3. Lab Procedure – Session 1

3.1 Handing live organisms (a reminder)

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 the study organism in this lab:

• 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).

Working with live organisms is challenging! Please remember that your study organism may not obviously respond to the stimuli you are presenting with them. There are a lot of subtle responses that may not be easily detected. So, be patient, observant, and always keep the biology of the organism in mind!

3.2 Defining behaviour and deciding on stimulus to test

The experiment should be based on existing knowledge about the species and on your own observations. Test out a few ideas during this lab period to see which one will work best. When designing and carrying out your experiment, you must ensure the ethical treatment of your study organisms. Please carefully read the reminder on handling live organisms above before proceeding.

1. In your team:

a) Discuss the observations you made from the video. One team member should start an inventory of behaviours/movements that you observed and write a definition for each – recall that these are operational definitions that will allow for consistency in scoring later on. This does not need to be a complete inventory, but starting it now while observations are fresh in your mind will help later on! Note: even if you are planning on a physiology study, having this information will be very helpful!

b) Discuss the stimuli that each team member thought would be relevant and interesting to test. Be sure to share the questions you came up with as well and why you think the stimulus/question would be interesting to investigate. Finally, decide as a team which stimulus your team will investigate.

c) Decide, as a team, whether you are most interested in looking at a physiological response (by measuring CO₂ production), the behavioural response, or both responses together.

d) Talk to your instructor to get approval before moving on.

2. (Read all of #2 before carrying out your observations!) Individually or in pairs, make observations (physiological and/or behavioural) of how your organism reacts when placed in a new environment. Go back to the stimulus your team identified as interesting and relevant to test and expose a few individuals to each stimulus. For each stimulus you test, try 2 different levels (e.g., hot and cold, dim and bright light). Each individual should make detailed observations in the lab notebook for discussion afterward. Be sure to think about how you could quantify the response in a relevant and unbiased manner. Some tips to get started:

• If looking at behaviour: As soon as you introduce the organism to the stimulus, start making observations and continue for at least 5 minutes. Each person should write out their own observations.
• If looking at physiology: allow for 4 minutes for the sensor to start accurately reading the environment before you start making the observations on CO₂. You will also want to be making behavioural observations to provide context for your CO₂ values, even if you are not directly analyzing the behavioural observations. You can decide specifically what you will be measuring when you design the experiment in 3.2 below. For now, I would recommend setting up SparkVue to collect data every second and go for at least 5 minutes.

3. Discuss as a team what you all observed, paying attention to whether you were all using consistent language in your descriptions, and whether you all understood what you were looking for. Decide which 2-4 levels of stimulus (i.e., IV) your team wants to test in the experiment, and how you will measure the response (i.e., DV).

4. Before you leave the lab, ensure that you have decided the following (take pictures if it is helpful!):

• what specific IV you are manipulating
• what your DV is
• how you will manipulate your IV
• what “arena” you will be using

3.2 Design your experiment (while still in the lab or immediately after)

1. Based on your ad libitum and further observations, your operational definitions as well as the natural history research you have done, formulate a question related to one stimulus you will test further.

2. Once your question is formulated, work on your preliminary hypothesis and prediction(s). As you learn more about your organism, you can adjust your hypothesis and provide relevant rationale.

3. Decide how to test your hypothesis and prediction – once you have your question, hypothesis, and prediction, decide on how you can test your prediction. This is a very important step to ensure that what you are testing is not harmful to the organism! Write down how you plan on carrying out your study/experiment. Address the following before moving on – you can always revise your plan as you go:

• Does your experimental design minimize the harm/discomfort of the subjects?
• Are the conditions you are testing simulating natural conditions? How many levels of stimulus are you testing?
• Is there a less complex way to answer your question? What are possible sources of bias? Could the animal be responding to a stimulus other than what you are testing (i.e., if testing response to light, could temperature also be involved)?
• Are you observing for an appropriate length of time? Be sure that your observation period is sufficiently long so that you will be able to observe numerous incidences of the behaviour. To maintain consistency in your data, observe each animal for the same length of time.
• How many replicates will you have? Keep in mind that individuals vary. What one does may not be typical. So repeat your experiments on several animals. Remember, you will have a full lab period to collect data – use your time effectively.
• If applicable, was acclimation time allowed for? Whenever you are exposing your organism to a new environment, you should allow some time for the organism to get used to that environment. That way when you expose them to the new stimuli, you are able to measure the response to the stimuli, not to the environment. Note: sometimes it is interesting to make observations during this acclimation period as well.
• What are you measuring and is the variable objective (i.e., will everyone be able to measure it the same way and come to a similar conclusion)? See the ideas for behavioural and physiological variables in the introduction.
• What kind of data (continuous, categorical) will you have at the end of your experiment? This will help you decide on which type of graph (bar or scatter) to use for your prediction graph.
• Draw/make a sample data collection table that you can use to collect data during your experiment. This can be on paper, in Word/Google Docs, or Excel/Google Sheets.