Biology investigation: temperature and Daphnia heart rate

What the standard rewards

• This is an internal standard: you plan and carry out your own investigation with your teacher's guidance, then hand in a report. You may change your method after a trial run, and you should say why you did.
• The report needs a hypothesis, a method, raw and processed data, a graph, a conclusion that answers the purpose, and findings from at least one other source.
• The step up at each grade is in how well you justify your choices and link the result to biology.

Purpose and hypothesis

• Daphnia (water fleas) are small freshwater crustaceans and ectotherms: their body temperature follows the water around them, so warmer water warms every cell.
• The heart's beat is driven by pacemaker cells whose ion pumps and channels, and the enzymes making ATP for the muscle, all work faster when molecules have more kinetic energy.
• A purpose written as a hypothesis names both variables, predicts the direction and gives the reason: as water temperature increases from 10 °C to 30 °C, the heart rate of Daphnia will increase, because metabolic reactions speed up with temperature.
• A null hypothesis is useful too: temperature has no effect on heart rate. Your data then either support rejecting it or do not.

Variables and a valid range

• The independent variable is what you change, the dependent variable is what you measure, and controlled variables are kept the same so they cannot explain the result.
• Pick a range that is wide enough to show a trend but safe for the organism: Daphnia live in ponds from about 5 °C to the mid 20s, so 10, 15, 20, 25 and 30 °C covers the realistic range with five evenly spaced values.
• A trial run tells you what is practical; one class found counting by eye was impossible above 250 beats per minute, so they filmed in slow motion on a phone through the eyepiece and counted from playback.

Try it: repeats and outliers

Controls, sample size and repeats

• Use a different Daphnia for each repeat, five at each temperature: counting one animal five times only shows how well you count, not how Daphnia vary.
• Assign animals to temperatures at random, by drawing them from the culture with a pipette without picking the biggest or most active.
• The microscope lamp heats the slide, so a slide reading taken after the lamp has been on for minutes is a hidden variable; switch the lamp off between counts or use an LED.
• A control is the 20 °C group, close to the culture's own temperature, which gives the normal rate the others are compared with.

Ethics of working with live animals

• The Animal Welfare Act 1999 covers vertebrates and some invertebrates such as crabs, crayfish and octopus; Daphnia are not covered, but the same principles of care apply.
• Use the fewest animals that give reliable data, keep each one out of its culture water for the shortest time, and do not go above 30 °C, where Daphnia show heat stress.
• Return each animal to a recovery container at room temperature, and never test drugs such as caffeine or alcohol on them without your teacher's approval.
• Say in your report what you did to look after the animals; it is part of a valid method.

Try it: the class's mean heart rates

Statistics: what error bars tell you

• Standard deviation measures the spread of the repeats; a large one means the individuals vary a lot or the measurement was hard to make.
• If the error bars of two groups overlap a lot, the difference between their means could easily be chance; if they do not overlap, the difference is probably real.
• A t-test makes that judgement with a number: for 25 °C against 30 °C, t is about 3.3, larger than the critical value of about 2.4 for this sample size, so the difference is unlikely to be chance.
• Five Daphnia per group is the bare minimum; ten would narrow the spread of the mean and make a small difference easier to detect.

Validity and reliability

• Validity asks whether you measured what you meant to: did the Daphnia's body actually reach the water temperature, and did the lamp warm the slide during counting?
• Reliability asks whether you would get the same result again: small standard deviations and agreement with other groups or published data show reliable data.
• Sources of error are specific: miscounting fast beats, the slide cooling or warming while you count, and animals of different ages or sizes.
• For Excellence, say how each choice in your method reduced one of these, and how much that error could have changed your conclusion.

The biology behind the trend

• Between 10 and 25 °C, extra kinetic energy means more successful collisions between enzymes and substrates, so respiration supplies ATP faster and the pacemaker cells reach their firing threshold sooner.
• Above about 25 °C the rise slows: some enzymes are near their optimum and the heart muscle cannot shorten and relax much faster, so the Q10 drops.
• The wider spread at 30 °C fits heat stress: some individuals cope better than others, which is why the investigation stopped there.
• Published work on Daphnia and other ectotherms shows the same shape, which lets you compare your Q10 with theirs and discuss why yours differs.