摘 要

昆虫与肠道微生物在长期的协同进化中，形成了相互选择，相互适应的共生关系。近年来，生物杀虫剂与肠道微生物之间的关系逐渐成为生物防治的研究热点，鉴于苏云金芽胞杆菌（Bt）的研究，发现昆虫肠道细菌在Bt杀虫中发挥了一定的作用，或是协同或是保护，但观点不一致。HcNPV作为一种优良的美国白蛾专性杀虫剂，其致病机理与Bt极为相似，本文通过生物测定探究肠道细菌在HcNPV杀虫活性中的作用，以期寻找对HcNPV有增效作用的肠道细菌。研究结果如下：

在人工饲料中添加氨苄青霉素，卡那霉素，庆大霉素，利福霉素，链霉素各500μg/ml饲养美国白蛾，采用可培养与不可培养方法均检测不到肠道细菌存在，且此抗生素混合物对幼虫生长发育无明显的不良影响，至此成功建立了美国白蛾无肠道细菌幼虫种群。以美国白蛾核型多角体病毒（HcNPV）对无肠道细菌幼虫进行生物测定，结果显示无肠道细菌的美国白蛾幼虫依然能够被HcNPV感染致死，这也说明肠道共生菌并非HcNPV杀虫必须的因素。进一步实验结果显示，无肠道细菌幼虫的LC₅₀为3.192x10⁵PIB·mL^-1，正常品系的LC₅₀为5.445×10⁴PIB·mL^{- 1}，这表明肠道细菌的缺失会导致美国白蛾对HcNPV的敏感性降低，并且这种降低效果在不同的HcNPV浓度时表现不同，在10⁵，10⁶PIB·mL^{- 1}时，肠道细菌对HcNPV杀虫作用有着显著的影响，无肠道细菌美国白蛾幼虫死亡率和正常品系相差约20%，而这种现象在低浓度和高浓度HcNPV生测时并未表现。在无肠道细菌幼虫的体内分别重建两种优势可培养细菌，其中HcM3对HcNPV有显著的增效作用，HcM7则具有一定的保护作用。

关键词：美国白蛾；肠道细菌；HcNPV；杀虫活性

Effect of Midgut Bacteria of Hyphantria Cunea on the Insecticidal Activity of HcNPV

ABSTRACT

In the long term of coevolution, insects and intestinal microbes have formed a symbiotic relationship of mutual selection and adaptation. In recent years, the relationship between biological insecticides and intestinal microbes has gradually become a research hotspot in biological control. Based on the study of Bacillus thuringiensis (BT), it was found that intestinal bacteria played a certain role in Bt insecticidal killing, either synergistic or protective, but their views were not consistent. HcNPV as an excellent specific insecticide for Hyphantria cunea, and its pathogenetic mechanism is very similar to that of BT. In this paper, the role of intestinal bacteria in the insecticidal activity of HcNPV was explored by bioassay in order to find the intestinal bacteria that had synergistic effect on HcNPV. The results were as follows:

500μ g/ml mixture of ampicillin, kanamycin, gentamicin, rifamycin and streptomycin in artificial diet for rearing Hyphantria cunea, both culturable and non-culturable methods could not detect the presence of intestinal bacteria, and the mixture of antibiotics had no obvious adverse effect on larval growth and development. The biological test of the HcNPV on the intestines free bacteria larvae showed that the larvae without intestinal bacteria could still be infected by HcNPV, which indicated that the intestinal symbiotic bacteria were not the necessary factors for HcNPV killing insects. The further experimental results showed that the LC₅₀ of non-intestinal bacterial larvae was 3.192x10⁵PIB mL^-1, the LC₅₀ of intestinal bacterial larvae was 5.445×10⁴PIB·mL^{- 1}. This suggests that the absence of intestinal bacteria leads to a decrease in the sensitivity of the Hyphantria cunea to HcNPV. Moreover, the effect of this decrease was different at different HcNPV concentrations. At 10⁵, 10⁶PIB·mL^{- 1}, intestinal bacteria had a significant effect on the insecticidal effect of HcNPV. There was a decrease between the mortality rate of non-intestinal bacterial larvae and the normal group by about 20 percent, but this phenomenon was not observed at low and high concentrations of HcNPV. In the absence of intestinal bacteria larvae, two dominant culturable bacteria were reconstructed respectively, of which HcM3 had a significant synergistic effect on HcNPV, while HcM7 had some protective effects.

Key words: Hyphantria cunea; Midgut bacteria; HcNPV; Insecticidal activity

目 录

第一章 文献综述…………………………………………………………………………………1

1 美国白蛾核型多角体病毒概述…………………………………………………………1

1.1 美国白蛾研究概况………………………………………………………………1

1.2 美国白蛾核型多角体病毒简介…………………………………………………2

1.3 美国白蛾核型多角体病毒杀虫机制……………………………………………3

1.4 美国白蛾核型多角体病毒的增效途径…………………………………………4

2 昆虫与肠道微生物………………………………………………………………………5

2.1 昆虫肠道微生物种类……………………………………………………………5

2.2 肠道微生物对宿主的影响………………………………………………………5

第二章 材料与方法………………………………………………………………………………8

1 实验材料…………………………………………………………………………………8

2 实验方法…………………………………………………………………………………8

2.1 供试昆虫饲养………………………………………………………………8

2.2 HcNPV室内活性测定………………………………………………………9

2.2.1 核型多角体病毒复壮………………………………………………………9

2.3 无肠道细菌美国白蛾幼虫的获取……………………………………………9

2.4 肠道细菌对HcNPV毒力的影响…………………………………………………9

2.5 数据处理与分析…………………………………………………………………10

第三章 结果与分析……………………………………………………………………………11

1 抗生素对美国白蛾生长发育影响………………………………………………………11

2 美国白蛾幼虫的生物测定………………………………………………………………11

2.1 HcNPV毒力测定…………………………………………………………………12