Tamotsu Nagumo

Previously unknown virus infects marine diatom.

ABSTRACT Diatoms are a major phytoplankton group that play important roles in maintaining oxygen levels in the atmosphere and sustaining the primary nutritional production of the aquatic environment. Among diatoms, the genus Chaetoceros is one of the most abundant and widespread. Temperature, climate, salinity, nutrients, and predators were regarded as important factors controlling the abundance and population dynamics of diatoms. Here we show that a viral infection can occur in the genus Chaetoceros and should therefore be considered as a potential mortality source. Chaetoceros salsugineum nuclear inclusion virus (CsNIV) is a 38-nm icosahedral virus that replicates within the nucleus of C. salsugineum. The latent period was estimated to be between 12 and 24 h, with a burst size of 325 infectious units per host cell. CsNIV has a genome structure unlike that of other viruses that have been described. It consists of a single molecule of covalently closed circular single-stranded DNA (ssDNA; 6,005 nucleotides), as well as a segment of linear ssDNA (997 nucleotides). The linear segment is complementary to a portion of the closed circle creating a partially double-stranded genome. Sequence analysis reveals a low but significant similarity to the replicase of circoviruses that have a covalently closed circular ssDNA genome. This new host-virus system will be useful for investigating the ecological relationships between bloom-forming diatoms and other viruses in the marine system. Our study supports the view that, given the diversity and abundance of plankton, the ocean is a treasury of undiscovered viruses.

Isolation and Characterization of a Single-Stranded RNA Virus Infecting the Marine Planktonic Diatom Chaetoceros tenuissimus Meunier

ABSTRACT Diatoms are important components of the biological community and food web in the aquatic environment. Here, we report the characteristics of a single-stranded RNA (ssRNA) virus (CtenRNAV01) that infects the marine diatom Chaetoceros tenuissimus Meunier (Bacillariophyceae). The ca. 31-nm virus particle is icosahedral and lacks a tail. CtenRNAV01 forms crystalline arrays occupying most of the infected hosts cytoplasm. By growth experiments, the lytic cycle and the burst size were estimated to be <24 h and ∼1 × 104 infectious units per host cell, respectively. Stationary-phase C. tenuissimus cultures were shown to be more sensitive to CtenRNAV01 than logarithmic-phase cultures. The most noticeable feature of this virus is its exceptionally high yields of ∼1010 infectious units ml−1; this is much higher than those of any other algal viruses previously characterized. CtenRNAV01 has two molecules of ssRNA of approximately 8.9 and 4.3 kb and three major proteins (33.5, 31.5, and 30.0 kDa). Sequencing of the total viral genome has produced only one large contig [9,431 bases excluding the poly(A) tail], suggesting considerable overlapping between the two RNA molecules. The monophyly of CtenRNAV01 compared to another diatom-infecting virus, Rhizosolenia setigera RNA virus, was strongly supported in a maximum likelihood phylogenetic tree constructed based on the concatenated amino acid sequences of the RNA-dependent RNA polymerase domains. Although further analysis is required to determine the detailed classification and nomenclature of this virus, these data strongly suggest the existence of a diatom-infecting ssRNA virus group in natural waters.

Isolation and characterization of a single-stranded RNA virus infecting the bloom-forming diatom Chaetoceros socialis.

ABSTRACT Diatoms are very significant primary producers in the worlds oceans. Various environmental factors affect the depletion of diatom populations. The importance of viruses as a potential mortality source has recently been recognized. We isolated and characterized a new diatom virus (Chaetoceros socialis f. radians RNA virus [CsfrRNAV]) causing the lysis of the bloom-forming species Chaetoceros socialis Lauder f. radians (Schütt) Proschkina-Lavrenko. The virus infectious to C. socialis f. radians was isolated from water samples collected in Hiroshima Bay. Here we show the physiology, morphology, and genome characteristics of the virus clone. Virions were 22 nm in diameter and accumulated in the cytoplasm of the host cells. The latent period and the burst size were estimated to be <48 h and 66 infectious units per host cell, respectively. CsfrRNAV harbors a single-stranded RNA (ssRNA) genome and encodes at least three polypeptides of 32.0, 28.5, and 25.0 kDa. Sequencing analysis shows the length of the genome is 9,467 bases, excluding a poly(A) tail. The monophyly of CsfrRNAV and other diatom-infecting RNA viruses, Rhizosolenia setigera RNA virus and Chaetoceros tenuissimus RNA virus, was strongly supported by phylogenetic analysis based on the amino acid sequence of the RNA-dependent RNA polymerase domains. This suggested a new ssRNA virus family, Bacillariornaviridae. This discovery of CsfrRNAV may aid in further understanding the ecological dynamics of the C. socialis f. radians population in nature and the relationships between ssRNA diatom viruses and their hosts.

Novel Sex Cells and Evidence for Sex Pheromones in Diatoms

Background Diatoms belong to the stramenopiles, one of the largest groups of eukaryotes, which are primarily characterized by a presence of an anterior flagellum with tubular mastigonemes and usually a second, smooth flagellum. Based on cell wall morphology, diatoms have historically been divided into centrics and pennates, of which only the former have flagella and only on the sperm. Molecular phylogenies show the pennates to have evolved from among the centrics. However, the timing of flagellum loss – whether before the evolution of the pennate lineage or after – is unknown, because sexual reproduction has been so little studied in the ‘araphid’ basal pennate lineages, to which Pseudostaurosira belongs. Methods/Principal Finding Sexual reproduction of an araphid pennate, Pseudostaurosira trainorii, was studied with light microscopy (including time lapse observations and immunofluorescence staining observed under confocal scanning laser microscopy) and SEM. We show that the species produces motile male gametes. Motility is mostly associated with the extrusion and retrieval of microtubule-based ‘threads’, which are structures hitherto unknown in stramenopiles, their number varying from one to three per cell. We also report experimental evidence for sex pheromones that reciprocally stimulate sexualization of compatible clones and orientate motility of the male gametes after an initial ‘random walk’. Conclusions/Significance The threads superficially resemble flagella, in that both are produced by male gametes and contain microtubules. However, one striking difference is that threads cannot beat or undulate and have no motility of their own, and they do not bear mastigonemes. Threads are sticky and catch and draw objects, including eggs. The motility conferred by the threads is probably crucial for sexual reproduction of P. trainorii, because this diatom is non-motile in its vegetative stage but obligately outbreeding. Our pheromone experiments are the first studies in which gametogenesis has been induced in diatoms by cell-free exudates, opening new possibilities for molecular ‘dissection’ of sexualization.

Isolation and Characterization of a Single-Stranded DNA Virus Infecting Chaetoceros lorenzianus Grunow

ABSTRACT Diatoms are one of the most significant primary producers in the ocean, and the importance of viruses as a potential source of mortality for diatoms has recently been recognized. Thus far, eight different diatom viruses infecting the genera Rhizosolenia and Chaetoceros have been isolated and characterized to different extents. We report the isolation of a novel diatom virus (ClorDNAV), which causes the lysis of the bloom-forming species Chaetoceros lorenzianus, and show its physiological, morphological, and genomic characteristics. The free virion was estimated to be ∼34 nm in diameter. The arrangement of virus particles appearing in cross-section was basically a random aggregation in the nucleus. Occasionally, distinctive formations such as a ring-like array composed of 9 or 10 spherical virions or a centipede-like array composed of rod-shaped particles were also observed. The latent period and the burst size were estimated to be <48 h and 2.2 × 104 infectious units per host cell, respectively. ClorDNAV harbors a covalently closed circular single-stranded DNA (ssDNA) genome (5,813 nucleotides [nt]) that includes a partially double-stranded DNA region (979 nt). At least three major open reading frames were identified; one showed a high similarity to putative replicase-related proteins of the other ssDNA diatom viruses, Chaetoceros salsugineum DNA virus (previously reported as CsNIV) and Chaetoceros tenuissimus DNA virus. ClorDNAV is the third member of the closed circular ssDNA diatom virus group, the genus Bacilladnavirus.

Taxonomic re‐examination of 17 species of Nitella subgenus tieffallenia (Charales, Charophyceae) based on internal morphology of the oospore wall and multiple DNA marker sequences (Vol. 41:195–211)

In an attempt to reconstruct the natural taxonomic system for Nitella, 17 species of Nitella subgenus Tieffallenia were reexamined using SEM observations of the internal morphology of the oospore wall (IMOW) and phylogenetic analyses of 4553 base pairs from multiple DNA markers (atpB, rbcL, psaB, and ITS‐5.8S rRNA genes). Our SEM observations identified three types of IMOW: homogeneous (HG), weakly spongy (W‐SG), and strongly spongy (S‐SG) types. Based on differences in the IMOW, species with reticulate or tuberculate oospore wall ornamentation in the external morphology of the oospore wall (EMOW) were subdivided into two distinct groups (characterized by the HG or S‐SG types of IMOW, respectively), which were robustly separated from each other in our molecular phylogenetic analyses. In our molecular phylogeny, the subgenus Tieffallenia consisted of four robust monophyletic groups—three clades of the HG type and a spongy (S‐SG and W‐SG) type clade—that were characterized by differences in the IMOW and EMOW. In addition, our SEM observations and sequence data verified the distinct status of five species (N. japonica Allen, N. oligospira A. Braun, N. vieillardii stat. nov., N. imperialis stat. nov., and N. morongii Allen) that R. D. Wood had assigned as infraspecific taxa. Moreover, our SEM observations of the IMOW also suggested that N. megaspora (J. Groves) Sakayama originally identified by LM includes at least two distinct species, characterized by W‐SG and S‐SG types of IMOW, respectively.

Observations on the valve structure of marine species of the diatom genus Cocconeis Ehr.

The valve structure of three marine diatom species, Cocconeis molesta var. crucifera, C. dirupta and C. pellucida was studied by transmission and scanning electron microscopy. In all Cocconeis examined, the areolae are occluded by hymenes located near the internal openings in the raphid valve and near the internal openings in the araphid valves.

AUXOSPORE FORMATION AND THE MORPHOLOGY OF THE INITIAL CELL OF THE MARINE ARAPHID DIATOM GEPHYRIA MEDIA (BACILLARIOPHYCEAE)1

Recent studies have led to a rapid increase in knowledge of auxospore formation in diatoms. However, these studies have been limited to centric and raphid pennate diatoms, and there is still very little information for the araphid pennate diatoms. Using LM and SEM, we studied the development of the auxospore and the initial cell of the marine epiphytic diatom Gephyria media Arnott. Auxospores were bipolar and curved in side view, as in many other pennate diatoms. SEM revealed many transverse perizonial bands, all of which were incomplete rings. There was an elongate, sprawling, silicified structure beneath the ventral suture of the transverse perizonial bands. This structure is presumably equivalent to the longitudinal perizonial band in other pennate diatoms, although we could not determine the homologous relationship between the two features. Scales were found both in the inner wall of the perizonium and around the primary perizonial bands. The presence or absence of scales may be of phylogenetic significance in diatoms, only during the final stages of auxospore formation because scales are found in early spherical stages. The distinctive finger‐like structures observed throughout all stage of G. media have not been observed before in the other diatom taxa.

Fine-structure of the vegetative frustule, perizonium and initial valve of Achnanthes yaquinensis (Bacillariophyta)

This study clarifies the fine structure of the vegetative and initial valves of Achnanthes yaquinensis and briefly compares them to other Achnanthes species. It also elucidates the structure of the perizonium, based on auxospore development in short-term cultures. The araphid valve has marginal ridges and terminal spines that allow connecting valves to form a chain. The terminal spines develop from the rapheless sternum. The complete cingulum consists of 3–5 split bands with two rows of areolae. These features can be used to discriminate species within the genus. Sexual reproduction is isogamous with two mother cells producing two auxospores, which are enclosed in mucilage. The perizonium develops on one side of an auxospore only, comprising one large central longitudinal band and four closed bands. There are no transverse perizonial bands. The raphid valve of the initial cell forms first, underneath the longitudinal perizonium, followed by the araphid valve, which is not covered by any perizonial bands. The araphid valve of the initial cells lacks a marginal spine, and the rapheless sternum lies more centrally than in the vegetative cell. The relationship of the genus Achnanthes to other monoraphid diatoms is discussed briefly.

Auxospore Fine Structure and Variation in Modes of Cell Size Changes in Grammatophora Marina (Bacillariophyta)

S. Sato, D.G. Mann, T. Nagumo, J. Tanaka, T. Tadano and L.K. Medlin. 2008. Auxospore fine structure and variation in modes of cell size changes in Grammatophora marina (Bacillariophyta). Phycologia 47: 12–27. DOI: 10.2216/07-34.1 Examination of Grammatophora marina from rough and clonal cultures showed that cell size changes were more flexible than is generally reported for diatoms. Allogamous sexual auxosporulation took place through copulation between small male cells and larger female cells, but only in mixed rough culture and never in clonal cultures. Auxospores were also formed without copulation in clonal cultures (‘uniparental auxosporulation’) and these, like sexual auxospores, developed through formation of a perizonium, which consisted of a series of transverse bands. All of these bands, including the primary band, were open. Circular scales were present in the auxospore wall before initiation of perizonium formation and irregular, elongate structures lined the suture of the transverse perizonium. Perizonium and scales resembled those of another araphid pennate diatom, Gephyria media. Initial cells were formed within the perizonium and consisted of an initial epivalve with a simplified structure, an initial hypovalve (formed beneath the perizonium suture) and a third, normally structured valve formed beneath the epivalve; the epivalve was then sloughed off. Initial cells of similar configuration but often aberrant morphology could also be formed through expansion from vegetative cells, without involvement of a perizonium. Vegetative cells were also capable of limited enlargement through simple expansion without formation of an initial cell, and abrupt size reduction. Cell size ranges in populations from different regions suggest that G. marina may contain pseudocryptic species.