BACKGROUND In holometabolous insects, the major developmental transitions – larval molting and pupation – are triggered by a pulse of 20‐hydroxyecdysone (20E) and coordinated by juvenile hormone. Methoxyfenozide (MF), an ecdysteroid agonist, represents a new class of insect growth regulators and is effective against lepidopteran pests. Fushi‐tarazu factor 1 (FTZ‐F1) is an ecdysone‐inducible transcription factor. To date, the effect of MF on 20E‐response genes remains unclear, and we speculate the involvement of FTZ‐F1 in MF's growth regulating effect. RESULTS MF at LC25 and LC10 caused severe ecdysis failure in Helicoverpa armigera, extended their larval duration, lowered their pupal weight, and reduced the respiratory, pupation and emergence rates. Furthermore, sublethal doses of MF inhibited ecdysteroidogenesis and lowered the intrinsic 20E titer, but showed an inductive effect on 20E‐response genes including HaFTZ‐F1. HaFTZ‐F1, predominantly expressed in larval epidermis, was markedly upregulated before or right after larval ecdysis, and maintained a high level in prepupal stage. Knockdown of HaFTZ‐F1 in 4th‐instar larvae severely impaired larval ecdysis, whereas its knockdown in final‐instar larvae caused abnormal pupation. Moreover, knocking down HaFTZ‐F1 downregulated three critical ecdysteroidogenesis genes, lowered 20E titer, and suppressed the expression of 20E receptors and 20E‐response genes. The introduction of 20E into HaFTZ‐F1‐RNAi larvae partly relieved the negative effects on the 20E‐induced signaling cascade. CONCLUSION Our findings reveal the adverse effects of sublethal doses of MF on the development of H. armigera and elucidate the resulting perturbations on the 20E‐induced signaling cascade; we propose that HaFTZ‐F1 regulates ecdysis and pupation by mediating 20E titer and its signaling pathway. © 2020 Society of Chemical Industry

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The brassica leaf beetle Phaedon brassicae is a notorious defoliator of cruciferous vegetables. However, few molecular studies of this pest have been conducted due to limited sequence data. Recently, RNA sequencing has offered a powerful platform to generate numerous transcriptomic data, which require RT-qPCR to validate target gene expression. The selection of reliable reference genes to normalize RT-qPCR data is a prerequisite for gene expression analysis. In the present study, the expression stabilities of eight candidate reference genes under biotic conditions (development stages and various tissues) and abiotic perturbations (thermal stress and pesticide exposure) were evaluated using four different statistical algorithms. The optimal suites of reference genes were recommended for the respective experimental conditions. For tissue expression analysis, RPL32 and EF-1α were recommended as the suitable reference genes. RPL19 and TBP were the optimal reference genes across different developmental stages. RPL32 and TBP were identified as the most suitable references for thermal stress. Furthermore, RPL32 and RPL19 were ranked as the best references for insecticide exposure. This work provides a systematic exploration of the optimal reference genes for the respective experimental conditions, and our findings would facilitate molecular studies of P. brassicae.

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