| The green peach aphid, Myzus persicae (Sulzer) (Hemiptera:Aphididae), is one of the most economically important crops pest insects worldwide. It causes economic damage both through direct feeding and virus transmission. The control of M. persicae has been primarily dependent on the application of insecticides in China. M. persicae has developed different levels of resistance to many insecticides such as organophosphates, carbamates, pyrethroids, and neonicotinoids due to their multiple and heavy applications.In this study, firstly, the insecticide resistance to various field populations of M. persicae in China has been monitored by bioassays continuously from 2011 to 2014 with ten insecticides. The resistance mechanism of field populations of M. persicae in China was detected by synergistic assays, biochemistry and molecular biological method. Secondly, the resistance variation of M. persicae to nine insecticides was accessed following spraying cypermethrin or acetamiprid in ten field trails. Thirdly, sublethal effects and resistance risk of sulfoxaflor on M. persicae were assessed using life table parameters. Lastly, the genetic diversity and variability of field populations of M. persicae in China were studied using microsatellites. The main results are as follows:1. Sixty-two field populations of M. persicae collected from 11 provinces or municipalities in China have been continuously monitored by leaf-dip bioassays with ten insecticides (beta-cypermethrin, cypermethrin, deltamethrin, bifenthrin, imidacloprid, acetamiprid, methomyl, pirimicarb, omethoate, and sulfoxaflor) from 2011 to 2014. Resistance factor (RF) is calculated based on the baseline data tested with a relative susceptible strain of M. persicae in our lab. The results showed that most field populations of M. persicae have produced high level resistance to beta-cypermethrin (RF=2.69-472) with more than 85.5% of populations exhibited high level resistance. High level resistance to cypermethrin (RF=15.3-123), deltamethrin (RF=4.1-139.3), bifenthrin (RF= 1.0-306.5) and pirimicarb (RF=0.09-102), omethoate (RF=6.6-279) and methomyl (RF=0.57-134.2) have produced in 72.1%, 55.2%,19.1%,50%,30% and 21.7% of field populations of M. persicae, respectively. For imidacloprid (RF=0.3-2853),32.8% of all field populations were susceptible,34.4% of field populations were in moderate level resistance, and 6.6% of field populations were in high and extremely high level resistance. Similarly, for acetamiprid (RF=0.03-168.7), more than 41.9% of field populations were susceptible, and the moderate level resistant populations account for 24.2%, and 11.3% of field populations exhibited high and extremely high level resistance. All of the field populations of M. persicae investigated in this study were susceptible to sulfoxaflor with RF<1. Each field population of M. persicae investigated in last four years has developed moderate or high levels of resistance to nine insecticides in total. However, there are some differences of insecticide resistance among different areas; on the whole, populations of M. persicae collected from East China and North China exhibited higher level of resistance than the populations collected from Southwest China and Northwest China.2. Biochemical and molecular resistance mechanisms of M. persicae were further studied by synergist experiments, individual esterase activity test, and detection of esterase genes amplification and L1014F mutation (kdr). The results are as follows:(1) According to synergistic assays in field populations of M. persicae, Piperonyl butoxide (PBO) has obvious synergism to β-cypermethrin, cypermethrin, omethoate, pirimicarb and imidacloprid and their highest synergic ratios were varied from 1.82 to 23.3. Diethyl maleate (DEM) increased pirimicarb toxicity 1.38 fold and S, S, S-tributyl phosphorotrithioate (DEF) increased β-cypermethrin toxicity 1.34 fold in a mixed field population (2012-Mix).(2) Individual esterase activity test in 32 field populations exhibited that esterase overexpression were detected in 30 field populations, and the average esterase activities of these populations (16.83-50.72 mOD/min/individual) were significantly higher than that in a susceptible population (11.39 mOD/min/individual). The esterase activities only in Beijing population (16.12 mOD/min/individual) and Gucheng population (16.11 mOD/min/individual), exhibited no significant differences as compared to that in the susceptible population.(3) All the 45 field populations and the susceptible population analyzed possess amplified FE4 gene only, no amplified E4 gene was detected in China by now. The frequencies of FE4 gene amplification in field populations (25-100%) were significantly higher than that in susceptible population (5%). All FE4 genes amplified in 29 field populations were found and the rest 16 field populations posses FE4 gene amplification frequencies of 25-97.5%.(4) The L1014F kdr mutations in sodium channel were detected in 45 field populations. Among them, ten field populations possess all genotypes of SS homozygotes without L1014F mutations,30 field populations possess L1014F mutations with 2.7-96.73% genotypic frequencies of RS heterozygotes, and five field populations possess L1014F mutations with 100% genotypic frequencies of RS heterozygotes. L1014F mutations of all individuals detected were heterozygous mutations, the genotypes were RS, and genotypic RR homozygous mutant was not found in these field populations.These results mentioned above showed that combined effects of multiple resistant mechanisms, such as enhancement of detoxifying enzyme activities of mixed function oxidase (MFO) and carboxylesterase, FE4 gene amplification, and L1014F kdr mutation, could result in field populations of M. persicae generated various resistance levels to pyrethroids, organophosphates and carbamates in China.3. The resistance variation of M. persicae to nine insecticides in ten field trails after cypermethrin or acetamiprid sprayed was assessed by leaf-dip method. The results indicated that a cypermethrin spraying in field has no significantly effect on the resistance of M. persicae to nine insecticides in Kuancheng, Dalian, Suizhong and Qingshen trail field populations. However, in the rest 6 trail fields, following spraying cypermethrin, an enhancement on the resistance of M. persicae to one to two insecticides was observed in 2012.4. Exposure of the parent generation of M. persicae to sublethal concentration (LC25) of sulfoxaflor has no significantly effects on the longevity and fecundity of adults in F0; similarly, the developmental period of each instars of nymph, longevity of adults and adult preoviposition period of F1 generation were not significantly stimulated. However, the exposure of the parent generation of M. persicae to sublethal sulfoxaflor induced increase of pre-adult and total preoviposition period of F1 generation, and finally induced increase in reproduction. Both Ro and GRR of aphids for treatment group were significantly higher than for the control in F1 generation, the mean generation time and TPOP were significantly postponed in treated group. These results suggest that the exposure of the parent generation of M. persicae to sublethal sulfoxaflor has no stimulate effect on the parent generation, whereas, a hormesis was induced by lower concentration of sulfoxaflor in the first progeny generation. It suggests that the sublethal concentration of sulfoxaflor possesses a potential to induce hormesis of M. persicae, and praying of sulfoxaflor in fields has potential risk of causing insecticide resistance and resurgence of M. persicae.5. In this study, ten pairs of microsatellites primers were screened to investigate the genetic variability of eight M. persicae geographic populations, which were collected from seven provinces in China. All investigated M. persicae populations exhibited high genetic diversity. The populations of Kuancheng, Qingdao and Xiqing showed highest diversity, the populations of Ningde, Yixing, Guiyang and Yancheng exhibited moderate diversity, and the lowest genetic diversity was detected in Chengdu population. Our results indicated that the genetic differentiation between eight populations was very high:Kuancheng and Yixing population showed the lowest genetic differentiation with a small pairwise FST value (0.0714). The pairwise FST value between the Qingdao and Kuancheng populations, Qingdao and Yixing populations were 0.1061 and 0.1090, respectively, and the genetic differentiation was moderate. The highest level of differentiation was detected between Chengdu and Yancheng populations (FST=0.1061). The genetic differentiation was mainly within individuals in eight populations, which accounted for more than 95.71%, and the average gene flow was lower than 1 (Nm=0.7343), which could not counteract the divergence caused by genetic drift. The highest Nei’s genetic distance (DA) was 0.6257, which was found between Chengdu and Yancheng populations, and the DA between Kuancheng and Yancheng populations was the lowest between eight populations (DA=0.1563). |
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