Name: dissection and observation of honey bee dorsal vessel for studies of cardiac function
Uploaded: 2016-12-12T13:00:00
Description: Watch this Scientific Journal Video about Dissection and Observation of Honey Bee Dorsal Vessel for Studies of Cardiac Function at JoVE.com

The authors thank Drs. Jeffrey Bloomquist and Daniel Swale for their technical comments and suggestions. This project was partially funded by the Department of Entomology and the College of Agriculture and Life Sciences at Virginia Tech.

1. Collection and Preparation of Test Subjects
<ol>
	<li>Collect the appropriate number of bees from the colony. 
	NOTE: The number needed depends upon not only the size and scope of the experiment, but also the skill of the investigator. For example, if there are 2 treatment groups with a desired sample size of 10 bees per group, a reasonably skilled investigator might collect at least 30 bees to account for unsuccessful dissections and end up with 20 useful videos to score.</li>
	<li>Minimize the amount of time ...

<ol>
	<li>Klowden, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulatory+Systems.">Circulatory Systems.</a> Physiological Systems in Insects, 3rd Edition. , 365-413 (2013).</li><li>League, G. P., Onuh, O. C., Hillyer, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+structural+and+functional+analysis+of+the+larval+and+adult+dorsal+vessel+and+its+role+in+hemolymph+circulation+in+the+mosquito+Anopheles+gambiae.">Comparative structural and functional analysis of the larval and adult dorsal vessel and its role in hemolymph circulation in the mosquito Anopheles gambiae.</a> J Exp Biol. 218 (Pt 3), 370-380 (2015).</li><li>Wasserthal, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oscillating+Hemolymph+Circulation+in+the+Butterfly+Papilio-Machaon+L+Revealed+by+Contact+Thermography+and+Photocell+Measurements.">Oscillating Hemolymph Circulation in the Butterfly Papilio-Machaon L Revealed by Contact Thermography and Photocell Measurements.</a> J Comp Physiol. 139 (2), 145-163 (1980).</li><li>Wasserthal, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+circulation+and+tracheal+ventilation+in+holometabolous+insects.">Interaction of circulation and tracheal ventilation in holometabolous insects.</a> Adv Insect Physiol. 26, 297-351 (1996).</li><li>Schwab, E. R., Chilson, R. A., Eddleman, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heartbeat+Rate+Modulation+Mediated+by+the+Ventral+Nerve+Cord+in+the+Honey-Bee,+Apis-Mellifera.">Heartbeat Rate Modulation Mediated by the Ventral Nerve Cord in the Honey-Bee, Apis-Mellifera.</a> J Comp Physiol B-Biochem Syst Environ Physiol. 161 (6), 602-610 (1991).</li><li>Kaiser, W., Weber, T., Otto, D., Miroschnikow, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+supply+of+the+heart+and+electrocardiogram+potentials+with+reversed+polarity+in+sleeping+and+resting+honey+bees.">Oxygen supply of the heart and electrocardiogram potentials with reversed polarity in sleeping and resting honey bees.</a> Apidologie. 45 (1), 73-87 (2014).</li><li>Papaefthimiou, C., Theophilidis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Octopamine--a+single+modulator+with+double+action+on+the+heart+of+two+insect+species+(Apis+mellifera+macedonica+and+Bactrocera+oleae):+Acceleration+vs.+inhibition.">Octopamine--a single modulator with double action on the heart of two insect species (Apis mellifera macedonica and Bactrocera oleae): Acceleration vs. inhibition.</a> J Insect Physiol. 57 (2), 316-325 (2011).</li><li>Castro, C. A., Hogan, J. B., Benson, K. A., Shehata, C. W., Landauer, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral-Effects+of+Vehicles+-+Dmso,+Ethanol,+Tween-20,+Tween-80,+and+Emulphor-620.">Behavioral-Effects of Vehicles - Dmso, Ethanol, Tween-20, Tween-80, and Emulphor-620.</a> Pharmacol Biochem Behav. 50 (4), 521-526 (1995).</li><li>Papaefthimiou, C., Papachristoforou, A., Theophilidis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biphasic+responses+of+the+honeybee+heart+to+nanomolar+concentrations+of+amitraz.">Biphasic responses of the honeybee heart to nanomolar concentrations of amitraz.</a> Pestic Biochem Phys. 107 (1), 132-137 (2013).</li><li>Roeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Octopamine+in+invertebrates.">Octopamine in invertebrates.</a> Prog Neurobiol. 59 (5), 533-561 (1999).</li><li>Johnson, E., Ringo, J., Dowse, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+Drosophila+heartbeat+by+neurotransmitters.">Modulation of Drosophila heartbeat by neurotransmitters.</a> J Comp Physiol B. 167 (2), 89-97 (1997).</li></ol>

The protocol presented here provides a simple and effective approach to testing pharmacological compounds for their effects on honey bee heart rate. As observed in prior experiments that either transect the ventral nerve cord of a living insect5 or dissect out the ventral nerve cord when exposing the dorsal vessel7, the loss of central nervous system regulation results in a stable, low frequency heartbeat. The low frequency of beats allows the investigator to visually assess heart rate without havin...

The overall goal of this methodology is to allow the investigator to quickly and easily observe and quantify the effect that a pharmacological agent has on the heart rate of honey bees. Bees, like other insects, have an open circulatory system that disseminates hemolymph, the insect equivalent of blood, throughout the body cavity, known as the hemocoel. The circulation of hemolymph is essential for the transport of nutrients, immune factors, waste products, as well neurohormones and other signaling molecules1. Circulation is facilitated by the dorsal vessel, which extends along the dorsal midline of the insect, as well as accessory pulsatile organs. The dorsal vessel is divided into two functionally distinct sections, designated the heart in the abdomen and the aorta in the thorax and head. Propagated contractions in the heart pump hemolymph towards the thorax and head, while accessory pulsatile organs ensure hemolymph flow to the extremities.
Insect cardiac function can be observed using a variety of methods, depending upon the size, physiology, or life stage of the insect. A common approach for observing heart rate in larvae or smaller insects is the use of intravital imaging2. This method is less useful in adult bees, however, as it can be difficult to clearly view the dorsal vessel through the abdominal wall. An established approach for recording heart rate in a variety of insects, including bees, is the use of contact thermography, which utilizes thermistors applied to the exterior of the insect to detect cardiac pulsations3,4. Heart rate in adult bees has also been recorded using an electrophysiological technique to measure an electrical impedance signal4,5. This technique requires the insertion of electrodes into the animal next to the heart and the use of an impedance converter to record heartbeats4. Similarly, electrocardiograms have been used to detect electrical signals produced by the heart and combined with video recording of the bee to observe changes in cardiac activity6. A distinct advantage to these approaches is that heart rate is assessed in an intact, living bee, rather than in a dissected specimen, which helps to ensure the availability of the full range of physiological responses in the subject. The challenges of these approaches include accounting for immobilization or anesthetization of the subject, the need to limit outside variables and stimuli that might alter heart rate, as well as determining an appropriate delivery method when testing pharmacological agents.
Another approach that has been used for studying bee cardiac activity is to partially dissect the insect in order to expose the heart, then measure dorsal vessel contractions using a force displacement transducer7. In this protocol, the heart is continually bathed with running physiological saline and test compounds can be dissolved in this solution for application to the subject7. A significant difference between this method and those previously described is that the ventral nerve cord is removed, eliminating the role that the central nervous system has been shown to play in modulating heart rate5. The result is that the baseline heartbeat, which is usually quite erratic, stabilizes at a much lower frequency and amplitude than is typically observed in a living insect5,7. What all of these methods have in common is that they require highly specialized and often expensive equipment, in addition to a certain level of expertise, in order to be conducted. Perhaps the greatest disadvantage is that none of these approaches are particularly well suited to experiments that involve testing a large number of subjects, such as screening a library of potentially cardiomodulatory compounds.
The greatest strength of the approach described here is its simplicity. The protocol is relatively easy to master, the setup requires little space, and only a minimal financial input is necessary. The method requires little more than some bees, a few surgical instruments, an isotonic solution, and either a digital microscope or a traditional microscope with a digital camera. Bees are dissected to visualize the dorsal vessel and digital videos are used to record heart rate before and after treatment with pharmacological agents. Although video recording is not actually necessary to observe changes in heart rate, it will greatly increase throughput (i.e., the number of subjects that can be processed in a given amount of time). The investigator can maximize efficiency by recording a large number of videos at once and then later scoring these videos at a more convenient time. Another advantage of this approach is that videos allow the investigator to start over, should the scoring process be interrupted, and make it easier for the viewer to be blinded to the treatment in order to reduce bias.

<table border="0" cellpadding="0" cellspacing="0" width="834">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:200px;">Dino-Lite Edge digital USB microscope</td>
			<td style="width:111px;">Dino-Lite</td>
			<td style="width:119px;">AM4815ZT</td>
			<td style="width:405px;">Any digital microscope or similar setup will suffice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:200px;">Microscope stand</td>
			<td style="width:111px;">Dino-Lite</td>
			<td style="width:119px;">RK-10</td>
			<td>Any stand appropriate for the digital microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:200px;">Laptop or PC</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td>Necessary for digital microscope</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:200px;">Microdissection scissors (Vannas, 8cm, Straight, 5mm Blades)</td>
			<td style="width:111px;">World Precision Instruments</td>
			<td style="width:119px;">14003</td>
			<td>Any similar scissors suitable for microdissection will suffice</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:200px;">Microdissecting Forceps, 10.2cm, Angled (2 pair)</td>
			<td style="width:111px;">World Precision Instruments</td>
			<td style="width:119px;">504482</td>
			<td>Any similar forceps suitable for microdissection will suffice</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:200px;">Ringers solution 1/4 strength tablets&#160;</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">96724-100TAB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:200px;">Dissecting tray</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td>Any surface suitable for microdissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:200px;">Single channel 10 &#181;l pipette</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td>Any device capable of accurately delivering 10 &#181;l volume</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:200px;">Pipette tips</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:200px;">Small beaker or container of water</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td>Used to rinse instruments between subjects</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:200px;">Hand tally counter</td>
			<td style="width:111px;">Office Depot</td>
			<td style="width:119px;">295033</td>
			<td>Any similar product will suffice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:200px;">Timer</td>
			<td style="width:111px;">Office Depot</td>
			<td style="width:119px;">644219</td>
			<td>Any similar product will suffice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:200px;">Deionized water</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td>Preparation of Ringers solution and rinsing instruments</td>
		</tr>
	</tbody>
</table>

dissection and observation of honey bee dorsal vessel for studies of cardiac function

The European honey bee, Apis mellifera L., is a valuable agricultural and commercial resource noted for producing honey and providing crop pollination services, as well as an important model social insect used to study memory and learning, aging, and more. Here we describe a detailed protocol for the dissection of the dorsal abdominal wall of a bee in order to visualize its dorsal vessel, which serves the role of the heart in the insect. A successful dissection will expose a functional heart that, under the proper conditions, can maintain a steady heartbeat for an extended period of time. This allows the investigator to manipulate heart rate through the application of cardiomodulatory compounds to the dorsal vessel. By using either a digital microscope or a microscope equipped with a digital camera, the investigator can make video recordings of the dorsal vessel before and after treatment with test compounds. The videos can then be scored at a time convenient to the user in order to determine changes in heart rate, as well as changes in the pattern of heartbeats, following treatment. The advantages of this protocol are that it is relatively inexpensive to set up, easy to learn, requires little space or equipment, and takes very little time to conduct.

Since many of the pharmacologically active compounds that might be tested using this protocol are not soluble in water, it is necessary to have a reliable solvent that will allow test compounds to be delivered via the isotonic solution used to bathe the dorsal vessel. Dimethyl sulfoxide (DMSO) is a solvent that is commonly used as a vehicle for delivering experimental drugs and other compounds in animals8, and it has been used successfully for this purpose in studies examining ...

Watch this Scientific Journal Video about Dissection and Observation of Honey Bee Dorsal Vessel for Studies of Cardiac Function at JoVE.com

Dissection and Observation of Honey Bee Dorsal Vessel for Studies of Cardiac Function

The abdominal dorsal vessel of the honey bee and other insects serves as the functional equivalent of the mammalian heart and plays an important role in nutrient transport, waste removal, immune function, and more. Here we describe a protocol for the visualization and pharmacological manipulation of bee heart rate.

The European honey bee, Apis mellifera L., is a valuable agricultural and commercial resource noted for producing honey and providing crop pollination ...

The overall goal of this protocol is to observe changes in bee heart rate, resulting from the direct application of cardiomodulatory agents to the dorsal vessel. This method can answer key questions about insect physiology, especially those relating to pollinator health. The main advantage of this technique is its simplicity which makes it cost effective and easy to perform.Collect the appropriate number of bees from the colony. A prudent measure would be to include a 50 percent surplus to account for failed dissections, which even skilled investigators must plan for. Provide the bees with a source of water and food while housed in the lab.For housing durations that are less than six hours, at a minimum provide access to a 50 percent sucrose solution. For longer periods, also provide access to honey. House the bees overnight at approximately 32 degrees celsius with 60 to 80 percent relative humidity.Freshly collected bees are easier to dissect, so proceed with dissecting as soon as possible. Prior to the dissections, anesthetize the bees just enough to reduce their movement by briefly exposing them to carbon dioxide. The depth of the anesthesia can make the dissection more difficult, so use as little gas as possible.The bees must be alive at the time of dissection. To begin, use forceps or micro-dissection scissors to remove the legs and wings. After each manipulation, rinse the tools in distilled water.Next, restrain the bee with forceps and use the scissors to cut laterally along the dorsal abdominal wall, between the first and second tergites. Then, lightly grip the posterior edge of the second tergite with the forceps and carefully cut longitudinally along each side of the bee from the initial incision to the stinger. Do not puncture the digestive tract.Next, use fine forceps to carefully separate the dorsal abdominal wall from the rest of the abdomen. Then, gently remove the stinger and carefully remove any portion of the digestive tract that remains attached to the dorsal abdominal wall. Don't release the gut's contents, as they can obscure visualization of the dorsal vessel.Now remove the final turgite in order to improve visualization of the dorsal vessel. The next step is to adjust the stage to view the dorsal vessel and trim away any excess abdominal wall that impedes its view. The shape of the dorsal abdominal wall should resemble a shallow cup or bowl when properly situated.Cover the dorsal vessel with 10 microliters of the recommended isotonic solution to maintain its phsyiological conditions, and remove any remaining membrane that impedes visualization of the dorsal vessel. Addition of the isotonic solution can cause loose membrane to float and obscure the view of the dorsal vessel. These membrane pieces can be removed with fine forceps, but use caution to avoid damaging the dorsal vessel.Initially, there may appear to be no heartbeat but in a few minutes in isotonic solution, the heart will usually resume beating. If kept bathed in solution, the heart can continue beating for hours. Allow the dorsal vessel to sit undisturbed until a stable, continuous heartbeat is achieved.This usually occurs within five minutes. Measure the baseline heart rate using a hand tally counter, or for more accuracy, make a video recording of the experiment and take the measurements later. Record at least one minute of stable heartbeats prior to the first treatment.Now, prepare to apply the potential cardiomodulators, which are ideally dissolved in the same isotonic solution as the bath. For this reason, the recommended solution contains some DMSO, which allows most compounds to be solubilized. After adding a test compound, wait at least one minute before measuring the change in heart rate.To get an accurate measure of the heart rate, at least a minute of data is needed, and several minutes of data are usually required. A variety of isotonic solutions containing a range of DMSO concentrations were tested for any effect on heart rate. Five percent and 10 percent DMSO significantly decreased the heart rate, whereas a one percent DMSO isotonic solution did not.So the one percent solution was used for experiments. Octopamine, a well characterized demodulator in invertebrates, was tested against the vehicle. Higher concentrations of octopamine significantly increased the heart rate, thus demonstrating the utility of the assay.After watching this video, you should have a good understanding of how to perform a dissection to visualize the dorsal vessel as well as how to test the effects of potential cardiomodulators on bee heart rate. Once this technique has been mastered, a dissection can be completed in a matter of minutes if performed properly.

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