ANALYSIS OF CLONAL CULTURES OF THE BROWN TIDE ALGAE AUREOCOCCUS AND AUREOUMBRA (PELAGOPHYCEAE) USING 18S rRNA, rbcL, AND RUBISCO SPACER SEQUENCES

Variability among clonal cultures of the brown tide algae Aureococcus anophagefferens Hargraves et Sieburth and Aureoumbra lagunensis Stockwell, DeYoe, Hargraves et Johnson was examined by DNA sequence comparisons. Nuclear-encoded 18S rRNA and plastid-encoded rbc L gene sequences were determined for six Aureococcus strains. RUBISCO spacer sequences were determined for 14 strains of Aureococcus. No differences among Aureococcus strains were found in the DNA regions examined. The rbc L and RUBISCO spacer sequences for three Aureoumbra strains were identical but differed from those of Aureococcus. These data indicate that blooms of these species are comprised of cells that are very similar and also imply that Aureococcus and Aureoumbra do not contain varieties or cryptic species. Separate and combined phylogenetic analyses of the 18S rRNA and rbc L gene sequences were performed. Results confirm that the brown-tide-causing algae of Long Island Sound, New York ( Aureococcus ), and Laguna Madre, Texas ( Aureoumbra ), are best classified in separate genera within the Pelagophyceae. Phylogenetic trees place Aureococcus and Aureoumbra within the Pelagomonadales and Sarcinochrysidales, respectively.     

Transport of the harmful bloom alga Aureococcus anophagefferens by oceangoing ships and coastal boats

It is well established that cyst-forming phytoplankton species are transported in ships' ballast tanks. However, there is increasing evidence that other phytoplankton species which do not encyst are also capable of surviving ballast transit. These species have alternative modes of nutrition (hetero- or mixotrophy) and/or are able to survive long-term darkness. In our studies of no-ballast-on-board vessels arriving in the Great Lakes, we tested for the presence of the harmful algal bloom species Aureococcus anophagefferens (brown tide) in residual (i.e., unpumpable) ballast water using methods based on the PCR. During 2001, the brown tide organism was detected in 7 of 18 ballast water tanks in commercial ships following transit from foreign ports. Furthermore, it was detected after 10 days of ballast tank confinement during a vessel transit in the Great Lakes, a significant result given the large disparity between the salinity tolerance for active growth of Aureococcus (>22 ppt) and the low salinity of the residual ballast water (approximately 2 ppt). We also investigated the potential for smaller, recreational vessels to transport and distribute Aureococcus. During the summer of 2002, 11 trailered boats from the inland bays of Delaware and coastal bays of Maryland were sampled. Brown tide was detected in the bilge water in the bottoms of eight boats, as well as in one live-well sample. Commercial ships and small recreational boats are therefore implicated as potential vectors for long-distance transport and local-scale dispersal of Aureococcus.     

Inhibition of harmful algal blooms caused by Aureococcus anophagefferens (Pelagophyceae) using native (Gracilaria tikvahiae) and invasive (Dasysiphonia japonica) red seaweeds from North America

Journal of Applied Phycology - Harmful algal blooms (HABs) caused by the pelagophyte Aureococcus anophagefferens have spread globally and are a threat to coastal ecosystems. Although some HAB...     

ULTRASTRUCTURE AND ECOLOGY OF AUREOCOCCUS ANOPHAGEFERENS GEN. ET SP. NOV. (CHRYSOPHYCEAE): THE DOMINANT PICOPLANKTER DURING A BLOOM IN NARRAGANSETT BAY,RHODE ISLAND,SUMMER 19851

Observations of a marked cessation of feeding in filter feeding animals maintained in flowing Narragansett Bay seawater in June 1985 drew our attention to a bloom of a golden alga 2 μm in diameter at unprecedented populations of 10⁹ cells. L^?1. This picoplankter lacked morphological features useful in discriminating it from other similar sized forms with either phase contrast or epifluorescence light microscopy. Natural populations of picoplankton, obtained from the height of the bloom until its decline, were examined in thin section with transmission electron microscopy. A cell with a single chloroplast, nucleus, and mitochondrion and an unusual exocellular polysaccharide-like layer was apparently the bloom alga. The ultrastructure of this alga is consistent with that of the Chrysophyceae, and a new genus and species, Aureococcus anophagefferens is described. Attempts to grow this previously unrecognized picoplanktonic alga as an obligate phototroph failed and only yielded cultures of other previously described picoalgae. Facultative and obligate phagotrophic protists with ingested cells of Aureococcus were only observed as the bloom waned and minute diatoms became common. Cells of A. anophagefferens with virus particles typical for picoalgae occurred throughout the bloom. Populations of the usually dominant photosynthetic picoplankter, the cyanobacterium Synechococcus Nägeli, were depressed during the bloom. This could be due in part to selective grazing on Synechococcus rather than Aureococcus by elevated populations of Calycomonas ovalis Wulff which accompanied the algal bloom.     

The role of viruses in the dynamics of phytoplankton blooms

Abstract

Viruses and virus-like particles (VLP) have been found, in most cases perchance, in about 20 marine phytoplankton species. However only in six of these have there been further investigations on this phenomenon. Different mechanisms of interaction have been hypothesized. These include continuous dynamic viral control on populations (Synechococcus spp.), at times suppressed by external environmental factors (Aureococcus anophagefferens), termination of a bloom caused by viral infection (Emiliania huxleyi and Micromonas pusilla) or by induction of lysogenic cells (Heterosigma akashiwo), and effects on the survivability of specific clones through genetic control (Aureococcus anophagefferens). These examples illustrate the complexity of virus-algae relationships and provide an indication that they may represent a key factor in the dynamics of phytoplankton blooms.     

Dissolved organic nitrogen hydrolysis rates in axenic cultures of Aureococcus anophagefferens (Pelagophyceae): comparison with heterotrophic bacteria.

The marine autotroph Aureococcus anophagefferens (Pelagophyceae) was rendered axenic in order to investigate hydrolysis rates of peptides, chitobiose, acetamide, and urea as indicators of the ability to support growth on dissolved organic nitrogen. Specific rates of hydrolysis varied between 8 and 700% of rates observed in associated heterotrophic marine bacteria.     

Dissolved Organic Nitrogen Hydrolysis Rates in Axenic Cultures of Aureococcus anophagefferens (Pelagophyceae): Comparison with Heterotrophic Bacteria

The marine autotroph Aureococcus anophagefferens (Pelagophyceae) was rendered axenic in order to investigate hydrolysis rates of peptides, chitobiose, acetamide, and urea as indicators of the ability to support growth on dissolved organic nitrogen. Specific rates of hydrolysis varied between 8 and 700% of rates observed in associated heterotrophic marine bacteria.     

Sterol Chemotaxonomy of Marine Pelagophyte Algae

Several marine algae of the class Pelagophyceae produce the unusual marine sterol 24‐propylidenecholesterol, mainly as the (24E)‐isomer. The (24Z)‐isomer had previously been considered as a specific biomarker for Aureococcus anophagefferens, the ‘brown tide’ alga of the Northeast coast of the USA. To test this hypothesis and to generate chemotaxonomic information, the sterol compositions of 42 strains of pelagophyte algae including 17 strains of Aureococcus anophagefferens were determined by GC analysis. A more comprehensive sterol analysis by HPLC and ¹H‐NMR was obtained for 17 selected pelagophyte strains. All strains analyzed contained 24‐propylidenecholesterol. In all strains belonging to the order Sarcinochrysidales, this sterol was found only as the (E)‐isomer, while all strains in the order Pelagomonadales contained the (Z)‐isomer, either alone or together with the (E)‐isomer. The occurrence of Δ²² and 24α‐sterols was limited to the Sarcinochrysidales. The first occurrence of Δ²²‐24‐propylcholesterol in an alga, CCMP 1410, was reported. Traces of the rare sterol 26,26‐dimethyl‐24‐methylenecholesterol were detected in Aureococcus anophagefferens, and the (25R)‐configuration was proposed, based on biosynthetic considerations. Traces of a novel sterol, 24‐propylidenecholesta‐5,25‐dien‐3β‐ol, were detected in several species.     

An immunofluorescent survey of the brown tide chrysophyte Aureococcus anophagefferens along the northeast coast of the United States

Surveys were conducted along the northeast coast of the USA.between Portsmouth, NH, and the Chesapeake Bay in 1988 and 1990,to determine the population distribution of Aureococcus anophagefferens,the chrysophyte responsible for massive and destructive ‘browntides’ in Long Island and Narragansett Bay beginning in1985. A species-specific immunofluorescent technique was usedto screen water samples, with positive identification possibleat cell concentrations as low as 10–20 cells ml^–1.Both years.A.anophagefferens was detected at numerous stationsin and around Long Island and Barnegat Bay, NJ, typically athigh cell concentrations. To the north and south of thus ‘center’,nearly half of the remaining stations were positive for A.anophagefferens,but the cells were always at very low cell concentrations. Manyof the positive identifications in areas distant from Long Islandwere in waters with no known history of harmful brown tides.The species was present in both open coastal and estuanne locations,in salinities between 18 and 32 practical salinity units (PSU).The observed population distributions apparently still reflectthe massive 1985 outbreak when this species first bloomed, giventhe number of positive locations and high abundance of A.anophagefferensin the immediate vicinity of Long Island. However, the frequentoccurrence of this species in waters far from this population‘center’ is disturbing. Aureococcus anophagefferensis more widely distributed than was previously thought. Numerousareas thus have the potential for destructive brown tides suchas those associated with the sudden appearance of the speciesin 1985.     

CHARACTERIZATION OF A LYTIC VIRUS INFECTIOUS TO THE BLOOM-FORMING MICROALGA AUREOCOCCUS ANOPHAGEFFERENS (PELAGOPHYCEAE)

Aureococcus anophagefferens Hargraves and Sieburth has caused recurring monospecific blooms in Long Island embayments since it was first described in 1985. It was termed the "brown tide," due to the resulting water color, and has had a devastating effect on Long Island's (New York) marine ecosystem. In 1992, a virus that was capable of causing lysis of A. anophagefferens was isolated and maintained in culture. We report on the further characterization of this virus, Aureococcus anophagefferens virus-1 (AaV-1), indicated by a buoyant density of 1.2776 g·mL⁻¹ in a CsCl equilibrium gradient. Electron microscopy revealed a phage with a hexagonal head and tail similar to previously described phages. By using adenovirus for calibration, the virus was found to have a head 50—55 nm wide and a tail 70–75 nm long. The viral band was infectious to A. anophagefferens after dialysis. The virus was composed of at least 16 distinct polypeptides ranging in molecular weight from 20 to 230 kDa. The adsorption coefficient for the virus was 7.2 × 10⁻⁹ mL·min⁻¹, and the burst size was calculated to be 9.4 viruses per A. anophagefferens cell at 20° C. Complete lysis of A. anophagefferens occurred with a titer as low as 893 viruses·mL⁻¹, and the lower limit of infectivity was 93 viruses·mL⁻¹. The virus lost its infectivity between 30° and 40° C. These results suggest that AaV-1 is highly infectious and that the role of the virus in preventing or ending A. anophagefferens blooms needs further investigation.     

BACTERIAL LYSIS OF AUREOCOCCUS ANOPHAGEFFERENS CCMP 1784 (PELAGOPHYCEAE)1

Water samples from Great South Bay, New York, contained biological agents capable of causing the lysis of the brown‐tide alga Aureococcus anophagefferens Hargraves et Sieburth. From these water samples, 32 bacterial isolates were tested for algicidal effects against the algae. Six of the isolates were shown to have algicidal effects against A. anophagefferens. Sequencing of the gene 16S rRNA revealed that the isolates were relatively diverse. Based on their similarity to sequences in repositories, the isolates seem to be closely related to Bacillus, Halomonas, Croceibacter atlanticus, Marinobacter, and an Arctic deep‐sea bacterium.     

VARIATION IN DCMU-ENHANCED FLUORESCENCE RELATIVE TO CHLOROPHYLL A: CORRELATION WITH THE BROWN TIDE BLOOM1

DCMU–enhanced fluorescence and extracted chlorophyll a were simultaneously measured in Narragansett Bay, Rhode Island and the MERL (Marine Ecosystems Research Laboratory) mesocosms during the 1985 brown tide bloom. Marked differences in the relationship between these variables were observed as the phytoplankton community shifted from dominance by picoalgae to diatoms. The fluorescence to chlorophyll a ratio was significantly (P< 0.05) higher in the mesocosms and the bay when the brown tide species (Aureococcus anophagefferens Hargraves et Sieburth) dominated the phytoplankton community compared with other taxa. Although several factors could have affected the relationship we believe the high ratios are related to the pigment composition and/or small size of the brown tide organism.     

DESCRIPTION AND CHARACTERIZATION OF THE ALGAL SPECIES AUREOUMBRA LAGUNENSIS GEN. ET SP. NOV. AND REFERRAL OF AUREOUMBRA AND AUREOCOCCUS TO THE PELAGOPHYCEAE1

The Texas brown tide alga (strain TBA-2) is described as Aureoumbra lagunensis Stockwell, DeYoe, Hargraves, et Johnson, gen. et sp. nov. Pigment composition, chloroplast structure, and 18s ribosomal RNA gene sequence data indicate that A. lagunensis and the east coast brown tide alga Aureococcus anophagefferens (originally placed in the Chrysophyceae) belong in the class Pelagophyceae. The new genus Aureoumbra with A. lagunensis as the type species differs from Aureococcus in 18s ribosomal RNA gene sequence, pyrenoid form, nitrogen physiology, and possession of basal bodies. The genus Aureococcus is placed in the order Pelagomonadates and family Pelagomonadaceae while ordinal placement of Aureoumbra is deferred.     

Long-term perspective on the dynamics of brown tide blooms in Long Island coastal bays

Brown tide, a bloom of the picoplankter Aureococcus anophagefferens, first appeared in eastern Long Island (Suffolk County) waters in the late spring of 1985, at about the same time it emerged, although to a lesser degree, in Narraganset Bay, RI. Since then, it has recurred sporadically in Suffolk County, and blooms have been reported in New Jersey, Delaware, Maryland, and only one other area of the world, Saldanha Bay, South Africa. Bloom initiation and maintenance within Suffolk County appear to be related to A. anophagefferens’ ability to use dissolved organic nitrogen (DON) during periods of limited dissolved inorganic nitrogen (DIN) availability. Factors controlling DIN availability include groundwater influx related to meteorological conditions, introduction of septic leachate from on-site wastewater treatment systems, and biological removal. The complexity of bloom dynamics is illustrated by a cascade of events in Great South Bay involving shellfish clearing rates, a macroalgal bloom, and microbial decomposition.     

Development and application of a monoclonal-antibody technique for counting Aureococcus anophagefferens,an alga causing recurrent brown tides in the Mid-Atlantic United States

A method was developed for the rapid detection and enumeration of Aureococcus anophagefferens, the cause of harmful algal blooms called "brown tides" in estuaries of the Mid-Atlantic United States. The method employs a monoclonal antibody (MAb) and a colorimetric, enzyme-linked immunosorbent assay format. The MAb obtained exhibits high reactivity with A. anophagefferens and very low cross-reactivities with a phylogenetically diverse array of other protists and bacteria. Standard curves are constructed for each 96-well microtiter plate by using known amounts of a preserved culture of A. anophagefferens. This approach allows estimation of the abundance of the alga in natural samples. The MAb method was compared to an existing method that employs polyclonal antibodies and epifluorescence microscopy and to direct microscopic counts of A. anophagefferens in samples with high abundances of the alga. The MAb method provided increased quantitative accuracy and greatly reduced sample processing time. A spatial survey of several Long Island estuaries in May 2000 using this new approach documented a range of abundances of A. anophagefferens in these bays spanning nearly 3 orders of magnitude.     

A TRANSIENT BLOOM OF OSTREOCOCCUS (CHLOROPHYTA, PRASINOPHYCEAE) IN WEST NECK BAY, LONG ISLAND, NEW YORK

The smallest known eukaryote, Ostreococcus tauri Courties et Chrétiennot-Dinet, was first reported as the dominant picoplankter in a French lagoon known for its diverse phytoplankton community and high oyster productivity. Long-term seasonal blooms of this picoeukaryote were observed in association with stable plankton communities. On 5 June 2001, a distinctive monotypic picoplankton bloom was detected by flow cytometry as part of an ongoing study of "brown tide" ( Aureococcus anophagefferens ) bloom initiation in Long Island bays. The bloom reached a concentration of 5 × 10⁵ cells·mL⁻¹ in West Neck Bay and lasted less than 2 weeks. Epifluorescence microscopy and TEM indicated that the bloom organism was an Ostreococcus -like picoalga, the first ever observed in a Long Island bay. Many cells of this alga contained numerous virus-like particles. The Ostreococcus -like picoalga, which resembles O. tauri , was rare in samples collected the following week. Instead, a substantial increase in the Synechococcus population was observed. Such rapid population changes have not previously been reported for Ostreococcus . Viral lysis and grazing by heterotrophic nanoflagellates may have contributed to the rapid decline of the Ostreococcus -like cells in West Neck Bay.     

Development and Application of a Monoclonal-Antibody Technique for Counting Aureococcus anophagefferens, an Alga Causing Recurrent Brown Tides in the Mid-Atlantic United States

A method was developed for the rapid detection and enumeration of Aureococcus anophagefferens, the cause of harmful algal blooms called “brown tides” in estuaries of the Mid-Atlantic United States. The method employs a monoclonal antibody (MAb) and a colorimetric, enzyme-linked immunosorbent assay format. The MAb obtained exhibits high reactivity with A. anophagefferens and very low cross-reactivities with a phylogenetically diverse array of other protists and bacteria. Standard curves are constructed for each 96-well microtiter plate by using known amounts of a preserved culture of A. anophagefferens. This approach allows estimation of the abundance of the alga in natural samples. The MAb method was compared to an existing method that employs polyclonal antibodies and epifluorescence microscopy and to direct microscopic counts of A. anophagefferens in samples with high abundances of the alga. The MAb method provided increased quantitative accuracy and greatly reduced sample processing time. A spatial survey of several Long Island estuaries in May 2000 using this new approach documented a range of abundances of A. anophagefferens in these bays spanning nearly 3 orders of magnitude.     

Molecular Cloning and Antiserum Development of Cyclin Box in the Brown Tide Alga Aureococcus anophagefferens

Cyclins can be useful cell cycle markers for growth rate studies on harmful algal blooms. In this study, a gene fragment corresponding to cyclin box was cloned for the brown tide alga Aureococcus anophagefferens. This algal gene fragment, designated as Btcycl1, was most similar to cyclin B. Oligopeptides based on the deduced amino acid sequence were synthesized and used to raise an antiserum that reacted on Western blots with a protein of about 63 kDa, the same size as cyclin B in other organisms. The cyclin B–like protein recognized by this antiserum, and the messenger RNA amplified using the primers, were more abundant in exponential cultures and decreased markedly in stationary cultures. This protein also appeared to be cell cycle dependent. Immunofluorescence labeling showed that this antiserum specifically stained a protein in Aureococcus cells and had no cross-reaction with bacteria that were present in the algal culture. The Btcycl1 sequence and the antiserum will provide a useful tool for studies on regulation of in situ growth rate for this brown tide alga. Received May 22, 2000; accepted July 13, 2000.