Inactivation of Amphidinium sp. in ballast waters using UV/Ag-TiO2+O3 advanced oxidation treatment

Ballast water poses a biological threat to the world’s waterways by transferring aquatic species from one body of water to another. This study investigates the use of combined ultraviolet (UV)/Ag-TiO₂ + ozone (O₃) processes for treating ballast water using Amphidinium sp. as an indicator microorganism. Sufficient Amphidinium sp. cells in ballast waters can be inactivated using O₃ alone, UV irradiation alone (with or without an Ag-TiO₂ coating), and combined treatments. For the low inactivation ratio (<40%) regime, the effects of ozonation and photocatalysis were observed to be cumulative. The combined UV/Ag-TiO₂ + O₃ treatment produced excess hydroxyl radicals and total residual oxidants (TROs), and readily damaged cell membranes to release intracellular substances. The comparison tests revealed that the combined treatments synergistically inactivate Escherichia coli in ballast waters. However, the combined process did not synergistically inactivate Amphidinium sp. cells. Inactivating different aqua species in ballast waters needs distinct treatment methods and dosages.     

Discovery of a novel type of body scale in the marine dinoflagellate,Amphidinium cupulatisquama sp. nov. (Dinophyceae)

A new species of Amphidinium, A. cupulatisquama Tamura et Horiguchi, from sand samples from Ikei Island, Okinawa Prefecture in subtropical Japan, is described based on light, scanning and transmission electron microscopy and the partial sequencing of the large subunit rDNA gene. The species has a typical morphology for the genus, but is distinguished from previously described species by having a combination of the following characteristics: (i) a relatively large cell (over 30 µm in length); (ii) possessing an eyespot on the dorsal side of the cingulum; (iii) the longitudinal flagellum emerging from a point close to the cingulum; (iv) cell division taking place in the motile phase; and (v) possessing body scales. This is the third species of this genus to possess body scales. The body scales of A. cupulatisquama are uniform and cup‐shaped in side view and elliptical in face view. Their dimensions are 136.4 nm by 91.0 nm by 81.8 nm high. In side view, the scale is seen to have a thick lower half and a thin upper half. This scale type is very different from those of previously reported Amphidinium species (HG114 and HG115). The molecular tree indicated that A. cupulatisquama and the two other strains of body scale‐bearing Amphidinium are distantly related within the Amphidinium clade.     

Taxonomy of some common Amphidinium species

The taxonomic status of several well known species belonging to the genus Amphidinium (Dinophyceae) has been re-examined. Studies in culture and observations on the micro-anatomy of these organisms suggest that the number of species can be reduced to two, A. klebsii and A. carterae. They may be distinguished on the basis of fundamental differences in chloroplast morphology.     

MORPHOLOGY AND MOLECULAR PHYLOGENY OF ANKISTRODINIUM GEN. NOV. (DINOPHYCEAE), A NEW GENUS OF MARINE SAND‐DWELLING DINOFLAGELLATES FORMERLY CLASSIFIED WITHIN AMPHIDINIUM1

The classical athecate dinoflagellate genera (Amphidinium, Gymnodinium, Gyrodinium) have long been recognized to be polyphyletic. Amphidinium sensu lato is the most diverse of all marine benthic dinoflagellate genera; however, following the redefinition of this genus ～100 species remain now of uncertain or unknown generic affiliation. In an effort to improve our taxonomic and phylogenetic understanding of one of these species, namely Amphidinium semilunatum, we re‐investigated organisms from several distant sites around the world using light and scanning electron microscopy and molecular phylogenetic methods. Our results enabled us to describe this species within a new heterotrophic genus, Ankistrodinium. Cells of A. semilunatum were strongly laterally flattened, rounded‐quadrangular to oval in lateral view, and possessed a small asymmetrical epicone. The sulcus was wide and characteristically deeply incised on the hypocone running around the antapex and reaching the dorsal side. The straight acrobase with hook‐shaped end started at the sulcal extension and continued onto the epicone. The molecular phylogenetic results clearly showed that A. semilunatum is a distinct taxon and is only distantly related to species within the genus Amphidinium sensu stricto. The nearest sister group to Ankistrodinium could not be reliably determined.     

AMPHIDINIUM REVISITED. II. RESOLVING SPECIES BOUNDARIES IN THE AMPHIDINIUM OPERCULATUM SPECIES COMPLEX (DINOPHYCEAE), INCLUDING THE DESCRIPTIONS OF AMPHIDINIUM TRULLA SP. NOV. AND AMPHIDINIUM GIBBOSUM. COMB. NOV.1

Amphidinium operculatum Claparède et Lachmann, the type species of the dinoflagellate genus Amphidinium, has long had an uncertain identity. It has been considered to be either difficult to distinguish from other similar species or a morphologically variable species itself. This has led to the hypothesis that A. operculatum represents a “species complex.” Recently, the problem of distinguishing A. operculatum from similar species has become particularly acute, because several morphologically similar species have been found to produce bioactive compounds of potential interest to the pharmaceutical industry. In this study, we cultured and examined existing cultures of several species of Amphidinium, most of which have been previously identified as A. operculatum or as species considered by some to be synonyms or varieties of A. operculatum. Thirty strains were examined using comparative LM, SEM, and partial large subunit (LSU) rDNA sequence data. Through morphological and molecular phylogenetic analyses, six distinct species were identified, including Amphidinium trulla sp. nov. and Amphidinium gibbosum comb. nov. Amphidinium operculatum was redescribed based on four cultures. Genetic variability within the examined Amphidinium species varied greatly. There was little difference among strains in partial LSU rDNA for most species, but strains of A. carterae and A. massartii Biencheler differed by as much as 4%. In both A. carterae and A. massartii, three distinct genotypes based on partial LSU rDNA were found, but no morphological differences among strains could be observed using LM or SEM. In the case of A. carterae, no biogeographically related molecular differences were found.     

The benthic dinoflagellate genus Amphidinium in south-eastern Australian waters,including three new species

Seventeen species of the mainly sand-dwelling genus Amphidinium are reported from south-eastern Australian waters. Three new species are described: A. boggayum, A. mootonorum and A. yuroogurrum. Additional features from previously described species are presented, including non-motile cell stages. Nine of these species are new records for Australia; however, these species are probably widespread and common. Two potentially ichthyotoxic species of Amphidinium were commonly found: A. carterae and A. cf. operculatum. A table of morphological characters is compiled as a step towards recognizing new characters with which to group species of this presently artificially defined unarmoured dinoflagellate genus.     

Effects of temperature,photosynthetic photon flux density,photoperiod and O<Subscript>2</Subscript> and CO<Subscript>2</Subscript> concentrations on growth rates of the symbiotic dinoflagellate, <Emphasis Type="Italic">Amphidinium</Emphasis> sp.

Symbiotic dinoflagellates of the species Amphidinium are expected to be pharmaceutically useful microalgae because they produce antitumor macrolides. A microalgae production system with a large number of cells at a high density has been developed for the efficient production of macrolide compounds. In the present study, the effects of culture conditions on the cellular growth rate of dinoflagellates were investigated to determine the optimum culture conditions for obtaining high yields of microalgae. Amphidinium species was cultured under conditions with six temperature levels (21–35°C), six levels of photosynthetic photon flux density (15–70 μmol photons m⁻² s⁻¹), three levels of CO₂ concentration (0.02–0.1%), and three levels of O₂ concentration (0.2–21%). The number of cells cultured in a certain volume of solution was monitored microscopically and the cellular growth rate was expressed as the specific growth rate. The maximum specific growth rate was 0.022 h⁻¹ at a temperature of 26°C and O₂ concentration of 5%, and the specific growth rate was saturated at a CO₂ concentration of 0.05%, a photosynthetic photon flux density of 35 μmol photons m⁻² s⁻¹ and a photoperiod of 12 h day⁻¹ upon increasing each environmental parameter. The results demonstrate that Amphidinium species can multiply efficiently under conditions of relatively low light intensity and low O₂ concentration.     

A survey of epiphytic dinoflagellates from the coastal waters of the island of Hawai‘i

Three hundred and sixty-nine macroalgal and non-algal samples were collected from six coastal sites around the island of Hawai‘i on a biweekly basis over a 1-year period to ascertain (1) the presence of potentially toxigenic benthic dinoflagellates, and (2) substrate and environmental preferences of the dinoflagellates. Twenty-six genera/species of dinoflagellates were encountered including the (potentially) toxigenic species Amphidinium sp., Coolia monotis, Gambierdiscus sp., Ostreopsis ovata, Prorocentrum concavum, Prorocentrum hoffmannianum, Prorocentrum lima, and Prorocentrum mexicanum. Twenty of the species are being reported for the first time as present in Hawaiian coastal waters. There was some evidence of macroalgal host preference (e.g., C. monotis on Tolypiocladia glomerulata), although host morphology preferences was greater (e.g., Gambierdiscus sp., and P. lima on filamentous turfs; O. sp. 1 on sheet-like macroblades). While some dinoflagellate groups were significantly correlated with nutrient concentrations (e.g., total dinoflagellates with nitrate + nitrite and phosphate concentrations), others were not (e.g., Gambierdiscus sp., Prorocentrum emarginatum, P. lima, and Sinophysis microcephalus). The presence of several potentially toxigenic dinoflagellate species in Hawai‘i merits future study on possible impacts of these dinoflagellates on coastal food webs and human health.     

Testudodinium gen. nov. (Dinophyceae), a new genus of sand‐dwelling dinoflagellates formerly classified in the genus Amphidinium

A new genus of sand‐dwelling photosynthetic dinoflagellate, Testudodinium Horiguchi, Tamura, Katsumata et A. Yamaguchi is proposed based on Testudodinium testudo (Herdman) Horiguchi, Tamura, Katsumata, et A. Yamaguchi comb. nov. (Basionym: Amphidinium testudo Herdman) and a new species in this new genus, Testudodinium maedaense Katsumata et Horiguchi sp. nov. is described. Amphidinium corrugatum is also transferred to this genus, making a new combination T. corrugatum (Larsen et Patterson) Horiguchi, Tamura et A. Yamaguchi. These three species are similar to the members of the genus Amphidinium in having an extremely small episome and a dorsoventrally flattened cell body. They are, however, distinguished from the genus Amphidinium seusu stricto by the possession of a distinct longitudinal furrow in the middle of ventral side of the episome. Phylogenetic trees based on small subunit (SSU) rDNA revealed that all three of these Testudodinium species formed a robust clade and, although statistical support is not high, the tree suggests Testudodinium clade is not closely related to Amphidinium seusu stricto clade. The morphological differences together with molecular data support the establishment of a new genus for A. testudo and its related species.     

Morphology and phylogenetic interpretation of a new Cambrian edrioasteroid (Echinodermata) from Spain

Abstract: The edrioasteroid, Aragocystites belli gen. nov. sp. nov. from the middle Cambrian Murero Formation of Spain, is described based on a small number of very well‐preserved specimens. Important anatomical characteristics include star‐shaped and pseudoclavate theca, rare or absent epispires, well‐developed interradially positioned oral plates and several unorganized cover plates associated with each widely exposed flooring plate. A phylogenetic analysis including several Cambro–Ordovician species shows it is more derived than Stromatocystites and Totiglobus but is a sister group to a clade comprising Cambraster and Edriodiscus. Ontogenetic observations based on juveniles of 5 mm in diameter suggest that this species changed thecal shape markedly during growth. A. belli gen. nov. sp. nov. probably lived in quiet environments where it attached directly to the sea floor on stabilized substrates.     

Genetic transformation of dinoflagellates (Amphidinium and Symbiodinium): expression of GUS in microalgae using heterologous promoter constructs

Genetic transformation of two dinoflagellates (Amphidinium sp., Symbiodinium microadriaticum) was achieved using plasmid constructs containing the neomycin phosphotransferase gene (nptII) fused to the Agrobacterium nos promoter, or the hygromycin B phosphotransferase gene (hpt) fused to the bidirectional Agrobacterium p1′2′ promoter. Gene transfer into intact (walled) dinoflagellate cells was achieved by agitation in the presence of silicon carbide (SiCa) whiskers. Transformation rates of 5–24 transformants per 10⁷ cells were obtained. Southern hybridization of transformants revealed stable integration of multiple copies of the constructs. Activity of integrated copies of the β-glucoronidase (GUS) reporter gene coupled to the cauliflower mosaic virus 35S promoter or the p1′2′ promoter was confirmed both histochemically and fluorometrically. This is the first report of successful application of heterologous and widely used promoter and reporter genes in microalgae, and is the first demonstration of transformation of a dinoflagellate. There appear to be no substantial barriers to transformation of Amphidinium and Symbiodinium, which must now be considered as the first of the dinoflagellate genera accessible to genetic manipulation.     

Bispinodinium angelaceum gen. et sp. nov. (Dinophyceae), a new sand‐dwelling dinoflagellate from the seafloor off Mageshima Island,Japan1

A new athecate dinoflagellate, Bispinodinium angelaceum N. Yamada et Horiguchi gen. et sp. nov., is described from a sand sample collected on the seafloor at a depth of 36 m off Mageshima Island, subtropical Japan. The dinoflagellate is dorsiventrally compressed and axi‐symmetric along the sulcus. The morphology resembles that of the genus Amphidinium sensu lato by having a small epicone that is less than one third of the total cell length. However, it has a new type of apical groove, the path of which traces the outline of a magnifying glass. The circular component of this path forms a complete circle in the center of the epicone and the straight “handle” runs from the sulcus to the circular component. Inside the cell, a pair of elongated fibrous structure termed here the “spinoid apparatus” extends from just beneath the circular apical groove to a point near the nucleus. Each of two paired structures consists of at least 10 hyaline fibers and this is a novel structure found in dinoflagellates. Phylogenetic analyses based on the SSU and LSU RNA genes did not show any high bootstrap affinities with currently known athecate dinoflagellates. On the basis of its novel morphological features and molecular signal, we conclude that this dinoflagellate should be described as a new species belonging to a new genus.     

Sterols of Testudodinium testudo (formerly Amphidinium testudo): Production of the Δ8(14) sterol gymnodinosterol and chemotaxonomic relationship to the Kareniaceae

Testudodinium testudo is a peridinin-containing dinoflagellate recently renamed from Amphidinium testudo. While T. testudo has been shown via phylogenetic analysis of small subunit ribosomal RNA genes to reside in a clade separate from the genus Amphidinium, it does possess morphological features similar to Amphidinium sensu stricto. Previous studies of Amphidinium carterae and Amphidinium corpulentum have found the sterols to be enriched in Δ⁸⁽¹⁴⁾ sterols, such as 4α-methyl-5α-ergosta-8(14),24(28)-dien-3β-ol (amphisterol), uncommon to most other dinoflagellate taxa and thus considered possible biomarkers for the genus Amphidinium. Here, we provide an examination of the sterols of T. testudo and show they are dominated not by amphisterol, but rather by a different Δ⁸⁽¹⁴⁾ sterol, (24R)-4α-methyl-5α-ergosta-8(14),22-dien-3β-ol (gymnodinosterol), previously thought to be a major sterol only within the Kareniaceae genera Karenia, Karlodinium, and Takayama. Also found to be present at low levels were 4α-methyl-5α-ergosta-8,14,22-trien-3β-ol, a sterol previously observed in Karenia brevis to be an intermediate in the production of gymnodinosterol, and cholesterol, a sterol common to many other dinoflagellates. The presence of gymnodinosterol in T. testudo is the first report of this sterol as the sole major sterol in a dinoflagellate outside of the Kareniaceae. The implication of this chemotaxonomic relationship to the Kareniaceae is discussed.     

Lipid synthesized by micro‐algae grown in laboratory‐ and industrial‐scale bioreactors

Tetraselmis sp. and Nannochloropsis oculata, cultivated in industrial‐scale bioreactors, produced 2.33 and 2.44% w/w lipid (calculated as the sum of fatty acid methyl esters) in dry biomass, respectively. These lipids contained higher amounts of neutral lipids and glycolipids plus sphingolipids, than phospholipids. Lipids of Tetraselmis sp. were characterized by the presence of eicosapentaenoic acid (that was located mainly in phospholipids), and octadecatetraenoic acid (that was equally distributed among lipid fractions), while these fatty acids were completely absent in N. oculata lipids. Additionally, lipids produced by 16 newly isolated strains from Greek aquatic environments (cultivated in flask reactors) were studied. The highest percentage of lipids was found in Prorocentrum triestinum (3.69% w/w) while the lowest in Prymnesium parvum (0.47% w/w). Several strains produced lipids rich in eicosapentaenoic and docosahexaenoic acids. For instance, docosahexaenoic acid was found in high percentages in lipids of Amphidinium sp. S1, P. parvum, Prorocentrum minimum and P. triestinum, while lipids produced by Asterionella sp. (?) S2 contained eicosapentaenoic acid in high concentration. These lipids, containing ω‐3‐long‐chain polyunsaturated fatty acids, have important applications in the food and pharmaceutical industries and in aquaculture.     

Further investigations on the PHAEOTHAMNIOPHYCEAE using a multigene phylogeny,with descriptions of five new species

We examined 12 strains representing eight species classified in the algal class Phaeothamniophyceae (Heterokontophyta). Based upon a five-gene molecular phylogeny (nuclear-encoded SSU rRNA and plastid-encoded psaA, psbA, psbC, and rbcL) and light microscopic observations, we describe five new species: Phaeoschizochlamys santosii sp. nov., Phaeoschizochlamys siveri sp. nov., Phaeothamnion wetherbeei sp. nov., Stichogloea dopii sp. nov. and Stichogloea fawleyi sp. nov. The Phaeothamniophyceae, as delimited here, form a natural group that is sister to the Aurearenophyceae. Molecular phylogenetic analyses proved more reliable than morphological characters for distinguishing species. Evolutionary trends with the SI clade of the heterokont algae are discussed.     

Emergence of Antagonism Against the Pathogenic Fungus Fusarium oxysporum by Interplay Among Non‐Antagonistic Bacteria in a Hydroponics Using Multiple Parallel Mineralization

The rhizosphere microbial community in a multiple parallel mineralization (MPM) system contributes to suppression of root‐borne diseases. We hypothesized this phenomenon can be attributed to the interplay of non‐antagonistic bacteria rather than to a single antagonistic microbe. In this study, we tested this hypothesis by investigating the potential roles of bacterial interplay in a subset of MPM microbiota in the suppression of the fungal phytopathogen Fusarium oxysporum. Bacterial strains isolated from the MPM system were subjected to in vitro and in planta tests on F. oxysporum. A community of seven bacterial strains (Kaistia sp. TBD58, Sphingopyxis sp. TBD84, Bosea sp. TBD101, Ancylobacter sp. TBD132, Cupriavidus sp. TBD162, Brevibacillus sp. TBD179 and Sphingopyxis sp. TBD181) suppressed F. oxysporum growth. None of the strains alone was antagonistic against F. oxysporum, whereas several pairs of those non‐antagonistic strains inhibited its growth. Morphological observations showed the formation of swollen F. oxysporum cells in the presence of these bacterial pairs. The same bacterial pairs also suppressed Fusarium wilt disease in Arabidopsis thaliana. These results indicate that a complex bacterial interplay among non‐antagonistic bacteria can significantly contribute to the development of antagonism against F. oxysporum in the context of the MPM system.     

Effects of manganese on the growth,photosystem II and SOD activity of the dinoflagellate <Emphasis Type="Italic">Amphidinium</Emphasis> sp.

Experimental ecology methods and chlorophyll fluorescence technology were used to study the effects of different concentrations of manganese (10⁻¹²– 10⁻⁴ mol L⁻¹) on the growth, photosystem II and superoxide dismutase (SOD) activity of Amphidinium sp. MACC/D31. The results showed that manganese had a significant effect on the growth rate, fluorescence parameters (maximal photochemical efficiency of PSII (F _v /F _m ), photochemical quenching (qP) and non-photochemical quenching (NPQ)) in the exponential stage (days 1–3) and SOD activity of Amphidinium sp. (P < 0.05). F _v/F _m in the exponential stage in 10⁻¹² mol L⁻¹ manganese concentration was significantly lower whilst qP and NPQ significantly higher than those in the other concentrations. F _v /F _m (days 6–9) in 10⁻⁴ mol L⁻¹ manganese was significantly higher than those in the other concentrations. F _v /F _m (days 3–6) increased with increased concentration of manganese from 10⁻¹² to 10⁻⁴ mol L⁻¹. The values of qP and NPQ decreased with decreased concentrations of manganese, except for those in days 4–6. F _v /F _m under each concentration increased earlier and decreased later with culture stage whilst NPQ decreased earlier and increased later. The SOD activity increased with increased concentration of manganese from 10⁻¹² to 10⁻⁸ mol L⁻¹. The SOD activity in 10⁻⁴ mol L⁻¹ manganese was significantly higher than those in the other concentrations and in 10⁻¹² mol L⁻¹ manganese, it was significantly lower than those in the other concentrations.