【作者】 黄诚华；

【导师】 叶恭银；

【作者基本信息】 浙江大学 ， 农业昆虫与害虫防治， 2006， 博士

【摘要】 二化螟Chilo suppressalis （Walker）和大螟Sesamia inferens Walker是水稻上两个重要害虫。自20世纪90年代以来,由于气候变暖、耕作制度改变、杂交稻种植面积扩大及害虫抗药性等原因,稻螟连年大爆发,对水稻的高产优质造成严重威胁。长期以来,防治稻螟主要依靠化学农药。由于大量用药,已导致二化螟对多种农药产生抗性。为了控制螟害,新农药不断被引进。然而,一种新农药可能对主要靶标害虫有效,但有时却可引起另一种害虫的猖獗。因此,在大面推广之前,对引进的新农药进行全面评估很有必要。本文就二化螟和大螟对氟虫腈和三唑磷的敏感性差异及其机理进行研究,主要结果如下: 1 二化螟的抗性监测 抗性监测结果表明,浙江省各地二化螟种群对杀虫单和三唑磷均已普遍产生高水平抗性,特别是浙东南沿海的瑞安、平阳、乐清和温岭等地的二化螟种群,其抗性倍数均在160倍以上。大部分种群对氟虫腈和阿维菌素仍然保持敏感,其抗性倍数在5倍以下,但监测发现瑞安二化螟种群对氟虫腈的抗性达6.5倍,为低水平抗性。药剂敏感性监测结果显示,二化螟和大螟的田间种群对氟虫腈的敏感性逐年下降,两种螟虫对氟虫腈抗性的发展趋势一致。相比而言,二化螟田间种群个体间的异质性逐年增加,而大螟种群个体异质性无明显变化。连续两年的田间调查结果表明,氟虫腈对水稻螟虫的控害能力优于三唑磷,但氟虫腈的使用易引起大螟种群数量的上升,而三唑磷则易引起二化螟田间种群的上升。 2 二化螟和大螟不同地区种群抗性相关酶系的分析 2.1 二化螟不同地区种群抗性相关酶系的活性与频率分布 从频率分布来看,天台、上虞、秀城和瑞安种群高羧酸酯酶（CarE）酶活性[>6.0μmol·min^-1·（mg pro）^-1)]个体的频率明显比其他种群要高。CarE的平均活性则以秀城和上虞两个种群的比活力最高,分为9.176和8.793μmol·min^-1·（mg pro）^-1,其他种群CarE的平均酶活性在2.543～7.165μmol·min^-1·（mg pro）^-1之间。相关性分析表明,二化螟不同种群的平均CarE活性与其对氟虫腈、三唑磷和杀虫单的抗性水平均没有显著相关,说明CarE在二化螟对这些杀虫剂的抗性形成中不是主导因子。 乐清、平阳、瑞安、秀洲4个种群的谷胱甘肽S-转移酶（GSTs）个体酶活性明显较高,大部分个体分布于200μmol·min^-1·（mg pro）^-1以上的酶活区段。GSTs的平均酶活性以温州地区的平阳、瑞安及乐清3个种群明显高于其他种群。不同地区二化螟种群GSTs活性与其对更多还原

【Abstract】 Striped stem borer (SSB), Chilo suppressalis (Walker) and purplish stem borer (PSB), Sesamia inferens Walker are two pests of rice. Since 1990s, there have been outbreaks in China because of the changes in climate, rice cultivation system, the extension of hybrid varieties and insecticide resistance. From past to now, control of rice stem borers mainly depended on pesticides. Because of extensive use of pesticides, SSB have developed resistances. Therefore, newly pesticides have to be introduced to control the populations of rice stem borers. However, a newly pesticide may effectively control one of pests but cause another pest outbreak. In this paper, the differential susceptibility and its mechanisms to fipronil and triazophos of SSB and PSB are studied. 1 Monitoring of insecticide resistance in SSBResults of monitoring showed that all populations of SSB collected from different counties or cities, Zhejiang province, had developed high level resistance (>40-fold) to monosultap and triazophos. Very high level of resistance to these two insecticides (> 160-fold) were detected in Rui’an, Pingyang, yueqi and Wenling populations collected from southeast Zhejiang province, where insecticides had been most extensively used. Low level of resistance to fipronil (6.5-fold) was detected in Rui’an population, but other populations tested remained susceptible (<5-fold) to this newly introduced insecticide though some susceptibility variation existed. No apparent resistance to abamectin had been detected after examining 12 populations collected from Zhejiang provinces, with susceptibility variation among them less than 2.8-fold.Dynamics of fipronil susceptibility in SSB and PSB were monitored continuously from 2003to 2005. The results indicated that the susceptibility to fipronil decreased year by year, and the trends of developing resistance to fipronil is consistent in these two rice stem borers. By comparison, the heterogeneity in population of SSB increased year by year, but vary little in PSB.The rate of deadheart and whitehead of rice and the amounts of populations of SSB and PSB were investigated continuously from 2004 to 2005. The results showed that fipronil could more effectively control the rate of deadheart and whitehead than triazophos in the paddy field. Meanwhile, it was found that the population of PSB increased significantly in fipronil-treated paddy field, while the population of SSB increased in triazophos-treated paddy field. 2 Biochemical characterization of insecticide-resistance related enzymes in different populations of SSB and PSB2.1 Activities and their frequencies of insecticide-resistance related enzymes in different populations of SSBFrequency distribution patterns of carboxylesterase (CarE) activity of Tiantai, Shanyu, Xiucheng and Rui’an populations had much more individuals possessing high activity [>6.0umol?nin"1?(pigpro)"1)] of CarE than other populations. The highest mean CarE activity were found in Xiucheng and Shangyu. Their specific activities were 9.176 and 8.793 umol?min"I?(rng pro)"1, respectively. The specific activities of other populations ranged 2.543 to 7.165 umol-min~1?(mg pro)"1. There is no significant correlation between the levels of insecticide resistance (LD50) and enzyme activity. It revealed that SSB developed resistance to fipronil, triazophos and monosultap was not due to CarE activity.Results showed that there were much more individuals possessing high activity [>200umol?min"1?(mg pro)"1)] of GSTs in Yueqing, Pingyang, Rui’an and Xiuzhou than other populations. Pingyang, Rui’an and Yueqing population collected from Wenzou, Zhejiang province, had higher mean GSTs activities compare with other populations. The correlation coefficient between GSTs activity and resistance levels was 0.667 to fipronil and 0.839 to monosultap. It suggested that GSTs activity had some relations to fipronil and monosultap resistance levels and it could be used as a reference index for biochemical detection in SSB.There was 55% individuals possessing high activity [>1.0 nmol?min"I?(mgpro)"1)] of MFOs in Yueqing, 35% in Tiantai, 30% in Rui’an and Changxing. The highest mean MFOs activity were found in Rui’an and its specific activity was 1.407 nmolinin"1 ? (mg pro)"1. The specific activities ofother populations ranged 0.510 to 1.063 nmolTnin"1 ?(mg pro)"1. The correlation coefficient between MFOs activity and resistance level was 0.794 to fipronil and 0.669 to monosultap. It revealed that MFOs activity had some relations to fipronil and monosultap resistance levels and it could be used as a reference index for biochemical detection in SSB.Results indicated that Yuhang, Dongxiang, Shaoxing, Pujiang and Changxing populations had more individuals with high activity of AChE [>1.5umol?min"1?(mg pro)"1] than other populations. The highest mean AChE activity was found in Yuhang and its specific activity was 2.511 umol?min"1?(mg pro)"1, specific activities of other populations ranged 0.989 to 1.976 umol?min"1?(mgpro)"1. The correlation coefficient between AChE activity and resistance level was -0.828 to triazophos. It suggested that AChE activity had some relations to triazophos resistance level. 2.2 The different susceptibility and its mechanisms to insecticides of SSB and PSBBioassay showed that SSB is susceptible to fipronil and resistant to triazophos, PSB is resistant to fipronil and susceptible to triazphos. Synergism experiments indicated that PBO had some antagonism to fipronil but some synergism to triazophos in both SSB and PSB.These results inferred that fipronil is metabolic activation in the rice stem borers. On the contrary, triazophos is metabolic detoxification.Biochemical analysis indicated that the activities and its frequencies of the insecticide-resistance related enzymes in SSB were all higher than those in PSB. Kinetic parameters between SSB and PSB varied greatly. These results implied that there were significant differences in both quantity and quality of the insecticide-resistance related enzymes between SSB and PSB.Inhibition tests in vitro showed that/50 of triazophos onAChE of SSB was 12.35-fold higher and the Kt of AChE in SSB was only 68% of that in PSB. Therefore, insensitive AChE may be a key mechanism responsible for the difference of triazophos susceptibility between SSB and PSB.Results also showed that the content of lipid in SSB was significant higher than that in PSB. The correlation between content of lipid and insecticide resistance was discussed. 3 Tissue and subcellular distribution of insecticide-resistance related enzymes in SSB and PSBOn the tissue distribution, MFOs and GSTs in the SSB and PSB were all distributed mostly infat body, integument and gut, and the lowest in haemolymph. The pattern of CarE tissue distribution in both SSB and PSB were similar, which the major portion (>85%) of CarE specific activities were found in gut and fat body, and a little portion (<15%) in integument and haemolymph. The highest specific activities of AChE in SSB was detected in gut, and relatively low specific activities in haemolymph, integument and fat body. The AChE activities of fat body in PSB was highest among the tissues tested, and the lowest was found in haemolymph.On the subcellular distribution, results showed that the major portions (>90%) of MFOs in both SSB and PSB located in cell nucleus and cell debris fraction, microsome fraction and mitochondrion fraction, and a little (<10%) with cytosol fraction. It revealed that MFOs was distributed mostly in membraneous subcells. The highest GSTs in SSB and PSB were found in cytosol fraction, relatively low level of GSTs in other subcellular fractions. The major portion (46.24%) of CarE in SSB located in the cytosol fraction, whereas the highest specific activity was found in the mitochondrion fraction. Result showed the great mass of of AChE in both SSB and PSB all located in membraneous subcells, include cell nucleus and cell debris fraction, microsome fraction and mitochondrion fraction.4 The sublethal effect of insecticides on the insecticide-resistance related enzymes in SSB and PSBThe sublethal effects of fipronil and triazophos on the insecticide-resistance related enzymes in SSB and PSB was investigated. The results indicated The CarE specific activities of SSB and PSB were induced by the sublethal dose of fipronil, whereas depressed by the sublethal dose of triazophos.No distinct changes were observed in GSTs activities after treatment with fipronil and triazophos in SSB and PSB, suggesting that there were no significant effects on the specific activities of GSTs by these two insecticides.The specific activities of MFOs in both SSB and PSB were induced significantly by the sublethal dose of fipronil. However, there were some differences in the effects on MFOs after treated with the sublethal dose of triazophos between SSB and PSB. Compare with the control, the MFOs activities in SSB induced obviously within 12h, but depressed slightly after 24h treatment. The MFOs activities in PSB treated with triazophos were higher than the control during the experiment, revealling that the MFOs was induced by triazophos.Results showed that the AChE activities varied little after treatment with the sublethal dose of fipronil in SSB, but depressed significantly in PSB. AChE activities were depressed obviously by the sublethal dose of triazophos.There were no significant differences in the specific activities of superoxide dismutase (SOD) and peroxidase (POD) between the group treated by fipronil and the control in SSB and PSB.5 Penetration of fipronil through the cuticles of larvae of SSB and PSBResults showed that the rates of fipronil penetration through the cuticle of larvae of SSB were higher than that of PSB within 4h, while no significant difference was observed after 6h treatment. The fitting cures of the rates of fipronil penetration to SSB and PSB were y=83.0594e"°0823 /x) (R2=0.9850) and y=9.3523x10124 (R2=0.9287), respectively. It was deduced that the median penetration times of fipronil to SSB and PSB were 2.1h and 5.2h, respectively. Therefore, it is obvious that the velocity of fipronil penetration through the cuticular of SSB was much faster than that of PSB.6 In vivo and in vitro metabolism of fipronil by larvae of SSB and PSBResults of in vivo metabolism indicated that fipronil-sulfone and fipronil-sulfide were detected in both SSB and PSB after topical application, and the amounts of fipronil-sulfone were much higher than that of fipronil-sulfide. Thus, it was concluded that fipronil oxidation to its sulfone metabolite is the major route of metabolic conversion in these two rice stem borers. The results of in vitro metabolism showed that fipronil-sulfone and fipronil-sulfide were also detected, further confirming that fipronil was degraded by oxidation and reduction in SSB and PSB. Comparably, the residue of fipronil-sulfone in SSB enzyme preparation was significantly higher than that of PSB, inferring that the level of fipronil oxidation in SSB enzyme preparation was higher than that of PSB. On the tissue distribution study, it was found that fipronil and fipronil-sulfone accumulated in the haemolymph of SSB, but not in PSB, suggesting that there was some difference in rates of fipronil transfer and conversion between SSB and PSB.7 Molecular detection of insecticide resistance-associated mutations in GABA receptor of rice stem borers by PCRGene fragment of the GABA receptor were amplified by PCR method from two populations of SSB and one population PSB. The fragment length of them were 152bp, 152bp and 145bp, respectively. The nucleotide sequences of conservative region fragmentsshare high similarity (100%) between the two populations of SSB, but only about 70.8 % similarity between SSB and PSB. It revealed that there was nucleotide polymorphisms among species. However, the deductive sequences of amino acid share high similarity (>88%) with GABA receptor genes from various insects. It was found that there was a amino acid substitution in SSB, which is homologous to Ala302~*-Ser mutation in Drosophila, but not in PSB. Therefore, Ala302—Ser point mutation was not suitable to be a molecular marker to detect frequency of insecticide-resistant gene in rice stem borers. Meanwhile, this point mutation also was not responsible for the differential susceptibility to fipronil between SSB and PSB.更多还原