Brief Summary of Minutes of Annual Meeting

The annual meeting of the Multi-State Project NE1043, Biology, Ecology and Management of Emerging Disease Vectors was held on March 1-2, 2011 at The Connecticut Agriculture Experiment Station, New Haven, CT. The following were in attendance: Allan (ARS-Gainesville), Anderson (CT), Andreadis (CT), Armstrong (CT), Becnel (ARS-Gainesville), Cortinas (NB), Fallon (MN), Federici (CA), Fonseca (NJ), Hardstone (CT), Kaufman (MI), Kline (ARS-Gainesville), Molaei (CT), Shepard (CT), Stafford (CT), Thomas (CT), and Vossbrinck (CT). Dr. Kirby Stafford presented an administrative report and indicated that the project had 24 members representing 12 states with additional representation from ARS/USDA. Mark Robinson (USDA-NIFA representative) was unable to attend due to travel restrictions. Future funding opportunities through NIFA remain uncertain pending budget reductions. A presentation was given by Dr. Goudarz Molaei (CAES) entitled Discriminating blood-feeding mosquitoes, their catholic counterparts, and transmission dynamics of West Nile virus in the US. Discussion on blood meal sources, role of nestlings and effect of virus infection on host choice followed. Updates were provided by each cooperator with active discussion ensuing. The group photo was taken. Several in depth presentations were made providing stimulus for further discussion and collaboration. Two discrete efforts that emerged were to develop databases on maintained mosquito colonies and cell culture lines with efforts headed by Allan (colonies) and Fallon (cell cultures). Possible expansion of the group was discussed to include representatives from other Experiment Stations with scientists involved in mosquito research and the US Department of Defense Armed Forces Pest Management Board. Elected officers remain in position for two years with no vacancies until next year. The next meeting will be at The Connecticut Agricultural Experiment Station at the same time of year. The format will remain the same with a 1.5 day meeting starting on Tuesday and featuring an invited speaker.

Objective 1. Strengthen basic and applied research on the mosquito, pathogen, hosts, and environmental factors that influence disease emergence. In an effort to evaluate the invasion success and impact of Aedes japonicus japonicus on populations of native container dwelling species, thirteen waste tire disposal sites and four natural rock pool habitats were sampled for mosquito larvae throughout Connecticut in 2005, and data were compared with results from prior surveys of similar sites made in 1987 and 1999. Ae. j. japonicus was the predominant species collected at the waste tire disposal sites regardless of surrounding landscape features, accounting for 55.9% of all larvae. A comparison with collections from prior surveys revealed a 90% reduction in the relative abundance of larval populations of Aedes triseriatus and significant reductions among larval populations of Aedes atropalpus and Culex restuans. Ae.. j. japonicus was also the most abundant mosquito collected in rock pool habitats, accounting for nearly 80% of all collected larvae, except where water temperatures exceeded 30oC. This was concomitant with significant declines in the relative abundance of Oc. atropalpus and Cx. restuans. We conclude that Oc. j. japonicus is a potentially effective competitor in rock pool and tire environments and may be responsible for reducing populations of several native species occupying these habitats through interspecific competition for limited resources. The exclusion of Oc. j. japonicus from warm water pools further suggests that a temperature barrier may exist for Oc. j. japonicus and that populations may not be able to colonize effectively regions of the United States with relatively high summer temperatures. (CT) A 3-yr study was undertaken to examine the parity status, survival and prevalence of West Nile virus (WNV) in overwintering populations of Culex pipiens collected from a hibernaculum located in a WNV endemic region in New York City. Nearly 6,000 females were collected from December through April. Parity rates were highest among females collected in December and January, ranging from 12.3% to 21.9% depending on the year. In each year of the study, the proportion of parous females declined significantly during the course of the winter with the percentage of parous females found in April ranging from 0.9% to 10%. Results provide unequivocal evidence that parous female Cx. pipiens from this region of the northeastern US enter hibernacula in the fall in comparatively high proportions not previously recognized for this species, and while these females experience significant mortality during the winter some survive to April to emerge in the spring. The absence of any detectible blood remnants in overwintering females reaffirm that blood feeding does not occur among diapusing females during the winter. The possibility that a portion of the diapausing population may be autogenous as a result of hybridization with sympatric below-ground populations of Cx. p. pipiens form molestus is discussed. A single isolation of WNV was obtained in Vero cell culture from a pool of 50 females collected on January 11, 2007 representing an infection prevalence of 0.07% in the overwintering population in 2007. No isolations of WNV were made from mosquitoes collected in 2008 or 2009). Findings provide further evidence for local overwintering of WNV in diapausing Cx. p. pipiens, albeit at very low rates, consistent with the paucity of WNV positive mosquitoes detected in June and early July despite the emergence of females from hibernacula in early May in this region. (CT) Eastern equine encephalitis virus (EEEV) is maintained in an enzootic cycle involving Culiseta melanura mosquitoes and avian hosts, whereas other mosquito species that feed opportunistically on mammals have been incriminated as bridge vectors to humans and horses. To evaluate the capacity of these mosquitoes to acquire, replicate, and possibly transmit EEEV, we estimated the infection prevalence and virus titers in mosquitoes collected in CT by cell culture, plaque titration, and quantitative RT-PCR. Cs. melanura was the most important source of EEEV (68% of 122 virus isolations) and the only species to support consistently high virus titers required for efficient transmission. Our findings suggest that Cs. melanura serves as the primary enzootic and epidemic vector of EEEV in this region, which may explain the relative paucity of human cases. More generally, this study emphasizes the importance of evaluating virus titers from field-collected mosquitoes to help assess their potential role as vectors. (CT) Southern California remains an important focus of WNV virus activity, with persistently elevated incidence after invasion by the virus in 2003 and subsequent amplification to epidemic levels in 2004. Ecoepidemiological studies of vectors-hosts-pathogen interactions are of paramount importance for better understanding of the transmission dynamics of WN virus and other emerging mosquito-borne arboviruses. We investigated vector-host interactions and blood feeding behavior of 4 competent mosquito vectors by using a PCR method targeting mitochondrial DNA to identify vertebrate hosts of blood-fed mosquitoes. Diagnostic testing by cell culture, real-time RT-PCR, and immunoassays were additionally used to examine WNV infection in blood-fed mosquitoes, mosquito pools, dead birds, and mammals. Analyses of engorged Culex quinquefasciatus revealed that this mosquito species acquired 88% of the blood meals from avian and 11.6% from mammalian hosts, including humans. Similarly, Cx. tarsalis fed 82% on birds and 18% on mammals. Culex erythrothorax fed opportunistically on both birds (59%) and mammals (41%). In contrast, Cx. stigmatosoma acquired all blood meals from avian hosts. House finches and a few other mostly passeriform birds served as the main hosts for the blood-seeking mosquitoes. Evidence of WN virus infection was detected in mosquito pools, wild birds, dead birds, and mammals, including humans with cases of fatalities during the study. These findings emphasize the important role of house finches and several other passeriform birds in the maintenance and amplification of WNV in southern CA, with Cx. quinquefasciatus acting as both the principal enzootic and bridge vector responsible for the spillover of WNV to humans. Cx. tarsalis and Cx. stigmatosoma, are important but less widely distributed (CT) Approvals have been obtained, allowing for the open release of Aedes albopictus males in Kentucky. Initial field trials have tested mark-release-recapture methods for assessing Ae. albopictus male dispersal and survival following a point release. Methods developed in Project Year 1 were trialed, including classical fluorescent dyes and molecular methods. One of the molecular methods was observed to show good persistence in the field with little fitness impact on treated males. A deficiency observed is the current inability to capture Ae. albopictus males. Collections were typically female biased (e.g. 61%, 56%) and less than 1% of the released males were recaptured, which complicates interpretation of the results. In an effort to increase male recapture rates, future MRR experiments will include additional collection methods and tools. (KY) Research revolved around analysis of the tree-hole ecosystem as a model for studies on larval mosquito nutritional relationships and resultant production of adult mosquitoes, and in particular larval mosquito/microbial interactions. Larval mosquitoes were shown to be strong top-down predators on the microbial community in tree holes, such that a trophic cascade was evident in the protozoan and bacterial compartments of the community. The cascade was most intense on flagellate and ciliate populations and dampened at the bacterial trophic level. However, as larval mosquito feeding pressure was experimentally released and protozoan populations rebounded, predation pressure on bacteria increased due to protozoans, such that a group of predation-resistant bacteria emerged as elongated filamentous forms. In related research, stable isotope analysis showed that larval Ae. triseriatus mosquitoes utilized primarily plant based detritus early in the development season but that animal detritus became relatively more important later in the season. One key finding, through stable isotope analysis, was the important of summer inputs of tree flower parts into the tree hole system as a nutrient pulse. (MI) Aedes japonicus was collected during summer 2010 in eastern Nebraska as part of the Nebraska Department of Health and Human Services WNV surveillance program. Specimens were collected using CDC-light traps baited with carbon dioxide. Specimens were confirmed by Harry Savage (CDC/OID/NCEZID). The mosquito was collected in at least four independent trapping visits to separate sites. These are the first recorded collections of Ae. japonicus in Nebraska. The importance of Ae. japonicus to vector disease ecology and epidemiology in eastern Nebraska is not known. (NE) We found that low concentrations of malathion (0.11 ppm) that are often detected in aquatic systems affect competition between two invasive mosquito species Ae. albopictus and Ae. japonicus. There were no survivors of Ae. japonicus larvae in malathion. There was a significant negative effect of Ae. japonicus density on Ae. albopictus survival but this effect was absent in the presence of malathion. These findings indicate that pesticide mediated alterations in competition and species-specific differences in susceptibility to pesticides could play a role in enhancing invasive Ae. albopictus potential. (NJ) Accomplishments have been made toward strengthening our understanding of how pathogens, hosts and environmental factors influence mosquito-borne disease. Our laboratory has examined how invasive plants affect container breeding mosquitoes in Florida, and has extended this work to invasive plant dominated ecosystems in the southern Midwest. In collaboration with colleagues from the University of Florida, we have examined how abiotic factors influence the distribution of vectors of dengue fever virus and chikungunya virus, how chikungunya virus affects adult longevity in Ae. aegypti and Ae. albopictus, and how abiotic conditions in larval environments impact vector competence. In addition, we completed one study on how combinations of leaf species affect competition in Ae. aegypti and Ae. albopictus (OK). Objective 2. Use knowledge of mosquito, pathogen, vertebrate reservoir, and environment interactions to enhance ability to predict conditions leading to disease. The emergence of arthropod vectors of human disease, and the pathogens and pathogen systems associated with them, is of primary concern in our modern era of rapid population movement, trade, and environmental change. The risk of human infection with West Nile virus (WNV) in urban areas of the upper midwestern US remains one of the distinct study topics in this project. Models of the effects of environmental factors on WNV disease risk have yielded conflicting outcomes, in part because of the temporal and spatial scales of investigation. We used spatial and statistical modeling techniques to analyze and forecast fine scale spatial (2000 m grid) and temporal (weekly) patterns of WNV mosquito infection relative to changing weather conditions in the urban landscape of the greater Chicago, Illinois. Increased air temperature was the strongest temporal predictor of increased infection in Culex pipiens and Culex restuans mosquitoes, with differences in cumulative high temperature differences being a key factor distinguishing years with higher mosquito infection and higher human illness rate from those with lower rates. Spatially, precipitation was the most important variable predicting mosquito infection; precipitation and temperature alone could explain the pattern of spatial variability better than could other environmental variables. Other spatial environmental factors that tended to be important, such as impervious surfaces and elevation, mediate the effect of rainfall on soils and in urban catch basins. Evolutionary studies of WNV from 2005 suggest moderate evolutionary change but significant local scale processes. We analyzed viral nucleotide sequences from mosquitoes collected in 2005, 2006, and 2007 from a known transmission hot spot in suburban Chicago. Within this small area, the viral envelope gene has increased approximately 0.1% per year in nucleotide-level genetic diversity. In each year, viral diversity was higher in residential sites characterized by dense housing than in urban green space sites such as cemeteries and parks. Phylodynamic analyses show an increase in viral effective population size around 2005, consistent with a higher-than-average peak in mosquito and human infection rates that year. Analyses of times to most recent common ancestor suggest that WNV in 2005 and 2006 may have arisen predominantly from viruses present during 2004 and 2005, respectively, but that WNV in 2007 had an older common ancestor, perhaps indicating a predominantly mixed or exogenous origin. (MI) From the environmental models developed, we conclude that finely grained temporal and spatial patterns of precipitation and air temperature have consistent and significant impacts on the timing and location of increased mosquito infection with WNV. Changes in weather patterns with global climate change make it especially important to improve our ability to predict how inter-related local weather and environmental factors affect vectors and vector-borne disease risk. However, we now have a distinctive set of models that predict with a high degree of accuracy the variation in WNV infection in vector mosquito populations with solely two variables (temperature and precipitation), providing simplicity to a complex system. Interestingly, viral evolutionary analyses show that the population of WNV in suburban Chicago is an admixture of viruses that are both locally derived and introduced from elsewhere, containing evolutionary information aggregated across a breadth of spatial and temporal scales. Our results suggest that the population of WNV in suburban Chicago is broadly representative of that on coarser spatial scales, but also that it contains population genetic and phylogenetic signatures of local ecological and epidemiological processes, such as urban landscape type and climate. Moreover, WNV in suburban Chicago appears to show substantial temporal variability, both in its propensity to increase in diversity across years and in its variable evolutionary dynamics from year to year. WNV in our small hot spot study area thus appears to represent an admixture of viruses that are both locally derived and introduced from elsewhere and that reflect transmission dynamics aggregated across a breadth of spatial and temporal scales. We suggest that further studies of WNV on fine spatial and temporal scales in other regions might help clarify the importance of micro-scale processes to the transmission and evolution of this and other emerging arboviral pathogens. (MI) Related research examined the application of sequential sampling methodology to the process of seasonal amplification of WNV in Cx. pipiens vector populations. The goal is to develop a system of early warning as infection rate in the vector population rises, so as to anticipate the timing and intensity of human cases. This was accomplished by analyzing through time series cross correlation analysis the time-lagged relationships between vector infection rate, scored as the number of positive pools found per week of season, with the number of human cases scored by date of onset. Neither percentage of positive pools nor classic MIR were as useful as sheer number of positive pools per week as the predictor variable. Lags were in the range of 3-4 weeks, suggesting that the rise in vector infection rate in most summers precedes the rise of the human epidemic curve. From these relationships, sampling and outbreak threshold lines were developed, incorporating data on sampling characteristics of the mosquito population. Trends were examined at the local, regional (county), and state level using long term data from metropolitan Chicago and Illinois, our main study sites. Results showed that a sequential sampling system of mosquito infection, based on the number of positive pools found per week, is indeed useful as a tool for anticipating the beginning of the human epidemic curve, and it was most sensitive at the local vs. regional or state spatial scales. (MI) We have made a number of accomplishments in predicting and preventing disease transmission in Oklahoma through two projects. First, a project funded by the Oklahoma Center for the Advancement of Science and Technology (OCAST) has examined the landscape ecology of mosquito assemblages in the Cross Timbers region of Oklahoma. We have determined a high degree of habitat fidelity in mosquito assemblages, suggesting targeting certain areas for control would be more efficient and effective for certain diseases. Also funded through OCAST is a project on response to temperature of Ae. albopictus. This study has found significant differences in response to temperature depending upon nutrient level and population origin. In addition, we found an apparent change in the allometric relationship between measurements of mosquito total growth as a function of temperature induced changes. We also have a project examining the potential transmission of dog heartworm in urban versus rural areas in OK, data from which are currently being analyzed. (OK) Objective 3. Develop strategies to control mosquito vectors. An aposymbiotic laboratory strain of Ae. albopictus has been compared with a recently established, wild caught strain. No differences were observed in male performance, including competition for mates. Experimental values for percent hatching broods did not differ statistically with predicted values through three replications. In anticipation of Wolbachia-based biopesticide experiments, a closed greenhouse mesocosm system has been developed for maintaining and monitoring closed populations of Ae. albopictus. Replicate populations were maintained and monitored for 50 days, recording eclosion rates, sex ratio, egg production and estimating hatch rates. The results support downstream large-scale experiments assessing proposed control strategies. (KY) We evaluated pellet formulations of a monomolecular film (Agnique®) and (S)-methoprene (Altosid®) against Ae. albopictus larvae and pupae. In the lab, Agnique® provided 80% control for 20 d, whereas Altosid® in combination with Agnique® provided 80% control for > 60 d. During field trials, the 1:1 pellet ratio of products provided > 95% control for at least 32 d and 50% control for at least 50 d. Altosid® remained effective after a 107 d lab-induced drought, suggesting the product may serve as a possible means of control during drought conditions. Agnique® and Altosid®, when used in tandem for difficult-to-treat locations, can provide long-term control and reduce the number of treatments. (NJ) A broad based approach to new toxicant discovery includes screening of conventional chemical libraries and structure activity relationship analysis, evaluation of registered compounds for mosquitocidal activity and bioassays of compounds derived from natural products. In addition, we are exploring gene silencing using RNAi technology as a method to knock down critical proteins in mosquitoes with possible applications for control. A new research agreement with an industry partner has been initiated that will focus on identification of effective gene targets, production of large quantities of dsRNA and evaluation of delivery methods. (ARS-Gainesville, FL). Host odor analysis to discover new attractants and attraction-inhibitors was continued in 2010. Odors from horses were analyzed by gas chromatography/mass spectrometry and further evaluations of the identified compounds are in progress. Research progress was also made in the development of novel repellents and insecticides by structure-activity modeling of compounds. The data for model development are taken from the USDA data archive of more than 30,000 compound activities. A model of carboxamide repellents was developed and these have been evaluated against Ae. aegypti, An. albimanus, and An. gambiae. In collaboration with ARS-Oxford researchers, natural product repellents have been discovered and reported. The emphasis of the ARS-Gainesville modeling work has shifted towards development of novel insecticides. Several lead compounds have been found, but models to develop novel compounds have been unsatisfactory. In conjunction with the Natick Soldier Center, the bite protection perfomance of permethrin-treated US Army combat uniforms was evaluated. Results from these studies indicated that Fire-Resistant Army Combat Uniforms (FRACUs) treated with permethrin at the factory level were highly efficacious at preventing bites of Ae. aegypti and An. albimanus through 50 standardized wash cycles. The US Army has transitioned to a FRACU stock supply that is 100% factory-level permethrin treated as of September 1, 2010. The US Marine Corps used this same procedure to transition to factory treated combat uniforms in 2007. (ARS-Gainesville, FL) The role of prallethrin as a component of the formulated product, Duet", was evaluated in a wind tunnel against female Cx. quinquefasciatus. By using commercially formulated product without each of the active ingredients (prallethrin, sumethrin, piperonyl butoxide), the impact of prallethrin on resting mosquitoes was evaluated. Prallethrin enhanced flight during the spray period and resulted in a larger number of droplets detected on the treated mosquitoes with higher subsequent mortality. Similar results were obtained with sand flies (Lutzomyia shannoni). On-going studies are examining the effects of prallethrin exposure on activation and flight of unfed and blood-fed Ae. aegypti and Ae. albopictus. (ARS-Gainesville, FL) Towards development of a mosquito control strategy based on the nectar feeding behavior of mosquitoes, a series of effective oral toxicants with different modes of action were identified against three genera of mosquitoes in laboratory assays. Further examination on the role of sugar and amino acid ingestion by Culex has underscored the critical need for sugar-feeding by male and female mosquitoes. Flowering plants attractive to Culex spp. in north Florida have been identified and studies are on-going to identify attractant chemicals from these plants that could be used for surveillance or with an attract-and-kill approach. (ARS-Gainesville, FL) Semi-field and field studies were conducted on the use of spatial repellents/barriers to reduce host-vector contact. Barriers were constructed of camouflage material. These barrier materials were sprayed with lambda-cyhalothrin. Traps baited with carbon dioxide and 1-octen-3-ol served as surrogate hosts. The traps were placed in the center of the treated barrier plots (either 5 x 5 ). Spatial repellents tested included S.C Johnsons Clip-On, ThermaCell and Conceal (linalool). Preliminary results indicate some protection from mosquitoes by the physical barrier alone; the addition of a spatial repellent usually resulted in increased protection, but not always. The effectiveness of the spatial repellent varied with species. (ARS-Gainesville, FL) Objective 4. Enhance surveillance technologies for mosquitoes and mosquito-borne pathogens. Statewide trapping and testing for mosquito-borne arboviruses was conducted in CT from June through October at 91 fixed collection sites. A total of 115,725 mosquitoes (10,654 pools) representing 35 species were trapped and tested. 220 isolations of WNV were made from 9 mosquito species: Cx. pipiens = 170, Cx. restuans = 25, Cx. salinarius = 15, Ae. japonicus = 4, Ae. vexans = 2, Cs. melanura = 1, Oc. taeniorhynchus = 1, Oc. triseriatus = 1, Ur. sapphirina = 1. The first positive mosquitoes were collected on Jun. 14, and the last on Oct. 7. The majority of virus activity was detected in densely populated urban and suburban regions in southwestern and central CT. Eight human cases of WNV were locally acquired with no fatalities (age range 50 - 81 yrs., date of onset Jun. 10  Sept. 18). Four isolations of EEE were made from Cs. melanura at a single location (Aug. 20  Sept. 30). Other mosquito-borne viruses isolated included: Cache Valley = 6 isolates from 4 species (Sept. 1  28); Highlands J = 1 isolate from 1 species (Oct. 26); Jamestown Canyon = 22 isolates from 11 species (Jun. 3  Aug. 17) (CT). The Delaware Dept. of Natural Resources and Environmental Control (DNREC) asked us to address this question  can NJ light trap mosquito collections be used for both population estimation and WNV detection purposes? The problem in past years has often been the overwhelming number of mosquitoes and also non-target species that need to be sorted with these trap collections. We selected 10 sites from which to keep DNREC-collected mosquitoes for an 8-week period from July 5 to September 3, 2010. Of the ten, only five had good numbers of Culex spp. mosquitoes. Because of the extreme drought, numbers were low throughout the period. However, we found one WNV-positive pool during the week of 15021 August at a site that has been endemic for WNV over the past five years. The MFIR was relatively high (5.94) compared to past years, most likely because of the drought and reductions in mosquito emergence during August. The problem is that although detections can be made, species identity is very hard to determine in these trap collections. (DE). In rural areas of Nebraska, dry ice availability can be a problem. Octenol has a stable shelf life (if sealed), does not need to be refrigerated and is commercially available. The Nebraska Department of Health and Human Services was interested in finding out if a widely-available octenol gel product (Nosquito octenol lure 24%) could be used in lieu of dry ice in CDC light traps. The study was conducted at ten different sites within Lancaster County, NE during the summer of 2010. At each site, an octenol-baited light trap, a dry ice-baited trap, and an unbaited trap (control) were used. Overall mosquito and Culex spp. capture using octenol was not statistically significant compared with the control. Culex spp. and total mosquito captures were significantly higher using dry ice compared to octenol and the control. These findings are in line with studies conducted in other regions of the US. (NE) The oviposition behavior of Ae. albopictus was examined to help engineer a gravid trap specifcally for this important vector. This OCAST funded project has shown that Ae. albopictus prefer containers with small surface area but greater depth, over containers of the same volume but larger. This experimental work will continue this summer with a focus on oviposition media, and the results will be applied to field collections at the end of the summer. (OK) Lactic acid, as a component of human skin and an effective attractant for Ae. aegypti in the presence of carbon dioxide, was examined as an attractant for Culex. Olfactometer studies indicated that it was attractive for Cx. quinquefasciatus (although less so than for Ae. aegypti) but decreased attraction of both Cx. tarsalis and Cx. nigripalpus. A blend of lactic acid, acetone and dimethyl disulfide was effective to Ae. aegypti but less so for Culex. This research is continuing with field evaluations and additional host odors. (ARS-Gainesville, FL) Objective 5. Develop strategies for sustainable mosquito control by including training at all levels. No specific reports submitted; however, training of students and postdocs is an integral component of nearly all of the above reports.

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