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
(1924), who
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(1934) reported his detailed results on the genetic analysis of the
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

162

T. KAJISHIMA

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 KAJISHIMA1960d), 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,
found albino goldfish in Favors Aquarium in New York,
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,
KAJISHIMAand HAMA1960). 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

COLOR MUTANTS I N GOLDFISH

163

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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T. KAJISHIMA

TABLE 1

Resulis of crosses between wakin and black moor goldfish

Number of offspring
Depigmented Pigmented

Crosses

Wakin

Fl
F,

x Black Moor (F,)

x F,
x Black Moor

478
185
62

Ratio

0
12
26

15:l
3:l

XZ

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)

First

Second

Third

No.

Number of offspring
Depigmented Pigmented

1
2
3
4
5

67
78
62
53
72

23
-

1
2
3
4.
5

62
87
47
59
132

1
2
3
4

21
47
31
24

black moor) and black moor

Ratio

XZ

3:l
-

0.62 (P=O.5-0.3)
-

21

3:l
3:l

0.36 (P=0.7-0.5)
0.07 (P=0.8-0.7)
-

19
55
29
30

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

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)

13

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

165

COLOR MUTANTS I N GOLDFISH

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 with wakin and black moor: The parental red
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

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

F,

x F,

Black Moor
X Red Telescope (1)
(B)
-__-- (2)
(3 )
Homo. Transparent
Wakin (F,)
(C)
F, X F,
F, X Black Moor

Number of offspring
Depigmented Pigmented

Ratio

X2

80

30

156
309

0
0

81
36
93

0
41
35

1:l
3:l

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

194
144
134

0
9
47

15:l
3:l

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

166

T. KAJISHIMA

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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COLOR MUTANTS IN GOLDFISH

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

Albino

x Wakin (F,)

F, x F,
(A)

F,

(St-21)
(St-27)
Albino (St-21)
(St-27)

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)

Number of offspring
Albino
Pigmented

0
137
65
133
32

381
427
991
151
104

Ratio

X2

1:3
1:15
1:l
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)

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

258

54
30
33

144.

425
101

1:3
1:15
1:3

0
1(32
80

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.
111. Analysis of recessive-transparent character:
(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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T. KAJISHIMA

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 x
Homo. Transparent (F,)

(A)

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

(B)

F,

Ratio

xz

22

3:l

0.10 (P=0.8-0.7)

390

0
19

67

3:l

0.39 (P=0.7-0.5)

253
287

0
91

3:l

x F,

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

Recessive
transparent

0.17 (P=0.7-0.5)

osnly in normal-scaled type.

+* Scored
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.

COLOR MUTANTS IN GOLDFISH

169

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.

170

T. KAJISHIMA

The albino goldfish is represented by mmss, and the genotypes of the latepigmented type (YAMAMOTOS
light type) as mm SS or mm Ss. YAMAMOTO
and
the present author agree that two genes are involved, but we differ somewhat
regarding the function of the two genes. YAMAMOTO
has postulated that both the
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 (KAJISHIMA1967).
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
fish were all lost by accident, so that it is impossible to
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.

COLOR M U T A N T S IN GOLDFISH

171

In previous experiments on albino goldfish (TAKEUCHI

and KAJISHIMA
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
and ANDERSON
(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
and HANSEN
( 1931) considered that the melanophore
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
and KAJISHIMA ( 1973) have
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 (FUKUI1927; GOODRICH
and HANSEN
1931) and gonadectomy (GOODRICH
and HANSEN1931) 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 (KAJISHIMA1975), 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.

172

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 ATKZN1966; OHNOet al. 1967; WOLFet al. 1969;
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

Wakin (Common goldfish)

Telescope-eyed fish
Black

Melanophore
Formation
Destruction

Iridophore
Formation Destruction

pp

cc

DPlDP, DP,DP,

GG

tt

pp

cc

dPldP, dP,dP,

GG

tt

PP

cc

______

(The Black Moor)

Red
Albino
Recessive trsnsparent-fish
Black
Red
Transparent-scaled fish
Homozygous fish
Heterozygous fish

PP cc

Dp,Dp, DP,DP,?

gg
gg

tt
tt

PP cc
PP cc

dP,dP, dP,dP,
~ P & P , dp,dp,

GG
GG

TT
Tt

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 HANSEN
tinguishable from the heterozygous type Tt (CHEN1928; GOODRICH
1931; MATSUI1934; KAJISHIMA1960a). 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-oYAMAMOTO
and
who kindly provided the opportunity to use the albino goldfish for this experiDR. H. TOMITA,
ment. I am also grateful to Mrs. C. A. MAHI, of The University of Hawaii for critical reading
of the manuscript.
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Corresponding editor: D. T. SUZUKI