RNA interference‐mediated knockdown of the Halloween gene Spookiest (CYP307B1) impedes adult eclosion in the western tarnished plant bug, Lygus hesperus

Abstract

Ecdysteroids play a critical role in coordinating insect growth, development and reproduction. A suite of cytochrome P450 monooxygenases coded by what are collectively termed Halloween genes mediate ecdysteroid biosynthesis. In this study, we describe cloning and RNA interference (RNAi)-mediated knockdown of the CYP307B1 Halloween gene (Spookiest) in the western tarnished plant bug, Lygus hesperus. Transcripts for Ly. hesperus Spookiest (LhSpot) were amplified from all life stages and correlated well with timing of the pre-moult ecdysteroid pulse. In adults, LhSpot was amplified from heads of both genders as well as female reproductive tissues. Heterologous expression of a LhSpot fluorescent chimera in cultured insect cells co-localized with a fluorescent marker of the endoplasmic reticulum/secretory pathway. RNAi-mediated knockdown of LhSpot in fifth instars reduced expression of ecdysone-responsive genes E74 and E75, and prevented adult development. This developmental defect was rescued following application of exogenous 20-hydroxyecdysone but not exogenous 7-dehydrocholesterol. The unequivocal RNAi effects on Ly. hesperus development and the phenotypic rescue by 20-hydroxyecdysone are causal proof of the involvement of LhSpot in ecdysteroid biosynthesis and related developmental processes, and may provide an avenue for development of new control measures against Ly. hesperus.

Characteristics of Halloween genes and RNA interference‐mediated functional analysis of LmCYP307a2 in Locusta migratoria

“…Apart from the research on DmCYP307a2 , the functional characteristics of CYP307a2 and CYP307b1 in ecdysteroidogenesis have only been confirmed in very few insect species (Feyereisen, 2005). Silencing of LhCYP307b1 prevented adult eclosion and increased nymphal mortality in L. hesperus , which could be rescued by the application of exogenous 20E, but not 7‐dehydrocholesterol (van Ekert et al ., 2016). Dietary introduction of ds LsCYP307a2 successfully knocked down the target gene in Laodelphax striatellus and caused nymphal lethality and delayed development, which could be almost completely rescued by ingestion of 20E (Jia et al ., 2015).…”

“…Most insect genomes contain at least one CYP307a1 , while Drosophilidae species carry one of the paralogs, CYP307a2 . The other paralog, CYP307b1 , has been identified in Pediculus humanus , Tribolium castaneum , and various species of plant hopper, mosquito, and honey bee (van Ekert et al ., 2016).…”

Molecular characterization and functional analysis of the vitellogenin receptor in the rice stem borer, Chilo suppressalis

“…The enhanced green fluorescent protein (EGFP) gene (GenBank accession no. : KC896843) was used as a negative control for the nonspecific effects in the RNAi experiments (Ekert, Wang, Miao, Brent, & Hull, ). The final dsRNA products were diluted with diethylpyrocarbonate (DEPC)‐treated water at the concentration of 3 μg/μl.…”

RNA interference‐mediated knockdown of eye coloration genes in the western tarnished plant bug (Lygus hesperus Knight)

“…Despite being key pests, molecular resources for L. hesperus have only recently been developed (Christie, Hull, Richer, Geib, & Tassone, ; Hull, Geib, Fabrick, & Brent, ; Hull et al, ; Tassone et al, ). While injection‐based RNAi has been successfully demonstrated for this species (Van Ekert, Wang, Miao, Brent, & Hull, ), the ineffectiveness of oral‐based RNAi (Allen & Walker, ) in Lygus limits the overall utility of the method for high throughput functional genomic approaches. Consequently, other techniques, such as the CRISPR/Cas9 system, could prove useful for demonstrating in vivo gene functionality.…”