Transcriptional responses of the marine diatom Chaetoceros tenuissimus to phosphate deficiency.

The planktonic diatom Chaetoceros tenuissimus sometimes forms blooms in coastal surface waters where dissolved inorganic phosphorus (P) is typically deficient. To understand the molecular mechanisms for survival under P-deficient conditions, we compared whole transcripts and metabolites with P-sufficient conditions using stationary growth cells. Under P-deficient conditions, cell numbers and photosynthetic activities decreased as cells entered the stationary growth phase, with downregulation of transcripts related to the Calvin cycle and glycolysis/gluconeogenesis. Therefore, metabolites varied across nutritional conditions. Alkaline phosphatase, phosphodiesterase, phytase, phosphate transporter, and transcription factor genes were drastically upregulated under dissolved inorganic P deficiency. Genes related to phospholipid degradation and nonphospholipid synthesis were also upregulated. These results indicate that C. tenuissimus rearranges its membrane composition from phospholipids to nonphospholipids to conserve phosphate. To endure in P-deficient conditions, C. tenuissimus modifies its gene responses, suggesting a potential survival strategy in nature.

Single-Cell Level Raman Molecular Profiling Reveals the Classification of Growth Phases of Chaetoceros tenuissimus.

Harmful algal blooms (HABs) are a natural phenomenon caused by outbreaks of algae, resulting in serious problems for aquatic ecosystems and the coastal environment. Chaetoceros tenuissimus (C. tenuissimus) is one of the diatoms responsible for HABs. The growth curve of C. tenuissimus can be observed from beginning to end of HABs: therefore, detailed analysis is necessary to characterize each growth phase of C. tenuissimus. It is important to examine the phenotype of each diatom cell individually, as they display heterogeneity even in the same growth phase. Raman spectroscopy is a label-free technique to elucidate biomolecular profiles and spatial information at the cellular level. Multivariate data analysis (MVA) is an efficient method for the analysis of complicated Raman spectra, to identify molecular features. Here, we utilized Raman microspectroscopy to identify the molecular information of each diatom cell, at the single-cell level. The MVA, together with a support vector machine, which is a machine learning technique, allowed the classification of proliferating and nonproliferating cells. The classification includes polyunsaturated fatty acids such as linoleic acid, eicosapentaenoic acid, and docosahexaenoic acid. This study indicated that Raman spectroscopy is an appropriate technique to examine C. tenuissimus at the single-cell level, providing relevant data to assess the correlation between the molecular details obtained from the Raman analysis, at each growth phase.

Chronological age-related metabolome responses in the dinoflagellate Karenia mikimotoi, can predict future bloom demise.

Karenia mikimotoi is a common harmful algal bloom (HAB)-forming dinoflagellate and has caused severe financial loss in aquaculture. There are limited metabolomic studies on dinoflagellate biology. Here, we examined alterations in metabolic profiles over the growth curve of K. mikimotoi under nitrogen or phosphorus deficiency and further explored a key criterion for the diagnosis of late stationary phase to identify when the dinoflagellate cells will enter bloom demise. The results demonstrate the differential expression of metabolites for coping with chronological aging or nutrient deprivation. Furthermore, an increase in the glucose to glycine ratio in the late stationary phase was indicative of dinoflagellate cells entering bloom demise; this was also detected in the cultured diatom, Chaetoceros tenuissimus, indicating that this may be the general criterion for phytoplankton species. Our findings provide insights regarding chronological aging and the criterion for the prediction of phytoplankton bloom demise.

Structural Insights into Common and Host-Specific Receptor-Binding Mechanisms in Algal Picorna-like Viruses.

Marnaviridae viruses are abundant algal viruses that regulate the dynamics of algal blooms in aquatic environments. They employ a narrow host range because they merely lyse their algal host species. This host-specific lysis is thought to correspond to the unique receptor-binding mechanism of the Marnaviridae viruses. Here, we present the atomic structures of the full and empty capsids of Chaetoceros socialis forma radians RNA virus 1 built-in 3.0 Å and 3.1 Å cryo-electron microscopy maps. The empty capsid structure and the structural variability provide insights into its assembly and uncoating intermediates. In conjunction with the previously reported atomic model of the Chaetoceros tenuissimus RNA virus type II capsid, we have identified the common and diverse structural features of the VP1 surface between the Marnaviridae viruses. We have also tested the potential usage of AlphaFold2 for structural prediction of the VP1s and a subsequent structural phylogeny for classifying Marnaviridae viruses by their hosts. These findings will be crucial for inferring the host-specific receptor-binding mechanism in Marnaviridae viruses.

Primordial Capsid and Spooled ssDNA Genome Structures Unravel Ancestral Events of Eukaryotic Viruses.

Marine algae viruses are important for controlling microorganism communities in the marine ecosystem and played fundamental roles during the early events of viral evolution. Here, we have focused on one major group of marine algae viruses, the single-stranded DNA (ssDNA) viruses from the Bacilladnaviridae family. We present the capsid structure of the bacilladnavirus Chaetoceros tenuissimus DNA virus type II (CtenDNAV-II), determined at 2.4-Å resolution. A structure-based phylogenetic analysis supported the previous theory that bacilladnaviruses have acquired their capsid protein via horizontal gene transfer from a ssRNA virus. The capsid protein contains the widespread virus jelly-roll fold but has additional unique features; a third ß-sheet and a long C-terminal tail. Furthermore, a low-resolution reconstruction of the CtenDNAV-II genome revealed a partially spooled structure, an arrangement previously only described for dsRNA and dsDNA viruses. Together, these results exemplify the importance of genetic recombination for the emergence and evolution of ssDNA viruses and provide important insights into the underlying mechanisms that dictate genome organization. IMPORTANCE Single-stranded DNA (ssDNA) viruses are an extremely widespread group of viruses that infect diverse hosts from all three domains of life, consequently having great economic, medical, and ecological importance. In particular, bacilladnaviruses are highly abundant in marine sediments and greatly influence the dynamic appearance and disappearance of certain algae species. Despite the importance of ssDNA viruses and the last couple of years' advancements in cryo-electron microscopy, structural information on the genomes of ssDNA viruses remains limited. This paper describes two important achievements: (i) the first atomic structure of a bacilladnavirus capsid, which revealed that the capsid protein gene presumably was acquired from a ssRNA virus in early evolutionary events; and (ii) the structural organization of a ssDNA genome, which retains a spooled arrangement that previously only been observed for double-stranded viruses.

Detection and Characterization of RNA Viruses in Red Macroalgae (Bangiaceae) and Their Food Product (Nori Sheets).

Persistent RNA viruses, which have been suggested to form symbiotic relationships with their hosts, have been reported to occur in eukaryotes, such as plants, fungi, and algae. Based on empirical findings, these viruses may also be present in commercially cultivated macroalgae. Accordingly, the present study aimed to screen red macroalgae (family Bangiaceae conchocelis and Neopyropia yezoensis thallus) and processed nori sheets (N. yezoensis) for persistent RNA viruses using fragmented and primer-ligated dsRNA sequencing (FLDS) and targeted reverse transcription PCR (RT-PCR). A Totiviridae-related virus was detected in the conchocelis of Neoporphyra haitanensis, which is widely cultivated in China, while two Mitoviridae-related viruses were found in several conchocelis samples and all N. yezoensis-derived samples (thallus and nori sheets). Mitoviridae-related viruses in N. yezoensis are widespread among cultivated species and not expected to inhibit host growth. Mitoviridae-related viruses were also detected in several phylogenetically distant species in the family Bangiaceae, which suggests that these viruses persisted and coexist in the family Bangiaceae over a long period of time. The present study is the first to report persistent RNA viruses in nori sheets and their raw materials.

Characterization of Chaetoceros lorenzianus-infecting DNA virus-derived promoters of genes from open reading frames of unknown function in Phaeodactylum tricornutum.

Promoters are key elements for the regulation of gene expression. Recently, we investigated the activity of promoters derived from marine diatom-infecting viruses (DIVs) in marine diatoms. Previously, we focused on potential promoter regions of the replication-associated protein gene and the capsid protein gene of the DIVs. In addition to these genes, two genes of unknown function (VP1 and VP4 genes) have been found in the DIV genomes. In this study, the promoter regions of the VP1 gene and VP4 gene derived from a Chaetoceros lorenzianus-infecting DNA virus (named ClP3 and ClP4, respectively) were newly isolated. ClP4 was found to be a constitutive promoter and displayed the highest activity. In particular, the 3' region of ClP4 (ClP4 3' region) showed a higher promoter activity than full-length ClP4. The ClP4 3' region might involve high-level promoter activity of ClP4. In addition, the ClP4 3' region may be useful for substance production and metabolic engineering of diatoms.

The genome of the diatom Chaetoceros tenuissimus carries an ancient integrated fragment of an extant virus.

Diatoms are one of the most prominent oceanic primary producers and are now recognized to be distributed throughout the world. They maintain their population despite predators, infections, and unfavourable environmental conditions. One of the smallest diatoms, Chaetoceros tenuissimus, can coexist with infectious viruses during blooms. To further understand this relationship, we sequenced the C. tenuissimus strain NIES-3715 genome. A gene fragment of a replication-associated gene from the infectious ssDNA virus (designated endogenous virus-like fragment, EVLF) was found to be integrated into each 41 Mb of haploid assembly. In addition, the EVLF was transcriptionally active and conserved in nine other C. tenuissimus strains from different geographical areas, although the primary structures of their proteins varied. The phylogenetic tree further suggested that the EVLF was acquired by the ancestor of C. tenuissimus. Additionally, retrotransposon genes possessing a reverse transcriptase function were more abundant in C. tenuissimus than in Thalassiosira pseudonana and Phaeodactylum tricornutum. Moreover, a target site duplication, a hallmark for long interspersed nuclear element retrotransposons, flanked the EVLF. Therefore, the EVLF was likely integrated by a retrotransposon during viral infection. The present study provides further insights into the diatom-virus evolutionary relationship.

ICTV Virus Taxonomy Profile: Marnaviridae 2021.

The family Marnaviridae comprises small non-enveloped viruses with positive-sense RNA genomes of 8.6-9.6 kb. Isolates infect marine single-celled eukaryotes (protists) that come from diverse lineages. Some members are known from metagenomic studies of ocean virioplankton, with additional unclassified viruses described from metagenomic datasets derived from marine and freshwater environments. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Marnaviridae, which is available at ictv.global/report/marnaviridae.

RNA Viruses in Aquatic Unicellular Eukaryotes.

Increasing sequence information indicates that RNA viruses constitute a major fraction of marine virus assemblages. However, only 12 RNA virus species have been described, infecting known host species of marine single-celled eukaryotes. Eight of these use diatoms as hosts, while four are resident in dinoflagellate, raphidophyte, thraustochytrid, or prasinophyte species. Most of these belong to the order Picornavirales, while two are divergent and fall into the families Alvernaviridae and Reoviridae. However, a very recent study has suggested that there is extraordinary diversity in aquatic RNA viromes, describing thousands of viruses, many of which likely use protist hosts. Thus, RNA viruses are expected to play a major ecological role for marine unicellular eukaryotic hosts. In this review, we describe in detail what has to date been discovered concerning viruses with RNA genomes that infect aquatic unicellular eukaryotes.

Chronological distribution of dinoflagellate-infecting RNA virus in marine sediment core.

A bivalve-killing marine dinoflagellate, Heterocapsa circularisquama, is susceptible to the infectious single-stranded RNA virus, Heterocapsa circularisquama RNA virus (HcRNAV). The ecological relationship between H. circularisquama and HcRNAV was intensively studied from 2001 through 2005; however, only limited data are available for the ecological dynamics of HcRNAV before 2001. In this study, we applied radiometric dating and reverse transcription PCR (RT-PCR) to determine the chronological distribution of HcRNAV in a marine sediment core sampled from the Uranouchi Inlet, Kochi, Japan, where H. circularisquama was first discovered. Our results show that HcRNAV had existed in the inlet long before its first bloom in 1988. Furthermore, five HcRNAV variants, phylogenetically distinguishable based on the nucleotide sequence of the major capsid protein (MCP) gene, were identified. These variants were found to be distributed throughout the core over time, suggesting that the HcRNAV sequences registered in the NCBI database are only a portion of the variants that have emerged in the history of HcRNAV diversification. Herein, we have verified the applicability of the retrospective approach for speculating the distribution of algal RNA viruses over time in aquatic environments.

Growth Rate-dependent Cell Death of Diatoms due to Viral Infection and Their Subsequent Coexistence in a Semi-continuous Culture System.

Viral infections are a major factor in diatom cell death. However, the effects of viruses on diatom dynamics remain unclear. Based on laboratory studies, it is hypothesized that virus-induced diatom mortality is dependent on the diatom growth rate. The present study aimed to elucidate the relationship between the diatom growth rate and virus-induced mortality using model systems of the marine planktonic diatom, Chaetoceros tenuissimus and its infectious viruses. We also examined the fate of diatom populations in a semi-continuous dilution culture system, in which host growth rates were controlled at 0.69, 2.08, and 3.47 day-1. Diatom populations gradually decreased following the viral inoculation of each culture system, and virus-induced mortality inversely correlated with the diatom growth rate. Furthermore, the viral burst size was slightly higher in lower growth rate cultures. These results suggested that the host physiological status related to the growth rate affected viral infection and proliferation. Diatom populations were not completely lysed or washed out in any of the dilution systems; they showed steady growth in the presence of infectious viruses. This may be partially explained by defective interference particles from viruses and cell debris. The present results indicate that diatoms in dilution environments maintain their populations, even under viral pressure. Moreover, diatom populations with a low growth rate may partially sustain higher growth populations through nutrient recycling following virus-induced cell death. The results of the present study provide insights into diatom dynamics in natural environments in the presence of infectious viruses.

The possibility of using marine diatom-infecting viral promoters for the engineering of marine diatoms.

Marine diatoms constitute a major group of unicellular photosynthetic eukaryotes. Diatoms are widely applicable for both basic studies and applied studies. Molecular tools and techniques have been developed for diatom research. Among these tools, several endogenous gene promoters (e.g., the fucoxanthin chlorophyll a/c-binding protein gene promoter) have become available for expressing transgenes in diatoms. Gene promoters that drive transgene expression at a high level are very important for the metabolic engineering of diatoms. Various marine diatom-infecting viruses (DIVs), including both DNA viruses and RNA viruses, have recently been isolated, and their genome sequences have been characterized. Promoters from viruses that infect plants and mammals are widely used as constitutive promoters to achieve high expression of transgenes. Thus, we recently investigated the activity of promoters derived from marine DIVs in the marine diatom, Phaeodactylum tricornutum. We discuss novel viral promoters that will be useful for the future metabolic engineering of diatoms.

Viral RNA Genomes Identified from Marine Macroalgae and a Diatom.

Protists provide insights into the diversity and function of RNA viruses in marine systems. Among them, marine macroalgae are good targets for RNA virome analyses because they have a sufficient biomass in nature. However, RNA viruses in macroalgae have not yet been examined in detail, and only partial genome sequences have been reported for the majority of RNA viruses. Therefore, to obtain further insights into the distribution and diversity of RNA viruses associated with marine protists, we herein examined RNA viruses in macroalgae and a diatom. We report the putative complete genome sequences of six novel RNA viruses from two marine macroalgae and one diatom holobiont. Four viruses were not classified into established viral genera or families. Furthermore, a virus classified into Totiviridae showed a genome structure that has not yet been reported in this family. These results suggest that a number of distinct RNA viruses are widespread in a broad range of protists.

Capsid Structure of a Marine Algal Virus of the Order Picornavirales.

The order Picornavirales includes viruses that infect different kinds of eukaryotes and that share similar properties. The capsid proteins (CPs) of viruses in the order that infect unicellular organisms, such as algae, presumably possess certain characteristics that have changed little over the course of evolution, and thus these viruses may resemble the Picornavirales ancestor in some respects. Herein, we present the capsid structure of Chaetoceros tenuissimus RNA virus type II (CtenRNAV-II) determined using cryo-electron microscopy at a resolution of 3.1 Å, the first alga virus belonging to the family Marnaviridae of the order Picornavirales A structural comparison to related invertebrate and vertebrate viruses revealed a unique surface loop of the major CP VP1 that had not been observed previously, and further, revealed that another VP1 loop obscures the so-called canyon, which is a host-receptor binding site for many of the mammalian Picornavirales viruses. VP2 has an N-terminal tail, which has previously been reported as a primordial feature of Picornavirales viruses. The above-mentioned and other critical structural features provide new insights on three long-standing theories about Picornavirales: (i) the canyon hypothesis, (ii) the primordial VP2 domain swap, and (iii) the hypothesis that alga Picornavirales viruses could share characteristics with the Picornavirales ancestor.IMPORTANCE Identifying the acquired structural traits in virus capsids is important for elucidating what functions are essential among viruses that infect different hosts. The Picornavirales viruses infect a broad spectrum of hosts, ranging from unicellular algae to insects and mammals and include many human pathogens. Those viruses that infect unicellular protists, such as algae, are likely to have undergone fewer structural changes during the course of evolution compared to those viruses that infect multicellular eukaryotes and thus still share some characteristics with the Picornavirales ancestor. This article describes the first atomic capsid structure of an alga Marnavirus, CtenRNAV-II. A comparison to capsid structures of the related invertebrate and vertebrate viruses identified a number of structural traits that have been functionally acquired or lost during the course of evolution. These observations provide new insights on past theories on the viability and evolution of Picornavirales viruses.

Metabolomics-based approach to explore growth phase-dependent markers in cultured diatom Chaetoceros tenuissimus.

Chaetoceros tenuissimus is a cosmopolitan marine diatom whose metabolism has been little investigated. In this study, we examined the alterations of the metabolite profile between different growth phases (exponential and stationary phase) in cultured C. tenuissimus and identified growth phase-dependent candidate marker metabolite. First, the preparation methodology was optimized, focusing on extraction solvent. Metabolites of the cultured diatom (exponential phase 2.4 × 106 cells/mL, stationary phase 3.1 × 106 cells/mL) were extracted using two solvents with different constituents (solvent 1, methanol:H2O:chloroform = 5:2:2; solvent 2, methanol:ethanol:chloroform = 1:3:1). Gas chromatography mass spectrometry (GC/MS)-based metabolomics successfully detected 43 water-soluble metabolites in both solvents. The metabolic features were dependent on the growth phase: amino acid levels were higher in the exponential phase, whereas sugars and alcohols were more abundant in the stationary phase. Solvent 1 was superior in the recovery of the candidate metabolite that had a retention time of 18.13 min and predominantly contributed to discrimination between the growth phases; the metabolite level was higher in the stationary phase than in the exponential phase. The candidate metabolite was identified as mannonic acid by using GC/MS and liquid chromatography tandem mass spectrometry. Higher levels of mannonic acid during the stationary phase were also observed in other three diatom species. This study provides further insight into the use of metabolomics in the evaluation of physiological conditions of diatoms and suggests that mannonic acid content is a potential biomarker of the growth phase in cultured diatom cells.

Visualization of a Dinoflagellate-Infecting Virus HcDNAV and Its Infection Process.

HcDNAV (a type species of Genus Dinodnavirus) is a large double-stranded DNA virus, which lytically infects the bloom-forming marine microalga Heterocapsa circularisquama Horiguchi (Dinophyceae). In the present study, detailed observation of the HcDNAV particle and its infection process was conducted via field emission scanning electron microscopy (FE-SEM) and epifluorescence microscopy (EFM). Each five-fold vertex of the icosahedral virion was decorated with a protrusion, which may be related to the entry process of HcDNAV into the host. The transverse groove of host cells is proposed to be the main virus entry site. A visible DAPI-stained region, which is considered to be the viroplasm (virus factory), appeared in close proximity to the host nucleus at 11 h post infection (hpi); the putative viral DAPI signal was remarkably enlarged at 11⁻30 hpi. It was kidney-shaped at 13⁻15 hpi, horseshoe-shaped at 20 hpi, doughnut-shaped at 30 hpi, and changed into a three-dimensionally complicated shape at 51⁻53 hpi, by which time most parts of the host cell were occupied by the putative viral DAPI signal. While the virions were within the viroplasm, they were easily distinguishable by their vertex protrusions by FE-SEM.

Development of a method to assess the ichthyotoxicity of the harmful marine microalgae Karenia spp. using gill cell cultures from red sea bream (Pagrus major ).

The present study reports the development of a method to investigate ichthyotoxicity of harmful marine microalgae using cultured red sea bream (Pagrus major) gill cells. The cultured gill cells formed adherent 1-2 layers on the bottom of the culture plate and could tolerate seawater exposure for 4 h without significant alteration in cell survival. The microalgae Karenia mikimotoi, Karenia papilionacea, K. papilionacea phylotype-I, and Heterosigma akashiwo were cultured, then directly exposed to gill cells. After K. mikimotoi and K. papilionacea phylotype-I exposure, live cell coverage was significantly lower than in the cells exposed to a seawater-based medium (control cells; P < 0.05). Toxicity of K. mikimotoi cells was weakened when cells were ruptured, and was almost inexistent when the algal cells were removed from the culture by filtration. Significant cytotoxicity was detected in the concentrated ruptured cells, and in the concentrated of ruptured cells after freezing and thawing though cytotoxicity was weakened; whereas, cytotoxicity almost disappeared after heat treatment. In addition, examination of the distribution of toxic substances from the ruptured cells showed that cytotoxicity mainly occurred in the fraction with the resuspended pellet after centrifugation at 3000×g.

Bothrosome Formation in Schizochytrium aggregatum (Labyrinthulomycetes, Stramenopiles) during Zoospore Settlement.

Labyrinthulomycetes are characterized by the presence of ectoplasmic nets originating from an organelle known as the bothrosome, whose evolutionary origin is unclear. To address this issue, we investigated the developmental process from a zoospore to a vegetative cell in Schizochytrium aggregatum. After disappearance of the flagellum during zoospore settlement, the bothrosome emerged at the anterior-ventral pole of the cells. A new Golgi body also appeared at this stage, and the bothrosome was positioned close to both the new and the old Golgi bodies. This observation suggested that the Golgi body is related to the formation of the bothrosome. Actin appeared as a spot in the same location as the newly appeared bothrosome, as determined by immunofluorescence labeling. An immunoelectron microscopic analysis revealed that actin was present in the ectoplasmic nets and in the cytoplasm around the bothrosome, indicating that the electron-dense materials of the bothrosome are not the polar center of F-actin. This suggests that actin filaments pull the endoplasmic reticulum to the bothrosome and induce the membrane to become evaginated within ectoplasmic nets.

Occurrence of Karenia papilionacea (Dinophyceae) and its novel sister phylotype in Japanese coastal waters.

Several species of the genus Karenia (Dinophyceae) form blooms and often cause the mortality of cultured and wild fish. In Japan, blooms caused by two species - namely Karenia mikimotoi and Karenia brevis - have been reported so far. On the basis of morphological and molecular-phylogenic examinations, the present investigation found Karenia papilionacea and its novel sister phylotype for the first time in the coastal waters of the various regions of Japan. Of 34 strains isolated from the coastal waters, 27 strains displayed the typical morphological characteristics of K. papilionacea and further showed consensus DNA sequences corresponding to those of the originally described K. papilionacea. The other 7 strains displayed the same morphological characteristics of K. papilionacea, but showed divergent DNA sequences, at a genetic distance of over 0.04 (Internal Transcribed Spacer regions) from those of the original phylotype of K. papilionacea. These divergent strains were characterized as a novel sister phylotype (phylotype-I) of K. papilionacea. In the coastal waters of Japan, K. papilionacea-like (K. papilionacea and/or its phylotype-I) formed blooms at 20.3-30.4°C and salinity 30.1-33.9. No K. brevis was identified in Japanese coastal waters during this study. These findings demonstrated that K. papilionacea occurs along the coasts of western Japan and possibly shares several coastal regions with K. mikimotoi and K. papilionacea phylotype-I. In order to assess the risks of Karenia blooms to aquaculture, it is essential that the growth physiology and ichthyotoxicity of K. papilionacea and its novel phylotype should be characterized.