MOSQUITOES AND ARBOVIRUS TRANSMISSION

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: ANIMAL HEALTH
• Reporting Frequency: Annual
• Accession No.: 0189558
• Project Start Date: Oct 1, 2006
• Project End Date: Sep 30, 2011
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671

Performing Department
Entomology and Nematology

Non Technical Summary
The goal of this project is to identify protein(s) associated with variation in ability of mosquitoes to transmit West Nile virus and to use that knowledge to develop a simple, rapid assay to detect those protein(s) that will help mosquito control personnel make informed, strategic decisions about how to most effectively apply mosquito and disease control efforts.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
721	3110	1101	25%
721	6099	1130	25%
722	3110	1101	25%
722	6099	1130	25%

Knowledge Area
722 - Zoonotic Diseases and Parasites Affecting Humans; 721 - Insects and Other Pests Affecting Humans;

Subject Of Investigation
6099 - People and communities, general/other; 3110 - Insects;

Field Of Science
1101 - Virology; 1130 - Entomology and acarology;

Keywords

west nile virus

equine disease

mosquitoes

vector competence

disease prevention

surveillance

Goals / Objectives
West Nile (WN) virus is maintained in a transmission cycle between infected wild birds and mosquitoes (Hayes 1989). Field populations of Culex mosquitoes in California-particularly Culex pipiens pipiens, Cx. pipiens quinquefasciatus, and Cx. tarsalis-have yielded isolates of WN virus or have transmitted it in laboratory vector competence assays (Anderson et al. 1999; Goddard et al. 2002; Sardelis et al. 2001; Turell et al. 2000, 2001). Initial viral dose, source of host blood meal, temperature, and incubation period are extrinsic factors that influence mosquito susceptibility to infection with an arbovirus like WN (Hardy et al. 1983, Reisen et al. 2005). Alternatively, innate susceptibility to infection can be influenced by mosquito midgut physiology, midgut receptor specificity to viral ligands, and tropisms at the level of the salivary gland or other target tissues, such as the fat body or ovaries (Weaver et al. 1990, Bennett et al. 2005). Innate differences frequently vary from one geographic population to another or, in the case of the Cx. pipiens complex, across regions of genetic introgression (Cornel et al. 2003, Urbanelli et al. 1997). Consistent with this pattern, laboratory vector competence assays of Cx. p. pipiens and of Cx. p. quinquefasciatus from California have yielded various WN virus infection and transmission values, depending on geographic origin of mosquito populations.

To design effective disease prevention strategies it is crucial to understand variation in vector competence among California mosquitoes. Knowledge regarding which mosquito species are most likely to transmit WN virus will facilitate meaningful control activities by targeting and suppressing specific mosquito populations. Successful control strategies based on vector competence will be important for preventing disease in wildlife, horses, and humans.

I propose to investigate the antiviral genes responsible for refractoriness in California Culex mosquitoes as markers in natural populations for predicting local risk of virus transmission. A genetic probe for field-caught mosquitoes could be applied by public health and veterinary officials and mosquito abatement districts to assess the potential for virus transmission by geographically and temporally different mosquito populations. Based on results, mosquito control personnel could make informed, strategic decisions about where and when to most effectively apply their mosquito and disease control efforts. Such an assay would be immediate and simple, without the biohazard, time, and expense associated with traditional mosquito vector competence and transmission studies.

My four objectives are:
1. Carry out vector competence assays for populations of Cx. p. quinquefasciatus in a north-south cline of California. 2. Using mass spectrometry, identify peptides induced by WNV infection in mosquitoes. 3. Using targeted gene-silencing, determine the function of identified peptides in limiting WNV proliferation and transmission in mosquitoes. 4. Develop genetic markers for rapid assessment of vector competence based on confirmed antiviral genes.

Project Methods
Testing variability in vector competence of California mosquitoes: Induction of immune peptides likely explains a portion of the geographic variation in vector competence of California Culex mosquitoes. Infection and transmission data on populations collected at different times and locations from throughout California will provide a state-wide picture of WN virus transmission risk and provide the material I will need to identify and characterize immune peptides. Mosquito examined for vector competence will be obtained in collaboration with mosquito abatement personnel from throughout the state. Methods for feeding, maintaining, and testing mosquitoes for WN virus infection and transmission were previously described by Goddard et al. (2002) and are routine in my laboratory. Briefly, female mosquitoes will be allowed to feed on a suspension of WN virus in defibrinated pig blood. Mosquito bodies and salivary expectorate will be collected after 7 and 14 days of incubation. Whole mosquito body homogenates and salivary expectorate will be tested for WN virus by plaque assay in cell culture. Infective dose of virus and insectary conditions (28 deg.C, 85% RH and 16:8 light:dark) will remain constant across species and populations. Transmission will be determined using an in vitro capillary method. For each population the percent of mosquitoes that become infected and percentage that transmit virus 7 and 14 days post infection will be recorded.

All experimental WN virus infections and manipulations will be carried out at the UC Davis Center for Vector-Borne Disease level-3 containment laboratory on Old Davis Road. I have over 30 years of experience working with level-2 and level-3 arboviral.

Proteomics and molecular cloning: To identify peptides induced in response to WN virus infection, I will analyze peptides from mosquitoes from the same collections that I assess for vector competence. I will follow a standard protocol of solubilization, 2D gel electrophoresis, and MALDI-TOF mass spectrometry (Levy et al. 2003, Vierstraete et al. 2004). Most of the proteomics analysis can be completed at the UC Davis Molecular Structure Facility. Once identified, antiviral genes will be silenced by targeted double-stranded RNA interference (Hannon 2002). Synthesis of double-stranded RNA for targeted gene-silencing in mosquitoes will follow standard protocols (Blandin et al. 2002, Goto et al. 2003) using Promega kits. Phenotypic loss-of-function mutants will be confirmed by vector competence studies, Northern hybridization to confirm mRNA presence, and mass spectrometry to confirm loss of the antiviral peptide. If knock-out analyses result in refractory mosquitoes becoming susceptible, mRNA will be isolated by triturating WNV-infected mosquitoes, and reverse-transcribed to produce antiviral cDNA. Frequency of the antiviral gene will indicate the relative refractoriness or susceptibility of tested populations. An assay based on genetic markers would not require biocontainment-handling virus is unnecessary-and thus would simplify predicting the potential for local mosquito populations to transmit WN virus.

Progress 10/01/06 to 09/30/11

Outputs
OUTPUTS: Most significant outputs from this project concern increased understanding of factors that influence mosquito (1) biting behavior and (2) infection with the viruses that they transmit to people and horses. A better understanding of mosquito blood feeding patterns and vector competence help define the threat that California mosquitoes pose to human health and their role in West Nile virus amplification. Results were disseminated to California mosquito abatement districts, which used this information to improved surveillance and prevention programs for prevention of West Nile virus disease in humans, wildlife, and domestic animals. PARTICIPANTS: Post-doctoral fellow, Rajev Vaidyanathan, in my laboratory carried out the mosquito vector competence study and was the lead author on the resulting publication. That research was, in part, support by the California Mosquito Research Program. A masters student, Mathew Montgomery, in my laboratory carried out the blood feeding study and was the lead author on the resulting publication. That research was, in part, supported by the Sacramento-Yolo Mosquito and Vector Control District. TARGET AUDIENCES: Results from studies on West Nile virus vectors are directly relevant to mosquito control activities carried out locally by the Sacramento-Yolo Mosquito and Vector Control District and statewide by the Mosquito and Vector Control Association of California. Results were conveyed to mosquito control personnel in personal meetings, talks at scientific meetings, and in scientific publications. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
We determined that the conditions under which immature mosquitoes develop effects their capacity to become infected with and subsequently transmit virus. Mosquitoes emerging from nutrient poor natural environments may be more likely to transmit virus than those reared on relatively nutrient rich laboratory diets. Results from laboratory studies may, therefore, underestimate the capacity of wild, free-ranging mosquitoes to transmit West Nile virus in California. Using molecular markers that identified the specific species of vertebrate from whom the mosquito Culex pipiens took its blood meals, we determined that in Yolo and Sacramento Counties this species almost always bites an avian host. None of the mosquitoes tested bit a person. Our results indicate that this mosquito in northern California is more important at transmitting and amplifying virus in bird populations than infecting human or equine hosts. Mosquito control directed at this species will, therefore, dampen overall virus transmission but will not directly protect people or horses from exposure and disease from West Nile virus.

Publications

• Montgomery, M.J., P. Macedo, D.A. Brown, T. Thiemann, and T.W. Scott. 2011. Blood feeding patterns of the Culex pipiens complex in Sacramento and Yolo Counties, California. J. Med. Entomol. 48: 398-404

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: Mosquitoes in the Culex pipiens complex are competent vectors of West Nile virus in the laboratory and field collected mosquitoes have tested positive for the virus in California and elsewhere. A better understanding of Cx. pipiens complex blood feeding patterns will help define the threat that these mosquitoes pose to human health and their role in West Nile virus amplification in northern California. We collected blood engorged Cx. pipiens complex mosquitoes from resting sites near and away from human habitation in Sacramento and Yolo Counties. Cytochrome c oxidase I gene sequences were used to identify the vertebrate species from which bloodmeals were taken. PARTICIPANTS: A masters student, Mathew Montgomery, in my laboratory worked on this project by studying the extent to which important mosquito vectors of West Nile virus bite people and, therefore, constitute a major risk factor for human West Nile epidemics. A manuscript on this topic was submitted for publication, following revision it was accepted, and galley proofs are in press for publication. TARGET AUDIENCES: Results from studies on West Nile virus vectors are directly relevant to mosquito control activities carried out locally by the Sacramento-Yolo Mosquito and Vector Control District and statewide by the Mosquito and Vector Control Association of California. Results were conveyed to mosquito control personnel in personal meetings, talks at scientific meetings, and in scientific publications. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
In this study we sought to determine the role that the Cx. pipiens complex plays in the transmission of West Nile virus in Sacramento and Yolo Counties by studying its host associations. We hypothesized that the Cx. pipiens complex feeds on mammalian and avian hosts, including humans, and, therefore, functions as both an amplifying and epidemic vector in this region.

Of 330 engorged mosquitoes collected at 28 sites from June through August 2007 and May through August 2008 over 99% fed on an avian host. Three mosquitoes contained bovine blood and none had fed on a human. American Robins were bitten most often and the proportion of American Robin bloodmeals increased significantly over the summer. Other important avian hosts included House Finches, Barn Swallows, Western Meadowlarks, and Mourning Doves. In rural areas Barn Swallows, Brewer's Blackbirds, and House Sparrows were frequent hosts. In settings near human habitation Mourning Doves and Western Meadowlarks were common hosts. Our data indicate that in north-central California mosquitoes in the Cx. pipiens complex are more important transmitting West Nile virus to birds than to humans.

The applied implication of our study is that mosquito control activities directed at Cx. pipiens complex will reduce overall amplification of West Nile virus in northern California, but it will not directly protect humans from infection during periods of elevated or epidemic virus transmission. At those times mosquito control should be directed at other mosquito species that frequently bite humans.

Publications

• No publications reported this period

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: We carried out 2 analyses. First, we performed a meta-analysis of studies across a range of mosquito-virus systems to determine whether arboviruses reduce the survival of their mosquito vectors, but that the magnitude of the effect depends on the vector/virus taxonomic groups and the mode of virus transmission. Second, to test the hypothesis that mosquito vectors play an important role in shaping dengue virus (DENV) genetic diversity we measure gene by gene interactions in a system representative of a natural dengue virus transmission situation in Thailand by challenging three inbreed mosquito families from field-derived populations with three contemporaneous isolates of dengue virus. PARTICIPANTS: A post-doc, Louis Lambrechts, in my laboratory worked on this project by studying the relationship between virulence of an arbovirus to its mosquito vector and whether the virus is transmitted horizontally or vertically. A masters student, Mathew Montgomery, in my laboratory worked on this project by studying the extent to which important mosquitoes vectors of West Nile virus bite people and, therefore, constitute a major risk factor for West Nile epidemics. We are about to submit a manuscript for publication on this topic. TARGET AUDIENCES: Results from studies with dengue virus are directly relevant to mosquito control activities carried out dengue endemic countries around the world. Because 2.5 billion people are at risk of dengue infection each year, there is a huge clientele that is interested in our activities in this project. We aim to influence ministries of health, local mosquito control districts, funding agencies, and international bodies like the World Health Organization and the Pan American Health Organization. Results are conveyed to mosquito control personnel and policy makers in personal meetings, commissioned reports, talks at scientific meetings, and in scientific publications. Results from studies on West Nile virus vectors are directly relevant to mosquito control activities carried out locally by the Sacramento-Yolo Mosquito and Vector Control District and statewide by the Mosquito and Vector Control Association of California. Results were conveyed to mosquito control personnel in personal meetings, talks at scientific meetings, and in scientific publications. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Regarding the impact of arboviruses on their mosquito vectors, we determined that alphaviruses were associated with highest virulence levels in mosquitoes. Horizontal transmission was correlated with significant virus-induced mortality, whereas a lack of adverse effect was found for Aedes mosquitoes infected transovarially by bunyaviruses, a group of viruses characterized by high natural rates of vertical transmission in their enzootic vectors. Our findings are consistent with the general prediction that vertically transmitted pathogens should be less virulent than those transmitted horizontally. We conclude that varying degrees of virulence observed among vector-virus systems probably reflect different selective pressures imposed on arboviruses that are primarily transmitted horizontally versus vertically. Regarding the impact of mosquito vectors on virus evolution we found evidence that vector competence of Ae. aegypti for dengue virus is likely governed to a large extent by gene by gene interactions in genetically diverse, natural populations. This result challenges the general relevance of conclusions from laboratory systems that consist of a single combination of mosquito and dengue virus genotypes. Combined with earlier evidence for fine-scale genetic structure of natural Ae. aegypti populations, our finding indicates that the necessary conditions for local of dengue virus adaptation to mosquito vectors are met.

Publications

• Lambrechts, L. and T.W. Scott. 2009. Mode of transmission and the evolution of arbovirus virulence in mosquito vectors. Proc. R. Soc. Lond. B. 276: 1369-1378.
• Lambrechts, L., C. Chevillon, R.G. Albright, B. Thaisomboonsuk, J.H. Richardson, R.G. Jarman, and T.W. Scott. 2009. Genetic specificity and potential for local adaptation between dengue viruses and mosquito vectors. BMC Evolutionary Biology. 9:160.

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: Vector competence studies with mosquitoes provided different diets demonstrated that environmental factors, other than intrinsic genetic determinants, are important determinants of whether or not a mosquito becomes infected with and transmits West Nile virus. Development of simple rapid assays for assessing the capacity of different mosquito populations to transmit virus will be more difficult than anticipated due to these important environmental effects. PARTICIPANTS: A masters student, Mathew Montgomery, in my laboratory is working on this project by studying the extent to which important mosquitoes vectors of West Nile virus bite people and, therefore, constitute a major risk factor for West Nile epidemics. TARGET AUDIENCES: Results are directly relevant to mosquito control activities carried out locally by the Sacramento-Yolo Mosquito and Vector Control District and statewide by the Mosquito and Vector Control Association of California. Results were conveyed to mosquito control personnel in personal meetings, talks at scientific meetings, and in scientific publications. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Most female mosquitoes ingest plant sugars for flight energy and survival, and they imbibe vertebrate blood for egg development. We evaluated the effect of different sucrose meals following a blood meal containing West Nile virus on Culex pipiens pipiens survival, nutritional status, and susceptibility to viral infection and transmission. Ten days after blood feeding, no mosquitoes survived on distilled water, 55% survived on 2% sucrose, 61% on 10% and 20% sucrose meals, and over 70% survived on 40% sucrose. Applying survival data from mosquitoes maintained in the laboratory would therefore bias estimates of vectorial capacity for West Nile virus, yielding an index of pathogen transmission risk with greater comparative than predictive value. With regard to West Nile virus infection and transmission we found that mosquitoes from all the sugar series were equally susceptible to infection, but nutritionally stressed mosquitoes were more likely to orally transmit West Nile virus. Excess sugar in laboratory-based studies may provide the energy required to inhibit infection, resulting in an inaccurate estimate of vector competence compared to what actually occurs in the field. Because we expect mosquitoes in the field to be nutritionally stressed, results from our studies indicate that wild mosquitoes may become infected with and transmit West Nile virus more efficiently than results from laboratory studies suggest.

Publications

• Vaidyanathan, R., A.E. Fleisher, S.L. Minnick, K.A. Simmons, and T.W. Scott. 2008. Nutritional stress affects mosquito survival and vector competence for West Nile virus. Vector-borne and Zoonotic Dis. 8: 727-732.

Progress 01/01/07 to 12/31/07

Outputs
Material on three initiatives was published this year. The first concerned a classification scheme for the different containers in which immature mosquitoes develop. This is necessary for development of community-based mosquito control programs for dengue prevention. The lab developed a classification method that consists of the shape (S), use (U) and material (M) of each container (SUM-method). It was determined that by targeting the four container classes that held the most pupae of the dengue mosquito vector, Aedes aegypti, adult mosquito production and the threat of dengue transmission could theoretically be reduced by 70%. The new classification strategy will (1) facilitate comparison of results among different ecologic and geographic settings and (2) simplify communication among vector control personnel and affected communities. The second initiative concerned the effect of human blood feeding and egg laying on mortality of the dengue virus mosquito vector, Ae. aegypti. This was a follow-up study to research reported last year in which mosquitoes were fed only sugar and, thus, did not lay eggs. Results were identical to those previously reported and demonstrate that reproductively active mosquitoes exhibit age-dependent mortality, which underscores the need to incorporate increasing mortality with advancing age into predictive pathogen transmission models and research on mosquito biology in general. The third initiative concerns ecological issues associated with the use of genetic strategies to prevent insect transmitted disease. The discussion focused on mosquitoes for which the greatest body of current knowledge exists; i.e., mosquito vectors of human malaria, dengue, and yellow fever. Four topics relevant to any genetically modified mosquito strategy and that require additional research attention were reviewed in detail: (1) mating behavior and male biology, (2) assessing fitness, (3) population biology, and (4) regulatory issues.

Impacts
Results from my activities during 2007 are directly relevant to improved arbovirus surveillance and mosquito control for prevention of arbovirus transmission. Publications on container classification, mosquito mortality, and genetically modified mosquitoes provide guidance for the development of novel mosquito control and disease prevention programs.

Publications

• Koenraadt, C.J.M., J.W. Jones, R. Sithiprasasna, and T.W. Scott. 2007. Standardizing container classification for immature Aedes aegypti surveillance in Kamphaeng Phet, Thailand. J. Med. Entomol. 44: 938-944.
• Styer, L.M., S.L. Minnick, A.K. Sun, and T.W. Scott. 2007. Mortality and reproductive dynamics of Aedes aegypti fed human blood. Vector-borne and Zoonotic Dis. 7: 86-98.
• Scott, T.W., L.C. Harrington, B.G.J. Knols, and W. Takken. 2008. Applications of mosquito ecology for successful insect transgenesis-based disease prevention programs. In: Transgenesis and the Management of Vector-Borne Disease. Ed: Serap Aksoy. Landes Bioscience and Springer Science+Business Media. pp: 151-168. http://eurekah.com/chapter/3650

Progress 01/01/06 to 12/31/06

Outputs
Material on four initiatives was published this year. The first concerned a knowledge, attitude and practice (KAP) survey in Kamphaeng Phet province, Thailand, to determine if correct dengue knowledge and practice reduce dengue vector populations. We found that there was no relationship between knowledge of dengue and adult mosquito reduction practices and that measures against adult mosquitoes were used only when people experience a significant mosquito nuisance problem. The second initiative concerned entomological surveillance in places other than the home. Results demonstrate that dengue surveillance and control programs need to consider non-residential sites separately from residential sites. Even though mosquito production in non-residential sites appeared to be significantly and epidemiologically relevant, it is lower than residential sites and vector control programs priorities should remain focused on homes. The third initiative was an examination of mosquito evolution and the fundamental assumptions of the idea that we can use genetically modified mosquitoes to prevent disease. Results from genetic studies of mosquitoes and a particular species of bacteria that infect them imply that under proper circumstances, the number of transgenic mosquitoes that must be released into a natural setting may be low and that desirable genes in those mosquitoes could spread in a relatively short period of time across an extensive geographical range. The fourth initiative concerns a collaborative program to develop new products and programs for controlling mosquito populations and preventing disease. Few new insecticides have been produced for public health vector control in developing countries over the last three decades, due to market constraints, and the available insecticides are often poorly deployed. The Innovative Vector Control Consortium will address these market failures by developing a portfolio of chemical and technological tools that will be directly and immediately accessible to populations in the developing world. The Bill and Melinda Gates Foundation has supported this new initiative to enable industry and academia to change the vector control paradigm for malaria and dengue and ensure that vector control, alongside drugs, case management and vaccines, can be better deployed to reduce disease

Impacts
Results from my activities during 2006 are directly relevant to improved arbovirus surveillance and mosquito control for prevention of arbovirus transmission. Publications on knowledge, attitude and practice; mosquito surveillance; genetically modified mosquitoes; and Innovative Vector Control Consortium provide guidance for the development of novel mosquito control and disease prevention programs.

Publications

• Koenraadt, C.M.J., W. Tuiten, R. Sithiprasasna, U. Kijchalao, J.W. Jones, and T.W. Scott. 2006. Dengue knowledge and practice and their impact on Aedes aegypti populations in Kamphaeng Phet, Thailand. Am. J. Trop. Med. Hyg. 74: 692-700.
• Morrison, A.C., M. Sihuincha, J.D. Stancil, E. Zamora, H. Astete, J.G. Olson, C. Vidal-Ore, and T.W. Scott. 2006. Aedes aegypti (Diptera:Culicidae) production from non-residential sites in the Amazonian city, Iquitos, Peru. Ann. Trop. Med. Parasit. 100: S73-S86.
• Rasgon, J.L., A.J. Cornel, and T.W. Scott. 2006. Evolutionary history of Wolbachia invasion revealed through mitochondrial DNA sequence analysis in the mosquito Culex pipiens. Proc. R. Soc. Lond. B. 273: 1603-1611.
• Hemingway, J., B.J. Beaty, M. Rowland, T.W. Scott, and B.L. Sharp. 2006. The Innovative Vector Control Consortium: Improved control of mosquito-borne diseases in and around the home. Trends in Parasitology. 22: 308-312.

Progress 01/01/05 to 12/31/05

Outputs
Material on four initiatives was published this year. The first concerns arthropod-borne virus (arbovirus) surveillance. My colleagues and I carried out detailed studies of dengue virus transmission that are relevant to prevention of West Nile virus in California. We assessed different sampling methodologies for (1) estimating mosquito population densities and (2) predicting patterns of adult mosquito production from larval, aquatic habitats. These efforts constitute the basis for recommendations on sampling strategies for assessing risk of arbovirus transmission and what factors constitute triggers for a response to a perceived public health threat. Often times the response is in the form of vector control. We also studied movement patterns of adult mosquitoes, which in part define the spatial dimensions of arbovirus transmission and thus the spatial scale over which vector control must be applied. The second initiative concerns the evaluation of a prospective arbovirus vaccine. We determined whether the vaccine could infect, replicate in, and be transmitted by potential mosquito vectors. The third initiative concerns the use of genetically modified mosquitoes to interfere with or prevent transmission of pathogens. As part of an expert panel for the World Health Organization (WHO), I participated in a workshop in Nairobi, Kenya on the transition of research from an emphasis on laboratory to more epidemiologically relevant field studies. I gave oral presentations and wrote manuscripts on the (1) integration of field and lab research and (2) operationally feasible ways to assess fitness of genetically modified mosquito vectors. Both articles were published in a book published by the WHO, which is expected to provide guidance to scientists, public health officials, and policy makers around the world. The forth initiative concerns containment of medically important arthropods. I wrote a paper as part of a series of articles on proper practices for laboratory containment. The article was published in a journal that focuses on animal care and safety. I expect that it will provide guidance to university, government, and private industry safety committees. As a result of the article I was asked to serve as a consultant on mosquito containment for a company that is developing malaria vaccines.

Impacts
Results from my activities during 2005 are directly relevant to improved arbovirus surveillance and mosquito control for prevention of arbovirus transmission. Publications on genetically modified mosquitoes provide guidance for the development of novel mosquito control and disease prevention programs. Containment guidelines are essential for safe and effective research with medically important arthropods and the pathogens that they transmit.

Publications

• Rasgon, J.L. and T.W. Scott. 2004. Crimson: A novel sex-linked eye color mutant of Culex pipiens. J. Med. Entomol. 41: 385-391.
• Morrison, A.C., H. Estete, F. Chapilliquen, G. Ramirez Prada, G. Diaz, A. Getis, K. Gray, and T.W. Scott. 2004. Evaluation of a sampling methodology for rapid assessment of Aedes aegypti (Diptera: Culicidae) infestation levels in Iquitos, Peru. J. Med. Entomol. 41: 502-510.
• Morrison, A.C., K. Gray, A. Getis, H. Estete, M. Sihuincha, D. Focks, D. Watts, and T.W. Scott. 2004. Temporal and geographic patterns of Aedes aegypti (Diptera: Culicidae) production in Iquitos, Peru. J. Med. Entomol. 41: 1123-1142.
• Rasgon, J.L. and T.W. Scott. 2004. Phylogenetic Characterization of Wolbachia Symbionts Infecting Cimex lectularius L. and Oeciacus vicarius Horvath (Hemiptera: Cimicidae). J. Med. Entomol. 41: 1175-1178
• Scott, T.W. 2005. Containment of arthropod disease vectors. ILAR Journal. 46: 53-61.
• Harrington, L.C., T.W. Scott, K. Lerdthusnee, R.C. Coleman, A. Costero, G.G. Clark, J.J. Jones, S. Kitthawee, P. Kittayapong, R. Sithiprasasna, and J.D. Edman. 2005. Dispersal of the dengue vector Aedes aegypti within and between rural communities I. Am. . Trop. Med. Hyg. 72: 209-220.
• Hanley, K.A., L.B. Goddard, L.E. Gilmore, T.W. Scott, J. Speicher, B.R. Murphy, and A.G. Pletnev. 2005. West Nile/Dengue type 4 chimeras show reduced infectivity for virus-specific, but not generalist, mosquito vectors. Vector-Borne Zoonotic Dis. 5: 1-10
• Bosio, C.F., L.C. Harrington, J. Jones, D.E. Norris, and T.W. Scott. 2005. Genetic structure of Aedes aegypti populations in Thailand using mtDNA. Am. J. Trop. Med. Hyg. 72: 434-442.
• Scott, T.W. 2005. Current thoughts about the integration of field and laboratory sciences in genetic control of disease vectors. In: Strategic Plan to Bridge Laboratory and Field Research in Disease Vector Control. eds. B.G.J. Knols and C. Louis, FRONTIS, Dordrecht, The Netherlands. pp. 67-76.
• Schneider, J.R., A.C. Morrison, H. Astete, T.W. Scott, and M.L. Wilson. 2004. Adult size and distribution of Aedes aegypti (Diptera: Culicidae) associated with larval habitats in Iquitos, Peru. J. Med. Entomol. 41: 534-542.
• Scott, T.W., J.L. Rasgon, W.C. Black IV, and F. Gould. 2005. Fitness studies: Developing a consensus methodology. In: Strategic Plan to Bridge Laboratory and Field Research in Disease Vector Control. eds. B.G.J. Knols and C. Louis, FRONTIS, Dordrecht, The Netherlands. pp. 171-181.

Progress 01/01/04 to 12/31/04

Outputs
Material on three initiatives was published this year. The first concerns arbovirus surveillance and the role of mosquitoes in the transmission of West Nile (WN) virus. As part of the California surveillance program, my colleagues and I tested specimens from humans, equines, mosquitoes, sentinel chickens and dead birds for evidence of mosquito-borne encephalitis virus activity. Results were reported by the California Department of Health Services to mosquito and vector control districts and were used to make decisions about application of mosquito control programs. I also was part of a team that determined by assaying horse sera that horses in California were commonly infected with arboviruses (i.e., Bunyaviruses) prior to 1998, but there was no evidence of equine St. Louis virus infections. From this we predicted that WN virus will have a wide distribution when it is fully established in California. Concerning mosquito research, our results on vertical transmission of WN virus indicates that mosquitoes infected vertically (from mother to her progeny) in the fall could serve as a mechanism for WN virus to survive in the winter when mosquitoes are inactive and then rapidly initiate virus transmission the following spring when mosquitoes become active and start biting again. The second initiative concerns the use of genetically modified mosquitoes to interfere with or prevent transmission of pathogens. In a series of computer simulation studies we concluded that understanding ecological factors is critical for designing transgenic vector-borne disease control strategies. Specifically, we determined that release of genetically modified mosquitoes into natural populations with age-structure will be difficult and depending on the circumstances may not be economically or logistically feasible. The third initiative concerns new ways of estimating the chronological age of mosquitoes. In a series of field studies we developed and validated new models for estimating a mosquitos age that will be useful for epidemiological studies and for assessing the impact of mosquito control interventions on disease prevention.

Impacts
Results from our studies on vertical transmission of WN virus and arbovirus surveillance in California are directly relevant to improved arbovirus surveillance and mosquito control for prevention of arbovirus transmission. Our studies on genetically modified mosquitoes provide a theoretical and empirical basis for the development of novel mosquito control and disease prevention programs. Our application of age-grading methods to wild mosquitoes allows one, for the first time, to determine the age distribution of natural mosquito populations, which will be a powerful new tool for assessing the effectiveness of mosquito control programs.

Publications

• Rasgon, J.L. and T.W. Scott. 2003. Wolbachia and cytoplasmic incompatibility in the California Culex pipiens mosquito species complex: Parameter estimates and infection dynamics in natural populations. Genetics. 165: 2029-2038.
• Goddard, L.B., A.E. Roth, W.K. Reisen, and T.W. Scott. 2003. Vertical transmission of West Nile virus by three California Culex (Diptera: Culicidae) species. J. Med. Entomol. 40: 743-746.
• Benedict, M.Q., W.J. Tabachnick, S. Higgs, A.F. Azad, C.B. Beard, J.C. Beier, A.M. Handler, A.A. James, C.C. Lord, R.S. Nasci, K.E. Olson, J.Y. Richmond, T.W. Scott, D.W. Severson, E.D. Walker, and D.M. Wesson. 2003. Arthropod Containment Guidelines. Vector-Borne Zoonotic Dis. 3: 57-98.
• Husted, S., V.L. Kramer, A.B. Houchin, R.E. Chiles, M. Jay, W.K. Reisen, B.F. Eldridge, C. Glaser, C. Cossen, E. Tu, W.C. Reeves, T.W. Scott, S. Quick-Miles, K.McCaughey, and M. Castro. 2004. Surveillance for mosquito-borne encephalitis virus activity and human disease in California, 2002. Proc. Mosq. Vector Control Assoc. Calif. 71: 38-42.
• Nelson, D.M., I.A. Gardner, R.F. Chiles, U.B. Balasuriya, B.F. Eldridge, T.W. Scott, W.K. Reisen, and N.J. Maclachland. 2004. Prevalence of antibodies against Saint Louis encephalitis and Jamestown Canyon viruses in California horses. Comp. Immunol. Microbio. Infect. Dis. 27: 209-215.
• Rasgon, J.L. and T.W. Scott. 2004. An initial survey for Wolbachia (Rickettsiales: Rickettsiaceae) infection in selected California mosquitoes (Diptera: Culicidae). J. Med. Entomol. 41: 255-257.
• Gerade, B.B., S.H. Lee, T.W. Scott, J.D. Edman, L.C. Harrington, S. Kitthawee, J.W. Jones, and J.M. Clark. 2004. Field validation of Aedes aegypti (Diptera: Culicidae) age estimation by analysis of cuticular hydrocarbons. J. Med. Entomol.41: 231-238.
• Rasgon, J.L. and T.W. Scott. 2004. Impact of population age structure on Wolbachia transgene driver efficacy: Ecological complex factors and release of genetically-modified mosquitoes. Insect Biochem. Molec. Biol. 34: 707-713.
• Chiles, R.E., E.N. Green, Y. Fang, L. Goddard, A. Roth, W.K. Reisen, and T.W. Scott. 2004. Blinded laboratory comparison of the in situ enzyme immunoassay, the VecTest wicking assay and an RT-PCR assay to detect mosquitoes infected with West Nile and St. Louis encephalitis viruses. J. Med. Entomol. 41: 539-544.

Progress 01/01/03 to 12/31/03

Outputs
Material on three initiatives was published this year. The first concerns the role that different mosquito species will play in the transmission mission of West Nile (WN) virus when it arrives in California. Interestingly, the virus arrived during late 2003, after our studies were published, and the species we identified turned out to be the key vectors. We evaluated the vector competence of ten California mosquito species; all became infected and were able to transmit WN at some level. Considering their behavior, ecology, laboratory vector competence, and role in WN transmission in the Old World and the eastern United States, Culex species were identified as likely to play the primary role in the maintenance and transmission of WN in California. Differences in vector competence within a genetically complex group of important mosquito vectors indicated that their role in WN transmission may vary from one geographic location to another. Our entomological results will be useful for vector control and indicated that if WN is introduced it will become established in California. The second initiative concerns the use of genetically modified mosquitoes to interfere with or prevent transmission of pathogens. In a booked edited by a colleague and myself the ecological issues associated with genetically modified mosquitoes were outlined and discussed in detail. In a series of studies, we examined empirically and theoretically use of the bacteria Wolbachia as a mechanism to drive transgenes for refractoriness into mosquito vector populations. The third initiative concerns identification of the person a mosquito bit and thus the people who are at greatest risk of infection with mosquito-borne pathogens. Feeding was non-random with a bias towards young adults and males receiving most of the bites. Our results confirm that feeding frequently on different people is an important component of a mosquito's role in dengue virus transmission.

Impacts
Our studies on WN provide crucial information that identities mosquito species responsible for virus transmission so that surveillance and mosquito control programs may target suspect vector species. Our studies on the ecology of transgenic mosquitoes help resolve key issues associated with the application of that novel technology as a public health tool.

Publications

• Cornel, A., R. McAbee, J. Rasgon, M. Stanich, T.W. Scott, and M. Coetzee. 2003. Differences in extent of genetic introgression between sympatric Cx. pipiens and Cx.
• Goddard, L.B., A.E. Roth, W.K. Reisen, and T.W. Scott. 2002. Vector competence of California mosquitoes for West Nile virus. Emerging Infectious Diseases. 8: 1385-1391.

Progress 01/01/02 to 12/31/02

Outputs
Material on two different research initiatives was published this year. The first concerns the use of different kinds of traps to monitor mosquito density as part of the California-wide surveillance system for detection of arthropod-borne viruses. To provide information on comparative sensitivity and ability to measure abundance over time and space, catch of mosquitoes in NJ light traps were compared to catch in CDC traps operated concurrently at 8 sites within the Coachella Valley, Kern, San Joaquin County and Sacramento-Yolo Mosquito and Vector Control Districts. CDC traps always collected more female mosquitoes than NJ traps; however, differences in sensitivity varied markedly over time and space precluding the calculation of a universal conversion factor. Regressions of the catch of female Culex tarsalis in CDC traps as a function of catch in NJ traps within districts indicated that the slopes varied markedly, again precluding the derivation of a universal function. My co-authors and I concluded that mosquito control districts that switch surveillance from NJ to CDC traps should operate both traps concurrently at several sites to derive regression functions to convert historical relative abundance data from NJLTs to equivalent counts in CDC traps for analysis. The second concerns the use of mosquitoes that have been genetically modified to render them refractory to pathogen infection as a way to control mosquito-borne diseases of humans and domestic animals. Two articles were published in Science on this topic. In the first, my colleagues and I outlined key ecological and population biology issues that must be addressed before genetically modified mosquitoes can be used safely and effectively for the control of vector-borne diseases. In brief, we developed a research agenda for ecological studies on this topic. The second article focused specifically on the risks, benefits, and research priorities for using genetically manipulated mosquitoes for the control of malaria.

Impacts
With regard to the trap comparison, assessing risk of virus infection in humans and domestic animals is essential for the most efficient use of limited resources to control disease. This is especially true with West Nile virus moving westward and the expectation that it will invade California during 2003. With regard to the use of genetically modified vector for disease control, we defined key issues associated with a novel approach for disease control and outlined a research agenda for addressing those topics.

Publications

• Reisen, W.K., B.F. Eldridge, T.W. Scott, R. Takahashi, T. Dhapin, K. Lorenzen, J. DeBenedictis, K. Boyce, S. Wright, and R. Swartzell. 2002. Comparison of dry ice baited CDC and NJ light traps for measuring mosquito abundance. J. Amer. Mosq. Control. Assoc. 18: 158-163.
• Scott, T.W., W. Takken, B.G.J. Knols, and C. Boete. 2002. The ecology of genetically modified mosquitoes. Science. 298: 117-119.
• Alphey, L, C.B. Beard, . T.W. Scott, et al. 2002. Malaria control with genetically manipulated insect vectors. Science. 298: 119-121.

Progress 01/01/01 to 12/31/01

Outputs
Research on two major projects was published this year. In the first, my colleagues and I examined the observation that adult female Aedes aegypti, the vector of dengue and yellow fever viruses, have an affinity for feeding on human blood and a tendency to forego feeding on sugar. This observation challenges two tenets of mosquito biology: (1) mosquitoes imbibe plant carbohydrates for synthesis of energy reserves and blood for reproduction and (2) egg production is reduced when mosquitoes feed on human blood compared with blood from other species. In a series of experiments we tested the hypothesis that feeding on human blood is associated with a selective advantage for Ae. aegypti and is an underlying reason for this mosquito's intimate and epidemiologically important relationship with human beings. When mosquitoes imbibed blood meals over a 7 to 10 day period and were not fed sugar, increased isoluencine concentration in host blood decreased energy reserves and did not increase egg production. Aedes aegypti took smaller but more frequent blood meals when feeding on a low-isoleucine human host daily compared with a high isoleucine mouse host. Previous reports that isoleucine enhances egg production wereconfirmed only when females were fed sugar, an unusual behavior for most dometic Ae. aegypti populations. Females fed human blood and water had greater survival and reproductive output than cohorts fed human blood plus sugar or isoleucine-rich mouse blood with or without access to sugar. In our second study we examined the assumption that the daily probability of survival of wild adult mosquitoes is independent of age. We conducted mark-release-recapture studies with mosquitoes to determine if estimated daily survival raes between two different age cohorts of the dengue vector, Aedes aegypti (L.), were the same. Survivorship was estimated with a new method (non-linear regression analysis using bootstraping to obtain estimaes of errors). Initial recapture success of teh younger cohort was greater than for the older cohort. Our analysis revealed a significantly greater survival rate for the younger cohort of females at one study site but not the other. for comparison, a traditional aproach for analyzing these tyupe of data (linear regression of log-transformed captures over time), was used to calculate the probability of daily survival based on slopes of linear regression lines for recaptured mosquitoes. With this method, the estimated daily survival rate of older females was significantly greater than survival of younger ones.

Impacts
The unique low isoleucine concentration of human blood is associated with Ae. aegypti's unusual propensity to feed preferentially and frequently on humans. this process increases the mosquito's fitness, synthesis of energy reserves, and contact with human hosts and is the primary reason why it is such an effective disseminator of human pathogens. We demonstrated that survival raes of adult Ae. aegypti may be age dependent and non-linear regression analysis is a sensitive approach for comparing patterns of mosquito survival based on mark-single release, multiple recapture data.

Publications

• Harrington, L.C., J.D. Edman, and T.W. Scott. 2001. Why do female Aedes aegypti (Diptera: Culicidae) feed preferentially and frequently on human blood? J. Med. Entomol. 38:411-422.
• Harrington, L.C., J.P. Buonaccorsi, J.D. Edman, A. Costero, G.G. clark, P. Kittayapong, and T.W. Scott. 2001. analysis of survival rates for two age cohorts of Aedes aegypti (L.) (Diptera:Culicidae): Results from Puerto Rico and Thailand. J. Med. Entomol. 38:537-547.