Knockdown of Ecdysone-Induced Protein 93F Causes Abnormal Pupae and Adults in the Eggplant Lady Beetle

SIMPLE SUMMARY: In several hemimetabolous, neometabolous and holometabolous insects, ecdysone-induced protein 93F (E93), a critical member among three key players (Kru ppel-homolog 1, Kr-h1; E93; Broad-complex, BrC) in the regulation of metamorphosis, exerts triple roles during post-embryonic development. It acts as a determinant for the penultimate instar juveniles to attain competence, enabling the final instars to metamorphose, a repressor of juvenile characters and a specifier of adult structures during metamorphosis. However, for Drosophila flies whose larval instars are fixed at three, E93 only serves as an adult specifier. In a polyphagous beetle, Henosepilachna vigintioctopunctata, whose larval instars are fixed to four, we performed RNA interference to determine the roles of HvE93. Our results suggest that HvE93 has dual functions, repressing larval characters and determining adult structures. Our findings provide a missing link in the evolutionary process in terms of the roles of E93. ABSTRACT: Ecdysone-induced protein 93F (E93) plays triple roles during post-embryonic development in insects whose juvenile instars are more than four. However, it only acts as a specifier of adult structures in Drosophila flies whose larval instars are fixed at three. In this study, we determined the functions of E93 in the eggplant lady beetle (Henosepilachna vigintioctopunctata), which has four larval instars. We uncovered that E93 was abundantly expressed at the prepupal and pupal stages. A precocious inhibition of the juvenile hormone signal by RNA interference (RNAi) of HvKr-h1 or HvHairy, two vital downstream developmental effectors, at the penultimate instar larval stage increased the expression of E93, Conversely, ingestion of JH by the third-instar larvae stimulated the expression of HvKr-h1 but repressed the transcription of either HvE93X1 or HvE93X2. However, disturbance of the JH signal neither drove premature metamorphosis nor caused supernumerary instars. In contrast, depletion of E93 at the third- and fourth-instar larval and prepupal stages severely impaired pupation and caused a larval-pupal mixed phenotype: pupal spines and larval scoli were simultaneously presented on the cuticle. RNAi of E93 at the pupal stage affected adult eclosion. When the beetles had suffered from a dsE93 injection at the fourth-instar larval and pupal stages, a few resultant adults emerged, with separated elytra, abnormally folded hindwings, a small body size and short appendages. Taken together, our results suggest the larval instars are fixed in H. vigintioctopunctata; E93 serves as a repressor of larval characters and a specifier of adult structures during the larval–pupal–adult transition.