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

Abstract

27Oenocytes are an insect cell type having diverse physiological functions ranging 28 from cuticular hydrocarbon (CHC) production to insecticide detoxification that may 29 impact their capacity to transmit pathogens. To develop functional genetic tools to 30 study Anopheles gambiae oenocytes, we have trapped an oenocyte enhancer to 31 create a transgenic mosquito Gal4 driver line that mediates tissue-specific 32 expression. After crossing with UAS-reporter lines, An. gambiae oenocytes are 33 fluorescently tagged through all life stages and demonstrate clearly the two 34 characteristic oenocyte cell-types arising during development. The driver was then 35 used to characterise the function of two oenocyte expressed An. gambiae cyp4g 36 genes through tissue-specific expression of UAS-RNAi constructs. Silencing of 37 cyp4g16 or cyp4g17 caused lethality in pupae of differing timing and penetrance. 38 Surviving cyp4g16 knockdown adults showed increased sensitivity to desiccation. 39 Total cuticular hydrocarbon levels were reduced by approximately 80% or 50% in 40 both single gene knockdowns when assayed in young pupa or surviving adults 41 respectively, indicating both genes are required for complete CHC production in An. 42 gambiae and demonstrate synergistic activity in young pupae. Comparative CHC 43 profiles were very similar for the two knockdowns, indicating overlapping substrate 44 specificities of the two enzymes. Differences were observed for example with 45 reduced abundance of shorter chain CHCs in CYP4G16 knockdowns, and reduction 46 in longer, branched chained CHCs in CYP4G17 knockdown adults. This is the first 47 time that two cyp4gs have both been shown to be required for complete CHC 48 production in an insect. Moreover, the generation of tagged cells and identification of 49 an enhancer region can expediate oenocyte specific transcriptomics. The novel 50 4 driver line can also be used to explore oenocyte roles in pheromone production, 51 mating behaviour and longevity in the malaria mosquito. 52 53 54

Isolation and transcriptomic analysis of Anopheles gambiae oenocytes enables the delineation of hydrocarbon biosynthesis

“…gambiae oenocytes, we expressed the red fluorescent marker m-cherry specifically in these cells using the GAL4/UAS system ( Lynd and Lycett, 2012 ). Two transgenic lines were crossed: 1) a homozygous UAS-mCD8: mCherry responder line ( Adolfi et al, 2018 ) with 2) a homozygous oenocyte enhancer-GAL4 driver line (Oeno-Gal4) ( Lynd et al, 2019 ). Progeny of this cross had the expected m-cherry fluorescent oenocytes throughout their development ( Lynd et al, 2019 ).…”

“…Two transgenic lines were crossed: 1) a homozygous UAS-mCD8: mCherry responder line ( Adolfi et al, 2018 ) with 2) a homozygous oenocyte enhancer-GAL4 driver line (Oeno-Gal4) ( Lynd et al, 2019 ). Progeny of this cross had the expected m-cherry fluorescent oenocytes throughout their development ( Lynd et al, 2019 ). To purify adult oenocytes, mosquitoes were dissected to expose the oenocytes that are dispersed throughout tissues attached to the ventral abdominal integument.…”

“…Here we report the isolation of oenocytes from adult An. gambiae mosquitoes using a transgenic line with fluorescently tagged oenocytes ( Lynd et al, 2019 ). RNAseq of the isolated oenocytes identified the key biological processes enriched in these cells and revealed candidate genes for each step of the CHC biosynthetic pathway.…”

Hemocyte-targeted gene expression in the female malaria mosquito using thehemolectinpromoter fromDrosophila

“…In An. gambiae, functional Gal4 lines have been established to drive transgene expression specifically in the midgut, in oenocytes or ubiquitously (Lynd and Lycett 2012, Adolfi, Pondeville et al 2018, Lynd, Balabanidou et al 2019. While hemocytes are important immune effectors, to date no hemocyte-specific Gal4 line has been established in mosquitoes.…”

Cuticular hydrocarbon biosynthesis in malaria vectors: insights from the adult oenocyte transcriptome

“…gambiae oenocytes, we expressed the red fluorescent marker m-cherry 84 specifically in these cells using the GAL4/UAS system (Lynd and Lycett, 2012). Two transgenic 85 lines were crossed: 1) a homozygous UAS-mCD8: mCherry responder line (Adolfi et al, 2018) 86 with 2) a homozygous oenocyte enhancer-GAL4 driver line (Oeno-Gal4) (Lynd et al, 2019). Figure 1C).…”