中黑盲蝽性信息素组分鉴定及田间应用技术研究
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摘要
中黑盲蝽Adelphocoris suturalis (Jackson)是长江流域棉田的主要害虫之一。自1997年转基因棉推广种植以来,靶标害虫棉铃虫得以控制,农药使用的减少使得中黑盲蝽由次要害虫上升为主要害虫,且为害呈现扩增趋势。中黑盲蝽主要取食棉株的幼嫩部位和繁殖器官,造成花、蕾脱落,果实畸形从而影响棉花产量和品质。传统的化学防治防治效果不佳,且存在污染环境、破坏生态、杀害天敌等问题。
中黑盲蝽具有寄主广泛,转株危害性强等特点,目前缺乏有效的防治手段。而性诱剂作为害虫综合治理的一种新型技术被推广使用。本课题提出利用性信息素作为预测预报和控制中黑盲蝽的手段。我们利用气相色谱(GC)、气相色谱-触角电位仪(GC-EAD)联用技术和气相色谱-质谱联用仪(GC-MS)对中黑盲蝽性信息素进行提取、鉴定和定量分析,确定了中黑盲蝽性信息素主要组分及其配比,且通过田间诱捕试验对所得到的结果进行验证,同时对性诱剂田间应用技术进行研究,使得中黑盲蝽性信息素达到最佳的诱杀和预测预报效果。主要结果如下：
1、中黑盲蝽性信息素释放节律的研究和性成熟R龄确定
通过Y型昆虫嗅觉仪和田间诱捕试验对中黑盲蝽性信息素释放节律和性成熟同龄进行研究,结果表明性成熟的雌性处女成虫仅对雄性成虫具有引诱效果,对雌性成虫不具有引诱效果；雄性成虫对雌性和雄性成虫都不存在引诱效果。田间诱捕试验发现4-18日龄的雌性处女成虫对雄性成虫具有引诱效果,性信息素释放节律研究表明雌性吸引雄性开始于暗期前一个小时左右,并在19：00-23：00达到高峰。
2、中黑盲蝽后胸腺体的显微结构观察
后胸腺体由一个发育良好的贮藏囊和一对侧腺构成。贮藏囊与侧腺之间通过导管连接,侧腺呈多管状。中黑盲蝽后胸腺体具有一对开口,大而呈椭圆形,位于中足基节和后足基节之间的侧板。两个开口各有一条细长的渠道延伸至后胸中部。释放口周围称为释放面,其表面覆盖大量的蘑菇状释放体和刚毛。刚毛仅分布于舌状释放面的外侧,其他部位覆盖蘑菇状突起。蘑菇状突起呈不规则结构,相互之间通过4-5根桥状结构连接,并且每个蘑菇状突起延伸出一些小的脊与其他蘑菇状突起相互连接。
3、中黑盲蝽后胸腺体挥发物对雄性成虫求偶行为的影响
利用GC-MS对雌、雄性成虫后胸腺体浸提物进行分析,发现雌性后胸腺体含有19种化学物质,雄性21种,其中相同成分为15种。利用Y型昆虫嗅觉仪观察中黑盲蝽雌性后胸腺体挥发物对雄性成虫求偶行为的影响,结果表明：用液体石蜡阻塞雌性成虫的腹部和不做处理的雌性对雄性成虫具有强烈引诱效果；若虫和用液体石蜡阻塞后胸腺体释放口的雌性成虫对雄性成虫不具有引诱效果。
4、活性物质的提取
利用不同有机溶剂对雌、雄性成虫不同部位进行性信息素活性物质提取,GC-EAD分析检测出8种活性物质。通过对比后胸腺体浸泡法和顶空吸附法对性信息素活性物质取样,吸附法仅仅收集到3种活性物质。
5、性信息素活性物质的鉴定以及定量分析
利用GC-MS对8种活性物质进行鉴定,分别为：1、反-2-已烯醛；2、4-氧代-反-2-已烯醛；3、乙酸已酯；4、丁酸戊酯；5、丙酸已酯；6、丁酸已酯；7、已酸已酯；8、Fumaric acid dec-4-enyl propyl ester。对活性物质进行定量分析,雌性成虫分别为：反-2-已烯醛0.178±0.085μg、4-氧代-反-2-已烯醛37.312±8.068μg、乙酸已酯0.698+0.332pμg、丁酸戊酯1.699±0.549μg、丙酸已酯0.23±0.032μg、丁酸已酯46.570±6.g93μg、已酸已酯14.025±1.981μg;雄性成虫分别为：4-氧代-反-2-已烯醛9.491±2.405μg、乙酸已酯0.043±0.027μg、丁酸戊酯2.609±0.549μg、丙酸已酯0.416±0.071、μg丁酸已酯73.147±25.110μg、已酸已酯0.147±0.064μg。
6、田间诱捕试验
通过对五种活性物质一元、二元和三元组合配成诱芯进行田间诱捕检测,结果表明单一组分诱捕中黑盲蝽雄性成虫数量少；在二元配比中发现4-氧代-反-2-已烯醛和已酸己酯配比对中黑盲蝽雄性成虫的诱捕效果较好,而且比较稳定；但是三元配比对雄性成虫诱捕效果不佳。
7、田间性诱剂使用技术研究
通过田间诱捕试验对诱捕器颜色、摆放位置、摆放数量、间距、形状和诱芯进行评估,结果表明：不同颜色、摆放数量、放置位置和间距对中黑盲蝽的诱捕效果不存在显著影响；诱捕器悬挂于稍高于棉株顶冠的位置诱捕效果明显好于其他位置；菱形诱捕器诱捕数量明显高于三角形和五边形,聚乙烯小瓶作为诱芯诱捕效果要优于天然橡胶诱芯。
The plant bug, Adelphocoris suturalis (Hemiptera:Miridae) is a major insect pest of cotton, Gossypium hirsutum L., in the Changjiang River Region of China. Broad-spectrum pesticide applications had substantial decreased since the commercial cultivation of transgenic cotton from1997because cotton bollworm, Helicoverpa armigera Hubner, was successfully under control. Both A. suturalis nymphs and adults feed on delicate plant organs and buds of cotton, which leads to devastating yield losses and quality decrease in cotton production. Conventional chemical control methods were time-consuming, and often led to undesirable results, such as the development of pesticide resistance and environmental pollution.
There is no effective measure to control A. suturalis because of its high mobility and broad host range. Pheromone-baits traps is an effective measure for control and monitoring agricultural and forest pest. We applied the methods of GC, GC-MS, GC-EAD and field trap to identify the main compounents and their proportion of sex pheromone of A. suturalis. Pheromone-baited traps are an effective tool for field control and monitoring A. suturalis, and also potentially to be useful in direct control through the disruption of mating or mass trapping. The main results showed as follows:
1. Sexually mature age and the circadian rhythm of sex pheromone
Y-tube bioassay results showed that only males possessed strong reponse for odors from sexually mature virgin females. Two-year field trapping experiments indicated that males were a effectively ttracted to4-18-day-old virgin females, and males were attracted to virgin females started17:00, with a peak between19:00-23:00.
2. Morphology of the Metathoracic Scent Glands System
Used scanning electron microscope (SEM), the morphological structure of the metathoracic scent glands (MTGs) of A. suturalis was observed. The result indicated that MTGs are consist of a reservoir and paired lateral glands. The MTGs possessed two openings extends from a usually depressed channel downwards the middle of metathorax, which known as evaporative area. It was a tongue-like structure covered by bristles and mushroom-shaped cuticular structures
3. Behavioral responses of males to volatiles produced in the metathoracic glands
In GC-MS investigation, differences were found in quantitative and qualitative compositions of the substances between two sexes of A. suturalis. Female were determined19different chemical substances, and21different chemical substances in male,15were found in both sexes. In Y-tube bioassay study, males A. suturalis were strongly attracted to odors from live sexually mature virgin females and females occluded abdomen with paraffin. However, the male gave no or little responses to odors from nymphs and virgin females occluded MTGs.
4. Extracts of the active compounds
The active compounds of sex pheromone of different parts were extracted by different solvents, our GC-EAD recordings showed that the antennae of males of A. suturalis respond to eight active compounds produced by the MTGs of females. But the extracts of headspace absorption were only three same components comparing with the extracts of MTGs.
5. Identification and quantitative analysis of the active compounds
Eight active compounds were identified using gas chromatography-mass spectrometry (GC-MS). The result showed that active compounds were (E)-2-Hexenal,4-oxo-(E)-2-Hexenal, Hexyl acetate, Pentyl butyrate, Hexyl propionate, Hexyl butyrate, Hexyl hexanoate, Fumaric acid dec-4-enyl propyl ester, respectively. The compounds of female were(E)-2-Hexenal,4-oxo-(E)-2-Hexenal, Hexyl acetate, Pentyl butyrate, Hexyl propionate, Hexyl butyrate, Hexyl hexanoate. The contente was0.178±0.085μg,37.312±8.068μg,0.698±0.332μg,1.699±0.549μg,0.23±0.032μg,46.57±6.893μg,14.025±1.981μg, respectively. However, the compounds of male were4-oxo-(E)-2-Hexenal, Hexyl acetate, Pentyl butyrate, Hexyl propionate, Hexyl butyrate, Hexyl hexanoate, which were9.491±2.405μg,0.0426±0.027μg,2.609±0.549μg,0.416±0.071μg,73.147±25.110μg,0.147±0.064μg respectively.
6. Field trap experiments
Field trap experiments conducted with five active compounds as single components, with binary blends of there compounds and3multi-components mixtures. However, small number of the male A. suturalis was attracted by single component. The components of4-oxo-(E)-2-hexenal and hexyl hexanoate within a certain range possessed stronger attractiveness to the male A. suturalis. Three multi-components mixtures attract small numbers of male and were unstable.
7. Application of the sex pheromone-baited traps in field
We estimated the influence of trap color, trap number, trap height, trap placement and distance of trap on the numbers of A. suturalis captured. We found that trap color, trap number, trap placement, and distance had no a significant effect on the numbers of A. suturalis female and male captured. The most A. suturalis were captured by the traps placed at height of150cm from the ground than other height. The study showed that the rhombic trap was more attractive than triangle traps and pentagonal, and catches in trap bait with the polyethylene vials were greater than those in trap bait with a rubber septum.
中黑盲蝽具有寄主广泛,转株危害性强等特点,目前缺乏有效的防治手段。而性诱剂作为害虫综合治理的一种新型技术被推广使用。本课题提出利用性信息素作为预测预报和控制中黑盲蝽的手段。我们利用气相色谱(GC)、气相色谱-触角电位仪(GC-EAD)联用技术和气相色谱-质谱联用仪(GC-MS)对中黑盲蝽性信息素进行提取、鉴定和定量分析,确定了中黑盲蝽性信息素主要组分及其配比,且通过田间诱捕试验对所得到的结果进行验证,同时对性诱剂田间应用技术进行研究,使得中黑盲蝽性信息素达到最佳的诱杀和预测预报效果。主要结果如下：
1、中黑盲蝽性信息素释放节律的研究和性成熟R龄确定
通过Y型昆虫嗅觉仪和田间诱捕试验对中黑盲蝽性信息素释放节律和性成熟同龄进行研究,结果表明性成熟的雌性处女成虫仅对雄性成虫具有引诱效果,对雌性成虫不具有引诱效果；雄性成虫对雌性和雄性成虫都不存在引诱效果。田间诱捕试验发现4-18日龄的雌性处女成虫对雄性成虫具有引诱效果,性信息素释放节律研究表明雌性吸引雄性开始于暗期前一个小时左右,并在19：00-23：00达到高峰。
2、中黑盲蝽后胸腺体的显微结构观察
后胸腺体由一个发育良好的贮藏囊和一对侧腺构成。贮藏囊与侧腺之间通过导管连接,侧腺呈多管状。中黑盲蝽后胸腺体具有一对开口,大而呈椭圆形,位于中足基节和后足基节之间的侧板。两个开口各有一条细长的渠道延伸至后胸中部。释放口周围称为释放面,其表面覆盖大量的蘑菇状释放体和刚毛。刚毛仅分布于舌状释放面的外侧,其他部位覆盖蘑菇状突起。蘑菇状突起呈不规则结构,相互之间通过4-5根桥状结构连接,并且每个蘑菇状突起延伸出一些小的脊与其他蘑菇状突起相互连接。
3、中黑盲蝽后胸腺体挥发物对雄性成虫求偶行为的影响
利用GC-MS对雌、雄性成虫后胸腺体浸提物进行分析,发现雌性后胸腺体含有19种化学物质,雄性21种,其中相同成分为15种。利用Y型昆虫嗅觉仪观察中黑盲蝽雌性后胸腺体挥发物对雄性成虫求偶行为的影响,结果表明：用液体石蜡阻塞雌性成虫的腹部和不做处理的雌性对雄性成虫具有强烈引诱效果；若虫和用液体石蜡阻塞后胸腺体释放口的雌性成虫对雄性成虫不具有引诱效果。
4、活性物质的提取
利用不同有机溶剂对雌、雄性成虫不同部位进行性信息素活性物质提取,GC-EAD分析检测出8种活性物质。通过对比后胸腺体浸泡法和顶空吸附法对性信息素活性物质取样,吸附法仅仅收集到3种活性物质。
5、性信息素活性物质的鉴定以及定量分析
利用GC-MS对8种活性物质进行鉴定,分别为：1、反-2-已烯醛；2、4-氧代-反-2-已烯醛；3、乙酸已酯；4、丁酸戊酯；5、丙酸已酯；6、丁酸已酯；7、已酸已酯；8、Fumaric acid dec-4-enyl propyl ester。对活性物质进行定量分析,雌性成虫分别为：反-2-已烯醛0.178±0.085μg、4-氧代-反-2-已烯醛37.312±8.068μg、乙酸已酯0.698+0.332pμg、丁酸戊酯1.699±0.549μg、丙酸已酯0.23±0.032μg、丁酸已酯46.570±6.g93μg、已酸已酯14.025±1.981μg;雄性成虫分别为：4-氧代-反-2-已烯醛9.491±2.405μg、乙酸已酯0.043±0.027μg、丁酸戊酯2.609±0.549μg、丙酸已酯0.416±0.071、μg丁酸已酯73.147±25.110μg、已酸已酯0.147±0.064μg。
6、田间诱捕试验
通过对五种活性物质一元、二元和三元组合配成诱芯进行田间诱捕检测,结果表明单一组分诱捕中黑盲蝽雄性成虫数量少；在二元配比中发现4-氧代-反-2-已烯醛和已酸己酯配比对中黑盲蝽雄性成虫的诱捕效果较好,而且比较稳定；但是三元配比对雄性成虫诱捕效果不佳。
7、田间性诱剂使用技术研究
通过田间诱捕试验对诱捕器颜色、摆放位置、摆放数量、间距、形状和诱芯进行评估,结果表明：不同颜色、摆放数量、放置位置和间距对中黑盲蝽的诱捕效果不存在显著影响；诱捕器悬挂于稍高于棉株顶冠的位置诱捕效果明显好于其他位置；菱形诱捕器诱捕数量明显高于三角形和五边形,聚乙烯小瓶作为诱芯诱捕效果要优于天然橡胶诱芯。
The plant bug, Adelphocoris suturalis (Hemiptera:Miridae) is a major insect pest of cotton, Gossypium hirsutum L., in the Changjiang River Region of China. Broad-spectrum pesticide applications had substantial decreased since the commercial cultivation of transgenic cotton from1997because cotton bollworm, Helicoverpa armigera Hubner, was successfully under control. Both A. suturalis nymphs and adults feed on delicate plant organs and buds of cotton, which leads to devastating yield losses and quality decrease in cotton production. Conventional chemical control methods were time-consuming, and often led to undesirable results, such as the development of pesticide resistance and environmental pollution.
There is no effective measure to control A. suturalis because of its high mobility and broad host range. Pheromone-baits traps is an effective measure for control and monitoring agricultural and forest pest. We applied the methods of GC, GC-MS, GC-EAD and field trap to identify the main compounents and their proportion of sex pheromone of A. suturalis. Pheromone-baited traps are an effective tool for field control and monitoring A. suturalis, and also potentially to be useful in direct control through the disruption of mating or mass trapping. The main results showed as follows:
1. Sexually mature age and the circadian rhythm of sex pheromone
Y-tube bioassay results showed that only males possessed strong reponse for odors from sexually mature virgin females. Two-year field trapping experiments indicated that males were a effectively ttracted to4-18-day-old virgin females, and males were attracted to virgin females started17:00, with a peak between19:00-23:00.
2. Morphology of the Metathoracic Scent Glands System
Used scanning electron microscope (SEM), the morphological structure of the metathoracic scent glands (MTGs) of A. suturalis was observed. The result indicated that MTGs are consist of a reservoir and paired lateral glands. The MTGs possessed two openings extends from a usually depressed channel downwards the middle of metathorax, which known as evaporative area. It was a tongue-like structure covered by bristles and mushroom-shaped cuticular structures
3. Behavioral responses of males to volatiles produced in the metathoracic glands
In GC-MS investigation, differences were found in quantitative and qualitative compositions of the substances between two sexes of A. suturalis. Female were determined19different chemical substances, and21different chemical substances in male,15were found in both sexes. In Y-tube bioassay study, males A. suturalis were strongly attracted to odors from live sexually mature virgin females and females occluded abdomen with paraffin. However, the male gave no or little responses to odors from nymphs and virgin females occluded MTGs.
4. Extracts of the active compounds
The active compounds of sex pheromone of different parts were extracted by different solvents, our GC-EAD recordings showed that the antennae of males of A. suturalis respond to eight active compounds produced by the MTGs of females. But the extracts of headspace absorption were only three same components comparing with the extracts of MTGs.
5. Identification and quantitative analysis of the active compounds
Eight active compounds were identified using gas chromatography-mass spectrometry (GC-MS). The result showed that active compounds were (E)-2-Hexenal,4-oxo-(E)-2-Hexenal, Hexyl acetate, Pentyl butyrate, Hexyl propionate, Hexyl butyrate, Hexyl hexanoate, Fumaric acid dec-4-enyl propyl ester, respectively. The compounds of female were(E)-2-Hexenal,4-oxo-(E)-2-Hexenal, Hexyl acetate, Pentyl butyrate, Hexyl propionate, Hexyl butyrate, Hexyl hexanoate. The contente was0.178±0.085μg,37.312±8.068μg,0.698±0.332μg,1.699±0.549μg,0.23±0.032μg,46.57±6.893μg,14.025±1.981μg, respectively. However, the compounds of male were4-oxo-(E)-2-Hexenal, Hexyl acetate, Pentyl butyrate, Hexyl propionate, Hexyl butyrate, Hexyl hexanoate, which were9.491±2.405μg,0.0426±0.027μg,2.609±0.549μg,0.416±0.071μg,73.147±25.110μg,0.147±0.064μg respectively.
6. Field trap experiments
Field trap experiments conducted with five active compounds as single components, with binary blends of there compounds and3multi-components mixtures. However, small number of the male A. suturalis was attracted by single component. The components of4-oxo-(E)-2-hexenal and hexyl hexanoate within a certain range possessed stronger attractiveness to the male A. suturalis. Three multi-components mixtures attract small numbers of male and were unstable.
7. Application of the sex pheromone-baited traps in field
We estimated the influence of trap color, trap number, trap height, trap placement and distance of trap on the numbers of A. suturalis captured. We found that trap color, trap number, trap placement, and distance had no a significant effect on the numbers of A. suturalis female and male captured. The most A. suturalis were captured by the traps placed at height of150cm from the ground than other height. The study showed that the rhombic trap was more attractive than triangle traps and pentagonal, and catches in trap bait with the polyethylene vials were greater than those in trap bait with a rubber septum.
引文
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