| The bird cherry-oat aphid, Rhopalosiphum padi(L.)(Hemiptera: Aphididae), is one of the most important wheat pests worldwide. This aphid damages through direct feeding or by transmitting the Barley yellow dwarf virus(BYDV). Both types of damage significantly reduce the quality and yield of wheat crops globally. Imidacloprid is a neonicotinoid insecticide with the characteristics of broad spectrum, high efficiency, low toxicity and low residue. Meanwhile, Imidacloprid has a strong toxicity, stomach poisoning and high active absorption on insect pests, especially shows high efficiency in control of sucking pests including aphids, planthoppers, leafhoppers and whiteflies. In China, Imidacloprid has been widely used in the control of wheat aphids. With the intensive use of this chemical, many insect pests have developed resistant to imidacloprid. From the results of recent insecticide resistance monitoring, R. padi showed resistance to imidacloprid in some regions of China. So far, resistance mechanisms of R. padi to imidacloprid have not been reported.In the current study, we set up R. padi strains which are relative susceptible or resistant to imidacloprid in laboratory. The relative fitness and cross-resistance of the resistance strain was analyzed. Detoxifying enzyme activities of susceptible and resistant strains were determined. Eight nicotinic acetylcholine receptor subunit genes were successfully cloned from R. padi. Expression level of nicotinic acetylcholine receptor subunit genes and P450 genes in R. padi strains with or without exposure to imidacloprid were analyzed. Polymorphisms of the nicotinic acetylcholine receptor subunit genes in the resistance strains and susceptible strains as well as in 12 field R. padi populations were also analyzed. The main results are as follows:1. Imidacloprid-resistance selection of R. padi and relative fitness of the resistant strainThe resistance ratio of R. padi to imidacloprid reached to 40.35-fold after 52 generations continuous selection with imidacloprid. The resistance development of R. padi to imidacloprid is relatively slow from F0~F24 generations, and developed faster after that. It is deduced that there is a risk of occurrence of resistance to imidacloprid after intensive use of the chemical to control R. padi in the field.Compared to the susceptible strain(Rp-S), the F28 and F52 of the resistant strain(Rp-R) had obvious disadvantages in the survival rate and reproduction. The development duration of nymph was prolonged, longevity of adult was shortened, the net reproduction rate(R0), the finite rate of increase(λ) and the intrinsic rate of increase(rm) of the resistant strain were lower than the susceptible strain. The the intrinsic rate of increase(rm) was used to measure the relative fitness of resistant strain. The result showed that the relative fitness of the resistant strains F28 and F52 were 0.994 and 0.886, respectively. The resistant strain showed reproduction and development disadvantage.2. Cross-resistance of the imidacloprid-resistant strain of R. padiThe cross-resistance of the R. padi Rp-R strain to six insecticides commonly used in the field was measured. The results showed that R. padi with 40.35-fold resistance to imidacloprid had obvious cross-resistance to the neonicotinoid insecticides thiamethoxam(the resistance ratios was 10.72-fold), acetamiprid(6.21-fold) and carbamate insecticide isoprocarb(6.30-fold), had low cross-resistance to organophosphorus insecticides malathion(3.42-fold) and chlorpyrifos(2.48-fold), but no obvious cross-resistance to lambda-cyhalothrin(1.35-fold).3. Biochemical mechanisms of R. padi resistance to imidaclopridThe biochemical mechanisms of imdacloprid resistance in R. padi were studied by testing the synergism effects of three enzyme inhibitors(TPP, DEM and PBO) on imidacloprid and the main detoxifying enzyme activity in F52 of Rp-R strain and the susceptible strain Rp-S. Both the results of synergism test and detoxifying enzyme activity analysis showed that enhanced cytochrome P450-monooxygenases detoxifieation and carboxylesterase might contribute to the resistance of R. padi to imidacloprid.4. Cloning of nAChR subunit genes from R. padiThe full lengths of seven α-subunit(Rpα1, Rpα2, Rpα3, Rpα4, Rpα5, Rpα7-1, and Rpα7-2) and one β-subunit(Rpβ1) genes from R. padi were obtained with RT-PCR and RACE techniques. Sequence analysis showed that these genes had all the characteristics of the nAChR gene family. Alternative splicing was detected in Rpα3 and Rpα5 subunits. Phylogenetic analysis indicated that the protein sequences of R. padi nAChR subunit genes were highly homologous with those of the corresponding protein sequences of nAChR genes from other insects, such as Acyrthosiphon pisum, Myzus persicae, Aphis gossypii and Sitobion avenae. There is no amino acid mutation nAChR genes in resistant strain of R. padi, indicating that the mutation of nAChR genes was not a main mechanism of the Rp-R strain resistance to imidacloprid.5. The polymorphisms analysis of the Rpβ1 subunit extracellular region in R. padi field populationsTo analyze the polymorphisms of the Rpβ1 subunit gene in different field samples, the extracellular regions of the Rpβ1 gene that contribute to imidacloprid binding were sequenced from 120 R. padi samples collect in 12 regions of 11 provinces in China. Seventeen single nucleotide polymorphism(SNP) sites in the extracellular regions of the Rpβ1 subunit were found in the field samples, seven of which led to amino acid polymorphisms(V53I, V53 G, N54 T, A60 T, F61 L, W79 C, and V83I). No nucleotide polymorphism(SNP) sites were found in the laboratory samples. Two(W79C and V83I) of the seven amino acid polymorphism sites were located in the loop D region of the nAChR β1 subunit. Further studies are needed to confirm whether the SNPs discovered in this study are associated with resistance to neonicotinoid insecticides in R. padi.6. The relative expression of nAChR genes in imidacloprid resistant and susceptible strain of R. padiThe expression levels of nAChRs genes in imidacloprid resistant and susceptible strains of R. padi were detected by the real-time quantitative PCR. The results showed that eight nicotinic acetylcholine receptor subunits genes was all expressed in both imidacloprid resistant strain and susceptible strain, but the expression levels was different. The expressions of Rpα1, Rpα2, Rpα3, Rpα7-2 and Rpβ1 subunits genes in the imidacloprid resistant strain of R. padi was significantly lower than that of the susceptible strain. The expression profiles post-exposure to LC50 imidacloprid for 12 h, 24 h, 36 h and 48 h were investigated using quantitative RT-PCR. The results showed that the expression levels of eight receptor subunit genes varied in changes when post-exposure to imidacloprid.7. The relative expression of P450 s genes in imidacloprid resistant strain and susceptible strain of R. padiThe relative expression levels of six P450 genes in imidacloprid resistant and susceptible strains of R. padi were detected by the real-time quantitative PCR. The results showed that CYP6CY3-1, CYP6CY3-2 and Unigene 10092(CYP4 family gene) was significantly up-regulated in imidacloprid resistance strain, with 2.24, 2.23 and 2.91 times of the expression level in the susceptible strain, respectively. The results suggested that the CYP6CY3-1, CYP6CY3-2 and Unigene 10092(CYP4 family gene) might play important roles in resistance of R. padi to imidacloprid. The expression profiles of six P450 genes(CYP6CY3-1, CYP6CY3-2, Unigene 12972, Unigene 3757, Unigene 3094 and Unigene 10092) of the apterous aphids post-exposure to LC50 of imidacloprid for 12 h, 24 h, 36 h and 48 h was investigated using real-time quantitative PCR. The results showed that the expression levels of the six P450 genes were all increased after exposure to LC50 of imidacloprid, and the transcription levels of six P450 genes reached their peak levels at 12 h. After 12 h, the expression of six P450 genes gradually fell. At 48 h, the transcription levels of six P450 genes were not significantly difference between the treatments and control. |
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