| The diamondback moth, Plutella xylostella (L.), has great impact on the yield and quality of cruciferousvegetables. Abamectin belongs to macrolides insecticides and is recommended as the major insecticidefor P. xylostella control. However, the field P. xylostella has developed high level of resistance toabamectin. Therefore, it is necessary to understand the resistance mechanisms of P. xylostella toabamectin. Glutamate-gated chloride channels (GluCls) or inhibitory glutamate receptor (IGluR) is themajor action target of avermectins. However, people have little knowledge of IGluR in insects. In thisstudy, the indoor resistance selection, RNA interference of the IGluR in P. xylostella, the relativeexpression comparison, sequence analysis and electrophysiology of GluClα between susceptible andresistant strains of P. xylostella were conducted with an objective to elucidate the function of IGluR andits role in the resistance, which would lay the foundation for prolonging the utilization of abamectin.1. Laboratory selection of abamectin resistance in P. xylostellaThe original strain of P. xylostella was collected in the field of Fujian and reared in our laboratory forseveral years. The resistant ratio increased from10.7-fold to95.2-fold after selection and anti-selectionby abamectin. Continuous selection of the resistant strain was performed and the LC50rose from3.11mg/L to5.71mg/L. The resistance increased slowly at the beginning, thus, the anti-selection wasconducted to increase the susceptibility. The concentrations used for anti-selection were0.1mg/L and0.05mg/L, respectively. The results showed that the LC50of the susceptible strain decreased from0.29mg/L to0.06mg/L, which indicates the susceptibility enhanced significantly. The above results indicatethat it’s effective to increase the resistant ratio in combination with selection and anti-selection.2. RNA interference of the inhibitory glutamate receptor in P. xylostellaBy means of injecting the double strand RNA of IGluR into the2nd and3rd susceptible instar larvaeand analyzing the silent condition of post transcription level of IGluR through quantitative real-timePCR (qRT-PCR). The results showed that the most suitable injection volumes of dsRNA for the2nd and3rd instar larvae were50.6nL and71.3nL, respectively. The results of qRT-PCR showed that the posttranscriptional level of IGluR gene in the2nd instar larvae decreased by32.67﹪at36h after RNAi andthat in the3rd instar larvae decreased by49.30%at24h after RNAi. The bioassay of larvae injectedwith double strand RNA indicated that the mortality of the treated2nd instar larvae was29.67%, whichwas significantly lower than that of the control group57%when the AVMs concentration was0.05mg/L,at the concentration of0.2mg/L, the mortality of the3rd instar larva was59.67%, significantly lowerthan the control group38.67%under the same concentration.The results revealed that the mortality ofthe larvae injected with dsRNA of IGluR was significantly lower than that of the control, which suggestthat the IGluR in P. xylostella is a potential target site for abamectin.3. The relative expression and sequence analysis of the GluClα gene The relative expression of GluClα in susceptible and resistant strains of P. xylostella was compared byqRT-PCR technique. The results showed that no significant difference was found between the twostrains and the biggest difference was about2.38-fold. By analyzing the GluClα sequence of20and18individuals from susceptible and resistant strains, we found that there were two sets of substitutionpatterns between61and82at the amino acid level. The substitution rate occurred in individuals andspots had no significant difference between the susceptible and resistance strains. Besides, one64bpfragment was inserted into the site of1307, which resulted in the early termination of transcription. Theinsertion rate of both individuals and spots from the resistant strain were lower than that of thesusceptible strain. In addition, a stable deletion of12amino acids (5′-K P L M R G A V L D S K-3′)was found between374and385at the amino acid level. Both the deletionrate of individuals (61.11%)and spots (32.20%) in resistant strains were much higher than that in the susceptible strains (6.25%and20.00%), respectively.4. Electrophysiological studies of IGluR in P. xylostellaThe normal and defective sequences of GluClα were injected into Xenopus oocyte to express thehomomeric IGluR channel. The electrophysiological responses to glutamate and abamectin were testedthrough the two-electrode voltage clamp. The results showed that the EC50of PxIGluR-N andPxIGluR-D were52.61uM and689.7uM to glutamate, respectively, which means PxIGluR-D was13.11-fold less sensitive to glutamate than PxIGluR-N. In terms of abamectin, the EC50of thePxIGluR-N and PxIGluR-D were118.4nM uM and1146uM, respectively, which resulted in a9.68-foldless sensitive of PxIGluR-D to abamectin than that of PxIGluR-N. The results indicated that abamectincan act on IGluR directly and the deletion of GluClα gene may play one of great roles in the resistanceto abamectin. |
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