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Based on environmental DNA surveys, it is widely held that phylogenetically diverse protists exist in chemosynthetic ecosystems. However, knowledge regarding the protists associated with the endemic animals inhabiting these environments is still very limited. In the present study, utilizing polymerase chain reaction (PCR) techniques, we detected fragments of the small subunit ribosomal RNA (SSU rRNA) gene and the internal transcribed spacer (ITS) region of the ribosomal RNA genes from a particular protist in the gills of the vesicomyid clam Phreagena okutanii (formerly described as Calyptogena okutanii), a representative animal in chemosynthetic ecosystems. Based on the phylogeny of the SSU rRNA gene, the organism in question belongs to the genus Perkinsus, which is exclusively composed of protistan parasites infecting mollusks. Intriguingly, based on the ITS phylogeny, this protist was not related to any known Perkinsus species and was deeply branched within the radiation of this genus, thus represents an undescribed species. In addition, the protist detected by PCR was localized to the intercellular spaces in the gills of the host clam with fluorescence in situ hybridization. Although the ecological significance of this novel deep-sea perkinsid remains unclear, our present findings may provide important insights into the diversity of the genus Perkinsus.

The origin of eukaryotes remains unclear1–4. Current data suggest that eukaryotes may have emerged from an archaeal lineage known as ‘Asgard’ archaea5,6. Despite the eukaryote-like genomic features that are found in these archaea, the evolutionary transition from archaea to eukaryotes remains unclear, owing to the lack of cultured representatives and corresponding physiological insights. Here we report the decade-long isolation of an Asgard archaeon related to Lokiarchaeota from deep marine sediment. The archaeon—‘Candidatus Prometheoarchaeum syntrophicum’ strain MK-D1—is an anaerobic, extremely slow-growing, small coccus (around 550 nm in diameter) that degrades amino acids through syntrophy. Although eukaryote-like intracellular complexes have been proposed for Asgard archaea6, the isolate has no visible organelle-like structure. Instead, Ca. P. syntrophicum is morphologically complex and has unique protrusions that are long and often branching. On the basis of the available data obtained from cultivation and genomics, and reasoned interpretations of the existing literature, we propose a hypothetical model for eukaryogenesis, termed the entangle–engulf–endogenize (also known as E3) model., Isolation and characterization of an archaeon that is most closely related to eukaryotes reveals insights into how eukaryotes may have evolved from prokaryotes.

The Methyloprofundus clade is represented by uncultivated methanotrophic bacterial endosymbionts of deep-sea bathymodiolin mussels, but only a single free-living species has been cultivated to date. This study reveals the existence of free-living Methyloprofundus variants in the Iheya North deep-sea hydrothermal field in the mid-Okinawa Trough. A clade-targeted amplicon analysis of the particulate methane monooxygenase gene (pmoA) detected 647 amplicon sequence variants (ASVs) of the Methyloprofundus clade in microbial communities newly formed in in situ colonization systems. Such systems were deployed at colonies of bathymodiolin mussels and a galatheoid crab. These ASVs were classified into 161 species-like groups. The proportion of the species-like groups representing endosymbionts of mussels was unexpectedly low. A methanotrophic bacterium designated as INp10, a likely dominant species in the Methyloprofundus population in this field, was enriched in biofilm formed in a methane-fed cultivation system operated at 10°C. Genomic characterization with the gene transcription dataset of INp10 from biofilm suggested traits advantageous to niche competition in environments, such as mobility, chemotaxis, biofilm formation, offensive and defensive systems, and hypoxia tolerance. The notable metabolic traits INp10 shares with some Methyloprofundus members are the use of lanthanide-dependent XoxF as the sole methanol dehydrogenase due to the absence of the canonical MxaFI, the glycolytic pathway using fructose-6-phosphate aldolase instead of fructose-1,6-bisphosphate aldolase, and the potential to perform partial denitrification from nitrate under oxygen-limited conditions. These findings help better understand ecological strategies of this possibly widespread marine-specific methanotrophic clade. Importance The Iheya North deep-sea hydrothermal field in the mid-Okinawa Trough is characterized by abundant methane derived from organic-rich sediments and diverse chemosynthetic animal species, including those harboring methanotrophic bacterial symbionts such as bathymodiolin mussels Bathymodiolus japonicus and "Bathymodiolus" platifrons and a galatheoid crab Shinkaia crosnieri. Symbiotic methanotrophs have attracted significant attention, yet free-living methanotrophs in this environment have not been studied in detail. We focused on the free-living Methyloprofundus spp. that thrive in this hydrothermal field and identified an unexpectedly large number of species-like groups in this clade. Moreover, we enriched and characterized a methanotroph whose genome sequence indicated it corresponds to a new species in the genus Methyloprofundus. This species might be a dominant member of the indigenous Methyloprofundus population. New information on free-living Methyloprofundus spp. suggests that the hydrothermal field is a promising locale to investigate the adaptive capacity and associated genetic diversity of Methyloprofundus.

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Marine ecosystems are continuously subjected to anthropogenic environmental pollution. Understanding the spread of pollution and the potential risks it poses to deep-sea ecosystems is important for developing better conservation measures. Here, we identified non-negligible levels of persistent organic pollutants in deep-sea chemosynthetic bivalves with limited or no filter feeding. The bivalves were collected from two sites: one located near a highly populated region and the other located relatively far from human activity. Analyses of samples collected nearly every decade in a period of 30 years suggested that environmental policy restrictions might be effective in reducing chemical pollution. However, the detection of contamination in deep-sea chemosynthetic animals suggests that the pollution could be spreading globally to chemosynthetic organisms with limited or no feeding. To protect these highly endemic and vulnerable deep-sea chemosynthetic ecosystems, our findings indicate that further research on chemical contamination and its effects on these ecosystems is required.

Deep-sea Bathymodiolus mussels are generally thought to harbour chemosynthetic symbiotic bacteria in gill epithelial cells called bacteriocytes. However, previously observed openings at the apical surface of bacteriocytes have not been conclusively explained and investigated as to whether the Bathymodiolus symbiosis is intracellular or extracellular. In this study, we show that almost all the membranous chambers encompassing symbionts in a single bacteriocyte of Bathymodiolus septemdierum are interconnected and have pathways connecting to the external environment. Furthermore, the symbiont population colonising a single bacteriocyte is mostly clonal. This study hypothesises on a novel model of cellular localization at the interface between extra- and intracellular symbiosis, and the cellular-level process of symbiont acquisition in Bathymodiolus mussels.

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.

The deep-sea is considered to be an ultimate sink for marine plastic debris. The abyssal (3500-6500 m) covers the bulk of the deep ocean floor yet little is known about the extent of plastic debris on the abyssal seafloor. We undertook a quantitative assessment of debris presents on the abyssal seafloor (5700-5800 m depth) beneath the Kuroshio Extension current system in the Northwest Pacific, which is one of the major transit points for massive amounts of debris sourced from Asia that are entering the North Pacific Ocean. The dominant type of debris was single-use plastics-mainly bags and food packaging. The density of plastic debris (average 4561 items km−2) in the abyssal zone was the highest recorded for an abyssal plain, suggesting that the deep-sea basin of the Northwest Pacific is a significant reservoir of plastic debris.

Echinoderms are an exceptional group of bilaterians that develop pentameral adult symmetry from a bilaterally symmetric larva. However, the genetic basis in evolution and development of this unique transformation remains to be clarified. Here we report newly sequenced genomes, developmental transcriptomes, and proteomes of diverse echinoderms including the green sea urchin (L. variegatus), a sea cucumber (A. japonicus), and with particular emphasis on a sister group of the earliest-diverged echinoderms, the feather star (A. japonica). We learned that the last common ancestor of echinoderms retained a well-organized Hox cluster reminiscent of the hemichordate, and had gene sets involved in endoskeleton development. Further, unlike in other animal groups, the most conserved developmental stages were not at the body plan establishing phase, and genes normally involved in bilaterality appear to function in pentameric axis development. These results enhance our understanding of the divergence of protostomes and deuterostomes almost 500 Mya., Li et al. investigate the evolution and genetic basis of the adult pentameral body plan in echinoderms using genomic, transcriptomic, and proteomic data. They determine that the last common ancestor of echinoderms contained an organized Hox cluster and endoskeleton genes, and suggest that cooption of bilateral development genes was involved in evolution of the pentameric body plan.

To elucidate the localization of oomycetes (water molds) and bacteria in a single egg of chum salmon, Oncorhynchus keta, eggs infected with oomycetes were examined by bright-field microscopy, scanning electron microscopy (SEM), and fluorescent in situ hybridization (FISH) using oomycete universal and bacterial species-specific probes. Furthermore, DNA barcoding for oomycetes and bacteria was performed on the chorion and cytoplasm of the eggs using genomic DNA extracted from the sections of eggs used for FISH analyses. Using bright-field microscopy and SEM, it was shown that oomycete hyphae penetrated the chorion and invaded the cytoplasm of the eggs. Based on DNA barcoding, two oomycetes (Pythium monospermum and Pythium sp. SE-OP1), and three bacterial species (Flavobacterium plurextorum, Flavobacterium sp. SE-BF1 and Undibacterium pigrum) were predominantly detected in the infected eggs. FISH analyses indicated that localization of the oomycetes was not noticeable different between the chorion and cytoplasm of the eggs. In contrast, FISH analyses using bacterial species-specific probes demonstrated clear differences in their localization. Flavobacterium sp. SE-BF1 and U. pigrum were predominantly found inside the chorion and around the oomycete hyphae invading the cytoplasm, respectively. These results are the first to indicate the possibility that U. pigrum has a specific relationship with oomycetes and coordinately invades eggs with oomycetes. However, it seems that Flavobacterium sp. SE-BF1 invades by a mechanism different from that of U. pigrum. Although this study is preliminary and the interaction between oomycetes and U. pigrum associated with chum salmon egg infection needs to be elucidated in detail, this finding may provide new insights into the infection mechanism of bacteria in salmon eggs, which is a longstanding problem in salmon hatcheries.

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.

Some deep-sea chemosynthetic invertebrates and their symbiotic bacteria can use molecular hydrogen (H2) as their energy source. However, how much the chemosynthetic holobiont (endosymbiont-host association) physiologically depends on H2 oxidation has not yet been determined. Here, we demonstrate that the Campylobacterota endosymbionts of the gastropod Alviniconcha marisindica in the Kairei and Edmond fields (kAlv and eAlv populations, respectively) of the Indian Ocean, utilize H2 in response to their physical and environmental H2 conditions, although the 16S rRNA gene sequence of both the endosymbionts shared 99.6% identity. A thermodynamic calculation using in situ H2 and hydrogen sulfide (H2S) concentrations indicated that chemosynthetic symbiosis could be supported by metabolic energy via H2 oxidation, particularly for the kAlv holobiont. Metabolic activity measurements showed that both the living individuals and the gill tissues consumed H2 and H2S at similar levels. Moreover, a combination of fluorescence in situ hybridization, quantitative transcript analyses, and enzymatic activity measurements showed that the kAlv endosymbiont expressed the genes and enzymes for both H2- and sulfur-oxidations. These results suggest that both H2 and H2S could serve as the primary energy sources for the kAlv holobiont. The eAlv holobiont had the ability to utilize H2, but the gene expression and enzyme activity for hydrogenases were much lower than for sulfur-oxidation enzymes. These results suggest that the energy acquisitions of A. marisindica holobionts are dependent on H2- and sulfur-oxidation in the H2-enriched Kairei field and that the mechanism of dual metabolism is controlled by the in situ H2 concentration.

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.

Microscopy techniques have been widely applied to observe cellular ultrastructure. Most of these techniques, such as transmission electron microscopy, produce high‐resolution images, but they may require extensive preparation, hampering their application for in vivo examination. Other approaches, such as fluorescent and fluorogenic probes, can be applied not only to fixed specimens but also to living cells when the probes are nontoxic. Fluorescence‐based methods, which are generally relatively easy to use, allow visual and (semi)quantitative studies of the ultrastructural organization and processes of the cell under natural as well as manipulated conditions. To date, there are relatively few published studies on the nearly ubiquitous marine protistan group Foraminifera that have used fluorescent and fluorogenic probes, despite their huge potential. The aim of the present contribution is to document the feasible application of a wide array of these probes to foraminiferal biology. More specifically, we applied fluorescence‐based probes to study esterase activity, cell viability, calcium signaling, pH variation, reactive oxygen species, neutral and polar lipids, lipid droplets, cytoskeleton structures, Golgi complex, acidic vesicles, nuclei, and mitochondria in selected foraminiferal species.

A Correction to this paper has been published: https://doi.org/10.1038/s42003-021-02005-4

Survival of deep-sea Calyptogena clams depends on organic carbon produced by symbiotic, sulfur-oxidizing, autotrophic bacteria present in the epithelial cells of the gill. To understand the mechanism underlying this symbiosis, the development of a long-term cultivation system is essential. We cultivated specimens of Calyptogena okutanii in an artificial chemosynthetic aquarium with a hydrogen sulfide (H2S) supply system provided by the sulfate reduction of dog food buried in the sediment. We studied morphological and histochemical changes in the clams' gills by immunohistochemical and energy-dispersive X-ray analyses. The freshly collected clams contained a high amount of elemental sulfur in the gill epithelial cells, as well as densely packed symbiotic bacteria. Neither elemental sulfur nor symbiotic bacteria was detected in any other organs except the ovaries, where symbiotic bacteria, but not sulfur, was detected. The longest survival of an individual clam in this aquarium was 151 days. In the 3 clams dissected on Days 57 and 91 of the experiment, no elemental sulfur was detected in the gills. The symbiotic bacteria content had significantly decreased by Day 57, and was absent by Day 91. For comparison, we also studied the deep-sea mussel Bathymodiolus septemdierum, which harbors a phylogenetically close, sulfur-oxidizing, symbiotic bacterium with similar sulfur oxidation pathways. Sulfur particles were not detected, even in the gills of the freshly collected mussels. We discuss the importance of the proportion of available H2S and oxygen to the bivalves for elemental sulfur accumulation. Storage of nontoxic elemental sulfur, an energy source, seems to be an adaptive strategy of C. okutanii.

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.

Hox cluster genes play crucial roles in development of the metazoan antero-posterior axis. Functions of Hox genes in patterning the central nervous system and limb buds are well known. They are also expressed in chordate endodermal tissues, where their roles in endodermal development are still poorly understood. In the invertebrate chordate, Ciona intestinalis, endodermal tissues are in a premature state during the larval stage, and they differentiate into the digestive tract during metamorphosis. In this study, we showed that disruption of a Hox gene, Ci-Hox10, prevented intestinal formation. Ci-Hox10-knock-down larvae displayed defective migration of endodermal strand cells. Formation of a protrusion, which is important for cell migration, was disrupted in these cells. The collagen type IX gene is a downstream target of Ci-Hox10, and is negatively regulated by Ci-Hox10 and a matrix metalloproteinase ortholog, prior to endodermal cell migration. Inhibition of this regulation prevented cellular migration. These results suggest that Ci-Hox10 regulates endodermal strand cell migration by forming a protrusion and by reconstructing the extracellular matrix.

Vesicomyid clams in deep-sea chemosynthetic ecosystems harbor sulfur-oxidizing bacteria in their gill epithelial cells. These symbionts, which are vertically transmitted, are species-specific and thought to have cospeciated with their hosts. However, recent studies indicate incongruent phylogenies between some vesicomyid clams and their symbionts, suggesting that symbionts are horizontally transmitted. To more precisely understand the evolution of vesicomyid clams and their symbionts, we compared the evolution of vesicomyid clams and their symbionts through phylogenetic analyses using multi-gene data sets. Many clades in the phylogenetic trees of 13 host species (Abyssogena mariana, Ab. phaseoliformis, Akebiconcha kawamurai, Calyptogena fausta, C. laubieri, C. magnifica, C. nautilei, C. pacifica, Isorropodon fossajaponicum, Phreagena kilmeri, Ph. okutanii, Ph. soyoae, and Pliocardia stearnsii) and their symbionts were well resolved. Six of the 13 host-symbiont pairs (C. fausta, C. magnifica, C. pacifica, Ph. kilmeri, Ph. okutanii, and Ph. soyoae, and their respective symbionts) showed topological congruence. However, the remaining seven pairs (Ak. kawamurai, Ab mariana, Ab. phaseoliformis, C. laubieri, C. nautilei, I. fossajaponicum, and Pl. stearnsii and their corresponding symbionts) showed incongruent topologies, which were supported by the approximately unbiased and Bayes factor tests. Coevolution analyses indicated that six pairs cospeciated, whereas host switching events occurred in the remaining seven pairs. Markedly, multiple host switching events may have occurred in the lineages from the common ancestral symbiont of C. pacifica and C. fausta. Our phylogenetic and coevolution analyses provide additional evidence for host switching during the evolution of vesicomyids.

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.

Comparative studies on expression patterns of developmental genes along the anterior–posterior axis of the embryonic central nervous system (CNS) between vertebrates and ascidians led to the notion of “tripartite organization,” a common ground plan of the CNS, consisting of the anterior, central and posterior regions expressing Otx, Pax2/5/8 and Hox genes, respectively. In ascidians, however, descriptions and interpretations about expression of the developmental genes regarded as region specific have become not necessarily consistent. To address this issue, we examined detailed expression of key developmental genes for the ascidian CNS, including Otx, Pax2/5/8a, En, Fgf8/17/18, Dmbx, Lhx3 and Hox genes, in the CNS around the junction of the trunk and tail of three different tailbud-stage embryos of Ciona intestinalis, employing double-fluorescence in situ hybridization, followed by staining with DAPI to precisely locate expressing cells for each gene. Based on these observations, we have constructed detailed gene expression maps of the region at the tailbud stages. Our analysis shows that expression of several genes regarded as markers for specific domains in the ascidian CNS changes dynamically within a relatively short period. This motivates us to revisit to the tripartite ground plan and the origin of the midbrain–hindbrain boundary (MHB) region.

Deep-sea vesicomyid clams, including the genus Phreagena (formerly Calyptogena), harbor thioautotrophic bacterial symbionts in the host symbiosome, which consists of cytoplasmic vacuoles in gill epithelial cells called bacteriocytes. The symbiont requires inorganic carbon (Ci), such as CO2, HCO3(-), and CO3(2-), to synthesize organic compounds, which are utilized by the host clam. The dominant Ci in seawater is HCO3(-), which is impermeable to cell membranes. Within the bacteriocyte, cytoplasmic carbonic anhydrase (CA) from the host, which catalyzes the inter-conversion between CO2 and HCO3(-), has been shown to be abundant and is thought to supply intracellular CO2 to symbionts in the symbiosome. However, the mechanism of Ci uptake by the host gill from seawater is poorly understood. To elucidate the influx pathway of Ci into the bacteriocyte, we isolated the genes related to Ci uptake via the pyrosequencing of cDNA from the gill of Phreagena okutanii, and investigated their expression patterns. Using phylogenetic and amino acid sequence analyses, three solute carrier family 4 (SLC4) bicarbonate transporters (slc4co1, slc4co2, and slc4co4) and two membrane-associated CAs (mcaco1 and mcaco2) were identified as candidate genes for Ci uptake. In an in situ hybridization analysis of gill sections, the expression of mcaco1 and mcaco2 was detected in the bacteriocytes and asymbiotic non-ciliated cells, respectively, and the expression of slc4co1 and slc4co2 was detected in the asymbiotic cells, including the intermediate cells of the inner area and the non-ciliated cells of the external area. Although subcellular localizations of the products of these genes have not been fully elucidated, they may play an important role in the uptake of Ci into the bacteriocytes. These findings will improve our understanding of the Ci transport system in the symbiotic relationships of chemosynthetic bivalves.

Background ParaHox and Hox genes are thought to have evolved from a common ancestral ProtoHox cluster or from tandem duplication prior to the divergence of cnidarians and bilaterians. Similar to Hox clusters, chordate ParaHox genes including Gsx, Xlox, and Cdx, are clustered and their expression exhibits temporal and spatial colinearity. In non-chordate animals, however, studies on the genomic organization of ParaHox genes are limited to only a few animal taxa. Hemichordates, such as the Enteropneust acorn worms, have been used to gain insights into the origins of chordate characters. In this study, we investigated the genomic organization and expression of ParaHox genes in the indirect developing hemichordate acorn worm Ptychodera flava. Results We found that P. flava contains an intact ParaHox cluster with a similar arrangement to that of chordates. The temporal expression order of the P. flava ParaHox genes is the same as that of the chordate ParaHox genes. During embryogenesis, the spatial expression pattern of PfCdx in the posterior endoderm represents a conserved feature similar to the expression of its orthologs in other animals. On the other hand, PfXlox and PfGsx show a novel expression pattern in the blastopore. Nevertheless, during metamorphosis, PfXlox and PfCdx are expressed in the endoderm in a spatially staggered pattern similar to the situation in chordates. Conclusions Our study shows that P. flava ParaHox genes, despite forming an intact cluster, exhibit temporal colinearity but lose spatial colinearity during embryogenesis. During metamorphosis, partial spatial colinearity is retained in the transforming larva. These results strongly suggest that intact ParaHox gene clustering was retained in the deuterostome ancestor and is correlated with temporal colinearity.

SummaryGenomic comparisons of chordates, hemichordates, and echinoderms can inform hypotheses for the evolution of these strikingly different phyla from the last common deuterostome ancestor [1–5]. Because hox genes play pivotal developmental roles in bilaterian animals [6–8], we analyzed the Hox complexes of two hemichordate genomes. We find that Saccoglossus kowalevskii and Ptychodera flava both possess 12-gene clusters, with mir10 between hox4 and hox5, in 550 kb and 452 kb intervals, respectively. Genes hox1–hox9/10 of the clusters are in the same genomic order and transcriptional orientation as their orthologs in chordates, with hox1 at the 3′ end of the cluster. At the 5′ end, each cluster contains three posterior genes specific to Ambulacraria (the hemichordate-echinoderm clade), two forming an inverted terminal pair. In contrast, the echinoderm Strongylocentrotus purpuratus contains a 588 kb cluster [9] of 11 orthologs of the hemichordate genes, ordered differently, plausibly reflecting rearrangements of an ancestral hemichordate-like ambulacrarian cluster. Hox clusters of vertebrates and the basal chordate amphioxus [10] have similar organization to the hemichordate cluster, but with different posterior genes. These results provide genomic evidence for a well-ordered complex in the deuterostome ancestor for the hox1–hox9/10 region, with the number and kind of posterior genes still to be elucidated.

The study of the pearl oyster Pinctada fucata is key to increasing our understanding of the molecular mechanisms involved in pearl biosynthesis and biology of bivalve molluscs. We sequenced ~1150-Mb genome at ~40-fold coverage using the Roche 454 GS-FLX and Illumina GAIIx sequencers. The sequences were assembled into contigs with N50 = 1.6 kb (total contig assembly reached to 1024 Mb) and scaffolds with N50 = 14.5 kb. The pearl oyster genome is AT-rich, with a GC content of 34%. DNA transposons, retrotransposons, and tandem repeat elements occupied 0.4, 1.5, and 7.9% of the genome, respectively (a total of 9.8%). Version 1.0 of the P. fucata draft genome contains 23 257 complete gene models, 70% of which are supported by the corresponding expressed sequence tags. The genes include those reported to have an association with bio-mineralization. Genes encoding transcription factors and signal transduction molecules are present in numbers comparable with genomes of other metazoans. Genome-wide molecular phylogeny suggests that the lophotrochozoan represents a distinct clade from ecdysozoans. Our draft genome of the pearl oyster thus provides a platform for the identification of selection markers and genes for calcification, knowledge of which will be important in the pearl industry.

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.

The Hox genes and its evolutionary sister, the ParaHox genes, are widely distributed among animals. Although it has been expected that hemichordates and echinoderms have a single set of Hox genes and most likely a single set of ParaHox genes, it is not known whether the ortholog of Hox8 is absent in hemichordates, and in turn, consensus view about Hox/ParaHox gene complements in hemichordates has not been established. In this study, we isolated either complete or nearly complete coding sequences of 12 Hox genes, including the ortholog of the Hox8 that has not been reported in the previous studies, and three ParaHox genes from the recently discovered indirect-developing acorn worm, Balanoglossus simodensis. Our data suggest that the ancestral hemichordate had intact complements of ambulacrarian prototypical Hox and ParaHox genes, consisting of 12 and three members, respectively.

Cephalochordates, urochordates, and vertebrates evolved from a common ancestor over 520 million years ago. To improve our understanding of chordate evolution and the origin of vertebrates, we intensively searched for particular genes, gene families, and conserved noncoding elements in the sequenced genome of the cephalochordate Branchiostoma floridae, commonly called amphioxus or lancelets. Special attention was given to homeobox genes, opsin genes, genes involved in neural crest development, nuclear receptor genes, genes encoding components of the endocrine and immune systems, and conserved cis-regulatory enhancers. The amphioxus genome contains a basic set of chordate genes involved in development and cell signaling, including a fifteenth Hox gene. This set includes many genes that were co-opted in vertebrates for new roles in neural crest development and adaptive immunity. However, where amphioxus has a single gene, vertebrates often have two, three, or four paralogs derived from two whole-genome duplication events. In addition, several transcriptional enhancers are conserved between amphioxus and vertebrates—a very wide phylogenetic distance. In contrast, urochordate genomes have lost many genes, including a diversity of homeobox families and genes involved in steroid hormone function. The amphioxus genome also exhibits derived features, including duplications of opsins and genes proposed to function in innate immunity and endocrine systems. Our results indicate that the amphioxus genome is elemental to an understanding of the biology and evolution of nonchordate deuterostomes, invertebrate chordates, and vertebrates.

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.