Insect Respiratory Physiology

Energy obtained from the oxidation of food is used by the cells of the animal to do work. The propagation of the nerve impulse, the contraction of muscle, and the secretion of cellular contents are all obvious examples of cellular work. However, essentially all processes in living cells require energy.

Since animals are heterotrophs, they receive all of their energy from the oxidation of food. When all forms of food are metabolized in the animal, oxygen is consumed and energy is released, along with water and carbon dioxide, as seen in the equation below:

Depending on the diet of the animal, at any given time its cells are metabolizing a mixture of carbohydrate, fat, and protein. For simplicity, we will assume the animals used in lab are oxidizing carbohydrate exclusively. (How accurate do you think this assumption is?) The complete oxidation of a mole of glucose releases 673 kilocalories of energy:

Notice that the amount of O₂ consumed is equal to the amount of CO₂ produced. Thus the rate of respiration of an animal oxidizing carbohydrate could be determined by measuring either the rate of O₂ consumption or the rate of CO₂ production.

Measuring Respiration in Insects

You will be measuring insect respiration with an infra-red gas analyzer (IRGA) that measures the amount of CO₂ in the air passing through it by detecting changes in the amount of infra-red radiation that the air absorbs. You must read the file "Measurement of Insect Respiration Using CO₂ Analysis" in the CO₂ Analysis Pack Folder or on the Web at www.grinnell.edu/courses/bio/bio135/ before proceeding with this experiment.

Materials

Qubit Systems infra-red gas analyzer

Insect cuvette

Appropriate insects (cockroaches, crickets, etc)

Procedure

1. Your experiment should take approximately 30 minutes to complete. Adjust the time axis on the computer display to show this value by clicking on the maximum value displayed and typing in 30.
   
   
2. To observe CO₂ production, you will need to set the Logger Pro display so that the maximum value on the y axis of the graph is 550 ppm and the minimum value is 350 ppm.
   
   
3. If the y axis requires adjustment, you may adjust the axes without stopping the run. If your trace goes off screen at any time during a run, you may use the slider control at the left side of the graph to alter the range of the y axis. Alternatively, you may select VIEW from the main menu, and then 'Autoscale' to bring your trace back on screen.
   
   
4. With Logger Pro running (start data collection by clicking on the Collect button at the top of the screen) attach the gas bag containing the reference gas to the inlet of the pump, and attach the outlet of the 100 ml/min flow restrictor to the inlet of the magnesium perchlorate drying column.
   
   
5. Attach the outlet of the magnesium perchlorate column to the inlet of the calibrated IRGA. If the IRGA is calibrated correctly, the stable CO₂ concentration shown on the digital display will match that shown numerically on the computer screen underneath the graph.
   
   
6. Record the CO₂ concentration in the gas bag in the Results and Discussion section. This is your "reference CO₂" concentration.
   
   
7. Seal one or two insects inside the chamber provided.
   
   
8. With the program still running attach the outlet of the insect chamber to the inlet of the drying column. Attach the outlet of the pump, with the 100 ml/min flow restrictor still attached, to the inlet of the insect chamber. Record the time at which you do this.
   
   
9. Observe the sharp increase in the CO₂ concentration of the gas leaving the chamber due to the build-up of CO₂ from insect respiration before it was attached to the system.
   
   
10. Measure respiration for approximately 10 minutes. There will be fluctuations in CO₂ concentration over time, most likely correlated with the relative activity of the insects in the chamber.
    
    
11. Record the maximum and minimum CO₂ concentrations during this 10 minute period. These are your "analysis CO₂" concentrations.
    
    
12. With the program still running bury the insect chamber in ice in the container provided, record the time at which you do this, and continue to measure CO₂ production for another 10-15 minutes or until a steady state is reached. Record the maximum and minimum CO₂ concentrations during this period.
    
    
13. Remove the chamber from the ice and warm it with your hand to return it to room temperature. Record the time at which you begin warming the chamber. Note what effect, if any, it has on CO₂ production. Did CO₂ production return to pre-cooling levels?
    
    
14. Stop data collection by clicking on the STOP button. Save your data by selecting Save As . . in the File menu. Give your data an appropriate file name and save it in your data folder.
    
    
15. Weigh the insect chamber with the insects in it. Return the insects to the "insect farm" and reweigh the chamber. The difference in weight should be the fresh weight of the insects. Record the weight in the Results and Discussion section.
    
    
16. If time permits, repeat this experiment with a different type of insect in order to compare their respiration rates.

Calculation of CO₂ Exchange Rate

Measurements of respiratory rates are usually expressed as rates of CO₂ exchange per unit weight per unit time. The units most commonly used are milliliters (ml) CO₂ per gram fresh weight per hour. To express your data in these units use the following calculations:

1. Calculate the difference between the CO₂ concentration in the reference and analysis gases. For example, if the experiment was conducted in air of 350 ppm CO₂, at a flow rate of 100 ml/min, the production of CO₂ due to insect respiration may result in an analysis gas CO₂ concentration of 410 ppm. The difference between the reference and analysis gas streams (DCO₂) in this example would be 60 ppm or 60 microliters/liter.
   
   
2. Multiply the dCO₂ value by the flow rate (in liters per min) used in your experiment to obtain a CO₂ exchange rate per minute. A flow rate of 100 ml/min is equivalent to 0.10 liters/min, so the CO₂ exchange rate in our example would be 6.0 mliters/min. Convert this to ml/min by dividing by 1000 (there are 1000 ml/ml). The exchange rate would then be 0.006 ml/min. Multiply this number by 60 to convert to ml/hour, giving you 0.36 ml/hr.
   
   
3. Express your CO₂ exchange rate on a gram fresh weight (gfw) basis by dividing the CO₂ exchange rate per hour by the fresh weight of the insects. The respiration rate in our example, if the fresh weight were 0.1 g, would therefore be 3.6 ml CO₂/gfw/hr.

If you failed to record any of the essential data for your calculations during the experiment, you may retrieve the data from your saved file using the following procedure:

• Open the file containing your data. Your data will appear on the screen exactly as it appeared when you saved it at the end of the experiment.
  
  
• Select Examine from the Analyze menu. A vertical line will appear on each of your graphs which can be moved along the data points on the graphs by moving the mouse. Boxes will also appear on each graph showing data values and time values for each run displayed. As you move the vertical line on a graph, the numerical display in the box will change to show you the exact data values and time value at the point on each graph where the line is situated. If the box obscures any part of the trace click and drag with the mouse to place the box in a convenient location.

Results and Discussion

1. Using your data, calculate the maximum and minimum rates of CO₂ production at room temperature. Did the peaks of production appear to correlate with the activity of the insects?
   
   
2. What happened to the rate of CO₂ production when you cooled the insect chamber with ice and then warmed it? Why did the insect’s metabolism respond in this way?
   
   
3. Estimate the metabolic rate of the animal by first assuming that the amount of CO₂ produced is equal to the amount of O₂ consumed. Convert the ml O₂ consumed/gfw/hr to liters O₂ consumed by dividing by 1000. Then multiply the liters O₂/gfw/hour by 5.01 kilocalories per liter of O₂ consumed. This will give you an estimate of the amount of energy (kilocalories/gfw/hr) produced as a result of the respiratory metabolism of the insect. This is energy that is available to the insect to do various kinds of work within its cells.