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Karyotype and fluorescence in situ hybridization analyses of the telomere sequence on chromosomes of the top shell Turbo (Batillus) cornutus (archaeogastropoda, Turbinidae).

ABSTRACT Although the top shell Turbo (Batillus) cornutus (Turbinidae) is a well-known and industrially important shellfish species in Japan, the diploid chromosome number and karyotype of this species have not been previously elucidated. In this study we investigated the diploid chromosome number and performed karyotype and fluorescence in situ hybridization (FISH) analyses of the telomere sequence on chromosomes of T. cornutus. A diploid chromosome number of 36 was identified in larval somatic cells, and the karyotype comprised 16 pairs of metacentric and 2 pairs of metacentric/submetacentric chromosomes. In the telomere FISH analysis using a vertebrate telomere probe, fluorescein isothiocyanate-positive hybridization signals were clearly observed on the telomeric regions of the chromosomes. This result suggests that the FISH technique is appropriate for other Turbinidae species and that the telomere sequence of T. cornutus is (TTAGGG)n, which is the same as that of vertebrates.

KEY WORDS: top shell, chromosome, karyotype, telomere, Turbo cornutus, Mollusca, invertebrate, fluorescence in situ hybridization

INTRODUCTION

The top shell Turbo (Batillus) cornutus (Turbinidae) is a well-known and industrially important shellfish species in Japan, where seed production and culture of this species are performed only in some localities. To increase the production of this top shell, genetic and breeding information is crucial; therefore, the study of chromosomes as the basis of genetics and breeding science is clearly important.

Chromosome analysis using meiotic spermatogonia has revealed that T. cornutus has a haploid chromosome number: 18 (Nishikawa 1962). Astralium haematragum and Lunella coronata coreensis, two other species of Turbinidae, were also found to have the haploid chromosome number 18, as determined from their meiotic spermatogonia (Nishikawa 1962). L. coronata coreensis, the only species to be karyotyped from somatic cells in this family, has a diploid chromosome number: 36 (Komatsu 1984, Nakamura 1986). Thus, within the Turbinidae family, chromosome studies of only 3 species have been carried out, and the diploid karyotype of only 1 species has been documented. However, even though T. cornutus is a very popular and important Turbinidae species in Japan, the diploid chromosome number and karyotype of this species have not yet been investigated. Therefore, in this study, we attempted to determine the diploid chromosome number and karyotype of T. cornutus.

The fluorescence in situ hybridization (FISH) technique is a useful tool for identifying specific DNA sites on chromosomes. FISH analyses of chromosomes of Turbinidae species, however, have not yet been reported. Because telomere DNA is composed of a simple repeat sequence that is more easily found on chromosomes by FISH than single-copy gene sequences, in the current study, we conducted FISH analysis of T. cornutus chromosomes by using a telomere probe. Thus, to introduce the FISH technique in Turbinidae species, we performed a straightforward telomere FISH analysis on chromosomes of this species.

MATERIALS AND METHODS

Artificial crosses were performed using parental T. cornutus reared in the Shika Fish Farming Center of the Ishikawa Prefecture Fisheries Research Center after collection of fish from wild populations in the coastal waters of Ishikawa Prefecture, Japan. Egg and sperm specimens were obtained by artificial spawning, and the eggs were fertilized artificially. After the fertilized eggs were transported to Kitasato University, School of Marine Biosciences, T. cornutus larvae were obtained approximately 24 h after fertilization and treated with 0.05% colchicine (at 20[degrees]C for 2 h) and 0.075 M KCl (at 20[degrees]C for 50 min). Larvae were then fixed in Carnoy solution (at -20[degrees]C for more than 3 h) according to the modified method of Arai et al. (1984). The metaphase chromosome preparations used for karyotyping and FISH analysis were made by the chopping method (Yamazaki et al. 1981), using 30-50 fixed larvae per slide. The slides were dried and maintained at room temperature until karyotyping and FISH analysis were performed.

For karyotype analysis, the slides were stained with Giemsa solution according to the method of Okumura et al. (1995) and were observed by light microscopy (BX50; Olympus, Tokyo, Japan). Of the many metaphase plates photographed, 65 well-spread metaphase plates on which chromosomes could be precisely counted were selected, and the chromosomes were counted. Ten well-spread metaphase plates on which the chromosome arm lengths could be determined were selected from among the 65 plates, and the short and long arm lengths of the chromosomes were then measured on the photographs. Relative lengths were calculated from the total chromosome length according to Thiriot-Quievreux (1984), and arm ratios were calculated according to the method of Levan et al. (1964). The arm ratios and relative lengths were used for morphological classification (Levan et al. 1964) and numbering of chromosomes, respectively.

For FISH analyses, hybridization using vertebrate telomere peptide nucleic acid (PNA) probes (Telomere PNA FISH Kit/ fluorescein isothiocyanate [FITC]; DakoCytomation, Glostrup, Denmark) was performed according to the manufacturer's instructions with some modifications and as described in detail by Sakai et al. (2005). In accordance with Sakai et al. (2005), the slides hybridized with the PNA probes were counterstained with propidium iodide, and the chromosomes bearing telomere FITC signals were observed and photographed with a confocal laser-scanning microscope (LSM510; Carl Zeiss, Jena, Germany).

RESULTS

A typical well-spread metaphase plate of T. cornutus is shown in Figure 1. The chromosome numbers of 65 well-spread plates ranged from 32-37, and the mode value was 36 (Table 1). Therefore, we determined that the diploid chromosome number of this species was 36. The mean values of the relative lengths and arm ratios of the 18 chromosome pairs were estimated from arm length measurements in 10 well-spread metaphase plates (Table 2). Chromosome pairs were numbered in order of their relative lengths (Table 2, Fig. 2). The mean arm ratios indicated that this species has 16 pairs of clearly metacentric chromosomes (chromosome pairs 1-16; Table 2). However, in 2 chromosome pairs (17 and 18), the mean arm ratios were close to 1.7, which is the borderline value between chromosomes classified as metacentric and submetacentric (Levan et al. 1964) (Table 2). Therefore, chromosome pairs 17 and 18 were identified as metacentric/submetacentric chromosomes (Table 2).

In telomere FISH analyses, FITC-positive hybridization signals were clearly observed in the telomeric regions of the chromosomes (Fig. 3).

DISCUSSION

In the current study, a diploid chromosome number, 36, was identified for the first time in somatic cells of T. cornutus. Our results support the former report of 18 haploid chromosomes in T. cornutus by Nishikawa (1962). As described previously in other species of Turbinidae, the haploid number of A. haematragum is 18 (Nishikawa 1962), and the diploid number of L. coronata coreensis is 36 (Komatsu 1984, Nakamura 1986). These numbers suggest a low variation in chromosome count among members of this family. In the order Archaeogastropoda, which includes the Turbinidae family, the haploid chromosome number varies widely from 9-21 (Nakamura 1986). However, families in which the haploid number is more than 18 are rare (only a few species in Trochidae) (Nishikawa 1962, Nakamura 1986). Among the majority of species of Archaeogastropoda, the maximum haploid number is 18 (Nakamura 1986). Therefore, Turbinidae species, all of which have thus far been observed to have a haploid number of 18, can be grouped with members of Archaeogastropoda, which have a relatively high number of chromosomes.

[FIGURE 1 OMITTED]

In this study, we found that the karyotype of T. cornutus comprised 16 pairs of metacentric chromosomes and 2 pairs of metacentric/submetacentric chromosomes. In another species of Turbinidae, L. coronata coreensis, which was the only species in the Turbinidae family karyotyped prior to the current study, 13 pairs of metacentric chromosomes, 3 pairs of submetacentric chromosomes, and 2 pairs of subtelocentric chromosomes were found (Komatsu 1984, Nakamura 1986). Therefore, especially with regard to the presence of subtelocentric chromosomes, there is a difference in karyotypes between T. cornutus and L. coronata coreensis. These results suggest that karyotypic evolution has occurred in the Turbinidae family even though the chromosome numbers are the same. In further studies analyzing more species, we could expect to learn more about the mechanisms of karyotypic evolution.

[FIGURE 2 OMITTED]

In the current study, FISH analysis of telomeres on chromosomes of T. cornutus was established. FISH analysis of telomeres or rDNA sites on the chromosomes of molluscs has been previously reported (Guo & Allen 1997, Insua et al. 1998, Zhang et al. 1999, Wang & Guo 2001, Sakai et al. 2005, Gallardo-Escarate et al. 2005); in addition, this is the first study that reports the successful FISH analysis of telomeres on chromosomes of a species belonging to Turbinidae. Because mapping of the telomeric sequence on the chromosomes of T. cornutus was successful, mapping of repetitive DNA in other Turbinidae species may also be successful. Moreover, this study establishes the basic technique of physical gene mapping in which the telomere-positive signals are obtained at hybridization by using a vertebrate telomere PNA probe. Our results suggest that the telomere sequence of T. cornutus is (TTAGGG)n, which is the same as that in Vertebrata (Meyne et al. 1989, Chew et al. 2002) and other phyla (Okazaki et al. 1993, Vitturi et al. 2000, Nomoto et al. 2001, Sakai et al. 2005, Sakai et al. 2007, Okumura et al. 2009).

[FIGURE 3 OMITTED]

ACKNOWLEDGMENTS

We are indebted to Takashi Waragaya and Takuji Oda for their expert technical assistance.

LITERATURE CITED

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KAZUMA KIMURA, (1) MIZUHO SAKAI, (1) YASUSHI NISHIO (2) AND SEI-ICHI OKUMURA (1) *

(1) Kitasato University, School of Marine Biosciences, Sanriku, Ofunato, Iwate 022-0101, Japan; (2) Ishikawa Prefecture Fisheries Research Center, Noto, Housu, Ishikawa 927-0435, Japan

* Corresponding author. E-mail: okumura@kitasato-u.ac.jp
TABLE 1.
Distribution of diploid chromosome numbers counted
in Turbo cornutus.

No. of chromosomes    <31   32   33   34   35   36   37   >38   Total
counted

No. of metaphase      --    2    1    9    7    44   2    --     65
plates

TABLE 2.
Chromosome arm length and chromosome morphological
classifications in Turbo cornutus.

Chromosome    Relative Length         Arm Ratio
Pair No.       (mean [+ or -]       (mean [+ or -]     Classification
                   SE *)                SE *)            ([dagger])

1            7.58 [+ or -] 0.48   1.16 [+ or -] 0.07          m
2            7.09 [+ or -] 0.25   1.21 [+ or -] 0.11          m
3            6.67 [+ or -] 0.17   1.14 [+ or -] 0.08          m
4            6.42 [+ or -] 0.16   1.14 [+ or -] 0.09          m
5            6.20 [+ or -] 0.22   1.22 [+ or -] 0.11          m
6            5.91 [+ or -] 0.19   1.24 [+ or -] 0.17          m
7            5.68 [+ or -] 0.15   1.51 [+ or -] 0.35          m
8            5.54 [+ or -] 0.13   1.49 [+ or -] 0.31          m
9            5.39 [+ or -] 0.08   1.42 [+ or -] 0.36          m
10           5.24 [+ or -] 0.08   1.34 [+ or -] 0.18          m
11           5.17 [+ or -] 0.08   1.42 [+ or -] 0.28          m
12           5.07 [+ or -] 0.09   1.39 [+ or -] 0.30          m
13           5.00 [+ or -] 0.10   1.37 [+ or -] 0.19          m
14           4.88 [+ or -] 0.13   1.40 [+ or -] 0.26          m
15           4.73 [+ or -] 0.11   1.51 [+ or -] 0.24          m
16           4.60 [+ or -] 0.09   1.34 [+ or -] 0.24          m
17           4.51 [+ or -] 0.15   1.65 [+ or -] 0.47        m/sm
18           4.32 [+ or -] 0.16   1.65 [+ or -] 0.52        m/sm

* Standard error.

([dagger]) metacentric chromosome; sm, submetacentric chromosome.
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Author:Kimura, Kazuma; Sakai, Mizuho; Nishio, Yasushi; Okumura, Sei-Ichi
Publication:Journal of Shellfish Research
Article Type:Report
Geographic Code:9JAPA
Date:Dec 1, 2010
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