title: 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.
authors.affiliation: USDA-ARS Arid Land Agricultural Research Center, Maricopa, AZ, USA.
authors.forename: E
authors.initials: E
authors.lastname: Van Ekert
authors.affiliation: Zhejiang University, Hangzhou, China.
authors.forename: M
authors.initials: M
authors.lastname: Wang
authors.affiliation: Zhejiang University, Hangzhou, China.
authors.forename: Y-G
authors.initials: YG
authors.lastname: Miao
authors.affiliation: USDA-ARS Arid Land Agricultural Research Center, Maricopa, AZ, USA.
authors.forename: C S
authors.initials: CS
authors.lastname: Brent
authors.affiliation: USDA-ARS Arid Land Agricultural Research Center, Maricopa, AZ, USA.
authors.forename: J J
authors.initials: JJ
authors.lastname: Hull
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D005260:Female; D055785:Gene Knockdown Techniques; D020062:Heteroptera / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008297:Male; D018978:Molting; D009758:Nymph / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D010802:Phylogeny; D034622:RNA Interference; D016415:Sequence Alignment
chemical_list: D018076:DNA, Complementary; D019476:Insect Proteins; D003577:Cytochrome P-450 Enzyme System
keywords: CYP307B1; Halloween gene; Lygus hesperus; RNA interference; Spookiest; western tarnished plant bug