Studys On The Mechanisms Of Imidacloprid Resistance In Cotton Aphid, Aphis Gossypii (Glover)

The cotton aphid, Aphis gossypii (Glover), is one of the worldwide pests on cotton. Because of the short generation time and the high fecundity of cotton aphid, coupled with the irrational use of pesticides in field, resistance of cotton aphid is complex. So far, serious resistance of A. gossypii to conventional insecticides has resulted in many problems in controlling of this pest in China. Since the early1990s, as the first commercialization product of neonicotinoid insecticides, imidacloprid was extensively used and had become the dominated pesticide to control cotton aphid. To delay the resistance to imidacloprid and prolong its service life in the field, the risk of high level resistance to imidacloprid was studied systematically with the laboratory selected strain. With selected resistant strain and susceptible strain, the biochemical and molecular mechanisms for the resistance were also analyzed. The results are as follows:1. The selection of imidacloprid-resistant strain of A. gossypii and the research of cross-resistanceThe resistance level of A. gossypii to imidacloprid reached to72.8-fold after selection of60generations with the pressure to kill60%to70%individuals of the population. From Fo to F54, the resistance of A. gossypii to imidacloprid developed slowly in the initial and middle stage of resistance selection. However, there was a sudden increase in F57, and the resistance ratio suddenly increased from47.0-fold to69.5-fold. By F57to F60, the resistance was elevated slightly. It proved that the development of resistance to imidacloprid of cotton aphid also showed "S" shaped curve similarly. The cross-resistance phenomena of RF60to nitenpyram (16.0-fold), acetamiprid (12.9-fold), buprofezin (11.6-fold), clothianidin (9.84-fold), dinotefuran (9.50-fold), carbosulfan (8.38-fold), lambda-cyhalothrin (6.17-fold), omethoate (6.13-fold), and chlorpyrifos (5.10-fold) were obviously, while the cross-resistance to pymetrozine and pyridaben at a low level (2.28-and4.52-fold, respectively). Biological research of imidacloprid between resistant and susceptible strains found that the honeydew excretion, boby weight, nymphal survival rate, net reproductive rate and intrinsic rate of natural had declined. In particular, the net reproductive rate of RF60was only47.7%compared with the susceptible strain. At the same time, the mean generation time extended about1.4days and the relative fitness decreased to0.71.The results of this study showed that the development of resistance to imidacloprid was relatively slow in the early stage, but the resistance of A. gossypii could increase suddenly after persistent imidacloprid selection, so the risk of high-level resistance still existed. Cross-resistance could significantly affect the effect of imidacloprid and most conventional insecticides, and only a small number of novel insecticides could use to be alternative pesticides. Imidacloprid conferred the fitness cost in A. gossypii, affecting its growth, development and reproduction, so the population growth of resistant strain could be inhibited.2. Studies of biochemical mechanisms of imidacloprid resistance in A. gossypiiThe biochemical mechanisms of imdacloprid resistance in A. gossypii were studied by testing the synergism of four enzyme inhibitors and the activity of related enzyme in resistant and susceptible strain of A. gossypii. The results showed that the synergism of PBO and TPP was singnificantly higher in the resistant strain than in the susceptible strain, especially when the synergism ratio of PBO was2.97. The synergism of SV1was relatively low, but DEM did not show any synergism on imidacloprid in both strains. Detoxifieation enzyme activity test in two strains showed that the activity of carboxylesterase (CarE) in resistant strain is much higher than the susceptible strain. However the activity of glutathione-S-transferase (GST) was similar in both strains. Furthermore, the activity of acetylcholinesterase (AChE) in resistant strain was a little higher than the susceptible strain. Thus, it was concluded that the enhanced carboxylesterase and cytochrome P450-monooxygenases detoxifieation might contribute to the high-level of imidacloprid resistance in A.gossypii. Acetylcholinesterase had also played a supporting role in the resistance.3. Studies of molecular mechanisms of imidacloprid resistance in A. gossypiiWe successfully cloned one carboxylesterase gene, two P450monooxygenase genes, five acetylcholine receptor a subunit and one β subunit from both resistant and susceptible strains. All genes we got were confirmed to belong to cotton aphid after homologous sequence alignment respectively. No mutation was found in the carboxylesterase and P450monooxygenase genes in the resistant strain, but the fluorescent quantitative analysis of three genes found that CYP6CY3-1and carboxylesterase gene overexpressed5.66-fold and 1.24-fold in resistant strain compared with the susceptible strain. Nevertheless, expression of CYP6CY3-2gene did not change obviously (only1.03-fold). The Aga2and Aga4subunit gene cDNA sequence we cloned in this study which contained the complete open reading frame were more complete than the A. gossypii nAChR a2and a4subunits cDNA sequences logged on NCBI previously. Comparison of the amino acid sequence of every subunit between the resistant and susceptible strains, a single mutation (R81T) in Agβ1subunit was observed from the resistant strain.Our results indicated that resistance to imidacloprid in the cotton aphid might be associated with an increase in cytochrome P450monooxygenase and carboxylesterase. The amino acid substitution (R81T) was the first time found in imidacloprid-resistant cotton aphids in this study, and the R81T was proven to significantly reduce the affinity between acetylcholine receptors and imidacloprid. Therefore, the R81T mutation led to the generation of high-level resistance in cotton aphid to imidacloprid directly.In summary, the risk of high-level imidacloprid-resistance in cotton aphid was determined in this study through laboratory selection. Studies of the resistance mechanisms confirmed that the mutation of target sites was the main reason for the high-level resistance. In addition, the increase of P450monooxygenase and carboxylesterase activity also played a role in the resistance to imidacloprid. Obviously these research results are significant for resistance management.