Reaction of planarians to pH, light, and temperature conditions

Introduction: (Initial Observation)

While studying about fresh water invertebrates, I came across planarians, small creatures that demonstrates unique and unusual properties. Planarians are free-living, flat bodied, freshwater creatures that exhibit the remarkable ability to regenerate their lost body parts. They live in lakes, streams, ponds, and other freshwater bodies. They can be conditioned to respond to stimuli, display the ability to master a two-choice maze, and can transfer the memory of training from one individual to another.

To learn more about planarians, I have decided to study their life cycle and find out how do they react to different environmental conditions such as light, pH, and temperature.

Information Gathering:

Find out about planarians and other invertebrates that live in a body of water such as a pond. Read books, magazines or ask professionals who might know in order to learn about possible effects of environmental factors such as pH, light, and temperature in such animals. Keep track of where you got your information from.

Following are samples of information that you may find:

Following are sample information that I gathered to be used in this project.

• Planaria belong to the phylum, Platyhelminthes, (flatworms).
• They are a free-living, flat bodied, freshwater creatures that exhibit the remarkable ability to regenerate their lost body parts.
• It lives in lakes, streams, ponds, and other freshwater bodies
• The planarian is non-parasitic, and eats decaying meat.
• The body includes:
  a simple nervous system that includes a ‘brain’
  muscle bundles
  an internal reproductive system
  a blind gut branching through the body
  an excretory system that includes specialized cells called ‘flame cells’
• They can be conditioned to respond to stimuli, display the ability to master a two-choice maze,
  and can transfer the memory of training from one individual to another.

To keep a planaria culture alive follow these tips:

Use only ‘spring water’ or ‘pond water’, not distilled water (it doesn’t contain any of the minerals and nutrients they need to survive) or tap water (the chlorine or flouride etc. that’s in tap water will kill them)
Feed them little bits of hard cooked ‘egg yolk’ every few days – to a week at most. Some varieties will eat liver or tubifex worms, but that’s very smelly and messy.

Don’t feed them at all during their mating season during February-March.

After you feed them (let them eat for about 30 minutes – 1 hour at most) make sure you change their water (rinse them off carefully) and add fresh water. This prevents any uneaten food from decaying and dirtying the culture. They also create a ‘slime’ that needs to be removed.

Keep them at a reasonable room temperature (68-72 degrees). Do not refrigerate them.

Do not expose them to harsh light. In fact, keep them in the dark for most of the time; maybe in the container they came in with a lid (loosely closed) and store it in a closed cupboard.

They are sensitive to extremes of light, temperature, and ph. Being such sensitive creatures, when you begin your experiments, any changes you may make in their environment should be the smallest that you can measure.

The Phylum Platyhelminthes contains a number of flat worms having three tissue layers and very diverse living conditions.

• Bilateral symmetry
• Flattened body design
• Three tissue layers
  • Ectoderm
  • Mesoderm
  • Endoderm
• Sac like digestive system – if any
• Ladder nervous system
• Some with sense organs

Planaria

• A freshwater, free living Platyhelminthes
• Lives on the under side of rocks and such – avoids light
• Feeds using a pharynx tube on its underside
• Uses eye spots to sense light and is photo phobic

Planaria are noted for their great ability to regenerate missing body parts. Below are two examples of experiments conducted on Planaria.

Question/ Purpose:

What do you want to find out? Write a statement that describes what you want to do. Use your observations and questions to write the statement.

The purpose of this project is to find out how changes in pH in a body of water affect the life of planarians living there.

Identify Variables:

When you think you know what variables may be involved, think about ways to change one at a time. If you change more than one at a time, you will not know what variable is causing your observation. Sometimes variables are linked and work together to cause something. At first, try to choose variables that you think act independently of each other.

This is a sample variable definition for the effect of pH on planarians.

Independent variable (also known as manipulated variable) is the pH of water.

Dependent variable is the rate of planarians that tolerate any specific pH and stay alive.

Controlled variables are (other environmental factors such as) water, light, and temperature.

Hypothesis:

Based on your gathered information, make an educated guess about what types of things affect the system you are working with. Identifying variables is necessary before you can make a hypothesis.

This is a sample hypothesis:

Adding 0.2% salicylic acid to the simulated pond water habitats will result in a drastic decline in pH that will have a detrimental effect on the test specimens. Secondly, it is hypothesized that the salicylic acid concentrations of .03%, .01% and .005% will be well tolerated by the planarians.

Experiment Design:

Design an experiment to test each hypothesis. Make a step-by-step list of what you will do to answer each question. This list is called an experimental procedure. For an experiment to give answers you can trust, it must have a “control.” A control is an additional experimental trial or run. It is a separate experiment, done exactly like the others. The only difference is that no experimental variables are changed. A control is a neutral “reference point” for comparison that allows you to see what changing a variable does by comparing it to not changing anything. Dependable controls are sometimes very hard to develop. They can be the hardest part of a project. Without a control you cannot be sure that changing the variable causes your observations. A series of experiments that includes a control is called a “controlled experiment.”

Experiment 1: Effect of pH on planarians

Introduction: Planarians and other invertebrates living in a body of water are members of a balanced ecosystem. In a balanced ecosystem each member has a role and change in population of one species may seriously affect the life in the entire ecosystem. Change in pH of a body of water may happen in different ways. Contaminants, biochemical reactions and use of algaecides are among the factors that may cause drastic changes in pH of water. Scientists often need to examine the effect of such changes to ensure a healthy ecosystem.

For example salicylic acid is an algaecide that may be used in ponds. Reduction of pH caused by Salicylic acid may be harmful to planarians, Daphnia and snail.

In this experiment we test the effect of pH on planarians.

Procedure:

1. Obtain live planarians for your experiment (Live specimens may be obtained from Boreal Laboratories.)
2. 4 liters of pond water are collected in a large plastic bucket.
3. The pond water must set for 24 hours to allow for
   • sedimentation of solids
   • the water to reach room temperature (22° C.)
4. 200ml of pond water is added to five – 480ml glass jars.
5. Jars are labeled – Control, .2% salicylic acid, .03%, .01% or .005%
6. Equal numbers of Planaria are added to the jars (I suggest 10 planarians per jar)
7. pieces of liver are added to each of these jars to feed the planarians

Note: If you are doing this experiment with pond snails, add lettuce leaves instead.

If you are doing this experiment on Daphnia, add several drops of a warm water yeast mixture.

8. All test specimens are observed for 24 hours in the new habitats.
9. Remaining liver (or lettuce if you are testing snails) is removed to prevent fouling the water.
10. The amount of salicylic acid to be added to each container is calculated.
11. Add different amounts of salicylic acid to the jars as follows:
a. Control = no salicylic acid added
b. .2% = 600mg
c. .03% = 90mg
d. .01% = 30mg
e. .005% = 15mg
12. The salicylic acid is weighed and measured on a digital scale and poured into labeled plastic vials.
13. Each vial of salicylic acid is first added to 100ml of pond water in a jar with a lid and shaken vigorously.
14. The salicylic acid solution is slowly poured into the corresponding container.
15. Daily observations are made on each test jar
16. 24 hours after addition of salicylic acid, the pH of each habitat is tested.
17. At 96 hrs. post-salicylic acid addition the number of live and dead specimens is counted and recorded.

Record your results in a table like this:

pH is one of the things you must measure. Above numbers for pH are from a sample experiment with Salicylic acid.

Experiment 2: Effect of Light on planarians

Introduction: By learning about the light condition favorable for planaria, we can determine what parts of an ecosystem planarians may live. In this experiment I will test the effect of light on planarians. Note that while testing the effect of light, temperature is our controlled variable.

Procedure:

1. Obtain live planarians for your experiment (Live specimens may be obtained from Boreal Laboratories.)
2. 4 liters of pond water are collected in a large plastic bucket.
3. The pond water must set for 24 hours to allow for
   • sedimentation of solids
   • the water to reach room temperature (22° C.)
4. 200ml of pond water is added to four – 480ml glass jars.
5. Jars are labeled – Control, low light, No light, High light
6. Equal numbers of Planaria are added to the jars (I suggest 10 planarians per jar)
7. pieces of liver are added to each of these jars to feed the planarians

Note: If you are doing this experiment with pond snails, add lettuce leaves instead.

If you are doing this experiment on Daphnia, add several drops of a warm water yeast mixture.

8. All test specimens are observed for 24 hours in the new habitats.
9. Remaining liver (or lettuce if you are testing snails) is removed to prevent fouling the water.
10. Use Aluminum foil to partially cover the jar labeled “Low Light”
11. Use Aluminum foil to fully cover the jar labeled “No Light”
12. Place a desk lamp above the jar labeled “High Light” and turn on the light.
13. Make daily observations on each test jar
14. At 96 hrs. after starting the light experiment the number of live and dead specimens is counted and recorded.

Record your results in a table like this:

Experiment 3: Effect of temperature on planarians

Introduction: By learning about the effect of temperature on planaria we can determine what will happen to the planarians in cold winters or hot summers. In this experiment we test the effect of temperature on planarians.

Note that while testing the effect of temperature, light must remain a controlled variable.

Procedure:

1. Obtain live planarians for your experiment
2. 4 liters of pond water are collected in a large plastic bucket.
3. The pond water must set for 24 hours to allow for
   • sedimentation of solids
   • the water to reach room temperature (22° C.)
4. 200ml of pond water is added to five – 480ml glass jars.
5. Jars are labeled – Hot, Warm, Control, Cold, Ice cold
6. Equal numbers of Planaria are added to the jars (I suggest 10 planarians per jar)
7. pieces of liver are added to each of these jars to feed the planarians

Note: If you are doing this experiment with pond snails, add lettuce leaves instead.

If you are doing this experiment on Daphnia, add several drops of a warm water yeast mixture.

8. All test specimens are observed for 24 hours in the new habitats.
9. Remaining liver (or lettuce if you are testing snails) is removed to prevent fouling the water.
10. Place the jar labeled “Hot” in a bath of 50ºC water
11. Place the jar labeled “Warm” in a bath of 30ºC water
12. Place the jar labeled “cold” in a bath of 10ºC water
13. Place the jar labeled “Ice cold” in a bath of 0ºC water
14. Make daily observations on each test jar
15. At 96 hrs. after starting the light experiment the number of live and dead specimens is counted and recorded.

Record your results in a table like this:

Note: If you don’t have instruments to control the bath water temperatures at exact amounts suggested above, do your best to be as close as possible to those numbers. Ice cold bath can be a mix of ice and water. You may be able to use some aquarium heaters for this experiment.

Materials and Equipment:

Combined list of material:

• Salicylic acid (buy from a local pharmacy or substitute it with white vinegar that contains 5% acetic acid)
• Planaria (or you can try other invertebrates such as Daphnia and Pond snails)
• Large plastic bucket
• Pond water
• 15ct. –10-dram dark plastic vials
• 16ct –8-dram plastic vials or test tubes
• Stir sticks or tongue depressors
• Metric measuring cup
• 16ct. –480ml clear glass jars
• 100ml-graduated cylinder
• Pipettes
• Digital gram scale
• Wide range pH test strips
• Liver
• Yeast (needed if you are doing this experiment on Daphnia)
• Lettuce (needed if you are doing this experiment on Snails)
• Metric ruler
• 12cc syringe barrel

Results of Experiment (Observation):

Experiments are often done in series. A series of experiments can be done by changing one variable a different amount each time. A series of experiments is made up of separate experimental “runs.” During each run you make a measurement of how much the variable affected the system under study. For each run, a different amount of change in the variable is used. This produces a different amount of response in the system. You measure this response, or record data, in a table for this purpose. This is considered “raw data” since it has not been processed or interpreted yet. When raw data gets processed mathematically, for example, it becomes results.

Sample experiment results indicate that the 0.2% salicylic acid did cause a drop in pH in all four habitats and had a deadly effect on the snails, Daphnia, and planaria.

Calculations:

Example calculation for the amount of Salicylic acid in order to make 300 ml 0f .2% solution:
a. .2% = 2mg/ml
b. 2mg/ml = Xmg/300ml
c. X = 600mg

Summary of Results:

Summarize what happened. This can be in the form of a table of processed numerical data, or graphs. It could also be a written statement of what occurred during experiments.

It is from calculations using recorded data that tables and graphs are made. Studying tables and graphs, we can see trends that tell us how different variables cause our observations. Based on these trends, we can draw conclusions about the system under study. These conclusions help us confirm or deny our original hypothesis. Often, mathematical equations can be made from graphs. These equations allow us to predict how a change will affect the system without the need to do additional experiments. Advanced levels of experimental science rely heavily on graphical and mathematical analysis of data. At this level, science becomes even more interesting and powerful.

Conclusion:

Using the trends in your experimental data and your experimental observations, try to answer your original questions. Is your hypothesis correct? Now is the time to pull together what happened, and assess the experiments you did.

Related Questions & Answers:

What you have learned may allow you to answer other questions. Many questions are related. Several new questions may have occurred to you while doing experiments. You may now be able to understand or verify things that you discovered when gathering information for the project. Questions lead to more questions, which lead to additional hypothesis that need to be tested.

Possible Errors:

If you did not observe anything different than what happened with your control, the variable you changed may not affect the system you are investigating. If you did not observe a consistent, reproducible trend in your series of experimental runs there may be experimental errors affecting your results. The first thing to check is how you are making your measurements. Is the measurement method questionable or unreliable? Maybe you are reading a scale incorrectly, or maybe the measuring instrument is working erratically.

If you determine that experimental errors are influencing your results, carefully rethink the design of your experiments. Review each step of the procedure to find sources of potential errors. If possible, have a scientist review the procedure with you. Sometimes the designer of an experiment can miss the obvious.

References:

List of References

www.planarians.org

http://www.okstate.edu/artsci/psych/abramson/plan.html

http://www.users.voicenet.com/~ginette/planaria.htm

Learning and Memory in Planaria