Genetic characterisation of candidate ecdysteroid kinases inDrosophila melanogaster

Abstract

Ecdysteroids are major hormones in insects and control moulting, growth, reproduction, physiology, and behaviour. The biosynthesis of ecdysteroids such as 20-hydroxyecdysone (20E) from dietary sterols is well characterised, but ecdysteroid catabolism is poorly understood. Ecdysteroid kinases (EcKs) mediate the reversible phosphorylation of ecdysteroids, which has been implicated in ecdysteroid recycling during embryogenesis and reproduction in various insects. However, to date only two EcK-encoding genes have been identified, in the silkwormBombyx moriand the mosquitoAnopheles gambiae. Previously, we identified two ecdysteroid kinase-like (EcKL) genesWallflower(Wall) andPinkman(pkm) in the model fruit flyDrosophila melanogasterthat are orthologs of the ecdysteroid 22-kinase geneBmEc22K. Here, using gene knockdown, knockout and misexpression, we exploreWallandpkm's possible functions and genetically test the hypothesis that they encode EcKs.Wallandpkmnull mutants are viable and fertile, suggesting they are not essential for development or reproduction, whereas phenotypes arising from RNAi and somatic CRISPR appear to derive from off-target effects or other artefacts. However, misexpression ofWallresults in dramatic phenotypes, including developmental arrest, and defects in trachea, cuticle and pigmentation.Wallmisexpression fails to phenocopy irreversible ecdysteroid catabolism through misexpression ofCyp18a1, suggesting Wall does not directly inactivate 20E. Additionally,Wallmisexpression phenotypes are not attenuated inCyp18a1mutants, strongly suggesting Wall is not an ecdysteroid 26-kinase. We hypothesise that the substrate of Wall in this misexpression experiment and possibly generally is an unknown, atypical ecdysteroid that plays essential roles inDrosophiladevelopment, and may highlight aspects of insect endocrinology that are as-yet uncharacterised. We also provide preliminary evidence thatCG5644encodes an ecdysteroid 22-kinase conserved across Diptera.