Laboratory Materials

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Topic 11.  Respiration.

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I. MATERIALS.

            A large pupal insect such as Manduca sexta, tobacco hornworm.

            Or any large insect which can be mounted to observe the spiracles.

            Mealworm larvae (Tenebrio molotior). Can be obtained as live fish bait.

            Microdroppers for water and oil.

            Sources of air, N₂, O₂ and CO₂.

            A respiratory chamber.

            Tubing for connections.

            Saline solution.

            Microscopes and lamps.

            Microscope slides.

            Plasticene.

            Dissection dishes.

II. LEARNING OBJECTIVES:

III. INTRODUCTION.

            Insects breathe by taking air directly into the tracheal system.  Air openings, called spiracles, are present as paired structures on each of the lateral (pleural) body segments.  Air is taken in and air saturated with CO₂ is exhausted to the outside in periodic bursts of activity.  Water is lost through the cuticle or in the exhaled air leaving the tracheal system.  Thus it is important that the spiracular openings be confined to a minimum in conditions where water is not available, or in insects which live in arid conditions.

            Coelopulse system.

            Ventilatory movements are said to be controlled by a central program situated in the mesothoracic ganglion.  From here, signals are sent simultaneously to the intersegmental muscles controlling movements of the body segments, and to the spiracles themselves.  Each time the spiracles open briefly to expel accumulated CO₂, the intersegmental muscles are given pulsed stimulations which cause a telescoping of the body segments to assist in actively ventilating the air.  These movements are too small to be seen by the naked eye, but can be detected by sensitive transducers.

            The muscles that control the spiracle valves are sensitive to CO₂ and acid saline both of which are said to cause the muscle to relax and the spiracle to open.  The readiness of spiracles to open under the influence of CO₂ depends on the state of hydration of the insects.  In desiccated insects, a larger concentration of CO₂ is needed to relax the spiracular muscle and cause the spiracle to open.  In well hydrated insects, lower concentrations of CO₂ will cause opening.

IV.  DIRECTIONS.

            A.  Effects of CO₂ and O₂ on respiratory movements.

            Selecting a fresh insect, place it in a cell with a glass tube leading into each end, and roofed with a microscope slide.  Observe the largest spiracle and time the frequency of its opening.  Note carefully any respiratory movements of the abdomen.  Now introduce a slow flow of air containing 1% CO₂ through the chamber.  Observe the rate of spiracle opening and respiratory movements as before.  Record all observations in suitable form.  Repeat using air with 5% CO₂. Summarize your conclusions regarding the effects of CO₂ on the control of respiration.

            To test the effects of oxygen lack, make up gas mixtures containing 10% and 5% of oxygen in nitrogen.  Expose your insect to these, allowing a "rest period" in normal air between trials.  Taking into account the normal habitat of the insect, evaluate the relative roles of excess CO₂ and lack of oxygen in controlling respiratory adjustments.

            B.  Dissection of the tracheal system (TA: Prepare a few for the class).

            Anesthetize an adult roach in water or CO₂ and dissect away the dorsal body wall under insect saline.  Examine the system of large air sacs and the air-filled tracheal tubes ramifying through the body.  Prepare suitable sketches and diagrams to record your observations.

            C.  Air and fluid in tracheoles.

            The tracheoles, the finest terminations of the tracheal system, are often very difficult to see because in many insects in a resting state they are filled with fluid. However, during exercise this fluid is withdrawn, allowing air to penetrate further along the tracheoles and presumably improving the oxygen supply of the active tissues. In practice this is not easy to observe.

            Place a small mealworm larva beneath a glass slide supported by three or four little pellets of Plasticene.  Press the cover down until the insect is firmly held and somewhat flattened.  By careful focusing, locate the fine air passages in the body or gut wall.  Make a sketch of the endings in a small selected area. Now, with a small pipette, flood the space under the cover-slip and around the larva with water, producing a state of asphyxiation against which the larva may struggle.  Observe the tracheal endings in the chosen area.  Sketch their extent. With an absorbent paper, draw away the water from around the larva.  Observe carefully the tracheal endings.  Sketch the final extent of air in the tracheal branches and compare with the original condition. (It might help to remove a ventral plate from the insect to see better).

V.  SUMMARY ANALYSIS.

            Describe what was learned in this exercise.

VI. REFERENCES.

            Keister, M.L. 1948. The morphogenesis of the tracheal system of Sciara. J. Morphol. 83:373-423.

            Wigglesworth, V.B. 1931. The extent of air in the tracheoles of some terrestrial insects. Proc. Roy. Soc. Lond. B. 109:354-359.

            Wigglesworth, V.B. 1953. "The Principles of Insect Physiology," 5th ed. , Meuthen, London. pp. 224-264.

            Wigglesworth, V.B. 1956. "Insect Physiology," 5th., Meuthen's Monographs on Biological Subjects.

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