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.
