Requirement of Leptinotarsa decemlineata gene within the 74EF puff for larval-pupal metamorphosis and appendage growth.

Two Drosophila melanogaster E-twenty-six domain transcription factor isoforms (E74A and E74B) act differentially at the start of the 20-hydroxyecdysone (20E) signalling cascade to regulate larval-pupal metamorphosis. In the present paper, we identified the two isoforms (LdE74A and LdE74B) in Leptinotarsa decemlineata. During the larval development stage, the mRNA transcript levels of the two LdE74 isoforms were correlated with circulating 20E titres. In vitro midgut culture and in vivo dietary supplementation with 20E revealed that the presence of 20E induced expression peaks of both LdE74A and LdE74B, with similar patterns observed for the two isoforms. Moreover, the mRNA transcript levels of both LdE74A and LdE74B isoforms were significantly downregulated in the L. decemlineata ecdysone receptor RNA interference (RNAi) specimens, but not in the LdE75 RNAi beetles. Ingestion of 20E reduced the larval fresh weights and shortened the larval development period, irrespective of knockdown of LdE74 or not. RNAi of LdE74 did not affect 20E-induced expression of the Ecdysone induced protein 75-hormone receptor 3-fushi tarazu factor 1 (E75-HR3-FTZ-F1) transcriptional cascade. Thus, it seems that LdE74 mediates 20E signalling independent of the E75-HR3-FTZ-F1 transcriptional cascade. Furthermore, silencing of both LdE74 isoforms caused failure of ecdysis. Most of the LdE74 RNAi beetles remained as prepupae. The LdE74 RNAi prepupae exhibited adult character-like forms underneath after removal of the apolysed larval cuticle. Their appendages such as antennae, legs and wings were shorter than those of control larvae. Only a few LdE74 RNAi larvae finally became deformed pupae, with shortened antennae and legs. Therefore, LdE74 is required for larval-pupal metamorphosis and appendage growth in L. decemlineata.