De Novo Sequencing Of The Transcriptome Of Spinosad-susceptible And-resistant Frankliniella Occidentalis(Pergande) And The Cloning Of NAChR α5 And α7 Subunits

The western flower thrips(WFT), Franklinella occidentalis(Pergande) is an important pest feeding on horticultural plants like vegetables, ornamental crops, and the fruit trees. The commonly used methods for WFT control include biological control, physical control and the widely adopted chemical control. However, because of the relatively short life cycle, high fecundity, the haploid diploid reproduction system, along with polyphagous habits, result in the WFT developing resistance to insecticides through different detoxification mechanisms. WFT is regarded one of the most serious insecticide resistant insect pests in the world. Although Spinosad is the most effective insecticides to control WFT, high levels of resistance to spinosad have been reported in different countries. The susceptibility to spinosad in China has been decreased as well. According to the documents, the resistance mechanisms in spinosad resistant WFT may vary in different populations. It is necessary to explore the resistance mechanism in the local population to devise resistant strategies more reasonably. In this paper, the development and reproduction of WFT under the conditions of low temperatures was conducted, the RNA-seq of spinosad-susceptible and –resistant of WFT was analysed, and the subunits of Fo.nAChRα5 and Fo.n AChRα7 were cloned. The results of the present study will provide more basic biological information of the WFT, make clear the resistance mechanisms in spinosad resistant WFT and also contribute to the effective control of WFT.The spinosad resistant strain of WFT is fed on the kidney bean being treated by spinosad formulation. Through the continuous selection pressure of spinosad, the laboratory resistant WFT has reached to an extremely higher level, with the LC50>3,000mg/L, and the resistance ratio maintains over 10,000-fold compared to the susceptible strain. The susceptible strain has never been exposed to any insecticide, and these two strains are kept separately, which lays a solid foundation for the subsequent study.The development and reproduction of WFT under two low temperatures of 5℃ and 10℃ were studied with an objective to clarify the effects of low temperatures on the these two biological characteristics of this pest species. The results showed that WFT could not complete a life cycle at 5℃. The eggs couldn’t hatch, the first instar nymphs were able to develop into the second instars but unable to develop into pupae. The pupae could develop into adults but the adults couldn’t lay eggs. When placed under the condition of 10℃, the thrips could complete a life cycle but showed a significant prolonged developmental time compared with that under 25℃. The results indicate that the low temperatures resulted in delayed development and decreased fecundity to WFT. However, the thrips returned to normal growth when transferred from low temperatures to room temperature, which indicates that WFT have strong tolerence to low temperatures..By the de novo sequencing of the transcriptome of spinosad-susceptible and –resistant WFT, a total of 68,358 unigenes were produced, among which 1,655 unigenes sorted into 20 genes families were classified. A number of 565 out of these 1,655 unigenes have hits to the NCBI-NR database. This is helpful to the large-scale discovery and utilization of genetic resources for WFT. Through the different expressed genes analysis, 2,618 unigenes were found to expresse differently between the two strains, among which 1,231 unigenes were up-regulated in the resistant strain, while the other 1,387 were down-regulated. In the resistant strain, the superoxide dismutase(SOD) were overexpressed, and the expression level was as high as 247-fold of that in the susceptible strain. In addition, the expression of some cytochrome P450 s genes in resistant strain was up-regulated while others were down-regulated. However, the up-regulated genes had a dominant role over that of down-regulated ones. The validation of qPCR showed that the results of differentially expressed genes from the RNA-seq was reliable, thus the data of RNA-seq could be used for the subsequent study on the resistance mechanisms of in spinosad resistant WFT.Based on results of RNA-seq above, the CDS of nACh Rα5 and nAChRα7 were cloned and the similarity alignment result showed that these two genes were the actural genes encoded the Fo.nACh Rα5 and Fo.nAChRα7. The expression level of nAChRα5 and nAChRα7 between susceptible and resistant strains were not significantly different. The sequence of nAChRα7 showed no difference between the two strains, while the nAChRα5 had more transcript isoforms in resistant strain than that in the susceptible strain. In the susceptible strain, nAChRα5 only had one isoform, named isoform A, while in resistant, five isoforms, named A – E were found. Compared with isoform A, the D and E isoforms had two short N-terminal respectively, while the B isoform were longer than that of isoform A with an insert of 11 amino acid residues – SGRSSIARCKN – from the 425(S) to the(N). Moreover, the isoform C had a deletion of 19 amino acid residues from 405(P) to 423(E) compared with A isoform, which is the only isoform in susceptible strain. These results suggest that the differences of nAChRα5 isoforms between the two strains may contribute to the resistance in spinosad resistant WFT, and its role in resistance needs further study.