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.

Blood-feeding mosquitoes, including the dengue and yellow fever vector Aedes aegypti, transmit many of the world's deadliest diseases. Such diseases have resurged in developing countries and pose clear threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for arthropod-borne disease control by genetic manipulation of vector insects. Targets of particular interest include genes that regulate development. However, although the Ae. aegypti genome project uncovered homologs of many known developmental regulatory genes, little is known of the genetic regulation of development in Ae. aegypti or other vector mosquitoes. This article provides an overview of the background, husbandry, and potential uses of Ae. aegypti as a model species. Methods for culturing, collecting and fixing developing tissues, analyzing gene and protein expression, and knocking down genes are permitting detailed analyses of the functions of developmental regulatory genes and the selective inhibition of such genes during Ae. aegypti development. This methodology, much of which is applicable to other mosquito species, is useful to both the comparative development and vector research communities.

Blood-feeding mosquitoes, including the dengue and yellow fever vector Aedes aegypti, transmit many of the world's deadliest diseases. Such diseases have resurged in developing countries and pose clear threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for arthropod-borne disease control by genetic manipulation of vector insects, and genes that regulate development are of particular interest. This protocol for whole-mount in situ hybridization can be used to analyze gene expression in Ae. aegypti embryos and larvae, a critical aspect of understanding developmental gene function in this vector mosquito.

BackgroundDespite the devastating global impact of mosquito-borne illnesses on human health, very little is known about mosquito developmental biology. In this investigation, functional genetic analysis of embryonic salivary gland development was performed in Aedes aegypti, the dengue and yellow fever vector and an emerging model for vector mosquito development. Although embryonic salivary gland development has been well studied in Drosophila melanogaster, little is known about this process in mosquitoes or other arthropods.ResultsMosquitoes possess orthologs of many genes that regulate Drosophila melanogaster embryonic salivary gland development. The expression patterns of a large subset of these genes were assessed during Ae. aegypti development. These studies identified a set of molecular genetic markers for the developing mosquito salivary gland. Analysis of marker expression allowed for tracking of the progression of Ae. aegypti salivary gland development in embryos. In Drosophila, the salivary glands develop from placodes located in the ventral neuroectoderm. However, in Ae. aegypti, salivary marker genes are not expressed in placode-like patterns in the ventral neuroectoderm. Instead, marker gene expression is detected in salivary gland rudiments adjacent to the proventriculus. These observations highlighted the need for functional genetic characterization of mosquito salivary gland development. An siRNA- mediated knockdown strategy was therefore employed to investigate the role of one of the marker genes, cyclic-AMP response element binding protein A (Aae crebA), during Ae. aegypti salivary gland development. These experiments revealed that Aae crebA encodes a key transcriptional regulator of the secretory pathway in the developing Ae. aegypti salivary gland.ConclusionsThe results of this investigation indicated that the initiation of salivary gland development in Ae. aegypti significantly differs from that of D. melanogaster. Despite these differences, some elements of salivary gland development, including the ability of CrebA to regulate secretory gene expression, are conserved between the two species. These studies underscore the need for further analysis of mosquito developmental genetics and may foster comparative studies of salivary gland development in additional insect species.