Fig. 2

Malathion penetration resistance is mediated by miR-994, which modulates cuticular thickness via CPCFC. A Expression analysis of miR-994 after injecting synthetic miR-994 or an inhibitor (n = 4). B B. dorsalis susceptibility to malathion after injecting synthetic miR-994 or an inhibitor into MR and MS strains, respectively. Survival rate was observed at 0–3 day after malathion exposure (n = 120). C Pronotum cuticular thickness after injecting synthetic miR-994 (n = 10), negative control (NC) miRNA (n = 9) or corresponding inhibitors (n = 10). The synthetic miR-994 and NC were injected into MR flies and the inhibitor into MS flies. Each thickness value is the mean of five randomly selected cross-sectional measurements. D Ultrastructure of the pronotum cuticle following the injection of synthetic miR-994 or an inhibitor. E Malathion penetration rate after injecting synthetic miR-994. Each eluant contained 30 5-day-old adult flies (n = 3). F Enrichment analysis (biotin–avidin RNA pull-down) following the binding of miR-994 to CPCFC (n = 4). (G) Expression of CPCFC after injecting synthetic miR-994 or an inhibitor (n = 4). H Expression profiles of miR-994 and CPCFC in adult flies at 3, 5, 7 and 9 days old (A3, A5, A7 and A9, n = 4). I Target site analysis, showing the interaction between miR-994 and CPCFC mRNA. Mutated CPCFC contained the sequence 5′-AGGAAT-3′ (red). J Luciferase activity indicating the targeting of CPCFC by miR-994 in vitro. CPCFC-wt and CPCFC-mut indicate HEK293-T cells containing the 3′ UTR of wild-type and mutated CPCFC, respectively (n =4)