Physiological response dynamics of the brown tide organism Aureococcus anophagefferens treated with modified clay

On the basis of experiences in mitigating harmful algal blooms (HABs) with modified clay (MC), a bloom does not continue after the dispersal of the MC, even though the density of the residual cells in the water remains as high as 20–30% of the initial cell density. This interesting phenomenon indicates that in addition to flocculation, MC has additional mechanisms of HAB control. Here, Aureococcus anophagefferens was selected as a model organism to study the physiological response dynamics of residual cells treated with MC, and RT-qPCR was used to measure the differential expression of 40 genes involved in anti-oxidation, photosynthesis, phospholipid synthesis, programmed cell death and cell proliferation at five time points. The results showed that every functional gene category exhibited a "V" shaped pattern with a turning point. It was reflected that there were two processes for MC inhibiting the growth of residual cells. One is the oxidative stress process (OSP) caused by ineffective collision with MC, whose effect weakened gradually; another is the programmed cell death process (PCDP) caused by the lysis of damaged residual cells, whose effect enhanced two days after MC treatment. In addition, the scanning electron micrographs verified that some of the residual cells were deformed or even lysed. Combined with the effects of OSP and PCDP in dynamics, the growth of residual cells was inhibited and was followed by gradual bloom disappearance. This study further elucidates the mechanism of MC controlling HABs at the molecular level and enable a more comprehensive understanding of HAB mitigation using MC.     

Flocculation and removal of the brown tide organism, Aureococcus anophagefferens (Chrysophyceae), using clays

Previous attempts to remove the brown tideorganism, Aureococcusanophagefferens, through flocculation withclays have been unsuccessful, in spite ofadopting concentrations and dispersalprotocols that yielded excellent cellremoval efficiency (RE >90%) with otherspecies, so a study was planned to improvecell removal. Four modifications in claypreparation and dispersal were explored: 1)varying the salinity of the claysuspension; 2) mixing of the clay-cellsuspension after clay addition; 3) varyingof concentration of the initial clay stock;4) pulsed loading of the clay slurry. Theeffect of salinity was dependent on theclay mineral type: phosphatic clay (IMC-P2)had a higher RE than kaolinite (H-DP) whenseawater was used to disperse the clay, butH-DP removed cells more efficiently whensuspended in distilled water prior toapplication. Mixing after dispersalapproximately doubled RE for both clayscompared to when the slurry was layeredover the culture surface. Lowering theconcentration of clay stock and pulsing theclay loading increased RE, regardless ofmineral type. However, this increase wasmore apparent for clays dispersed inseawater than in distilled water. Ingeneral, application procedures thatdecrease the rate of self-aggregation amongthe clay particles and increase thecollision frequency between clay particlesand A. anophagefferens achieve highercell removal efficiency. These empiricalstudies demonstrated that clays might be animportant control option for the brown tideorganism, given the proper attention topreparation, dispersal methods,environmental impacts, and the hydrodynamicproperties of the system being treated. Implications for the treatment of browntides in the field are discussed.     

Elevated organic nutrient ratios associated with brown tide algal blooms of Aureococcus anophagefferens (Pelagophyceae)

Natural assemblages of Aureococcus anophagefferens collectedfrom the coastal bays of Maryland and Long Island, New Yorkoccurred under water quality conditions higher in dissolvedorganic carbon and phosphorus (relative to nitrogen) than thosesame stations when Aureococcus was not present in significantnumbers. Additional work, both in culture and in the field,confirmed the importance of a heterotrophic nutritional modein this brown tide organism.     

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.     

Emergence of brown tides caused by Aureococcus anophagefferens Hargraves et Sieburth in China

Large-scale blooms suspected to be “brown tides” occurred in early summer for three consecutive years from 2009 to 2011 in the coastal waters of Qinhuangdao, China, and had significant negative impacts on the shellfish mariculture industry. To identify the causative species of the blooms, phytoplankton samples were collected from regions with and without bloom in the coastal waters of Qinhuangdao in 2011, and clone libraries were built using eukaryote-specific 18S ribosomal RNA gene (18S rDNA). Altogether 50 clones, including 17 clones from bloom area and 33 clones from nearby regions without bloom were amplified. Blasted in GenBank, 17 clones amplified from the bloom area were assigned to Pelagophyceae (8 clones), Mediophyceae (2 clones), Cryptophyta (2 clones), Dinophyceae (2 clones) and unidentified eukaryotic species (3 clones). Those from the non-bloom site were assigned to Cryptophyta, Eustigmatophyceae, Prasinophyceae, Coscinodiscophyceae, Mediophyceae, Raphidophyceae and Dinophyceae, but not Pelagophyceae. All 8 pelagophyte clones from the bloom area were 99.7–100% similar to a single species, Aureococcus anophagefferens Hargraves et Sieburth, the causative species of brown tides on the east coast of USA. For nearly the entire length of the 18S rDNA, there were 0–6 base pair differences between the 8 amplicons and those of A. anophagefferens from USA. Furthermore, all of the 8 clones were clustered into the same well-supported clade with A. anophagefferens (posterior probability = 0.99) in a phylogenetic tree established for pelagophytes and other related microalgae. In our previous studies, the causative species of the bloom was tentatively identified as a pelagophyte, haptophyte or silicoflagellate, based on the pigment profile of the size-fractioned phytoplankton samples. Based on this study, we conclude that blooms in the coastal waters of Qinhuangdao of the Bohai Sea were brown tides caused by A. anophagefferens. As far as we know, this is the first report of brown tide events caused by A. anophagefferens in China, which is the third country in the world reporting A. anophagefferens blooms in addition to USA and South Africa.     

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.     

Black Scoter habitat use along the southeastern coast of the United States

While the Atlantic Coast of the United States and Canada is a major wintering area for sea ducks, knowledge about their wintering habitat use is relatively limited. Black Scoters have a broad wintering distribution and are the only open water species of sea duck that is abundant along the southeastern coast of the United States. Our study identified variables that affected Black Scoter (Melanitta americana) distribution and abundance in the Atlantic Ocean along the southeastern coast of the United States. We used aerial survey data from 2009 to 2012 provided by the United States Fish and Wildlife Service to identify variables that influenced Black Scoter distribution. We used indicator variable selection to evaluate relationships between Black Scoter habitat use and a variety of broad‐ and fine‐scale oceanographic and weather variables. Average time between waves, ocean floor slope, and the interaction of bathymetry and distance to shore had the strongest association with southeastern Black Scoter distribution.     

Immunofluorescence Flow Cytometry Technique for Enumeration of the Brown-Tide Alga, Aureococcus anophagefferens

A new immunologically based flow cytometry (IFCM) technique was developed to enumerate Aureococcus anophagefferens, a small pelagophyte alga that is the cause of “brown tides” in bays and estuaries of the mid-Atlantic states along the U.S. coast. The method utilizes a monoclonal antibody conjugated to fluorescein isothiocyanate (FITC-MAb) to label the surface of A. anophagefferens cells which are then detected and enumerated by using a flow cytometer. Optimal conditions for FITC-MAb staining, including solution composition, incubation times, and FITC-MAb concentrations, were determined. The FITC-MAb method was tested for cross-reactivity with nontarget, similarly sized, photoautotrophic protists, and the method was compared to an enzyme-linked immunosorbent assay (ELISA) using the same MAb. Comparisons of the IFCM technique to traditional microscopy enumeration of cultures and spiked environmental samples showed consistent agreement over several orders of magnitude (r² > 0.99). Comparisons of the IFCM and ELISA techniques for enumerating cells from a predation experiment showed a substantial overestimation (up to 10 times higher) of the ELISA in the presence of consumers of A. anophagefferens, presumably due to egested cell fragments that retained antigenicity, using the ELISA method, but were not characterized as whole algal cells by the IFCM method. Application of the IFCM method to environmental “brown-tide” samples taken from the coastal bays of Maryland demonstrated its efficacy in resolving A. anophagefferens abundance levels throughout the course of a bloom and over a large range of abundance values. IFCM counts of the brown-tide alga from natural samples were consistently lower than those obtained using the ELISA method and were equivalent to those of the polyclonal immunofluorescence microscopy technique, since both methods discriminate intact cells. Overall, the IFCM approach was an accurate and relatively simple technique for the rapid enumeration of A. anophagefferens in natural samples over a wide range of abundance values (10³ to 10⁶ cells ml⁻¹).     

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 (~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.     

3,000 km and 1,500‐year presence of Aureococcus anophagefferens reveals indigenous origin of brown tides in China

The nonmotile, spherical, picoplanktonic (2‐μm‐sized) pelagophyte Aureococcus anophagefferens has caused numerous harmful blooms (“brown tides”) across global marine ecosystems. Blooms have developed along the east coast of the USA since 1985, a limited number of times in South Africa around 1997, and frequently in China since 2009. As a consequence, the harmful blooms have caused massive losses in aquaculture and coastal ecosystems, particularly mortalities in cultured shellfish. Therefore, whether A. anophagefferens was recently introduced to China via natural/artificial transport of resting stage cells or has been an indigenous species has become a question of profound ecological significance and broad interest, which motivated our extensive investigation on the geographic and historical presence of this species in the seas of China. We applied a combined approach of extensive PCR‐based detection and sequencing, germination experiments and monoclonal antibody staining of germlings to samples of surface sediment and sediment core (dated via combined isotopic measurements) collected from all four seas of China, and searched the supplementary data set of a recent Science publication. We discovered that A. anophagefferens does have a resting stage in the sediment, but it also has a wide geographic distribution both in China (covering a range of ~30° in latitude, ~15.7° in longitude and 2.5–3,456 m in water depth; temperate to tropical and coastal to open oceans) and in almost all oceans of the world and a historical presence of >1,500 years in the Bohai Sea, China. The work revealed that A. anophagefferens is not a recently introduced, but an indigenous species in China and has in fact a globally cosmopolitan distribution.     

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.     

Larval Sulcascaris sulcata from calico scallops, Argopecten gibbus, along the southeast coast of the United States

Adductor muscles of calico scallops, Argopecten gibbus, collected off the southeastern coast of the United States from May 1982 to December 1984 were examined for the presence of larvae of the parasitic nematode, Sulcascaris sulcata. Infection intensity, prevalence, and larval length were all positively correlated (P less than 0.001) with scallop shell height (age). Prevalence was generally highest in December and lowest in August as a result of yearly trends in the age structure of the scallop population. Prevalence was not influenced by latitude or depth over the ranges examined. The rate of infection of marine molluscs by S. sulcata is generally related to the degree and length of exposure to third-stage larvae, whose distribution is essentially that of the marine turtle definitive host. Thus, over the wide geographic area of the east coast of the U.S., overall rates of infection decrease with increasing latitude.     

Seaweed biogeography of the mid-Atlantic coast of the United States

The northern boundary of the warm temperate region of the mid-Atlantic coast of the United States is set at Cape Hatteras; the southern boundary lies at Cape Canaveral. There is some spillover of cool temperate species south of Cape Hatteras into North Carolina and spillover of warm temperate species south of Cape Canaveral toward Palm Beach. Elements of the warm temperate flora also extend into the northern Gulf of Mexico, but precise limits to the flora cannot be drawn there. Thirty-one species are endemic to the warm temperate flora. The inshore waters of North Carolina include approximately equal numbers of species with northern and southern centres of distribution; the species of the offshore waters have predominantly southern affinities, but also include most of the endemic species. Seasonal changes in the shallow water flora of North Carolina reflect eurythermal cool temperate and tropical elements in winter and summer respectively and a year-round warm temperate element. These groupings have been verified by experimental studies in which light and temperature were varied. The deep water flora is a summer flora dominated by perennial species. The inshore, eurythermal cool temperate and tropical species have a variety of cryptic stages by which they persist throughout the year.Paper presented at the Seaweed Biogeography Workshop of the International Working Group on Seaweed Biogeography, held from 3–7 April 1984 at the Department of Marine Biology, Rijks-universiteit Groningen (The Netherlands). Convenor: C. van den Hoek.     

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.     

The central role of selenium in the biochemistry and ecology of the harmful pelagophyte,Aureococcus anophagefferens

The trace element selenium (Se) is required for the biosynthesis of selenocysteine (Sec), the 21st amino acid in the genetic code, but its role in the ecology of harmful algal blooms (HABs) is unknown. Here, we examined the role of Se in the biology and ecology of the harmful pelagophyte, Aureococcus anophagefferens, through cell culture, genomic analyses, and ecosystem studies. This organism has the largest and the most diverse selenoproteome identified to date that consists of at least 59 selenoproteins, including known eukaryotic selenoproteins, selenoproteins previously only detected in bacteria, and novel selenoproteins. The A. anophagefferens selenoproteome was dominated by the thioredoxin fold proteins and oxidoreductase functions were assigned to the majority of detected selenoproteins. Insertion of Sec in these proteins was supported by a unique Sec insertion sequence. Se was required for the growth of A. anophagefferens as cultures grew maximally at nanomolar Se concentrations. In a coastal ecosystem, dissolved Se concentrations were elevated before and after A. anophagefferens blooms, but were reduced by >95% during the peak of blooms to 0.05 nℳ. Consistent with this pattern, enrichment of seawater with selenite before and after a bloom did not affect the growth of A. anophagefferens, but enrichment during the peak of the bloom significantly increased population growth rates. These findings demonstrate that Se inventories, which can be anthropogenically enriched, can support proliferation of HABs, such as A. anophagefferens through its synthesis of a large arsenal of Se-dependent oxidoreductases that fine-tune cellular redox homeostasis.     

Reconstructing the range expansion and subsequent invasion of introduced European green crab along the west coast of the United States

The European green crab, Carcinus maenas, was first documented in San Francisco Bay in 1989, and has since spread north along the west coast of North America. The spread of this invasion has not been a smooth expansion, which has raised questions about the underlying causes of variation in recruitment. We modeled larval development and transport along the West Coast by employing an individual-based model that incorporated oceanographic model output of water temperature and ocean currents at fine spatial and temporal scales. The distance that larvae were advected depended primarily on the timing of larval release. However, the effect of seasonal ocean currents varied across latitude and years. Our results imply that the furthest northern transport from California occurs when larvae are released from Humboldt Bay during the fall of an El Niño year, making this a particularly risky time for invasion to Oregon and Washington estuaries. To precisely predict future spread and potential impacts of green crab, we recommend further empirical research to determine the precise timing of larval release and seasonal abundance of green crab larvae from North American west coast populations.     

Life history and habitat use of the speckled worm eel, Myrophis punctatus, along the east coast of the United States

Many species of fishes along the east coast of the United States have complex life histories, especially those that move over hundreds of kilometers across ocean and estuarine habitats. To further unravel the life history of one of these, the speckled worm eel, Myrophis punctatus we examined samples from extensive time series and discrete samples collected in the ocean and estuaries between Florida and Massachusetts. We now surmise spawning occurs between fall and early winter in the ocean south of Cape Hatteras, North Carolina and in the vicinity of the Bahamas. The pelagic leptocephalus larvae (10?C80 mm Total Length [TL]) are transported north in the Gulf Stream and across the continental shelf to arrive at estuarine inlets at ages of 53 ?C 110 days. Their estuarine immigration and abundance varies along the east coast, with higher levels occurring at inlets in South Carolina (North Inlet), and North Carolina (Beaufort Inlet), during the winter and early spring. Much lower abundances occur in New Jersey (Little Egg Inlet) in winter and spring and again in the summer. These ingressing individuals were euryodontic leptocephali and metamorphic stages and were shrinking to lengths of 76?C52 mm TL as these stages progressed. Metamorphic individuals and glass eels subsequently settle and burrow in estuarine sediments, as do all subsequent stages, and thereby become relatively unavailable to many sampling gears. In estuaries they attained sizes up to 440 mm TL. Later, they presumably enter the ocean to spawn because that is where the smallest larvae are found.