2. Lab Procedure

2.1 Handing live organisms

Whenever we study organisms, living or dead, we need to ensure that we are handling them with care and respect. In Canada, the Canadian Council on Animal Care (CCAC) stresses that any animals used in research (and teaching) must be handled with humane care and have housing enrichments. This organization only works with higher order organisms (vertebrates and cephalopods), however, we should apply this to any living organism. At the University of Alberta, we must go through the animal care process to ensure that we are being intentional about how any living organisms are used in teaching, regardless of the classification. Ethically, we are responsible for the humane treatment of all living organisms, and when performing experiments with them, for minimizing pain and discomfort. When measuring behaviour, it is also important to recognize that how we handle the organisms can strongly influence their behaviour. Therefore, use the following guidelines in handling Armadillidium vulgare in this and future labs:

• Minimize handling in general, and when it is required, be gentle
• Know the biology of the organism and do not expose them to conditions that will create undue stress (e.g., extremely hot or cold conditions)
• Minimize environmental disturbance to the animals (e.g., minimize bumping or banging items on the bench).

2.2 Measuring phenotypic traits^[1]

In this part of the study, you will measure the extent of phenotypic variation for a set of behavioural, physiological and morphological traits in adult pillbugs (Armadillidium vulgare).

There are 5 traits you will be measuring (listed below; traits can be measured in whichever order your group decides). We are only interested in the biological variation of the traits, rather than artificial variation through measuring and observational techniques, therefore it is essential that all students in a group are familiar with the particular method used for measuring each trait. Ensure you keep track of the identity/number of each individual as you go through measuring. Two ideas: 1) use the tray with numbered positions and measure one trait at a time for all individuals, 2) measure all traits for one individual at a time and place in a second petri dish. Record values in the provided data collection tables and write observations in your lab notebook. Throughout all of these measurements, ensure that the pillbugs are kept in a moist environment (e.g., put a damp paper towel over the holding container).

Note: Before you get started with the pillbugs, turn on the CO₂ sensor, connect it to SparkVue and let sit on your bench while you get the pillbugs ready for measuring

• Number of times picked up and response when picked up – make a note of each time a person picks up an individual pillbug and record whether it rolls up. For example, you could decide to use a code of “F” for “flat” and “R” for “rolled” then record the frequency and order of each (RFFFRRRR); this would allow us to determine the percentage of handlings that resulted in rolling, and whether is a relationship between previous handlings and response. Keep brief notes about what circumstances the roll-ups happened (e.g., did you accidentally drop the individual, were they in dry air prior, etc.).
• Total length and width – Measure the total length and width at the widest part in millimeters using callipers. Tips: You may need a dissecting microscope for this. How will you keep the individuals still enough and in the same position to take accurate measurements?
• Mass – Measure the mass of each individual to the nearest milligram (i.e., 0.001 g). Be sure to tare the weighing device before measuring your pillbug.
• CO₂ Production – You are going to measure the amount of CO₂ produced by each individual pillbug. Procedure for measuring CO₂ below (section 2.2.1).

2.2.1 Measuring CO₂ Production

1. Place the pillbug into the test chamber – make sure the pillbug is not on its back. Then insert the CO₂ sensor. Make sure the sensor does not wobble.

2. Lay the sensor and chamber on its side, making sure it is as level as possible.

3. Allow the pillbugs to acclimate and the sensor to equilibrate to the environment for 3 minutes

4. After 3 minutes of acclimate, begin recording CO₂ data:

a) Press “Start” in SparkVue and set a timer for 3 minutes (ensure your screen does not go to sleep in this time because the sensor may disconnect from your device!)

b) Record the initial CO₂ value (ppm) in your data table

c) Watch the pillbug and record observations in your lab notebook. Some helpful observations would be what the pillbug is doing while the test is going – for example, is it walking around the container, climbing onto sensor, rolled, upside down, etc. These observations will be useful when you look at the variability in CO₂ production between individuals

d) When the 3 minutes is up, “Stop” SparkVue.

e) Record the final CO₂ value (ppm) in your data table.

f) Remove the sensor and return pillbug to its holding container or move it on to the next measurement.