Morphology and gene analysis of hybrids between two congeneric sea stars with different modes of development.
Most marine invertebrates have a characteristic larval stage, and their larval morphology and habitats are extremely diverse (Levin and Bridges, 1995; Young, 2002). Many echinoderms have pelagic feeding larvae, but nonpelagic and nonfeeding larvae have also been described in numerous species (e.g., Hayashi and Komatsu, 1971; Komatsu et al., 1979; McEuen and Chia, 1991; Amemiya and Emlet, 1992; Komatsu and Shosaku, 1993). In addition, facultative feeding larvae, which are able to feed but can complete development without food, have also been found in two sea urchins (Emlet, 1986; Hart, 1996). Development in sea stars is categorized by the larval forms. Oguro et al. (1976) distinguished between sea stars with only planktotrophic bipinnaria larvae and those that pass through both bipinnarian and brachiolarian stages. They referred to the former group, which includes Astropecten and Luidia species, as non-brachiolarian. Furthermore, barrel-shaped non-feeding pelagic larvae, with the external form of a gastrula, occur in some species of the family Astropectinidae, such as Astropecten latespinosus (Komatsu, 1975), A. gisselbrechti (Komatsu and Nojima, 1985), and Ctenopleura fisheri (Komatsu, 1982). On the basis of what was known of larval morphology and the systematic position of sea stars, Oguro (1989) and McEdward and Miner (2001) inferred that barrel-shaped larvae evolved from non-brachiolarian bipinnarian larvae. However, they did not consider the changes in larval morphology involved in the evolution of barrel-shaped larvae from bipinnariae, such as loss of ciliary bands and loss of a complete gut.
Raff and co-workers were the first investigators who utilized hybrid larvae in experiments to shed light on the evolutionary modifications of development in echinoderms. They studied hybrids of two sea urchins of the genus Heliocidaris to investigate the evolution of species with contrasting modes of development and found that fertilization of the eggs of the derived species. H. erythrogramma, with the sperm of the ancestral species, H. tuberculata, could restore ancestral larval features lost during evolution (Raff et al., 1999; Nielsen et al., 2000). In this study, to investigate evolutionary changes in the development of asteroids, we performed reciprocal crosses between two Astropecten sea stars with different larval forms. Eggs of Astropecten scoparius are translucent and small (230 [micro]m in diameter), and they develop into feeding bipinnariae (Oguro et al., 1976). Eggs of Astropecten latespinosus are semitranslucent and larger than those of A. scoparius (300 [micro]m in diameter), and they develop into nonfeeding barrel-shaped larvae (Komatsu, 1975). Here we describe the development from egg to juvenile in both cross-fertilizations.
Although many cross-fertilizations have been reported in echinoderms (e.g., Lucas and Jones, 1976; Byrne and Anderson, 1994; Raff et al., 1999; Rahman et al., 2001; Brandhorst and Davenport, 2001), few studies have directly examined the presence of parental genomes in the hybrids. To determine whether the juveniles resulting from our crosses were hybrids or not, we analyzed their genetic features by comparing nucleotide sequences between the juveniles and the parents. We used 18S rDNA as a gene marker because it is present in nuclear DNA and would be expected to lack intraspecific sequence variation because of its slow evolutionary rate. This approach enabled us to make two quite unexpected observations. First, hybrid larvae in each cross-fertilization were always bipinnaria-like and exhibited morphological variation in their gut. Second, parthenogenetic development occurred in the cross-fertilization between A. latespinosus eggs and A. scoparius sperm, but not in the reciprocal cross-fertilization.
Materials and Methods
Adults of Astropecten scoparius Valenciennes and Astropecten latespinosus Meissner were collected from Toyama Bay, Toyama Prefecture, and the coast of Tsuyazaki, Fukuoka Prefecture, Sea of Japan, by snorkeling or scuba diving. The sea stars were collected during their breeding season (from late June to early August) and then maintained in aquaria for several days at the University of Toyama. To obtain mature eggs, mature ovaries were isolated from females of both species by dissection, and treated with [10.sup.-6]mol [l.sup.-1] 1-methyladenine (Kanatani, 1969). We prepared at least 18,000 fertilizable eggs of A. scoparius and 500 of A. latespinosus for each experiment. They were each divided into two groups and inseminated artificially with either a conspecific or a heterospecific sperm suspension. Although we did not calculate the concentration of the sperm suspensions, it was sufficient to give 100% fertilization in conspecific combinations. Reciprocal cross-fertilizations were carried out three times. For each fertilization the gametes from one female and one male were used. For simplicity, cross-fertilization between eggs of A. scoparius and sperm of A. latespinosus is expressed as As X Al, and the reciprocal cross-fertilization as Al X As.
Observations on development
Embryos and larvae of A. scoparius, A. latespinosus, As X Al, and Al X As were reared at 25[degrees]C, as in previous laboratory studies of these species (Komatsu, 1975; Oguro et al., 1976). To assess the morphological differences between larvae of conspecific and heterospecific crosses, we carefully observed bipinnarian arms, digestive system, and ciliary bands, and measured the lengths of living larvae (Table 1). We classified larvae of the heterospecific crosses by their morphologies and reared each type separately.
Table 1 Comparison of developmental characteristics between conspecific and heterospecific fertilizations ofAstropecten scoparius (AS) and Astropecten latespinosus (A1) As x Al Characteristics As type type 1 2 Bipinnarian + + + arms Stomodeum + + + Complete + + - gut Ciliary + + + bands Max length 1061 678 646 ([mu]m) [+ or -] [+ or -] [+ or -] 13.6 17.3 8.4 (n = 15) (n = 15) (n = 15) Time to adult 9 8 6 rudiment (d) Time to end of 17 15 11 metamorphosis (d) Al x As Characteristics type 1 type 2 type 3 Al Bipinnarian + + - - arms Stomodeum + + - - Complete gut + - - - Ciliary bands + + - - Max length 834 793 686 706 ([mu]m) [+ or -] [+ or -] [+ or -] [+ or -] 14.2 18.3 9.2 5.6 (n = 10) (n = 10) (n = 11) (n = 15) Time to adult 4.5 4.5 2 2 rudiment (d) Time to end of 7 7 5 5 metamorphosis (d) +,present; -, absent. Max lengths (mean [+ or -] SE) are obtained for 5 larvae from each fertilization, and so n = 15. However, in the case of fewer than 5 larvae (e.g., type 2 in the first experiment of Al x As; see Table 2), all resultant larvae were measured.
The entire process of development was observed using light microscopy. Percentage fertilization was calculated by counting the number of eggs with fertilization membranes in a random sample of more than 100 from each experiment. Embryos of each experiment were reared in petri dishes until hatching, and free-swimming larvae were kept in glass beakers with a plastic propeller rotating at 30 rpm. Larvae indicating the bipinnarian external form (larvae of A. scoparius and As X Al, and those of types 1 and 2 in Al X As, as described later in this paper) were provided a mixture of four algae: Dunaliella tertiolecta, Chaetoceros gracilis, Skeletonema costatum, and Isochrysis galbana. The Mann-Whitney test was used to assess whether the larval lengths were significantly different between two experiments, and the Kruskal-Wallis test was used for testing equality of larval lengths among more than three experiments.
Newly metamorphosed juveniles from each experiment and tube feet from adults of the parental species were fixed in absolute ethanol for gene analysis. The numbers of juvenile samples for this analysis are shown in Table 2. Total DNA was extracted from the fixed samples using the phenol-chloroform method (Sambrook and Russell, 2001).
Table 2 Total number of resultant larvae from each cross (LAR),total number of juveniles that completed metamorphosis (JUV), and number of juveniles used for gene analysis(GA) in crosses between Astropecten scoparius (AS) and Astropecten Latespinosus (A1) As x Al type 1 type 2 Experiment LAR JUV GA LAR JUV GA 1 13 10 5 20 15 5 2 18 9 5 62 39 5 3 23 20 5 71 30 5 Al x As type 1 type 2 type 3 Experiment LAR JUV GA LAR JUV GA LAR JUV GA 1 9 4 4 2 1 1 1 0 0 2 16 0 0 9 4 4 5 1 1 3 0 0 0 3 1 1 31 21 5 Maximum size of specimens for gene analysis was 5 per type of each experiment. For example, in larvae of type 1 of As x Al, we collected 5 individuals from each experiment, thus analyzing 15 individuals. If fewer than 5 juveniles were produced, all resultant juveniles were used (e.g., type 1 in the first experiment of Al x As).
On the basis of the complete sequence of 18S rDNA of A. latespinosus (Matsubara et al., 2004), we amplified DNA fragments at the 3' end of 18S rDNA of adult A. latespinosus by polymerase chain reaction (PCR) with the following primers: RIB3S (forward) 5'-GTTCAGCCACGCGAGAT-TGAGCA-3' and RIB3AS (reverse) 5'-ACTTCCTCTA-AATGATCAAGTTCGATCG-3'. PCR was carried out in a 50-[micro]l reaction mixture using Blend Taq DNA polymerase (TOYOBO). The temperature regime for PCR was 1 min at 94[degrees]C, 1 min at 55[degrees]C, and 1 min at 72[degrees]C for 40 cycles. The homologus DNA fragments of adult A. scoparius and all juveniles were amplified with the same primers and PCR programs. These DNA fragments were purified using a QIAquick gel extraction kit (QIAGEN), after electrophoresin in 1.5% agarose gel. Purified fragments were used for cycle sequencing reactions with primer RIB3S. Cycle sequencing was performed in a 20-[micro]l reaction mixture using a BigDye Terminator cycle sequencing kit (Applied Bio-systems). The temperature regime for cycle sequencing was 30 s at 96 [degrees]C, 15 s at 50 [degrees]C, and 4 min at 60 [degrees]C for 25 cycles. Sequencing was carried out on a DNA sequencer (ABI prism 310). To determine whether the juveniles from cross-fertilizations were hybrid or not, we compared the nucleotide sequences among samples.
Percentage fertilizations in both conspecific crosses, Astropecten scoparius and Astropecten latespinosus, were nearly 100% (Table 3). A.scoparius eggs developed into feeding bipinnarian larvae (Fig. 1A), as previously reported (Oguro et al ., 1976; McEdward et al., 2002). Fully grown bipinnariae had ciliary bands divided into two loops; five conspicuous pairs of bipinnarian arms, which are hollow extensions of the body wall with ciliary bands arranged along the edge; and a complete gut formed by the fusion of the stomodeum to the anterior tip of the archenteron. They averaged 1061 [micro]m long (Table 1). We fed them algae, and observed that the larvae accumulated the algae the along into their stomach. On the otherhand, A. latespinosus eggs developed into barrel-shaped larvae (Fig. 1B), which had no body processes no cilairy bands, and an incomplete gut, as described by Komatsu (1975) and Komatsu et al. (1988). Fully grown barrel-shaped larvae averaged 706 [mu]m long (Table 1). A. scoparius took 17 days to complete metamorphosis, and A. latespinosus took 5 days. Larval characteristics of both species are shown in Table 1.
Table 3 Percentage fertilization (mean [+ or -] SE) in conspecific and heterospecific crosses Combinations Fertilization (%) As 99.90 [+ or -] 0.10 Al 99.83 [+ or -] 0.18 As x Al 0.42 [+ or -] 0.25 Al x As 38.9 [+ or -] 5.88
[FIGURE 1 OMITTED]
As X Al
Fertilized eggs were initially observed 1 h after insemination, and the mean percentage fertilization was 0.42% (Table 3). At 2 h after insemination, almost 100% of the eggs with fertilization membranes started to cleave. Embryos hatched as free-swimming early gastrulae 12 h after insemination. Early development from fertilized egg to early gastrula of this cross-fertilization was quite similar to that of the conspecific A. scoparius fertilization.
The gastrulae developed into bipinnaria-like larvae 36 h after insemination. Small processes on the side of the body appeared at the part corresponding to the bipinnarian arms of bipinnaria of A. scoparius. We observed ciliary bands divided into two loops, the pre-oral and post-oral bands, along the edges of the processes. We concluded that these processes were homologous to the bipinnarian arms. However, the larvae were roundish in external form, and their bipinnarian arms were shorter than in bipinnariae of A. scoparius (Fig. 1A, C, D). We divided the larvae into two types by the kind of gut (Table 1). One type had a complete gut, as in the bipinnariae (Fig. 1C). In this type, the stomodeum fused to the anterior tip of the archenteron and then the archenteron differentiated into intestine, stomach, and esophagus. In the other type of larva, the archenteron seemed to differentiate into intestine, stomach, and esophagus, but did not connect to the stomodeum, and no pharynx was formed (Fig. 1D, E). The morphology of this nonfunctional gut appeared to be intermediate between that of the bipinnaria of A. scoparius and that of the barrel-shaped larva of A. latespinosus. In this study, we refer to development with a complete gut as "type 1" and development with an incomplete gut as "type 2." In As X Al, 28.79% [+ or -] 5.33% (mean [+ or -] SE) of the offspring developed into type 1 larvae (Table 2).
Three days after insemination, both types of larvae had adoral ciliary bands in which cilia swept toward the mouth. Maximum lengths of larvae were 678 [mu]m (type 1) and 646 [mu]m (type 2) (Table 1), which were significantly different from the 1061 [mu]m of the bipinnariae of A. scoparius (Mann-Whitney test, P < 0.01), but not significantly different from that of the barrel-shaped larvae of A. latespinosus (Mann-Whitney test, P < 0.05). Type 1 larvae formed an adult rudiment at the posterior end 8 days after insemination, whereas type 2 larvae formed it at 6 days. In each type, the anterior region of the larva was subsequently absorbed into the posterior region, after which the type 1 larvae settled as juveniles at 14 days after insemination, and type 2 larvae at 10 days. The mouth opened a day later in both types, an indication that metamorphosis was complete.
Al X As
In the crosses of Al X As, the mean percentage fertilization was 38.9% (Table 3). Fertilized eggs first appeared 1 h after insemination, but then fewer than 10% of them divided. Early development of Al X As was quite similar to that of the conspecific A. latespinosus fertilization, and the early gastrula hatched 12 h after insemination.
After hatching, gastrulae developed into three types of larvae, distinguishable by their morphology (Table 1). Types 1 and 2 were bipinnaria-like and similar to those of As X Al (Fig. 1F, G), and were formed 2 days after insemination. Type 3 larvae were barrel-shaped (Fig. 1H) and were formed only 15 h after hatching. The frequencies of occurrence of these three types differed markedly in each experiment: more than 50% of larvae were type 1 in the first and second experiments, but no type 1 larvae appeared in the third experiment (Table 2).
Type 3 larvae lacked bipinnarian arms, a mouth, a complete gut, and ciliary bands (Fig, 1H). The mean length of fully grown larvae was 686 [mu]m (Table 1), not significantly different from that of the barrel-shaped larvae of A. latespinosus (Mann-Whitney test, P > 0.05), but significantly different from that of bipinnariae of A. scoparius (Mann-Whitney test, P < 0.01). Two days after insemination, type 3 larvae formed an adult rudiment at the posterior end, and the absorption of the anterior region started the next day. Type 3 juveniles settled 4 days after insemination, and their mouths opened at 5 days.
On the other hand, in types 1 and 2 larvae, the stomodeum formed at the anterior ventral region within a day of insemination. Two days after insemination, type 1 larvae had a complete gut (Fig. 1F), and type 2 larvae had an incomplete gut (Fig. 1G). The archenteron of both types differentiated into intestine, stomach, and esophagus. Both types had prominent pre-oral and post-oral ciliary bands divided into two loops, and an adoral band. In the larvae of As X Al, the adoral band of these larvae produced a current toward the mouth. Moreover, these larvae had small processes on their sides corresponding to the bipinnarian arms of A. scoparius. These arms were shorter than those of bipinnariae of A. scoparius, and the external form of the larvae was rounded. Three days after insemination, fully grown types 1 and 2 larvae were 834 [mu]m and 793 [mu]m long, respectively (Table 1). Both lengths were intermediate between those of A. scoparius and those of A. latespinosus, and were significantly different from both (Kruskal-Wallis test, P < 0.001). Larvae of both types began to metamorphose 5 days after insemination, and began to settle at 6 days. Their mouths opened 1 day later, signaling the completion of metamorphosis.
Genetic characteristics of parental species
We performed a genetic analysis to determine whether the larvae were genuine hybrids. The PCR products corresponded to the region from nucleotides 1388 to 1749 (362nt) of the complete sequence of 18S rDNA of A. latespinosus (GenBank Accession No. AB084546). The homologous region in A. scoparius lacked nucleotide 1487 (C) of A. latespinosus, but was otherwise identical (Fig. 2A, B). Therefore, we regarded the nucleotides at position 1487 as the species-specific gene marker. The 18S rDNA of newly metamorphosed juveniles of A. scoparius and A. latespinosus had the same nucleotide sequence as their respective parents.
To confirm the hybrid sequence patterns, we mixed the total DNA of both species in equal volumes and analyzed the 18S rDNA nucleotide sequence. Because this mixed sample contained the genomes of two species, it consequently showed "double-peaks" from ntl487 in its sequencing data (Fig. 2C).
Comparison of nucleotide sequences between parents and juveniles from crosses
We used 4 to 15 juveniles per type of both crosses for gene analysis (Table 2). All juveniles of the same type had the same nucleotide sequence. In As X Al, all the juveniles of both types had nucleotide sequences with double-peaks, resembling the pattern of the mixed sample (Fig. 2D, E). Consequently, it is highly probable that these juveniles were hybrids, having two different genomes, one from each parent. In Al X As, the juveniles of types 1 and 2 produced sequences with double-peaks, suggesting that they too were hybrid (Fig. 2F, G). However, the juveniles of type 3 had the same nucleotide sequence as the maternal species, A. latespinosus, suggesting parthenogenesis (Fig. 2H).
Experimental cross-fertilizations have been used as an aid to understanding species diversity and evolution in echinoderms (reviewed by Williamson, 2001; Byrne and Voltzow, 2004; Raff and Byrne, 2006), and hybridization studies using echinoids have been particularly informative. In relation to asteroids, Lucas and Jones (1976) were the first investigators to hybridize two species in the laboratory, Acanthaster planci and A. brevispinus, which do not interbreed in the field. Because of the success of the hybridization between the two sea stars, the authors suggested that the speciation had been recent. Although other sea stars have been cross-fertilized (Byrne and Anderson, 1994; Harper and Hart, 2005), no studies on hybridization using species with different modes of development have previously been reported. The present study shows that hybrid larvae were produced in the reciprocal cross-fertilizations between Astropecten scoparius and Astropecten latespinosus, a result that is demonstrated by the gene analysis as well as morphological observation. This is the first report concerning hybrids resulting from cross-fertilizations between two sea stars with different modes of development.
Interestingly, these hybrid larvae generally showed morphological characteristics of the bipinnariae of A. scoparius, such as the formation of body processes (i.e., bipinnarian arms), two ciliary bands, and a stomodeum. Barrel-shaped larvae are believed to be derived from bipinnariae (Oguro, 1989; McEdward and Miner, 2001), and the occurrence of many characteristics of bipinnariae in reciprocal hybrids between A. scoparius and A. latespinosus may be regarded as restoration to the ancestral condition. This hypothesis is consistent with previous reports on hybridization between two ascidians, Molgula oculata, which has a conventional tadpole larva, and M. occulta, which has a derived tailless larva (Swalla and Jeffery, 1990, 1996): most larvae of reciprocal cross-fertilizations had tadpole features. Raff et al. (1999) reported similar results with two Heliocidaris sea urchins. Our hybrid embryos, therefore, may show the restored bipinnarian form that is reported as the ancestral form.
In our hybrids, morphological variations in the larval gut were more striking than those in the bipinnarian arms and ciliary bands. Some hybrid larvae had a complete gut, as in the bipinnariae of A. scoparius, while others had a nonfunctional gut lacking a connection between the anterior tip of the archenteron and the stomodeum. Such remarkable differences were seen in hybrids from both cross-fertilizations. These results probably mean that the intensity of expression of a gene or genes promoting formation of the complete gut differed among the hybrid individuals, and thus that the expression patterns are easily altered. Hybrid larvae can be interesting models, contributing to the elucidation of genetic modifications causing evolutionary loss of the larval features in derived species.
In Heliocidaris sea urchins, all embryonic axes of H. erythrogramma (direct developer) are probably pre-established maternally (Bisgrove and Raff, 1989; Henry and Raff, 1990; Henry et al., 1990; Emlet, 1995), whereas the dorsoventral and lateral axes of H. tuberculata (indirect developer) are determined zygotically (McCain and McClay, 1994). When eggs of H. tuberculata were fertilized with H. erythrogramma sperm, their development was arrested at gastrulation because of the lack of paternal factors to determine the embryonic axes (Raff et al., 1999). In contrast, eggs of both A. scoparius and A. latespinosus completed development in our study when fertilized with the sperm of the other species. Published information is lacking on the embryonic axes and other developmental programs of Astropecten, but considering the previous reports on Heliocidaris and our results, it is probable that the developmental programs of the two Astropecten sea stars is more similar than those of the two Heliocidaris sea urchins, and their interaction is more harmonious in the Astropecten hybrids than in the Heliocidaris hybrids. This developmental harmony in our hybrid larvae implies considerable similarity of developmental gene regulation processes between A. scoparius and A. latespinosus in spite of the different developmental modes.
Average lengths of hybrid larvae were remarkably different between As X Al and Al X As, but were not significantly different between types 1 and 2 within the same cross-fertilization. These results seem to reflect the differences in the characteristics of the eggs between the parental species. As mentioned before, eggs of A. scoparius are translucent and 230 [mu]m in diameter, whereas those of A. latespinosus are semitranslucent and 300 [mu]m in diameter (Komatsu, 1975; Oguro et al., 1976). The quality of an egg is generally determined by the quality and quantity of nutriment, such as protein and lipid, which affect larval growth. It is to be expected, therefore, that eggs of A latespinosus would hold much more nutriment for larval growth than those of A. scoparius. Moreover, in As X Al, both types of larvae could complete metamorphosis without food in spite of the obligate feeding by bipinnariae of A. scoparius (data not shown); hybrids might develop without food. This also suggests that the larval lengths of hybrids are due mainly to the characteristics of the eggs. Thus, both types of hybrid larvae from Al X As grew markedly better than those from As X Al
In addition to hybridization between these two Astropecten species, we also found parthenogenesis in Al X As. The parthenogenetic individuals showed the same embryonic development and genetic characteristics as the maternal species, A. latespinosus. Previous papers have reported that artificial parthenogenesis in sea stars can be induced--for example, by treatment with thymol or methylxanthine (Obata and Nemoto, 1984; Washitani-Nemoto et al., 1994). However, we did not use such inducers, and Picard and Doree (1983) reported that [10.sup.-6] mol [1.sup.-1] 1-methyladenine, which is the concentration we used, could not induce parthenogenetic development. Therefore, it is clear that the parthenogenetic development in Al X As was not induced by 1-methyladenine. Furthermore, only species with feeding larvae were used in the previous studies. Our results suggest that parthenogenetic development of the eggs of a sea star with nonfeeding larvae can be also induced. Given the occurrence of parthenogenesis only in Al X As, differences in characteristics of eggs or in modes of development might relate to the degree of susceptibility of eggs to parthenogenetic development. More studies of parthenogenesis with species having nonfeeding larvae should be undertaken.
Both observation of development and genetic analysis using direct sequencing revealed hybridization and parthenogenesis in the reciprocal cross-fertilization between two Astropecten species with different modes of development. It is interesting that only hybrid larvae appear in As X Al, whereas both hybrid and parthenogenetic larvae occur in Al X As. Presumably this parthenogenesis is due to stimulation of A. latespinosus eggs by A. scoparius sperm.
We are grateful to Dr. G. Hendler, Natural History Museum of Los Angeles County; Dr. D. Williamson, Port Erin, Isle of Man IM9 6NH, UK; and Professor Emeritus C. Oguro, University of Toyama, for critical reading of the manuscript. We wish to express our gratitude to Messrs. M. Kinoshita, M. Tsujigawa, and the members of Fishery Research Laboratory, Kyushu University, for collecting animals. We also greatly appreciate the many pieces of advice from Drs. T. Ota, T. Kurihara, Y. Ishigaki, and Ms. M. Maeda, Kanazawa Medical University.
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Received 9 April 2007; accepted 20 February 2008.
* To whom correspondence should be addressed. E-mail: email@example.com
Abbreviations: As X Al, cross-fertilization between eggs of Astropecten scoparius and sperm of Astropecten latespinosus; Al X As, cross-fertilization between eggs of Astropecten latespinosus and sperm of Astropecten scoparius.
KAORI WAKABAYASHI (1) *, MIEKO KOMATSU (1), MANABU MURAKAMI (2), ISAO HORI (3), AND TSUTOMU TAKEGAMI (2)
(1) Graduate School of Science and Engineering University of Toyama, 3190 Gofuku, Toyama 930-8555, Japan; (2) Division of Molecular Oncology and Virology, Kanazawa Medical University, Uchinada, Ishikawa 920-0293, Japan; and (3) Division of Biology, Kanazawa Medical University, Uchinada, Ishikawa 930-0293, Japan
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|Author:||Wakabayashi, Kaori; Komatsu, Mieko; Murakami, Manabu; Hori, Isao; Takegami, Tsutomu|
|Publication:||The Biological Bulletin|
|Date:||Aug 1, 2008|
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