Camp. Biochem. Phvsiof. Vol.

Printed in Great B&in 8X, No. 1, pp. l-7, 1986 0306.4492/86 $3.00 + 0.00

IC 1986 Pergamon Press Ltd

CONTROL OF CHROMATOPHORE MOVEMENTS IN DERMAL CHROMATIC UNITS OF BLUE

DAMSELFTSH-41. THE MOTILE IRIDOPHORE

HIROAKI KASUKAWA, NORIKO O~HIh~A and Rvozo FUJII*

Deportment of Biology, Faculty of Science, Toho University, Miyama, Funabashi, Chiba 274, Japan. Telephone: (0474) 72-1141

(Received 2 1 Maq’ 1985)

Abstract-l. The mechanism regulating the movements of the unique motile iridophores of the blue damselfish, Chrysiptera cyanea, was studied.

2. The reaction in which the cells become reflective to light rays of longer wavelength, i.e. from the near U.V. region to the green region, was designated as the “coloring response”, while the reverse process was labeled the “clearing response”.

3. Both nervous stimuli and adrenergic agonists gave rise to the coloring response, which could be antagonized by alpha adrenolytic agents.

4. The clearing response was accelerated by adenosine and inhibited by theophylline. 5. None of the hormonal substances tested had any effect on the motile response of the cells. 6. It was concluded that the motile iridophores are solely under the control of the sympathetic

adrenergic system, and that the co-transmitter, adenosine, may function to antagonize quickly the true transmitter-induced colored state of the cells.

INTRODUCTION

Light-reflecting chromatophores are classified into two categories, i.e. leucophores and iridophores. The leucophores are dendritic cells, like melanophores and other common light-absorbing chromatophores, but contain a number of small light-scattering globules, called leucosomes. In response to nervous or hormonal cues, these organelles move back and forth within the dendritic processes to increase or decrease the reflectance of the skin. Apparently, therefore, the leucophores play an active role in the physiological color changes of many poikilotherms, including some teleostean species (Miyoshi, 1953; Iga et al., 1977; Fujii and Miyashita, 1979; Menter et al.,

1978; Yamada, 1982; Oshima and Fujii, 1985). On the other hand, the iridophores, which contain

a stack or stacks of large crystalline light-reflecting platelets, are generally known to take part in pro- ducing silvery or whitish tones of the skin, and have never been assumed to be motile (Parker, 1948; Bagnara, 1966; Denton and Nicol, 1966; Land, 1972).

Quite recently, however, we have found “motile” iridophores in the skin of the popular blue damselfish (Oshima et al., 1985a). These cells were predomi- nantly responsible for the various colors displayed by the fish through the multilayered thin-film interfer- ence phenomenon of “non-ideal” type, which takes place in the stacks of very thin retlecting platelets in their cytoplasm (Oshima et al., 1985a; Kasukawa et al., unpublished data). In other words, the distance between adjoining platelets changes simultaneously in the cells, leading to a shift in the spectral reflect- ance of the skin. The cellular motility of this entirely new category provides an explanation for the extra-

*Author to whom correspondence should be addressed.

ordinarily rapid changes of hue commonly observ- able in the damselfish, although the melanophores, which are the counterparts of the reflectile cells constituting the simple dermal chromatophore unit, function to modify the tones.

Using the same fish, we have lately reported that the regulatory mechanisms for the meianophores were fundamentally identical with those known hitherto in many fish species (Kasukawa ef al., 1985). Since the mechanism controIIing these interesting light-reflecting cells has not been investigated, on the other hand, we have attempted to elucidate it. The rather strange outcome was that these cells were found to be solely under the control of the sym- pathetic nervous system, entirely unaffected by the endocrine system.

MATERIALS AND METHODS

Preparation

The damselfish, Chrysiprera qaneu (Quay and Gaimard), varying in body length from 35 to 60 mm, was used for this study. Specimens were obtained from a commercial source, and were reared in our laboratory for at least a few days before sacrifice.

Split dorsal fin preparations were made in physiological saline of the following composition (mM): NaCl 125.3, KC1 2.7, CaCl, 1.8, MgCl, 1.8.-n-glucose 5.6, Tris-HCI buffer 5.0 (pH 7.2), according to a method nreviouslv described ._ (Kasukawa et al., 1985).

In experiments in which the response following neural stimuli was studied, fin pieces excised from chemically sympathectomized fish were used, since in such pieces the state attained by catecholamine action could more easily be maintained. The chemical denervation was done by injecting the animals i.p. with 80-16Opg/g body wt of 6-hydroxy- dopamine hydrobromide (Sigma Chemical, St. Louis) 2-4 days before sacrifice. This method has lately been described by Iga and Takabatake (I 982). who denervated the melano- phores of a cyprinid, Zncco.

1 C.BP *3,v--A

2 HIROAKI KAXJKAWA et al.

Optical and recording apparatuses

The cells we studied were “light-reflecting” due to the multilayered thin-film interference phenomenon, As will be pointed out later under Results, however, it proved reason- able for the present purpose to employ the same optical apparatus used in assessing the responses of leucophores (Fujii and Miyashita, 1979; Oshima and Fujii, 1984). Thus, we used the same dark-field epi-illumination microscope system described elsewhere (Oshima and Fujii, 1984; Opti- phot XF-BD with CF-BD objective lenses, Nikon, Tokyo).

An S1226-5BK silicon photodiode (Hamamatsu Pho- tonics, Hamamatsu) was adopted as the photoelectric trans- ducer. Whenever near U.V. light had to be measured, a u.v.-sensitive article (S1226-5BQ, Hamamatsu Photonics) was employed. The circular skin area to be measured for chromatophore responses was restricted to 80pm in di- ameter. Thus, the activities of about 25 iridophores were recorded.

In some experiments, the correlation between iridophore activity and that of the underlying melanophores was studied by using an apparatus which could record the responses of both cell types simultaneously (Oshima et al., 1984). Again, the diameter of the circular area to be recorded for the chromatophore responses was 80 pm.

In our spectral reflectance study of iridophores, a u.v.~ vis. light range monochromator (High-intensity Grating Monochromator; Shimadzu-Bausch & Lomb, Kyoto) was adopted as the incident light source, replacing the halogen lamp illuminator with which the microscope was originally equipped.

Stimulation qf chromatic neroes

The method for stimulating nerves controlling chromato- phores was essentially the same as that described elsewhere (Fujii and Miyashita, 1975). In the present study, however, a Nihon Kohden (Tokyo) SEN-3201 electronic stimulator was employed.

Drugs used

In addition to 6_hydroxydopamine, the following drugs were used: norepinephrine hydrochloride (Sankyo, Tokyo), epinephrine (Tokyo Chemical Ind., Tokyo), isoproterenol hydrochloride (Nikken Chemicals, Tokyo), metaproterenol sulfate (Daiichi Seiyaku, Tokyo), isoxsuprine hydrochlor- ide (Daiichi Seiyaku), phenoxybenzamine hydrochloride (Nakarai Chemicals, Kyoto), phentolamine mesylate (Ciba- Geigy, Basel), propranolol hydrochloride (ICI, St. Louis), adenosine (Kohjin, Tokyo), theophylline (Tokyo Chemical Ind.), alpha-melanophore stimulating hormone (alpha- MSH; Sigma Chemical, St. Louis) and melatonin (Nakarai Chemicals). Synthetic salmon melanin-concentrating hor- mone (MCH) was a gift from Dr M. E. Hadley of the University of Arizona. These drugs were appropriately diluted with physiological saline immediately before use.

All experiments were performed at room temperatures between 18 and 26°C.

RESULTS

Recording of iridophore responses

The changes in optical or reflectivity characteristics in damselfish due to the motile activities of irido- phores are entirely different from those known to date for motile chromatophores of other sorts. As briefly mentioned in the Introduction, these changes may now be explained as a shift of the waveband of reflected light produced by the multiple thin-film interference phenomenon (Oshima et al., 1985a). Further explanations of optical events within the iridophore will be dealt with in detail elsewhere (Kasukawa et al., unpublished data). In brief, the

spectral peak of the reflected light stood at around 380 nm when an excised skin piece was equilibrated in physiological saline, and it moved towards longer wavelengths up to green (ca 530 nm) when stimulated by some means. This process by which near U.V. ray-reflecting or practically transparent cells become variously colored, and sometimes maximally to green, was designated as the “coloring response”. The reverse process, i.e. colored cells becoming trans- parent (peak: ca 380 nm), such as when they are equilibrated in physiological saline, was labeled the “clearing response”.

In the first stage of our study, therefore, it seemed to us that iridophore responses might not be assess- able simply by using conventional photoelectric methods, in which either light transmittance through or the reflectance from the skin is measurable in terms of light intensity.

Using a u.v.-vis. light monochromator as the incident light source, we first measured the absorp- tion spectral characteristics of the epi-illumination microscope system. The objective lens employed was a CF-BD Plan 20X. As the standard light-reflecting plane for calibration, a piece of white board (average reflectance: 90%) supplied from the Japan Color Research Institute (Tokyo) was employed. The sur- face of the board was focused, and reflectance was measured from 350 to 800 nm.

As shown in Fig. 1, the output current of the photodiode was maximal around 650-700 nm. It decreased gradually until fading out occurred in the near U.V. region. The progressive decrease in output should naturally be due to the absorption of shorter wavelength light rays by the optical glasses constitut- ing the microscope system.

As stated above, the coloring response of the iridophore implied a shift of the waveband up to about 530 nm. Thus, the shift of the band due to iridophore movements was deservedly transformed into a change in output current, although correlation

100

s r

Wave Icngth 01 incident light, nm

Fig. 1. Spectral characteristics of the dark-field epi-illumi- nation microscope system employed in the present study. Ordinate, output current of the photo-sensor. Abscissa, wavelength of the incident light from a monochromator. Along with the progress of the coloring response of the motile iridophores of the blue damselfish, ChrysiDtera cyanea, the spectral peak of the light reflected from the cells moved from ca 380 to ca 530nm. Thus. there was a progressive increase in the current output of the sensor. For a detailed explanation of the measuring method see the text.

Control of damselfish motile iridophores

Fig. 2. Simultaneous recording of the coloring response of iridophores (upper trace) and the pigment aggregating response of melanophores (lower trace) to electrical nervous stimulation (NS) at a low frequency (0.14 Hz). Biphasic square pulses of 1OV in strength and l.Omsec in duration were applied on the proximal part of a split dorsal fin preparation. The maximal level of chromatophore responses (1OO’A) was attained by applying 5 x 1O-6 M norepinephrine

for 5 min, following the recording shown here.

of the degree of the response to the actual change in the value of the waveband shift was by no means linear. We therefore assessed the progress of the coloring response of iridophores as the increase in output current, and employed the same photoelectric recording method as described by Oshima and Fujii (1984) for assessing quantitatively the motile responses of light-scattering or reflecting chromato- phores.

In the present report, the magnitude of the coloring response was expressed as a percentage of the maxi- mal response (peak: cu 530nm), taking the reading for the fully transparent state (ca 380 nm) as zero. All figures illustrated in later sections were traced by this method.

EfSect of neruous stimulation

Electrical stimulation of chromatic nerves effec- tively induced a coloring response in the iridophores in the same way as pigment aggregation did in the

4 o”i 02 0.5 i 1 i 2 i 5 I 10 , 20 ,

Frequency of stlmulatlon, Hz

Fig. 4. Relationship between the frequency of supermaximat electrical nervous stimulation and the magnitude of the coloring response of the iridophores. Biphasic square pulses of 10 V in strength and 1 .O msec in duration were applied for 3 min. Frequencies varied from 0.1 to 20 Hz. Abscissa, frequency in Hz (logarithmic scale). Ordinate, magnitude of response as a percentage of the maximal level of the response attained by a 3-min application of 5 x 1O-6 M norepinephrine at the end of each series of measurements. Each point is the mean of seven measurements on different

animals. Vertical lines indicate SE.

melanophores (Figs 2 and 3). Even a single electrical shock aroused a small response. The summation of these unit responses due to a low frequency volley, which gave rise to a larger response, may be clearly seen in Fig. 2. It should be noted that the small responses expressed as small upward deflections in both traces corresponded well with each other.

As shown in Fig. 4, the level of the coloring re- sponse attained during the same period of stimulation increased upon an increase in the stimulus frequency. Up to about 1 Hz, the frequency increase resulted in a rapid increase in the level of the response. The maximal level of the response was attained at about 5 Kz. With further increases in the frequency, the magnitude of the response tended to fall slightly.

5 ” 0L-l u # L-__..J i Nf a a &f&J & &

PP , 3X10-sMI

Ph ,3x10-614,

NE ,5x IO-su,

Fig. 3. Typical recording of the motile response of the iridophores, showing the coloring response-inducing action of electrical nervous stimulation (NS), and the effect of propranolol (PP) and phentolamine (PA) on the nervous action. The maximal level of the coloring response for reference was finally attained by

application of 5 x lo-” M norepinephrine (NE).

4 HIROAKI KASUKAWA et al.

PP , 3x10-et4 ,

PA , 3x10-% ,

Fig. 5. Typical recording showing the coloring response-inducing action of norepinephrine (NE), and the effects on it of a beta adrenergic blocking agent, propranolol (PP), and an alpha one, phentolamine (PA).

Effects of sympathomimetic amines

Norepinephrine and epinephrine were employed as agents having alpha agonistic action. These sub- stances were likewise effective in inducing the color- ing response in the iridophores. As seen in Figs 3 and 5, for instance, application of a 5 x 10m6 M nor- epinephrine solution for 2 min brought about a full response.

Figure 6 indicates the relationship between the concentration of norepinephrine and the magnitude of the coloring response, indicating that the action of the amine was dose-dependent. The minimal effective concentration needed to arouse a discernible coloring response was about 5 x 10m9 M. From 2 x lOm8 M to 5 x lo-‘M, the magnitude of the response rapidly increased. At about lO-6 M, the maximal level was attained.

Concentration of NE, M

Fig. 6. Relationship between the concentration of norepi- nephrine (NE) and the magnitude of the coloring response of the iridophores. Amine solutions of various concentra- tions were applied for 3 min. The treatment was immediately followed by another 3-min application with 5 x 10m6 M NE to bring about the full level of the response. Abscissa, molar concentration of NE (logarithmic scale). Ordinate, magni- tude of the response as a percentage of the level of the full response. Each point is the mean of seven measurements on

different animals. Vertical lines indicate SE.

Beta adrenergic amines have recently been shown to have a pigment-dispersing effect on fish melano- phores (Reed and Finnin, 1972; Miyashita and Fujii, 1975). Lately, the melanophores of the present material have also been shown to possess beta adrenoceptors which mediate melanosome dispersion (Kasukawa et al., 1985). Thus, we have naturally been obliged to investigate the presence of adreno- ceptors of this type in the reflectile cells.

In the present study, three synthetic beta agonists, isoproterenol, metaproterenol and isoxsuprine, were tested, and none of them were found to possess the action required to reverse the colored state or to induce the clearing response in the cells. This absence of any response to metaproterenol is illustrated in Fig. 7a.

Conversely, these amines showed a rather potent effect in arousing the coloring response: isoprotere- nol, metaproterenol and isoxsuprine were effective at concentrations above 3 x 10e6, 10m5 and 5 x 10m5 M, respectively.

Fig. 7. Typical recordings showing the possible clearing response-inducing effect of (a) metaproterenol (MP), (b) adenosine (AS) and (c) alpha-melanophore stimulating hor- mone (MSH) on chemically sympathectomized iridophores. In each trace, the cells were pretreated with 5 x 10m6 M norepinephrine (NE) solution to induce a moderate and

sustained coloring response.

Control of damsei~sh motile iridophores 5

Eflects of adrenolytic agents

Phenoxybenzamine and phentolamine were used as alpha adrenolytic agents, while propranolol was selected as a beta antagonist. In Fig. 3 may be seen a recording in which the action of electrical nervous stimulation was antagonized by phentolamine, but not by propranolol. The coloring action of nor- epinephrine was also blocked by phentolamine, but not by propranolol (Fig. 4). The responses caused by isoproterenol, metaproterenol or isoxsuprine were also blocked by phentolamine. These results indicate that the coloring responses to sympathomimetic amines were exclusively mediated by alpha adreno- ceptors.

Efsects of adenosine and its inhibition

In contrast to sympathomimetic amines, adenosine was found to have an action which reversed the colored state of the iridophores (Fig. 7b). In solution above lo-* M it aroused a pronounced response. Theophylline, a specific blocker of adenosine recep- tors, effectively inhibited the clearing response in- duced by the nucleoside. This blockade, however, was reversible. When the blocking agent was washed out, the response to the stimulant soon recovered, as shown on the right side of Fig. 8.

Efects of pigment-motor hor~to~es

The pineal hormone, melatonin, and a pituitary one, melanin-concentrating hormone (MCH), both known to bring about potent pigment aggregation in damselfish melanophores (Oshima et al., 1985b; Kasukawa et al., 1985), did not arouse the coloring response of the iridophores. Moreover, MSH, which has also been found to act potently on damselfish melanophores to disperse their pigment (Kasukawa ef al., 1985), had no influence on the iridophores, as is shown in Fig. 7c.

DfSCUSSION

In the dermis of the present material, Chrysiptera cyanea, a melanophore and the overlying small irido- phores in a monolayered sheet constitute a simple dermal chromatophore unit (Oshima et al., 1985). Lately, we have shown that the rapid motile activities

or

of the characteristic three-dimensionally developed melanophores are primarily controlled by the sym- pathetic nervous system, being mediated by alpha adrenoceptors (Kasukawa et al., 1985), like those of flat melanophores observable in many other fish species studied to date (Parker, 1948; Fujii, 1961, 1969; Healey and Ross, 1966; Fujii and Novales, 1972; Fujii and Miyashita, 1975).

Histochemical and biochemical analyses have pro- vided additional evidence to support the conclusion that melanophores are supplied with adrenergic fibers (Jacobowitz and Laties, 1968; Falck Ed al., 1969; Kumazawa and Fujii, 1984; Yamada et af., 1984). Recently, non-melanophore fish chromatophores, i.e. erythrophores, xanthophores or leucophores, have also been shown to be under the control of adrenergic post-ganglionics (Ozato, 1977; Matsumoto et al., 1978; Fujii and Miyashita, 1979; Iga, 1983; Miyata and Yamada, 1985).

In the present studies, it has been shown that the motile activities of the round, non-dendritic irido- phores are likewise controlled by sympathetic post- ganglionic fibers through mediation by alpha adreno- ceptors on the effector cell membrane. Since motility in iridophores was first observed in the cells of the damselfish (Oshima et at., 1985a), they were naturally the first to be reported receiving motor innervation.

We are now aware that both cell types constituting the chromatophore unit are similarly controlled by an autonomic nervous supply of the same category, Actually, electron microscopic observations have re- vealed that nervous elements exist in close connection with the iridophores, and further that some of these even form synaptic contacts with those two types of chromatophores at the same time (Oshima et al., 1985a). In the present study, it was further shown that the coloring response of the iridophores and the melanin-aggregating response of the melanophores to nervous stimuli proceeded in a well synchronized manner.

Leucophores of the medaka, Oryzias latipes (Miyoshi, 1953; Fujii and Miyashita, 1979; Iwata et al., 1981), and of the killifish, Fund&us heteroclitus (Menter et al., 1978), are known to exist in close association with melanophores: a leucophore is usually located beneath a melanophore concen- trically. Leucophore inclusions, however, became

--l

100 NE

Ii 5X10%l l_l 1 5X10-%4 , ,5x10%

~-~ 5x10-%., SXJO%

A5 & a a

TO I lo-% J

Fig. 8. Typical recording of the response of the iridophores, showing the clearing response-inducing action of adenosine (AS) and the effect on it of theophylline (TO), an adenosine-receptor blocker. A weak norepinephrine (NE, 5 x IO-” M) solution was used to induce a moderate coloring response of the cells.

The maximal level of response was finally attained by application of 5 x 10m6M NE.

6 HIROAKI KASUKAWA et al.

aggregated into the perikarya when the skin piece is equilibrated in physiological solution, and disperse in response to a nervous volley or to norepinephr~ne. It is known that Oryzias leucophores are also adrenergically controiled. Being different from other dendritic chromatophores, however, their response was mediated by adrenoceptors of beta type (Iga et al., 1977; Iwata et al., 1981; Iga, 1983). Furthermore, observations of the synchrony existing in the Ba2+- induced pulsations of the combined melanophore and leucophore of the same species led Iwata et al. (198 1) to conclude that these two chromatophore species are innervated by branches of an adrenergic fiber, and that they belong to a single motor unit. That is, the modes of neural regulation clarified in the damselfish chromatic unit and in the melanophore-leucophore combination are quite similar.

Working on the nervous mechanism controlling tilapia melanophores, we lately showed that ATP is released as the co-transmitter from the chromatic adrenergic fibers along with the true transmitter, norepinephrine (Kumazawa et af., 1984). As the metabolite of ATP through the relevant exoenzymes in the synaptic cleft, adenosine may interact with adenosine receptors on the melanophore membrane to induce pigment dispersion within the cells (Kumazawa and Fujii, 1984). Using the melano- phores of the present material, the blue damselfish, we have lately found further evidence to support this conclusion (Kasukawa et al., 1985).

In the present study on the i~dophore, we also found that adenosine possessed a potent action for arousing the clearing response, and that this action was inhibited by theophylline, a specific blocker of the receptor involved. It may safely be assumed, therefore, that adenosine, as the co-transmitter of the adrenergic controlling fiber, may also play an impor- tant role in nervous control of the iridophores: that is, it functions to help iridophores recover from the effect of the true transmitter, norepinephrine, thus enabling fish to change their hue very rapidly.

When we equilibrated fin pieces in physiological saline, the iridophores maintained a transparent state, while the melanosomes in the melanophores were dispersed. Such a state apparently corresponds to that occurring in the skin of darkened fish. In contrast, live blue damselfish commonly display a cobalt-blue color, as the common name of the fish signifies. The question thus arises as to how this brilliant blue coloration is produced and maintained. Using an excised fin preparation, a hue identical with that seen in live fish could be experimentally induced and maintained by stimulating the chromatic nerves with supermaximal electrical pulses at frequencies of 0.2-0.3 Hz. Based on these observations, the mech- anism of production of normal skin coloration and the process of darkening in vim may be considered to be as follows. Periodic post-ganglionic nerve impulses at low frequencies maintain a moderate coloring response in the iridophores, i.e. the cobalt-btue color- ation of the skin, When the nervous impulses cease, a longer lasting co-transmitter, adenosine, brings about a quick reversal of the colored state or clearing of the iridophores, leading to the darkening of the skin. The sustained darkness observable in certain ethological conditions or which commonly takes

place during handling of the fish may be the result of quiescence or sustained inhibition of the chromatic nervous system. Even when nerve impulses are absent, there should be a spontaneous release of transmitter substances, as discussed elsewhere in reference to the meianophore-controlling system (Kumazawa and Fujii, 1984). In that system, the effect of quickly disappearing norepinephrine would not become apparent, but lasting adenosine might act to keep the cells clear.

In addition to the neural control, a humoral one has also been widely accepted to exist among the chromatophores of fishes (Parker, t 948; Pickford and Atz, 1957; Fujii, 1969). Actually, the melanophores of the present material have recently been found to be sensitive to some hormonal peptides known to affect chromatic effector cells: melanophore-stimulating hormone (MSH), a hypophyseal intermediate lobe principle, dispersed melanosomes very effectively, while melatonin from the pineal was potent enough in inducing reverse movement of the cells (Kasukawa et al., 1985). melanin-con~ntrating hormone (MCH) also acted quite effectively on those cells (Oshima ef al., 1985b). Beta agonistic amines, which have lately been shown to possess pigment-dispersing action on melanophores in some teleosts (Reed and Finnin, 1972; Miyashita and Fujii, 1975) have also been confirmed to have the same action on damselfish melanophores, being mediated by adrenoceptors of beta nature (Kasukawa et al., 1985). The endogenous catecholamines involved in this process have been presumed to originate in the adrenal chromafhn cells (Miyashita and Fujii 1975).

Although the motile iridophores of damselfish are closely associated with the melanophores, they have only now been disclosed to be entirely refractory to these hormonal substances, being solely regulated by nerves; that is, the motile activities of the iridophores and the melanophores are differentially controlled, and the former cells may play the principal role in the extraordinarily rapid changes of hue from blue to dark violet and vice versa. Such rapid changes of color can be realized due to the existence of special chromatic effector cells with machinery entirely different from that seen in ordinary chromatophores (Oshima et al., 1985; Kasukawa et al., unpublished data). In the ordinary chromatophores, as is well known, chromatosomes must migrate centripetally or centrifugally in file through thin dendritic processes. The subtle and delicate changes in hue observable in live fish may at least partly be due to such a differential regulation of chromatophore species.

The damselfish sometimes displays a greenish or yellowish coloration. It was possible to produce these same tints in vivo by injecting epinephrine or nor- epinephrine i.p., and in vitro by applying an amine solution at a concentration of above 10e6M to an excised skin piece. Thus, we regard these hues as the “excitement pallor”, sometimes observable among poikilothermal vertebrates. We know that sym- pathetic postganglionic neurones and adrenal chromaffin cells, from which catecholamines burst out, are ontogenetically homologous. Therefore, we may safely say that the color change of this type is the result of “semi-neural” control of the chromato- phores. Indeed, the responses both to catecholamines

Control of damselfish motile iridophores 7

Kumazawa T. and Fujii R. (1984) Concurrent releases of norepinephrine and purines by potassium from adre- nergic meianosome-aggregating nerve in tiiapia. Camp. Bioc~lem. Physiof. 78C, 263-266.

Kumazawa T., Oshima N., Fujii R. and Miyashita Y. (1984) Release of ATP from adrenergic nerves controlling pigment aggregation in tilapia melanophores. Comp. Biochem. Physioi. 78C, 14.

Kasukawa H., Sugimoto M., Oshima N. and Fujii R. (1985) Control of chromatophore movements in dermal chro- matic units of blue damselfish-I. The melanophore. Comp. Biochem. Physiol. MC, 253-251.

Land M. F. (1972) The physics and biology of animal reflectors. Prog. Biophys. molec. Biol. 24, 75106.

Matsumoto J., Watanabe Y., Obika M. and Hadley M. E. (1978) Mechanisms controlling pigment movements within swordtail (Xiphophorus helleri) erythrophores in primary cell culture. Comp. Biochem. Physiol. 61A, 509-5 17.

Menter D. G.. Obika M., Tchen T. T. and Taylor J. D. (1978) Leucophores and iridophores of Fundulus hetero- clitus: biophysical and ultrastructural properties. J. ~orphol. 160, 103-120.

Miyashita Y. and Fujii R. (1975) Receptor mechanisms in fish chromatophores-Ir. Evidence for beta adreno- ceptors mediating melanosome dispersion in guppy melanophores. Comp. B&hem. Physiol. 51C, 179-187.

Miyata S. and Yamada K. (1985) Pattern of adrenergic innervation to scale erythrophores of the swordtail, Xiphophorus helleri. Zool. Sci. 2, 49-51.

Miyoshi S. (1953) Response of iridocytes in isolated scales of the medaka (Or_vzias Iaripes) to chlorides. Annomes zooI. jap. 25, 21-29.

Oshima N. and Fujii R. (1984) A precision photoelectric method for recording chromatophore responses in &TO. Zool. Sci. 1, 545-552.

Oshima N. and Fujii R. (1985) Calcium requirement for MSH action on non-melanophoral chromatophores of some telcosts. Zool. Sci. 2, 127-129.

Oshima N., Fujii R. and Kasukawa H. (1984) Simultaneous recording of motile responses of light-absorbing and reflecting chromatophores in oitro. Zool. Sci. 1, 7 1 t-7 17.

Oshima N., Sato M., Kumazawd T., Okeda N., Kasukawa H. and Fujii R. (1985a) Motile iridophores play the leading role in damse~fish coloration. In Pigment Cell 1985: Biological, MoIecuIar and CIinicai Aspects of Pig- mentation (Edited by Bagnara J., Klaus S. N., Paul E. and Schartl M.), pp. 241-246. University of Tokyo Press, Tokyo.

and to nervous stimuli appear to be mediated by common receptors, adrenoceptors of alpha nature.

Skin coloration and rapid and subtle changes in skin coloration are certainly important signals of survival strategies and of mutual communication in many fishes. This also seems to hold true in the damselfish. For a thorough understanding of the ethological significance of these phenomena, how- ever, observations at organismal levels should care- fully be performed along with further intensive studies of the characteristics of motile activities and their controlling systems.

Ackno~~edgemenf.s-We thank Professor M. E. Hadley of the Department of Anatomy, University of Arizona, for the gift of synthetic melanin-concentrating hormone. This work was partly supported by grants from the Ministry of Education, Science and Culture of Japan, and also by a grant from the Ito Foundation for the Promotion of Ichthyological Research.

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Denton E. J. and Nicol J. A. C. (1966) A survey of reflectivity in silvery teleosts. J. mar. biol. Ass. U.K. 46, 685-722.

Falck B., Miinzing J. and Rosengren A. M. (1969) Adre- nergic nerves to the dermal melanophores of the rainbow trout, Salmo guirdneri. Z. Zel~orsch. 99, 430434.

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