Name: electroantennographic bioassay as a screening tool for host plant volatiles
Uploaded: 2012-05-06T14:00:00
Description: Watch this Scientific Journal Video about Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles at JoVE.com

This research was conducted under USDA-ARS CRIS Project 5325-42000-037-00D and with results from CRADA 58-3K95-7-1198 and TFCA 58-5325-8-419. The authors gratefully acknowledge Suterra for the gift of the (Z,Z)-11,13-hexadecadienal, B. Higbee for productive discussions, and J. Baker for technical assistance.

1. Preparation of Volatiles Detected from the Host Plant for EAG Screening
<ol>
 <li>After appropriate identification and authentication of all volatiles via GC-MS, perform EAG puff analysis of each available volatile. Initial screening can be a low replicate number of antennal responses (N=3-5) for each sex in order to achieve an indication of relative chemoreceptivity in a short amount of time (Table 1).</li>
 <li>Prepare a solution of each volatile at a 5 mg/mL concentration in pentane. Tightly seal...

<ol>
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Use of electroantennogram recordings as a bioassay to determine chemoreception responses of a target insect is fairly common and numerous studies utilizing EAG as a detector for effluent from a gas chromatogram (GC-EAD) can be found in the literature.9,10 The method demonstrated will provide a rapid screening of equivalent amounts of volatile components with high replications for confident assignment of the relative responsiveness. The AutoSpike program in the Syntech software is a good program for screening v...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Acetophenone</td>
			<td>Alfa-Aesar</td>
			<td>A12727</td>
			<td>Female positive control</td>
		</tr>
		<tr>
			<td>(Z,Z)-11,13-Hexadecadienal</td>
			<td>Suterra</td>
			<td>&nbsp;</td>
			<td>Male positive control</td>
		</tr>
		<tr>
			<td>&alpha;-Humulene</td>
			<td>Aldrich</td>
			<td>53675</td>
			<td>Sesquiterpene</td>
		</tr>
		<tr>
			<td>2-Undecanone</td>
			<td>Aldrich</td>
			<td>U1303</td>
			<td>Fatty acid derivative</td>
		</tr>
		<tr>
			<td>2-Phenylethanol</td>
			<td>Aldrich</td>
			<td>77861</td>
			<td>Benzenoid</td>
		</tr>
		<tr>
			<td>Pentane</td>
			<td>EMD</td>
			<td>PX0167-1</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>4-Channel acquisition controller</td>
			<td>Syntech</td>
			<td>IDAC-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EAG probe, pre-amplifier</td>
			<td>Syntech</td>
			<td>Type PRG-2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Antenna holder</td>
			<td>Syntech</td>
			<td>For PRG-2</td>
			<td>Fork electrode</td>
		</tr>
		<tr>
			<td>Stimulus controller</td>
			<td>Syntech</td>
			<td>CS-55</td>
			<td>Air flow and puffs</td>
		</tr>
		<tr>
			<td>Spectra Electrode Gel</td>
			<td>Parker</td>
			<td>12-02</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bioassay discs</td>
			<td>Whatman</td>
			<td>2017-006</td>
			<td>6 mm</td>
		</tr>
		<tr>
			<td>Pasteur pipets</td>
			<td>VWR</td>
			<td>14673-010</td>
			<td>5 &frac34;" (14.6 cm)</td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Bemis</td>
			<td>PM-992</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

electroantennographic bioassay as a screening tool for host plant volatiles

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host plant.1,2 When a host plant is an important agronomic commodity feeding damage by insect pests can inflict serious economic losses to growers. Accordingly, kairomones can be used as attractants to lure or confuse these insects and, thus, offer an environmentally friendly alternative to pesticides for insect control.3 Unfortunately, plants can emit a vast number volatiles with varying compositions and ratios of emissions dependent upon the phenology of the commodity or the time of day. This makes identification of biologically active components or blends of volatile components an arduous process. To help identify the bioactive components of host plant volatile emissions we employ the laboratory-based screening bioassay electroantennography (EAG). EAG is an effective tool to evaluate and record electrophysiologically the olfactory responses of an insect via their antennal receptors. The EAG screening process can help reduce the number of volatiles tested to identify promising bioactive components. However, EAG bioassays only provide information about activation of receptors. It does not provide information about the type of insect behavior the compound elicits; which could be as an attractant, repellent or other type of behavioral response. Volatiles eliciting a significant response by EAG, relative to an appropriate positive control, are typically taken on to further testing of behavioral responses of the insect pest. The experimental design presented will detail the methodology employed to screen almond-based host plant volatiles4,5 by measurement of the electrophysiological antennal responses of an adult insect pest navel orangeworm (Amyelois transitella) to single components and simple blends of components via EAG bioassay. The method utilizes two excised antennae placed across a "fork" electrode holder. The protocol demonstrated here presents a rapid, high-throughput standardized method for screening volatiles. Each volatile is at a set, constant amount as to standardize the stimulus level and thus allow antennal responses to be indicative of the relative chemoreceptivity. The negative control helps eliminate the electrophysiological response to both residual solvent and mechanical force of the puff. The positive control (in this instance acetophenone) is a single compound that has elicited a consistent response from male and female navel orangeworm (NOW) moth. An additional semiochemical standard that provides consistent response and is used for bioassay studies with the male NOW moth is (Z,Z)-11,13-hexdecadienal, an aldehyde component from the female-produced sex pheromone.6-8

Watch this Scientific Journal Video about Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles at JoVE.com

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

A method to rapidly screen host plant volatiles by measurement of the electrophysiological response of adult navel orangeworm (Amyelois transitella) antennae to single components and blends via electroantennographic analysis is demonstrated.

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host ...

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