ZHAO Yi, XU Huachao, MA Yan, et al. Oxidase and detoxifying enzyme activity of Apriona swainsoni (Hope) with diflubenzuron and flubenzuron[J]. Journal of Zhejiang A&F University, 2018, 35(1): 174-177. DOI: 10.11833/j.issn.2095-0756.2018.01.023
Citation: ZHAO Yi, XU Huachao, MA Yan, et al. Oxidase and detoxifying enzyme activity of Apriona swainsoni (Hope) with diflubenzuron and flubenzuron[J]. Journal of Zhejiang A&F University, 2018, 35(1): 174-177. DOI: 10.11833/j.issn.2095-0756.2018.01.023

Oxidase and detoxifying enzyme activity of Apriona swainsoni (Hope) with diflubenzuron and flubenzuron

DOI: 10.11833/j.issn.2095-0756.2018.01.023
  • Received Date: 2017-01-17
  • Rev Recd Date: 2017-04-07
  • Publish Date: 2018-02-20
  • To research the influence of tebufenozide and chlorbenzuron on the physiological mechanism of Apriona swainsoni (Hope), A. swainsoni larvae were fed with sawdust treated by chlorbenzuron (T1) and tebufenozide (T2). These were then sampled to determine enzymatic activity of catalase (CAT), superoxide dismutase (SOD), glutathione S transferases (GSTs), and carboxylesterase (CarE) in larvae at intervals of 12 h to 72 h in succession. Results showed that after treatment with chlorbenzuron, the enzymatic activity of CAT increased first and then decreased 24 h later. Enzymatic activity of SOD maintained a high level. Also, after treatment by tebufenozide, the enzymatic activity of CAT decreased first with enzymatic activity of SOD being higher than the control. After treatment by two types of the pesticides, enzymatic activity of GSTs was higher than the control with no differences of CarE activity with the control. Therefore, enzymatic activity of GSTs in A. swainsoni was assumed to be associated with resistance generated in the larva and was likely to be a resistance marker of the larvae.
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Oxidase and detoxifying enzyme activity of Apriona swainsoni (Hope) with diflubenzuron and flubenzuron

doi: 10.11833/j.issn.2095-0756.2018.01.023

Abstract: To research the influence of tebufenozide and chlorbenzuron on the physiological mechanism of Apriona swainsoni (Hope), A. swainsoni larvae were fed with sawdust treated by chlorbenzuron (T1) and tebufenozide (T2). These were then sampled to determine enzymatic activity of catalase (CAT), superoxide dismutase (SOD), glutathione S transferases (GSTs), and carboxylesterase (CarE) in larvae at intervals of 12 h to 72 h in succession. Results showed that after treatment with chlorbenzuron, the enzymatic activity of CAT increased first and then decreased 24 h later. Enzymatic activity of SOD maintained a high level. Also, after treatment by tebufenozide, the enzymatic activity of CAT decreased first with enzymatic activity of SOD being higher than the control. After treatment by two types of the pesticides, enzymatic activity of GSTs was higher than the control with no differences of CarE activity with the control. Therefore, enzymatic activity of GSTs in A. swainsoni was assumed to be associated with resistance generated in the larva and was likely to be a resistance marker of the larvae.

ZHAO Yi, XU Huachao, MA Yan, et al. Oxidase and detoxifying enzyme activity of Apriona swainsoni (Hope) with diflubenzuron and flubenzuron[J]. Journal of Zhejiang A&F University, 2018, 35(1): 174-177. DOI: 10.11833/j.issn.2095-0756.2018.01.023
Citation: ZHAO Yi, XU Huachao, MA Yan, et al. Oxidase and detoxifying enzyme activity of Apriona swainsoni (Hope) with diflubenzuron and flubenzuron[J]. Journal of Zhejiang A&F University, 2018, 35(1): 174-177. DOI: 10.11833/j.issn.2095-0756.2018.01.023
  • 锈色粒肩天牛Apriona swainsoni为鞘翅目Coleoptera天牛科Cerambycidae昆虫,主要寄生于国槐Sophora japonica,云实Caesalpinia decapetala等豆科Leguminosae植物[1-3],是危害较为严重的蛀干害虫之一。灭幼脲(chlorbenzuron)是保幼激素的类似物[4-6],由咽侧体分泌,对昆虫的生长、发育和生殖起着重要作用[7]。成虫饲喂灭幼脲后体质量减轻,飞行能力减弱,产卵量和孵化率下降,寿命缩短等[8-9],近年来被广泛用于双翅目Diptera和鳞翅目Lepidoptera等害虫的防治,在鞘翅目昆虫防治上也取得了一定成效[10],但在天牛幼虫的防治上研究较少。虫酰肼(flubenzuron)是昆虫蜕皮激素类似物,常见蜕皮激素类似物类杀虫剂有虫酰肼(RH5992)、米满(methoxyfenozide,RH2485)和氯虫酰肼(halofenozide,RH-0345)等3种,有报道称RH5992和RH2485只对鳞翅目昆虫有活性,RH-0345只对鞘翅目有活性[5],但也有文献指出虫酰肼对双翅目摇蚊属Chironomus昆虫同样有效[11]。机体正常生理功能多是通过酶的调控实现的,抗氧化酶和解毒酶是机体内广泛存在的2类酶。McCORD等[12]发现超氧化物歧化酶(SOD)可以使自由基发生歧化反应,谷胱甘肽S转移酶(GSTs)能催化还原型谷胱甘肽形成硫醚氨酸[13],羧酸酯酶(CarE)可以催化羧酸酯生成酸和醇,从而降低细胞毒性,保护细胞膜。本研究以虫酰肼和灭幼脲处理后木屑喂饲锈色粒肩天牛幼虫,通过测定虫体内4种酶的变化,反映药物处理后天牛的生理状态,为天牛的防治提供理论依据。

  • 锈色粒肩天牛由上饶云实农业开发有限公司惠赠,为半生态饲养的3龄幼虫(龄期评定参照LE等[14]、王小艺等[15]、张海滨等[16]方法),质量为0.80~0.85 g·头-1。过氧化氢酶(CAT),超氧化物歧化酶(SOD),谷胱甘肽S转移酶(GSTs),羧酸酯酶(CarE)的试剂盒均购自上海酶联生物公司。质量分数20%的虫酰肼悬浊液购自济南天邦化工有限公司,质量分数25%的灭幼脲购自潍坊华诺生物科技有限公司。BioTek synergyH1多功能酶标仪由美国Biotek公司生产。云实木屑由天台山1年生云实枝条粉碎制成。蒸馏水稀释虫酰肼和灭幼脲至100 mg·L-1后,按V(稀释药液):m(云实木屑)=0.2 L:1.0 kg均匀混合后备用。

  • 处理好的云实木屑饲喂天牛幼虫。60头·组-1,重复3次。对照用同量的蒸馏水处理木屑。饲喂12,24,36,48,60和72 h后分别随机取10头,液氮中研磨后称取0.5 g,放入装有磷酸缓冲液(PBS,pH 7.4)的50 mL离心管中,4 ℃下6 000 r·min-1离心20 min,取上清液,即为酶的提取液。提取液分装4管,-20 ℃保存备用。取样完毕后按试剂盒说明书操作,BioTek synergyH1多功能酶标仪测定酶活性。

    用SPSS进行双因素方差分析;用Excel软件分析药物处理后天牛体内酶的变化趋势。

  • 表 1可以看出:灭幼脲处理组(T1组)过氧化氢酶活性与对照组无显著差异;虫酰肼处理组(T2组)过氧化氢酶活性下降,相较于对照组以及T1组差异显著。就各时段而言,对照组在72 h内各相邻时段之间无明显差异。T1组过氧化氢酶活性先上升,24 h后开始下降。T2组过氧化氢酶活性在12 h内已低于对照组,并且有持续下降的趋势。无论哪种药物处理,处理组超氧化物歧化酶活性均高于对照组,且差异显著;灭幼脲处理12 h虫体超氧化物歧化酶活性已高于对照组,48 h后继续上升;虫酰肼处理24 h后虫体超氧化物歧化酶活性迅速上升,之后下降,与对照组差异显著。观测羧酸酯酶活性,处理组和对照组并无显著差异。与对照相比,处理组虫体谷胱甘肽S转移酶活性均显著增高,但处理组间差异不显著;虫酰肼处理后谷胱甘肽S转移酶活性在24 h时达到顶峰,之后下降;而灭幼脲处理后虫体谷胱甘肽S转移酶酶活性在12 h时已显著升高,之后下降。

    处理 t/h 酶活性/(×16.67 μkat.L-1)
    CAT SOD CarE GSTs
    对照 12 26.06±1.22aA 47.31±2.49aA 35.71±7.13aA 21.71±1.24aA
    24 25.59±0.28aA 45.53±2.95aA 34.83±5.02aA 21.98±2.14aA
    36 24.24±2.58aA 48.92±3.01aB 30.78±8.89 aA 21.70±2.10aA
    48 23.41±1.38aA 52.46±6.21aC 34.95±7.32aA 21.97±0.43aA
    60 23.76±2.53aA 50.34±2.99aC 31.51±6.01aA 21.47±1.53aA
    72 21.96±3.14aA 49.88±4.40aC 28.76±3.28aA 22.32±2.25aA
    T1 12 28.05±4.76aB 53.62±4.85bD 35.93±14.56aA 24.81±0.97bB
    24 36.49±3.16aC 52.34±5.40bD 37.37±16.25 aA 23.08±1.45bC
    36 20.26±1.75aD 53.67±3.90bD 36.52±17.06aA 24.51±0.61bD
    48 23.59±3.69aE 53.25±1.23bD 34.69±5.87aA 23.78±1.37bD
    60 25.54±5.63aE 49.92±1.22bE 32.41±8.95aA 24.78±0.96bD
    72 17.58±0.80aF 55.22±1.29bF 35.36±9.25aA 24.55±0.68bD
    T2 12 19.86±1.12bG 54.67±2.51bG 35.31±13.91aA 22.79±2.70bE
    24 19.64±5.13bG 60.09±8.00bH 37.52±1.61aA 25.62±4.87bF
    36 16.35±2.45bH 52.46±4.77bI 37.75±5.93 aA 24.27±1.60bF
    48 18.16±0.66bH 52.55±0.92bI 35.40±5.63aA 23.10±1.10bF
    60 14.37±1.35bI 51.82±2.27bI 33.90±4.55aA 23.54±1.34bF
    72 12.93±4.57bI 45.47±6.61bJ 36.19±6.78aA 24.03±4.02bF
    说明:表中数据为平均值±标准差;不同小写字母表示2种处理之间有显著差异(P<0.05),不同大写字母表示同一处理不同时间段间有显著差异(P<0.05)。

    Table 1.  Effects of 4 enzymatics activities under different treatments

  • 研究发现:用灭幼脲处理昆虫,虫体内2种氧化酶的活性升高、下降并不是同步的,原因可能是灭幼脲刺激使虫体在短时间内产生的大量自由基激活氧化酶,氧化酶应对速度和持续作用时间存在差异。本试验中,锈色粒肩天牛幼虫被喂饲灭幼脲处理的木屑,虫体氧化酶被激活并与自由基迅速结合;但在分离时过氧化氢酶较超氧化物歧化酶更为迅速,即在虫体内作用时间较短,最终表现为过氧化氢酶活性在24 h后迅速降低,而超氧化物歧化酶一直处于较高水平,持续灭活多余的自由基。用虫酰肼处理虫体后,过氧化氢酶活性低于对照组;超氧化物歧化酶活性高于对照组,但与灭幼脲处理组差异不显著(P<0.05)。由此猜测,虫酰肼可能会抑制虫体过氧化氢酶的激活,而对超氧化物歧化酶的激活并没有太大的影响,说明虫酰肼刺激下的天牛幼虫体内过氧化氢酶没有参与多余自由基的中和作用。因此,在灭活自由基的过程中,超氧化物歧化酶的作用更为广泛,虫体对虫酰肼产生伤害的抵抗力低于对灭幼脲,用虫酰肼防治锈色粒肩天牛的效果可能优于灭幼脲。

    DHADIALLA等[17]研究表明,用蜕皮激素处理虫体时,虫体会出现拒食、蛋白质合成增多等情况。徐希宝[18]指出在棉铃虫体内谷胱甘肽S转移酶活性和抗性形成有关。本研究发现,不管是用虫酰肼还是灭幼脲刺激锈色粒肩天牛幼虫,虫体内的谷胱甘肽S转移酶活性都明显高于对照组,因此猜测锈色粒肩天牛体内谷胱甘肽S转移酶活性升高也是一种自我调控产生抗性的过程;谷胱甘肽S转移酶活性的提高可以加快虫体内有害物质的代谢,SMAGGHE等[19]也指出虫酰肼代谢的加快是抗性产生的重要原因。谷胱甘肽S转移酶和羧酸酯酶都是昆虫体内的重要解毒酶,但是,在用药物处理后,羧酸酯酶活性和对照组相比并无显著差异,这可能从侧面说明虫体对虫酰肼和灭幼脲的抗性是由谷胱甘肽S转移酶直接体现的,换而言之,谷胱甘肽S转移酶可能是昆虫抗性的重要标志物。

    虫酰肼和灭幼脲是昆虫激素类似物,对于昆虫的机体调控作用大于直接的毒杀作用,且该调控作用有一个长期的过程。因此,在此之后还需要观察这2种药物对锈色粒肩天牛整个的生长发育过程,从而为防治天牛提供更多的理论基础。

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