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Genetic control of bluish shell color variation in the Pacific abalone, Haliotis discus hannai.

ABSTRACT Shell color variation is one of the more interesting phenomena in abalone breeding. Especially, the identification of inheritable shell color will be a useful visual genetic marker. Bluish shell colored individuals were discovered in one full-sib family A out of nine families (A to I) in the Pacific abalone, Haliotis discus hannai. The segregation of the variant (bluish) type and the normal (greenish) type was 13:27, suggesting the existence of a recessive allele at a single locus. To clarify the genetic control of shell color variation, mating experiments were designed for the F2 generation among the bluish-type and greenish-type individuals within the full-sib family A. All individuals were of the greenish type in the three full-sib families between greenish-type female and greenish-type male, whereas all individuals were of the bluish type in the six full-sib families between bluish-type female and bluish-type male. All individuals were of the greenish type in the four full-sib families between bluish-type female and greenish-type male, whereas segregation of the greenish type and the bluish type was observed in the two full-sib families between greenish-type females and bluish-type males. The observed numbers showed no significant deviation from expected numbers calculated under Mendelian segregation (1:1) in each family. These results indicate that the bluish and greenish shell color variant types are controlled by a recessive allele (b) and a dominant allele (G) at a single locus. Therefore, the genotype of parental individuals (f1 and m1) of the full-sib family A is estimated as G/b. To elucidate the genetic control of shell color variation, further mating experiments were designed for the F2 generation within and between full-sib families D and G in which the male parent (m1) is common with the family A. As expected, segregation of the bluish and greenish types was observed in the two full-sib families out of 13 families. The observed numbers showed no significant deviation from expected numbers calculated under Mendelian segregation (3:1). These results support the elucidation of the genetic control of shell color variations described in the present study.

KEY WORDS: abalone, Haliotis discus hannai, shell color variation, mating experiments

INTRODUCTION

Shell color variation is one of the most interesting phenomena in shellfish aquaculture. The genetic basis of shell color variation was reported in Urosalpinx cinerea (Cole 1975), Mytilus edulis (Newkirk 1980), Thais emarginata (Palmer 1985), Pinctada fucata martensii (Wada & Komaru 1990), Fulvia mutica (Fujiwara 1995), and Ruditapes philippinarum (Kishioka et al. 1997). Cole (1975) reported that in mating experiments a single-locus genetic model controlled three types of shell color variation in Urosalpinx cinerea with complete dominance. Similarly, Newkirk (1980) reported that at least two types of shell color variants occurs in Mytilus edulis and is determined by a simple genetic mechanism.

The Pacific abalone (Haliotis discus hannai) is a commercially valuable species. The creation of artificial culture strains has been expected to allow discrimination from the wild individuals. Visual genetic markers, such as color variation, are, therefore, desired. Shell color variation has been reported in a number of abalone species (Ino 1952, Sakai 1962, Ogino & Ohta 1963, Sibui 1971), however, the variation has been shown to be dependent on dietary differences. In the Pacific abalone, Haliotis discus hannai, Sakai (1962) observed bluish-green, green, greenish-brown, and brown shell colors produced by different alga diets. Ogino and Ohta (1963) reported that a green colored shell may be obtained through an artificial diet, however there has been limited research to determine the genetic basis of shell color variation in the Pacific abalone.

In an attempt to underpin the genetic control of traits such as color variation, the discovery of color variation and detection of color types are first needed, followed by segregation of phenotypes in offspring produced by systematic mating experiments under the same rearing conditions. In the present study, bluish shell color-variant individuals were discovered in one full-sib family (one male crossed with one female). Systematic mating experiments were then undertaken and segregation of the color types were analyzed in the offspring of several mating experiments in an attempt to understand the genetic control of shell color variation in the Pacific abalone.

MATERIALS AND METHODS

Detection of the Color Types

Shell color types were visually divided into two groups, one of which was a greenish type (green to bluish-green but not light and purplish blue) and the other a bluish type (light-blue to purplish-blue with greenish color) as shown in Figure 1. Determination of shell color types of the offspring in experiments I, II, and III were carried out using individuals of more than 17 mm of shell length at the ages of 229, 222, and 245 days after fertilization, respectively. Because, shell color types are clearly divided into two types at more than 17-mm shell length. Any changes of shell color types were not observed after the detection.

[FIGURE 1 OMITTED]

Mating Experiments

Figure 2 shows the mating experiment system used in the present study. Two populations were collected from the coastal water of Aomori (41[degrees]N, 141[degrees]E) and Iwate (40[degrees]N, 142[degrees]E) Prefectures in the northeastern part of Japan. Aomori population was stocked during two generations, and Iwate population was stocked during three generations before using. One greenish female (Pf) of the Aomori population and one greenish male (Pm) of the Iwate population were used for producing the parental generation (P1) during 1996. Three greenish females and three greenish males of the P1 generation were used for producing the nine full-sib families of the first offspring generation (F1) in September 1999 as shown in mating experiment I.

[FIGURE 2 OMITTED]

During mating experiment II, fifteen pair-crosses were produced in August 2001 using the 6 females (3 individuals of the bluish-type and 3 of the greenish-type) and 5 males (3 individuals of the bluish-type and 2 of the greenish-type) from the full-sib family A in which bluish and greenish color types were segregated. Combinations of the crossed individuals were as follows (Fig. 2): bluish-type female x bluish-type male (6 pair), bluish-type female x greenish-type male (4 pair), greenish-type female x bluish-type male (2 pair), and greenish-type female x greenish-type male (3 pair).

To elucidate the genetic control of shell color variation, mating experiment III was designed for the F2 generation within and between full-sib families D and G in which male parents are common with full-sib family A. Thirteen full-sib families were produced in November 2000 using five females and three males as shown in Figure 2.

Breeding and Rearing

Individuals of parents for mating were set into the separate containers before spawning. Spawning was artificially induced with ultraviolet light-irradiated seawater. The spawned eggs were separated into several containers for each pair-mating, and fertilized with the sperm of a different male, respectively. After hatching, larvae were reared in the meshed bottom containers for 4 days. Settlements were conducted with wavy-plates, which were covered with Ulvella lens, and placed into clear plastic 20-L containers. They were reared in a sunny place allowing juveniles to feed on diatoms, which grew on the wavy-plates and the surfaces of the containers. An artificial diet (Cosmo Kaihatsu K.K., Standard type), which usually produced greenish color shells on the Pacific abalone, was given after their shell lengths reached about l0 mm. They were reared in running seawater whose temperature was maintained at almost 20[degrees]C during the experiments.

Statistic Analysis

Expected numbers were calculated under Mendelian segregation, assuming that the bluish type was controlled by a recessive allele at a single locus. The X2 test was performed for comparison between the observed number and the expected one.

RESULTS

In the present study, the bluish color shell was formed after the artificial diet provided, although it was observed that diatoms and an artificial diet normally formed a greenish color shell. The bluish-type individuals initially formed green shells until their shell length reached about 10-15 mm, therefore around an apex of their shells remained greenish color (Fig. 1). The ranges of shell length determined the shell color types in the mating experiment I, II, and III were 17-36 mm, 17-46 mm, and 17-37 mm. The shell's bluish pigment appeared only on the outer shell surface, though the hues of the pearl layer were not changed.

The results of the mating experiment I are shown in Table 1. Nine full-sib families were obtained and only the greenish type was observed in all families except one. In full-sib family A, phenotypic segregation was observed; that is, 27 were of the greenish type, whereas 13 were the bluish type. The results showed no significant deviation from expected numbers calculated under Mendelian segregation (3:1).

Three females and three males of bluish type, and three females and two males of greenish type were then picked up from full-sib family A, and a total of 15 full-sib families of the F2 generation were produced. The results of the mating experiment II are shown in Table 2. All offspring individuals were of the greenish type in the three full-sib families (AA 13, AA 14, and AA 15) produced by mating between greenish-type females and greenish-type males and in the four full-sib families (AA4, AA5, AA8, and AA9) between bluish-type females and greenish-type males. On the other side, all offspring individuals were of the bluish type in the six full-sib families (AA1, AA2, AA3, AA6, AA7, and AA10) produced by mating between bluish-type females and bluish-type males. Phenotypic segregation of the greenish type and bluish type was observed in the two full-sib families (AA11 and AA12) between greenish-type females and bluish-type males. The observed numbers showed no significant deviation from expected numbers calculated under Mendelian segregation (1:1) in each family. These results suggest that the bluish and greenish shell color variant types are controlled by recessive allele (b) and dominant allele (G) at a single locus, and the genotype of parental individuals in the pair-cross A are estimated as G/b.

To elucidate the genetic control of shell color variation, further mating experiments (mating experiment III) were designed for the F2 generation within and between full-sib families D and G in which the male parent (ml) is the same as that of the full-sib family A (Table 3). All individuals in all full-sib families were of the greenish type except the two families. The bluish type appeared in the DG3 and GG3 families. The observed numbers of greenish to bluish individuals each of the DG3 (56:14) and GG3 (85:24) families showed no significant deviation from the expected numbers calculated under Mendelian segregation (3:1).

DISCUSSION

Shell color variations reported in the Pacific abalone are caused by environmental factors, such as natural and/or artificial dietary food. In the mating experiment I of the present study, however, bluish-type individuals were discovered along with the greenish type in one family, even though all individuals in the family have been reared in the same tank (same food and same environment). This suggests that color variations are caused by genetic factors. Moreover, the segregation rate of greenish and bluish types was not significantly different from 3:1, suggesting an existence of dominant (G) and recessive (b) alleles at a single locus. Also, the genotypes of both parents, fl and ml, are considered to be G/b.

To elucidate this assumption, mating experiments should be conducted between any combinations of color types within family A. Because an abalone spawns a high number of eggs, all combinations could be designed in the same generation and in the same family as in the present study. In mating experiment II, the results of segregation indicated that the assumptions of genotypes are elucidated without any exception. For example, the genotypes of G/G and G/b would be included in the greenish-type individuals of family A at the proportion of 1:1 in this assumption. As a matter of course, the genotypes of bluish-type parental individuals are estimated as b/b. Then, the genotypes of maternal greenish-type individual of family AA11 and AA12 are estimated as G/b, because, both families segregated no significant deviation from 1:1. Similarly, the genotypes of G/G and G/b would be included in the B, C, D, and G families at the proportion of 1:1. The results of mating experiment III show that all segregations in 13 combinations from the D and G families are not contradicted under the Mendelian Law assuming a single locus. From the earlier results, genetic color variations observed in the Pacific abalone population, and these color variations are controlled by dominant (G) and recessive (b) alleles at a single locus.

The color variation of the bluish type has not been observed or reported in the natural resources of abalone around Japan. It could be caused by a low frequency of the allele (b), by a newly arisen mutation of allele (b), and/or by the low adaptation (low viability) of an individual of homozygote (b/b). The clarification of this issue provides a subject for future investigation.

In the present abalone culture, the development of new artificial strains is expected and can be distinguished at a glance. The bluish type shell color variation will be useful for future abalone culture as a visual genetic marker for several breeding studies.

ACKNOWLEDGMENTS

This study was supported in part by Grant-in-Aid from the Ministry of Agriculture, Forestry and Fisheries (Development of Fundamental Technologies for Effective Genetic Improvement of Aquatic Organism).

LITERATURE CITED

Cole, T.J. 1975. Inheritance of juvenile shell colour of the oyster drill Urosalpinx cinerea. Nature 257:794-795.

Fujiwara, M. 1995. Inheritance of yellow coloration of the shell in the cockle Fulvia mutica. Bull. Jpn. Soc. Sci. Fish. 61:927-928.

Ino, T. 1952. Biological studies on the propagation of Japanese abalone (genus Haliotis). Bull. Tokai Reg. Fish. Res. Lab. 5:1-102.

Kishioka, M., T. Tateishi, H. Sakai, H. Kito, H. Ideo & S. Matuno. 1997. Inheritance of the banded purple shell type in the short neck clam Ruditapes philippinarum, and related selection experiments. Fish Genet. Breed. Sci. 25:91-97.

Newkirk, G. F. 1980. Genetics of shell color in Mytilus edulis L. and the association of growth rate with shell color. J. Exp. Mar. Biol. Ecol. 47:89-94.

Ogino, C. & E. Ohta. 1963. Studies on the nutrition of abalone. Feeding trials of abalone, Haliotis discus Reeve, with artificial diets. Bull. Jpn. Soc. Sci. Fish. 29:691-694.

Palmer, A. R. 1985. Genetic basis of shell variation in Thais emarginata (Prosobranchia, Muricacea). 1. Banding in populations from Vancouver Island. Biol. Bull. 169:638-651.

Sakai, S. 1962. Ecological studies on the abalone, Haliotis discus hannai Ino. Experimental studies on the food habit. Bull. Jpn. Soc. Sci. Fish. 28:766-779.

Sibui, T. 1971. Studies on the transplantation of red abalone and its growth and development. Bull. Jpn. Soc. Sci. Fish. 37:1168-1172.

Wada, T. K. & A. Komaru. 1990. Inheritance of white coloration of the prismatic layer of shells in the Japanese peal oyster Pincada fucata martensii and its importance in the pearl culture industry. Bull. Jpn. Soc. Sci. Fish. 56:1787-1790.

TOSHIMASA KOBAYASHI, (1) IKUE KAWAHARA, OSAMU HASEKURA (2) AND AKIHIRO KIJIMA (3)

(1) Iwate Fisheries Technology Center Heita Kamaishi Iwate 026-0001 Japan; (2) Iwate Inland Fisheries Technology Center Yoriki Matuo Iwate 028-7302 Japan; (3) Laboratory of Integrate Aquatic Biology, Marine Field in Field Science Center, Graduate School of Agricultural Science, Tohoku University Onagawa Miyagi 986-2242 Japan
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Author:Kijima, Akihiro
Publication:Journal of Shellfish Research
Date:Dec 15, 2004
Words:2555
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