IP Teaching Resources

Return to Table of Contents Instructions

Topic 9. Circulation.

________________

(Name)

I. MATERIALS.

American cockroach, Periplaneta americana.

Grasshopper or cricket (pet store food for lizards?)

Pupal tobacco hornworm, Manduca sexta.

Impedance converter and electrodes.

Brush recorder.

Heart saline.

Dissecting microscopes and lamps.

Dissecting dishes and pins.

Dissecting tools.

Stop watch.

II. LEARNING OBJECTIVES.

Each student should:

Observe the flow of hemolymph in the wings.

Identify accessory pulsatile organs.

Identify systolic and diastolic contractions of the dorsal vessel.

Understand how hemolymph is circulated.

How:

Mount and observe blood flow in wing veins.

Observe accessory pulsatile organ in pharate adult Manduca sexta.

Record abdominal heartbeat of cockroach and compare it to grasshopper or cricket.

III. INTRODUCTION.

Insects and some of their near relatives have developed a tracheal system to deliver air directly to all the cells of the body. The tracheal system bypasses the hemolymph; therefore, hemoglobin proteins are rare in insects, occurring only in larvae that live in low oxygen environments such as bot fly larvae or chironomid midge larvae. Thus one of the main functions of the cardiovascular system in mammals, oxygenation of tissues, is absent in insects.

Nevertheless, the circulatory system of insects is vital to their survival. It provides a medium in which energy reserves are mobilized and delivered and waste products taken away from all tissues. All internal hormones are delivered by the hemolymph to their target organs. The hemolymph also provides a medium in which wound repair and defensive reactions against invading microorganisms takes places, most of which is the function of specialized blood cells or hemocytes.

IV. DIRECTIONS.

A. Observation of circulation in the living insect.

Construct a cell large enough to contain a live adult American cockroach with Plasticene or wax. Pin the animal down with two strips of paper, one across the thorax, the other across the body beneath the wings. Slip a piece of tinfoil or glazed white paper beneath the forewing, and by adjusting a good light source so as to give a combination of reflected and transmitted illumination, the flow of blood in the wing may be observed with a dissecting microscope. Note the direction of flow in the various parts of the wing, and the moving blood cells. Repeat with the hindwing. (Note: If using a focused light, let it shine through a vessel of water to reduce the heat, or use fiber optics.)

Draw an outline of the wing with major wing veins and indicate the direction of hemolymph flow with small arrows:

Wings of the American cockroach, Periplaneta americana, with major wing veins. Blood flow is indicated by arrows.

B. Activity of the dorsal vessel.

To see the beating heart, pin the insect's wings out to the side, and observe the midline area of the abdomen, which is transparent. In a good preparation the dorsal vessel in the abdomen may be seen with bits of material sometimes flowing through it. What is the direction of blood flow? ___________

________________. Can you tell how the blood enters the heart?_______________

_______________________________________________________________________.

C. Accessory pulsatile organs.

Hemolymph is directed to all appendages in the insect. Because the dorsal vessel conducts hemolymph forward or rearward in the major body segments only, additional pulsatile organs or diaphragms are positioned to force hemolymph into the appendages or direct hemolymph to vital organs. The most obvious accessory pulsatile organs (APOs) are the antennal APOs, however, these are difficult to see in whole insects. Instead, we will view the APOs of the wing of Manduca sexta.

Obtain an adult, male or female, tobacco hornworm. After anesthesia, remove the scales from the rear of the thorax using watchmaker’s forceps or a razor blade. The cuticle underneath has a brown tint, but is somewhat transparent. Notice the wing APO near the back of the thorax midline. This appears as rhythmic tissue movements of around 60 beats/min (one per second).

Watch the APO activity for a short period of time, determine the beat rate with a stop watch and describe the overall activity you see. Does the activity change appreciably over several minutes?

__________________________________________________________________

________________________________________________________________________

_______________________________________________________________________.

__________________________________________________________________

________________________________________________________________________

_______________________________________________________________________.

D. Impedance recording of cockroach heartbeat.

Dissect a large American cockroach and an adult grasshopper or cricket. Pin the dorsal abdomens up-side-down in a wax dissection dish in a shallow depression pre-scooped out of the wax. Flush and then leave moistened with saline. Leave the lateral aspects of the abdominal tissues out of the saline. This will help retain air in the major tracheal tubes. Mount two insulated copper wires of about 20 gauge, onto a convenient rod holder. Connect one end to the input of an impedance converter, and cut off the copper ends that will go in the preparation. This removes any corrosion that might be present from pervious use, and provide the best signal.

Micromanipulate the electrodes to the side of the dorsal vessel so that the bare copper ends are fully submerged in the saline and near the dorsal vessel, but not touching. Be very careful not to touch the moving dorsal vessel tissues with the wires.

Notice that the transparent dorsal diaphragm forms a stiff, flat surface covering the middle of the largest part of the dorsal vessel and restricts direct access to the side of the dorsal vessel, unless it is penetrated; whereas, the area adjacent to the ostial valves is only loosely covered by the diaphragm affording easy access. Disturbing the dorsal diaphragm should not interfere with the heartbeat of the dorsal vessel unless the disturbance alters the suspension of the dorsal vessel. To see these structures better, you may want to apply a few drops of Janus Green dye to the preparation which should not interfere with the heartbeat.

Once the electrodes are in place, turn on the impedance converter. If the meter on the front panel shows rhythmic movements, plug the converter into a Brush recorder and obtain a recording of the heartbeat. Be careful to write, in ball-point pen, the rate of speed of the recorder drive motor if a time calibration mark is not already provided by a time marker pen. Also write the voltage range used.

After a small section of heartbeat record is obtained, paste it in this workbook with the diastolic and systolic direction of heartbeat indicated. This may take some coordination with a partner describing the heartbeat in the microscope and you noticing the corresponding movement on the pen recorder.

Attach labeled heartbeat record here:

Draw a diagram of the heart preparation. Find the segmental vessel in the four middle segments of the abdominal dorsal vessel, and indicate the position of the ostial valves in relation to the segmental vessels. It may be helpful to add a few drops of Janus Green dye to the preparation, then flush.

Draw the cockroach heart preparation:

Be sure to include a length calibration.

E. Dorsal vessel of grasshopper or cricket.

Repeat exactly the same steps above except use a cricket or grasshopper. Either a large nymph or adult will do. Attach a portion of the heartbeat record, properly labeled with name, date, time and voltage calibration and title: Impedance conversion record of abdominal heartbeat of __________________.

Attach heartbeat record here:

Describe the differences between the two records obtained:

__________________________________________________________________

________________________________________________________________________

_______________________________________________________________________.

V. SUMMARY ANALYSIS.

What was learned from this exercise?

VI. REFERENCES.

Welsh, J. H., Smith, R. I, and Kammer, A. E. 1963. Laboratory Exercises in Invertebrate Physiology, 3rd edn. Burgess Pub. Co, Minneapolis, MN

Jones, J. C. 1977. The Circulatory System of Insects. Charles C. Thomas, Springfield, IL.

Gillott, C. 1995. Entomology, 2nd edn., Plenum Press, New York, NY. See Chapter 17, pp. 493-511

_________________

(Name)

Topic 10