The Insect Ecdysone Receptor is a Good Potential Target for RNAi-based Pest Control

RNA interference (RNAi) has great potential for use in insect pest control. However, some significant challenges must be overcome before RNAi-based pest control can become a reality. One challenge is the proper selection of a good target gene for RNAi. Here, we report that the insect ecdysone receptor (EcR) is a good potential target for RNAi-based pest control in the brown planthopper Nilaparvata lugens, a serious insect pest of rice plants. We demonstrated that the use of a 360 bp fragment (NlEcR-c) that is common between NlEcR-A and NlEcR-B for feeding RNAi experiments significantly decreased the relative mRNA expression levels of NlEcR compared with those in the dsGFP control. Feeding RNAi also resulted in a significant reduction in the number of offspring per pair of N. lugens. Consequently, a transgenic rice line expressing NlEcR dsRNA was constructed by Agrobacterium- mediated transformation. The results of qRT-PCR showed that the total copy number of the target gene in all transgenic rice lines was 2. Northern blot analysis showed that the small RNA of the hairpin dsNlEcR-c was successfully expressed in the transgenic rice lines. After newly hatched nymphs of N. lugens fed on the transgenic rice lines, effective RNAi was observed. The NlEcR expression levels in all lines examined were decreased significantly compared with the control. In all lines, the survival rate of the nymphs was nearly 90%, and the average number of offspring per pair in the treated groups was significantly less than that observed in the control, with a decrease of 44.18-66.27%. These findings support an RNAi-based pest control strategy and are also important for the management of rice insect pests.     

Metamorphic changes of wild type and mutant Drosophila tissues induced by 20-hydroxy ecdysone in vitro

Wild type (Oregon R) and non-pupariating as well as late-pupariating mutant larval tissues were cultured in vitro up to 5 weeks with and without 20-hydroxy ecdysone (1 μg/ml). The following responses were elicited by the hormone: in the case of wild type tissues detachment of the larval epidermis and muscles from the cuticle; puparial tanning and sclerotization of the larval cuticle; dissociation of the fat body into single cells; inhibition of the movement of the hind intestine. Most of these responses developed within 1 week of culturing. Of the 4 mutants tested, 3 behaved like the wild type. In cultures of ?(1)npr-1, however, puparial tanning, disc evagination, and inhibition of the movement of the hind intestine was abnormally weak and the dissociation of fat body was not observed at all. Detachment of the epidermis and muscles as well as formation of the pupal cuticle by disc tissue occurred normally. The results are discussed with respect to the ecdysteroid-induced metamorphosis of the tissues and the autonomy of mutant gene action.     

Ecdysone 20-hydroxylation in imaginal wing discs of Pieris brassicae (lepidoptera): Correlations with ecdysone and 20-hydroxyecdysone titers in pupae

Ecdysone 20-hydroxylase activity has been detected in pupal wing discs of Pieris brassicae. This activity is due to an enzyme system located in microsomal fractions. Its apparent Km is 58 nM for ecdysone. The enzyme is inhibited by the reaction product 20-hydroxyecdysone with an apparent Ki of 2.6 μM. Its activity varied during pupal-adult development with a maximum on day 4, when ecdysone levels are the highest in the animal. Although low, the peak activity is sufficient to assure 25% of the conversion of endogenous ecdysone into 20-hydroxyecdysone in pupae. Ecdysone and 20-hydroxyecdysone levels were measured in hemolymph and whole animals; ecdysone appears to be mainly located in hemolymph, whereas 20-hydroxyecdysone seems to be equally distributed between hemolymph and tissues. All these findings are discussed in relation to the roles of ecdysone and 20-hydroxyecdysone during pupal-adult development.     

Allelochemical stimulation of ecdysone 20-monooxygenase in fall armyworm larvae

The influence of dietary allelochemical on ecdysone 20-monooxygenase activity was studied in the fall armyworm, Spodoptera frugiperda (J.E. Smith). Feeding the indoles (indole-3-carbinol, indole-3-acetonitrile), flavonoids (flavone, β-naphthoflavone), monoterpenes (menthol, menthone, peppermint oil), and a coumarin (xanthotoxin) to the larvae stimulated midgut microsomal ecdysone 20-monooxygenase activity from 28 to 200% as compared with the controls. β-Naphthoflavone was the most potent inducer among those tested. Phenobarbital, a well-known cytochrome P450 inducer, also caused a 2-fold increase in the microsomal ecdysone 20-monooxygenase activity. Ecdysone 20-monooxygenase activity was 2.7-fold higher in the microsomal fraction than in the mitochondrial fraction isolated from larval midguts. Microsomal ecdysone 20-monooxygenase activity was highest in the fat body, followed by the midgut and Malpighian tubules. Tissue localization and enzyme inducibility were different between ecdysone 20-monooxygenase and xenobiotic-metabolizing cytochrome P450 monooxygenases, including aldrin epoxidase, biphenyl hydroxylase, methoxyresorufin O-demethylase, 7-ethoxycoumarin O-deethylase, p-chloro-N-methylaniline N-demethylase, and phorate sulfoxidase in fall armyworm larvae. © 1995 Wiley-Liss, Inc.     

豌豆蚜翅分化相关miRNA及其预测靶基因对蜕皮激素的应答及miR-92a-1-p5靶基因的验证

[目的]蜕皮激素对孤雌蚜翅两型性分化具有重要调控作用.在前期研究中我们发现5个微小RNA(microRNA,miRNA)在豌豆蚜Acyrthosiphon pisum翅两型性分化中也发挥关键作用,但蜕皮激素是否与miRNA互作参与蚜虫翅型分化未知.本研究旨在探索蜕皮激素对5个miRNA及其预测靶基因表达的影响,揭示蜕皮...     

Ectopic expression of ecdysone oxidase impairs tissue degeneration in Bombyx mori

Metamorphosis in insects includes a series of programmed tissue histolysis and remolding processes that are controlled by two major classes of hormones, juvenile hormones and ecdysteroids. Precise pulses of ecdysteroids (the most active ecdysteroid is 20-hydroxyecdysone, 20E), are regulated by both biosynthesis and metabolism. In this study, we show that ecdysone oxidase (EO), a 20E inactivation enzyme, expresses predominantly in the midgut during the early pupal stage in the lepidopteran model insect, Bombyx mori. Depletion of BmEO using the transgenic CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases) system extended the duration of the final instar larval stage. Ubiquitous transgenic overexpression of BmEO using the Gal4/UAS system induced lethality during the larval–pupal transition. When BmEO was specifically overexpressed in the middle silk gland (MSG), degeneration of MSG at the onset of metamorphosis was blocked. Transmission electron microscope and LysoTracker analyses showed that the autophagy pathway in MSG is inhibited by BmEO ectopic expression. Furthermore, RNA-seq analysis revealed that the genes involved in autophagic cell death and the mTOR signal pathway are affected by overexpression of BmEO. Taken together, BmEO functional studies reported here provide insights into ecdysone regulation of tissue degeneration during metamorphosis.     

Ultrastructural changes accompanying secretion and cell death in the molting glands of an insect (Oncopeltus)

During the fifth (last) larval instar of Oncopeltus fasciatus, morphological changes in the molting glands associated with ecdysone secretion include an increase in cytoplasmic volume relative to that of the nucleus, increased amounts of rough endoplasmic reticulum and mitochondria, and the formation of deep infoldings of the plasma membrane. On the sixth day of the fifth instar large electron-lucent areas become apparent beneath the basement membrane; however, the glands remain intact until the seventh (last) day of the instar when a dramatic fragmentation of the cytoplasm, and condensation and fragmentation of the nucleus are observed. It is likely that such changes occur rapidly, just prior to the time of ecdysis to an adult. Cell death in the molting glands of Oncopeltus is markedly different from that described for the molting glands of other insect species in that autophagic vacuoles are not observed prior to a complete loss of cellular integrity.     

Phenol oxidases from Rhodnius prolixus: Temporal and tissue expression pattern and regulation by ecdysone

Rhodnius prolixus 5th instar nymphs have significant PO enzymatic activity in the anterior midgut, fat body and hemolymph. The tissue with the major amount of PO activity is the anterior midgut while those with higher specific activities are the fat body and hemolymph. In this work the temporal pattern of PO enzymatic activity in different tissues was investigated. In fat body, PO peaks occur at 7, 12 and 16 days after a blood meal. In hemolymph, PO diminishes until day 7, and then recovers by day 14. In the anterior midgut tissue, PO peaks on day 9 and just before ecdysis; a similar pattern was observed in the anterior midgut contents. Some of these activities are dependent on the release of ecdysone, as feeding blood meal containing azadirachtin suppresses them and ecdysone treatment counteracts this effect. These results suggest that during the development of the 5th instar, the insect has natural regulating cycles of basal PO expression and activation, which could be related to the occurrence of natural infections. The differences in temporal patterns of activity and the effects of azadirachtin and ecdysone in each organ suggest that, at least in R. prolixus, different tissues are expressing different PO genes.