BdmiR-994 Targeting BdCPCFC Mediated Malathion Penetration Resistance Of Bactrocera Dorsalis

Bactrocera dorsalis is an important agricultural pest.Due to its wide host range,strong adaptability and high reproduction capability,it has developed resistance to many insecticides including malathion in the field.Although many studies have found that up-regulated expression of detoxification metabolic enzyme genes plays a role in the resistance of B.dorsalis to malathion,it is not clear whether there are other resistance mechanisms involved in malathion resistance of B.dorsalis.Penetration resistance is an effective means for insects to resist insecticides.It blocks the penetration of insecticides by changing the structure and composition of the insect cuticle,and reduces the susceptibility to insecticides.It is also one of the reasons for cross-resistance of pests in field.Therefore,malathion susceptible(MS)and resistant(MR)strains were used in the current study to clarify the malathion penetration resistance mechanism of B.dorsalis,which will help to extend the service life of pesticides and reduce the cost of control of B.dorsalis.Through a variety of technique and method,such as transmission electron microscopy(TEM),high performance liquid chromatography(HPLC)and different insecticide applications,the penetration resistance to malathion of B.dorsalis was uncovered,in which the thickening of the pronotum cuticle blocked the penetration of malathion.On this basis,high-throughput transcriptome sequencing,bioinformatics analysis,biotin-streptavidin RNA pull-down,dual luciferase reporter system,fluorescence in situ hybridization,immunohistochemistry,heterologous expression and RNAi were comprehensively used to clarify the molecular mechanism by which BdmiR-994regulates the cuticle protein gene BdCPCFC to mediate the resistance of B.dorsalis to malathion penetration.The results of this study could provide research ideas and theoretical basis for the development of novel pesticides.The main findings were as follows:1 Physical and chemical analysis of the malathion penetration resistance of B.dorsalisAfter several consecutive selections with malathion which was applied on the pronotum of B.dorsalis,MR strain was obtained.The LD₅₀ were 3452.5 ng/fly and64.3 ng/fly in MR and MS,respectively.Compared with the malathion application which exposed malathion on the pronotum of B.dorsalis,the LD₅₀ of MR and MS were reduced to 1344.1 ng/fly and 44.1 ng/fly by injecting malathion through pronotum,respectively.These results showed that the malathion might increase toxicity by directly delivering to insects,which indicated that the cuticle is an important barrier against malathion.Further penetration investigation found that the malathion penetration ratios in MR strains were 60.3%,70.7%,79.9%and 84.6%,post malathion exposure 1,3,6 and 9 h,respectively.While malathion penetration ratios in MS strains were 83.1%,96.9%,98.0%and 98.5%,respectively.These results indicated that penetration of malathion in MR strain was significantly blocked.Subsequently,the ultrastructural observation of the pronotum cuticle showed that the thickness of pronotum cuticle in MR strain significantly increased with 4.5?m.It demonstrated that the cuticle thickening of pronotum block the penetration of malathion,result in the reduced susceptibility to malathion and eventually formed malathion penetration resistance in B.dorsalis.2 Analysis the genes related to malathion resistance in B.dorsalisBased on the RNA-sequencing data,157 up-regulated and 266 down-regulated genes were identified in MR strain compared to MS strain.Annotation analysis found that 18 cuticle protein genes up-regulated in MR strain,which belonged to the RR-1,RR-2,and CPCFC families.The RT-q PCR validation showed that the 16 cuticle protein genes up-regulated with 0.9-to 11.6-fold in the MR strain,which was consistent with the trend of the RNA-seq results.According to the RT-q PCR results,Bd RR1-2,Bd RR1-32,Bd RR1-35 and BdCPCFC were significantly up-regulated in 7day-old adults of the MR strain,suggested that these 4 genes might play important roles in the change of cuticle structure.The tissues expression pattern results showed that Bd RR1-2,Bd RR1-35 and BdCPCFC significantly up-regulated with 6.0-,1.8-and1.0-fold in the pronotum cuticle of MR strain,respectively.These results suggested that these three cuticle protein genes might play important roles in the pronotum cuticle of the MR strain of B.dorsalis.Based on the comparison of miRNA high-throughput transcriptome sequencing between MR and MS strains of B.dorsalis,75 known miRNAs and 372 novel miRNAs were identified.Further analysis revealed that the five known miRNAs were down-regulated in MR strain.Four of down-regulated miRNAs that TPM values more than 100 were investigated on the relative expression level in MR and MS strains.Results of RT-q PCR showed that BdmiR-994,BdmiR-318,BdmiR-6 and BdmiR-286might have a strong relationship with malathion resistance of B.dorsalis.Meanwhile,BdmiR-994 significantly down-regulated in MR strain of whole body and pronotum cuticle with 4.0-and 2.1-fold,respecitively,suggested that BdmiR-994 might regulate suscepitibility to malathion via affecting the related functional genes on pronotum cuticle.3 Target regulation of BdmiR-994 on the cuticle protein gene BdCPCFCAccording to the RNA-seq of BdmiR-994 mimic injection and the RNA-seq of pronotum cuticle,multiple cuticle protein genes were obtained.It provided research ideas for the subsequent screening of downstream functional genes which might be regulated by BdmiR-994.Based on conjoint analysis of the four softwares of miRNA target gene prediction and the RNA-seq results,four candidate target genes of BdmiR-994 were obtained.Using biotin-streptavidin RNA pull-down analysis,the cuticle protein gene BdCPCFC was the only one significantly enriched candidate target gene,suggesting that BdmiR-994 could bind BdCPCFC directly in B.dorsalis.The results of in situ hybridization showed that the fluorescent signals of BdmiR-994 and BdCPCFC were enriched in the cuticle of the pronotum simultaneously,that was consistent with the results of the previous RT-q PCR analysis of BdmiR-994 and BdCPCFC.After BdmiR-994 mimic treatment,the results of RT-q PCR and Western blot showed that the relative expression of BdmiR-994 up-regulated with nearly64-fold,the relative expression of BdCPCFC was reduced with 56.8%.Similarly,the protein of BdCPCFC obviously reduced in B.dorsalis.While treating with BdmiR-994inhibitor,the relative expression of BdmiR-994 down-regulated with 90.0%,and the relative expression of BdCPCFC up-regulated with 2.1-fold.The protein of BdCPCFC also obviously increased in B.dorsalis.Subsequently,the results of dual luciferase reporter system showed that BdmiR-994 negatively regulated the expression of BdCPCFC in vitro,through binding with sequences TCCTA of the BdCPCFC 3'untranslated region(UTR).These evidences proved that the BdmiR-994 could regulate the expression of BdCPCFC in B.dorsalis.4 Molecular mechanism of penetration resistance to malathion meditated by BdmiR-994/BdCPCFC in B.dorsalisBased on the expression pattern of BdmiR-994,the MR and MS strains of B.dorsalis were treated with BdmiR-994 mimic and inhibitor,respectively.After treating with BdmiR-994 mimic against MR strain,the cuticle thickness of the pronotum significantly reduced with 3.1?m,and the susceptibility to malathion was significantly increased with 30.0%.After treating with BdmiR-994 inhibitor against MS strain,the cuticle thickness of the pronotum was significantly increased with 1.1?m,and the susceptibility to malathion was significantly reduced with 23.3%.These results showed that BdmiR-994 regulates the pronotum cuticle thickness of B.dorsalis and affects the susceptibility to malathion,and play important roles in the malathion penetration resistance of B.dorsalis.The open reading frame of BdCPCFC contained 489 bp which encoded 162amino acids.Bioinformatics analysis revealed that it possessed 3 CPCFC family-specific C-X(5)-C conserved domains.The expression pattern analysis showed that the BdCPCFC significantly up-regulated in whole body of the MR strain with2.4-fold.It suggested that BdCPCFC might be related to the malathion resistance of B.dorsalis.Analysis of immunohistochemistry and immunogold staining revealed that the BdCPCFC enriched in the cuticle of the pronotum.Meanwhile,the chitin binding assay showed that BdCPCFC could effectively bind chitin resin directly,suggested that BdCPCFC might combine with chitin in the cuticle of the pronotum.After injection of ds BdCPCFC,the relative expression of BdCPCFC reduced with 43.5%,and the protein of BdCPCFC in B.dorsalis obviously reduced.After effectively silencing BdCPCFC,the cuticle thickness of the pronotum was significantly reduced with 2.1?m,and the mortality of B.dorsalis significantly increased with 30.0%post treatment with malathion 72 h.Taken together,BdmiR-994 modulated the pronotum cuticle thickness and participated in the penetration resistance of malathion via regulating the expression of BdCPCFC.This study enriches the research of insecticide penetration resistance,and provides ideas and theoretical basis for the development of new insecticides and divise strategy for resistant control of B.dorsalis in the field.