| Nilaparvata lugens is well known as a serious cosmopolitan pest on rice crop.The annually Chinese associated N.lugens losses was approximate an average of one million tons of paddy in recent years and N.lugens has been viewed as major yield constraints to rice production.Thus,insecticides have been viewed as necessary inputs to obtain more yield of rice.Due to intensive and extensive application of insecticides,it has evolved different levels of resistance to 31 compounds including organochlorines,organophosphates,carbamates,pyrethroids,insect growth regulator,neonicotinoids,phenylpyrazoles,and pyridine azomethine groups of insecticides.Currently,the number of case of insecticide resistance was 396 in N.lugens listed in the Arthropod Pesticide Resistance Database(APRD).Thus,insecticide resistance has been a major factor influencing N.lugens control and management.Nitenpyram belong to neonicotinoid insecticides,which is the third commercialized neonicotinoid nicotinic acetylcholine receptor(nAChR)competitive modulators.At present,nitenpyram is the primary insecticide for N.lugens control in paddy fields.Due to intensive application of this insecticide,moderate level of resistance to nitenpyram has rapidly evolved in field populations of N.lugens from China.Thus,a study of resistance monitoring,the discriminating concentration,cross-resistance,as well as inheritance mode,stability and metabolic mechanisms in laboratory-selected nitenpyram resistance strain was made to manage the resistance issues in N.lugens.1 Resistance monitoring and mechanism of field populations of N.lugensIn the present study,the LC50 values and the activities of the detoxifying enzymes of fifty-eight representative field populations of N.lugens were determined.The results showed that LC50 values of field populations of N.lugens varied from 0.45 to 6.44 mg/L,revealing that N.lugens has developed a moderate level of resistance(resistance ratio,RR= 2.4-33.9-fold)to nitenpyram.More importantly,the resistance levels of N.lugens to nitenpyram showed an increasing trend in the period 2011-2015.The activities of esterase(EST),glutathione S-transferase(GST),and cytochrome P450 monooxygenase(P450)were significantly different among the field populations of N.lugens from 2011 to 2015.The esterase activities varied from 1.29 ± 0.24(WX-2013)to 9.08 ± 1.41(TC-2012)?mol/min/mg protein,resulting in 7.0-fold variation among the N.lugens populations.The glutathione S-transferase activities also displayed 4.5-fold variation among the N.lugens populations,which varied from 4.42 ± 0.39(JS-2013)to 19.94 ± 1.72(ZJ-2015)nmol/min/mg protein.The activities of cytochrome P450 monooxygenase ranged from 0.37 ± 0.03(TM-2013)to 1.41 ± 0.35(FY-2015)nmol/min/mg protein with 3.8-fold variation.Furthermore,the pair-wise correlation analysis indicated significant positive correlations between the LC50 values of nitenpyram and the activities of detoxifying enzymes including the activity of cytochrome P450 monooxygenase(r= 0.394,P= 0.002)and esterase(r= 0.274,P= 0.037).Moreover,piperonyl butoxide(PBO)showed obvious synergism(Synergism ratio,SR= 1.6-2.1-fold)in the collected field populations.Obvious regional variation in nitenpyram susceptibility was detected among the field populations of N.lugens,suggesting that nitenpyram resistance has occurred in field populations of N.lugens in China,and the detoxification enzyme cytochrome P450 monooxygenase are likely a contributing factor to nitenpyram resistance in field populations of N.lugens.2 Biological characteristics study of resistance strain of N.lugens to nitenpyramThe Resistance strain of N.lugens was derived in the laboratory from a susceptible strain following 27 generations of continuous nitenpyram selection.After 27 generations of continuous selection,the resistance strain had evolved 144.7-fold resistance to nitenpyram.Stopping selection with nitenpyram led to a rapid decrease of resistance from 144.7-to 10.6-fold after 17 generations.After constant selections for 27 generations with nitenpyram,LC50 increased from 0.40 mg/L to 27.39 mg/L and the slope value was increased from 1.76 to 2.00 in the resistant strain.The estimated realized heritability(h2)of nitenpyram resistance was 0.12 in the resistant strain of N.lugens.Number of generations needed to increase 10-and 100-fold LC50 of nitenpyram at different selection pressure were expected to be 21?17?15?12?10 and 42?35?29?24?19,respectively.The result indicated that there is a risk to develop resistance to nitenpyram.Compared with the susceptible strain,the resistance strain showed a high level of cross-resistance to the five insecticides including clothianidin,dinotefuran,acetamiprid,sulfoxaflor and chlorpyrifos.No significant cross-resistance to other insecticides(imidacloiprid,thiamethoxam,thiacloprid,isoprocarb,buprofezin,etofenprox)was found.We investigated and compared the life-table of a high level of resistance strain(F27),a moderate level of resistance,an original strain and a susceptible strain of N.lugens,using the age-stage,two-sex life table approach.Compared to the susceptible strain,number of eggs,survival rate of egg,egg incubation period,the first,third and fourth instar nymph period,APOP,TPOP,longevity,Ro,r,l and the mean generation time(T)were significantly different in the strain of high level of resistance.In constrast,the duration of the egg stage,nymph stage(the first,second,fourth and fifth instar),survival rate of egg,APOP,TPOP,number of eggs,oviposition days,Ro,r,l and the mean generation time(T)were not significantly different in the moderately resistant strain.The strain of high level of resistance had a relative fitness of 0.567.The result of fitness cost illustrated nitenpyram resistance is not stable,and resistance may decrease after removal of the selection pressure.Thus,insecticide application tactics such as insecticide rotation,mixture of insecticides and reducing of insecticide application frequencies in controlling this devastating pest should be adopt to manage nitenpyram resistance.The genetic basis of inheritance of nitenpyram resistance was also studied by crossing the nitenpyram resistant and laboratory susceptible populations.Results revealed an autosomal and incompletely dominant mode of inheritance for nitenpyram resistance in the resistant population of N.lugens.Chi-square analyses of self-bred and backcross progenies(F2,F2' and BC respectively)rejected the hypothesis for a single gene control of the resistance.The results showed that nitenpyram resistance in N.lugens was autosomal and incompletely dominant trait,probably controlled by multiple genes.3 Transcriptome analysis of resistant and susceptible strains of N.lugensIn the study,transcriptomic databases of resistant and susceptible strains of N.lugens was established.Approximate 316469538 clean reads were obtained and then they were assembled into 122619 unigenes with mean size of 756 bp.These genes were annotated based on the databases including Nr(NCBI non-redundant protein sequences),Nt(NCBI non-redundant nucleotide sequences),Pfam(Protein family),KOG/COG(Clusters of Orthologous Groups of proteins),Swiss-Prot(A manually annotated and reviewed protein sequence database),KO(KEGG Ortholog database),GO(Gene Ontology)with gene description,gene ontology and clusters of orthologous group terms.146 unigenes of cytochrome P450 monooxygenase and 33 unigenes of nAChR were detected in the transcriptome database.52 P450 gene sequences and 3 nAChR gene sequences were identified using the tBLASTX.Gene expression levels were estimated by RSEM for each sample,and expression levels of 20 P450 genes were up-regulated and 22 P450 genes showed decreased expression.Moreover,only one subunit of nAChR(?6)had lower expression in resistant strain compared with susceptible strain.4 Genome-scale screen of nitenpyram resistance related P450 genes in N.lugensAssay of metabolic enzyme activities indicated that esterase specific activity of the resistant strain was 1.02 ?mol/mg pro/min,with a 2.0-fold higher than that of the susceptible strain(0.51 ?mol/mg pro/min).The cytochrome P450 monooxygenase activities of the resistant strain were 4.2-fold greater than those of the susceptible strain.GST activity of the resistance strain was significant 2.0-fold higher than that of the susceptible strain.The synergistic effects of PBO,TPP and DEM with nitenpyram against resistant and susceptible strains are performed for further demonstration of which detoxification enzyme contributed to nitenpyram resistance in N.lugens.A significant synergism was seen for PBO in the resistant strain(2.7-fold),but there was a no positive synergism effect in the susceptible strain.No synergy was seen for DEM and TPP in either the resistant or the susceptible strain.Thus,it showed that cytochrome P450 monooxygenases may be an important biochemical mechanism for nitenpyram resistance in N.lugens.The mRNA expression levels of cytochrome P450 monooxygenases were analysed and compared between susceptible and resistant strains of nitenpyram by qRT-PCR.The result indicated that the expression levels of CYP 6FL4,CYP 6CW1,CYP 439A1,CYP 4C78 and CYP 4DC1 in the resistant strain were significant higher than that in the susceptible strain.Thus,these genes may be associated with nitenpyram resistance in N.lugens. |
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