Color Control in Shrimp
Halifax, Nova Scotia B3H 4J1
Mary-Jane O'Halloran is a Senior Instructor in the Biology Department, Dalhousie University. She received her B.Sc. (1963) from Southampton University, England, and B.Ed. (1978) and M.Sc. (1986) from Dalhousie University. Teaching interests include animal physiology and biochemistry.
© 1990 Dalhousie University
[ABLE's Copyright Policy]
|Reprinted from: O'Halloran, M.J. 1990.
Color control in shrimp. Pages 15-26, in Tested studies for laboratory
teaching. Volume 11. (C. A. Goldman, Editor). Proceedings of the 11th
Workshop/Conference of the Association for Biology Laboratory Education
(ABLE), 195 pages.
Although the laboratory exercises in ABLE proceedings volumes have been tested and due consideration has been given to safety, individuals performing these exercises must assume all responsibilities for risk. The Association for Biology Laboratory Education (ABLE) disclaims any liability with regards to safety in connection with the use of the exercises in its proceedings volumes.
The six experiments on crustacean color change presented here are relatively simple and inexpensive and are suitable for use in introductory biology or more advanced animal physiology or behavior classes. They introduce students to an interesting physiological phenomenon that can be measured quantitatively by assessing the degree of pigment dispersion in chromatophores. The large number of experiments described here would not all fit into a standard 3 hour laboratory period, but instructors can select experiments that are appropriate to their students needs and the available time.
The marine sand shrimp, Crangon septemspinosus, are used in these experiments as they are fairly abundant in the inter-tidal waters of Atlantic Canada. However, many other marine crustacean species could be used instead, such as isopods, shrimps, and crabs. Addresses of possible suppliers are given in Appendix A. Lower vertebrates such as small fish (perch, minnows, mummichogs (Fundulus) or tadpoles also exhibit color change and have easily visible chromatophores. The number of animals used in each experimental group can easily be altered depending on the availability of animals, time constraints, etc. You may wish to increase the number of shrimp per group if your students are going to statistically analyze the data.
These experiments lend themselves well to independent research projects as well as more structured formal laboratories. I give my third-year comparative animal physiology students a list of references and ask them to design experiments to investigate the answer to the hypotheses described in Parts A, B, and C, leaving them about 5 weeks to do the reading, experimental work, and submit a report. There are many variables that should be controlled or taken into consideration during the experimental design: including temperature, salinity, background color, light intensity and wavelength, circadian and tidal rhythms, sex, size, molt stage, and reciprocal effect of other animals. The following experiments will try to control for as many of these variables as possible.
In Part A, students examine the effect of several environmental factors (background color, light intensity, light wave-length, and temperature) on pigment dispersion. In Part B, students locate the site of the receptors that bring about color change to determine whether the control is primary (direct action on chromatophores) or secondary (involves other receptors). Finally, in Part C they determine the type of communication (hormonal or nervous) between the receptors and the effectors (the chromatophores). Ideas for additional experiments are also provided.
All materials given are per experimental group.
Experiment 1: Background Color
16 shrimp; 5 1-liter glass beakers; sand-colored, yellow, red, and white paper to cover sides and bottom of beakers; dissecting microscope; thermometer; petri dish; scissors, tape; sea-water.
Experiment 2: Light Intensity
16 shrimp pre-adapted to sand-colored background; 4 1-liter glass beakers; sand-colored paper to cover sides and bottom of 4 beakers; light meter (photographic type); dissecting microscope; thermometer; petri dish; scissors, tape; sea-water.
Experiment 3: Light Wavelength
16 shrimp pre-adapted to sand-colored background; 4 1-liter glass beakers; white paper; 4 dissecting microscopes with colored filters (transparent plastic, available from art suppliers, taped over the light source); dissecting microscope; 4 large boxes to cover dissecting microscopes; thermometer; 4 petri dishes; sea-water.
Experiment 4: Effect of Temperature
12 shrimp pre-adapted to sand-colored background; 3 1-liter glass beakers; clear plastic bags to surround 1-liter glass beakers and elastic bands; sand-colored paper; 3 water baths set for 5, 15, and 25C (ice can be used for 5 and 15C if cooling baths are unavailable; four groups can share 1 water bath); dissecting microscope; scissors, tape; 3 thermometers; petri dish; sea-water.
Experiment 5: Painting Eyes to Block Light Entry
16 shrimp pre-adapted to a sand-colored background; 3 1-liter beakers; white and black paper; white typewriter correction fluid (Liquid Paper); dissecting microscope; thermometer; scissors, tape; petri dishes; sea-water.
Experiment 6: Injection of Crude Extracts of Color Control Hormones
8 intact shrimp (pre-adapted to sand-colored background); 8 shrimp with eyes covered with Liquid Paper; 2 1-liter beakers; sand-colored paper; 8 1-ml disposable syringes (25-gauge needle); crustacean saline (see Appendix B); crude eyestalk extract from shrimp (see Appendix B); dissecting microscope; thermometer; scissors, tape; petri dish.
Crustaceans are able to change their color or shading in response to numerous environmental changes, and can exhibit a great variety of pigment colors (for reviews see Fingerman, 1970; Florey, 1966; Hoar, 1983; Prosser, 1973). This ability to match body color with their background environment provides a useful mechanism for avoiding predators. We will be studying color change in the marine sand shrimp, Crangon septemspinosus, as it is fairly abundant in the inter-tidal waters of Atlantic Canada and its color change mechanisms have been well studied. However, many other marine crustacean species could be used instead.
The ability to change body color to match environmental changes (physiological color change) is brought about by pigment movements within the chromatophores in crustaceans. There are four primary pigment colors (yellow, red, silver, and black) observable in the chromatophores of Crangon septemspinosus, but the predominant and easiest to stage are the black-brown ommochromes. We will use a 5-stage chromatophore index to quantify the degree of brown-black pigment dispersion in the ommochromes which produces the changes in color and shading (Figure 2.1).
Figure 2.1. (A) Diagram of a chromatophore from the uropod of the prawn, Leander serratus, with the pigment fully dispersed. (B) The 5-stage chromatophore index showing the stages of pigment dispersion, from Stage 1 (fully concentrated) to Stage 5 (fully dispersed) (Highnam and Hill, 1977). Reprinted with permission.
In the following experiments we will examine the effect of several environmental factors (background color, light intensity, light wave length, and temperature) on pigment dispersion or color change (Part A). In Part B we will perform an experiment to locate the site of the receptors that bring about color change to determine whether the control is primary (direct action on chromatophores) or secondary (involves visual receptors). Finally, in Part C we will perform an experiment to determine the type of communication (hormonal or nervous) between the receptors and the effectors (the chromatophores).
There are many variables that should be controlled in these experiments: including temperature and salinity of sea-water, background color, light intensity and wavelength, circadian and tidal rhythms, sex, size, molt stage, and reciprocal effect of other animals. We will try to control for as many of these variables as possible during the following experiments. By doing the experiments at the same time of day circadian and tidal rhythms should be negated. Any changes in temperature should be noted during the experiments, but they will probably be minimal. It is best to use animals of approximately the same size as larger animals have more dispersed chromatophores. Determining sex and molt stage is too time consuming, but avoid using egg-bearing females and keep records of shrimp that molt during the experiments.
Many environmental factors are known to affect pigment dispersion in the chromatophores of shrimp. Background color, light intensity, light wave-length, and temperature will be studied in these experiments. Alterations in pigment dispersion with changes in background color or shading are known as albedo responses. They depend on the ratio of incident to reflected light, so that the dark pigments disperse on a dark background as it reflects less light, while the reverse happens on white backgrounds. Decreases in light intensity will decrease the amount of incident and reflected light and will probably cause dispersion of the dark pigments as the reduced light will make the background appear darker. Light of different wavelengths should have no effect on the albedo response, but red and yellow light causes red and yellow pigments to appear in the center of each chromatophore. At higher temperatures the dark pigments usually concentrate in the center of the chromatophore so the shrimp appear lighter and will reflect more light from their body surface and therefore absorb less heat.
Experiment 1: Background Color
Table 2.1. Calculation of total chromatophore number. The total chromatophore number for 20 chromatophores is 35.
Experiment 2: Light Intensity
NOTE: If you have access to a darkened room, this experiment can be performed using a bench lamp as a light source and placing glass tanks containing shrimp at different distances from the light source to give variations in light intensity. If larger containers are used the water will not warm up as much. A tank containing water only can be placed immediately in front of the lamp to absorb radiated heat.
Experiment 3: Light Wavelength
NOTE: If you have access to a dark room this experiment can be set-up easily with all four dissecting microscope lights set up around the room, as there will be little interference from these low intensity bulbs. The sea-water temperature will not increase as much as when the lights are enclosed in a box.
Experiment 4: Effect Of Temperature
Additional Experiments on Environmental Factors and Color Change
The albedo response can also be studied easily by comparing the degree of pigment dispersion of shrimp that have been kept in containers whose interiors have been painted with (a) shiny, gloss, reflective paint of different colors or (b) matte, flat, non-gloss paint, as this will vary the amount of light that is reflected off the background onto the lower surface of the eye.
Circadian rhythms can be examined by keeping groups of shrimp for 5 days under different light cycles and staging the chromatophores at regular 6 hour intervals. Studies have found that the chromatophores have a diurnal rhythm of darkening at night and lightening in the day (Fingerman and Lowe, 1970). Different groups of shrimp could be kept in total darkness, total light, reversed photoperiod (dark in the day, light at night), or other abnormal light/day cycles and compared with shrimp kept under normal photoperiods. Since Crangon septemspinosus survive longer at temperatures below 15C, glass tanks should be set up in a temperature controlled room if possible. Lamps set on timers that automatically turn on and off are convenient. All of the experimental groups can be set up in one temperature controlled room if they are partitioned from each other using clamp stands and black plastic sheets.
The eyes are known to be the receptors responsible for color change in shrimp, but various experiments can be done to prove this and to check whether other areas of the body are also involved. Chromatophores that respond directly to changes in illumination are classified as primary responses. Chromatophore responses that involve visual receptors and pathways are classified as secondary responses. The following experiment involves painting the eyes to block light entry and then placing the shrimp on different color backgrounds to see if the ability to change color ceases.
Experiment 5: Painting Eyes To Block Light Entry
Additional Experiments on Receptor Location
Various parts of the body can be painted or shielded from light and the shrimp placed on different backgrounds to observe whether the chromatophores in the covered region have the same amount of pigment dispersion as the ones on the uncovered regions. Any differences would indicate that light reacts directly on the chromatophores (primary receptors) rather than through the eyes (secondary receptors). To shield parts of the body from light the shrimp can be placed in shrimp-sized black tubing, or placed in narrow glass vials that have one half covered with black tape. The shrimp can be easily removed from the tubing or vials to enable counting of the chromatophores from the covered regions. The chromatophores in the tail region should be studied independently since they are thought to be controlled by different hormones than the body region.
Color change in shrimp is brought about by hormones (Carlisle and Knowles, 1959; Fernlund, 1970; Fernlund and Josefsson, 1972; Fingerman, 1985) and there are thought to be one set of hormones responsible for body lightening and darkening, and another set for the tail region. Color change in other species (cephalopods) is nervous and brought about by the contraction of muscles surrounding an elastic sac. The sinus gland at the base of the eyestalk is known to be the immediate source of chromatophore hormones (chromatophorotropins) in crustaceans. The hormones are thought to arise from the neurons of the x-organ, brain, or ventral ganglia and are then stored in the sinus gland at the base of the eyestalk (Figure 2.2). In the following experiment crude hormone extracts (prepared by grinding eyestalks in crustacean saline; see Appendix B) will be injected into shrimp to see if any changes in pigment dispersion occur in the chromatophores. More sophisticated solvent extractions can be performed to further purify these peptide hormones (Fernlund and Josefsson, 1972; Fingerman, 1985). Commercially prepared hormones are not available.
Experiment 6: Injection of Crude Extracts of Color Control Hormones
Figure 2.2. Diagram of the crustacean sinus gland neurosecretory system at the base of the eyestalk (from Waterman, 1961). Reprinted with permission.
Additional Experiments to Determine Type of Control
Classical experiments first reported by Koller (1929) can be performed but often result in mortalities as it is difficult to extract blood from small shrimp. Koller extracted blood from shrimp adapted to black backgrounds and injected it into shrimp on white backgrounds and darkening occurred. The ventral nerve cord can be cut to see if this prevents color change when shrimp are placed on a different background. However, a major blood vessel lies next to the ventral nerve cord making the operation very difficult. Different concentrations of possible crustacean neurotransmitters (5-hydroxy-tryptamine, adrenaline, acetylcholine) can be injected into shrimp to see if they play a part in relaying light changes received by the receptors in the eyes to the sinus gland x-organ complex at the base of the eyestalk.
Carlisle, D.B., and F. Knowles. 1959. Colour change. (Chapter 3). Pages 40-69, in Endocrine control in crustaceans. Cambridge University Press, England, 119 pages.
Fernlund, P. 1970. Chromactivating hormones of Pandalus borealis; isolation and purification of a light-adapting hormone. Biochimica et. Biophysica Acta, 237:519-529.
Fernlund, P., and L. Josefsson. 1972. Crustacean color change hormone: amino acid sequence and chemical synthesis. Science, 177:173-175.
Fingerman, M. 1970. Comparative physiology: chromatophores. Annual Review of Physiology, 32:345-372.
------. 1985. The physiology and pharmacology of crustacean chromatophores. American Zoologist, 25:233-252.
Fingerman, M., and M. Lowe. 1970. Influence of time upon the chromatophore systems of two crustaceans. Physiological Zoology, 30:216-231.
Florey, E. 1966. Color change and the chromatophores. (Chapter 15). Pages 345-374, in An introduction to general and comparative animal physiology. W. B. Saunders, Philadelphia, 713 pages.
Highnam, K.C., and L. Hill. 1977. Endocrine mechanisms in Crustacea-III. (Chapter 11). Pages 237-254, in The comparative endocrinology of the invertebrates (Second Edition). University Park Press, Baltimore, Maryland, 357 pages.
Hoar, W.S. 1983. Electric discharges, light production, and color change. (Chapter 10). Pages 365-406, in General and comparative physiology (Third Edition). Prentice Hall, Englewood Cliffs, New Jersey, 851 pages.
Koller, G. 1929. Die inner sekretion bei wirbellosen tieren. Biological Reviews and Biological Proceedings of the Cambridge Philosophical Society, 4:269-306.
Prosser, C.L. 1973. Chromatophores and color change. (Chapter 23). Pages 915-950, in Comparative animal physiology (Third Edition). W. B. Saunders, Philadelphia, 966 pages.
Waterman, T. H. 1961. Neurohumors and neurosecretion. (Chapter 8). Pages 281-311, in The physiology of Crustacea (Volume 2). Academic Press, New York, 681 pages.
Atlantic Biological Company
St. Andrews, New Brunswick E0G 2X0
(Crangon septemspinosus listed in catalogue for $8.05 CDN per 12 shrimp)
Gulf Specimen Company
P.O. Box 237
Woods Hole Marine Biological Laboratory
Woods Hole, Massachusetts 02543
Marine Crustacean Saline
Also add buffer (17.6 ml of 0.5 M boric acid and 0.956 ml of 0.5 M NaOH) per liter of solution.
Crude Hormone Extract
Quantity required per experimental group = 0.5 ml (injecting eight shrimp with 0.05 ml each).
Anaesthetize eight (8) shrimp in a solution of MS-222 (tricaine methanesulphonate; approximately 0.1 g/liter) until movement ceases. Place the shrimp on a petri dish under and dissecting microscope and remove the eyes as close to the base as possible with fine forceps or a scalpel. Place the eyes plus eyestalk into a small petri dish, add 0.5 ml of crustacean saline, and grind for a few minutes until a smooth homogenous solution is obtained.
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