A New Species of Sexually Dimorphic Brittle Star of the Genus Ophiodaphne (Echinodermata: Ophiuroidea).

We describe a new species of sexually dimorphic brittle star, Ophiodaphne spinosa, from Japan associated with the irregular sea urchin, Clypeaster japonicus based on its external morphology, and phylogenetic analyses of mitochondrial COI (cytochrome c oxidase subunit I). Females of this new species of Ophiodaphne are characterized mainly by the presence of wavy grooves on the surface of the radial shields, needle-like thorns on the oral skeletal jaw structures, and a low length-to-width ratio of the jaw angle in comparison with those of type specimens of its Ophiodaphne congeners: O. scripta, O. materna, and O. formata. A tabular key to the species characteristics of Ophiodaphne is provided. Phylogenetic analyses indicate that the new species of Ophiodaphne, O. scripta, and O. formata are monophyletic. Our results indicate that the Japanese Ophiodaphne include both the new species and O. scripta, and that there are four Ophiodaphne species of sexually dimorphic brittle stars with androphorous habit.

Reproductive behaviors of the sexually dimorphic brittle star Ophiodaphne formata (Koehler, 1905) (Echinodermata: Ophiuroidea).

We investigated Ophiodaphne formata, an exceptional sexually dimorphic ophiuroid, in situ using scuba, and in the laboratory. Solitary males, solitary females, and pairs of coupled individuals were found associated with the irregular echinoid sand dollar Astriclypeus manni. We observed that coupled females, solitary males, and solitary females always hold their aboral surface against the oral surface of the host. Coupled males and females with disk diameters > 0.63 mm and > 3.7 mm, respectively, are considered to be ripe for reproduction. In order to couple, a solitary male approaches the margin of a female's disk, quickly inverts, interdigitates its arms with the female's, and keeps its mouth pressed against that of the female. At first, solitary males and females attach to their host singly and, secondly, couple with each other in order to engage in reproductive behavior. Solitary and coupled individuals are capable of changing their positions on the host, and transferring from one host to another. Coupled individuals separate after a period of attachment on the host.

Life cycle of the multiarmed sea star Coscinasterias acutispina (Stimpson, 1862) in laboratory culture: sexual and asexual reproductive pathways.

The multiarmed sea star Coscinasterias acutispina generally has 7-10 arms and 2-5 madreporites. It is known to be able to reproduce by asexual fission, and we have previously observed that this species also has the ability to reproduce sexually; however, there has been no report until now of spawning in this species. We succeeded in establishing a long-term culture of juveniles produced by artificial fertilization. Twelve months after the completion of metamorphosis, three individuals had six arms of the same length and a madreporite. At this time, fission occurred in two of these individuals, while the remaining individual underwent fission four months later. Each sea star divided into two halves, provided with three arms each. Thereafter, four or five new arms and two or four madreporites were formed anew in each of the six daughter sea-stars, so that by 30 days after the first fission the number of arms and madreporites in each was similar to that in adults. A second fission occurred in four of these six individuals, four or five months after the first fission, and in three of them the plane of division was the same as that of the first fission. The original three individuals eventually proliferated to 12 by undergoing fission. All individuals had fully developed gonads by 1-3 months after the second fission. Some of them eventually spawned under laboratory culture, and the resulting larvae metamorphosed into juveniles. Our observations demonstrate that individuals of C. acutispina possess the potential for both sexual and asexual reproduction.

Functional evolution of Ets in echinoderms with focus on the evolution of echinoderm larval skeletons.

Convergent evolution of echinoderm pluteus larva was examined from the standpoint of functional evolution of a transcription factor Ets1/2. In sea urchins, Ets1/2 plays a central role in the differentiation of larval skeletogenic mesenchyme cells. In addition, Ets1/2 is suggested to be involved in adult skeletogenesis. Conversely, in starfish, although no skeletogenic cells differentiate during larval development, Ets1/2 is also expressed in the larval mesoderm. Here, we confirmed that the starfish Ets1/2 is indispensable for the differentiation of the larval mesoderm. This result led us to assume that, in the common ancestors of echinoderms, Ets1/2 activates the transcription of distinct gene sets, one for the differentiation of the larval mesoderm and the other for the development of the adult skeleton. Thus, the acquisition of the larval skeleton involved target switching of Ets1/2. Specifically, in the sea urchin lineage, Ets1/2 activated a downstream target gene set for skeletogenesis during larval development in addition to a mesoderm target set. We examined whether this heterochronic activation of the skeletogenic target set was achieved by the molecular evolution of the Ets1/2 transcription factor itself. We tested whether starfish Ets1/2 induced skeletogenesis when injected into sea urchin eggs. We found that, in addition to ectopic induction of mesenchyme cells, starfish Ets1/2 can activate some parts of the skeletogenic pathway in these mesenchyme cells. Thus, we suggest that the nature of the transcription factor Ets1/2 did not change, but rather that some unidentified co-factor(s) for Ets1/2 may distinguish between targets for the larval mesoderm and for skeletogenesis. Identification of the co-factor(s) will be key to understanding the molecular evolution underlying the evolution of the pluteus larvae.

Morphology and gene analysis of hybrids between two congeneric sea stars with different modes of development.

The sea star Astropecten scoparius has feeding bipinnarian larvae, whereas its congener Astropecten latespinosus has nonfeeding barrel-shaped larvae. To investigate evolutionary changes in the development of asteroids, we performed reciprocal crosses between these two species with different larval forms. In the cross between A. scoparius eggs and A. latespinosus sperm, embryos developed into bipinnaria-like larvae. The larvae exhibited either a functional digestive system (a maternal feature) or a nonfunctional digestive system with the tip of the archenteron not connected to the stomodeum (a paternal characteristic). However, in the reciprocal cross between A. latespinosus eggs and A. scoparius sperm, barrel-shaped larvae resembling those of A. latespinosus were produced, in addition to bipinnaria-like larvae, some with functional digestive systems and some with nonfunctional ones. Juveniles were produced from all types of crosses. 18S rDNA was used as a gene marker in cycle sequencing analysis to investigate the genetic features of these juveniles. The sequences of juveniles from bipinnaria-like larvae showed double-peak nucleotide signals, indicating a biparental genome. On the other hand, juveniles from barrel-shaped larvae from A. latespinosus eggs and A. scoparius sperm showed the same sequence as A. latespinosus juveniles. This suggests that bipinnaria-like larvae of both crosses are always hybrids, whereas barrel-shaped larvae develop parthenogenetically.

Anatomy and ecology of the shell-less endoparasitic gastropod Asterophila japonica Randall and Heath, 1912 (Mollusca: Eulimidae).

The shell-less, endoparasitic gastropod, Asterophila japonica , was collected from two species of sea star, Leptychaster anomalus Fisher, 1906 and Ctenodiscus crispatus (Retzius, 1805) in Toyama Bay, Japan. All observed individuals were located on the aboral side of the host's disk (except one specimen parasitizing the arm) between the epidermis and the coelomic epithelium. More than one large individual frequently co-occur on a single host. The body plan of A. japonica is surprisingly modified from that of general gastropods; organs unrelated to digestion and reproduction are greatly reduced, simplified or completely lost. Dimorphism of body size is striking between males and females: males are much smaller than females and are attached to the surface of the pseudopallium of females. Females deposit and brood an egg mass(es) in the pseudopallial cavity until the eggs develop to veliger larvae. At the late developmental stage, brooded larvae reduce the velum and develop the foot for crawling, suggesting lecithotrophic development with or without a short planktonic stage. It is uncertain as to how the larvae can find and parasitize the next generation of the host. The systematic placement of Asterophila in the family Eulimidae is supported by three characters, viz. parasitism on echinoderms, smooth hydrophobic protoconch, and the enclosure of the visceral mass with the pseudopallium.

Patterns of asexual reproduction in the fissiparous seastar Coscinasterias acutispina (Asteroidea: Echinodermata) in Japan.

The morphological and anatomical characteristics of the fissiparous seastar Coscinasterias acutispina were investigated at 23 sites around Japan in order to clarify the environmental factors determining asexual reproduction. The frequency of recent fission among the sites differed, although no significant differences were evident among the three major sea areas around Japan (the Sea of Japan, Seto Inland Sea, and Pacific Ocean), indicating that the spatial variability in incidence of fission might be determined on a local scale. Morphological observations revealed that the seastars endoparasitized by Dendrogaster okadai had not undergone fission recently, suggesting that fission was suppressed by parasitism. Stepwise multiple regression analysis of the frequency of seastars that had recently undergone fission revealed that nutrient conditions affected the incidence of fission. The relationships between the pyloric caeca index and arm length ratio at the individual level suggested that good nutritional conditions were required before the occurrence of fission. Additionally, regression analysis showed that high temperature was one of the triggers of fission in C. acutispina. Thus, the process of asexual reproduction in C. acutispina appears to be governed by intricate interactions among several factors, including parasitism by D. okadai, nutrient conditions, and high temperature.

A gastric-brooding asteroid, Smilasterias multipara.

The gastric-brooding asterinid sea star, Smilasterias multipara, broods from late August to early November in the shallow sublittoral zone of southeastern Australia. We observed males and females spawning in the laboratory. They shed gametes through gonopores on the sides of the arms. The eggs were orange, about 1.0 mm in diameter, and heavier than seawater. They were externally fertilized by sperm, and placed into the stomach of the female by the tube feet. Twenty-four hours after fertilization, the first cleavage occurred. Cleavage was equal, total, and radial. Development via a non-feeding lecithotrophic brachiolaria was direct, there being no planktrotrophic bipinnaria or brachiolaria larva. Embryos developed, through wrinkled blastula and gastrula stages, into brachiolariae with arms. All of the surfaces of the brachiolaria were covered by cilia. At metamorphosis, a starfish rudiment appeared on the posterior portion of the larval body, while the anterior portion of the larval body was absorbed. Two months after fertilization, metamorphosis was complete. After metamorphosis, juveniles in the stomach grew six pairs of tube feet in each arm. Juveniles, 3 mm in diameter, emerged from the mouth of the mother in early November. Developmental evidence suggests that this asteroid has evolved mechanisms for the protection of larvae and juveniles from gastric digestion.

Morphological diversity of blastula formation and gastrulation in temnopleurid sea urchins.

Embryos of temnopleurid sea urchins exhibit species-specific morphologies. While Temnopleurus toreumaticus has a wrinkled blastula and then invaginates continuously at gastrulation, others have a smooth blastula and their invagination is stepwise. We studied blastula and gastrula formation in four temnopleurids using light and scanning electron microscopy to clarify the mechanisms producing these differences. Unlike T. toreumaticus, blastomeres of mid-blastulae in T. reevesii, T. hardwickii and Mespilia globulus formed pseudopods. Before primary mesenchyme cells ingressed, embryos developed an area of orbicular cells in the vegetal plate. The cells surrounding the orbicular cells extended pseudopods toward the orbicular cell area in three Temnopleurus species. In T. toreumaticus, the extracellular matrix was well-developed and developed a hole-like structure that was not formed in others. Gastrulation of T. reevesii, T. hardwickii and M. globulus was stepwise, suggesting that differences of gastrulation are caused by all or some of the following factors: change of cell shape, rearrangement, pushing up and towing of cells. We conclude that (1) many aspects of early morphogenesis differ even among very closely related sea urchins with indirect development and (2) many of these differences may be caused by the cell shape and structure of blastomeres or by differences in extracellular matrix composition.

WRINKLED BLASTULA OF THE SEA-STAR, ASTERINA MINOR HAYASHI.

In naturally raised embryos of Asterina minor, the changes of internal structure during the wrinkled blastula stage were followed. Eight hours after spawning, blastomeres on the surface of the coeloblastula drop into the blastocoel and eventually form multiple invaginations of the blastodermal layer. Four and a half hours after the commencement of wrinkling, the blastocoel appears to be filled by the developed invaginations. The invaginations then decrease in number and complexity, and 11.5 hr thereafter the wrinkled blastula stage ends, although gastrulation takes place before the complete disappearance of the wrinkle-invaginations.

DEVELOPMENT OF THE SEA-STAR, ASTERINA BATHERI GOTO.

Using natural spawning and artificial fertilization, the entire process of development from eggs to juveniles was observed in the sea-star, Asterina batheri Goto. The breeding season of this animal in Tsukumo Bay and Toyama Bay is estimated to be late summer. The spawned eggs are approximately 430 µm in diameter and float near the surface of sea water. They develop, through a wrinkled blastula stage by holoblastic. radial cleavage, into a pear-shaped brachiolaria bearing 3 blunt brachiolar arms. Metamorphosis takes place while the brachiolariae are swimming. Ten days after fertilization, metamorphosis is complete; the resulting juveniles are about 800 µm in diameter and colored pale brown with a green tint. They bear 2 pairs of tube-feet and a terminal tentacle in each arm. Development of this species is thus of the direct type, and very similar in every respect to that of Asterina coronata japonica, which is closely related to the present species.

Chromosomes of Japanese starfishes.

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.

Close relationship between Asterina and Solasteridae (Asteroidea) supported by both nuclear and mitochondrial gene molecular phylogenies.

Phylogenetic relationships among asteroids remain to be extremely controversial in spite of many morphological and molecular studies have been applied to this issue. In the present study, especially focusing on resolving the relationship of Asterina and Solasteridae, we reconstructed the molecular phylogenetic tree of asteroids using nuclear 18S rDNA. A close relationship between Asterina and Solasteridae, which has been suggested from analyses of mitochondrial 12S rDNA and 16S rDNA, is supported here by the nuclear 18S rDNA dataset. The support is even stronger when the sequences of mitochondrial rDNAs and nuclear 18S rDNA are combined as a total dataset. The independent support from both nuclear 18S rDNA and mitochondrial rDNAs strongly argues for a close relationship between the Asterina and Solasteridae.

Reproduction and development of the conspicuously dimorphic brittle star Ophiodaphne formata (Ophiuroidea).

Ophiodaphne formata is a conspicuously dimorphic ophiuroid; the disk diameters are approximately 1 mm for males and 5 mm for females. The dwarf male clings to the larger female, with the oral surfaces and bursae of the paired ophiuroids closely appressed. Moreover, the female of each pair adheres aborally to the oral surface of a host sand dollar, Astriclypeus manni. Spawning and external fertilization occur in August, at Tsuruga Bay, Sea of Japan. Development of the dimorphic brittle star O. formata is described for the first time, from spawning through metamorphosis, with special attention to the formation of the skeletal system and the external morphology of early juveniles. Fertilized eggs are about 90 microm in diameter, pale pink, and negatively buoyant. The embryos undergo equal, total, and radial cleavage, and the larval skeleton first forms as a pair of tetraradiate spicules. Larval development proceeds to an 8-armed planktotrophic ophiopluteus, with skeletal elements that consist of a body rod and two recurrent rods. Three weeks after fertilization, all the pluteal arms, except for the postero-lateral arms, are absorbed, and the metamorphosing larvae sink to the bottom. Metamorphosis is completed 21.5 days after fertilization, and the resulting juvenile is pentagonal and approximately 270 microm in diameter. The smallest specimen (480 microm in disk diameter) collected by field sampling exhibited male features on the skeletal plates of the jaw and disk. Sexual dimorphism, the peculiar pairing behavior, and the close relationship with the host sand dollar may have evolved as distinct reproductive characteristics in this ophiuroid with its typical ophiopluteus larvae.

Development of the nervous system in the brittle star Amphipholis kochii.

There are several studies of neural development in various echinoderms, but few on ophiuroids, which develop indirectly via the production of pluteus larvae, as do echinoids. To determine the extent of similarity of neuroanatomy and neural development in the ophiuroids with other echinoderm larvae, we investigated the development of the nervous system in the brittle star Amphipholis kochii (Echinodermata: Ophiuroidea) by immunohistochemistry. Immunoreactive cells first appeared bilaterally in the animal pole at the late gastrula stage, and there was little migration of the neural precursors during A. kochii ontogeny, as is also the case in echinoids and holothuroids. On the other hand, neural specification in the presumptive ciliary band near the base of the arms does occur in ophiuroid larvae and is a feature they share with echinoids and ophiuroids. The ophiopluteus larval nervous system is similar to that of auricularia larvae on the whole, including the lack of a fine network of neurites in the epidermis and the presence of neural connections across the oral epidermis. Ophioplutei possess a pair of bilateral apical organs that differ from those of echinoid echinoplutei in terms of relative position. They also possess coiled cilia, which may possess a sensory function, but in the same location as the serotonergic apical ganglia. These coiled cilia are thought to be a derived structure in pluteus-like larvae. Our results suggest that the neural specification in the animal plate in ophiuroids, holothuroids, and echinoids is a plesiomorphic feature of the Ambulacraria, whereas neural specification at the base of the larval arms may be a more derived state restricted to pluteus-like larvae.

Neural architecture of the brachiolaria larva of the starfish, Asterina pectinifera.

The nervous system of the brachiolaria larva of the starfish, Asterina pectinifera, was characterized using immunohistochemistry with the neuron-specific monoclonal antibodies 1E11 and 1F9 and an anti-serotonin antibody. The antigen recognized by 1F9 was determined by immunoprecipitation, peptide identification by mass spectrometry, and cDNA cloning as a novel START (steroidogenic acute regulatory protein [StAR]-related lipid transfer) domain-containing protein. Nerve cells are prominent in the brachiolar arms, ciliary bands, and adult rudiment. The brachiolar arms contain sensory-like nerve cells in the adhesive papillae, flask-shaped nerve cells in the adhesive disk, and bundles of fibers with branches interconnecting them. In the ciliary bands, nerve cells are interconnected with axon bundles along the ciliary bands and some neurons send fibers toward the oral and aboral epidermis. These neural components of the ciliary bands are regionally modified to form masses such as lateral and oral ganglia. The future aboral epidermis of the adult rudiment forms a nerve plexus with cell bodies enriched over spicules. Serotonergic nerve cell bodies are found throughout the nervous system except in the adhesive disk, the bipinnaria arms, and the adult rudiment. In addition, there are neural components in the esophagus and in the coelom where nerve fibers or bundles have distinct orientations with respect to the muscle fibers. The neuroanatomy of the brachiolaria suggests how it may function in controlling larval physiology and identifies intriguing problems on the origin of larval and adult nerves.

Adhesive papillae on the brachiolar arms of brachiolaria larvae in two starfishes, Asterina pectinifera and Asterias amurensis, are sensors for metamorphic inducing factor(s).

It has been hypothesized by Barker that starfish brachiolaria larvae initiate metamorphosis by sensing of metamorphic inducing factor(s) with neural cells within the adhesive papillae on their brachiolar arms. We present evidence supporting Barker's hypothesis using brachiolaria larvae of the two species, Asterina pectinifera and Asterias amurensis. Brachiolaria larvae of these two species underwent metamorphosis in response to pebbles from aquaria in which adults were kept. Time-lapse analysis of A. pectinifera indicated that the pebbles were explored with adhesive papillae prior to establishment of a stable attachment for metamorphosis. Microsurgical dissections, which removed adhesive papillae, resulted in failure of the brachiolaria larvae to respond to the pebbles, but other organs such as the lateral ganglia, the oral ganglion, the adhesive disk or the adult rudiment were not required. Immunohistochemical analysis with a neuron-specific monoclonal antibody and transmission electron microscopy revealed that the adhesive papillae contained neural cells that project their processes towards the external surface of the adhesive papillae and they therefore qualify as sensory neural cells.

The phylogenetic status of Paxillosida (Asteroidea) based on complete mitochondrial DNA sequences.

One of the most important issues in asteroid phylogeny is the phylogenetic status of Paxillosida. This group lacks an anus and suckers on the tube feet in adults and does not develop the brachiolaria stage in early development. Two controversial hypotheses have been proposed for the phylogenetic status of Paxillosida, i.e., Paxillosida is primitive or rather specialized in asteroids. In this study, we determined the complete mitochondrial DNA nucleotide sequences from two paxillosidans (Astropecten polyacanthus and Luidia quinaria) and one forcipulatidan (Asterias amurensis). The mitochondrial genomes of the three asteroids were identical with respect to gene order and transcription direction, and were identical to the previously reported mitochondrial genomes of Asterina pectinifera (Valvatida) and Pisaster ochraceus (Forcipulatida) in this respect. Therefore, the comparison of genome structures was uninformative for the purposes of asteroid phylogeny. However, molecular phylogenetic analyses based on the amino acid sequences and the nucleotide sequences from the five asteroids supported the monophyly of the clade that included the two paxillosidans and Asterina. This suggests that the paxillosidan characters are secondarily derived ones.