Regulatory cocktail for dopaminergic neurons in a protovertebrate identified by whole-embryo single-cell transcriptomics.

The CNS of the protovertebrate Ciona intestinalis contains a single cluster of dopaminergic (DA) neurons, the coronet cells, which have been likened to the hypothalamus of vertebrates. Whole-embryo single-cell RNA sequencing (RNA-seq) assays identified Ptf1a as the most strongly expressed cell-specific transcription factor (TF) in DA/coronet cells. Knockdown of Ptf1a activity results in their loss, while misexpression results in the appearance of supernumerary DA/coronet cells. Photoreceptor cells and ependymal cells are the most susceptible to transformation, and both cell types express high levels of Meis Coexpression of both Ptf1a and Meis caused the wholesale transformation of the entire CNS into DA/coronet cells. We therefore suggest that the reiterative use of functional manipulations and single-cell RNA-seq assays is an effective means for the identification of regulatory cocktails underlying the specification of specific cell identities.

scRNA-seq analysis of cells comprising the amphioxus notochord.

Cephalochordates occupy a key phylogenetic position for deciphering the origin and evolution of chordates, since they diverged earlier than urochordates and vertebrates. The notochord is the most prominent feature of chordates. The amphioxus notochord features coin-shaped cells bearing myofibrils. Notochord-derived hedgehog signaling contributes to patterning of the dorsal nerve cord, as in vertebrates. However, properties of constituent notochord cells remain unknown at the single-cell level. We examined these properties using Iso-seq analysis, single-cell RNA-seq analysis, and in situ hybridization (ISH). Gene expression profiles broadly categorize notochordal cells into myofibrillar cells and non-myofibrillar cells. Myofibrillar cells occupy most of the central portion of the notochord, and some cells extend the notochordal horn to both sides of the ventral nerve cord. Some notochord myofibrillar genes are not expressed in myotomes, suggesting an occurrence of myofibrillar genes that are preferentially expressed in notochord. On the other hand, non-myofibrillar cells contain dorsal, lateral, and ventral Müller cells, and all three express both hedgehog and Brachyury. This was confirmed by ISH, although expression of hedgehog in ventral Müller cells was minimal. In addition, dorsal Müller cells express neural transmission-related genes, suggesting an interaction with nerve cord. Lateral Müller cells express hedgehog and other signaling-related genes, suggesting an interaction with myotomes positioned lateral to the notochord. Ventral Müller cells also expressed genes for FGF- and EGF-related signaling, which may be associated with development of endoderm, ventral to the notochord. Lateral Müller cells were intermediate between dorsal/ventral Müller cells. Since vertebrate notochord contributes to patterning and differentiation of ectoderm (nerve cord), mesoderm (somite), and endoderm, this investigation provides evidence that an ancestral or original form of vertebrate notochord is present in extant cephalochordates.

A single-cell RNA-seq analysis of early larval cell-types of the starfish, Patiria pectinifera: Insights into evolution of the chordate body plan.

Ambulacrarians (echinoderms and hemichordates) are a sister group to chordates; thus, their larval cell-types may provide clues about evolution of chordate body plans. Although most genic information accumulated to date pertains to sea urchin embryogenesis, starfish embryogenesis represents a more ancestral mode than that of sea urchins. We performed single-cell RNA-seq analysis of cell-types from gastrulae and bipinnarial larvae of the starfish, Patiria pectinifera, and categorized them into 22 clusters, each of which is composed of cells with specific, shared profiles of development-relevant gene expression. Oral and aboral ectoderm, apical plate, hindgut or archenteron, midgut or intestine, pharynx, endomesoderm, stomodeum, and mesenchyme of the gastrulae, and neurons, ciliary bands, enterocoel and muscle of larvae were characterized by expression profiles of at least two relevant transcription factor genes and signaling molecular genes. Expression of Hox2, Hox7, Hox9/10, and Hox11/13b was detected in cells of clusters that form the larval enterocoel. By comparing homologous gene expression profiles in chordate embryos, we discuss and propose how the chordate body plan evolved from a deuterostome ancestor, from which the echinoderm body plan also evolved.

Impact of Direct Measurement of Small Dense Low-Density Lipoprotein Cholesterol for Long-Term Secondary Prevention in Patients with Stable Coronary Artery Disease.

BACKGROUND: This study investigated whether directly measured small dense low-density lipoprotein cholesterol (D-sdLDL-C) can predict long-term coronary artery disease (CAD) events compared with low-density lipoprotein cholesterol (LDL-C), non-high-density lipoprotein cholesterol (non-HDL-C), apolipoprotein B (apoB), and estimated small dense low-density lipoprotein cholesterol (E-sdLDL-C) determined by the Sampson equation in patients with stable CAD. METHODS: D-sdLDL-C measured at Showa University between 2010 and 2022, and E-sdLDL-C were evaluated in 790 male and 244 female patients with stable CAD. CAD events, defined as sudden cardiac death, onset of acute coronary syndrome, and/or need for coronary revascularization, were monitored for 12 years. Cutoff lipid levels were determined by receiver operating characteristic curves. RESULTS: CAD events were observed in 238 male and 67 female patients. The Kaplan-Meier event-free survival curves showed that patients with D-sdLDL-C ≥32.1 mg/dL (0.83 mmol/L) had an increased risk for CAD events (P = 0.007), whereas risk in patients with E-sdLDL-C ≥36.2 mg/dL (0.94 mmol/L) was not increased. In the group with high D-sdLDL-C, the multivariable-adjusted hazard ratio (HR) was 1.47 (95% CI, 1.15-1.89), and it remained significant after adjustment for LDL-C, non-HDL-C, or apoB and in patients treated with statins. HRs for high LDL-C, non-HDL-C, or apoB were not statistically significant after adjustment for high D-sdLDL-C. Higher D-sdLDL-C was associated with enhanced risk of high LDL-C, non-HDL-C, and apoB (HR 1.73; 95% CI, 1.27-2.37). CONCLUSIONS: Higher D-sdLDL-C can predict long-term recurrence of CAD in stable CAD patients independently of apoB and non-HDL-C. D-sdLDL-C is an independent risk enhancer for secondary CAD prevention, whereas E-sdLDL-C is not.UMIN-CTR Clinical Trial Number: UMIN000027504.

A single-cell RNA-seq analysis of Brachyury-expressing cell clusters suggests a morphogenesis-associated signal center of oral ectoderm in sea urchin embryos.

Brachyury is a T-box family transcription factor and plays pivotal roles in morphogenesis. In sea urchin embryos, Brachyury is expressed in the invaginating endoderm, and in the oral ectoderm of the invaginating mouth opening. The oral ectoderm is hypothesized to serve as a signaling center for oral (ventral)-aboral (dorsal) axis formation and to function as a ventral organizer. Our previous results of a single-cell RNA-seq (scRNA-seq) atlas of early Strongylocentrotus purpuratus embryos categorized the constituent cells into 22 clusters, in which the endoderm consists of three clusters and the oral ectoderm four clusters (Foster et al., 2020). Here we examined which clusters of cells expressed Brachyury in relation to the morphogenesis and the identity of the ventral organizer. Our results showed that cells of all three endoderm clusters expressed Brachyury in blastulae. Based on expression profiles of genes involved in the gene regulatory networks (GRNs) of sea urchin embryos, the three clusters are distinguishable, two likely derived from the Veg2 tier and one from the Veg1 tier. On the other hand, of the four oral-ectoderm clusters, cells of two clusters expressed Brachyury at the gastrula stage and genes that are responsible for the ventral organizer at the late blastula stage, but the other two clusters did not. At a single-cell level, most cells of the two oral-ectoderm clusters expressed organizer-related genes, nearly a half of which coincidently expressed Brachyury. This suggests that the ventral organizer contains Brachyury-positive cells which invaginate to form the stomodeum. This scRNA-seq study therefore highlights significant roles of Brachyury-expressing cells in body-plan formation of early sea urchin embryos, though cellular and molecular mechanisms for how Brachyury functions in these processes remain to be elucidated in future studies.

Left-Right Reversal Recurrently Evolved Regardless of Diaphanous-Related Formin Gene Duplication or Loss in Snails.

Bilateria exhibit whole-body handedness in internal structure. This left-right polarity is evolutionarily conserved with virtually no reversed extant lineage, except in molluscan Gastropoda. Phylogenetically independent snail groups contain both clockwise-coiled (dextral) and counterclockwise-coiled (sinistral) taxa that are reversed from each other in bilateral handedness as well as in coiling direction. Within freshwater Hygrophila, Lymnaea with derived dextrality have diaphanous related formin (diaph) gene duplicates, while basal sinistral groups possess one diaph gene. In terrestrial Stylommatophora, dextral Bradybaena also have diaph duplicates. Defective maternal expression of one of those duplicates gives rise to sinistral hatchlings in Lymnaea and handedness-mixed broods in Bradybaena, through polarity change in spiral cleavage of embryos. These findings led to the hypothesis that diaph duplication was crucial for the evolution of dextrality by reversal. The present study discovered that diaph duplication independently occurred four times and its duplicate became lost twice in gastropods. The dextrality of Bradybaena represents the ancestral handedness conserved across gastropods, unlike the derived dextrality of Lymnaea. Sinistral lineages recurrently evolved by reversal regardless of whether diaph had been duplicated. Amongst the seven formin gene subfamilies, diaph has most thoroughly been conserved across eukaryotes of the 14 metazoan phyla and choanoflagellate. Severe embryonic mortalities resulting from insufficient expression of the duplicate in both of Bradybaena and Lymnaea also support that diaph duplicates bare general roles for cytoskeletal dynamics other than controlling spiralian handedness. Our study rules out the possibility that diaph duplication or loss played a primary role for reversal evolution.

An environmental DNA metabarcoding survey reveals generic-level occurrence of scleractinian corals at reef slopes of Okinawa Island.

Coral reefs have the highest biodiversity of all marine ecosystems in tropical and subtropical oceans. However, scleractinian corals, keystone organisms of reef productivity, are facing a crisis due to climate change and anthropogenic activities. A broad survey of reef-building corals is essential for worldwide reef preservation. To this end, direct observations made by coral-specialist divers might be supported by another robust method. We improved a recently devised environmental DNA (eDNA) metabarcoding method to identify more than 43 scleractinian genera by sampling 2 l of surface seawater above reefs. Together with direct observations by divers, we assessed the utility of eDNA at 63 locations spanning approximately 250 km near Okinawa Island. Slopes of these islands are populated by diverse coral genera, whereas shallow 'moats' sustain fewer and less varied coral taxa. Major genera recorded by divers included Acropora, Pocillopora, Porites and Montipora, the presence of which was confirmed by eDNA analyses. In addition, eDNA identified more genera than direct observations and documented the presence of previously unrecorded species. This scleractinian coral-specific eDNA method promises to be a powerful tool to survey coral reefs broadly, deeply and robustly.

Transcriptomic evidence for Brachyury expression in the caudal tip region of adult Ptychodera flava (Hemichordata).

Most metazoans have a single copy of the T-box transcription factor gene Brachyury. This gene is expressed in cells of the blastopore of late blastulae and the archenteron invagination region of gastrulae. It appears to be crucial for gastrulation and mesoderm differentiation of embryos. Although this expression pattern is shared by most deuterostomes, Brachyury expression has not been reported in adult stages. Here we show that Brachyury of an indirect developer, the hemichordate acorn worm Ptychodera flava, is expressed not only in embryonic cells, but also in cells of the caudal tip (anus) region of adults. This spatially restricted expression, shown by whole-mount in situ hybridization, was confirmed by Iso-Seq RNA sequencing and single-cell RNA-seq (scRNA-seq) analysis. Iso-Seq analysis showed that gene expression occurs only in the caudal region of adults, but not in anterior regions, including the stomochord. scRNA-seq analysis showed a cluster that contained Brachyury-expressing cells comprising epidermis- and mesoderm-related cells, but which is unlikely to be associated with the nervous system or muscle. Although further investigation is required to examine the roles of Brachyury in adults, this study provides important clues for extending studies on Brachyury expression involved in development of the most posterior region of deuterostomes.

The crown-of-thorns starfish genome as a guide for biocontrol of this coral reef pest.

The crown-of-thorns starfish (COTS, the Acanthaster planci species group) is a highly fecund predator of reef-building corals throughout the Indo-Pacific region. COTS population outbreaks cause substantial loss of coral cover, diminishing the integrity and resilience of reef ecosystems. Here we sequenced genomes of COTS from the Great Barrier Reef, Australia and Okinawa, Japan to identify gene products that underlie species-specific communication and could potentially be used in biocontrol strategies. We focused on water-borne chemical plumes released from aggregating COTS, which make the normally sedentary starfish become highly active. Peptide sequences detected in these plumes by mass spectrometry are encoded in the COTS genome and expressed in external tissues. The exoproteome released by aggregating COTS consists largely of signalling factors and hydrolytic enzymes, and includes an expanded and rapidly evolving set of starfish-specific ependymin-related proteins. These secreted proteins may be detected by members of a large family of olfactory-receptor-like G-protein-coupled receptors that are expressed externally, sometimes in a sex-specific manner. This study provides insights into COTS-specific communication that may guide the generation of peptide mimetics for use on reefs with COTS outbreaks.

In Vitro Phagocytosis of Different Dinoflagellate Species by Coral Cells.

Coral-dinoflagellate symbiosis is a unique biological phenomenon, in which animal cells engulf single-celled photosynthetic algae and maintain them in their cytoplasm mutualistically. Studies are needed to reveal the complex mechanisms involved in symbiotic processes, but it is difficult to answer these questions using intact corals. To tackle these issues, our previous studies established an in vitro system of symbiosis between cells of the scleractinian coral Acropora tenuis and the dinoflagellate Breviolum minutum, and showed that corals direct phagocytosis, while algae are likely engulfed by coral cells passively. Several genera of the family Symbiodiniaceae can establish symbioses with corals, but the symbiotic ratio differs depending on the dinoflagellate clades involved. To understand possible causes of these differences, this study examined whether cultured coral cells show phagocytotic activity with various dinoflagellate strains similar to those shown by intact A. tenuis. We found that (a) A. tenuis larvae incorporate Symbiodinium and Breviolum, but not Cladocopium, and very few Effrenium, (b) cultured coral cells engulfed all four species but the ratio of engulfment was significantly higher with Symbiodinium and Breviolum than Cladocopium and Effrenium, (c) cultured coral cells also phagocytosed inorganic latex beads differently than they do dinoflagellates . It is likely that cultured coral cells preferentially phagocytose Symbiodinium and Breviolum, suggesting that specific molecular mechanisms involved in initiation of symbiosis should be investigated in the future.

Eighteen Coral Genomes Reveal the Evolutionary Origin of Acropora Strategies to Accommodate Environmental Changes.

The genus Acropora comprises the most diverse and abundant scleractinian corals (Anthozoa, Cnidaria) in coral reefs, the most diverse marine ecosystems on Earth. However, the genetic basis for the success and wide distribution of Acropora are unknown. Here, we sequenced complete genomes of 15 Acropora species and 3 other acroporid taxa belonging to the genera Montipora and Astreopora to examine genomic novelties that explain their evolutionary success. We successfully obtained reasonable draft genomes of all 18 species. Molecular dating indicates that the Acropora ancestor survived warm periods without sea ice from the mid or late Cretaceous to the Early Eocene and that diversification of Acropora may have been enhanced by subsequent cooling periods. In general, the scleractinian gene repertoire is highly conserved; however, coral- or cnidarian-specific possible stress response genes are tandemly duplicated in Acropora. Enzymes that cleave dimethlysulfonioproprionate into dimethyl sulfide, which promotes cloud formation and combats greenhouse gasses, are the most duplicated genes in the Acropora ancestor. These may have been acquired by horizontal gene transfer from algal symbionts belonging to the family Symbiodiniaceae, or from coccolithophores, suggesting that although functions of this enzyme in Acropora are unclear, Acropora may have survived warmer marine environments in the past by enhancing cloud formation. In addition, possible antimicrobial peptides and symbiosis-related genes are under positive selection in Acropora, perhaps enabling adaptation to diverse environments. Our results suggest unique Acropora adaptations to ancient, warm marine environments and provide insights into its capacity to adjust to rising seawater temperatures.

Genomic analysis of a reef-building coral, Acropora digitifera, reveals complex population structure and a migration network in the Nansei Islands, Japan.

Understanding the structure and connectivity of coral populations is fundamental for developing marine conservation policies, especially in patchy environments such as archipelagos. The Nansei Islands, extending more than 1000 km in southwestern Japan, are characterized by high levels of biodiversity and endemism, supported by coral reefs, which make this region ideal for assessing genetic attributes of coral populations. In this study, we conducted population genomic analyses based on genome-wide, single-nucleotide polymorphisms (SNPs) of Acropora digitifera, a common species in the Nansei Islands. By merging newly obtained genome resequencing data with previously published data, we identified more than 4 million genome-wide SNPs in 303 colonies collected at 22 locations, with sequencing coverage ranging from 3.91× to 27.41×. While population structure analyses revealed genetic similarities between the southernmost and northernmost locations, separated by >1000 km, several subpopulations in intermediate locations suggested limited genetic admixture, indicating conflicting migration tendencies in the Nansei Islands. Although migration networks revealed a general tendency of northward migration along the Kuroshio Current, a substantial amount of southward migration was also detected, indicating important contributions of minor ocean currents to coral larval dispersal. Moreover, heterogeneity in the transition of effective population sizes among locations suggests different histories for individual subpopulations. The unexpected complexity of both past and present population dynamics in the Nansei Islands implies that heterogeneity of ocean currents and local environments, past and present, have influenced the population structure of this species, and similar unexpected population complexities may be expected for other marine species with similar reproductive modes.

Coral larvae suppress heat stress response during the onset of symbiosis decreasing their odds of survival.

The endosymbiosis between most corals and their photosynthetic dinoflagellate partners begins early in the host life history, when corals are larvae or juvenile polyps. The capacity of coral larvae to buffer climate-induced stress while in the process of symbiont acquisition could come with physiological trade-offs that alter behaviour, development, settlement and survivorship. Here we examined the joint effects of thermal stress and symbiosis onset on colonization dynamics, survival, metamorphosis and host gene expression of Acropora digitifera larvae. We found that thermal stress decreased symbiont colonization of hosts by 50% and symbiont density by 98.5% over 2 weeks. Temperature and colonization also influenced larval survival and metamorphosis in an additive manner, where colonized larvae fared worse or prematurely metamorphosed more often than noncolonized larvae under thermal stress. Transcriptomic responses to colonization and thermal stress treatments were largely independent, while the interaction of these treatments revealed contrasting expression profiles of genes that function in the stress response, immunity, inflammation and cell cycle regulation. The combined treatment either cancelled or lowered the magnitude of expression of heat-stress responsive genes in the presence of symbionts, revealing a physiological cost to acquiring symbionts at the larval stage with elevated temperatures. In addition, host immune suppression, a hallmark of symbiosis onset under ambient temperature, turned to immune activation under heat stress. Thus, by integrating the physical environment and biotic pressures that mediate presettlement event in corals, our results suggest that colonization may hinder larval survival and recruitment under projected climate scenarios.

Transcriptomes of Giant Sea Anemones from Okinawa as a Tool for Understanding Their Phylogeny and Symbiotic Relationships with Anemonefish.

The relationship between anemonefish and sea anemones is one of the most emblematic examples of mutualistic symbiosis in coral reefs. Although this is a textbook example, the major aspects of this symbiosis are still not fully understood in mechanistic terms. Moreover, since studies of this relationship have usually been focused on anemonefish, much less is known about giant sea anemones, their similarities, their phylogenetic relationships, and their differences at the molecular level. Since both partners of the symbiotic relationship are important, we decided to explore this well-known phenomenon from the perspective of giant sea anemones. Here, we report reference transcriptomes for all seven species of giant sea anemones that inhabit fringing reefs of Okinawa (Japan) and serve as hosts for six species of local anemonefish. Transcriptomes were used to investigate their phylogenetic relations, genetic differences and repertoires of nematocyte-specific proteins. Our data support the presence of three distinct groups corresponding to three genera: Entacmaea, Heteractis, and Stichodactyla. The basal position among the three groups belongs to Entacmaea, which was the first to diverge from a common ancestor. While the magnitude of genetic difference between the representatives of Entacmaea and Stichodactyla is large, intra-specific variation within Stichodactyla is much smaller and seems to result from recent speciation events. Our data reconfirms that Heteractis magnifica belongs to the genus Stichodactyla, despite an overall morphological similarity with representatives of the genus Heteractis. The availability of reference transcriptomes will facilitate further research into the fascinating relationship between sea anemones and anemonefish.

New azodyrecins identified by a genome mining-directed reactivity-based screening.

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.

Identification of functional cytochrome P450 and ferredoxin from Streptomyces sp. EAS-AB2608 by transcriptional analysis and their heterologous expression.

Bioconversion using microorganisms and their enzymes is an important tool in many industrial fields. The discovery of useful new microbial enzymes contributes to the development of industries utilizing bioprocesses. Streptomyces sp. EAS-AB2608, isolated from a soil sample collected in Japan, can convert the tetrahydrobenzotriazole CPD-1 (a selective positive allosteric modulator of metabotropic glutamate receptor 5) to its hydroxylated form at the C4-(R) position. The current study was performed to identify the genes encoding the enzymes involved in CPD-1 bioconversion and to verify their function. To identify gene products responsible for the conversion of CPD-1, we used RNA sequencing to analyze EAS-AB2608; from its 8333 coding sequences, we selected two genes, one encoding cytochrome P450 (easab2608_00800) and the other encoding ferredoxin (easab2608_00799), as encoding desirable gene products involved in the bioconversion of CPD-1. The validity of this selection was tested by using a heterologous expression approach. A bioconversion assay using genetically engineered Streptomyces avermitilis SUKA24 ∆saverm3882 ∆saverm7246 co-expressing the two selected genes (strain ES_SUKA_63) confirmed that these gene products had hydroxylation activity with respect to CPD-1, indicating that they are responsible for the conversion of CPD-1. Strain ES_SUKA_63 also showed oxidative activity toward other compounds and therefore might be useful not only for bioconversion of CPD-1 but also as a tool for synthesis of drug metabolites and in optimization studies of various pharmaceutical lead compounds. We expect that this approach will be useful for bridging the gap between the latest enzyme optimization technologies and conventional enzyme screening using microorganisms. KEY POINTS: • Genes easab2608_00800 (cyp) and easab2608_00799 (fdx) were selected by RNA-Seq. • Selection validity was evaluated by an engineered S. avermitilis expression system. • Strain ES_SUKA_63 showed oxidative activity toward CPD-1 and other compounds.

Integrated omics unveil the secondary metabolic landscape of a basal dinoflagellate.

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.

Comparative genomics of four strains of the edible brown alga, Cladosiphon okamuranus.

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.

ORTHOSCOPE: An Automatic Web Tool for Phylogenetically Inferring Bilaterian Orthogroups with User-Selected Taxa.

Identification of orthologous or paralogous relationships of coding genes is fundamental to all aspects of comparative genomics. For accurate identification of orthologs among deeply diversified bilaterian lineages, precise estimation of gene trees is indispensable, given the complicated histories of genes over millions of years. By estimating gene trees, orthologs can be identified as members of an orthogroup, a set of genes descended from a single gene in the last common ancestor of all the species being considered. In addition to comparisons with a given species tree, purposeful taxonomic sampling increases the accuracy of gene tree estimation and orthogroup identification. Although some major phylogenetic relationships of bilaterians are gradually being unraveled, the scattering of published genomic data among separate web databases is becoming a significant hindrance to identification of orthogroups with appropriate taxonomic sampling. By integrating more than 250 metazoan gene models predicted in genome projects, we developed a web tool called ORTHOSCOPE to identify orthogroups of specific protein-coding genes within major bilaterian lineages. ORTHOSCOPE allows users to employ several sequences of a specific molecule and broadly accepted nodes included in a user-specified species tree as queries and to evaluate the reliability of estimated orthogroups based on topologies and node support values of estimated gene trees. A test analysis using data from 36 bilaterians was accomplished within 140 s. ORTHOSCOPE results can be used to evaluate orthologs identified by other stand-alone programs using genome-scale data. ORTHOSCOPE is freely available at https://www.orthoscope.jp or https://github.com/jun-inoue/orthoscope (last accessed December 28, 2018).

Heterologous Expression of the Biosynthetic Gene Cluster for Verticilactam and Identification of Analogues.

Verticilactam and the new geometric isomers, verticilactams B and C, were produced by heterologous expression of the biosynthetic gene cluster for verticilactam using the Streptomyces avermitilis SUKA17 strain. Only verticilactam, a compound with a characteristic ß-ketoamide unit within a 16-membered polyketide macrolactam conjugated with an octalin skeleton, had been previously reported having been isolated from Streptomyces spiroverticillatus JC-8444. In this report, minor verticilactam derivatives were isolated from the transformed strain, and their structures elucidated by spectral analysis. Verticilactam B was a geometric isomer at Δ17 and Δ19, and verticilactam C was the Δ19 and Δ21 isomer. In addition, the absolute configuration of verticilactam was confirmed by ECD analysis and NMR chemical shifts. The stereochemistry assignments of the hydroxy groups at C-10 and C-12 were supported by the domain organization of the polyketide synthase identified in the verticilactam gene cluster. Verticilactam showed moderate activity against the malaria parasite Plasmodium falciparum 3D7 strain with no significant cytotoxicity or antimicrobial effects.