Investigation on the functionality of genes of pathogen-vectors mosquitoes

Abstract

The contemporary profusion of descriptive data of genomes of medically-interesting insects have reinforced the demand for the knowledge of the function of their genes. The investigation of the genetic features of mosquito vectors have received particular attention because it can enhance vector control methods and the development of genetically-modified mosquitoes. In this context, we propose to study two main genetic mechanisms of the reproductive physiology of vector mosquitoes (Culicidae): the "laccase 2" and the "Grauzone" genes. As biological models, we will use two species of higher epidemiological relevance mosquitoes in the world: Aedes aegypti, vector of Dengue and Chikungunya viruses, and Culex quinquefasciatus, the vector of WNV virus and filaria. Operationally, this proposal aims to support two newly-initiated projects of our team (FAPESP Processes 2013/26017-7 and 2013/26014-8). Accordingly, the objectives, the experimental design and the expected results of those projects are, in essence, reproduced in this proposal. We already show preliminary results demonstrating the feasibility of this proposal, which is organized into two sub-projects: 1) Activity "expressional characterization of laccase gene 2 in Aedes aegypti" We will quantitatively describe the expression profile of the laccase gene 2 in Aedes aegypti, as well as the effects that its silencing may have on females viability / survival of eggs. The gene encoding the enzyme laccase 2 has its major function in the sclerotization of eggs and insect body cuticle. When the expression of the laccase gene 2 is silenced in adult Aedes albopictus (a sister species of Ae. Aegypti) their females produce hyaline and inviable eggs. In theory, this is a good candidate gene to be manipulated to produce genetically-modified mosquitoes or to design biological control solutions. A homologous gene to laccase 2 was already sequenced from the genome of Ae. aegypti, (which is the main vector of dengue virus in Brazil) but despite the promising scenario, it has not been subjected to functional studies in the species. The proposed experimental approach to the laccase gene 2 promises to reduce this gap of knowledge. 2) Activity "Influence of Grauzone gene in the reproductive fitness of Culex quinquefasciatus infected with Wolbachia." We will test whether the expression of a Grauzone-homologous gene is present in neotropical Cx. quinquefasciatus mosquitoes infected and non-infected by Wolbachia endosymbiont bacteria. We will test also if the silencing of this gene in infected females promotes changes in the fitness and/or the cytoplasmic incompatibility (CI) of the mosquito. It is known that bacteria can suppress or alter the host gene expression in order to produce favorable ambience for its maintenance as endosymbiont. Because of this potential manipulating skills, Wolbachia has been tested as a "tool" for population control of insect vectors of parasites. The mosquito Cx. Quinquefasciatus, an important vector of several pathogens that affect humans, is naturally infected with Wolbachia in the Neotropics. Our research team has shown that the presence of bacteria changes the mosquito fitness, promoting cytoplasmic incompatibility, temporal changes in oogenesis, and changes in reproductive fecundity and fertility. It is known that the Grauzone gene of Cx. Quinquefasciatus, regulator of cell cycles of oogenesis and spermatogenesis, may have its expression altered by Wolbachia action. However, in the neotropical model Wolbachia-Cx. quinquefasciatus, the genetic basis of phenotypes "fitness changed" and CI still remains unknown. To investigate this issue as we want here, to answer if Grauzone gene influences the fitness of the mosquito is a essential step for the possible development of a new biological control method which combines Cx. Quinquefasciatus, Wolbachia and the Grauzone gene. (AU)