Duration: 08/01/2009 to 09/30/2014

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

Infectious diseases are on the rise worldwide and account for a quarter of all human mortality and morbidity despite extraordinary medical advances. Diseases once thought to menace only remote tropical inhabitants are now spreading everywhere, fueled by international travel.

The mosquito could be considered the most dangerous creature on earth. Diseases transmitted by mosquitoes have killed more people than all the wars in history. Each year there are 300 - 500 million cases of malaria reported, resulting in up to 2.7 million deaths, mostly children. By contrast, the AIDS virus afflicts <6 million people annually.

Arboviruses are the most significant cause of mosquito-borne disease in the U.S. Arboviral infections often result in encephalitis, a brain inflammation which can result in death or severe neurologic after-effects. U.S. mosquitoes transmit several serious endemic encephalitic viruses including St. Louis, LaCrosse, and eastern and western equine encephalitis. There are no vaccines, antibiotics, or treatments for viral encephalitis. Mitigation centers on controlling mosquitoes that transmit the diseases.

The introduction and rapid dispersal of mosquito-borne West Nile virus (WNV) has effectively demonstrated the infectious threat posed by arboviruses. WNV is merely the latest in a series of infectious viruses of national public health significance to be introduced into the United States. This virus swept across the country following its appearance in New York in 1999, and the disease is now endemic in the continental 48 states. The outbreak constituted the largest documented epidemic of mosquito-borne meningoencephalitis in the history of the western hemisphere. Although principally a disease of birds (explaining its swift dispersal), nearly 24,000 people in the U.S. have become infected with WNV with 900 deaths recorded to date. The elderly and children are at particular risk of developing serious illness.

The U.S. will continue to experience the arrival of new arboviral diseases. Consider chikungunya virus, a deadly infectious disease that causes crippling arthritic damage to survivors. The virus has infected more than 1.5 million people in the Indian Ocean region in a massive eruption over the past 24 months. Nearly a dozen U.S. states have reported cases of infected travelers returned from Asia and East Africa. Ultimately infectious patient will meet competent vector and the disease will undergo establishment, amplification, and dispersal. The U.S. not only is a travel and immigrant destination for large numbers of South Asians, but also has a highly competent vector -- Aedes albopictus. Or consider dengue, the predominant mosquito-borne viral disease affecting humans. The disease is now endemic in >100 countries and infects 100 million persons each year with 2.5 billion at risk. The World Health Organization has reported a 30-fold incidence increase in dengue over the past 50 years. Dengue reemerged in Hawaii in 2001; four cases were reported in Philadelphia this year. Other important potential invasive pathogens transmitted by mosquitoes include Rift Valley fever virus (devastating livestock and human disease, competent vectors in the US, 2006-2007 outbreak in Africa), Japanese encephalitis virus (widespread in pigs throughout eastern Asia and the cause of universal childhood vaccination in some countries), and Ross River virus (common infection in Australia). Although these viruses have been spread inadvertently, their potential introduction as agents of bioterrorism is a very real threat.

The economic impact of mosquito-borne illness is also devastating. The cost of treating WNV infections has been immense, with Louisiana estimating $70 million for 2002 alone. The estimated cost per human case of eastern equine encephalitis (EEE) is $3 million. EEE and WNV both threaten the nation's multi-billion dollar equine industry. The mortality rate of horses infected with WNV is 34 percent; the rate for those with EEE is 100 percent. In 2000, the estimated loss in New Jersey due to equine cases of WNV was $6 million. Tourism, which increases human exposure to mosquitoes, is similarly impacted by outbreaks of mosquito-borne disease. This was effectively demonstrated in 1959 in New Jersey, when 21 people died during an EEE outbreak, resulting in tourism coming to a near standstill.

Effective interventions based on sound medical entomology have had tremendous success in America against diseases caused by mosquito-borne pathogens including malaria, filariasis, yellow fever, and dengue. However, the 1999 epidemic of West Nile virus disease was a strong reminder that the U.S. remains susceptible to these kinds of threats. The lessons learned from WNV and the threat posed by additional new and emerging arboviruses serve as an impetus for bolstering U.S. vigilance. The U.S. requires research and outreach capabilities that provide answers to preventing and controlling outbreaks of arboviruses and other pathogens transmitted by mosquitoes. Improving the effectiveness and environmental safety of entomological interventions continues to be a priority outcome for research.

Related, Current and Previous Work

One way to outline the state of the art of mosquito abatement is within the structure of applied entomology known as Integrated Pest Management (IPM), which provides the basic organization for mosquito research. Developed during the 1960s and 1970s (Metcalf and Luckman 1975), IPM drew on the previous hundred years of accumulated entomological wisdom to produce a methodology that would guide research, education, and application toward solutions that were effective, economical, sustainable, and safe.

Risk assessment/biology is the first necessary step for any IPM program. Background information on the identification (systematics and taxonomy), distribution (spatial and temporal), behavior (particularly potential for causing damage), and bionomics of the pest defines the problem and suggests strategies for its control. Research on almost any aspect of the pest's biology can contribute toward the practical goals of risk assessment. In a sense, biological studies contribute toward risk assessment by improving our ability to "know thy enemy." Further, when the damage is caused by a pathogen transmitted by the pest (i.e., vector), an understanding of the pathogenesis and epidemiology is also important. This knowledge of the relationship between the vector and the pathogen can lead to a much better understanding of the problem. Particularly useful tools for risk assessment include geographic information systems and spatial analysis, especially when they are applied at a sufficiently local level to direct the next steps in IPM.

Mosquitoes are one of the best studied groups of insects, but important, large gaps still exist in our knowledge of their biology. In contrast to other parts of the world (Rongnoparut et al. 2006), the taxonomy of North American mosquitoes is relatively complete, though surprises still exist, as demonstrated by the discovery that Anopheles quadrimaculatus is actually a group of five fully distinguishable species (Reinert et al. 1997), each with its own geographic and behavioral characteristics. Genetic associations between species and even strains of mosquitoes are assisting in the task of evaluating risk from specific viruses (Vazeille et al. 2007, Mayer et al. 2004). Tools for evaluating geographic distribution and for predicting spatial presence of mosquitoes and pathogens would be very useful for predicting outbreaks and targeting resources (Anyamba et al. 2006, Sithiprasasna et al. 2005). For example, a model for predicting Rift Valley fever occurrence based on ocean temperatures and their effects on rainfall in East Africa (Anyamba et al. 2006b) resulted in the prediction of an outbreak in 2006 fully four months before cases were detected. Applied to the United States, similar models could predict where a mosquito introduced from Africa would have a chance of establishing the infection in America. Other kinds of models based on temperature (Zou et al. 2006) or remote sensing (Zou et al. 2006b) can assist in determining where it is necessary to concentrate operational assets for control of, for example, West Nile virus. Finally, the United States has experienced an accelerating rate of introduction and establishment of exotic mosquitoes. We would be much better prepared to prevent such establishment if we had a more precise idea of which species were the most likely invasives (Bram et al. 2002, Chretien et al 2006). What appear to be basic biological studies can have strong implications for control procedures once the results of the studies are understood in context. For example, movement of mosquitoes and other insects vertically (Anderson et al. 2004) or along distinct corridors (Tomberlin et al. 2006) imply that surveillance and control efforts might be concentrated in order to save time, funds, and to minimize pesticide use. Information on the sources of mortality of mosquitoes (Kobylinski et al. 2007) can prevent the application of vigorous control measures to life stages of the mosquitoes that would probably be decimated by natural factors under certain conditions.

Surveillance is the measurement of factors that inform the IPM program about where to concentrate control measures. Examples include detection and enumeration of the pest species, its damage, and occurrence of infection. Surveillance also includes measurement of correlates such as soil moisture or canopy density that are related to pest population. In addition, trap development and interpretation of trapping results are important aspects of research on surveillance.

Mosquitoes are among the easiest insects to collect for surveillance purposes. Adult mosquitoes seek hosts and therefore expose themselves through their movement and centralization. Larval mosquitoes are restricted to well-defined aquatic habitats, for which many inventive methods have been developed for sampling (Welch et al. 2006). Standard trapping methods are constantly being modified to become more efficient, easier, or to increase the number of individuals collected (Kline et al. 2006). One trend is toward development of specific attractant chemicals that can be integrated into trap operation to increase yield, sensitivity, and knowledge of what segment of the population is being sampled. For example, specific attractants for gravid mosquitoes (Allan and Kline 2004, Allan et al. 2005, Trexler et al. 2003), human-seeking mosquitoes (Allan et al. 2006, Allan et al 2007, Galimard et al. 2007), and avian-seeking mosquitoes (Allan et al 2006b) have been developed. These efforts are refinements that should continue, but the new developments may come from entirely different research directions. For example, remote sensing of mosquitoes directly with radar or passive detection of sound are based on good principles, but require more development to be routinely useful. The accuracy of sampling methods, in the sense of knowing how to relate results to actual population levels, is in need of much work (Strickman et al. 2000), and may be affected by what appear to be subtle aspects of biology (Williams et al. 2006, Wiwatanaratanabutr et al. 2007). During an IPM campaign, operational assets are completely dependent on surveillance to guide their control efforts. If eradication of an invasive species is the objective, it becomes even more important to know whether absence of a species in a trap indicates absence of the species in the environment.

Control includes all measures that might be taken to prevent damage from a pest. Conventional classifications of techniques include cultural, mechanical, physical, and chemical control. Development of new methods often requires coordination with industry and extension to translate results into action. The other major developmental challenge is the consideration of biology in order to integrate control methods efficiently. Careful timing of each kind of treatment can make a big difference in effectiveness, efficiency, and safety of an IPM program. Each control program must balance risk and benefit, but the admonition to do no harm to the environment, applicator, and consumer should be at the foundation of all research on this aspect of IPM.

Professional mosquito abatement programs often do an exemplary job of integration of methods. Using education they enlist the help of the public to eliminate sources of larvae, using civil engineering they remove large larval habitats that would otherwise require frequent chemical retreatment, using biopesticides they kill larvae without harming other kinds of insects or other animals, and using adulticides they protect the public from those mosquitoes that escaped other control measures. Each of these techniques could be improved through refinement and, in the case of chemical control, through the discovery of additional compounds for particular situations (Chauhan and Raina 2005, Grieco et al. 2005, Lloyd et al. 2002). Formulation of existing (Xue et al. 2003, 2006) and new toxicants would also improve mosquito abatement, for example, by combining them with baits (Xue and Barnard 2003) that reduce the total amount of active ingredient required. With the exception of bacterial toxins and limited use of fish, biological control has been disappointing in U.S. mosquito abatement. Development of new biological control agents (Andreadis et al. 2003, Becnel et al. 2003, Perera et al. 2006, Shapiro et al. 2004) or at least a better understanding of the role of natural biological control agents (Becnel et al. 2002, Becnel et al. 2005, Micieli et al. 2003, Reyes-Villanueva et al. 2003, Slothouber et al. 2004), would improve our ability to target other control methods. Recent years have seen the development of specific attractants that have proven to be very useful for surveillance. Wider use might also be helpful when combined with toxicants. Sterile male release was used successfully in the 1970s against two species of mosquitoes, but has only recently enjoyed revived interest thanks to the possibility of genetically sterile strains that avoid the complications of chemical or radiation based sterilization (Benedict and Robinson 2003). Individuals in communities often need to exercise their own actions to prevent mosquito bites, either through application of repellents (Debboun et al. 2007, Debboun et al. 2005b) or attempts to use traps for control (Kline 2006). Attempts to improve continue repellents continue despite the relatively small market, probably because of hope that the right product will be accepted and used much more widely. New testing methods based on scientific evidence (Barnard and Xue 2004, Barnard 2005) are important as more different active ingredients come on the market and as regulatory agencies attempt to standardize labeling. New active ingredients have been based on botanical discovery (Bernier et al. 2005, Cantrell et al. 2005, Chauhan et al. 2005), chemical modeling (Katritzky et al. 2006, Natarajan et al. 2005), and studies of the mixture of attractants and repellents that are naturally produced by hosts (Bernier et al. 2003). Traditional comparisons of repellents help define their effectiveness (Klun et al. 2003, 2004, 2005, 2006, 2006b). Entirely new methods for control of mosquitoes or their diversion from human hosts (Debboun et al. 2005, Carlson et al. 2006) are possible.

Monitoring a pest to ensure sustainable, successful IPM has proven very difficult for the field of entomology. Farmers or other applicators may lose interest in the problem once it has disappeared. Monitoring for sustainability requires systems that can accurately detect the reappearance of the pest, its damage, or disease caused by a transmitted pathogen. In general, entomological research has concentrated its efforts more on surveillance for control rather than monitoring for sustainability, with the result that many successful IPM programs have eventually failed as operational resources were diverted to other problems. In many cases, the technical tools of surveillance will be the same as those for monitoring, but the deployment of those tools will be different.

Medical entomology has some glaring historical examples of successful control programs that were not sustained. During the 1940s, Aedes aegypti, the species that transmits yellow fever and dengue viruses, was completely eliminated from Brazil (Soper et al. 1943) and subsequently was eradicated from 11 additional Latin American countries (Kerr et al. 1964). Unfortunately, the programs were not continued and Aedes aegypti returned to the parts of South and Central America from which it had been eliminated. This sort of problem is as much social as it is entomological, involving public awareness, economic feasibility, and simple community desire to accomplish the objective. The solution to dissipation of preventive programs once they are successful is not clear, but some programs seem to be sustained through careful monitoring, establishment of institutions that are large enough to seek political preservation [e.g., the screwworm eradication program (Wyss 2006)], or periodic failure that stimulates the public to fund the effort. The means to solve this serious problem are not clear, but there is a great need to develop them.

Problem Statement: Priority species of mosquito (e.g., Aedes aegypti, Aedes albopictus, floodwater mosquitoes, Culex vectors of viruses, Anopheles) are often controlled inefficiently because appropriate tools either do not exist or are not used in the most effective manner.

Related Multistate Projects: A search of the national multistate website shows only one other project involving mosquitoes: S-1029 "Improved Methods to Combat Mosquitoes and Crop Pests in Rice". This project has an exclusively agricultural and economic focus, whereas NE-507 has a medical and public health focus. This is clearly shown in the disparate objectives. Moreover, NE-507 involves different mosquito species than those of interest to S-1029 members. Most telling, there is no overlap among participants.

Objectives

1. Strengthen basic and applied research on the mosquito, pathogen, hosts, and environmental factors that influence disease emergence.
   
2. Use knowledge of mosquito, pathogen, vertebrate reservoir, and environmental interactions to enhance ability to predict conditions leading to disease.
   
3. Develop strategies to control mosquito vectors.
   
4. Enhance surveillance technologies for mosquitoes and mosquito-borne pathogens.
   
5. Develop strategies for sustainable mosquito control by including training at all levels.
   

Methods

Objective 1 - Strengthen basic and applied research on the mosquito, pathogen, hosts, and environmental factors that influence disease emergence.

Proactive integrated pest management protocols must be based on accurate measures of disease risk. The best measures of disease risk combine epidemiological and vector distribution/infection information. Although measures of vector infection rates have become standard in arboviruses such as the West Nile (WN) virus, there are high levels of spatial heterogeneity and the degree to which infected mosquitoes disperse from hot-spots is extremely unclear. This is particularly so when the vector is an introduced species with consequent low degree of local adaptation and low genetic diversity. We propose to develop a coordinated comparison of standard and vanguard methodologies to permit comparative analyses of mosquito dispersal. They will include mark-recapture using rare-earth methods and real-time high definition molecular methods using average distances between siblings. Species of interest will include invasives, expanders, and vectors of West Nile virus and Rift Valley Fever. Developing high-resolution molecular markers and the background methodology to implement sibling based dispersal analyses will be central.

Field dispersal and survival of marked and released male Aedes albopictus will be studied using MRR experiments. Additional effort will be devoted to comparing marking methods for persistence in the field and potential impacts on male survivorship and dispersal. Young (d48 hrs old) Aedes albopictus males will be marked and field released. A backpack aspirator will sample around the collection zone following releases. Collections will be made in zones, separated based upon distance from the release site and habitat type. Samples will be brought back to the lab for detection of markers. Comparisons of longevity, dispersal rate and survivorship will be used to determine the advantages/disadvantages with the differing approaches. The experimental design (e.g. collection times, types of habitat) and mathematical interpretation of results will be coordinated with other participants. Pending appropriate regulatory approval, males infected with differing Wolbachia infection types may also be compared using the developed approaches.

The entire WN viral genome for up to 1000 WN virus isolates obtained from mosquitoes collected from 1999-present will be sequenced by high-throughput technologies. Nucleotide and encoded amino acid sequences will be compared to track evolutionary change, determine viral population structure and dynamics, and identify genetic correlates of disease emergence. (2.) Vertical and horizontal transmission of La Crosse (LAC) virus variants by Ochlerotatus triseriatus mosquitoes. We will compare the ability of geographic variants of LAC virus to infect, replicate, and be transmitted by Oc. Triseriatus mosquitoes to determine their relative fitness in vector competence assays. (3.) Infection patterns and genetic diversity of Cache Valley (CV) virus. We will evaluate the prevalence and genetic diversity of CV viruses. The identity, geographic distribution, and seasonal abundance of infected mosquitoes will be analyzed to identify potential mosquito vectors and discern major features of virus transmission.

We propose to examine the biology, ecology and behavior of native and exotic mosquito vectors in relation to transmission and persistence of arboviruses of public health importance in the northeastern US. 1) The involvement of various bird species in overwintering and amplification of the eastern equine encephalitis (EEE) virus in the northeastern U.S. is unknown, as is the role these mosquito species may play in epidemic transmission to humans and other mammals. To better understand the role of mosquito species and various bird species in the epidemiology of EEE, studies will be undertaken to identify the source of blood meals. We will focus on mosquitoes collected from EEE foci where recent outbreaks of human disease have taken place. 2) The manner in which WN virus overwinters in the northeastern US is poorly understood. Studies will be initiated to examine overwintering populations of Cx. Pipiens collected from hibernacula in New York City where there has been a history of virus activity. Parity rates will be determined through dissection and microscopic examination of ovarian tracheoles, and attempts will be made to isolate virus from females collected monthly from December through April. 3) Culex pipiens is the primary mosquito vector of WN virus in the eastern US but is known to exist as a complex that exhibits substantially different behavioral and physiologic characteristics. In an effort to gain a better understanding of regional populations in the eastern US, we will examine the genetic structure of urban and rural populations using microsatellite markers along an east coast transect. 4) To assess the invasion success and impact of Ochlerotatus japonicus japonicus on native species, we will study populations in waste tire disposal sites and natural rock pool habitats. Data will be compared with results from surveys of similar sites made prior to this species introduction and establishment. We will additionally examine interspecific competition mechanisms under ecologically realistic conditions involving Oc. j. japonicus and native resident species.

Larval habitat has generally been considered the principal determinant of mosquito distribution. We will focus on understanding the distribution of larval habitat in a landscape, variation in habitat quality, and consequent effects on mosquito populations.

Objective 2 - Use knowledge of mosquito, pathogen, vertebrate reservoir, and environmental interactions to enhance ability to predict conditions leading to disease.

Human risk of acquiring a vector-borne zoonotic disease is highly variable in space and time and is associated with variation in the intensity of enzootic transmission and the abundance of vectors serving as transmission bridges. If vectors preferentially feed on one competent reservoir host species, this concentration of feedings will increase transmission intensity but few human cases will occur unless another vector acts as a 'bridge'. More generalist vectors may be less able to amplify pathogen transmission as many feedings will be on non-reservoir competent hosts, but generalists may naturally bridge infection to humans. Neither scenario is mutually exclusive as vector and reservoir host species may undergo population and behavioral changes throughout the transmission season, potentially changing vector-host interactions and transmission patterns. West Nile virus constitutes an ideal model system to study these heterogeneities given the wide diversity of vector and host species involved in transmission. We will to develop a model to explore how these scenarios result in optimal enzootic amplification and transmission to humans. We will identify factors driving amplification by a combination of field, lab and behavioral studies to be conducted along an urban-suburban-forest gradient. Test sites are populated by different vector and host communities, and we will quantify spatiotemporal heterogeneities among vector and host abundance and vector feeding behavior. We hypothesize that transmission will be amplified in areas with high enzootic vector/host ratio and higher vector specialization on American robins, the preferred reservoir host of mosquito vectors of WNV. We will test two alternative hypotheses to explain late season transmission of WNV to humans: (1) that roosting areas act as WNV foci and a source of transmission to potential bridge vectors, and (2) that most bridging occurs in breeding territories due to a switch in vector feeding behavior to include more mammalian hosts, including humans.

We propose mapping of disease vectors, associated habitat and climatic variables in urban and rural areas. These data, along with laboratory studies examining the development thresholds for importance disease vector mosquitoes, will be used to develop mathematical, predictive models of mosquito abundance and distribution across the landscape. Mosquito abundance is an important determinant of disease transmission, both empirically and theoretically.

Objective 3 - Develop strategies to control mosquito vectors.

For the urban mosquito Aedes albopictus, the Asian tiger mosquito, an area-wide approach to control is needed because its populations are highly diffuse. We will develop a multidisciplinary approach which incorporates extensive public education and involvement, with focused application of established biological and chemical control interventions. First, we will perform an assessment of Ae. Albopictus abundance, dispersal, population structure, and insecticide resistance status, as well as of its current social and economic impact. Second, we will compare Ae. Albopictus abundance and associated human behavior in two pairs of experimental and control plots of 1000 homes each. Third, we will expand our control efforts county-wide, targeting first source populations. The costs of all control procedures will be documented and a detailed economic analysis will be conducted. If successful and our strategy is economically sound, we will expand the initiative nationally by recruiting 12 geographically diverse mosquito control agencies to apply our methodology.

A female-specific promoter combined with tetracycline-repressible transcription control ('tet-off') is being developed that generates female-specific flightlessness in a transgenic strain of Ae. Albopictus for control using the sterile insect technique. We intend to focus on the possible fitness costs of this transgenesis. We will assess whether a transgenic tetracycline-repressive construct of Ae. Albopictus performs as predicted with respect to adult survival and female flightlessness. We will further compare the longevity, and mating and larval competitiveness of transgenic and wild type strains.

The sterile insect technique will also be examined using a Wolbachia-based microbial biopesticide approach against Ae. Albopictus. Tests will be conducted in a greenhouse mesocosm divided to form two similar zones. Each zone will contain nutrient sources and resting habitats. Virgin males will be pre-released into each of the greenhouse cages. Virgin Ae. Albopictus females will be exposed to varying ratios of males infected with different Wolbachia types or uninfected. Subsequently, females will be removed from the cage and brought into the lab where they will be blood fed. Females will be separated two days later and placed in individual cups for oviposition. Following oviposition, females will be dissected and their spermatheca checked for insemination. Broods from females found to be not inseminated will be excluded from the dataset. Each brood of eggs will be hatched following egg maturation. Fertilized females that produce unhatching broods will be scored as "mated with incompatible males." Females that produce hatching broods will be scored as "mated with compatible males." Comparison of the percent "incompatibly mated" females with the percent incompatible males will allow assessment of male competitiveness for mates. Ratios of incompatible and compatible will be performed in replications of three. Males from a variety of sources (e.g. laboratory strains, hybrid strains, wild caught) will be tested.

Studies will be initiated to evaluate the effectiveness of several new biological agents for control of Culex mosquitoes developing in catch basins. The first will include VectoMaxTM, a unique biological larvicide that combines two bacteria, Bacillus thuringiensis israelensis and Bacillus sphaericus in one homogeneous formulation. These investigations will be conducted in residential settings and a primary objective will be to determine the length of residual activity and how frequently catch basins would need to be retreated to achieve a relatively high level of control throughout the course of the season. We also will attempt to correlate larval and pupal abundance with titers of the two bacteria in treated and untreated catch basins throughout the summer using selective media. Other agents that will be similarly evaluated include spinosad, a naturally occurring product of the fermentation of the bacterium Saccharopolyspora spinosa; and Strelkovirmermis spiculatus, a naturally occurring nematode parasite from Argentina.

New toxicants for adult and immature mosquitoes can be discovered through bioassay and structural activity relationship (SAR) analysis of existing and experimental chemical compounds, natural products and microbes. High throughput bioassays will screen candidate compounds from existing libraries of patented and new synthetic compounds, microbes and natural products. Screening will use an established high throughput larval Ae. Aegypti assay and categorizes activity based on 24 hr mortality. Active compounds will be evaluated with topical assays against adult female Ae. Aegypti and/or larval assays with 4th instars to determine relative toxicity based on 24 hr mortality. Toxicity data from the above assays will be modeled by quantitative structure activity relationship (QSAR) to identify characteristics of efficacious toxicants and lead to the prediction of novel structures. Active compounds will be evaluated for efficacy and modes of action across several mosquito species, including susceptible and insecticide-resistant Ae. Albopictus, Cx. Quinquefasciatus, An. Albimanus and An. Gambiae.

New toxicants will be developed using RNAi technology (gene silencing) to suppress expression of specific critical proteins in target species (Aedes aegypti, Ae. Albopictus, Cx. Quinquefasciatus, An. Albimanus and An. Gambiae). Critical target proteins that have been identified include inhibitors of apoptosis protein (IAPs) that regulates programmed cell death (apoptosis) and others will be identified. Double stranded RNA molecules targeting different critical genes/proteins will be synthesized and evaluated in vivo against mosquitoes through topical/residual application, per os and by micro-injection. When required, constructs will also be evaluated in vitro against mosquito cell lines. Quantitative PCR will be used to evaluate effectiveness of RNAi molecules in knock down of targeted proteins in both systems.

We will focus on the basic molecular biology of insecticidal bacteria, with emphasis on understanding the genetic regulatory elements that control the synthesis of mosquitocidal proteins. Knowledge from this basic research will be used to manipulate these elements with recombinant DNA technology to construct more cost-effective bacterial strains than those currently employed in commercial bacterial larvicides. An advantage to this strategy compared to generic strategies that target individual mosquito species is that new bacterial larvicides emerging from this research would be useful against a wider range of vector mosquitoes, including key species of Aedes, Culex, and Anopheles. Initial constructs have been shown to be at least ten-fold more larvicidal to important Culex and anopheline vectors such as Cx. pipiens quinquefasciatus and An. Gambiae than the Bacillus thuringiensis subsp. israelensis (Bti) and B. sphaericus (Bs) strains used in, respectively, VectorBacTM and VectoLexTM. In essence, these recombinants combine the best properties of Bti and Bs in novel strains. Further, we will focus on removing the selectable markers used to develop these recombinant bacteria to make them more environmentally sound, and combining a range of new mosquitocidal proteins to construct strains more effective against a wider range of Aedes species.

We propose to demonstrate a reduction in dengue virus transmission by area-wide deployment of a prototype attractant-baited lethal ovitrap (ALOT) as a component of community-based dengue management programs. We will finalize structural and toxicant components of the ALOT and validate under field conditions; complete characterization of oviposition attractants and stimulants; develop delivery systems and validate under field conditions; optimize the ALOT containing oviposition attractants and stimulants and validate activity under field conditions; demonstrate the ALOT efficacy (reduction in mosquito density and dengue incidence) in an experimental field trial; and work with public health officials and vector control experts to establish standards and benchmarks for use of lethal ovitraps. We propose a large scale evaluation of this device in dengue endemic areas in Latin America and in South Asia, where we will measure changes in mosquito populations and trends in new human dengue infections. We expect that area-wide deployment of the ALOT will result in a measurable decrease in abundance of physiologically old mosquitoes and in new human infections.

Objective 4 - Enhance surveillance technologies for mosquitoes and mosquito-borne pathogens.

Mosquito surveillance for arboviruses that are known to cause human disease in the northeastern US will be undertaken from June through October in an effort to provide information necessary for the prevention and control of human infections. The focus of these activities will be to provide: 1) early evidence of local sustained arbovirus activity; 2) information on the identity, abundance and distribution of potential mosquito vectors; and 3) data on the prevalence of arboviral infection in these mosquito vectors. Mosquito trapping will be conducted daily at specified locations using light and gravid traps. Mosquitoes will be tested for West Nile, Eastern equine encephalitis, Jamestown Canyon, Cache Valley, LaCrosse, Trivittatus, Highlands J, and Potosi viruses using Real Time (TaqMan) PCR

New trap designs and attractants will be developed and evaluated against Aedes albopictus as well as other mosquito species particularly Culex spp. Trap size, shape, color combinations and placement of port-of-entry will be varied. Initial studies will be conducted under semi-field conditions (large outdoor cages) where video tracking equipment will be utilized to record behavioral responses to the various trap designs. Laboratory (olfactometer and electrophysiology), semi-field and field studies will also be conducted to isolate and identify volatiles from oviposition infusions, flowering plants and other nectar sources, and vertebrate hosts. The best combination of factors i.e., new chemical and physical attractants and trap design will then be evaluated under semi-field conditions (laboratory reared insects), followed by field evaluations against natural populations. The ultimate goal is to develop a trap capable of attracting and capturing adult mosquitoes in all physiological states.

Research is proposed to investigate the importance of allochthonous plant input into larval habitats of the invasive mosquito Aedes albopictus to determine both productivity of the habitat (e.g. adults emerging) and oviposition choice by gravid female. The data collected on oviposition behavior will be used to design an Aedes specific gravid trap, which will assist in surveillance of these mosquitoes for disease transmission.

Objective 5 - Develop strategies for sustainable mosquito control by including training at all levels.

Training will occur at the undergraduate, graduate and postdoctoral levels in all of the above objectives. In addition, we will develop a grant proposal to train the next generation of scientists and vector borne disease control professionals by offering practical hands-on training courses for graduate and post graduate students as well as mosquito control personnel. Accordingly, special effort will be made to include mosquito control practioners to supplement conventional academic instructors. These training programs will introduce attendees to new technology and methodology, ecological and evolutionary theory, data management and analysis. In addition, training through the development of short courses and seminars will provide attendees with a background in classical vector ecology methods. An additional benefit of this program will be to bring together those involved in vector borne diseases research and control providing networking opportunities and strengthening future collaborations.

Measurement of Progress and Results

Outputs

• Development of an integrated control strategy and specific recommendations for area-wide control of the Asian tiger mosquito.
• Development of models that predict risk for key mosquito-borne diseases.
• Improved methodologies for performance and analysis in mark-and-recapture studies.
• Development of efficient, inexpensive traps for operational mosquito surveillance and control.
• Assess the feasibility of initiating field cage experiments on the sterile insect technique.
• Discovery of novel, environmentally benign, and cost effective mosquito toxicants and pathogens with modes of action effective against resistant species.
• Development of a cadre of trained medical entomologists.

Outcomes or Projected Impacts

• An interactive and interdependent network of scientific expertise to deal with new mosquito-borne disease outbreaks.
• Impact on the general public by understanding, assessing, and mitigating the threat posed by mosquitoes of public health importance.
• Enhanced capacity to detect, predict and respond to outbreaks of vectors and associated human and livestock diseases.
• Provide for and encourage environmentally sound, scientifically based, and professional control by mosquito control agencies.
• Increased understanding of plant material as a provision for larval mosquitoes and how this impacts adult behavior.
• New, marketable, vector control tools and products for public health.

Milestones

(2010): <ul><li>Establish markers to assess adult movement. <li>Establish protocol for genomic sequencing of west nile virus. <li>Screen 1st group of experimental compounds and develop database of activity; refine RNAi-IAP constructs; Initiate tests of toxins/pathogens against diverse mosquito species. <li>Establish BSL-2 insectary for recombinant mosquitoes. <li>Establish test plots for area-wide management of Ae. albopictus; initiate operational control. <li>Initiate program screening novel mosquito toxins. <li>Initiate structural and toxicant studies for attractant-baited lethal ovitraps. <li>Initiate development of statistical models to predict mosquito-borne disease outbreaks.</ul>

(2011): <ul><li>Field test and refine mark-and-recapture methodologies. <li>Conduct QSAR analysis on year 1 toxicants, design and synthesize analogues; screen 2nd group of experimental compounds and develop database of activity; evaluate new RNAi-IAP constructs for activity. <li>Continue to screen new genes/proteins for novel mosquitocidal activity. <li>Construct new strains useful against <i>Aedes</i> species combining <i>B. sphaericus</i> mosquitocidal proteins secreted during vegetative growth with the key <i>B. thuringiensis</i> subsp. <i>israelensis</i> proteins. <li>Initiate lab studies to assess the fitness of mosquitoes modified for sterile insect method. <li>Design new adult mosquito surveillance traps. <li>Complete characterization of oviposition attractants. <li>Develop mathematical models to predict mosquito abundance and distribution. <li>Develop operational control strategy for <i>Ae. Albopictus</i> in area-wide plots. <li>Truth-testing of predictive models.</ul>

(2012): <ul><li>Continue field testing of mark-and-recapture studies. <li>Conduct QSAR analysis on year 2 compounds, design and synthesize analogues; screen analogues and 3rd group of experimental compounds and develop database of activity; identify and select best RNAi constructs for large quantity production and evaluate for larvicidal activity. <li>Continue to construct new strains useful against <i>Aedes</i> species combining <i>B. sphaericus</i> larvicidal proteins secreted during vegetative growth with the key <i>B. thuringiensis</i> subsp. <i>israelensis</i> proteins. <li>Initiate small cages studies to assess effectiveness of sterile insect method. <li>Expand operational control study for <i>Ae. Albopictus</i> on a county-wide basis. <li>Field test and optimize adult mosquito surveillance traps. <li>Optimize attractant-baited lethal ovitrap. Initiate field tests. <li>Place predictive models on internet for end-user access.</ul>

(2013): <ul><li>Conduct QSAR analysis on year 3 compounds; design and synthesize analogues; screen analogues and identify best candidate compounds; optimize parameters (<i>e.g.</i> dose response, mortality, specificity) of RNAi constructs. <li>Develop improved fermentation media for new recombinants, and field test commercial formulations of these being developed to determine efficacy and cost-effectiveness. <li>Initiate small scale field testing of new recombinant bacterial larvicides. <li>Large scale deployment of ovitrap in dengue endemic areas. <li>Continue to field test and optimize adult mosquito surveillance traps. <li>Continue to test operational control strategy for <i>Ae. Albopictus</i> county-wide.</ul>

(2014): <ul><li>Large scale tests of practical area-wide mosquito management in multiple mosquito abatement districts. <li>Expanded deployment of ovitrap in dengue endemic areas. <li>Large field cage and greenhouse tests of sterile insect approach. <li>Large scale field testing of new toxicants/pathogens.</ul>

Projected Participation

View Appendix E: Participation

Outreach Plan

The key function of the project's annual meeting is to promote cooperation among participants that leads to enhanced research opportunities. Results of our meetings and activities will be available to all interested parties via the NIMSS website. All publications, both refereed and non-refereed, will be listed on NIMSS. Informational meetings will be held to brief appropriate state and county mosquito control personnel on our progress, particularly via oral presentations at national (e.g., American Mosquito Control Association) and local (e.g., New Jersey Mosquito Control Association) meetings that are designed to together scientists and mosquito control practitioners. Further, we will create educational posters and extension bulletins to transfer applied research results to stakeholders. Journal publications will be made easily available to the public by providing downloadable pdf files from websites.

Note: This proposal has purposefully been written to define the project in broad research terms. This is intended to best position NE-507 to cast a broad net to secure an expanded membership, and therefore expanded opportunities for community building and collaboration. First, an expansion of membership will be sought geographically so NE-507 can become a truly national project rather than being almost exclusively northeastern in focus. Second, the intended expansion will be aimed at securing participants beyond the traditional agricultural experiment station and ARS-UDSA mosquito biologists to involve mathematical modelers, community ecologists and others with special skills regardless of whether or not they study mosquitoes.

Organization/Governance

The multistate research project will be established in accordance with the format suggested in the Manual for Cooperative Regional Research. One person at each participating institution or agency will be designated, with approval of the institutions or agencys director, as the voting member of the Technical Committee. Other individuals and interested parties are encouraged to participate as non-voting members of the committee. There will be elections of a Chair, Chair-elect, and a Secretary. All officers are to be elected for two-year terms to provide continuity. Administrative guidance will be provided by an assigned Administrative Advisor and a CSREES Representative.

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Attachments

Land Grant Participating States/Institutions

CT, DE, KY, MA, MD, MI, MN, NE, NJ, NY, OH, TX, WI

Non Land Grant Participating States/Institutions

USDA ARS, USDA-ARS/Florida, USDA, ARS, USDA/ARS Grain Marketing and Production Research Center