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 13, 2012 at The Connecticut Agriculture Experiment Station, New Haven, CT. The following were in attendance: Federici (CA), Andreadis (CT), Anderson (CT), Armstrong (CT), Molaei (CT), Shepard (CT), Thomas (CT), Vossbrinck (CT); Leisnham (MD), Fallon (MN), Marcombe (NJ), Harrington (NY), Kline (ARS-Gainesville), Stafford (Administrative Advisor-CT), and Robinson (USDA-NIFA). Dr. Theodore Andreadis (Chair) reviewed the agenda, local arrangements and format for the meeting. He circulated the Annual Report and noted that reports were submitted from 9 participating agencies (CT, MD, MN, NJ, NY, OK, WI, USDA-Gainesville). No reports were received or participation at the annual meeting from DL, KY, MA, NE, or TX. He indicated that there are currently 26 official members of the project representing 13 Experiment Stations/Universities and 2 USDA laboratories, the Medical and Veterinary Entomology Laboratory (Gainesville, FL) and the Grain Marketing and Production Research Center (Manhattan, KS). Notification of the meeting was sent to individuals within the CDC and US Military (Armed Forces Pest Management Board and Navy Marine Corps Public Health Center Detachment) but no representatives were able to attend. He announced that his 2yr-term as chair would terminate with this meeting and that elections for a new slate of officers (chair, vice-chair, and secretary) would be held at the final business meeting. Dr. Mark Robinson (USDA-NIFA representative) circulated his report and reviewed the FY2012 NIFA budget and AFRI research priority areas for 2012. He announced that the RFAs would be out this spring and would notify the chair of funding opportunities that pertain to members of the project. He noted that members could receive electronic notification of funding opportunities by registering at http://www.nifa.usda.gov/funding/notification.html. There was concern expressed by members that the current research priorities established by AFRI do not reflect the research interests and objectives of the project. Dr. Kirby Stafford (Administrative Advisor) presented his administrative report and indicated that the current project will terminate on September 30, 2014. He presented a timetable for preparing a new project. New proposals require two years for approval. A request to re-write should be submitted in March 2013 and the reviewed proposal in March 2014. A presentation was given by Dr. Philip Armstrong, Center for Vector Biology & Zoonotic Diseases, CAES entitled: Molecular Evolution of West Nile Virus in a Northern Temperate Region: Connecticut 1999-2008. Brief research updates were given by each cooperator with active discussion ensuing. A group photo was taken. Several in-depth presentations were made providing stimulus for further discussion and collaboration. 1.) Dr. Brian Federici (CA): current research on recombinant bacteria, Bacillus thuringiensis isrealensis Bacillus sphearicus, and a new research iniative to examine the genomics of fungal Coelomoyces parasites of mosquitoes; 2.) Dr. John Anderson (CT): research on the efficacy of a novel chemical attractant lure for Ochlerotatus japonicus, and vector competence evaluating the comparative efficiency of West Nile virus genotypes NY99 and WN02 transmission by Culex tarsalis and Culex salinarius 3.) Dr. Theodore Andreadis (CT): research on a novel microsporidian parasite imported from Japan on the invasive mosquito Ochlerotatus j. japonicus. Nominations for new officers were solicited at the final business meeting. It was unanimously decided that the current Vice-Chair, Dr. Edward Walker (MI) would assume the Chair position. Dr. Daniel Kline (USDA-Gainesville) was elected Vice-Chair. It was suggested that next years meeting could be held in conjunction with the Annual Meeting of the American Mosquito Control Association that will be held February 24-28, 2013 in Atlantic City, NJ. However, it was decided the date and location of next years annual meeting would be at the discretion of the newly elected Chair who is responsible for coordinating such.

Objective 1. Strengthen basic and applied research on the mosquito, pathogen, hosts, and environmental factors that influence disease emergence. To determine whether West Nile virus (WNV) overwinters locally or is reseeded annually, we examined the patterns of viral lineage persistence and replacement in CT over 10 consecutive transmission seasons by phylogenetic analysis. We compared the full protein coding sequence among WNV isolates to search for evidence of convergent and adaptive evolution. Viruses sampled from CT segregated into a number of well-supported subclades by year of isolation with few clades persisting e2 years. Similar viral strains were dispersed in different locations across the state and divergent strains appeared within a single location during a single transmission season, implying widespread movement and rapid colonization of virus. Numerous amino acid substitutions arose in the population but only one change, VàA at position 159 of the envelope protein, became permanently fixed. Several instances of parallel evolution were identified in independent lineages, including one amino acid change in the NS4A protein that appears to be positively selected. Results suggest that annual reemergence of WNV is driven by both reintroduction and local-overwintering of virus. Despite ongoing diversification of WNV, most amino acid variants occurred at low frequencies and were transient in the virus population (CT). Cx. tarsalis is a superior horizontal and vertical vector of WNV compared with Cx. salinarius. Cx. salinarius transmitted WNV genotype NY99 (CT 2741-99 strain) horizontally to suckling mice at significantly lower rates than Cx. tarsalis and Cx. salinarius transmitted WNV genotype NY99 to offspring at a lower vertical transmission infection rate than Cx. tarsalis. Cx. tarsalis transmitted WNV genotypes NY99 and WN02 (CT S0084-08 strain) with equal efficiency. Daily percent horizontal transmission of genotype NY99 by Cx. tarsalis -infected per os and by intra-thoracic infection was not significantly different from daily transmission of genotype WN02. Findings do not support the previously published hypothesis that genotype NY99 was replaced in the New World by WN02 because of a shorter extrinsic incubation of WN02 (CT). A survey of mosquito larvae infected with microsporidia was conducted from 2005-08 in the Tomsk, Kemerovo and Novosibirsk regions of western Siberia, Russia. Twenty-one morphologically and genetically unique species of microsporidia were isolated from 9 species of mosquitoes including 14 new species of Amblyospora, a new genus and species, Novothelohania ovalae and 6 species of Amblyospora, Parathelohania and Trichoctosporea from which gene sequences had not been previously obtained. Detailed ultrastructure of meiospores revealed unique cytological features associated with the length, arrangement and ratio of broad to narrow coils of the polar filament, comparative thickness of the exospore and endospore, and overall size of each species reaffirming their value in distinguishing taxonomic relationships. SSU rDNA sequences were unique when compared with GenBank entries. Phylogenetic trees yielded similar topologies with a high degree of congruence between parasite and host at the generic level. Species that parasitize Aedes/Ochlerotatus and Culex mosquitoes segregate into distinct monophyletic groupings mirroring their host phylogeny, while species from Anopheles mosquitoes group as a sister clade basal to the entire group of mosquito-parasitic microsporidia as their Anopheles hosts cluster as a sister clade to the entire group of culicine mosquitoes. This provides strong evidence for host-parasite coevolution by descent at the generic level and limited host lineage switching between unrelated taxa. Among parasites of Aedes/Ochlerotatus and Anopheles mosquitoes, we found several instances where a single mosquito species serves as a host for two or more related species of microsporidia, an observation consistent with host switching and independent parasite speciation. Among the microsporidian parasites of Culex mosquitoes, we found only one parasite per host indicating a higher degree of host specificity and less host switching among parasites of this genus. Findings suggest a degree of host-parasite co-speciation with host switching occurring occasionally when the normal host is unavailable in the aquatic ecosystem. Frequency of host switching seems to be occurring in proportion to host relatedness and does not cross generic boundaries in this system (CT). The spread of exotic mosquito species into new environments can introduce shifts in mosquito populations and potentially alter public health risks to mosquito-borne diseases. The successful establishment of exotic species may occur due to their competitive advantage over other cohabitating species. We hypothesized that the recently introduced exotic mosquito Ae. japonicus would be a more effective competitor than Ae. atropalpus and Ae. triseriatus, and an equal competitor to Cx. pipiens based on larval abundance data within tire habitats. Impacts of competition were measured using the larval developmental rate and survival of larvae, adult mortality, wing length, and sex ratio. We found that intraspecific competition acted strongest against Ae. japonicus versus the other three resident mosquito species by delaying larval development and increasing adult mortality. Interspecific competition was generally weak and significant main effects were only detected for species and density. Results show that larval competition between Ae. japonicus and the three resident species was weak when present, indicating that other ecological or behavioral factors may be influencing the invasion success for Ae. japonicus in North America (CT). Life-table experiments were conducted to test for differences in the adult survival and reproductive schedules of Ae. albopictus females from three populations from the northern and southern extremes of the species distribution in North America. There were consistent differences between northern and southern populations in incidence of photoperiodically-induced egg diapause. Under short daylength, diapause eggs constituted twice the proportion of total viable eggs from northern females than southern females. There were no consistent differences between northern and southern populations in resource allocation between reproduction and maintenance, reproduction over time, and reproductive investment among offspring, and no apparent trade-offs between diapause incidence with reproduction or longevity. Results suggest that the main response of North American Ae. albopictus to unfavorable winter climates is via the life history strategy of producing diapausing eggs, rather than quantitative variation in reproduction, and that there are no detectable costs to adult survival (MD). A study was conducted to characterize mosquito communities among different habitats in historically ditched tidal salt marshes and adjacent wooded areas in a wetland management area on the Maryland Delmarva Peninsula. Wooded habitats had more total mosquitoes, were more frequently occupied by mosquitoes, and had higher densities of mosquitoes. The majority of larvae at the control site were Ae. sollicitans in marsh pannes while Cx. salinarius, An. bradleyi, Ae. cantator, and Ae. sollicitans were collected in high numbers from ditches at the experimental site. We found a difference in the proportion of marsh pannes occupied by Ae. sollicitans but not total mosquitoes sampled 45 days after spring tide events than on other occasions (MD). Carbon-based secondary or structural plant compounds usually increase when they are exposed to elevated atmospheric CO2, and may differentially affect container mosquito species that feed on litter that fall into water-filled containers. Competition between Ae. albopictus and Ae. triseriatus was tested in laboratory microcosms provisioned with litter grown in either elevated (1,000 ppm) or ambient CO2 (385 ppm). In additional microcosms, single-species cohorts of either Ae. albopictus or Ae. triseriatus were provided with increasing amounts of one of the two litter types or concentrations of commercial tannic acid to test potential toxicity effects of leaf-derived tannins. We found no differences in mosquito competition between litter types, with Ae. albopictus consistently superior to Ae. triseriatus. Single-species microcosms provisioned with elevated leaf litter had slower male development time than microcosms with ambient (lower-tannin) litter. Ae. albopictus and Ae. triseriatus also experienced similarly higher mortality and slower development with increasing commercial tannic acid concentrations. These results suggest that possible litter grown in high CO2 conditions may suppress mosquito production by increasing the toxicity of leaf litter, but that it would not affect population-level competition between Ae. albopictus and Ae. triseriatus (MD). We conducted yard surveys for mosquito larval habitats paired with knowledge, attitude, and practice questionnaires administered to residents among six neighborhoods in Washington DC that vary in socioeconomic status. Household income was related to overall knowledge and attitudes of residents towards mosquitoes, but that knowledge and attitudes were not related with source reduction as measured through questionnaire responses or actual numbers of water-holding containers in their yard. Numbers of water-holding containers was strongly positively related to exposure to Ae. albopictus, Cx. pipiens and pupae but not related to whether or not households practiced source reduction. Households that reported practicing source reduction had lower Cx. pipiens infestation and abundances but no difference in Ae. albopictus or pupal infestation or abundance. Results suggest large households are a greater source of vector mosquitoes and should be targeted in education campaigns, and that self-perceived source reduction of residents may not reduce urban mosquito habitat (MD). The risk presented to humans by circulation of WNV in the Culex bird enzootic cycle established in metropolitan areas of the upper Midwest was continued. Culex Flavivirus was positively associated with WNV infection in a case-control study of mosquito pools collected from an endemic focus of WNV transmission in Chicago. This study provides direct evidence that insect-specific flaviviruses may influence human health risk by enhancing WNV infection in mosquitoes that are epidemic bridge vectors to human hosts (MI). Analysis of molecular genetic diversity in 9 marker regions of 5 genes within the bacteriophage WO region of the Wolbachia pipentis genome revealed high diversity of wPip in the population of Cx. pipiens sampled in metropolitan Chicago, IL. Wolbachia endosymbionts may modulate propensity to infection with this virus and may affect population structure of their mosquito hosts. Multiple regression showed that markers associated with gene Gp2d were significantly associated with ancestry of individuals to form molestus or form pipiens, as determined by prior microsatellite allele frequency analysis. Data suggest that certain wPip molecular genetic types are associated with genetic substructuring in the Cx. pipiens complex of Chicago, and that the association extends to host preference. The influence of host diversity on multi-host pathogen persistence and transmission is confounded by the large number of species and complex biological interactions that comprise the transmission system. Although diversity and composition of host communities have been hypothesized to affect intensity of transmission, the effect of host community structure on the transmission of vector-borne pathogens remains largely unknown. Results suggest that host community measures, such as species diversity, may not be useful indicators of transmission risk at fine spatial scales in vector-borne disease systems (MI). Culex mosquitoes carry an intracellular symbiont, W. pipientis, a bacterium which can potentially be manipulated to control vector populations by spreading a reproductive distortion called cytoplasmic incompatibility. We obtained a Cx. pipiens colony from Ohio State University and, for comparative purposes, have developed a sister colony cured of Wolbachia infection. By comparing protein bands from dissected testes, we identified proteins produced in response to Wolbachia infection. Efforts are underway to clone two Wolbachia DNA binding proteins that are implicated in the reproductive distortion known as cytoplasmic incompatibility. Parallel investigations with Wolbachia-infected cell lines are used to identify Wolbachia-inducible protein expression in undifferentiated cells that do not engage in gamete production. We are investigating methods to modulate expression of these proteins by testing effects of specific components, including the DNA synthesis inhibitor mimosine and the mitochondrial inhibitor, paraquat, in the culture medium. We are finalizing methodology for quantifying Wolbachia infections using fluorescence activated cell sorting (MN). Ae. japonicus, which was first collected during summer 2010, was again collected during summer 2011 in eastern NE. Specimens were collected using CDC-light traps baited with carbon dioxide. Mosquitoes were collected in four sites within Lincoln, NE. The importance of Ae. japonicus to vector disease ecology and epidemiology in eastern NE is not known. (NE). Using a panel of seven microsatellite loci, we confirmed the existence of two abundant genetic forms in specimens originally collected in 19992000 that matches the disjunctive distribution of mitochondrial haplotypes. To examine the distribution of the two genetic types across PA we created a fine scale genetic map of Ae. japonicus using specimens collected from 54 Pennsylvania counties in 20022003. We also made direct comparisons between collections in 19992000 and new collections made in 20042005 obtained from the same areas in the northeastern US. We observed that the strong association between mtDNA haplotype and microsatellite signature seen in 19992000 had weakened significantly by 2002 across PA, a trend continued to some extent in 20042005 in PA, NJ, and NY, indicating that once easily distinguishable separate introductions are merging. The two expanding genetic forms create a complex correlation between spatial and genetic distances. The existence of multiple introductions would be obscured without sampling early and across time with highly polymorphic molecular markers. Results provide a high resolution analysis of the spatial and temporal dynamics of a newly introduced disease vector and argue that successive introductions may be a common pattern for invasive mosquitoes (NJ) We documented the potential importance of eastern red cedar, Juniperus virginiana leaves and fruit as a larval resource for the invasive Ae. albopictus in OK. We examined how different leaf species commonly found in FL interact to affect competition within and between Ae. albopictus and Aedes aegypti. We have also continued investigations into the effects of temperature and food on growth in larval mosquitoes (OK). Laboratory and field tests of potential biological control agents were conducted for control of Cx. pipiens. The key goals of this project are to test locally adapted fathead minnows and copepods for control of Cx. pipiens/restuans. Fish are used in aboveground breeding sites while copepods are used in the underground catch basin system. Fish were not released in 2011 because of extended dry conditions which limited the extent of targeted wet sites and the occurrence of larvae in those sites. Belowground sites did begin to generate toward the end of the season and these were used for trials of copepod releases. We tested the efficacy of copepods as predators of Culex or Anopheles larvae. Acanthocyclops trajani and Macrocyclops albidus were tested against An. stephensi and Cx. pipiens. M. albidus females killed 50% of Culex larvae within 24 hr while A. trajani killed only 35% of larvae. Results for An. stephensi were similar. We tested whether releases of M. albidus could affect larval Culex in catch basins (WI). We released M. albidus, into storm drain catch basins in Madison, WI at the beginning of August. We monitored these sites along with untreated control sites until the end of September. The catch basins were sampled every week using a mosquito dipper, and immature mosquitoes were collected, counted, and reared to adulthood for identification of the species. All adult mosquitoes collected were identified as Cx. pipiens. Copepods were collected during the sampling and identified as M. albidus or A. vernalis or inconsequential species. Catch basins were infested with mosquito larvae. M. albidus failed to establish in all but one location. This single location, however, maintained and multiplied the number of M. albidus with many being collected every week. M. albidus colonized an adjacent catch basin after approximately 1 month and their numbers continued to increase throughout the survey. Citywide water main flushing after our introductions took place may have resulted in the flushing of M. albidus from the test sites. Heavy rainfall events may have also flushed these copepods from their new habitats on more than one occasion. The A. vernalis colony was created from the catch basin samples and these copepods were frequently found in our sites. They appear better adapted to the catch basin environment than M. albidus (WI). Objective 2. Use knowledge of mosquito, pathogen, vertebrate reservoir, and environment interactions to enhance ability to predict conditions leading to disease. Seasonal epizootics of vector-borne pathogens infecting multiple species are ecologically complex and difficult to forecast. Pathogen transmission potential within the host community is determined by the relative abilities of host species to maintain and transmit the pathogen and by ecological factors influencing contact rates between hosts and vectors. Increasing evidence of strong feeding preferences by a number of vectors suggests that the host community experienced by the pathogen may be very different from the local host community. We developed an empirically informed transmission model for WNV in 4 sites using Cx. pipiens and preferred and non-preferred avian hosts. We measured strong feeding preferences for American robins (Turdus migratorius) by Cx. pipiens, quantified as the proportion of Cx. pipiens blood meals from robins in relation to their abundance (feeding index). The model accurately predicted WNV prevalence in Cx. pipiens at 3 of 4 sites. Sensitivity analysis revealed feeding preference was the most influential parameter on intensity and timing of peak WNV infection in Cx. pipiens and a threshold feeding index for transmission was identified. Findings indicate host preference induced contact heterogeneity is a key mediator of vector-borne pathogen epizootics in multi-species host communities, and should be incorporated into multi-host transmission models (CT). We studied the impacts of landscape heterogeneity and climatic variability on the richness and diversity patterns of mosquitoes and on the abundance and WNV infection rate of Cx. pipiens. Heterogeneity in the landscape was the best predictor of both mosquito species richness and diversity, with the most heterogeneous landscapes harboring the largest number of species. In general there were no changes in species richness over the years that could be associated with weather patterns and climatic variability. Although MIR was independent of mosquito diversity, it was associated with overall mosquito abundance, which had a convex association with species richness (i.e. abundance increases to a point after which it decreases as function of species richness). Results highlight the importance of considering dominant vector species as part of a community of vectors, whose biodiversity patterns can directly or indirectly impact the risk of infectious disease transmission (MI). We examined vector competence of Cx. pipiens and Cx. restuans mosquitoes for WNV collected over 2 years from 16 local sites across 2 states to measure spatial and temporal variation in vector competence. We found extreme variation in vector competence with 0-52% of mosquitoes transmitting WNV at a single site between different sampling periods, and similar variation across populations. We also found that vector competence within a smaller geographic range (counties within a state) tended to vary somewhat synchronously, supporting environmental or population genetic drivers of variability in vector competence. In support of genetic influences, we found significant genetic variance between mosquitoes that became infected with WNV after feeding on infected blood, and those that did not. These results highlight the spatio-temporal variability in vector competence in mosquito populations and indicate that temporal variability may be influenced by local processes, such as population genetics and environmental factors. It is important that consideration of vector competence in risk estimates take this variability into account when interpreting any single measurement (NJ). Chikungunya (CHIK) fever is a mosquito-borne viral infection that has started to invade temperate countries showing a surprising potential for geographic expansion. We developed a model for disease introduction based on virus introduction by one individual. Our study combines a climate-based mosquito population dynamics stochastic model with an epidemiological model to identify temporal windows that have epidemic risk. We ran this model with temperature data from different US locations (combined with strain-specific Ae. albopictus development and CHIK virus transmission rates) to study the geographic sensitivity of epidemic potential. We found that in locations with marked seasonal variation in temperature there also was a season of epidemic risk matching the period of the year in which mosquito populations survive and grow. I n these locations controlling mosquito population sizes might be an efficient strategy. But, in other locations where the temperature is enough for mosquito development all year around the epidemic risk is high and (practically) constant. In these locations, mosquito population control alone might not be an efficient strategy control and other strategies should be implemented to complement it. Results suggest that in the event of an introduction and establishment of CHIK in the US endemic and epidemic regions would emerge initially, primarily defined by environmental factors controlling annual mosquito population cycles. These regions should be identified to plan different intervention measures. In addition, reducing vector:human ratios can lower the probability and magnitude of outbreaks for regions with strongly marked seasonal temperature patterns (NY). We continued extending our work on the landscape ecology of mosquito assemblages, transmission of dog heartworm, larval ecology, and mosquito oviposition behavior. We found highly significant segregation of mosquito species assemblages at scales of less than 100m, with different groups of species found in pasture, deciduous forest, and pasture invaded with eastern red cedar J. virginiana. We found significantly different rates of dog heartworm in vectors in urban compared to rural sites, and variable rates of dog heartworm and other parasites in coyotes across Oklahoma (OK). Objective 3. Develop strategies to control mosquito vectors. Catch basins are a major source of Cx. pipiens, Cx. restuans, and Ae. japonicus in northeastern U.S. VectoBac® CG (ai. Bacillus thuringiensis israelensis (Bti),VectoLex® CG (ai, Bacillus sphaericus (Bs), and VectoBac® 12AS (ai, Bti), each applied at maximum label rates (VectoBac CG, 1.8g per catch basin, VectoLex CG, 1.8g per catch basin, and VectoBac 12 AS, 0.193 ml per catch basin) significantly reduced numbers of larvae for one week. The dosages on the labels for treatment of mosquito larvae in catch basins, where mosquito breeding is continuous, are not adequate for providing long-term control in northeastern US without the need for frequent retreatment. When applied at 3 times the maximum label rate, VectoLex CG, VectoBac 12AS, and VectoBac CG significantly reduced numbers of larvae for 5, 4, and 2 weeks, respectively. A single application of VectoMaxTM WSP (ai, Bti + Bs) per catch basin significantly reduced numbers of 3rd and 4th instar larvae and healthy pupae in catch basins but numbers of 3rd and 4th instars in treated catch basins at 21 days post-treatment had increased to 40% of the numbers in untreated catch basins. A second treatment of 1 pouch per catch basin reduced the numbers of 3rd and 4th instars and healthy pupae to near zero. VectoMax applied as 1 pouch per catch basin on July 1 and again on August 18 significantly reduced numbers of healthy pupae throughout the summer until the end of September. A second application of VectoMax to catch basins is likely needed during summer, when rainfall averages 13.7 inches during June through September, to keep numbers of Culex and Ae. japonicus significantly reduced to lower risk of human exposure to WNV. The application of one NatularTM XRT tablet, each weighing approximately 40.5 g (ai, 6.25% spinosad), to individual catch basins in significantly reduced total numbers of larvae for 5 weeks (CT). Funded by the Area-wide management of the Asian tiger mosquito (USDA-ARS) we have tested, optimized, and shared with the mosquito control community multiple methodologies to control Ae. albopictus. We have made multiple presentations at AMCA, ESA, NJMCA, ASTMH (NJ, ARS-Gainesville, FL). 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 (ARS-Gainesville, Fl). Studies on mosquito pathogenic viruses and microsporidia are focused on pathogen/host interactions. A cell culture system for the mosquito iridescent IIV-3 has been developed for conducting functional genomic investigations. Studies investigating the immune response in mosquitoes challenged with a baculovirus (CuniNPV) have found that a reaper ortholog (mx) is induced but apoptosis is blocked in permissive hosts by an unknown mechanism. Complete genomic sequences for three mosquito microsporidia are nearly completed. Sequencing of the genomes of Edhazardia aedis and Vavria culicis are completed and annotation is in the final stages. Anncallia algerae sequencing is underway. The annotation of all three species is expected to be completed in late 2012 (ARS-Gainesville, FL). Objective 4. Enhance surveillance technologies for mosquitoes and mosquito-borne pathogens. Mosquitoes transmit a number of distinct viruses including important human pathogens such as WNV, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. We describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing d50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures. Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in CT, and among these, 5 are known to cause human disease. Three of these viruses (WNV Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population (CT). In rural areas, dry ice availability can be a problem. Octenol has a stable shelf life, does not need to be refrigerated and is commercially available. Last year, we compared a widely-available octenol gel product (Nosquito octenol lure 24%) versus dry ice in CDC light traps. The findings demonstrated that overall mosquito and Culex spp. capture was not statistically significant using octenol and Culex spp. and total mosquito captures were significantly higher using dry ice compared to the octenol lure. We also continued studies assessing the use of octenol as a chemoattractant. Our objective was to assess if different concentrations and forms of the compound influence Culex spp. collections. Treatments include single vs. multiple octenol gels, octenol gel vs. liquid octenol, and low vs. high diffusion of liquid octenol (NE). We used two mitochondrial loci (nicotinamide adenine dinucleotide dehydrogenase subunit 4 and cytochrome oxidase II) and a nuclear locus (28S-D2 spacer) for a total of 1337 bp to evaluate the relationships among the four subspecies of Ae. japonicus which was recently introduced into the US and has been expanding rapidly. We included in our analysis a morphologically very closely related species, Ae. koreicus, as well as 3 more distantly related species: Ae. togoi, Ae. hatorii, and Ae. vexans. We found that the 4 subspecies in the Ae. japonicus complex are genetically distinct but form a monophyletic group that includes Ae. koreicus, suggesting the need for a taxonomic reconsideration of the group. We found the 2 southern subspecies are more closely related to each other than to any of the remaining subspecies or to Ae. koreicus and may indicate an ancient north9south split of the lineage. Considering the overlap between Ae. j. japonicus and Ae. koreicus, but the stronger association between Ae. koreicus and humans, we are surprised it also has not expanded from its original range. As a proactive reaction to this possibility, we designed and tested a DNA-based rapid assay to differentiate Ae. koreicus from some of the species with which it may be confused in the US. Belgian and Italian researchers have used our rapid assay to conclude that Ae. koreicus has expanded to Belgium and Italy. These Aedes are putative vectors of several important viral encephalitides (NJ). We developed new methodology to identify the bloodmeals of Ae. albopictus using blocking primers to avoid nuclear copies and developed a new high throughput qPCR methodology to identify large numbers of container Aedes eggs (NJ). We examined the importance of physical size characteristics of larval habitats in oviposition choice in Ae. albopictus. This information can be applied to the generation of species specific gravid trapping (OK). We continued our phonological survey of the flowering plants of North Central FL. Naturally occurring, landscape and nursery plants were all included in this survey. Olfactometer studies using both male and female Ae. albopictus were conducted utilizing intact flowers of many different flowering plants. Positive responses were obtained from many of the plants, but the two which resulted in the best responses were the butterfly bush (Buddleja davidi) and golden rod (Solidago spp.). Additional olfactometer studies were conducted with these two plant types utilizing intact flowers, solvent extracts of whole flowers and individual compounds/blends that were isolated and identified by gas chromatography/mass spectrometry (ARS-Gainesville, FL). Objective 5. Develop strategies for sustainable mosquito control by including training at all levels. Andrea Egizi, graduate student, obtained an Ecology and Evolution Graduate Program Small Grants Award, Understanding the successful establishment of a temperate mosquito on a tropical island and received a Buell Award, Department of Ecology, Evolution and Natural Resources Opening the black box: Microbial metagenomics of larval mosquito feeding. George Condon, graduate student, started in September 2011 and submitted an NSF Graduate Research Fellowship entitled "High resolution spatial and temporal population genetics of invasive species". Rafael Valentin, undergraduate student will start as a graduate student in the fall of 2012. He is a co-author on a paper using qPCR for high throughput container Aedes egg identification. Rebekah Heiry, undergraduate student has been working with Dr. Fonseca since June 2011. She is now a student technician. Talks by Dr. Fonseca outside of the scientific meeting circuit aimed at enhancing understanding of vector-borne diseases and mosquito control. May, 2011: Cervia, Ravenna, Italy, Meeting entitled "Emerging vector borne disease: the role of Aedes mosquitoes". Talk entitled "The Asian tiger mosquito in the United States: past, present, and future". May 17 and May 25, 2011: two 3-hour presentations entitled "Enlightened mosquito control" to the Rutgers Environmental Stewards, at the Atlantic County Utility Authority, NJ and the Duke Farms, Hillsborough, NJ. August, 2011: presented two talks in Ghana, Africa during the workshop on Genetics and Diseases Control (NJ). Dr. Sebastien Marcombe, a postdoc funded by the Area-wide management of the Asian tiger mosquito, Ae. albopictus started May 2011 and has uncovered extensive patterns of insecticide resistance and reduced susceptibility in populations of Ae. albopictus (NJ). Dr. Jiawu Xu funded in part by AW-ATM and in part by a new NIH-R21, has optimized 8 microsatellite loci for the Asian tiger mosquito. The NextGen sequencing project of Ae. albopictus to develop a high resolution SNP array is progressing, now funded by an NIH R21 (NJ). One masters student was finished in OK in 2011, Ms. Kelsey Paras. Ms. Paras is continuing her education at the Ohio State University, pursuing a DVM. Dr. Valerie OBrien continued her post-doctoral position in mosquito ecology during 2011 (OK). We co-instructed an all-day short course with Chris Lesser (Manatee County, FL, Mosquito Abatement District) on The Asian Tiger Mosquito: Biology & Control, at the Florida Mosquito Control Associations Dodd Short Courses (USDA-ARS, Gainesville, FL).

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