Evolutionary Diversification and Functional Characterization of Acyl-CoA Thioesterase 9 in Diptera and the Black Soldier Fly (Hermetia illucens)

Acyl-CoA thioesterase 9 (ACOT9), a member of the type II hot-dog fold thioesterase superfamily, play important roles in regulating the balance between intracellular acyl-CoA and free fatty acids. Although its functions have been extensively studied in vertebrates, its evolutionary diversification and physiological roles in Diptera, particularly in the economically important insect black soldier fly (Hermetia illucens), remain largely unclear. In this study, ACOT9 homologs were identified from 93 representative dipteran species, and H. illucens was used as a model to investigate phylogenetic re-lationships, synteny, domain architecture, expression profiles, and RNA interference (RNAi)-based functional analysis. The results showed that ACOT9 underwent lineage-specific duplications in the suborder Brachycera, giving rise to four paralogs (ACOT9.1–9.4), accompanied by lineage-specific gene losses. Synteny and conserved domain analyses suggested potential functional divergence among these paralogs. In contrast, the ancestral single-copy ACOT9 retained a catalytically con-served domain (e.g., d1lo7a) shared between vertebrates and insects. We cloned the full-length open reading frame (ORF) of H. illucens ACOT9. Transcript abundance peaked on day 12 of larval devel-opment (pre-pupation), with higher expression in males than in females, and predominant localiza-tion in the Malpighian tubules. RNAi-mediated knockdown in 6- to 7-day-old larvae significantly altered fatty acid composition: total saturated fatty acids increased by 13.35%, while monounsatu-rated and polyunsaturated fatty acids decreased by 5.39% and 14.27%, respectively, with relative abundances of lauric acid and linoleic acid changing by more than 10%. Transcriptomic analysis revealed that ACOT9 knockdown significantly upregulated genes involved in de novofatty acid synthesis (FAS1/2/3, ACC, MAT, KAS, ACP) and acyl-CoA activation (ACSM1/2, ACSL4/6), down-regulated the lipolysis gene LIP, and induced expression of the mitochondrial β-oxidation trans-porter genes CPT1/2. These findings indicate that ACOT9 deficiency triggers compensatory lipid synthesis and β-oxidation activation, ultimately leading to saturated fatty acid accumulation. In summary, this study elucidates the evolutionary diversification of ACOT9 in Diptera and demon-strates its pivotal role in lipid homeostasis in H. illucens, providing important insights into insect thioesterase function and endogenous lipid metabolic networks.