Bruce A. Keafer

DETECTION OF THE TOXIC DINOFLAGELLATE ALEXANDRIUM FUNDYENSE (DINOPHYCEAE) WITH OLIGONUCLEOTIDE AND ANTIBODY PROBES: VARIABILITY IN LABELING INTENSITY WITH PHYSIOLOGICAL CONDITION

 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.

Burial of living dinoflagellate cysts in estuarine and nearshore sediments

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.

Historic 2005 toxic bloom of Alexandrium fundyense in the western Gulf of Maine : 2. Coupled biophysical numerical modeling

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 blooms 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.

Suppression of the 2010 Alexandrium fundyense bloom by changes in physical, biological, and chemical properties of the Gulf of Maine

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. fundyenses 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. fundyenses 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.

Investigation of the 2006 Alexandrium fundyense bloom in the Gulf of Maine: In-situ observations and numerical modeling

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 models 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 blooms 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.