RNA interference targeting ecdysone receptor blocks the larval-pupal transition in Henosepilachna vigintioctopunctata

Henosepilachna vigintioctopunctata is a serious insect pest which attacks a large number of nightshades and cucurbits in Asian countries,Brazil and Australia.Prolonged application of traditional pesticides has caused environmental pollution and exerted deleterious effects on human health.Finding new approaches with high target specificity and low environmental contamination has become an urgent task.RNA interference(RNAi)induced by double-stranded RNA(dsRNA)is expected to be applicable to managing this pest.Here we evaluated the effects of Escherichia co/Z-expressed dsRNAs targeting ecdvsone receptor(EcR)gene via dietary delivery in laboratory and foliar spraying in a greenhouse.The target transcript was successfully knocked down when the 4th-instar larvae had fed on potatofoliage dipped with dsEcR in a laboratory bioassay.Around 85%of the HvEcR RNAi larvae remained as prepupae or became abnormal pupae,and failed to emerge into adults.Ingestion of ds£c7?-immersed foliage by the 3rd-instar larvae effectuated a comparable RNAi response and brought about more severe defects:all the resultant larvae arrested development,remained as prepupae and finally died.For assay in the greenhouse,a ds£c7?-contained E.coli suspension was directly sprayed to the foliage of greenhouse-growing potato plants and the 3rd-and 4th-instar larvae were transferred to the leaves.High RNAi efficacy was obtained and identical RNAi phenotypes were observed in treated larvae.In addition,spraying dsEcR reduced leaf damage.Our results indicate a possibility of practical application of dsEcR as an environmentally friendly RNA pesticide to control H.vigintioctopunctata larvae.

Ecdysone receptor isoforms play distinct roles in larval–pupal–adult transition in Leptinotarsa decemlineata

A heterodimer of two nuclear receptors,ecdysone receptor(EcR)and ultraspiracle,mediates 20‐hydroxyecdysone(20E)signaling to modulate many aspects in insect life,such as molting and metamorphosis,reproduction,diapause and innate immunity.In the present paper,we intended to determine the isoform‐specific roles of EcR during larval–pupal–adult transition in the Colorado potato beetle.Double‐stranded RNAs(dsRNAs)were prepared using the common(dsEcR)or isoform‐specific(dsEcRA,dsEcRB1)regions of EcR as templates.Ingestion of either dsEcR or dsEcRA,rather than dsEcRB1,by the penultimate(3rd)and final(4th)instar larvae caused failure of larval–pupal and pupal–adult ecdysis.The RNA interference(RNAi)larvae remained as prepupae,or became deformed pupae and adults.Determination of messenger RNA(mRNA)levels of EcR isoforms found that LdEcRA regulates the expression of LdEcRB1.Moreover,silencing the two EcR transcripts,LdEcRA or LdEcRB1 reduced the mRNA levels of Ldspo and Ldsad,and lowered 20E titer.In contrast,the expression levels of HR3,HR4,E74 and E75 were significantly decreased in the LdEcR or LdEcRA RNAi larvae,but not in LdEcRB1 depleted specimens.Dietary supplement with 20E did not restore the expression of five 20E signaling genes(USP,HR3,HR4,E74 and E75),and only partially alleviated the pupation defects in dsEcR‐or dsEcRA‐fed beetles.These data suggest that EcR plays isoform‐specific roles in the regulation of ecdysteroidogenesis and the transduction of 20E signal in L.decemlineata.

A new binding mode for ecdysteroid analog tebufenozide as a multitargeted IGR with chitinase

Tebufenozide,an efficient insect growth regulator(IGR)against lepidopteran pests,presents a novel mode of action with minimal non-target impact.By competing with ecdysteroids for ecdysone receptor(EcR)binding,it regulates insect growth precisely.This study explores tebufenozide's potential as a multitarget IGR,targeting both EcR and Ostrinia furnacalis chitinase I(OfChtI).The inhibitory activity against OfChtI is comparable to that of substrates(GlcNAc)5,with an IC50 of 45.77μM.Our computational findings indicate that tebufenozide binds at the subsite1 to t1 of OfChtI through various interactions.Notably,tebufenozide establishes a pi-pi interaction with the flipped sidechain of Trp107,enabling tebufenozide to deeply penetrate into the S1 pocket,thereby obstructing substrate binding to OfChtI.These insights highlight the potency of multitarget strategies,laying the groundwork for innovative IGR designs that offer comprehensive pest management solutions.

EcR/USP-1-mediated ecdysteroid signaling regulates wolf spider(Pardosa pseudoannulata) development and reproduction

Lycosidae females demonstrate meticulous maternal care of offspring by carrying egg sacs and juvenile spiderlings during the reproductive stage. Nuclear receptors(NRs), especially the ecdysone receptor(EcR) and ultraspiracle(USP), have attracted considerable attention in the regulation of arthropod development and reproduction due to their pivotal roles in ecdysteroid signaling cascades. In the present study, 23 NRs, including one EcR and two USPs, were identified in the genome of the predatory wolf spider Pardosa pseudoannulata. RNA interference(RNAi) targeting EcR and USP-1inhibited spiderling development and resulted in nonviable eggs in the egg sacs. EcR and USP-1responded to changes in ecdysteroid levels, and interference in ecdysteroid biosynthesis led to similar phenotypes as ds EcR and ds USP-1 treatments.These findings suggest that EcR/USP-1-mediated ecdysteroid signaling regulates P. pseudoannulata development and reproduction. The P.pseudoannulata females with suppressed ecdysteroid signaling proactively consumed their non-viable egg sacs, resulting in a 7.19 d shorter first reproductive cycle than the controls. Termination of the failed reproductive cycle enabled the spiders to produce a new egg sac more rapidly. This reproductive strategy may partially rescue the reduction in population growth due to non-viable eggs and compensate for the physiological expenditure of wasted maternal care, which would be beneficial for the conservation of P.pseudoannulata populations and their natural control of insect pests.

Expression, subcellular localization and protein-protein interaction of four isoforms of EcR/USP in the common cutworm

Ecdysone receptor （EcR） and ultraspiracle （USP） form heterodimers to mediate ecdysteroid signaling during molting and metamorphosis. Various EcR/USP heterodimers have been reported. However, it is unclear what kind of EcR/USP combination is adopted by lepidopteran insects during the larval-pupal metamorphosis and whether the EcR/USP heterodimer varies among different tissues. To address these questions, two isoforms of each EcR and USP were cloned from the common cutworm, their messenger RNA expression patterns were examined by real-time quantitative polymerase chain reaction in different tissues during the larval-pupal metamorphosis and in the midgut in response to hormonal induction. Furthermore, their subcellular localization and protein-protein interaction were explored by transient expression and far-western blotting, respectively. All the four genes were significantly up-regulated in prepuae and/or pupae. The expression profiles of EcRB1 and USP1 were nearly identical to each other in the epidermis, fat body and midgut, and a similar situation also applied to EcRA and USP2. The three genes responded to 20-hydroxyecdysone （20E） induction except for USP2, and USP1 could be up-regulated by both 20E and juvenile hormone. The four proteins mainly localized in the nucleus and the nuclear localization was promoted by 20E. The protein-protein interaction between each EcR and USP was found in vitro. These results suggest that two types of EcR/USP heterodimer （EeRA/USP2 and EcRB 1/USP1） may exist simultaneously in the common cutworm, and the latter should play more important roles during the larval-pupal metamorphosis. In addition, the types of EcR/USP heterodimer do not vary in the tissues which undergo histolysis and regeneration during metamorphosis.

Molting process revealed by the detailed expression profiles of RXR1/RXR2 and mining the associated genes in a spider mite, Panonychus citri

Spider mites have one ecdysone receptor(EcR)and multiple retinoid X receptors(RXRs).However,the function of these RXRs in spider mite development is unknown.Here,we screened the expression dynamics of two PcRXR isoforms at 4 h intervals in the deutonymphal stage of Panonychus citri.The results showed that PcEcR had an expression pattern similar to that of PcRXR2.For PcRXR1,its expression remained at a certain high level,when there was a decrease of both PcEcR and PcRXR2.In situ hybridization showed that PcRXR2 was detected in the central nervous mass,while the ecdysteroid biosynthesis gene PcSpo was mainly expressed at the edge of the central nervous mass.RNAi-based silencing of PcRXR1 or PcRXR2 showed the same phenotype as in mites with that of silencing PcEcR.Furthermore,RNA-seq was used to mine the genes associated with the expression dynamics of PcRXR1 or PcRXR2,which revealed that the heterodimer of EcR-RXR2 in spider mites might be linked with the cell autophagy and tissue remodeling during apolysis,and RXR1 might be linked with new epicuticle and exocuticle secretion during ecdysis.Taken together,these results increase our understanding of the regulation mechanism of ecdysteroid signal pathway in spider mite development.