The Fatty Acid Biosynthesis And Metabolism Pathways Mediate The Refractoriness To RNAi And Humoral Immunity In Bactrocera Dorsalis

The oriental fruit fly,Bactrocera dorsalis(Hendel),one of the most destructive pests throughout South East Asia,causes severe economic loss to over 250 fruit crops.RNA interference(RNAi)is a conserved regulatory mechanism that is triggered by dsRNA;it functions in a remarkable variety of organisms.As RNAi can be achieved easily in many eukaryote species,it has great potential in many areas of scientific research such as functional genetic,medical and agricultural studies.Even though a number of progess of RNAi has been achieved in the studies of agricultural pests,there still remains many problems need to be solved till this technology could be applied in production.For instance,feeding dsRNA can not indce RNAi effect in Drosophila melanogastera and B.dorsalis can develop an refractoriness to RNAi.In this study we illuminated the molecular mechanism of the refractoriness to RNAi in B.dorsalis and revealed a role of the fatty acid biosynthesis and metabolism pathways in regulation of the refractoriness to RNAi.We also explored the function of the key gene on the the fatty acid biosynthesis and metabolism pathways,noa,in the innate immunity of B.dorsalis.1.Hemolymph of ds-rpl19 challenged flies can induce RNAi blocking.Hemolymph was collected from the challenged flies,which have been successfully induced RNAi blocking by feeding ds-rpl19.After injecting hemolymph of the challenged(Ch)flies into untreated flies,feeding ds-rpl19 could not induce down-regulation of target gene while the target gene appeared a decrease of 51% after feeding ds-rpl19 followed by injecting hemolymph from na?ve(Nv)flie.The hemolymph could still induce the blockage of RNAi in untreated flies after being kept at 25 °C for 3 days.Low temperature(-20 °C for 24 h)had no effect on the ability of the hemolymph to block RNAi,while hemolymph heated to 100 °C for 10 min did not influence the RNAi effect.These results indicates that there exist an ?inducing factor? in the hemolymph from challenged B.dorsalis which was unstable at high temperature and can induce RNAi blocking in untreated flies.2.Comparative metabolomic analysis revealed that the content ratio of linoleic acid(LA)to arachidonic acid(AA)in the hemolymph of the Ch group was significantly higher than that in the Nv group.A total of 37 different metabolites were identified and displayed at different levels between the Ch and Nv hemolymph samples.17 kinds of metabolites showed significantly higher accumulations in the Ch samples and the contents of the other 20 kinds of metabolites were higher in the Nv samples.Among the metabolites,13 types of phospholipids and 12 types of fatty acids were identified,meaning that 67.57% of the metabolites were lipids.We found that the LA:AA ratio in the hemolymph of the Ch group was 11.5:1,which was significantly higher than the 9.8: 1 ratio observed for the Nv group.This result indicated that the relative content of AA to LA was decreased in the hemolymph of the Ch group.Each of the up-regulated metabolites of the Ch group was injected into untreated flies and we found that only the injection of linoleic acid could induce a similar blockage of RNAi with that seen form the injection of the hemolymph from the Ch group.The inducing ability of LA was concentration relate.After injecting 200 ?M LA,the RNAi blocking effect lasted for 2 days.We next tested the mRNA accumulation of genes that have been reported to be responsible for the cellular entry of dsRNA after injecting LA.The results indicated that the expression of bet3?pi3k?nina c?vha16-1?vha-sfd?arf72a and ap50 appeared decreases varied from 25%-65%.Fluorescence microscopy revealed that injecting LA blocked the entry of dsRNA into the midgut cells of B.dorsalis.We injected AA of 50 ?M?100 ?M and 200 ?M into Ch group flies prior to a secondary exposure to ds-rpl19 and as expected,the target gene showed down-regulation of 50%?67% and 46% in the secondary exposre,respectively.The mRNA accumulation of genes involved in the cellular entry of dsRNA was also monitored afterinjecting AA and most were found to be increased in AA-injecting flies as compared to DM-injecting flies up-regulated.Previous research has demonstrated that feeding dsRNA to Drosophila does not induce an RNAi effect.Baesd on our reslts,we injected AA into Drosophila flies before letting the Drosophila flies feed on ds-rpl19 from D.melanogaster(ds-Dmrpl19).qPCR analysis showed that feeding ds-Dmrpl19 could induce a decrease of 44% to the mRNA accumulation of the Dmrpl19 in AA-injected fruit flies.Fluorescence microscopy indicated that AA injection facilitated dsRNA entry into D.melanogaster midgut cells.3.Results of transcriptomic and proteomic analysis showed that most genes and proteins involved in the fatty acid biosynthesis and metabolism pathways play important roles in the regulation of refractoriness to RNAi in B.dorsalis.There were 1204 genes and 72 proteins were identified up-regulated while 1343 gens and 43 proteins were down-regulated in the early samples.844 genes and 68 proteins appeared to be up-regulated while 1119 genes and 79 proteins were down-regulated in the late samples.The result of GO enrichment analysis showed 265 GO terms that were overrepresented in the early set samples and 132 GO terms were overrepresented in the late samples in the biological processes category.GO terms overrepresented in the early set were mainly classified into 14 clusters including development process,macromolecule metabolic process and fatty acids metabolic process;the overrepresented GO terms were mainly classified into12 clusters in the late samples including phospholipid metabolic process,transmembrane transport and nitrogen compound metabolic process.Silencing the key gene fasn in the fatty acid biosynthesis and metabolism pathways could block the immune response to dsRNA targeting endogenous genes.4.The essential gene in the fatty acid biosynthesis and metabolism pathways,noa,which encoding a very long chain fatty acid elongase,can mediate the activation of innate immune pathways of B.dorsalis.we cloned the noa gene of B.dorsalis,which yielded a sequence of 1608 bp,containing a 990 bp ORF.The gene encoded a 329 amino acid protein.The expression profiles indicated that the transcriptional level of noa was high at the egg stage and in the testis tissue.Silencing of noa would influence the expression of immune related genes,including MyD88 and defensin in the Toll pathway and relish and diptericin in the Imd pathway.noa expression was up-regulated after Listeria monocytogenes,Staphylococcus aureus and Escherichia coli infection.Moreover,infection with L.monocytogenes and S.aureus after feeding ds-noa,the expression of MyD88 and defensin down-regulated significantly in ds-noa group compared with in ds-egfp group.Meanwhile,infection with L.monocytogenes and E.coli,which activated the Imd pathway,do not cause increase of the mRNA levels of relish and diptericin in ds-noa group as severely as in ds-egfp treatment.The median time to death(MTD)caused by L.monocytogenes infection was approximately 4 days in the ds-egfp group,while the MTD was approximately 2 days in the ds-noa group.All these results indicated that silencing noa could influence the activation of Toll/Imd pathways.