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Abstract Coral polyps are composed of four tissues; however, their characteristics are largely unexplored. Here we report biological characteristics of tentacles (Te), mesenterial filaments (Me), body wall (Bo), and mouth with pharynx (MP), using comparative genomic, morpho-histological, and transcriptomic analyses of the large-polyp coral, Fimbriaphyllia ancora. A draft F. ancora genome assembly of 434 Mbp was created. Morpho-histological and transcriptomic characterization of the four tissues showed that they have distinct differences in structure, primary cellular composition, and transcriptional profiles. Tissue-specific, highly expressed genes (HEGs) of Te are related to biological defense, predation, and coral-algal symbiosis. Me expresses multiple digestive enzymes, whereas Bo expresses innate immunity and biomineralization-related molecules. Many receptors for neuropeptides and neurotransmitters are expressed in MP. This dataset and new insights into tissue functions will facilitate a deeper understanding of symbiotic biology, immunology, biomineralization, digestive biology, and neurobiology in corals.

Abstract Background The Tunicata or Urochordata is the only animal group with the ability to synthesize cellulose directly and cellulose is a component of the tunic that covers the entire tunicate body. The genome of Ciona intestinalis type A contains a cellulose synthase gene, CesA, that it acquired via an ancient, horizontal gene transfer. CesA is expressed in embryonic epidermal cells and functions in cellulose production. Ciona CesA is composed of both a glycosyltransferase domain, GT2, and a glycosyl hydrolase domain, GH6, which shows a mutation at a key position and seems functionless. Interestingly, the Ciona genome contains a glycosyl hydrolase gene, GH6-1, in which the GH6 domain seems intact. This suggests expression and possible functions of GH6-1 during Ciona embryogenesis. Is GH6-1 expressed during embryogenesis? If so, in what tissues is the gene expressed? Does GH6-1 serve a function? If so, what is it? Answers to these questions may advance our understanding of evolution of this unique animal group. Results Quantitative reverse transcription PCR and in situ hybridization revealed that GH6-1 is expressed in epidermis of tailbud embryos and in early swimming larvae, a pattern similar to that of CesA. Expression is downregulated at later stages and becomes undetectable in metamorphosed juveniles. The GH6-1 expression level is higher in the anterior-trunk region and caudal-tip regions of late embryos. Single-cell RNA sequencing analysis of the late tailbud stage showed that cells of three clusters with epidermal identity express GH6-1, and that some of them co-express CesA. TALEN-mediated genome editing was used to generate GH6-1 knockout Ciona larvae. Around half of TALEN-electroporated larvae showed abnormal development of adhesive papillae and altered distribution of surface cellulose. In addition, three-fourths of TALEN-electroporated animals failed to complete larval metamorphosis. Conclusions This study showed that tunicate GH6-1, a gene that originated by horizontal gene transfer of a prokaryote gene, is recruited into the ascidian genome, and that it is expressed and functions in epidermal cells of ascidian embryos. Although further research is required, this observation demonstrates that both CesA and GH6-1 are involved in tunicate cellulose metabolism, impacting tunicate morphology and ecology.

Mesophotic coral ecosystems (MCEs) are light-dependent tropical or subtropical communities occurring at depths of 30–150 m. Broader surveys of MCEs are needed to better understand stony corals, the keystone species of coral-reef ecosystems. While MCEs have been studied by professional SCUBA divers and with deep-sea robots, comprehensive surveys of MCEs are required. An eDNA metabarcoding method has recently been used to survey scleractinian corals in shallow reefs. We tested whether MCEs might be more comprehensively surveyed by collecting seawater samples using an underwater mini-remote operated vehicle (mini-ROV). Seawater was collected 1–2 m above reef tops at depths of 20–80 m at 24 sites in six locations around the Zamami Islands (Okinawa, Japan). Water samples were then subjected to coral-specific eDNA amplification. Metabarcoding analyses of amplicons showed that except for one site, coral-specific eDNA from approximately 0.5 l seawater samples was sufficient to identify genera. The proportion of Acropora eDNA was higher at shallow reefs and upper ridges of slopes, while the proportion of Porites increased at mesophotic sites. Although further technical improvements are required, this study suggests that it may be possible to monitor mesophotic corals to the generic level using eDNA collected using mini-ROVs.

Abstract The Ogasawara Islands are a highly isolated oceanic archipelago in the Pacific Ocean that possess unique faunal and floral biodiversity with a high level of endemism. As historically more focus has been put on the terrestrial realm in examining diversification and evolutionary processes on oceanic islands, publicly accessible and spatially resolved data of marine reef ecosystems remain scarce. To address this issue, we conducted the first environmental DNA (eDNA) metabarcoding surveys of the actinopterygian (ray‐finned) and elasmobranch fishes and of Scleractinia coral assemblages in the waters of the Ogasawara Islands. We detected a total of 124 unique taxa of fish and 38 unique taxa of scleractinian corals. Overall, our eDNA results confirmed that the Ogasawara Islands host a rich variety of coral and fish fauna and underline the strength of eDNA surveys in rapidly obtaining targeted multi‐taxa data using seawater samples, requiring comparatively little effort and a lack of requirement for in situ taxonomic expertise. We anticipate that continued biomonitoring using eDNA with high sampling effort will add to and complement the body of knowledge regarding species distributions, invasive species, and biodiversity hotspots within oceanic archipelagos.

Background The crown-of-thorns starfish (COTS; Acanthaster species) is a slow-moving corallivore protected by an extensive array of long, sharp toxic spines. Envenomation can result in nausea, numbness, vomiting, joint aches and sometimes paralysis. Small molecule saponins and the plancitoxin proteins have been implicated in COTS toxicity. Methods Brine shrimp lethality assays were used to confirm the secretion of spine toxin biomolecules. Histological analysis, followed by spine-derived proteomics helped to explain the source and identity of proteins, while quantitative RNA-sequencing and phylogeny confirmed target gene expression and relative conservation, respectively. Results We demonstrate the lethality of COTS spine secreted biomolecules on brine shrimp, including significant toxicity using aboral spine semi-purifications of >10 kDa (p > 0.05, 9.82 µg/ml), supporting the presence of secreted proteins as toxins. Ultrastructure observations of the COTS aboral spine showed the presence of pores that could facilitate the distribution of secreted proteins. Subsequent purification and mass spectrometry analysis of spine-derived proteins identified numerous secretory proteins, including plancitoxins, as well as those with relatively high gene expression in spines, including phospholipase A2, protease inhibitor 16-like protein, ependymin-related proteins and those uncharacterized. Some secretory proteins (e.g., vitellogenin and deleted in malignant brain tumor protein 1) were not highly expressed in spine tissue, yet the spine may serve as a storage or release site. This study contributes to our understanding of the COTS through functional, ultrastructural and proteomic analysis of aboral spines.

Only a few azoxy natural products have been identified despite their intriguing biological activities. Azodyrecins D–G, four new analogs of aliphatic azoxides, were identified from two Streptomyces species by a reactivity-based screening that targets azoxy bonds. A biological activity evaluation demonstrated that the double bond in the alkyl side chain is important for the cytotoxicity of azodyrecins. An in vitro assay elucidated the tailoring step of azodyrecin biosynthesis, which is mediated by the S-adenosylmethionine (SAM)-dependent methyltransferase Ady1. This study paves the way for the targeted isolation of aliphatic azoxy natural products through a genome-mining approach and further investigations of their biosynthetic mechanisms.

ABSTRACT Aims/Introduction Diabetic kidney disease (DKD) exacerbates dyslipidemia and increases the incidence of atherosclerotic cardiovascular disease. DKD is a concept that includes typical diabetic nephropathy and an atypical phenotype without proteinuria. We investigated dyslipidemia in different DKD phenotypes that have not been fully studied. Materials and Methods Fasting plasma was obtained from 1,073 diabetes patients enrolled in the regional diabetes cohort (ViNA cohort). Non‐proteinuric and proteinuric DKD were defined as an estimated glomerular filtration rate 300 mg/g. Novel lipid risk factors, low‐density lipoprotein (LDL) triglyceride (TG) and small dense LDL cholesterol were measured using our established homologous assay. Results The proportion of atherosclerotic cardiovascular disease patients was higher in non‐proteinuric DKD and even higher in proteinuric DKD than in non‐DKD. Increased estimated glomerular filtration rate grade and albuminuric stage were independently correlated with higher TG, TG‐rich lipoprotein cholesterol and apolipoprotein CIII. Therefore, proteinuric DKD had the highest of these levels. Small dense LDL cholesterol and LDL‐TG were higher in the proteinuria without renal dysfunction group in the lipid‐lowering drug‐free subset. Lipoprotein(a) was higher in DKD regardless of proteinuria. Conclusions Proteinuria was associated with an atherogenic subspecies of LDL, whereas renal dysfunction was associated with increased lipoprotein(a). Proteinuria and renal dysfunction independently exacerbated TG‐rich lipoprotein‐related dyslipidemia. This is in good agreement with the results of large‐scale clinical studies in which proteinuria and renal dysfunction synergistically increased the risk of atherosclerotic cardiovascular disease in populations with diabetes.

Summary: Various parasitic flatworms infect vertebrates for sexual reproduction, often causing devastating diseases in their hosts. Consequently, flatworms are of great socioeconomic and biomedical importance. Although the cessation of parasitic flatworm sexual reproduction is a major target of anti-parasitic drug design, little is known regarding bioactive compounds controlling flatworm sexual maturation. Using the planarian Dugesia ryukyuensis, we observed that sex-inducing substances found in planarians are also widespread in parasitic flatworms, such as monogeneans and flukes (but not in tapeworms). Reverse-phase HPLC analysis revealed the sex-inducing substance(s) eluting around the tryptophan retention time in the fluke Calicophoron calicophorum, consistent with previous studies on the planarian Bipalium nobile, suggesting that the substance(s) is likely conserved among flatworms. Moreover, six of the 18 ovary-inducing substances identified via transcriptome and metabolome analyses are involved in purine metabolism. Our findings provide a basis for understanding and modifying the life cycles of various parasitic flatworms.

Recurring outbreaks of crown-of-thorns starfish (COTS) severely damage healthy corals, especially in the Western Pacific Ocean. To obtain a better understanding of population genetics of COTS and historical colonization across the Pacific Ocean, complete mitochondrial genomes were sequenced from 243 individuals collected in 11 reef regions. Our results indicate that Pacific COTS (Acanthaster cf. solaris) comprise two major clades, an East-Central Pacific (ECP) clade and a Pan-Pacific (PP) clade, separation of which was supported by high bootstrap value. The ECP clade consists of COTS from French Polynesia, Fiji, Vanuatu and the Great Barrier Reef (GBR). The Hawaii population is unique within this clade, while California COTS are included in EPC clade. On the other hand, the PP clade comprises multiple lineages that contain COTS from Vietnam, the Philippines, Japan, Papua New Guinea, Micronesia, the Marshall Islands, GBR, Vanuatu, Fiji and French Polynesia. For example, a lineage of the PP clade, which has the largest geographic distribution, includes COTS from all of these locations. These results suggest two alternative histories of current geographic distributions of COTS in the Pacific Ocean, an ECP clade ancestry or Western Pacific clade ancestry. Although further questions remain to be explored, this discovery provides an evolutionary context for the interpretation of COTS population structure which will aid future coral reef research in the Pacific Ocean, and ultimately improve reef management of COTS.

Abstract Molluscan shells are organo-mineral composites, in which the dominant calcium carbonate is intimately associated with an organic matrix comprised mainly of proteins and polysaccharides. However, whether the various shell matrix proteins (SMPs) date to the origin of hard skeletons in the Cambrian, or whether they represent later deployment through adaptive evolution, is still debated. In order to address this issue and to better understand the origins and evolution of biomineralization, phylogenetic analyses have been performed on the three SMP families, Von Willebrand factor type A (VWA) and chitin-binding domain-containing protein (VWA-CB dcp), chitobiase, and carbonic anhydrase (CA), which exist in both larval and adult shell proteomes in the bivalves, Crassostrea gigas and Pinctada fucata. In VWA-CB dcp and chitobiase, paralogs for larval and adult SMPs evolved before the divergence of these species. CA-SMPs have been taken as evidence for ancient origins of SMPs by their presumed indispensable function in biomineralization and ubiquitous distribution in molluscs. However, our results indicate gene duplications that gave rise to separate deployments as larval and adult CA-SMPs occurred independently in each lineage after their divergence, which is considerably more recent than hitherto assumed, supporting the “recent heritage and fast evolution” scenario for SMP evolution.

Various Hydra species have been employed as model organisms since the 18th century. Introduction of transgenic and knock-down technologies made them ideal experimental systems for studying cellular and molecular mechanisms involved in regeneration, body-axis formation, senescence, symbiosis, and holobiosis. In order to provide an important reference for genetic studies, the Hydra magnipapillata genome (species name has been changed to H. vulgaris) was sequenced a decade ago (Chapman et al., 2010) and the updated genome assembly, Hydra 2.0, was made available by the National Human Genome Research Institute in 2017. While H. vulgaris belongs to the non-symbiotic brown hydra lineage, the green hydra, Hydra viridissima, harbors algal symbionts and belongs to an early diverging clade that separated from the common ancestor of brown and green hydra lineages at least 100 million years ago (Schwentner and Bosch 2015; Khalturin et al., 2019). While interspecific interactions between H. viridissima and endosymbiotic unicellular green algae of the genus Chlorella have been a subject of interest for decades, genomic information about green hydras was nonexistent. Here we report a draft 280-Mbp genome assembly for Hydra viridissima strain A99, with a scaffold N50 of 1.1 Mbp. The H. viridissima genome contains an estimated 21,476 protein-coding genes. Comparative analysis of Pfam domains and orthologous proteins highlights characteristic features of H. viridissima, such as diversification of innate immunity genes that are important for host-symbiont interactions. Thus, the H. viridissima assembly provides an important hydrozoan genome reference that will facilitate symbiosis research and better comparisons of metazoan genome architectures.

Abstract Background Some dinoflagellates cause harmful algal blooms, releasing toxic secondary metabolites, to the detriment of marine ecosystems and human health. Our understanding of dinoflagellate toxin biosynthesis has been hampered by their unusually large genomes. To overcome this challenge, for the first time, we sequenced the genome, microRNAs, and mRNA isoforms of a basal dinoflagellate, Amphidinium gibbosum, and employed an integrated omics approach to understand its secondary metabolite biosynthesis. Results We assembled the ~ 6.4-Gb A. gibbosum genome, and by probing decoded dinoflagellate genomes and transcriptomes, we identified the non-ribosomal peptide synthetase adenylation domain as essential for generation of specialized metabolites. Upon starving the cells of phosphate and nitrogen, we observed pronounced shifts in metabolite biosynthesis, suggestive of post-transcriptional regulation by microRNAs. Using Iso-Seq and RNA-seq data, we found that alternative splicing and polycistronic expression generate different transcripts for secondary metabolism. Conclusions Our genomic findings suggest intricate integration of various metabolic enzymes that function iteratively to synthesize metabolites, providing mechanistic insights into how dinoflagellates synthesize secondary metabolites, depending upon nutrient availability. This study provides insights into toxin production associated with dinoflagellate blooms. The genome of this basal dinoflagellate provides important clues about dinoflagellate evolution and overcomes the large genome size, which has been a challenge previously.

Crown-of-thorns starfish, Acanthaster planci (COTS), are common in coral reefs of Indo-Pacific Ocean. Since they are highly fecund predators of corals, periodic outbreaks of COTS cause substantial loss of healthy coral reefs. Using complete mitochondrial DNA sequences, we here examined how COTS outbreaks in the Ryukyu Archipelago, Japan are reflected by the profile of their population genetics. Population genetics of the blue starfish, Linckia laevigata, which lives in the Ryukyu Archipelago, but not break out and the northern Pacific sea star, Asterias amurensis, which lives in colder seawater around the main Islands of Japan, were also examined as controls. Our results showed that As. amurensis has at least two local populations that diverged approximately 4.7 million years ago (MYA), and no genetic exchanges have occurred between the populations since then. Linckia laevigata shows two major populations in the Ryukyu Archipelago that likely diverged ∼6.8 MYA. The two populations, each comprised of individuals collected from coast of the Okinawa Island and those from the Ishigaki Island, suggest the presence of two cryptic species in the Ryukyu Archipelago. On the other hand, population genetics of COTS showed a profile quite different from those of Asterias and Linckia. At least five lineages of COTS have arisen since their divergence ∼0.7 MYA, and each of the lineages is present at the Okinawa Island, Miyako Island, and Ishigaki Island. These results suggest that COTS have experienced repeated genetic bottlenecks that may be associated with or caused by repeated outbreaks.

Abstract Background The brown alga, Cladosiphon okamuranus (Okinawa mozuku), is one of the most important edible seaweeds, and it is cultivated for market primarily in Okinawa, Japan. Four strains, denominated S, K, O, and C, with distinctively different morphologies, have been cultivated commercially since the early 2000s. We previously reported a draft genome of the S-strain. To facilitate studies of seaweed biology for future aquaculture, we here decoded and analyzed genomes of the other three strains (K, O, and C). Results Here we improved the genome of the S-strain (ver. 2, 130 Mbp, 12,999 genes), and decoded the K-strain (135 Mbp, 12,511 genes), the O-strain (140 Mbp, 12,548 genes), and the C-strain (143 Mbp, 12,182 genes). Molecular phylogenies, using mitochondrial and nuclear genes, showed that the S-strain diverged first, followed by the K-strain, and most recently the C- and O-strains. Comparisons of genome architecture among the four strains document the frequent occurrence of inversions. In addition to gene acquisitions and losses, the S-, K-, O-, and C-strains possess 457, 344, 367, and 262 gene families unique to each strain, respectively. Comprehensive Blast searches showed that most genes have no sequence similarity to any entries in the non-redundant protein sequence database, although GO annotation suggested that they likely function in relation to molecular and biological processes and cellular components. Conclusions Our study compares the genomes of four strains of C. okamuranus and examines their phylogenetic relationships. Due to global environmental changes, including temperature increases, acidification, and pollution, brown algal aquaculture is facing critical challenges. Genomic and phylogenetic information reported by the present research provides useful tools for isolation of novel strains.

Abstract In the open ocean without terrain boundaries, marine invertebrates with pelagic larvae can migrate long distances using ocean currents, suggesting reduced genetic diversification. Contrary to this assumption, however, genetic differentiation is often observed in marine invertebrates. In the present study, we sought to explain how population structure is established in the western Pacific Ocean, where the strong Kuroshio Current maintains high levels of gene flow from south to north, presumably promoting genetic homogeneity. We determined the population structure of the pearl oyster, Pinctada fucata, in the Indo‐Pacific Ocean using genome‐wide genotyping data from multiple sampling localities. Cluster analysis showed that the western Pacific population is distinct from that of the Indian Ocean, and that it is divided into northern (Japanese mainland) and southern (Nansei Islands, China, and Cambodia) populations. Genetic differentiation of P. fucata can be explained by geographic barriers in the Indian Ocean and a local lagoon, and by environmental gradients of sea surface temperature (SST) and oxygen concentration in the western Pacific. A genome scan showed evidence of adaptive evolution in genomic loci, possibly associated with changes in environmental factors, including SST and oxygen concentration. Furthermore, Bayesian simulation demonstrated that the past population expansion and division are congruent with ocean warming after the last glacial period. It is highly likely that the environmental gradient forms a genetic barrier that diversifies P. fucata populations in the western Pacific. This hypothesis helps to explain genetic differentiation and possible speciation of marine invertebrates.

Hemichordate enteropneust worms regenerate extensively in a manner that resembles the regeneration for which planaria and hydra are well known. Although hemichordates are often classified as an extant phylogenetic group that may hold ancestral deuterostome body plans at the base of the deuterostome evolutionary line leading to chordates, mammals, and humans, extensive regeneration is not known in any of these more advanced groups. Here we investigated whether hemichordates deploy functional homologs of canonical Yamanaka stem cell reprogramming factors, Oct4, Sox2, Nanog, and Klf4, as they regenerate. These reprogramming factors are not expressed during regeneration of limbs, fins, eyes or other structures that represent the best examples of regeneration in chordates. We first examined Ptychodera flava EST libraries and identified Pf-Pou3, Pf-SoxB1, Pf-Msxlx, and Pf-Klf1/2/4 as most closely related to the Yamanaka factors, respectively. In situ hybridization analyses revealed that all these homologs are expressed in a distinct manner during head regeneration. Furthermore, Pf-Pou3 partially rescued the loss of endogenous Oct4 in mouse embryonic stem cells in maintaining the pluripotency gene expression program. Based on these results, we propose that hemichordates may have co-opted these reprogramming factors for their extensive regeneration or that chordates may have lost the ability to mobilize these factors in response to damage. The robustness of these pluripotency gene circuits in the inner cell mass and in formation of induced pluripotent stem cells from mammalian somatic cells shows that these programs are intact in humans and other mammals and that these circuits may respond to as yet unknown gene regulatory signals, mobilizing full regeneration in hemichordates.

Coral reefs, the most biodiverse habitats in the ocean, are formed by anthozoan cnidarians, the scleractinian corals. Recently, however, ongoing climate change has imperiled scleractinian corals and coral reef environments are changing drastically. Thus, convenient, high-density monitoring of scleractinian corals is essential to understand changes in coral reef communities. Environmental DNA (eDNA) metabarcoding is potentially one of the most effective means of achieving it. Using publicly available scleractinian mitochondrial genomes, we developed high-specificity primers to amplify mitochondrial 12S ribosomal RNA (12S) and cytochrome oxidase-1 (CO1) genes of diverse scleractinian corals, which could be used for genus-level metabarcoding analyses, using next-generation sequencing technologies. To confirm the effectiveness of these primers, PCR amplicon sequencing was performed using eDNA isolated along the seashore of Okinawa, Japan. We successfully amplified all eDNA samples using PCR. Approximately 93 and 72% of PCR amplicon sequences of 12S and CO1 primers originated from scleractinian 12S and CO1 genes, respectively, confirming higher specificities for coral mitochondrial genes than primers previously used for coral eDNA metabarcoding. We also found that hierarchical clustering, based on the percentage of mapped reads to each scleractinian genus, discriminates between sampling locations, suggesting that eDNA surveys are sufficiently powerful to reveal differences between coral communities separated by

AbstractThe kuruma shrimp Marsupenaeus japonicusM. japonicusM. japonicusM. japonicusM. japonicus

Among chordate taxa, the cephalochordates diverged earlier than urochordates and vertebrates; thus, they retain unique, primitive developmental features. In particular, the amphioxus notochord has muscle-like properties, a feature not seen in urochordates or vertebrates. Amphioxus contains two Brachyury genes, Bra1 and Bra2. Bra2 is reportedly expressed in the blastopore, notochord, somites, and tail bud, in contrast to a low level of Bra1 expression only in notochord. To distinguish the expression profiles of the two Brachyury genes at the single-cell level, we carried out single-cell RNA-seq (scRNA-seq) analysis using the amphioxus, Branchiostoma japonicum. This scRNA-seq analysis classified B. japonicum embryonic cells into 15 clusters at developmental stages from midgastrula to early swimming larva. Brachyury was expressed in cells of clusters 4, 5, 8, and 9. We first confirmed that cluster 8 comprises cells that form somites since this cluster specifically expresses four myogenic factor genes. Cluster 9 contains a larger number of cells with high levels of Bra2 expression and a smaller number of cells with Bra1 expression. Simultaneous expression in cluster 9 of tool-kit genes, including FoxA, Goosecoid, and hedgehog, showed that this cluster comprises cells that form the notochord. Expression of Bra2, but not Bra1, in cells of clusters 4 and 5 at the gastrula stage together with expression of Wnt1 and Caudal indicates that clusters 4 and 5 comprise cells of the blastopore, which contiguously form the tail bud. In addition, Hox1, Hox3, and Hox4 were highly expressed in Bra2-expressing clusters 4, 5, 8, and 9 in a temporally coordinated manner, suggesting roles of anterior Hox genes in specification of mesodermal organs, including somites, notochord, and tail bud. This scRNA-seq analysis therefore highlights differences between the two Brachyury genes in relation to embryonic regions in which they are expressed and their levels of expression. Bra2 is the ancestral Brachyury in amphioxus, since expression in the blastopore is shared with other deuterostomes. On the other hand, Bra1 is a duplicate copy and likely evolved a supplementary function in notochord and somite formation in the Branchiostoma lineage.

Coral reefs are the biodiversity hot spots of the oceans, but they have suffered from increasing environmental stresses caused principally by anthropogenic global warming. The keystone species of coral reefs are scleractinian corals, which maintain obligatory symbiotic relationships with photosynthetic dinoflagellates. Understanding cellular and molecular mechanisms of symbiosis is therefore essential for future preservation of coral reefs. To date, however, almost no in vitro experimental systems have been devised to illuminate such mechanisms. To this end, our previous study established stable in vitro cell culture lines, including IVB5, originating from planula larvae of the scleractinian coral, Acropora tenuis. Here, we show that soon after mixture with the dinoflagellate, Breviolum minutum, flattened amorphous coral cells with endodermal properties exhibited elevated locomotor activity using filopodia and lamellipodia and interacted with dinoflagellates. Several minutes thereafter, coral cells began to incorporate B. minutum, and in vitro symbiosis appeared to have been accomplished within 30 min. Nearly a half of the coral cells had incorporated algal cells within 24 h in a reproducible manner. Coral cells that harbored algal cells gradually became round and less mobile, and the algal cells sometimes settled in vacuole-like structures in coral cell cytoplasm. This symbiosis state was maintained for at least a month. The IVB5 line of A. tenuis therefore provides an experimental system to explore cellular and molecular mechanisms involved in coral-dinoflagellate symbiosis at the single-cell level, results of which may be useful for future preservation of coral reefs.

Abstract Metazoans have evolved a great variety of life histories in response to environmental conditions. A unique example is encountered in dicyemid mesozoans. In addition to a highly simplified adult body comprising only ~ 30 cells, dicyemids exhibit a parasitic lifestyle that includes nematogens (asexual reproductive adults), rhombogens (sexual reproductive adults), vermiform larvae generated by nematogens, and infusoriform larvae generated by rhombogens. However, due to the difficulties of observing microscopic endoparasites, the complex life cycle and biological functions of life-cycle stages of dicyemids have remained mysterious. Taking advantage of the recently decoded genome of Dicyema japonicum, we examined genes that undergird this lifestyle. Using stage-specific gene expression profiles, we found that biological processes associated with molecular transport, developmental regulation, and sensory response are specified at different stages. Together with the expression of potential neurotransmitters, we further suggest that apical cells in infusoriform larva probably serve sensory functions, although dicyemids have no nervous system. Gene expression profiles show that more genes are expressed in free-living infusoriform larvae than in the other three stages, and that some of these genes are likely involved in locating new hosts. These data provide molecular information about the unique lifestyle of dicyemids and illustrate how an extremely simplified endoparasite adapted and retained gene sets and morphological characters to complete its life cycle.

Summary: Whole-genome duplication (WGD) has been recognized as a significant evolutionary force in the origin and diversification of multiple organisms. Acropora, a speciose reef-building coral genus, is suspected to have originated by polyploidy. Yet, there is no genetic evidence to support this hypothesis. Using comprehensive phylogenomic and comparative genomic approaches, we analyzed six Acroporid genomes and found that a WGD event likely occurred ∼31 million years ago in the most recent common ancestor of Acropora, concurrent with a worldwide coral extinction. We found that duplicated genes were highly enriched in gene regulation functions, including those of stress responses. The functional clusters of duplicated genes are related to the divergence of gene expression patterns during development. Some proteinaceous toxins were generated by WGD in Acropora compared with other cnidarian species. Collectively, this study provides evidence for an ancient WGD event in corals, which helps explain the origin and diversification of Acropora. : Genetics; Evolutionary Biology; Bioinformatics; Omics Subject Areas: Genetics, Evolutionary Biology, Bioinformatics, Omics

Abstract Background Spiral cleavage is a feature of non-ecdysozoan protostomes, in which left-right reversal frequently evolved in gastropod molluscs. In pulmonate gastropods, maternal molecules are responsible for chirality patterning, on which the polarities of visceral and coiling asymmetries depend. In the pond snail, Lymnaea stagnalis (the clade Hygrophila), a frame-shift mutation of one of tandem-duplicated, diaphanous-related formin genes (diaph) resulted in incomplete reversal from dextral to sinistral cleavage. Is this mechanism of chirality regulation common to, or shared with other pulmonates? To answer this question, we examined genes involved in chirality patterning in the land snail, Bradybaena similaris which belongs to the clade Stylommatophora. Results Both dextral and sinistral siblings develop from progeny of a racemic mutant of B. similaris. Differences in maternal mRNAs between the two strains were searched by transcriptome analyses. We found fifty maternal transcripts that exhibited less expression in early embryos of the mutant strain. The most conspicuous was a homolog of diaph. The diaph gene was duplicated in the stylommatophoran ancestor (diaph-a and diaph-b), as in the case of the ancestor of Lymnaea (Lsdiaph1 and Lsdiaph2). The quantity of maternal diaph-b mRNA was drastically reduced in early embryos of the racemic mutant compared to wild-type, while diaph-a expression was at nearly the same level in both strains. Unlike the case of Lsdiaph2, which is frame-shifted to produce truncated peptides in the mutant of L. stagnalis, however, Bsdiaph-b mRNA in the mutant strain is not frame-shifted and most probably produces normal Diaph-b protein. These results suggest the presence of regulatory mechanisms of gene expression for chirality patterning in pulmonate gastropods, although genomic analyses are required for confirmation. Conclusions Heterochirality resulting from the loss of polarity control in spiral cleavage does not require mutation of the diaph gene in B. similaris. The determination of left-right polarity instead depends on the expression of this diaph gene, which is duplicated in stylommatophoran Bradybaena, as well as in hygrophilan Lymnaea. Our results provide an avenue to identifying a regulatory mechanism that controls the direction of spiral cleavage in gastropods.

Molluscan shells are among the most fascinating research objects because of their diverse morphologies and textures. The formation of these delicate biomineralized structures is a matrix-mediated process. A question that arises is what are the essential components required to build these exoskeletons. In order to understand the molecular mechanisms of molluscan shell formation, it is crucial to identify organic macromolecules in different shells from diverse taxa. In the case of bivalves, however, taxon sampling in previous shell proteomics studies are focused predominantly on representatives of the class Pteriomorphia such as pearl oysters, edible oysters and mussels. In this study, we have characterized the shell organic matrix from the crocus clam, Tridacna crocea, (Heterodonta) using various biochemical techniques, including SDS-PAGE, FT-IR, monosaccharide analysis, and enzyme-linked lectin assay (ELLA). Furthermore, we have identified a number of shell matrix proteins (SMPs) using a comprehensive proteomics approach combined to RNA-seq. The biochemical studies confirmed the presence of proteins, polysaccharides, and sulfates in the T. crocea shell organic matrix. Proteomics analysis revealed that the majority of the T. crocea SMPs are novel and dissimilar to known SMPs identified from the other bivalve species. Meanwhile, the SMP repertoire of the crocus clam also includes proteins with conserved functional domains such as chitin-binding domain, VWA domain, and protease inhibitor domain. We also identified BMSP (Blue Mussel Shell Protein, originally reported from Mytilus), which is widely distributed among molluscan shell matrix proteins. Tridacna SMPs also include low-complexity regions (LCRs) that are absent in the other molluscan genomes, indicating that these genes may have evolved in specific lineage. These results highlight the diversity of the organic molecules – in particular proteins – that are essential for molluscan shell formation.

Genes encoding snake venom toxins have been studied extensively. However, genes involved in the modification and functioning of venom proteins are little known. Protobothrops is a genus of pit vipers, which are venomous and inhabit the Nansei (Southwest) islands of Japan, Taiwan China, Vietnam, Thailand, Myanmar, Nepal, Bhutan, and India. Our previous study decoded the genome of Protobothrops flavoviridis, a species endemic to the Nansei Islands, Japan, and revealed unique evolutionary processes of some venom genes. In this study, we analyzed genes that are highly expressed in venom glands to survey genes for candidate enzymes or chaperone proteins involved in toxin folding and modification. We found that, in addition to genes that encode venom proteins and ribosomal proteins, genes that encode protein disulfide isomerase (PDI) family members (orthologs of human P4HB and PDIA3), Selenoprotein M (SELENOM), and Calreticulin (CALR) are highly expressed in venom glands. Since these enzymes or chaperones are involved in protein modification and potentially possess protein folding functions, we propose that P4HB, SELENOM, CALR, and PDIA3 encode candidate enzymes or chaperones to confer toxic functions upon the venom transcriptome.

Coral reefs are under threat and innovative management strategies are urgently required. However, discoveries from innovative fields of coral reef research are rarely transposed in practical conservation actions. This is mainly due to the difficulties in knowledge exchange between scientists and conservation stakeholders. The ManaCo consortium (http://manaco.ird.nc/) is an international network federating conservation stakeholders and researchers in a common effort to preserve the coral reefs. The focus is on using modern tools to build a bridge between indigenous knowledge and scientific innovation. ManaCo aims to orientate research toward relevant conservation needs and to facilitate the transposition of research into concrete management strategies. This will allow to coordinate a collaborative response against coral reef decline. We invite anyone sharing the same interests in joining us.

Abstract The group Vertebrata is currently placed as a subphylum in the phylum Chordata, together with two other subphyla, Cephalochordata (lancelets) and Urochordata (ascidians). The past three decades, have seen extraordinary advances in zoological taxonomy and the time is now ripe for reassessing whether the subphylum position is truly appropriate for vertebrates, particularly in light of recent advances in molecular phylogeny, comparative genomics, and evolutionary developmental biology. Four lines of current research are discussed here. First, molecular phylogeny has demonstrated that Deuterostomia comprises Ambulacraria (Echinodermata and Hemichordata) and Chordata (Cephalochordata, Urochordata, and Vertebrata), each clade being recognized as a mutually comparable phylum. Second, comparative genomic studies show that vertebrates alone have experienced two rounds of whole-genome duplication, which makes the composition of their gene family unique. Third, comparative gene-expression profiling of vertebrate embryos favors an hourglass pattern of development, the most conserved stage of which is recognized as a phylotypic period characterized by the establishment of a body plan definitively associated with a phylum. This mid-embryonic conservation is supported robustly in vertebrates, but only weakly in chordates. Fourth, certain complex patterns of body plan formation (especially of the head, pharynx, and somites) are recognized throughout the vertebrates, but not in any other animal groups. For these reasons, we suggest that it is more appropriate to recognize vertebrates as an independent phylum, not as a subphylum of the phylum Chordata.

Abstract Background The striped catfish, Pangasianodon hypophthalmus, is a freshwater and benthopelagic fish common in the Mekong River delta. Catfish constitute a valuable source of dietary protein. Therefore, they are cultured worldwide, and P. hypophthalmus is a food staple in the Mekong area. However, genetic information about the culture stock, is unavailable for breeding improvement, although genetics of the channel catfish, Ictalurus punctatus, has been reported. To acquire genome sequence data as a useful resource for marker-assisted breeding, we decoded a draft genome of P. hypophthalmus and performed comparative analyses. Results Using the Illumina platform, we obtained both nuclear and mitochondrial DNA sequences. Molecular phylogeny using the mitochondrial genome confirmed that P. hypophthalmus is a member of the family Pangasiidae and is nested within a clade including the families Cranoglanididae and Ictaluridae. The nuclear genome was estimated at approximately 700 Mb, assembled into 568 scaffolds with an N50 of 14.29 Mbp, and was estimated to contain ~ 28,600 protein-coding genes, comparable to those of channel catfish and zebrafish. Interestingly, zebrafish produce gadusol, but genes for biosynthesis of this sunscreen compound have been lost from catfish genomes. The differences in gene contents between these two catfishes were found in genes for vitamin D-binding protein and cytosolic phospholipase A2, which have lost only in channel catfish. The Hox cluster in catfish genomes comprised seven paralogous groups, similar to that of zebrafish, and comparative analysis clarified catfish lineage-specific losses of A5a, B10a, and A11a. Genes for insulin-like growth factor (IGF) signaling were conserved between the two catfish genomes. In addition to identification of MHC class I and sex determination-related gene loci, the hypothetical chromosomes by comparison with the channel catfish demonstrated the usefulness of the striped catfish genome as a marker resource. Conclusions We developed genomic resources for the striped catfish. Possible conservation of genes for development and marker candidates were confirmed by comparing the assembled genome to that of a model fish, Danio rerio, and to channel catfish. Since the catfish genomic constituent resembles that of zebrafish, it is likely that zebrafish data for gene functions is applicable to striped catfish as well.

The coevolution of the animal gut mucosa and the gut microbiota is poorly understood. Here, Nakashima et al. identify intestinal chitinous membranes in basal chordates and ray-finned fish, and propose that the loss of this chitin barrier allowed mucus layers to become colonized by microbes in mammals.

Abstract Evolution of novel traits is a challenging subject in biological research. Several snake lineages developed elaborate venom systems to deliver complex protein mixtures for prey capture. To understand mechanisms involved in snake venom evolution, we decoded here the ~1.4-Gb genome of a habu, Protobothrops flavoviridis. We identified 60 snake venom protein genes (SV) and 224 non-venom paralogs (NV), belonging to 18 gene families. Molecular phylogeny reveals early divergence of SV and NV genes, suggesting that one of the four copies generated through two rounds of whole-genome duplication was modified for use as a toxin. Among them, both SV and NV genes in four major components were extensively duplicated after their diversification, but accelerated evolution is evident exclusively in the SV genes. Both venom-related SV and NV genes are significantly enriched in microchromosomes. The present study thus provides a genetic background for evolution of snake venom composition.

Abstract Background The marine dinoflagellate, Symbiodinium, is a well-known photosynthetic partner for coral and other diverse, non-photosynthetic hosts in subtropical and tropical shallows, where it comprises an essential component of marine ecosystems. Using molecular phylogenetics, the genus Symbiodinium has been classified into nine major clades, A-I, and one of the reported differences among phenotypes is their capacity to synthesize mycosporine-like amino acids (MAAs), which absorb UV radiation. However, the genetic basis for this difference in synthetic capacity is unknown. To understand genetics underlying Symbiodinium diversity, we report two draft genomes, one from clade A, presumed to have been the earliest branching clade, and the other from clade C, in the terminal branch. Results The nuclear genome of Symbiodinium clade A (SymA) has more gene families than that of clade C, with larger numbers of organelle-related genes, including mitochondrial transcription terminal factor (mTERF) and Rubisco. While clade C (SymC) has fewer gene families, it displays specific expansions of repeat domain-containing genes, such as leucine-rich repeats (LRRs) and retrovirus-related dUTPases. Interestingly, the SymA genome encodes a gene cluster for MAA biosynthesis, potentially transferred from an endosymbiotic red alga (probably of bacterial origin), while SymC has completely lost these genes. Conclusions Our analysis demonstrates that SymC appears to have evolved by losing gene families, such as the MAA biosynthesis gene cluster. In contrast to the conservation of genes related to photosynthetic ability, the terminal clade has suffered more gene family losses than other clades, suggesting a possible adaptation to symbiosis. Overall, this study implies that Symbiodinium ecology drives acquisition and loss of gene families.

Abstract Background In the previous paper published in 2017, we described the structure of Hox gene cluster of the ascidian, Halocynthia roretzi, and discussed the scenario for the disintegration of Hox gene clusters during evolution of ascidians. The description about the Hox gene cluster structure still represents the latest information, hence it has been left unchanged. In contrast, some points in Discussion, the description on the phylogenetic relationships among tunicates and the theoretical scenario for the disintegration of Hox gene cluster during evolution of ascidians, should be changed because the phylogenetic relationships among tunicates have recently been updated. The above mentioned points were made in accordance with the phylogenetic tree for tunicates based on the mitochondrial DNA sequences, which was the latest at the time of publication. In 2018, however, Kocot et al. and Delsuc et al. proposed new phylogenetic trees for tunicates based on a large number of nuclear gene sequences. The trees obtained by the two groups are essentially the same and different from the previous one in the phylogenetic positions of Appendicularia and Thaliacea, which leads to a change in the order of the emergence of ascidians and the Hox gene cluster disintegration during evolution of ascidians or tunicates. Results We add here a note to update the previous description on the phylogenetic relationships among tunicates and the theoretical scenario, including one Figure, so as to coincide with the new phylogenetic relationships among tunicates based on the nuclear gene sequences. Conclusion The previous summarized conclusion remains unchanged: we suggest that the Hox gene cluster of the ancestral ascidian experienced extensive genome shuffling during the course of evolution to Hr and Ci. Nevertheless, some features are shared in Hox gene components and gene organization on the chromosomes, suggesting that Hox gene cluster disintegration in ascidians involved early events common to all ascidians and later lineage-specific events.

Abstract Background Xenoturbella is a group of marine benthic animals lacking an anus and a centralized nervous system. Molecular phylogenetic analyses group the animal together with the Acoelomorpha, forming the Xenacoelomorpha. This group has been suggested to be either a sister group to the Nephrozoa or a deuterostome, and therefore it may provide important insights into origins of bilaterian traits such as an anus, the nephron, feeding larvae and centralized nervous systems. However, only five Xenoturbella species have been reported and the evolutionary history of xenoturbellids and Xenacoelomorpha remains obscure. Results Here we describe a new Xenoturbella species from the western Pacific Ocean, and report a new xenoturbellid structure - the frontal pore. Non-destructive microCT was used to investigate the internal morphology of this soft-bodied animal. This revealed the presence of a frontal pore that is continuous with the ventral glandular network and which exhibits similarities with the frontal organ in acoelomorphs. Conclusions Our results suggest that large size, oval mouth, frontal pore and ventral glandular network may be ancestral features for Xenoturbella. Further studies will clarify the evolutionary relationship of the frontal pore and ventral glandular network of xenoturbellids and the acoelomorph frontal organ. One of the habitats of the newly identified species is easily accessible from a marine station and so this species promises to be valuable for research on bilaterian and deuterostome evolution.

Abstract Background Hox gene clusters with at least 13 paralog group (PG) members are common in vertebrate genomes and in that of amphioxus. Ascidians, which belong to the subphylum Tunicata (Urochordata), are phylogenetically positioned between vertebrates and amphioxus, and traditionally divided into two groups: the Pleurogona and the Enterogona. An enterogonan ascidian, Ciona intestinalis (Ci), possesses nine Hox genes localized on two chromosomes; thus, the Hox gene cluster is disintegrated. We investigated the Hox gene cluster of a pleurogonan ascidian, Halocynthia roretzi (Hr) to investigate whether Hox gene cluster disintegration is common among ascidians, and if so, how such disintegration occurred during ascidian or tunicate evolution. Results Our phylogenetic analysis reveals that the Hr Hox gene complement comprises nine members, including one with a relatively divergent Hox homeodomain sequence. Eight of nine Hr Hox genes were orthologous to Ci-Hox1, 2, 3, 4, 5, 10, 12 and 13. Following the phylogenetic classification into 13 PGs, we designated Hr Hox genes as Hox1, 2, 3, 4, 5, 10, 11/12/13.a, 11/12/13.b and HoxX. To address the chromosomal arrangement of the nine Hox genes, we performed two-color chromosomal fluorescent in situ hybridization, which revealed that the nine Hox genes are localized on a single chromosome in Hr, distinct from their arrangement in Ci. We further examined the order of the nine Hox genes on the chromosome by chromosome/scaffold walking. This analysis suggested a gene order of Hox1, 11/12/13.b, 11/12/13.a, 10, 5, X, followed by either Hox4, 3, 2 or Hox2, 3, 4 on the chromosome. Based on the present results and those previously reported in Ci, we discuss the establishment of the Hox gene complement and disintegration of Hox gene clusters during the course of ascidian or tunicate evolution. Conclusions The Hox gene cluster and the genome must have experienced extensive reorganization during the course of evolution from the ancestral tunicate to Hr and Ci. Nevertheless, some features are shared in Hox gene components and gene arrangement on the chromosomes, suggesting that Hox gene cluster disintegration in ascidians involved early events common to tunicates as well as later ascidian lineage-specific events.

Abstract Population genetics of the coral genus Pocillopora have been more intensively studied than those of any other reef-building taxon. However, recent investigations have revealed that the current morphological classification is inadequate to represent genetic lineages. In this study, we isolated and characterized novel microsatellite loci from morphological Pocillopora meandrina (Type 1) and Pocillopora acuta (Type 5). Furthermore, we characterized previously reported microsatellite loci. A total of 27 loci (13 novel loci) proved useful for population genetic analyses at two sites in the Ryukyu Archipelago, in the northwestern Pacific. Clonal diversity differed in each genetic lineage. Genetic structure suggested by microsatellites corresponded to clusters in a phylogenetic tree constructed from a mitochondrial open reading frame (mtORF). In addition, we found an unknown mitochondrial haplotype of this mtORF. These microsatellite loci will be useful for studies of connectivity and genetic diversity of Pocillopora populations, and will also support coral reef conservation.

Abstract Background In marine organisms, and in particular for benthic invertebrates including echinoderms, olfaction is a dominant sense with chemosensation being a critical signalling process. Until recently natural product chemistry was the primary investigative approach to elucidate the nature of chemical signals but advances in genomics and transcriptomics over the last decade have facilitated breakthroughs in understanding not only the chemistry but also the molecular mechanisms underpinning chemosensation in aquatic environments. Integration of these approaches has the potential to reveal the fundamental elements influencing community structure of benthic ecosystems as chemical signalling modulates intra- and inter-species interactions. Such knowledge also offers avenues for potential development of novel biological control methods for pest species such as the predatory Crown-of-Thorns starfish (COTS), Acanthaster planci which are the primary biological cause of coral cover loss in the Indo-Pacific. Results In this study, we have analysed the COTS sensory organs through histological and electron microscopy. We then investigated key elements of the COTS molecular olfactory toolkit, the putative olfactory rhodopsin-like G protein-protein receptors (GPCRs) within its genome and olfactory organ transcriptomes. Many of the identified Acanthaster planci olfactory receptors (ApORs) genes were found to cluster within the COTS genome, indicating rapid evolution and replication from an ancestral olfactory GPCR sequence. Tube feet and terminal sensory tentacles contain the highest proportion of ApORs. In situ hybridisation confirmed the presence of four ApORs, ApOR15, 18, 25 and 43 within COTS sensory organs, however expression of these genes was not specific to the adhesive epidermis, but also within the nerve plexus of tube feet stems and within the myomesothelium. G alpha subunit proteins were also identified in the sensory organs, and we report the spatial localisation of Gαi within the tube foot and sensory tentacle. Conclusions We have identified putative COTS olfactory receptors that localise to sensory organs. These results provide a basis for future studies that may enable the development of a biological control not only for COTS, but also other native pest or invasive starfish.

Abstract Background Obtaining phylogenomic data for enigmatic taxa is essential to achieve a better understanding of animal evolution. Dicyemids have long fascinated biologists because of their highly simplified body organization, but their life-cycles remain poorly known. Based on the discovery of the dicyemid DoxC gene, which encodes a spiralian peptide, it has been proposed that dicyemids are members of the Spiralia. Other studies have suggested that dicyemids may have closer affinities to mollusks and annelids. However, the phylogenetic position of dicyemids has remained a matter of debate, leading to an ambiguous picture of spiralian evolution. Results In the present study, newly sequenced transcriptomic data from Dicyema japonicum were complemented with published transcriptomic data or predicted gene models from 29 spiralian, ecdysozoan, and deuterostome species, generating a dataset (Dataset 1) for phylogenomic analyses, which contains 348 orthologs and 58,124 amino acids. In addition to this dataset, to eliminate systematic errors, two additional sub-datasets were created by removing compositionally heterogeneous or rapidly evolving sites and orthologs from Dataset 1, which may cause compositional heterogeneity and long-branch attraction artifacts. Maximum likelihood and Bayesian inference analyses both placed Dicyema japonicum (Dicyemida) in a clade with Intoshia linei (Orthonectida) with strong statistical support. Furthermore, maximum likelihood analyses placed the Dicyemida + Orthonectida clade within the Gastrotricha, while in Bayesian inference analyses, this clade is sister group to the clade of Gastrotricha + Platyhelminthes. Conclusions Whichever the case, in all analyses, Dicyemida, Orthonectida, Gastrotricha, and Platyhelminthes constitute a monophyletic group that is a sister group to the clade of Mollusca + Annelida. Based on present phylogenomic analyses, dicyemids display close affinity to orthonectids, and they may share a common ancestor with gastrotrichs and platyhelminths, rather than with mollusks and annelids. Regarding spiralian phylogeny, the Gnathifera forms the sister group to the Rouphozoa and Lophotrochozoa, as has been suggested by previous studies; thus our analysis supports the traditional acoeloid–planuloid hypothesis of a nearly microscopic, non-coelomate common ancestor of spiralians.

Duckweeds are small, fast growing, and starch- and protein-rich aquatic plants expected to be a next generation energy crop and an excellent biomaterial for phytoremediation. Despite such an importance, very little is known about duckweed–microbe interactions that would be a key biological factor for efficient industrial utilization of duckweeds. Here we first report the duckweed growth promoting ability of bacterial strains belonging to the phylum Acidobacteria, the members of which are known to inhabit soils and terrestrial plants, but their ecological roles and plant–microbe interactions remain largely unclear. Two novel Acidobacteria strains, F-183 and TBR-22, were successfully isolated from wild duckweeds and phylogenetically affiliated with subdivision 3 and 6 of the phylum, respectively, based on 16S rRNA gene sequence analysis. In the co-culture experiments with aseptic host plants, the F-183 and TBR-22 strains visibly enhanced growth (frond number) of six duckweed species (subfamily Lemnoideae) up to 1.8–5.1 times and 1.6–3.9 times, respectively, compared with uninoculated controls. Intriguingly, both strains also increased the chlorophyll content of the duckweed (Lemna aequinoctialis) up to 2.4–2.5 times. Under SEM observation, the F-183 and TBR-22 strains were epiphytic and attached to the surface of duckweed. Taken together, our findings suggest that indigenous plant associated Acidobacteria contribute to a healthy growth of their host aquatic plants.

The mollusk class, Bivalvia, plays an important role in the formation of calcium carbonate in the ocean. The bivalve hinge ligament is a hard but pliant tissue that resists the stress placed on the hinge during opening and closing. The ligament comprises a fine microstructure of fibrous aragonite crystals surrounded by a dense organic matrix. The matrix consists of organic fibers that are aligned with the fibrous aragonite crystals. Previous studies identified a tissue inhibitor of metalloproteinase (Pf-TIMP: Pinctada fucata-Tissue Inhibitor of Metalloproteinase) in the organic fibers of P. fucata ligaments. This enzyme exhibited strong inhibition of matrix metalloproteinase (MMP) activity in vitro, suggesting that MMPs also play a role in formation of the organic fibers of the ligament. Using transcriptome data, we identified MMP genes from the mantle isthmus, which is a soft tissue attached to the ligament. To investigate the function of MMP genes in vivo, we performed RNA interference experiments. The expression of MMP14973 and MMP07860 genes was inhibited after injection of each dsRNA. Cumulative injection of MMP07860 dsRNA induced aggregated aragonite fiber orientation, whereas the injection of MMP14973 showed minor effects. When the decalcified ligament was incubated in a solution saturated with calcium carbonate, aragonite fibers aligned along the surface. When the decalcified ligament was treated with recombinant human (hr) MMP-13, the precipitation of calcium carbonate was inhibited. To investigate general MMP functions in calcium carbonate crystallization in detail, we precipitated aragonite crystals in collagen gels treated with or without recombinant human (hr) MMP-1. Treatment with hrMMP-1 increased the interaction between collagen gels and calcium carbonate. These results imply that Pinctada fucata (Pf)-MMPs degrade extracellular matrices in the ligament to produce the fine organic fibers that regulate the orientation of fibrous aragonite crystals.

Frequent, high-density coral monitoring is essential to understand coral reef ecosystems. For this purpose, we developed a novel method for simultaneous monitoring of Acropora corals and their symbiont, Symbiodinium, from environmental DNA (eDNA) in seawater using next generation sequencing technology (NGS). We performed a tank experiment with running seawater using 19 Acropora species. Complete mitochondrial genomes of all the Acropora species were assembled to create a database and major types of their Symbiodinium symbionts were identified. Then eDNA was isolated by filtering inlet and outlet seawater from the tanks. Acropora and Symbiodinium DNA were amplified by PCR and sequenced. We detected all of the tested Acropora types from eDNA samples. Proportions and numbers of DNA sequences were both positively correlated with masses of corals in the tanks. In this trial, we detected DNA sequences from as little as 0.04 kg of Acropora colony, suggesting that existence of at least one adult Acropora colony (~30 cm diameter = 1 kg) per m2 at depths < 10 m could be detected using eDNA in the field. In addition, we detected major types of Symbiodinium within host corals from seawater, suggesting that it should be possible to detect major coral symbiont types if Acropora corals exist nearby, and possible free-living state Symbiodinium cells from eDNA in seawater. eDNA abundance of Symbiodinium types did not correlate well with frequencies of major Symbiodinium types in the corals, suggesting that quantification of Symbiodinium is difficult at this stage. Although this is the initial attempt to detect coral and Symbiodinium simultaneously from eDNA in seawater, this method may allow us to perform high-frequency, high-density coral reef monitoring of both corals and their symbionts in the near future.

Mass mortality that is acompanied by reddish browning of the soft tissues has been occurring in cultured pearl oyster, Pinctada fucata martensii. The disease is called Akoya oyster disease (AOD). Although spreading pattern of the disease and transmission experiments suggest that the disease is infectious, the causative agent has not yet been identified. We used shotgun and 16S rRNA-based metagenomic analysis to identify genes that are present specifically in affected oysters. The genes found only in diseased oysters were mostly bacterial origin, suggesting that the causative agent was a bacterial pathogen. This hypothesis was supported by the inhibition of AOD development in naïve oysters injected with the hemolymph of diseased animals followed immediately with penicillin bath-administration. Further analyses of the hemolymph and mantle specifically and universally detected genes of bacteria that belong to phylum Spirochaetes in diseased pearl oysters but not in healthy oysters. By in situ hybridization or immunostaining, a Brachyspira-like bacterium was observed in the smears of hemolymph from affected oysters, but not from healthy oysters. Phylogenetic analysis using 16S rRNA sequences showed that the presumptive causative bacterium was outside of but most closely related to family Brachyspiraceae. We propose 'Candidatus Maribrachyspira akoyae' gen. nov, sp nov., for this bacterium.

After publication of Nakano et al. (2017) [1], the authors became aware of the fact that the new species-group name erected for the two specimens of a Japanese xenoturbellid species in the article is not available because Nakano et al. (2017) [1] does not meet the requirement of the amendment of Article 8.5.3 of the International Code of Zoological Nomenclature (the Code) [2]. The authors therefore describe the two xenoturbellids as a new species again in this correction article. Methods for morphological observation, DNA extraction and sequencing were as described in Nakano et al. (2017) [1]. The holotype and paratype specimens are deposited in the National Museum of Nature and Science, Tsukuba (NSMT), Japan. The DNA sequences obtained were deposited in the International Nucleotide Sequence Database (INSD).

Snake venoms are complex mixtures of toxic proteins encoded by various gene families that function synergistically to incapacitate prey. A huge repertoire of snake venom genes and proteins have been reported, and alternative splicing is suggested to be involved in the production of divergent gene transcripts. However, a genome-wide survey of the transcript repertoire and the extent of alternative splicing still remains to be determined. In this study, the comprehensive analysis of transcriptomes in the venom gland was achieved by using PacBio sequencing. Extensive alternative splicing was observed in three venom protein gene families, metalloproteinase (MP), serine protease (SP), and vascular endothelial growth factors (VEGF). Eleven MP and SP genes and a VEGF gene are expressed as a total of 81, 61, and 8 transcript variants, respectively. In the MP gene family, individual genes are transcribed into different classes of MPs by alternative splicing. We also observed trans-splicing among the clustered SP genes. No other venom genes as well as non-venom counterpart genes exhibited alternative splicing. Our results thus indicate a potential contribution of mRNA alternative and trans-splicing in the production of highly variable transcripts of venom genes in the habu snake.

Despite the importance of stony corals in many research fields related to global issues, such as marine ecology, climate change, paleoclimatogy, and metazoan evolution, very little is known about the evolutionary origin of coral skeleton formation. In order to investigate the evolution of coral biomineralization, we have identified skeletal organic matrix proteins (SOMPs) in the skeletal proteome of the scleractinian coral, Acropora digitifera, for which large genomic and transcriptomic datasets are available. Scrupulous gene annotation was conducted based on comparisons of functional domain structures among metazoans. We found that SOMPs include not only coral-specific proteins, but also protein families that are widely conserved among cnidarians and other metazoans. We also identified several conserved transmembrane proteins in the skeletal proteome. Gene expression analysis revealed that expression of these conserved genes continues throughout development. Therefore, these genes are involved not only skeleton formation, but also in basic cellular functions, such as cell-cell interaction and signaling. On the other hand, genes encoding coral-specific proteins, including extracellular matrix domain-containing proteins, galaxins, and acidic proteins, were prominently expressed in post-settlement stages, indicating their role in skeleton formation. Taken together, the process of coral skeleton formation is hypothesized as: 1) formation of initial extracellular matrix between epithelial cells and substrate, employing pre-existing transmembrane proteins; 2) additional extracellular matrix formation using novel proteins that have emerged by domain shuffling and rapid molecular evolution and; 3) calcification controlled by coral-specific SOMPs.

The reef-building, scleractinian coral, Galaxea fascicularis, is classified into soft and hard types, based on nematocyst morphology. This character is correlated with the length of the mitochondrial non-coding region (mt-Long: soft colony type, and nematocysts with wide capsules and long shafts; mt-Short: hard colony type, and nematocysts with thin capsules and short shafts). We isolated and characterized novel polymorphic microsatellite markers for G. fascicularis using next-generation sequencing. Based upon the mitochondrial non-coding region, 53 of the 97 colonies collected were mt-Long (mt-L) and 44 were mt-Short (mt-S). Among the 53 mt-L colonies, 27 loci were identified as amplifiable, polymorphic microsatellite loci, devoid of somatic mutations and free of scoring errors. Eleven of those 27 loci were also amplifiable and polymorphic in the 44 mt-S colonies; these 11 are cross-type microsatellite loci. The other 16 loci were considered useful only for mt-L colonies. These 27 loci identified 10 multilocus lineages (MLLs) among the 53 mt-L colonies (NMLL/N = 0.189), and the 11 cross-type loci identified 7 MLLs in 44 mt-S colonies (NMLL/N = 0.159). Significant genetic differentiation between the two types was detected based on the genetic differentiation index (FST = 0.080, P = 0.001). Bayesian clustering also indicated that these two types are genetically isolated. While nuclear microsatellite genotypes also showed genetic differentiation between mitochondrial types, the mechanism of divergence is not yet clear. These markers will be useful to estimate genetic diversity, differentiation, and connectivity among populations, and to understand evolutionary processes, including divergence of types in G. fascicularis.