The RNA-Seq approach to studying the expression of mosquito mitochondrial genes.

In this study, we used extensive expressed sequence tag evidence obtained through 454 and Solexa next-generation sequencing to explore mtDNA transcription in male and female first instar larvae of Aedes aegypti and adults of Aedes aegypti, Anopheles gambiae, and Anopheles quadrimaculatus. Relative abundances of individual transcripts differed considerably within each sample, consistent with the differential stability of messenger RNA species. Large differences were also observed between species and between larval and adult stages; however, the male and female larval samples were remarkably similar. Quantitative PCR analysis of selected genes, cox1, l-rRNA and nd5, in larvae and adults of Ae. aegypti and in An. gambiae adults was consistent with the RNA-Seq-based quantification of expression. Finally, the absence of a conserved mtDNA region involved in transcriptional control in other dipterans suggests that mosquitoes have evolved a distinct mechanism of regulation of gene expression in the mitochondrion.

Anopheles gambiae P450 reductase is highly expressed in oenocytes and in vivo knockdown increases permethrin susceptibility.

We describe an in vivo model for investigation of detoxification mechanisms of the mosquito Anopheles gambiae, important for the development of malaria control programmes. Cytochrome P450s are involved in metabolic insecticide resistance and require NADPH cytochrome P450 reductase (CPR) to function. Here we demonstrate that the major sites of adult mosquito CPR expression are oenocytes, mid-gut epithelia and head appendages. High CPR expression was also evident in Drosophila oenocytes indicating a general functional role in these insect cells. RNAi mediated knockdown drastically reduced CPR expression in oenocytes, and to a lesser extent in mid-gut epithelia; the head was unaffected. These flies showed enhanced sensitivity to permethrin, demonstrating a key role for abdominal/mid-gut P450s in pyrethroid metabolism, aiding the development of insecticides.

Genetic manipulation of insect vectors as a strategy for the control of vector-borne disease.

A variety of very effective methods have been employed for suppressing insect vector populations, including the application of biological control agents and the elimination of breeding sites, with a continuing and heavy reliance on the use of chemical insecticides. However, the development of insecticide resistance by vector insects, the cost of developing and registering new insecticidal compounds, and the increase in legislation to combat the detrimental effects of insecticidal residues on the environment, have emphasized the need to assess alternative strategies for vector control. What is required is a completely novel approach to either suppress vector populations, or to alter their ability to transmit disease-causing organisms in such a way as to have a profound and long-lasting effect on disease transmission. Genetic manipulation of insect vectors may provide just such an approach. The major requirements for genome manipulation in insects and the progress which has been made to create transgenic vector insects are reviewed. The potential applications of this methodology are then explored in the context of its future use for the control of vector-borne diseases.

Stable transformation of mosquito cell lines using a hsp70::neo fusion gene.

Mosquito cell culture transfection will allow the advancement of genetic studies of these important disease-transmitting insects. Towards this end, we report the generation of stably transformed Aedes aegypti Mos20 cells using a plasmid construct containing the Tn5 neo gene, the Drosophila melanogaster hsp70 promoter, an SV40 intron and poly adenylation sequence, and a pBR 322 backbone. The apparent frequency of transfection, as measured by transient resistance of cell colonies to Geneticin (G418), ranged between 1 x 10(-4) and 1 x 10(-5), whereas the mean frequency of transformation, as assessed by establishment of cloned lines, was 3.3 x 10(-6). The stable cell lines display typical characteristics common to mammalian cell lines transformed with plasmids, including stable resistance to G418 after removal of selection, and co-transformation with unlinked plasmids. However, in contrast to the report of transformation of Ae. albopictus cells [Monroe et al., Proc. Natl. Acad. Sci. USA 89 (1992) 5725-5729], the plasmids within transformed Ae. aegypti cells have a wide range of copy number (3 to 5000), are extensively rearranged, and are only found integrated into the chromosome.