We have identified 242 Anopheles gambiae genes from 18 gene families implicated in innate immunity and have detected marked diversification relative to Drosophila melanogaster . Immune-related gene families involved in recognition, signal modulation, and effector systems show a marked deficit of orthologs and excessive gene expansions, possibly reflecting selection pressures from different pathogens encountered in these insects' very different life-styles. In contrast, the multifunctional Toll signal transduction pathway is substantially conserved, presumably because of counterselection for developmental stability. Representative expression profiles confirm that sequence diversification is accompanied by specific responses to different immune challenges. Alternative RNA splicing may also contribute to expansion of the immune repertoire.

Female Aedes aegypti mosquitoes infect more than 400 million people each year with dangerous viral pathogens including dengue, yellow fever, Zika and chikungunya. Progress in understanding the biology of mosquitoes and developing the tools to fight them has been slowed by the lack of a high-quality genome assembly. Here we combine diverse technologies to produce the markedly improved, fully re-annotated AaegL5 genome assembly, and demonstrate how it accelerates mosquito science. We anchored physical and cytogenetic maps, doubled the number of known chemosensory ionotropic receptors that guide mosquitoes to human hosts and egg-laying sites, provided further insight into the size and composition of the sex-determining M locus, and revealed copy-number variation among glutathione S-transferase genes that are important for insecticide resistance. Using high-resolution quantitative trait locus and population genomic analyses, we mapped new candidates for dengue vector competence and insecticide resistance. AaegL5 will catalyse new biological insights and intervention strategies to fight this deadly disease vector.

The role of cuticle changes in insecticide resistance in the major malaria vector Anopheles gambiae was assessed. The rate of internalization of ¹⁴C deltamethrin was significantly slower in a resistant strain than in a susceptible strain. Topical application of an acetone insecticide formulation to circumvent lipid-based uptake barriers decreased the resistance ratio by ∼50%. Cuticle analysis by electron microscopy and characterization of lipid extracts indicated that resistant mosquitoes had a thicker epicuticular layer and a significant increase in cuticular hydrocarbon (CHC) content (∼29%). However, the CHC profile and relative distribution were similar in resistant and susceptible insects. The cellular localization and in vitro activity of two P450 enzymes, CYP4G16 and CYP4G17, whose genes are frequently overexpressed in resistant Anopheles mosquitoes, were analyzed. These enzymes are potential orthologs of the CYP4G1/2 enzymes that catalyze the final step of CHC biosynthesis in Drosophila and Musca domestica, respectively. Immunostaining indicated that both CYP4G16 and CYP4G17 are highly abundant in oenocytes, the insect cell type thought to secrete hydrocarbons. However, an intriguing difference was indicated; CYP4G17 occurs throughout the cell, as expected for a microsomal P450, but CYP4G16 localizes to the periphery of the cell and lies on the cytoplasmic side of the cell membrane, a unique position for a P450 enzyme. CYP4G16 and CYP4G17 were functionally expressed in insect cells. CYP4G16 produced hydrocarbons from a C18 aldehyde substrate and thus has bona fide decarbonylase activity similar to that of dmCYP4G1/2. The data support the hypothesis that the coevolution of multiple mechanisms, including cuticular barriers, has occurred in highly pyrethroid-resistant An. gambiae. Significance Malaria incidence has halved since 2000, with 80% of the reduction attributable to the use of insecticides, which now are under threat of resistance. Understanding the mechanisms of insecticide resistance is a key step in delaying and tackling the phenomenon. This study provides evidence of a cuticular mechanism that slows the uptake of pyrethroids, contributing to the resistance phenotype and potentially broadening resistance to multiple insecticide classes, thus providing additional challenges to resistance management. Quantitative modification of cuticular hydrocarbons is associated with increased expression of a 4G cytochrome P450 enzyme, CYP4G16, which catalyzes epicuticular hydrocarbon biosynthesis. This work improves our understanding of insecticide resistance and may facilitate the development of insecticides with greater specificity to mosquitoes and greater potency.

Female-fatal product of the Yob gene: Mechanisms of sex determination are highly variable and complex in invertebrates. For vector-borne disease control strategies, it is important to understand the reproductive biology of the organisms involved. Krzywinska et al. have studied sex determination in species of Anopheles mosquitoes, whose females suck blood and can transmit malaria parasites (see the Perspective by Sinkins). One gene called Yob is found only on the Y chromosome of males. Yob is expressed in male embryos within 2 hours of eggs being laid, and transcription continues lifelong. If Yob is injected into embryos younger than 2 hours, only male mosquitoes emerge, because Yob 's presence is lethal to female embryos. Science , this issue p. 67 ; see also p. 33