1 and cytochrome P450 gene families in Drosophila melanogaster by integrating 2 evolutionary and transcriptomic data 3 4Abstract: The capacity to detoxify toxic compounds is essential for adaptation to the 22 ecological niches of many organisms, especially insects. However, detoxification in 23 insects is often viewed through the lens of mammalian detoxification research, even 24 though the organ and enzyme systems involved have diverged for over half a billion 25 years. Phosphorylation is a non-canonical phase II detoxification reaction that, 26 among animals, occurs near exclusively in insects, but the enzymes responsible 27 have never been cloned or otherwise identified. We propose the hypothesis that 28 members of the arthropod-specific ecdysteroid kinase-like (EcKL) gene family 29 encode detoxicative kinases. To test this hypothesis, we annotated the EcKL gene 30 family in 12 species of Drosophila and explored their evolution within the genus. 31 Many ancestral EcKL clades are evolutionarily unstable and have experienced 32 repeated gene gain and loss events, while others are conserved as single copy 33 orthologs. Leveraging multiple published gene expression datasets from D. 34 melanogaster, and using the cytochrome P450s-a canonical detoxification family-35 as a test case, we demonstrate relationships between xenobiotic induction, 36 detoxification tissue-enriched expression and evolutionary instability in the EcKLs 37 and the P450s. We also found previously unreported genomic and transcriptomic 38 variation in a number of EcKLs and P450s associated with toxic stress phenotypes 39 using a targeted phenome-wide association study (PheWAS) approach. Lastly, we 40 devised a systematic method for identifying candidate detoxification genes in large 41 gene families that is concordant with experimentally determined functions of P450 42 genes in D. melanogaster. Applying this method to the EcKLs suggested a 43 significant proportion of these genes play roles in detoxification, and that the EcKLs 44 may constitute a detoxification gene family in insects. Additionally, we estimate that 45 between 11-16 uncharacterised D. melanogaster P450s are strong detoxification 46 candidates. 47 48 Highlights 49 50• The poorly characterised ecdysteroid kinase-like (EcKL) gene family is 51 hypothesised to encode enzymes responsible for detoxification by 52 phosphorylation in insects. 53• An integrative 'detoxification score' method accurately categorises the known 54 functions of a canonical detoxification family, the cytochrome P450s, and 55 suggests many EcKLs are also involved in detoxification. 56• A targeted phenome-wide association study finds novel associations between 57 EcKL/P450 variation and a number of toxic stress phenotypes, such as two 58 unlinked EcKL paralogs that are both associated with developmental 59 methylmercury resistance. 60 61 1983 131 132The identification of detoxification genes and enzymes is an important part of 133 bridging the gap between toxicology, chemical ecology and functional genomics in ...