Topical Application And Injection Based RNAi Efficiency In Acyrthosiphon Pisum And The Mechanism Of DsRNA Uptake

RNAi is an important technology to study the gene's function of eukaryotic organisms and it is also considered as a potential insect pest control strategy.Acyrthosiphon pisum is an important agricultural pest as well as model organism in fundamental study.Together with the direct sucking of plant nutrients and the secretion of honeydew causing sooty blotch,A.pisum is also a key vector for transmission of various plant viruses.RNAi efficiency of aphids is considered to be variable.Therefore,it is scientifically important to study the delivery method of dsRNA,mechanism dsRNA uptake and RNAi efficiency in aphids.To parallel comparison of the two dsRNA delivery systems(Topical application and injection)of A.pisum,we evaluated time-and dosage-dependent RNAi efficiency and detected the induction activity of core genes of the siRNA pathway upon dsRNA treatment of the two systems.In addition,RNAi efficiency based on the two systems were also evaluated in different populations of A.pisum.The relationship between the body size of aphids,frequency of virus infection,degradation rate of hemolymph to dsRNA and the RNAi efficiency of different populations in A.pisum were further explored.Finally,the "RNAi of RNAi" reporter system was used to reveal the molecular mechanism of dsRNA uptake by Apsid–1 and endocytosis in A.pisum.The main results of this study are as follow: 1 Topical is better than injection based RNAi in A.pisumThe dose-and time-effects of the indicator gene,hunchback of A.pisum(Aphb),were evaluated the injection and the topical application methods.In the dose-dependent RNAi effect,there was no significant silencing efficiency of Aphb at 36 h upon the injection of 60 ng dsAphb.The highest silencing efficiency of Aphb(73.3%)was detected by an injection of 600 ng dsAphb at 36 h.However,the silencing efficiency of Aphb was 80.4% at 36 h when 60 ng ds Aphb was administered by topical application.and the highest silencing efficiency of Aphb was 99.5% at 36 h when 540 ng dsAphb was employed.In the time-dependent effect,the significant silencing efficiency could be detected at 2 h by both the injection and topical application.The silencing efficiency could still be detected until 96 h.The RNAi efficiencies of the six different target genes(ApAChE–1,ApC002,ApCHS,ApJHBP,Apsod–2 and Apsid–1)were tested by the two dsRNA delivery systems,and the silencing efficiency of the target genes by the topical application was higher than the injection.Detecting the fluorescence intensity of Cy3 under the treatment of two dsRNA delivery systems,we observed that although the topical application employed 10-folds dsRNA dose of the injection based RNAi,the Cy3 fluorescence signal could not be detected in the aphid body.The results further indicated that the with low level of dsRNA uptake by topical RNAi,it induced higher RNAi efficiency than the injection.2 Induction of the siRNA pathway activity in A.pisum by exogenous dsRNAThere was one copy of core gene(ApDcr-2,ApAgo-2,and ApR2d2)was identified in the siRNA pathway of A.pisum.By the injection,when 600 ng dsRNA was delivered,the expression of core genes were significantly up-regulated at 12 h;and when 1,200 ng dsRNA was delivered,the expression of core genes was significantly up-regulated at 36 h.By the topical application,when the dose of dsRNA was 1,200 ng,the expression of core genes was significantly up-regulated in the epidermis at 3 h,and then the significant up-regulation of core genes were detected in the gut at 12 h.In addition,the injection of 60 ng of ds Aphb,following the pre-injection of the 600 ng dsGFP,led to the significant silencing of Aphb by 33% at 36 h.Similarly,after the pre-injection of 600 ng ds GFP,the injection of 600 ng of dsAphb induced a significant silencing of Aphb by 81% at 36 h.The above results indicated that the siRNA pathway activity of A.pisum could induced under the treatment of exogenous dsRNA.3 RNAi sensitivity among different populations of A.pisum is variableRNAi efficiency of four target(ApAChE-1,ApC002,Aphb,and Apsod-2)in four populations of the A.pisum(populations include: GS–1,GS–2,NY–1,and NY–2)was evaluated by two dsRNA delivery system.In all tested populations of the pea aphid,gene silencing was achieved by two dsRNA delivery system.In addition,that the topical application achieved higher efficiency than injection.The NY–1 was the most sensitive population in RNAi.Factors,such as frequency of viral infection,the difference of body size and the rate of degradation of dsRNA by hemolymph were also evaluated in four population to correlate with sensitivity of RNAi,while the significant correlation was not identified.4 Transmembrane channel protein Apsid–1 and endocytosis are involved in the uptake of exogenous dsRNA in A.pisumWe identified a single sid–1 homologous gene in A.pisum.The expression of Apsid–1 was induced under the treatment of exogenous dsRNA,which suggested that Apsid–1 may be involved in dsRNA uptake.Using the "RNAi of RNAi" reporting system to analyze the function of Apsid–1,it is found that the inhibition of Apsid–1 expression resulted in a decreased RNAi efficiency of Aphb.The results demonstrate that Apsid–1 may be one of the channel for the uptake of exogenous dsRNA.Two genes,Apchc and Apvha16,being the core genes of endocytosis,were conserved in A.pisum.The expression of endocytosis related genes could be induced by the treatment of exogenous dsRNA.This indicated that the endocytosis of A.pisum was responsive for exogenous dsRNA.When the expression of Apchc and Apvha16 was inhibited by RNAi,the RNAi efficiency of Aphb was reduced by using the "RNAi of RNAi" reporting system.In addition,the endocytosis inhibitor treatment also resulted in a decreased RNAi efficiency of Aphb.These results demonstrate that endocytosis of pea aphid may also be an involved in the uptake of exogenous dsRNA.In conclusion,the topical application based RNAi efficiency of A.pisum was better than the injection based RNAi efficiency,and the transmembrane channel protein Apsid–1 and endocytosis may be two important ways for uptake of exogenous dsRNA by A.pisum's cell.