198. Studies on the Chromatophores of Oryzias latipes (Teleostean Fish) : Behavior of the Pteridine, Fat and Carotenoid during Xanthophore Differentiation in the Color Varieties*)

Materials and methods.
The four homozygous strains (wild, blue, orange-red, and white) were used. The developmental stage was defined by the whole length of the embryo or larva. The chorion was removed by the treatment of Pronase P (Kaken-Kagaku Co., Ltd.). The following tests were tried under the high magnification on the definite xanthophore which was located along the head cartilage near the eyeball.

Fat test. The fat was identified by the red color staining by Sudan III, IV, and the bluish black by Sudan black B.

Carotenoid test. The carotenoid was identified by the positive reaction of two kinds of reagents, viz., the concentrated sulphuric acid (H2SO4) and the iodine-potassium iodine (I-KI). The former showed the sequential color change of green, violet and finally blue, and the latter a green color.

Identitication of pteridines. The pteridines were identified principally by the paperchromatography. Details were previously reported.2)

Results
1. Normal development of the xanthophores in the four color varieties.
The xanthophores were first found in the 2.0-2.5 mm long embryo before hatch (hatching embryo, 4.5-5.0 mm). The xanthophores were fillled up with the deep yellow granules of almost equal size, followed with the deposition of yellow fat droplets. This aspect was the same among the four color varieties. After this stage, the xanthophore differentiation was different depending on the color varieties. In the rr strains (blue and white), the deep yellow color of granules became light with development, and finally the adult xanthophore was composed of the yellow colored droplets and non-colored granules which filled the cell. In the RR strains (wild and orange-red), the orange-red dots appeared at the time of hatch or before that. These larvae beared a pale yellow coloration on their whole body surface. The deep yellow color of granules became light in the 6.0-7.0 mm long larvae, and very faint in the 12 mm long adult. In reverse to this, the orange-red dots increased until the maximum coloration of the adult xanthophore. The adult xanthophores were composed of orange-red dots, yellow droplets, and yellowish or non-colored granules filled up within the cell.

2. Localization of sepiapterins in the larval xanthophore.
The isolation of larval xanthophores alone was difficult in Oryzias. The eyeball at the time of hatch had the xanthophores, melanophores and iridophores. It has been known that the pteridines in the latter two chromatophores were scarcely found (e.g. Bagnara 1961).3) This standed for Oryzias. The kinds of pteridines of eyeball were as follows: hynobius-blue, 2-Amino-4-hydroxypteridine, isoxanthopterin, xanthopterin, dihydroxanthopterin, and sepiapterins (sepiapterin and isosepiapterin). Thus, it may be said that these pterins were of the larval xanthophores. These pterins were also in some relation to the metabolism of sepiapterins.4)

3. Transformation of sepiapterinosome.
The following results were reported in our previous paper.2) The sepiapterins and drosopterins (drosopterin, isodrosopterin, and neodrosopterin) were found much in the hatched larvae. However, these pterins decreased their amounts in the 6.0-7.0 mm long larvae, and disappeared almost in the adult. As described above, the xanthophore color resulted from the deposition of fat or/and carotenoid pigments after the first yellow granulation. The real color of granules was clarified by eliminating the fact and carotenoid. The xanthophores at various developmental stages were treated with the organic solvents e.g. petroleum ether, acetone, chloroformCethanol, methanol, benzol or carbon bisulphide. The yellow color at the onset of xanthophore was not at all changed with the treatment of organic solvents, that is, treated xanthophore was filled up with the deep yellow granules alone. The deep yellow color of granules became faint in the 6.0-7.0 mm long larvae and very faint in the 12 mm long adult. The adult xanthophores were filled up with the faintly yellowish or almost non-colored granules which were previously named medaka-pterinosomes.2) The kinds of pteridines at various stages were examined in those which were extracted with dilute ammoniacal alcohol after the pretreatment of organic solvents. The result indicated that the decrease of sepiapterins was in parallel with that of yellow color of granules during xanthophore differentiation. Concerning the pterin pattern, no difference was found among the four kinds of color varieties. From the above facts, the properties of the yellow granules of larval xanthophore seemed to be transformed gradually into those of non-colored ones of adult type. Here, we suggest the "sepiapterinosome" for the deep yellow granules of the larval xanthophore. This name seemed to be responsible for the almost all kinds of the larval xanthophores in the fish and amphibians.5)

4. The two types of carotenoid and acarotenoid xanthophores.
Fat test. The result was summarized in Table I. As indicated in Table I, the fat test was positive in about 3.0 mm long larvae. The shape of xanthophore was clearly detected by the intracellular distribution of stained red or bluish black granules. The state of distribution and the dendritic shape of xanthophores in the blue and white color varieties were almost the same compared with those of xanthophores in the wild and orangered.6) This is why the yellow pigment cells in the rr strains belong to the category of the xanthophre regardless the lack of carotenoids.

Carotenoid test. a. Blue and white color varieties. The xanthophores were all negative in carotenoid test through the whole developmental stage.

b. Wild and orange-red color varieties. The result was summarized in Table II.

The carotenoid test of H2SO4 seemed to be more sensitive than the iodine test. The carotenoid test became positive in about 3.5 mm long larvae. In Table II, the orange-red dots containg xanthophores were unmistakably positive in carotenoid test. However, the xanthophores before the deposition of orange-red dots were also positive in carotenoid test, that is, a faint blue coloration was seen diffusely within the xanthophore. Again, close examination was done paying attention to the fragile blue color. The result was summarized in Table III.


The Table showed that the carotenoids appeared evenly within the xanthophore so early before the appearence of orangeied dots. A possibility of the simultaneous appearence of fat and carotenoid was thought. According to Takeuchi,7) the xanthophore carotenoids of Oryzias is transferred from the yolk carotenoids and the food, and the quantity of yolk carotenoids plays an important role for the carotenoid deposition within the xanthophore. There was no relation with food in our experiment since many of the above tests were carries out before hatch. However, the blood circulation was already taken place in the 2.0-2.5 mm long larvae, so that the carotenoid transfer is to be possible in the materials used. The kinds of carotenoids of larval xanthophore is unknown. At any rate, regardless the presence of yolk carotenoid, no carotenoids were found in the blue and white color varieties. The carotenoid lacking xanthophore of these rr strains may be called the "acarotenoid xanthophore". Contrary to this, the carotenoid containg xanthophore of the wild and orange-red ones (RR strains) the "carotenoid xanthophore" or xanthophore. The gene action of R and r may be ascribed to the presence and absence of capacity which make the carotenoid appear within the xanthophore respectively.

5. Relation among the pterins, fat and carotenoids during xanthophore differentiation.
The first sign of the onset of xanthophore was pointed out in 1963 by one of the authors to be the yellow granules which contained sepiapterins in the fish and amphibians.5) These yellow granules were called the sepiapterinosomes in this paper. The differentiationg processes of xanthophore were concluded as follows:

In this scheme, the second step adopts a different metabolic pathway according to the acarotenoid and carotenoid xanthophores.

a. Acarotenoid xanthophore of the blue and white color varieties.

In the overall scheme (1 and 2), the first step proceeds rapidly, and the second step expresses the gradual decrease of sepiapterins within the xanthophore. In the adult xanthophore, a mechanism in which the sepiapterins, if they were synthesized, would be rapidly excreated was assumed, for the sepiapterins were always found in the adult skin and scales by the use of a large amounts of materials. A simmilar phenomenon was seen in the case of leucophore. The erythrophore laden with drosopterinosomes, that is, larval leucophore was transformed into the adult leucophore by the gradual excretion of reddish orange drosopterins, and the ceaseless excretion of drosopterins was demonstrated to be taken place in the adult leucophore9) (unpublished data).

b. Carotenoid xanthophore of the wild and orange-red color varieties.


In this overall scheme (1 and 3), a possibility was thought that the first and second steps may occur at the same time.