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Symbiotic associations with beneficial microorganisms endow a variety of host animals with adaptability to the environment. Stable transmission of symbionts across host generations is a key event in the maintenance of symbiotic associations through evolutionary time. However, our understanding of the mechanisms of symbiont transmission remains fragmentary. The deep-sea clam Phreagena okutanii harbors chemoautotrophic intracellular symbiotic bacteria in gill epithelial cells, and depends on these symbionts for nutrition. In this study, we focused on the association of these maternally transmitted symbionts with ovarian germ cells in juvenile female clams. First, we established a sex identification method for small P. okutanii individuals, and morphologically classified female germ cells observed in the ovary. Then, we investigated the association of the endosymbiotic bacteria with germ cells. We found that the symbionts were localized on the outer surface of the cell membrane of primary oocytes and not within the cluster of oogonia. Based on our findings, we discuss the processes and mechanisms of symbiont vertical transmission in P. okutanii.

Deep-sea vesicomyid clams, including the genus Phreagena, harbor obligate sulfur-oxidizing symbiotic bacteria in gill epithelial cells. Difficulty in maintaining Phreagena clams in rearing tanks has been a major obstacle to achieving a better understanding of their unique biology. To improve the method of rearing Phreagena clams, here we reared them in an artificial chemosynthetic aquarium and evaluated the effects of the aquarium system on long-term clam rearing. We compared the survival of clams reared in the artificial chemosynthetic tank with the survival of those in the normal tank, and analyzed the symbiont abundance using semi-quantification of fluorescent in situ hybridization signals. Our results indicate that the artificial chemosynthetic aquarium system had specific effects on symbiont abundance and possibly on host survival. Furthermore, transmission electron microscopic observations of sulfur globules in the symbiont cells and expression analyses of the dsrA gene of the symbiont indicated that stocked elemental sulfur could be consumed as an energy source to reduce sulfide shortages. We discuss the importance of higher and more stable sulfide concentrations and the proportions of available O2 and CO2 in driving appropriate metabolic functions of the symbiont and improving the survival of the clams.

In a previous report, we analyzed the stomach contents of juvenile chum salmon Oncorhynchus keta by morphological observation and also by molecular identification using the mitochondrial cytochrome c oxidase subunit I (COI) region. However, one of the most frequently detected COI sequences could not be assigned to any specific taxon, even at the phylum level. In the present study, we conducted in situ hybridization (ISH) on the stomach contents of juvenile chum salmon using the COI sequence and polymerase chain reaction amplification of a 18S ribosomal RNA gene from the tissue sections where ISH signals were detected. As a result, the organism that was enigmatic at the phylum level was found to be an appendicularian. Moreover, Oikopleura longicauda collected from the bay where the juvenile chum salmon samples were obtained was shown to have the same COI sequences as this taxonomic “orphan” COI sequence from the stomach contents. The present results suggest that the COI sequences previously deposited in public databases for “Oikopleura” are actually derived from taxonomic groups other than appendicularians, and that this may have hampered our understanding of prey richness in the stomach or gut of certain marine animals based on DNA barcoding.

Sterols are key cyclic triterpenoid lipid components of eukaryotic cellular membranes, which are synthesized through complex multi-enzyme pathways. Similar to most animals, Bathymodiolus mussels, which inhabit deep-sea chemosynthetic ecosystems and harbor methanotrophic and/or thiotrophic bacterial endosymbionts, possess cholesterol as their main sterol. Based on the stable carbon isotope analyses, it has been suggested that host Bathymodiolus mussels synthesize cholesterol using a sterol intermediate derived from the methanotrophic endosymbionts. To test this hypothesis, we sequenced the genome of the methanotrophic endosymbiont in Bathymodiolus platifrons. The genome sequence data demonstrated that the endosymbiont potentially generates up to 4,4-dimethyl-cholesta-8,14,24-trienol, a sterol intermediate in cholesterol biosynthesis, from methane. In addition, transcripts for a subset of the enzymes of the biosynthetic pathway to cholesterol downstream from a sterol intermediate derived from methanotroph endosymbionts were detected in our transcriptome data for B. platifrons. These findings suggest that this mussel can de novo synthesize cholesterol from methane in cooperation with the symbionts. By in situ hybridization analyses, we showed that genes associated with cholesterol biosynthesis from both host and endosymbionts were expressed exclusively in the gill epithelial bacteriocytes containing endosymbionts. Thus, cholesterol production is probably localized within these specialized cells of the gill. Considering that the host mussel cannot de novo synthesize cholesterol and depends largely on endosymbionts for nutrition, the capacity of endosymbionts to synthesize sterols may be important in establishing symbiont-host relationships in these chemosynthetic mussels.

In symbiotic systems in which symbionts are transmitted horizontally, hosts must accept symbionts from the environment while defending themselves against invading pathogenic microorganisms. How they distinguish pathogens from symbionts and how the latter evade host immune defences are not clearly understood. Recognition of foreign materials is one of the most critical steps in stimulating immune responses, and pattern recognition receptors (PRRs) play vital roles in this process. In this study, we focused on a group of highly conserved PRRs, peptidoglycan recognition proteins (PGRPs), in the deep-sea mussel, Bathymodiolus septemdierum, which harbours chemosynthetic bacteria in their gill epithelial cells. We isolated B. septemdierum PGRP genes BsPGRP-S and BsPGRP-L, which encode a short- and a long-type PGRP, respectively. The short-type PGRP has a signal peptide and was expressed in the asymbiotic goblet mucous cells in the gill epithelium, whereas the long-type PGRP was predicted to include a transmembrane domain and was expressed in gill bacteriocytes. Based on these findings, we hypothesize that the secreted and transmembrane PGRPs are engaged in host defence against pathogenic bacteria and/or in the regulation of symbiosis via different cellular localizations and mechanisms.

Chemosynthetic symbiosis is one of the successful systems for adapting to a wide range of habitats including extreme environments, and the metabolic capabilities of symbionts enable host organisms to expand their habitat ranges. However, our understanding of the adaptive strategies that enable symbiotic organisms to expand their habitats is still fragmentary. Here, we report that a single-ribotype endosymbiont population in an individual of the host vent mussel, Bathymodiolus septemdierum has heterogeneous genomes with regard to the composition of key metabolic gene clusters for hydrogen oxidation and nitrate reduction. The host individual harbours heterogeneous symbiont subpopulations that either possess or lack the gene clusters encoding hydrogenase or nitrate reductase. The proportions of the different symbiont subpopulations in a host appeared to vary with the environment or with the host's development. Furthermore, the symbiont subpopulations were distributed in patches to form a mosaic pattern in the gill. Genomic heterogeneity in an endosymbiont population may enable differential utilization of diverse substrates and confer metabolic flexibility. Our findings open a new chapter in our understanding of how symbiotic organisms alter their metabolic capabilities and expand their range of habitats.

Symbiont transmission is a key event for understanding the processes underlying symbiotic associations and their evolution. However, our understanding of the mechanisms of symbiont transmission remains still fragmentary. The deep-sea clam Calyptogena okutanii harbours obligate sulfur-oxidizing intracellular symbiotic bacteria in the gill epithelial cells. In this study, we determined the localization of their symbiont associating with the spawned eggs, and the population size of the symbiont transmitted via the eggs. We show that the symbionts are located on the outer surface of the egg plasma membrane at the vegetal pole, and that each egg carries approximately 400 symbiont cells, each of which contains close to 10 genomic copies. The very small population size of the symbiont transmitted via the eggs might narrow the bottleneck and increase genetic drift, while polyploidy and its transient extracellular lifestyle might slow the rate of genome reduction. Additionally, the extracellular localization of the symbiont on the egg surface may increase the chance of symbiont exchange. This new type of extracellular transovarial transmission provides insights into complex interactions between the host and symbiont, development of both host and symbiont, as well as the population dynamics underlying genetic drift and genome evolution in microorganisms.

Despite the enormous ecological and economic importance of coral reefs, the keystone organisms in their establishment, the scleractinian corals, increasingly face a range of anthropogenic challenges including ocean acidification and seawater temperature rise1, 2, 3, 4. To understand better the molecular mechanisms underlying coral biology, here we decoded the approximately 420-megabase genome of Acropora digitifera using next-generation sequencing technology. This genome contains approximately 23,700 gene models. Molecular phylogenetics indicate that the coral and the sea anemone Nematostella vectensis diverged approximately 500 million years ago, considerably earlier than the time over which modern corals are represented in the fossil record (~240 million years ago)5. Despite the long evolutionary history of the endosymbiosis, no evidence was found for horizontal transfer of genes from symbiont to host. However, unlike several other corals, Acropora seems to lack an enzyme essential for cysteine biosynthesis, implying dependency of this coral on its symbionts for this amino acid. Corals inhabit environments where they are frequently exposed to high levels of solar radiation, and analysis of the Acropora genome data indicates that the coral host can independently carry out de novo synthesis of mycosporine-like amino acids, which are potent ultraviolet-protective compounds. In addition, the coral innate immunity repertoire is notably more complex than that of the sea anemone, indicating that some of these genes may have roles in symbiosis or coloniality. A number of genes with putative roles in calcification were identified, and several of these are restricted to corals. The coral genome provides a platform for understanding the molecular basis of symbiosis and responses to environmental changes.

In animals, region specific morphological characters along the anteroposterior axis are controlled by a number of developmental genes, including Hox genes encoding homeodomain transcription factors. Although Hox genes have been regarded to play a key role in the evolution of morphological diversity, as well as in the establishment of the body plan, little is known about the function of Hox genes in invertebrates, except for in insects and nematodes. The present study addresses the role of Hox genes in body patterning during the larval development of the ascidian Ciona intestinalis conducting knockdown experiments of the seven Hox genes expressed during embryogenesis. Experimental results have demonstrated that Ci-Hox12 plays an important role in tail development through the maintenance of expression of Ci-Fgf8/17/18 and Ci-Wnt5 in the tail tip epidermis. Additionally, it has been shown that Ci-Hox10 is involved in the development of GABAergic neurons in the dorsal visceral ganglion. Surprisingly, knockdown of Ci-Hox1, Ci-Hox2, Ci-Hox3, Ci-Hox4 and Ci-Hox5 did not give rise to any consistent morphological defects in the larvae. Furthermore, expression of neuronal marker genes was not affected in larvae injected with MOs against Ci-Hox1, Ci-Hox3 or Ci-Hox5. In conclusion, we suggest that the contribution of Hox genes to the larval development of the ascidian C. intestinalis might be limited, despite the fact that Ci-Hox10 and Ci-Hox12 play important roles in neuronal and tail development.

Hox genes have been regarded to play a central role in anterior-posterior patterning of the animal body. Variations of Hox genes among animal species in the number, order on a chromosome, and the developmental expression pattern may reflect an evolutionary history. Therefore, it is definitely necessary to characterize Hox genes of wide variety of animal species, especially the species occupying key positions in the animal phylogeny. Ascidians, belonging to the subphylum Urochordata, are one of the sister groups of vertebrates in the phylum Chordata. Recent studies have shown that nine Hox genes of Ciona intestinalis, an ascidian species, are present on two chromosomes in the genome. In this review, we discuss the present state of Hox genes in ascidians, focusing on their novel chromosomal organization and expression pattern with unique features and how the novel organization has evolved in relation to the unique body plan of ascidians.

Ascidians, belonging to the subphylum Urochordata, the earliest branch from the lineage to the vertebrates, exhibit a prototypical morphogenesis of chordates in the larval development, although they subsequently metamorphose into adults with a unique body structure. Recent draft genome analysis of the ascidian Ciona intestinalis has identified 9 Hox genes, which, however, have been located on five scaffolds. Similarly, expression patterns of Ciona Hox genes are largely unknown, although some data have been available for a few Hox member genes. Thus, the cluster structure and colinearity of Hox genes are still an enigma in C. intestinalis . To address these issues, we used fluorescence in situ hybridization and whole-mount in situ hybridization techniques and examined the genomic organization and spatiotemporal expression of all Hox as well as extended Hox member genes (Evx and Mox) of C. intestinalis . We found that seven of nine Ciona Hox genes are located on a single chromosome with some ordering exceptions, and the other genes, including Evx and Mox, are located on three other chromosomes. Some Ciona Hox genes, if not all, exhibited spatially coordinated expression within the larval central nervous system and the gut of the juvenile. In light of these observations, we suggest that the cluster organization and colinearity of the Hox genes are under dispersing and disintegrating conditions in C. intestinalis .

The draft genome of the ascidian Ciona intestinalis has been sequenced. Mapping of the genome sequence to the Ciona 14 haploid chromosomes is essential for future studies of the genome-wide control of gene expression in this basal chordate. Here we describe an efficient protocol for fluores-cent in situ hybridization for mapping genes to the Ciona chromosomes. We demonstrate how the locations of two BAC clones can be mapped relative to each other. We also show that this method is efficient for coupling two so-far independent scaffolds into one longer scaffold when two BAC clones represent sequences located at either end of the two scaffolds.