Chromatophores and Color Changes: Background Color Adaptations
By
Casey Rurode
Department of Biology, University of Delaware, Newark, Delaware. U.S.A.
Biology 316
INTRODUCTION
Physiological color changes differ from morphological in that they are rapid occurrences caused by the movement of existing pigment granules within the chromatophores driven by motor proteins. Morphological changes are long term processes that are the result of decreased or increased pigment amounts and/or changes in the number of pigment cells (Green, 1967). Three types of dermal chromatophores are well researched in poikilotherms melanophores, xanthophores, and iridophores. The three types of chromatophores synthesize and store different pigments: melanophores produce melanin (brown to black pigments), xanthophores produce carotenoid, pteridine (yellow to orange pigments), and iridophores produce guanine and purine in reflecting platelets.
Over the last century, and even prior to that, scientists have been examining the color changes in poikilotherms. After such extensive study it has been determined that the darkening processes of Fundulus heterclitus and Anolis carolinesis are rapid, physiological processes which are the result of stimulation of the dermal chromatophores (Parker, 1944). As of 1944 there was never a darkening period for either animal that persisted for longer than 10 minutes (Parker, 1944). Connolly performed much research on Fundulus and determined that light does not directly affect the color changing process, since temporary blinding did not affect integumentary color changes. Parker reported that the average darkening times of Fundulus were 45 seconds while the average paling time was 60 seconds. Carlton thoroughly examined the histology and integumentary of the Anolis and reported that pigment changes that coincide with the color changes in the skin occur only in the melanophores. The structure of the melanophore is such that there is a cell body with 6 or 7 projections through which pigment granules can travel to disperse through the cell (Carlton, 1903; Green, 1967). Due to the intertwining nature of the projections of the melanophores and their invasion into the layer immediately under the epidermis, when the pigment granules are dispersed the skin of the anole appears brown. When the granules densely fill the body of the cell the skin appears to have a pea-green color (Carlton, 1903). The average darkening process in Anoles was reportedly takes 4 minutes, and that the green color rarely persisted for great lengths of time in anoles exposed to light (Carlton 1903).
The purpose of this experiment was to observe the time course of the darkening and paling processes in Fundulus heteroclitus, making observations about the visible patterns of the processes, to determine the length of time required for each process to occur and ultimately to determine the trends in which the process occurs—linear, exponential, sigmoidal. The purpose of the second part of the experiment was to observe the darkening process in Anolis carolinesis for an hour, noting visible patterns in the process; to determine the length of time required to reach plateau (3 identical readings) in skin color index (SCI), or to observe the progress of the process over a one hour time course; and to determine the trend in which the process occurs. Based on previous research it was hypothesized that in part one the darkening process in the Fundulus heteroclitus would take 45-60 seconds and yield a sigmoidal data trend. It was also speculated that the paling process would take 60-90 seconds and yield a sigmoidal data trend. For part two of the experiment, it was hypothesized that the darkening process of the Anolis carolinesis would take 4-6 minutes.
MATERIALS AND METHODS
Experimental animals
The killifish, Fundulus heterclitus, were purchased from a local bait shop. Prior to testing, the fish were maintained in the lab for two weeks at a temperature of 21oC ± 2 oC, under LD 12:12 (light from 8am-8pm) conditions, and fed TetraMin Tropical fish food one time per day. The fish were kept in an aquarium with an off-white-gray colored background; off-white, black, and tan colored pebbles in the bottom; and a salinity of 15ppt.
The Anolis carolinesis were purchased from the Carolina Biological Company. Prior to testing they were maintained in the lab for two weeks under LD 12:12 (light from 8am-8pm) conditions at a temperature of 21oC ± 2 oC.
Skin color index
The method used to determine the progress in the darkening and paling processes was the skin color index (SCI). This scale has values of 1-5, 5 being the darkest and 1 the lightest. All of the negative fish that remained in the white dishpans and the anole prior to placement on the black background were level 1.
Time course for color change in Fundulus heteroclitus
Eight killifish were randomly chosen from the main tank and transferred using the small fishnet into a white square dishpan that was filled halfway with water from the main tank. Then 6 fish were transferred, using the same fishnet, from the original dishpan to 3 identical white dishpans, putting 2 fish in each ensuring one fish in each dishpan was noticeably larger than the other so the experimental and negative fishes were easily identifiable. The negative fish have a skin color index of 1.The four white dishpans were arranged in a 2x2 square in an attempt to keep the light exposure as close to identical as possible for each dishpan. The fish were left in their dishpans for 20 minutes during which time two aerators were passed between dishpans 1 and 3 and dishpans 2 and 4 every 5 minutes. One black dishpan, identical to the white, was filled halfway with water from the main tank and placed centered between dishpans 2 and 4 located 3 inches away from white dishpans. After 20 minutes one aerator was moved to the black dishpan and the second aerator was moved to the dishpan from which the experimental fish was taken. The experimental fish was then transferred from white dishpan 1 into the black dishpan using the small fishnet at time 0 and observed until 9 consecutive identical skin color index values were recorded. Readings were taken every 10 seconds. The fish was left the black dishpan for a total of 5 minutes. Then it was retransferred back to dishpan 1 using the small fishnet so the paling process could be observed. During the paling process, readings were taken every 10 seconds until the fish reached a skin color index of 1. The aerator from dishpan 1 was then transferred to dishpan 2 and the experimental fish from dishpan 2 was transferred to the black dishpan using the small fishnet at time 0 where the darkening and paling processes were observed and recorded just as they were for the fish1. To observe the paling process the experimental fish was returned to its respective dishpan just like fish 1. This procedure was repeated for all four fish over 3 trials. Figure 1 shows the setup of the white dishpans and indicates which size fish was the experimental specimen for each dishpan.
Dishpan 1Smaller fish was tested / Dishpan 2
Larger fish was tested
Dishpan 3
Larger fish was tested / Dishpan 4
Smaller fish was tested
Figure 1. Experimental setup of the four white dishpans indicating which specimen was tested for darkening and paling time.
Time course for color change in Anolis carolinesis
One Anole was randomly chosen from the main terrarium and placed in a small clear plastic container housed in a white dishpan for 20 minutes in order to establish the most paled color (SCI 1). The clear tank was then transferred to a black background at time 0. For 1 hour the dorsal skin color of the anole was observed every 5 minutes and assigned a skin color index (1-5, 5 being the darkest). The animals were not disturbed during the observation period but the top of the container was covered at 30 minutes to prevent direct white light from reaching the anole.
RESULTS
The SCI values of the darkening process in Fundulus heteroclitus were recorded in Figure 2. None of the trials showed linear trends however more testing must to be done to determine whether the curves show a sigmoid or exponential trend, since it cannot definitively determined based on our data. All three average trials also reached a plateau at SCI values of 5 and never decreased. Trial 1 had the largest darkening duration and trial 2 had the smallest duration. Until a time of 80 seconds the averages of all three trials displayed similar darkening patterns and SCI values—starting at SCI values of 1, increasing by 1 SCI approximately every 20-30 seconds, and reaching an SCI of 4.25 at 80 seconds. After 80 seconds trial 1’s average SCI remained unchanged for 20 seconds, then increased 0.25 SCI values and remained unchanged for 40 seconds, then increased another 0.25 SCI values remaining unchanged for 20 seconds before finally completing the darkening process (SCI 5) at 180 seconds. Trial 2’s SCI values changed rapidly after 80 seconds reaching and SCI of 4.75 at 90 seconds and finally reaching a level 5 at 100 seconds. Trial 3’s darkening process from 80 seconds to completion was between the other trials remaining unchanged for 20 seconds at a SCI of 4.25 for 20 seconds then at 4.75 for 10 seconds before completely darkening at 120 seconds.
Figure 2. Time course of integumentary darkening in Fundulus heteroclitus. Four killifish were observed one at a time until 9 consecutive identical skin color index values were recorded, after being transferred from a white to black dishpan, to determine the duration of the darkening process. Each trial was averaged as shown above.
The skin color index values of the paling process in Fundulus heteroclitus were recorded in Figure 3. None of the trials showed linear trends however more testing must to be done to determine whether the curves show a sigmoid or exponential trend, since it cannot definitively determined based on our data. All three average trials also reached a plateau at SCI values of 1 and never increased. Trial 1 had the largest paling duration and trial 3 the smallest paling duration. All three trials showed the same general paling process and average SCI values for the first 60 seconds changing from SCI values of 5 to 4 in 10 seconds, 4 to 3 in 20 seconds, and 3 to 2 in 25 seconds. After a total of 55 seconds, however the trials became more distinct. Trial 1 required nearly 2 minutes to decrease from a SCI of 2 to 1, dropping to a SCI of 1.75 and remaining unchanged for 30 seconds, then to a 1.50 for 10, and finally to a 1.25 remaining unchanged for 110 seconds before finally reaching a SCI of 1 at 200 seconds. Trial 2’s paling process from 60 seconds to completion was between trials 1 and 3 changing to 1.75, then to 1.5 and remaining unchanged for 30 seconds, then to 1.25 for 50 seconds before completely paling at 160 seconds. Trial 3’s average SCI values at some point were 1.75, 1.50, and 1.25 before reaching a SCI of 1 at 130 seconds but never persisted for longer than 20 seconds.
Figure 3. Time course of integumentary paling in Fundulus heteroclitus. Four killifish were observed one at a time once every 10 seconds until they reached a skin color index of 1, after being transferred from a black to white dishpan, to determine the duration of the paling process. Each trial was averaged as shown above.
A few other numerical trends in data not reported in graphs and tables. During the darkening process, the 2 relatively larger fish, animals 2 and 3, took 123 and 130 seconds to reach SCI values of 5 respectively. The smaller fish, animals 1 and 4, took 83 and 87 seconds to darken respectively. Overall, the darkening time from trial 1 to trial 3 decreased by about 30-60 seconds in ¾ animals; the fourth animal showed a 20 second increase in darkening. During the paling process no general trends based on size were observed. Also the paling took anywhere from 67-176 seconds to complete. From trial 1 to trial 3 the required paling time for ¾ animals increased by 10-40 seconds; the fourth animal showed an overall 70 second decrease in paling time. The average total time required for each process was nearly identical, darkening taking 106 seconds and paling 105 seconds.
Figure 4 shows the SCI values for the darkening process in Anolis carolinesis. The results for all four specimens showed significantly different darkening patterns over the observed time and had no visible trend. All four anoles started at SCI values of 1, remained relatively unchanged for the first 5 minutes (after which time each animal showed a distinct darkening pattern), and showed a general increase in SCI over the time course. Also, ¾ animals also reached a plateau for 3 consecutive recordings at the end of the time course. The largest increase in SCI was shown by anole 2 which achieved a SCI of 3.5. The animal that changed the least in SCI was anole 1 which reached a level 1.5. Anoles 3 and 4 fell in between reaching SCI values of 3 and 1.85 respectively. The decrease in SCI at 25 minutes for anole 1 was unexpected and cannot be explained. After 30 minutes a cover was placed over the lid of Anole 1’s set up to prevent direct white light from shining into the clear tank.
Figure 4. Time course of integumentary darkening in the Anolis carolinesis. Four anoles were individually observed, in a clear container against a black background, once every 5 minutes for 1 hour to determine the duration of the darkening process.