We developed a method for preparing starfish chromosomes from embryos. Blastulae were treated with colchicine (0.2–4.0 mg/ml), dissociated into single blastomeres by pippeting, swollen with 7% sodium citrate, and fixed with methanol: acetic acid (3:1). The fixed cells were dropped on a slide and air-dried. We examined the chromosome number in five species of asteroids belonging to 4 families (Luidiidae, Astropectinidae, Asterinidae, and Asteriidae), and all had a diploid number of 44. We analyzed the karyotype in 4 of the species, and all were different. We visualized the nucleolus organizer regions of an Asterina species and an Asterias species and found them to be quite different from each other.
About 1800 species of starfishes have been identified (Hendler et al., 1995; McEdward et al., 2002). While extensive morphological (Fisher, 1911, 1928, 1930; Hayashi, 1940, 1943, 1973; Spencer and Wright, 1966; Fell, 1963; Heddle, 1967; Blake, 1987; Downey, 1973; Clark and Downey, 1992), developmental (Oguro et al., 1976; Komatsu et al., 1979; McEdward, 1992; Chia et al., 1993; Byrne and Cerra, 1996), biochemical (Kubo, 1961; Schopf and Murphy, 1973; Mochizuki and Hori, 1980; Matsuoka, 1981, Matsuoka et al., 1994), and molecular (Lafay et al., 1995; Wada et al., 1996; Knott and Wray, 2000) studies have been systematically carried out on starfishes, cytogenetic studies have not yet been applied to asteroid phylogeny.
Starfish chromosomes have been examined in early embryo sections since the start of the 20th century (Delage, 1901; Tennent and Hogue, 1906; Tennent, 1907; Jordan, 1910). In Japan, Makino and Niiyama (1947), examining testis sections showed that the diploid chromosome number of 4 species ranged from 38 to 50. Later studies with testes showed that the chromosome numbers of 8 species belonging to 4 families were 44 (Delobel, 1971; Colombera, 1974; Colombera et al., 1977; Colombera and Venier, 1980; Colombera and Tagliaferri, 1986). The constancy in chromo-some number necessitates karyotypic comparison, but chromosomes from starfish testes are too small to permit karyo-type analysis. Since I have been able to obtain clear chromosome preparations from sea urchin embryos by the air-drying method (Saotome, 1987), we thought it might be possible to modify the method and apply it to starfish embryos.
In this paper, we describe a method for preparing starfish chromosomes from embryos, and the results of karyo-type and NOR analysis.
MATERIALS AND METHODS
We used 5 species belonging to 4 families of asteroids and one species of ophiuroid (Table 1). We removed testes and ovaries from adults and immersed them in artificial sea water (Jamarin U, Jamarin Laboratory, Osaka, Japan). We treated the ovaries with 1–2 μM 1-methyladenine for 30 min (Sigma, St Louis, Mo., USA) to induce breakdown of germinal vesicles and obtain mature eggs (Kanatani, 1969). When the motility of sperm released in sea water was poor, we improved it by adding L-histidine (0.1 mM) (Wako Pure Chemical Industries, Osaka, Japan) to the sperm suspension. Eggs were inseminated before extrusion of the second polar body. The embryos (2000 per ml sea water) were cultured at room temperature and only Asterias amurensis embryos at 10–13°C. In Asterina minor and Amphipholis kochii, the several adults cultured at room temperature were transferred to small beakers (200–300 ml), kept at 4–10°C for 1–2 hr, and then returned to room temperature (Yamashita, 1983). We harvested the embryos, which were naturally born during overnight.
Asteroid and ophiuroid species analyzed in this study.
Embryos (1×104) at the early blastula stage (250–500 cell stage) or wrinkled blastulae (20–30 embryos) in A. minor were treated with colchicine (0.2–4.0 mg/ml) (Sigma, St Louis, Mo., USA), sampled 4–5 times at 5–30 min intervals before or after a division, collected by centrifugation (300 ×g×5 min), suspended in 1M urea, and dissociated into their component blastomeres by pipetting. The fertilization membranes were simultaneously removed. The colchicine concentration, sampling interval time, and sampling numbers varied with species. A. minor embryos, which have large eggs containing a lot of yolk, were suspended in 0.5 ml sea water in 35 mm dishes, beaten slightly with a bamboo spit (2 mm in diameter), and dissociated into their blastomeres. The dissociated blastomeres were collected by centrifugation, swollen with 7% sodium citrate for 10 min, fixed with methanol: acetic acid (3:1), and washed twice with the fixative. The fixed cells were air-dried (Saotome, 1982a), and the preparations were stained for 10 min with 3% Giemsa solution (Merck, Whitehouse Station, NJ, USA) diluted with phosphate buffer (pH 6.9). We used more than 6 pairs of adults and prepared chromosomes through 2–5 breeding seasons. Since we used only one pair and prepared chromosomes for only one season for Luidia maculata and A. kochii, their data were shown as preliminary ones.
We visualized the nucleolus organizer regions (NORs) by the silver-staining method of Howell and Black (1978), with slight modifications. The preparations stained with Giemsa were destained with methanol after observation, and then stained with 50% silver nitrate solution for 2–3 min at 50–57°C (Saotome, 1991).
We classified chromosomes by arm ratio on the basis of the nomenclature of Levan et al. (1964) into metacentric (m), submetacentric (sm), subtelocentric (st) and telocentric (t) chromosomes. We divided chromosomes into large-sized ones (L) and mediumsized ones (M).
We examined stage of preparation, concentration of colchicine and hypotonic solution, dissociation into single blastomeres, and composition of fixative to establish preparing conditions. Good chromosome preparations could be obtained, when blastulae were treated with colchicine (0.2–4.0 mg/ml), dissociated into single blastomeres by pippeting, swollen with 7% sodium citrate, and fixed with methanol: acetic acid (3:1).
Fig. 1a–e show typical metaphase chromosomes from starfish embryos of 5 species. The chromosomes spread over one layer, which made it possible to count them and to analyze the karyotype. The chromosomes were small, ranging in length from 1 μm to 5 μm. Fig. 1f indicates meiotic chromosomes from Asterina minor testes treated with colchicine by air-drying.
The modal diploid chromosome numbers in all 4 starfish species were 44 (Fig. 2a–d); the modal haploid number in A. minor testes was 22 (Fig. 2e). Preliminary data in Luidia maculata indicated that 39 (53%) out of 73 cells also had a diploid chromosome number of 44.
Astropecten scoparius was characteristic in having 2 pairs of large subtelocentric chromosomes. The karyotype consisted of 9 pairs of metacentric, 7 pairs of submetacentric, and 6 pairs of subtelocentric chromosomes (Fig. 3). Asterina pectinifera had one pair of medium-sized metacentric, 10 pairs of metacentric, and 9 pairs of submetacentric chromosomes, and one pair each of subtelocentric and telocentric chromosomes (Fig. 4a). A. minor showed one pair of medium-sized metacentric, 14 pairs of metacentric, 6 pairs of submetacentric, and one pair of telocentric chromosomes (Fig. 4b). Asterias amurensis had 12 pairs of metacentric, 7 pairs of submetacentric, and 3 pairs of telocentric chromosomes (Fig. 5). Preliminary data for L. maculata showed that it had about 6 pairs of metacentric, two pairs of large submetacentric, about 6 pairs of submetacentric and about 8 pairs of subtelocentric chromosomes.
We tried to prepare chromosomes from several ophiuroid species but succeeded only with Amphipholis kochii (Fig. 6). We made only three preparations because of the difficulty in getting mature adults and harvesting well-developed embryos. The preliminary results indicated that 42 (72%) out of 58 cells had a diploid chromosome number of 42, which had three pairs of metacentric, three pairs of submetacentric, 7 pairs of subtelocentric, and 8 pairs of telocentric chromosomes (Fig. 7).
The NORs of A. pectinifera occurred on two regions. One was large (long arrow) and located on the long arm of a metacentric chromosome pair, while the other was small (short arrow) and located on a terminal position of a telocentric chromosome pair (Fig. 8a). The NORs of A. amurensis were located on a terminal position of three telocentric chromosome pairs (arrows in Fig. 8b).
In this paper, we reported a method for preparing starfish chromosomes from embryos. The timing of the preparation was critical because colchicine inhibited cytoplasmic division but did not stop nuclear division. Indeed, we observed many polyploid cells after long colchicine treatment, and were not able to obtain metaphase plates in those shortly after the stoppage of cleavage. Many metaphase plates could be prepared during the M phase at the early blastula stage (250 or 500 blastomeres). In Asterina minor eggs, which are large and contain a lot of yolk, wrinkled blastulae were good for preparation. Though we do not understand why colchicine did not stop nuclear division, we were not able to obtain good metaphase plates without it. Colchicine may affect chromosome morphology. We could prepare well-spread chromosomes from A. minor testes (Fig. 1f) and count clearly chromosome number of 22 as a haploid number (Fig. 2e), but not analyze karyotype owing to chromosome smallness. Though karyotype analysis might be also difficult in the metaphase plates from testes of other species (Colombera, 1974; Colombera et al., 1977; Colombera and Venier, 1980; Colombera and Tagliaferri, 1986), we found that chromosome preparations from embryos were good to analyze karyotype.
The diploid chromosome number was 44 in all 5 asteroid species we studied. Since all 8 species (Astropecten bispinosus, Astropecten arantiacus, Ophidiaster ophidianus, Hacelia attenuata, Asterina gibbosa, Asterina pectinifera, Asterina coronata japonica, and Echinaster sepositus) belonging to 4 families reported until now also had a diploid chromosome number of 44, (Delobel, 1971; Colombera, 1974; Colombera et al., 1977; Colombera and Venier, 1980; Colombera and Tagliaferri, 1986), the basic diploid chromo-some number for asteroids may be 44. The high aneuploidy frequency (17%–22%) we observed may have been caused by contamination of embryos showing polyspermy, which induces irregular cleavage and results in abnormal chromo-some separation (Saotome, 1982b). When adults matured fully, good chromosome preparations could be obtained owing to the high rate of germinal vesicle breakdown and fertilization, and synchronous cleavage. When they did not mature fully, however, the fertilization rate was low and cleavage was not synchronous. In those cases, the addition of more concentrated sperm solution was apt to induce polyspermy. The cases showing polyspermy, of course, K. Saotome and M. Komatsu were not used for chromosome preparations.
The karyotypes from 5 species were compared on the basis of chromosome size and arm ratio. The karyotype of Astropecten scoparius (chromosome formula; (9 m+7 sm+(2 L+4) st) belonging to Astropectinidae, differed from the karyotypes of Asterina pectinifera ((1M+10) m+9 sm+1 st+1 t) and Asterina minor ((1M+14) m+6 sm+1 t) (both Asterinidae) as well as Asterias amurensis (12 m+7 sm+3 t) (Asteriidae) (Fig. 3, 4, 5). Astropectinidae is distinct from Asterinidae and Asteriidae morphologically (Hayashi, 1974) and immunologically (Mochizuki and Hori, 1980), which is consistent with the karyological difference. The A. scoparius karyotype may be close to the karyotype of L. maculata (about 6 m+(2L+about 6) sm+about 8 st), belonging to Luidiidae. Paleontological evidence suggests that the order Platyasterida (Luidiidae) comprises the more primitive starfish from which the Paxillosida (Astropectinidae) diverged (Fell, 1963; Spencer and Wright, 1966). Moreover, A. scoparius resembles L. maculata in its nonbrachiolarian mode of development (Oguro et al., 1976). These relationships may be consistent with the similarity in their karyotypes, but clear karyotype analysis is needed to clarify them. The karyotypes of A. pectinifera and A. minor were similar but differed in number of meta- and submetacentric chromosomes. Though A. pectinifera and A. minor belong to the same genus, they differ in the size and yolk content of eggs and in type of larval development–indirect (A. pectinifera; Komatsu, 1984) VS. direct (A. minor ; Komatsu et al. 1979). Immunological data indicate that A. pectinifera differs considerably from A. minor compared with other species belonging to genus Asterina (Mochizuki and Hori, 1980; Matsuoka, 1981). Those developmental and immunological differences may be correlated with karyological differences. Structural changes, such as inversion may have occurred between meta- and submetacentric chromosomes in the course of evolution. The karyotype of A. amurensis was quite different from that of other three species we examined. Asteriidae is a well- characterized family, and well-defined by the possession of both straight and crossed pedicellariae and compressed ambulacrals, by which it is distinguished from all other asteroids (Fisher, 1911; Clark and Downey, 1922; Hyman, 1955; Spencer and Wright, 1966; Blake, 1987; Gale, 1987). Asteriidae is also well differentiated from the other families immunologically (Mochizuki and Hori, 1980). These morphological and immunological differences are consistent with the karyological one. A. pectinifera and A. amurensis are similar in larval development (indirect type), but differ morphologically. We were also able to distinguish them by karyotype and NORs (Fig. 8). We could visualize NORs in only 2 species out of 5 asteroid species. There was variation in number of NORs. 10 cells (45.5%) out of 22 cells had 4 Ag-NOR sites in A. pectinifera and cells of 57% (4/7 cells) had 6 Ag-NOR sites in A. amurensis. The NORs on one pair of telocentric chromosomes were common to A. pectinifera and A. amurensis, but NORs on one pair of metacentric chromosomes was specific for A. pectinifera. Further data about NORs in other species may make it possible to discuss asteroid phylogeny. We, furthermore, have to compare karyotypes in many species belonging to each order to discuss phylogeny at the order level.
The karyotype figures in this paper show paired chromosomes but we also observed a pair of heterotypic chromosomes, which might represent sex chromosomes. In A. pectinifera, about half of karyotypes examined had heterotypic chromosomes (data are not shown). More detailed examination by several banding and FISH methods is needed to determine whether the asteroids indeed have sex chromosomes.
Some palentological evidence suggests that the ophiuroids are more closely related to the asteroids than to the echinoids (Fell, 1963; Spencer and Wright, 1966). Our preliminary data showed that the ophiuroid A. kochii (Amphiuridae) had a diploid chromosome number of 42 (Fig. 6). Since diploid chromosome number of Ophiomyxa pentagona (Ophiomyxidae) and Macrophiothrix longipeda (Ophiotrichidae) has been reported to be 42, and that of Ophiothrix fragilis (Ophiotrichidae), Ophioderma longicauda (Ophiodermatidae), and Ophiocomina nigra (Ophiocomidae) is 44 (Colombera, 1974; Colombera and Tagliaferri, 1986), chromosome numbers of 42 and 44 may exist in ophiuroids. A. kochii had many subtelocentric and telocentric chromosomes (Fig. 7), which contrasted with the karyotypes of asteroids. Since we found considerable difference in karyo-type between asteroids and ophiuroids, karyotypic comparison may be an important phylogenetic tool.
To elucidate chromosome evolution in asteroids and ophiuroids, we need chromosomal data from many species. This method described here is applicable to species from which we can consistently obtain mature eggs and sperm, fertilized eggs, and well-developed embryos.
We are grateful to the staff of the Misaki Marine Biological Station, University of Tokyo, Kanagawa Prefecture, the Tateyama Marine Biological Station, Ochanomizu University, Chiba Prefecture, and the Amakusa Marine Biological Laboratory, Kyushu University, Kumamoto Prefecture, for kindly supplying materials.