Euichi Hirose

Genome streamlining and chemical defense in a coral reef symbiosis

Secondary metabolites are ubiquitous in bacteria, but by definition, they are thought to be nonessential. Highly toxic secondary metabolites such as patellazoles have been isolated from marine tunicates, where their exceptional potency and abundance implies a role in chemical defense, but their biological source is unknown. Here, we describe the association of the tunicate Lissoclinum patella with a symbiotic α-proteobacterium, Candidatus Endolissoclinum faulkneri, and present chemical and biological evidence that the bacterium synthesizes patellazoles. We sequenced and assembled the complete Ca. E. faulkneri genome, directly from metagenomic DNA obtained from the tunicate, where it accounted for 0.6% of sequence data. We show that the large patellazoles biosynthetic pathway is maintained, whereas the remainder of the genome is undergoing extensive streamlining to eliminate unneeded genes. The preservation of this pathway in streamlined bacteria demonstrates that secondary metabolism is an essential component of the symbiotic interaction.

Two new species of diplosoma (Ascidiacea: Didemnidae) bearing prokaryotic algae prochloron from Okinawajima (Ryukyu Archipelago, Japan).

Abstract Two new species of didemnid ascidians, Diplosoma ooru sp. nov. and Diplosoma simileguwa sp. nov., are described from coral reefs on Okinawajima (Ryukyu Archipelago, Japan). These two species form green colonies, having a symbiotic association with a prokaryotic alga Prochloron sp. The former species was found at the reef edges in the subtidal zone and the latter was found in a shallow reef lagoon. In these species, the colonies are thinner and the zooids are smaller than those of any other Prochloron-bearing Diplosoma species so far described. Moreover, each of the present new species has a unique combination of stigmatic numbers: 5 stigmata in the first and third rows, 6 in the second row, and 4 in the fourth in D. ooru; 4 stigmata in the first and third rows, 5 in the second row, and 3 in the fourth in D. simileguwa. In both of the new species, the retractor muscle emerges from the underside of the thorax. Larval morphology of D. ooru is also described.

Plant Rake and Algal Pouch of the Larvae in the Tropical Ascidian Diplosoma similis: An Adaptation for Vertical Transmission of Photosynthetic Symbionts Prochloron sp

Abstract The embryos of Diplosoma similis are brooded within the thick walled tunic of the colony in isolation from the symbiotic algae Prochloron sp., which are in the cloacal cavity of the parent colony. Prior to the spawning, the plant rake, a tassel-like structure, protrudes from the postero-dorsal end of the larval trunk and extends into the cloacal cavity. The algal cells in the cloacal cavity adhere to the plant rake. When the larvae are spawned, the trunk tunic extends posteriorly and forms a pouch entirely covering the plant rake. The algal cells are packed in the pouch (algal pouch) enveloping the basal part of the tail. The cell density of the algae in the pouch is much higher than that in the colony, suggesting that the plant rake functions for gathering and concentrating the symbionts into the algal pouch. In the course of metamorphosis, the algal pouch expands and turns into the cloacal cavity of the young colony. The high density of algal cells in the pouch would ensure that the young colony possesses the symbiotic algae of appropriate cell density in the cloacal cavity, and the colony can sufficiently receive benefits from the symbionts just after the settlement.

Tunic morphology and cellulosic components of pyrosomas, doliolids, and salps (Thaliacea, Urochordata)

The morphology and cellulosic composition of the tunic was studied in pelagic tunicates (3 pyrosomas, 2 doliolids, and 13 salps). The tunic is transparent and gelatinous, consisting of an electron-dense cuticular layer with a fibrous tunic matrix. The thickness and density of the cuticular layer and of the tunic matrix differ from species to species. In some salps, the cuticular layer has numerous minute protrusions that are structurally identical to those found in several ascidians. Free mesenchymal cells (tunic cells) are distributed in the tunic. Whereas the number of tunic cells in the pyrosomas is similar to that in ascidians, there are many fewer tunic cells in doliolids and salps. These differences may be caused by the different functions of the tunic in each group. The existence of cellulose in the tunic was confirmed using electron diffraction in all of the species studied thus far. Their diffractograms indicate that the cellulose microfibrils consist of nearly pure I{beta} of the allomorph. These results show that tunic morphology and cellulosic composition are similar in ascidians and thaliaceans (pyrosomas, doliolids, and salps). The tunic is considered to be a homologous tissue in these animals, and their most recent common ancestor would have possessed this tissue.

Cellulose in the house of the appendicularianOikopleura rufescens

SummaryBy electron diffraction analysis, highly crystalline cellulose Iβ was found in the house (a special structure in which the tunicate lives) of the appendicularianOikopleura rufescens. Cellulose microfibrils 20 nm in width were observed in a random array or highly organized with rectangular spacing of 2 to 10 (im in the house. The bundled cellulose microfibrils formed in the inlet filters, which are highly ordered meshwork structures. This paper provides the first account of the existence of cellulose in the house of an appendicularian. Our findings showed that the house and tunic are homologous tissues among the tunicates, and that the common ancestor of the tunicates (ascidians, thaliaceans, and appendicularians) already possessed cellulose-biosynthetic ability.

Complete nucleotide sequences of mitochondrial genomes of two solitary entoprocts, Loxocorone allax and Loxosomella aloxiata: Implications for lophotrochozoan phylogeny

The complete nucleotide sequences of the mitochondrial (mt) genomes of the entoprocts Loxocorone allax and Loxosomella aloxiata were determined. Both species carry the typical gene set of metazoan mt genomes and have similar organizations of their mt genes. However, they show differences in the positions of two tRNA(Leu) genes. Additionally, the tRNA(Val) gene, and half of the long non-coding region, is duplicated and inverted in the Loxos. aloxiata mt genome. The initiation codon of the Loxos. aloxiata cytochrome oxidase subunit I gene is expected to be ACG rather than AUG. The mt gene organizations in these two entoproct species most closely resemble those of mollusks such as Katharina tunicata and Octopus vulgaris, which have the most evolutionarily conserved mt gene organization reported to date in mollusks. Analyses of the mt gene organization in the lophotrochozoan phyla (Annelida, Brachiopoda, Echiura, Entoprocta, Mollusca, Nemertea, and Phoronida) suggested a close phylogenetic relationship between Brachiopoda, Annelida, and Echiura. However, Phoronida was excluded from this grouping. Molecular phylogenetic analyses based on the sequences of mt protein-coding genes suggested a possible close relationship between Entoprocta and Phoronida, and a close relationship among Brachiopoda, Annelida, and Echiura.

Algivore or phototroph? Plakobranchus ocellatus (Gastropoda) continuously acquires kleptoplasts and nutrition from multiple algal species in nature.

The sea slug Plakobranchus ocellatus (Sacoglossa, Gastropoda) retains photosynthetically active chloroplasts from ingested algae (functional kleptoplasts) in the epithelial cells of its digestive gland for up to 10 months. While its feeding behavior has not been observed in natural habitats, two hypotheses have been proposed: 1) adult P. ocellatus uses kleptoplasts to obtain photosynthates and nutritionally behaves as a photoautotroph without replenishing the kleptoplasts; or 2) it behaves as a mixotroph (photoautotroph and herbivorous consumer) and replenishes kleptoplasts continually or periodically. To address the question of which hypothesis is more likely, we examined the source algae for kleptoplasts and temporal changes in kleptoplast composition and nutritional contribution. By characterizing the temporal diversity of P. ocellatus kleptoplasts using rbcL sequences, we found that P. ocellatus harvests kleptoplasts from at least 8 different siphonous green algal species, that kleptoplasts from more than one species are present in each individual sea slug, and that the kleptoplast composition differs temporally. These results suggest that wild P. ocellatus often feed on multiple species of siphonous algae from which they continually obtain fresh chloroplasts. By estimating the trophic position of wild and starved P. ocellatus using the stable nitrogen isotopic composition of amino acids, we showed that despite the abundance of kleptoplasts, their photosynthates do not contribute greatly to the nutrition of wild P. ocellatus, but that kleptoplast photosynthates form a significant source of nutrition for starved sea slugs. The herbivorous nature of wild P. ocellatus is consistent with insights from molecular analyses indicating that kleptoplasts are frequently replenished from ingested algae, leading to the conclusion that natural populations of P. ocellatus do not rely on photosynthesis but mainly on the digestion of ingested algae.

Subcuticular Rejection: an Advanced Mode of the Allogeneic Rejection in the Compound Ascidians Botrylloides simodensis and B. fuscus

Allogeneic rejection between colonies (colony specificity) was described by electron microscopy in two compound ascidians, Botrylloides simodensis and B. fuscus. When two incompatible colonies are brought into contact at their growing edges, the tunic cuticle dissolves and the tunics of the colonies partially fuse. Alloreactive, humoral factors may diffuse to the opposite colony through the partially fusing tunic, and the tunic cells (free cells distributed in the tunic) possibly recognize these factors and induce a rejection reaction. Then, blood cells--mainly morula cells--infiltrate into the tunic, while tunic cells are disintegrating near where the partial fusion of the tunic is occurring. The infiltrating blood cells aggregate, disintegrate, and discharge electron-dense materials in the tunic at the subcuticular regions where the tunics have partially fused. Since the rejection lesion is restricted to the subcuticular area, some regulatory systems may be involved in this restriction. At the end, new walls are formed in the tunic matrix to separate the rejection lesion from the contacting colonies. The new wall is a continuous layer composed of electron-dense fibers and is structurally identical to the regenerating tunic cuticle. The mode of occurrence of colony specificity (Hirose et al., 1994) and the present results indicate that tunic cells are the only allorecognition sites in B. fuscus.

Intracellular Symbiosis of a Photosynthetic Prokaryote, Prochloron sp., in a Colonial Ascidian

Cells of a symbiotic prokaryote, Prochloron sp., in colonies of a tropical ascidian, Lissoclinum punctatum, occur not only outside but also inside cells of the host. Cells of these photosynthetic symbionts of ascidians have previously been reported only extracellularly. The intracellular and extracellular symbionts do not differ morphologically. The host cells carrying the symbionts are freely distributed in the ascidian tunic. They probably endocytize the symbionts and then retain them within a vacuole. Since the intracellular prokaryotes showed no evidence of rejection or degeneration, this association between tunic phagocytes of L. punctatum and cells of Prochloron sp. seems to constitute a stable symrnbiosis, comparable to the postulated ancestral association between heterotrophic cells and the photosynthetic prokaryotes which gave rise to chloroplasts. Additional key words: algae, cyanobacteria, plastid evolution, prochlorophytes, tunicates Chloroplasts are photosynthetic organelles found in all photosynthetic eukaryotes. There is now convincing evidence from molecular biological studies on 16S-rRNA and RNA polymerase subunit (rpo Cl) and ribulose-bisphosphate carboxylase genes (Seewaldt & Stackebrandt 1982; Palenik & Haselkorn 1992; Urbach et al. 1992; Shimada et al. 1995) that these organelles originated from prokaryotic photosynthetic endosymbionts (cyanobacteria) engulfed and retained by heterotrophic host cells (Lewin 1981; Margulis 1981). Symbiotic photosynthetic prokaryotes are therefore of interest in relation to the evolution of chloroplasts. Prochloron is a genus of unicellular prokaryotes with the same chlorophyll pigments, chl a and b, as those in the chloroplasts of green algae and all other green plants (Lewin 1976). Prochlorophyta/Prochlorales was originally established (Lewin 1976, 1977; Florenzano et al. 1986) for prokaryotes that bear chl. a and b, lack bilin pigments, and generate oxygen in photosynthesis; molecular phylogenetic studies, however, indicate that this is a polyphyletic group whose members arose within the cyanobacterial radiation and should be treated as members of Cyanophyta/Cyanobacteria (Palenik & Haselkorn 1992; Urbach et al. 1992; Shimada et al. 1995). Cells of Prochloron occur in coral reef areas, almost exclusively as symbionts of colonial didemnid ascidians (Lewin & Cheng 1989). There the symbionts are normally associated with external or internal colony surfaces, but outside the host cells. Prochloron didemni LEWIN 1977 was originally described from the outer surfaces of didemnid colonies; but since no prochloron cells have been cultured in vitro, and specific distinctions remain to be established, we will refer to the symbionts in this paper simply as Prochloron sp. We present here the first report of Prochloron sp. as an intracellular symbiont in a didemnid ascidian, Lissoclinum punctatum KOTT 1977. Recent molecular biological data (Palenik & Haselkorn 1992; Urbach et al. 1992) indicate that the phylogenetic affinities of chloroplasts are closer to other cyanobacteria than to Prochloron, but we suggest that intracellular Prochloron can be regarded as a model of the ancestral green plastid.

Ultrastructures and Classification of Circulating Hemocytes in 9 Botryllid Ascidians (Chordata: Ascidiacea)

Abstract Ultrastructures of circulating hemocytes were studied in 9 botryllid ascidians. The hemocytes are classified into five types: hemoblasts, phagocytes, granulocytes, morula cells, and pigment cells. These five types are always found in the 9 species. They should represent the major hemocyte types of the circulating cells in the blood. Hemoblasts are small hemocytes having a high nucleus/cytoplasm ratio. There are few granular or vacuolar inclusions in the cytoplasm. Phagocytes have phagocytic activity and their shape is variable depending on the amount of engulfed materials. In granulocytes, shape and size of granules are different among the species. Morula cells are characterized by several vacuoles filled with electron dense materials. In pigment cells, the bulk of the cytoplasm is occupied by one or a few vacuoles containing pigment granules. We also described some other hemocyte types found in particular species. Furthermore, we encountered free oocytes circulating in the blood in two species, Botryllus primigenus and Botrylloides lentus.