltamethrin was chosen for testing because it could be purchased nearby and in preliminary trials some of the released mosquitoes were found dead in the box making collection of mosquitoes easier. Comparison of Accuracy of Resting box/Resting Net to Hand Collection A comparison of accuracy of the collection methods was performed in Lwanda Nyamasare village from April 13 th to June 30 th 2004.

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20 A. B. C) Figure 3-4. Components of the experimental tests A) Resting box hung in experimental hut. B) Experimental hut in modified greenhouse. C) Modified greenhouse. Preliminary trials suggested that a double size blue bednet (Supanet ) hung from the highest point inside a dwelling and then spread out and tied to the top part of the eaves could facilitate in capturing resting mosquitoes out of reach from the technician performing hand catches. To aid in collection this resting net could be lowered within reach of the technician when needed. All dwellings were made with mud walls; two had thatched roofs, the other had a roof composed of corrugated iron sheets. Three dwellings of similar size and make were chosen to compare the efficacy of three methods of indoor resting collection in a Latin Square design. The three collection methods compared were (Fig. 3.5): 1. A cloth resting box (described in previous semi-field trials) and a resting net

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21 2. A plain cardboard box of similar size to the cloth resting box (42 cm by 21.5 cm by 25 cm) and resting net 3. Aspiration for 15 minutes using a plastic tube aspirator. The time (15 min) chosen for hand collec tion was based on the results from the previous survey and recommended in WH O (1992). When trials involved hand collection, the resting net wa s tied in a knot and placed out of the way for the homeowner. Both the cardboard box and be dnets (for resting nets) are items that can easily be found in Lwanda. The blue cloth re sting box was made from materials found in Mbita (12 km away). Immediately after mo squito collections at each dwelling, a PSC was performed (as described previously). Fo llowing the completion of spray catches at each house the collection methods were rotated. Dwellings were tested every 3 days to allow ample time for the insecticide spraye d during spray catches to dissipate. The number of days was chosen based on prelim inary data from the Latin square. Trials showed that numbers of mosquitoes caught by PSC increased from the previous spray catch after two days. The sex and gonotrophi c status of all captured mosquitoes was noted. The categories for gonotrophic status (bloodfed, half gravid, gravid, and unfed) was taken from Service and Townson (2002). The mosquitoes collected were identified as either An. gambiae s.l., An. funestus Culicine, or unidentif ied Anopheline. The Latin square was run for a total of 27 experimental days in 3 complete rotations. The time taken to complete sampling of a dwelling usi ng the resting box (cloth or cardboard) with a resting net was also noted.

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22 A) B) C) D) Figure 3-5. Treatments used in the Latin Square. A) Cloth resting box hanging in dwelling B) Cardboard box hanging in dwelling C)Diagram of a resting net spread out in a dwelling D) Expert collecting mosquitoes by aspiration. Development of an Alternative to Cloth Box in Semi-Field Conditions Field Trials To evaluate the possibility of using a wicker basket (30 cm tall with an opening of 28 cm in diameter) with an attractive black cotton cloth lining inside (Bidlingmayer 1994) as method to sample indoor resting mosquitoes, a preliminary field trial was run from April 23 rd to June 30 th 2004 in the village of Lwanda Nyamasare (Fig. 3.6). The resting basket was hung approximately one meter from the floor in a corner of a bedroom in a single family dwelling. The opening was faced toward the middle of the room. The dwelling had walls made of mud and stick with an iron corrugated sheet roof.

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23 Two to five people slept there nightly. Collections from the basket were made once at approximately 10:00 hr at least every three days. The number of mosquitoes collected was noted. Semi-Field From May 15 to July 13, 2004 trials were run in semi-field conditions using a resting basket of equal size and make to the one tested in the field. The basket was hung approximately 50 cm from the ground placed in the far right corner opposite the door of an experimental hut made of plywood (3.2 m x 2.7 m x 1.7 m) (Fig. 3.6) inside a modified screen-walled 11.4 m x 7.1 m x 4.2 m greenhouse (Cambridge Glass House Co. Ltd., U.K.). Both hut and greenhouse are described in Mathenge et al., (2002). Each night one of three different densities of female An. gambiae s.s. mosquitoes (low = 50, medium = 100; and high = 200, as in Mathenge et al. 2002) from the ICIPE Mbita strain colony was released into the greenhouse at approximately 21:00 hr. The method for testing the recapture rate of the resting basket was the same as the previously described trials using the cloth resting box. For both low (50 females) and high (200 females) densities, ten trials were run while thirty-two trials were run for the medium (100 densities). A) B) Figure 3-6. Alternative to the cloth resting box. A) Cloth resting box and resting basket B) Resting basket hanging in dwelling

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CHAPTER 4 RESULTS AND DISCUSSION Results Survey of Indoor Resting Habitats of Mosquitoes Dwellings typically held one family. Some dwellings were part of a single large structure which consisted of at least three or more dwellings (rooms with doors to the outside) connected under the same roof (Fig. 4.1). All style of dwellings in the study area remained quite constant among single and multiple family structures. Dwellings consisted of a rectangular room divided by a hanging cloth sheet, reed mat, or mud wall. The sleeping area was always on the opposite side of the divider from the door. Structures containing a single family tended to be larger in area and had a higher roof than dwellings in multiple family structures (Table 4.1). In the dwellings mosquitoes were collected off a variety of different substrates with around a quarter of those found in areas hidden from plain view (Table 4.2). The category Hidden (protected) was noted in addition to the substrate. For example, a mosquito collected from a plastic item was found in an area on the item that was hidden from plain view of the collector. A General Linear Model procedure using a Wilcoxon rank sum test and a Kruskal-Wallis test was performed to analyze the percent of mosquitoes caught at low, mid, or high heights in the dwellings (SAS 2001). There was no significant difference between the heights the mosquitoes were found (Table 4.3). 24

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25 A. B. Figure 4-1. Layout of dwellings. A) Single Family B) Multiple Family Table 4-1. Mean area and height of dwellings in study area Structure Mean area ( Std) Mean Height ( Std) Number Single Family 14.4 4.2 m 2 3.4 0.4 m 2 13 Multiple Family 9.5 2.5 m 2 2.7 0.4 m 2 14 Representative Accuracy of Hand Collection Out of the 531 mosquitoes caught in 31 dwellings by both hand collection and the PSC performed afterwards, hand collection caught 32.2% of the total of the two methods. Development of Cloth Indoor Resting Box in Semi-Field Conditions Trials using a treated box were stopped once it became apparent that the percent of the mosquitoes released that were captured was less than the untreated. The mean percentages for each of the densities tested on the untreated box were similar (Table 4.4).

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26 The total mean percent of mosquitoes recaptured from all trials of the untreated box (Table 4.4) was similar to the percent caught by hand collection in 31 dwellings (32.2%). Table 4-2. Percent of total actual number of mosquitoes (437) collected from each location. Wall/Floor/Door 34% Furniture (more permanent) 30% Cloth/Bednet 24% Temporary items 8% Plastic items 4% Hidden (protected) 26%* *The category Hidden was noted separately. Table 4-3. Total actual number of mosquitoes collected at each height. Note: Numbers followed by the same letter are not significantly different (P0.05), df = 2, Chi-square = 0.97. Height dwelling where collected (in cm) Number of mosquitoes caught 0 to 80 Low 176 a 81 to 160 Medium 111 a 161 to 240 High 119 a Table 4-4. The mean percent of mosquitoes recaptured in semi-field conditions using a resting box either treated or untreated with deltamethrin. (Mean Standard Error ) Type of box Density-50 females Density-100 females Density-200 females Total percentage Untreated 39.4% 10.6 N=10 38.3% 8.9 N=10 30.7% 8.5 N=10 36.1% 9.9 N=30 Treated* 15.3% 3.0 N=3 25.3% 10.9 N=3 27.3% 6.2 N=3 22.6% 9.6 N=9 Note: (N=number of trials performed). *Trials with the treated box were stopped early once the it was shown that the it caught fewer mosquitoes than the treated. Comparison of Representative Accuracy of Resting Box/Resting Net to Hand Collection The total number of mosquitoes collected during the Latin Square experiment using each method (hand collection, cloth resting box and resting net, and cardboard box and resting net) was calculated (Table 4.5). For each method, the total number of mosquitoes caught by a method was added to the numbers caught by Pyrethrum Spray Catch (PSC) performed after the same method giving an actual overall total (Table 4.5). The percent

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27 of the overall total number of mosquitoes collected using each method was found by dividing the total actual number mosquitoes caught a method by the overall total number of mosquitoes caught for each method (Table 4.5) Table 4-5. Comparison of actual number of mosquitoes collected by the three methods tested. N=27 Hand Collection Blue resting box and resting net Cardboard box and resting net Overall total mosquitoes caught (PSC + method) 1166 1020 1060 Total mosquitoes caught using method 166 282 256 Percent of overall number of mosquitoes collected using method 14.2% 27.6% 24.1% Note: (N=number of trials performed). The cloth resting box/resting net caught 1.7 times the number of mosquitoes and almost twice the percentage of the total mosquitoes as hand of collection (Table 4.5). The cardboard box/resting net caught 1.5 times the number of mosquitoes and almost 10% more of the percentage of the total mosquitoes as hand collection (Table 4.5). The percentages captured by each of the methods on each test day were transformed to achieve linear model assumptions by: arsin x where x is the percentage of mosquitoes captured by a method on each test day. The results were then analyzed using the Tukey Multiple Comparison Procedure (SAS 2001). The catches from the cloth resting box/resting net method were significantly different than hand collection (significance was set at P0.05) (Table 4.6). There was no significant difference between the cloth resting box/resting net and cardboard box/resting

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28 net methods nor between the cardboard box/resting net and hand collection. The time taken to complete sampling using a resting box/resting net method was slightly shorter than the 15 minute time suggested by WHO (1992) for hand collection (Table 4.7). The results of the identification to species, sex, and gonotrophic status showed An. gambiae s. l. to be the most collected of the four types of mosquitoes (An. gambiae s.l., An. funestus, Culicine, and an unidentified anopheline) identified from the Latin Square catches (Tables 4.8-4.11). The highest numbers of An. gambiae s.l. mosquitoes found in the dwellings were bloodfed (882 mosquitoes) and unfed females (884 mosquitoes) (Table 4.8). This trend was also seen with An. funestus (Table 4.9). For both Culicine and unidentified Anopheline mosquitoes, males were the most collected in the dwellings (Tables 4.10 and 4.11) Development of an Alternative to Cloth Box in the Semi-Field The resting basket caught a mean of 4 mosquitoes daily in one dwelling in the field (Table 4.12). A General Linear Model procedure was performed on the percentages of mosquitoes recaptured using a Type III sum of squares test for the total percent of mosquitoes recaptured (SAS 2001). There was no significant difference (P0.05) in the total percent recaptured between the two methods (Table 4.13). There was no interaction between the density of mosquitoes and the methods used but a difference was found among the densities of mosquitoes. For further analysis, a Tukey-Kramer adjustment for multiple comparisons was performed on the pooled (resting box and resting basket) percents of mosquitoes recaptured by each density (SAS 2001). Results showed a significant difference between low and high densities (Table 4.14

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29 Table 4-6. Comparison of the accuracy (percent of the overall number of mosquitoes found in each dwelling) over the 27 test dates for the three methods tested. Methods followed by the same letter are not significantly different (P0.05). Note: F= 3.37, df = 2, 50. Date Hand Collection a Cloth Box/ Resting net b Cardboard Box/Resting net ab 13-Apr 6.7% 56.0% 2.2% 16-Apr 9.6% 12.0% 85.7% 19-Apr 48.1% 34.6% 18.9% 22-Apr 14.7% 14.2% 9.3% 25-Apr 14.5% 25.0% 20.5% 28-Apr 28.5% 28.7% 11.9% 1-May 9.0% 63.2% 5.2% 4-May 0.0% 25.0% 14.2% 7-May 8.3% 0.0% 31.6% 10-May 4.6% 35.7% 0.0% 13-May 0.0% 0.0% 23.6% 16-May 37.0% 10.7% 21.5% 19-May 13.3% 38.4% 3.2% 22-May 17.2% 17.3% 42.3% 25-May 34.7% 20% 45.6% 28-May 3.8% 20% 15.0% 31-May 14.8% 23.2% 48.0% 3-Jun 28.8% 21.4% 20.8% 6-Jun 14.4% 66.6% 18.7% 9-Jun 5.1% 19.4% 30.7% 12-Jun 11.1% 0.0% 17.0% 15-Jun 33.3% 60.7% 12.5% 18-Jun 4.1% 32.5% 37.5% 21-Jun 33.3% 10% 37.5% 24-Jun 22.8% 10.5% 9.0% 27-Jun 6.6% 29.7% 46.1% 30-Jun 30% 14.2% 16.6% Mean percentage for each method SE 15.9 12.9% a 27.1 18.5% b 23.3 18.6% ab Note: F= 3.37, df = 2, 50 Table 4-7. Comparison of time taken to complete sampling using a resting box (cloth or cardboard) and resting net to hand collection. Number of trials using a resting box and resting net Mean time taken to complete sampling using resting box and resting net Standard Error Time suggested by WHO (1992) to complete a search with hand collection 31 8.0 3.9 min 15 min

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30 Table 4-8. Actual number of An. gambiae mosquitoes caught by method, including sex and gonotrophic status, during the 27 test days of the Latin Square. Method Bloodfed Gravid Half Gravid Unfed Male Total Cloth box 18 4 8 7 9 46 Cardboard box 6 4 4 3 0 17 Hand Collection 60 21 10 30 12 133 Resting Net 163 53 26 103 35 380 PSC 635 265 138 741 243 2022 Total 882 347 186 884 299 2598 Note: For the resting net method, the numbers from both nets used in the Latin Square were calculated together. Table 4-9. Actual number of An. funestus mosquitoes caught by method, including sex and gonotrophic status, during the 27 test days of the Latin Square Method Bloodfed Gravid Half Gravid Unfed Male Total Cloth box 1 0 3 0 0 4 Cardboard box 0 0 2 0 0 2 Hand Collection 4 1 2 1 1 9 Resting Net 7 2 5 2 2 18 PSC 26 16 8 29 14 93 Total 38 19 20 32 17 126 Note: For the resting net method, the numbers from both nets used in the Latin Square were calculated together.

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31 Table 4-10. Actual number of Culicine mosquitoes caught by method, including sex and gonotrophic status, during the 27 test days of the Latin Square. Method Bloodfed Gravid Half Gravid Unfed Male Total Cloth box 0 1 0 0 0 1 Cardboard box 0 0 0 0 0 0 Hand Collection 0 0 0 0 1 1 Resting Net 0 2 2 2 3 9 PSC 8 27 4 17 24 80 Total 8 30 6 19 28 91 Note: For the resting net method, the numbers from both nets used in the Latin Square were calculated together. Table 4-11. Actual number of unidentified anopheline mosquitoes caught by method, including sex and gonotrophic status, during the 27 test days of the Latin Square. Method Bloodfed Male Unfed Total Cloth box 0 0 0 0 Cardboard box 0 0 1 1 Hand Collection 1 0 0 1 Resting Net 0 0 1 1 PSC 4 15 5 24 Total 5 15 7 27 Note: For the resting net method, the numbers from both nets used in the Latin Square were calculated together. Table 4-12. Mean number of mosquitoes caught daily by a resting basket placed in a house in the village of Lwanda. Mean number of mosquitoes caught Number of days tested 3.9 3.3 33 Table 4-13. Comparison of the resting basket to the cloth resting box of the total percentage of mosquitoes recaptured in semi-field conditions. Type of trap Total mean percent of mosquitoes recaptured ( SE) Resting Basket 33.8% 8.9 a N=52 Resting Box 36.1% 9.9 a N=30 Note: N=Number of trials run for each method, percentages followed by the same letter are not significantly different (P0.05), F= 0.51, df = 1, 76)

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32 Table 4-14. Mean percent of mosquitoes recaptured by each density used for both the resting box and resting basket. Density Mean Percent of Mosquitoes Recaptured High 31.3% a Low 39.1% b Medium 35.6% ab Note: N=Number of trials run for each method, percentages followed by the same letter are not significantly different (P0.05), F= 41.47, df = 1, 76) Discussion Indoor Resting Places of Mosquitoes The most important malaria vector in Africa, An. gambiae, will readily rest in the dwellings of rural villages (Service and Townson 2002). A common method for sampling populations of these resting mosquitoes is a search of a dwelling by a trained technician using an oral aspirator (Service 1993, WHO 1992). A properly trained technician performing the search is essential as the mosquitoes are difficult to find and catch. This idea is reinforced by the results of this study. Since it is also generally thought that both male and female mosquitoes are attracted to darker areas indoors, one would expect to find mosquitoes resting at low heights around the dwelling since they would be further away from a main light source, the eaves. Although a larger percent of mosquitoes were found at heights from 0 to 80 cm, the number of catches was not significantly different from those higher than 80 cm. About a quarter of the mosquitoes (26%) were also found in an area not in plain view of the collector. The results of this study would suggest that it would be difficult to accurately pinpoint an area in a dwelling to focus the aspiration as mosquitoes. The mosquitoes were found on a broad variety of substrates at various heights with many resting in areas that would require the collector to search under, above, or around household items. Many times during the study the collector needed to move furniture, pots, clothes, and other household items to be able to

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33 reach the mosquitoes. This emphasizes the need for adequate training for anyone attempting to sample resting mosquitoes by hand collection using an oral or mechanical aspirator. Comparison of Sampling Techniques The local expert technician had eight years experience with hand catches, likely more than most people attempting that method. This would suggest a high quality of collection in comparison to other hand collectors. Since the expert was given as much time needed to complete a search of each dwelling during the initial part of the study, it was assumed that the majority of the mosquitoes collected in the PSC, performed after the search, were resting above the reach and out of the sight of the collector. Since the expert caught only approximately a third (32%) of the total number found, it was decided that any sampling method developed should account for mosquitoes resting on the exposed underside of roofs (made of thatch or corrugated iron sheets) as this would be a likely area to find them (Lindsay et al. 1995, Schofield and White 1983). Resting boxes have been a well established method for sampling mosquitoes (Service 1993). Few, however, have been used indoors. The results from the semi-field trials suggest that the cloth resting box could collect close to the same percentage of mosquitoes (36.1%) as the expert hand collector in the field (32.2%). However, it is likely that a cloth resting box placed near a similar location in a dwelling could not accurately sample mosquitoes that prefer to rest in the underside of the roof. This assumption is based on the idea that since the expert technician was not able to sample mosquitoes resting so high, a stationary trap would also have the same difficulty. For this reason, a resting net was tested with the cloth resting box. The results of the Latin Square trial suggest that the cloth resting box/resting net method could catch a significantly

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34 higher percentage of the total population resting in a dwelling than that of an expert performing hand catches using an oral aspirator. To increase the practicality of the method the resting basket was tested in the semi-field. Because there was no significant difference in recapture rates between the resting basket and the cloth resting box, the resting basket could be an acceptable alternative to the cloth resting box. The cloth resting box (or resting basket) and resting net method has a number of advantages over hand collection: 1. It can more accurately sample the population resting in a dwelling (a higher percentage of mosquitoes will be collected). 2. The materials needed for construction are inexpensive and can be easily found in many rural areas as opposed to the time and money to needed to train and/or hire a technician to perform hand catches. 3. It requires less training, not only because mosquitoes are easier to see in the black cloth of the box and in the bright blue of the net, but also because the investigator only needs to search a set area (the trap and the net) rather than the whole house. 4. The time needed to complete a search is shorter than 15 minutes, which is commonly assigned for hand catches (WHO 1992). The use of resting boxes and nets also has advantages over the PSC technique. Although a greater percentage of mosquitoes can be caught with spray catches, the spray technique is too expensive and impractical for use on a regular basis for local control programs. Pyrethrum spray catches also expose the investigators and the members of the tested household to potentially harmful chemicals that may take several days to dissipate (Service 1993). This study showed that cloth resting boxes or resting baskets used with resting nets can be a practical method for local malaria control programs to sample indoor resting mosquitoes.

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35 Summary Malaria continues to be one of the worlds most important human diseases. Although, has been eradicated in some areas, it continues to be a threat to 40% of the worlds population. Many of those people affected by the disease live in some of the poorest nations in the world. The lack of resources and infrastructure has produced major obstacles to malaria control in those countries. Because malaria affects many people living in impoverished nations, many of the more expensive and technical methods utilized by developed countries for mosquito and disease control are not feasible. An initiative to create sustainable malaria control programs at a community level has been implanted by international organizations such as the WHO (2000). As local malaria control programs become more accepted, technology is needed to keep such programs sustainable. Developing new methods and tools for malaria surveillance and control, which are both user-friendly and economically viable, are essential for this to happen. Because the most important vector of malaria in Africa, An. gambiae s.l., readily rests indoors after feeding, developing practical ways to sample populations of those mosquitoes would be valuable. The sampling technique tested in this study, was developed specifically for malaria control programs based at community levels. The materials needed for the technique can be found inexpensively in rural villages where community malaria control programs are run. The technique provides a more user-friendly alternative to both PSC and hand catches, both of which are commonly used in malaria control programs and research (Service 1993, WHO 1992). Members of control programs sampling with the new technique have only to collect from a defined area (box or basket and net) compared to hand catches where a complete

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36 search of a dwelling is required. Mosquitoes can also be seen easier on the black cloth in the box or basket and on the blue cloth of the net than on the walls, thatch, and other items in the dwellings. Since mosquitoes can be found and collected more easily than hand catches, less training and experience is needed to effectively sample a dwelling. Although sampling with PSC captures most, if not all, mosquitoes in a dwelling, the cost of the materials needed (insecticide, sheets, protection against chemicals) is often beyond the means of community malaria control programs. The use of such a method could not be sustained for very long due to its expense and the constant need for insecticide. The safe use and storage of the chemicals needed for PSC is also an important issue for the safety of the people doing the sampling and the homeowners. Since no chemicals are needed with the new technique, it can be performed safely by members of control programs with no danger to the homeowners. Because the materials needed for the new technique can be inexpensively purchased once, it can be used indefinitely. Further research should focus on developing more inexpensive and practical methods of malaria surveillance and control that are accessible to the people facing the greatest disease load. Such research should be broadly based in multiple disciplines due the complexity of the malaria problem. Things like access to healthcare, economic stability, and cultural behavior also play important roles in the transmission of malaria and need to be considered. Even though much of the worlds malaria control stems from international organizations, a larger global effort is needed to have a lasting effect on malaria control.

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CHAPTER 5 NOTES ON THE DEVELOPMENT OF A PRACTICAL ANOPHELES GRAVID TRAP Introduction The ability to sample the populations of host-seeking mosquitoes is essential to the evaluation of malaria control programs. The exposure to potentially infectious bites on people sampling host-seeking mosquitoes makes many such sampling methods ethically questionable. As an alternative to sampling host-seeking adults, gravid traps can provide similar data (reproductive capacity of mosquito populations and the dispersal patterns of mosquitoes as they seek larval habitats after biting humans) without the risks of exposure. Such information is of great importance to vector control programs as many programs focus on and have seen success with larvicide application in potential or known larval habitats. Gravid traps that have been tested and used in research and mosquito control programs, sample only Culicine mosquitoes (Service 1993, Ritchie et al. 2004). Many of these traps focus specifically on collecting gravid Culex mosquitoes (Service 1993, Mboera et al. 2000). Although a variety of methods exist to sample the eggs of malaria-carrying Anopheline mosquitoes, no technique has been devised to effectively trap and sample these mosquitoes solely when they are gravid (Service 1993). Gravid traps, like those used to sample Culicine mosquitoes, would become problematic in many malaria endemic regions because they require a significant degree of skill and maintenance to ensure they are working correctly. The commonly used, CDC Gravid Trap (J. W. Hock Co. Gainesville, FL.), requires a net collection bag, motorized 37

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38 trap with a fan, pan with water, a battery, and a battery charger. They would also be quite costly to programs, particularly in African countries where resources available for control are usually very limited. This study was undertaken to develop a much more practical gravid trap that effectively samples egg-laying adults. A simple trap would have a number of advantages over present traps: 1. It would not require skilled personnel for sampling mosquitoes 2. It would be far less expensive than current gravid traps 3. Data collected from traps will most likely reflect a more realistic composition and behaviour of the egg-laying mosquito population since no artificial attractants will be used. 4. It could be easily replicated and therefor e be more likely to be used in mosquito surveillance programs; since collected adults and eggs would be disposed, it could directly aid malaria control programs. The objective of this investigation was to develop a practical gravid trap using materials readily available in rural areas where malaria is endemic. Such a trap would be beneficial to malaria control programs at a community level where resources are often scarce. Preliminary Semi-Field Trials in Florida Semi-field trials were conducted weekly from June 8 th to October 1 st 2004 at the United States Department of Agricultures (USDA) Center for Medical, Agricultural, and Veterinary Entomology (CMAVE) located in Gainesville, Florida. Traps were tested singly in screened cages (2m x 2m x 2m). Prior to placement of the traps, 70 gravid Anopheles albimanus females were released into each cage and allowed to acclimatize for 3-4 hours. The females were left in each cage with a trap for 24 to 48 hours after which the number of females caught was noted. After collections were made the cage was left open for at least four days to release any females left in the cage.

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39 One design was tested and modified from oviposition basins from CDC Gravid Traps (J. W. Hock Co. Gainesville, FL.), plastic funnels, and plastic 40 ounce peanut butter jars. All designs were spray-painted with a matte finish primarily of black and white (Fig. 5.1). Spray-painted traps were left outside for at least six weeks prior to testing to remove any odors. Some variations of trap designs had modifications such as the addition of black plastic mesh, the presence of cotton soaked in sugar water, and various amounts of hay infusion (Fig. 5.2). All designs tested had approximately 2 liters of well water placed in the basin. A) B Figure 5-1. Design of oviposition trap. A) Full view. B) Collecting cartridge

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40 A) B) C) Figure 5-2. Modifications to design. A) with clothes hangers B) with black mesh C) with vial with sugar solution. Semi-Field Trials in Kenya From February 17 th to March 11 th 2004, 1 to 3 color variations of 4 trap designs were tested daily in a screened cage (3m x 3m x 3m) at the ICIPE, Mbita Point Biological Station at Mbita Point, western Kenya. A black plastic bucket (35cm x 30cm) was placed in a hole in the middle of the screened cage. The bucket was placed so that the top was even with the ground level. Some of the soil removed from the hole was placed back into the bucket and water from Lake Victoria (of varying amounts depending on the trap design) was also placed in the bucket. The water was changed daily. Traps were made of 2cm thick plain galvanized wire, white mosquito netting and either black or blue colored cotton cloth. Daily, fifty gravid An. gambiae s.s. females were collected from the ICIPE Mbita strain colony and released into the screened cage at 6 pm. The following day from 6:30 to 12:00 hr, traps were checked for any females. From14:00 to 16:00 hr. the remaining females were aspirated out of the screened cage. Following colony protocol all gravid females had been allowed to blood feed on the investigators arm for ten minutes daily for three consecutive days prior to their release. A colony egg trap was

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41 placed in the colony cage from which the tested gravid females were taken to note the ovipositional success of that batch of females. The first experimental day, no trap was placed on the bucket and eggs were noticed the following morning. On February 21 st the bucket was removed from the screened cage, fresh lake water placed in it and then positioned in a sunlit area for 6 hours to cook. The bucket was checked for any mosquito eggs that may have been laid by wild mosquitoes and then returned to the screen cage prior to the release of that days colony mosquitoes. This cooking was done for each of the remaining trials. On February 23 rd the soil in the bucket was replaced by mud from a known Anopheles larval site in Mbita that was not subject to larval control. On March 5 th dark blue and black cotton cloth was draped on the outside of the screened cage and water was poured on it prior to release of mosquitoes to increase the humidity. This was again continued daily. On March 7 th filter paper used in the colony egg traps was attached to the lower part of one of the traps and positioned half in the water to entice the females into ovipositing. On March 8 th the screened cage was moved into a modified greenhouse (Mathenge et al. 2002). On March 12 th a miniature version of the black trap (Fig. 5.3) was placed in a colony cage over a standard egg trap used for maintaining colonies. Only one gravid female was captured during all semi-field trials. Field Trials in Kenya From February 19 th to March 17 th 2004 field trials of the traps designs were conducted. Field sites were chosen in the Kamasengre district of Rusinga Island, about 12 km away by road from Mbita. The trap bucket was placed in a hole dug in the ground approximately 4m from a single family dwelling. Soil from the hole and fresh lake water were added to the bucket. Four days later mud from a nearby larval site was placed

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42 in the bucket instead of soil from the hole. The design placed on or in the bucket was checked daily at approximately 7:00 hr. A week after these preliminary trials two more sites were added; one had a bucket placed approximately a meter from a large larval site (about 10m long) and another placed far from any larval sites or homes. This location was considered a control. Mud from the larval site was placed in both of these new buckets as well as lake water. The lake water was changed daily. Trap designs were rotated daily among the three field sites and the semi-field site. On March 17 th the field sites were closed. Results Florida Trials Out of 31 traps tested from June 8th to October 1st, 14 traps captured mosquitoes. Out of those 14 traps, an average of 2.36 1.74 mosquitoes was caught. The mean recapture ( SE) rate of the mosquitoes released for the 14 traps was 3.3 2.3%. Kenya Trials In the semi-field experiment only one gravid female was captured. This was by the emergence design with black cloth. Only one mosquito (a Culicine) was caught in trials in the field. The emergence design with blue cloth placed near the larval habitat caught the mosquito. Discussion The description of larval habitats for An. gambiae s.l. has been well studied (Warrell and Gilles, 2002). Larvae are typically found in sunlit temporary pools often associated with humans. They develop in roadside ditches, wheel ruts, hoof prints from wild and domestic animals, concrete holes, and village pots. Larvae can even to survive close to five days on damp soil (Koenraadt et al. 2003).

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43 A) B) C) D) Figure 5-3. Trap designs in Kenya. A) Mbita (two variations) B) Emergence C) Florida D) Cone (two variations). Most larval sites contain fresh water without much debris. Preliminary trials conducted by other investigators in Mbita Point, Kenya showed that An. gambiae will readily oviposit in plastic tubs and buckets used for washing clothes and commonly found around the town and nearby communities (Pers.comm. U. Filinger, University of Durham, England). Although the identification of larval sites can be accomplished without much difficulty, the identification of oviposition attractants has yet to be determined.

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44 One of the obstacles faced by testing a practical gravid trap out in the field is that because An. gambiae will lay eggs in such a wide variety of habitats that are commonly found in great numbers around the area, the trap would need to be at least as attractive to females as the natural habitats. By placing a trap over a bucket or other potential larval habitat the site no longer becomes sunlit and therefore probably less attractive. Further investigations into oviposition chemical and visual attractants would be needed to overcome the deterrent of shade created by the trap. A problem faced by semi-field experiments is that gravid colony females that have emerged from the same batch of colony pupae may not oviposit at the same time if at all. If fifty gravid females of the same age and exposed to the same amount of blood are released into a semi-field cage only a small number of females if any may actually try to oviposit. More time spent rearing test mosquitoes should be taken to insure that conditions are created to enhance the number gravid females that attempt to oviposit each day. Although a practical method for sampling gravid Anopheles has yet to be developed, such a tool would be of great use to malaria control programs. A practical gravid trap could be a safer alternative to other sampling methods involving chemicals or sampling host-seeking mosquitoes. Identifying oviposition attractants for Anopheles mosquitoes could not only aid the development and use of gravid traps but provide insight to the ecology of the mosquito and create more options for mosquito monitoring programs.

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LIST OF REFERENCES Bidlingmayer, W.L. 1994. How mosquitoes see traps: role of visual responses. J. Am. Mosq. Control Assoc. 10(2):272-279. Bogh, C., Pedersen, E.M., Mukoko, D.A., and J.H. Ouma. 1998. Permethrin-impregnated bednet effects on resting and feeding behaviour of lymphatic filariasis vector mosquitoes in Kenya. Med Vet Entomol. 12: 52-59. Centers Disease Control and Prevention (CDC). April 23, 2004a. CDCs originsand malaria. www.cdc.gov/malaria/history/eradication_us.htm Accessed September 2004. Centers for Disease Control and Prevention (CDC). April 23, 2004b. Eradication of malaria in the United States (1947-1951). www.cdc.gov/malaria/history/eradication_us.htm Accessed September 2004. Conteh, L., Sharp, B., Streat, E., Barreto, A., and S. Konar. 2004. The cost and cost-effectiveness of malaria vector control by residual insecticide house-spraying in southern Mozambique: a rural and urban analysis. Trop. Med. Int. Health. 9(1):125-132. Crans, W. J. 1989. Resting boxes as mosquito surveillance tools. from Proceedings of the Eighty-Second Annual Meeting of the New Jersey Mosquito Control Association, Inc., pp.53-57. http://www.rci.rutgers.edu/~insects/restbox.htm Accessed September 2004. Das, P.K., Sivagnaname, N., and D.D. Amalraj. 1997. A comparative study of a new insecticide-impregnated fabric trap for monitoring adult mosquito populations resting indoors. Bull. Ent. Res. 87: 397-403. Deressa, W., Ali A., and F. Enqusellassie. 2003. Self-treatment of malaria in rural communities, Butajira, southern Ethiopia. Bulletin WHO 81(4): 261-268. Durden L. and G. Mullen. 2002. Introduction. Medical and Veterinary Entomology. Mullen, G. and L. Durden Eds. Academic Press. San Diego. pp. 1-13. Edman, J., Kittayapong, P., Linthicum, K., and T. Scott. 1997. Attractant resting boxes for rapid collection and surveillance of Aedes aegypti (L.) inside houses. J. Am. Mosq. Control Assoc. 13(1):24-27. 45

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46 Foster, W. and E. Walker. 2002. Mosquitoes (Culicidae). Medical and Veterinary Entomology. Mullen, G. and L. Durden Eds. Academic Press. San Diego. pp. 203-262. Fullerton, H., and E. Bishop. 1933. Improved rural housing as a factor in malaria control. South. Med. J. 26(5):465-8. Gallup, J., and J. Sachs. 2001. The economic burden of malaria. Am. J. Trop. Med. Hyg. 64(1, 2) S: 85-96. Gamage-Mendis, A., Carter, R., Mendis, C., De Zoysa, A., Herath, P., and K. Mendis. 1991. Clustering of malaria infections within an endemic population: risk of malaria associated with the type of housing construction. Am. J. Trop. Med. Hyg. 45(1):77-85. Garfield, R. 1999. Malaria control in Nicaragua: social and political influences on disease transmission and control activities. Lancet 354: 414-18. Geissler, P., Nokes, K., Odhiambo, R., Aagard-Hansen, J, and J. Ouma. 2000. Children and medicines: self-treatment of common illnesses among Luo schoolchildren in western Kenya. Soc. Sci. Med. 50: 1771-1783. Gilles, H. 2002. Historical outline. Essential Malariology 4 th ed. Warrell, D.and H.Gilles eds. Oxford University Press Inc. New York. pp. 1-7. Githeko, A., Service, M., Mbogo, C., Atieli, F., and F. Juma. 1994. Origin of blood meals in indoor and outdoor resting malaria vectors in western Kenya. Acta Trop. 58: 307-316. Harwood, R. and M. James. 1979. Entomology in Human and Animal Health. 7 th ed. Macmillan Publishing Co. New York. Honigsbaum, M. 2001. The Fever Trail. Farrar, Straus, and Giroux. New York. Kager, P. 2002. Malaria control: constraints and opportunities. Trop. Med. Int. Health 7(12):1042-1046. Killeen, G., Fillinger, U., Kiche, I., Gouagna, L., and B. Knols. 2002. Eradication of Anopheles gambiae from Brazil: lessons for malaria control in Africa? Lancet. Infect. Dis. 2(6): 18-27. Kenya Medical Research Institute. 2001. Approval from the 87 th meeting of the Kenya Medical Research Institute and National Ethical Review Committee for screen-house mosquito rearing and semi-field experimental environments for risk-free malaria transmission studies. Nairobi, Kenya.

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47 Koenraadt, C., Paaijmans, K., Githeko, A., Knols, B., and W. Takken. 2003. Egg hatching, larval movement and larval survival of the malaria vector Anopheles gambiae in desiccating habitats. Malaria J. 2:20. Konradsen, F., Amerasinghe, P., Van Der Hoek, W., Amerasinghe, F., Perera, D., and M. Piyaratne. 2003. Strong association between house characteristics and malaria vectors in Sri Lanka. Am. J. Trop. Med. Hyg. 68(2):177-181. Knols, B., Njiru, B., Mathenge, E., Mukabana, W., Beier, J., and G. Killen. 2002. MalariaSphere: A greenhouse-enclosed simulation of a natural Anopheles gambiae (Diptera: Culicidae) ecosystem in western Kenya. Malaria J. 1:19. Komar, N., Pollack, R., and A. Spielman. 1995. A nestable fiber pot for sampling resting mosquitoes. J. Am. Mosq. Control Assoc. 11(4):463-467. Kroeger, A., Ordonez-Gonolez, J., and A. Avina. 2002. Malaria control reinvented: Health sector reform and strategy development in Colombia. Trop. Med. Int. Health (7)5: 450-8. Lindblade, K., Walker, E., and M. Wilson 2000. Early warning of malaria epidemics in African highlands using Anopheles (Diptera: Culicidae) indoor resting density. J. Med. Entomol. 37(5):664-674. Lindsay, S., Jawara, M., Paine, K., Pinder, M., Walraven, G. and P. Emerson 2003. Changes in house design reduce exposure to malaria mosquitoes. Trop. Med. and Int. Health 8(6) :512-517. Lindsay, S., Armstrong, J., Amstrong Schellenberg, J., Zeiler, H., Daly, R., Salum, F., and H. Wilkins. 1995. Exposure of Gambian children to Anopheles gambiae malaria vectors in an irrigated rice production area. Med. Vet. Entomol. 9:50-58. Magnussen, P., Ndawi, B., Sheshe, A., Byskov, J., and K. Mbwana. 2001. Malaria diagnosis and treatment administered by teachers in primary schools in Tanzania. Trop. Med. Int. Health 6(4):273-279. Marquadt, W., Demaree, R., and R. Grieve. 2000. Parasitology and Vector Biology. Academic Press. San Diego. Mathenge, E., Killeen, G., Oulo, D., Irunga, L., Nfegwa, P., and B. Knols. 2002. Development of an exposure-free bednet trap for sampling afrotropical malaria vectors. Med. Vet. Entomol. 16: 67-74. Mboera, L., Takken, W., Mdira, K. and J. Pickett. 2000. Sampling gravid Culex quinquefasciatus (Diptera: Culicidae) in Tanzania with traps baited with synthetic oviposition pheromone and grass infusions. J. Med. Entomol.37(1):172-176.

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48 Muirhead-Thomson, E. 1982.Behaviour patterns of blood-sucking flies. Pergamon Press. Oxford. Okech, B., Gouagna, L., Killen, G., Knols, B., Kabiru, E., Beier, J., Yan, G., and J. Githure. 2003. Influence of sugar availability and indoor microclimate on survival of Anopheles gambiae (Diptera: Culicidae) under semifield conditions in western Kenya. J. Med. Entomol. 40 (5): 657-663. Pal, R., Nair, C., Ramalingham, S., Patil, P., and B. Ram. 1960. On the bionomics of vectors of human filariasis in Ernakulam (Kerala), India. Ind. J Malariol. 14: 595-604. Ritchie, S., Long, S., Smith, G., Pike, A., and T. Knox. 2004. Entomological investigations in a focus of dengue transmission in Cairns, Queensland, Australia, by using sticky ovitraps. J. Med. Entomol. 41(1):1-4. SAS Institute. 2001. SAS system for Windows, release 8.2. SAS Institute. Cary, NC. Schofield, C. and G. White. 1983. Engineering against insect-borne diseases in thedomestic environment: House design and domestic vectors of disease. Am. J. Trop. Med. Hyg. 78: 285-292. Service, M. 1993. Mosquito Ecology. Field Sampling Methods. 2 nd Ed. ElsevierScience Publishers, Essex, UK. Service, M. 2002 Characteristics of some major Anopheles vectors of human malaria. Essential Malariology 4 th ed. Warrell, D.and H.Gilles eds. Oxford University Press Inc. New York. pp. 326-332 Service, M and H. Townson. 2002. The Anopheles vector. Essential Malariology 4 th ed. Warrell, D.and H.Gilles eds. Oxford University Press Inc. New York. pp. 59-84. Sexton, J., Ruebush, T., Brandling-Bennett, A., Breman, J., Roberts, J., Odera, J., and J. Were. 1990. Pyrethrin-impregnated curtains and bed-nets prevent malaria in western Kenya. Am. J. Trop. Med. Hyg. 43:11-18. Shulman C. and E. Dorman. 2002. Clinical features of malaria in pregnancy. Essential Malariology 4 th ed. Warrell, D.and H.Gilles eds. Oxford University Press Inc. New York. pp. 218-235 Smith, G.E. 1942. The keg shelter as a diurnal resting place of Anopheles quadrimaculatus. Am. J. Trop. Med. 58: 307-316.

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49 Tan, D., Upshur, R., and N. Ford. 2003. Global plagues and the Global Fund: Challenges in the fight against HIV, TB, and malaria. BMC Int.Health Human Rights. 3:2. Warrell, D. 2002. Clinical features of malaria. Essential Malariology 4 th ed. Warrell, D.and H.Gilles eds. Oxford University Press Inc. New York. pp. 191-218. Wattal, B. and N. Kalra. 1960. Studies on Culicine mosquitoes: Preferential indoor resting habits of Culex fatigans Wiedmann, 1828, near Ghaziabad, Utter Pradesh. India. Ind. J Malariol. 14: 605-616. World Health Organization (WHO). 1992. Entomological Field Techniques for Malaria Control: Part 1 Learners Guide. World Health Organization, Geneva. World Health Organization (WHO) 1995. Technical Report Series 857. Vector control for malaria and other mosquito borne diseases. World Health Organization, Geneva. World Health Organization (WHO). 2000. Technical Report Series 892. WHO Expert Committee on Malaria. World Health Organization, Geneva. World Health Organization (WHO). 2003. Africa malaria report 2003. World Health Organization, Geneva. World Health Organization (WHO). 2004. Fact sheet 94, Roll Back Malaria InfoSheet. World Health Organization, Geneva. http://www.who.int/mediacentre/factsheets/fs094/en/print.html fact sheet. Accessed September 2004. Yasuno, M., Rajagopalan, P.K., Kazmi, S.J., and G.C. La Brecque. 1977. Seasonal change in larval habitats and population density of Culex fatigans in Delhi villages. Ind. J. Med. Res. 65: 52.

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BIOGRAPHICAL SKETCH Justin Eric Harbison was born in St. Paul, Minnesota, on September 1, 1977, to Kent and Judy Harbison. He graduated from St. Paul Academy in St. Paul, Minnesota, in 1996. Justin graduated from Macalester College in May 2000 with a Bachelor of Arts degree. Prior to pursuing his masters degree at the University of Florida, Justin spent nine months as a field technician in the Organization for Tropical Studies Palo Verde field station in the Guanacaste region of Costa Rica. 50


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DEVELOPMENT OF A PRACTICAL TECHNIQUE FOR SAMPLING THE
AFROTROPICAL MALARIA VECTORS Anopheles gambiae S.L. AND An. funestus















By

JUSTIN ERIC HARBISON


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
2005

































This document is dedicated to my parents, Kent and Judy Harbison.















ACKNOWLEDGMENTS

I would like to thank my parents, Kent and Judy Harbison, for their unwavering

support. I thank Dr. Jonathan Day, Dr. Dan Kline, Dr. Roxanne Rutledge-Connelly, and

Dr. Sandra Allan for their help in Florida. I thank Dr. Richard Mukabana, Dr. Evan

Mathenge, Dr. Bart Knols, Dr. Gerry Killeen, and Dr. Ulrike Fillinger for their

willingness to help out an American student. I thank everyone else back in Kenya not

only for helping me complete this project, but for their willingness to teach me about

their great country. I thank Debbie Hall for all the administrative help. I thank Marinela

Capanu for statistical assistance and Scott Weihman for the use of his photos. Finally,

special thanks go to Camille Francisco for her undying patience while I was in Africa.
















TABLE OF CONTENTS

page


A C K N O W L E D G M E N T S ................................................................................................. iii

LIST OF TABLES ........... .. ............. ................................ vi

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

A B STR A C T ..................... ................................... ........... ................. viii

1 IN TR OD U CTION ............................................... .. ......................... ..

A approaches to M alaria Control. ...................................................... .....................1
H history of M alaria Control ..................................................................................... 2
Future of M alaria C control ............................................... ...... .............. ...............
Role of Rural Housing in Malaria Transmission...................................................3
Sam pling M ethods for M alaria Vectors Indoors ........................................ ...............4
Indoor Sampling of Resting Mosquitoes ........................................ ...............5

2 LITER A TU R E REV IEW ............................................................. ....................... 7

H historical O overview .................... .................................. .... ........ ................. 7
Im portance of the D disease W orldw ide ................................. ..................................... 8
D description of D disease Transm mission .................................. ............... ............... 8
Im portance of M alaria in A frica............................................................................... 10
Im portant V ector Species in Africa................................ .................................... 11
S u m m a ry .......................................................................................................1 2

3 M ATERIALS AND M ETHOD S ........................................ ......................... 14

Site Descriptions ...................................... .......................... ............... 14
Survey of Indoor Resting Habitats of M osquitoes ....................................................14
Representative Accuracy of Hand Collection........................................ ...............17
Development of Cloth Indoor Resting Box in Semi-Field Conditions..................... 18
Comparison of Accuracy of Resting box/Resting Net to Hand Collection ..............19
Development of an Alternative to Cloth Box in Semi-Field Conditions....................22
F ield T rials ............................................... .... ......... ...... 2 2
S e m i-F ie ld ...................................................................... 2 3









4 RESULTS AND DISCU SSION ........................................... .......................... 24

Results .................. ............................. ............ ...............24
Survey of Indoor Resting Habitats of Mosquitoes ............................................24
Representative Accuracy of Hand Collection ..................................................25
Development of Cloth Indoor Resting Box in Semi-Field Conditions ..............25
Comparison of Representative Accuracy of Resting Box/Resting Net to Hand
C collection ................. ...................................... .. ... .... ................. 26
Development of an Alternative to Cloth Box in the Semi-Field.........................28
D iscu ssion ..... ............... ....................................32
Indoor Resting Places of Mosquitoes................... .. ......... .............32
Com prison of Sam pling Techniques ...................................... ............... 33
Sum m ary ............. ............................................... .... ... ...... .. 35

5 NOTES ON THE DEVELOPMENT OF A PRACTICAL Anopheles GRAVID
T R A P ................................................................................3 7

Introduction .................................... .. ....... ..................... 37
Prelim inary Sem i-Field Trials in Florida......................................... ............... 38
Sem i-F ield T rials in K eny a .............................................................. .....................40
Field Trials in K enya .................................. .. ... ... ..... ............ 41
Results ........... ............. ........ .................................42
F lo rid a T ria ls ................................................................................................. 4 2
K en y a T rials ................................................................4 2
D isc u ssio n ............................................................................................................. 4 2

L IST O F R E FE R E N C E S ............. ................. ..................................................45

B IO G R A PH IC A L SK E T C H ....................................................................................... 50























v















LIST OF TABLES


Table page

4.1. M ean area and height of dwellings in study area.....................................................25

4.2 Percent of total actual number of mosquitoes (437) collected from each location......26

4.3 Total actual number of mosquitoes collected at each height.......................................26

4.4 The mean percent of mosquitoes recaptured in semi-field conditions using a resting
box either treated or untreated with deltamethrin. ............. ................................... 26

4.5 Comparison of actual number of mosquitoes collected by the three methods tested. .27

4.6 Comparison of the accuracy (percent of the overall number of mosquitoes found in
each dwelling) over the 27 test dates for the three methods tested ........................29

4.7 Comparison of time taken to complete sampling using a resting box (cloth or
cardboard) and resting net to hand collection. ................................. ............... 29

4.8 Actual number of An. gambiae mosquitoes caught by method, including sex and
gonotrophic status, during the 27 test days of the Latin Square. ..........................30

4.9 Actual number of An. funestus mosquitoes caught by method, including sex and
gonotrophic status, during the 27 test days of the Latin Square .............................30

4.10 Actual number of Culicine mosquitoes caught by method, including sex and
gonotrophic status, during the 27 test days of the Latin Square. ...........................31

4.11 Actual number of unidentified anopheline mosquitoes caught by method,including
sex and gonotrophic status, during the 27 test days of the Latin Square. ...............31

4.12 Mean number of mosquitoes caught daily by a resting basket placed in a house in
the village of Lwanda. ........................ ...... ................ ..1... ... 31

4.13 Comparison of the resting basket to the cloth resting box of the total percentage of
mosquitoes recaptured in semi-field conditions ................................. ............... 31

4.14 Mean percent of mosquitoes recaptured by each density used for both the resting
box and resting basket. ........................... .................... .. .. .. .... ........... 32















LIST OF FIGURES


Figure p

3.1 Map of Kenya showing the study sites. .......................... ..................... 15

3.2 Dwellings in Lwanda. A) Single family structure B) Multiple family structure.........16

3.3 Objects in dwellings on which mosquitoes can be found resting.............................17

3.4 Components of the experimental tests A) Resting box hung in experimental hut. B)
Experimental hut in modified greenhouse. C) Modified greenhouse. ...................20

3.5 Treatments used in the Latin Square. A) Cloth resting box hanging in dwelling B)
Cardboard box hanging in dwelling C)Diagram of a resting net spread out in a
dwelling D) Expert collecting mosquitoes by aspiration. ......................................22

3.6 Alternative to the cloth resting box. A) Cloth resting box and resting basket B)
Resting basket hanging in dwelling. ............................................. ............... 23

4.1 Layout of dwellings. A) Single Family B) Multiple Family ....................................25

5.1 Design of oviposition trap. A) Full view. B) Collecting cartridge ...........................39

5.2 Modifications to design. A) with clothes hangers B) with black mesh C) with vial
w ith sugar solution. ....................... ...................... ................... .. .....40

5.3 Trap designs in Kenya. A) Mbita (two variations) B) Emergence C) Florida D) Cone
(tw o variations). .................................................... ................. 43















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

DEVELOPMENT OF A PRACTICAL TECHNIQUE FOR SAMPLING THE
AFROTROPICAL MALARIA VECTORS Anopheles gambiae S.L. AND An. funestus


By

Justin Eric Harbison

May 2005

Chair: Jonathan Day
Major Department: Entomology and Nematology

Malaria, transmitted by Anopheline mosquitoes, is not only the most important

insect-borne disease but one of the top three infectious diseases in the world. Resting

boxes are a well-established method for sampling Anopheline mosquitoes. Few resting

box designs, however, are used indoors. A practical method, utilizing cloth resting boxes

and "resting nets" for sampling indoor malaria vectors of Africa, was developed in semi-

field (a modified greenhouse) and field conditions. In semi-field conditions, a cloth

resting box recaptured 36.1 9.9% (mean SE) of three different densities of An.

gambiae s.s. (Giles) females of varying gonotrophic status. The accuracy method then

was compared to the collection of mosquitoes using an oral aspirator in a rural Kenyan

village. The developed method caught a significantly higher percentage (P=0.05) of

resting mosquitoes than hand catches with an oral aspirator and required slightly less time

to complete (8.0 + 3.9 minutes versus 15.0 + 0.0 minutes). A "resting basket" was also

developed as an even more practical alternative to the cloth resting box. The resting









basket was tested under the same semi-field conditions as the resting box. No significant

difference (P=0.47) was found in recapture rates between the resting basket and box,

suggesting that it could be used as an alternative to the cloth resting box. The materials

used in the method, which requires little training to implement, can be easily obtained in

rural settings where malaria is of great concern.














CHAPTER 1
INTRODUCTION

Approaches to Malaria Control.

Historically, there are two general approaches (vertical and horizontal) to control of

diseases such as malaria. Vertical approaches call for centralized national programs that

act as an independent entity in national health care systems. Horizontal approaches

involve broad based local programs emphasizing basic needs such as health education,

safe water, and adequate food supply (Tan et al. 2003). Positive aspects to vertical

approaches are a government commitment to standardized health care and attention given

to the control of all aspects of the disease (such as prevention and treatment). Also, due

to clear hierarchical structures typically found in such programs, there are fewer

organizational complications compared to those observed in decentralized systems

(Kroeger et al. 2002). Vertical programs, however, are criticized for focusing solely on

one health problem and failing to build local capacities with wider health benefits (Tan et

al. 2003). In some cases vertical programs must be initiated by larger international

organizations when national governments do not have the needed resources.

Horizontal control programs are strongly based in decentralized health systems and

in community mobilization. They are geared to become more focused to the needs of

each specific region. Problems that arise from horizontal approaches are that often

decentralized health systems do not have the resources and support held in vertical

programs, and in many rural regions people cannot access basic health facilities (Deressa

et al. 2003, Garfield 1999, Killeen et al. 2002).









History of Malaria Control

In the 1950s, the World Health Organization (WHO) realized the severity of

malaria in terms of human morbidity and mortality and the resulting reduction of

agriculture and industry on a global scale. In 1956, the Global Malaria Eradication

campaign was launched. The campaign was a relatively worldwide vertical effort based

on interrupting transmission through indoor residual spraying (and other mosquito control

measures), management of marshes, creation of agricultural land, and modification of

human lifestyle (i.e., window screens, air conditioning, and television). The success of

the National Malaria Eradication Program in the United States was, in part, due to

vertical disease programs from the Tennessee Valley Authority and the Communicable

Disease Center (previously the Office of Malaria Control in War Areas) (CDC 2004a,

CDC 2004b). Malaria eradication was realized in much of the world's temperate areas,

but failed or was not attempted in many tropical areas. In Africa, eradication was only

attempted in Zimbabwe, South Africa, and Ethiopia since malaria was considered too

great a problem in the other countries (Kager 2002). In response to the anticipation that

national primary health care systems would take over comprehensive malaria information

systems, aiding in management of malaria, the vertical approach programs of the WHO

were dismantled in the 1980s. International interest and funding in malaria research

waned during this time. Since then malaria transmission in many of the affected regions

has increased dramatically (Gilles 2002).

Future of Malaria Control

In the past 15 years, global efforts have been made to combat the increasing

morbidity and mortality associated with the disease. Initiatives such as Roll Back

Malaria (RBM), Multilateral Initiatives on Malaria (MIM), and the African Initiative on









Malaria (AIM) have been created to reestablish malaria as a global priority, to increase

support from wealthier nations, and to develop sector-wide approaches to malaria control.

This global push to create regional programs involves not only peripheral health care

systems, but educating and empowering community members of the affected areas

(Conteh et al. 2004, Deressa et al. 2003, Magnussen et al. 2001). Because such

horizontal approaches still have shortcomings, it is likely that large scale malaria control

will only be achieved through the integration of aspects from both horizontal and vertical

programs (Kroeger et al. 2002).

Role of Rural Housing in Malaria Transmission

The physical condition of rural houses plays an important part in the epidemiology

of malaria, especially when transmitted by endophagic and endophilic vectors like

Anopheles gambiae (Giles). High levels of malaria transmission are usually associated

with vectors that prefer to feed on humans indoors (Harwood and James 1979).

Successful eradication of malaria in many regions of the world is due in part to mosquito-

proofing houses (i.e., screened windows and air conditioning). Many people living in

regions where malaria causes the highest morbidity and mortality do not have access to

such luxuries.

Rural houses and villages (especially those of poor construction) are considered the

main foci for malaria transmission in these regions (Muirhead-Thomson 1982, Konradsen

et al. 2003, Fullerton and Bishop 1933, Gamage-Mendis et al. 1991). In Kenya, Githeko

et al. (1994) and Bogh et al. (1998) found that 74% and 99%, respectively, of An.

gambiae sensu lato (Giles) collected resting indoors had fed on human blood. Open eaves

provide a ready entry point into rural houses and increase the chance of finding higher

densities of mosquitoes resting indoors (Lindsay et al. 2003, Schofield and White 1983).









Houses built without ceilings also have been shown to increase human exposure to

malaria vectors. (Lindsay et al. 1995, Schofield and White 1983). Houses also provide

suitable habitats for vectors of human filariasis (Culex spp), yellow fever, and dengue

(Aedes aegypti L.).

Sampling Methods for Malaria Vectors Indoors

Although there are various methods for sampling the eggs and larvae of

mosquitoes, the most commonly used approach is to sample adults (Service 1993). There

exist numerous methods to sample adults, especially host-seeking females. Many of the

techniques used to sample mosquitoes seeking human blood are conducted indoors or in

artificial shelters. Such methods include bednet traps, hand net collections, drop net

collections, and aspirating mosquitoes attempting to bite a sleeping human subject

(Service 1993). However, ethical issues arise when studies attempt to quantify the

numbers of mosquitoes actively trying to bite humans. Studies involving methods such

as counting the number of mosquitoes landing on a human (human landing catches) and

human-baited traps can potentially expose human subjects to malaria through a bite from

an infected mosquito. These studies are often conducted during the time of day when

many female mosquitoes are actively seeking blood. Ways to circumvent using human

subjects include the use of carbon dioxide and light traps, as well as animal baited traps.

The accuracy of these methods varies, but their cost and practicality are often beyond the

means of community level control programs in developing countries. Also, mosquitoes

may be attracted to mechanical devices for reasons other than host-seeking and

mosquitoes attempting to feed on animals may not behave in the same manner as

anthropophagic species.









Collecting adult resting mosquitoes, however, is a method that is considered to be

more representative of mosquito populations than collections made by trapping adults in

flight. This is because a wider range of mosquitoes, in terms of bloodmeal status, age,

sex, and gonotrophic cycle, are taken in resting collection (Service 1993). Resting

collections are often made from outdoor natural shelters, such as vegetation and tree-

holes, but a few important vector species can be found in man-made structures. Because

of this, sampling a population of vectors resting indoors can be valuable to surveillance

and control programs (WHO 1992, Lindblade et al. 2000).

Indoor Sampling of Resting Mosquitoes

Hand catches with a plastic tube or mechanical aspirator and Pyrethrum Spray

Catches (PSC) are two commonly used methods for collecting mosquitoes resting indoors

(WHO 1992). However, depending on the ability and willingness of the collector, human

error can reduce the accuracy of counts made by hand catches. Pyrethrum spray catches

are not only expensive but can cause unnecessary exposure to chemicals. A wide variety

of artificial resting places have been tested to sample outdoor resting mosquitoes, but

relatively few artificial resting places have been used indoors. Those tested indoors have

seen limited success (Service 1993, Komar et al.1995, Smith 1942). Yasuno et al. (1977)

tested the use of plywood boxes as a method to sample indoor mosquitoes, but found the

boxes worked only in high densities of mosquitoes and low humidities. Resting boxes

made of cardboard and black muslin cloth caught 30-60% of all Aedes aegypti collected

indoors by hand catches (Edman et al. 1997). Das et al. (1997) developed an insecticide-

impregnated fabric (IIF) trap for use indoors, but this is an impractical tool in much of the

rural settings in Africa where many of the materials needed for construction are not

readily available. In Kenya, Sexton et al. (1990) used a 5 x 6 ft reed ceiling mat from






6


which to make weekly hand catches of indoor resting densities of Anophelines but did

not test this method against other kinds of indoor resting collections














CHAPTER 2
LITERATURE REVIEW

Historical Overview

Malaria has long been a plague of mankind. Evidence of this is seen in the high

host specificity of the four malaria species that infect humans, suggesting a long

association between humans and the parasites (Gilles 2002). Archeological evidence

suggests that human malaria existed in the eastern Mediterranean region as early as the

beginning of the Neolithic Period (9,500 BC) (Harwood and James 1979). In the fifth

century, Hippocrates was the first to describe the clinical picture and some complications

of malaria. The English word "malaria" is derived from the Italian mal aria meaning

"bad air." The French word "paludisme" and the Spanish "paludismo" also refer to

malaria and come from the Latinpalus meaning "swamp." Malaria, paludisme, and

paludismo have all originated from the idea that malaria was the result of inhaling "bad

air" from swamps (Foster and Walker 2002, Gilles 2002, Harwood and James 1979).

The first description of the malaria parasites in human red blood cells was from the

French army surgeon Laveran in 1880. Seventeen years later Ronald Ross, a physician in

India, discovered a developing form of malaria parasite in the body of an infected

mosquito, greatly aiding the understanding of malaria transmission. In the late 1950s the

WHO launched a global campaign that successfully eradicated malaria in much of the

world's temperate zones including parts of North America, Europe, and Australia.

Control of malaria was not as successful in many tropical areas. In fact, in the past

twenty-five years there has been a significant increase of malaria incidence in tropical









areas such as southeastern Asia and tropical Africa. In 1998, the WHO stated malaria to

be returned to its list of top priorities and introduced a new initiative called, "Roll Back

Malaria." The success of this program has yet to be evaluated and reported.

Importance of the Disease Worldwide

Today, malaria is the most important insect-borne disease in public health (Durden

and Mullen 2002, Foster and Walker 2002). Malaria, along with AIDS and tuberculosis,

are the three most important infectious diseases in the world. Malaria affects around 40%

of the world's population, much of which live some of the poorest countries in the world

(WHO 2004, WHO 1995). In 1995, countries with intense malaria transmission had

income levels equal to roughly a third of those in countries without malaria (Gallup and

Sachs 2001). In Africa alone, it is estimated that the annual costs of malaria exceed US

$2 billion (WHO 2000). Malaria not only affects poor people, but it keeps them poor.

The disease is found mainly in subtropical and tropical climates. In the past,

malaria was more ubiquitous and was found in temperate regions including areas in the

United States (Gilles 2002, Honigsbaum 2001). In the early 1900s, 6 to 7 million cases of

malaria were reported in the continental United States annually (Harwood and James

1979). Now malaria is responsible for more than 300-500 million illnesses and a least

one million deaths each year (WHO 2004, WHO 1995).

Description of Disease Transmission

Human malaria is caused by one of four species of parasite in the genus

Plasmodium, family Plasmodiidae, suborder Haemosporiidae, order Coccidae (Sinden

and Gilles 2002, Marquadt et al 2000). The life cycle of Plasmodium species is complex,

including an exogenous phase (a cycle in mosquitoes) and an endogenous phase (a cycle

in humans) (Foster and Walker 2002). The four species of Plasmodium infecting humans









are P. falciparum, P. vivax, P. ovale, and P. malariae. These parasites are transmitted to

humans by mosquitoes of the genus Anopheles. The first sexual developmental stage of

the parasites, the gametocytes, are ingested along with asexual stages by the mosquito in

the bloodmeal of an infected human. After fertilization occurs a diploid zygote is formed.

The zygote (or ookinete) moves through the peritrophic membrane of the mosquito gut.

Depending on the species of parasite, 8 to 16 days later the ookinete ruptures, releasing

sporozoites that make their way to the mosquito's salivary glands where they can be

passed on to another human upon a subsequent bloodmeal (Sinden and Gilles 2002,

Foster and Walker 2002).

The severity of disease is dependent on the species of parasite and the general

health and immune status of the infected person (WHO 1995). Cases of mixed infections

of two or more Plasmodium species are not uncommon (Sinden and Gilles 2002).

Nonfatal infections that are left untreated can last more than five months depending on

the immune system of the individual (Foster and Walker 2002). Common symptoms are

febrile paroxysm (violent attack of fever), myalgia (muscle pain), headache, nausea,

diarrhea, and vomiting.

In areas of stable malaria transmission (little variation in transmission over several

years) some degree of acquired immunity is common in adults who survived bouts of

malaria as children. This means that infected adults may be asymptomatic or exhibit only

slight clinical symptoms. Effects of immunity to malaria on individual are; 1) prevention

of infection with the same species of parasite, 2) reduction in parasite multiplication, 3)

destruction of parasite, and 4) aid in tissue repair (Marquadt et al. 2000). This immunity

is no doubt beneficial to the individual, but typically in such cases proper treatment is not









sought and the infected immune person can serve as a suitable host for the disease. In

areas of unstable malaria transmission (much variation in transmission over several

years), epidemics become a problem since people in these regions aren't exposed to the

disease long enough to develop immunity.

Children under the age of five and pregnant women are the most vulnerable to

malaria infection. Mortality in children results from three main presentations of malaria.

One being an acute infection (often presented as seizures or coma) killing the infected

child quickly. The second is the development of severe anemia from repeated infections

of malaria parasites. The final is a low birth weight often the consequence of the mother

being infected with malaria during her pregnancy. This last presentation is a major risk

factor during the child's first month of life. Malaria can also cause children to become

more susceptible to other infections including respiratory illnesses, diarrhea, and other

common childhood illnesses. In regions of unstable malaria transmission, pregnant

women are at extremely high risk of maternal and perinatal death. In regions of stable

malaria transmission, infection is usually asymptomatic due to some acquired immunity,

but commonly causes severe maternal anemia and babies with a low birth weight. A

curious occurrence that results from malaria during pregnancy is that there is often a

heavy infection of parasites concentrated in the placenta, impairing fetal nutrition. The

reason for this is poorly understood (Shulman and Dorman 2002).

Importance of Malaria in Africa

Of at least one million annual deaths attributed to malaria, 90% occur in sub-

Saharan Africa with the majority (90%) of these deaths occurring in children (WHO

2003). For many of the sub-Saharan countries more than a quarter of hospital admissions

are due to malaria. Data suggests that the number of malaria cases have increased over









the last decade (WHO 2004). Plasmodiumfalciparum is most common in Africa and is

responsible for the majority of malaria deaths worldwide (WHO 2003).

The appearance of a P. falciparum infection can range from almost asymptomatic,

to an acute febrile, illness, to severe life-threatening cerebral malaria sometimes resulting

in coma. Cerebral malaria can also cause anemia (lower than normal amount of

hemoglobin or red blood cells), hemoglobinuria (presence of red blood cells in the urine),

jaundice, hypoglycemia (low blood sugar), renal dysfunction (improperly functioning

kidneys), psychosis, shock, and pulmonary edema (fluid in the lungs) (Warrell 2002).

Important Vector Species in Africa

The most important and well-studied vectors of malaria are found in the An.

gambiae complex. The complex consists of seven described species: An. gambiae sensu

strict (s.s.), An. arabiensis, An. merus, An. melas, An. quadriannulatus, An.

quadriannulatus species B, andAn. bwambae (Service 2002). Their behavior, vector

status, and distribution differ in various aspects. Anopheles quadriannulatus and An.

quadriannulatus species B feed mainly on cattle and are not vectors of malaria.

Anopheles bwambae is a rare mosquito found only to breed in the Semiliki forest in

Uganda and is not considered an important malaria vector. Anopheles merus is a malaria

vector found in lagoons and mangrove swamps along the coast of West Africa. This

species breeds only in salt-water. Anopheles melas is considered to be the East African

equivalent to An. merus breeding in salt-water lagoons and swamps.

The two most important species in the complex are An. arabiensis and An. gambiae

s.s. The first is found in regions of dry savannah. The second is found in more humid

climates and readily becomes anthropophilic and endophilic (preferring to feed on

humans both outdoors and indoors and to rest indoors). Because most of its time is spent









indoors close to people, it is considered a highly efficient vector for malaria. Anopheles

arabiensis also exhibits some endophily but will also rest and feed outdoors, sometimes

feeding on domestic animals. Often An. gambiae s.s. will feed at least two times before

beginning its gonotrophic cyle (Foster and Walker 2002). The extra blood meals act as a

substitute for sugar (Foster and Walker 2002). Anopheles gambiae s.s. is found in almost

all sub-Saharan countries. Collectively all the species in the An. gambiae complex are

referred to as An. gambiae sensu lato (s.l).

The most important vector in Africa after An. gambiae s.s. and An. arabiensis is

An. funestus. It has a widespread distribution south of the Sahara and feeds

predominately indoors. The larvae are found in more permanent waters associated with

vegetation such as swamps and marshes. Anophelesfunestus feeds both indoors and

outdoors and prefers to rest indoors after feeding.

Summary

Malaria has been, and continues to be, one of the world's most important diseases.

Despite extensive attempts at eradication and control, the malaria situation has worsened

over the past two decades and parasites are re-emerging in areas where they had been

successfully or nearly eradicated, such as the Republic of Korea, Iraq, and Turkey (WHO

2000). Because malaria affects many of the world's poorest nations, control of the

disease has been difficult due to a lack of a solid health infrastructure. Sub-Saharan

Africa is most affected by disease and is home to An. gambiae, one of the world's most

efficient malaria vectors. It is considered a highly efficient vector because it prefers to

feed on humans and will take multiple blood meals (Foster and Walker 2002, Service and

Townson 2002).









African malaria vector control and research programs often focus on sampling adult

populations of An. gambiae s.l. (Giles), An. funestus (Giles), and other important vectors.

A major constraint in many developing countries where malaria is rampant is the lack of

funds available to such programs. Commonly used methods for sampling adult

mosquitoes include hand catches with oral or mechanical aspirators, Pyrethrum Spray

Catch, and CDC light traps. These methods can become costly and labor-intensive,

especially for community-based malaria control programs. The goal of this study was to

develop a technique for sampling adult malaria mosquitoes for use in research and

community control programs. To accomplish this, the following five objectives were

delineated:

1. Test the efficacy of simple resting boxes for sampling An. gambiae s.s. mosquitoes
resting indoors in semi-field conditions (screen-walled greenhouse). The design of
the semi-field test structure is described by Mathenge et al. (2002).

2. Identify the preferred natural indoor resting sites for common malaria vectors, An.
gambiae s.l. and An. funestus.

3. Assess the representative accuracy of the sampling of mosquito specimens through
hand catches by an experienced collector using an oral aspirator.

4. Compare the accuracy of sampling common malaria vectors using a technique
utilizing indoor resting boxes and bednets to 15 minutes of hand catches by an
experienced collector using an oral aspirator.

5. Test the efficacy of resting baskets for sampling An. gambiae mosquitoes indoors
in semi-field conditions (screen-walled greenhouse).














CHAPTER 3
MATERIALS AND METHODS

Site Descriptions

All study sites were located on the shores of Lake Victoria in the Suba District,

within Nyanza Province, Western Kenya (altitude 1100-1300m)(Fig. 3.1). All semi-field

trials were conducted at the International Centre for Insect Physiology and Ecology's

(ICIPE) Biological Station in the town of Mbita Point. Fields trials were conducted in the

nearby village ofLwanda Nyamasare. The two sites are within approximately 9 km of

each other. The area has two rainy seasons from March to June and October to

December with an average annual rainfall of 700-1800mm. Average temperatures range

from 160 to 340 Celsius. Malaria is considered holoendemic in the region. Suba District

is home to around 156,000 people with the majority of these people belonging to the

patrilineal Luo ethnic group. Luos make a living primarily through fishing and

subsistence agriculture. Description of the area is given in Mathenge et al. (2002),

Geissler et al. (2000), Okech et al. (2003), and Knols et al. (2002).

Survey of Indoor Resting Habitats of Mosquitoes

From March 27th to April 18th2004 at varying times of the day (8:25 to 16:45 hr.),

thirty-two dwellings were visited for inspection by a local expert mosquito collector with

eight years experience in collecting resting mosquitoes with a plastic tube oral aspirator.

This method for sampling adult mosquitoes resting indoors, often referred to as hand

collection or hand catches, is a widely used method and provides a suitable technique to

quantify the resting site of each collected mosquito (Service 1993, WHO 1992).



















Lake Victoria


Suba Disrict /


e kaL Victoria


bita (ICIPE) KENYA




-'-- -- /r y

4r U'





.. 4 0/'


/... -. |
J '--" 3^-

J /- '' .
4 / 0d



4 ___ 0 __4 __ aKoftr


Figure 3-1. Map of Kenya showing the study sites.

The technician thoroughly searched each dwelling (Fig. 3.2) collecting mosquitoes

and aspirating them into paper cups covered with netting. Searching involved not only

walking around the dwelling and collecting visible mosquitoes, but also looking under

and behind furniture, behind curtains, around and under pots and pans, etc. Cups

containing live collected specimens were placed in a sealable container with cotton balls

soaked with ethyl acetate to prepare the specimens for processing. Nine dwellings were

revisited five to ten days after the first collection. Two separate rooms, each of similar

size to a single dwelling, were checked in the same large house. The technician was given


s
i
1
r









as much time needed to collect all mosquitoes seen. The time spent collecting at each

house was noted.

Dwellings were made of wood and mud with a thatch or a corrugated iron sheet

roofs or made completely out of wood and corrugated iron sheets. All dwellings were

built without ceilings with the underside of the roof exposed. Dwellings were all

rectangular except for one circular house. The lengths of two adjacent walls inside all

rectangular dwellings were measured to find the total area inside. For the circular

dwelling, the diameter was measured to find the area. The height from the floor to the

highest point of all dwellings was also measured from the inside.







IA ) B)


Figure 3-2. Dwellings in Lwanda. A) Single family structure B) Multiple family
structure.

The height where each mosquito was collected was recorded as high, medium, or

low. A mark of "high" required the technician to lift his arm above his shoulder (roughly

161 to 240 cm from the ground). "Medium" was designated as any collection made from

the technician's shoulders to his waist (approximately 81 to 160 cm from the ground).

Mosquitoes caught below the waist of the technician, causing him to bend over or kneel,

were designated as "low" (0 to about 80 cm from the ground). The object (wall, jug,

pots, hanging shirt, etc.) the mosquito was collected from was also recorded. The objects









on which the mosquitoes were collected were later divided into 5 different categories

(Fig. 3.3):

1. wall/floor/door
2. plastic (plastic items such as bottles and basins)
3. cloth (items such as clothes and bed-nets)
4. furniture (more permanent items less likely to be moved around such as a bed, a
cabinet, chairs, etc.)
5. temporary (found on items likely to be disturbed or moved such as lanterns or
bicycles).

Finally, the number of all mosquitoes that were collected in an area deemed

"hidden" or protected was noted (Fig. 3.3). These categories are similar to the studies

completed by Pal et al. (1960) and Wattal and Kalra (1960).












Figure 3-3. Objects in dwellings on which mosquitoes can be found resting.

Representative Accuracy of Hand Collection

Twenty-one dwellings of similar size and make were visited by the expert collector

and the investigator from April 1st to April 16th 2004. The walls were constructed of

either wood and mud or wood and corrugated iron sheets. The roofs were made from

either corrugated iron sheets or thatch. All dwellings were without ceilings. Fourteen

were in multiple family units and seven were single family units. Mosquitoes were

collected by hand using a plastic tube oral aspirator by the collector until an entire search

of the house was completed (no more mosquitoes found). The time needed to hand

collect all mosquitoes was noted. The time of day the collection took place ranged from









8:25 to 16:30 hr. Immediately after finishing a search of the dwelling, a Pyrethrum Spray

Catch (PSC) was performed. A commercial-grade pyrethroid insecticide (Doom Fast

KillTM, manufactured by Mortein, Australia) in 300g/494 mls containers were substituted

for pyrethrum because it could be easily purchased and was safer (higher dilution of

chemicals than the industrial grade) to use for the investigator and families living in the

dwellings. The active ingredients of the insecticide were d-phenothrin (Pyrethroid) 1.0

g/kg and imiprothrin (Pyrethroid) 0.4 g/kg. For each dwelling the combined number of

mosquitoes collected from the PSC and hand collection was found giving a total

complete catch for each dwelling. From this combined number, the percentage collected

by hand was found.

Development of Cloth Indoor Resting Box in Semi-Field Conditions

Semi-field trials were conducted from April 3rd to June 15th, 2004 at the ICIPE,

Mbita Point Biological Station at Mbita Point, western Kenya (000 25'S, 340 13'E). A

30cm x 30cm x 30cm cloth resting box, similar to the design described by Crans (1989)

was tested for its ability to sample indoor resting mosquitoes. The box was made of a

plain 2 cm thick galvanized wire frame with blue cotton cloth. Blue cloth was sewn to

cover the outside of the box with black cotton cloth sewn to cover the inside. The

attractiveness of dark colors to mosquitoes is discussed in Bidlingmayer (1994). A flap

made of mosquito netting (mesh size 196) with a sleeve was sewn to the top of the box.

This was closed to facilitate capture when the number of mosquitoes in the box was

relatively high.

The box was hung approximately 50 cm from the ground in the opposite right

corner from the door of an experimental hut made of plywood (3.2m x 2.7m x 1.7m)

inside a modified screen-walled 11.4m x 7.1 m x 4.2m greenhouse (Cambridge Glass









House Co. Ltd., U.K.) (Fig. 3.4). Both hut and greenhouse are described by Mathenge

(2002). The box was hung from the far right corner because it was the opposite corner

from the door, a large source of light in the hut. Each night one of three different

densities of female An. gambiae s.s. mosquitoes from Mbita strain colony at ICIPE (low

= 50, medium = 100, and high = 200, as in Mathenge et al. 2002) was released into the

greenhouse at approximately 21:00 hr. For each density, mosquitoes were allowed to

feed on 10% sugar solution for at least 24 hours after emergence. Half of the females

were not allowed a bloodmeal and half were allowed to feed on blood for ten minutes

daily for three nights prior to release. This adult rearing protocol is approved by the

Kenya Medical Research Institute and the Kenyan National Ethical Review Committee

(2001). The resting box was checked hourly from 7:00 to 12:00 hr and again at 15:00 hr.

All mosquitoes caught in the box were counted and removed each time. The number of

mosquitoes collected each hour was noted. After the final collection of the day, two

technicians searched and collected the remaining females in the greenhouse and then

disposed of them. One similarly designed resting box treated with deltamethrin (the cloth

soaked in 2 liters of water with a 25% m/m concentration) was also tested in preliminary

tests but was discarded when almost 15% fewer mosquitoes were caught. The

deltamethrin was chosen for testing because it could be purchased nearby and in

preliminary trials some of the released mosquitoes were found dead in the box making

collection of mosquitoes easier.

Comparison of Accuracy of Resting box/Resting Net to Hand Collection

A comparison of accuracy of the collection methods was performed in Lwanda

Nyamasare village from April 13th to June 30th 2004.
























A ,i B.










,- ." *'. C )
Figure 3-4. Components of the experimental tests A) Resting box hung in experimental
hut. B) Experimental hut in modified greenhouse. C) Modified greenhouse.

Preliminary trials suggested that a double size blue bednet (Supanet TM) hung from the

highest point inside a dwelling and then spread out and tied to the top part of the eaves

could facilitate in capturing resting mosquitoes out of reach from the technician

performing hand catches. To aid in collection this resting net could be lowered within

reach of the technician when needed. All dwellings were made with mud walls; two had

thatched roofs, the other had a roof composed of corrugated iron sheets. Three dwellings

of similar size and make were chosen to compare the efficacy of three methods of indoor

resting collection in a Latin Square design. The three collection methods compared were

(Fig. 3.5):

1. A cloth resting box (described in previous semi-field trials) and a resting net









2. A plain cardboard box of similar size to the cloth resting box (42 cm by 21.5 cm by
25 cm) and resting net

3. Aspiration for 15 minutes using a plastic tube aspirator.

The time (15 min) chosen for hand collection was based on the results from the

previous survey and recommended in WHO (1992). When trials involved hand

collection, the resting net was tied in a knot and placed out of the way for the

homeowner. Both the cardboard box and bednets (for resting nets) are items that can

easily be found in Lwanda. The blue cloth resting box was made from materials found in

Mbita (12 km away). Immediately after mosquito collections at each dwelling, a PSC

was performed (as described previously). Following the completion of spray catches at

each house the collection methods were rotated. Dwellings were tested every 3 days to

allow ample time for the insecticide sprayed during spray catches to dissipate. The

number of days was chosen based on preliminary data from the Latin square. Trials

showed that numbers of mosquitoes caught by PSC increased from the previous spray

catch after two days. The sex and gonotrophic status of all captured mosquitoes was

noted. The categories for gonotrophic status (bloodfed, half gravid, gravid, and unfed)

was taken from Service and Townson (2002). The mosquitoes collected were identified

as either An. gambiae s.l., An. funestus, Culicine, or unidentified Anopheline. The Latin

square was run for a total of 27 experimental days in 3 complete rotations. The time

taken to complete sampling of a dwelling using the resting box (cloth or cardboard) with

a resting net was also noted.
























A B)











IC) D)
Figure 3-5. Treatments used in the Latin Square. A) Cloth resting box hanging in
dwelling B) Cardboard box hanging in dwelling C)Diagram of a resting net
spread out in a dwelling D) Expert collecting mosquitoes by aspiration.

Development of an Alternative to Cloth Box in Semi-Field Conditions

Field Trials

To evaluate the possibility of using a wicker basket (30 cm tall with an opening of

28 cm in diameter) with an attractive black cotton cloth lining inside (Bidlingmayer

1994) as method to sample indoor resting mosquitoes, a preliminary field trial was run

from April 23rd to June 30th 2004 in the village of Lwanda Nyamasare (Fig. 3.6). The

'resting basket' was hung approximately one meter from the floor in a corner of a

bedroom in a single family dwelling. The opening was faced toward the middle of the

room. The dwelling had walls made of mud and stick with an iron corrugated sheet roof.









Two to five people slept there nightly. Collections from the basket were made once at

approximately 10:00 hr at least every three days. The number of mosquitoes collected

was noted.

Semi-Field

From May 15 to July 13, 2004 trials were run in semi-field conditions using a

resting basket of equal size and make to the one tested in the field. The basket was hung

approximately 50 cm from the ground placed in the far right corer opposite the door of

an experimental hut made of plywood (3.2 m x 2.7 m x 1.7 m) (Fig. 3.6) inside a

modified screen-walled 11.4 m x 7.1 m x 4.2 m greenhouse (Cambridge Glass House Co.

Ltd., U.K.). Both hut and greenhouse are described in Mathenge et al., (2002). Each

night one of three different densities of female An. gambiae s.s. mosquitoes (low = 50,

medium = 100; and high = 200, as in Mathenge et al. 2002) from the ICIPE Mbita strain

colony was released into the greenhouse at approximately 21:00 hr. The method for

testing the recapture rate of the resting basket was the same as the previously described

trials using the cloth resting box. For both low (50 females) and high (200 females)

densities, ten trials were run while thirty-two trials were run for the medium (100

densities).





r_| "" .




A) B)
Figure 3-6. Alternative to the cloth resting box. A) Cloth resting box and resting basket
B) Resting basket hanging in dwelling














CHAPTER 4
RESULTS AND DISCUSSION

Results

Survey of Indoor Resting Habitats of Mosquitoes

Dwellings typically held one family. Some dwellings were part of a single large

structure which consisted of at least three or more dwellings (rooms with doors to the

outside) connected under the same roof (Fig. 4.1).

All style of dwellings in the study area remained quite constant among single and

multiple family structures. Dwellings consisted of a rectangular room divided by a

hanging cloth sheet, reed mat, or mud wall. The sleeping area was always on the

opposite side of the divider from the door. Structures containing a single family tended

to be larger in area and had a higher roof than dwellings in multiple family structures

(Table 4.1).

In the dwellings mosquitoes were collected off a variety of different substrates with

around a quarter of those found in areas hidden from plain view (Table 4.2). The category

"Hidden (protected)" was noted in addition to the substrate. For example, a mosquito

collected from aplastic item was found in an area on the item that was "hidden" from

plain view of the collector. A General Linear Model procedure using a Wilcoxon rank

sum test and a Kruskal-Wallis test was performed to analyze the percent of mosquitoes

caught at low, mid, or high heights in the dwellings (SAS 2001). There was no significant

difference between the heights the mosquitoes were found (Table 4.3).











Dugout hole
for cooking

Single bed
or reed mat O



Corrugated iron 0
sheet door
sheet oor Reed mat, cloth,
I or mud divider
0l


*Most collection done
on this side of divider,
away from light of door*

Window





Chairs, jugs,
' pots, etc.


Table and chairs

Figure 4-1. Layout of dwellings. A) Single Family B) Multiple Family

Table 4-1. Mean area and height of dwellings in study area
Structure Mean area ( Std) Mean Height ( Std) Number
Single Family 14.4 + 4.2 m2 3.4 0.4 m2 13
Multiple Family 9.5 2.5 m2 2.7 0.4 m2 14

Representative Accuracy of Hand Collection

Out of the 531 mosquitoes caught in 31 dwellings by both hand collection and the

PSC performed afterwards, hand collection caught 32.2% of the total of the two methods.

Development of Cloth Indoor Resting Box in Semi-Field Conditions

Trials using a treated box were stopped once it became apparent that the percent of

the mosquitoes released that were captured was less than the untreated. The mean

percentages for each of the densities tested on the untreated box were similar (Table 4.4).









The total mean percent of mosquitoes recaptured from all trials of the untreated box

(Table 4.4) was similar to the percent caught by hand collection in 31 dwellings (32.2%).

Table 4-2. Percent of total actual number of mosquitoes (437) collected from each
location.
Wall/Floor/Door 34%
Furniture (more permanent) 30%
Cloth/Bednet 24%
Temporary items 8%
Plastic items 4%
Hidden (protected) _26%*
*The category "Hidden" was noted separately.

Table 4-3. Total actual number of mosquitoes collected at each height. Note: Numbers
followed by the same letter are not significantly different (P<0.05), df = 2,
Chi-square = 0.97.
Height dwelling where collected (in cm) Number of mosquitoes caught
0 to 80 "Low" 176a
81 to 160 "Medium" 111a
161 to 240 "High" 119a

Table 4-4. The mean percent of mosquitoes recaptured in semi-field conditions using a
resting box either treated or untreated with deltamethrin. (Mean Standard
Error )
So Density-50 Density-100 Density-200 Total
Type of box
T ffemales females females percentage
39.4% + 10.6 38.3% + 8.9 30.7% + 8.5 36.1% + 9.9
Untreated
N=10 N=10 N=10 N=30
15.3% + 3.0 25.3% + 10.9 27.3% + 6.2 22.6% + 9.6
Treated*
N=3 N=3 N=3 N=9
Note: (N=number of trials performed). *Trials with the treated box were stopped early
once the it was shown that the it caught fewer mosquitoes than the treated.

Comparison of Representative Accuracy of Resting Box/Resting Net to Hand
Collection

The total number of mosquitoes collected during the Latin Square experiment using

each method (hand collection, cloth resting box and resting net, and cardboard box and

resting net) was calculated (Table 4.5). For each method, the total number of mosquitoes

caught by a method was added to the numbers caught by Pyrethrum Spray Catch (PSC)

performed after the same method giving an actual overall total (Table 4.5). The percent









of the overall total number of mosquitoes collected using each method was found by

dividing the total actual number mosquitoes caught a method by the overall total number

of mosquitoes caught for each method (Table 4.5)

Table 4-5. Comparison of actual number of mosquitoes collected by the three methods
tested. N=27
Hand Collection Blue resting box and Cardboard box and
resting net resting net
Overall total
mosquitoes caught 1166 1020 1060
(PSC + method)
Total mosquitoes
caught using 166 282 256
method
Percent of overall
number of
mosquitoes 14.2% 27.6% 24.1%
collected using
method
Note: (N=number of trials performed).

The cloth resting box/resting net caught 1.7 times the number of mosquitoes and

almost twice the percentage of the total mosquitoes as hand of collection (Table 4.5).

The cardboard box/resting net caught 1.5 times the number of mosquitoes and almost

10% more of the percentage of the total mosquitoes as hand collection (Table 4.5). The

percentages captured by each of the methods on each test day were transformed to

achieve linear model assumptions by:

arsin X

where x is the percentage of mosquitoes captured by a method on each test day. The

results were then analyzed using the Tukey Multiple Comparison Procedure (SAS 2001).

The catches from the cloth resting box/resting net method were significantly different

than hand collection (significance was set at P<0.05) (Table 4.6). There was no

significant difference between the cloth resting box/resting net and cardboard box/resting









net methods nor between the cardboard box/resting net and hand collection. The time

taken to complete sampling using a resting box/resting net method was slightly shorter

than the 15 minute time suggested by WHO (1992) for hand collection (Table 4.7).

The results of the identification to species, sex, and gonotrophic status showed An.

gambiae s. 1. to be the most collected of the four types of mosquitoes (An. gambiae s.l.,

An. funestus, Culicine, and an unidentified anopheline) identified from the Latin Square

catches (Tables 4.8-4.11). The highest numbers of An. gambiae s.l. mosquitoes found in

the dwellings were bloodfed (882 mosquitoes) and unfed females (884 mosquitoes)

(Table 4.8). This trend was also seen with An. funestus (Table 4.9). For both Culicine

and unidentified Anopheline mosquitoes, males were the most collected in the dwellings

(Tables 4.10 and 4.11)

Development of an Alternative to Cloth Box in the Semi-Field

The resting basket caught a mean of 4 mosquitoes daily in one dwelling in the field

(Table 4.12). A General Linear Model procedure was performed on the percentages of

mosquitoes recaptured using a Type III sum of squares test for the total percent of

mosquitoes recaptured (SAS 2001). There was no significant difference (P<0.05) in the

total percent recaptured between the two methods (Table 4.13). There was no interaction

between the density of mosquitoes and the methods used but a difference was found

among the densities of mosquitoes. For further analysis, a Tukey-Kramer adjustment for

multiple comparisons was performed on the pooled (resting box and resting basket)

percent of mosquitoes recaptured by each density (SAS 2001). Results showed a

significant difference between low and high densities (Table 4.14










Table 4-6. Comparison of the accuracy (percent of the overall number of mosquitoes
found in each dwelling) over the 27 test dates for the three methods tested.
Methods followed by the same letter are not significantly different (P<0.05).
Note: F= 3.37, df= 2, 50.


Date Hand Collectiona

13-Apr 6.7%
16-Apr 9.6%
19-Apr 48.1%
22-Apr 14.7%
25-Apr 14.5%
28-Apr 28.5%
1-May 9.0%
4-May 0.0%
7-May 8.3%
10-May 4.6%
13-May 0.0%
16-May 37.0%
19-May 13.3%
22-May 17.2%
25-May 34.7%
28-May 3.8%
31-May 14.8%
3-Jun 28.8%
6-Jun 14.4%
9-Jun 5.1%
12-Jun 11.1%
15-Jun 33.3%
18-Jun 4.1%
21-Jun 33.3%
24-Jun 22.8%
27-Jun 6.6%
30-Jun 30%
Mean percentage for a
each method SE
Note: F= 3.37, df= 2, 50


Cloth Box/
Resting netb
56.0%
12.0%
34.6%
14.2%
25.0%
28.7%
63.2%
25.0%
0.0%
35.7%
0.0%
10.7%
38.4%
17.3%
20%
20%
23.2%
21.4%
66.6%
19.4%
0.0%
60.7%
32.5%
10%
10.5%
29.7%
14.2%
27.1 18.5%b


Cardboard
Box/Resting neta
2.2%
85.7%
18.9%
9.3%
20.5%
11.9%
5.2%
14.2%
31.6%
0.0%
23.6%
21.5%
3.2%
42.3%
45.6%
15.0%
48.0%
20.8%
18.7%
30.7%
17.0%
12.5%
37.5%
37.5%
9.0%
46.1%
16.6%
23.3 18.6%ab


Table 4-7. Comparison of time taken to complete sampling using a resting box (cloth or
cardboard) and resting net to hand collection.
Mean time taken to
Number of Time suggested by WHO
complete sampling using
trials using a resting box complete sampling using (1992) to complete a search
d r g n resting box and resting net with hand collection
and resting net with hand collection
+ Standard Error
31 8.0 3.9 min 15 min









Table 4-8.


Actual number of An. gambiae mosquitoes caught by method, including sex
na d gonotrophic status during the 27 te e


Half
Method Bloodfed Gravid af Unfed Male Total
Gravid
Cloth box 18 4 8 7 9 46
Cardboard
Cardboard 6 4 4 3 0 17
box
Hand
nd 60 21 10 30 12 133
Collection
Resting 163 53 26 103 35 380
Net
PSC 635 265 138 741 243 2022
Total 882 347 186 884 299 2598
Note: For the resting net method, the numbers from both nets used in the Latin Square
were calculated together.

Table 4-9. Actual number of An. funestus mosquitoes caught by method, including sex
and gonotrophic status, during the 27 test days of the Latin Square
Half
Method Bloodfed Gravid af Unfed Male Total
Gravid
Cloth box 1 0 3 0 0 4
Cardboard
Cardboard 0 0 2 0 0 2
box
Hand
nd 4 1 2 1 1 9
Collection
Resting 7 2 5 2 2 18
Net
PSC 26 16 8 29 14 93
Total 38 19 20 32 17 126
Note: For the resting net method, the numbers from both nets used in the Latin Square
were calculated together.









Table 4-10. Actual number of Culicine mosquitoes caught by method, including sex and
gonotrophic status, during the 27 test days of the Latin Square.
Method Half
Bloodfed Gravid Hf Unfed Male Total
Gravid
Cloth box 0 1 0 0 0 1
Cardboard
Cardboard 0 0 0 0 0 0
box
Hand
and 0 0 0 0 1 1
Collection
Resting 0 2 2 2 3 9
Net
PSC 8 27 4 17 24 80
Total 8 30 6 19 28 91
Note: For the resting net method, the numbers from both nets used in the Latin Square
were calculated together.

Table 4-11. Actual number of unidentified anopheline mosquitoes caught by method,
including sex and gonotrophic status, during the 27 test days of the Latin
Square.
Method Bloodfed Male Unfed Total
Cloth box 0 0 0 0
Cardboard box 0 0 1 1
Hand
Collection
Resting Net 0 0 1 1
PSC 4 15 5 24
Total 5 15 7 27
Note: For the resting net method, the numbers from both nets used in the Latin Square
were calculated together.

Table 4-12. Mean number of mosquitoes caught daily by a resting basket placed in a
house in the village of Lwanda.
Mean number of mosquitoes caught Number of days tested
3.9 3.3 33


Table 4-13. Comparison of the resting basket to the cloth resting box of the total
percentage of mosquitoes recaptured in semi-field conditions.
Type of trap Total mean percent of mosquitoes
Typerecaptured ( SE)
_________J __________________recaptured ( SE )____


Resting Basket

Resting Box


33.8% + 8.9a
N=52
36.1% + 9.9a
N=30


Note: N=Number of trials run for each method, percentages followed by the same letter
are not significantly different (P<0.05), F= 0.51, df = 1, 76)










Table 4-14. Mean percent of mosquitoes recaptured by each density used for both the
resting box and resting basket.
Density Mean Percent of Mosquitoes Recaptured
High 31.3%a
Low 39.1%b
Medium 35.6%ab
Note: N=Number of trials run for each method, percentages followed by the same letter
are not significantly different (P<0.05), F= 41.47, df = 1, 76)

Discussion

Indoor Resting Places of Mosquitoes

The most important malaria vector in Africa, An. gambiae, will readily rest in the

dwellings of rural villages (Service and Townson 2002). A common method for

sampling populations of these resting mosquitoes is a search of a dwelling by a trained

technician using an oral aspirator (Service 1993, WHO 1992). A properly trained

technician performing the search is essential as the mosquitoes are difficult to find and

catch. This idea is reinforced by the results of this study. Since it is also generally

thought that both male and female mosquitoes are attracted to darker areas indoors, one

would expect to find mosquitoes resting at low heights around the dwelling since they

would be further away from a main light source, the eaves. Although a larger percent of

mosquitoes were found at heights from 0 to 80 cm, the number of catches was not

significantly different from those higher than 80 cm. About a quarter of the mosquitoes

(26%) were also found in an area not in plain view of the collector. The results of this

study would suggest that it would be difficult to accurately pinpoint an area in a dwelling

to focus the aspiration as mosquitoes. The mosquitoes were found on a broad variety of

substrates at various heights with many resting in areas that would require the collector to

search under, above, or around household items. Many times during the study the

collector needed to move furniture, pots, clothes, and other household items to be able to









reach the mosquitoes. This emphasizes the need for adequate training for anyone

attempting to sample resting mosquitoes by hand collection using an oral or mechanical

aspirator.

Comparison of Sampling Techniques

The local expert technician had eight years experience with hand catches, likely

more than most people attempting that method. This would suggest a high quality of

collection in comparison to other hand collectors. Since the expert was given as much

time needed to complete a search of each dwelling during the initial part of the study, it

was assumed that the majority of the mosquitoes collected in the PSC, performed after

the search, were resting above the reach and out of the sight of the collector. Since the

expert caught only approximately a third (32%) of the total number found, it was decided

that any sampling method developed should account for mosquitoes resting on the

exposed underside of roofs (made of thatch or corrugated iron sheets) as this would be a

likely area to find them (Lindsay et al. 1995, Schofield and White 1983).

Resting boxes have been a well established method for sampling mosquitoes

(Service 1993). Few, however, have been used indoors. The results from the semi-field

trials suggest that the cloth resting box could collect close to the same percentage of

mosquitoes (36.1%) as the expert hand collector in the field (32.2%). However, it is

likely that a cloth resting box placed near a similar location in a dwelling could not

accurately sample mosquitoes that prefer to rest in the underside of the roof. This

assumption is based on the idea that since the expert technician was not able to sample

mosquitoes resting so high, a stationary trap would also have the same difficulty. For this

reason, a resting net was tested with the cloth resting box. The results of the Latin Square

trial suggest that the cloth resting box/resting net method could catch a significantly









higher percentage of the total population resting in a dwelling than that of an expert

performing hand catches using an oral aspirator.

To increase the practicality of the method the resting basket was tested in the semi-

field. Because there was no significant difference in recapture rates between the resting

basket and the cloth resting box, the resting basket could be an acceptable alternative to

the cloth resting box. The cloth resting box (or resting basket) and resting net method has

a number of advantages over hand collection:

1. It can more accurately sample the population resting in a dwelling (a higher
percentage of mosquitoes will be collected).

2. The materials needed for construction are inexpensive and can be easily found in
many rural areas as opposed to the time and money to needed to train and/or hire a
technician to perform hand catches.

3. It requires less training, not only because mosquitoes are easier to see in the black
cloth of the box and in the bright blue of the net, but also because the investigator
only needs to search a set area (the trap and the net) rather than the whole house.

4. The time needed to complete a search is shorter than 15 minutes, which is
commonly assigned for hand catches (WHO 1992).

The use of resting boxes and nets also has advantages over the PSC technique.

Although a greater percentage of mosquitoes can be caught with spray catches, the spray

technique is too expensive and impractical for use on a regular basis for local control

programs. Pyrethrum spray catches also expose the investigators and the members of the

tested household to potentially harmful chemicals that may take several days to dissipate

(Service 1993). This study showed that cloth resting boxes or resting baskets used with

resting nets can be a practical method for local malaria control programs to sample indoor

resting mosquitoes.









Summary

Malaria continues to be one of the world's most important human diseases.

Although, has been eradicated in some areas, it continues to be a threat to 40% of the

world's population. Many of those people affected by the disease live in some of the

poorest nations in the world. The lack of resources and infrastructure has produced major

obstacles to malaria control in those countries. Because malaria affects many people

living in impoverished nations, many of the more expensive and technical methods

utilized by developed countries for mosquito and disease control are not feasible.

An initiative to create sustainable malaria control programs at a community level

has been implanted by international organizations such as the WHO (2000). As local

malaria control programs become more accepted, technology is needed to keep such

programs sustainable. Developing new methods and tools for malaria surveillance and

control, which are both user-friendly and economically viable, are essential for this to

happen. Because the most important vector of malaria in Africa, An. gambiae s.l., readily

rests indoors after feeding, developing practical ways to sample populations of those

mosquitoes would be valuable.

The sampling technique tested in this study, was developed specifically for malaria

control programs based at community levels. The materials needed for the technique can

be found inexpensively in rural villages where community malaria control programs are

run. The technique provides a more user-friendly alternative to both PSC and hand

catches, both of which are commonly used in malaria control programs and research

(Service 1993, WHO 1992).

Members of control programs sampling with the new technique have only to collect

from a defined area (box or basket and net) compared to hand catches where a complete









search of a dwelling is required. Mosquitoes can also be seen easier on the black cloth in

the box or basket and on the blue cloth of the net than on the walls, thatch, and other

items in the dwellings. Since mosquitoes can be found and collected more easily than

hand catches, less training and experience is needed to effectively sample a dwelling.

Although sampling with PSC captures most, if not all, mosquitoes in a dwelling,

the cost of the materials needed (insecticide, sheets, protection against chemicals) is often

beyond the means of community malaria control programs. The use of such a method

could not be sustained for very long due to its expense and the constant need for

insecticide. The safe use and storage of the chemicals needed for PSC is also an

important issue for the safety of the people doing the sampling and the homeowners.

Since no chemicals are needed with the new technique, it can be performed safely by

members of control programs with no danger to the homeowners. Because the materials

needed for the new technique can be inexpensively purchased once, it can be used

indefinitely.

Further research should focus on developing more inexpensive and practical

methods of malaria surveillance and control that are accessible to the people facing the

greatest disease load. Such research should be broadly based in multiple disciplines due

the complexity of the malaria problem. Things like access to healthcare, economic

stability, and cultural behavior also play important roles in the transmission of malaria

and need to be considered. Even though much of the world's malaria control stems from

international organizations, a larger global effort is needed to have a lasting effect on

malaria control.














CHAPTER 5
NOTES ON THE DEVELOPMENT OF A PRACTICAL ANOPHELES GRAVID TRAP

Introduction

The ability to sample the populations of host-seeking mosquitoes is essential to the

evaluation of malaria control programs. The exposure to potentially infectious bites on

people sampling host-seeking mosquitoes makes many such sampling methods ethically

questionable. As an alternative to sampling host-seeking adults, gravid traps can provide

similar data (reproductive capacity of mosquito populations and the dispersal patterns of

mosquitoes as they seek larval habitats after biting humans) without the risks of exposure.

Such information is of great importance to vector control programs as many programs

focus on and have seen success with larvicide application in potential or known larval

habitats.

Gravid traps that have been tested and used in research and mosquito control

programs, sample only Culicine mosquitoes (Service 1993, Ritchie et al. 2004). Many of

these traps focus specifically on collecting gravid Culex mosquitoes (Service 1993,

Mboera et al. 2000). Although a variety of methods exist to sample the eggs of malaria-

carrying Anopheline mosquitoes, no technique has been devised to effectively trap and

sample these mosquitoes solely when they are gravid (Service 1993).

Gravid traps, like those used to sample Culicine mosquitoes, would become

problematic in many malaria endemic regions because they require a significant degree of

skill and maintenance to ensure they are working correctly. The commonly used, CDC

Gravid Trap (J. W. Hock Co. Gainesville, FL.), requires a net collection bag, motorized









trap with a fan, pan with water, a battery, and a battery charger. They would also be quite

costly to programs, particularly in African countries where resources available for control

are usually very limited. This study was undertaken to develop a much more practical

gravid trap that effectively samples egg-laying adults. A simple trap would have a

number of advantages over present traps:

1. It would not require skilled personnel for sampling mosquitoes

2. It would be far less expensive than current gravid traps

3. Data collected from traps will most likely reflect a more realistic composition and
behaviour of the egg-laying mosquito population since no artificial attractants will
be used.

4. It could be easily replicated and therefore be more likely to be used in mosquito
surveillance programs; since collected adults and eggs would be disposed, it could
directly aid malaria control programs.

The objective of this investigation was to develop a practical gravid trap using

materials readily available in rural areas where malaria is endemic. Such a trap would be

beneficial to malaria control programs at a community level where resources are often

scarce.

Preliminary Semi-Field Trials in Florida

Semi-field trials were conducted weekly from June 8th to October 1st 2004 at the

United States Department of Agriculture's (USDA) Center for Medical, Agricultural, and

Veterinary Entomology (CMAVE) located in Gainesville, Florida. Traps were tested

singly in screened cages (2m x 2m x 2m). Prior to placement of the traps, 70 gravid

Anopheles albimanus females were released into each cage and allowed to acclimatize for

3-4 hours. The females were left in each cage with a trap for 24 to 48 hours after which

the number of females caught was noted. After collections were made the cage was left

open for at least four days to release any females left in the cage.










One design was tested and modified from oviposition basins from CDC Gravid

Traps (J. W. Hock Co. Gainesville, FL.), plastic funnels, and plastic 40 ounce peanut

butter jars. All designs were spray-painted with a matte finish primarily of black and

white (Fig. 5.1). Spray-painted traps were left outside for at least six weeks prior to

testing to remove any odors. Some variations of trap designs had modifications such as

the addition of black plastic mesh, the presence of cotton soaked in sugar water, and

various amounts of hay infusion (Fig. 5.2). All designs tested had approximately 2 liters

of well water placed in the basin.

Plastic jar
collecting cartridge Holes poked through
jar to allow for air flow
Funnel suspended
with pieces of basin Ild
Larce funnel (black
outsidewhite inside) *


Small clear
plastic funnel



Plastic basin (from CDC gravid trap)
painted black on outside and
white on inside A) B
Figure 5-1. Design of oviposition trap. A) Full view. B) Collecting cartridge















Vial holding cotton soaked
in 10% sugar solution





Funnel held up with clothes hanger wire A) Blak plastic esh B) C)
Figure 5-2. Modifications to design. A) with clothes hangers B) with black mesh C) with
vial with sugar solution.

Semi-Field Trials in Kenya

From February 17th to March 11th 2004, 1 to 3 color variations of 4 trap designs

were tested daily in a screened cage (3m x 3m x 3m) at the ICIPE, Mbita Point Biological

Station at Mbita Point, western Kenya. A black plastic bucket (35cm x 30cm) was placed

in a hole in the middle of the screened cage. The bucket was placed so that the top was

even with the ground level. Some of the soil removed from the hole was placed back

into the bucket and water from Lake Victoria (of varying amounts depending on the trap

design) was also placed in the bucket. The water was changed daily. Traps were made

of 2cm thick plain galvanized wire, white mosquito netting and either black or blue

colored cotton cloth. Daily, fifty gravid An. gambiae s.s. females were collected from the

ICIPE Mbita strain colony and released into the screened cage at 6 pm. The following

day from 6:30 to 12:00 hr, traps were checked for any females. Froml4:00 to 16:00 hr.

the remaining females were aspirated out of the screened cage. Following colony

protocol all gravid females had been allowed to blood feed on the investigator's arm for

ten minutes daily for three consecutive days prior to their release. A colony egg trap was









placed in the colony cage from which the tested gravid females were taken to note the

ovipositional success of that batch of females.

The first experimental day, no trap was placed on the bucket and eggs were noticed

the following morning. On February 21st the bucket was removed from the screened

cage, fresh lake water placed in it and then positioned in a sunlit area for 6 hours to

"cook." The bucket was checked for any mosquito eggs that may have been laid by wild

mosquitoes and then returned to the screen cage prior to the release of that day's colony

mosquitoes. This "cooking" was done for each of the remaining trials. On February 23rd

the soil in the bucket was replaced by mud from a known Anopheles larval site in Mbita

that was not subject to larval control. On March 5th, dark blue and black cotton cloth was

draped on the outside of the screened cage and water was poured on it prior to release of

mosquitoes to increase the humidity. This was again continued daily. On March 7th,

filter paper used in the colony egg traps was attached to the lower part of one of the traps

and positioned half in the water to entice the females into ovipositing. On March 8th, the

screened cage was moved into a modified greenhouse (Mathenge et al. 2002). On March

12th a miniature version of the black trap (Fig. 5.3) was placed in a colony cage over a

standard egg trap used for maintaining colonies. Only one gravid female was captured

during all semi-field trials.

Field Trials in Kenya

From February 19t to March 17th 2004 field trials of the traps designs were

conducted. Field sites were chosen in the Kamasengre district of Rusinga Island, about

12 km away by road from Mbita. The trap bucket was placed in a hole dug in the ground

approximately 4m from a single family dwelling. Soil from the hole and fresh lake

water were added to the bucket. Four days later mud from a nearby larval site was placed









in the bucket instead of soil from the hole. The design placed on or in the bucket was

checked daily at approximately 7:00 hr. A week after these preliminary trials two more

sites were added; one had a bucket placed approximately a meter from a large larval site

(about 10m long) and another placed far from any larval sites or homes. This location

was considered a control. Mud from the larval site was placed in both of these new

buckets as well as lake water. The lake water was changed daily. Trap designs were

rotated daily among the three field sites and the semi-field site. On March 17th the field

sites were closed.

Results

Florida Trials

Out of 31 traps tested from June 8th to October 1st, 14 traps captured mosquitoes.

Out of those 14 traps, an average of 2.36 1.74 mosquitoes was caught. The mean

recapture ( SE) rate of the mosquitoes released for the 14 traps was 3.3 2.3%.

Kenya Trials

In the semi-field experiment only one gravid female was captured. This was by the

emergence design with black cloth. Only one mosquito (a Culicine) was caught in trials

in the field. The emergence design with blue cloth placed near the larval habitat caught

the mosquito.

Discussion

The description of larval habitats for An. gambiae s.l. has been well studied

(Warrell and Gilles, 2002). Larvae are typically found in sunlit temporary pools often

associated with humans. They develop in roadside ditches, wheel ruts, hoof prints from

wild and domestic animals, concrete holes, and village pots. Larvae can even to survive

close to five days on damp soil (Koenraadt et al. 2003).












Entry hole diameter
3 cm with a cinch
Bucket tie to close the hole
Height:
35 cm Mud from larval habitat

Entry hole
Lake water Sleeve Is 6.5 cm
Lake watery for removal diameter
changed daily of caught
Bucket diameter: 30 cm mosquitoes A)

13cm 9 cm 52.5 cm 13 cm

62.5 cm- ..-Diameter. 19.5 cm
Entry /7 cn

5.5 cm

B) C)
9.5 cm
Trap
with
... black
44 cm cloth
cover





D)
Figure 5-3. Trap designs in Kenya. A) Mbita (two variations) B) Emergence C) Florida
D) Cone (two variations).

Most larval sites contain fresh water without much debris. Preliminary trials conducted

by other investigators in Mbita Point, Kenya showed that An. gambiae will readily

oviposit in plastic tubs and buckets used for washing clothes and commonly found

around the town and nearby communities (Pers.comm. U. Filinger, University of

Durham, England). Although the identification of larval sites can be accomplished

without much difficulty, the identification of oviposition attractants has yet to be

determined.









One of the obstacles faced by testing a practical gravid trap out in the field is that

because An. gambiae will lay eggs in such a wide variety of habitats that are commonly

found in great numbers around the area, the trap would need to be at least as attractive to

females as the natural habitats. By placing a trap over a bucket or other potential larval

habitat the site no longer becomes sunlit and therefore probably less attractive. Further

investigations into oviposition chemical and visual attractants would be needed to

overcome the deterrent of shade created by the trap.

A problem faced by semi-field experiments is that gravid colony females that have

emerged from the same batch of colony pupae may not oviposit at the same time if at all.

If fifty gravid females of the same age and exposed to the same amount of blood are

released into a semi-field cage only a small number of females if any may actually try to

oviposit. More time spent rearing test mosquitoes should be taken to insure that

conditions are created to enhance the number gravid females that attempt to oviposit each

day.

Although a practical method for sampling gravid Anopheles has yet to be

developed, such a tool would be of great use to malaria control programs. A practical

gravid trap could be a safer alternative to other sampling methods involving chemicals or

sampling host-seeking mosquitoes. Identifying oviposition attractants for Anopheles

mosquitoes could not only aid the development and use of gravid traps but provide

insight to the ecology of the mosquito and create more options for mosquito monitoring

programs.
















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BIOGRAPHICAL SKETCH

Justin Eric Harbison was born in St. Paul, Minnesota, on September 1, 1977, to

Kent and Judy Harbison. He graduated from St. Paul Academy in St. Paul, Minnesota, in

1996. Justin graduated from Macalester College in May 2000 with a Bachelor of Arts

degree. Prior to pursuing his master's degree at the University of Florida, Justin spent

nine months as a field technician in the Organization for Tropical Studies' Palo Verde

field station in the Guanacaste region of Costa Rica.