A) Enriched gene pathways and differentially expressed genes (DEGs) of w1118 male and female flies during larvae-pupae transition; B) Differentially expressed microRNAs of w1118 male and female flies in the larvae-pupae transition; C) Screening of potential microRNA candidates that can regulate sesquiterpenoid pathway gene candidates picked up by genome-wide in silico prediction; D) MicroRNA pull-down assay showing interactions of miR-277-3p and Kr-h1 and AstC-R1 transcripts in Drosophila S2 cells and 3rd instar larvae in vivo; E-F) Expression profiles of miR-277-3p, miR-277-5p, miR-34-3p and miR-34-5p read counts in fly embryos, larval and pupal stages; G-H) Expression level of Kr-h1 and AstC-R1 at larval and pupal stages and pupae stages.

A) Mortality of heterozygous miR-277 and miR-34 double knockout and homozygous miR-277 and miR-34 double knockout flies after eclosion within 5 days; B) Kr-h1 and C) AstC-R1 relative expression level in larvae male (LM), larvae female (LF), pupae male (PM) and pupae female (PF) of w1118, miR-277-KO, and miR-34-KO flies; D) i. JHB3, ii. JH-III, iii. FA and iv. 20E hormone titre of w1118, miR-277-KO and miR-34-KO for LM, LF, PM and PF; E) Differentially expressed genes showing up-(green) or down-regulation (red) in the sesquiterpenoid hormone biosynthesis pathway in w1118, miR-277-KO, and miR-34-KO flies; G) Differentially expressed genes showing up-(green) or down-regulation (red) in the ecdysteroid hormone biosynthesis pathway in w1118, miR-277-KO, and miR-34-KO flies.

A) Expression level of transcripts in the miR-277/miR-34 locus retrieved from Flybase (version FB2023_0); B) Enriched gene pathways in miR-277-KO and miR-34-KO flies when comparing to w1118 at different stages in male and female; C) Differentially expressed genes showing up-(green) or down-regulation (red) in dorsal-ventral axis formation pathway in miR-277-KO and miR-34-KO in males and females at larvae and pupae stages. C) Genes showing downregulation in the FoxO signalling, MAPK signalling, Hippo signalling, and dorsal-ventral axis formation pathways in miR-277-KO and miR-34-KO in males and females at larvae and pupae stages.

A) Enriched gene pathways in males and females of miR-277-KO and miR-34-KO that are unique to one sex during larvae-pupae transition; B) Schematic summary of gene pathways enriched with differentially expressed genes during larvae-pupae transition.