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云斑天牛Batocera horsfieldi属鞘翅目Coleoptera天牛科Cerambycidae沟胫天牛亚科Lamiinae白条天牛属Batocera,又名白条天牛,是在中国广泛分布的一种丰富且具有破坏性的天牛。幼虫蛀食活树树干的内皮和边材部分,导致树容易断裂,其基本生物学特性与亚科其他天牛相似,生命周期通常在2 a内完成[1]。成虫出现在初夏,主要以山核桃Carya cathayensis的枝条为食。云斑天牛性成熟,雌雄成虫交配后,雌虫将卵存放在寄主树的树皮下度过整个夏天。孵化后的幼虫立即进入内部树皮,化蛹前至少喂食20个月[2]。据报道,在浙江省杭州市临安区,多数山核桃被天牛钻蛀,其种类主要有桑天牛Apriona germari,星天牛Anoplophora chinensis和云斑天牛[3]。云斑天牛幼虫主要生活在寄主植物树干内,而成虫会飞行,鞘翅坚硬且耐药性强,一般的物理和化学方法很难防控。许多植食性昆虫利用气味作为寄主定位的线索,无论是营养、配偶地点还是存放其后代[4-8]。有研究显示:植物挥发性气体能引起天牛显著的行为反应[9-10]。植物挥发性气体是多种微浓度的挥发性化合物组成的混合物[11],可以调控天牛取食、产卵和交配等行为[12]。如ZHUGE等[13]发现:云斑天牛可能依赖特定比例的挥发物来寻找定位寄主植物;寄主植物挥发物中的壬醛对云斑天牛有较好的林间诱捕效果[14]。植物在自然状态、受到天牛攻击或机械损伤时释放的挥发物在种类和浓度上存在差异[15]。天牛利用识别植物挥发物的化学成分和相对含量来甄别植物的状态,进而做出反应[16],如光肩星天牛Anoplophora glabripennis对受到天牛攻击后的复叶槭Acer negundo的挥发物有显著驱避效果,而对健康复叶槭的挥发物有显著引诱效果[17]。近年来,许多国内外学者通过研究昆虫和植物挥发物之间的化学联系,明确昆虫和植物挥发物互作机制,探究如何利用有益昆虫或防控有害昆虫,可以采用类似方法来探索防控云斑天牛的防治方法。本研究运用动态顶空套袋吸附法法收集不同受害状态下山核桃释放的挥发物,并通过气相色谱-触角电位测量系统(GC-EAD)技术和Y型嗅觉仪技术,检测能引起云斑天牛显著电生理和行为反应的挥发物,为筛选云斑天牛的植物源引诱剂或驱避剂的成分和比例提供理论基础。
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试验所用云斑天牛成虫和山核桃样树来自于浙江省杭州市临安区玲珑街道雅园村的山核桃苗圃基地(30°12′N,119°38′E,平均海拔113 m)。山核桃苗圃基地有少量苦楝Melia azedarach,林下除杂草外无其他植被,林地地势较平坦。其中山核桃树龄5~20年生,树高为3~15 m,胸径为8~25 cm,林冠郁闭度约0.5。虫源利用云斑天牛的假死性进行人工捕捉。
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在山核桃林中随机选取健康状态以及被天牛取食、产卵和钻蛀受害状态的山核桃(树龄为5~10年生,树高为5~15 m,胸径为10~15 cm)各3株并标记,其中各株样树仅1种受害状。采用活体植物动态顶空吸附法在同一时间收集样树不同受害状态下山核桃释放的挥发物,采样6 h(9:00-16:00),重复3次。为消除空气中的气体对采集样品的污染,各种受害状态设空白对照样2份。具体的收集方法和步骤参见文献[18]。
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在中国科学院动物研究所实验室进行雌、雄云斑天牛成虫对不同受害状态下山核桃枝条挥发物洗脱液的触角电位反应实验。GC-EAD分析采用Agilent 6890N气相色谱,色谱柱为HP-5MS,载气:高纯度氮气,流速1.0 mL·min-1;进样量2.0 μL,无分流进样,进样口温度220 ℃,氢火焰离子检测器,检测器温度280 ℃。柱温升温程序:40 ℃恒温1 min,然后以4 ℃·min-1的速度升温到220 ℃恒温3 min,共运行49 min。测试的样品为不同受害状态下山核桃枝条挥发物洗脱液和对照正己烷溶剂,试验用虫均为活体,重复5次,GC-EAD与色谱图上的化合物的峰形和保留时间进行对比,从而鉴定出对雌、雄云斑天牛触角有触角电位反应的物质。具体测定方法和步骤参见文献[18]。
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据云斑天牛成虫对不同受害状态下山核桃挥发物的触角电位反应,选择有强烈电生理反应的8种挥发物(表 1),用1.0 mL液体石蜡作溶剂将各化合物配制成浓度为1.0 mol·L-1的溶液[19]。Y型嗅觉仪生物测定装置的规格和嗅觉反应的实验方法和步骤参见文献[18]。
表 1 8种标准挥发物的名称、纯度和来源
Table 1. Name, purity and source of the eight standard volatiles
化合物 纯度/% CAS号 来源 蒎烯(1R)-(+)-alpha-pinene 99 7785-70-8 阿拉丁 萜品烯g-terpinene 98 99-85-4 南京草本源生物科技有限公司 壬醛nonanal 96 124-19-6 阿拉丁 α-萜品醇α-terpineol ≥96 98-55-5 上海源叶生物科技有限公司 五甲基苯pentamethyl phenyl ≥98 700-12-9 阿拉丁 丙烯酸异辛酯2-ethylhexyl acrylate ≥99 103-11-7 阿拉丁 2, 6-二甲基萘2, 6-dimethylnaphthalene 98 581-42-0 阿拉丁 正十六烷hexadecane 98 544-76-3 阿拉丁 -
试验数据用SPSS 19.0软件进行统计分析。比较云斑天牛成虫对不同化合物行为反应的差异显著性通过卡方检验来分析[20-21],其中选择率计算公式如下:选择率=(气味臂内的总虫数或对照臂的总虫数/测试的总虫数)×100%。
Electroantennogram and behavioral responses of Batocera horsfieldi (Coleoptera:Cerambycidae) to volatiles released from the Carya cathayensis in different damaged states
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摘要: 为探索能影响云斑天牛Batocera horsfieldi行为反应的山核桃Carya cathayensis挥发物,采用活体植物动态顶空套袋吸附法收集不同受害状态(健康、天牛取食、天牛产卵和天牛钻蛀后)山核桃释放的挥发物,并运用气相色谱-触角电位测量系统(GC-EAD)及Y型嗅觉仪技术分析鉴定出对云斑天牛有电生理及行为反应的挥发物。结果表明:云斑天牛成虫对不同受害状态下山核桃挥发物中蒎烯、萜品烯、壬醛、α-萜品醇、五甲基苯、丙烯酸异辛酯、2,6-二甲基萘及正十六烷8种挥发物均产生电生理反应。嗅觉反应结果显示:萜品烯对云斑天牛雄成虫产生显著的引诱作用(P < 0.05);壬醛对云斑天牛雌成虫产生极显著的引诱作用(P < 0.01),对其雄成虫产生显著的引诱作用(P < 0.05);而蒎烯对云斑天牛雌成虫产生极显著的驱避作用(P < 0.01);α-萜品醇则对其雌成虫产生极显著的引诱作用(P < 0.01),对其雄成虫产生显著的驱避作用(P < 0.05)。研究结果说明:α-萜品醇和壬醛对云斑天牛雌成虫产生较强的引诱作用,而蒎烯对其雌成虫产生较好的驱避作用;萜品烯和壬醛对云斑天牛雄成虫产生较强的引诱作用,而α-萜品醇则对其雄成虫产生较好的驱避作用。Abstract: To explore the volatiles from the Carya cathayensis, which affect the behavioral response of the brown mulberry longhorn beetle, Batocera horsfieldi, and to provide a theoretical basis for screening plant-originated attractants or repellents of this insect. In the C. cathayensis of Lin'an Yayuan Village, 3 C. cathayensis trees were vandamly selected from different damaged states[healthy (ck), as well as feeding, oviposition and boring damage by longicorn beetle]. The volatiles were collected by using dynamic headspace adsorption living plants, and those causing electrophysiological and behavioral responses in B. horsfieldi were identified by gas chromatography-electrophysiological antennal detecting system (GC-EAD) and Y-tube olfactometer. Results showed that the B. horsfieldi adults had significant electrophysiological responses to eight volatiles of C. cathayensis volatiles with different damaged states, including (1R)-(+)-alpha-pinene, g-terpinene, nonanal, α-terpineol, pentamethyl phenyl, 2-ethylhexyl acrylate, 2, 6-dimethylnaphthalene and hexadecane. Female and male adults had the strongest electrophysiological responses to nonanal and α-terpineol followed. The olfactory reaction showed that g-terpinene had a significant attractivity to male adults of B. horsfieldi (P < 0.05). Also nonanal had an highly significant attractivity to female adults (P < 0.01) and a significant attractivity to male adults (P < 0.05). Meanwhlie, (1R)-(+)-alpha-pinene had an highly significant repellent activity to female adults (P < 0.01). In addition α-terpineol had a highly significant attractivity to female adults (P < 0.01), but a significant repellent activity to male adults (P < 0.05). These results indicate that α-terpineol and nonanal showed strong attractivity to female adults of B. horsfieldi, while (1R)-(+)-alpha-pinene showed good repellent activity, g-terpinene and nonanal showed strong attractivity to male adults, while α-terpineol showed good repellent activity.
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Key words:
- forest protection /
- Batocera horsfieldi /
- Carya cathayensis /
- volatiles /
- GC-EAD /
- Y-tube olfactory
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图 1 云斑天牛雌成虫对不同受害状态下山核桃释放的挥发物的触角电位反应图谱
A.健康; B.取食受害; C.产卵受害; D.钻蛀受害。
1.蒎烯(1R)-(+)-alpha-pinene; 2.萜品烯g-terpinene;3.壬醛nonanal; 4. α-萜品醇α-terpineol; 5.五甲基苯pentamethyl phenyl; 6.丙烯酸异辛酯2-ethylhexyl acrylate; 7. 2, 6-二甲基萘2, 6-dimethylnaphthalene; 8.正十六烷hexadecaneFigure 1 Electroantennogram responses pattern of female Batocera horsfieldi adults to volatiles released from the Carya cathayensis in different damaged states
表 1 8种标准挥发物的名称、纯度和来源
Table 1. Name, purity and source of the eight standard volatiles
化合物 纯度/% CAS号 来源 蒎烯(1R)-(+)-alpha-pinene 99 7785-70-8 阿拉丁 萜品烯g-terpinene 98 99-85-4 南京草本源生物科技有限公司 壬醛nonanal 96 124-19-6 阿拉丁 α-萜品醇α-terpineol ≥96 98-55-5 上海源叶生物科技有限公司 五甲基苯pentamethyl phenyl ≥98 700-12-9 阿拉丁 丙烯酸异辛酯2-ethylhexyl acrylate ≥99 103-11-7 阿拉丁 2, 6-二甲基萘2, 6-dimethylnaphthalene 98 581-42-0 阿拉丁 正十六烷hexadecane 98 544-76-3 阿拉丁 -
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链接本文:
https://zlxb.zafu.edu.cn/article/doi/10.11833/j.issn.2095-0756.2019.03.002