From drugs to climate change: hands-on experiments with Daphnia as a model organism Teach article

Author(s): Hugo Miguel Faria, António Pedro Fonseca

How do social drugs affect metabolism? How is toxicity measured? How does climate change affect water ecosystems? Promote active learning by investigating these questions with Daphnia.

Introduction

Daphnia, a freshwater microcrustacean keystone species, is a model organism with many practical advantages: simplicity of culture under laboratory conditions, small body size (2–5 mm), short life cycle, easy handling, high fecundity, parthenogenetic reproduction, and low maintenance costs.⁠[1,2] Due to their translucent chitinous exoskeleton, it is possible to observe Daphnia’s internal organs under a microscope or binocular magnifying glasses.[3] Since they are very sensitive animals, and because they have fundamental biological responses very similar to those of humans, they are used as models to test the effects of various ‘social drugs’ (also called recreational drugs), such as coffee, tobacco, and alcohol, and are used to evaluate water quality.[2] Freshwater ecosystems, which are among the most threatened in the world, provide drinking and irrigation water, food, climate regulation, erosion prevention, and recreation for human society. Climate change is increasing the salt concentration of freshwater, with drastic effects on the health and survival of freshwater organisms.

Daphnia Pulex on dark background. The internal organs are visible.
Daphnia pulex
Image: Paul Hebert/Reference [4], CC BY 4.0

The hands-on activities outlined in this article are suitable for students aged 14–19 and are a valuable way to evaluate the effects of social drugs on the cardiovascular system and the influence of salinization on Daphnia survival. By investigating these effects, students learn to record and manage experimental data to draw their own conclusions. From the conclusions, students can consider the implications for human health, and understand the effects of climate change on freshwater ecosystems.

How to culture Daphnia

Cultures can be maintained in containers with a maximum of 30 Daphnia per litre in chlorine-free water with a pH of 7–8, at 18–25°C, over a controlled photoperiod of 16 h and fed 2–3 times per week with microalgae or yeast (5 drops/litre). Low levels of dissolved oxygen are lethal.

How to prepare the yeast suspension

To prepare a yeast (unicellular fungus) suspension, add chlorine-free water to a 1 litre container and stir in 2 g of baker’s yeast. Store the suspension in a refrigerator (4°C) and always agitate before use to resuspend the yeast. Add 5 drops/litre of this food to Daphnia cultures 2–3 times per week.

How to culture Microalgae

Microalgae are microscopic photosynthetic organisms. They play a central role in life on Earth, as they form the basis of the food web in many aquatic ecosystems. Scenedesmus is a nonmotile green microalgae, typical of freshwater phytoplankton, that is easy to culture and maintain in the laboratory and is routinely used to feed Daphnia.

Scenedesmus
Image courtesy of the authors

Cultures of Scenedesmus require the use of chlorine-free water with a pH of 7–8, at a minimum temperature of 16°C (18 to 25°C), and with a minimum photoperiod of 12 h provided by an LED (or fluorescent) aquarium light. The microalgae culture should be kept in an aerated container (bioreactor) to provide the CO2 required for photosynthesis and prevent sedimentation of microalgae in the bottom of the vessel, which greatly limits their reproduction. Every 15 days, the culture should be supplemented with mineral salts from 3–5 drops/litre of commercial liquid plant fertilizer.

The algae bioreactor
Image courtesy of the authors

Activity 1: The rise and fall of a heartbeat

Social-drug consumption causes changes to the central nervous system, resulting in physiological and/or behavioural changes. Stimulant drugs (caffeine, nicotine) are characterized by increasing body metabolism, whereas depressor drugs (alcohol) decrease it. The heart rate can be monitored as an optimal indicator of these changes.

Observing how drugs affect a living organism in real time allows students to establish a relationship with the possible effects of drugs in their body. The transparent body of Daphnia allows measurement of different physiological parameters using noninvasive optical methods. A bioassay is a procedure that uses living organisms to determine the effects of a chemical. The activity requires around 2 hour to complete but can be done in 1 h if the solutions are prepared beforehand.

Daphnia magna. Antenna, eye, midgut, mouth, hearth, eggs, leg and anus are indicated.
Female Daphnia magna from a laboratory culture of a clone originating in lake Oued Mellah (Morocco)
Image courtesy of the authors

Materials

  • Aged/dechlorinated tap water
  • Jar containing living, young Daphnia (aged less than 48 h)
  • Stereo- or optical microscope
  • Concave slide
  • Plastic pipettes (3 ml) with the tips cut off
  • Cotton wool
  • Filter paper
  • Beakers
  • Alcohol (6%, 12%, and 20%)
  • Coffee 30% (70 ml distilled water + 30 ml instant coffee)
  • Tobacco
Materials: concave slide, plastic pipettes (with and without the tip cut off), cotton wool, and filter paper
Image courtesy of the authors

Preparation of tobacco solution

  1. Weigh 2 g of tobacco, add distilled water to bring it to a volume of 100 ml, and leave to macerate (12 h), stirring at least 4 times.
  2. Filter through filter paper. The tobacco solution lasts for 2–3 days.

Procedure

On the provided worksheet (see sample table), students predict the effects of the social drugs on the heart rate of Daphnia. Before starting experimental work, watch this video to learn how to find the Daphnia heart and how to count heartbeats.

  1. Add 2 drops of water from the Daphnia culture medium (enough to immerse the animal) on the concave slide.
  2. Use a pipette (with the tip cut off) to carefully place a Daphnia on the concave slide.
  3. Place some cotton threads in the depression of the slide (just enough to immobilise the Daphnia).
  4. Using a stereo- or an optical microscope, determine the basal heart rate of each Daphnia (control). Make three measures of 30 s each.
  5. Remove the concave slide from the microscope and add one drop of the social-drug solution (experiment).
  6. After a minute, repeat step 4 and determine the heart rate. Make three measures of 30 s each.
  7. Repeat the procedure with the other social-drug samples using a new Daphnia each time.
  8. Record and discuss the results.
  9. Construct and present a scientific poster.
The optical microscope and solutions (alcohol, tobacco, coffee)
Image courtesy of the authors

Notes

Caution: if excess cotton is used, it may be difficult to observe the Daphnia heart.

Prediction
(according to key below)		Control heart rate
(bpm)		Experimental heart rate
(bpm)
Tobacco
Alcohol (6%)
Alcohol (12%)
Alcohol (20%)
Coffee

Key:

+++big increase in heart rate
++increase in heart rate
+small increase in heart rate
0no change in heart rate
–small decrease in heart rate
—small decrease in heart rate
—small decrease in heart rate
xheart stops

Control counts below 200 beats per minute (bpm) or above 300 bpm should be repeated.

Safety notes

Care must be taken to ensure that experimental solutions do not come into contact with students’ skin, eyes, or mouth.

At the end of the activity, students must wash their hands.

Discussion

The transparent bodies of Daphnia allows visual observation of internal organs and measurement of physiological characteristics, such as heart rate. They can be used as a model system for toxicology because they are highly sensitive to environmental disturbances and exhibit rapid physiological responses when exposed to water contamination.[5]

The use of live animals, as part of authentic research experiences, has been shown to increase understanding of scientific concepts. After the experiment, ask students the following questions to promote their understanding of the influence of social drugs on the nervous system using Daphnia as a model organism:

What is the purpose of having some Daphnia in water only?
Why is the average of three heartbeat counts used?
Why is a new Daphnia used for each experimental solution (social drug) tested?
What is the advantage/importance of using clones?
Compare predictions with experimental data.
Discuss how Daphnia is a suitable biological model for studying multiple stressors.

Activity 2: Climate change: how does salt exposure affect Daphnia?

Salinization of freshwater ecosystems is an increasing problem due to human activities such as irrigation, resource extraction, accelerated weathering of rocks by acid rain, use of agricultural fertilizers and road salts, mining, and urban construction. This is further exacerbated by climate change due to decreased rainfall, increased evaporation, and an increased need for irrigation.[6,7] The use of bioassay investigations can develop students’ understanding of the effects of salinization on Daphnia survival, thus promoting a conceptual connection between the survival of simple organisms and the effects on freshwater ecosystems. These organisms need to maintain an osmotic balance between the ion concentration within their cells and their body fluids, and this is strongly influenced by the salinity of the surrounding water.

In this activity, students determine the toxicity of NaCl for Daphnia by calculating the LC50 (lethal concentration, 50%) value, which is defined as the toxicant concentration that kills 50% of the exposed organisms. This experiment should take around 20 min.[1]

Materials

  • Aged/dechlorinated tap water
  • Jar containing young Daphnia (aged less than 48 h)
  • NaCl solutions (0.5%, 1%, 1.5%, and 2%)
  • Graduated cylinder (10 ml)
  • 5 small beakers or cups for mixing solutions
  • 5 test tubes, 10 ml minimum (or exposure chambers and test tube racks or small cups)
  • plastic pipettes (3 ml) with the tips cut off
Test tubes, NaCl solutions, graduated cylinder, and pipettes
Image courtesy of the authors

Procedure

  1. Prepare solutions with different salt concentrations (0.5%, 1%, 1.5%, and 2% NaCl).
  2. Label five test tubes and add aged water or one of the salt solutions (10 ml).
  3. Use a pipette (with the end cut off) to carefully place 10 Daphnia in each test tube.
  4. After 5 min, 10 min, and 20 min, observe the behaviour of the living Daphnia and count the number of Daphnia that have died (are immobilized).
  5. Record the results in a table.
  6. Graph data on Daphnia mortality (percentage of Daphnia that died after 20 min) versus concentration of salt solution.
  7. Determine the LC50 value of salt for Daphnia.
  8. Construct and present a scientific poster.

Note

In the control, the mortality cannot be higher than 10%.

NaCl (%)Number of Daphnia that died after% mortality (after 20 min)Observations of Daphnia behaviour
5 min10 min20 min
0
0.5
1
1.5
2

Note

Do not use detergent to clean the glass material. Detergent residues may be toxic to Daphnia.

Discussion

After the experiment, discuss the results to promote an understanding of the influence of climate change on the behaviour and survival of Daphnia and other aquatic organisms.[8,9] Questions could include the following:

  • What is the purpose of having some Daphnia in water only?
  • Calculate the LC50 of sodium chloride for Daphnia.
  • What do you think the long-term effect of salinization would be on Daphnia populations?
  • In what ways can climate change affect freshwater ecosystems?
  • Can you think of any implications of this research for human populations?

This activity could also be the basis for a discussion on the ethics of using live animals in research. The use of very simple organisms like Daphnia is fairly uncontroversial, but where would students draw the line? More information on why the use of animals in research can be important and how we can minimise it can be found in this article.

References

[1] Cahill K (2006) Bioassay investigations with Daphnia. My Environment, My Health, My Choices. University of Rochester.

[2] OECD (2004) Test No. 202: Daphnia sp. Acute Immobilisation Test, OECD Guidelines for the Testing of Chemicals, Section 2. OECD Publishing, Paris. doi: doi.org/10.1787/9789264069947-en

[3] Tkaczyk A et al. (2021) Daphnia magna model in the toxicity assessment of pharmaceuticals: a review. Science of the Total Environment 763: 143038. doi: 10.1016/j.scitotenv.2020.143038

[4] Gewin V (2005) Functional Genomics Thickens the Biological Plot. PLOS Biology 3: e219. doi:10.1371/journal.pbio.0030219

[5] Gleichsner AM, Butler SR, Searle CL (2019) Dynamic Daphnia: an inquiry-based research experience in ecology that teaches the scientific process to first-year biologists. CourseSource. doi: 10.24918/cs.2019.2

[6] Kaushal SS et al. (2021) Freshwater salinization syndrome: from emerging global problem to managing risks. Biogeochemistry 154: 255–292. doi: 10.1007/s10533-021-00784-w

[7] Jeppesen E et al. (2020) Salinization increase due to climate change will have substantial negative effects on inland waters: a call for multifaceted research at the local and global scale. The Innovation 1: 100030. doi: 10.1016/j.xinn.2020.100030

[8] Martinez D et al. (2022) Daphnia magna and mixture toxicity with nanomaterials – current status and perspectives in data-driven risk prediction. nanotoday 43: 101430. doi: 10.1016/j.nantod.2022.101430

[9] Müller M et al. (2018) Temperature-driven response reversibility and short-term quasi-acclimation of Daphnia magna. PLOS ONE 13: e0209705. doi: 10.1371/journal.pone.0209705

Resources

Author(s)

Hugo Miguel Faria has been a science teacher for 32 years. His main research interest is in the use of model organisms (Daphnia, Planaria, Lymnaea) in K-12 teaching, concept mapping, and computer adaptive assessment. He is a collaborator at Coastal and Marine Environmental Toxicology (CIIMAR-Universidade do Porto).

António Pedro Fonseca has been a science teacher and professor for 33 years with a MSc in biomedical engineering and a PhD in biomedicine. He is a PhD collaborator at CIIMAR-Universidade do Porto. He has supervised pre-/postgraduate work in the areas of biofilms, knowledge engineering, and active-learning biological models.

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