INHERITANCE of ALBINISM in the GOLDFISH, CARASSIUS AURATUS TOKI-0 YAMAMOTO Biological Institute, Faculty of Science, Nagoya Univ

JAPAN. J. GENETICS Vol. 48, No. 1: 53-64 (1973)

INHERITANCE OF ALBINISM IN THE GOLDFISH, CARASSIUS AURATUS

TOKI-0 YAMAMOTO

Biological Institute, Faculty of Science, Nagoya University, Nagoya 464 and Zoological Laboratory, Faculty of Agriculture, Meijo University, Nagoya 468

Received December 2, 1972

Matsui-renowned goldfish geneticist-has been searching for albino goldfish for fifty odd years since 1914, but it has been fruitless (cf. Makino and Matsui 1970). Taking into consideration of tremendous numbers of the goldfish yearly produced, it is remarkable that the albino has not been found in Japan. In 1956, a report entitled "Rare albino goldfish" appeared in a hobbyist's magazine (The Aquarium, 1956, pp. 348-349) in the U.S.A. Judging from the illustration, the fish seem to be albino fantails (Ryukin). According to the news, they were descendants from some albinos originally found in Singapore. We find also, a color photograph of an albino fantail (Photo by R. Law) in a booklet by Teitler (1967). It is not certain whether or not this fish is related to the stock of the albinos above mentioned. At any rate, the mode of inheritance of albinism in the goldfish has not hitherto been known. The present paper deals with inheritance of the albino globe-eyed goldfish (demekin) originally found in New York by the writer several years ago. Recently, Kajishima (1972) using materials provided by the writer, presented a short account on the inheritance of the albino goldfish in a book (Kingyo Taikan) edited by Matsui. Using mates of different varieties of the goldfish, both Kajishima and the writer arrived at the conclusion that albinism of the goldfish is not a simple Mendelian character but is represented by the double homozygote of two non-linked autosomal recessive genes since the F2 show the 15:1 ratio for non-albino and albino types like the case of duplicate genes. Closer observation on advanced embryos and larvae in the present study revealed, further, that the dark (normal), the light and the albino types appear in the 12:3:1 ratio characteristic for gene interaction in dominant epistasis. The made of gene interaction in the present case may be called, therefore, as a dominant epistasis akin to duplicate genes. 54 T. YAMAMOTO

MATERIALS AND METHODS

While staying in New York in 1965, the writer and his collaborator H. Tomita found and purchased a lot (about 30) of young albino globe-eye goldfish (demekin) from Favor's Aquarium, Brooklyn, New York. The dealer told us that they came from Hong Kong. We carried them alive to Nagoya University in the next year and the stock has since been kept in our institute. About ten of original albinos were supplied to Kajishima for research on these rare materials separately. The original albinos and inbred progeny (Plate 1, A) have the following characteristics: pink eyed, body shape like red globe-eye (Aka demekin) and black moor (Kuro demekin), normal metallic scales, double caudal and anal fins. Most original fish were orange, while a few were pale red in body color. Albinos with orange body color were used in the present study. Melanin is completely absent not only in the eye but everywhere in the body. The present report deals with the results obtained from two crosses Globe-eye nacreous calico (Sanshiki demekin) X Albino globe-eye and Shubunkin matt pink x Albino globe-eye. Genetics of scale transparency in the goldfish has been worked out by Chen (1928) and Matsui (1933,1934) . We adopt the gene symbols used by the latter. As to scaleness, however, we follow the following nomenclature developed by the Goldfish Society of Great Britain (1949) ; Metallic : normal scales with guanine-laiden reflective tissue (Matsui's normal scales, genotype being tt), Matt : typically, transparent or flat coloration without reflective tissue in the scales, opercula and eyeball (Matsui's totally transparent scales, genotype being T T) , Nacreous : a hybrid of the two, showing signs of both (Matsui's mosaic transparent scales, the genotype being Tt). Calico has originally been referred to nacreous fish but is recommended to refer to fish of any group which has three or more colors scattered over the body haphazardly, usually including blue (cf. Aflleck 1952; Teitler 1967). It may be remarked, however, that some fish known to be TT from their pedigree, may possess reflective tissue in two or three scales or in a part of opercula and eyeball (unpublished). In describing mates to albinos, we write type or form firstly, scaleness secondly and color thirdly as suggested by Teitler (personal communication). However, original albino globe-eye metallic orange goldfish is simply expressed as albino globe-eye for the sake of brevity. In each cross, a single female was mated with a single male.

RESULTS

1. Albino globe-eye x Albino globe-eye Offspring from four matings per se were all albinos without exceptions. Albinos lack melanin everywhere including eyes which are pink (Plate 1, A). The situation is the same in embryonic, larval stages. This is in strong contrast to ordinary black- INHERITANCE OF ALBINISM IN THE GOLDFISH 55 eyed varieties in which embryos and larvae invariably possess melanophores like those of their ancestral form, the crucian carp. Of 325 mature fish, 158 were males and 167 were females. 2. Globe-eye nacreous calico (Sanshiki demekin) x Albino globe-eye Table 1 gives characteristics of the Fi adults from this cross. Eye color was all black and eye shape was all globe-eyed with a single exception which was normal. Since albino male parent was belonged to the metallic scale group (tt) and mated to a nacreous (Tt) female, the two scale types appeared in the Fl offspring. Body color of metallic fish were mostly red or red and white, while that of nacreous ones were red and pink or red and white. Noteworthy fact is that no calico (with three or more colors) fish appeared even in the Fl Tt fish. An Fi nacreous female was mated with an Fl nacreous male to produce the F2 generation.

Table 1. Fl from Globe-eye nacreous calico (Sanshiki demekin) X Albino globe-eye, scored on 2-year-old mature fish

Table 2, F2 from Globe-eye nacreous calico (Sanshiki demekin) X Albino globe-eye, scored on advanced embryos and newly hatched fry. Proposed symbols for genes are given in parentheses

Table 2 gives the F2 offspring scored on advanced embryos and larvae. In embryos of ordinary black-eyed varieties of the goldfish, eye pigment begins to form at the stage 21 (cf. Kajishima's normal developmental stages, 1960). This stage is reached 36 hours after spawning when reared at 21°C. Embryonic melanophores first appear at the stage 22 (50 hours, 21°C) and heart pulsates and blood circulation begins at the stage 23 (60 hours, 21°C). It was found that three phenotypes, dark pigmented (normal), light pigmented and albino types appear in the F2 embryos at the middle stage between 22 and 23 stages (Fig. 1) at which heart begins to pulsate but blood circulation is not yet established. This is reached 2-days after spawning at uncontrolled room tempera- ture ranged from 22 to 26°C. 56 T. YAMAMOTO

In the dark embryos (Fig. 1, A), melanin is already deposited in the eye and normal embryonic melanophores appear in the body and on the yolk sac, especially gathering on the prospective area of the Cuvierian ducts. These characters are exactly same as those of other black-eyed varieties of the goldfish. In the light embryos (Fig. 1, B), the eye pigment is not yet formed and smaller and fewer melanophores appear in the body and on the yolk sac. In albino ones (Fig. 1, C), no melanin is formed in the eye and no embryonic melanophores appear.

Fig. 1. Dark (A), light (B), and albino (C) F2 embryos (stage 22-23) from the cross Globe-eye nacreous calico (Sanshiki demekin) X Albino globe-eye metallic orange.

In Fig. 2, the dark, the light and the albino fry on the day of hatching are illustrated. In the dark fry, the eye is deeply pigmented and normal melanophores are distribut: d in the body mainly in two pairs of lines, one along dorsal side and the other along the cardinal veins. On the york sac, melanophores are distributed densely on the Cuvierian ducts and sparsely on the other parts. Their size and numbers are the same as those of ordinary black-eyed varieties. In the light fry at this stage, the eye is only faintly pigmented and larval melanophores are smaller in size and fewer in number. In albinos, no melanin is deposited in the eye and no melanohores appear. A large part of the F2 fry were reared together, including the dark and the light fry excluding albinos and a small part of fry were separately raised in three groups, i.e., the dark, the light and the albino ones. The F2 mature fish reared together excluding albinos, scored at the age of 2.5 years old are tabulated in Table 3. The result is far from satisfactory. Since the initial number of fry was great and examination was performed too late, mortality was very high. In respect to staleness, metallic fish predominate, nacreous ones are few, and no matt fish are found. This may be due to selection for strong type (tt) owing to overclouding. However, it is worth while noting that a considerable numbers of the black moor appeared among other color types. INHERITANCE OF ALBINISM IN THE GOLDFISH 57

• • • fI f f Fig. 2. Dark (A), light (B), and albino (C) F2 fry (newly hatched) from the cross Globe-eye nacreous calico (Sanshiki demekin) X Albino globe-eye metallic orange.

Table 3. Non-albino F2 mature fish (2.5 years old) from the cross Globe-eye nacreous calico (Sanshiki demekin) X Albino globe-eye, developed from the non-separated group including dark and light types. Parents used were both Fi nacreous (Tt) fish in scaleness 58 T. YAMAMOTO

Table 4. F3 offspring from Globe-eye nacreous calico (Sanshiki demekin) X Albino globe-eye, scored on advanced embryos and fry. Parents used were F2 fish developed from the light (m, S) fry, separately reared and an F2 albino male. Superscripts denote fish No. Proposed symbols and established genotypes of ~-parents are also given

In the separately raised groups, only a few fish survived and reached maturity. Using these F2 fish, three crosses were made to obtain the F3 generation (Table 4). An F2 female and an F2 male developed from light fry produced abundant all light offspring (Cross 1). The same female mated with an F2 albino male (Plate 1, B) produced small numbers of fry which were also all light (Cross 2). Another F2 female mated with the same albino male, on the other hand, produced the light and the albino fry in the 1:1 ratio (Cross 3). 3. Shubunkin matt pink x Albino globe-eye This cross produced only a few Fl fry, of which only one male and one female reached maturity. Both were black-eyed nacreous (Tt) pink and red. The eye shape was normal and the caudal and the anal fins were single. The two fish were used to produce the F2 generation. Table 5 gives the F2 data scored on advanced embryos and larvae. The dark, the light and the albino types appeared in the 12:3:1 ratio as in the F2 results previously mentioned. Characteristics of the three types at the stage 22-23 and hatching time are the exactly the same as those illustrated in Fig. 1 and Fig. 2, respectively. In Fig. 3, photographs of 5-day-old dark, light and albino larvae are presented. In the dark type, the eye is fully pigmented and size and number of melanophores are normal. In the light type, the eye pigmentation is approaching that of the dark one, but melanophores are still smaller in size and fewer in number. In albinos, no melanin is formed in the eye and the pupil looks pink. There is no melanophores in all parts of the body. The eyeball of the all three types is silvery owing to development of the reflective tissue (iridocytes). As development proceeds, the degree of phenotypic divergence between the dark and the light types gradually decreases. When fish reach the young fish, no differ- ence in melanophore condition can be observed. The dark and the light fry were reared separately from hatching time and their INHERITANCE OF ALBINISM IN THE GOLDFISH 59

Fig. 3. Dark (A), light (B), and albino (C) F2 fry (5-days after hatching, total length 6.8 mm) from the cross Shubunkin matt pink X Albino globe-eye metallic orange. 60 T. YAMAMOTO

Table 5. F2 offspring from Shubunkin matt pink x Albino globe-eye, scored on advanced embryos and newly hatched fry. Proposed symbols for genes are given in parentheses

Table 6. Scaleness and colors of non-albino F2 immature offspring (0.5 year old) from the cross Shubunkin matt pink X Albino globe-eye, developed from the dark and the light fry separately raised. Parents used were an F1 female and F1 male both having normal shaped black eyes and nacreous (Tt) scaleness

characters were compared after a half year. The results are tabulated in Table 6. It is apparent that all color conditions are found in the immature fish developed from fry of both types.

DISCUSSION

Observed and expected frequencies of three types in advanced embryos as well as larvae as shown in Tables 2 and 5 indicate that the albino type is represented by the double recessive genes assorting independently. Evidently, two pairs of nonlinked autosomal genes are controlling coloration of embryos and larvae of the goldfish. The following symbols of genes responsible for coloration are proposed. M: Controls normal melanin formation and normal melanophore development in embryos as well as larvae. Its recessive allele is m. S : Governs slower melanin formation and slower melanophore development. Its recessive allele is s. The albino type is represented by m, s. Hence, non-albino and albino types appear in INHERITANCE OF ALBINISM IN THE GOLDFISH 61 the 15:1 ratio in the F2 generation. Closer observation on the F2 advanced embryos and larvae revealed that non-albino fraction can be subdivided into the dark and the light phenotypes in the 12:3 ratio. In the dark type, melanin pigmentation in the eye and melanophore development are normal. In the light type., initial pigmentation in the eye is delayed and melanophores in embryos and larvae are smaller and fewer because of slower melanophore development. The F2 ratio scored on advanced embryos and larvae is the 12: 3:1 for the dark, the light and the albino classes, characteristic of the F2 segregation of two pairs of genes interacting in dominant epistasis. The ratio appeared in the F2 embryos and larvae seems to be 12 (9M, S and 3M, s) : 3 (m, S) :1 (m, s) for the dark, the light and the albino classes. Because of epistatic action of M to S, s, the effect of S is masked when it combines with M. Thus, M, S and M, S become the same phenotype. The albino genes, m, s, not only eliminates melanin in the eye, but also in all other parts of the body. The eye pigmentation and the size and the numbers of melanophores of the light type (m, S) gradually approach those of the dark type (M, S and M, s) during post larval development. By the stage of young fish, the eye pigment and melanophores of the two types become practically the same. Two genotypes, homozygous m/m, S/S and heterozygous m/m, S/s, are expected to occur among the light type (m, 5). Of two F2 females, developed from the m, S fry, that were tested by an F2 albino male mlm, s/s (Plate 1, B), one proved to be homozygous and the other heterozygous (Table 4). Thus, the occurrence of the expected genotypes was actually realized. If a heterozygous female were mated with a heterozygous male, the F3 offspring produced would be in the 3:1 ratio for the light and the albino types. Such result was actually obtained by Kajishima (personal communication). At this point, it is worth remarking that terminology on the modified F2 ratios are sometimes varied and confusing. Darlington and Mather (1961) presented appropriate terms. However, Fasoulas' article (1971) is by far the most comprehensive presentation on definitions, terms and symbols. The 15:1 ratio for triangular and ovoid capsules in the shepherd's purse Capsella has been attributed to duplicate genes (Skull 1914). In this famous example, A and B (in general symbols) substitutes one another completely. They are called by Fasoulas as isoepistatic and symbolized as A=B. His symbolism for the 12:3:1 type of dominant epistasis is A> B. The 12:3:1 ratio was observed in colors of the summar squash (Sinnott and Durham 1922) and in bulb colors of the onion (Clark, Jones and Little 1944). In both cases, the epistatic gene A seems to be a dominant inhibitor or an inactive gene, so that A, B and A, b are colorless and the hypostatic B produces a visible effect only in a, B. The 13:3 type of dominant epistasis was observed in colors of the fowl (Bateson et a1.1905- 1908; Hadley 1915; Sinnott et a1.1950) . White leghorn fowls (AA, BB) owe their absence of color to a dominant inhibitor A which is, epistatic to other color genes, e.g., B just like the above examples. In the F2 from the cross with white Wyandotte (aa, bb), 13 62 T. YAMAMOTO white (A, B; A, b; and a,b) and 3 colored (a, B) appeared. In the goldfish, however, the epistatic gene A (M) is neither inhibitor nor inactive gene but it controls normal melanin deposition and normal melanophore development. The hypostatic gene B(S), also, governs melanin formation and melanophore development but its action is slow. In embryos of the dark type (M, S and M, s), the onset of melanin formation in the eye and melanophore development are rapid (normal) . In embryos of the light type (m, S), the onset and rate of melanin formation in the eye as well as melanophore development are slow. This results in smaller size and fewer numbers in embryonic and larval melanophores. The degree of phenotypic divergence between the dark and the light types is gradually obliterated as development proceeds. This fact holds true in both eye pigmentation as well as melanophores. Final size and number of melanophores in immature fish developed from both the dark and the light fry become eventually similar. Since both the M and the S genes act in the same sense in controlling melanin deposition and melanophore development, the two are allied to duplicate genes. Strictly speaking, however, the two are not equal in action since the M is epistatic and the S is hypostatic (M> S) . So that the mode of the non-allelic gene-interaction in the present case may be termed as a dominant epistasis akin to duplicate genes. In the present research, mates to albinos were the globe-eye nacreous calico (San- shiki demekin) and the Shubunkin matt pink. Kajishima (1972) using the common goldfish (Wakin) and the black moor (Kuro demekin) as mates, obtained also the 15:1 F2 ratio. Although his symbols are different from ours, it seems to the writer that all these varieties of the goldfish possess the M and the S genes pertaining to melanin and melanophores. Albinism appeared in fishes have been analyzed in the paradise fish, Macropodus opercularis (Kosswig 1935; Goodrich and Smith 1937), the carp, Cyprinus carpio (Matsui 1936), the swordtail, Xiphophorus hellerii (Kosswig 1935; Gordon 1942, 1948), the guppy, Poecilia reticulata (Haskins and Haskins 1948; Dzwillo 1959), the Mexican blind cave fish, Anoptichthys atrobius (Sadoglu 1957a, b) and the medaka, Oryzias latipes (Yamamoto 1969). All these are shown to be the simple Mendelian recessive. Besides albino gene (ab), Sadoglu and McKee (1959) reported the second one, brown (bw), that affects larval eye and body color. Brown-eyed (+ab, bw) larvae have melanophores smaller in size and fewer in number. In passing it may be remarked that the 9:3:4 ratio in the F2 dihybrid, characteristic for two pairs interacting in recessive epistasis, has been found in the swordtail (Gordon 1942, 1948), the medaka (Yamamoto 1969) and the blind cave fish (Sadoglu and McKee 1959). Mutation must have occurred at two loci in the different autosomes instead of one locus before appearance of albinism in the goldfish. This may be the reason why the albino has not been appeared for long years inspite of enormous numbers of yearly INHERITANCE OF ALBINISM IN THE GOLDFISH 63 production of the goldfish.

SUMMARY

Albinism appeared in the globe-eyed goldfish (demekin) is not a simple Mendelian character but is the double recessive of two independently assorting autosomal genes since non-albino and albino types appear in the 15:1 ratio in the Fz generation. When scored on advanced embryos and larvae, however, the dark (normal), the light and the albino types appear in the 12:3:1 ratio, related in action of dominant epistasis. Symbol M is proposed for the epistatic dominant gene responsible for normal melanin deposition as well as normal melanophore development, S for the hypostatic dominant gene governing slower melanin formation and slower melanophore development. The effect of the S is masked in the combination M, S, because of epistasis of the M to the S (M> S) . The dark type, thus, is represented by either M,S or M,s, the light one by m, S and the albino by m, s. The F2 ratio scored on embryos and larvae becomes 12 dark (M, S and M, s) : 3 light (m, S) :1 albino (m, s). In the light embryos and larvae, eyes darken slowly and melanophores are smaller in size and fewer in number, i.e., in earlier developmental stage. The degree of phenotypic divergence between the dark and the light types is gradually obliterated as development proceeds. Final melanin deposition as well as final state of melanophores become eventually the same since the two genes differ only in the time-relationships in the developmental process. The M and the S act in the same sense like duplicate genes. Since, however, M> S in epistasis but not M=S, the mode of non-allelic gene interaction in this case may be termed as a dominant epistasis akin to duplicate genes. The presence of the M and the S genes pertinent to melanin as well as melanophores in a number of the ordinary black-eyed varieties of the goldfish is pointed out.

ACKNOWLEDGMENT

The author is greatly obliged to Dr. Hideo Tomita. Without his efficient coopera- tion, the writer should not have been able to carry through the investigation here presented.

LITERATURECITED

Aflleck, R. I., 1952 The nacreous (mottled) group of the goldfish (Carassius auratus L.), with an analysis of the colour seen in these fish. Austr. J. Mar. Freshw. Res. 3: 126-139. Bateson, W., E. R. Saunders, and R. C. Punnet, 1905-1908 Experimental studies in the physiology of heredity: poultry and sweet peas. Rep. Evol. Committ. Roy. Soc. Nos. 2, 3 and 4. Chen, S. C., 1928 Transparency and mottling, a case of Mendelian inheritance in the goldfish (Car- assius auratus). Genetics 13: 434-452. 64 T. YAMAMOTO

Clarke, A. E., H. A. Jones, and T. M. Little, 1944 Inheritance of bulb color in the onion. Gene- tics 29: 569-575. Darlington, C. D., and K. Mather, 1961 The elements of genetics. George Allen & Unwin Ltd., London, The MacMillan Comp., New York. Dzwillo, M., 1959 Genetische Untersuchungen an domestizierten Stammen von Lebistes yeticulatus (Peters). Mitt. Hamburg Zool. Mus. u. Inst. 57: 143-186. Fasoulas, A., 1971 Teaching allelic and non allelic gene action and interaction in elementary genetics. Thessaloniki, Greece. Goodrich, H. R., M. A. Smith, 1937 Genetics and histology of the color pattern in the normal and albino paradise fish, Macropodus opercularis L. Biol. Bull. 73: 527-534. Gordon, M., 1942 Mortality of albino embryos and aberrant Mendelian ratios in certain broods of Xiphophorus hellerii. Zoologica 27: 73-74. Gordon, M., 1948 Effects of five primary genes on the site of melanomas in fishes and influence of two other genes on their pigmentation. Special Publ. New York Acad. Sci. 4: 216-268. Hadley, P. B., 1915 The white leghorn. J. Hered. 6: 147-151. Haskins, C. P., and E. F. Haskins, 1948 Albinism, a semilethal autosomal mutation in Lebistes yeticulatus. Heredity 2: 251-262. Kajishima, T., 1960 The normal developmental stages of the goldfish, Carassius auratus. Japan. J. Ichthy. 8: 20-28. Kajishima, T., 1972 Inheritance of albinism (in Japanese). In " Kingyo Taikan " (Y. Matsui, ed.), pp: 39-40. Midori Shobo, Tokyo. Makino, S., and Y. Matsui, 1970 Tropical fishes and the goldfish (in Japanese). Hoikusha, Osaka. Matsui, Y., 1933 A preliminary note on the inheritance of scale transparency in gold-fish of Japan. Proc. Imp. Acad. (Tokyo) 9: 424-427. Matsui, Y., 1934 Genetical studies on gold-fish of Japan. 3. On the inheritance of the scale transparency of gold-fish. J. Imp. Fish. Inst. 30: 47-66. Matsui, Y., 1936 On the genetical studies in carp. A preliminary note (in Japanese). Japan. J. Genetics 12: 44-47. Sadoglu, P., 1957a A Mendelian gene for albinism in natural cave fish. Experientia 13: 394. Sadoglu, P., 1957b Mendelian inheritance in the hybrids between the Mexican cave fish and their overground ancestor. Ver. Deut. Zool. Ges., Graz, 1957, 432-439. Sadoglu, P., and A. McKee, 1969 A second gene that affects eye and body color in Mexican blind cave fish. J. Hered. 60: 1-14. Shull, G. H., 1914 Duplicate genes for capsule form in Bursa bursa-pastoris. Zeit. Ind. Abst. Vererb. 12: 265-302. Sinnot, E. W., L. C. Dunn, and Th. Dobzhansky, 1950 Principles of genetics. McGraw-Hill Book Comp. Inc. New York, Toronto, London. Sinnott, E. W., and G. H. Durham, 1922 Inheritance in the summer squash. J. Hered. 13: 177-186. Teitler, N., 1967 Know your goldfish. The Pet Library, Ltd., New York. Yamamoto, T., 1969 Inheritance of albinism in the medaka, Oryzias latipes, with special reference to gene interaction. Genetics 62: 797-809. Plate 1

A Albino globe-eye metallic (tt) orange (0) from the cross of original albinos. B Albino globe-eye nacreous (Tt) pink (0). An F2 from the cross Globe-eye nacreous (Tt) calico (Sanshiki demekin)xAlbino globe-eye metallic (tt) orange. Parents were both black-eyed nacreous (Tt) Fl fish.