Potential impacts of brown tide, Aureococcus anophagefferens, on juvenile hard clams, Mercenaria mercenaria, in the Coastal Bays of Maryland, USA

The rate of growth of juvenile hard clams, Mercenaria mercenaria, was studied in the Coastal Bays of Maryland during an outbreak of the brown tide, Aureococcus anophagefferens. Brown tide dominated the plankton community during the month of June 2002, with cell densities at several sites reaching category 3 (>200,000 cells ml⁻¹) levels. Temperatures during the bloom were 18.6–27.5 °C. Nutrient conditions preceding and during the bloom were conducive for the proliferation of A. anophagefferens: while inorganic nitrogen and phosphorus were <1 μg at N or P l⁻¹, urea was elevated during bloom development. Organic nitrogen, phosphorus and carbon were in the range of levels observed in previous brown tide blooms and increased following the collapse of the bloom. Growth rates of juvenile clams were significantly lower during the period of the brown tide bloom than following its collapse. Growth rates of M. mercenaria were found to be negatively impacted at brown tide densities as low as 20,000 cells ml⁻¹, or category 1 levels. The low growth rates of M. mercenaria could not be explained by temperature, as the lowest growth rates were found when water temperatures were at levels previously found to be optimal for growth.     

HPLC pigment records provide evidence of past blooms of Aureococcus anophagefferens in the Coastal Bays of Maryland and Virginia, USA

Concentrations of the accessory phytoplankton pigment 19′-butanoyloxyfucoxanthin (but-fuco), derived from archived high performance liquid chromatography (HPLC) data from the Atlantic coastal bays of Maryland and Virginia (1993–1995 and 1999–2002), were used to determine the presence of Aureococcus anophagefferens at 18 stations. Paired data of direct cell counts of A. anophagefferens and but-fuco concentration data from 2000 to 2002 were linearly regressed (R² = 0.78). This regression was used to estimate historical cell densities from 1994 to 1995 and to improve the spatial resolution from 1999 to 2002. Although the HPLC method used did not permit quantification of but-fuco before 1994, the records indicate that qualitatively A. anophagefferens was present in 1993. Quantitative measurements grouped into bloom index categories showed that annually, peak densities occurred in May to July. Severe Category 3 blooms (>200,000 cells ml⁻¹) were found in 1995, 2001, and 2002. Spatially, concentrations of but-fuco were higher in the northern extent of the study region than in the lower Chincoteague Bay, and along the western shore of Chincoteague Bay than on the eastern side. On an interannual basis, it appeared that A. anophagefferens became more geographically widespread and blooms more intense throughout the study period.     

Ecology of phytoplankton communities dominated by Aureococcus anophagefferens: the role of viruses, nutrients, and microzooplankton grazing

We investigated the impact of viruses, nutrient loading, and microzooplankon grazing on phytoplankton communities in two New York estuaries that hosted blooms of the brown tide alga Aureococcus anophagefferens during 2000 and 2002. The absence of a bloom at one location during 2002 allowed for the fortuitous comparison of a bloom and non-bloom year at the same location as well as a comparison of two sites experiencing bloom and non-bloom conditions during the same year. During the study, blooms were found at locations with high levels of dissolved organic nitrogen and lower nitrate concentrations compared to a non-bloom location. Experimental additions of inorganic nitrogen and phosphorus yielded growth rates within the total phytoplankton community which significantly exceeded control treatments in 83% of experiments, while A. anophagefferens experienced significantly increased growth during only 20% of experimental inorganic nutrient additions. Consistent with prior research, these results suggest brown tides are not caused by eutrophication, but instead are more likely to occur when sources of labile DOM are readily available. Microzooplankton grazing rates on the total phytoplankton community during a bloom were lower than grazing rates at a non-bloom site, and grazing rates on A. anophagefferens were lower than grazing rates on the total community on some dates, suggesting that reduced grazing mortality may also promote brown tides. Mean densities of viruses during blooms (3 × 10⁸ ml⁻¹) were elevated compared to most estuarine environments and were twice the levels found at a non-bloom site. Experimental enrichment of the natural viral densities yielded a significant increase in A. anophagefferens growth rates relative to control treatments when background levels of viruses were low (<1.7 × 10⁸ ml⁻¹), suggesting that viruses may promote bloom occurrence by regenerating DOM or altering the composition of microbial communities.     

Pico- and nanoplankton dynamics during bloom initiation of Aureococcus in a Long Island, NY bay

The entire microbial plankton community was quantified on a weekly basis April through June of 2000 in Quantuck Bay as part of an ongoing study to identify factors contributing to the initiation of blooms of Aureococcus anophagefferens (brown tide) in Long Island, NY bays. We used flow cytometry, imaging cytometry, fluorescent antibody cell counts, and traditional visual cell counting to quantify the picophytoplankton, heterotrophic bacteria, nanophytoplankton, heterotrophic protists, and microplankton prior to, and during the initiation of a brown tide bloom. Cells passing through a 5 μm mesh dominated the total chlorophyll concentration (>80%) for most of the spring study period. The A. anophagefferens bloom occurred in the context of a larger pico/nanophytoplankton bloom where A. anophagefferens accounted for only 30% of the total cell count when it was at its maximum concentration of 4.8 × 10⁵ mL⁻¹. Levels of dissolved organic nitrogen were enriched during the bloom peak relative to pre-bloom levels and heterotrophic bacteria also bloomed, reaching abundances over 10⁷ mL⁻¹. A trophic cascade within the heterotrophic protist community may have occurred, coinciding with the A. anophagefferens bloom. Before the onset of the bloom, larger grazers increased in abundance, while the next smaller trophic level of grazers were diminished. These smaller grazers were the likely water column predators of A. anophagefferens, and the brown tide bloom initiated when they were depleted. These results suggest that this bloom initiated due to interactions with other pico/nano algae and release from grazing pressure through a trophic cascade.     

Characterization, dynamics, and ecological impacts of harmful Cochlodinium polykrikoides blooms on eastern Long Island, NY, USA

We report on the emergence of Cochlodinium polykrikoides blooms in the Peconic Estuary and Shinnecock Bay, NY, USA, during 2002–2006. Blooms occurred during late summer when temperatures and salinities ranged from 20 to 25 °C and 22 to 30 ppt, respectively. Bloom patches achieved cell densities exceeding 10⁵ ml⁻¹ and chlorophyll a levels exceeding 100 μg l⁻¹, while background bloom densities were typically 10³–10⁴ cells ml⁻¹. Light, scanning electron and ultrathin-section transmission electron microscopy suggested that cells isolated from blooms displayed characteristics of C. polykrikoides and provide the first clear documentation of the fine structure for this species. Sequencing of a hypervariable region of the large subunit rDNA confirmed this finding, displaying 100% similarity to other North American C. polykrikoides strains, but a lower similarity to strains from Southeast Asia (88–90%). Bioassay experiments demonstrated that 24 h exposure to bloom waters (>5 × 10⁴ cells ml⁻¹) killed 100% of multiple fish species (1-week-old Cyprinodon variegates, adult Fundulus majalis, adult Menidia menidia) and 80% of adult Fundulus heteroclitus. Microscopic evaluation of the gills of moribund fish revealed epithelial proliferation with focal areas of fusion of gill lamellae, suggesting impairment of gill function (e.g. respiration, nitrogen excretion, ion balance). Lower fish mortality was observed at intermediate C. polykrikoides densities (10³–10⁴ cells ml⁻¹), while fish survived for 48 h at cell densities below 1 × 10³ cells ml⁻¹. The inability of frozen and thawed-, or filtered (0.2 μm)-bloom water to cause fish mortality suggested that the thick polysaccharide layer associated with cell membranes and/or a toxin principle within this layer may be responsible for fish mortality. Juvenile bay scallops (Argopecten irradians) and American oysters (Crassostrea virginica) experienced elevated mortality compared to control treatments during a 9-day exposure to bloom water (5 × 10⁴ cells ml⁻¹). Surviving scallops exposed to bloom water also experienced significantly reduced growth rates. Moribund shellfish displayed hyperplasia, hemorrhaging, squamation, and apoptosis in gill and digestive tissues with gill inflammation specifically associated with areas containing C. polykrikoides cells. In summary, our results indicate C. polykrikoides blooms have become annual events on eastern Long Island and that bloom waters are capable of causing rapid mortality in multiple species of finfish and shellfish.     

Application and assessment of a nutrient pollution indicator using eelgrass (Zostera marina L.) in Barnegat Bay-Little Egg Harbor estuary, New Jersey

Eutrophication degrades numerous estuaries worldwide and a myriad of assessment metrics have been developed. Here, we apply an example of a previously developed metric (Lee et al., 2004) designed to indicate incipient estuarine eutrophication to validate this technique in an already eutrophic estuary end-member, Barnegat Bay-Little Egg Harbor, New Jersey. The metric, termed ‘Nutrient Pollution Indicator’ (NPI) uses eelgrass (Zostera marina L.) as a bioindicator and is calculated as the ratio of leaf nitrogen content (%N) to area normalized leaf mass (mg dry wt cm⁻²). Eelgrass samples were collected along the entire length of the Barnegat Bay-Little Egg Harbor from June to October 2008 to determine if leaf chemistry and morphology reflect eutrophication status and a north-south gradient of nitrogen loading from the Barnegat Bay watershed. Nitrogen content, area normalized leaf mass, and NPI values all significantly (p < 0.05) varied temporally but not spatially. NPI values did not significantly correspond to the north-south gradient of nitrogen loading from the Barnegat Bay watershed. The NPI metric is therefore not deemed to reliably indicate estuarine eutrophic status. Differences between sampling effort (number of stations) and replication did not bias the overall conclusions.     

Impacts and potential effects due to Prorocentrum minimum blooms in Chesapeake Bay

The dinoflagellate Prorocentrum minimum (P. minimum) can be found in all seasons and over a broad range of habitat conditions in the Chesapeake Bay and its tributaries. Blooms (>3000 cells ml⁻¹), locally referred to as ‘mahagony tides’, were restricted to salinities of 4.5–12.8 psu, water temperatures of 12–28 °C, and occurred most frequently in April and May. P. minimum blooms have been detected at routine water quality monitoring stations located in the main channel of the Bay and tidal tributaries. Nearshore investigations of bloom events, however, have accounted for the majority of events recorded in excess of 10⁵ cells ml⁻¹. Mahogany tides were associated with widespread harmful impacts including anoxic/hypoxic events, finfish kills, aquaculture shellfish kills and submerged aquatic vegetation losses. We summarize the state of knowledge regarding physical and chemical factors related to P. minimum blooms, their abundance, distribution and frequency, and ecological effects in Chesapeake Bay.     

Karlotoxin mediates grazing by Oxyrrhis marina on strains of Karlodinium veneficum

Karlodinium veneficum is a common member of temperate, coastal phytoplankton assemblages that occasionally forms blooms associated with fish kills. Here, we tested the hypothesis that the cytotoxic and ichthyotoxic compounds produced by K. veneficum, karlotoxins, can have anti-grazing properties against the heterotrophic dinoflagellate, Oxyrrhis marina. The sterol composition of O. marina (>80% cholesterol) renders it sensitive to karlotoxin, and does not vary substantially when fed different algal diets even for prey that are resistant to karlotoxin. At in situ bloom concentrations (10⁴–10⁵ K. veneficum ml⁻¹), grazing rates (cells ingested per Oxyrrhis h⁻¹) on toxic K. veneficum strain CCMP 2064 were 55% that observed on the non-toxic K. veneficum strain MD5. At lower prey concentrations typical of in situ non-bloom levels (<10³ cells ml⁻¹), grazing rates (cells ingested per Oxyrrhis h⁻¹) on toxic K. veneficum strain CCMP 2064 were 70–80% of rates on non-toxic strain MD5. Growth of O. marina was significantly suppressed when fed the toxic strain of K. veneficum. Experiments with mixed prey cultures, where non-toxic strain MD5 was fluorescently stained, showed that the presence of toxic strain CCMP 2064 inhibited grazing of O. marina on the co-occurring non-toxic strain MD5. Exogenous addition of a sub-lethal dose (100 ng ml⁻¹) of purified karlotoxin inhibited grazing of O. marina by approximately 50% on the non-toxic K. veneficum strain MD5 or the cryptophyte S. major. These results identify karlotoxin as an anti-grazing compound for those grazers with appropriate sterol composition (i.e., desmethyl sterols). This strategy is likely to be an important mechanism whereby growth of K. veneficum is uncoupled from losses due to grazing, allowing it to form ichthyotoxic blooms in situ.     

High-performance liquid chromatographic analysis of AQ4N, an alkylaminoanthraquinone N-oxide

A simple, highly selective and reproducible reversed-phase high-performance liquid chromatography method has been developed for the analysis of the new anti-cancer pro-drug AQ4N. The sample pre-treatment involves a simple protein precipitation protocol, using methanol. Chromatographic separations were performed using a HiChrom HIRPB (25 cm×4.6 mm I.D.) column, with mobile phase of acetonitrile–ammonium formate buffer (0.05 M) (22:78, v/v), with final pH adjusted to 3.6 with formic acid. The flow-rate was maintained at 1.2 ml min⁻¹. Detection was via photodiode array performed in the UV range at 242 nm and, since the compounds are an intense blue colour, in the visible range at 612 nm. The structurally related compound mitoxantrone was used as internal standard. The validated quantification range of the method was 0.05–10.0 μg ml⁻¹ in mouse plasma. The inter-day relative standard deviations (RSDs) (n=5) ranged from 18.4% and 12.1% at 0.05 μg ml⁻¹ to 2.9% and 3.3% at 10.0 μg ml⁻¹ for AQ4N and AQ4, respectively. The intra-day RSDs for supplemented mouse plasma (n=6) ranged from 8.2% and 14.2% at 0.05 μg ml⁻¹ to 7.6% and 11.5% at 10.0 μg ml⁻¹ for AQ4N and AQ4, respectively. The overall recovery of the procedure for AQ4N was 89.4±1.77% and 76.1±7.26% for AQ4. The limit of detection was 50 ng ml⁻¹ with a 100 μl sample volume. The method described provides a suitable technique for the future analysis of low levels of AQ4N and AQ4 in clinical samples.     

Detection of Karenia brevis blooms on the west Florida shelf using in situ backscattering and fluorescence data

Using shipboard data collected from the central west Florida shelf (WFS) between 2000 and 2001, an optical classification algorithm was developed to differentiate toxic Karenia brevis blooms (>10⁴ cells l⁻¹) from other waters (including non-blooms and blooms of other phytoplankton species). The identification of K. brevis blooms is based on two criteria: (1) chlorophyll a concentration ≥1.5 mg m⁻³ and (2) chlorophyll-specific particulate backscattering at 550 nm ≤ 0.0045 m² mg⁻¹. The classification criteria yielded an overall accuracy of 99% in identifying both K. brevis blooms and other waters from 194 cruise stations. The algorithm was validated using an independent dataset collected from both the central and south WFS between 2005 and 2006. After excluding data from estuarine and post-hurricane turbid waters, an overall accuracy of 94% was achieved with 86% of all K. brevis bloom data points identified successfully. Satisfactory algorithm performance (88% overall accuracy) was also achieved when using underway chlorophyll fluorescence and backscattering data collected during a repeated alongshore transect between Tampa Bay and Florida Bay in 2005 and 2006. These results suggest that it may be possible to use presently available, commercial optical backscattering instrumentation on autonomous platforms (e.g. moorings, gliders, and AUVs) for rapid and timely detection and monitoring of K. brevis blooms on the WFS.     

Growth of dinoflagellates, Ceratium furca and Ceratium fusus in Sagami Bay, Japan: The role of temperature, light intensity and photoperiod

Seasonal changes of field populations and growth rates of two dinoflagellates, Ceratium furca and Ceratium fusus, were examined in the temperate coastal water of Sagami Bay, Japan. Weekly field sampling was conducted from August 2002 to August 2003, and laboratory experiments were also carried out to investigate effects of temperature, irradiance and photoperiod on the growth rates of these two Ceratium species. In the field, the abundances of both species increased significantly from April to August 2003, were gradually decreased from November 2002 and were not observed in January 2003. C. fusus was able to increase at lower temperatures in February 2003 compared to C. furca. In the laboratory, the two species did not grow at <10 °C or >32 °C. The highest specific growth rate of C. furca was 0.72 d⁻¹ at 24 °C and 600 μmol m⁻² s⁻¹. Optimum growth rates (>0.4 d⁻¹) of C. furca were observed at temperatures from 18 to 28 °C and at irradiances from 216 to 796 μmol m⁻² s⁻¹. The highest growth rate of C. fusus was 0.56 d⁻¹ at 26 °C and 216 μmol m⁻² s⁻¹. Optimum growth rates of C. fusus were observed at the same irradiance rage of C. furca, whereas optimum temperature range was narrower (26–28 °C). The growth curves of both species indicated saturation of the growth rates when light intensity was above 216 μmol m⁻² s⁻¹, and did not show photoinhibition at irradiances up to 796 μmol m⁻² s⁻¹. The specific growth rates of both Ceratium species were clearly decreased at L:D = 10:14 relative to those at L:D = 14:10 and L:D = 12:12. The present study indicates the two Ceratium species can adapt to a wide range of temperature and irradiance.     

Stimulation of the brown tide organism, Aureococcus anophagefferens, by selective nutrient additions to in situ mesocosms

The influence of nutrient additions and sediment exchange on Aureococcus anophagefferens growth was studied using 200 l mesocosms deployed in situ at the Southampton College Marine Science Center in Long Island, New York. A. anophagefferens cell density increased in mesocosms with separate additions of the following materials: urea + glucose and desiccation-stressed Enteromorpha tissue. A decrease in A. anophagefferens cell density was observed in mesocosms with either no additions (control) or with added nitrate. A treatment containing a sediment layer exhibited an increase in average cell densities, but the increase was not statistically significant (P = 0.15). In the 9 day experiment, net growth of A. anophagefferens was only observed during the last 3 days, which corresponded to a period of high solar irradiation. Total chlorophyll concentration declined in all treatments during the first 6 days, which corresponded to relatively low daily irradiance, suggesting low-light stress on the phytoplankton assemblage during the initial phase of the experiment. During the ensuing sunny period, a 4–5-fold increase in chlorophyll concentration was observed in the nitrate and urea treatments with lesser increases in the other treatments. A. anophagefferens density increased relative to total phytoplankton biomass (Chl basis) in the urea + glucose and Enteromorpha treatments. Results are consistent with a prevailing hypothesis that organic nitrogen nutrients favor the growth of A. anophagefferens. Specifically, our evidence indicates that A. anophagefferens exhibited net population growth under organic N, but not inorganic N nutrient (specifically NO₃⁻) loading.     

Toxic activity from cultures of Karlodinium micrum (=Gyrodinium galatheanum) (Dinophyceae)—a dinoflagellate associated with fish mortalities in an estuarine aquaculture facility

The goal of this study was to test for, and partially characterize, toxic activity associated with the dinoflagellate Karlodinium micrum. Since 1996, three fish kill events associated with blooms of K. micrum have occurred at HyRock Fish Farm, an estuarine pond aquaculture facility raising hybrid striped bass on the Chesapeake Bay, MD, USA. Using an assay based on the lysis of rainbow trout erythrocytes, cultures of a Chesapeake Bay isolate of K. micrum have been shown to produce toxic substances which are released upon cell disturbance or damage. The LC₅₀ for hemolysis of a sonicated cell suspension was 2.4×10⁴ cells ml⁻¹, well within the range of cell concentrations observed associated with fish kills. The toxic activity from K. micrum cells and culture filtrates was traced to two distinct fractions that co-elute with polar lipids. The LC₅₀ for hemolysis of the larger of these two fractions (Tox A) was 284 ng ml⁻¹ while the LC₅₀ of the second, smaller, fraction (Tox B) was 600 ng ml⁻¹. For comparison, the LC₅₀ for the standard hemolysin saponin was 3203 ng ml⁻¹. At concentrations of 800 and 2000 ng ml⁻¹, respectively, Tox A was further shown to be ichthyotoxic to zebrafish (Danio rerio) larvae (80% mortality), and cytotoxic to a mammalian GH(4)C(1) cell line (100% LDH release). At a concentration of 600 ng ml⁻¹ Tox B was shown to be cytotoxic to a mammalian GH(4)C(1) cell line (>30% LDH release), but not ichthyotoxic to zebrafish (D. rerio) larvae up to a concentration of 250 ng ml⁻¹. Although treatment with either algicidal copper or potassium permanganate caused significant lysis of K. micrum cells (>70%), toxic activity was released after treatment with copper and eliminated following treatment with potassium permanganate. This observation in cultures is consistent with observations made at HyRock Fish Farm where significantly higher mortality was observed following treatment of a K. micrum bloom with copper sulfate compared to treatment with potassium permanganate. This study represents the first direct evidence of the toxicity of K. micrum isolated from the Chesapeake Bay.     

Ingestion of the dinoflagellate, Pfiesteria piscicida, by the calanoid copepod, Acartia tonsa

The dinoflagellate, Pfiesteria piscicida, can form harmful algal blooms in estuarine environments. The dominant copepod species usually found in these waters is Acartia tonsa. We tested the ability of A. tonsa to graze the non-toxic zoospore stage of P. piscicida and thus serve as a potential biological control of blooms of this algal species. A. tonsa grazed the non-toxic zoospore stages of both a non-inducible P. piscicida strain (FDEPMDR23) and a potentially toxic strain (Tox-B101156) at approximately equal rates. Ingestion of P. piscicida increased with cell concentration and exhibited a saturated feeding response. Both the maximum number of cells ingested (I_max) and the slope of the ingestion curve (α) of A. tonsa feeding on P. piscicida were comparable to these ingestion parameters for A. tonsa fed similar-sized phytoplankton and protozoan species. When these laboratory ingestion rates were combined with abundance estimates of A. tonsa from the Pocomoke Estuary and Chesapeake Bay, we found that significant grazing control of the non-toxic zoospore stage of P. piscicida by A. tonsa would only occur at high copepod abundances (>10 copepods L⁻¹). We conclude that under most in situ conditions the potential biological control of blooms of P. piscicida is exerted by microzooplankton grazers. However, in the less saline portions of estuaries where maximum concentrations of copepods often occur with low abundances of microzooplankton, copepod grazing coefficients can be similar to the growth rates of P. piscicida.     

Biodeterioration of some herbal raw materials by storage fungi and aflatoxin and assessment of Cymbopogon flexuosus essential oil and its components as antifungal

Deterioration of raw materials of six medicinal plants viz. Terminalia arjuna, Acorus calamus, Rauvolfia serpentina, Holarrhena antidysenterica, Withania somnifera and Boerhaavia diffusa was examined. Some of the contaminated raw materials were found to be deteriorated by toxigenic strains of Aspergillus flavus and contain aflatoxin B₁ (41.0–95.4 μg kg⁻¹) which is above the permissible limit. Essential oil of Cymbopogon flexuosus and its components was found efficient in checking fungal growth and aflatoxin production. C. flexuosus essential oil absolutely inhibited the growth of A. flavus and aflatoxin B₁ production at 1.3 μl ml⁻¹ and 1.0 μl ml⁻¹ respectively. The individual oil components were more efficacious than the Cymbopogon oil as such which emphasizes masking of their efficacy when combined together. Eugenol exhibited potent antifungal and aflatoxin inhibitory activity at 0.3 μl ml⁻¹ and 0.1 μl ml⁻¹ respectively. Eugenol was found superior over some prevalent synthetic antimicrobials and exhibited broad fungitoxic spectrum against some biodeteriorating moulds. Prospects of exploitation of the oil and its components as acceptable plant based antimicrobials in qualitative as well as quantitative control of biodeterioration of herbal raw materials have been discussed.     

Influence of humic, fulvic and hydrophilic acids on the growth, photosynthesis and respiration of the dinoflagellate Prorocentrum minimum (Pavillard) Schiller

Blooms of the dinoflagellate Prorocentrum minimum often occur in coastal regions characterized by variable salinity and elevated concentrations of terrestrially derived dissolved organic carbon (DOC). Humic, fulvic and hydrophilic acid fractions of DOC were isolated from runoff entering lower Narragansett Bay immediately after a rainfall event and the influence of these fractions upon P. minimum growth, cell yield, photosynthesis and respiration was examined. All organic fractions stimulated growth rates and cell yields compared with controls (no organic additions), but the extent of stimulation varied with the fraction and its molecular weight. Greatest stimulations were observed with humic and fulvic acids additions; cell yields were more than 2.5 and 3.5 times higher than with hydrophilic acid additions while growth rates were 21 and 44% higher, respectively. Responses to additions of different molecular weight fractions of each DOC fraction suggest that growth rate effects were attributable to specific molecular weight fractions: the >10,000 fraction of humic acids, both the >10,000 and <500 fractions of fulvic acids and the <10,000 fraction of hydrophilic acids. The form and concentration of nitrogen (as NO₃⁻ or NH₄⁺) present also influenced P. minimum response to DOC; 10–20 μg ml⁻¹ additions of fulvic acid had no effect upon growth rates in the presence of NH₄⁺ but significantly increased growth rates in the presence of NO₃⁻, a relationship probably related to fulvic acid effects upon trace metal bioavailability and subsequent regulation of the biosynthesis of enzymes required for NO₃⁻ assimilation. The influence of DOC additions on P. minimum respiration and production rates also varied with the organic fraction and its concentration. Production rates ranged from 1.1 to 3.4 pg O₂ cell⁻¹ h⁻¹, with highest rates observed upon exposure to fulvic and hydrophilic acid concentrations of >10 μm ml⁻¹. Low concentrations (5–10 μg ml⁻¹) of humic acid had no statistically significant effect upon production, but exposure to concentrations >25 μg ml⁻¹ resulted in a 30% decrease in O₂ evolution, probably due to light attenuation by the highly colored humic acid fraction. Respiration rates ranged from 1.2 to 2.7 pg O₂ cell⁻¹ h⁻¹ and were elevated upon exposure to both fulvic and hydrophilic acids, but not to humic acid. These results demonstrate that terrestrially derived DOC fractions play an active role in stimulation of P. minimum growth via direct effects upon growth, yield and photosynthesis as well as via indirect influences such as interactions with nitrogen and effects upon light attenuation.     

Feeding by ciliates on two harmful algal bloom species, Prymnesium parvum and Prorocentrum minimum

We have focused on ciliates as potential grazers on toxic phytoplankton because they are major herbivores in aquatic food webs. Ciliates may exert top down control on toxic phytoplankton blooms, potentially suppressing or shortening the duration of harmful algal blooms (HABs). We measured the growth rates of several ciliate species on uni-algal and mixed diets of both HAB and non-HAB algae. The tintinnids Favella ehrenbergii, Eutintinnus pectinis and Metacylis angulata and the non-loricate ciliates Strombidinopsis sp. and Strombidium conicum were isolated from Long Island Sound (LIS), and fed HAB species including the prymnesiophyte Prymnesium parvum (strain 97-20-01) and the dinoflagellate Prorocentrum minimum (strains Exuv and JA 98-01). Ciliates were fed algal prey from cultures at various growth phases and at varying concentrations. We observed no harmful effects of P. minimum (Exuv) on any of the ciliates. However in a comparison of strains, P. minimum (Exuv) supported high growth rates, whereas P. minimum (JA 98-01) supported only nominal growth. P. parvum was acutely toxic to ciliates at high concentrations (2×10⁴–3×10⁴ cells ml⁻¹). At low concentrations (5×10³–1×10⁴ cells ml⁻¹), or in culture filtrate, ciliates survived for at least several hours. In mixed diet experiments, as long as a non-toxic alga was available, ciliates survived and at times grew well at concentrations of P. parvum (5×10²–3×10⁴ cells ml⁻¹) that would otherwise have killed them. The present study suggests that prior to the onset of toxicity and bloom formation ciliates may exert grazing pressure on these HAB species, potentially contributing to the suppression or decline of P. minimum and P. parvum blooms.     

Blooms of Pseudo-nitzschia and domoic acid in the San Pedro Channel and Los Angeles harbor areas of the Southern California Bight, 2003–2004

Abundances of Pseudo-nitzschia spp. and concentrations of particulate domoic acid (DA) were determined in the Southern California Bight (SCB) along the coasts of Los Angeles and Orange Counties during spring and summer of 2003 and 2004. At least 1500 km² were affected by a toxic event in May/June of 2003 when some of the highest particulate DA concentrations reported for US coastal waters were measured inside the Los Angeles harbor (12.7 μg DA L⁻¹). Particulate DA levels were an order of magnitude lower in spring of 2004 (February and March), but DA concentrations per cell at several sampling stations during 2004 exceeded previously reported maxima for natural populations of Pseudo-nitzschia (mean = 24 pg DA cell⁻¹, range = 0–117 pg DA cell⁻¹). Pseudo-nitzschia australis dominated the Pseudo-nitzschia assemblage in spring 2004. Overall, DA-poisoning was implicated in >1400 mammal stranding incidents within the SCB during 2003 and 2004. Ancillary physical and chemical data obtained during our regional surveys in 2004 revealed that Pseudo-nitzschia abundances, particulate DA and cellular DA concentrations were inversely correlated with concentrations of silicic acid, nitrogen and phosphate, and to specific nutrient ratios. Particulate DA was detected in sediment traps deployed at 550 and 800 m depth during spring of 2004 (0.29–7.6 μg DA (g sediment dry weight)⁻¹). The highest DA concentration in the traps was measured within 1 week of dramatic decreases in the abundances of Pseudo-nitzschia in surface waters. To our knowledge these are the deepest sediment trap collections from which DA has been detected. Sinking of the spring Pseudo-nitzschia bloom may constitute a potentially important link between DA production in surface waters and benthic communities in the coastal ocean near Los Angeles. Our study indicates that toxic blooms of Pseudo-nitzschia are a recurring phenomenon along one of the most densely populated coastal stretches of the SCB and that the severity and magnitude of these events can be comparable to or greater than these events in other geographical regions affected by domoic acid.     

Dinoflagellate cysts from surface sediments of Saldanha Bay, South Africa: an indication of the potential risk of harmful algal blooms

The distribution and abundance of dinoflagellate cysts from recent coastal sediments in Saldanha Bay, was investigated, and compared to the cyst assemblages of the adjacent coastal upwelling system as reflected in the sediments off Lambert's Bay on the southern Namaqua shelf. Twenty-two cyst types were identified from three sample sites off Lambert's Bay with recorded abundances between 1726 and 1863 cysts ml⁻¹ wet sediment. At least 21 distinctive cyst types were identified from 32 sample sites within Saldanha Bay. Cyst abundance in Saldanha Bay was relatively low, averaging 116 cysts ml⁻¹ wet sediment. The region off Lambert's Bay is especially susceptible to the formation of harmful algal blooms attributed to high biomass dinoflagellate blooms. Owing to these blooms and the retentive circulation characteristics of this area, cyst formation and deposition is high. Blooms can be advected into Saldanha Bay, but their development and duration in the Bay is restricted by the system of exchange that operates between the Bay and the coastal upwelling system, in that there is a net export of surface waters from the Bay. Consequently, fewer cysts are formed and deposited within the Bay thereby reducing the likelihood of in situ bloom development initiated from the excystment of cysts.     

Ammonium excretion by a mutant of the nitrogen-fixing cyanobacterium Anabaena siamensis

Anabaena siamensis isolated from rice fields in Thailand is a fast growing cyanobacterium with a high nitrogen-fixing activity. Mutant strains resistant to the l-glutamate analogue, l-methionine sulfoximine (MSX) were isolated by ethyl methanesulfonate mutagenesis. A stable mutant named A. siamensis SS1, which released ammonium to the medium, was studied further. In batch cultures the rate of ammonium production peaked at the early log phase and gradually decreased until the 4th day of growth when the cultures reached a density of 90 μg chl ml⁻¹. To obtain constant release of ammonium by SS1, continuous culture experiments were performed at a cell density of 5 μg chl ml⁻¹ and the following results were obtained: (1) growth rate as the parent (μ:0·123 h⁻¹) in the presence and absence of 500 μm MSX; (2) 48% GS transferase activity when compared with the parent; (3) ammonium excretion at a rate of 8 μmol (mg chl)⁻¹ h⁻¹ as measured up to 20 generations (120 h); (4) depressed nitrogenase activity; and (5) 30% higher nitrogenase activity than that of the parent. SS1 immobilized in alginate beads (5 μg chl ml⁻¹) exhibited values of glutamine synthetase and nitrogenase activity similar to those of free cells. However, ammonium excretion at the rate of 11·61 μmol (mg chl)⁻¹ h⁻¹ was obtained only up to 20 h after loading in bioreactors, due to the fast growth of SS1 as also occurred in batch cultures.