COLOUR CHANGE IN XENOPUS TADPOLES

Introduction

Rapid colour changes in animals, for camouflage or courtship, involve cells in the skin called chromatophores which contain pigments. Colour change occurs because the pigments can be expanded to fill the cell or contracted so they are less visible - the chromatophore itself does not change shape. Colour change is most highly developed in cephalopods such as octopus and cuttlefish, which can undergo rapid colour change in under a second. Pigment changes are neurally controlled in these animals, by nerves running to the chromatophores. Similar neurally-controlled colour change is found in some teleost fish, such as cichlids, many reef-fish, and flatfish. Many more lower vertebrates (agnathans, elasmobranches, teleosts, amphibians, and reptiles) show a slower colour change, mediated by hormones, which allows some camouflage. Here the pigmented cells usually contain only the dark brown pigment melanin, contained in melanophores, and colour change is limited to lightening or darkening of the general body tone to suit the background. The animal detects the colour of the background through its eyes and this information is passed to the anterior pituitary gland which can secrete a hormone called melanocyte stimulating hormone (MSH). This causes dispersal of the melanin in the melanophores and thus a darkening of the body colour.

Xenopus (clawed toad) is a permanently aquatic anuran amphibian from Africa. The tadpoles look and behave very differently from the familiar tadpoles of European frogs and toads. They hover in the water and feed by filtering small particles. The purpose of this practical is to observe colour changes, quantify the results in a simple way, and get an idea of the speed with which hormonally-controlled events can occur. The tadpoles have been kept on either dark or light backgrounds. You will transfer a small group of those from a black background to a white one, and vice versa, and observe the colour change. This is done by removing each animal at intervals of 20 minutes after changing the background, to a dish under a microscope, and observing the malanophores. The state of the melanophores can be assigned a number from 1 to 5, called the melanophore index, by comparison with a set of photographs provided. You can then plot the results on a graph and analyse them.

• Work in pairs for this practical
• Remember that you are dealing with living animals. They are not as delicate as they appear, but treat them with great care.
• If you need extra water, use the aerated warmed water provided in the laboratory. Do not put the tadpoles in tapwater.
• The colour changes only occur if the animals can see the background colour clearly. Keep a bright light shining on them, but do not overheat them.

Procedure

1. Look at killed tadpoles from a white and a black background under the microscope, to become familiar with the appearance of the melanophores. You will then be able to make observations very rapidly in later experiments with living tadpoles.
2. Note that the melanophores occur in patches, which may have different states of expansion. Always make your assessments on the same area of the body, which you choose at this stage and keep to later. To get the best results choose an area where there is the largest difference in melanophores between the dead tadpoles from dark and light backgrounds.

3. Discard the dead tadpoles and assess the state of the melanophores in six living ones from a white background. Then transfer them immediately to a black container. Reassess the state of the melanophores at intervals of 20 minutes, looking at each individual under the microscope and returning it to the black container.

4. Conduct exactly the same experiment with animals transferred from a black to a white background, beginning 10 minutes after you started the first group so that all the observations can be fitted in. Return these tadpoles to the white container after each observation.

5. Plot your results on graph paper as you proceed, and do a statistical test on the final results. Continue until there is no further change (after 80-120 minutes).

6. Obtain the tails from two anaesthetised and freshly killed tadpoles. Place each in a watch glass with 1 ml of Xenopus saline and assess the melanophore index as for the living tadpoles. Place 0.1 ml of the solution of melatonin in one watch glass, and 0.1ml of the solution of melanocyte stimulating hormone (MSH) in the other. Observe changes in the melanophores at intervals of 5 minutes for 30 minutes. Do not keep the watch glasses under bright light during this period.

Statistical analysis

The melanophore index is a score rather than a measurement, and so unsuitable for the standard type of statistics that you may already be familiar with. We will use a simple nonparametric statistic, the Mann-Whitney test. This is equivalent to a t test comparing two sets of data, to ask the question: what is the probability that the two sets are measuring the same thing, any difference being just due to chance? You can do four tests, comparing:

• The initial and final scores of tadpoles moved from light to dark.
• The initial and final scores of tadpoles moved from dark to light.
• The initial scores of the two groups.
• The final scores of the two groups.

Calculation

For example to compare the data sets 2, 2, 2.5, 3, 3, 4 and 3.5, 4, 4.5, 4.5, 4.5, 5

1. Combine the melanophore index scores of the two data sets and write them down in numerical order, underlining ties:

   2 2 2.5 3 3 3.5 4 4 4.5 4.5 4.5 5

2. Replace each score with the rank, i.e. from 1 to 12:

   1 2 3 4 5 6 7 8 9 10 11 12

3. Ties are given average ranks. The numbers then become:

   1.5 1.5 3 4.5 4.5 6 7.5 7.5 10 10 10 12

4. Add up the ranks of the two data sets to give the sum of each set, R:

   R₁ = 1.5+ 1.5 + 3 + 4.5 + 4.5 + 7.5 = 22.5 and R₂ = 6 +7.5 +10 +10 +10 +12 = 55.5.

5. Calculate U₁ = 57 - R₁ (to compare two groups of six). In this case U₁ = 57 - 22.5 = 34.5.
6. Calculate U₂ = 36 - U₁ (where both groups are of six). Whichever of U₁ or U₂ is the smaller is taken as the test statistic U. In this case U₂ = 36 - 34.5 = 1.5, so U = 1.5.
7. If U is lower than 7 (where both groups are of six), then the result is significant at P = 0.05. This means that there is a small probability (<0.05 or 1 in 20) that the two data sets are really the same - in other words, the melanophore index scores of the two groups are significantly different.
8. In the example U = 1.5. We can conclude that the two sets of melanophore index scores are significantly different. We would predict this result, because there was little overlap between them (e.g. point 1 above).

Writing your report

Present your graph of the changes of melanophore index in the living tadpoles over time, using different symbols for the two groups. Show the calculation of the four statistical tests. What do these tell you about the colour changes of the two groups of tadpoles? Give a descriptive account of the experiment with tadpole tails exposed to melatonin and MSH, and the conclusions you reach about hormonal control in living tadpoles. Apart from larger sample sizes, how would you improve the experiment with tadpole tails - for example what controls could be used?