Name: using single sensillum recording to detect olfactory neuron responses of bed bugs to semiochemicals
Uploaded: 2016-01-18T15:00:00
Description: Watch this Scientific Journal Video about Using Single Sensillum Recording to Detect Olfactory Neuron Responses of Bed Bugs to Semiochemicals at JoVE.com

The project was supported by Award AAES 461Hatch/Multistate Grants ALA08-045 and ALA015-1-10026 to N.L.

1. Preparation of Instruments, Stimuli Solutions, and Bed Bugs
<ol>
	<li>Prepare a 50% KNO2 solution (w/v) in a 20 ml bottle.</li>
	<li>Sharpen two tungsten microelectrodes in KNO2 solution at 5 V by repeatedly dipping the tungsten electrodes in and out of the solution.
	<ol>
		<li>Roughly sharpen the tungsten wire by dipping about 10 mm of the tungsten wire in and out of the KNO2 solution at the speed of 2 dips/sec&#160;for about 5 min, which can greatly consume the front end of the tu...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant+reception+in+insects:+roles+of+receptors,+binding+proteins,+and+degrading+enzymes.">Odorant reception in insects: roles of receptors, binding proteins, and degrading enzymes.</a> Annu Rev Entomol. 58, 373-391 (2013).</li><li>Den Otter, C. J., Behan, M., Maes, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+response+in+female+Pieris+brassicae.+(Lepidoptera:+Pieridae)+to+plant+volatiles+and+conspecific+egg+odours.">Single cell response in female Pieris brassicae. (Lepidoptera: Pieridae) to plant volatiles and conspecific egg odours.</a> J Insect Physiol. 26 (7), 465-472 (1980).</li><li>Levinson, H. Z., Levinson, A. R., Muller, B., Steinbrecht, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+of+sensilla,+olfactory+perception,+and+behavior+of+the+bed+bug,+Cimex+lectularius.,+in+response+to+its+alarm+pheromone.">Structural of sensilla, olfactory perception, and behavior of the bed bug, Cimex lectularius., in response to its alarm pheromone.</a> J Insect Physiol. 20 (7), 1231-1248 (1974).</li><li>Harraca, V., Ignell, R., L&#246;fstedt, C., Ryne, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+antennal+olfactory+system+of+the+bed+bug+(Cimex+lectularius).">Characterization of the antennal olfactory system of the bed bug (Cimex lectularius).</a> Chem Senses. 35 (3), 195-204 (2010).</li><li>Liu, F., Haynes, K. F., Appel, A. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odor+coding+in+the+Drosophila+antenna.">Odor coding in the Drosophila antenna.</a> Neuron. 30 (2), 537-552 (2001).</li><li>Qiu, Y. T., Loon, J. J. A., Takken, W., Meijerink, J., Smid, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+coding+in+antennal+neurons+of+the+malaria+mosquito,+Anopheles+gambiae.">Olfactory coding in antennal neurons of the malaria mosquito, Anopheles gambiae.</a> Chem Senses. 31 (9), 845-863 (2006).</li><li>Ghaninia, M., Ignell, R., Hansson, B. 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The Single Sensillum Recording technique has been extensively used in testing the neural responses of insects such as fruit flies, mosquitoes and bed bugs to different chemical stimuli in the environment. These chemical stimuli are often dissolved and diluted in a common solvent in order to prepare different doses of treatments. However, different solvents can produce quite different release rates for the stimuli. Previous studies on some extensively studied insects such as Drosophila melanogaster, Anopheles...

The common bed bug Cimex lectularius L (Hemiptera: Cimicidae), as a temporary ectoparasite, is an obligated blood-sucking insect, which means their survival, development, and reproduction require blood sources from hosts, including both humans and animals1,2. Although virus transmission has rarely been reported due to C. lectularius, the biting nuisance generated by an infestation seriously affects hosts both physically and psychologically3. The introduction and widespread use of chemical insecticides, especially DDT, lowered the risk of infestations and by the end of the 1950s infestations were at such a low level that they were no longer a serious public concern. However, a number of possible factors have led to resurgence in bed bug populations worldwide, such as the reduced use of insecticides, a decline of public awareness, increased traveling activity, and the development of resistance to insecticides4-9.
Chemical cues in the environment are detected and recognized by insects through olfactory organs such as antennae and maxillary palps. The olfactory sensilla on the insect antennae play a crucial role in detecting these chemical cues. The chemical molecules enter the antennal cuticle through pores on the cuticle surface. Odorant binding proteins in the antennal lymph bind to these chemical molecules and transport them onto the odorant receptors10. The odorant receptors and their co-receptor from the non-selective cation ion channel on the neural membrane, which will be depolarized once these chemical molecules are recognized by the odorant receptors11.
Single sensillum recording (SSR) was developed to detect the extracellular change in the action potential caused by the application of either chemical or non-chemical stimuli. By inserting a recording electrode into the sensillum lymph and a reference electrode into some other part of the insect body (usually either the compound eyes or the abdomen), the firing rate of the neurons in response to stimuli can be recorded12. Changes in the number of spikes represent the sensitivity of the insect to specific stimuli. Chemical stimuli of different identities and concentration will elicit different neural responses, with different firing rates and temporal structures, and can thus be used to investigate the coding process of the insect to specific chemicals.
For the common bed bug, both sexual forms share the same pattern of olfactory sensilla on the antennae: nine grooved peg C sensilla, 29 hair-like E (E1 and E2) sensilla, and one pair each of D&#945;, D&#946;, D&#947; smooth peg sensilla 13,14. As multiple neurons have been identified in each type of sensillum, it is not easy to distinguish the action potentials from different neurons housed in the same sensillum, so for this experiment the total numbers of action potentials were counted off-line for a 500 msec period before and after stimulation. The number of action potentials after stimulation was then subtracted from the number of action potentials before stimulation and multiplied by two in order to quantify the changes in the firing rate in each individual sensillum in spikes per second15.

<table border="0" cellpadding="0" cellspacing="0" width="1064">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">Tungsten wire</td>
			<td style="width:139px;">A-M SYSTEMS</td>
			<td style="width:117px;">#716500</td>
			<td style="width:511px;">Used for preparing the electrode</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KNO2</td>
			<td>Sigma</td>
			<td>#310484</td>
			<td>Used for sharpening the tungsten wire</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AC Power Supply</td>
			<td>BK Precision</td>
			<td>1653A</td>
			<td>Providing the voltage in sharpening the tungsten wire</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Leica Z6 APO Microscope</td>
			<td>Leica</td>
			<td>10447424</td>
			<td>Used for observing the sensilla on antennae</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Simulus controller</td>
			<td>Syntech</td>
			<td>CS-55</td>
			<td>Used for controlling the stimulus application</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4-Channel USB Acquisition Controller</td>
			<td>Syntech</td>
			<td>IDAC-4</td>
			<td>Real-time on screen display of all signals before and during recording</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Light Source</td>
			<td>SCHOTT</td>
			<td>A20500</td>
			<td>Providing light sources for observation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micromanupulator</td>
			<td>Leica</td>
			<td>115378</td>
			<td>Used for minor movement of electrode</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Speaker</td>
			<td>Juster</td>
			<td>95a</td>
			<td>Connected with Acquisition Controller IDAC-4 and providing sound for the signal</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnetic stand</td>
			<td>Narishige</td>
			<td>GJ-1</td>
			<td>Used to hold the reference electrode, stablized bed bug and stimulus delivery tube</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TMC Vibration Isolation Table</td>
			<td>TMC</td>
			<td>63-500</td>
			<td>Used for isolating the vibration from the equipments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Coverslip</td>
			<td>Tedpella</td>
			<td>2225-1</td>
			<td>Used for holding the bed bug</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Double-sided Tape</td>
			<td>3M</td>
			<td>XT6110</td>
			<td>Used for stablizing the bed bug on the coverclip</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dental Wax</td>
			<td>Dentakit</td>
			<td>DK-R012</td>
			<td>Used for supporting the coverclip where bed bug is stablized&#160;</td>
		</tr>
	</tbody>
</table>

using single sensillum recording to detect olfactory neuron responses of bed bugs to semiochemicals

The insect olfactory system plays an important role in detecting semiochemicals in the environment. In particular, the antennal sensilla which house single or multiple neurons inside, are considered to make the major contribution in responding to the chemical stimuli. By directly recording action potential in the olfactory sensillum after exposure to stimuli, single sensillum recording (SSR) technique provides a powerful approach for investigating the neural responses of insects to chemical stimuli. For the bed bug, which is a notorious human parasite, multiple types of olfactory sensillum have been characterized. In this study, we demonstrated neural responses of bed bug olfactory sensilla to two chemical stimuli and the dose-dependent responses to one of them using the SSR method. This approach enables researchers to conduct early screening for individual chemical stimuli on the bed bug olfactory sensilla, which would provide valuable information for the development of new bed bug attractants or repellents and benefits the bed bug control efforts.

Single sensillum recording is a powerful investigative technique used in studies of insect chemical ecology and neural physiology. Investigating the neural responses of insects to different volatile compounds, especially those thought to be ecologically related to the survival and development of the insects, not only gives us invaluable insights into the insect olfaction process, but also opens up promising new avenues that could potentially lead to the development of useful new reagents ...

Watch this Scientific Journal Video about Using Single Sensillum Recording to Detect Olfactory Neuron Responses of Bed Bugs to Semiochemicals at JoVE.com

Using Single Sensillum Recording to Detect Olfactory Neuron Responses of Bed Bugs to Semiochemicals

Bed bugs rely on olfactory receptor neurons housed in their antennal olfactory sensilla to detect semiochemicals in the environment. Utilizing single sensillum recording, we demonstrate a method to evaluate bed bug response to semiochemicals and explore the coding process involved.

The insect olfactory system plays an important role in detecting semiochemicals in the environment. In particular, the antennal sensilla which house ...

Research

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