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Author Posting. © The Author(s), 2006. This is the author's version of the work. It is posted here by permission of Geological Society for the Micropalaeontological Society for personal use, not for redistribution. The definitive version was published in Biological and Geological Perspectives of Dinoflagellates, edited by Jane Lewis, F. Marret, L.Bradley, :141-147. London: Geological Society for the Micropalaeontological Society, 2013. ISBN: 9781862393684.

Paralytic Shellfish Poisoning (PSP) toxins are annually recurrent along the Massachusetts coastline (USA), which includes many small embayments and salt ponds. Among these is the Nauset Marsh System (NMS), which has a long history of PSP toxicity. Little is known, however, about the bloom dynamics of the causative organism Alexandrium fundyense within that economically and socially important system. The overall goal of this work was to characterize the distribution and dynamics of A. fundyense blooms within the NMS and adjacent coastal waters by documenting the distribution and abundance of resting cysts and vegetative cells. Cysts were found predominantly in three drowned kettle holes or salt ponds at the distal ends of the NMS – Salt Pond, Mill Pond, and Town Cove. The central region of the NMS had a much lower concentration of cysts. Two types of A. fundyense blooms were observed. One originated entirely within the estuary, seeded by cysts in the three seedbeds. These blooms developed independently of each other and of the A. fundyense population observed in adjacent coastal waters outside the NMS. The temporal development of the blooms was different in the three salt ponds, with initiation differing by as much as 30 days. These differences do not appear to reflect the initial cyst abundances in these locations, and may simply result from higher cell retention and higher nutrient concentrations in Mill Pond, the first site to bloom. Germination of cysts accounted for a small percentage of the peak cell densities in the ponds, so population size was influenced more by the factors affecting growth than by cyst abundance. Subsurface cell aggregation (surface avoidance) limited advection of the vegetative A. fundyense cells out of the salt ponds through the shallow inlet channels. Thus, the upper reaches of the NMS are at the greatest risk for PSP since the highest cyst abundances and cell concentrations were found there. After these localized blooms in the salt ponds peaked and declined, a second, late season bloom occurred within the central portions of the NMS. The timing of this second bloom relative to those within the salt ponds and the coastal circulation patterns at that time strongly suggest that those cells originated from a regional A. fundyense bloom in the Gulf of Maine, delivered to the central marsh from coastal waters outside the NMS through Nauset Inlet. These results will guide policy decisions about water quality as well as shellfish monitoring and utilization within the NMS and highlight the potential for “surgical” closures of shellfish during PSP events, leaving some areas open for harvesting while others are closed.

A coupled physical/biological modeling system was used to hindcast the 2005 Alexandrium fundyense bloom in the Gulf of Maine and investigate the relative importance of factors governing the bloom’s initiation and development. The coupled system consists of a state-of-the-art, free-surface primitive equation Regional Ocean Modeling System (ROMS) tailored for the Gulf of Maine (GOM) using a multi-nested configuration, and a population dynamics model for A. fundyense. The system was forced by realistic momentum and buoyancy fluxes, tides, river runoff, observed A. fundyense benthic cyst abundance, and climatological nutrient fields. Extensive comparisons were made between simulated (both physical and biological) fields and in-situ observations, revealing that the hindcast model is capable of reproducing the temporal evolution and spatial distribution of the 2005 bloom. Sensitivity experiments were then performed to distinguish the roles of three major factors hypothesized to contribute to the bloom: 1) the high abundance of cysts in western GOM sediments; 2) strong northeaster storms with prevailing downwelling-favorable winds; and 3) a large amount of fresh water input due to abundant rainfall and heavy snowmelt. Results suggested that the high abundance of cysts in western GOM was the primary factor of the 2005 bloom. Wind forcing was an important regulator, as episodic bursts of northeast winds caused onshore advection of offshore populations. These downwelling favorable winds accelerated the alongshore flow, resulting in transport of high cell concentrations into Massachusetts Bay. A large regional bloom would still have happened, however, even with normal or typical winds for that period. Anomalously high river runoff in 2005 resulted in stronger buoyant plumes/currents, which facilitated the transport of cell population to the western GOM. While affecting nearshore cell abundance in Massachusetts Bay, the buoyant plumes were confined near to the coast, and had limited impact on the gulf-wide bloom distribution.

Observations of harmful algal blooms (HABs) of the dinoflagellate Alexandrium fundyense in an estuary over multiple years were used to assess drivers of their spatial and temporal variability. Nauset Estuary on Cape Cod, Massachusetts has a recurrent, self-seeding A. fundyense population that produces paralytic shellfish poisoning toxins and leads to nearly annual closure to shellfishing. Weekly surveys of the entire estuary were made in 3 of 4 consecutive years, with surveys of a subembayment during the intervening year. Major A. fundyense blooms were observed all 4 years, with maximum concentrations >106 cells L−1. Concentrations were greatest in three salt ponds at the distal edges of the estuary. The bloom timing varied among the salt ponds and among years, although the blooms had similar durations and maximum cell concentrations. Nutrient concentrations did not correlate with the growth of the bloom, but differences in water temperature among years and ponds were significant. Net growth rates inferred from the surveys were similar to those from laboratory experiments, and increased linearly with temperature. A growing degree day calculation was used to account for effects of interannual variability and spatial gradients in water temperature on population development. The approach collapsed variability in the timing of bloom onset, development, and termination across years and among ponds, suggesting that this relatively simple metric could be used as an early-warning indicator for HABs in Nauset and similar areas with localized, self-seeding blooms.

Measurements of the DNA content of different protist populations can shed light on a variety of processes, including cell division, sex, prey ingestion, and parasite invasion. Here, we modified an Imaging FlowCytobot (IFCB), a custom-built flow cytometer that records images of microplankton, to measure the DNA content of large dinoflagellates and other high-DNA content species. The IFCB was also configured to measure fluorescence from Cy3-labeled rRNA probes, aiding the identification of Alexandrium fundyense (syn. A. tamarense Group I), a photosynthetic dinoflagellate that causes paralytic shellfish poisoning (PSP). The modified IFCB was used to analyze samples from the development, peak and termination phases of an inshore A. fundyense bloom (Salt Pond, Eastham, MA, USA), and from a rare A. fundyense ‘red tide’ that occurred in the western Gulf of Maine, offshore of Portsmouth, NH (USA). Diploid or G2 phase (‘2C’) A. fundyense cells were frequently enriched at the near-surface, suggesting an important role for aggregation at the air-sea interface during sexual events. Also, our analysis showed that large proportions of A. fundyense cells in both the Salt Pond and red tide blooms were planozygotes during bloom decline, highlighting the importance of sexual fusion to bloom termination. At Salt Pond, bloom decline also coincided with a dramatic rise in infections by the parasite genus Amoebophrya. The samples that were most heavily infected contained many large cells with higher DNA-associated fluorescence than 2C vegetative cells, but these cells' nuclei were also frequently consumed by Amoebophrya trophonts. Neither large cell size nor increased DNA-associated fluorescence could be replicated by infecting an A. fundyense culture of vegetative cells. Therefore, we attribute these characteristics of the large Salt Pond cells to planozygote maturation rather than Amoebophrya infection, though an interaction between infection and planozygote maturation may also have contributed. The modified IFCB is a valuable tool for exploring the conditions that promote sexual transitions by dinoflagellate blooms but care is needed when interpreting results from samples in which parasitism is prevalent.

Cysts residing in benthic nepheloid layers (BNLs) documented in the Gulf of Maine have been proposed as a possible source of inoculum for annual blooms of a toxic dinoflagellate in the region. Herein we present a spatially extensive data set of the distribution and thickness of benthic nepheloid layers in the Gulf of Maine and the abundance and inventories of suspended Alexandrium fundyense cysts within these near-bottom layers. BNLs are pervasive throughout the gulf and adjacent Bay of Fundy with maximum layer thicknesses of 50–60 m observed. Mean BNL thickness is 30 m in the eastern gulf and Bay of Fundy, and 20 m in the western gulf. Cyst densities in the near-bottom particle resuspension layers varied by three orders of magnitude across the gulf with maxima of 105 cysts m−3. An important interconnection of elevated BNL cyst densities is observed between the Bay of Fundy, the Maine Coastal Current and the south-central region of the gulf. BNL cyst inventories estimated for the eastern and western gulf are each on the order of 1015 cysts, whereas the BNL inventory in the Bay of Fundy is on the order of 1016. Although BNL cyst inventories in the eastern and western gulf are 1–2 orders of magnitude smaller than the abundance of cysts in the upper 1 cm of sediment in those regions, BNL and sediment-bound cyst inventories are comparable in the Bay of Fundy. The existence of widespread BNLs containing substantial cyst inventories indicates that these near-bottom layers represent an important source of germinating A. fundyense cysts in the region.

Cysts of Alexandrium fundyense, a dinoflagellate that causes toxic algal blooms in the Gulf of Maine, spend the winter as dormant cells in the upper layer of bottom sediment or the bottom nepheloid layer and germinate in spring to initiate new blooms. Erosion measurements were made on sediment cores collected at seven stations in the Gulf of Maine in the autumn of 2011 to explore if resuspension (by waves and currents) could change the distribution of over-wintering cysts from patterns observed in the previous autumn; or if resuspension could contribute cysts to the water column during spring when cysts are viable. The mass of sediment eroded from the core surface at 0.4 Pa ranged from 0.05 kg m−2 near Grand Manan Island, to 0.35 kg m−2 in northern Wilkinson Basin. The depth of sediment eroded ranged from about 0.05 mm at a station with sandy sediment at 70 m water depth on the western Maine shelf, to about 1.2 mm in clayey–silt sediment at 250 m water depth in northern Wilkinson Basin. The sediment erodibility measurements were used in a sediment-transport model forced with modeled waves and currents for the period October 1, 2010 to May 31, 2011 to predict resuspension and bed erosion. The simulated spatial distribution and variation of bottom shear stress was controlled by the strength of the semi-diurnal tidal currents, which decrease from east to west along the Maine coast, and oscillatory wave-induced currents, which are strongest in shallow water. Simulations showed occasional sediment resuspension along the central and western Maine coast associated with storms, steady resuspension on the eastern Maine shelf and in the Bay of Fundy associated with tidal currents, no resuspension in northern Wilkinson Basin, and very small resuspension in western Jordan Basin. The sediment response in the model depended primarily on the profile of sediment erodibility, strength and time history of bottom stress, consolidation time scale, and the current in the water column. Based on analysis of wave data from offshore buoys from 1996 to 2012, the number of wave events inducing a bottom shear stress large enough to resuspend sediment at 80 m ranged from 0 to 2 in spring (April and May) and 0 to 10 in winter (October through March). Wave-induced resuspension is unlikely in water greater than about 100 m deep. The observations and model results suggest that a millimeter or so of sediment and associated cysts may be mobilized in both winter and spring, and that the frequency of resuspension will vary interannually. Depending on cyst concentration in the sediment and the vertical distribution in the water column, these events could result in a concentration in the water column of at least 104 cysts m−3. In some years, resuspension events could episodically introduce cysts into the water column in spring, where germination is likely to be facilitated at the time of bloom formation. An assessment of the quantitative effects of cyst resuspension on bloom dynamics in any particular year requires more detailed investigation.

As part of the NOAA ECOHAB funded Gulf of Maine Toxicity (GOMTOX) 1 project, we determined Alexandrium fundyense abundance, paralytic shellfish poisoning (PSP) toxin composition, and concentration in quantitatively-sampled size-fractionated (20–64, 64–100, 100–200, 200–500, and >500 μm) particulate water samples, and the community composition of potential grazers of A. fundyense in these size fractions, at multiple depths (typically 1, 10, 20 m, and near-bottom) during 10 large-scale sampling cruises during the A. fundyense bloom season (May–August) in the coastal Gulf of Maine and on Georges Bank in 2007, 2008, and 2010. Our findings were as follows: (1) when all sampling stations and all depths were summed by year, the majority (94%±4%) of total PSP toxicity was contained in the 20–64 μm size fraction; (2) when further analyzed by depth, the 20–64 μm size fraction was the primary source of toxin for 97% of the stations and depths samples over three years; (3) overall PSP toxin profiles were fairly consistent during the three seasons of sampling with gonyautoxins (1, 2, 3, and 4) dominating (90.7%±5.5%), followed by the carbamate toxins saxitoxin (STX) and neosaxitoxin (NEO) (7.7%±4.5%), followed by n-sulfocarbamoyl toxins (C1 and 2, GTX5) (1.3%±0.6%), followed by all decarbamoyl toxins (dcSTX, dcNEO, dcGTX2&3) (

As part of the Gulf of Maine Toxicity (GOMTOX) project, we determined Alexandrium fundyense abundance, paralytic shellfish poisoning (PSP) toxin levels in various plankton size fractions, and the community composition of potential grazers of A. fundyense in plankton size fractions during blooms of this toxic dinoflagellate in the coastal Gulf of Maine and on Georges Bank in spring and summer of 2007, 2008, and 2010. PSP toxins and A. fundyense cells were found throughout the sampled water column (down to 50 m) in the 20-64 μm size fractions. While PSP toxins were widespread throughout all size classes of the zooplankton grazing community, the majority of the toxin was measured in the 20-64 μm size fraction. A. fundyense cellular toxin content estimated from field samples was significantly higher in the coastal Gulf of Maine than on Georges Bank. Most samples containing PSP toxins in the present study had diverse assemblages of grazers. However, some samples clearly suggested PSP toxin accumulation in several different grazer taxa including tintinnids, heterotrophic dinoflagellates of the genus Protoperidinium, barnacle nauplii, the harpacticoid copepod Microsetella norvegica, the calanoid copepods Calanus finmarchicus and Pseudocalanus spp., the marine cladoceran Evadne nordmanni, and hydroids of the genus Clytia. Thus, a diverse assemblage of zooplankton grazers accumulated PSP toxins through food-web interactions. This raises the question of whether PSP toxins pose a potential human health risk not only from nearshore bivalve shellfish, but also potentially from fish and other upper-level consumers in zooplankton-based pelagic food webs.

For the period 2005–2009, the abundance of resting cysts in bottom sediments from the preceding fall was a first-order predictor of the overall severity of spring-summer blooms of Alexandrium fundyense in the western Gulf of Maine and southern New England. Cyst abundance off mid-coast Maine was significantly higher in fall 2009 than it was preceding a major regional bloom in 2005. A seasonal ensemble forecast was computed using a range of forcing conditions for the period 2004–2009, suggesting that a large bloom was likely in the western Gulf of Maine in 2010. This did not materialize, perhaps because environmental conditions in spring-summer 2010 were not favorable for growth of A.fundyense. Water mass anomalies indicate a regional-scale change in circulation with direct influence on A. fundyense’s niche. Specifically, near-surface waters were warmer, fresher, more stratified, and had lower nutrients than during the period of observations used to construct the ensemble forecast. Moreover, a weaker-than-normal coastal current lessened A. fundyense transport into the western Gulf of Maine and Massachusetts Bay. Satellite ocean color observations indicate the 2010 spring phytoplankton bloom was more intense than usual. Early-season nutrient depletion may have caused a temporal mismatch with A. fundyense’s endogenous clock that regulates the timing of cyst germination. These findings highlight the difficulties of ecological forecasting in a changing oceanographic environment, and underscore the need for a sustained observational network to drive such forecasts.

Zooplankton grazing is often a significant loss term for phytoplankton populations, including harmful algae, impacting the development and decline of blooms. However, detecting and quantifying predation on phytoplankton is often challenging, particularly during early bloom stages when phytoplankton cell concentrations are low. In this study, we used polymerase chain reaction (PCR) to detect ingestion of toxic dinoflagellates of the Alexandrium tamarense species complex by two copepods, Acartia hudsonica (laboratory population) and Calanus finmarchicus (field population). Recent ingestion of Alexandrium fundyense cells was indicated by positive amplification of an LSU rDNA fragment specific to A. fundyense from whole copepod extracts. In laboratory experiments, A. fundyense DNA was detectable for 2―4 h post-ingestion in A. hudsonica fed A. fundyense, but not detected in animals fed other phytoplankton, or starved. In field samples, ingestion of A. fundyense by C. finmarchicus was confirmed by PCR, including at four stations where the A. fundyense concentration was ≤ 14 cells L ―1 . At these low prey concentrations, ingestion rates on A. fundyense may have been as low as 1 cell copepod ―1 day ―1 . Nevertheless, simulations of A. fundyense population growth suggest that a few predators L ―1 have the potential to curb the early development of a slow-growing bloom, even if ingestion rates are extremely low. Low predation rates can still have a large impact when prey populations are small.

In-situ observations and a coupled bio-physical model were used to study the germination, initiation, and development of the Gulf of Maine (GOM) Alexandrium fundyense bloom in 2006. Hydrographic measurements and comparisons with GOM climatology indicate that 2006 was a year with normal coastal water temperature, salinity, current and river runoff conditions. A. fundyense cyst abundance in bottom sediments preceding the 2006 bloom was at a moderate level compared to other recent annual cyst survey data. We used the coupled bio-physical model to hindcast coastal circulation and A. fundyense cell concentrations. Field data including water temperature, salinity, velocity time series and surface A. fundyense cell concentration maps were applied to gauge the model's fidelity. The coupled model is capable of reproducing the hydrodynamics and the temporal and spatial distributions of A. fundyense cell concentration reasonably well. Model hindcast solutions were further used to diagnose physical and biological factors controlling the bloom dynamics. Surface wind fields modulated the bloom's horizontal and vertical distribution. The initial cyst distribution was found to be the dominant factor affecting the severity and the interannual variability of the A. fundyense bloom. Initial cyst abundance for the 2006 bloom was about 50% of that prior to the 2005 bloom. As the result, the time-averaged gulf-wide cell concentration in 2006 was also only about 60% of that in 2005. In addition, weaker alongshore currents and episodic upwelling-favorable winds in 2006 reduced the spatial extent of the bloom as compared with 2005.

As part of the NOAA ECOHAB funded Gulf of Maine Toxicity (GOMTOX)1 project, we determined Alexandrium fundyense abundance, paralytic shellfish poisoning (PSP) toxin composition, and concentration in quantitatively-sampled size-fractionated (20–64, 64–100, 100–200, 200–500, and > 500 μm) particulate water samples, and the community composition of potential grazers of A. fundyense in these size fractions, at multiple depths (typically 1, 10, 20 m, and near-bottom) during 10 large-scale sampling cruises during the A. fundyense bloom season (May–August) in the coastal Gulf of Maine and on Georges Bank in 2007, 2008, and 2010. Our findings were as follows: (1) when all sampling stations and all depths were summed by year, the majority (94% ± 4%) of total PSP toxicity was contained in the 20–64 μm size fraction; (2) when further analyzed by depth, the 20–64 μm size fraction was the primary source of toxin for 97% of the stations and depths samples over three years; (3) overall PSP toxin profiles were fairly consistent during the three seasons of sampling with gonyautoxins (1, 2, 3, and 4) dominating (90.7% ± 5.5%), followed by the carbamate toxins saxitoxin (STX) and neosaxitoxin (NEO) (7.7% ± 4.5%), followed by n-sulfocarbamoyl toxins (C1 and 2, GTX5) (1.3% ± 0.6%), followed by all decarbamoyl toxins (dcSTX, dcNEO, dcGTX2&3) (< 1%), although differences were noted between PSP toxin compositions for nearshore coastal Gulf of Maine sampling stations compared to offshore Georges Bank sampling stations for 2 out of 3 years; (4) surface cell counts of A. fundyense were a fairly reliable predictor of the presence of toxins throughout the water column; and (5) nearshore surface cell counts of A. fundyense in the coastal Gulf of Maine were not a reliable predictor of A. fundyense populations offshore on Georges Bank for 2 out of the 3 years sampled.

Embayments and salt ponds along the coast of Massachusetts can host localized blooms of the toxic dinoflagellate Alexandrium fundyense. One such system, exhibiting a long history of toxicity and annual closures of shellfish beds, is the Nauset Marsh System (NMS) on Cape Cod. In order to measure net growth rates of natural A. fundyense populations in the NMS during spring 2012, incubation experiments were conducted on seawater samples from two salt ponds within the NMS (Salt Pond and Mill Pond). Seawater samples containing natural populations of grazers and A. fundyense were incubated at ambient temperatures. Concentrations of A. fundyense after incubations were compared to initial abundances to determine net increases from population growth, or decreases presumed to be primarily due to grazing losses. Abundances of both microzooplankton (ciliates, rotifers, copepod nauplii and heterotrophic dinoflagellates) and mesozooplankton (copepodites and adult copepods, marine cladocerans, and meroplankton) grazers were also determined. This study documented net growth rates that were highly variable throughout the bloom, calculated from weekly bloom cell counts from the start of sampling to bloom peak in both ponds (Mill Pond range = 0.12–0.46 d−1; Salt Pond range = −0.02 to 0.44 d−1). Microzooplankton grazers that were observed with ingested A. fundyense cells included polychaete larvae, rotifers, tintinnids, and heterotrophic dinoflagellates of the genera Polykrikos and Gymnodinium. Significant A. fundyense net growth was observed in two incubation experiments, and only a single experiment exhibited significant population losses. For the majority of experiments, due to high variability in data, net changes in A. fundyense abundance were not significant after the 24-h incubations. However, experimental net growth rates through bloom peak were not statistically distinguishable from estimated long-term average net growth rates of natural populations in each pond (Mill Pond = 0.27 d−1 and Salt Pond = 0.20 d−1), which led to peak bloom concentrations on the order of 106 cells l−1 in both ponds. Experimental net growth rates from the incubations underestimated the observed natural net growth rates at several time intervals prior to bloom peak, which may indicate that natural populations experienced additional sources of vegetative cells or periods of reduced losses that the 24-h incubation experiments did not capture, or that the experimental procedure introduced containment artifacts.

The Casco Bay region, an embayment adjacent to the Kennebec River, has been suggested as a source region for Alexandrium fundyense bloom development in the western Gulf of Maine (GOM). In this study, shipboard observations were acquired within Casco Bay and the nearby coastal waters during the spring of 1998 and 2000. In the early bloom season, low A. fundyense abundances ( −1 ) were observed within the bay, sometimes isolated from A. fundyense populations observed in adjacent coastal waters. When high abundances of A. fundyense (>500 cells l −1 ) were observed within Casco Bay, they were contiguous with coastal populations observed within the Kennebec/Penobscot river plume and within offshore waters of the western segment of the Maine Coastal Current (WMCC). This general distributional pattern occurred during both study years. Wind directly affected the pathway of the incoming coastal populations. Downwelling-favorable winds generally facilitated bloom formation (and outbreaks of shellfish toxicity) within Casco Bay by enhancing the connection with offshore populations via alongshore and onshore transport of cells from the upstream coastal waters. In contrast, persistent upwelling-favorable winds were associated with low A. fundyense cell abundances (and shellfish toxicity) in Casco Bay by slowing the advance of the coastal population and shifting it offshore with the Kennebec plume front. The striking difference between late season (June) population abundances of the two study years can be explained by a combination of the wind pre-history and interannual differences in large-scale (Gulf-wide) circulation patterns, as evidenced by higher salinities in the coastal waters in 2000 vs. 1998. Advection of A. fundyense cells into Casco Bay and retention, not local growth within the Bay, are likely the dominant processes that typically result in the accumulation of high populations and shellfish toxicity in the Bay. A variety of mechanisms (e.g., circulation underneath or steerage around the Kennebec plume) promote the transfer of cells across the Kennebec/Penobscot plume barrier and into the Bay. These dynamics are complex given the variability of the wind, river inputs, and the Maine Coastal Current structure. Nonetheless, general distributional patterns of A. fundyense and the associated hydrography clearly demonstrate that populations within Casco Bay are not isolated, but instead are part of the large-scale coastal populations that inhabit the western GOM, likely originating from further upstream in the coastal flow. With that knowledge, the ultimate goal of predicting outbreaks of shellfish toxicity in the western GOM based on meteorological and hydrographic conditions may become a reality.

The dinoflagellate Alexandrium fundyense is a common, recurring harmful algal bloom (HAB) species in the Gulf of Maine. To date, most physiological measurements of phytoplankton in the field provide data on the entire community, yet efforts to obtain species-specific data are particularly important for understanding the ecological and physiological dynamics of HAB species, such as, Alexandrium. Alexandrium spp., do not usually dominate the planktonic community in the Gulf of Maine, but are of great interest due to the potent toxins produced. In order to determine the nutritional status of Alexandrium spp. in natural populations, indicators of nutrient deprivation need to be identified that are specific to that one species. To date, the saxitoxin content of A. fundyense is known to vary under different environmental conditions such as nitrogen and phosphorous limitation. However, in batch culture the composition of the toxin (the relative amounts of each saxitoxin derivative per cell) appears to be a stable quantity and thus is sometimes viewed as a biochemical marker of individual strains. In more recent studies, toxin composition has been shown to vary during progressive N- and Plimitation, once the cells are given time to achieve steady state in semi-continuous, nutrient-limited cultures. Using both the absolute toxin concentrations and relative proportion (mole % total toxin) of each toxin derivative, N- and P-limitation can be distinguished based on the observed trends in the different saxitoxin derivatives. In this study, we examine the toxin content and composition in natural A. fundyense populations during a spring bloom in Casco Bay, ME from April–June of 1998. This allows us to examine whether A. fundyense populations in the western Gulf of Maine are sufficiently homogenous to permit the detection of toxin composition and toxin content differences through time and space, and if so, to determine whether those changes are indicative of a particular nutritional state (e.g., N-limitation). Using both toxin composition and toxin ratios determined from field samples during an A. fundyense bloom, the ratios generally correlated with N-limitation in the Casco Bay region. r 2005 Elsevier Ltd. All rights reserved.

Three different molecular methods were used with traditional brightfield microscope techniques to enumerate the toxic dinoflagellate Alexandrium fundyense in samples collected in the Gulf of Maine in 1998, 2000, 2001, and 2003. Two molecular probes were used in fluorescent whole-cell (WC) microscopic assays: a large-subunit ribosomal RNA (LSU rRNA) oligonucleotide probe (NA1) and a monoclonal antibody probe thought to be specific for Alexandrium spp. within the tamarense / catenella / fundyense complex. Cell abundance estimates also were obtained using the NA1 oligonucleotide probe in a semi-quantitative sandwich hybridization assay (SHA) that quantified target rRNA in cell lysates. Here we compare and contrast the specificity and utility of these probe types and assay approaches. WC counts of the 1998 field samples demonstrated that A. fundyense cell densities estimated using the antibody approach were higher than those using either the NA1 oligonucleotide or brightfield microscopy due to the co-occurrence of A. ostenfeldii with A. fundyense , and the inability of the antibody to discriminate between these two species. An approach using cell size and the presence or absence of food vacuoles allowed more accurate immunofluorescent cell counts of both species, but small cells of A. ostenfeldii that did not contain food vacuoles were still mistakenly counted as A. fundyense . For 2001, a dual-labeling procedure using two oligonucleotide probes was used to separately enumerate A. ostenfeldii and A. fundyense in the WC format. In addition, the SHA was used in 2001 and 2003 to enumerate A. fundyense . Some agreement was observed between the two oligonucleotide methods, but there were differences as well. Not including samples with cell numbers below empirically determined detection limits of 25 cells l –1 , good correlation was observed for surface samples and vertical profiles in May 2001 and June 2003 when the SHA estimates were, on average, equivalent, and 1.5× the WC counts, respectively. The worst correlations were for virtually all samples from the June 2001 cruise where the SHA both over- and under-estimated the WC counts. Some differences were expected, since the SHA and the WC assays measure different, but related parameters. The former quantifies intact cells and particulate material that might contain non-viable cells or fragments, whereas the latter measures only intact cells that survive sample processing and are visible in a sample matrix. A variety of factors can thus affect results, particularly with the WC method, including variable uptake of the oligonucleotide probe due to cell permeability changes, cell lysis during sampling, preservation and processing; variable rRNA content or accessibility due to nutritional or environmental factors; and the variable detection of intact cells or cell fragments in fecal pellets and detritus. The SHA offers dramatic increases in sample throughput, but introduces uncertainties, such as those due to sample matrix effects (non-specific labeling and cross-reactions), variable rRNA levels in intact cells or to the possible presence of target rRNA in cell fragments, fecal pellets, or detritus. Molecular probes are powerful tools for monitoring and research applications, but more work is needed to compare and refine these different cell enumeration methods on field samples, as well as to assess the general validity of brightfield or fluorescent WC approaches.

The transfer of marine algal toxins involving a range of phytoplanktivorous vectors is well documented as a means of exposing organisms at higher trophic levels (including humans) to these naturally occurring yet harmful compounds. While previous studies have examined the potential for, and dynamics of, algal toxin accumulation by individual zooplankton species, few have attempted to distinguish the contribution of various grazer size classes to toxin trophic transfer in natural communities and characterize some of the factors that can influence this process. The current investigation was aimed at describing the size-fractioned (64–100, 100–200, 200–500, >500 μm) accumulation of paralytic shellfish poisoning (PSP) toxins by zooplankton in Casco Bay and the adjacent coastal waters of the Gulf of Maine during a series of cruises from April to June 1998. Several variables, including the abundance of PSP toxin-producing Alexandrium fundyense , in-water toxin concentrations associated with this dinoflagellate, and algal toxin cell quotas, were measured and their relationship to zooplankton toxin accumulation assessed. A principal finding of this work was the ability of any grazer size class examined (including grazers present in the 20–64 μm A. fundyense -containing fraction) to serve as an initial vector for introducing PSP toxins into the Casco Bay food web at various times during the sampling period, thereby providing multiple potential routes of toxin trophic transfer. In addition, trends observed in the coincident mapping of A. fundyense cells and their associated toxin were generally in agreement, yet did not remain closely coupled at all times. Therefore, although A. fundyense abundance can be a reasonable indicator of PSP toxin presence in the phytoplankton, this relationship can vary considerably and lead to situations where elevated toxin levels occur at low cell concentrations and vice versa. The uncoupling of A. fundyense cell and in-water toxin concentrations in the 20–64 μm, A. fundyense -containing size fraction implied fluctuations in the algal toxin cell quota, which ranged from ca. 10 to 2000 fmol STX equiv. cell −1 . Some of this variability may reflect the changing presence in this size fraction of grazers (e.g., tintinnids) capable of toxin accumulation, causing an upward bias in A. fundyense toxin cell quota estimates. Overall, the extent of PSP toxin transfer into zooplankton will be determined by a complex interaction among several factors, including A. fundyense and grazer abundance, algal toxin cell quota, and zooplankton community composition. An ability to predict zooplankton toxin accumulation will require further investigation of the relationships between these and other factors, aimed specifically at modeling the process of toxin trophic transfer to grazers and ultimately to their predators.

During spring blooms of the toxic dinoflagellate Alexandrium fundyense in Casco Bay, Maine in 1998, we investigated vectorial intoxication of various zooplankton size fractions with PSP toxins, including zooplankton community composition from quantitative zooplankton samples (>102 μm), as well as zooplankton composition in relation to toxin levels in various size fractions (20–64, 64–100, 100–200, 200–500, >500 μm). Zooplankton abundance in 102 μm mesh samples was low (most values 102 μm) were dominated by copepod nauplii, and Oithona similis copepodites and adults at most locations except for those furthest inshore. At these inshore locations, Acartia hudsonica copepodites and adults were usually dominant. Larger copepods such as Calanus finmarchicus, Centropages typicus, and Pseudocalanus spp. were found primarily offshore, and at much lower abundances than O. similis. Rotifers, mainly present from late April to late May, were most abundant inshore. The marine cladoceran Evadne nordmani was sporadically abundant, particularly in mid-June. Microplankton in 20–64 μm size fractions was generally dominated by A. fundyense, non-toxic dinoflagellates, and tintinnids. Microplankton in 64–100 μm size fractions was generally dominated by larger non-toxic dinoflagellates, tintinnids, aloricate ciliates, and copepod nauplii, and in early May, rotifers. Some samples (23%) in the 64–100 μm size fractions contained abundant cells of A. fundyense, presumably due to sieve clogging, but most did not contain A. fundyense cells. This suggests that PSP toxin levels in those samples were due to vectorial intoxication of microzooplankters such as heterotrophic dinoflagellates, tintinnids, aloricate ciliates, rotifers, and copepod nauplii via feeding on A. fundyense cells. Dominant taxa in zooplankton fractions varied in size. Samples in the 100–200 μm size fraction were overwhelmingly dominated in most cases by copepod nauplii and small copepodites of O. similis, and during late May, rotifers. Samples in the 200–500 μm size fraction contained fewer copepod nauplii, but progressively more copepodites and adults of O. similis, particularly at offshore locations. At the most inshore stations, copepodites and adults of A. hudsonica were usual dominants. There were few copepod nauplii or O. similis in the>500 μm size fraction, which was usually dominated by copepodites and adults of C. finmarchicus, C. typicus, and Pseudocalanus spp. at offshore locations, and A. hudsonica inshore. Most of the higher PSP toxin concentrations were found in the larger zooplankton size fractions that were dominated by larger copepods such as C. finmarchicus and C. typicus. In contrast to our earlier findings, elevated toxin levels were also measured in numerous samples from smaller zooplankton size fractions, dominated by heterotrophic dinoflagellates, tintinnids and aloricate ciliates, rotifers, copepod nauplii, and smaller copepods such as O. similis and, at the most inshore locations, A. hudsonica. Thus, our data suggest that ingested PSP toxins are widespread throughout the zooplankton grazing community, and that potential vectors for intoxication of zooplankton assemblages include heterotrophic dinoflagellates, rotifers, protozoans, copepod nauplii, and small copepods.

Toxic Alexandrium fundyense blooms in the western Gulf of Maine (GOM) are a common occurrence, causing paralytic shellfish poisoning (PSP) each spring. In contrast, high A. fundyense abundances and PSP toxicity commonly occur later in the summer in the eastern GOM and the Bay of Fundy. The objective of this study was to determine if the bloom dynamics of the two areas are linked early in the bloom season when initial outbreaks of toxicity are reported. A. fundyense cell abundance and hydrographic data were acquired during three cruises in May and June, 2001 spanning areas of the western and eastern GOM. Surface drifters also were released into the nearshore coastal flow of the eastern GOM. These data provide a coherent view of the springtime evolution of toxic A. fundyense blooms in the GOM and the influence of both small- and large-scale circulation. Early in the bloom season (May), the bulk of the A. fundyense population was consistently observed in an alongshore band of cells in the eastern GOM associated with relatively low-salinity water (o32) that likely originated from inputs further upstream in the coastal flow, predominantly the St. John River in the Bay of Fundy. At that time, the western GOM was virtually devoid of cells. In June, the population was bifurcated, with one branch extending alongshore into the western GOM and the other into the offshore waters of the interior GOM. This pattern was consistent with circulation models of the coastal GOM that unambiguously revealed a bifurcated flow with the branch nearest the coast directed alongshore to the western GOM. The most significant finding of this study is that A. fundyense populations along the eastern Maine coast were delivered along an ‘‘inside track’’ relative to the core of the eastern segment of the Maine Coastal Current. The transport pathway carried cells across the mouth of Penobscot Bay and into the western GOM coincident with outbreaks of nearshore PSP toxicity. The transport is influenced by wind. In particular, some of the cells within the nearshore flow may be lost to the interior GOM when upwelling-favorable winds transport them offshore and into the large-scale circulation dominated by the cyclonic flow of the Jordan Basin Gyre. Downwelling-favorable winds keep the cells close to the coast and rapidly transport them into the western GOM. This study unequivocally demonstrates the linkage between early season A. fundyense bloom formation in the eastern GOM and blooms in the western GOM. It also indicates that the lack of toxicity along the mid-Maine coast commonly referred to as the ‘‘PSP sandwich’’ may be an artifact of the lack of shellfish sampling along the outer remote islands of Penobscot Bay, as populations sufficient to cause toxicity can be found adjacent to the coastline. The association of the A. fundyense

The flux of cells from germinated cysts is critical in the population dynamics of many dinoflagellates. Here, data from a large-scale cyst survey are combined with surveys in other years to yield an Alexandrium fundyense cyst distribution map for the Gulf of Maine that is massive in geographic extent and cyst abundance. The benthic cyst population extends nearly 500 km alongshore. Embedded within it are several distinct accumulation zones or “seedbeds,” each 3000–5000 km 2 in area. Maximal cyst abundances range from 2–20×10 6 cysts m −2 . Cysts are equally or more abundant in deeper sediment layers; nearshore, cysts are fewer by a factor of 10 or more. This cyst distribution reflects sedimentary dynamics and the location of blooms in overlying surface waters. The flux of germinated cells from sediments was estimated using a combination of laboratory measurements of cyst germination and autofluorescence and observations of cyst autofluorescence in the field. These measurements constrained a germination function that, when applied to the cyst distribution map, provided an estimate of the germination inoculum for a physical/biological numerical model. In the laboratory studies, virtually all cysts incubated at different temperatures and light regimes became autofluorescent, but the rate of development was slower at lower temperatures, with no difference between light and dark incubations. Germination rates were highest at elevated temperatures, and were 2-fold greater in the light than in the dark. Laboratory and field fluorescence measurements suggest that>70% of the cysts in the top cm of sediment would germinate over a 60–90 day period in offshore waters. The combination of laboratory germination experiments and numerical modeling predicts nearly 100% germination of cysts in the top cm of sediment and resulting early season cell concentrations that are comparable in magnitude to observed cell distributions. It cannot account for late-season peaks in cell abundance that are heavily influenced by vegetative growth. Cyst germination flux from deep-water (>50 m) cyst seedbeds is 14X the flux in shallow waters. A conceptual model is proposed that is consistent with observed and modeled A. fundyense cyst and motile cell distributions and dynamics in the Gulf of Maine. Cysts germinate within the Bay of Fundy seedbed, causing localized, recurrent blooms that are self-seeding and “propagatory” in nature, supplying cells that populate the eastern segment of the Maine Coastal Current (MCC) and eventually deposit cysts offshore of Penobscot and Casco Bays. These cysts serve as a seed population for western Maine blooms that are transported to the west by the western segment of the MCC, where cells are removed either by mortality or advection from the region. Without the localized, “incubator” characteristic of the Bay of Fundy bloom zone, A. fundyense populations in the Gulf of Maine should diminish through time. Their persistence over many decades highlights the effectiveness of the mechanisms described here.

From May to July, 2005, an extensive bloom of Alexandrium fundyense occurred along the coast of southern New England. The outbreak eventually closed shellfish beds from central Maine to Massachusetts, including Nantucket Island and portions of Martha’s Vineyard, and resulted in the closure of 40,000 km 2 of offshore federal waters as well. The coastal Alexandrium bloom was exceptional in several ways: high toxin levels were measured farther south than ever before in New England; levels of toxicity in many locations were higher than previously observed at those stations; for the first time toxicity at some locations was above quarantine levels; cell concentrations far exceeded those observed in the coastal waters of southern New England in the past; and for the first time in the region the governors of Maine and Massachusetts officially declared the red tide to be a disaster, clearing the way for federal assistance. Initial observations suggest that several factors contributed to this bloom. Abundant rainfall and heavy snowmelt substantially increased the amount of fresh water entering the Gulf of Maine. Combined with other freshwater inputs, we hypothesize that this provided macro- and micro-nutrients, a stratified water column, and a transport mechanism that led to high cell abundances and broad, region-wide dispersal of the organism. Warm temperatures in western waters also would have favored A. fundyense growth. In addition, several storms with strong winds out of the northeast occurred at times when cells were abundant and in locations where the winds could advect them into Massachusetts and Cape Cod Bays and keep them there, leading to high cell concentrations and toxicity. Another contributing factor may have been the high abundance of newly deposited cysts in western Gulf of Maine sediments, as documented in a fall 2004 survey. Here, we evaluate this bloom and the patterns of toxicity in light of the conceptual models for A. fundyense dynamics developed during the Ecology and Oceanography of Harmful Algal Blooms (ECOHAB)–Gulf of Maine (GOM) program. Several features of the 2005 bloom conform to the mechanisms proposed in those models, including the alongshore transport of cells in major water masses and episodic intrusions of cells toward shore due to downwelling-favorable wind forcings. The

Inorganic nutrients and organic nitrogen were measured in April–June of 1998 and 2000 near Casco Bay, Maine and the adjacent coastal waters as part of the Ecology and Oceanography of Harmful Algal Blooms—Gulf of Maine (ECOHAB-GOM) program. The samples were collected during development of toxic Alexandrium fundyense blooms [ Keafer, B.A., Churchill, J.H., Anderson, D.M., 2005 . Blooms of the toxic dinoflagellate, Alexandrium fundyense in the Casco Bay region of the western Gulf of Maine: advection from offshore source populations and interactions with the Kennebec River plume. Deep Sea Research II, this issue [ doi:10.1016/j.dsr2.2005.06.017 ]] and shellfish toxicity outbreaks [ Bean, L.L., McGowan, J.D., Hurst Jr., J.W., 2005 . Annual variations of paralytic shellfish poisoning in Maine, USA 1997–2001. Deep Sea Research II, this issue [ doi:10.1016/j.dsr2.2005.06.023 ]] along the western Maine coastline. Nutrient data were correlated with cell abundance of the toxic dinoflagellate A. fundyense and hydrographic data. There was a general trend of decreasing dissolved inorganic nitrogen (DIN) concentrations with increasing A. fundyense cell density for both years. Greater DIN concentrations in surface waters in early May of 2000 may partially explain why A. fundyense populations were larger in 2000 compared to 1998. Average DIN concentrations in the surface were less than 1 μM during peak A. fundyense cell abundance with N:P ratios typically less than 4:1. Silicate concentrations were high relative to inorganic nitrogen concentrations, with average Si:DIN ratios greater than 5:1 in water associated with A. fundyense blooms. Dissolved organic nitrogen (DON) was the dominant form of nitrogen in areas of high cell abundance, with concentrations typically 5–10 times higher than DIN. It is possible that A. fundyense may have utilized dissolved organic nitrogen forms to some extent; however, no correlation was observed between DON and A. fundyense blooms in either year. Since the A. fundyense abundance did not reach bloom concentrations (>100 cells/L) until water temperatures exceeded approximately 6 °C, low DIN concentrations at the on-set of bloom initiation may be the result of uptake by an earlier cold-adapted, spring diatom bloom. It is likely that A. fundyense populations in the western Gulf of Maine are habitually nitrogen-limited each season which may partially explain why relatively small cell concentrations are usually recorded in this region.

The plume advection hypothesis links blooms of the toxic dinoflagellate Alexandrium fundyensein the western Gulf of Maine (GOM) to a buoyant plume derived from river outflows. This hypothesis was examined with cruise and moored-instrument observations in 1993 when levels of paralytic shellfish poisoning (PSP) toxins were high, and in 1994 when toxicity was low. A coupled physical‐biological model simulated hydrography and A. fundyense distributions. Initial A. fundyense populations were restricted to low-salinity nearshore waters near Casco Bay, but also occurred in higher salinity waters along the plume boundary. This suggests two sources of cells—those from shallow-water cyst populations and those transported to shore from offshore blooms in the eastern segment of the Maine coastal current (EMCC). Observations confirm the role of the plume inA. fundyense transport and growth. Downwelling-favorable winds in 1993 transported the plume and its cells rapidly alongshore, enhancing toxicity and propagating PSP to the south. In 1994, sustained upwelling moved the plume offshore, resulting in low toxicity in intertidal shellfish. A. fundyense blooms were likely nutrient limited, leading to low growth rates and moderate cell abundances. These observations and mechanisms were reproduced by coupled physical‐biological model simulations. The plume advection hypothesis provides a viable explanation for outbreaks of PSP in the western GOM, but should be refined to include two sources for cells that populate the plume and two major pathways for transport: one within the low-salinity plume and another where A. fundyense cells originating in the EMCC are transported along the outer boundary of the plume front with the western segment of the Maine coastal current.

Observations in the Gulf of Maine, USA, were used to characterize the freshwater transport, temporal variability and dynamics of the western Maine coastal current. These observations included moored measurements, multiple hydrographic surveys, and drifter releases during April–July of 1993 and 1994. There is a strong seasonal signal in salinity and along-shore velocity of the coastal current, caused by the freshwater inputs of the rivers entering the western Gulf. Surface salinity within the coastal current during the spring freshet is typically 2 psu below ambient, and along-shore currents in the surface layer are directed southwestward at speeds of 0.10–0.20 ms � 1 , occasionally reaching 0.50 ms � 1 . The plume thickness is typically 10–20 m in water depths of 50–100 m, thus it is well isolated from the bottom over most of its areal extent. The along-coast freshwater transport within the plume varies considerably due to variations in wind stress, but on time scales of weeks to months it follows the variations of riverine input, with a time lag consistent with the advective velocity. Less than half of the transport of the coastal current is explained by the baroclinic gradient; the barotropic forcing associated with the larger-scale dynamics of the Gulf of Maine accounts for about 60% of the transport. The volume of freshwater transport in the coastal current exceeds the local riverine input of fresh water by 30%, suggesting a significant contribution of freshwater transport fromthe St. John River, 500 kmnortheastward. The measurements within the western Maine coastal current, however, indicate a significant decrease in the baroclinic transport of fresh water along the coast, with an e-folding scale of approximately 200 km. r 2004 Elsevier Ltd. All rights reserved.

A combination of observations and model results suggest a mechanism by which coastal blooms of the toxic dinoflagellate Alexandrium fundyense can be initiated from dormant cysts located in offshore sediments. The mechanism arises from the joint effects of organism behavior and the wind-driven response of a surface-trapped plume of fresh water originating from riverine sources. During upwelling-favorable winds, the plume thins vertically and extends offshore; downwelling winds thicken the plume and confine it to the nearshore region. In the western Gulf of Maine, the offshore extent of the river plume during upwelling conditions is sufficient to entrain upward-swimming A. fundyense cells germinated from offshore cyst beds. Subsequent downwelling conditions then transport those populations towards the coast.

A method to isolate cells of the toxic dinoflagellate Alexandrium fundyense from sea- water samples using magnetic beads was evaluated to determine whether it could be used to obtain species-specific physiological measurements. Two isolation procedures were tested. The direct tech- nique used an Alexandrium-specific primary antibody coupled directly to magnetic beads, which were then bound to the target cells. With the indirect technique, the primary antibody (Ab) was bound to the target cells, and this Ab-cell complex was then exposed to the beads. Four different bead types, varying in size and coating, were evaluated. The bead isolation method was tested on live cells and on cells preserved with either 0.23 N sulfuric acid or 4% formalin. Under optimal condi- tions using either live or preserved cells, ca. 80% recovery was achieved when the indirect technique was applied with either M-280 Streptavidin or M-280 sheep anti-mouse beads. However, with the di- rect technique, the highest recoveries were only ca. 20% with live samples and ca. 50% with acid- preserved cells. After bead isolation, ca. 70% of the recovered live cells were intact; 30% were com- pletely or partially broken. A variety of post-isolation measurements were conducted on the separated cells, including nucleic acids, total proteins, chlorophyll a (chl a), C-2 toxin, saxitoxin (STX), neosaxitoxin (NEO), organic carbon (C) and nitrogen (N), and 13 C/ 12 C and 15 N/ 14 N. These measurements were normalized to the number of intact cells. For live samples, there were no statisti- cally significant differences between the control cells and the bead-recovered cells for cell quotas of nucleic acids, total proteins, chl a, STX, C-2 toxin, and NEO. Similarly, no differences were found in these parameters between the live samples and the acid-preserved samples, except for the nucleic acid measurements where the acid interfered with the measurements. However, there were signifi- cant differences between the live cells and formalin-preserved, bead-recovered cells for all para- meters measured. This was not due to the bead isolation procedure but to the preservative. Signifi- cant C, N, C/N, 13 C/ 12 C, and 15 N/ 14 N differences between live cells and bead-recovered cells were introduced by the immunomagnetic separation protocols, due to the chemical and isotopic content of the beads. These artifacts need to be removed if monospecific elemental and isotopic abundance measurements are to be attempted using this isolation method. Overall, immunomagnetic cell sorting is potentially a valuable tool for taxon-specific isolation, allowing physiological, chemical, and isotopic characterization of naturally occurring phytoplankton populations. However, each measurement must be evaluated to insure that the cell components do not change due to the isolation procedure.

Measurements of the DNA content of different protist populations can shed light on a variety of processes, including cell division, sex, prey ingestion, and parasite invasion. Here, we modified an Imaging FlowCytobot (IFCB), a custom-built flow cytometer that records images of microplankton, to measure the DNA content of large dinoflagellates and other high-DNA content species. The IFCB was also configured to measure fluorescence from Cy3-labeled rRNA probes, aiding the identification of Alexandrium fundyense (syn. A. tamarense Group I), a photosynthetic dinoflagellate that causes paralytic shellfish poisoning (PSP). The modified IFCB was used to analyze samples from the development, peak and termination phases of an inshore A. fundyense bloom (Salt Pond, Eastham, MA USA), and from a rare A. fundyense ‘red tide’ that occurred in the western Gulf of Maine, offshore of Portsmouth, NH (USA). Diploid or G2 phase (‘2C’) A. fundyense cells were frequently enriched at the near-surface, suggesting an important role for aggregation at the air-sea interface during sexual events. Also, our analysis showed that large proportions of A. fundyense cells in both the Salt Pond and red tide blooms were planozygotes during bloom decline, highlighting the importance of sexual fusion to bloom termination. At Salt Pond, bloom decline also coincided with a dramatic rise in infections by the parasite genus Amoebophrya. The samples that were most heavily infected contained many large cells with higher DNA-associated fluorescence than 2C vegetative cells, but these cells’ nuclei were also frequently consumed by Amoebophrya trophonts. Neither large cell size nor increased DNA-associated fluorescence could be replicated by infecting an A. fundyense culture of vegetative cells. Therefore we attribute these characteristics of the large Salt Pond cells to planozygote maturation rather than Amoebophrya infection, though an interaction between infection and planozygote maturation may also have contributed. The modified IFCB is a valuable tool for exploring the conditions that promote sexual transitions by dinoflagellate blooms but care is needed when interpreting results from samples in which parasitism is prevalent.

Quantification of Alexandrium cyst fluxes through the Gulf of Maine water column is central to understanding the linkage between the source and fate of annual Alexandrium blooms in the offshore waters. These blooms often lead to paralytic shellfish poisoning (PSP) and extensive closures of shellfish beds. We report here on time-series sediment trap deployments completed at four offshore locations in the gulf between 2005 and 2010 as components of two ECOHAB–GOM field programs. Data presented documents the substantial spatial and temporal fluctuations in Alexandrium fundyense cyst fluxes in the gulf. Cyst delivery out of the euphotic zone peaked primarily between July and August following annual spring–summer Alexandrium blooms and was greatest in the western gulf. At all sites, cyst flux maxima to the subsurface waters were rarely coincident with seasonal peaks in the total mass export of particulate material indicating that cyst delivery was primarily via individually sinking cysts. Where persistent benthic nepheloid layers (BNLs) exist, significant sediment resuspension input of cysts to the near-bottom water column was evidenced by deep cyst fluxes that were up to several orders of magnitude greater than that measured above the BNL. The largest cyst fluxes in the BNL were observed in the eastern gulf, suggesting greater resuspension energy and BNL cyst inventories in this region. Temporal similarities between peak cyst export out of the upper ocean and peak cyst fluxes in the BNL were observed and document the contribution of seasonal, newly formed cysts to the BNL. The data however also suggest that many Alexandrium cells comprising the massive, short-lived blooms do not transition into cysts. Time-series flow measurements and a simple 1D model demonstrate that the BNL cyst fluxes reflect the combined effects of tidal energy-maintained resuspension, deposition, and input of cysts from the overlying water column.

A major goal in harmful algal bloom (HAB) research has been to identify mechanisms underlying interannual variability in bloom magnitude and impact. Here the focus is on variability in Alexandrium fundyense blooms and paralytic shellfish poisoning (PSP) toxicity in Maine, USA, over 34 years (1978–2011). The Maine coastline was divided into two regions – eastern and western Maine, and within those two regions, three measures of PSP toxicity (the percent of stations showing detectable toxicity over the year, the cumulative amount of toxicity per station measured in all shellfish (mussel) samples during that year, and the duration of measurable toxicity) were examined for each year in the time series. These metrics were combined into a simple HAB Index that provides a single measure of annual toxin severity across each region. The three toxin metrics, as well as the HAB Index that integrates them, reveal significant variability in overall toxicity between individual years as well as long-term, decadal patterns or regimes. Based on different conceptual models of the system, we considered three trend formulations to characterize the long-term patterns in the Index – a three-phase (mean-shift) model, a linear two-phase model, and a pulse-decline model. The first represents a “regime shift” or multiple equilibria formulation as might occur with alternating periods of sustained high and low cyst abundance or favorable and unfavorable growth conditions, the second depicts a scenario of more gradual transitions in cyst abundance or growth conditions of vegetative cells, and the third characterizes a ”sawtooth” pattern in which upward shifts in toxicity are associated with major cyst recruitment events, followed by a gradual but continuous decline until the next pulse. The fitted models were compared using both residual sum of squares and Akaike's Information Criterion. There were some differences between model fits, but none consistently gave a better fit than the others. This statistical underpinning can guide efforts to identify physical and/or biological mechanisms underlying the patterns revealed by the HAB Index. Although A. fundyense cyst survey data (limited to 9 years) do not span the entire interval of the shellfish toxicity records, this analysis leads us to hypothesize that major changes in the abundance of A. fundyense cysts may be a primary factor contributing to the decadal trends in shellfish toxicity in this region. The HAB Index approach taken here is simple but represents a novel and potentially useful tool for resource managers in many areas of the world subject to toxic HABs.

Phytoplankton toxins undergo trophic transport and accumulation in marine food webs, causing vectorial intoxication of upper-level consumers such as fishes, seabirds, and marine mam- mals. An entry point for phytoplankton toxins into these pelagic trophic pathways is frequently the herbivorous zooplankton. During the 1995 spring-summer red tide season in Massachusetts Bay, we examined accumulation of paralytic shellfish poisoning (PSP) toxins from the dinoflagellate Alexan- drium spp. in various plankton size fractions (20-64, 64-100, 100-200, 200-500, and >500 µm), and identified the relative composition of the zooplankton in these size fractions. Toxin levels were esti- mated by both high-performance liquid chromatography (HPLC) and a receptor-binding assay, the latter based on sample toxic potency. Although no PSP toxicity was detected in nearshore shellfish by routine monitoring programs using the mouse bioassay, positive responses were detected in zoo- plankton size fractions with the more sensitive HPLC and the receptor assay methods. The toxin signal was disproportionately concentrated in the larger zooplankton size fraction, frequently domi- nated by large copepods such as Calanus finmarchicus and Centropages typicus, which comprised only a small portion of total zooplankton abundance in quantitative samples obtained with 100 µm mesh nets. By comparison, signal levels were low or undetectable in the smaller size fraction, which contained the overwhelmingly most-abundant zooplankters such as protists, copepod nauplii and copepodites and adults of small copepods such as Oithona similis, Paracalanus parvus, and Pseudo- calanus spp. The larger toxin-accumulating copepods could provide a direct trophic linkage for vectorial intoxication of baleen whales that are known to feed upon such copepods.

The toxic dinoflagellate Alexandrium fundyense Balech was grown under temperature- and nutrient-limited conditions, and changes in labeling intensity on intact cells were determined for two probe types: an oligonucleotide probe targeting rRNA and a monoclonal antibody (MAb) targeting a cell surface protein. In nutrient-replete batch culture, labeling with the rRNA probe was up to 400% brighter during exponential phase than during stationary phase, whereas MAb labeling did not change significantly with growth stage at the optimal growth temperature. In cultures grown at suboptimal, low temperatures, there was a significant difference between labeling intensity in stationary versus exponential phase for both probe types, with exponential cells labeling brighter with the rRNA probe and slightly weaker with the MAb. The decrease in rRNA probe labeling with increasing culture age was likely due to lower abundance of the target nucleic acid, as extracted RNA varied in a similar manner. With the MAb and the rRNA probes, slower growing cultures at low, nonoptimal temperature labeled 35% and 50% brighter than cells growing faster at warmer temperatures. Some differences in labeling intensity per cell disappeared when the data were normalized to surface area or volume, which indicated that the number of target antigens or rRNA molecules was relatively constant per unit area or volume, respectively. Slow growth accompanying phosphorus and nitrogen limitation resulted in up to a 400% decrease in labeling intensity with the rRNA probe compared to nutrient-replete levels, whereas the MAb labeling intensity increased by a maximum of 60%. With both probes, labeling was more intense under phosphorus limitation than under nitrogen limitation, and for all conditions tested, labeling intensity was from 600% to 3600% brighter with the MAb than with the rRNA probe. Thus, it is clear that significant levels of variability in labeling intensity can be expected with both probe types because of the influence of environmental conditions and growth stage on cellular biochemistry, cell size,rRNA levels, and the number or accessibility of cell surface proteins. Of the two probes tested, the rRNA probe was the most variable, suggesting that in automated, whole-cell assays, it can be used only in a semiquantitative manner. For manual counts, the human eye will likely accommodate the labeling differences. The MAb probe was less variable, and thus should be amenable to both manual and automated counts.

Here we document Alexandrium fundyense cyst abundance and distribution patterns over nine years (1997 and 2004-2011) in the coastal waters of the Gulf of Maine (GOM) and identify linkages between those patterns and several metrics of the severity or magnitude of blooms occurring before and after each autumn cyst survey. We also explore the relative utility of two measures of cyst abundance and demonstrate that GOM cyst counts can be normalized to sediment volume, revealing meaningful patterns equivalent to those determined with dry weight normalization. Cyst concentrations were highly variable spatially. Two distinct seedbeds (defined here as accumulation zones with > 300 cysts cm-3) are evident, one in the Bay of Fundy (BOF) and one in mid-coast Maine. Overall, seedbed locations remained relatively constant through time, but their area varied 3-4 fold, and total cyst abundance more than 10 fold among years. A major expansion of the mid-coast Maine seedbed occurred in 2009 following an unusually intense A. fundyense bloom with visible red-water conditions, but that feature disappeared by late 2010. The regional system thus has only two seedbeds with the bathymetry, sediment characteristics, currents, biology, and environmental conditions necessary to persist for decades or longer. Strong positive correlations were confirmed between the abundance of cysts in both the 0-1 and the 0-3 cm layers of sediments in autumn and geographic measures of the extent of the bloom that occurred the next year (i.e., cysts → blooms), such as the length of coastline closed due to shellfish toxicity or the southernmost latitude of shellfish closures. In general, these metrics of bloom geographic extent did not correlate with the number of cysts in sediments following the blooms (blooms → cysts). There are, however, significant positive correlations between 0-3 cm cyst abundances and metrics of the preceding bloom that are indicative of bloom intensity or vegetative cell abundance (e.g., cumulative shellfish toxicity, duration of detectable toxicity in shellfish, and bloom termination date). These data suggest that it may be possible to use cyst abundance to empirically forecast the geographic extent of the forthcoming bloom and, conversely, to use other metrics from bloom and toxicity events to forecast the size of the subsequent cyst population as the inoculum for the next year's bloom. This is an important step towards understanding the excystment/encystment cycle in A. fundyense bloom dynamics while also augmenting our predictive capability for this HAB-forming species in the GOM.

A series of oceanographic surveys on Georges Bank document variability of populations of the toxic dinoflagellate Alexandrium fundyense on time scales ranging from synoptic to seasonal to interannual. Blooms of A. fundyense on Georges Bank can reach concentrations on the order of 104 cells l-1, and are generally bank-wide in extent. Georges Bank populations of A. fundyense appear to be quasi-independent of those in the adjacent coastal Gulf of Maine, insofar as they occupy a hydrographic niche that is colder and saltier than their coastal counterparts. In contrast to coastal populations that rely on abundant resting cysts for bloom initiation, very few cysts are present in the sediments on Georges Bank. Bloom dynamics must therefore be largely controlled by the balance between growth and mortality processes, which are at present largely unknown for this population. Based on correlations between cell abundance and nutrient distributions, ammonium appears to be an important source of nitrogen for A. fundyense blooms on Georges Bank.

13 pages, 10 figures, 1 table, Paralytic Shellfish Poisoning (PSP) toxins are annually recurrent along the Massachusetts coastline (USA), which includes many small embayments and salt ponds. Among these is the Nauset Marsh System (NMS), which has a long history of PSP toxicity. Little is known, however, about the bloom dynamics of the causative organism Alexandrium fundyense within that economically and socially important system. The overall goal of this work was to characterize the distribution and dynamics of A. fundyense blooms within the NMS and adjacent coastal waters by documenting the distribution and abundance of resting cysts and vegetative cells. Cysts were found predominantly in three drowned kettle holes or salt ponds at the distal ends of the NMS – Salt Pond, Mill Pond, and Town Cove. The central region of the NMS had a much lower concentration of cysts. Two types of A. fundyense blooms were observed. One originated entirely within the estuary, seeded by cysts in the three seedbeds. These blooms developed independently of each other and of the A. fundyense population observed in adjacent coastal waters outside the NMS. The temporal development of the blooms was different in the three salt ponds, with initiation differing by as much as 30 days. These differences do not appear to reflect the initial cyst abundances in these locations, and may simply result from higher cell retention and higher nutrient concentrations in Mill Pond, the first site to bloom. Germination of cysts accounted for a small percentage of the peak cell densities in the ponds, so population size was influenced more by the factors affecting growth than by cyst abundance. Subsurface cell aggregation (surface avoidance) limited advection of the vegetative A. fundyense cells out of the salt ponds through the shallow inlet channels. Thus, the upper reaches of the NMS are at the greatest risk for PSP since the highest cyst abundances and cell concentrations were found there. After these localized blooms in the salt ponds peaked and declined, a second, late season bloom occurred within the central portions of the NMS. The timing of this second bloom relative to those within the salt ponds and the coastal circulation patterns at that time strongly suggest that those cells originated from a regional A. fundyense bloom in the Gulf of Maine, delivered to the central marsh from coastal waters outside the NMS through Nauset Inlet. These results will guide policy decisions about water quality as well as shellfish monitoring and utilization within the NMS and highlight the potential for “surgical” closures of shellfish during PSP events, leaving some areas open for harvesting while others are closed, This work was supported by NOAA Grant NA06OAR4170021, NPS Grant H238015504 and by the Woods Hole Center for Oceans and Human Health through NSF Grants OCE-0911031 and OCE-0430724 and NIEHS Grant 1P50-ES01274201. B.G.C. was supported by a Xunta de Galicia Ángeles Alvariño fellowship and the Stanley W. Watson Chair for Excellence in Oceanography under a Postdoctoral program at the Woods Hole Oceanographic Institution.[SS]

Abst ract. Surveys were conducted along the northeast coast of the USA, be1ween Po rtsmouth, NH, and I he Chesapeake Bay in 1988 and 1990, to determine the population distribulion of Aureococcus anoplwgefferens, lhe chrysophyte responsible for massive and destructive 'brown tides' in Long Island and Narraganselt Bay beginning in 1985. A species-specific immunofluorescent technique was used to screen water samples, with positive identification possible at cell concentrations as low as 1020 cells ml • Bolh years, A.anophagefferens was detected at numerous stations in and around Long Island and Barnegat Bay, J , typically at high cell concentrations. To the north and south of this 'center·, nearly half of the remaining stations were positive for A.anophagefferens, but the cells were always at very low cell concentrations. Many of the positive identifications in areas distant from Long Island were in waters with no known history of harmful brown tides. The species was present in both open coastal and estuarine locations, in salinities between 18 and 32 prac1ical salinity units (PSU). The observed population distributions apparently still reflect the massive 1985 outbreak when this species first bloomed, given the number of positive locations and high abundance of A.anophagefferens in the immediate vicinity of Long Island. However, the frequent occurrence of I his species in waters far from this populalion 'center· is disturbing. Aureococcus anophagefferens is more widely distributed than was previously thought. Numerous areas lhus have the potential for destructive brown lides such as those associated with the sudden appearance of lhe species in 1985.

Harmful algal blooms (HABs) are a global problem that affects both human and ecosystem health. One of the most serious and widespread HAB poisoning syndromes is paralytic shellfish poisoning, commonly caused by Alexandrium spp. dinoflagellates. Like many toxic dinoflagellates, Alexandrium produces resistant resting cysts as part of its life cycle. These cysts play a key role in bloom initiation and decline, as well as dispersal and colonization of new areas. Information on cyst numbers and identity is essential for understanding and predicting blooms, yet comprehensive cyst surveys are extremely time- and labor-intensive. Here we describe the development and validation of a quantitative real-time PCR (qPCR) technique for the enumeration of cysts of A. tamarense of the toxic North American/Group I ribotype. The method uses a cloned fragment of the large subunit ribosomal RNA gene as a standard for cyst quantification, with an experimentally determined conversion factor of 28,402±6152 LSU ribosomal gene copies per cyst. Tests of DNA extraction and PCR efficiency show that mechanical breakage is required for adequate cyst lysis, and that it was necessary to dilute our DNA extracts 50-fold in order to abolish PCR inhibition from compounds co-extracted from the sediment. The resulting assay shows a linear response over 6 orders of magnitude and can reliably quantify ?10 cysts/cm3 sediment. For method validation, 129 natural sediment samples were split and analyzed in parallel, using both the qPCR and primulin-staining techniques. Overall, there is a significant correlation (p

The deposition and burial of living dinoflagellate cysts was studied in two different environments: the nearshore waters of the southern Gulf of Maine and a small shallow shat pond on Cape Cod, Massachusetts (Perch Pond). Vertical profiles of cysts and two naturally occuring radionuclides (210Pb, 234Th) differed significantly between the two environments. At 160 m depths in the Gulf of Maine, cyst profiles in the sediment often showed a subsurface peak in abundance 6–8 cm below the surface. The 210Pb profiles were consistent with a rapidly mixed surface layer (2–6 cm thick) above another region (6 at least 12 cm thick) where mixing was slower but still dominant over sediment deposition. The sediment mixing coefficient (Db) ranged from 15 to 26 cm2 y−1 in this lower region. The radiotracer profiles and modelling results both suggest that the subsurface peaks in cyst abundance are not the result of a pulse input in one year followed by burial (via bioturbation or sediment deposition). Instead, we hypothesize that they arise from a combination of germination from the surface mixed layer and mortality at depth. In contrast, the cyst and radiotracer profiles in the shallow Perch Pond embayment are consistent with a single thin (2 cm) mixed layer at the sediment surface. Burial of cysts below this level is due to a relatively high rate of sediment deposition (2.9 mm y−1), with little or no biological mixing. This lack of mixing is consistent with reports of seasonal anoxia in Perch Pond, a recurrent process which kills benthic animals before they reach the size or community composition needed for deep bioturbation. Opportunistic, recolonizing species are only capable of mixing the top 1 or 2 cm. Resuspension and redeposition of cysts by wind and storms appears to be limited by the small size and somewhat protected location of the pond. The lack of deep mixing allows us to compare the survival of cysts of different species by modelling the decrease in cyst abundance below 2 cm. A simple exponential decay equation fits the data well, and indicates that the living cysts of some species (e.g., Gyrodinium uncatenum, Gonyaulax polyedra) are more susceptable to mortality in the deeper anoxic sediments than are Gonyaulax verior and Alexandrium (formerly Protogonyaulax) tamarense.

A coupled physical/biological modeling system was used to hindcast the 2005 Alexandrium fundyense bloom in the Gulf of Maine and investigate the relative importance of factors governing the bloom's initiation and development. The coupled system consists of a state-of-the-art, free-surface primitive equation Regional Ocean Modeling System (ROMS) tailored for the Gulf of Maine (GOM) using a multi-nested configuration, and a population dynamics model for A. fundyense. The system was forced by realistic momentum and buoyancy fluxes, tides, river runoff, observed A. fundyense benthic cyst abundance, and climatological nutrient fields. Extensive comparisons were made between simulated (both physical and biological) fields and in-situ observations, revealing that the hindcast model is capable of reproducing the temporal evolution and spatial distribution of the 2005 bloom. Sensitivity experiments were then performed to distinguish the roles of three major factors hypothesized to contribute to the bloom: 1) the high abundance of cysts in western GOM sediments; 2) strong northeaster storms with prevailing downwelling-favorable winds; and 3) a large amount of fresh water input due to abundant rainfall and heavy snowmelt. Results suggested that the high abundance of cysts in western GOM was the primary factor of the 2005 bloom. Wind forcing was an important regulator, as episodic bursts of northeast winds caused onshore advection of offshore populations. These downwelling favorable winds accelerated the alongshore flow, resulting in transport of high cell concentrations into Massachusetts Bay. A large regional bloom would still have happened, however, even with normal or typical winds for that period. Anomalously high river runoff in 2005 resulted in stronger buoyant plumes/currents, which facilitated the transport of cell population to the western GOM. While affecting nearshore cell abundance in Massachusetts Bay, the buoyant plumes were confined near to the coast, and had limited impact on the gulf-wide bloom distribution.

Alexandrium ostenfeldii is a thecate, mixotrophic dinoflagellate recently linked to a novel suite of toxins called spirolides. This study provides the first description of the regional distribution of A. ostenfeldii in the Gulf of Maine (GOM), and the first report and analysis of spirolide toxicity in A. ostenfeldii in waters south of Nova Scotia. Morphological examination of cells in field samples and of clonal cultures isolated from several stations in the GOM confirmed the presence of A. ostenfeldii. A genus-specific antibody probe, and an A. ostenfeldii species-specific oligonucleotide probe labeled these cells; a probe specific for the North American A. fundyense/tamarense/catenella species complex did not label A. ostenfeldii cells. Cell size ranged from 20 to nearly 90 μm, and most cells contained food vacuoles, with a total vacuole size from 1 to 48 μm. The hydrographic forcings controlling the distribution of A. ostenfeldii in the GOM are quite similar to those acting on the A. fundyense population at the same time of the year. The highest concentrations of A. ostenfeldii were observed nearshore, to the east of Penobscot Bay, at times with an offshore-turning branch of high cell concentration to the south of Penobscot Bay. Casco Bay appears to be an area of accumulation for A. ostenfeldii cells advected toward shore from the core of the population to the northeast. Concentrations of A. ostenfeldii were generally higher at the surface than deeper, except at locations where the pooling of lower-salinity water at the surface may have led to the subduction of the population flowing in from the east. PSP toxins were detected in field populations containing A. ostenfeldii and A. fundyense , but not in A. ostenfeldii cultures isolated from the GOM. Spirolide toxins were found in 36 of 60 field samples. More than 83% of samples containing A. ostenfeldii cells had one or more of spirolide congeners A, B, C2 and D2. The total concentration of spirolides per cell at each station where A. ostenfeldii was detected ranged from non-detectable to 282 fmol cell −1 . A. ostenfeldii cultures originating from different locations showed a great diversity in spirolide content and composition. All cultures contained spirolides, ranging from 28 to 113 fmol cell −1 . Spirolide congeners desMeC, C and D were present in some cultures, but were not detected in any of the field samples. Based on differences and similarities between their toxin profiles, five toxin phenotypes were identified. The highest per cell spirolide contents in the cultures were nearly two times lower than the highest levels observed in field samples. Currently there is no routine monitoring for spirolide toxins in shellfish in the region, but it may be necessary eventually to expand ongoing toxin monitoring in the GOM to include analysis for spirolides.

Alkaline phosphatase (AP) activity in marine and freshwater phytoplankton has been associated with phosphorus (P) limitation whereby the enzyme functions in the breakdown of exogenous organic P compounds to utilizable inorganic forms. Current enzyme assays to determine the P status of the phytoplankton measure only the AP activity of the whole community and do not yield information on individual species. A new insoluble fluorogenic substrate for AP, termed ELF (Enzyme-Labeled Fluorescence), yields a stable, highly fluorescent precipitate at the site of enzyme activity and thus has the capability to determine the P status of individual cells. In this study, ELF was utilized for in situ detection and quantification of AP in marine phytoplankton cultures and a comparison was made between the insoluble ELF substrate and several soluble AP substrates [3-O-methylfluorescein phosphate (MFP), 3,6-fluorescein diphosphate (FDP) and Attophos]. Non-axenic batch cultures of Alexandrium fundyense, Amphidinium sp. and Isochrysis galbana were grown in different media types using orthophosphate as an inorganic source and sodium-glycerophosphate as an organic source, with final phosphate concentrations ranging from 38.3 to 3 μM (i.e. f/2, f/40, f/80, plus ambient P). Epifluorescence microscopy was used to determine if and where the cells were labeled with ELF, while flow cytometry was used to quantify the amount of ELF retained on individual cells. The detection of the soluble substrates utilized a multiwell fluorescence plate reader (Cytofluor™). Only cells grown in low phosphate concentrations (f/40, f/80) exhibited the bright green fluorescence signal of the ELF precipitate. This signal was always observed for P-starved Amphidinium sp. and I. galbana cells, but was seen in some A. fundyense cells only during the late stationary phase. Cells grown in high phosphate concentrations (i.e. at f/2 levels) showed no ELF fluorescence. Slightly positive soluble substrate assays suggest that these species may have produced small amounts of AP constitutively that were not detected with the precipitable substrate. Similar results were obtained when the cultures were analyzed by flow cytometry. Except for A. fundyense, cells grown in low phosphate concentrations showed high ELF fluorescence. However, no positive ELF fluorescence was detected with the Cytofluor for all 3 species due to lack of instrument sensitivity. Comparable analysis using the soluble substrates MFP, FDP, and Attophos™ on the Cytofluor showed little activity for A. fundyense, but high fluorescence for P-starved Amphidinium sp. and I. galbana. Insoluble ELF thus provides a means to detect and quantify AP in individual cells using visual observations or flow cytometry. This technique offers a new level of resolution and sensitivity at the single cell level that can provide insights into the P nutrition of phytoplankton and other microorganisms in natural waters., Sí

A novel method was developed for isolating cells of the toxic dinoflagellate Alexandrium fundyense from preserved natural seawater samples using paramagnetic beads and a monoclonal antibody against the surface antigens of the dinoflagellate. 'Direct' and 'indirect' approaches to bead/cell attachment were tested as well as 3 types of bead coatings (streptavidin, and 2 secondary antibodies: sheep anti-mouse, and goat anti-mouse), and 2 blocking agents [normal goat serum (NGS) and bovine serum albumin (BSA)]. Optimal 'indirect' bead attachment protocols, where the species-specific primary antibody is first bound to the target cells before bead attachment, utilized either 2.8 μm streptavidin-coated beads with NGS blocking or sheep anti-mouse-coated beads with BSA blocking. Although there were undoubtably some cell losses during the initial antibody-labeling and washing procedures, ca 90% of the labeled A. fundyense cells in unialgal cultures were removed using these bead procedures. Non-specific binding was low, as only 5 to 10% of the A. fundyense cells were recovered when the primary antibody was omitted. The 'direct' approach, where the species-specific primary antibody is first bound to the beads, was most effective (80% recovery) using sheep anti-mouse-coated beads (2.8 μm) with BSA blocking, and the non-specific binding remained very low (, Sí

Blooms of the toxic dinoflagellate Gonyaulax tamarensis (synonyms Protogonyaulax tamarensis1 and Alexandrium tamarense2) cause outbreaks of paralytic shellfish poisoning (PSP) in coastal waters throughout the world. In the Gulf of Maine, episodes occur between April and November, a seasonally due in part to life-cycle alternations between motile, vegetative cells and resting cysts which overwinter in bottom sediments3,4. Newly formed cysts have a mandatory 2–6 month dormancy period during which germination is not possible5, but once mature, the resting state will continue if temperatures are unfavourable5 or oxygen is unavailable6. We now report another factor controlling germination of cysts of G. tamarensis from deep coastal waters—an endogenous annual clock that can override an otherwise favourable environment for germination. Similar annual variability in germination has not been observed for cysts of this species from shallow estuaries. These results represent the first conclusive demonstration of an endogenous circannual rhythm in a marine plant. They are evolutioiiarily and ecologically significant because an endogenous annual clock can lead to the release of motile cells into deep and relatively invariant bottom waters at those times when temperature and light at the surface are suitable for growth. In shallow waters where seasonal variability is large and extends to bottom sediments, a strategy similar to that of the seeds of terrestrial plants would be more appropriate, namely a direct coupling between germination and the external environment.