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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L., Leffler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bed+bug+infestations+in+the+news:+a+picture+of+an+emerging+public+health+problem+in+the+United+States.">Bed bug infestations in the news: a picture of an emerging public health problem in the United States.</a> J Environ Health. 70 (9), 24-27 (2008).</li><li>Boase, C., Robinnson, W., Bajomi, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bed+bugs+(Hemiptera:+Cimicidae):+an+evidence-based+analysis+of+the+current+situation.">Bed bugs (Hemiptera: Cimicidae): an evidence-based analysis of the current situation.</a> Sixth international conference on urban pests. OOK-Press Kft. , (2008).</li><li>Doggett, S. L., Geary, M. J., Russell, R. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+molecular+analysis+of+deltamethrin+resistance+in+the+common+bed+bug+(Hemiptera:+Cimicidae).">Biochemical and molecular analysis of deltamethrin resistance in the common bed bug (Hemiptera: Cimicidae).</a> J Med Entomol. 45 (6), 1092-1101 (2008).</li><li>Wang, L., Xu, Y., Zeng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resurgence+of+bed+bugs+(Hemiptera:+Cimicidae)+in+mainland+China.">Resurgence of bed bugs (Hemiptera: Cimicidae) in mainland China.</a> Fla Entomol. 96 (1), 131-136 (2013).</li><li>Haynes, K. F., Potter, M. F., Ishaaya, I., Palli, S. R., Horowitz, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+bed+bug+management.">Recent progress in bed bug management.</a> Advanced technologies for managing insect pests. , 269-278 (2013).</li><li>Carey, A. F., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+olfaction+from+model+systems+to+disease+control.">Insect olfaction from model systems to disease control.</a> Proc Natl Acad Sci. 108 (32), 12987-12995 (2011).</li><li>Leal, W. 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. G., Liu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antennal+olfactory+sensilla+responses+to+insect+chemical+repellents+in+the+common+bed+bug,+Cimex+lectularius.">Antennal olfactory sensilla responses to insect chemical repellents in the common bed bug, Cimex lectularius.</a> J Chem Ecol. 40 (6), 522-533 (2014).</li><li>Olson, J. F., Moon, R. D., Kells, S. A., Mesce, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology,+ultrastructure+and+functional+role+of+antennal+sensilla+in+off-host+aggregation+by+the+bed+bug,+Cimex+lectularius.">Morphology, ultrastructure and functional role of antennal sensilla in off-host aggregation by the bed bug, Cimex lectularius.</a> Arthropod Struct Dev. 43 (2), 117-122 (2014).</li><li>Bruyne, M., Foster, K., Carlson, J. 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. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+classification+and+central+nervous+projections+of+olfactory+receptor+neurons+housed+in+antennal+trichoid+sensilla+of+female+yellow+fever+mosquito,+Aedes+aegypti.">Functional classification and central nervous projections of olfactory receptor neurons housed in antennal trichoid sensilla of female yellow fever mosquito, Aedes aegypti.</a> Eur J Neurosci. 26 (6), 1611-1623 (2007).</li><li>Hill, S. R., Hanson, B. S., Ignell, R. <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+antennal+trichoid+sensilla+from+female+southern+house+mosquito,+Culex+quinquefasciatus+Say.">Characterization of antennal trichoid sensilla from female southern house mosquito, Culex quinquefasciatus Say.</a> Chem Senses. 34 (3), 231-252 (2009).</li></ol>

The authors have nothing to disclose.

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 ...

The overall goal of the single sensillum recording is to investigate neuronal responses of the antennal olfactory sensillum to various odorants. This method can help to answer the key questions in the field of chemical ecology such as if the insect can detect the chemical odorants from their environment and how they process the chemical odorants. The main advantage of this technique is that it allows mirroring the single neuron activity of the function.It provides more precise measurement of the sensitivity of neurons to the chemical odorants. The implication of this technique extended toward the development of attractants or repellants in the insect control. Because this technique can be used large amount of chemicals that are biologically important for the insect.Begin this procedure by anesthetizing bed bugs on ice for two to three minutes. Then fix the insects antennae and body on a microscope cover slip with double sided tape and remove the legs with fine scissors. Use a small pin to gently adjust the antennae on the tape.Next, rest the cover slip against a small ball of dental wax to facilitate manipulation and adjust it to a 90 degree angle for placing the recording electrode. Once secured, place the bed bug under a stereo microscope. Turn on the light source and adjust the intensity of illumination until the antennae is clearly presented.Then focus the microscope on the second flagellum of the bed bug's antennae at a high magnification. In this step, connect the pre-amplifier with a signal acquisition controller, which is connected with the computer for signal recording and visualization. Next, open the software and click record from the menu bar.Then choose wave to start recording the wave signals. Turn on the speaker connected to the pre-amplifier to present the tone of the neuronal responses from the antennal sensillum. Now insert the reference electrode into the abdomen of the stabilized bed bug.After the reference electrode has been connected to the bed bug's abdomen, move the recording electrode towards the posterior end of the bed bug's antennae. When the recording electrode is in contact with the tip of the right antennae, locate the electrode at low magnification. Then adjust the recording electrode while gradually increasing the magnification until both the electrode and the antennal sensillum are in the same plane and clearly visible under the microscope.After that, insert the recording electrode into the shaft of the sensillum using the micro-manipulator. Lower it deeper if the background noise is too high. Once clear action potentials are observed from the recorded sensillum, fill a micro pipette with botanical stimulus.Use the micro pipette to deposit a 10 microliter aliquot of the stimulus onto a filter paper strip inside a glass pastier pipette. Accurately and directly recording electrodes to the sensillum is the key to the success of the experiment. Background noise can be partially reduced after inserting the electrodes a little deeper.Next, connect the loaded pipette to the outlet of the stimulus flow tube and place the pipette tip into the small hole in the tube oriented towards the antennae. When all the connections are stabilized, depress the foot switch of the stimulus controller to deliver a stimulus into the continuous humidified air stream and start recording. The recording process will last for 10 seconds starting one second before the stimulation.Count the action potentials offline for two 500 millisecond periods, one before, and one after the stimulation. To present another stimulus, label a new pipette with 001%eucalyptol to be tested. Apply 10 microliters of the stimulus before placing a small piece of filter paper onto it.Then wait for two to five minutes until the stimulus is completely vaporized in the glass pipette. Attach the pipette onto the outlet of the stimulus flow tube. Subsequently, insert the pipette tip into the small hole of the tube oriented towards the antennae.Depress the foot switch and start the 10 second recording. After that, disconnect the pipette and prepare another pipette with 01%eucalyptol. Test all the rest of the doses of eucalyptol on the antennal sensilla to observe the most dependent responses.In this figure, the signal trace shows the typical neural response of an olfactory sensillum to the solvent which is used as the control in the single sensillum recording. Signal recording started one second before the 0.5 second of puff stimulus. The signal was recorded continuously for 10 seconds after initiating the stimulus puff.This signal trace shows the extremely strong neural response of an olfactory sensillum to a botanical stimulus 10%positive betapynene. After the puff of stimulus is delivered to the olfactory sensillum, the olfactory sensory neurons inside this sensillum fire with high frequency into a long-lasting temporal dynamic. For in this procedure are the message that behavior by an insect can be performed in order to answer additional questions like what are the behavioral responses of insects to certain chemicals with strong neuron responses.After watching this video, you should have a better understanding of how to record the insect olfactory neuron responses to the chemical stimulus.

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Neuroscience

9.7K vistas.  Auburn University. The overall goal of the single sensillum recording is to investigate neuronal responses of the antennal olfactory sensillum to various odorants. This method can help to answer the key questions in the field of chemical ecology such as if the insect can detect the chemical odorants from their environment and how they process the chemical odorants. The main advantage of this technique is that it allows mirroring the single neuron activity of the function.It provides more precise measurem...