The stagnation of our fight against malaria in recent years, mainly due to the development of mosquito insecticide resistance, argues for the urgent development of new weapons. The dramatic evolution of molecular tools in the last few decades led to a better understanding of parasite-mosquito interactions and coalesced in the development of novel tools namely, mosquito transgenesis and paratransgenesis. Here we provide a historical view of the development of these new tools and point to some remaining challenges for their implementation in the field.

RNA interference is widely used to analyze gene functions via phenotypic knockdown of target transcripts in mosquitoes, which transmit numerous mosquito-borne diseases. Functional analysis of mosquito genes is indispensable to understand and reduce transmission of mosquito-borne diseases in mosquitoes. Intrathoracic injection of double-stranded RNA (dsRNA) remains the simplest and most customizable method in mosquitoes for functional analysis of the genes of interest. However, achieving consistent and effective knockdown by dsRNAi is often elusive and may require extensive optimization. We tested the effectiveness of gene silencing by intrathoracic injection of four different quantities of dsRNA targeting two genes, cysteine desulfurylase (Nfs1) and short-chain dehydrogenase (SDH). We found that Nfs1 gene has a lower expression level upon silencing than SDH gene. In the case of the gene that is easier to silence, Nfs1 gene expression was significantly silenced by all four tested quantities of dsRNA up to 21 d.p.i., but silencing of SDH, the gene that is difficult to silence, was less effective, with knockdown lasting up to 9 d.p.i. only when 1,000 ng of dsRNA was used. Based on our observation, intrathoracic injection of 500 ng of dsRNAs per mosquito is recommended to achieve effective knockdown for well-silenced transcripts such as Nfs1 for up to 3 weeks. This includes most bioassays involving arboviral infections in . The estimated quantities of dsRNA described in this study should be applicable to most dsRNAi studies and thus provide a guideline to develop efficient dsRNAi in other experimental investigations.

Insects are responsible for the transmission of major infectious diseases. Recent advances in insect genomics and transformation technology provide new strategies for the control of insect borne pathogen transmission and insect pest management. One such strategy is the genetic modification of insects with genes that block pathogen development. Another is to suppress insect populations by releasing either sterile males or males carrying female-specific dominant lethal genes into the environment. Although significant progress has been made in the laboratory, further research is needed to extend these approaches to the field. These insect control strategies offer several advantages over conventional insecticide-based strategies. However, the release of genetically modified insects into the environment should proceed with great caution, after ensuring its safety, and acceptance by the target populations.