RNA interference-mediated silencing of a Halloween gene spookier affects nymph performance in the small brown planthopper Laodelphax striatellus

Post-embryonic development of insects is highly dependent on ecdysteroid hormone 20-hydroxyecdysone. Halloween gene spookier （spok, cyp307a2） has been documented to be involved in ecdysteroidogenesis in Drosophila melanogaster and Bombyx mori. We describe here the cloning and characterization of Halloween gene spookier （Lsspok, Lscyp307a2） in the small brown planthopper Laodelphax striatellus, a hemipteran insect species. LsSPOK has three insect-conserved P450 motifs, that is, Helix-K, PERF motif and heme-binding domain. Temporal and spatial expression patterns of Lsspok were evaluated by quantitative polymerase chain reaction. Through the fouth- instar and the early fifth-instar stages, Lsspok showed two expression peaks in the second- and fifth-day fourth-instar nymphs, and two troughs in the first-day fourth and fifth in- stars. On day 5 of the fourth-instar nymphs, Lsspok clearly had a high transcript level in the thorax where prothoracic glands were located. Dietary introduction of double-stranded RNA of Lsspok in the nymph stage successfully knocked down the target gene, decreased expression level ofecdysone receptor （LsEcR） gene, caused nymphal lethality and delayed development. Ingestion of 20-hydroxyecdysone in Lsspok-dsRNA-exposed nymphs did not increase Lsspok expression level, but almost completely rescued the LsEcR mRNA level and relieved the negative effects on survival and development. Thus, our data suggest that the ecdysteroidogenic pathway is conserved in insects and LsSPOK is responsible for specific steps in ecdysteroidogenesis in L. striatellus.

Molecular cloning and characterization of the putative Halloween gene Phantom from the small brown planthopper Laodelphax striatellus

Ecdysteroid hormone 20-hydroxyecdysone plays fundamental roles in in- sect postembryonic development and reproduction. Several cytochrome P450 mono- oxygenases （CYPs）, encoded by the Halloween genes, have been documented to be involved in ecdysteroidogenesis in representative insects in Diptera, Lepidoptera and Orthoptera. Here the putative Halloween gene Phantom （Phm, cyp306al） from a hemipteran insect species, the small brown planthopper Laodelphax striatellus, was cloned. LsPHM shows five insect conserved P450 motifs, that is, Helix-C, Helix-I, Helix-K, PERF and heme- binding motifs. Temporal and spatial expression patterns of LsPhm were evaluated by quantitative polymerase chain reaction. Through the fourth-instar and the early fifth-instar stages, LsPhm showed two expression peaks in day 2 and days 4-5 fourth-instar nymphs, and three troughs in day 1 and 3 fourth instars and day 1 fifth instars. On day 5 of the fourth-instar nymphs, LsPhm clearly had a high transcript level in the thorax where the prothoracic glands were located. Dietary introduction of double-stranded RNA （dsRNA） of LsPhm at the nymph stage successfully knocked down the target gene, decreased expres- sion level ofecdysone receptor （LsEcR） gene and caused a higher nymphal mortality rate and delayed development. Ingestion of 20-hydroxyecdysone on LsPhm-dsRNA-exposed nymphs did not increase LsPhm expression level, but almost completely rescued the LsEcR mRNA level, and relieved the negative effects on survival and development. Thus, our data suggest that the putative LsPhm encodes a ftmctional 25-hydroxylase that catalyzes the biosynthesis of ecdysteroids in L. striatellus.