INTRODUCTION

Body coloration of animals is important for their behaviors (Edmunds, 1974; Endler, 1978). The body color of lower vertebrates is determined by the types of chromatophores in the skin (Bagnara et al., 1979). According to Bagnara (1966), melanophores appear first in the dorsal integument of a larva during development. Xanthophores, which have pteridines and/or carotenoids, appear second; and iridophores, containing purine crystals, or the so-called reflecting platelets, appear last. There are two ways for formation of the body coloration. One is that body color is produced by auto-synthesis of pigments. In fact, Ziegler et al. (2000) showed an active metabolic pathway of pteridine synthesis in the zebrafish. The other occurs by the uptake of pigments directly from foods. In amphibians, 3 kinds of pigment cells, i.e., melanophores, xanthophores and iridophores, are seen in the ventral skin of the juvenile after metamorphosis (Bagnara, 1998). In a previous study, we described the ultrastructure of carotenoid granules in xanthophores in the ventral skin of the wild Japanese adult newt C. pyrrhogaster, and found many rcv in them (Matsui et al., 2002). This suggests that the body coloration of the Japanese newt depends on the number and the carotenoid content of carotenoid granules (hcg and rcv) in these xanthophores in the ventral skin. However, it still remains unknown whether the size and the number of hcg and rcv and also the carotenoid content in the ventral skin change during the growth of this animal. Thus, histological and biochemical studies were carried out to understand the formation of the red coloration of the ventral skin of the adult Japanese newt C. pyrrhogaster.

MATERIALS AND METHODS

RESULTS

Fig. 1 shows the color of the skin of a larva at stage 59 (a), a juvenile newt at metamorphosis (b), and small (left), medium (middle) and large (right) wild juvenile newts (c). As can be seen in Fig. 1, the color of the ventral skin of a larva at stage 59 and a small juvenile was creamy, whereas that of medium and large juveniles was red. Then, we examined hcg in the xanthophores in the skin of newts at different stages. As shown in Fig. 2a, a few hcg were observed in the creamy skin of larvae at stage 59, as indicated by the black arrowhead. Their average size and the number were 0.78±0.07 μm (mean±SEM) and 0.99±0.62/100 μm² of a xanthophore (mean±SEM), respectively. Only hcg were also observed in the creamy skin of newts at metamorphosis (Fig. 2b); and their average size and the number were 0.85±0.05 μm and 1.35±0.58/100 μm², respectively. In the skin of small, medium and large juveniles, only hcg were seen (Fig. 2d, 2e). Sizes of the hcg decreased dramatically to 0.35–0.45 μm, but the number dramatically increased to 99–101/100 μm². However, in the skin of adult newts, many rcv and a few hcg were found (Fig. 2f). As shown in Table 1, average size of carotenoid granules (rcv and hcg) in the red skin of adult newts was larger (0.46 μm) than that of hcg in the creamy skin of small juveniles (0.35 μm) (p<0.05). However, the number of rcv and hcg in the skin between adult newts and small juveniles was not different (117±17 vs 101±13/100 μm²), as shown in Table 1 and Fig. 3. In addition, average sizes of hcg in the ventral skins of larvae at stages 44 to 59 and juveniles at metamorphosis were relatively large (0.70–0.85 μm), but dropped significantly in the creamy skin of small juveniles (0.35 μm) (P<0.05) (Table 1). Then, the size of carotenoid granules increased in the red skin of medium and large juveniles and adult newts (0.45–0.47 μm) (p<0.05) (Table 1). Moreover, total area of carotenoid granules was 0.77 μm²/100 μm² of a xanthophore in the ventral skin of juveniles at metamorphosis when the out line of carotenoid granules was assumed to be circular, but significantly increased to 9.76 μm² in small juveniles and then to 19.49 μm² in adult newts (P<0.01) (Table 1).

Fig. 1

Color of the ventral skin of a larva, a juvenile at metamorphosis, and juveviles of 3 sizes of the Japanese newt Cynops pyrrhogaster. (a) A larva at stage 59. (b) A newt at metamorphosis. (c) Small (left), medium (middle) and large (right) juveniles. Bar=1 cm.

Fig. 2

Ultrastructures of hcg and rcv in a xanthophore in the newt skin. (a) A larva at stage 59. (b) A juvenile at metamorphosis. (c) A small juvenile. (d) A medium juvenile. (e) A large juvenile. (f) An adult newt. Black and white arrowheads indicate hcg and rcv, respectively. A pterinosome is indicated by the white arrow. Bar=5 μm.

Table 1

Number (N) and Size (S) of carotenoid granules in the ventral skin of growing newts at different stages. Total area (μm²) of caro-tenoid granules was calculated by π(S/2)²x N, assuming a circular outline of carotenoid granules in Fig. 2. The letter n means the number of animals. Values are presented by the mean±SEM.

Fig. 3

Relationship between the number and size of carotenoid granules. Dotted and solid lines indicate the number and size of carotenoid granules, respectively. Numbers of granules in several areas of 100 μm² in the sections are counted and plotted in the figure. Vertical lines incicate the mean±SEM. M, a juvenile at metamorphosis. S-j, a small juvenile. M-j, a medium juvenile. L-j, a large juvenile. Ad, an adult newt.

Finally, we analyzed the contents of carotenoids in the whole ventral skin of a juvenile and an adult. Contents of total carotenoids in the ventral skin were determined by the methods of TLC and HPLC. The results are shown in Fig. 4. The content of total carotenoids was low in the creamy skin of small juveniles (<2 μg/total ventral skin), but was very high in the red skin of the adult newts (approximately 35 μg/total ventral skin) (Fig. 4). The coefficient correlation was y =0.049 · e(0.12x) (r=0.84).

Fig. 4

Change in the content of total carotenoids in the ventral skin of newts. SVLs of newts at different stages are shown on the right side of the panel. Abbreviations at different stages are the same as described in the legend to Fig. 3. A regression curve was obtained according to Beyer (1976). (○) The carotenoid content measured by the TLC method. (•)The carotenoid content measured by the HPLC method.

DISCUSSION

The ventral skin of the wild Japanese newt C. pyrrhogaster is creamy at metamorphosis, but turns red as the animal matures. Previously we described the ultrastructure of carotenoid granules in the red ventrum of wild and laboratory-reared Japanese adult newts (Matsui et al., 2002). In the electron-microscopic study we found many rcv (85%) and a few hcg (15%) in the red skin of wild adult newts. The red skin of a wild adult newt contained a large amount of carotenoids, but the yellow skin of a laboratory-reared adult newt did not (Matsui et al., 2002). Therefore, the amount of carotenoids in the ventral skin seems to be very critical for producing the red ventral coloration of the Japanese newt C. pyrrhogaster. This hypothesis is supported by the present findings that 3 factors (size, number and carotenoid content of carotenoid granules in the ventral skin) change during the growth of the Japanese newt C. pyrrhogaster. It is well-known that the body coloration of amphibians is determined by chromatophores (melanophores, xanthophores, and iridophores) in the body skin. Xanthophores in the ventral skin of the newt cannot synthesize carotenoids (Britton, 1983; Bagnara, 1998). Therefore, the cells must uptake carotenoids through foods to produce the red ventral color. However, as the mechanism is still unclear, it will be important to investigate what kinds of foods juvenile newts eat routinely and what sorts of pigments are contained in these foods.

It should be noted that only hcg were seen in the creamy skin of small juveniles but that many rcv and a few hcg were observed in the red skin of the adult newts. The average size of rcv and hcg in the red skin of adult newts is larger than that of hcg in the creamy skin of small juveniles (Table 1 and Fig. 3). Furthermore, the number of rcv and hcg/100 μm² of a xanthophore in the red skin of adult animal was very similar to that of hcg/100 μm² of a xanthophore in the creamy skin of the small juvenile, indicating that the number of rcv is increased whereas that of hcg is decreased. However, this does not mean that hcg are replaced by rcv. Also, the amount of carotenoids in the red skin was much greater than that in the creamy skin. Thus, the size and the number of carotenoid granules, as well as the carotenoid content in the granules, are important factors for the red coloration of the ventral skin of the Japanese newt. As reported previously (Matsui et al., 2002), β-carotene and lutein were retained on the filter paper, but astaxanthin was not, when carotenoids on the paper were treated with OsO₄ and propylene oxide. It is evident that the amount of carotenoids in rcv and hcg in the red skin of the adult newt was much greater than that in hcg in the red skin of the large juvenile (see Fig. 2e, 2f, Fig. 4). It is, therefore, very likely that rcv were formed from hcg after some carotenoids in the hcg had been extracted during TEM processing. It should also be noted that hcg in small juveniles appeared before carotenoids began to accumulate. As far as we know, this is the first report showing that the appearance of carotenoid granules in a pigment cell precedes the accumulation of carotenoids in them during the growth of the newt.

The Japanese newt C. pyrrhogaster has pterinosomes containing pteridines in xanthophores in the ventral skin. A few kinds of pteridines have been found in the skin of larvae, but not adults, of the Japanese newt C. pyrrhogaster (Obika, 1963). The skin of the adult newt instead contains a large amount of riboflavin (Obika, 1963). As it can be seen in Fig. 2, many pterinosomes were present in the skin of larval, juvenile and adult newts. However, we do not know at the present time how pterinosomes are involved in producing the red coloration of the ventral skin of the newt. The color of the ventral skin of the wild axolotl Ambystoma mexicanum is creamy. In the skin of this animal, no carotenoid was detected (Frost et al., 1984). The American newt Notophthalmus viridescense and the toad Bombina orientalis have xanthophores (erythrophores) in their skin, in which many carotenoid granules are present (Forbes et al., 1973; Frost and Robinson, 1984). Further studies are required to explain how carotenoids and pteridines are involved in the red coloration of the ventral skin of the Japanese newt C. pyrrhogaster. Nevertheless, the size, the number and the carotenoid content of hcg and rcv need to be increased for the red body coloration of the Japanese newt C. pyrrhogaster.