Brief Summary of Minutes of Annual Meeting

The annual meeting of Multi-State Project NE1043, "Biology, Ecology & Management of Emerging Disease Vectors" was held on February 25, 2010 at the Rutgers University Center for Vector Biology in New Brunswick, New Jersey. The following were in attendance: Allan (ARS-Gainesville), Andreadis (CT), Cortines (NE) Dobson (KY), Gingerich (DE), Fallon (MN), Federici (CA), Fefferman (NJ), Fonseca (NJ), Gaugler (NJ), Kaufman (MI), (OK), Paskewitz (WI), Robinson (USDA-NIFA), Ruiz-Moreno (NY), Stafford (CT) Randy Gaugler (Chair) reported that NE1043 has been formally approved and will run from August 1, 2009 through June 30 2014. Kirby Stafford (CT) is the new Administrative Advisor and Mark Robinson will remain as the USDA/NIFA (formally CSREES) representative. We currently have 22 members representing 11 Agricultural Experiment Stations with representatives from ARS/USDA. Mark Robinson, (USDA-NIFA representative) discussed the new Farm Bill and the reorganization of CSREES into the National Institute for Food & Agriculture. He noted that there will be more emphasis on the direction of Hatch funding into Multi-State projects from 25% to 30% with a strong emphasis on research. He reviewed the Federal FY2011 budget highlights and announced that $400 million in increased funding will support the following 5 high priority issues with a focus on multi state/agency and interdisciplinary approaches: bioenergy, global climate change, global food security, nutrition and health, and food safety. A discussion ensued as to how the current project may fit into these priority areas. Nina Fefferman (Rutgers Center for Vector Biology) presented a seminar entitled "Risk assessment models for West Nile virus and eastern equine encephalitis" which stimulated much open discussion on the importance of various factors that should be included in any model. Brief research and project updates with accompanying discussion were presented by each cooperator. The following individuals were elected to serve 2 yr terms as officers for the project: Theodore Andreadis (CT) - chair Edward Walker (MI) - vice-chair Sandra Allan (ARS-Gainesville) - secretary The meeting was adjourned one day early as a result of a snowstorm. The next meeting is scheduled for late February or early March 2011 at The Connecticut Agricultural Experiment Station, New Haven, CT. The participants agreed to hold a 1.5 day meeting with a similar venue and to include invited research seminar on a topic to be announced. The next meeting should also provide opportunities for 'cooperator reports' by objective, emphasizing presentations that show promise for collaboration.

Objective 1. Strengthen basic and applied research on the mosquito, pathogen, hosts, and environmental factors that influence disease emergence. An introduction of Aedes albopictus was documented at a commercial tire recycling plant in northeastern CT. Adult females were collected from late Jul  Oct at the plant. Host-seeking females attempting to alight on human subjects and larvae hatching from eggs collected in ovitraps placed in the woodlands surrounding the tire plant, were detected from mid-Aug - Oct denoting seasonal establishment. However, no larvae were recovered from eggs collected in ovitraps placed in the surrounding woodlands or in traps placed 1.6 km away, nor were females detected by human subjects the following season, indicating that the species did not survive winter conditions. The failure of Ae. albopictus to overwinter was likely due to winter egg mortality and/or interspecific competition from Ae. triseriatus and Ae. japonicus. (CT) Blood-engorged female Ae. j. japonicus were collected and blood meals identified by sequencing portions of the cytochrome b gene of mitochondrial DNA. Mammalian hosts included white-tailed deer (53%), humans (36%), fallow deer (5%), horse (3%), and opossum (3%). No avian, amphibian, reptilian or mixed blood meals were identified. (CT, NJ) Engorged An. quadrimaculatus and An. punctipennis mosquitoes were collected from EEE virus foci in central NY, and throughout NJ and their blood meals were identified. Analysis of An. quadrimaculatus and An. punctipennis from NY revealed 97.7% and 97.2% acquired blood from mammalian hosts, respectively. An. quadrimaculatus and An. punctipennis from NJ revealed 100% and 96% mammalian-derived blood meals. Individual mosquitoes containing mixed-blood meals from both avian and mammalian hosts were detected in 1.6% of An. quadrimaculatus in NY, and 2.8% and 4.0% of An. punctipennis from NY and NJ, respectively. White-tailed deer constituted the most common vertebrate host, comprising 85.8% to 97.7% of all blood meals. One horse- and two human-derived blood meals were detected from An. quadrimaculatus collected in NJ. EEE virus was isolated from one An. punctipennis collected in NY. Limited avian-derived blood meals were detected from mourning dove, sharp-shinned hawk, and house finch. Occasional feeding on avian hosts suggests that these mosquitoes may participate as epidemic/epizootic bridge vectors of EEE virus from viremic birds to horses and humans. Predominance of white-tailed deer as a source of blood meals supports enzootic amplification of deer-associated arboviruses in this region, including Jamestown Canyon, Cache Valley, and Potosi viruses. (CT, NJ, NY). We examined the genetic variations of Cx. pipiens mosquitoes from Chicago, IL that were determined to be principally ornithophilic but exhibited a higher inclination for mammalian hosts including humans. Microsatellite analysis of 10 polymorphic markers was performed on Cx. pipiens specimens with identified avian or mammalian blood meals. There were no significant differences in allelic richness, the pattern of conformity to Hardy-Weinberg equilibrium and linkage disequilibrium, nor was there overall genetic differentiation between specimens with avian- and mammalian-derived blood meals. However, Cx. pipiens form pipiens with mammalian- (including human-) derived blood meals had significantly higher ancestry and proportion of hybrids from the Cx. pipiens form molestus (population from NYC) than did those with avian-derived blood meals. By contrast, there were no significant differences in the ancestry and the proportion of hybrids from Cx. quinquefasciatus (TX). No temporal genetic variation was detected in accordance with the observation that there was no shift in blood feeding from birds to mammals. The results suggest that the probability of genetic ancestry from Cx. pipiens f. molestus may predispose mosquitoes to feed more readily on mammals, however the genetic mechanisms are unknown. (CT, MI) We have completed initial investigations of dispersal and survival of marked male Ae. albopictus in the field 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. 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. A large greenhouse mesocosm has been used to examine a Wolbachia-based microbial biopesticide approach against Ae. albopictus. Additional tests will compare the percent 'incompatibly mated' females with the incompatible males and allow assessment of male competitiveness for mates. (KY) Operational research was conducted in preparation for a large scale study of Culex pipiens dispersal from larval habitats (catch basins in urban Chicago). The research focused on the development and application of 13C and 15N stable isotope labeling methods of larval foods, in particular either in solution or incorporated into algae. The concept is to amend the larval habitats with these materials which results in 'marked' adults at a ca. 10-fold higher ratio of 13C/12C or 15N/14N than natural occurs. Mass spectroscopy is then used to detect the isotopic signature in individual, or in pooled, mosquitoes captured at the study site. Additional research was continued on the distribution and abundance of Oc. japonicus in Michigan, and new studies on the significance of algae to larval nutrition of this species. (MI) High resolution spatial population genetics has revealed two independent introductions of the invasive species Ae. japonicus, that are now mixing. We have obtained specimens from multiple locations in Japan for this analysis. In a study on sibling analysis of Cx pipiens populations, we have finished analysis of the proportion of egg masses fertilized by multiple males by examining offspring from successive egg masses from females mated in captivity. We have also dissected the spermatheca of females caught in the wild and will examined these next. We have completed inheritance analyses on real-time rates of dispersal of insect populations. A panel of 17 microsatellites were developed for Cx restuans. We have confirmed that, unlike Cx pipiens, this species over winters in large numbers even in forested areas with few underground human-made structures. Also, bioassays to examine Cx. pipiens and Ae. albopictus resistance to organophosphates and pyrethroids have been developed. (NJ) While examining hybrid populations in the Culex pipiens complex in Asia we identified and characterized a system where males are genetically distinguishable from females at the DNA level using a simple PCR- based assay. This unusual but very useful condition resulted from an old hybridization event coupled with suppressed recombination on chromosome 1, as ascertained using Single Nucleotide Polymorphisms (SNPs). Since suppressed recombination is often associated with critical sex determining genes, and the MDL has been linked to several genetic markers we have co-located in this region, we assert that the elusive MDL is located within this region of about 1,000,000 bp. An initial scan of the genes present has identified the testis development protein (TDP), a candidate gene in the sex determination pathway. (NJ) A knowledge, attitudes, and practices (KAP) questionnaire combined with entomological surveys of residential mosquito breeding sites were conducted in two neighborhoods. We tested the hypothesis that "correct" West Nile virus (WNV) knowledge and perceptions correspond with the use of practices that prevent mosquitoes from breeding and biting. Our results demonstrate that perceptions of WNV relate to the number of positive containers in yards and the use of mosquito preventive measures. In contrast, WNV knowledge was not related. Culex pipiens and Cx. restuans were common species found breeding in containers. Ochlerotatus japonicus was the most abundant species in 77% of positive containers (buckets, flower pots and birdbaths). This new invasive mosquito together with the Culex species identified in this study represents significant potential as disease vectors. This is the first study to directly investigate the relationship between KAP and breeding of WNV vectors in residential yards. (NY) Behavioral analysis of oviposition behavior by Aedes albopictus and Aedes aegypti revealed differences between the two species and provide the basis for subsequent evaluations of potential oviposition repellents or toxicants. (ARS-Gainesville) Objective 2. Use knowledge of mosquito, pathogen, vertebrate reservoir, and environment interactions to enhance ability to predict conditions leading to disease. Field studies suggest that Cx. pipiens feeds preferentially on American robins. To determine the contribution of innate preferences to observed preference patterns in the field, we conducted host preference trials with adult female Cx. pipiens in outdoor cages comparing the relative attractiveness of American robins with two sympatric bird species, European starling and house sparrow. Host seeking Cx. pipiens were 3x more likely to enter robin-baited traps when paired with a starling and almost twice more likely when paired with a house sparrow. There was no difference in the probability of trap entry when two robins were offered. Logistic regression analysis determined that the age, sex and weight of the birds, the date of the trial, starting-time, temperature, humidity, wind-speed and age of the mosquitoes had no effect on the probability of a choosing a robin over an alternate bird. Findings indicate that preferential feeding by Cx. pipiens mosquitoes on certain avian hosts is likely to be inherent. (CT) The optimal method for early prediction of West Nile virus (WNV) infection risk remains controversial. We analyzed the predictive utility of risk factor data for human WNV over a six-year period in CT. Using multiple logistic regression, the 30-day risk of human WNV infection by town was modeled using environmental variables as well as mosquito and wild bird surveillance. Using only environmental variables or animal sentinel data was less predictive than a model that considered all variables. In the final parsimonious model, population density, growing degree-days, temperature, WNV positive mosquitoes, dead birds and WNV positive birds were significant predictors of human infection risk, with an ROC value of 0.75. A real-time model using climate, land use, and animal surveillance data to predict WNV risk appears feasible. The dynamic patterns of WNV infection suggest a need to periodically refine such prediction systems (CT) WNV infection rate in C. pipiens is highly seasonal, being virtually zero in early summer, rising rapidly in mid-summer, then declining. The rise in infection rate mid-summer is strongly correlated with the appearance of fledgling birds, suggesting that age structure of the bird populations being fed upon influenced viral infection dynamics. However, results of a field study did not uphold the hypothesis that variation in avian community species diversity influenced the intensity of transmission, nor were nestling birds found to have higher exposure to infectious mosquito bites. In the latter case, nestling birds were not concluded to be the major age class promoting virus transmission; rather, post-nestling birds (fledglings) are likely the important age class whose appearance seasonally promotes annual, mid-summer epizootics. Blood meal analysis of C. pipiens, the primary vector in the study area, revealed that few bird species contribute to most of the estimated amplification fraction based on an ecological index estimator, namely, American robin, house finch, and blue jay. Further, C. pipiens fed at a higher than anticipated frequency on mammals, in particular humans (~16%). The tendency to feed on avians versus mammals was found to have an association with genetic ancestry within this mosquito population, where those individuals that had fed on mammals had a significantly higher ancestry for the unusual molestus form, whilst the avian feeding individuals lacked these genetic markers. Additional research on molecular genetic diversity of WNV over three year epidemic period showed that the virus is slowly evolving under a model of neutral, stabilizing selection with an increase of approximately 0.1% per yr in nucleotide-level genetic diversity. Analysis of mosquito infection among sites and years showed a strong relationship to temperature; cooler temperatures resulted in lower and seasonally delayed infection rates. (MI) A multivariate model was developed using both climatic and biotic variables in predicting the number of weekly New Jersey human cases using data from 2002-2006 data. We were able to account for greater than 70 percent of the variability. The model featured variables that were lagged to include time from being bitten by an infected mosquito to showing symptoms (i.e., incubation time up to 14 days). (NJ) We are developing a mathematical model that explores how behavioral influences of feeding preferences could cause disease amplification within a vector species without associated amplification being a necessary consequence in any of the host species. Additionally, a predictive model is being developed to explore hotspots for the potential evolution of newly arising insecticide resistance. This work will set the foundation for the construction of strategies for pesticide use which will enable long-term disease control rather than merely short-term effects which can lose efficacy as vector populations develop resistance. (NJ) Due to clear differences in rates of human infections between high/low latitude and mid latitude eastern seaboard states, in collaboration with Dr. Kramer we are analyzing the effect of underlying genetic make-up on the WNV vector competence of the northern Cx. pipiens, tropical/southern Cx quinquefasciatus, and hybrids between the two. We have optimized diapause markers for Cx pipiens in our lab to examine how the interplay of temperature and genetics affects vector competence in the Culex pipiens complex across the United States. (NJ) The behavioral role of acoustics in mosquito mating was explored, with male and female Aedes converging flight tone harmonics to match each other. We further demonstrated that this interaction occurs most frequently when the potential mate represents the optimal physical conditions to maximize reproductive success. Consequently, this acoustic behavior may be used in mate assessment. Further studies are investigating potential approaches for acoustic disruption of this essential pre-copulatory behavior. (NY) Studies been aimed at identifying male reproductive proteins that are transferred to females and may have significant impacts on female physiology. These proteins represent potential and novel targets for mosquito control. A novel method of reverse isotope labeling followed by proteomic analysis was used to examine proteins transferred to females. Sperm enriched proteins were also identified and analyzed. A total of 50 male seminal fluid proteins and 38 sperm proteins have been identified using this approach. The seminal fluid protein classes suggested roles in sperm storage and protection from oxidative stress, ecdysteroidogenesis, and protein activation. Many of the sperm proteins were homologous to Drosophila sperm proteins, suggesting conservation of sperm-related function across Diptera. (NY) Objective 3. Develop strategies to control mosquito vectors. Previous studies have shown that several species of anophelines are sensitive to the Bacillus sphaericus (Bs) binary toxin (Bin toxin). Thus, we initiated studies on the efficacy and resistance management properties of our two best recombinant larvicidal stains of B. thuringiensis subsp. israelensis (Bti) against Anopheles gambiae. Both of these strains have been genetically engineered to produce large amounts of the Bs Bin toxin. The best strain, referred to here as Bti/BsBin, produces the Bs Bin in combination with the four major endotoxins of Bti, which are Cry4A, Cry4B, Cry11A and Cyt1A. When tested against fourth instars of An. gambiae, this strain was nine-fold more effective at the LC95 level than the strains of Bti and Bs used in current commercial products. Interestingly and importantly, preliminary selection studies with the Bti/BsBin recombinant show no resistance development in An. gambiae after nine generations of selection. These results are similar to what we have observed in selection studies with the same Bti/BsBin strain against Cx. quinquefasciatus, which is known to develop high levels of resistance quickly under field conditions when only treated with B. sphaericus formulations. (CA) Factors that directly impact horizontal transmission of the microsporidium Amblyospora albifasciati to its intermediate copepod host, Mesocyclops annulatus were examined in laboratory bioassays in relation to life history strategies that facilitate persistence of the parasite in natural populations of its definitive mosquito host, Oc. albifasciatus. A moderately high quantity of meiospores from mosquito larvae was required to infect adult female copepods (IC50 = 3.6 x 10 4 meiospores/ml). Meiospore infectivity following storage at 25C was detected up to 30 days, while meiospores stored at 4C remained infectious to copepods for 17 months. The pathological impact of A. albifasciati infection on M. annulatus resulted in a 30% reduction in survivorship after 7 days followed by gradual progressive mortality up to 40 days. Infected female copepods survived in sediment under desiccation up to 30 days. The susceptibility of late stage copepodid M. annulatus to meiospores of A. albifasciati and transstadial transmission of infection to adult females was established. (CT) Our goal is to establish research collaborations related to mosquito reproduction, mosquito cell cultures, and maintenance of the bacterium Wolbachia pipientis, for eventual modification as an agent for manipulation of mosquito populations. (MN) In an Asian tiger mosquito area-wide management project, we achieved 50%-60% reductions in intervention sites in Mercer and Monmouth counties. Ae. albopictus populations in the 3 Monmouth sites increased very slowly (compared to both the 2008 season and Mercer) and therefore significant reduction occurred only in late August and September when the populations peaked. Education efforts resulted in a significant reduction in Ae. albopictus populations in Mercer but not in Monmouth. Dramatic socioeconomic differences between the two counties had unexpected results. That is, it was considerably easier to access teachers and their students in inner-city Mercer than in suburban Monmouth. Overall, Mercer sites were easier to work in, both for the education and the control teams. The number and nature of Ae. albopictus larval sources in the two counties was also different and impacted both the type and outcome of education and management measures. (NJ, ARS-Gainesville) We are examining the potential of biological control to supplement current mosquito control efforts in southern Wisconsin. We determined that fathead minnows serve as good predators of Culex spp. in the lab and as excellent biocontrol tools in a pilot study of three field sites. In 2009, we expanded field introductions to nine locations and measured ecological parameters that affected fish persistence. In 6 of the 9 sites, control of Culex spp. was nearly absolute for most of the summer. In three sites, fish were not retained. Depth and energy dissipaters were key factors in persistence. Further, we are examining the effectiveness of copepods as predators. These crustaceans are more effective against mosquitoes than any other invertebrate predator and they can be mass produced inexpensively. We collected copepods from catch basins in Fall of 2009, identified three species, and began culturing them in the lab. Preliminary trials using Macrocyclops albidus indicated that local isolates are capable of feeding on early instar Culex spp. Current work focuses on optimizing culture conditions in the lab for scaled up production and releases into catch basins. (WI) Emphasis has been on examination of host odors for attractant discovery, use of structure-activity models to develop new repellents and toxicants, and characterization of barrier sprays. Odors from chickens were identified by gas chromatography/mass spectrometry and bioassays of these compounds are in progress. Research with repellents and toxicants is drawn from data generated by the USDA screening program from 1942-1977. From a set of structurally similar acylpiperdine compounds, a successful model was developed, and novel efficacious repellents were predicted, synthesized and verified by bioassay. Some compounds from this set are being evaluated against other arthropods. The current emphasis of modeling work is on the development of new toxicants. Collaborative projects with barrier sprays involved comparison of sprayers and comparison of efficacy duration for three pyrethroid active ingredients. (ARS-Gainesville) Landing responses of mosquitoes (Aedes albopictus) and sand flies (Lutzomyia shannoni, Phlebotomus papatsi) on wax myrtle leaves treated with commercial residual pesticides (maximum label rate) were video-taped and analyzed to determine effect on landing frequency and duration. Significant differences existed in landing and resting responses between mosquito species (Aedes aegypti, Anopheles quadrimaculatus and Culex quinquefasciatus) exposed to sub-lethal rates of pyrethroids and better knowledge of the differences helps optimize efficacy of treatment against targeted species. Lab evaluation of components of Duet® ULV spray using Culex quinquefasciatus and Lutzomyia shannoni indicated that prallethrin enhanced flight activity resulting in more droplets and greater mortality. Species of plant, application method, rain exposure and sunlight significantly decreased efficacy of bifenthrin-treated leaves for eliciting mortality of Ae. aegypti determined in laboratory assays. (ARS-Gainesville) In addition to studies on structure activity relationships of compounds for new toxicant discovery, we are using gene silencing using RNAi technology as a method to knock down critical proteins in mosquitoes with possible applications for control. An inhibitor of apoptosis protein 1 gene in Aedes aegypti (AaeIAP1) was chosen as a target for the development of molecular pesticides and when topically applied was shown to cause mortality in adult Aedes aegypti. Other potential targets and new constructs are being evaluated. (ARS-Gainesville) Semi-field and field studies were conducted on the use of barrier sprays to reduce host-vector contact. Barriers were constructed of either treated vegetation or camouflage fabric. These barrier materials were sprayed with one of several candidate synthetic pyrethroid compounds. 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 or 7 x 7). Preliminary results indicate effective protection from mosquitoes for 3-6 weeks depending on the mosquito species, type of insecticide treatment, barrier material and/or environmental conditions. (ARS-Gainesville) Objective 4. Enhance surveillance technologies for mosquitoes and mosquito-borne pathogens. Statewide mosquito trapping and testing for mosquito-borne arboviruses was conducted from Jun - Oct in CT. A total of 291,641 mosquitoes (16,895 pools) representing 38 species were trapped and tested. 35 isolations of WNV were made from: Cx. pipiens (25), Cx. restuans (3), Cx. salinarius (1) and Oc. stimulans (1), collected at 19 sites in 5 counties (Fairfield, Hartford, Middlesex, New Haven, and New London) from Jul 15 - Oct 8. Widespread eastern equine encephalitis (EEE) activity was detected. 123 isolations of EEE were made from: Cs. melanura (84), Oc. canadensis (10), Ae. cinereus (6), Ur. sapphirina (4), An. punctipennis (4), Cx. salinarius (3), Ae. vexans (2), An. quadrimaculatus (2), An. walkeri (2), Oc. trivittatus (2), Cx. restuans (1), Oc. cantator (1), Oc. triseriatus (1), and Ps. ferox (1) collected at 25 sites in 7 counties from Aug 17 - Oct 27. One horse case and infections in 2 commercial pheasant flocks were reported. Other mosquito-borne viruses isolated included: Jamestown Canyon (43), Cache Valley (1), Highlands J (60), Trivittatus (29), and Flanders (1). (CT) New Jersey surveillance for EEE indicated high activity levels in the enzootic vector Culiseta melanura with 57 positives of 371 pools submitted from the traditional resting box sites -- a five-fold increase from the previous year. Dissemination of the virus was widespread and began earlier (15 July 2009 versus early August of 2008). Non-melanura species were also positive for the first time in several years, including first time positive pools for Culex erraticus and Aedes japonicus. Six horses and two alpaca were EEE positive. Surveillance for WNV found 322 positive pools from the 9,965 pools submitted, mostly in Culex pipiens (primary enzootic vector), Culex restuans or mixed Culex pools. One horse case and three human cases developed. Positive mosquitoes appeared well before the first positive dead bird, indicating that the value of dead bird reports have decreased. Surveillance for SLE and LAC viruses was negative. (NJ) We compare the efficiency and sensitivity of the Rapid Analyte Measurement Platform (RAMP) to reverse transcriptase polymerase chain reaction (RT-PCR) from 2005 to 2008 to detect West Nile virus (WNV). Overall, 316 pools tested negative and 115 pools tested positive for WNV. Eighty-nine pools tested positive using RAMP and all were confirmed by RT-PCR; 26 pools were WNV-negative using RAMP but positive using RT-PCR. False-positives from RAMP were not detected. (NJ) More Ae. albopictus females were collected at 1 m compared to 6 m above ground with significantly more collected in suburban compared to sylvatic habitats. Optimal placement of traps will increase surveillance sensitivity. Propane powered commercial traps producing CO2 were as effective as CDC traps that required a separate CO2 source for Ae. albopictus surveillance efforts and these types of traps may provide additional surveillance tools. (ARS-Gainesville) 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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