Identification of FOXO targets that generate diverse features of the diapause phenotype in the mosquito Culex pipiens

Significance Diapause is an alternative developmental pathway exploited by insects and other invertebrates to survive inimical seasons. Like many insects that enter an adult diapause, the mosquito Culex pipiens responds to the short day lengths of autumn by feeding extensively on sugar, stockpiling huge fat reserves, halting reproduction, suppressing metabolism, boosting defense responses, and migrating to protected sites where it can safely bridge the winter months. In the experiments presented here, we propose that many of the diverse features of the diapause phenotype are the consequence of activating forkhead transcription factor (FOXO), a transcription factor downstream of insulin and juvenile hormone signaling. Our experimental results reveal how these upstream hormonal signaling pathways act through FOXO to generate the complex phenotype known as diapause. Insulin and juvenile hormone signaling direct entry of the mosquito Culex pipiens into its overwintering adult diapause, and these two critical signaling pathways appear to do so by converging on the regulation of forkhead transcription factor (FOXO). Diapause is a complex phenotype, and FOXO emerges as a prime candidate for activating many of the diverse physiological pathways that generate the diapause phenotype. Here, we used ChIP sequencing to identify direct targets of FOXO. The nearest gene in a 10-kb region surrounding a predicted binding site was extracted for each binding site, resulting in a dataset containing genes potentially regulated by FOXO. By selecting candidate genes based on their functional relevance to diapause, we identified five gene categories of potential interest, including stress tolerance, metabolic pathways, lifespan extension, cell cycle and growth regulation, and circadian rhythms. Twelve targets were prioritized for further analysis, 10 of which were validated by ChIP-quantitative PCR and quantitative real-time PCR. These 10 genes activated by FOXO are highly up-regulated during diapause and are thus strong candidates for implementation of the diapause syndrome.