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.

The actions of neonicotinoid insecticides on nicotinic acetylcholine subunits Ldα1 and Ldα8 of Leptinotarsa decemlineata and assembled receptors

The insect nicotinic acetylcholine receptor(nAChR)is a pentameric channel protein and also a target for neonicotinoids.There are few reported studies on the molecular interactions of Leptinotarsa decemlineata nAChRs with neonicotinoids.In this study,we analyzed the response of acetylcholine and neonicotinoids(thiamethoxam[TMX],imidacloprid[IMI],and clothianidin[CLO])on hybrid receptors constructed by nAChRα1 andα8 subunits of L.decemlineata(Ldα1 and Ldα8)co-expressed with ratβ2 subunit(rβ2)at different capped RNA(cRNA)ratios in Xenopus oocytes.In addition,we evaluated the expression changes of Ldα1 and Ldα8 after median lethal dose of TMX treatment for 72 h by quantitative polymerase chain reaction(qPCR).The resulting functional nAChRs Ldα1/rβ2 and Ldα1/Ldα8/rβ2 showed different pharmacological characteristics.The neonicotinoids tested showed lower agonist affinity on Ldα1/Ldα8/rβ2 compared to Ldα1/rβ2 at same ratios of subunit cRNAs.The sensitivities of neonicotinoids tested for Ldα1/rβ2 and Ldα1/Ldα8/rβ2 at cRNA ratios of 5:1,1:1 and 5:5:1,1:1:1,respectively,were lower than those for nAChRs at ratios of 1:5 and 1:1:5,respectively,whereas the values of maximum response(Imax)varied.For Ldα1/Ldα8/rβ2,a reduction of Lda8 cRNA resulted in increased sensitivity to IMI and decreased sensitivity to TMX.The expression of Ldα1 and Ldα8 significantly decreased in adults by 82.12%and 47.02%,respectively,while Ldα8 was significantly upregulated by 2.44 times in 4th instar larvae after exposure to TMX.We infer that Ldα1 and Ldα8 together play an important role in the sensitivity of L.decemlineata to neonicotinoids.