A Halloween gene shadow is a potential target for RNA-interference-based pest management in the small brown planthopper Laodelphax striatellus.

BACKGROUND: Laodelphax striatellus is an economically important rice pest in China. Ecdysteroid hormone 20-hydroxyecdysone regulates insect development and reproduction. The cytochrome P450 monooxygenase Shadow (Sad) plays a critical role in ecdysteroidogenesis. Here, tests were conducted to establish whether Lssad was a potential target gene for RNA-interference-based management of L. striatellus. RESULTS: Lssad was cloned and characterised. LsSad had Helix-C, Helix-I, Helix-K, PERF and haem-binding motifs. Lssad is expressed at a higher level in the thorax, where prothoracic glands are located, compared with the level in the head or abdomen. It showed two expression peaks in day 2 and day 4-5 fourth-instar nymphs, and two troughs in day 1 fourth and fifth instars. Oral delivery of double-stranded RNA (dsRNA) of Lssad at the nymph stage successfully knocked down the expression of the target gene, reduced the expression level of ecdysone receptor (LsEcR) gene, caused nymphal lethality and delayed development in a dose-dependent manner. Ingestion of 20-hydroxyecdysone in Lssad-dsRNA-exposed nymphs did not increase Lssad expression level, but almost completely rescued the LsEcR mRNA level and relieved the negative effects on survival and development. CONCLUSIONS: The ecdysteroidogenic pathway is conserved in L. striatellus. Lssad can serve as a possible target for dsRNA-based pesticides for planthopper control.

The putative Halloween gene phantom involved in ecdysteroidogenesis in the white-backed planthopper Sogatella furcifera.

Postembryonic development of insects is highly dependent on ecdysteroid hormones ecdysone (E) and 20-hydroxyecdysone (20E). A cytochrome P450 monooxygenase CYP306A1, the product of the Halloween gene phantom (phm), is involved in the ecdysteroidogenesis in representative insects in Diptera, Lepidoptera and Orthoptera. In the present paper, Sfphm was cloned from a hemipteran insect species, the white-backed planthopper Sogatella furcifera. SfPHM has five insect conserved P450 motifs, i.e., Helix-C, Helix-I, Helix-K, PERF and heme-binding motifs. Temporal and spatial expression patterns of Sfphm were evaluated by q-PCR. Sfphm showed three expression peaks in late second-, third- and fourth-instar stages. In contrast, the expression levels were lower and formed three troughs in the newly-molted second-, third- and fourth-instar nymphs. The relative 20E levels exhibited similar temporal patterns to Sfphm expression levels. On day 3 of the fourth-instar nymphs, Sfphm clearly had a high transcript level in the thorax where PGs were located. Dietary introduction of double-stranded RNA of Sfphm into the second instars successfully knocked down the target gene, and greatly reduced 20E level and ecdysone receptor (EcR) expression level. Moreover, knockdown of Sfphm caused lethality and slowed down ecdysis during nymphal stages. Furthermore, ingestion of 20-hydroxyecdysone did not alter Sfphm expression level, but almost completely rescued SfEcR expression level, and relieved the negative effects on nymphal survival and ecdysis in Sfphm-dsRNA-exposed planthoppers. Thus, our results suggest that Sfphm plays a critical role in ecdysteroidogenesis in S. furcifera.

RNA interference depletion of the Halloween gene disembodied implies its potential application for management of planthopper Sogatella furcifera and Laodelphax striatellus.

Sogatella furcifera and Laodelphax striatellus are economically important rice pests in China by acting as vectors of several rice viruses, sucking the phloem sap and blocking the phloem vessels. Ecdysteroid hormone 20-hydroxyecdysone regulates insect development and reproduction. A cytochrome P450 monooxygenase CYP302A1 (22-hydroxylase), encoded by the Halloween gene disembodied (dib), plays a critical role in ecdysteroidogenesis. The objective of this study is to test whether dib genes are potential targets for RNA interference-based management of S. furcifera and L. striatellus. We cloned and characterized Sfdib and Lsdib. The open reading frame regions of dib genes were generated and used for designing and constructing dsRNA fragments. Experiments were conducted using oral delivery of dsdib to investigate the effectiveness of RNAi in S. furcifera and L. striatellus nymphs. Real-time quantitative reverse transcriptase-PCR analysis demonstrated that continuous ingestion of dsdib at the concentration of 0.01, 0.05 and 0.50 mg/ml diminished Sfdib expression levels by 35.9%, 45.1% and 66.2%, and ecdysone receptor (SfEcR) gene mRNA levels by 34.0%, 36.2% and 58.5% respectively in S. furcifera, and decreased Lsdib expression level by 18.8%, 35.8% and 56.7%, and LsEcR mRNA levels by 25.2%, 46.8% and 68.8% respectively in L. striatellus. The reduction in dib and EcR transcript abundance resulted in observable phenotypes. The development of nymphs was impaired and the survival was negatively affected. Our data will enable the development of new insect control strategies and functional analysis of vital genes in S. furcifera and L. striatellus nymphs.