GENETIC AND DEVELOPMENTAL ANALYSIS OF SOME NEW COLOR MUTANTS IN THE GOLDFISH, CARASSZUS AURATUS

TAKA0 KAJISHIMA
Biological Institute, Faculty of Science, Nagoya University, Nagoya 464, Japan

Manuscript received August 31, 1976 Revised copy received January 4 1977 ,
ABSTRACT

The genotypes of three color mutants i n goldfish: a depigmentation character of larval melanophores, albinism and a recessive-transparent character, were analyzed by crossing experiments. The depigmentation character in the common goldfish is controlled by two dominant multiple genes, Dp, and Dpd, and only fish with double recessive alleles dp,dp, dp,dp, can retain larval melanophores throughout life. Albinism is also controlled by double autosomal genes, p and c. The genotype of an albino fish is represented by p p cc; the non-albino fish is PP CC. Fish with either a p p CC or p p Cc genotype are albino when scored at the time of melanosome differentiation in the pigment retina, but after the time of skin melanophore differentiation, they change to the nonalbino type under the control of the C gene. The recessive-transparent character is controlled by a single autosomal gene, g . The mechanisms of gene expression of these characters were proposed as a result of observation and/or experimental data on the differentiation processes of their phenotypes, and the genotypes of these color mutant goldfish were considered in relation to the "gene duplication hypothesis in the Cyprinidae."

HE goldfish is one o l the most popular animals used as a n experimental

subject in many laboratories. Although it has many varieties of color and form, only a few genetic analyses of these characters have been carried out. One of the reasons or this is that a great deal of time and space is required to raise a sufficient number of off spring. Furthermore, as has been pointed out, many characters of the goldfish are controlled by multiple factors, so that it is difficult to get invariablc and regular ratios that fit Mendelian expectations (CHEN 1934). The first report on the genetics of the goldfish was that of HANCE 924), who (1 reported the recessiveness of the telescope-eye to the normal-eye, although no experimental data were shown. The first well-founded case of a Mendelian character in the goldfish was reported by BERNDT(1925) and CHEN (1925, 1928) on the transparency and mottling character of the scales. A few years later, MATSUI 934) reported his detailed results on the genetic analysis of the (1 goldfish varieties involving scale-transparency and telescopeeye. I n the same year, CHEN (1934) reported on the inheritance of blue and brown coloration in the goldfish, and concluded that there are four pairs of independent factors controlling the production of these characters, and only when all of these pairs were in the homozygous recessive condition was the brown coloration produced.
Genetics 86: 161-174 May, 1977

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When rearcd at 21 C, the common goldfish begins to differentiate the melanosomes in its retinal pigment cells about 36 hours after spawning (Stage-21, developmental stage from KAJISHIMA 1960d), and more than 14 hours later three kinds of skin chromatophores begin to differentiate successively; at first the melanophores (50 !iours after spawning, St-22) ,then iridophores (80 hours after spawning, St-24) and finally xanthophores (50 hours after hatching, 150 hours after spawning, St-26). Then the fry usually have black eyes and blackishbrown coloration. However, as is well known, the larval melanophores of the common goldfish begin to depigment about two to three months after hatching, and within a month the fish becomes xanthic to deep orange in coloration. On the other hmd, the black moor (black telescope-eyed goldfish) usually retains its melanophores throughout life. I n the present paper, the depigmentation and nondepignien tation character of larval melanophores is analysed first. Albinism is a common mutation, which has been reported in many animals, including the fish. In the goldfish, however, it was not reported until 1956 in an American hobbyists magazine, The Aquarium. According to the report, albino goldfish were offspring of an albino fish found in Singapore, but unfortunately and all of the fish died without any genetic investigations. In 1967, YAMAMOTO his colleague, TOMITA, albino goldfish in Favors Aquarium in New York, found and the following year they brought the live fish back to our laboratory. These fish did not seem to be related to the 1956 strain, because the eye character of the 1956 fish secmed to be genetically dominant (normal-eye) in type, whereas the present animals are all homozygous recessive (telescope-eye) in type. The original fish and their progeny lack melanosomes not only in the skin, but also in the retinal pigment cells from the earliest developmental stage; they are orange in color and have pink eyes. The results of genetic analyses of albino goldfish that were carried out by crossing albinos with various genetically defined, color-mutant fish are described, and the processes of the phenotypic expression of the albino genes, including the mechanisms of their functions, are discussed. From 1957 to 1960 we were engaged in experiments to clarify the mechanisms of gene function of the dominant transparent-scaled character T (KAJISHIMA 1960a,b,c; MATSUMOTO, KAJISHIMA HAMA and 1960). During that time, a new recessive transparent mntant was discovered. The results of the gene analysis of this mutant are also reported here. Recently, OHNO and his co-workers have proposed the hypothesis that some of the teleosts of the family Cyprinidae duplicated their chromosome numbers during the evolutionary process, and the genomes of these fish are tetraploid and not diploid (OHNO 1970). The data obtained from the genetic analyses of color mutants in the goldfish will be considered in relation to this hypothesis.
MATERIALS AND METHODS

The fish used in these experiments were of the following varieties: The common goldfish, wakin: The wakin acquires its mature coloration following destruction of the larval melanophores. The fish used in these experiments were inbred more than ten

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generations by brother-sister matings in our laboratory, and all of the offspring had depigmented melanophores within a year. T h e red telescope-eyed goldfish: The red telescope fish also loses its skin melanophores during its life, but the time of depigmentation varies extensively, usually occurring at a n age of more than two years. Until that time, it cannot be distinguished from the black moor. However, after depigmentation it has a deep orange coloration and usually does not develop melanophores again. The fish used in these experiments were progenies which were inbred more than five generations. The black telescope-eyed goldfish, black moor: The black moor usually does not show depigmentation of its melanophores throughout its entire life and produces more of them during development. The fish used in these expeiiments were also inbred in our laboratory more than ten generations and during that period none of the individuals lost their melanophores. The transparent-scaled goldfish: The adult homozygous transparent-scaled goldfish is pinkish in coloration and has no pigment except in the retinal pigment cells. At the time of hatching, however, it has the same three kinds of chromatophores as the common goldfish, i.e., melanophores, iridophores and xanthophores. But when the fry reaches six to seven mm in length (St-27+), about ten days after hatching, all three kinds of chromatophores begin to depigment and within a month they acquire the final coloration. The transparent-scaled character ( T T ) is incompletely dominant to the normal-scaled character ( I t ) , and heterozygous F, fish show a characteristic calico pattern (CHEN1928; MATSUI 1934) following partial depigmentation of the three kinds of chromatophores. The F, offspring segregate into the homozygous transparent type, heterozygous transparent type (calico) and normal type i n the ratio of 1:2:1. The homozygous fish used i n these experiments were also inbred more than ten generations. The albino goldfish: The albino goldfish used in these experiments were the inbred offspring of the albino goldfish discovered in Favors Aquarium. Albino goldfish have no melanosomes in the skin or the retinal pigment cells, and they have a n orange body color and pink eyes. As mentioned above, in the nonalbino goldfish (wakin, black moor, transparent-scaled goldfish, etc.) the retinal pigment granules become visible a t St-21, and the skin melanophores begin to appear at St-22. In the analysis of albino genes, the scoring of phenotype was carried out in two different stages; first in St-21, soon after the differentiation of retinal pigment granules, and then in St-27, when the skin melanophores are completely developed. T h e recessiue transparent-scaled goldfish: Fish carrying this character were isolated in our laboratory in 1960 from the F, offspring of homozygous transparent fish and wakin. In one of these matings, six new mutant fish appeared, which almost completely lacked pigment granules in their iridophores. These fish were apparently different from the ordinary transparent-scaled fish, because they did not differentiate the pigment granules from the time of iridophore differentiation (St-24), although some of the fish later synthesized them incompletely. The melanophores and xanthophores differentiated as usual in the embryonic stage, and they did not depigment a t the time the pigment cells degenerate i n the ordinary transparent-scaled goldfish (St-27-k). Two of them, however, lost their melanophores two to three months after hatching, at the time of depigmentation of larval melanophores in the common goldfish. The remaining four have retained their melanophores throughout their lives. The former type was designated red recessive-transparent fish (simply, red transparent fish) and the latter was black recessive-transparent fish (simply, black transparent fish) . All of the matings were carried out between a single male and a single female under natural breeding conditions. The specific matings are presented in the results. There was no evidence of sex linkage for any of the mutations in any of the experiments, and it is assumed that all of the mutations are autosomal.
RESULTS

I. Analysis of the depigmentation character of larval melanophores:

( A ) Crosses of wakin with black moor: Results of crosses between wakin and

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TABLE 1

Resulis of crosses between wakin and black moor goldfish

Crosses Number of offspring Depigmented Pigmented

Ratio

XZ

Wakin

x Black Moor (F,)

x F, x Black Moor

Fl F,

478 185 62

0 12 26

15:l 3:l

0.9 (P=O.5-0.3) 0.97 (P=O.5-0.3)

black moor F,, F, X F, fish, and F, fish backcrossed to black moor fish are given in Table 1. It is apparent that the character f o r depigmentation of the melanophores in the wakin is dominant to the character for nondepigmentation in the black moor. The results of the F, X F, and F, x black moor are consistent with the assumption that the depigmentation character of wakin (nondepigmentation character of black moor) is controlled by two multiple genes. The results presented in Table 1 are the final phenotypes of the fish after four years; however, not all fish that were eventually scored as depigmented started to lose their pigment at two to three months after hatching, as do the wakin goldfish. Some of the fish in the F, x F, crosses and F, x black moor cross did not depigment until their third year. To ascertain the genotypes of the depigmented F, individuals, the fish that depigmented in the first year, second year and third year were backcrossed to the black moor. As shown in Table 2, backcross offspring of four of the five fish which depigmented in their first year all depigmented completely, while offspring of the fifth fish segregated in the ratio of 3: 1 depigmented to pigmented. Three crosses out of five involving second-year depigTABLE 2

Result of back crosses between depigmenied F , (wakin

Age of depigmentation (year)

black moor) and black moor

XZ

No.

Number of offspring Depigmented Pigmented

Ratio

First

1 2 3 4 5 1 2 3 4. 5

67 78 62 53 72 62 87 47 59 132
21 47 31 24

23 -

3:l -

13

3:l 3:l

0.62 (P=O.5-0.3) -

Second

19 55 29 30

21

0.36 (P=0.7-0.5) 0.07 (P=0.8-0.7) 0.10 ( P ~ 0 . 8 - 0 . 7 ) 0.62 (P=O.5-0.3) 0.06 (P=0.9-0.8) 0.66 (P=0.5-0.3)

Third

1 2 3 4

1:l 1:l 1:l 1:l

The numbers represent the total number of depigmented or nondepigmented offspring for four years.

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mented F, produced all depigmented progeny, and in the other two crosses the segregation ratio of the depigmented and pigmented types was 3: 1. All the offspring of third-year depigmented F, segregated in the ratio of 1: 1 depigmented to pigmented. The differences in segregation ratios in these crosses can be explained by the differences in the number and combination of dominant and recessive genes. Those parental fish that produced all depigmented offspring may be homozygous dominant for one or both depigmentation genes, and those fish producing the ratio of 1:1 may be homozygous recessive at one locus and heterozygous a t the other. The fish whose offspring segregated in the ratio of 3:1 must have both dominant genes in the heterozygous condition, because the same ratio was observed in the backcross of wakin and black moor F, to black moor (Table 1 ) . These results indicate that the onset of depigmentation is related to the number of depigmentation and/or nondepigmentation genes. (B) Crosses of red telescqpe fish w t wakin and black moor: The parental red ih telescope-eyed fish were inbred for five generations in our laboratory. However, the offspring of these crosses always included some of the nondepigmentation type (Table 3A) that could not be distinguished from the black moor. I n the depigmented offspring, more than two-thirds of the individuals lost their melanophores in the second to fourth year, thereby acquiring their final phenotype. From these results it became apparent that the red telescope fish are not homozygous, but heterozygous for the depigmentation genes. The results of crosses between red telescope fish and wakin (F,) and F, X F, are shown in Table 3A. All of the F, and F, offspring lost their melanophores.
TABLE 3

( A ) Results of inbreeding of red telescope fish and of crosses between red telescope fish and wakin ( B ) Results of crosses between black moor and red telescope fish ( C ) Results of crosses between homozygous transparent-scaled fish and wakin and between F , and black moor
Crosses Number of offspring Depigmented Pigmented Ratio

X2

Red Telescope x Red Telescope (A) Red Telescope x Wakin (F,)

80
156 309 81 36 93
194 144 134

30
0 0 0 41 35 0 9 47

F,

x F,

1:l 3:l

Black Moor X Red Telescope (1) (B) -__-- (2) (3 )

Homo. Transparent Wakin (F,) (C) F, X F, F, X Black Moor

0.3 (P=O.7-0.5) 0.37 (P=0.7-0.5)

15:l 3:l

0.56 (P=0.7-0.5) 0.09 (P=0.8-0.7)

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This result indicates that the early depigmentation character of the wakin is dominant to the later depigmentation character of the red telescope fish. When the red telescope fish were crossed with the black moor, the segregation ratios for the depigmented and nondepigmented type were divided into three types: (1) all of the offspring had depigmented melanophores within four years, (2) the ratio of the depigmented and nondepigmented type was 1:1, and (3) the experimental ratio was 3: 1 (Table 3B). These results may reasonably be explained by assuming that the parental red telescope fish had different genotypes for the depigmentation character. (C) Crosses of wakin with homozygous transparent-scaled fish: The F, offspring of this cross depigment part of their chromatophores during an early developmental stage, and the remaining melanophores began to depigment under the control of the depigmentation genes of the wakin more than two to three months after hatching. The F, offspring produced by crosses between F, individuals segregated into three types according to scale character, i.e., the homozygous transparent type 1: heterozygous transparent type (calico) 2: normal type 1. The final ratio of the depigmented and pigmented types in these normal-scaled fish was approximately 15: 1 (Table 3C) . The F, heterozygous transparent fish were mated to the black moor. The offspring segregated into the heterozygous transparent type and normal type in the ratio of 1: 1. The ratio of the depigmented and pigmented types to those with normal type was approximately 3: 1 (Table 3C). From these results, it was concluded that the homozygous transparent-scaled fish carries a double recessive genotype for the depigmentation character of the melanophores, and that the scale transparency and depigmentation characters are not linked to each other.

11. Analysis of albinism:

(A) Crosses of albino with wakin: All of the F, offspring were nonalbino in type, having black eyes and skin melanophores, though the latter depigmented within a year. These results suggested that albinism is recessive to the wakin, and that albino goldfish carry the double depigmentation genes. As shown in Table 4A, in one fourth of the F, offspring the melanosomes in the retinal pigment cells did not differentiate at the usual time of melanosome formation in the pigment retina (St-21). However, when the embryos reached the differentiation stage of skin melanophores (St-22) , in three fourths of the initially albino fish, melanosomes in the retinal pigment cells began to differentiate along with the skin melanophores. Although at first the number of melanosomes in the melanophores and pigment retina of the late-melanizing fish was less than in normally pigmented fish, the number increased gradually so that these fish became indistinguishable from normally pigmented fish in a few days, and the final segregation ratio of pigmented and albino fish was 15:l (Table 4A). The segregation ratio of offspring resulting from backcrosses of F, fish with albino was 1: 1 when scored at St-21, and the ratio of albino to pigmented was 1:3 after the stage of skin melanophore differentiation (Table 4A).

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TABLE 4

Results of crosres ( A ) between albino and wakin, ( B ) between albino and black moor and ( C ) between albino and homozygous transparent-scaledgoldfish, scored at St-21 and St-27
Crosses

Number of offspring Albino Pigmented

Ratio

X2

Albino (A)

F, x F,
F,

x Wakin (F,)
(St-21) (St-27) Albino (St-21) (St-27)

0 137 65 133 32
0

381 427 991 151

104

1:3 1:15 1:l 1:3 1:3 1:15 1:3

0.15 (P=O.7-0.5) 0.02 (P=O.9-0.8) 1.08 (P=0.3-01.2) 0.15 (P=0.7-0.5)

Albino x Black Moor (F,) F, X F, (St-21) (B) __-(St-27) F, x Albino (St-27) Albino x Transparentscaled fish (F,) (C) F, X F, (St-21) -_-_ (St-27)

258
144.

0.53 (Pz0.5-0.3) 0.10 (Py0.8-0.7) 0.10 (P=O.8-0.7)

54 30 33
0 1(32 80

425 101 563 3 02 1256

1:3 1:15

0.01 (P=0.95-0.9) 0.15 (Pz0.7-0.5)

(B) Crosses of albino with black moor: All of the F, offspring were pigmented, and the larval melanophores in the skin were depigmented during their lifespan. This suggests that the albino character in the goldfish is recessive to the black moor, though the albino carries the dominant depigmentation genes. The segregation ratios of the albino and nonalbino types in the F, offspring are shown in Table 4B, and the ratio of the albino and nonalbino types in offspring resulting from F, backcrossed with albino was 1:3. (C) Crosses of albino with homozygous transparent-scaled fish: The F, fish had the characteristic calico pattern, and in the F, offspring (both albino and nonalbino) three kinds of scale types segregated in the ratio of 1:2:1. The ratio of the albino and nonalbino types was 1:15 (Table 4C). Offspring exhibiting both the homozygous transparent and albino types, with no pigment at all in the skin or pigment retina, segregated in the ratio of 1:63. From these results it was ascertained that the albino and scale transparency character are not linked to each other.
1 1 Analysis of recessive-transparent character: 1.
(A) Crosses of recessive-transparent fish with homozygous transparent-scaled fish: All of the F, offspring were heterozygous transparent in type, having three kinds of pigment cells in a complicated calico pattern. This indicates that the recessive-transparent fish is homozygous normal-scaled in type and is recessive to the ordinary transparent-scaled type. The F, offspring segregated into the following three types: homozygous transparent-scaled type, heterozygous transparent-scaled type and normal-scaled type in the ratio of 1:2: 1; and in the normal-scaled type, the recessive-transparent type segregated in the ratio of 1 :3 (Table 5A).

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TABLE 5
Results of crosses ( A ) beiween recessive-iramparent fish and homozygous transparent-scaled fish, ( B ) beiween recessive-transparent fish and wakin and ( C ) between recessive-transparent fish and albino goldfish
Number of offspring
Crosses
Normal

Recessive transparent

Ratio

xz

Recessive-transparent x Homo. Transparent (F,)

3:l

0.10 (P=0.8-0.7)
0.39 (P=0.7-0.5) 0.17 (P=0.7-0.5)

(A)

F, X F , '
Recessive-transparent Wakin (F,)

22

7
0 19 0 91

x
390

3:l

(B)

F,

x F,
x

67 253 287

Recessive-transparent Albino (F,) (C) F, x F,t

3:l

* + Scored osnly in normal-scaled type. Scored only in non-albino type.

(B) Crosses of recessive-transparent fish with wakin: In the first mating a red transparent male fish was used, and in the second a black transparent female was used. I n both cases the F, offspring were all normal (wakin) type. But the depigmentation character of the larval melanophores was different iE the two matings. I n the first cross, all of the F, fish lost their larval melanophores within a year, but in the second, some of the offspring retained their larval melanophores for more than two years. These differences seemed to be caused by a difference in the genotypes of the depigmentation genes. The segregation ratio of normal and recessive-transparent type in the F, offspring is shown in Table 5B. From these results it became apparent that the wakin is dominant to the recessivetransparent type, and the latter character is controlled by a single gene. (C) Crosses of recessive-transparent fish with albino: All of the offspring were normal in type. The segregation ratio of the F, offspring is shown in Table 5C. From these results it became apparent that the albino goldfish is dominant for the recessive-transparent character, although the fish is recessive for the albino character.
DISCUSSION

Depigmentation character of the melanophore: I n the present experiments it became apparent that depigmentation of the larval melanophores in the wakin is dominant to nondepigmentation in the black moor, and that the character is controlled by two autosomal dominant multiple genes. When the two genes are both homozygous recessive, the fish retains its larval melanophores throughout its life. The symbols Dp, and Dp2 are proposed for the depigmentation type, and dp, and dp9 for the nondepigmentation type. The genotype of the wakin is represented by Dp, Dp, Dp2 Dp, and the black moor by dpl dpl dp, dp2.

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As mentioned in the previous section, red telescope fish always produced some nondepigmented offspring upon inbreeding, and it was concluded that the genotype of these fish is not homozygous dominant for the depigmentation character. The recessive transparent fish (both red and black) also did not seem to be homozygous for the depigmentation genes, although a detailed analysis was not carried out on this character. On the other hand, the segregation ratios in the F, and F, of crosses of homozygous transparent-scaled fish with wakin and of F, with black moor (Table 3C) were in complete agreement with the ratios in the crosses of black moor with wakin (Table 1 ) . From these results it became apparent that the homozygous transparent-scaled fish carries the same genotype as the black moor for the depigmentation character of larval melanophores. The segregation ratios of albino to wakin (Table 4A) lead to the conclusion that the fish carries the homozygous dominant genes for the depigmentation character. I n the backcross of F, with wakin and black moor with black moor, the segregation ratios of depigmented and nondepigmented type separated into three types (Table 2). These results led to the following conclusions: fish that depigmented within a year may carry the dominant depigmentation genes in the homozygous condition, and the later it occurs, the smaller the number of dominant alleles. Though the initiation of depigmentation in melanophores may be related to the number of depigmentation alleles, no qualitative nor quantitative differences between the functions of the two genes were found in the present experiments. Albinism: Albinism in the goldfish is not a simple Mendelian character, but is represented by the double homozygous condition of two recessive genes. A final segregation ratio of 1: 15 in the F, offspring resulting from crosses between the albino and non-albino types supports this conclusion. Moreover, developmental observations on the course of phenotypic expression revealed that three-fourths of the initially albino fish changed to the pigmented type in an advanced embryonic stage. Thus, the precise phenotypes in the F, offspring are in a ratio of 1 :3: 12 for the albino, late-pigmented and pigmented types. This ratio is characteristic of duplicate gene interaction in dominant epistasis, and the following symbols are proposed for the genes for melanin synthesis in goldfish (KAJISHIMA 1972).

P: Controls primarily the normal pigmentation of the pigment retina. The

homozygous recessive allele p causes pink eyes. C: Controls primarily the color formation in the skin melanophores and is epistatic to P and p . The homozygous recessive allele c causes a colorless condition in melanophores. The genotype of an albino is represented by pp cc, and the genotype of wakin, black moor, homozygous transparent-scaled fish and recessive transparent fish are all represented by PP CC. YAMAMOTO (1973) has proposed the symbols M and 5' for normal pigmented goldfish. The M gene controls normal melanin formation and normal melanophore develcpment; and S governs slower melanin and melanophore formation.

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The albino goldfish is represented by mmss, and the genotypes of the latepigmented type (YAMAMOTOS type) as mm SS or mm Ss. YAMAMOTO light and the present author agree that two genes are involved, but we differ somewhat regarding the function of the two genes. YAMAMOTO postulated that both the has M and S genes act on the same melanophores but in different developmental stages, but in the present experiments it appears that the P gene acts chiefly on the differentiation of melanosomes in the pigment retina, and the C gene controls melanosome formation in both the retinal pigment cells and skin melanophores. Recessive-transparent character: Genetic analysis of the recessive-transparent character made it apparent that the character is controlled by a single gene, not linked with the melanophore depigmentation genes Dpl and Dps, the albino genes p and c or with the transparent-scaled gene T , because in the F, offspring the recessive-transparent type segregated in the ratio of 1 :3 independently from these genes. The symbol g is proposed for this gene; the dominant allele G controls guanine synthesis in the iridophores and g lacks this potency (KAJISHIMA 1967). As shown in the crossing experiments with wakin, this character is further divided into two types, red transparency and black transparency, but these different types result from a difference in the genotype for the depigmentation character of the melanophores. MATSUI (1934) has found a net transparent-scaled goldfish which lacks iridophores in a network pattern in each scale. He called this character the netlike transparent-scaled type, and proposed the symbol n. He considered that the gene n and its dominant allele N are hypostatic against the transparent-scaled gene T , because only the tt nn individuals express the net-like transparent character. The phenotype of this character resembles very much the recessivetransparent character. I n the present experiments, however, it became apparent that the T and g genes are inherited independently and become functional at different developmental stages; G acts on guanine synthesis in an early pigment cell differentiation stage and T o n the degradation stage of the iridophores after assumptions. It was dishatching. These facts, however, do not deny MATSUIS appointing that MATSUIS were all lost by accident, so that it is impossible to fish analyze the relationship of the n gene with T and g. Developmental analysis of color mutant genes: The initiation of gene expression is restricted to certain developmental periods, and the color mutant genes analyzed in these experiments are good evidence for this statement. At first, one of the albino genes, P, becomes functional in the pigment retina soon after the differentiation of the optic cup, and 14 hours later the melanosomes begin to be synthesized in skin melanophores under the control of the C gene. During this interval, the albino or nonalbino phentoypes of the F, individuals are controlled only by the single gene P, and the segregation ratio represents a monohybrid ratio of 3:l. But when the embryos reach St-22, the time of skin melanophore differentiation, the C gene becomes functional and three-fourths of the initially albino fish begin to synthesize melanosomes in their melanophores and pigment retina, so that the segregation ratio finally changes to 15 :1.

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In previous experiments on albino goldfish (TAKEUCHI KAJISHIMA and 1974), we observed numerous granules containing various quantities of electron-dense material in the retinal pigment cells, and when the fish reached St-22, partially pigmented granules also differentiated in the melanophores. These granules increased in number gradually until the fish reached St-27, about a week after hatching, but thereafter they began to decrease in number until they completely disappeared. Because of these results, we considered that the albino genes may carry information for synthesizing pigment granules that are abnormal in structure and/or constitution. I n the present experiment, it become apparent that the C gene can repair the defect caused by the p gene in late-pigmenting fish. If this is true, both the C and P genes may code for similar information on melanosome formation, though the time of gene expression is somewhat different in each. The recessive-transparent gene, g, also becomes functional at the time of iridophore differentiation (St-24), and homozygous fish almost completely lack pigment granules in the iridophores. These results seem to lead to the assumption that the dominant allele G, controls the synthesis of pigment granules in iridophores, but g lacks this potency. About ten days after hatching, the transparent-scaled gene, T ,begins to express its phenotype following the destruction of the three kinds of chromatophores. The gene functions of this character have been studied by several authors. I n the heterozygous transparent fish, GOODRICH ANDERSON and (1939) observed that the melanophores, which remained heakhp without being destroyed by the transparent gene, begin to depigment about eight months after hatching. Prior to this report, GOODRICH HANSEN1931) considered that the melanophore and ( destruction in the transparent type might be caused by the same mechanisms as the depigmentation of melanophores in the common goldfish, and that the progression of gene function is proportional to the quantity of the controlling gene substance. The depigmentation of larval melanophores in the common goldfish usually occurs more than two to three months after hatching, and is caused by cellular destruction of the skin melanophores. I n electron microscopic studies on the process of melanophore depigmentation, TAKEUCHI KAJISHIMA ( 1973) have and observed melanophages, which ingested the melanosome debris of destroyed melanophores. Thus the depigmentation of larval melanophores in young goldfish may be caused by genetically programmed melanophore death. The initiation mechanisms of melanophore death have been studied by several authors. Experiments involving blinding (FUKUI 1927; GOODRICH HANSEN and 1931) and gonadectomy (GOODRICH HANSEN and 1931) have indicated no relationship between these factors and depigmentation. TERAO (1922) and BLACHER 1927) have observed melanophore depigmentation after feeding ( large doses of thyroid, but MULLER (1953) has observed that the effect of thyroid hormone is restricted to a shortening of the period of depigmentation. In previous experiments (KAJISHIMA 1975), it was ascertained that the initiation of depigmentation is controlled by an extrinsic factor(s), but the thyroid hormone and ACTH were excluded as possibilities for this triggering factor.

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T. K A J I S H I M A

As a result of the present experiment, it becomes plausible that the time of initiation of depigmentation is controlled by the number of depigmentation genes. When the dominant genes are in the homozygous condition, depigmentation usually occurs within a year, and the lesser the number of dominant genes, the later it occurs. Finally, in the homozygous recessive condition, the larval melanophores do not depigment throughout the life of the fish. These facts, however, do not contradict the assumptions of the previous paper. The quantity of gene products which carry out the melanophore destruction must be proportional to the gene dosage, and the production of such substances may be initiated by the triggering factor(s). But the nature of the gene product, and the reason its quantity affects the time of melanophore destruction, remain in question. Color mutant genes and Gem-duplication hypothesis: Recently OHNOand his coworkers proposed the Gene-duplication hypothesis in the fish family Cyprinidae (OHNOand ATKZN 1966; OHNOet al. 1967; WOLF al. 1969; et OHNO 1970). This hypothesis is chiefly attributed to the following three observations: (1) In some species of Cyprinidae, including the goldfish, the number of chromosomes are duplicated. (2) The comparative DNA content per cell is increased relatively along with the chromosome number. (3) The number of gene loci which control some of the dehydrogenase isozyme patterns are duplicated in tetraploid fish. If this is true, the number of gene loci which control the pigmentation of chromatophore and pigment retina may also be duplicated in the goldfish. The genotypes which have been analyzed in the present experiment are summarized in Table 6. It is remarkable that half of the characters in this table are governed by multiple gene pairs; especially the depigmentation character of melanophores in which two multiple genes, Dp, and Dp,,seem to be identical in function, and the albino genes, in which the information of the P and C genes seems to resemble each other. Though it is not cited in this table, CHEN (1934)
TABLE 6

The genotypes of color mutants in the goldfish

Gene function Mutants Melanophore Formation Destruction Iridophore Formation Destruction

Wakin (Common goldfish) Telescope-eyed fish Black (The Black Moor) Red Albino Recessive trsnsparent-fish Black Red Transparent-scaled fish Homozygous fish Heterozygous fish

pp
pp

cc cc

DPlDP, DP,DP,
dPldP, dP,dP,

GG GG

tt tt

PP

PP cc

cc

______
Dp,Dp, DP,DP,?
dP,dP, dP,dP, ~ P & P , dp,dp,

gg gg

tt tt TT Tt

PP cc PP cc

GG GG

COLOR MUTANTS I N GOLDFISH

173

has reported tetrahybrid inheritance in the brown coloration in the goldfish. These facts seem to support OHNOS hypothesis. On the other hand, the remaining two mutants, which are both related to the iridophore character, are each controlled by a single gene. However, for the transparent-scaled character, some question remains as to whether o r not it is actually controlled by a single gene. It has been pointed out that the results of the early experiments show that the homozygous transparent-scaled type TT does not always lose the iridophores completely, and frequently some of the fish retain a considerable number of chromatophores (not only the iridophores, but also the melanophores and xanthophores) so that sometimes the fish are not easily disand tinguishable from the heterozygous type Tt (CHEN1928; GOODRICH HANSEN 1931; MATSUI 1934; KAJISHIMA 1960a). These facts cannot be explained by the incomplete dominance of gene T. An hypothesis which can explain these situations is as follows: scale transparency is a character that is controlled by two multiple genes; one the T allele and the other an unknown wild-type allele. If this is true, the genotype of homoand the phenotype of these zygous transparent fish is represented by TT fish may be difficult to distinguish from that of the heterozygous fish Tt From 1957 to 1960, we examined more than fifty thousand F, fish resulting from crosses between the homozygous transparent-scaled fish and wakin, but were unable to get any specimens which verify the above hypothesis, so that it still remains in doubt at the present time. On the other hand, it was ascertained in the present experiment that the recessive-transparent character is apparently controlled by a single recessive gene. The same conclusion has been reported by MATSUI(1934) for the inheritance of the telescope-eyed character. To explain these exceptional cases, OHNO (personal communication) has proposed the hypothesis that the redundant copy of a gene can either be a new gene o r become degenerate, joining the rank of nonfunctional DNA base sequences. At the present time, however, we have no evidence to test this hypothesis, but a detailed analysis of these problems may be expected in the future.

++,

++.

The author wishes to express his hearty thanks to EMERITUS PROF. TOKI-o YAMAMOTO and who DR. H. TOMITA, kindly provided the opportunity to use the albino goldfish for this experiment. I am also grateful to Mrs. C. A. MAHI, of The University of Hawaii for critical reading of the manuscript.
LITERATURE CITED

BERNDT, W., 1925 Vererbungsstudien am Goldfischrassen. Zeits. Indik. Abst. Vererd. 36: 161-349. L. n BLACHER, J., 1927 The role of the hypophysis and of the thyroid gland i the cutaneous pigmentary function of amphibian and fishes. Trans. Lab. exp. Biol. Zoopark, Moscow 3: 79-81. CHEN, S . C., 1925 Variation in external characters of goldfish, Carassius auratus. Contri. Biol. Lab. Sci. Soc. China 1: 1-651. __, 1928 Transparency and mottling, a case of Mendelian inheritance i n the goldfish, Carassius auratus. Genetics 13: 434452. 1934 , The inheritance of blue and brown colors in the goldfish, Carassius auratus. J. Genet. 29: 61-74.

174
Japonica 5: 257-302.

T KAJISHIMA .

FUKUI, 1927 On the color pattern produced by various agents in the goldfish. Folia. Anat. K.

H. 1939 Variations of color pattern i n hybrids of the goldGOODRICH, B. and P. L. ANDERSON, fish, Carassius aumtus. Biol. Bull. 77: 184-191.
GOODRICH, B. and I. B. HANSEN, H. 1931 The postembryonic development of Mendelian characters in the goldfish, Carassius auratus. J. exp. Zool. 59: 337-358. HANCE, T., 1924 Heredity in goldfish. A note on the inheritance of dove-tail and telescopeR. eyes in goldfish. J. Heredity 15: 177-182.

KAJISHIMA, T., 1960a Analysis of gene action in the transparent-scaled goldfish, Carassius auratus. I. On the gene action in the disappearance of guanophoers. Embryoolgia 5: 107, 1960b Analysis of gene action in the transparent-scaled goldfish, Carasius 126. aura!us. 11. The effects of pituitary and thyroid on gene action. Embryologia 5 : 127-138.

-_ ,

1960c Analysis of gene action in the transparent-scaled goldfish, Carassius auralus.

1 1 Guanine metabolism in the developing larvae. Annot. Zool. Japon. 33: 245-248. 1.

1960d The normal developmental stages of the goldfish, Carassius auralus. Jap. Jour. Ichthy. 8 : 20-28. , 1967 Inheritance of a new recessive transparent fish i n the goldfish. Jap. J. Genet. 42: 418. (abstract) --, 1972 Genetics and embryological , studies of albino goldfish, Jap. J. Genet. 48: 351-352. (abstract) - 1975 In vitro analysis of gene depression in goldfish choroidal melanophores. J. exp. Zool. 191 : 121-126.

Y., MATSUI, 1931. Genetical studies on goldfish of Japan. J. Fish. Inst. Tokyo 30: 1-96.
MATSUMOTO, T. KAJISHIMA J., and T. HAMA, 1960 Relation between the pigmentation and pterin derivatives of chromatophores during development i n the normal black and transparent scaled type of goldfish (Carassius auratus). Genetics 45: 1177-1189. MULLER,J., 1953 Uber die Wirkung von Thyroxin und Thyrotropem Hormon auf den Stoffwecksel und die Farbung des Goldfisches. Zeit. Vergl. Physiol. 35: 1-12. OHNO,S., 1970 Evolution by gene duplication. Springer-Verlag, Berlin. OHNO,S. and N. B. ATKIN, 1966 Comparative DNA values and choromosome complements of eight species of fishes. Chromosoma (Berl.) 18: 455-466.

L. and OHNO,S., J. MURAMOTO, CHRISTIAN N. B. ATKIN, 1967 Diploid-tetraploid relationship : among old-world members of the fish family Cyprinidae. Chromosoma (Berl.) 7 245-250. TAKEUCHI, and T . KAJISHIMA, I. K. 1973 Fine structure of goldfish melanophages appearing in the depigmentation process. Annot. Zool. Japon. 46: 77-84. , 1974 Pigment cell differentiation in albino goldfish I. Ultrastructural differentiation of retinal pigment epithelium. Cell Tissue Res. 155: 383-398.

TERAO, 1922 Effect of feeding thyroid on goldfish. J. Fish. Tokyo 17: 257-259. A., WOLF, H. RITTER,N. B. ATKIN and S. OHNO, 1969 Polyploidization in the fish family U., Cyprinidae, order Cypriniformes, I. DNA-content and chromosome sets in various species
of Cyprinidae. Humangenetik 7:240-244. T., YAMAMOTO, 1973 Inheritance of albinism in the goldfish, Carassius auratus. Jap. J. Genet. 4 : 53-64. 8 Corresponding editor: D. T. SUZUKI