| Brown planthopper is one of the main pests on rice in China and other Asian countries.Because of its easy migration and strong reproductive ability,brown planthoppers directly cause a reduction of about 1.2 million tons in rice production in China each year.At present,the control of brown planthoppers is mainly chemical,but with the long-term and large-scale use of insecticides,brown planthoppers have developed resistance to most pesticides.With the fipronil being banned in rice fields in 2009 due to the high toxicity to bees,pymetrozine has become the preferred agent for controlling brown planthoppers.It has poor quick-acting properties in the field,but it has a long duration of effectiveness,and its control effect on brown planthoppers can reach more than a month.However,with the extensive use of this agent,the monitoring results with the rice stem dipping method found that the brown planthopper had a high level of resistance to pymetrozine in 2012,and the control effect of the field also declined significantly.Unlike the toxicological mechanism of most pesticides before commercial application,the target of pymetrozine(Nanchung-Inactive complex protein)was not discovered until 2015.However,the specific toxicity mechanism of pymetrozine has not been completely revealed.What kind of resistance mechanism is involved in the resistance shown by the short-term control effect of pymetrozine on brown planthopper?These issues need to be further studied.The research contents were as follows:1.The effects of pymetrozine on reproductive behavior of brown planthoppersPymetrozine,as the main agent for the control of brown planthoppers,shows the effect of inhibiting the population of the next generation of brown planthoppers in the field,but the specific toxicological mechanism of pymetrozine for controlling the population of pests is unknown.In this chapter,the No.005 method recommended by the Insecticide Resistance Action Committee(IRAC)is used to confirm that pymetrozine can inhibit the fecundity of brown planthoppers.The number of offsprings of brown planthoppers treated with pymetrozine obviously decreased,and the inhibitory effect grew when the concentration of pymetrozine increased,which showed a certain dose effect between the concentration of pymetrozine and the inhibitory effect and the inhibitory effect grew when the concentration of pymetrozine increased,which showed a certain dose effect between the concentration of pymetrozine and the inhibitory effect.In order to determine whether pymetrozine affects the mating mechanism of brown planthoppers,we systematically divided the mating behavior of brown planthoppers in 8 steps:abdominal vibration,following,positioning,wing extension,attempted copulation,copulation,terminated copulation and leaving.Further analysis showed that PM mainly inhibited courtship initiation such that 83.3%of males do not initiate courtship and 76.7%of females do not vibrate the abdomen within the 30 min observation time.In addition,the number of eggs laid by mated brown planthoppers treated with pymetrozine also decreased significantly,to 42%of untreated females.As a model insect,Drosophila melanogaster has been widely used in the study of pesticide toxicology and resistance.So that we also observed the effect of pymetrozine on it,its male courtship index,female receptivity and fecundity were significantly reduced after treated with pymetrozine.Compared with wild-type Drosophila,the courtship index of male nan36a mutant Drosophila males,female receptivity and fecundity also decreased significantly.After we heterologously expressed the brown planthopper NlNan gene in the nan36a mutant fruit fly,the reproductive behavior defect of the mutant fruit fly was rescued,but pymetrozine could interfere with its reproductive behavior again.These results confirm that pymetrozine reduces the number of offsprings by interfering with mating and spawning behavior,and also shows that the NINan gene plays a crucial role in the reproductive behavior of brown planthoppers.2.Study on the target resistance mechanism of brown planthopper to pymetrozineDue to the unique insecticidal activity,pymetrozine became the leading agent of brown planthopper control after BPH developed resistance to imidacloprid.With the large-scale use of pymetrozine,the effect of pymetrozine on the brown planthopper field population in 7 days was reduced by more than 10 times in 2012,and the test insects at the same dose almost survived for 7 days after 2013,indicating that the brown planthopper in the field has developed resistance to pymetrozine.This chapter attempts to explore the target resistance mechanism of brown planthopper to pymetrozine.First,we cloned pymetrozine target genes(NlNan and NlIav)from different populations of brown planthoppers.By comparing the target gene sequences,we found that there is an amino acid substitution on the NlNan gene(the 504th amino acid in the coding region is mutated from arginine to glutamine).The mutation frequency of this site in susceptible strain is 0,and this mutation site is found in the pymetrozine-selected strain and other field populations.After screening of pymetrozineresistant strain,the frequency of homozygous mutation of NlNan-504Q increased from 20.83%to 43.24%.To further verify whether brown planthopper NlNan-R504Q is involved in resistance to pymetrozine,we co-expressed NlNan-504R or NlNan-504Q with Iav in Xenopus oocytes,and the electrophysiological techniques were used to compare the current differences between NlNan-504R and NlNan-504Q after stimulation of pymetrozine.The results showed that their EC50 were 55 nM(33-91 nM)and 95 nM(52-170 nM),respectively.In addition,we used genetic methods to express brown planthoppers NlNan-504R and NlNan504Q in nan36a mutant flies,and evaluated the effects of pymetrozine on the climbing ability of the fruit flies.The results showed that the IC50 of pymetrozine for them was 10.18 ?M and 9.12 ?M,respectively.Our experimental results show that although brown planthoppers are resistant to pymetrozine,both in vivo and in vitro results have confirmed that NlNan-R504Q is only a single nucleotide polymorphism,and no effective mutation site has been found to mediate the resistance to pymetrozine.3.Transcriptome analysis of pymetrozine-resistant populations of brown planthopperThe above studies indicate that no effective amino acid substitutions were found to mediate resistance to pymetrozine in brown planthoppers during the detection of target mutations.However,the short-term control effect of pymetrozine on brown planthoppers has decreased significantly,so there must be other resistance mechanisms.The development of transcriptomics has provided new ideas for the study of insecticide resistance mechanism.In order to study the resistance mechanism of brown planthopper to pymetrozine,we have performed high-throughput sequencing on brown planthopper susceptible strain,pymetrozine-selected strain,and two field populations.After the original sequencing data being filtered,the sequencing error rate of reads was as low as 0.03;the base recognition rate of about 97%of the sample data was as high as 99%;in addition,the GC content of each sample remained at 45%,which remained basically unchanged.Through Pearson correlation and principal component analysis,we found that the brown planthopper samples of different populations had good repeatability,and there were obvious differences between the groups.During the differential expression gene screening process,compared with the susceptible strain,the pymetrozine-selected strain had 549 genes up-regulated and the 516 genes downregulated;the SG18 population had 438 genes up-regulated and the 323 genes downregulated;the TC18 population had 471 genes up-regulated and the 557 genes downregulated.The heat map shows that the biological expression of the differentially expressed genes in the same population is very good.In addition,compared with the differentially expressed genes in the susceptible strain,the fold change of the differential genes in other populations is very significant whether they are up-regulated or down-regulated.GO and KEGG functional enrichment analysis showed that these differential genes were mainly enriched in metabolic pathways.Subsequently,q-PCR technology was used to confirm that three P450 genes(CYP301B1?CYP304H1v4 and CYP6CS1),two solute carrier genes(SLC1 and SLC2),two cuticular protein genes(CPR95 and CPR96),one esterase gene,and one unknown functional gene(LOC111060640)were overexpressed in the pymetrozine-selected strain,SG18 and TC18 field populations.In summary,the transcriptomics analysis not only described the expression of different genes of brown planthopper,but also provided clues for the potential genes involved in pymetrozine resistance.4.Study on the biochemical resistance mechanism of brown planthopper to pymetrozineThrough transcriptome analysis,we found that three P450 genes were up-regulated in the pymetrozine-selected strain.Studies have shown that P450s are involved in insect resistance to a variety of insecticides,among which CYP6CM1 in Bemisia tabaci can hydroxylate pymetrozine.Whether metabolic resistance exists is still unknown.The metabolic resistance mechanism of BPH to pymetrozine was investigated in this chapter,we first evaluated the synergistic effects of three synergists(PBO,DEM,and TPP)on pymetrozine in brown planthopper susceptible strain and pymetrozine-selected strain.The inhibitor PBO had a significant synergistic effect on the pymetrozine-selected strain of brown planthopper,with a synergistic ratio of 2.83;DEM(SR=1.98)also had certain synergistic effect.Enzyme activity measurement showed that the activity of the multifunctional oxidase of the pymetrozine-selected strain was 1.7 times higher than that of the susceptible strain,suggesting that the resistance mechanism of the brown planthopper to the pymetrozine may involve multifunctional oxidase.In order to investigate which P450 genes may be involved in resistance to pymetrozine,we then compared the expression of P450 genes in the two brown planthoppers at the mRNA level by q-PCR technology.In the pymetrozine-selected strain,13 P450s genes were significantly up-regulated.Six P450 genes showed extremely significant differences(P<0.001).They were CYP301B1,CYP304H1v4,CYP4C61,CYP4C62,CYP6CS1,and CYP6ER1.The two genes,CYP6CS1 and CYP301B1,had the most significant up-regulation,which were 6.1 and 6.7 times higher than that of the susceptible strain,respectively.CYP6CS1 was also significantly upregulated in different field populations,including GXNN19,GDSG19 and JXSG19,with expression levels exceeding three times higher than that of the susceptible strain.When the P450 genes of brown planthopper were overexpressed in Drosophila melanogaster by GAL48/UAS expression system,the transgenic fruit fly overexpressing CYP6CS1 significantly reduced its sensitivity to pymetrozine,which was 2.27 times that of its control;The transgenic Drosophila overexpressing Bemisia tabaci CYP6CM1 was only 1.64 times that of its control;however,there was no significant difference between the transgenic Drosophila with overexpression of CYP301B1 and the control group.In order to further explore the function of CYP6CS1,we used RNAi technology to silence the CYP6CS1 gene of the pymetrozine-selected strain and found that the survival rate of brown planthoppers injected with dsNlCYP6CS1 after treatment with pymetrozine significantly decreased.To sum up,multifunctional oxidase mediates resistance of brown planthopper to pymetrozine,and CYP6CS1 participates in the detoxification metabolism of pymetrozine. |
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