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ZOOLOGICAL SCIENCE 15: 425–431 (1998) © 1998 Zoological Society of Japan
* Corresponding author: Tel. +81-5996-6-1830;
FAX. +81-5996-6-1962.
Transparent-Scaled Variant of the Rosy Bitterling,
Rhodeus ocellatus ocellatus
(Teleostei: Cyprinidae)
Kouichi Kawamura
1
*, Kazumi Hosoya
2
and Masanari Matsuda
3
1
National Research Institute of Aquaculture, Nakatsuhamaura, Nansei,
Watarai, Mie 516-0193, Japan
2
National Research Institute of Fisheries Science, Komaki 1088,
Ueda, Nagano 386-0031, Japan
3
Lake Biwa Museum, Oroshimo, Kusatsu, Shiga 525-0001, Japan
ABSTRACT—Transparent-scaled variant (TSV) of the rosy bitterling
Rhodeus ocellatus ocellatus
(Kner)
was observed on both morphology and heredity. Compared with the normal-scaled type (NST), TSV is
characterized by the blackish coloration in both eyes and peritoneum, and the luminescent one over the
whole body. Histologically, the density of guanophores containing reflecting platelets was conspicuously low,
especially in scale, iris, choroid and peritoneum, while the increase in the number of goblet cells (mucous
cells) was recognized all over the dermal/epidermal regions. The heredity of TSV was recessive and sup-
posed to be controlled by a single pair of genes unrelated to sex, judging from the result of crossbreeding
experiments between NST and TSV. In growth and reproduction, no difference was seen between these two
phenotypes. Transparent-scaled variant of the rosy bitterling can be competent for a genetic marker in
experimental and developmental biology.
INTRODUCTION
The rosy bitterling
Rhodeus ocellatus ocellatus
is one of
the bitterling forms, widely distributed in the temperate regions
of Euracia (Nichols, 1943; Okada, 1960). Originally it was not
distributed in Japan, but its subspecies
R. o. kurumeus
,
endemic to Japan, was distributed. However,
R. o. ocellatus
was accidentally introduced into Japan from China during the
World War II, contaminated in the seedlings of the grass
carp
Ctenopharyngodon idellus
and the silver carp
Hypophthalmichthys molitrix
(Nakamura, 1955). In conse-
quence of expulsion of
R. o. kurumeus
,
R. o. ocellatus
inhab-
its many small lakes, ponds and creeks all over Japan (Nagata
et al
., 1996)
Rhodeus o. ocellatus
is a colorful ornamental fish, which
is excellent in growth and reproduction, in addition to the less
susceptibility to disease. Owing to the extensive distribution
in Japan, it is easily obtainable in field. As an experimental
material in fishes, the medaka
Oryzias latipes
and the zebra-
danio
Brachydanio rerio
are well-known and used widely in
biological study. These fishes are excellent especially in growth
and reproduction. Contrarily
R. o. ocellatus
has an inherent
and great property in reproduction, lacking in these fishes. It
is easily reproduced by artificial insemination, and ovulation
timing in female is exactly presumed by the length of oviposi-
tor (Ohta, 1982). The developmental and morphological fea-
tures in
R. o. ocellatus
were described in detail by Nakamura
(1969).
Rhodeus o. ocellatus
seems suited not only for ap-
preciation but also for laboratory research, especially in de-
velopmental biology (Ueda
et al
., 1990; Kawamura, 1991).
A color variant of
R. o. ocellatus
was found in a small
pond in the northern part of Osaka, Japan. The color variant
was easily discriminated from the normal color type in ap-
pearance. Color variants have often been found in many cyp-
rinid fishes. However, the morphological trait and the heredi-
tary basis have not been well studied except for some orna-
mental fishes, e.g. the goldfish
Carassius auratus
(Kajishima,
1977) and
O. latipes
(Iwamatsu, 1993). In this paper, the
morphological and hereditary traits in the color variant of
R. o.
ocellatus
were examined and the possibility of its utility in bio-
logical study was also discussed.
MATERIALS AND METHODS
Color variant of
R. o. ocellatus
used in this study (Fig. 1), was
sampled in a small pond adjacent to the Expo ’70 commemoration
park in Suita City, located in the northern part of Osaka, Japan. Matsui
(1934) called the same color variant in the goldfish “transparent-
scaled”, based upon its feature in appearance. Then, the color vari-
ant of
R. o. ocellatus
was termed transparent-scaled variant (TSV),
contrary to the normal-scaled type (NST) in this paper.
For the clarification of the morphological trait in TSV, histological
observation was carried out over the whole body using NST and TSV,
both of which were in the same clutch and at the same age of three
months (young). Samples were fixed in Bouins solution. Each body
K. Kawamura
et al
.426
part was dehydrated, embedded in wax, and sectioned to a thickness
of 7 mm. The sections were stained with hematoxylin and eosin. Chro-
matophores in scale were observed by optical microscopy after dip-
ping scales in 0.1 M NaCl or 0.1 M KCl for a few minutes, according
to Ohta (1983). The heredity of TSV was inferred by the crossbreed-
ing between NST and TSV, which was composed of the following five
experiments: 1) TSV ¥ TSV, 2) NST ¥ TSV, 3) F
1
¥ NST, 4) F
1
¥ TSV
and 5) F
1
¥ F
1
(Fig. 2). F
1
were all offspring between NST (♂) and
TSV (♀). The rearing experiments were performed in fish tanks (45
cm in width ¥ 30 cm in depth ¥ 30 cm in height) under the conditions
at the water temperature of 21-24∞C and the photoperiod of 14:10 hr
LD cycle. Survival rate, sex ratio and phenotype in offspring were
examined once a week.
RESULTS
Field observation
Transparent-scaled variant of
R. o. ocellatus
was sampled
together with NST and the Japanese minnow
Pseudorasbora
parva
with fish net. They inhabited a small pond, which was
20 m and 2 m at the largest diameter and depth. Approxi-
mately, the appearance rate of TSV was below 2% in the cap-
tured samples of
R. o. ocellatus
. Transparent-scaled variant
was easily distinguished from NST, based upon the differ-
ence in external coloration. The intermediate phenotype be-
tween the two color was not recognized at all.
Morphology
In appearance, TSV was primarily characterized by its
specific coloration (Fig. 1). The body surface was so lumines-
cent that the branchial arches and the air bladder could be
clearly seen without dissection. In matured male, nuptial col-
oration was prominent, especially the reddish color on the
unpaired fins. Contrary to the luminescence of the body sur-
face, the coloration on the orbital and abdominal regions was
conspicuous in black. Concerning the condition of the body
surface, TSV was softer and slimier than NST. In behavior,
TSV seemed somewhat slower and timider with the tendency
of settlement on the bottom.
Histological traits of TSV were recognized in the orbital,
abdominal, dermal/epidermal regions and scale.
Orbital region: In NST, iris and choroid, which were sil-
very in color and easily distinguished from the neighboring
connective tissues, were developed between the inner retina
and the outer cartilaginous sclera (Fig. 3A). In TSV, iris was
hardly recognized, and the development of choroid was re-
markably poor (Fig. 3B).
Abdominal region: In the transverse section of the ab-
dominal region in NST, the peritoneum in black color was lined
between the abdominal cavity and the musculature (Fig. 3C).
In TSV, the thickness of peritoneum was thinner than that of
NST (Fig. 3D).
Dermal/epidermal regions: Both the two phenotypes had
goblet cells, secreting mucus over the whole body. In TSV,
the density and the size of goblet cell were much higher and
larger than those in NST (Fig. 3E, F).
Scale: Four kinds of chromatophores (melanophore,
xanthophore, erythrophore and guanophore), as known in
teleostei, were recognized in both the two phenotypes. The
density of guanophore in TSV is much lower than that in NST
Fig. 1. Color variant of
Rhodeus ocellatus ocellatus
. Normal-scaled type (NST) (A, C) and transparent-scaled variant (TSV) (B, D). Scale bar
indicates 10 mm.
Color Variant of Rosy Bitterling 427
(Fig. 3G, H).
Hereditary examination
The results in the crossbreeding between NST and TSV
are as follows (Table 1). The discrimination in both phenotype
and sex was possible only in appearance at 100th day after
hatching, when all reared fish reached maturation. In all cross-
breeding, no intermediate phenotype between the two was
seen at all.
TSV ¥ TSV: Offspring were all TSV, the same as their
parents, not containing even a single NST in four experiments
(Table 1A). Hatchability was high and ranged from 66.7% (a-
2) to 100.0% (a-3) (84.0 ± 15.8%, Mean ± SD). The survival
rate was 45.3 ±16.7% (Mean ± SD). The sex ratio in offspring
was almost 1:1 (♂:♀) except for a-3 (only two females). The
low survival rate in a-1 and 2 was owing to a white spot dis-
ease, caused by protozoon
Ichthyophthirius
.
NST ¥ TSV: Offspring were all NST in the reciprocal cross-
breeding (Table 1B). Hatchability was high (79.1 ± 25.4%,
Mean ± SD), independent of the phenotype of parents, ex-
cept for b
1
-4 (25%). The survival rate at 100th day was 49.0 ±
17.6% (Mean ± SD). The sex ratio in offspring was about 1:1
(♂:♀) in almost all experiments, except for b
1
-2 (only three
females).
F
1
¥ NST: Offspring were all NST (Table 1C). TSV did not
appear in the least. Hatchability ranged from 63.3% (c-1) to
Fig. 2. Schematic illustration of crossbreeding between NST and TSV of
R. o. ocellatus
. (A) TSV ¥ TSV, (B) NST ¥ TSV, (C) F
1
¥ NST, (D) F
1
¥ TSV and (E) F
1
¥ F
1
. T, TSV; N, NST; F
1
, offspring between NST (♂) and TSV (♀). “A” means a gene determinant to NST, while “a” to TSV.
K. Kawamura
et al
.428
Fig. 3. Morphological comparison between NST and TSV of
R. o. ocellatus
. (A, B) Sagittal sections of orbital region. (C, D) Sagittal sections of
abdominal region. (E, F) Sagittal sections of dermal/epidermal regions. (G, H) Scale. A, C, E and G from NST, while B, D, F and H from TSV. C,
choroid; E, erythrophore; G, goblet cell; I, iris; Gp, guanophore; M, melanophore; P, peritoneum. Scale bars indicate 60 mm.
Color Variant of Rosy Bitterling 429
Table 1. Results in crossbreeding between NST and TSV of
R. o. ocellatus
, as shown in Fig. 2
Fish at 100th day after hatching
Eggs treated Embryos hatched (%)* NST TSV Total (%)***
♂(%)** ♀(%)** ♂(%)** ♀(%)**
a-1 18 17 (94.4) 0 (0.0) 0 (0.0) 3 (50.0) 3 (50.0) 6 (35.3)
A
TSV ¥ TSV a-2 36 24 (66.7) 0 (0.0) 0 (0.0) 3 (42.9) 4 (57.1) 7 (29.2)
(♂)(♀) a-3 4 4 (100) 0 (0.0) 0 (0.0) 0 (0.0) 2 (100.0) 2 (50.0)
a-4 16 12 (75.0) 0 (0.0) 0 (0.0) 4 (50.0) 4 (50.0) 8 (66.7)
b
1
-1 12 10 (83.3) 2 (40.0) 3 (60.0) 0 (0.0) 0 (0.0) 5 (50.0)
NST ¥ TSV b
1
-2 12 10 (83.3) 0 (0.0) 3 (100.0) 0 (0.0) 0 (0.0) 3 (30.0)
(♂)(♀)b
1
-3 13 11 (84.6) 2 (50.0) 2 (50.0) 0 (0.0) 0 (0.0) 4 (36.4)
Bb
1
-4 28 7 (25.0) 2 (66.7) 1 (33.3) 0 (0.0) 0 (0.0) 3 (42.9)
TSV ¥ NST
b
2
-1 8 8 (100.0) 1 (33.3) 2 (66.7) 0 (0.0) 0 (0.0) 3 (37.5)
b
2
-2 27 21 (77.8) 7 (46.7) 8 (53.3) 0 (0.0) 0 (0.0) 15 (71.4)
(♂)(♀)
b
2
-3 8 8 (100.0) 3 (50.0) 3 (50.0) 0 (0.0) 0 (0.0) 6 (75.0)
F
1
**** ¥ NST
c-1 11 7 (63.3) 2 (40.0) 3 (60.0) 0 (0.0) 0 (0.0) 5 (71.4)
C c-2 34 29 (85.3) 6 (42.9) 8 (57.1) 0 (0.0) 0 (0.0) 14 (48.3)
(♂)(♀)
c-3 21 18 (85.7) 5 (45.4) 6 (54.6) 0 (0.0) 0 (0.0) 11 (61.1)
F
1
**** ¥ TSV
d-1 32 24 (75.0) 2 (16.7) 1 (8.3) 4 (33.3) 5 (41.7) 12 (50.0)
D d-2 42 11 (26.2) 1 (33.3) 1 (33.3) 0 (0.0) 1 (33.3) 3 (27.3)
(♂)(♀)
d-3 45 34 (75.6) 3 (33.3) 1 (11.1) 3 (33.3) 2 (11.1) 9 (26.5)
E
F
1
**** ¥ F
1
**** e-1 40 16 (40.0) 3 (50.0) 1 (16.7) 1 (16.7) 1 (16.7) 6 (37.5)
(♂)(♀) e-2 36 24 (66.7) 6 (50.0) 4 (33.3) 1 (8.3) 1 (8.3) 12 (50.0)
* Embryos hatched (eggs treated).
** Ratio of each sex to total individuals at 100th day after hatching.
*** Total individuals at 100th day after hatching (embryos hatched).
**** Offspring between NST (♂) and TSV (♀) in experiment b
1
.
85.7% (c-3) (78.1 ± 12.8%, Mean ± SD). The survival rate at
100th day was 60.3 ± 11.6% (Mean ± SD). The sex ratio in
offspring was about 1:1 (♂:♀).
F
1
¥ TSV: Contrary to the result in crossbreeding between
F
1
and NST (Table 1C), not only NST but also TSV was ob-
served in this crossbreeding (Table 1D). Hatchability and the
survival rate at 100th day were 58.9 ± 28.3% and 34.6 ± 13.3%
(Mean ± SD) respectively. The ratio between NST and TSV
was 1:3 (d-1), 2:1 (d-2) and 4:5 (d-3). The sex ratio ranged
from 1:1 (♂:♀) (d-2) to 3:1 (d-3) in NST and from 3:2 (d-3) to
4:5 (d-1) in TSV. The low survival rates in all the crossbreed-
ing were caused by white spot disease.
F
1
¥ F
1
: Both NST and TSV appeared (Table 1E). Hatch-
ability was 40.0% (e-1) and 66.7% (e-2). The survival rate at
100th day was 37.5% (e-1) and 50.0% (e-2). The ratio be-
tween NST and TSV was 2:1 (e-1) and 5:1 (e-2). In the sex
ratio, though male and female appeared in both NST and TSV,
NST male dominantly outnumbered the opposite sex or the
other phenotype (50% in both e-1 and 2).
DISCUSSION
Morphological traits in TSV of the rosy bitterling
Kajishima (1960a-c, 1977) tried to clarify the morphologi-
cal trait in TSV of the goldfish. He found in TSV, that not only
the reflecting guanine layer in eye but also melanophores and
xanthophores degenerate over the whole body at the early
developmental stage. Especially, the decrease in the number
of guanophores was conspicuous in iris, peritoneum and oper-
culum. In
R. o. ocellatus
, the discrimination of phenotype was
impossible in larvae just after hatching (at second day after
fertilization at 20∞C). However, in juvenile at about 20th day
after hatching, the phenotype was visibly discriminated. This
finding suggests that, in TSV of
R. o. ocellatus
as well as the
goldfish, the degeneration of each chromatophore occurs
during the larval stage. In the morphological observation in
TSV of the adult
R. o. ocellatus
, three characteristics were
recognized, which were the degeneration of iris, choroid
and peritoneum, the increase in the number of goblet cell and
the low density of guanophores in scales (Fig. 3). In NST,
iris, choroid and peritoneum remarkably contain many
guanophores, where there are many reflecting platelets of
guanine, giving fish a shiny appearance (Kajishima, 1960a;
Hawks, 1983). Therefore, the low content of guanine on the
body surface is considered to create the transparent body
coloration, as well as the blackish color in eye and perito-
neum.
Contrary to the goldfish, however, such a remarkable dif-
ference was not seen in the other chromatophores except
guanophore in
R. o. ocellatus
. The increase in the number of
goblet cell on the dermal/epidermal regions in TSV has not
been reported in other fishes so far. It remains unknown
whether this trait was caused by either genetic or environ-
mental factors. As one of the possibilities, the inner body parts
K. Kawamura
et al
.430
in TSV are directly exposed to the influence of UV rays, be-
cause of the low content of guanine. Therefore, on behalf of
guanine, many goblet cells develop and excrete much mucus
for the purpose of body protection. In behavior, TSV of
R. o.
ocellatus
seemed cowardlier and stagnanter than NST. Pos-
sibly, such a behavior in TSV has some relation with the de-
generation of the iris and choroid in eye.
Hereditary traits in TSV of the rosy bitterling
Berndt (1925) considered that the character “hypo-
lepidose” (transparent-scaled) in the goldfish is dominant
against the normal character. However, Chen (1928) demon-
strated that the genetic dominance of this character is imper-
fect. He also distinguished the homozygous and the heterozy-
gous types in TSV of the goldfish. The former is all transpar-
ent and the latter mosaic transparent. In the genetic and de-
velopmental analyses of the goldfish, Kajishima (1960a, 1977)
concluded that the recessive transparent-scaled character is
incompletely dominant to the normal-scaled character and
determined by a single autosomal gene.
In the crossbreeding experiments of
R. o. ocellatus
, the
transparent-scaled character is obviously recessive to the
normal-scaled character, because the offspring between NST
and TSV were all NST (Table 1B). In the backcross between
F
1
and TSV and the crossbreeding in F
1
, however, TSV ap-
peared again (Table 1D, E), while in the backcross between
F
1
and NST, only NST did (Table 1C). The ratio between TSV
and NST is about 1:1 in the backcross between F
1
and TSV,
and about 1:3 in the crossbreeding in F
1
. Judging from the
results in crossbreeding, the transparent-scaled character in
R. o. ocellatus
is thought to be controlled by a single gene (“a”
in Fig. 2), which is recessive to a gene determinant to the
normal-scaled character (“A” in Fig. 2). In addition, the fact
that the sex ratio always converges on 1:1 in both TSV and
NST in all crossbreeding, indicates that a gene determinant
to the transparent-scaled character is unrelated to sex.
The hereditary manner of the transparent-scaled charac-
ter in
R. o. ocellatus
is very similar to that in the goldfish
(Kajishima, 1977). In both fishes, the transparent-scaled char-
acter is controlled by a single gene, unrelated to sex.
R. o.
ocellatus
differs from the goldfish in that the former has only
one type in TSV, contrary to two types (all transparent and
mosaic transparent-scaled) in TSV of the latter. This means
that, in
R. o. ocellatus
, a gene determinant to the transparent-
scaled character is completely recessive, not incompletely
dominant to the normal-scaled character, different from that
of the goldfish.
Utility in TSV of the rosy bitterling
The color variant in fish has often been reported in many
freshwater fishes. The well-known examples are the medaka
(Iwamatsu, 1993), the Guppy
Poecilia resticulata
(Winge,
1927), the platyfish
Xiphophorus maculatus
(Borowski, 1984)
and the goldfish (Matsui, 1934). As for TSV of
R. o. ocellatus
,
the most outstanding morphological trait is the low density of
guanophores in the whole body. The same color variant as
seen in
R. o. ocellatus
has already been reported in other
fishes e.g.
O. latipes
(Takeuchi, 1969) and
C. auratus
(Chen,
1928). Therefore, the occurrence of TSV in fish does not seem
to be so rare and restricted to certain taxa.
In general, color variants in fishes, including albino, are
inferior to the normal type in growth and reproduction
(Kirpichnikov, 1981). In TSV of
R. o. ocellatus
, however, such
deficiency has not seen at all in this study. In addition, the
simple heredity and the conspicuous morphological charac-
ter in TSV of
R. o. ocellatus
suggest that TSV in
R. o. ocellatus
,
which is excellent in growth and reproduction, can be a good
material in experimental biology. Particularly, the recessive
transparent-scaled character is useful as a genetic marker for
experimental and developmental biology and genetics.
ACKNOWLEDGMENTS
Dr. M. Okauchi (NRIA) and Dr. A. Komaru (Mie Univ.) critically
read the manuscript and offered valuable suggestions. Dr. K. T. Wada
(NRIA) gave us valuable comment on the early draft. We would like
to express the sincere thanks to these investigators. This study was
supported in part by a grant from the Fisheries Agency of Japan for
Research on Breeding of Aquatic Organisms.
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