Morphological studies on the bathyal ascidian,Megalodicopia hians Oka 1918 (Octacnemidae,Phlebobranchia), with remarks on feeding and tunic morphology

Megalodicopia hians Oka is a solitary ascidian belonging to the family Octacnemidae inhabiting the bathyal /abyssal zone as well as other octacnemid ascidians. The phylogenetic relationship of octacnemids is open to argument because of its extraordinary morphological features due to habitat adaptation, e.g., a pharynx lacking ciliated stigmata. Aggregations of M. hians were discovered by the manned submersible Shinkai 2000 in the bathyal seafloor of Toyama Bay, Japan Sea, in 2000; this was the first in situ observation of M. hians in the Japanese coastal waters. In 2001, a total of 36 M. hians specimens were collected from the bay (592 to approximately 978 m deep). In situ observation indicated that M. hians usually opens its large oral apertures to engulf the drifting food particles in the water current. Microscopical observation of the gut contents also showed that M. hians is a non selective macrophagous feeding on small crustaceans, diatoms, detritus, and so on. Along with the position of the intestinal loop and gonad, the morphological characteristics of the tunic (integument of ascidians) suggest that M. hians is closely related to Cionidae and/or Corellidae. Some symbiotic/parasitic organisms were occasionally found in the tunic, including rod-shaped bacteria, fungi-like multicellular structure, and spawns of unknown animals.     

Complete nucleotide sequence of the mitochondrial genome of Doliolum nationalis with implications for evolution of urochordates

The evolutionary history of the diverse lifestyles adopted by urochordates has attracted intense interest because it may effect the evolutionary history of vertebrates. Here, we report the complete mitochondrial (mt) DNA sequence of the pelagic thaliacean doliolid Doliolum nationalis. The doliolid mt genome shares the unusual tRNAs of trnM(uau) and trnG(ucu) with other ascidians, such as Halocynthia and Ciona. On the other hand, the gene order of the doliolid mt genome is significantly different from that of any ascidian species or vertebrate reported to date. Phylogenetic analyses of the amino acid sequences of 12 protein-coding genes strongly support the sister-grouping of doliolids and the Phlebobranch ascidian Ciona, with the Stolidobranch ascidian alocynthia as the outgroup, thereby providing strong support for the paraphyly of ascidians, as has been suggested by 18S rDNA studies. Given the paraphyletic nature of ascidians, it seems likely that the common ancestor of ascidians and thaliaceans was sessile, as are the present-day ascidians, and that the thaliaceans subsequently evolved a pelagic lifestyle.     

Expression of a muscle determinant gene, macho-1, in the anural ascidian Molgula tectiformis

In anural (tailless) ascidian species, functional embryonic muscle is not formed. In urodele (tailed) ascidians, macho-1 functions as a maternally supplied factor for embryonic muscle formation. The failure of embryonic muscle development in anural ascidians may be due to the suppression of macho-1 expression. In this paper, however, we report the expression of macho-1 in embryos of an anural ascidian, Molgula tectiformis. Although M. tectiformis has lost the developmental potential to form functional embryonic muscle, macho-1 was expressed in a very similar manner as in urodele ascidians. This result, together with those of previous studies, strongly suggests that in M. tectiformis the upstream genetic cascade responsible for muscle formation is intact, while the downstream cascade including the expression of muscle structural genes is severely affected.Electronic Supplementary Material Supplementary material is available for this article at     

A genome-wide survey of the genes for planar polarity signaling or convergent extension-related genes in Ciona intestinalis and phylogenetic comparisons of evolutionary conserved signaling components

Non-canonical Wnt signals similar to planar cell polarity (PCP) signaling in the fly control convergent extension (CE) of the dorsal mesoderm during gastrulation in vertebrates. Using the Ciona complete genome sequence and EST sequence data, we present here an initial and exhaustive search in non-vertebrate chordates, Ciona intestinalis for the family members as well as homologs or orthologs that are involved in PCP/CE signaling cascades. We clarified 7 cardinal gene families, including the MAPK, STE20 group kinase, Rho small GTPase, STAT, Glypican, Fz and Wnt gene families, as well as gene homologs or orthologs for known PCP/CE signaling components with their phylogenetic nature. As a result, we characterized 62 Ciona component genes. Among them, 59 genes were novel and functional genes which were supported by EST expressions and 15 genes belonged to PCP/CE component orthologs of other organisms or common ancestor genes. Moreover, from the phylogenetic point of view, we compared these components genome-widely with the PCP signaling components of fly and the CE signaling components of vertebrates. We then discovered not only that ascidians contain the basic ancestral signaling pathway components in chordates but also that several signaling components have not found in ascidian, indicating that ascidian CE pathway might have several gaps from vertebrate CE pathway. The present study provides an initial step for the subsequent analysis of CE in the non-vertebrate chordates, ascidians. In addition, this phylogenetic approach will help to facilitate understanding of the relationship between fly PCP signaling and the vertebrate CE pathway.     

Ascidian molecular phylogeny inferred from mtDNA data with emphasis on the Aplousobranchiata

We explored the usefulness of mtDNA data in assessing phylogenetic relationships within the Ascidiacea. Although ascidians are a crucial group in studies of deuterostome evolution and the origin of chordates, little molecular work has been done to ascertain the evolutionary relationships within the class, and in the studies performed to date the key group Aplousobranchiata has not been adequately represented. We present a phylogenetic analysis based on mitochondrial cytochrome c oxidase subunit I (COI) sequences of 37 ascidian species, mainly Aplousobranchiata (26 species). Our data retrieve the main groups of ascidians, although Phlebobranchiata appeared paraphyletic in some analyses. Aplousobranch ascidians consistently appeared as a derived group, suggesting that their simple branchial structure is not a pleisiomorphic feature. Relationships between the main groups of ascidians were not conclusively determined, the sister group of Aplousobranchiata was the Stolidobranchiata or the Phlebobranchiata, depending on the analysis. Therefore, our data could not confirm an Enterogona clade (Aplousobranchiata+Phlebobranchiata). All of the tree topologies confirmed previous ideas, based on morphological and biochemical characters, suggesting that Cionidae and Diazonidae are members of the clade Aplousobranchiata, with Cionidae occupying a basal position within them in our analyses. Within the Aplousobranchiata, we found some stable clades that provide new data on the evolutionary relationships within this large group of ascidians, and that may prompt a re-evaluation of some morphological characters.     

Urochordates are monophyletic within the deuterostomes

Understanding the phylogenetic relationships of the three major urochordate groups within the deuterostomes is central to understanding the evolution of the chordates. We have prepared a detailed phylogenetic analysis of urochordates based on comparisons of 10 new urochordate 18S ribosomal DNA sequences with other urochordate sequences in GenBank. Maximum parsimony, neighbor-joining, minimum evolution, and maximum likelihood analyses of this large urochordate data set are consistent with a topology in which the urochordates are monophyletic within the deuterostomes and there are four separate clades of urochordates. These four distinct clades--styelid + pyurid ascidians, molgulid ascidians, phlebobranch ascidians + thaliaceans, and larvaceans--are mostly consistent with traditional morphological hypotheses and classifications. However, we find that the ascidians may not be a monophyletic group (as they have been considered traditionally) but instead appear paraphyletic. Another disparity with traditional classification is that the thaliaceans do not form a separate urochordate clade but rather cluster with the phlebobranch ascidians. Larvaceans have long branch lengths, which can be problematic for molecular phylogenetic methods, and their position within the urochordates cannot be unequivocally determined with 18S rDNA. This is important because the tadpole morphology of larvacean and ascidian larvae is the key trait of interest that distinguishes urochordates as chordates. Nevertheless, the present data set resolves at least three clades of urochordates and suggests strongly that urochordates form a monophyletic clade within the deuterostomes.     

Decoding cis-regulatory systems in ascidians

Ascidians, or sea squirts, are lower chordates, and share basic gene repertoires and many characteristics, both developmental and physiological, with vertebrates. Therefore, decoding cis-regulatory systems in ascidians will contribute toward elucidating the genetic regulatory systems underlying the developmental and physiological processes of vertebrates. cis-Regulatory DNAs can also be used for tissue-specific genetic manipulation, a powerful tool for studying ascidian development and physiology. Because the ascidian genome is compact compared with vertebrate genomes, both intergenic regions and introns are relatively small in ascidians. Short upstream intergenic regions contain a complete set of cis-regulatory elements for spatially regulated expression of a majority of ascidian genes. These features of the ascidian genome are a great advantage in identifying cis-regulatory sequences and in analyzing their functions. Function of cis-regulatory DNAs has been analyzed for a number of tissue-specific and developmentally regulated genes of ascidians by introducing promoter-reporter fusion constructs into ascidian embryos. The availability of the whole genome sequences of the two Ciona species, Ciona intestinalis and Ciona savignyi, facilitates comparative genomics approaches to identify cis-regulatory DNAs. Recent studies demonstrate that computational methods can help identify cis-regulatory elements in the ascidian genome. This review presents a comprehensive list of ascidian genes whose cis-regulatory regions have been subjected to functional analysis, and highlights the recent advances in bioinformatics and comparative genomics approaches to cis-regulatory systems in ascidians.     

Mini-review: Impact and dynamics of surface fouling by solitary and compound ascidians

Globally, ascidians are a significant contributor to benthic marine fouling communities, but have remained poorly studied in this context. In some cases, such as in shellfish and finfish aquaculture, ascidians are the most problematic of all fouling organisms. The disproportionate impact of ascidian fouling in some specific geographic locations has been related directly to anthropogenic translocation of these organisms around the globe. In the case of ascidians, therefore, the economic issue of biofouling and the ecological ramifications of invasion are inextricably linked. This mini-review briefly discusses the introduction of non-native ascidians to areas where they have subsequently proven to be a significant fouling pest. The elements of ascidian reproductive ecology that support their aggressive fouling character are discussed and the scant information pertaining to their adhesion and adhesives is presented. Finally, strategies for mitigating ascidian fouling are examined. It is suggested that sufficient working knowledge currently exists to support the inclusion of one or more common ascidian species as ‘standard’ test organisms used for evaluation of novel fouling-resistant surfaces.     

Studies on Japanese botryllid ascidians. II. A new species of the genus Botryllus from the vicinity of Shimoda

Morphology and life history of a new species of the genus Botryllus belonging to the family Botryllidae were described in detail. This ascidian was collected from the stony shore in the vicinity of Shimoda (Shizuoka prefecture, Japan). The arrangement of ovary and testis in this ascidian was the same as that in other species of the genus Botryllus, while the embryo developed in a brood pouch formed from the invagination of peribranchial epithelium, as in the other genus Botrylloides. The processes and features of the allorecognition reaction of this ascidian were observed. The reaction showed the same processes as that in the species of the genus Botrylloides. Therefore, this ascidian has both features of the two genera of the family Botryllidae, which strongly suggests the necessity of reconsidering on the classificatory criteria of botryllid ascidians.     

Species specificity of symbiosis and secondary metabolism in ascidians

Ascidians contain abundant, diverse secondary metabolites, which are thought to serve a defensive role and which have been applied to drug discovery. It is known that bacteria in symbiosis with ascidians produce several of these metabolites, but very little is known about factors governing these ‘chemical symbioses''. To examine this phenomenon across a wide geographical and species scale, we performed bacterial and chemical analyses of 32 different ascidians, mostly from the didemnid family from Florida, Southern California and a broad expanse of the tropical Pacific Ocean. Bacterial diversity analysis showed that ascidian microbiomes are highly diverse, and this diversity does not correlate with geographical location or latitude. Within a subset of species, ascidian microbiomes are also stable over time (R=−0.037, P-value=0.499). Ascidian microbiomes and metabolomes contain species-specific and location-specific components. Location-specific bacteria are found in low abundance in the ascidians and mostly represent strains that are widespread. Location-specific metabolites consist largely of lipids, which may reflect differences in water temperature. By contrast, species-specific bacteria are mostly abundant sequenced components of the microbiomes and include secondary metabolite producers as major components. Species-specific chemicals are dominated by secondary metabolites. Together with previous analyses that focused on single ascidian species or symbiont type, these results reveal fundamental properties of secondary metabolic symbiosis. Different ascidian species have established associations with many different bacterial symbionts, including those known to produce toxic chemicals. This implies a strong selection for this property and the independent origin of secondary metabolite-based associations in different ascidian species. The analysis here streamlines the connection of secondary metabolite to producing bacterium, enabling further biological and biotechnological studies.     

p68, a DEAD-box RNA helicase, is expressed in chordate embryo neural and mesodermal tissues

The p68 DEAD-box RNA helicases have been identified in diverse organisms, including yeast, invertebrates, and mammals. DEAD-box RNA helicases are thought to unwind duplexed RNAs, and the p68 family may participate in initiating nucleolar assembly. Recent evidence also suggests that they are developmentally regulated in chordate embryos. bobcat, a newly described member of this gene family, has been found in eggs and developing embryos of the ascidian urochordate, Molgula oculata. Antisense RNA experiments have implicated this gene in establishing basic chordate features, including the notochord and neural tube in ascidians (Swalla et al. 1999). We have isolated p68 homologs from chick and Xenopus in order to investigate their possible role in vertebrate development. We show that embryonic expression of p68 in chick, frog, and ascidian embryos is high in the developing brain and spinal cord as well as in the sensory vesicles. In frog embryos, p68 expression also marks the streams of migrating cranial neural crest cells throughout neural tube development and in tailbud stages, but neural crest expression is faint in chick embryos. Ascidian embryos also show mesodermal p68 expression during gastrulation and neurulation, and we document some p68 mesodermal expression in both chick and frog. Thus, as shown in these studies, p68 is expressed in early neural development and in various mesodermal tissues in a variety of chordate embryos, including chick, frog, and ascidian. Further functional experiments will be necessary to understand the role(s) p68 may play in vertebrate development.     

Microbial diversity associated with ascidians: a review of research methods and application

This paper reviews research in microbial diversity associated with ascidians (commonly known as sea squirts). The application of culture-dependent and culture-independent techniques is introduced in detail and these methods are analyzed for their advantages and limitations. Because of the limitations of available media and cultivation conditions, culture-dependent methods can only reveal a limited portion of the microorganisms in ascidians. However, the acquisition of typical microbial community members in culture remains a valuable resource for exploring their bioactive potential and relationships with the ascidian hosts. The application of metagenomic library methods has greatly accelerated ascidian metabolites studies. The next-generation sequencing techniques have led to the acquisition of an unprecedented quantity of ascidian microorganism data, providing the most comprehensive information about ascidian microbial diversity. Ascidians provide unique ecological niches that harbor an unexpected diversity of microorganisms different from planktonic bacteria in the local seawater. Microbial communities associated with ascidians tend to be species-specific and tissue-specific. Different tissue of the same ascidian may be associated with different microbial communities.     

Expression and functional analysis of Cititf1, an ascidian NK-2 class gene, suggest its role in endoderm development.

In solitary ascidians the fate of endoderm is determined at a very early stage of development and depends on cytoplasmic factors whose nature has not been determined. We have isolated a member of the NK-2 gene family, Cititf1, from the ascidian Ciona intestinalis, showing high sequence homology to mammalian TITF1. The Cititf1 gene was expressed in all endodermal precursors at the pregastrula and gastrula stages, and is thus the first specific regulatory endodermal marker to be isolated from an ascidian. Cititf1 expression was downregulated at the end of gastrulation to reappear at middle tailbud and larval stages in the most anterior and ventral parts of head endoderm, regions which give rise, after metamorphosis, to the adult endostyle, where Cititf1 mRNA was still present. Microinjection of Cititf1 mRNA into fertilized eggs resulted in tadpole larvae with abnormalities in head-trunk development consequent to the formation of excess endoderm, perhaps due to recruitment of notochord precursors to an endodermal fate. These data suggest that Cititf1 plays an important role in normal endoderm differentiation during ascidian embryogenesis.     

Phylogenetic diversity of bacteria associated with ascidians in Eel Pond (Woods Hole, Massachusetts, USA)

Recent studies have revealed that many marine invertebrates are closely associated with diverse microorganisms, frequently resulting in the production of compounds of biomedical interest. Thus far, ascidians have not been widely examined for the presence of bacterial associations, although the production of secondary metabolites is well documented. In the present study, we examined the gonad of Molgula manhattensis and the tunic surfaces of Botryllus schlosseri, Didemnum sp., and Botrylloides violaceus for the presence of associated bacteria. These ascidians are common inhabitants of the coastal ocean of Cape Cod, Massachusetts. We used denaturing gradient gel electrophoresis (DGGE) as well as cloning and sequencing of 16S rDNA to analyze the microbial communities. There is a strong evidence that spiroplasma-like bacteria colonize the gonad of the solitary ascidian M. manhattensis. The bacteria might be vertically transmitted and may be involved in the production of secondary metabolites that deter predators of the ascidians. The bacterial communities found on the tunic surfaces of the colonial ascidians were found to be more diverse. However, in all cases the bacterial communities were predominated by alpha-proteobacteria. Alpha-proteobacteria related to the obtained sequences have been identified as symbionts in a variety of hosts, suggesting a specific role for these bacteria. However, based on our data it is difficult to differentiate between persistently and only transiently associated bacteria. Overall, this study demonstrates that ascidian species are associated with diverse bacterial populations. Future studies will aim to elucidate the precise relationships between bacteria and ascidians and to identify bioactive compounds that might be produced as a result of these relationships.     

Ascidians as excellent chordate models for studying the development of the nervous system during embryogenesis and metamorphosis

The swimming larvae of the chordate ascidians possess a dorsal hollowed central nervous system (CNS), which is homologous to that of vertebrates. Despite the homology, the ascidian CNS consists of a countable number of cells. The simple nervous system of ascidians provides an excellent experimental system to study the developmental mechanisms of the chordate nervous system. The neural fate of the cells consisting of the ascidian CNS is determined in both autonomous and non-autonomous fashion during the cleavage stage. The ascidian neural plate performs the morphogenetic movement of neural tube closure that resembles that in vertebrate neural tube formation. Following neurulation, the CNS is separated into five distinct regions, whose homology with the regions of vertebrate CNS has been discussed. Following their larval stage, ascidians undergo a metamorphosis and become sessile adults. The metamorphosis is completed quickly, and therefore the metamorphosis of ascidians is a good experimental system to observe the reorganization of the CNS during metamorphosis. A recent study has shown that the major parts of the larval CNS remain after the metamorphosis to form the adult CNS. In contrast to such a conserved manner of CNS reorganization, most larval neurons disappear during metamorphosis. The larval glial cells in the CNS are the major source for the formation of the adult CNS, and some of the glial cells produce adult neurons.     

Evolution of Chordate Actin Genes: Evidence from Genomic Organization and Amino Acid Sequences

The origin and evolutionary relationship of actin isoforms was investigated in chordates by isolating and characterizing two new ascidian cytoplasmic and muscle actin genes. The exon–intron organization and sequences of these genes were compared with those of other invertebrate and vertebrate actin genes. The gene HrCA1 encodes a cytoplasmic (nonmuscle)-type actin, whereas the MocuMA2 gene encodes an adult muscle-type actin. Our analysis of these genes showed that intron positions are conserved among the deuterostome actin genes. This suggests that actin gene families evolved from a single actin gene in the ancestral deuterostome. Sequence comparisons and molecular phylogenetic analyses also suggested a close relationship between the ascidian and vertebrate actin isoforms. It was also found that there are two distinct lineages of muscle actin isoforms in ascidians: the larval muscle and adult body-wall isoforms. The four muscle isoforms in vertebrates show a closer relationship to each other than to the ascidian muscle isoforms. Similarly, the two cytoplasmic isoforms in vertebrates show a closer relationship to each other than to the ascidian and echinoderm cytoplasmic isoforms. In contrast, the two types of ascidian muscle actin diverge from each other. The close relationship between the ascidian larval muscle actin and the vertebrate muscle isoforms was supported by both neighbor-joining and maximum parsimony analyses. These results suggest that the chordate ancestor had at least two muscle actin isoforms and that the vertebrate actin isoforms evolved after the separation of the vertebrates and urochordates. Received: 20 June 1996 / Accepted: 16 October 1996     

Cell lineage and timing of fate restriction, determination and gene expression in ascidian embryos

Tadpole larvae of ascidians show the basic body plan of chordates. An ascidian larva consists of only few types of cells and has a relatively small number of cells. Cell lineages are simple and invariant among individuals and have been described in detail. The clonal restriction of developmental fate takes place considerably early in development. I review here the temporal relationship between fate restriction, determination and initiation of lineage-specific gene expression during ascidian embryogenesis. In several cases, determination and initiation of gene expression precede fate restriction and occur during the last cell cycle before fate restriction. Such a phenomenon contradicts the traditional view of fate specification and has several important implications for the understanding of the way in which cells execute the developmental pathway.     

Evidence for the Heparin-Binding Ability of the Ascidian Xlink Domain and Insight into the Evolution of the Xlink Domain in Chordates

The vertebrate Xlink domain is found in two types of genes: lecticans and their associated hyaluronan-and-proteoglycan-binding-link-proteins (HAPLNs), which are components of the extracellular matrix, and those represented by CD44 and stabilins, which are expressed on the surface of lymphocytes. In both types of genes, Xlink functions as a hyaluronan binding domain. We have already reported that protochordate ascidians possess only the latter type of gene. The present analysis of the expression of ascidian Xlink domain genes revealed that these genes function in blood cell migration and apoptosis. While the Xlink domain is found in various metazoans, including ascidians and nematodes, hyaluronan is believed to be specific for vertebrates. In comprehensive genome surveys for hyaluronan synthase (HAS), we found no HAS gene in ascidians. We also established that hyaluronan is absent from the ascidian body biochemically. Therefore, ascidians possess the Xlink domain, but they lack HA. We recovered one ascidian Xlink domain gene that encoded a heparin-binding protein, although it shows no affinity for hyaluronan. Based on these findings, we conclude that in invertebrates, the Xlink domain serves as heparin-binding protein domain and functions in blood cell migration and apoptosis. Its binding affinity for HA might have been acquired in the vertebrate lineage.