Chromatophore Behavior in 1. resecata. p. Introduction The alteration of body color in an isopod through the concentration and dispersal of pigments in chromatophores was first seen in Idotea tricuspidata by Matzdorff (1883). Menke (1914) later found this species to exhibit a diurnal rhythm of color change, the chro¬ matophore pigments being dispersed during the day and punctate at night. Oguro (1959) demonstrated a persistent diurnal rhythm in chromatophores in the high intertidal species Idotea japonica held in constant darkness. The present study was undertaken to determine the behavior of chromatophores of a subtidal isopod, Idotea resecata, under different conditions of illumination and background coloration. Idotea resecata occurs in two color varieties, green and brown (lee and Gilchrist 1972). Only the brown form, which occurs exclusively on the brown alga Macrocystis, was used. Animals were collected from the kelp beds off Mussel Point in Monterey Bay, California, and maintained until used on the kelp Macrocystis in large outdoor tanks at the Hopkins Marine Station. The brown chromatophores of the dorsal side of the pleotelson are easily seen with the aid of a dissecting microscope, and were therefore chosen for study. Degree of dispersal of pigment in the chromatophores was rated using the 5 point scale of Hogben and Slome (1931), where stage I indicates maximum concentration of pigment, stage 5 indicates maximum dispersion, and stages 2, 3, and 4 represent intermediate conditions (fig. 1b). Chromatophores of the pleotelson were generally all in the same stage of dispersion and rating therefore represents all the chromatophores of the pleotelson. Values of 1.5. 2.5. 3.5. and 4.5 were assigned in cases where chromatophores were at stages Chromatophore Behavior in 1. resecata. p. 3. intermediate between those defined by Hogben and Slome (1931). In cases where chromatophores in different stages of dispersion were present at the same time, a subjective judgement was made as to the relative abundance of each stage in the posterior region of the pleotelson, and intermediate values were again used. Increments smaller than 0.5 were not used. Ratings for a sample of 15 animals could be completed in less than 5 minutes. The black chromatophores which were often present around the edge of the pleotelson and occasionally found around spots on the pleotelson which contained no chromatophores, were not rated. White chromatophores which were occasionally found on the pleotelson were also not rated. All light intensities were measured with a Photovolt 200M light meter. For intensities over 2 ft-c., a 25% neutral density filter was placed over the meter. Both male and female isopods, ranging in length from 15mm to 3Omm, were used in the experiments. Behavior of chromatophores in a natural light regime. Initial experiments were made to determine the behavior of chromatophores under normal day and night light condition. Lots of twelve animals each were placed in; (1) a black enamaled pan, (2) a white enamaled pan, and (3) a pan with a gray background. The pans were exposed to daylight but not direct sunlight. Animals were not fed during the experiment, and sea water in the pans was changed every 4 hours. The temperature of the water remained constant at 14 C. Chromatophores were observed at intervals of 4 to 6 hours over a 3 day period. Figure I shows the results: under a normal day/night light regime, chromatophore pigments were Chromatophore Behaviori in. resecata. p. 4. dispersed during the day and concentrated at night. During the day pigments were significantly more dispersed in animals on a black background than in those on a white background; animals on the gray substratum were intermediate in pigment dispersion. Behavior of chromatophores under constant condition of illumination The following experiments were carried out to determine whether expansion and contraction of chromatophore pigments persists under conditions of constant light or darkness. Three pans, each containing 12 animals, were kept in the dark for a total of 72 hours. During this time, chromatophores of all animals were examined with the aid of a dissecting scope and low intensity light every 4 hours. Temperature ranged from 14°C to 17°C during the experiment. The results (fig. 2c) show that pigments were punctate at night and dispersed during the day. These results are in good agreement with the observations of Oguro (1959) on Idotea japonica and Kleinholz (1937) on Ligia baudiniana. A second group of isopods was kept under conditions of constant low intensity illumination provided by a 15 watt incandescent light bulb. Fifteen animals were placed in a shallow black pan and another 15 in a white pan. Light intensity was held at 0.04 ft-c. except when the chromatophores were being observed. Animals were checked every 4 to 8 hours over a 60 hour period. The results (fig. 2a) show that on both backgrounds the chromatophore rhythm persisted. This experiment was repeated once so that a total of 30 animals were looked at on each colored substrate. A third group of animals was kept under conditions of constant Chromatophore Behavior in 1. resecata. p. 5. strong light. Twelve isopods were placed in a black pan and another 12 in a white pan, and both pans illuminated at 40 ft-c. by a 60 watt incandescent light bulb. Chromatophores were observed every 4 hours. The results (fig. 2a) show no diurnal rhythm in movement of chromatophore pigments; chromatophores remain expanded, those on the black background exhibiting greater pigment dispersal than those on the white background. Evidence of an endogenous rhythm in chromatophore pigment dispersal The preceding experiment shows that the rhythmic movement of pigments in chromatophores persists in the absence of a light stimulus, suggesting an endogenous rhythm is involved. If this is indeed the case, one might expect that under prolonged monotonous conditions, the biological clocks of individual isopods would begin to get out of phase with one another. In order to determine whether this occurred, ten isopods were selected and placed in pairs into petri dishes containing seawater. Each dish contained one animal in the 20-30mm range and one in the 15-20mm size range. The dishes were wrapped with aluminum foil and placed in a light¬ tight box whose inside temperature remained relatively constant at 14°C throughout the experiment. The chromatophores of these isopods were examined in dim light every 4 hours for a 4 day period, and quickly returned to the dark. No significant asynchrony developed during this period (fig. 3a) but the maximum degree of dispersion of chromatophore pigments increased on successive days. A gradual decrease in the degree of pigment concentration at night was also indicated. Chr lore Behavior in resecata. p. 6. In order to see what might occur in more prolonged periods of darkness, a total of 36 animals was kept in the dark in 3 containers holding 12 isopods each. The containers were made light-tight with aluminum foil, and placed under a light tight box. Water In each container, and a small piece of Macrocystis added for food, was changed every 24 hours. Animals were observed briefly in dim light only at noon and midnight, and their chromatophores rated. The results (fig.3b) show a rise in the amount of pigment dispersion at 1200 hours for the first 4 days, while all chromato¬ phores were essentially punctate at night during this period. From day 7, the amount of dispersion at 1200 hours stabilized at approximately the 3.0 level while a gradual decrease in the degree of concentration of pigment at 2400 hours was observed. By the 19th day the mean chromatophore ratings at 1200 and 2400 hours were observed to level off in the 2.5 to 3.5 range. Twelve animals died during the course of this experiment, mainly due to canibalism. At the end of the 19th day, 8 of the surviving animals were selected. placed in separate petri dishes wrapped in aluminum foil, placed under a light-tight box, and observed every 4 to 6 hours for a 24 hour period. The results for each individual (fig. 4) show that the reason the mean chromatophore ratings at 1200 and 2400 hours tended to level off (fig. 3b) was because the endogenous rhythms of the individual isopods had become out of phase, and also because in some animals the chromatophores did not exhibit complete contraction and/or expansion. Since individuals in this experiment had been examined and hence illuminated in dim light at least every 12 hours, a second Unromatophore Behavior in 1. resecata. p. 7 experiment was conducted in order to determine if individual biological clocks get out of phase under conditions of uninterrupted darkness. Fourteen individuals, all in the 20-30mm length range, were placed in separate petri dishes wrapped with aluminum foil. and placed in a light-tight box for 9 days. Food and water were changed in the absence of light every 24 hours. Temperature was constant at 14'C through the course of the experiment. On the tenth day, the chromatophores of each individuals were examined in dim light at intervals of approximately 2 hours for a 24 hour period; between observations animals were returned to darkness. The results (fig. 5, 6a) show that in continued darkness the chromatophore rhythm of individuals became asynchronous. Both the time of initiation of pigment dispersal, and the duration of the period spent in the dispersed state varied between individuals. Asynchrony is much greater than that exhibited by isopods maintained on a normal day/night light cycle as controls. Effect of different intensities of light on chromatophores. Observations on chromatophore changes during ordinary day/night light conditions suggest that the chromatophores of l. resecata respond differently to different light intensities. In its natural environment 1. resecata is subjected to frequent changes in light intensity even during the day due to movement of the kelp canopy with waves and tides, and to movement of the isopods up and down kelp stipes. The next experiment was designed to test the response of the chromatophores of 1. resecata to various intensities of light. All tests were conducted between 2200 and 0200 hours Chromatophore Behavior in 1. resecata. p. 8. because chromatophores are maximally contracted between those times. The isopods used were dark-adapted for 4 hours in a plastic pan of seawater which was wrapped in aluminum foil and placed in a light-tight box at 14 C. Macrocystis was added to the pan for food. Immediately before use isopods were checked for maximal concentration of chromatophore pigments, and only animals with a stage I concentration of pigments were selected for this experiment. The selected animals were placed in black finger bowls in lots of 7, exposed to a particular intensity of light for I hour, and then observed for chromatophore stage. Illumination was provided by a 15 watt incandescent light bulb; adjustments in intensity were made by varying the distance between the light source and the isopods, and by varying the voltage used. Individual animals were used only once, and the experiment was repeated at each intensity 3-7 times depending on the extent of variation between individuals. Temperature remained at 12-14°C throughout the one hour period. The results (fig. 7) show that chromatophore pigments are more dispersed at higher light intensities than at lower intensities. Rate of chromatophore expansion and contraction at different times of day Menke (1911) claimed that in 1. balthica chromatophores expanded or contracted at different rates at different times of the day or night, and that adaptation leading to a more punctate state was faster at night than during the day because chromatophores tended toward contraction at night. The following experiment was designed to determine 1. resecata's ability to adapt to different Chromatophore Behavior in 1. resecata. p. 9. times of the diel cycle. Four hours prior to each experiment, 30 animals were placed in a pan of seawater which was wrapped in aluminum foil and placed in a light-tight box. Macrocystis was placed in the pan for food. Temperature was held constant at 14°0. At the start of the experiment, 15 animals were rated for chromatophore stage and were then placed in a shallow black bowl. The other 15 individuals were similarly rated and placed in a white bowl. Both were then placed under a 60 watt incandescent bulb providing 40 ft-c. of illumination, and the chromatophores of each animal were staged every fifteen minutes through the course of the experiment. When two successive ratings on the animals in a bowl produced the same results, the backgrounds were reversed such that animals from the black bowls were placed in white bowls and vice versa. Animals were again staged every 15 minutes until successive ratings were the same. This experiment was conducted twice at each of the following times: 0600, 1200, 1800, and 2400 hours. Water in the pans was changed at the end of I hour. The results are shown in figures 8a and 8b. Chromatophore adjustment from a dark adapted (punctate) state to a black back¬ ground under the experimental conditions takes between 30 and 45 minutes at each time of day (fig. 8a), with the initial rates of pigment dispersal being the same. For a white background, a similar adaptation appears to be complete within 15 minutes. Adaptation after a subsequent switching of backgrounds occurred within 15 minutes in all cases. Thus it appears that there is no difference in rate of chromatophore expansion or contraction at different times of day though the time needed to adapt may vary depending on the experimental conditions and the initial and final Chromatophore Behavior in 1. resecata. p. 10. chromatophore stages. resecata appears to be able to disperse or concentrate chromatophore pigments with equal speed. Effect of constant illumination on rate of background adaptation The diurnal rhythm leading to dispersion of chromatophore pigments during the day appears to be a mechanism which maintains body pigmentation in a state so that the animal is at least partially protectively colored at any given time. The preceeding experiment indicates that 1. resecata can disperse or contract pigments of its chromatophores equal speed. A partial dispersion of chromato¬ phore pigments during the day, regardless of whether the animal is exposed to daylight or not prepares the animal should it suddenly come our from a dark hiding place into the light, by reducing the amount of dispersion or contraction of chromatophores which might be necessary. A sample of 75 1. resecata at 1600 hours yielded a mean rating of 3.06 with a standard deviation of 1. These isopods were taken directly from the field and rated as quickly as possible. The question might also be asked, what is the advantage of concentrating pigment in chromatophores to a fully punctate state at night? There seems no obvious advantage from the stand point of protective coloration. Perhaps a daily movement of chromatophore pigments to the punctate stae is necessary in order to maintain an optimal rate of adaptation, i.e. perhaps chromatophores "need exercise" to keep in good condition. This hypothesis was tested in a preliminary way by placing 15 animals in a shallow black pan and another 15 animals in a white pan and Chromatophore Behavior in 1 resecata. p. 11. exposing them to a constant illumination of 40 ft-c. for the entire experiment. No food was placed in the pans and the water was changed at approximately 8 hour intervals. Temperature in the pans ranged 14-17 C. Isopods were maintained on their respective backgrounds for 48 hours, after which time their chromatophores were rated and their backgrounds switched. Animals were observed at 15 minute intervals thereafter. Backgrounds were switched when two successive ratings were the same, which was at O. 90. and 120 minutes. The experiments was repeated a second time so that each line in figure 8c represents 30 animals. It appears that chromatophore contraction in animals going from a black to a white background after 48 hours of constant illumination procedes rather more slowly than is normal. Subsequent reswitching from a black to white backgrounds at 120 minutes resulted in a normal rate of pigment concentration, and adaptation was essentially completed within fifteen minutes. These results while not conclusive suggest the possibility that a secondary function of the diurnal rhythm of chromatophores is related to a need for daily contraction or "exercise". Alternatively the results might be explained by the possible effects of constant illumination on the isopod eye. Animals illuminated on the white background for 48 hours did not adapt significantly more slowly to a black background than did the animals in previous experiments. Summar 1. Chromatophore pigments in the isopod Idotea resecata are dispersed during the day and punctate at night under normal day/night light condition, in constant darkness, and in constant low intensity Chromatophore Be light. 2. Under constant strong illumination there is no diurnal rhythm of movement of chromatophore pigments. 3. Under conditions of prolonged constant darkness individual sopods become asynchronous in rhythmic chromatophore changes, and the degree of pigment dispersal and concentration is decreased in some animals. 4. The degree of expansion or contraction of chromatophores depends on both background color and light intensity. 5. Rates of change in chromatophores accompanying changes in background color are constant at all times of the day or night. 6. After constant strong illumination for 48 hours on a black background, the rate at which isopods adapted to a white back ground was decreased, while the accompanying switch from white to black background is unchanged. Acknowledgments I wish to thank the entire Hopkins Marine Station staff for their help and advice. I would especially like to thank Dr. Donald P. Abbott for his patience and encouragement. Chromato 1 resecata. Captions for igures Fig. la. The diurnal cycle of chromatophore change in 1. resecata in a normal day and night light regime on a black, a white, and a gray background. Twelve animals were placed on each colored substrate. Fig. Ib. Hogben and Slome (1931) Stages illustrating the degree of dispersion of chromatophore pigments (after Hogben and Slome, 1931). Fig. 2a. Mean chromatophore ratings for animals under constant bright illumination (40 ft-c.) on a black and on a white background. Twelve isopods were placed in each colored pan. Fig. 2b. Mean diurnal rhythm of chromatophore change in 1. resecata keot on a black and on a white background under constant low illumination (0.04 ft-c.). Each line represents results of lE isopods. Fig. 2c. Mean diurnal rhythm of chromatophore change in 1. resecata kept in constant darkness."A total of 36 isopods were used. Vertical bars show 95% confidence limits of the mean. Fig. 3a. Mean chromatophore ratings and 95% confidence limits (vertical bars) for 10 1. resecata keot in constant darkness. Chromatophore Behavior in 1 resecata. p. 15. Fig. 3b. The diurnal cycle of chromatophore change in L resecata kept in constant darkness and examined only at 1200 and 2400 hours, Mean chromatophore ratins and 95% confidence levels for the mean are shown. N=36. Fig. 4. Diel cycle of chromatohpore change in 8 individual 1. resecata atter being kept in constant darkness for 19 days (fig. 3b). Fig. 5. Diel chromatophore rhythms of 5 1. resecata after 9 days in constant darkness withou observation. Isopods were kept in individual dishes. Cycles are clearly asynchronous. Fig. 6a. Chromatophore rating of individual 1. resecata kept in a normal day/night light regime on a black background. Size of rectangle is proportional to number of individuals. Fig. 6b. Chromatophore ratings of 14 1 resecata in constant darkness for 9 days kept in separate containers. Size of rectangle is proportional to number of individuals; scatter of readings indicates degree of asynchrony. Fig. 7. Chromatophore ratings for 1 resecata after ! hour of illumination at a particular light intensity on a black back- ground. Animals were dark adapted for 4 hours prior to testing All tests were conducted between 2200 and 0200 hours so chromato¬ phores of all animals were at stage I at the start of the experiment. Mean chromatophore ratings and 95% confidence leyels Chromatophore Behavior in 1 resecata. p. 16. for the mean are shown. Fig 8a. Rates of concentration and dispersion of chromatophore pigments in 1. resecata at different times of day. Animals were dark adapted for 4 hours, observed, then placed on a black background and illuminated at 40 ft-c. When adaptation for the black background was completed, animals were transferred to a white background. The experimnet was done twice at 0600, 1200. 1800, and 2400 hours. Each line represents a mean for 30 animals. Fig. 8b. Rates of concentration and dispersion of chromatophore pigments in L resecata at different times of day. Animals were dark adapted for 4 hours, observed, then placed on a white background and illuminated at 40 ft-c. When adaptation for the white background was completed, animals were transferred to a black background. The experiments was done twice at 0600, 1200. 1800, and 2400 hours. Each line represents a mean for 30 animals. Fig. 8c. Rates of concentration and dispersion of chromatophore pigments in 1. resecata after 48 hours of illumination at 40 ft-c. on a black and on a white substrate. Isopods were placed on backgrounds opposite to the ones to which they were adapted at 0 minutes. Substratum colors were switched at 90 and 120 minutes. Chromate Bibliography Armitage, K., 1960. Chromatophore behavior of the isopod Ligia occidentalis Dana 1853. Crustaceana 1:193-207. Hogben, L. and Slome, D., 1931. The pigmentary effector system. VI. The dual character of endocrine coordination in amphibian colour change. Proc. Roy. Soc., Lond. (B) 108:10-53. Kleinholz, L., 1937. Studies in the pigmentary system of Crustacea. 1. Color changes and diurnal rhythm in Ligia baudiniana. Biol. Bull. 72:24-36. Lee, W., and B. Gilchrist. 1972. Pigmentation, color change and the ecology of the marine isopod Idotea resecata. J. Exp. Mar. Biol. Ecol. 10:1-27. Matzdorff, C., 1883. Über die Farbung von Idothea tricuspidata Desm. Jena. Zeitschr. Naturwiss. 16:1. Menke, H., 1911. Periodische Bewegung und ihr Zusammenhang mit Licht und Stoffwechsel. Pflug. Arch. Physiol. 140:37-91. Oguro, c., 1959. On the physiology of melanophores in the marine isopod, ldotea japonica 1. Endocrinol. Japon. 6:246- 252. Pieron, H., 1914. Recherches sur le comportement chromatique des Invertebres et en particulier des Isopodes. Bull. Sci. France Belg. 48:30. — BLACK 5.0 4.0 3.0 2.0 0 2——X GRAY g b TIME OF DAY 9 ----- WHITE F16 1 g E16. 2 — BLACK •---- WHITE ggs — — BLACK ---- WHITE o 2 5 4 E B O —2400 1200 2400 1200 2400 00 D 12400 - 1200 2400 - So 1) ZOVIS BAOHdOIVWO SHO O O L — 5 5 1L — — — 5 L L 5. N 3 — L P16. 4 F16.5 5. 3 L 5. 3 P LP t 5 31 P P ILL 5 3 JL 5 3 L TIME OF DAY F16 6 - BS5 E EDE 5 s .E a BE- 41 . . . . L TAA abhbLa 5 .E L- n6 4 . . . . . . . 34 a..... . . . . . . . . L. 21= = . . - 11 L L IE R TIME OF DAY 50 4.0 30 20 1O N A I — L O INTENSITY (ft.c.) El6 7 L —---— K — —-- — t set 2 — BLACK ADAPTED ----- WHITE ADAPTED 5 J Y 24 L o TIME IN MINUTES 21 P16.8 — 2400 hrs. --- 1200 hrs. —.— 1800 hrs. 0600 hrs. • BLACK WHITE 2400 hrs. E ——- 1200 hrs. —.— 1800 hrs. 0600 hrs. • BLACK WHITE • BLACK WHITE +