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Topic 3. Ecdysis triggering hormone

________________

(Name)

I. MATERIALS.

Dissecting microscopes and lamps.

Dissecting tools, dissecting dishes, pins.

Janus Green dye in saline (10% solution).

Saline solution in dropper bottle, or squeeze bottle.

Adult houseflies and large nymph or adult American cockroaches.

100 ml beakers for waste saline, tissue papers, waste basket, a wash basin with hand towels, disposable gloves.

II. LEARNING OBJECTIVES.

Each student should be able to:

Identify the major parts of the insect nervous system.

Understand the sensory and motor nerve connections.

Identify parts of the neurosecretory nervous system.

How:

A slide session will show how the nervous system is internally organized.

Compare the nervous systems in cockroach and housefly by labeling the enclosed drawings. Identify the median nervous system and hyperneural muscles in cockroach.

III. INTRODUCTION.

The insect central nervous system is thought to be derived from paired ganglia in each segment. In most cases the ventral ganglia have become fused, but in most cases the paired connectives attaching each of the ganglia in adjacent segments are still present. In some cases, such as the house fly, even this pairing is lost. The ganglia in adjacent segments have evolved into fused structures. The terminal abdominal ganglion in the American cockroach is comprised of three fused segmental ganglia, and the housefly thoracic ganglion is comprised of all the thoracic and all the abdominal ganglia fused together as an extreme case.

Ganglionic organization is uniform. An outer acellular layer, the neural lamella is secreted by the cellular perineurial layer underneath. These layers represent a protective covering to all ganglion structures. Under the perineurium are the motor and interneuronal cells bodies that are positioned in discrete around the central core of neuropile comprised of synaptic connections and bungles of axons in major tracks traversing the ventral nerve cords, besides the incoming sensory axons and outgoing motor axons.

Each ganglion also has a complement of neurosecretory neurons. This extra nervous system is best seen in the abdomen of the American cockroach as the median nervous system, but neurosecretory neurons accompany all elements of the insects nervous system. Including pockets of neurosecretory neurons making up neurohemal organs outside the central nervous system such as the corpora cardiaca in the retrocerebral complex, and the aforementioned median nervous system.

IV. DIRECTIONS.

Cockroach:

Adults can be immobilized by cold or by being exposed to carbon dioxide gas. Remove the legs of cockroach close to the body. Remove the wings, if it is an adult. Cut down the middle of the dorsum from tail to head. Push one pin temporarily through the rear of the abdomen off center and into the wax to hold the body in place in the middle of the wax dish. Place a second pin into the pronotal shield of the prothorax. Before pushing this second pin into the wax, pull gently on the body to stretch it out slightly. Now reach into the middle of the abdomen and grasp the midgut gently by forceps, pull it up and out of the body cavity and lay it to the side. The gut will unfold somewhat allowing you to pull it free of the abdomen.

At this point, moisten the exposed body cavity in the abdomen with a few drops of saline. Do not flood the cavity, just use a few drops. Now, take two large pins, place one at the front of the abdomen in the sectioned opening. Move the dorsal flap on one side laterally with the tip of the pin and push the pin into the wax at the lateral-most part of the anterior abdomen. Do the same with the second pin on the opposite side. Repeat the pinning process with two move pairs of pins in the middle of the abdomen and at the back of the abdomen, replacing the temporary pin used above.

At this point it should be obvious if the thorax has been cut properly. The gut should be removed from the tail end of the abdomen to the head. If it is not, dissect the thorax carefully to free the foregut. Once the main part of the gut is free, carefully cut it behind the head and at its terminal attachment to the anus. Try no to spill any gut contents into the exposed body cavity.

Before going any further, discard all dissected body parts and tissues including the gut, and flush the exposed body cavity with fresh saline. Use forceps to hold the tissues and body parts. Don't use your bare hands. It may be necessary to decant the saline into a waste beaker, or if convenient, flush the dissection dish at a sink. Do not submerge the preparation in excess saline. use only a few drops to keep the tissues moist and try to keep the lateral abdomen out of the water so the main tracheal trunks remain filled with air (this makes it easier to see tissues).

If you did not use disposable gloves during the dissection, stop now and wash your hands before continuing. If you touched the grease on the cuticle of the cockroach with your bare hands during the dissection (it has a powerful odor) it is best to remove it promptly. You will be unlikely to come in contact with any body parts or tissues from this point on.

Examine the exposed body in a dissecting microscope. If the tissues are fresh, you may see the hyperneural muscles contracting slowly. If you stretched the abdomen, the ventral nerve cord (VNC) in the middle of the ventrum of the abdomen should be in the exact middle of the abdomen. Two large tracheal trunks lie on either side of the VNC with segmental tracheal projecting to join these main trunks in each segment of the abdomen.

Decant all of the saline by holding the dissection dish and attached tissues vertically for a few seconds, and then lay it flat again and carefully add several drops of Janus Green saline to the abdominal cavity starting with the thorax. Wait several seconds to a minute, then flush with fresh saline. The Janus Green dye will be taken up by several tissues and the resulting contrast should reveal the VNC and median nervous system. It should also reveal the hyperneural muscle for the first time which is ordinarily transparent. It is important to decant the saline so that the dye comes in contact with all of the tissues you wish to stain. The staining only works once with each preparation. You will not be able to repeatedly stain because the tissues become saturated with the dye the first time they are exposed.

Notice how the hyperneural muscle bands are connected to each of the ganglia of the VNC, and the connectives of the VNC project through the large spaces in the middle of the hyperneural muscle bands that lie on either side of the midline of the ventrum. Identify the three conspicuously larger thoracic ganglia in the thorax. Now starting to the rear of the metathoracic ganglion, count the number of abdominal ganglia. There should be five anterior ones and a conspicuously larger sixth or terminal abdominal ganglion.

If the dye staining has been successful, you should be able to detect the large segmental nerve trunks that leave the rear lateral aspects of each of the ganglia. These segmental nerve trunks then travel to the rear a short way and turn laterally. If it is difficult to see structures in the thoracic area, dissect away more of the leg muscle tissue and flush with fresh saline again. See if you can draw the segmental nerve leaving the metathoracic ganglion and innervating the first abdominal segment. This nerve bundle will be coming from the abdominal ganglion that supplies the first abdominal segment which has fused with the metathoracic ganglion, so the first abdominal ganglion you identified above is actually the second one. See if you can find the segmental nerve leaving the first abdominal ganglion. It should project to the second abdominal segment.

You should now be able to see the median nervous system by looking carefully at the rear of one of the abdominal ganglia. It is exactly in the mid line of the body, connects successive ganglia, and sends conspicuous branches left and right from some point in the median nerve. If your staining has been particularly good, you may be able to see connections between these lateral branches and the hyperneural muscle. Otherwise, the lateral branches run directly up the side of the abdominal ventrum.

Now find the terminal abdominal ganglion (TAG). Notice that the paired connectives joining the TAG with the fourth abdominal ganglion are very uniform in size from one end of the abdomen to the other. Find two large nerve bundles at the rear of TAG supplying the cerci, the two largish hair-covered appendages at the rear of the abdomen. It may be necessary to trim some more of the overlying tissues to see where the terminal nerve bundles are going. Convince yourself that the TAG terminal nerve bundles are actually entering the cerci. These large bundles leaving the rear of the TAG are full of sensory axons coming from nerve cells associated with the cercal hairs which are sensitive to air movements. By tracing segmental nerves, try to determine how many ganglia are fused into the TAG (_____________).

Very carefully, place a pin through the very front of the head from the top of the head in front of the eyes, until it comes out the front of the mount parts. Before inserting this pin in the wax, tip the head forward so as to stretch the neck region. As carefully as possible, separate the membranes on the back of the neck with a pin or sharp object so as to expose the underlying tissues. The retrocerebral complex lies immediately under the soft cuticle in this area, so if may be best to pull these soft cuticular membranes away by pulling from the side and not disturbing the midline region. Try to locate the bulbous retrocerebral complex and label the component corpora cardiaca and corpora allata.

Now try to remove the back of the head so as to expose the brain. Follow the connections of the retrocerebral complex forward. It may be necessary to watch this being done by the TA first.

Before discarding the preparation, with two pairs of forceps grasp the top of the TAG and tear the tissue. Once the tissue is torn, it should pull back and your should be able to see this if there is enough dye left staining the tissues. This is the perineurium.

In the space below draw the cockroach central nervous system on the right and connect the names on the left to the appropriate structures.

Scientific name of insect:________________________________

Date:____________________

Indicate relative size by providing a calibration mark in millimeters here:

Show the hyperneural muscle with dotted lines on the figure above.

Housefly:

Obtain either a housefly adult, or a blowfly adult. Immobilize by freezing. Remove the legs close to the thorax. Remove the wings and place ventrum down in a shallow groove in a wax dissection dish. With a razor blade, cut down the exact middle of the thorax from the neck to the waist. Be sure to cut in the exact middle. Push one pin through each side of the thorax, about in the middle, pry the thorax apart exposing the underlying. If you expose the gut running from the head end to the abdomen, you have been successful.

Now try cutting down the middle of the soft dorsum of the abdomen. Pin this out with a couple pairs of pins similar to the thorax. Moisten the tissues with a drop of saline. The gut should be moving somewhat which should help identify it. Try to clear the connection between the thorax and abdomen of overlying cuticle. Once this is done, the gut can be snipped at the front and rear and discarded. Try decanting the fresh saline or daubing with a tissue and staining the exposed tissues with the Janus Green saline, as above. Use only a drop or two and do not leave too long, only 5-10 seconds.

Label the attached drawing outline with the central nervous tissue elements observed in the adult fly preparation.

Notice it is impossible to discern a paired connective entering the front of the thoracic ganglion mass. If your preparation is especially clean, you may be able to see large axons traveling down the cervical connective. They should appear as clear spaces bordered by parallel lines, but may be only visible as shades of difference in opaqueness.

Now, very carefully, try to open the head to expose the brain by cutting the cuticle away from the area between the rear of the compound eyes down to the neck region. It may be helpful to crisscross pins through the lateral aspects of the compound eyes and tip the head forward as above with the cockroach. Try following the cervical connective forward, exposing cuticle and tissues as you go. If this proves too difficult, observe it in another preparation or have the TA prepare one. Draw the connection between the cervical connective and the brain. Be sure to include a scale on the drawing.

In the drawing below, connect the labels to the proper structures.

Housefly ventral preparation.

Date: ____________; Name of insect:________________________

Brain. [

Cervical connective. [

Thoracic ganglion mass. [

Abdominal nerve bundles. [

Apply a calibration scale to your drawing as before.

IV. SUMMARY ANALYSIS.

Describe what you have learned from this exercise.

V. REFERENCES.

Huber, I., E. P. Masler and B. R. Rao. (eds.) 1990. Cockroaches as Models for Neurobiology: Applications in Biomedical Research. Vol. I. See especially chapter 3, Anatomy of the cockroach, A. P. Gupta, Y. T. Das, and B. R. Rao, pp. 33-61, figure 22, page 57.

Guthrie, D. M. and A. R. Tindall. 1968. The Biology of the Cockroach. Edward Arnold, Ltd, London. See especially, Fig. 8.27, page 203.

Topic 5

Brain

Suboesophageal ganglion

Prothoracic ganglion

Mesothoracic ganglion

Metathoracic ganglion (T3)

Ganglion I

Ganglion II

Median nerves (draw in)

Ganglion III

Ganglion IV