Molecular Mechanism Of Amitraz Resistance In Panonychus Citri

Monitoring and management of citrus pest mites is a key issue for citrus production regions around the world each year.Frequent outbreaks of citrus red mite,Panonychus citri（Mc Gregor）are particularly prominent in China,which is partially due to lack of effective monitoring and forewarning.In addition,effectiveness of chemical acaricides has been weakened as a result of mite resistance is a crucial reason.Resistance reports worldwide have suggested that P.citri has developed resistance to different classes of chemical acaricides,such as spirodiclofen,bifenazate,hexythiazox,pyridaben,abamectin,fenpropathrin,and azocyclotin.At present,the slow development of new acaricide with high selectivity has not meet the consistent demands of growers,which has led growers touse older acaricides to control P.citri.Amitraz is a broad-spectrum insecticide/acaricide against aphids,psyllids,ticks,and mites.In recent years,it has been widely used to control P.citri.According to the Insecticide Resistance Action Committee（IRAC）classification system,amitraz is considered to be the octopamine receptor.The latest research showed that amitraz resistance can include multiple mechanisms,such as mutations inβ-adrenergic octopamine receptor and octopamine/tyramine receptor and changes in the enzymes activity of monoamine oxidases（MO）or ATP binding cassette transporter（ABC transporters）.In order to better monitor and delay amitraz resistance in P.citri and provide the theoretical basis for resistance management,a amitraz-resistant strain of P.citri was obtained in indoors.Resistance mechanism was further studied with biochemistry,genetics and molecular biology.The main results obtained in this study are as follows:1.Inheritance patterns of amitraz resistance in P.citriAn amitraz-resistant strain of P.citri was obtained by indoor selection.The amitraz-resistant strain of P.citri had an LC₅₀ value of 2361.45 mg L^-1.The resistance ratio was 81.35 times higher in the resistant strain of P.citri compared with the sensitive strain.Crossing experiments between the sensitive and resistant strains of P.citri were conducted,resulting in a D value of 0.11 for F_{1SS♀×RS♂}and 0.06 for F_{1 RS♀×SS♂}.Reciprocal cross experiments showed that the dose-mortality curves for the F₁generations coincided,indicating that the resistance trait was not affected by cytoplasmic inheritance.The dose-expected response relationship was evaluated in the backcross generation and a significant difference was observed compared with the actual value.The above results indicated that the inheritance of resistance trait was incompletely dominant,governed by polygenes on the chromosome.2.Whole genome sequencing of P.citriIn this study,the genome survey map of P.citri was completed by the second-generation Illumina Hiseq sequencing platform,and the high-quality assembly and annotation of the P.citri whole genome was also finished with the third generation Nanopore sequencing platform.The total assembly size of the genome is about 83.97Mb（Gene Bank Accession Number:JAAABK000000000）,which contains 144 Contigs.Contig N50 is about 1.81 Mb,GC content is 31.33%,and Gap total length is 0.Since the Contig N50 is large enough,it is not performed Scaffold assembly.The sequencing reads alignment from Illumina Hiseq,BUSCO integrity assessment,and CEGMA integrity assessment revealed that the genome assembly was of good quality.11,577genes were assembled,with an average gene length of 3924.13bp,an average exon length of 2003.09bp,and an average intron length of 1921.04bp.There are 10,940 genes were annotated in GO,KOG,KEGG,Swissprot,nr and other databases.3.Bulked segregant analysisThe genome contains five octopamine receptor-related genes:EVM0004493.1,EVM0000696.1,EVM0005310.1,EVM0003022.1,and EVM0010902.1.Phylogenetic analysis showed that they were clustered with three types of octopamine receptors:α-adrenergic-like,β-adrenergic-like and OCT/Tyr.No I/F nonsynonymous mutation reported in the literature were found in the receptor from sensitive and resistant strains of P.citri,but EVM0010902.1 and EVM0004493.1 genes were differentially expressed among them,and the expression levels in resistant strains were lower than those in sensitive strains.The bulked segregant analysis was performed on parents and offspring which with extreme traits through genomic resequencing technology.A total of 76.86Gbp data（SRA accession:PRJNA600137）was obtained,with an average coverage depth of 205.50×.There were 66,718 SNPs were found between sensitive and resistant parents and 68,285 SNPs were found between sensitive and resistant offspring.The association analysis between SNPs and traits was performed by two methods,SNP-index and ED.38 trait-related SNPs shared by parents and offspring were screened,7 of that at contig00002 and 31 at contig00033.These SNPs contained 29NON＿SYNONYMOUS＿CODING and 9 UTR＿5＿PRIME.Among them,Among them,the SNP most relevant to the resistant trait was located in the EVM0004493.1 gene.Gene annotation revealed that the gene wasβ-2R octopamine receptor,the SNP was located at the 752 base of the 5′UTR region,and the T base was mutated to the C base（T752C）.The prediction of the secondary structure of m RNA revealed that,after the base mutation,one long stem loop changes to three short hairpin structures in the upstream region ofβ-2R octopamine receptor m RNA.The T752C mutation also existed in the field population of P.citri,and there was a correlation between the resistant ratio and the mutation frequency,with a correlation coefficient of 94.40%.4.Screening of detoxification-related genes in amitraz-resistant strain of P.citriSynergism studies demonstrated that cytochrome P450s and esterase may play important roles in the detoxication of amitraz and glutathione S-transferases（GSTs）have little effect on that.The metabolism-related genes were screened at the genome level.It was found that the genome of P.citri contains 54 P450s,20 GSTs,46carboxylesterase family genes,92 ABC transporters,80 major facilitator superfamily,16 lipocalins and 39 uridine diphosphate glucuronyl transferases.Based on differential gene analysis,23 metabolism-related genes of P.citri were identified,including P450s,GSTs,CCEs,and ABC transporters.No GSTs were found,which isconsistent with the results of synergism studies.Real-time PCR verification implied that P4504C1（c22254.graph＿c0）,ABCG23（c23311.graph＿c0）,and Ach E4（c17984.graph＿c0）might influence detoxification of amitraz in P.citri.5.Functional verification of candidate metabolic genesFunctional verification of three metabolic candidate genes,P4504C1,Ach E4 and ABCG23,was performed by RNAi and eukaryotic protein expression systems.The results showed that the relative expression level of P4504C1,Ach E4,and ABCG23m RNA was decreased by 42.90%,33.37%and 70.56%,respectively,compared with the control group,and the corrected mortality rate of P.citri increased by 15.07%,7.59%,and 16.26%,respectively,suggesting that these genes have played a certain role in the metabolism and detoxification of amitraz.Two expression vectors were successfully constructed and two proteases,P4504C1 and Ach E4,were successfully synthesized in vitrothrough the Sf21 cell-free expression system of Spodoptera frugiperda,which can be used for in vitro metabolism experiments of amitraz in the future.