Molecular Mechanism Of MafB Regulating The Susceptibility Of Bactrocera Dorsalis (Hendel) To Avermectin

Bactrocera dorsalis?Hendel?,a destructive agricultural insect pest,is widely distributed in tropical and subtropical areas.The major management method is chemical control.Because of unreasonable insecticides application,the resistance of B.dorsalis against different insecticides has developed quickly.Recent studies have shown that the insecticide resistance is mainly related to the elevated expression of detoxification enzymes including glutathione s-transferases,cytochrome P450s,carboxylesterases and other metabolic enzymes.It has been well documented that transcription factors are important regulators of genes expression,regulating the expression of metabolic enzymes related to the insects sensitivity to insecticides.In the current study,the transcription factor BdMafB were identified based on the transcriptome and genome data of B.dorsalis.The BdMafB full-length was cloned.Real-time quantitative PCR?RT--qPCR?was used to analyze the temporal and spatial expression patterns of BdMafB gene and various detoxification enzyme genes.The expression patterns of BdMafB and various detoxification metabolic enzymes were further analyzed after avermectin treatment.RNAi and bioassay were used to explore the effect of BdMafB on the expression of detoxification enzymes and the susceptibility to avermectin.Our results are as follows:1 Molecular cloning and expression profiling of BdMafB in B.dorsalis1.1 Molecular cloning and sequence analysis of BdMafBAccording to the transcriptome database of B.dorsalis,the open reading frame of BdMafB was cloned and sequenced.The result showed that BdMafB ORF contained1323 bp and codes for 440 amino acids.The phylogenetic tree indicated that BdMafB was closely related to Mediterranean fruit fly and olive fruit fly.Structures and function domains of the deduced amino acid sequence of BdMafB was the predicted on NCBI web site.The results showed that BdMafB has two conservative domains of transcription factor Basic leucine and zipper?bZIP?family.There is a dimerization domain consist of nineteen residues and a DNA-binding site composed of thirteen residues,which can form a dimer and bind to the gene promoter to initiate transcription..1.2 Developmental and tissue expression patterns of BdMafBRT-qPCR was used to analyze the expression patterns of BdMaf B in different development stages and tissues of the fruit fly.BdMafB was expressed from egg stage to adult stage.With the age increased,transcription level of the gene increased continuously and the highest expression was observed in the 7-day-old adult.Meanwhile,BdMaf B expression levels were low in midgut,malpighian tubule but high in fat body with significantly differences.Fat body is the main tissue of detoxification,metabolism and energy supply.A large number of detoxification enzyme genes are expressed in fat body,which suggestes that BdMaf B may be involved in regulation of the detoxification enzyme gene in fruit flies.2 Avermectin treatment and functional analysis of BdMafB based on RNAi2.1 BdMafB responsive expression after avermectin treatmentThe 4-day-old adults were treated with avermectin at LC₄₀,and RNA was extracted at 12 h after the treatment.The expression level of BdMafB gene was detected by RT-qPCR.The results showed that the expression of BdMafB was significantly upregulated at 12 h after the treatment with abamectin LC₄₀.Howerer,the expression level of BdMafB was not significantly different from the control at 24 h and36 h.2.2 Susceptibility of B.dorsalis to avermectin after BdMafB konckdownAfter injecting dsBdMafB into the abdomen of the B.dorsalis,the expression of BdMafB was significantly downregulated at 24 h with the highest silenced efficiency of 88.4%.The mortality significantly increased to 50.4%at 24 h after BdMafB konckdown,while the mortality of control group was 36.9%.It suggested that BdMaf B was involved in the susceptibility to avermectin.3 Identification of BdMafB gene regulating downstream detoxification metabolic enzyme3.1 Spatial and avermectin induced expression patterns of detoxification enzymesThirty metabolic enzyme genes that may be regulated by BdMafB were considered as candidates of the downstream target genes.RT-qPCR was used to analyze the expression patterns of 30 metabolic enzyme genes in the fat body,malpighian tubule and midgut.The results showed that the transcription level of GSTd6 was the lowest in malpighian tubules among the tested tissues.The expression patterns of BdGSTz2?BdGSTe6?BdCYP437A3?BdCYP4AC4 and BdaE6 were consistent with BdMaf B,which are highest in fat body.Therefore,it was speculated that the expressions of BdGSTz2?BdGSTe6?BdCYP437A3?BdCYP4AC4 and BdaE6were related to BdMafB.After avermectin treatment,only the expression of BdGSTz2and BdCYP437A3 significantly increased at 36 h.The results suggested that the expression of BdGSTz2 and BdCYP437A3 might be regulated by BdMafB.3.2 Susceptibility of B.dorsalis to avermectin after BdGSTz2 and BdCYP437A3knockdownRNAi was used to silence BdMafB.RT-qPCR was used to investigate the transcription levels of BdGSTz2 and BdCYP437A3.After silencing BdMafB,the expression of BdGSTz2 and BdCYP437A3 were significantly downregulated at 24 h.Furthermore,silencing BdGSTz2 resulted in a significant increase of the susceptiblity of the fly to avermectin compared to the control,while silencing BdCYP437A3 did not.Our data demonstrated that BdMafB was involved in the modulation of the susceptibility of B.dorsalis to avermectin by regulating the of BdGSTz2.