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Feb 2021
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Preference Test of Plutella xylostella Larvae upon DMNT Treatment
小菜蛾幼虫对DMNT处理的偏好试验   

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Abstract

We describe a method to test the preference of insects in response to (3E)-4,8-dimethyl-1,3,7-nonatriene (DMNT). We use a device that includes a horizontal glass tube, two grooves (with activated carbon), air flow, rubber stoppers/tubes, transparent glass containers (optional), and a holder for the glass tube (optional). Equal amounts of activated carbon in the groove (removable) are placed at both ends to avoid air contamination. The air flow is generated by an air pump. In the closed device, different samples are placed at each end of the glass tube. The air pump at the top of the glass tube forms an air flow that converges to the middle site of the glass tube. In each test, insect larvae are located in the middle of the glass test tube. If the test samples release DMNT that can be sensed by insects, the insects will selectively move to one specific end of the glass tube. The number of insects that move to each end will be recorded for further studies. This method can also be used to test the preference of insects in response to other volatile compounds.

Keywords: Insect preference test (昆虫偏好测试), Device (装置), Volatile compound (挥发性化合物), DMNT (DMNT), Insect larvae ( 昆虫幼虫), P. xylostella (小菜蛾)

Background

Insects have the ability to sense certain volatile compounds and show a differentiated response. However, this response is easily influenced by the surrounding environment. To make the data reliable for scientific research, it is necessary to set up a device to test the response more accurately. Previous research has showed that the preference of insects in response to volatile compounds can be tested by a Y tube (Adhikary et al., 2014; Ndomo-Moualeu et al., 2016). Here, we provide a schematic representation of a choice test system to check the behavior of insects affected by volatile DMNT or other compounds released from different samples.

Materials and Reagents

  1. DMNT standard was synthesized according to previous research (Huang and Yang, 2007; Chen et al., 2021).

  2. Paraffin oil (Sigma, catalog number: M1180)

Equipment

  1. Air pump (Beijing Municipal Institute of Labour Protection, model: QC-1S)

  2. Activated carbon (Sangon Biotech, catalog number: A600287-0001)

  3. Glass tube (Length: 35 cm; Inner diameter of glass tube: 3.8 cm)

  4. Rubber stoppers/tubes (Tansoole, catalog numbers: 02036109 [8#]; 02025735 [6#]; 02025732 [3#])

  5. Holder for glass tube (Height: 15 cm, optional)

  6. Glass containers (Length: 4.2 cm; Width: 4.2 cm; Height: 4.2 cm)

Procedure

  1. As shown in figure 1, the test samples are located at each end of the glass tube and both ends of the tube are closed with rubber stoppers. The groove (activated carbon included) is connected at both ends of the horizontal glass tube (activated carbon is used to filter the gases in the experimental environment, which may interfere with insect response).



    Figure 1. A schematic representation of insect choice test system (modified from Chen et al., 2021).


  2. When this system is used to test plants that may release DMNT, the plants should be placed in a transparent glass container, which is connected to the glass test tube via plastic tubes. The plants can be kept in the glass containers for a few hours to enrich the environment for DMNT before proceeding to the next steps.

  3. Turn on the pump (1 ml/min) for 5 min to create a clean and stable air flow inside the glass tube.

  4. Place insect larvae in the port, right in front of the glass tube, and close the tube with a rubber stopper (Video 1).


    Video 1. Setup of the device to test the preference of P. xylostella larvae in response to DMNT.


  5. After 3-10 min, the number of insects that moved towards each end of the glass tube is recorded (The standard of choice is for larvae to move 2 cm away from the middle of the glass test tube to each end, Video 2).


    Video 2. P. xylostella larvae are repelled by DMNT.


  6. To avoid interference from the surrounding environment, the test and control samples should swap places during experiments.

  7. Clean all the parts of the device and repeat the above procedures several times to obtain reliable results.

Data analysis

We counted the number of larvae that crawled toward each end of the glass test tube and calculated the percentage of larvae at each end of the glass tube.

Notes

  1. Before and after different experiments, use a fragrance-free cleaner to clean the whole device, rinse it thoroughly with double distilled water, and dry it in a drying oven to avoid the influence of residual odor or contamination.

  2. The air extraction rate, as controlled by the pump, should be slow (approximately 1 ml/min).

  3. This device is suitable to test the choice of small insect larvae such as Plutella xylostella and Ostrinia furnacalis.

  4. Activated carbon should be changed regularly between different tests.

  5. The device can be extended to test volatile compounds released from plant materials. The plants tested can be placed into closed tanks, which are connected to the two ends of the glass test tube with rubber tubes.

  6. All the rubber stoppers and rubber tubes should be made of high standard rubber materials without odor. All the rubber parts should be cleaned and dried thoroughly before experiments.

Acknowledgments

This work was supported by the National Key Research and Development Program of China (2017YFD0301301, 2016YFD0101803) and the Natural Science Foundation of China (31670264). This protocol was adapted from the publication by Chen et al. (2021; Doi: 10.7554/eLife.63938).

Competing interests

The authors declare no conflicts of interest.

References

  1. Adhikary, P., Mukherjee, A. and Barik, A. (2014). Role of surface wax alkanes from Lathyrus sativus L. seeds for attraction of Callosobruchus maculatus (F.) (Coleoptera: Bruchidae). J Stored Prod Res 59: 113-119.
  2. Chen, C., Chen, H., Huang, S., Jiang, T., Wang, C., Tao, Z., He, C., Tang, Q. and Li, P. (2021). Volatile DMNT directly protects plants against Plutella xylostella by disrupting the peritrophic matrix barrier in insect midgut. Elife 10: e63938.
  3. Huang, H. J. and Yang, W. B. (2007). Synthesis of moenocinol and its analogs using BT-sulfone in Julia-Kocienski olefination. Tetrahedron Lett 48(8): 1429-1433.
  4. Ndomo-Moualeu, A. F., Ulrichs, C. and Adler, C. (2016). Behavioral responses of Callosobruchus maculatus to volatile organic compounds found in the headspace of dried green pea seeds. J Pest Sci 89(1): 107-116.

简介

[摘要]我们描述了一种测试昆虫对 (3E)-4,8-二甲基-1,3,7-壬三烯 (DMNT)。我们使用的设备包括一个水平玻璃管、两个凹槽(带有活性炭)、气流、橡胶塞/管、透明玻璃容器(可选)和支架用于玻璃管(可选)。凹槽内等量活性炭(可拆卸)放置两端,避免空气污染。气流由气泵产生。在封闭装置中,不同的样品放置在玻璃管的每一端。玻璃管顶部的气泵形成会聚到玻璃管中间部位的气流。在每次测试中,昆虫幼虫位于玻璃试管中间。如果测试样品释放 DMNT 可以通过昆虫,昆虫会选择性地移动到玻璃管的特定一端。昆虫的数量将记录到每一端的移动以供进一步研究。这个方法也可以用来测试昆虫对其他挥发性化合物的偏好。

[背景]昆虫具有感知某些挥发性化合物的能力,并表现出有区别的回复。但是,这种反应很容易受到周围环境的影响。为了使科学研究的数据可靠,需要设置一个装置来更准确地测试响应。先前的研究表明,昆虫对挥发性化合物的偏好可能是由 Y 管测试(Adhikary等人,2014 年;Ndomo-Moualeu等人,2016 年)。在这里,我们提供一个示意图选择测试系统的代表,以检查受挥发性 DMNT 影响的昆虫的行为或从不同样品中释放的其他化合物。

关键字:昆虫偏好测试, 装置, 挥发性化合物, DMNT, 昆虫幼虫, 小菜蛾

材料和试剂

1. DMNT 标准品是根据之前的研究合成的(Huang and Yang, 2007; Chen等。, 2021)

2.石蜡油(Sigma,目录号:M1180

设备

1、气泵(北京市劳动保护研究所,型号:QC-1S

2.活性炭(Sangon Biotech,目录号:A600287-0001

3. 玻璃管(长度:35 cm;玻璃管内径:3.8 cm

4. 橡胶塞/管(Tansoole,目录号:02036109 [8#]02025735 [6#]02025732[3#])

5. 玻璃管支架(高度:15 cm,可选)

6. 玻璃容器(长:4.2 厘米;宽:4.2 厘米;高:4.2 厘米)

程序

1、如图1所示,测试样品位于玻璃管的两端和两端管子用橡胶塞封闭。凹槽(含活性炭)连接在水平玻璃管的两端(活性炭用于过滤管内的气体)实验环境,这可能会干扰昆虫的反应)。

1. 昆虫选择测试系统的示意图(修改自 Chen等阿尔。, 2021)

2. 当本系统用于测试可能释放 DMNT 的植物时,应将植物置于透明玻璃容器,通过塑料管与玻璃试管相连。这植物可以在玻璃容器中保存几个小时,以丰富 DMNT 的环境在继续下一步之前。

3. 开启泵 (1 ml/min) 5 分钟,在玻璃管内产生干净稳定的气流。

4. 将昆虫幼虫放在玻璃管正前方的端口中,并用橡皮密封管塞子(视频 1

视频 1. 测试小菜蛾幼虫偏好的设备设置DMNT

5. 3-10分钟后,记录向玻璃管两端移动的昆虫数量(选择的标准是幼虫离玻璃试管中间2cm到每一端,视频 2)

视频 2. 小菜蛾幼虫被 DMNT 排斥。

6. 为避免周围环境的干扰,测试和控制样品应实验时交换位置。

7. 清洁设备的所有部件并重复上述步骤数次以获得可靠的结果。

数据分析

我们计算了爬向玻璃试管两端的幼虫数量并计算出玻璃管两端的幼虫百分比。

笔记

1. 不同实验前后,使用无香味清洁剂对整个设备进行清洁,用双蒸水彻底冲洗干净,然后在烘箱中烘干,以免影响残留气味或污染。

2. 由泵控制的抽气速度应该很慢(大约 1 毫升/分钟)。

3.本装置适用于测试小昆虫幼虫的选择如小菜蛾和Ostrinia furnacalis

4、活性炭在不同试验之间应定期更换。

5. 该装置可扩展用于测试植物材料释放的挥发性化合物。这被测试的植物可以放入封闭的水箱中,连接到玻璃的两端带橡胶管的试管。

6、所有胶塞、胶管均采用高标准橡胶材料无异味。实验前应彻底清洁并干燥所有橡胶部件。

致谢

这项工作得到了国家重点研发计划的支持(2017YFD0301301, 2016YFD0101803) 和国家自然科学基金 (31670264)。该协议改编自 Chen等人的出版物。(2021 年;Doi10.7554/eLife.63938)。

参考

1. Adhikary, P.Mukherjee, A. Barik, A. (2014)。 来自Lathyrus的表面蜡烷烃的作用sativus L. 种子用于吸引Callosobruchus maculatus (F.) (鞘翅目: Bruchidae)J存储产品 Res 59113-119

2. Chen, C., Chen, H., Huang, S., Jiang, T., Wang, C., Tao, Z., He, C., Tang, Q. Li, P. (2021)。挥发性 DMNT通过破坏周围营养直接保护植物免受小菜蛾的侵害昆虫中肠的基质屏障。Elife 10e63938

3. Huang, HJ Yang, WB (2007)。使用 BT-砜合成莫诺西诺及其类似物Julia-Kocienski 烯化。Ť etrahedron快报488):14291433年。

4. Ndomo-Moualeu, AFUlrichs, C. Adler, C. (2016)。的行为反应Callosobruchus maculatus对干绿色顶部空间中发现的挥发性有机化合物豌豆种子。J Pest Sci 89(1): 107-116

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Copyright Chen et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Chen, C., Tao, Z., Wang, C., Wang, T., Chen, H., Jiang, T. and Li, P. (2021). Preference Test of Plutella xylostella Larvae upon DMNT Treatment. Bio-protocol 11(21): e4208. DOI: 10.21769/BioProtoc.4208.
  2. Chen, C., Chen, H., Huang, S., Jiang, T., Wang, C., Tao, Z., He, C., Tang, Q. and Li, P. (2021). Volatile DMNT directly protects plants against Plutella xylostella by disrupting the peritrophic matrix barrier in insect midgut. Elife 10: e63938.
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