The ultimate goal of this research was to perform a domain exchange between a computer identified insect specificity region of the mosquito larvicidal protein Cry IVB and a previously identified domain in a related protein toxin which targets lepidopteran insect larvae. If the insect specificity domain has been correctly identified, an exchange of DNA in this manner transfers the toxicity of one peptide to another by an exchange of the insect specificity domains. New, chimeric peptides may be designed which will target a larger spectrum of insect larvae.In previous research a domain exchange was performed between the two genes carried on plasmid vectors in E. coli and low levels of toxicity to mosquito larvae were observed. Initial efforts of this research attempted to identify these recombinants. However, stability was not achieved by sequential colony screens. Furthermore, a recently published three-dimensional structural model for all the B. thuringiensis crystalline toxins became available and it was quickly determined that the first exchanges excluded most of the f3-sheet domain that is responsible for insect cell receptor binding, the feature that gives the toxins their specificity. Therefore, it was decided that a larger, more inclusive region of Cry IVB DNA must be exchanged between the two toxins.Extensive computer analyses of the Cry IVB sequence and retroactive comparison of these sequences to the three-dimensional model yielded a fragment of DNA that encoded more than 60% of the putative insect specificity domain. Oligonucleotide primers were subsequently designed to flank this region so that the polymerase chain reaction could be employed to amplify the region. Additionally, the primers were engineered to contain terminal restriction endonuclease sites to ease in the exchange of the domain encoding region into Cry IA(c). The region of Cry IVB DNA flanked by the oligonucleotide primers was successfully amplified by the PCR and cloned into the plasmid vector pUC 19 as a reservoir for a future domain exchange. / Department of Biology

Twenty-six of the 49 mosquito species reported from Virginia were collected in James City County, Virginia during 1985 and 1986. The most abundant species were Psorophora columbiae, Anopheles quadrimaculatus, and Culex species. P. columbiae accounted for 50% and 16% of the light trap collections in 1985 and 1986, respectively. The most predominant mosquitoes collected during 1986 were Culex species. Mosquitoes had an impact on the amount of time residents and campers spent outdoors. Forty-seven percent of the residents and 28% of the campers surveyed had their time outdoors limited by mosquitoes. There was no significant association between the area where the respondent was brought up as a child and their perception of the mosquito problem. No economic impact could be attributed to mosquitoes but the benefits the county's residents received from the mosquito control program annually was estimated to exceed the programs costs by two times. The human tolerance level for mosquito bites was determined to be 5 per night. James City County could adopt and benefit from an integrated mosquito management program. The components of the program are seasonal monitoring of mosquito populations, periodic public attitude surveys, public education programs, physical, chemical, and biological control tactics, and periodic program evaluation. / Master of Science

Background: Mosquito-borne diseases are a growing concern for temperate regions including the northeastern US. There the two primary mosquito vectors, Cx. pipiens and Ae. albopictus are widespread, endemic circulation of West Nile virus causes sporadic outbreaks, and imported arboviruses such as dengue, chikungunya, and Zika are on the rise. With temperate mosquito-borne disease outbreaks likely to increase in frequency, it is critical to reduce mosquito populations in the northeastern US. Community-based source reduction is heralded as the most sustainable component of integrated mosquito management. Yet mosquitoes develop rapidly, requiring weekly maintenance of mosquito habitat. This is onerous and community commitment flags. The development of predictive models to inform focused vector-control efforts is therefore of great utility. Objectives and Methods: The overarching objective of this research is to make robust predictive modeling frameworks based on empirically derived relationships of the ecology and epidemiology of mosquito-borne disease systems in the northeastern US. We aim to quantify the relationships between local environmental and meteorological conditions and mosquito vectors. In Chapters 2 and 4 we use lengthy surveillance records to develop models and use model ensembles to generate predictions based on out-of-sample data. For chapter 3 we use more spatially refined data to investigate the influence of intra-urban heterogeneities and how climatic conditions influence mosquito populations across these defined differences. Results: In Chapter 2, we model and forecast WNV infection rates among mosquito vectors using meteorological and hydrological conditions. We show that real-time climate information can predict WNV Culex infection rates prior to when human risk is greatest. In Chapter 3, we link infrastructure degradation and vegetation patterns with Ae. albopictus infestation levels as well as the interactive effect of precipitation across these environmental conditions. In Chapter 4, we identify key land use characteristics and meteorological conditions associated with annual Ae. albopictus abundance. Further we use imported chikungunya cases to delineate areas of high arboviral importation and, in combination with areas of high Ae. albopictus abundance, areas at heightened risk for arboviral transmission. Conclusions: While temperate outbreaks are often self-limiting they may be increasing in frequency and severity. Due to the multitude of invasive vectors and arboviruses, vector control techniques that work for multiple mosquito species are likely more effective and sustainable. Here we build build empirical models that accurately predict mosquito dynamics before populations peak which is critical for vector control. We recommend integrating predictive modeling into mosquito management guidelines as this could focus valuable resources to when and where mosquito-borne transmission risk is greatest. Further we find social and ecological determinants of mosquito dynamics, supporting further study that combine socio-ecological processes into model frameworks.

Graduation date: 1996

No description available.

Bacillus sphaericus 2362 is a mosquito pathogenic bacterium. Its greatest industrial potential may be in developing countries where mosquitos are often vectors for diseases. This strain is typical of the species in that it is unable to grow using carbohydrates as a sole source of carbon. The goal of this research was to determine the metabolic deficiency(s) responsible for the inability of this organism to grow on carbohydrates. Compounds that supported light growth of this organism on an agar-solidified, defined medium included acetate, glycerol, and gluconate. Growth in a defined liquid medium with acetate as the source of carbon was much slower than growth in a complex, protein-based broth. B. sphaericus grew poorly in a defined, liquid medium with glycerol or gluconate as the carbon source. Activity of enzymes responsible for the initiation of metabolism of some substrates was not detected in cell extracts. These enzymes were: glucokinase, hexokinase, beta-galactosidase, and amylase. Growth of this bacterium on glycerol as a sole source of carbon implies the presence of the enzymes from the lower half of the Embden-Meyerhof-Parnas (EMP) pathway. Two enzymes of the upper half of the EMF pathway, phosphofructokinase and fructose diphosphate aldolase, were undetected in cell extracts. In addition, glucose dehydrogenase activity was not detected. The inability to form glucose-6-phosphate from glucose prevents the catabolism of this and related substrates via the Entner-Doudoroff (ED), hexose monophosphate (HMP), and EMF pathways. Oxygen uptake studies indicated that B. sphaericus oxidized gluconate slightly but only when the cells were grown in a complex, protein-based medium supplemented with gluconate. Although gluconokinase activity was detected in cell extracts, no activity was detected for the key enzymes of the ED (phosphogluconate dehydratase/KDPG aldolase), or HMP (6-phosphogluconate dehydrogenase) pathway. It is unclear how B. sphaericus grows on a defined medium with gluconate as the sole source of carbon. In addition to enzymatic deficiencies, whole cells were unable to accumulate [¹⁴C]glucose or [¹⁴C]sucrose. / Master of Science

Media for the selective recovery of the mosquito pathogens Bacillus sphaericus 1593 and B. thuringiensis serovar. israelensis were developed. Streptomycin at 100 µg/ml and 500 µg/ml in NY agar (nutrient agar with 0.05% yeast extract) successfully selected for B. sphaericus, and allowed counts equivalent to those obtained on the nutrient medium NY without antibiotics. The medium containing 100 µg/ml of streptomycin (NYST) was used to recover B. sphaericus 1593 from a mixed microbial population in pond water. Sodium chloride, penicillin G, and pH adjustment of the medium were found to be unsatisfactory selective agents. Two selective media for the recovery of B. thuringiensis serovar. israelensis gave counts equivalent to those obtained on the nonselective NY medium. One medium contained 100 µg/ml of polymyxin with 1.0 µg/ml chloramphenicol (NYPC), and the other contained 500 µg/ml of polymyxin alone. The use of the higher level of polymyxin with chloramphenicol reduced the number of viable B. thuringiensis serovar. israelensis. NYPC was used to recover B. thuringiensis serovar. israelensis from a mixed microbial population in pond water. The selective media reduced the number of pond water microorganisms on plates by 90 to 99%. A heat treatment of 50ºC for 10 minutes also reduced pond water microbiota by approximately one log. The use of heat treatment plus either NYST or NYPC reduced the pond water microbiota further. The heat treatment had little effect on sediment microbiota. A selective-differential medium for B. thuringiensis serovar. israelensis was developed for use when heat treatment of samples would be undesirable. This medium, PEMBAC, permitted the observation of peptone deamination and hydrolysis of lecithin, which are characteristic of B. thuringiensis serovar. israelensis. The medium contained 50 µg/ml of polymyxin and 1.0 µg/ml chloramphenicol as selective agents. The parasporal crystals of B. thuringiensis serovar. israelensis are the site of the mosquito larval toxin. Because the crystals are not viable, another method for their enumeration was examined. Antisera to whole crystals and to solubilized crystal antigens were prepared in rabbits for use in the indirect fluorescent antibody technique. Because of the small size and irregular shape of the parasporal crystals of B. thuringiensis serovar. israelensis, the crystals were difficult to distinguish from other small fluorescing particles. The antisera prepared precipitated several antigens in solubilized crystals, but did not adsorb to the majority of the antigens in whole crystals. / Master of Science

The objectives of this study were to provide a comprehensive review of the risks and benefits of using adulticides to reduce risk of mosquito-borne disease (particularly West Nile Virus) transmission to humans, as well as to decrease annoyance from nuisance mosquitoes. The study was designed with two major research components, including: 1) an extensive literature review to determine the efficacy of adulticide use, the adverse effects of adulticide use, the impact of mosquitoes on community livability, and the risks and benefits of pesticide use in controlling mosquitoes; and 2) interviews with selected vector districts in seven states to determine effective and ineffective practices in mosquito management. This study has demonstrated that an integrated mosquito management program may be beneficial in reducing risk of disease transmission and mosquito annoyance when performed appropriately. The contribution of adulticiding to reducing mosquito-borne disease transmission, however, is unknown. Research is needed to: 1) further assess the ecological and human impacts of adulticides using the dose and exposure rates realistic to an adulticide program; 2) gain an understanding of the human and ecological impacts of aggregate and cumulative exposures to pesticides, especially for special populations, such as children; and 3) determine the contribution of adulticiding in interrupting or reducing the enzootic amplification of arboviruses, as well as the transmission of WNV to humans. / Graduation date: 2004