By H. Tomita
At present, 37 mutant genes on 34 loci are established in the medaka. They are concerned with body colors and morphogenetic deformities. One is an incomplete dominant mutant and others are all recessive mutants. As most of them are recently found in our laboratory, the genetic analyses of them have not yet been completed. The outlines of genetic characters of these mutants are shown in this chapter. All mutant genes mentioned in this chapter are keeping in our laboratory. It is very desirable to receive any information concerning with mutants other than the ones kept in our laboratory.
Mutants of the medaka known hitherto have been the b and r alleles concerning with body colors. Their origins are entirely unknown. The fused (fused vertebrae) and wavy (curved vertebral column) were found by Aida (1930), and the color interferer (ci) was found by Nakabori and analyzed by Aida (unpublished) and Takeuchi (1969). Mutants known hitherto were listed up by Tanaka (1943) and Egami (1956). When we start to analyze the genetic characters of the medaka, only these five alleles are known as a simple Mendelian characters though the inheritance of wavy is somewhat complex. The 31 mutant genes on 29 loci were collected in our laboratory. They are found in the wild populations, fish stocks and the offspring of test crosses, except one received from Takahashi at Izumo, Shimane Prefecture in 1971.
The body colors mainly depend on the combinations of states of chromatophores and iridocytes. The chromatophores are melano-phores, xanthophores and leucophores. In the wild type, melanophores have black melanin granules, xanthophores contain an orange-red pigment and leucophores have pigment granules which are white to orange in the reflect light. Aida (1921) proposed the following genetic symbols: wild (brown-black) BR, orange-red bR, white br, bluish black Br, orange-red variegated with black B'R, white variegated with black, B'r. The pigmentation of melanophores is controlled by the b alleles (B>B'> b). The B gene produces black melanin granules, the b gene induces colorless melanophores and the B' gene causes black variegation. The r alleles (R> r) govern the deposition of the orange-red pigment in xanthophores. The r and R genes produce colorless and orange-red xanthophores, respectively. When the wild type (BR) are reared in the white background and in clean water indoors, their body colors become pale like light blue. The orange-red type (bR) also becomes pale yellow. The phenotypic expression of body colors depends not only on genotypes but also environmental factors such as backgrounds, food and culture media. The orange-red type (bR) looks red to someones whereas it looks yellow to others. It is apparent that the phenotypic names of body colors are conventional and flexible. There are so many color phenotypes that it is almost impossible to describe color names of all color types of the medaka.
The chromatophores are effector cells. The activities of chromatophores are the principal ones involved in the response to light and darkness and to the black and white backgrounds. The melanophores and xanthophores in isolated skin disperse in the isotonic NaCl solution and concentrate in the isotonic KCl solution. On the contrary, the leucophores disperse in the isotonic KCl solution and concentrate in the isotonic NaCl solution.
The chromatophores are present in the dermis of skin both outside and beneath the scales. They are often found in the peritoneum, eye balls and even deeply around the brain and spinal cord. Leucophores appear on the edges of both anal and caudal fins during breeding season in adult males. Iridocytes are present on the edges of anal fin at young, and disappear at adult stages when leucophores appear. Iridocytes are present on the eye balls, peritoneum, beneath the scales and on the gill covers.
The 26 genes on 23 loci are related to the expression of body colors in the medaka. As many color genes have pleiotropic actions on the state of chromatophores, the genic expression is very complex in body colors. The combinations of these genes are very numerous. If linkages are neglected, the combination may reach to 12 ´ 2 exp21 (over 25,000,000). Therefore a color name does not always correspond to one phenotype, but it must express analogous types of body colors. As slight color differences are impossible to check in external body colors, the color name must be used with genetic symbols, for example, orange-red (bR), orange-red (blfR), orange-red (bRwl), orange-red (bRsm) and so on. The phenotypic name, orange-red corresponds to many types caused by the genic interaction. Therefore, it is necessary in experimental materials to express body colors with genetic symbols and to exclude ambiguous phenotypic color names. For example the genetic symbol BR may stand for brown, brown-black or black.
As mentioned above, Aida (1921) used the genetic symbols, B> B'>b and R>r. It is important to note that the genes B and R are not dominant mutants but they are wild types (standard types). The B' gene belongs to the b alleles. The B>B'>b correspond to +b>b'>b and R>r to +r>r. But the genetic symbols B, B' and R are used in this chapter in memory of Aida's famous works.
The morphogenetic deformities are the dwarf (fused), the reduced scales, the reduced fins and so on. The six kinds of dwarfs are difficult to distinguish each other externally. Deformed individuals are often less viable. The expressions of deformities are so variable that in extreme case, it can be detected by external characters but slight deformities can only be detected by dissection.
The chromosome number of diploid are 48 and sex chromosomes are male heterogametic type (XY). Therefore, the numbers of linkage groups are 25. The r alleles are sex linked, and autosomal linkages are detected in three groups such as the ci and i alleles, the co and dx-2 alleles, and the di and wl alleles. As mentioned above, 34 loci are found in this fish and some linkages may be added, following future genetic analyses. At present, it has finished only one fifth of planned genetic analyses.
The plural genetic symbols are usually arranged in an order of alphabet or of the linkage group. In the medaka, the plural sequences of genetic symbols are expressed as the order of alphabet.
In our laboratory, all mutant genes are keeping by means of mass matings. The population size is usually 50 to 100 individuals except special cases, and therefore, to make isogenic strains it is necessary to make matings for 500 to 1,000 generations. This means that the isogenic strains may not be established by mass matings of the large population. The pair mating or the mass matings of a small size are necessary to make the isogenic strains. The pair mating is the best for establishing and keeping the pure strains. To each mutant stocks in our laboratory, we have informations only concerned with the mutant gene in question and have not informations about other genetic characters. For example, the bR type is homozygous for the b and R genes, but the state of other genes in this stock are unknown. For experimental materials, the isogenic strains are useful tools for the biological analyses. We wish to establish many kind of isogenic strains for each purpose in many laboratories using the medaka. To our regret, we have no isogenic strains in our laboratory, although we are attempting to make them.
Most of mutant genes in our laboratory are established in recent ten years. Morphological and developmental studies of genetic characters are carrying and the genetic analyses are finished only about one fifth. The description in this chapter will be rewritten in future elsewhere.
Mutant genes
* Aida (1921) gave no name to the b gene, but he stated that the B gene is able to produce black pigment or melanophores uniformly through the whole body.
B': Black variegated*, recessive to B and dominant to b, autosomal (Aida, 1921). The origin is unknown. Homozygotes are black variegated with dark and light regions in body colors. They possess black melanophores and colorless melanophores at dark and light regions, respectively. These melanophores show normal color changes. It is questionable that black and colorless melanophores of the B' type show the same form, the same size and the same degree of pigmentation as those of the wild (BR) and orange-red (bR) types, respectively. The colorless melanophores have tyrosinase activity. The B'R type, having a few black melanophores are almost similar to the body color of the orange-red type (bR), while ones, having many black melanophores, reduce the orange-red color and are like to the wild type (BR). In the white background, the variegation of the B'R type become obscure because melanophores concentrate to punctate states. The B' gene has no effect on the pigmentation of leucophores and the physiological response of xanthophores and leucophores. The B'dmR type possess dispersed black and colorless melanophores and dispersed leucophores, and the black variegation is clear even in the white background. The B'cmR type is often alike to the orange-red type (bR) in the body color except fins.
* Aida (1921) gave no name to the B' gene, but he stated that it controls production of black pigment in mottled state.
* In 1971, the orange-reddish brown (BfmR) stock, one male, and three females, and immatured two fish (all females after maturation) were received to our laboratory from Takahashi. The BfmR stock was not homozygous in the b allele. The offspring of mass mating of these fishes were the BfmR, bR and new color (yellowish gray) types. Genetic analyses show that this new color is dependent on the combination of genes, B, fm, R and ci. According to Takahashi, the ci gene had never been introduced to his fish stock, and he had never found this new color type containing homozygous ci gene. New combination may occur by genetic drift. In 1971, there were heavy snow at Izumo and many medaka died in his pond. Only four adults were survived in this mutant stock. The sudden decrease of population size may give the chance of finding this gene. As his pond is narrow, and the population size was always small this gene might not be detected by him.
Da: Double anal fins, incomplete dominant, autosomal (Tomita, 1969a). Found in progeny of the wild medaka collected at Toyokawa, Aichi Prefecture (1966) and established in our laboratory (1967). Homozygotes (BBDaDaRR) have an anal fin in place of a dorsal fin. It has two anal fins at ventrum and dorsum symmetrically. In adult males, both anal fins possess the papillar processes on fin rays of posterior parts as secondary sexual characters. The xanthophores and melanophores are abundant on the anal fin at dorsum but are a few on the anal fin at ventrum. The anal fin at dorsum shows color feature like as a dorsal fin. The iridocytes are abundant on the surface of dorsal region between head and anal fin at dorsum. The caudal fin in this type shows the rohomic form consisting of two parts, above and below, and the caudal fin is symmetrical externally and internally. The vertebral column extends stright back to the tip of the body with the fin developed symmetrically above and below.
Heterozygotes (BBDa+RR) have the large dorsal fin (7-14 fin rays), while the normal dorsal fin has 5-7 fin rays (mode is 6). In adult males, papillar processes often yield on posterior fin rays of the dorsal fin. The caudal fin is normal in the form and in the size. The bDaiR type is albino having double anal fins. The BciDaR type is gray and has double anal fins.
de: Decreased melanophores, recessive, autosomal (Tomita, 1971). Found in the progeny of the wild medaka collected at Nagakute, near Nagoya (1967) and established in our laboratory (1967). Homozygotes (BBdedeRR) are orange-reddish brown in the body color. Especially, the orange-reddish colors are remarkable at head region and caudal fin. Expression is very variable. This character may depend on the decrease of melanophores in number and on the increase of the orange-red pigment. The newly hatched larvae resemble to the wild type (BR). The mutant characters appear at about 10 mm body length. At first, the region behind head becomes orange-reddish color. The de gene has no effects on the physiological color change of chromatophores. The bdeR type is orange-red similar to the bR type in body colors. The de gene affects on the carotenoid content in the presence of the B gene. Double recessive BcmdeR type is like to the orange-red type (bR) except fins. The BcideR type is yellowish gray in body colors.
di: Dispersed xanthophore, recessive, autosomal (Tomita, 1966), linked with wl (Tomita, 1970). Found in the orange-red stock of a fish dealer at Yatomi near Nagoya (1963) and established in our laboratory(1964)*. Again, found in the wild populations at Toyokawa, Aichi Prefecture (1965) and Nagakute near Nagoya (1967), and in the orange-red stock at Yamagata, Yamagata Prefecture (1966)#. Homozygotes (bbdidiRR) have dispersed xanthophores which do not concentrate to punctate state by various treatments. The cellular boundary of dispersed xanthophores are obscure. This character begins to appear on the differentiation of xanthophores in embryos. The di gene may give no effects on the pigmentation and the physiological color change of melanophores and leucophores. The dispersed xanthophores in isolated skin are not concentrated in the isotonic KCl solution. The BdiR type shows yellowish brown in body colors. The xanthophores of the bcodiR type show intermediate characters between those of the bcoR and bdiR types and are obscure in the cellular boundary, and red pigment granules scatter in xanthophores. Their body colors are bright orange-red.
* The fish stocks at Yatomi often contain the di gene in low frequency. When the white type (br) is selected and established from fish stock at Yatomi, the gene di is rarely contained in the established white type, as the white type (br) is not distinguished from the white type (bdir) externally. In our laboratory, white stocks keeping by mass mating for 15 generations have the di gene in low frequency. And it is also certain that the white fused (bfur) type keeping in our laboratory contains this di gene in low frequency. As the di gene may distribute in orange-red stocks of fish dealers in our country, it is necessary to check the di gene for the study of xanthophores.
dx-1: Dilute xanthophore-1, recessive, autosomal (Tomita, 1969a). Found in the wild population at Sugashima, Toba, Mie Prefecture at low frequency (0.5%) (1957) and established in our laboratory (1959). Homozygotes (BBdx-1dx-1RR) are dilute brown in the body color and have dilute orange-red xanthophores. The carotenoid content of this mutant is one eighth to one forth of that of the normal ones. The dx-1 gene shows no effect on the pigmentation of melanophores and leucophores. The physiological color change of chromatophores are normal in this mutant. The body color of the Bdx-1R type is dilute brown alike to that of the blue type (Br). The bdx-1R type is dilute orange-red like to the white type (br). The bcodx-1R type has concentrated dilute orange-red xanthophores. The bdidx-1R type has the dispersed dilute orange-red xanthophores (dilute yellowish orange-red). The Bcmdx-1R type has the concentrated melanophore and dilute orange-red xanthophore, and shows dilute blond in the body color.
dx-2: Dilute xanthophore-2, recessive, autosomal (Tomita, 1969a), linked with co (Tomita, 1970). Found in the wild population at Tame, Toyohashi, Aichi Prefecture (1965) and established in our laboratory (1966). Homozygotes (BBdx-2dx-2RR) are dilute brown in the body color alike to the blue type (Br) and have dilute orange-red xanthophores. The phenotypic differences between the Bdx-1R and Bdx-2R types are very small. The dx-2 gene has no effect on the pigmentation of melanophores and leucophores, and on the physiological color changes of chromatophores. The offspring of crosses between the Bdx-1R and Bdx-2R types are all the wild type, and F2 progeny segregates the wild and dilute brown types in a ratio of 9:7. The double recessive Bdx-1dx-2R type is phenotypically similar to the Bdx-1R and Bdx-2R types. The dx-2 gene links with the co gene but the dx-1 gene is independent from the co gene.
fl: A few leucophores, recessive, autosomal (Tomita, unpublished). Found in the offspring of the wild medaka collected at Idaka, Nagoya (1968) and established in our laboratory (1968). Adult homozygotes (BBflflRR) are brown in the body color and can not be distinguished from the wild type (BR) externally. The embryos and larvae just after hatching have a few leucophores on the dorsal trunk of body and around the brain. They often show pale color (whitish) at these stages. Mutants are easily checked. The segregation ratio must be scored at these stages. The adult mutants have a small number of leucophores. The adult male mutants have a few leucophores on the edges of anal and caudal fins in breeding season. Expression is very variable. The fl gene has no effects on the pigmentation of melanophores and xanthophores and on the physiological color changes of chromatophores.
fm: A few melanophores, recessive, autosomal (Tomita, unpublished). Found in the wild population at Daito, Ohara, Shimane Prefecture, by Koji Takahashi (1965)*. Established in our laboratory in 1971. Homozygotes (BBfmfmRR) have small numbers of melanophores which are often small in the size, and are orange-reddish brown at the head region and caudal fin. They resemble the orange-reddish type (BdeR) phenotypically but are easily distinguished from the wild type (BR). At the embryonic and larval stages, a few small melanophores differentiate and often arrange on dorsum of mid trunk forming parallel lines. The bfmR type is orange-red in body colors and is not distinguished from the orange-red type (bR) externally. The mutant character expresses itself in the presence of the B gene. The number and size of melanophores are very variable at larval and adult stages. The fm gene has no effect on the physiological color change of chromatophores.
fs: Fused interhemal spins, recessive, autosomal (Tomita, 1967). Found in offspring of the wild medaka collected at Idaka, Nagoya (1964) and established in our laboratory (1965). Homozygotes (BBfsfsRR) have the small anal and dorsal fins. The interhemal spins fuse to a few pieces. The base of anal fin is short (about one half to two third length of normal ones). The some fin rays do not differentiate fully and have no segments. But the decrease of numbers of fin rays and interneural spins does not occur. The same effects occur in interneural spins, and the dorsal fin is also small in size. As the dorsal fin has only 5 to 7 fin rays (mode is 6), the fusion of interneural spins give no serious effects on the form of dorsal fin. The BDafsR type has two small anal fins at dorsum and ventrum.
* K. Takahashi, former teacher of Daito High School, found a female mutant in the wild medaka collected at Daito by students in 1965, and then he started to mate for test-crosses and to keep this mutant in his private pond. According to his preliminary test, this mutant belongs to the b alleles. This mutant was offered to our laboratory from Takahashi in 1971. As result of re-examination in our laboratory, this mutant does not belong to the b alleles and may not link with the b alleles.
* Aida used no genetic symbol of this mutant. The genetic symbol f was occasionally used in Japan. The fu symbol was used by Egami (1956).
fu-3: Fused-3, recessive, autosomal (Tomita, 1967). Found in the offspring of the wild population at Idaka, Nagoya (1964) and established in our laboratory (1965). Secondly found in the offspring of the wild medaka collected at Tame, Toyohashi, Aichi Prefecture (1965) and established (1966). Homozygotes (BBfu-3fu-3RR) show short body (dwarf). Expression is very variable. In extreme case, vertebrae fuse into a few pieces. The presumed site of fusion are not detected after hatching by the method used in the fu and fu-2 types. The mechanism of fusion may be different from that of the fu and fu-2 types. The fusion may occur at later stages than those of the fu and fu-2 types.
fu-4: Fused-4, recessive, autosomal (Tomita, unpublished). Found in the offspring of the wild population collected at Nagakute near Nagoya (1970) and established (1970). Secondly, found in the offspring of test crosses between the bciR and the bR types (1972) and established (1973). Homozygotes (BBfu-4fu-4RR) show short body (dwarf). They have fused vertebrae. Expression is very variable. The presumed site of fusion can not be checked at a few days after hatching by the method used in the fu type. The mode of fusion may be different from the fu and fu-2 types.
fu-5: Fused-5, recessive, autosomal (Tomita,unpublished). Found in the offspring of the wild medaka collected at Toyokawa, Aichi Prefecture (1970) and established in our laboratory (1971). Homozygotes (BBfu-5fu-5RR) show short body (dwarf). They have fused vertebrae. The presumed site of fusion can not be detected as in the case of the fu-3 type. The mode of fusion may be similar to that of the fu-3 type. Expression is very variable.
fu-6: Fused-6, recessive, autosomal (Tomita, unpublished). Found in the offspring of test crosses of the wild medaka collected at Nagakute near Nagoya (1972) and established in our laboratory (1973). Homozygotes (BBfu-6fu-6RR) are short body (dwarf) and sometimes show fused wavy. At embryonic stages, the notochord are bent wavily on both side, and above and below. In extreme case, they can hatch out but can not swim. This wavy character is often lost with growth. Some adults are short body with wavy vertebral column and fused vertebrae and others with fused vertebrae. Expression is very variable. This character is detected easily before hatching externally. The probable phenocopy has already been obtained with treatments of various chemicals such as phenylthiourea, semicarbazide and so on, at embryonic stages (Tomita and Matsuda, 1961; Yamamoto, Tomita and Matsuda, 1963).
gl: Guanineless, recessive, autosomal (Tomita,unpublished). Found in the offspring of the wild medaka collected at Idaka, Nagoya (1968) and established in our laboratory (1968). They are keeping in heterozygous conditions. The embryos of homozygotes (BBglglRR) develop and hatch out normally. But all larvae die till reaching 10 mm body length. The embryos and larvae have small quantity of guanine on eye balls and peritoneal wall. They have black eye balls with a small amount of guanine. They are lethal. The similar types are often found in various kind of fish stocks, although they have not yet been analyzed.
i: Albino, recessive, autosomal (Yamamoto, 1969), linked with ci (Yamamoto and Oikawa, 1968, 1973). Found in the offspring of dilute orange-red (brd) type selected from fish stock at Yatomi near Nagoya (1961) and established in our laboratory (1961). At second chance, found in the offspring of the orange-red male collected at Anjo, Aichi Prefecture (1967)* and established (1967) (Tomita, unpublished). Mammalian albino are devoid of all body colors, as they have only melanocytes and no other chromatophores. But the albino medaka (BiR) are dilute orange-red to extreme dilute orange-red color in body colors, as they have colored xanthophores and leucophores. The first albinos are segregated in the F2 Offspring of crosses between dilute orange-red (brd) and white (br) types. This dilute orange-red female is bbi+irrd in the genotype. The albino (BiR) have amelanotic melanophores, dilute orange-red xanthophores and numerous well developed leucophores. Expression of xanthophores and leucophores is very variable. The amelanotic melanophores show almost no tyrosinase activity. The second albino was segregated in the F2 offspring of crosses between orange-red females and orange-red male which was collected by Ohta. The first and second albino belong to the same alleles but it is not certain whether they are the same gene or multiple alleles. The larvae of the first albino have often a few black melanophores on eyes and in skins of body trunk, but these black melanophores in skins disappear at adult stages. In rare case, a few black melanophores remain in skin at adult stage. The larvae of the second albino have usually many slightly colored melanophores. They also disappear at adult stages. It is unknown the relation between the black melanophores of the first albino and the many slightly colored melanophores of the second albino. The expression of melanophores at larval stages is very variable and complex.
* An orange-red male among the wild medaka was found in paddy field at Sakurai, Anjo, Aichi Prefecture, by Masafumi Ohta in early summer of 1966, and it was offered to our laboratory by Hideko Tsuzuki. This fish is bbi+iRR in the genotype.
il-1: Iridocyteless-1, recessive, polymeric, autosomal (Tomita, unpublished).
il-2: Iridocyteless-2, recessive, polymeric, autosomal (Tomita, unpublished). Found in the offspring of orange-red stock bought at Yamagata (1971) and established in our laboratory (1972). Homozygotes (bbil-1il-1il-2il-2RR) are lacking of iridocytes on gill covers, beneth scales and in the skin. The gill covers are transparent and red blood color are seen through them. It is not yet clear whether guanineless iridocytes are present on these regions devoid of guanine or not. The iridocytes are present on eyes and peritoneum as well as those of the wild type (BR). At larval stages, iridocytes on gill covers and in the skin do not differentiate in this mutant. It is not true that iridocytes differentiate and then disappear in definite regions. This character may be dependent on the loss of migrating ability of iridoblasts to definite regions or guanineless iridocytes can not accumulate guanine at definite regions.
At larval to young stages, normal fish have iridocytes on the edges of the anal fin, and in adults iridocytes disappear and leucophores appear in males. In this mutant, the iridocytes are not present the edge of the anal fin, but in adult males leucophores appear as those of the wild type.
This il-1 and il-2 genes have no effect on the pigmentation and physiological color changes of chromatophores. The Bil-1il-2R type is.iridocyteless brown and red color of blood are seen through transparent gill covers. The bdiil-1il-2R has dispersed xanthophores and are iridocyteless. The biil-1il-2R type is iridocyteless albino.
lf: Leucophore-free, recessive, autosomal (Tomita, unpublished). Found in the offspring of the wild medaka collected at Toyokawa, Aichi Prefecture (1970) and established in our laboratory (1971). Adult homozygotes (BBlflfRR) have no visible leucophores. Adult wild males (BR) have many leucophores on the edges of the anal and caudal fins in breeding season. This mutant shows no this feature. In embryos, the leucophores of the wild type (BR) appear around brain at the beginning of heart beat, and then develop on the embryonic body. The embryos of this mutant have no leucophores. Newly hatched larvae (BlfR) and (blfR) are pale in body color and easily distinguished from the wild (BR) and orange-red (bR) types. As the leucophore-free character gives no effect on the external body color at adult, the adult BlfR and blfR types are almost similar to the wild (BR) and orange-red (bR) types, respectively. The Il gene has no effect on the pigmentation of melanophores and xanthophores and on the physiological color changes of chromatophores.
The lf gene is epistatic to the ci, dm, fl, i, wl and other genes which concern with the state of leucophores. All types containing homozygous lf gene have no leucophores. For example, the bilfR type is leucophore-free albino, and the BcilfR type is leucophore-free gray.
ml-1: Many leucophores-1, recessive, polymeric, autosomal (Tomita, unpublished).
ml-2: Many leucophores-2, recessive, polymeric, autosomal (Tomita, unpublished). Found in the offspring of test crosses of iridocyteless stock (bil-1il-2R) (1972) and established in our laboratory (1972). The embryos and larvae of homozygotes (BBml-1ml-1ml-2ml-2RR) have leucophores about three times as many as those of the wild type (BR). As the mutant larvae show reddish color for larval leucophores are orange-red in color, they are distinguished from the wild type (BR) externally. This mutant embryos are semi-lethal. Many embryos die during the differentiation of embryonic body. The segregation ratio is necessary to score at the embryonic stages at which leucophores begin to differentiate. Larvae and adult fish are also weak and occasionally die. Adult mutant often have numerous small leucophores. Melanophores are normal in size and number, and show normal response of color changes. The color of xanthophores are often reduced to pale orange-red. The expression of chromatophore is very variable.
mm: Mixed melanophores, recessive, autosomal (Tomita, unpublished). Found in the progeny of the wild medaka collected at Nagakute near Nagoya (1970) and established in our laboratory (1970). Homozygotes (BBmmmmRR) are black variegated with light and dark color regions. The light color regions possess undifferentiated melanophores and leucophores (concentrated melanophores and leucophores). The dark regions have normal dendritic melanophores and leucophores. As the normal melanophores disperse and undifferentiated ones concentrate, the black variegation is remarkable in the black background.
The mm gene is hypostatic to the cm gene. The double recessive BcmmmR type is blond in the body color except fins. The BcimmR type is variegated gray having undifferentiated melanophores and leucophores partially. The BcommR type is variegated reddish brown having concentrated xanthophores, and has undifferentiated melanophores and leucophores partially.
r: Colorless xanthophore*, recessive (Toyama, 1916; Ishiwara 1917), sex linked (Aida, 1921). The origin is unknown. Homozygotes (bbrr) have colorless xanthophores and are white in the body color. The colorless xanthophores contain almost no orange-red pigment. The size and structures of colorless xanthophores are similar to those of the orange-red type (bR). The deposition of the orange-red pigment in xanthophores are mainly controlled by the r alleles. The r gene has no effects on the pigmentation of melanophores and leucophores and on the physiological color change of chromatophores. The r gene is epistatic to the co, di, dx-1, dx-2 and other genes concerning with the state of xanthophores. Therefore, the bcor, bdir, bdx-1r and bdx-2r types are all white in body colors and can not be distinguished from white type (br) externally.
* Aida (1921) gave no name to the r gene, but he stated that the R controls production of yellow pigment or xanthophores.
rf: Reduced fins, recessive, autosomal (Tomita, unpublished). Found in the offspring of the wild medaka collected at Tame, Toyohashi, Aichi Prefecture (1967) and established in our laboratory (1968). The fins of this mutant differentiate normally, and then the tips of fin rays bent irregularly and begin to reduce in size. In extreme case, the fins loss wholly and fish can not swim normally. Mutant females and males are almost sterile. Only a few females are fertile. Occasionally, vertebral column bents wavily. Most of them have the deformed scales with irregular ridges. The distribution of melanophores is also irregular in the skin beneath and outside scales. The melanophores often arrange around scales as a hexagonal pattern. The BcmRrf type is blond in the body color and has reduced fins. The BciRrf type is gray in the body color and has reduced fins.
rs: Reduced scale, recessive, autosomal (Tomita, 1969b). Found in the offspring of the wild medaka collected at Nagakute near Nagoya (1966) and established in our laboratory (1967). In the wild medaka, whole body except the head and fins is covered by scales. These scales overlap each other like tiles of a roof. This mutant (BRrs) has small scales of irregular shape. The differentiation and growth of scales are delayed in this mutant. In extreme case, the scales are small and scatter on the body separately each other. Chromatophores distribute irregularly in the reduced scale. Melanophores in the inner side of the scale form a hexagonal pattern whereas those on the outer side of the scale cover whole surface of it. Expression is very variable. The BciRrs type is gray in the body color and has reduced scales. The BDaRrs type is brown with a hexagonal pattern, and has double anal fins and reduced scales.
sm: Slow response melanophore, recessive, autosomal (Tomita 1969b). Found in the wild population at Nagakute near Nagoya (1965) and established in our laboratory (1966). This mutant is often found in paddy fields of Nagakute in low frequency. As paddy fields are too wide to determine the gene frequency, we have never checked it exactly. The melanophores of this mutant usually disperse in a natural condition. They concentrate partially in a white background for a few hours, while normal melanophores concentrate wholly for a few minutes. The physiological change of melanophores of this mutant takes a long times. Melanophores in isolated skin are dispersed in the isotonic KCl solution, while normal melanophores concentrate. The injection of adrenaline to body also does not induce concentration of melanophores while the normal fish causes to pale the body color in a few minutes. The form and size of melanophores are the same as those of the wild type (BR). The BcmRsm type shows blond in the body color and has dispersed melanophores on fins. The BdmRsm type shows black brown and the sm character is masked by the dm gene.
vc: Variegated chromatophores, recessive, autosomal (Tomita, unpublished). Found in the wild population at Nagakute near Nagoya (1969) and established in our laboratory (1970). Homozygotes (BBRRvcvc) show black variegation in body colors. Melanophores are present in a dark color area and absent in a light color area. They are present on the same layers as those of wild type (BR). Leucophores distribute variegately in the dermis beneath scales and are absent in the pigment layer of the skin outside scales. The caudal and anal fins have not leucophores on the edges, whereas in normal males, many leucophores appear on edges of the caudal and anal fins for a breeding season. The sizes of melanophore and leucophore are often larger in the area located a few chromatophores than those of the wild type (BR). Expression is very variable. The BdmRvc type is black variegated in the body color and has dispersed melanophores and leucophores. The BcoRvc type is reddish black variegated and has concentrated xanthophores.
wl: White leucophore, recessive, autosomal, linked with di (Tomita 1970). Found in the offspring of white (br) stock in our laboratory (1968) and established (1968). The color of leucophores in this mutant is wholly white in the reflect light, as normal leucophores show orange to orange-red color at embryonic and larval stages. As the wl gene is no effect on the external body color, the body colors of the BRwl and bRwl types are brown and orange-red similar to those of the wild (BR) and orange-red (bR) types, respectively. The color of leucophores at adult stage is difficult to determine by interfering with other chromatophores, especially xanthophores. The physiological color change of leucophores is normal in this mutant. At embryonic and larval stages, this mutant shows whitish color and easily distinguishes from the wild type (BR). The segregation ratio must be scored at these stages, as it is difficult at adult stages. The wl gene has no effects on the pigmentation and physiological color changes of melanophores and xanthophores. The biRwl type is albino having white leucophores. The bDaRwl type has double anal fins and white leucophores.
wy*: Wavy, recessive, autosomal (Aida, 1930). Found in fish stock by Aida and were analyzed (Aida, 1930). This mutant is often found in various kind of fish stock and wild populations. The vertebral column curves dorso-ventrally and forms two wavy crests. The external body form shows a more or less wavy outline dorsally as well as ventrally according to the undulation of vertebral column. The wavy character is selective to normal and wavy. This means the presence of modifiers of the major gene (Takeuchi 1960, 1966).
* Aida gave no genetic symbol to this mutant. The symbol w was occasionally used in Japan. The wy symbol was used by Egami (1956).
In: "MEDAKA(killifish) : Biology and Strains"
Yamamoto, T. (ed.) , Keigaku Pub. Co., Tokyo, 1975, pp. 251-272.
b: Colorless melanophore*, recessive, autosomal (Toyama, 1916; Ishiwara, 1917; Aida, 1921). The origin is unknown. Homozygotes have colorless melanophores which contain slightly pigmented melanin granules. These melanophores are invisible in the dispersed conditions. When they concentrate to the punctate state, the black melanophores become visible under the ordinary microscope. The form of colorless melanophore is dendritic as black melanophores of the wild type (BR), and shows normal physiological color changes. The b gene has no effects on the pigmentation of leucophores and on the color changes of xanthophores and leucophores. The content of an orange-red pigment of the wild type (BR) is one third to half of that of the orange-red type (bR). The b alleles have effects on the pigmentation of xanthophores. Embryos and larvae of the b type have invisible and visible melanophores which contain various degree of black pigment. The pigmented melanophores at the larval stage disappear at the adult stage. In colorless melanophores, tyrosinase activity is easily demonstrated by histochemical method (Hishida, Tomita and Yamamoto, 1961; Tomita and Hishida 196la, b). The black melanophores of the wild type (BR) differentiate on the yolk sac at early embryonic stage before beginning of heart beat and the differentiation of somites. The difference of characters between the genes B and b are clear at all stages of the development. This allele is one of useful markers. Expressions of colorless melanophores are very variable in embryos and larvae.
The bR and br types are orange-red and white in body colors. The bcmR type has colorless concentrated melanophores and the bdmR has colorless dispersed melanophores and dispersed leucophores. But both bcmR and bdmR types are orange-red in body colors. As the b alleles are epistatic to the cm, de, dm, fm, mm, sm and vc alleles which control the states of melanophores, the bcmR, bdeR, bdmR, bfmR, bmmR, and bRvc types are all orange-red in the body color and cannot be distinguished from the orange-red types (bR) externally.
bd: Dilute black, recessive to B and dominant to B' and b, autosomal (Tomita, 1971). Found in a stock of the orange-red type from a fish dealer at Yatomi near Nagoya (1970) and established in our laboratory (1970). Homozygotes (bdbdRR) show dark orange-red to brown in body colors. Most adults (bdR) can not be distinguished from the wild type (BR) in body colors externally. The body colors of embryos, larvae and youngs (bdR) are similar to those of the orange-red type (bR). They have colorless melanophores and never black melanophores like as the wild type (BR). At about 10 mm body length, the colorless melanophores begin to darken. At this changing phase, some melanophores are already black and others remain in the colorless state. This darkening has never occurred uniformly. The fish (bd) shows temporary variegation. The bd gene gives no effect on the color changes of chromatophores and the pigmentation of leucophores. The color of xanthophores is reduced by progress of darkening melanophores. The adult bdcmR type is alike to the orange-red type (bR) in the body color, while the BcmR type is blond in the body color. The bdcmR type has concentrated light black colored melanophores. The bddmR type has dispersed light black colored melanophores and dispersed leucophores. Heterozygotes (B'bdRR) show black variegation till about 10 mm body length and then become brown color (bdR type), as colorless melanophores begin to darken.
ci: Color interferer, recessive, autosomal (Aida, unpublished; Takeuchi, 1969), linked with i (Yamamoto and Oikawa, l968, 1973). Found in the wild population at Kyoto by Eiji Nakabori (1951) and analyzed by Aida (unpublished) and Takeuchi (1969). Recently found in the offspring of the BfmR stock received from Takahashi at Izumo, Shimane Prefecture (1971) and established in our laboratory (1972) (Tomita, unpublished).* Homozygotes (BBciciRR) are gray in body colors and have a decreased number of melanophores and xanthophores, and well developed numerous leucophores. This gene has pleiotropic effects on all kind of chromatophores. Expressions of numbers of chromatophores are very variable. The ci gene has no effect on the physiological color changes of chromatophores. The bciR type is cream, and the Bcir and bcir types are pale blue and milky in body colors, respectively. The BcilfR type is dilute brown and has no leucophores. The BcidmR type has dispersed melanophores and leucophores and shows dark gray in body colors. The new ci and old ci genes belong to the same alleles but it is not yet certain whether they are the same gene or multiple alleles. The melanophores and leucophores in the new ci type may be more numerous in number and smaller in size than those of the old ci type. The old ci type (BciR) often has larger melanophores in the skin beneath scales. The new ci type has not this feature.
cm: Concentrated melanophore, recessive, autosomal (Tomita, 1966). Found in the wild population at Idaka, Nagoya (1961) and established in our laboratory (1962). Homozygotes (BBcmcmRR) have concentrated melanophores (punctate state) on the body regions and nearly normal melanophores on the fins. The concentrated melanophores in isolated skins do not disperse fully in the isotonic NaCl solution, in which normal melanophores disperse and show the dendritic form. A few small stellate melanophores are often present in the skin of body among concentrated melanophores. These small stellate melanophores show the weak response of physiological color changes. These melanophores may be occurred by incomplete expression of this gene. The melanophores on the fins have normal responsibility for physiological color changes. The xanthophores and leucophores show the normal response for color changes. The BcmR type is blond in body colors and the content of carotenoid increases more than that of the wild type (BR). But the bcmR type is orange-red in body colors and cannot be distinguished from the orange-red type (bR) externally. The cm gene affects on the carotenoid content of xanthophores in presence of the B gene. The BcmcoR type has concentrated melanophores and xanthophores, and is reddish blond in body colors. The BcmdeR and BcmfmR types are orange-red except fins in the body color in spite of presence of the gene B. They have a few small concentrated black melanophores except fins.
co: Concentrated xanthophore, recessive, autosomal (Tomita, 1966), linked with dx-2 (Tomita, 1970). Found in the wild population at Idaka, Nagoya (1963) and established in our laboratory (1964). Homozygotes (BBcocoRR) have concentrated xanthophores with reddish color. These xanthophores in isolated skins do not disperse in the isotonic NaCl solution, and contain clear orange-red pigment granules which are not found in normal xanthophores under the ordinary microscope. The co gene may affect on the structures of xanthophores and pigment granules. The physiological color changes of melanophores and leucophores are not effected by the co gene. The concentrated xanthophores are detected in the punctate state at late embryonic stages, while the normal xanthophores are difficult to be detected before hatching. The BcoR type is reddish brown and the bcoR shows reddish orange-red in body colors. The double recessive Bcodx-1R and Bcodx-2R types have concentrated dilute orange-red xanthophores. Their body colors are dilute reddish brown. The bcoiR type is albino having concentrated orange-red xanthophores (reddish albino). The BcicoR type is reddish gray in body colors.
# In 1966, ten orange-red medaka were bought at Yamagata, when we collected the wild medaka at Yonezawa and Yamagata. These medaka were transferred from Tokyo according to dealers. One of them has dispersed xanthophores. The fu-2 (1966), il-1, il-2 (1971) and mi-1, ml-2 (1972) gene are found in the offspring of mass mating of them.
dm: Dispersed melanophore, recessive, autosomal (Tomita, 1966). Found a mutant in the wild medaka at Tsukude, Minamishidara, Aichi Prefecture (1962) and established in our laboratory (1963). Homozygotes (BBdmdmRR) are dark brown (black velvet) in body colors and have dispersed melanophores and leucophores. The dispersed melanophores in the isolated skin do not concentrate in the isotonic KCl solution, and have more slender processes than those of the wild type (BR). The structure and form of melanophores in this mutant are different from those of the wild type (BR). This character is incomplete at embryonic stages. Leucophores in adult fish also disperse in the black background, and do not concentrate in the isotonic NaCl solution. This character of leucophore begins to appear at about 10 mm body length. The embryonic and larval leucophores show the normal response to chemicals and enviromental changes. In adult fish, the movement of pigment granules occurs to all directions at some processes of melanophores and leucophores but the regular movement to one direction is not observed. The dm gene gives no effect on the physiological color change of xanthophores. Adult fishes (BdidmR) have all dispersed chromatophores. The BdmiR type is albino having dispersed leucophores. The BcmdmR type is dark blond in body colors and shows intermediate form of melanophores between those of the BcmR and BdmR types.
fu*: Fused, recessive, autosomal (Aida, 1930; Yamamoto, Tomita and Matsuda, 1963). Found in the fish stock and established by Aida (1930). Homozygotes (bbfufurr) have fused vertebrae and show dwarf form. Expression is very variable. In extreme case, the vertebrae fuse into a few pieces and the fish shows a strong dwarf. When only a few vertebrae fuse, the character is difficult to detect externally, and is recognized only after examination by dissection. At a few days after hatching, the presumed site of fusion, showing a trapezoid profil are detected by staining with alizaline (Ogawa, 1965).
fu-2: Fused-2, recessive, autosomal (Tomita, 1967). Found in the offspring of orange-red stock of a fish dealer at Yatomi near Nagoya (1962) and established in our laboratory (1964). Secondly found in the offspring of orange-red stock obtained at Yamagata (1966) and established (1967). Homozygotes (bbfu-2fu-2RR) show short body (dwarf). Expression is very variable. The mode of fusion are similar to that of the fu mutant. The presumed site of fusion can be detected at a few days after hatching as well as in the case of the fu mutant. The offspring of crosses between the fu and fu-2 types are all normal. The F2 offspring comprise normal and fused in a ratio of 9 : 7. The double recessive bfufu-2r is detected by test crossess. They are almost similar to the fu and fu-2 types.
The i gene is epistatic to the b, cm, dm and other genes concerning with the state of melanophores. The BiR and biR types are dilute orange-red albino in the body color, and the bir type is white albino. The BdmiR type has dispersed leucophores, and it can be distinguished from other albino under ordinary microscope at adult stage. The bilfR type is dilute orange-red albino having no leucophores, and the bcoiR type is slightly reddish albino having concentrated xanthophores,< and the bdiiR type is slightly yellowish albino having dispersed xanthophores.
rd: Dilute orange-red, recessive to R and dominant to r, sex linked (Tomita, unpublished). Found in orange-red stocks of a fish dealer at Yatomi near Nagoya (1959) and established in our laboratory (1960). This type (bbrdrd) has dilute orange-red xanthophores with carotenoid content about half of that of the orange-red type (bR). The XrXrd types are often found in fish stocks at Yatomi but the XrYrd type is rare. The r alleles are R > rd > r.
The author also wishes to thank the staffs of Sugashima Marine Station of Nagoya University and to Professor M. Kusa of Yamagata University who afforded facilities in collection of the wild medaka in Yamagata Prefecture. The writer is thankful to many owners of paddy fields who broadmindly have given him tacit permission to collect the wild medaka for about 15 years.
The maintenance of these mutant genes in our laboratory is supported financially by the Ministry of Education of Japan.
dl: Dilute melanophore, recessive, autosomal. Found in the offspring of the test crosses between the ci and i types (1973). The body color of the homozygote (BBdldlRR) is an intermediate color (dark orange-red) between brown (wild type) and orange-red (bR type). This mutant has dilute pigmented melanophores. The embryos and larvae of this type have uniformly slight pigmented melanophores (visible), while in the bR type, they have colorless (invisible) and slight pigmented (visible) ones. This is a new modifier alleles for the expression of melanophores.
Si: Spotless iridocyte, dominant, autosomal. Found in the orange-red stock from a fish dealer at Yatomi near Nagoya (1974). The wild (BR) type has a pair of semicircular spots of iridocytes on head region (above brain and behind eye balls). The mutant (bRSi) has not these spots and the region behind head shows somewhat transparent, as the iridocytes on peritoneum are partially absent. The homozygote and heterozygote for this gene show the same appearance.
lc: Light color, recessive. Found in the offspring of the test crosses of the wild type collected at Nagakute near Nagoya (1974). This mutant (BlcR) has a small number of leucophores and melanophores on the body and a few melanophores on the eye balls at embryonic stages. The larvae show a whitish gray in the body color. They die within about two weeks after hatching. This type is keeping in the heterozygous condition.