Despite the devastating impact of mosquito-borne illnesses on human health, surprisingly little is known about mosquito developmental biology, including development of the olfactory system, a tissue of vector importance. Analysis of mosquito olfactory developmental genetics has been hindered by a lack of means to target specific genes during the development of this sensory system. In this investigation, chitosan/siRNA nanoparticles were used to target semaphorin-1a (sema1a) during olfactory system development in the dengue and yellow fever vector mosquito Aedes aegypti. Immunohistochemical analyses and anterograde tracing of antennal sensory neurons, which were used to track the progression of olfactory development in this species, revealed antennal lobe defects in sema1a knockdown fourth instar larvae. These findings, which correlated with a larval odorant tracking behavioral phenotype, identified previously unreported roles for Sema1a in the developing insect larval olfactory system. Analysis of sema1a knockdown pupae also revealed a number of olfactory phenotypes, including olfactory receptor neuron targeting and projection neuron defects coincident with a collapse in the structure and shape of the antennal lobe and individual glomeruli. This study, which is to our knowledge the first functional genetic analysis of insect olfactory development outside of D. melanogaster, identified critical roles for Sema1a during Ae. aegypti larval and pupal olfactory development and advocates the use of chitosan/siRNA nanoparticles as an effective means of targeting genes during post-embryonic Ae. aegypti development. Use of siRNA nanoparticle methodology to understand sensory developmental genetics in mosquitoes will provide insight into the evolutionary conservation and divergence of key developmental genes which could be exploited in the development of both common and species-specific means for intervention.

SHORT ABSTRACT Here we describe a procedure for inhibiting gene function in disease vector mosquitoes through the use of chitosan/interfering RNA nanoparticles that are ingested by larvae. LONG ABSTRACT Vector mosquitoes inflict more human suffering than any other organism—and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae and A. aegypti larvae. Following larval feedings, knockdown, which is verified through qRT-PCR or in situ hybridization, can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field.

Bone morphogenic protein-4 (BMP4) and fibroblast growth factor-8 (FGF8) are thought to have opposite roles in defining epithelial versus neurogenic fate in the developing olfactory/vomeronasal system. In particular, FGF8 has been implicated in specification of olfactory and gonadotropin releasing hormone-1 (GnRH) neurons, as well as in controlling olfactory stem cell survival. Using different knock-in mouse lines and Cre-lox-mediated lineage tracing, Fgf8 expression and cell lineage was analyzed in the developing nose in relation to the expression of Bmp4 and its antagonist Noggin (Nog). FGF8 is expressed by cells that acquire an epidermal, respiratory cell fate and not by stem cells that acquire neuronal olfactory or vomeronasal cell fate. Ectodermal and mesenchymal sources of BMP4 control the expression of BMP/TGF␤ antagonist Nog, whereas mesenchymal sources of Nog define the neurogenic borders of the olfactory pit. Fgf8 hypomorph mouse models, Fgf8 neo/neo and Fgf8 neo/null , displayed severe craniofacial defects together with overlapping defects in the olfactory pit including (1) lack of neuronal formation ventrally, where GnRH neurons normally form, and (2) altered expression of Bmp4 and Nog, with Nog ectopically expressed in the nasal mesenchyme and no longer defining the GnRH and vomeronasal neurogenic border. Together our data show that (1) FGF8 is not sufficient to induce ectodermal progenitors of the olfactory pit to acquire neural fate and (2) altered neurogenesis and lack of GnRH neuron specification after chronically reduced Fgf8 expression reflected dysgenesis of the nasal region and loss of a specific neurogenic permissive milieu that was defined by mesenchymal signals.

Genome sequencing projects have presented the opportunity for analysis of developmental genes in three vector mosquito species: Aedes aegypti, Culex quinquefasciatus, and Anopheles gambiae. A comparative genomic analysis of developmental genes in Drosophila melanogaster and these three important vectors of human disease was performed in this investigation. While the study was comprehensive, special emphasis centered on genes that 1) are components of developmental signaling pathways, 2) regulate fundamental developmental processes, 3) are critical for the development of tissues of vector importance, 4) function in developmental processes known to have diverged within insects, and 5) encode microRNAs (miRNAs) that regulate developmental transcripts in Drosophila. While most fruit fly developmental genes are conserved in the three vector mosquito species, several genes known to be critical for Drosophila development were not identified in one or more mosquito genomes. In other cases, mosquito lineage-specific gene gains with respect to D. melanogaster were noted. Sequence analyses also revealed that numerous repetitive sequences are a common structural feature of Drosophila and mosquito developmental genes. Finally, analysis of predicted miRNA binding sites in fruit fly and mosquito developmental genes suggests that the repertoire of developmental genes targeted by miRNAs is species-specific. The results of this study provide insight into the evolution of developmental genes and processes in dipterans and other arthropods, serve as a resource for those pursuing analysis of mosquito development, and will promote the design and refinement of functional analysis experiments.

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.