Pill Bug Investigation[1]

Pill bugs are terrestrial isopods that serve as models for studying invertebrate animal behavior. The goal of this lab is to begin to answer the following question: Can pill bugs learn?

In order to answer this question, we’re going to utilize techniques first used by Pavlov, who famously taught dogs to associate the ringing of a bell with being fed. After learning to associate the two stimuli – the bell and the food – the dogs would salivate when the bell was rung, whether or not they were being fed. A stimulus (pl. stimuli) is anything that elicits a behavioral response.

There are several types of stimuli we’ll be using for this lab:

  • Unconditioned stimuli – What pill bugs should instinctually respond positively to. Food, certain environmental conditions, certain smells, etc. In Pavlov’s experiment, the unconditioned stimulus is the food, since dogs instinctually want to eat.
  • Conditioning stimuli – What pill bugs should be able to detect (Smell? Color? Vibrations?), but instinctually respond neither positively nor negatively to. In Pavlov’s experiment, the unconditioned stimulus is the bell, since dogs can hear it, but don’t respond necessarily positively or negatively to.

We will be investigating pill bugs’ response to stimuli through their movements. Since pill bugs cannot communicate to us which stimuli they instinctually respond to, positively or negatively, we use their movements as an indicator of their responses.

  • Orientation behavior: The animal responds, through taxis, to a stimulus/set of stimuli that place it in its most favorable environment for a set of circumstances.
  • Taxis: The animal moves toward or away from a stimulus.
  • Kinesis: The animal moves randomly and does not result in orientation with respect to a stimulus.
  • Agonistic behavior: When animals respond to each other through aggressive or submissive responses, often as display.
  • Mating behavior: A complex series of activities that facilitate finding, courting, and mating with a member of the same species.

We will be using pill bugs positive and negative taxis – moving toward or away from a specific stimulus – as an indication of their instinctive response. Pill bugs will be put in “choice chambers,” two connected dishes, each with a specific stimulus. For instance, one chamber may be wet and one may be dry. Pill bugs can then move between the chambers depending on their instinctual choice. The number of pill bugs in each chamber can be tracked for specific time intervals.

Discerning taxis from kinesis can be difficult: If 10 pill bugs are used for the experiment, and five are in one chamber and five in the other, for all the time intervals, then we can reasonably conclude that the pill bugs are neutral to the stimuli. However, if six pill bugs are in one chamber, on average, and four in the other, can we draw the same conclusion? If seven are in one chamber, on average, and three in the other, can we draw the same conclusion?

Procedure

We will be conducting five separate, but interconnected, experiments for this lab.

Experiment One: Negative control – One Trial

Two choice chambers (Chamber A and Chamber B) are exactly the same. Put five pill bugs in each chamber and count the number in Chamber A every 30 seconds for four minutes. Graph data as time for the X-axis and number of pill bugs for the Y-axis.

Experiment Two: Unconditioned Stimulus (Instinctual choice) – Two Trials

Chamber A should contain the stimulus expected to attract pill bugs. Put five pill bugs in each chamber and count the number in Chamber A every 30 seconds for four minutes. Graph data as time for the X-axis and number of pill bugs for the Y-axis.

Experiment Three: Conditioning Stimulus (Detected but no preference) – Two Trials

Chamber A should contain a stimulus that pill bugs can detect (vibrations, smells, color, etc.) but will neither prefer nor avoid. Put five pill bugs in each chamber and count the number in Chamber A every 30 seconds for four minutes. Graph data as time for the X-axis and number of pill bugs for the Y-axis.

Experiment Four: Training (Bothe stimuli in Chamber A) – Three Trials

Chamber A should contain both unconditioned and conditioning stimuli. Put five pill bugs in each chamber and count the number in Chamber A every 30 seconds for four minutes. Graph data as time for the X-axis and number of pill bugs for the Y-axis.

Experiment Five: Testing (Only conditioning stimulus) – Two Trials

Chamber A should contain only the conditioning stimulus (as in Experiment 3). Put five pill bugs in each chamber and count the number in Chamber A every 30 seconds for four minutes. Graph data as time for the X-axis and number of pill bugs for the Y-axis.

For this lab, you will complete the lab using the LAB REPORT FORMAT distributed at the beginning of the year.

Pre-lab and analysis questions specific to this lab are found below.

Pre-lab questions:

Complete the following readings in Campbell and Reese.

Crustaceans (692)

Kinesis and Taxis (1122)

Associative learning (1127-1128)

More information on pill bugs (optional, but informative, especially for conditioning stimuli)

  1. How many pairs of appendages can we expect to see on pill bugs?
  2. Pill bugs have gills. What are these used for?
  3. How do pill bugs rid their bodies of nitrogenous waste (similar to urine)?
  4. Describe what you expect to see if a pill bug performs taxis. Describe what you expect to see if a pill bug performs kinesis.
  5. Distinguish between classical and operant conditioning. Which are we going to be performing on the pill bugs? How do you know?

Analysis: Answer the following questions.

  1. Did the pill bugs display taxis or kinesis in EACH of your experiments (one through five)? Explain how you know for each. (You may use a series of bullet points.)
  2. Did pill bugs show a new behavior toward the conditioning stimulus after the training? Why or why not?
  3. Is there a significant difference between the results for experiment #3 and experiment #5? Perform a t-test using Microsoft Excel to determine if your results were in fact significantly different. See the ‘Hand Size and T-Tests’ handout for more specific instructions on how
  4. For the t-test, use the number of pill bugs in ONE chamber (either A or B) during experiment #3 and experiment #5.
  5. Set the data up as two columns within Excel, with one column titled Experiment 3 and the other titled Experiment 5.
  6. Find the mean number of pill bugs in each chamber, and the standard deviation of each.
  7. Perform a t-test (=ttest(A2:A(n+1),B2:B(n+1),2,1)), and then determine if results are significant by performing the following logic test: =if(A(cell with your t-test results)>0.05,”Same”,”Different”).
  8. If your answer is ‘same,’ then your learning difference is NOT significant, since your results before and after are the same. You have failed to reject your null hypothesis.
  9. If your answer is ‘different,’ then your learning difference is significant, since and you have successfully rejected your null hypothesis.
  10. Did the pill bugs show associative learning? How do you know?
  11. What could the limits of any pill bug learning (regardless of your results) be? How could the experiment be redesigned to test how long pill bugs can learn for?

[1] Adapted from Bill Wallace’s pillbug lab and the AP Biology: Animal Behavior lab.