Fig S2.docx from MicroRNAs regulate the sesquiterpenoid hormonal pathway in <i>Drosophila</i> and other arthropods

Arthropods comprise the majority of all described animal species, and understanding their evolution is a central question in biology. Their developmental processes are under the precise control of distinct hormonal regulators, including the sesquiterpenoids juvenile hormone (JH) and methyl farnesoate (MF). The control of the synthesis and mode of action of these hormones played important roles in the evolution of arthropods and their adaptation to diverse habitats. However, the precise roles of non-coding RNAs, such as microRNAs, controlling arthropod hormonal pathways are unknown. Here, we investigated the microRNA regulation of the expression of the juvenile hormone acid methyltransferase gene (JHAMT), which encodes a rate-determining sesquiterpenoid biosynthetic enzyme. Loss of function of the microRNA bantam in the fly <i>Drosophila melanogaster</i> increased JHAMT expression, while overexpression of the bantam repressed JHAMT expression and resulted in pupal lethality. The male genital organs of the pupae were malformed, and exogenous sesquiterpenoid application partially rescued the genital deformities. The role of the bantam in the regulation of sesquiterpenoid biosynthesis was validated by transcriptomic, qPCR and hormone titre (JHB3 and JH III) analyses. In addition, we found a conserved set of microRNAs that interacted with JHAMT, and the sesquiterpenoid receptor Methoprene-tolerant (Met) in different arthropod lineages, including insects (fly, mosquito and beetle), crustaceans (water flea and shrimp), myriapod (centipede) and chelicerate (horseshoe crab). This suggests that these microRNAs might have conserved roles in the post-transcriptional regulation of genes in sesquiterpenoid pathways across the Panarthropoda. Some of the identified lineage-specific microRNAs are potential targets for the development of new strategies in aquaculture and agricultural pest control.