Mosquitoes are vectors of parasitic and viral diseases of immense importance for public health. The acquisition of the genome sequence of the yellow fever and Dengue vector, Aedes aegypti ( Aa ), has enabled a comparative phylogenomic analysis of the insect immune repertoire: in Aa , the malaria vector Anopheles gambiae ( Ag ), and the fruit fly Drosophila melanogaster ( Dm ). Analysis of immune signaling pathways and response modules reveals both conservative and rapidly evolving features associated with different functional gene categories and particular aspects of immune reactions. These dynamics reflect in part continuous readjustment between accommodation and rejection of pathogens and suggest how innate immunity may have evolved.

Closing the Vector Circle: The genome sequence of Culex quinquefasciatus offers a representative of the third major genus of mosquito disease vectors for comparative analysis. In a major international effort, Arensburger et al. (p. 86 ) uncovered divergences in the C. quinquefasciatus genome compared with the representatives of the other two genera Aedes aegypti and Anopheles gambiae . The main difference noted is the expansion of numbers of genes, particularly for immunity, oxidoreductive functions, and digestive enzymes, which may reflect specific aspects of the Culex life cycle. Bartholomay et al. (p. 88 ) explored infection-response genes in Culex in more depth and uncovered 500 immune response-related genes, similar to the numbers seen in Aedes , but fewer than seen in Anopheles or the fruit fly Drosophila melanogaster . The higher numbers of genes were attributed partly to expansions in those encoding serpins, C-type lectins, and fibrinogen-related proteins, consistent with greater immune surveillance and associated signaling needed to monitor the dangers of breeding in polluted, urbanized environments. Transcriptome analysis confirmed that inoculation with unfamiliar bacteria prompted strong immune responses in Culex . The worm and virus pathogens that the mosquitoes transmit naturally provoked little immune activation, however, suggesting that tolerance has evolved to any damage caused by replication of the pathogens in the insects.

Juvenile hormone (JH) governs a great diversity of processes in insect development and reproduction. It plays a critical role in controlling the gonadotrophic cycles of female mosquitoes by preparing tissues for blood digestion and egg development. Here, we show that in femaleAedes aegyptimosquitoes JH III control of gene expression is mediated by a heterodimer of two bHLH-PAS proteins—the JH receptor methoprene-tolerant (MET) and Cycle (CYC, AAEL002049). We identifiedAedesCYC as a MET-interacting protein using yeast two-hybrid screening. Binding of CYC and MET required the presence of JH III. In newly eclosed female mosquitoes, the expression of two JH-responsive genes,Kr-h1andHairy, was dependent on both the ratio of light to dark periods and JH III. Their expression was compromised by in vivo RNA interference (RNAi) depletions ofCYC,MET, and the steroid receptor coactivatorSRC/FISC. Moreover, JH III was not effective in induction ofKr-h1andHairygene expression in vitro in fat bodies of female mosquitoes with RNAi-depletedCYC,METorSRC/FISC.A sequence containing an E-box–like motif from theAedes Kr-h1gene promoter specifically interacted with a protein complex, which included MET and CYC from the female mosquito fat body nuclear extract. These results indicate that a MET/CYC heterodimer mediates JH III activation ofKr-h1andHairygenes in the context of light-dependent circadian regulation in female mosquitoes during posteclosion development. This study provides an important insight into the understanding of the molecular basis of JH action.

Significance: Mosquitoes transmit some of the most dangerous human diseases. Microarray analysis of developmental gene dynamics in the femaleAedes aegyptifat body has demonstrated that 6,146 genes are differentially expressed during juvenile hormone-dependent posteclosion development. These genes show striking temporal and functional separation. A RNAi microarray screen revealed a differential action of Met in the down- and up-regulation of genes expressed during early and late PE, respectively. Thus, this study provides substantial evidence of a central role for insect-specific JH and its receptor Met in the regulation of female mosquito reproductive biology.

To overcome burden of mosquito-borne diseases, multiple control strategies are needed. Population replacement with genetically modified mosquitoes carrying antipathogen effector genes is one of the possible approaches for controlling disease transmission. However, transgenic mosquitoes with antipathogen phenotypes based on overexpression of a single type effector molecule are not efficient in interrupting pathogen transmission. Here, we show that co-overexpression of two antimicrobial peptides (AMP), Cecropin A, and Defensin A, in transgenic Aedes aegypti mosquitoes results in the cooperative antibacterial and anti Plasmodium action of these AMPs. The transgenic hybrid mosquitoes that overexpressed both Cecropin A and Defensin A under the control of the vitellogenin promoter exhibited an elevated resistance to Pseudomonas aeruginosa infection, indicating that these AMPs acted cooperatively against this pathogenic bacterium. In these mosquitoes infected with P. gallinaceum , the number of oocysts was dramatically reduced in midguts, and no sporozoites were found in their salivary glands when the mosquitoes were fed twice to reactivate transgenic AMP production. Infection experiments using the transgenic hybrid mosquitoes, followed by sequential feeding on naive chicken, and then naive wild-type mosquitoes showed that the Plasmodium transmission was completely blocked. This study suggests an approach in generating transgenic mosquitoes with anti Plasmodium refractory phenotype, which is coexpression of two or more effector molecules with cooperative action on the parasite.