| Rhopalosiphum padi has many hosts and short generation cycle,and is widely distributed in various wheat areas in our country.Studies have shown that DIMBOA has an effect on the fecundity and survival rate of wheat aphids,but few researchers have reported the effect of DIMBOA on the feeding behavior of R.padi and its mechanism.In this study,wheat seedlings were treated with DIMBOA to feed the aphid,in order to test the effects of DIMBOA on the biological characteristics of the aphid,such as,longevity,fecundity,position of reproduction of aphid and feeding position.The change of feeding behavior of R.padi after treatment with DIMBOA when feeding on wheat stems were detected by using the EPG technology.Differentially expressed genes of R.padi after treatment with DIMBOA were analyzed by transcriptomics,and the RNAi test was conducted for candidate genes to verify the function of the genes.The research results will provide new ideas for the control of wheat aphids and lay a theoretical foundation for the green control technology of pests.The main results are as follows:1.Effects of DIMBOA treatment on the fecundity and longevity of R.padiThrough the statistical analysis of the fecundity of R.padi reared in the 5 mg/L and 20mg/L DIMBOA treatment solutions and the control group,it was found that the average total reproduction of R.padi in each group was 52.3 aphids,41.1 aphids and 63.1 aphids,respectively.There was significant difference between the 20 mg/L DIMBOA treatment group and the control group.At the same time,the average longevity of the three groups of aphids was 15.9 d,12.7 d and 18.1 d,respectively.Among them,the treatment of 20mg/L DIMBOA significantly reduced longevity of R.padi.2.Effect of DIMBOA treatment on the position of reproduction and feeding of R.padi.According to the statistics of the total reproduction of each aphid in different parts of wheat seedlings,it was found that the average total reproduction of R.padi in stems and leaves after treatment with 20 mg/L DIMBOA solution was 12.0 aphids and 29.1 aphids respectively,with significant differences.The number of R.padi feeding on the stems of the control group was higher than that on the leaves,and the number of R.padi feeding on the leaves of the 5 mg/L solution treatment group was higher than that on the stems in the first seven days.indicating a tendency to feed on the leaves of wheat seedlings.The number of R.padi feeding in the group treated with 20 mg/L DIMBOA solution was overall higher in the leaves than in the stems,presumably due to the higher concentration of DIMBOA solution,which affected the feeding behavior of adult of R.padi.3.Detection of feeding behavior of R.padi by EPG.The results showed that the duration of np wave of R.padi in the stems of wheat seedlings treated with 20 mg/L DIMBOA was significantly longer than that in the stems of control group.The duration of E wave was significantly lower than that in the stems of the control group,indicating that the feeding duration of R.padi on the stems of wheat seedlings was significantly reduced after treatment with DIMBOA,which influenced the feeding behavior of R.padi.4.Screening candidate genes in response to DIMBOA treatment of R.padi based on transcriptome.Based on transcriptomics data,three candidate genes of R.padi,alpha-tocopherol transfer protein(Rp ATT),COP9 signalosome subunit 5(Rp CSN5)and aspartate aminotransfer(Rp AST),were selected for functional study.The first-instar nymphs of R.padi were selected and fed with 5 mg/L DIMBOA to treat the stems and leaves of wheat seedlings and the stems and leaves of control group,respectively.RNA was extracted after feeding for five days and fluorescent quantitative analysis was performed on three candidate genes.The results showed that the expression levels of Rp ATT gene in four groups had no significant difference.when R.padi feeding on the stem of the control group,the expression levels of Rp CSN5 gene were the highest,with significant differences from the other three groups.The expression level of Rp AST gene was highest in the stem of the control group,and there was a significant difference in expression level compared to the leaves of the control group and the stem treated with 5 mg/L DIMBOA,but there was no significant difference in expression level compared to the leaves treated with 5 mg/L DIMBOA.The first-instar nymphs of R.padi were selected and treated with20 mg/L DIMBOA to treat the stems and leaves of wheat seedling and the stems and leaves of control group,respectively.RNA was extracted after five days of feeding and fluorescence quantitative analysis was performed on three candidate genes.The results showed that the expression levels of Rp ATT gene also had no significant difference among the four groups.In addition,when R.padi feeding on the stem of the control group,the expression levels of Rp CSN5 and Rp AST genes were the highest,with significant differences compared to the other three groups.5.Analysis of the Functions of Rp ATT,Rp CSN5,and Rp AST genes in R.padi based on RNAi.After feeding on the ds RNA of Rp ATT,Rp CSN5 and Rp AST genes,the expression levels of the genes were all down-regulated,and the silencing efficiency of Rp ATT,Rp CSN5 and Rp AST genes was 70.7%,82.1% and 95.6% respectively.It shows that the target genes can be effectively silenced after introducing ds RNA into aphids by feeding method.After feeding on the wheat seedlings treated with ds GFP,ds Rp ATT,ds Rp CSN5 and ds Rp AST solutions for 48 h,the mortality rates of R.padi were 16.7%,40%,66.7%and 23.3%,respectively.The mortality rate of ds Rp CSN5 was the highest,which was significantly different from that of ds GFP.Moreover,R.padi completely climbed to the leaves of wheat seedlings,suggesting that Rp CSN5 might have a lethal effect on R.padi.The statistic of R.padi among feeding on stems of wheat seedlings treated with ds Rp ATT and the stems of wheat seedlings of the control group showed that the reproduction in the stems of wheat seedlings treated with ds Rp ATT solution was significantly reduced.6.The effects of Rp ATT,Rp CSN5,and Rp AST genes on the response of R.padi to DIMBOA Treatment.The third-instar nymphs of R.padi fed on the wheat seedlings treated with ds RNA solution of three candidate genes and DIMBOA together,and set the wheat seedlings treated with DIMBOA solution as the control group.After 48 h,the mortality rates of the three genes to R.padi was observed.the results showed that the mortality rates of DIMBOA treatment group,DIMBOA and ds GFP co-treatment group,DIMBOA and ds Rp ATT co-treatment group,DIMBOA and ds Rp CSN5 co-treatment group and DIMBOA and ds Rp AST co-treatment group were 12.5%,29.2%,41.7%,50%,37.5%respectively.Only the co-treatment group with DIMBOA and ds Rp CSN5 had significant difference from the control group,and all aphids crawled to the leaves of wheat seedlings to feed.Moreover,there was a significant difference in the fecundity between the ds Rp CSN5 group with DIMBOA and the control group with DIMBOA and the ds GFP group with DIMBOA. |
