siRNA-Mediated Gene Targeting in Aedes aegypti Embryos Reveals That Frazzled Regulates Vector Mosquito CNS Development

Abstract

Although mosquito genome projects uncovered orthologues of many known developmental regulatory genes, extremely little is known about the development of vector mosquitoes. Here, we investigate the role of the Netrin receptor frazzled (fra) during embryonic nerve cord development of two vector mosquito species. Fra expression is detected in neurons just prior to and during axonogenesis in the embryonic ventral nerve cord of Aedes aegypti (dengue vector) and Anopheles gambiae (malaria vector). Analysis of fra function was investigated through siRNA-mediated knockdown in Ae. aegypti embryos. Confirmation of fra knockdown, which was maintained throughout embryogenesis, indicated that microinjection of siRNA is an effective method for studying gene function in Ae. aegypti embryos. Loss of fra during Ae. aegypti development results in thin and missing commissural axons. These defects are qualitatively similar to those observed in Dr. melanogaster fra null mutants. However, the Aa. aegypti knockdown phenotype is stronger and bears resemblance to the Drosophila commissureless mutant phenotype. The results of this investigation, the first targeted knockdown of a gene during vector mosquito embryogenesis, suggest that although Fra plays a critical role during development of the Ae. aegypti ventral nerve cord, mechanisms regulating embryonic commissural axon guidance have evolved in distantly related insects.

Physical features and chitin content of eggs from the mosquito vectors Aedes aegypti, Anopheles aquasalis and Culex quinquefasciatus: Connection with distinct levels of resistance to desiccation

“…Up to date this approach is yet not feasible for mosquito embryos. There do exist a few RNAi studies with mosquitoes aiming the egg but these are directed toward maternal genes that affect early embryogenesis (e.g., Wu et al, 2013) or siRNA directed silencing aiming late zygotic genes (e.g., Clemons et al, 2011). In both approaches, egg viability is compromised and therefore these methods can not be employed to address genes with the potential to affect mosquito egg viability under dehydrating conditions.…”

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

“…Perform and analyze qRT-PCR assays with three biological replicates on at least 10 pooled control vs. experimental larvae as described 11,19,20 , or according to standard lab procedure. Representative results are included below.…”

“…27 protocol for confirmation of knockdown as described previously 11,20 and discussed in the representative results section below.…”

“…For a detailed history and comparison of the use of these three techniques in insects, please refer to Yu et al 8 . We have successfully used microinjection 10 as a means of delivering siRNAs to target developmental genes in A. aegypti embryos, larvae, and pupae 11–14 . However, this labor-intensive delivery strategy requires both a microinjection setup and a skilled hand.…”

Developmental neurogenetics of sexual dimorphism in Aedes aegypti

“…Magnusson et al (2011) assessed sex-specific transcriptomes throughout Anopheles gambiae development and characterized the functions of several testis- and ovary-specific genes during gonad development. Functional genetic analysis of nervous system development has been performed in A. aegypti (Clemons et al, 2011; Haugen et al, 2011; Sarro et al, 2013; Mysore et al, 2013, 2014a, 2014b), an emerging model for vector mosquito development studies (Clemons et al, 2010a). A recent functional genetic study explored the development of sexual dimorphism in the A. aegypti pupal nervous system (Tomchaney et al, 2014).…”