Phylogenomics of the ecdysteroid kinase-like (EcKL) gene family in insects highlights roles in both steroid hormone metabolism and detoxification

Abstract

The evolutionary dynamics of large gene families can offer important insights into the functions of their individual members. While the ecdysteroid kinase-like (EcKL) gene family has previously been linked to the metabolism of both steroid moulting hormones and xenobiotic toxins, the functions of nearly all EcKL genes are unknown and there is little information on their evolution across all insects. Here, we perform comprehensive phylogenetic analyses on a manually annotated set of EcKL genes from 140 insect genomes, revealing the gene family is comprised of at least 13 subfamilies that differ in retention and stability. Our results show the only two genes known to encode ecdysteroid kinases belong to different subfamilies and therefore ecdysteroid metabolism functions must be spread throughout the EcKL family. We also provide comparative phylogenomic evidence that EcKLs are involved in detoxification across insects, with positive associations between family size and dietary chemical complexity, and also find similar evidence for the cytochrome P450 and glutathione S-transferase gene families. Unexpectedly, we find that the size of the clade containing a known ecdysteroid kinase is positively associated with host plant taxonomic diversity in Lepidoptera, possibly suggesting multiple functional shifts between hormone and xenobiotic metabolism. This work provides a robust framework for future functional studies into the EcKL gene family and opens promising new avenues for exploring the genomic basis of dietary adaptation in insects, including the classically studied co-evolution of butterflies with their host plants.

Genetic characterisation of candidate ecdysteroid kinases inDrosophila melanogaster

“…The UAS- CG5644 line was further crossed to the phm-GAL4 driver at 25 °C, resulting in 66 misexpression adults surviving compared to 115 balancer adults (proportion = 0.365, 95% CI: 0.295– 0.439, p = 0.0003, binomial test), suggesting PG misexpression of CG5644 can also disrupt development. These data are consistent with the hypothesis that CG5644 encodes an ecdysteroid 22-kinase, previously proposed based on 1:1 orthology to AgEcK2 in Anopheles gambiae (Peng et al 2022; Scanlan & Robin 2023). The lack of developmental arrest seen in tub>CG31300 and tub>JhI-26 individuals is consistent with previously published experimental data (Liu et al 2014; Scanlan et al 2022).…”

“…It is possible Wall and/or pkm null mutants have subtle or environmentally specific phenotypes we were unable to detect in our experiments, although the lack of expression of either gene in reproductive tissues (Leader et al 2018) makes this unlikely for reproductive functions. The lack of strict conservation of the EcKL clades containing pkm and Wall (referred to as Dip9 and Dip10 in Scanlan & Robin 2023) is consistent with non-essential functions of these genes in Drosophila and other dipteran taxa.…”

“…However, data from our misexpression experiments is consistent with CG5644 encoding an EcK, especially the developmental arrest seen upon PG-specific misexpression of this gene. Our previous phylogenetic analyses of the EcKL gene family determined that CG5644 is a 1:1 ortholog of AgEcK2 , which encodes an ecdysteroid 22-kinase in A. gambiae (Peng et al 2022; Scanlan & Robin 2023). While CG5644 is predominantly expressed in the adult nervous system (Leader et al 2018), it also appears expressed in the migratory border cells in the ovary (Borghese et al 2006), which might make it responsible for the presence of E 22-phosphate in this tissue (Pis et al 1995; Grau et al 1995).…”

“…We recently performed a comprehensive phylogenomic analysis of the EcKL family in 140 insect genomes, classifying them into 13 subfamilies. BmEc22K and AgEcK2 belong to the A and D subfamilies, respectively, making other genes in these clades strong a priori candidates for encoding EcKs (Scanlan & Robin 2023). D. melanogaster contains two subfamily A genes: CG13813 —which we name Wallflower ( Wall ) in this study—and Pinkman ( pkm ), previously named CG1561 .…”

“…D. melanogaster contains two subfamily A genes: CG13813 —which we name Wallflower ( Wall ) in this study—and Pinkman ( pkm ), previously named CG1561 . Both genes have been lost 2–3 times in Diptera but are otherwise conserved as single-copy orthologs in most species (Scanlan & Robin 2023), and they are more closely related to each other than they are to any lepidopteran subfamily A EcKL (Figure S1).…”