Investigating factors affecting the heart rate of Daphnia
In the water flea Daphnia, the single, small heart is easily visible when viewed under transmitted light under a low power microscope. The heart rate (which can be up to 300 beats per minute) can be monitored and counted in different conditions – for example changing water temperature, or changing the type and concentration of chemicals added to the water. A change in Daphnia heart rate may not be a predictor of a similar change in human (or vertebrate) heart rate under the same conditions, but the procedure provides an interesting technique for investigating the effects of different chemicals on a metabolic process.
Thanks to the British Pharmacological Society for providing the teaching notes on this practical.
With modifications made by Prof Richard Handy, University of Plymouth
This will depend on access to a healthy culture of Daphnia and on the number of microscopes you have. Students can readily follow this procedure working in pairs. Because of the variability of results between individual Daphnia, it is not appropriate to draw conclusions from one set of results; each pair (or group) of students should carry out more than one investigation to contribute to the class set.
One option is to record a live video of a sample Daphnia, during a time period in which students count the heart beats. Then you replay the video in slow motion and count the heart beats again. This allows students to consider the accuracy of their counting.
If your time or access to chemicals is limited, you could allow the students to work through the procedure in order to evaluate it and then use the example results provided for analysis.
Apparatus and Chemicals
For each group of students:
Microscope – low power, transmission
Small piece of cotton wool
Pasteur pipette (for water from the Daphnia culture tank)
Chemicals that may affect the heart rate – at low concentrations (Note 4)
For the class – set up by technician/ teacher:
Culture of water flea – Daphnia (Note 1)
Water from Daphnia culture tank at different temperatures – 0 °C (in an ice bath), 10 °C (by adding ice to a water bath), 20 °C, 30 °C and 40 °C (in water baths) (Note 2)
Ethanol, 1% and 10%, 10 cm3 of each (Note 3)
Health & Safety and Technical notes
With Daphnia cultured in the laboratory, fed on yeast, Liquifry No.1, Spirulina or egg-yolk medium, there are no significant hazards associated with this procedure. With pond water culture, or other sources of food, more careful hygiene precautions are necessary.
1 Keeping live cultures of Daphnia: These notes are based on information in the CLEAPSS Laboratory Handbook. You will find more details in section L56. Daphnia are crustaceans, commonly found in ponds and lakes and widely sold as live fish food. These animals are fascinating objects for observation and study in their own right. They feed by filtering minute particles such as bacteria and algae, from the fresh water in which they live.
Daphnia can be kept in any watertight container containing tap water that has been allowed to stand for a few days. Keeping a few Daphnia is not difficult, but cultivating a vigorous, dense colony requires some care. A good supply of oxygen is necessary, either by aeration or by using a large shallow tank to ensure that a large surface area of water is exposed to the air. Warming the water to about 15 °C also ensures rapid growth of the colony.
You can purchase live cultures from suppliers, including pet shops and local aquarists. Some scientific suppliers sell viable dried Daphnia eggs and culture kits. Alternatively, you can collect adult Daphnia by pond dipping; in this case you must observe strict hygiene procedures, since pollutants and the bacteria causing Weil’s disease may contaminate pond water. Stock purchased from aquarists is usually free from this hazard.
The safest, most hygienic and most convenient ways to provide the necessary food for a colony of Daphnia is to feed them on a few drops of a suspension of fresh yeast or of egg-yolk medium (made by blending a hard-boiled egg in 500 cm3 of water). Alternatively, you can buy food such as Liquifry No 1 or Spirulina powder from aquarists or scientific suppliers.
Small, regular supplies of food are required. Provide only sufficient to cause the water to turn faintly cloudy. After a few days the Daphnia will have filtered out the suspended particles of food, making the water clear once more, which is your cue to add more food. Clear scum from the surface of the water; but leave debris that sinks to the bottom – it may contain Daphnia eggs.
2 Instead of heating water in a water bath, you could surround the Daphnia in the Petri dish with a circular heating coil connected to a 6V battery. This will gradually heat the water in the dish, and the cardiac frequency can be estimated at 5 °C or 10 °C intervals. An additional, larger dish outside the small one could also be filled with water at the appropriate temperature to help reduce heat loss from the experimental chamber.
3 Ethanol (IDA) Hazcard 40A is highly flammable and harmful because of the presence of methanol. Once diluted to 10% and 1%, this is low hazard for the students using the liquid.
4 Physiologically-active compounds: (Refer to Hazcard 3C) Each compound will have different hazards and associated risk control measures. Acetylcholine is an irritant (to eyes, respiratory system and skin) and is used at a concentration of 1 g in 1000 cm3 of water. L-adrenaline (epinephrine) is toxic by inhalation, in contact with the skin and if swallowed. Used by students at a concentration of 1 g in 1000 cm3 of water it is low hazard. Caffeine is harmful if swallowed (!). 0.3 g in 1000 cm3 of water is similar to the concentration of caffeine in an ordinary cup of coffee or a cola drink and so is low hazard for the students (see also Hazcard 103). Aspirin (o-acetylsalicylic acid) is harmful if swallowed, but a soluble tablet dissolved according to the manufacturers’ instructions would give a suitable concentration to use in the investigation at low hazard to the students. In each case, add one drop to 5 cm3 of water before applying to the Daphnia.
5 Heating due to the microscope lamp: When working with organisms under a microscope, the effects of heating due to the microscope lamp itself can be significant. Turning the lamp on only when observing the Daphnia will help, and LED microscopes produce less heat than those with incandescent lamps.
Teachers should be careful to introduce these animals in a way that promotes a good ethical attitude towards them and not a simply instrumental one. Although they are simple organisms that may not 'suffer' in the same way as higher animals, they still deserve respect. Animals should be returned promptly to the holding tank after being examined. This supports ethical approaches that are appropriate to field work where pond animals are returned to their habitat after observations have been made.
Take care handling any chemicals that might affect the heart rate of Daphnia.
Observe normal, good laboratory hygiene practices when completing the practical.
a Take a small piece of cotton wool, tease it out and place it in the middle of a small Petri dish.
b Select a large Daphnia and use a pipette to transfer it onto the cotton wool fibres.
c Immediately add pond water to the Petri dish until the animal is just covered by the water.
d Place the Petri dish on the stage of a microscope and observe the animal under low power. The beating heart is located on the dorsal side just above the gut and in front of the brood pouch (see diagram). Make sure that you are counting the heart beats, and not the flapping of the gills or movements of the gut. The heart must be observed with transmitted light if it is to be properly visible.
e Use a stopwatch to time 20 seconds, and count the number of heart beats in several periods of 20 seconds. The heart beat of Daphnia is very rapid, so count the beats by making dots on a piece of paper in the shape of a letter S. Count the dots and express heart rate as number of beats per minute.
f At the end of the investigation, return the Daphnia to the stock culture.
Investigating the effect of temperature
g Record the temperature of the water in the Petri dish.
h Add pond water at a different temperature to the Petri dish. Allow the Daphnia some time to acclimatise, but keep a check on the temperature of the water in the dish and add more hot or cool pond water if necessary to adjust the temperature.
i Record the heart rate again as in step e.
j Plot a graph of mean frequency of heart beats per minute against temperature.
Investigating the effect of chemicals
k Take a large Daphnia from the stock culture and record its heart beat at room temperature in pond water (as in step e).
l Add one drop of 1% ethanol to 5 cm3 of pond water in a beaker. Mix well. Draw the pond water off the Daphnia with a pipette and replace it with 2 or 3 cm3 of the water containing ethanol (Note 3). Record the rate of heart beat again.
m Repeat step l using 10% ethanol in place of 1%.
n Repeat with other chemicals such as acetylcholine, L-adrenaline (epinephrine), caffeine or aspirin (Note 4).
Daphnia is poikilothermic, which means that its body temperature and therefore its metabolic rate are affected directly by the temperature of the environment. The change in metabolic rate is reflected in the rate at which the heart beats (cardiac frequency).
The effect of temperature on a metabolic activity may be expressed in terms of the temperature coefficient (Q10). This is the ratio of the rate of activity at one temperature to its rate at a temperature 10 degrees higher.
Within a range of 10 °C above and below ‘normal’ environmental temperatures, the rate of a metabolic process is expected to double for every 10 °C rise in temperature. Daphnia heart rate has a more complex relation to temperature than a single enzyme-controlled reaction, so Q10 = 2 is not expected. Above 40 °C and 50 °C, the relation between the two rates will not hold because of the deleterious effects of extreme temperature.
There will be considerable variation in the data gathered. Class results for the heart beat at any temperature should be recorded and mean results (and standard deviation) calculated.
Background information: chemicals and the heart
Acetylcholine: In humans and many other animals, heart rate is slowed by the parasympathetic nervous system (neurotransmitter: acetylcholine) via activation of cell surface receptors in the sinoatrial node (pacemaker) called acetylcholine muscarinic receptors. This occurs after feeding, during sleep, and during breath-holding and swimming underwater. A slowed heart rate and the associated fall in the rate of ejection of blood from the heart is sufficient to maintain body function during rest, and conserves energy in the heart under conditions where its supply (and the supply of oxygen in the blood) are diminished. A drug that slows heart rate is called a negative chronotrope; this is demonstrated in this experiment, where acetylcholine is used to slow the rate of the Daphnia's heart.
Noradrenaline and adrenaline: In contrast, heart rate is increased by the sympathetic nervous system (neurotransmitter: noradrenaline) and the hormone adrenaline circulating in the blood via activation of cell surface receptors in the sinoatrial node - pacemaker) (called beta-1 adrenoceptors). This occurs during exercise or fear. The effect is to increase the rate of ejection of blood by the heart. This means that there will be more blood flow to skeletal muscle (in which exercise causes dilatation of blood vessels), so the skeletal muscle cells are supplied with more oxygen and respiratory substrates used to generate energy in respiration where it is needed. A drug that increases heart rate is called a positive chronotrope, and this is demonstrated in this experiment when adrenaline is used to increase heart rate in Daphnia.
One of the ways adrenaline increases heart rate is through the action of what is known as a 'second messenger' or 'transduction component', in this case it is a chemical made in the cell known as cyclic adenosine monophosphate (cAMP). Transduction is the process that follows the action of a drug, hormone or neurotransmitter at a receptor. Thus, when adrenaline activates the beta-1 adrenoceptor in the sinoatrial node, this leads to an increase in cAMP in the sinoatrial node and the result is an increase in heart rate.
Caffeine: Caffeine mimics some of the effects of adrenaline and noradrenaline in the heart. By a different mechanism not involving beta-1 adrenoceptors, caffeine also increases the amount of cAMP in the sinoatrial node. Then cAMP levels increase and this increases the electrical activity of the sinoatrial node, making it depolarize and 'beat' faster. Caffeine has additional effects on the heart. Like adrenaline and noradrenaline, it can affect the main pumping chambers (ventricles), leading to an increase in the rate of contraction and relaxation of each heart beat. This means that, as well as beating faster, the heart's individual beats are associated with an increased volume of blood ejected into the circulation per unit time. This is called increasing cardiac output. Two or three cups of strong coffee or tea contain enough caffeine (and a similar acting compound called theobromine) to cause an increase in human heart rate of 5-20 beats/min.
Ethanol: Ethanol slows heart rate. At the concentrations used in this experiment, ethanol depresses the nervous system by acting as what is known as a non-selective neurodepressant. The amounts of ethanol necessary to achieve this effect in humans would also be sufficient to depress the respiratory centres of the brain, rather like the effect of an overdose of general anaesthetic, resulting in death.
Aspirin: Aspirin has no effect on heart rate. Despite this, aspirin has beneficial effects in the heart. By reducing the ability of platelets to adhere to damaged blood vessel walls, aspirin reduces the chance of coronary artery thrombosis, the event that precipitates a heart attack. People who are take aspirin long-term for medical reasons (because they have cardiovascular disease or diabetes) may have a lower heart rate than controls, simply because they experience less coronary and peripheral thrombosis and thus have a better lubricated cardiovascular system.
Some words of caution
The Daphnia’s heart differs from the human heart in many respects. In terms of heart rate, the Daphnia sinoatrial node is actually a collection of spontaneously active nerves in a body called the cardiac ganglion. This means that it would be risky to extrapolate heart rate findings from Daphnia directly to humans without first validating the model.
Model validation requires examination of a range of positive and negative controls for their effects in the model. To achieve this, the type and extent of the effect in humans at the same drug concentration (the human template) must be known. It is not always possible to obtain such a human template; this is why the outcome of a novel non-human experimental study is of only provisional clinical relevance. Proof of model validity emerges only once human data sets are available.
Health & Safety checked, May 2009
Observing the effects of exercise on the human body
Compare the results of this experiment with the results of the investigation into the effects of exercise on human heart rate.