| The western flower thrips(WFT),Frankliniella occidentalis(Pergande),a world spread agricultural pest,is the most efficient vector for Tomato spotted wilt orthotospovirus(TSWV).Due to the long-term and overuse of insecticides to control the thrips,WFT has developed resistance to a variety of insecticides.Spinosyn is the most effective type of insecticide for thrips control.However,the highly dependent use of spinosyn has made WFT developed high leves of resistance in many regions of the world.Insecticide resistance is a common issue in pest management.The interactions between insecticide resistance in insect vector and the transmission of plant viurs,however,is largely unknown.Infection with TSWV is beneficial to the growth and development of thrips,but the effect of TSWV on the offspring of thrips is not clear.In the process of co-evolution between WFT and TSWV,the two develop molecular and ecological interactions.But the proteins that interact with the TSWV in thrips are not known.In order to clarify questions mentioned above,the following experiments were conducted in the present study.The effects of spinosad resistance on TSWV transmission by WFT were assessed using a pair of near-isogenic lines of susceptible(Ivf03)and resistant(NIL-R)F.occidentalis.The differences in the transcriptome changes of Ivf03 and NIL-R infected with TSWV or not were compared.The effects of TSWV on the population and sex ratio of progeny of F.occidentalis were studied.Yeast two hybrid techniques were used to screen the interactors from F.occidentalis associated with transmission of TSWV.The main results are as follows:1.The vector competence of spinosad-resistant thrips was higher than that of the susceptible thrips,however,the number of spinosad-resistant adults that transmit TSWV effectively were less than that of susceptible one.When given a shorter acquisition access period(AAP)of 6 h,virus replication and transmission efficiency of NIL-R were significantly higher than that of Ivf03.And a longer AAP of 96 h led to a similar virus acquisition and transmission efficiency for both strains,although TSWV replication was greater in NIL-R.The NIL-R had a prolonged pre-adult stage(7.55 d)than Ivf03(6.21 d),but a shorter male life span(6.5 d)than Ivf03(10.6 d).The pupation rate in NIL-R(57.6%)was significantly reduced in comparison to Ivf03(96.3%).2.The differences in the transcriptome changes of Ivf03 and NIL-R infected with TSWV or not were significant.Totally,36 genes were up-regulated and 39 genes were down-regulated in both strains.Specifically,27 genes were up-regulated in Ivf03 but down-regulated in NIL-R,and 116 genes were down-regulated in Ivf03 but up-regulated in NIL-R.There were 355 genes differentially expressed in NIL-R but not in Ivf03,and310 genes differentially expressed in Ivf03 but not in NIL-R.3.TSWV can promote its spread through mediating the development and reproduction of its vector WFT.We found that TSWV-infected thrips,in general,had a significantly longer adult longevity than that of non-infected individuals.And adults is the main stage associated with virus dispersal.TSWV-infected F.occidentalis produced substantially more progeny than that of non-infected thrips.Compared to the offspring of non-infected thrips,when the TSWV-infected parental sex ratio was 3:1,both male(2.8-to 5.2-fold)and female(1.9-to 2.3-fold)progenies were increased.While under the 1:1 parental sex ratio,the male progeny of TSWV-infected thrips increased approximately 1.5-fold than that of non-infected thrips,and there was no significant difference between female proeny of TSWV-infected and non-infected thrips.Most of the increase in progeny came from an increase in males,the sex ratio of progeny shifted from 1.3-7:1 to 0.6-1:1,and male thrips has a greater dispersal and virus transmission capability.4.The western flower thrips yeast library and TSWV membrane protein bait vector pBT3-STE-GN of split-ubiquitin based mambrane yeast two hybrid system were successfully constructed.The cDNA library had a storage capacity of 3.2×10~6 cfu,and recombination rate was 100%,the average amplification sizes of inserts fragment of cDNA library were above 1 kb.Finally obtained 74 proteins interacting with TSWV GN by NCBI blast for sequence alignment.The GO pathway annotation results showed that these host factors were involved in metabolic processes,biological regulation processes,immune system processes,stimuli response,detoxification,cell proliferation,and growth and development processes and so on.The western flower thrips yeast library and TSWV membrane protein bait vectors pGNKT7-GN and pGBKT7-GC based on GAL4 yeast two hybrid systm were successfully constructed.The capacity of the western flower thrips cDNA library was 5.6×10~6 cfu,and the average library recombination rate was about 100%and the average amplification sizes of inserts fragment of c DNA library were above 1 kb.The transformation ratio and titer of the Y187 yeast library was 5×10~6 cfu/?g and 2.97×10~8.The constructed pGNKT7-Gn and pGBKT7-Gc bait vectors were well expressed in yeast and had no self-activation and toxicity. |
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