Constructing a nectar delivery system for mosquito control using Impatiens walleriana.

Mosquitoes are vectors of numerous human pathogens. These pathogens are spread when female mosquitoes blood feed to lay eggs. Since males do not feed on blood and females mostly need blood to produce eggs, both consume plant nectar as a primary source of energy. Previously, a study of mosquito nectar preference showed that extrafloral nectar from Impatiens walleriana is an attractive food source for mosquitoes and has potential to become a platform for delivering targeted peptides to mosquitoes. However, since this study was conducted indoors, the assumption that impatiens nectar will attract mosquito feeding outdoors needs to be tested. This dissertation tests three components necessary for the development of mosquitocidal nectar technology: (1) the assumption that mosquitoes will feed on impatiens nectar outdoors; (2) the development of mosquito-specific peptides; and (3) the development of a nectar expression model in Impatiens walleriana. The first component of outdoor nectar feeding was tested by measuring the nectar feeding of 3 mosquito species in outdoor, simulated gardens. This work demonstrated that the nectar of Impatiens walleriana is highly attractive to mosquitoes in an outdoor setting despite the presence of potentially compounding factors. To develop mosquito-targeted peptides for the second component, an external loop of Domain III from the Dengue virus type 2 envelope glycoprotein was attached to the fluorescent protein EGFP and spider toxin Hv1a and fed to Aedes aegypti and Culex quinquefasciatus mosquitoes. The midgut binding of the targeted EGFP and specific oral toxicity of the targeted Hv1a in Ae. aegypti but not Cx. quinquefasciatus indicates that the addition of the targeting domain conferred species-specific targeting to these proteins. For the third technology component, to develop a nectar expression model in Impatiens walleriana, nectary promoters and a nectar-specific signal peptide were identified from Impatiens walleriana using RNA, DNA and protein sequencing. These methods identified promoters for phylloplanin and SWEET14 homologs and a nectar-specific signal peptide from the phylloplanin homolog. These elements will be used alongside other non-native promoters and signal peptides to see which promoter/signal peptide combination achieves the highest level of foreign protein expression in transgenic impatiens nectar. The first component of outdoor nectar feeding was tested by measuring the nectar feeding of 3 mosquitoes species in outdoor, simulated gardens. This work demonstrated that the nectar of Impatiens walleriana is highly attractive to mosquitoes in an outdoor setting despite the presence of potentially compounding factors. To develop mosquito-guided peptides for the second component, an external loop of Domain III from the Dengue virus type 2 envelope glycoprotein was attached to the fluorescent protein EGFP and spider toxin Hv1a and fed to Aedes aegypti and Culex quinquefasciatus mosquitoes. The midgut binding of the guided EGFP and specific oral toxicity of the guided Hv1a in Ae. aegypti but not Cx. quinquefasciatus indicates that the addition of the guiding domain conferred species-specific targeting to these proteins. For the third technology component, to develop a nectar expression model in Impatiens walleriana, nectary promoters and a nectar-specific signal peptide were identified from Impatiens walleriana using RNA, DNA and protein sequencing. These methods identified promoters for phylloplanin and SWEET14 homologs and a nectar-specific signal peptide from the phylloplanin homolog. These elements will be used alongside other non-native promoters and signal peptides to see which promoter/signal peptide combination achieves the highest level of foreign protein expression in transgenic impatiens nectar.