Knockdown of ecdysis-triggering hormone gene with a binaryUAS/GAL4RNA interference system leads to lethal ecdysis deficiency in silkworm

Abstract

Ecdysis-triggering hormone (ETH) is an integration factor in the ecdysis process of most insects, including Bombyx mori (silkworm). To understand the function of the ETH gene in silkworm, we developed an effective approach to knockdown the expression of ETH in vivo based on RNA interference (RNAi) and a binary UAS/GAL4 expression system that has been successfully used in other insect species. Two kinds of transgenic silkworm were established with this method: the effector strain with the ETH RNAi sequence under the control of UAS and the activator strain with the GAL4 coding sequence under the control of Bombyx mori cytoplasmic actin3. By crossing the two strains, double-positive transgenic silkworm was obtained, and their ETH expression was found to be dramatically lower than that of each single positive transgenic parent. Severe ecdysis deficiency proved lethal to the double-positive transgenic silkworm at the stage of pharate second instar larvae, while the single positive transgenic or wild-type silkworm had normal ecdysis. This UAS/GAL4 RNAi approach provides a way to study the function of endogenous silkworm genes at different development stages.

Advanced technologies for genetically manipulating the silkwormBombyx mori, a model Lepidopteran insect

“…For example, heat-inducible promoters such as Dmhsp70 have been used to control transcription of dsRNA expression-cassettes in B. mori [47]. Likewise, Gal4-mediated expression of UAS-regulated dsRNA expression-cassettes has been used to efficiently regulate gene expression in specific tissue and temporal patterns [48]. Collectively, these results demonstrate that the transgenic RNAi system can serve as a powerful tool when used alone or jointly with other technologies for gene expression analyses that benefit from the creation of hypomorphic or loss-of-function genotypes and phenotypes.…”

Ecdysis triggering hormone peptide in the African malaria mosquito Anopheles gambiae: The peptide structure for receptor activation

“…Additional functions of the ETH have recently been uncovered for the regulation of another crucial endocrine factor, juvenile hormone (JH) in Dipteran insects (Areiza et al ., 2014 ; Nouzova et al ., 2018 ) as well as for the control of reproduction (Areiza et al ., 2014 ; Nouzova et al ., 2018 ; Shi et al ., 2019 ). Most of all, disruption of this signaling system has been demonstrated to be lethal to many species of insects (Park et al ., 2002a ; Dai et al ., 2008 ; Lenaerts et al ., 2017 ; Shi et al ., 2017 ; Shen et al ., 2021 ), offering a potential target system for development of an insecticidal strategy.…”

Host-delivered RNAi mediated silencing of HaAce1, encoding the major isoform of acetylcholinesterase, imparts resistance against the polyphagous insect-pest, Helicoverpa armigera

“…Silencing of the chitin synthase gene by RNAi has been found to disrupt the larval development of Spodoptera exigua (Chen et al 2008). Whereas knockdown of the ecdysis regulatory gene with a binary UAS/GAL4 RNAi system led to lethal ecdysis deficiency in silkworm (Dai et al 2008). Host-delivered hpRNA-mediated resistance to pod borer was few studies reported AChE2 as the primary catalytic enzyme in some other insect species (Kim and Lee 2013).…”