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Electrical Penetration Graph Recording (Whitefly)
电渗透图记录(粉虱)   

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Abstract

Electrical penetration graph (EPG) was first developed by Mclean and Kinsey (1964) and later modified by Tjallingii (1978 and 1985). The EPG system turns a phloem-sucking insects and its host plant into part of an electrical circuit that is completed when the insect’s mouthparts penetrate the plant. The electrical signal is amplified by an amplifier and digitized by a converter. Fluctuations in voltage and electrical resistance are recorded and can be matched to specific feeding events. Current protocol is an efficient use of the EPG system to record whitefly feeding behavior on plants.

Materials and Reagents

  1. Plant (e.g., 2-3 true leaf stage)
  2. Newly-emerged (e.g., 2-5 d old) female whiteflies

Equipment

  1. Ice-bag or box
  2. Glass dish (Diameter = 6 cm)
  3. Gold wire (Diameter = 12.5 μm, http://www.epgsystems.eu/products.php)
  4. Silver glue (Vial of 2ml glue, http://www.epgsystems.eu/products.php)
  5. Faraday cage (self-made cage with10 mesh wire net, length = 60 cm, width = 60 cm, height = 70 cm)
  6. Insect-proof cage (self-made cage with 80 mesh nylon screening, length = 40 cm, width = 40 cm, height = 60 cm)
  7. Transparent tubes (self-made tube with bottle, 3 cm long, inner diameter = 5 mm)
  8. Small ice-chilled glass dish
  9. Giga-8 DC-EPG system with 109 Ohm input resistance (Wageningen University, http://www.epgsystems.eu/products.php)
  10. DI710-UL analogue-to-digital converter (DATAQ Instruments, http://www.epgsystems.eu/products.php)
  11. Computer
  12. Dissecting microscope

Software

  1. PROBE 3.4 software (Wageningen University, advanced version can be downloaded from http://www.epgsystems.eu/downloads.php)

Procedure

  1. The age of newly-emerged whiteflies should be estimated precisely. All adults are used within 2-5 d of their emergence.
    1. They are obtained by picking a plant leaf infested with fourth-instar nymphs, removing any adults, and putting the infested leaf on an uninfested plant (of the same species) in an insect-proof cage.
    2. After 24 h, remove the nymph-infested leaf and any excess nymphs; the only whiteflies remaining in the cage are adults that have emerged in the past 24 h. After 48 h, 2-3 d-old whitefly adults can be randomly picked for use in the experiment.
    3. After 120 h, any remaining whiteflies are discarded.
  2. Only female whiteflies are used because sexes might differ in their probing behavior. Adults are sexed by using transparent bottle tubes (3 cm long, inner diameter = 5 mm) to pick up individual whiteflies, with one whitefly per tube. Then a dissecting microscope is used to examine each whitefly in its tube in order to determine its sex.
  3. A newly-emerged (2-5 d) female whitefly is immobilized in a small ice-chilled glass dish which is put on an ice bag or ice box beforehand.
  4. Attach a 1.5 cm x 12.5 μm gold wire to the whitefly's dorsum using a droplet of water-based silver glue (Figure 1).
  5. The wired whitefly is then connected to the input of the Giga-8 probe and place on the lower surface of the bottom leaf of the appropriate host plant (Figure 1).
  6. Each replicate whitefly-plant-probe combination is placed into an electrically-grounded Faraday cage to shield the setup from electrical noise.
  7. Six to eight hours should be recorded according to the respective experiment objectives.
  8. EPG signals are digitized with a DI710-UL analogue-to-digital converter and acquired with PROBE 3.4 software.
  9. All experiments are carried out at 26 ± 2 °C, 70% relative humidity, and under artificial light (1,500 lux) with a 14:10 h L: D regime.


    Figure 1. Schematic diagram of DC-EPG, especially for the whitefly-plant-probe combination when attaching the gold wire with silver glue (Liu et al., 2013)

Acknowledgments

This protocol is adapted from Liu et al. (2013).

References

  1. Liu, B., Preisser, E. L., Chu, D., Pan, H., Xie, W., Wang, S., Wu, Q., Zhou, X. and Zhang, Y. (2013). Multiple forms of vector manipulation by a plant-infecting virus: Bemisia tabaci and tomato yellow leaf curl virus. J Virol 87(9): 4929-4937.
  2. Mclean, D. and Kinsey, M. (1964). A technique for electronically recording aphid feeding and salivation. Nature 202(4939): 1358-1359.
  3. Tjallingii, W. (1978). Electronic recording of penetration behaviour by aphids. Entomologia Experimentalis et Applicata 24(3): 721-730.
  4. Tjallingii, W. (1985). Electrical nature of recorded signals during stylet penetration by aphids. Entomologia Experimentalis et Applicata 38(2): 177-186.

简介

电穿透图(EPG)由Mclean和Kinsey(1964)首先开发,并且随后由Tjallingii(1978和1985)修改。 EPG系统将韧皮部吸虫和其宿主植物变成当昆虫的口器穿透植物时完成的电路的一部分。 电信号由放大器放大并由转换器数字化。 记录电压和电阻的波动,并可与特定的进料事件相匹配。 当前的方案是EPG系统有效地使用记录植物上的粉虱摄食行为

材料和试剂

  1. 植物(如,2-3真叶阶段)
  2. 新出现的(例如,2-5天龄)雌flies

设备

  1. 冰袋或盒子
  2. 玻璃皿(直径= 6cm)
  3. 金线(直径= 12.5μm, http://www.epgsystems.eu/products.php
  4. 银胶(2ml胶水小瓶, http://www.epgsystems.eu/products.php
  5. 法拉第笼(具有10目网丝的自制笼,长度= 60cm,宽度= 60cm,高度= 70cm)
  6. 防虫笼(自制笼,80目尼龙筛网,长度= 40cm,宽= 40cm,高= 60cm)
  7. 透明管(具有瓶的自制管,3cm长,内径= 5mm)
  8. 小冰冷玻璃皿
  9. 具有10 9欧姆输入电阻的Giga-8 DC-EPG系统(Wageningen University, http ://www.epgsystems.eu/products.php
  10. DI710-UL模拟到数字转换器(DATAQ Instruments, http://www.epgsystems.eu/products。 php
  11. 电脑
  12. 解剖显微镜

软件

  1. PROBE 3.4软件(瓦赫宁根大学,高级版本可以从 http://www.epgsystems.eu/downloads下载。 php

程序

  1. 新出现的粉虱的年龄应该精确估计。 所有成年人在出现的2-5天内使用。
    1. 它们通过挑取被第四龄期若虫侵染的植物叶子,除去任何成虫,并将被侵染的叶子放在未防治的植物(同一物种)上的昆虫防护笼中而获得。
    2. 24小时后,除去若虫侵染叶和任何多余的若虫; 在笼子里剩下的唯一的粉虱是在过去24小时出现的成虫。 48小时后,可以随机抽取2-3天的粉虱成虫用于实验
    3. 120小时后,丢弃任何剩余的粉虱。
  2. 只有女性粉虱被使用,因为性别可能不同的探测行为。成年人通过使用透明瓶管(3cm长,内径= 5mm)进行性别化以拾取单独的粉虱,每管一只粉虱。然后使用解剖显微镜检查其管中的每只粉虱,以确定其性别
  3. 将新出现的(2-5天)雌蝇固定在预先放在冰袋或冰盒上的小的冰冷玻璃皿中。
  4. 使用一滴水基银胶将1.5cm×12.5μm的金丝附着在粉虱的背部(图1)。
  5. 然后将有线粉虱连接到Giga-8探针的输入端,并放置在适当宿主植物的底叶下表面(图1)。
  6. 将每个重复的粉虱 - 植物 - 探针组合置于电接地的法拉第笼中,以使装置免受电噪声。
  7. 应根据各自的实验目标记录六至八小时
  8. EPG信号通过DI710-UL模拟到数字转换器进行数字化,并使用PROBE 3.4软件采集。
  9. 所有实验在26±2℃,70%相对湿度下和在人工光(1,500lux)下以14:10h L:D状态进行。


    图1. DC-EPG的示意图,特别是在用银胶连接金线时的粉虱 - 植物 - 探针组合 (Liu et ,2013)

致谢

该协议改编自Liu et al。(2013)。

参考文献

  1. Liu,B.,Preisser,E.L.,Chu,D.,Pan,H.,Xie,W.,Wang,S.,Wu,Q.,Zhou,X.and Zhang, 植物感染病毒的多种形式的载体操纵: Bemisia tabaci 和番茄黄叶病毒。 J Virol 87(9):4929-4937。
  2. Mclean,D。和Kinsey,M。(1964)。 电子记录蚜虫喂养和唾液分泌的技术 < em> Nature 202(4939):1358-1359。
  3. Tjallingii,W。(1978)。 电子记录蚜虫的穿透行为。 Entomologia Experimentalis et Applicata 24(3):721-730。
  4. Tjallingii,W。(1985)。 记录的电气性质 通过蚜虫穿透探针期间的信号。 Entomologia Experimentalis et Applicata 38(2):177-186。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Liu, B., Zhou, X. and Zhang, Y. (2014). Electrical Penetration Graph Recording (Whitefly). Bio-protocol 4(5): e1063. DOI: 10.21769/BioProtoc.1063.
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