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Inheritance of the red color in tilapias
L. Reich, J. Don & R. R.Avtalion Laboratory offish Immunology and Genetics, Department of Life Sciences, Bar-Ilan University, Ramat-Gan Israel Received31.5.1989 Accepted revisedform 27.1.1990 in

Abstract The inheritance of the red color was studied in two different varieties of tilapia which are both considered as hybrids of Oreochromis mossambicus. Crosses between red tilapia from the Philippines (PRT) and Sarotherodon galilaeus, or Oreochromis aureus gave a 1:1 ratio of red: normal and crosses between F~ black fish gave only black offspring. On the other hand crosses between the F~ red fish gave a 3:1 ratio of red:black and crosses between F~ red and black offspring gave a 1:1 ratio. These results lead to the conclusion that red color is dominant over the normal black color and controlled by a single autosomal gene (R). A unique phenotype named 'albino with black eyes' was observed among offspring of PRT and a presumed model of inheritance of this trait is proposed. Genetic analysis of a second variety of red tilapia (derived from an unknown origin) showed the following results: crosses between parents and between their Fl offspring consistently gave 100% red fish and crosses between this red tilapia and Oreochromis aureus gave 100% black offspring. The crosses between red and black F~ of these last two crosses gave a 1:1 ratio and crosses carried out between the black Ft offspring gave a 1:3 ratio of red:black. It may be concluded from these results that the black color is dominant in this strain and that this color is controlled by a single autosomal gene (B). The presumed mode of action of the dominant gene (R) as well as of the recessive gene (b) are discussed.

Introduction The culture of red tilapia is of some importance because its red color (a blend of red, pink, yellow and gold) is more appreciated in some markets than black tilapia. It has been reported that this fish has a fast growth rate and the ability to grow in both brackish and salt water (Sipe, 1979; Liao & Chang, 1983; Galman & Avtalion, 1983). The origin and the history of the red tilapia originating in Taiwan and the Philippines is not well documented. It is considered as a hybrid of O. mossambicus X O. hornorum with O. niloticus. In fact various morphological traits of these species (e.g. shape of snout, width to width of head)could be observed in individual different red tilapia (Galman & Avtalion, 1983). Electrophoretic analysis of transferrins and esterases also revealed high polymorphism, and no specific electrophoretic markers could be found.

The genetic determination of color and pigmentation is not yet well understood. A number of models for the inheritance of body color and pigmentation in red tilapia have been suggested (Mires, 1987; Scott et al., 1987; McAndrew et al., !988) and in the present work some of these models have been considered in order to explain the experimental results.

Materials and methods Fish The following tilapias were used in this study: (1) Sarotherodon galilaeus (Gal) originally from the sea of Galilee in Israel; (2) red tilapia originally from the Philippines (PRT) showing red (rPRT) and black (bPRT) phenotypes; (3) F~ hybrids of Gal )< rPRT; (4) a strain of red tilapia of unknown origin considered by the farmers as a red mutant of Oreochromis mossam-

UK). Artificial fertilization Artificial fertilization of the eggs was carried out in Petri dishes using modified Eagle's medium (MEM).1M Sorensen buffer (pH=7. (5) O. The water temperature was 26-28°C during the spawning season and 24-26°C in winter. galilaeus or with O. The eggs were gently agitated and then transferred after 10 min to Zuger-bottles for incubation (Don et al. 0.2-7.5-0. 10-20 days old embryos. . The families were kept in aquaria of 280 liters. Immediately before spawning. 1987). (B) red. A bPRT X bPRT cross gave a . and examined by elecron microscopy (TEM 100SK.2). thin sectioned using Ultratome III (LKB). The fish were divided into families of 4-5 females and 1-2 males each.2 and 1. Embryos were kept in the Zuger-bottles until complete yolk sac absorption. The eggs were washed with UV-sterilized water from Zuger-bottles before being fertilized with sperm. stained with uranyl acetate and lead citrate Results Crosses carried out between 7 pairs of rPRT. each equipped with a closed circulating system (Koiller & Avtalion. 1) were determined at the embryonic age of 8 days by microscopic examination (X 20).9-0.2). 1.(C) red with black eyes (X20). red or blackeyed.196 bicus (MRT). consistently gave a ratio of 3:1. The temperature of the water was kept constant at 26°C.5-0. the females were gently caught and the eggs were stripped out into a small Petri dish. respectively).5% glutaraldehyde in 0. (6) Ft hybrids of Au X MRT. the tissues were dehydrated in an alcohol series and then embedded in spures (Polaron. Jeol. A ratio of 1:1 (red:normal) was obtained in 3 crosses of rPRT X bPRT or when an rPRT female was crossed with S. Fig.. In order to avoid the influence of differential mortality at the early ages.4). Sperm was stripped out of the male in a similar way and diluted with MEM (pH 7. were fixed in 2. The fish were fed with pellets containing 40% protein. Electron microscopy Small pieces ofiridal tissue from normal.7. After washing in buffer and fixing with 1% osmium tetroxide in the same buffer. the ratios between red and black phenotypes (Fig. containing 5% carp serum. aureus (P=0. Colored phenotypes of 8-10 day old embryos: (A) black. 1985). red:black (P=0. Japan). as described by Yeheskel and Avtalion (1988). aureus (Au). The observed segregation ratios were tested by the X2 test for acceptance or rejection of the proposed hypothetical model. The water was mechanically filtered through activated charcoal and sterilized by passage through a UV irradiator.

insert (×100O0).5-0. .5 56 7 10 7. red with black eyes. N. red. black. N. Ultrastructure of iridophores of the golden tissue of normal eye.5-0.197 ratio of 0:1.5-0.1 -0.7-0. N.1 0.2 8 6 9 32 Total: 8 90 6.2 21:3:8 21:3:8 21:3:8 21:3:8 0. The segregation results showed 3 different ratios: 9:3:4 (in 2 pairs. PRT Tag No.5%) among offspring of 7 different rPRT × rPRT crosses (Rr × Rr).1 0.2 0.7-0. P=0.5-0.2 0. nucleus. aureus. B. lr.5) for the following 3 phenotypes: red. red. BE. G. galilaeus and O. B. RP.5-0. BE and black.9-0. Q (3 Q 84 rr 72 rr 148 rr 64 Rr rr Rr Rr rr rr Rr bPRT bPRT 2 G PRT 2 A PRT 2 9 5 2 0 84 0:l 39 33 1:1 74 74 1:1 30 34 1:1 105 112 1:1 25 18 1:1 160 164 682 248 156 98 190 28 221 239 90 56 23 76 10 66 1:1 3:1 3:1 3:1 3:1 3:1 3:1 3:1 1 0. Fig.2-0.2 6 4 1 7 1 1 217 Rr 43 rr Total: 8 8 6 6 32 9 23 7 16 7 16 16 7 16 6 2 2 2 2 1 1 921 338 212 121 266 38 287 Rr Rr Rr Rr Rr Rr Rr Rr Rr Rr Rr Rr Rr Rr Total: 0.5 0.7-0. at different rates (5-19. are homozygous (rr) for a recessive gene (r) (Table 1).95 0.phenotype with bright color and black eyes (BE) was observed.7-0.2). The segregation results for body and eye Table 2.5 9:3:4 0. Results of rPRT × PRT crosses showing segregation for red.99-0.5 0.1 45:3:16 0. P=0. and 45:3:16 (in 1 pair. Results of crosses showing red and black phenotype segregations in PRT. bPRT. and presumed parental genotypes.2 0.2 0.2-0.9 76 10 232 239 8 5 21:3:8 0.2 -0.5-0. offspring number.5-0. aureus. reflecting platlets (X500). S. A. cate N Color R BE B Ratio %BE R/ Q 6 23 ~ 16 16 2 1 121 287 80 18 165 56 245 74 16 7 7 16 2 2 1 2 338 226 22 212 141 15 38 25 3 266 163 27 555 67 7 6 921 636 46 23 66 89 BE/B 15 9:3:4 0. Replicate N Genotype Color of parents R B Ratio R/B P A unique . galilaeus. R.7 17 9:3:4 0.9-0.7-0.5 Total: 1623 560 3:1 R.5 0. red: normal (P= 1). black PRT. PRT. These results suggest that rPRT is heterozygous (Rr) for a red controlling gene (R) and that bPRT. Table 1.2 0. black.5-0. S. P=0. offspring number. O. 21:3:8 (in 4 pairs. respectively (Table 2).7 0. Iridophores.05 19. black eyes and black.2 -0.7 PRT RepliTag No.1 0.5-0.1).2 1 0.9-0. Red tilapia from the Philippines. 2.5-0.

Meridionai section of pupiliary border of the iris of tilapia embryo: (A) black. iridophore layer.05 492 1521 1:3 0.5 18 1:1 0. seems to be a recessive homozygote (bb).1) was shown in FI hybrids of 3 different crosses of a MRT female with an O.198 Fig. PC. respectively of black offspring was obtained.7-0. ML. red.7-0.7-0. O. 3. R. anterior chamber. (B) red with black eyes . Crosses carried out between 5 pairs of MRT gave a ratio of 1:0 red:black (P=I). Replicate N Genotype of parents Color R B Ratio R/B Q A 45 20 42 0 0 ~ 41 41 4 3 3 4 3 4 1 1 1 1 492 1531 44 200 105 39 Q BB bb bb bb bb bb c3 bb bb bb bb bb bb 0 492 0:1 1 1531 44 200 105 39 0 1:0 1 0 I:0 1 0 1:0 1 0 1:0 1 0 1:0 1 0 1:0 1 Total: 1919 42 20 0 39 39 39 1 1 1 38 62 39 bb bb bb Bb Bb Bb 19 33 21 73 19 1:1 1 29 1:1 0.5 Total: A. Table 3. aureus. melanophore layer. Three crosses carried out between a black hybrid male and 3 different MRT females gave rise to a ratio of 1:1 (P=0. which gave 100% red offspring. N. 12) which seem to be associated to the R gene and can only be expressed in its presence (discussed below). offspring number. posterior chamber. color point to the possibility that this phenotype is controlled by two recessive homozygous genes (Is.5 Total: 2 2 39 32 5 2 1350 663 Bb Bb Bb Bb 348 1002 1:3 0. . in the Ft black hybrid crosses (Bb) and in the crosses of MRT with O. aureus male.7-0. and a ratio of 0:1 red:black (P~. cornea 0 < 1000). Genetic analysis clearly showed that in this fish the red color is recessive to the black color. ' Light and electron microscopy examinations showed that in normal fish an external tissue layer of the iris (golden tissue) contains structures characteristic of pigment cells called iridophores (Fig.7-0.5). A small proportion of these fish (5-10%) were able to reach a weight of 4-5 g before dying. 3). This layer was found to be completely absent in the iridal tissue of the black-eyed embryos which contain only the black melanophore tissue supported by the inner epithelium (Fig. IL.5) (Table 3). IE.7-0. MRT Tag No.5 144 519 1:3 0. This phenotype seems to be associated with a lethal trait because most of the fish died during the embryonic period before the feeding stage was reached. inner epithelium. Therefore MRT. black. Seven crosses carried out between 2 pairs of hybrids yielded a ratio of 1:3 red:black (P----0. B. 2). Hence. aureus (BB) 75% and 100%. C. Results of crosses showing red and black phenotype segregation in MRT and its presumed genotypes.AC.5 66 1:1 0.7-0.

A similar phenomenon was reported in carp by Katasonov (1978). red homozygous individuals. the latter authors suggested that in their blond phenotype there is a partial block of melanin granule formation. which gave 100% red offspring when crossed with heterozygotic red (Rr) or normal black fish (rr). It also seems to us that this phenomenon is associated with the R gene and can only be expressed in its presence. The latter named it 'albino with black eyes'. This point should be clarified by further studies to be carried out by our group in the next spawning season. These results suggest that the black color is determined by a dominant gene B and its recessive allele b is responsible for the red color. because a dominance of the black color over red color was shown in crosses of MRT (red) with O. Red PRT are characterized by black spots of different sizes. This has also been reported by Fitzgerald (1979) and Galman et al. We observed that these spots develop at the post-embryonic stage. According to this model it is possible to divide the F 2 red fish into two genotypes: homozygous red (RR) and heterozygous red (Rr). It was then reported that only the pink color is homozygous while the red is heterozygous. 3). develops during early embryonic development of PRT. By using electron microscopy. red with black eyes (R-il il i2 i2). it could be suggested that in MRT the red color depends on a recessive gene (b) which might be involved in the inhibition of melanin synthesis. Identification of the homozygote (RR) was attempted by performing testcrosses between homozygotes (RR) and normal black fish (rr) which were predicted to give 100% red offspring. while the second.199 Discussion The results of this study show that red color of PRT is a dominant trait which seems to be controlled by a single autosomal gene. we observed that the golden tissue which is lacking in the black eyes contains structures characteristic of iridophores (Fig. this m gene was found to enhance the differentiation of melanophores. with complete inhibition of iridophore differentiation including the eyes. We would suggest that the corresponding genotypes are as follows: Black (rr). there exists a color mutation controlled by the melanoid (m) gene. where one type of melanophores was found to be dominated by the R genes whereas the other type was not. Genetic results of the segregation ratios of body and eye color point to the possibility that this phenotype is controlled by two recessive homozygous genes (11.R_il i112 -) for the three ratios of 9:3:4. In contrast to PRT. orange and albino). and similar to that described by Scott et al. niloticus strain originating in Uganda (Mires. and. . R_I 1_i 2 i 2. 1988). Therefore we have come to the conclusion that two types of melanophores which develop at two different ontogenetic stages.. 2. was obtained in our group (Galman et al. which develops a few months later. 45:3:16 (red:red with black eyes: black) observed (Table 2). could be selected from a stock of O. Because of the occurrence of skin melanophores as seen by electron microscopy. (1988) for the inheritance of blond color in another strain of O. Unfortunately. (1987) and by McAndrew et al. the few F2 red fish we examined in the frame of this work were not identified as homozygotes (not shown). 1987) in PRT following three generations of inbreeding of the pink phenotype and by others in Taiwanese red tilapia (Huang et ai. especially on the head. aureus (black) and in crosses between two different black hybrids of this cross which gave a ratio of 1:3 red:black (Table 3). susceptible to the R regulating gene. This model is in agreement with the results obtained in a red O.12). no melanophores at all could be observed in our MRT either with light or electron microscopy (not shown).. It is still not clear to us whether the lack of iridophore formation in the black-eyed tilapia exists all over the body or is "limited to the eyes only. dispersed over different parts of the skin. In this animal. niloticus originating from Egypt (McAndrew et al. 1988). However a pink phenotype yielding 100% pink offspring. In contrast. The color inheritance in MRT was found to be different from PRT. Red (R_I 1__12_. as reported in the Mexican axolotl (Benjamin. to a lesser extent inhibition of the formation of xanthophores. niloticus originating in Egypt.. is not susceptible to the suppressive action of this gene. In contrast. are present in adult fish: the first. 21:3:8. An unusual phenotype characterized by bright color and black eyes wgtsobserved by us. 1987) which showed three different phenotypes (red. 1970). (1987). Therefore it is possible that a genetic defect related to the formation of the iridophores is the cause of the black eye phenotype. However.

The biochemical effects of the d. International Symposium on Tilapiain Aquaculture. J. 1979. Koiller. I.. rPRT. M. Tel-Aviv Univ. niloticus from two African origins. O. Devel. C. rrBB. 3. Colloq. R. Fishelson & Z. & Avtalion... 6: 69-74. C. Israel. 1978. 237-241. T. ICLARM Edit. 1985. pp. Engin. R. & Mc Ewen. Aquaculture News..R. Press. Press.R. Maclean (eds). Cheng. rrB_. 23: 62-85. pp. Yeheskel. 169-175. J. Maclean (eds).200 Therefore we suggest the following genotypes for the different tilapias studied: Oreochromis aureus. Pullin. 291-301. M. C. M. Mires. Sipe. 1988. A. Roberts. Moreu. Aquacult. Artificial fertilization in tilapia . Galman. 1987. Yaron (eds). In: L. R__BB. respectively. Chang. 1983...R..a useful new genetic marker in tilapia O. We wish to thank Mrs. & Avtalion. R. Fish Farming 6: 26-27. laboratory scale recycling water unit for Tilapia breeding. O.S. Golden perch. J. Roubal. Biol. 524-533. Liao. . International Symposium on Tilapia in Aquaculture. S. Aquaculture 74: 227-232. S. & Liao. M. Yeheskel. 1988. F. Genetica 76: 127-137. where Rr and Bb are the genes which we suggest control the red and black colours. O. rrBB. Single gene inheritance of red body coloration in Thaiwanese red tilapia. Ya. A. Koiller. H.. In: R. 1979. R. Skibinski. Aquacult. References Benjamin. In: L. W. 'Blond' . C. Commercial Fish Farmer. Don. INRA 44: 169-175. 4: 235-246. Pullin. J. 1987. Increased Tilapia embryo viability using ultraviolet irradiation in closed recirculating Zuger-bottle system. Fish Manag. 5..R. & Avtalion. Tonguthaiand J. Susan Weingarten for reviewing this manuscript. Inheritance of blue and orange color types. Sarotherodon galilaeus.A preliminary study. A. P. Tel-Aviv Univ.R. ICLARM Edit. G. M. In: R. Fishelson and Z. O. & Avtalion.. R. S.V. L. & Chang. pp. L. I. K. Scott. The genetics and histology of red blond and associated color variants in O. L. 18: 159-165. bMRT.56.8526) from the Bar-Ilan research and development company. Mair. (Manila and Bangkok). (Manila and Bangkok). growth performance and implications for aquaculture of Philippine red tilapia. 1988. D. 1988. niloticus (L) Aquacult. R. The inheritance of black pigmentation in O... A preliminary investigation of the characteristics of red tilapia from the Philippines and Taiwan. pp. L. B. M. Fitzgerald. Bhukaswan. Katasonov. Acknowledgements This research was supported by a grant (FR. 1970. K. Bhukaswan. C. V. Israel. J. rMRT. R. Huang. F.. Bullock. rrbb. G. V. D. m and a genes on pigment cell differentiation in the Axolotl. The red-orange tilapia. bPRT. J. T. a hybrid that could become a world favourite. McAndrew. 1988. R. Tonguthai and J.. Genetika 14: 2185-2192. 1983. R. niloticus. J. & Beardmore. Studies on the feasibility of the red tilapia culture in saline water. Galman. I. A. rrBB. Yaron (eds).. Color in hybrids common and ornamental (Japanese) carp. & Avtalion. Engin. Breeding characteristics. O.