Resistance Mechanisms To Chlorpyrifos In Apolygus Lucorum And The Construction Of CRISPR/Cas9 Gene Knockout System

Apolygus lucorum(Heteroptera:Miridae)is an important agricultural pest,which causes serious loses in economic crops including cotton,vegetables and fruit trees.Mirids have historically been considered as the minor pests of cotton and other crops in China,but since the adoption of Bt-cotton they have gradually become the major pests of cotton.The control of mirids in Bt-cotton relies heavily on the use of insecticides.Chlorpyrifos is an organophosphorus insecticide with contact and stomach action.It has been widely used to control A.lucorum due to its effective and broad spectrum of activity.However,the long-term and large-scale over-utilization of chemical pesticides causes insecticide resistance.To clarify the resistance mechanism to chlorpyrifos in A.lucorum,the target and metabolic resistance mechanism to chlorpyrifos were investigated in this study.The contribution of ace-1 A216S mutation to chlorpyrifos resistance was clarified and several potential detoxification enzyme genes associated with chlorpyrifos resistance were screened.These results are helpful to the improvement of resistance monitoring technology and the optimization of resistance management strategy for delaying resistance evolution of A.lucorum to chlorpyrifos.Moreover,functional genomic studies on A.lucorum are seriously constrained by lack of genetic tools.Here,the eye pigmentation gene Al-white was successfully knocked out by CRISPR/Cas9 technology in A.lucorum and thus provides basis for the research of functional genomics in non-model insects of Miridae.The results are as follows:1.Field resistance monitoring of A.lucorumTo investigate insecticide resistance levels and development trend of resistance in different A.lucorum populations,the monitoring of field resistance to 6 chemical insecticides was conducted.Samples of A.lucorum populations were collected from the Yellow River region and the Yangtze River region.During 2015 to 2018,the populations from the Yellow River region have developed low to moderate levels of resistance to chlorpyrifos and beta-cypermethrin and the populations from the Yangtze River region are still susceptible or have low level of resistance to the two insecticides.In 2015,A.lucorum field populations were still susceptible to emamectin benzoate,but most populations have developed low to moderate levels of resistance to emamectin benzoate from 2016 to 2018,among which,the resistance ratio of Binzhou population from Shandong province(SD-BZ)were 12.4 and 14.4 in 2017 and 2018.During 2015 to 2017,A.lucorum field population were still susceptible to fipronil.During 2016 to 2018,except the Chuzhou population from Anhui province was still susceptible to imidacloprid,four other populations were all at the low to moderate resistance level,among which,Binzhou population from Shandong province(SD-BZ)and Yancheng population from Jiangsu province(JS-YC)had 10-fold resistance to imidacloprid.Most populations were still susceptible to sulfoxaflor,except e Anyang population from Henan province which has developed low level of resistance to sulfoxaflor in 2016 and 2018.The results above provide an important basis for the precise insecticide selection in field control of A.lucorum.2.Target mechanisms for resistance to chlorpyrifos in A.lucorumBZ-R is a resistance strain of A.lucorum to chlorpyrifos,which was developed from a field-collected strain(BZ)by continuous selection with chlorpyrifos.BZ-R showed 21and 58-fold resistance to chlorpyrifos at the 18th generation compared with BZ-S strain derived from BZ and the susceptible strain SLF.The resistance levels gradually increased to 41-and 113-fold at the 31st generation compared with BZ-S and SLF.Meanwhile BZ-R also showed low level cross-resistance to trichlorfon,malathion and carbosulfan.Acetylcholinesterase(AChE),as the target of chlorpyrifos,were investigated in this study.No sequence and mRNA expression level difference in AChE2(Al-ace2)were found to be associated with the resistance in BZ-R.However,the frequencies of an alanine to serine substitution at position 216 of AChE1(Al-acel)were 100%in BZ-R strain and 23%in BZ strain,respectively.After the treatment with the discriminating dosage of chlorpyrifos to BZ strain,frequency of the serine substitution increased to 64%from 23%,it means the correlation between Al-acel A216S mutation and resistance to chlorpyrifos.Genetic linkage analysis showed that A216S mutation was closely linked with resistance to chlorpyrifos in BZ-R strain.The mutant and wild-type Al-acel were expressed by baculovirus expression system in vitro.I50 of chlorpyrifos oxon to recombinant mutant protein AChE1 was higher about 5-fold than the wild-type.The result means the mutant is less sensitive to chlorpyrifos oxon.All these results above indicate that the target resistance to chlorpyrifos was mediated by A216S mutation of Al-ace1 in A.lucorum.The correlation analysis between chlorpyrifos resistance and S216 allele and genotype frequencies of Al-ace1 were conducted in 25 field populations from 5 geographic locations of China from 2014 to 2018.There is a statistically significant and positive correlation(0.81<r<0.87;p<0.01)between the resistant level in field populations and S216 allele and genotype frequencies of Al-ace1.It means that A216S mutation plays an important role in chlorpyrifos resistance in field populations of A.lucorum.3.Detoxification mechanisms for resistance to chlorpyrifos in A.lucorumThe decreased target sensitivity due to A216S mutation of Al-ace1 was the main reason for the chlorpyrifos resistance in BZ-R strain,but other minor factors were also involved.The detoxification metabolic enzymes were inspected in BZ-R strain.The synergistic effect detection showed that cytochrome P450 monooxygenase inhibitor PBO and esterase inhibitor DEF had no obvious synergism on chlorpyrifos in susceptible strain SLF and background strain BZ-S,PBO and DEF had significant synergism on chlorpyrifos in the resistant strain BZ-R,and glutathione S-transferase inhibitor DEM had no synergistic effect in these three strains.Enzyme activity test showed that cytochrome P450 monooxygenase and esterase activity in resistant strain BZ-R were significantly increased.Compared to susceptible strain SLF and BZ-S,the cytochrome P450 monooxygenase activity were increased 7.4-and 6.1-fold and esterase activity were increased 2.0-and 1.7-fold in BZ-R strain,respectively.There were no significant difference of glutathione S-transferase activity in three strains.Furthermore,several potential detoxification enzyme genes associated with chlorpyrifos resistance were identified from the transcriptome data of A.lucorum.The mRNA expression levels of detoxification genes between SLF,BZ-S and BZ-R were compared by the quantitative RT-PCR.Three P450 genes CYP395L1,CYP3092C4,CYP6X2 and three esterase genes were significantly overexpressed.Combining synergism and enzyme activity data,it is suggested that the over-expression of multiple detoxification genes plays a role in chlorpyrifos resistance A.lucorum.4.The construction of CRISPR/Cas9 gene knockout system in A.lucorumCRISPR/Cas9 technology is a powerful tool for the functional genomic studies.However,application of this technology has not been reported in A.lucorum.Based on the transcriptome,an eye pigmentation gene Al-white was cloned,and then been as the target gene to be knocked out using CRISPR/Cas9 system in A.lucorum.This study showed that two sgRNAs of the Al-white gene of A.lucorum can separately induced insertion and deletion(indels)in the G0 generation,there were 26-30%individuals with mosaic eyes consisting of colorless area in compound eye as result of white and lightly pigmented ommatidia.Then the mutations induced by one sgRNA were heritably transmitted to the following G1 generation.This study found that there is no knockout homozygote of Al-white in G1 and G2,but 28-31%individuals of heterozygote with inserting 2 bp at exon2 showed mosaic white eye phenotype.These results show that CRISPR/Cas9-mediated gene editing is achievable in A.lucorum,offering a technological reserve for the study of functional genomics in mirid insects.