Development of a functional genetic tool for Anopheles gambiae oenocyte characterisation: application to cuticular hydrocarbon synthesis

Oenocytes are an insect cell type having diverse physiological functions ranging from cuticular hydrocarbon (CHC) production to insecticide detoxification that may impact their capacity to transmit pathogens. To develop functional genetic tools to study Anopheles gambiae oenocytes, we have trapped an oenocyte enhancer to create a transgenic mosquito Gal4 driver line that mediates tissue-specific expression. After crossing with UAS-reporter lines, An. gambiae oenocytes are fluorescently tagged through all life stages and demonstrate clearly the two characteristic oenocyte cell-types arising during development. The driver was then used to characterise the function of two oenocyte expressed An. gambiae cyp4g genes through tissue-specific expression of UAS-RNAi constructs. Silencing of cyp4g16 or cyp4g17 caused lethality in pupae of differing timing and penetrance. Surviving cyp4g16 knockdown adults showed increased sensitivity to desiccation. Total cuticular hydrocarbon levels were reduced by approximately 80% or 50% in both single gene knockdowns when assayed in young pupa or surviving adults respectively, indicating both genes are required for complete CHC production in An. gambiae and demonstrate synergistic activity in young pupae. Comparative CHC profiles were very similar for the two knockdowns, indicating overlapping substrate specificities of the two enzymes. Differences were observed for example with reduced abundance of shorter chain CHCs in CYP4G16 knockdowns, and reduction in longer, branched chained CHCs in CYP4G17 knockdown adults. This is the first time that two cyp4gs have both been shown to be required for complete CHC production in an insect. Moreover, the generation of tagged cells and identification of an enhancer region can expediate oenocyte specific transcriptomics. The novel driver line can also be used to explore oenocyte roles in pheromone production, mating behaviour and longevity in the malaria mosquito.