运用α-氰代酯荧光底物检测抗性棉蚜羧酸酯酶代谢活性

为了研究抗性和敏感棉蚜Aphis gossypii品系对菊酯类药剂代谢的差异, 本实验合成了溴氰菊酯和高效氯氰菊酯报告荧光底物, 应用这两种底物水解后生成具有荧光化合物的特性,测定了不同品系棉蚜羧酸酯酶的代谢活性。结果表明: 氧化乐果棉蚜抗性和敏感品系羧酸酯酶对溴氰菊酯报告荧光底物的代谢活性分别为10.0和3.4 pmol/min·mg; 对高效氯氰菊酯报告荧光底物的代谢活性分别为4.0和2.4 pmol/min·mg, 抗性品系羧酸酯酶对溴氰菊酯和高效氯氰菊酯报告荧光底物的代谢活性分别为敏感品系的2.9和1.7倍; 溴氰菊酯棉蚜抗性和敏感品系羧酸酯酶对溴氰菊酯报告荧光底物的代谢活性分别为7.6和6.2 pmol/min·mg; 对高效氯氰菊酯报告荧光底物的代谢活性分别为9.3和5.2 pmol/min·mg, 抗性品系羧酸酯酶对溴氰菊酯和高效氯氰菊酯报告荧光底物的代谢活性分别为敏感品系的1.2和1.8倍。这种衍生的报告荧光底物能够用来检测抗性棉蚜羧酸酯酶的水解活性, 表明羧酸酯酶可能参与棉蚜对溴氰菊酯和氧化乐果抗性的形成。     

Differential mRNA expression levels and gene sequences of carboxylesterase in both deltamethrin resistant and susceptible strains of the cotton aphid, Aphis gossypii

Extensive use of insecticides on cotton has prompted resistance development in the cotton aphid, Aphis gossypii (Glover) in China. A deltamethrin‐selected population of cotton aphids from Xinjiang Uygur Autonomous Region, China with 228.59‐fold higher resistance to deltamethrin was used to examine how carboxylesterase conferred resistance to this pyrethroid insecticide. The carboxylesterase activity in the deltamethrin‐resistant strain was 3.67‐, 2.02‐ and 1.16‐fold of the susceptible strain when using α‐naphthyl acetate (α‐NA), β‐naphthyl acetate (β‐NA) and α‐naphthyl butyrate (α‐NB) as substrates, respectively. Carboxylesterase cDNA was cloned and sequenced from both deltamethrin‐resistant and susceptible strains. The cDNA contained 1581 bp open reading frames (ORFs) coding a 526 amino acid protein. Only one amino acid substitution (Val⁸⁷‐Ala) was observed between deltamethrin‐resistant and susceptible strains but it is not genetically linked to resistance by the catalytic triad and signature motif analysis. The real‐time polymerase chain reaction analysis indicated that the resistant strain had a 6.61‐fold higher level of carboxylesterase mRNA than the susceptible strain. The results revealed that up‐regulation of the carboxylesterase gene, not modified gene structure, may be responsible for the development of resistance in cotton aphids to deltamethrin.     

Resistance of aphis gossypii (Homoptera: Aphididae) to fenvalerate and imidacloprid and activities of detoxification enzymes on cotton and cucumber

Resistance of two strains of cotton aphid, Aphis gossypii Glover, to fenvalerate and imidacloprid were determined on cotton (Gossypium hirsutum L.) and cucumber (Cucumis sativa L.) after resistance selection of one strain to fenvalerate for 16 consecutive generations, and of a second strain to imidacloprid for 12 consecutive generations on cotton in greenhouses. Dose-response and activities of detoxication enzymes of the fenvalerate-resistant strain (R-fenvalerate), the imidacloprid-resistant strain (R-imidacloprid), and a susceptible strain (S) were determined. After 16 consecutive generations of selection, resistance of A. gossypii to fenvalerate increased >29,000-fold and to imidacloprid 8.1-fold. On cucumber. resistance of the R-fenvalerate strain to fenvalerate increased 700-fold and to imidacloprid 3.6-fold. However, the most significant finding in this study was that the R-imidacloprid strain exhibited cross-resistance to fenvalerate, with a resistance ratio of 108.9-fold on cotton and 3:3.5-fold on cucumber, whereas the R-fenvalerate strain did not show significant cross-resistance to imidacloprid on either plant species. Both resistant strains of A. gossypii were more resistant to fenvalerate on cotton than on cucumber, whereas their susceptibility to imidacloprid on otton and cucumber were not significantly different. The response of the S strain to fenvalerate and imidacloprid were similar on cotton and Cucumber. Activities of acetylcholinesterase (AChE) and alpha-naphthylacetate (alpha-NA) esterases of A. gossypii were significantly different among the three strains, with the R-fenvalerate strains having the highest, followed by the R-imidacloprid strain, and the S strain the lowest. The activities of the AChE and alpha-NA esterases for all three strains were also significantly higher on cotton than on cucumber. The resistance mechanism and resistance management strategies for the R-fenvalerate and R-imidacloprid strains of A. gossypii to fenvalerate and imidacloprid on cotton and cucumber are discussed.     

应用酶标仪动力学方法监测棉蚜的抗药性

用酶标仪动力学测定法对3个抗性水平不同的棉蚜品系（R1、R2和R3）和1个敏感品系（S）的羧酸酯酶进行了研究,S、R1、R2和R3品系对α-乙酸萘酯（α-NA）的平均比活力分别为57．10、1171．69、1236．14和3293．00μmol·mgpro－1·min-1（分光光度计终点测定法）和38．24、85．27、198．14和762．25mOD·min－1·aphid-1（酶标仪动力学法）。终点测定法结果显示出不同品系间最大相差达60倍;酶标仪动力学测定法研究表明,4个棉蚜品系羧酸酶活性与其抗药性程度显著相关。通过对这两种方法的比较,酶动力学方法的测定结果更可靠。     

棉蚜抗氧化乐果品系的羧酸酯酶基因突变

用氧化乐果对室内敏感品系棉蚜Aphis gossypii (Glover)进行抗性选育,经24代筛选,抗性指数达到124.7倍。以α-乙酸萘酯（α-NA）为底物,比较了氧化乐果敏感和抗性品系棉蚜羧酸酯酶的比活力,发现抗性品系羧酸酯酶比活力明显小于敏感品系。对这两个品系的羧酸酯酶基因进行了克隆,通过对抗性和敏感品系羧酸酯酶基因核苷酸序列及推导的氨基酸序列比较,发现抗性品系有4个氨基酸残基发生了替代 (His¹⁰⁴→Arg, Ala¹²⁸→Val, Thr³³³→Asp, Lys⁴⁸⁴→Arg)。对其蛋白质三维结构分析推测只有His¹⁰⁴→Arg的替代是位于其活性中心。棉蚜氧化乐果敏感和抗性品系羧酸酯酶基因cDNA全长的GenBank登录号分别为AY485216和AY485214。     

棉蚜啶虫脒抗性种群交互抗性和增效剂增效作用的研究

【目的】明确棉蚜Aphis gossypii Glover啶虫脒抗性品系与其它杀虫剂的交互抗性现状以及增效剂的增效作用,为延缓和治理棉蚜对啶虫脒的抗性提供依据。【方法】采用单头反选育和群体汰选的方式,获得了棉蚜啶虫脒敏感和抗性品系;采用叶片药膜法测定了13种杀虫剂对啶虫脒的交互抗性以及增效剂对啶虫脒的增效作用。【结果】经过室内棉蚜敏感和抗性品系的筛选,获得了相对抗性倍数为82.33倍的棉蚜啶虫脒抗性品系。棉蚜啶虫脒抗性品系的交互抗性谱的研究表明,交互抗性倍数小于5的药剂为:吡蚜酮,甲基阿维菌素;交互抗性倍数在5~10倍的药剂为:噻虫嗪,联苯菊酯,毒死蜱,马拉硫磷,丙溴磷,辛硫磷;交互抗性倍数在10~15倍的药剂为:硫丹,阿维菌素,高效氯氰菊酯,三唑磷,氧化乐果;交互抗性倍数大于1 5倍的药剂为:吡虫啉。增效剂实验表明,TPP和PBO在啶虫脒敏感品系中增效作用不明显,但在抗性品系中增效作用显著。在啶虫脒抗性品系中的增效比为1.77、1.61,在啶虫脒敏感品系中的增效比为1.02、1.03。DEM在啶虫脒抗性、敏感品系中的增效作用均不明显,增效比为1.04、1.02。TPP和PBO对啶虫脒有很好的增效作用。以室内棉蚜敏感品系(LC_(50)为0.180 mg/L)为基础,对新疆各主要棉区的棉蚜种群进行了啶虫脒药剂的抗性调查,结果表明新疆各主要棉区棉蚜对啶虫脒的相对抗性倍数为6.1~22.0倍。【结论】由此说明新疆主要棉区棉蚜对啶虫脒具有一定的抗性风险,生产中可以利用无交互抗性的吡蚜酮和甲基阿维菌素来治理抗性棉蚜种群。     

棉蚜抗氧化乐果品系及敏感品系羧酸酯酶性质的比较

在室内用氧化乐果逐代筛选的棉蚜抗性品系,相对于敏感品系的抗性倍数是17。用α-乙酸萘酯（α-NA）、α-丁酸萘酯（α-NB）、α-磷酸萘酯（α-NP）和β-磷酸萘酯（β-NP）作底物比较研究了氧化乐果抗性和敏感品系棉蚜Aphis gossypii羧酸酯酶的比活力、米氏常数（K_m）和最大反应速度V_max）等有关的动力学常数。以α-NA和α-NB作底物时,抗性品系棉蚜的比活力显著低于敏感品系的;以α-NP和β-NP作底物时,两个品系棉蚜的比活力、K_m和V_max没有明显差异。用α-NA、β-NA作底物染色做酯酶同工酶电泳,抗性品系棉蚜的酯酶同工酶染色比敏感品系棉蚜的浅。     

棉蚜抗杀灭菊酯品系的某些生物学特性

1908-1990年抗性监测表明,河南棉区棉蚜Aphis gossypii Glover对杀灭菊酯、氧化乐果和 久效磷的抗性水平明显增长,特别是对杀灭菊酯的抗性已达较高水平。使用杀灭菊酯对敏感棉蚜连续选择8次,棉蚜对杀灭菊酯抗性上升1392.67倍(浸溃法测定),并表现对久效磷和氧化乐果有3-4倍的正交互抗性。对4个不同抗性品系棉蚜的实验种群生命表分析表明,杀灭菊酯抗性棉蚜的适合度并不降低,抗性棉 蚜的繁殖力不表现降低趋势。利用棉田常用8种有机磷、菊酯类和脒类农药的单剂及混剂测定了对高抗棉蚜的毒力水平,DDVP表现对高抗杀灭菊酯棉蚜有较强毒力;脒类和菊酯类农药混配有显著的增效作用, 但与DDVP混用则有拮抗作用。     

棉花抗蚜性与苯丙氨酸解氨酶活性的关系

为探讨苯丙氨酸解氨酶活性与棉花抗蚜性的关系,在室内人工接蚜危害和田间自然蚜群2种条件下,测定不同抗蚜性棉花品种受棉蚜Aphis gossypii Glover危害后叶片的PAL活性。结果表明:未受棉蚜危害时,抗、感品种PAL活性无明显差异;棉蚜危害胁迫棉花PAL活性升高,抗蚜品种受到棉蚜危害诱导所产生的PAL活性远比感蚜品种高。田间有蚜株率与棉花叶片苯丙氨酸解氨酶活性的呈显著的负相关关系。研究说明PAL酶活性对棉花的抗蚜性有一定的影响。     

棉蚜抗吡虫啉品系和敏感品系主要解毒酶活性比较

通过生物测定和生物化学方法比较了棉蚜 Aphis gossypii 对吡虫啉抗性（约为7倍）和敏感品系几种主要解毒酶的活性。结果表明：氧化胡椒基丁醚对两个品系均无明显增效作用。抗性品系中羧酸酯酶和谷胱甘肽S-转移酶的比活力均明显高于敏感品系,抗性品系中羧酸酯酶的K_m值也显著高于敏感品系,说明抗性品系羧酸酯酶与底物的亲和力明显高于敏感品系。上述结果证明羧酸酯酶和谷胱甘肽S-转移酶活力增强在棉蚜对吡虫啉的抗性中起重要作用。     

INSECTICIDE RESISTANCE OF COTTON APHID IN NORTH CHINA

Abstract  The cotton aphid ( Aphis gossypii Glover) is one of the most important pests infesting cotton in the cotton areas of North China. Since 1953 organophosphorus insecticides such as parathion and systox have been used to control the aphids for keeping up good yield of cotton. After several years. the concentration and the amount of spray were increasing in the field. In the early 1980's highly effective pyrethroid insecticides such as decis and sumicidin were largely imported into China. When first used 2. 5% decis emulsion was diluted in the ratio from 1:10000 to 1: 12000. However in 1985 the resistance of cotton aphid to pyrethroids increased by 171 times in general, 3230 times in some cotton fields. Thus it has prompted us to investigate the mechanism of resistance to insecticides and to search for the strategy to control the resistant aphids.
Experiments showed that the use of synergists including SV₁ (O, O-diethyl, O-phenyl phos-phorothionate) and PB has given evidence indicating mixed function oxidases (MFO). α-NA esterases and α-NA carboxylesterase are involved in the formation of resistance. The results also showed that the sensitivity of AChE to paraoxon in resistant aphids was lower than that in susceptible aphids.
Experiments showed that SV₁ was particularly synergistic to organophosphorus or pyrethroid insecticides and had played an excellent role in overcoming the resistance of cotton aphids to insecticides.     

Purification and characterization of a carboxylesterase involved in malathion-specific resistance from Tribolium castaneum (Coleoptera: Tenebrionidae)

Specific resistance to malathion in a strain of Tribolium castaneum is due to a 44-fold increase in malathion carboxylesterase (MCE) activity relative to a susceptible strain, whereas non-specific esterase levels are slightly lower. Unlike the overproduced esterase of some mosquito and aphid species, MCE in Tribolium castaneum accounts for only a small fraction (0.033-0.045%) of the total extractable protein respectively in resistant and susceptible strains. The enzyme was purified to apparent homogeneity from these two strains and has a similar molecular weight of 62,000. However, preparative isoelectricfocusing indicated that resistant insects possess one MCE with pI of 7.3, while susceptible insects possess a MCE with a pI of 6.6. Purified MCE from both populations had different K(m) and V(m) values for hydrolysis of malathion as well as for alpha-naphthyl acetate. The kinetic analysis suggests that MCE of resistant insects hydrolyses malathion faster than the purified carboxylesterase from susceptible beetles and that this enzyme has greater affinity for malathion than for naphthyl esters. Malathion-specific resistance is due to the presence of a qualitatively different esterase in the resistant strain.     

抗吡虫啉棉蚜种群对啶虫脒和噻虫胺的交互抗性及相关酶学机理

【目的】通过对乙酰胆碱受体β1亚基突变后的抗吡虫啉棉蚜Aphis gossypii (Glover)种群的继续筛选, 明确该种群的抗性发展规律以及对其他新烟碱类杀虫剂啶虫脒和噻虫胺的交互抗性及相关酶学机理。【方法】采用浸渍法连续对抗吡虫啉棉蚜进行室内筛选、 测定噻虫胺和啶虫脒对抗吡虫啉棉蚜种群的毒力; 选择LC₂₀剂量吡虫啉、 啶虫脒和噻虫胺处理抗性棉蚜, 采用生化分析法测定其体内羧酸酯酶、 谷胱甘肽-S-转移酶和乙酰胆碱酯酶的活性变化, 并观察其生物学特性的变化。【结果】本研究对抗性棉蚜突变种群用吡虫啉继续筛选至75代, 抗性倍数达到72.6倍, RF75停止用药筛选12代（RF₇₅₊₁₂）, 抗性仍达72.0倍。且RF₇₅₊₁₂对噻虫胺和啶虫脒的交互抗性可分别达11.9倍和20.1倍。噻虫胺对抗吡虫啉棉蚜的蜜露分泌和体重的抑制作用均大于吡虫啉和啶虫脒。噻虫胺对RF₇₅₊₁₂的羧酸酯酶、 谷胱甘肽-S-转移酶和乙酰胆碱酯酶均具有明显的抑制作用, 而啶虫脒的抑制作用较小。【结论】结果表明乙酰胆碱受体基因突变棉蚜种群对吡虫啉的抗性水平不仅升高, 且停止用药后其抗性可稳定遗传; 第二代新烟碱类的噻虫胺在抗吡虫啉棉蚜靶标突变种群的治理中具有较大的应用价值。     

USING FILTER PAPER TEST FOR DETECTING INHIBITORY FREQUENCY OF ORGANOPHOSPHATE TO ESTERASES IN COTTON APHIDS, APHIS GOSSYPII (GLOVER)

Abstract The individual esterase activity which is measured by filter paper test (FPT) method may determine the resistance of cotton aphids Aphis gossypii (Glover) against organophosphorus (OP) insecticides. For testing accurately resistant level caused by different insecticides, we applied FPT method for measuring inhibitory action of methyl-parathion, monocrotophos and omethoate to α-NA esterase of individual cotton aphids, and compared the inhibitory frequencies of these three insecticides to susceptible population (BCA) and resistant population (GCA). Results showed that their inhibitory frequencies of the susceptible population were evidently higher than that of the resistant population. The inhibitory rate of α-NA esterase in F1 generation individual cotton aphids by monocrotophos was low when the cotton aphid population had been treated in advance with monocrotophos, but it got to 75%-90% when the cotton aphid population had been untreated in advance with monocrotophos. Besides, the differences in esterase activity were not obvious between them. In same region when cotton aphids were treated with insecticides the inhibitory frequency of esterases in individual by the insecticides was lower than counterparts in individual cotton aphids which were not treated with insecticides. All these demonstrated that inhibitory frequency of α-NA esterase in individual cotton aphids by OP insecticides could be used as a technique of forecasting pest resistance.     

抗性品系棉蚜乙酰胆碱酯酶和羧酸酯酶的变异

用浸叶法测定了采自我国不同地区(泰安、莱阳、南京、北京和安阳)的棉蚜品系Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ对久效磷、甲胺磷、抗蚜威和灭多威等杀虫剂的抗性水平,各棉蚜品系对杀虫剂的抗性依次为Ⅴ>Ⅳ>Ⅲ,Ⅱ>Ⅰ。进一步研究表明,Ⅴ和Ⅳ品系棉蚜乙酰胆碱酯酶对杀虫剂的敏感性显著下降,Ⅱ品系次之,Ⅲ和Ⅰ品系接近于敏感品系。Ⅴ和Ⅳ品系乙酰胆碱酯酶的Km值显著下降,表明酶发生了质的变化。不同棉蚜抗性品系的酯酶（全酯酶和羧酸酯酶）活性均显著升高,其中Ⅲ品系的酯酶活力为Ⅱ品系的2倍。Ⅴ品系羧酸酯酶Km值达2460.4 μmol/L,而Ⅳ品系仅为84.4 μmol/L,该两个品系羧酸酯酶发生了质的变化。研究结果表明,不同抗性程度的棉蚜品系均存在代谢抗性和靶标抗性。低抗水平的棉蚜品系,以代谢抗性为主,靶标抗性为辅;中抗水平的棉蚜品系,抑或由于解毒代谢酶的活性显著增强,也可能由于靶标的敏感性显著下降;而高抗水平的棉蚜品系,依赖于代谢抗性和靶标抗性的联合作用。     

Different exposure methods to neonicotinoids influenced biochemical characteristics in cotton aphid,Aphis gossypii Glover (Hemiptera:Aphididae)

We measured lipid, carbohydrate and protein content in three strains of cotton aphid, Aphis gossypii (Hemiptera: Aphididae) from very resistant to neonicotinoids up to semi sensitive and sensitive strains in different exposure method of spraying to neonicotinoids. We observed changes in energy source rates at which each substrate was metabolised under starvation, selection and residue stress method spraying that assist in metabolisation of their biochemical parameters. These particular exposure methods influenced some of biochemical parameters in cotton aphid. Results indicated that Sugar, glycogen, total protein and lipid in aphids have significant changes. Studies showed that among three strains called Ag-R, Ag-M and Ag-D which have different susceptibilities to neonicotinoids, strain Ag-R was the most tolerated aphids in counter of imidacloprid and thiametoxam, strain Ag-D was somewhat more tolerated to these insecticides and strain Ag-M was the most sensitive strain which has the resistance factor of nearly 890 in starvation method and was different depending on the method of exposure. Among energy sources, the total lipid in susceptible strain were decreasing more than resistant strain, whereas total proteins were increasing in resistant strain compared with sensitive strain. Total Glycogen was affected significantly in the stress condition which caused an increase and was the most resistant strain.     

Aspects of insecticide resistance in the melon and cotton aphid, Aphis gossypii (Hemiptera: Aphididae)

Strains of the aphid Aphis gossypii, taken from cucumber in UK glasshouses were tested for resistance to pirimicarb, diazinon and heptenophos. Compared to a susceptible strain, resistance factors to diazinon of up to 659 were found in populations collected between 1987 and 1989. Compared to the same susceptible strain, diazinon-resistant populations also showed a reduced response to heptenophos. All field populations tested against pirimicarb were resistant. Host plant preference tests showed that all strains (including stock cultures) tested could readily breed on cotton. However strains originally collected from chrysanthemum did not transfer to cucumber and likewise strains from cucumber would not colonise chrysanthemum. Polyacrylamide gel electrophoresis of four of the aphid strains showed differences in migration rates and intensity of staining of esterase bands. Intensity of staining was correlated with the degree of diazinon resistance.     

Assessment of cotton aphids, Aphis gossypii, and their natural enemies on aphid-resistant and aphid-susceptible wheat varieties in a wheat–cotton relay intercropping system

The cotton aphid, Aphis gossypii Glover (Homoptera: Aphididae), is an important cotton pest in northern China, especially in the seedling stage of cotton. After large scale commercial use of transgenic Bt cotton, cotton aphids became one of the most important cotton pests. A 2‐year study was conducted to evaluate the role of four winter wheat varieties that were resistant or susceptible to wheat aphid, Sitobion avenae Fabricius (Homoptera: Aphididae), in conserving arthropod natural enemies and suppressing cotton aphids in a wheat–cotton relay intercropping system in northern China. The results indicated that wheat–cotton intercropping preserved and augmented natural enemies more than a monoculture of cotton. The density of natural enemies in cotton was significantly different among relay‐intercropping fields with different wheat varieties. The highest density of natural enemies and low cotton aphid populations were found in the treatment of cotton in relay intercropped with the wheat variety Lovrin10, which is susceptible to wheat aphid. The lowest density of predators and parasitoids associated with high cotton aphid populations were found with the wheat variety KOK1679, which is resistant to wheat aphid. The results showed that wheat varieties that are susceptible or moderately resistant to wheat aphid might reduce cotton aphids more effectively than an aphid‐resistant variety in the intercropping system by enhancing predators to suppress cotton aphids during the cotton seedling stage.     

Biochemical mechanisms of resistance in strains of Oryzaephilus surinamensis (Coleoptera: Silvanidae) resistant to malathion and chlorpyrifos-methyl.

The acetylcholinesterase, carboxylesterase, and cytochrome P450 monooxygenase activities of three strains of Oryzaephilus srinamensis (L.) were examined to better understand biochemical mechanisms of resistance. The three strains were VOS49 and VOSCM, selected for resistance to malathion and chlorpyrifos-methyl, respectively, and VOS48, a standard susceptible strain. Cross-resistance to malathion and chlorpyrifos-methyl was confirmed in VOS49 and VOSCM. Acetylcholinesterase activity was not correlated to resistance among these strains. VOS49 and VOSCM showed elevated levels of carboxylesterase activity based on p-nitrophenylacetate, alpha-naphthyl acetate, or beta-naphthyl acetate substrates. PAGE zymograms showed major differences in caboxylesterase isozyme banding among strains. VOSCM had one strongly staining isozyme band. A band having the same Rf-value was very faint in VOS48. The VOS49 carboxylesterase banding pattern was different from both VOSCM and VOS48. Cytochrome P450 monooxygenase activity was based on cytochrome P450 content, aldrin epoxidase activity, and oxidation of organophosphate insecticides, all elevated in resistant strains. The monooxygenase activity varied with insecticide substrate and resistant strain, suggesting specific cytochromes P450 may exist for different insecticides. The monooxygenase activity of the VOS49 strain was much higher with malathion than chlorpyrifos-methyl as substrates, whereas VOSCM monooxygenase activity was higher with malathion than chlorpyrifos-methyl as substrates. Results are discussed in the context of resistance mechanisms to organophosphate insecticides in O. surinamensis.