INTRODUCTION TO BIOLOGY 161

INVESTIGATING FACTORS AFFECTING THE HEART RATE OF DAPHNIA

Lab summary
In this experiment, you will be observing Daphnia magna (also known as the water flea) and the effects that
ethanol and caffeine have on Daphnia heart rate.

Purposes of this lab exercise:

• To gain experience formulating hypotheses and predictions.
• To become acquainted with Daphnia as a model organism.
• To determine if/how caffeine and ethanol affect the heart rate of Daphnia.

Introduction
Daphnia magna are small small freshwater crustaceans
and members of the same phylum as insects and arachnids
(Arthropoda). Their bodies are transparent, which makes them
ideal for use in the laboratory. Additionally, since Daphnia
quickly respond to environmental changes they can be used to
assess changes in water quality in their native habitats. These
animals are poikilothermic, which means that their metabolic
rate is tied to their body temperature
Daphnia generally reproduce via a type of asexual
reproduction, parthenogenesis, and make only female
offspring. In times of environmental stresses, such as the
approach of winter or under drought conditions, male young
are made and males mate with females to produce fertilized
eggs that are more resistant to environmental conditions. One
or more juvenile animals are nurtured in the brood pouch
insed to the carapace (“shell” or “covering”) of the adult. The
the daphnium molts the young are released. You may be able
to observe either eggs or even live young in this pouch!
In this lab, the effect of drugs that are mammalian stimulants and depressants on the physiology of
Daphnia will be investigated. You will be able to see the heart beating under a dissecting microscope. The
heart is located on the back of the organism (see figure). Students will observe and calculate the heart rate of
an animal in spring water-, ethanol-, and caffeine-containg environments and determine the impact of these
chemicals on heart rate.

Hypothesis and prediction:

A hypothesis is a statement that proposes and explanation for something and can be tested and
falsified. Hypotheses generate predictions, but should not be considered the same as predictions. Predictions
are stated in an “if/then” format, always including comparisons between experimental and control groups
One must be very careful to not allow these predictions to introduce assumptions when collecting data and
making observations.
In this particular experiment, the following hypotheses and predictions can be made:
o Hypothesis: Exposure to ethanol changes Daphnia heart rate and, even more specifically, it
decreases heart rate. Our reasoning for this is that ethanol has been shown to decrease heart rate
in other animals.
o Prediction: If ethanol decreases heart rate in Daphnia, then Daphnia exposed to ethanol will have
slower haer rates than those not exposed to ethanol.

Prelab assignment: (answer these questions in your lab notebook prior to coming to lab)
1) From a hypothesis for the Daphnia heart rate with the caffeine treatment

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2) Make a prediction based on this hypothesis.
3) What is the basis for your hypothesis?

Also be sure you understand how heart rate will be calculated, and where the heart is located in the animal.

Procedure
Materials:
live Daphnia, concavity (deep well) glass slides, cotton, transfer pipettes
spring water (AKA rearing water), 5% ethanol solution, 51 mM caffeine solution,
handheld clickers, dissecting microscope

Methods (for this week, enter your procedure in your notebook in a flow chart. Pick out important
information, rather than re-writing the whole procedure. This is the first lab, so your instructor will
give you some aid in lab).
1. Using a modified transfer pipette with a wide opening, remove an animal (singular = daphnium)
from the growth culture and add it to your prepared slide. Allow the animal to acclimate to its new
environment for 2 min before observing with the microscope. (Note: never put Daphnia in deionized
(DI) water or tap water. This is fatal.)
2. Place the slide on the stage of the dissection scope and focus on the daphnium. If the daphnium is
moving around too much for you to observe its heart easily, add a small amount of cotton to the
depression. This should slow it down so that you are able to see the heart.
3. Mark the time you put the slide on the microscope stage – you will measure the heart rate after the
animal has been in the slide for 15 minutes total. During this time, check on the animal every 2-3
minutes to make sure it is still alive. (Questions for thought – when would you want to turn on the microscope
light? When should it be off?)
4. After 15 minutes, measure heart rate:
a. One lab partner will use the stopwatch or wall clock to keep track of 20 sec intervals.
Another lab partner will determine the heart rate by counting the number of beats per 20
sec. The Daphnia heart rate is quite rapid and it may be easier to make dots on a slip of
paper or use a handheld “clicker” for the 20 sec intervals determinations. Decide on one
method for your group.
b. To determine an average heart rate, measure the heart rate 3 different times successively
(everyone in the group should get at least one chance to count the heart rate). You will
want to be efficient as your Daphnia will also be affected by the heat being generated from
the light of the microscope! This average heart rate in normal spring water/rearing water
will count as your control.
5. Return this animal to the “rest home” container and wipe off your slide with a kimwipe.
6. Select a new daphnium. Replace the water in the animal’s environment with the 5% ethanol solution,
and allow the animal to acclimate to its new environment for 2 min before observing with the
microscope.
7. Repeats step 2-5 with this new animal. (Note: you should also observe and record whether you see
any differences in regularity of beating with this treatment. This is a qualitative assessment, versus the
quantitative assessment you are making with the heart rate counting).
8. Again select a new daphnium. Replace the water in the animal’s environment with the caffeine
solution, and allow the animal to acclimate to its new environment for 2 min before observing with
the microscope.
9. Repeats steps 2-5 with this new animal. (Note: again you should also observe and record whether
you see any differences in regularity of beating with this treatment.)
10. Return this animal to the “rest home” container, discard the cotton, rinse the slide with DI water and
wipe off your slide with a kimwipe.

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 11. Repeat these heart rate measurements (control, ethanol, caffeine) for as many animals as the lab time
allows. You may also wish to be working with a couple of different microscopes and animals at a
time, staggering your measurements.
12. Before you leave: enter your data directly into an excel worksheet as described by your instructor.
We will combine all this data to analyze next week.

Clean up: At the end of the experiment, remove the cotton from the deep well slides, rinse the slides with
distilled water, and return to your instructor. DO NOT DISPOSE OF THE CONCAVITY SLIDES!
Also please put the clickers and the stopwatches at the front bench. You may leave the caffeine and
ethanol solutions by your station.

Questions (please answer these in your lab notebook):

1. Were the effects what you predicted initially? Is this what you saw?
2. Can you draw conclusions from this data as to what other arthropods might experience when their
bodies are exposed to elevated concentrations of ethanol and caffeine? Why or why not?

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 INTRODUCTION TO BIOLOGY 161
FACTORS AFFECTING THE HEART RATE OF DAPHNIA: EXPERIMENTAL DESIGN

Summary
You observed the effects that ethanol and caffeine, have on Daphnia heart rate. Other chemicals or
concentrations of ethanol and caffeine could also be tested using this model organism. We will be returning
to Daphnia in a few weeks to do “the next experiment.”

Assignment:

1. Working with your lab partners, you should fill out the hypothesis, prediction worksheet.

2. You will need to design another experiment to test your hypothesis. What will you have to
control for? Will you have just one control group and one experimental group?

3. Make a flow chart or sketch to show your experimental design in your notebook. Be sure to
think about any solutions or chemicals you will need to make, make a list to show to your
instructor. (We must know the chemicals needed in case we have to order them).

Your instructor will review your experimental design to make sure you are ready to proceed.

Database searches: supplemental information

Typically, scientists read published primary literature, which describes other scientists’ research, as a way to
help them develop new hypotheses or compare their data to others working in the field. For the Daphnia
experiment, you can use the primary scientific literature get new ideas about things to test. To find relevant
articles, can search through the published literature. The National Library of Medicine and National Institutes
of Health maintain a database called PubMed that contains citations and abstracts for many life science
journals. Alternatively, the Thompson Reuters database Web of Knowledge searches a broader group of
science, social science, and humanities journals. Both of these are good choices to begin your search.

First, think about a keyword or keywords to submit to the database to identify research on the effects of
chemicals on the heart rate of either Daphnia or other organisms. Search the databases using these keywords.
What keywords did you use? How many articles did you find?

If you feel like you got too many articles, how might you limit your search? One suggestion is to add
additional keywords or limits (ie: review articles only, publication dates).

PubMed and Web of Knowledge both have features that can help you search for related articles or articles
that cite or are cited by the article you have already found. When looking at an abstract of interest in one of
these databases, take note of the sidebars that lead to other articles, as they may also be useful for your
research.

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