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

Summary

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.

Abstract

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.

Introduction

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 dor....

Protocol

1. Collection and Preparation of Test Subjects

1. 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.
2. Minimize the amount of time that passes between collection and dissection.
   NOTE: Although bees can be housed in the lab for days prior to dissection, the success rate of disse....

Results

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 .......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dino-Lite Edge digital USB microscope	Dino-Lite	AM4815ZT	Any digital microscope or similar setup will suffice
Microscope stand	Dino-Lite	RK-10	Any stand appropriate for the digital microscope
Laptop or PC			Necessary for digital microscope
Microdissection scissors (Vannas, 8 cm, Straight, 5 mm Blades)	World Precision Instruments	14003	Any similar scissors suitable for microdissection will suffice
Microdissecting Forceps, 10.2 cm, Angled (2 pair)	World Precision Instruments	504482	Any similar forceps suitable for microdissection will suffice
Ringers solution 1/4 strength tablets	Sigma-Aldrich	96724-100TAB
Dissecting tray			Any surface suitable for microdissection
Single channel 10 µl pipette			Any device capable of accurately delivering 10 µl volume
Pipette tips
Small beaker or container of water			Used to rinse instruments between subjects
Hand tally counter	Office Depot	295033	Any similar product will suffice
Timer	Office Depot	644219	Any similar product will suffice
Deionized water			Preparation of Ringers solution and rinsing instruments