Novel dichromatic chromatophores in the integument of the mandarin fish synchiropus splendidus.
The variety of colors, patterns, and spectacular color changes the animals can exhibit is a subject of much interest. The generation of color and color changes is important to many species because it provides protection and assists in survival in their habitats. The delicate changes in hues and patterns can also be used to communicate with conspecifics. Such colors in the integument of animals are generated as a result of the absorption by pigment of incident light rays of certain wavelengths, and by the scattering and reflection of light by intracellular structures with reflective indices different from those of the cytoplasm.
In teleosts, chromatophores in the integument possess pigments, light-reflecting structures, or both in the cytoplasm, and generate colors and patterns (1, 2). The chromatophores of teleosts are generally classified into seven categories on the basis of their color: melanophore (black), erythrophore (red), xanthophore (yellow), cyanophore (blue), leucophore (white), iridophore (iridescent color), and erythro-iridophore (reddish violet) (1-4). The colors of melanophores, erythrophores, xanthophores, and cyano-phores are generated by light absorbance on biogenic pigmentary substances that are contained in organelles (chromatosomes). In contrast, leucophores and iridophores have light-reflecting substances in organelles that allow for scattering or interference phenomena of the incident light (1-3). The erythro-iridophores have a mixed color due to the presence of two coloration schemes, an interference phenomenon on light-reflecting substances and light absorption by biogenic pigments (4). These chromatophores are responsible not only for generating color, but also for changes in the coloration of the skin of animals.
Polychromatic chromatophores, on the other hand, which contain multiple types of chromatosomes in the cytoplasm, are rare in vertebrates and invertebrates. One example, the ovary of the glass shrimp Palaemonetes vulgaris, has polychromatic chromatophores with four kinds of chromatosomes: erythrosomes, leucosomes, xanthosomes, and blue chromatosomes (5). In amphibian chromatophores cultured in vitro, melanosomes and reflecting platelets or xantho-somes are observed in a single chromatophore (6, 7). In the case of teleosts, it was reported in the 1960s that the erytnro-phores of Xiphophorine fish, such as the swordtail (Xiphopho-rus helleri) and platyfish (X. maculates), have two kinds of pigmentary organelles, brownish-red pterinosomes and yellow carotenoid vesicles (8). No polychromatic chromatophores have since been reported in teleost species.
Studies of the physiological and morphological features of the bluish colorations of tropical fish have shown that the integumental bluish hues are generated by a multi-layered interference phenomenon of the "non-ideal" type in piles of extremely thin-film reflecting platelets formed inside the iridophores (9-13). In contrast, in 1995, we reported novel light-absorbing chromatophores that revealed bluish hues (3) in the integument of two species of callionymid fish, the mandarin fish Synchiropus splendidus and the psychedelic fish S. picturatus, belonging to the family Callionymidae (Gobiesociformes). These chromatophores were named "cyanophores" and included the pigmentary organelles "cyanosomes." Cyanophores have not been found in teleost species other than the two callionimid species. In the present study, we used mandarin fish with a body length between 80 and 100 mm obtained from local dealers in the Tokyo and Chiba Prefectures (Fig. 1A). In nature, mandarin fish dwell in coral reefs, crawling on the bottom and frequently hovering among the corals. For slower movement, they mainly use their pectoral fins. Their integument has no scales, but the whole surface is covered with a thick layer of mucus, and it displays various extraordinary hues: green, orange, yellow, brown, and blue. Obvious differences in the colors and patterns between the two sexes are not recognizable by the naked eye; the clue to distinguishing the sexes is the shape of the dorsal fin.
In our experiments, fish, irrespective of sex, were acclimatized in our aquarium tank for at least 1 week prior to decapitation for the study. After decapitation, the skin of the trunk and several fins was rapidly excised and immersed in physiological saline comprising (in millimoles): NaC1 (125.3), KC1 (2.7), Ca[C1.sub.2] (1.8), Mg[C1.sub.2] (1.8), D-(+)-glucose (5.6), and Tris-HC1 buffer (5.0; pH 7.2). Split pectoral-fin preparations were made in physiological saline for light microscopic observations according to the method of Fujii (14). To investigate the morphological features and motile activities of the chromatophores, we used a standard transmission light microscope (Optiphot, Nikon) equipped with a 35-mm camera (FX-35A; Nikon) and an automatic exposure system (UFX; Nikon). The physiological experiments were performed at room temperature between 19 [degrees]C and 24 [degrees]C.
Excised skin specimens were fixed in a solution of 2.5% glutaraldehyde and 1% paraformaldehyde in 0.1 mol phosphate buffer (pH 7.2) at room temperature for 2 h. During fixation, the skins were cut into small pieces, approximately 2 [mm.sup.3], and rinsed in 0.1 mol [1.sup.-1] phosphate buffer (pH 7.2). The pieces were then post-fixed in a solution of 1% osmium tetroxide in 0.1 mol [1.sup.-1] phosphate buffer (pH 7.2) at 4 [degrees]C for 30 min. The fixed samples were dehydrated in a graded ethanol series, treated with methyl glycidyl ether, and embedded in epoxy resin (Quetol 812; Nisshin EM). The embedded skin specimens were cut on an MT-1 ultramicrotome with a diamond knife and mounted on Formvar-coated grids. These ultrathin sections were stained with 3% uranyl acetate for 15 min and with Reynolds' lead citrate for 7 min. Stained sections were examined using a transmission electron microscope (JEOL, Tokyo) operated at 80 kV.
Dermal cyanophores with slender dendrites were observed in the light-blue regions of the pectoral fins (Fig. 1B, C). A few iridophores that displayed a small amount of reflected and scattered incident light were observed in the blue region under epi-illumination optics (data not shown). Around the edge of the blue regions in the dermis, we found novel dichromatic chromatophores that displayed both bluish and reddish hues (Fig. 1D). These novel dichromatic chromatophores had slender dendrites like the dermal cya-nophores observed in the pectoral fins. Two tints, red and blue, from the dichromatic chromatophores were observed either separately or together in the cytoplasm. The diversity of the morphological features of the chromatophores contributed to generate various hues in the integument of the mandarin fish.
Electron microscopic observations at high magnification revealed many cyanosomes in dermal cyanophores containing entangling fibrous materials, as indicated by the white arrows in Figure 2A and B. The cyanosomes were about 500 nm in diameter with a rather irregular configuration, and contained fibrous material (Fig. 2B). Usually, erythrophores are the dendritic chromatophores that contribute the reddish components to the integumental coloration (2). Organelles containing reddish pigments in erythrophores are normally called "erythrosomes," which look like cytoplasmic oil droplets in electron microscopy. In our electron microscopy, the cytoplasm of the novel dichromatic chromatophores contained two kinds of pigment granules, cya-nosomes and oil droplets (erythrosomes) (Fig. 2C, D). The morphological features of the cyanosomes in the dichromatic chromatophores did not differ from those in the cyanophores observed in the pectoral fins.
Aggregation of pigment perikaryon and dispersion throughout the cytoplasm are the bases of the motile activities of an ordinary dendritic light-absorbing chromatophore. In teleosts, the motile activities are usually regulated by the endocrine or the adrenergic nervous systems in vivo (2), and we generally observe pigment dispersion of the chromatophores by immersion in physiological saline in vitro. Here, to investigate the motile activities of the dichromatic chromatophores, we performed pharmacological experiments with skin pieces excised from pectoral fins. The photomicrographs in Figure 3 show that treatment with 2.5 [micro]mol [1.sup.-1] norepinephrine induced the aggregation of both chromatosomes--cyanosomes and erythrosomes--in the novel dichromatic chromatophores (Fig. 3A-D); whereas treatment with acetylcholine, which induces the aggregation of melanosomes in the melanophores of some fish species belonging to the family Siluridae (order Siluri-formes) (15, 16), did not affect the chromatophores (data not shown). Aggregation of both chromatosomes at the perikarion of the novel dichromatic chromatophores displayed a very dark color due to the additive color mixture of the two hues (Fig. 3D). These observations suggest that the novel dichromatic chromatophores are motile like the other dendrhic light-absorbing chromatophores in teleosts. Further, these observations show a possibility that adrenergic stimulation is a cue to control the motile activities of the dichromatic chromatophores.
Here we found novel dichromatic chromatophores containing both cyanosomes and erythrosomes in the cytoplasm and displaying two hues, blue and red. To date, these novel dichromatic chromatophores have been found only in the mandarin fish. It is quite likely, however, that other species, either in the same genus or in other genera (for example, the psychedelic fish Synchiropus picturatus) also possess novel dichromatic chromatophores for displaying integumental color.
This work was supported by the Japan New Energy and Industrial Technology Development Organization (NEDO).
(1.) Bagnara, J. T., and M. E. Hadley. 1973. Chromatophores and Color Change. Prentice-Hall, Englewood Cliffs, NJ.
(2.) Fujii, R. 1993. Coloration and chromatophores. Pp. 535-562 in The Physiology of Fishes, D. H. Evans, ed. CRC Press, Boca Raton, FL.
(3.) Goda, M., and R. Fujii. 1995. Blue chromatophores in two species of callionymid fish. Zool. Sci. 12: 811-813.
(4.) Goda, M., M. Ohata, H. Ikoma, Y. Fujiyoshi, M. Sugimoto, and R. Fujii. 2011. Integumental reddish-violet coloration owing to novel dichromatic chromatophores in the teleost fish. Pseudochromis dia-demo. Pigment Cell Melanoma Res. 24: 614-617.
(5.) Robinson, W. G., Jr., and J. S. Charlton. 1973. Microtubules, microfilaments. and pigment movement in the chromatophores of Palaemotzetes vulgaris (Crustacea). J. Exp. Zool. 186: 279-304.
(6.) Ide, H., and T. Hama. 1976. Transformation of amphibian irido-phores into melanophores in clonal culture. Dev. Biol. 53: 297-302.
(7.) Ide, H. 1978. Transformation of amphibian xanthophores into melanophores in clonal culture.J. Exp. Zool. 203: 287-294.
(8.) Matsumoto, J. 1965. Studies on fine structure and cytochemical properties of erythrophores in swordtail. Xiphophorus helleri, with special reference to their pigment granules (pterinosomes). J. Cell Biol. 27: 493-504.
(9.) Lythgoe. J. N., and J. Shand. 1982. Changes in spectral rellexions from the iridophore of neon tetra. J. Physiol. 325: 23-34.
(10.) Kasukawa, H.. N. Oshima. and R. Fujii. 1987. Mechanism of light reflection in blue damselfish motile iridophores. Zool. Sci. 4: 243-257.
(11.) Oshima. N., H. Kasukawa, and R. Fujii. 1989. Control of chromatophore movements in the blue-green damselfish. Chromis viridis. Comp. Biochem. Physiol. 93C: 239-245.
(12.) Goda, M., J. Toyohara, M. A. Visconti, N. Oshima, and R. Fujii. 1994. The blue coloration of the common surgeonfish, Paracan-thurus hepatus. I. Morphological features of chromatophores. Zool. Scl. 11: 527-535.
(13.) Goda, M., and R. Fujii. 1998. The blue coloration of the common surgeonfish. Paracanthurus hepatus. II. Color revelation and color changes. Zool. Scl. 15: 323-333.
(14.) Fujii, R. 1959. Mechanism of ionic action in the melanophore system of fish. I. Melanophore-concentrating action of potassium and some other ions. Antwt. Zool. Jpn. 32: 47-59.
(15.) Fujii, R., and N. Oshima. 1986. Control of chromatophore movements in teleost fishes. Zool. Sci. 3: 13-47.
(16.) Fujii, R., and N. Oshima. 1994. Factors influencing motile activities of fish chromatophores. Pp. 1-54 in Advances in Comparative and Environmental Physiology, Vol. 20, R. Gilles, ed. Springer-Verlag, Berlin.
MAKOTO GODA (1), YOSHINORI FUJIYOSHI (1), MASAZUMI SUGrMOT0 (2), AND RYOZO FUJII (2)
(1.) Cellular and Structural Physiology Institute (CeSPI), Nagoya University, Furo-cho, Chikusa, Nagoya 464-8601, Japan; and (2.) Department of Biomolecular Science, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan
Received 24 April 2012; accepted 26 November 2012.
* To whom correspondence should be addressed. E-mail: email@example.com
|Printer friendly Cite/link Email Feedback|
|Author:||Goda, Makoto; Fujiyoshi, Yoshinori; Sugimoto, Masazumi; Fujii, Ryozo|
|Publication:||The Biological Bulletin|
|Date:||Feb 1, 2013|
|Previous Article:||Developmental modification of synaptic NMDAR composition and maturation of glutamatergic synapses: matching postsynaptic slots with receptor pegs.|
|Next Article:||Ferulic acid: a natural antioxidant against oxidative stress induced by oligomeric A-beta on sea urchin embryo.|