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Myosin Myo20 plays vital roles in the morphogenesis of wings and legs among insects, but the function and signalling of Myo20 remain unclear. We show that Myo20 regulates wing cell division, ecdysteroid and amino acid metabolism, and gene expression in Tribolium castaneum. By RNA-seq, we identified 582 differentially expressed genes (DEGs) between control and ds- Myo20 larvae of T. castaneum. Of these DEGs, silencing Myo20 significantly decreased the mRNA and protein levels of lowfat. During development, lowfat has the highest expression in early pupae and the lowest level in 1-day embryos. Tissue-specific analysis indicated that lowfat was abundantly expressed in the head, fat body and epidermis of late-stage larvae and in wings and legs of 1, 2 and 5-day pupae. Likewise, knockdown of lowfat affected wing and leg morphogenesis, ecdysteroid and amino acid metabolism, and gene expression in T. castaneum. Silencing Myo20 or lowfat activated CYP18A1 to degrade ecdysteroids, stimulated amino acids catabolism to increase the transcription of 4E-BP but reduce S6K and cycE expression. These results suggest that Lowfat works downstream of Myo20 to employ target of rapamycin (TOR) signalling for wing and leg morphogenesis in insects. [Display omitted] • Myo20 regulates wing cell proliferation, ecdysteroid and amino acid metabolism, and gene expression. • Lowfat functions downstream of Myo20. • Myo20 and lowfat work upstream of TOR signalling to regulate wing morphogenesis. [ABSTRACT FROM AUTHOR]