Prova de Conceito: plantas cultivadas com dsRNA como forma efetiva de controle do mosquito Aedes aegypti (Diptera: Culicidae)

Abstract

The Aedes aegypti mosquito is a vector of important arboviruses that have triggered epidemics throughout history. Mosquitoes feed on sugars to maintain metabolism, however, females need blood intake to produce eggs. During the blood meal, infection and viral transmission occurs. Without effective and safe vaccines or therapies for most transmitted arboviruses, the most effective way to prevent their spread is through vector control of A. aegypti. The control of this mosquito has been done mainly through chemical insecticides, but the excessive use of these substances has contributed to the emergence of resistant populations. Thus, it is necessary to create new control measures. With regard to insect control, RNA interference (RNAi) technology is considered ecological and specific, compared to others already used. The chitin polymer (N-acetylglucosamine) is present in the trachea, ovary, eggs and eggshells of insects, it is the main compound in the cuticle of arthropods. Therefore, enzymes that act in the metabolism of this polymer, such as chitin synthases, are considered good targets for insect control. In most insects, including the A. aegypti mosquito, two chitin synthase genes, CHSA and CHSB, are known. The CHSA genes are expressed in epidermal cells and related ectodermal cells, whereas CHSB genes are expressed only in midgut epithelial cells. The aim of this work was to propose a new method of delivering double-stranded RNA molecules (dsRNA) directed to the CHSA and B genes of A. aegypti, simultaneously. And to evaluate the phenotypic changes, the parental effects and the effectiveness of gene silencing, compared to the control condition. The dsRNA molecules targeting the green fluorescent protein gene (GFP) (control) and those targeting the A. aegypti CHSs genes (experimental) were produced by heterologous expression in E. coli bacteria, the production of dsRNA fragments was confirmed by polymerase chain reaction (PCR). The dsRNA constructs inside the plants were administered in breeding water containing 2nd and 4th instar A. aegypti larvae, separately. The treated group showed sluggishness, decreased body mass and developmental delay compared to the control group. Adult mosquitoes derived from the treatment of 4th instar larvae with dsRNACHS or dsRNAGFP were fed with blood. Females in the experimental group had lower egg laying, which over 20 days showed dryness, crushing, holes on their surface and reduced CHSA transcripts phenotype, resulting in reduced hatching rate compared to the control condition dsRNAGFP. The silencing of both CHSs genes in adult mosquitoes, coming from the larvicide assay with dsRNACHS, was confirmed by quantitative PCR technique (qPCR).