C.5.2.9 Process studies

Year 4 of the project will be devoted to detailed studies of important processes and mechanisms involved in Alexandrium population dynamics in both habitats.

The WMCC Region:

Rationale: To move beyond relating distributions of Alexandrium to physical and chemical fields, we will examine the physiology and dynamics of Alexandrium in unprecedented detail within discrete patches of cells. Planned activities are outlined below; others will be incorporated, based on experience from the larger surveys, and as new techniques are developed.

Approach: Two intensive studies are planned for the Casco Bay region, where annually recurrent toxicity ensures that blooms will be found, and where hydrographic features exist to maintain patches (e.g., Fig 4C) for continuous study over 4-5 days or longer. First, a patch of cells will be followed in early May when the WMCC is "seeded" with cells from Casco Bay. The study will examine bloom dynamics at a time when advection of cells into the coastal current by winds or runoff is likely. The second study will focus on a patch of cells at a time (late May, early June),when transport is reduced.

For the study of bloom entrainment, data from Years 1 & 3 will help identify likely locations for accumulation of Alexandrium, presumably at a hydrographic feature such as a salinity or plume front (Fig. 4B,C). A small vessel survey with near-real time rRNA probe detection of cells will locate a suitable patch which will be "tagged" with Rhodamine dye, injected by pump in the near-surface water (0-2 m) and tracked with a flow-through fluorometer and GPS surface drifters. Surveys in and around the patch will provide estimates of horizontal and vertical dispersion and a shipboard ADCP will measure the vertical shear that may influence the transport of cells beneath the patch. The vertical and horizontal distribution of Alexandrium cells and nutrients will be determined, as will the composition of the zooplankton community. Net tows will be taken to document toxin in the food web. Physiological measurements will be made on Alexandrium (see below), but the emphasis of this study will be the mechanisms for bloom transport and delivery into the WMCC.

The second process study will focus on a patch of Alexandrium cells (e.g. Fig. 4C) during late spring when runoff and other forcings are reduced and cells tend to be retained in Casco Bay. A hydrographic survey will be conducted to explore the mechanisms underlying the population accumulation and to keep track of the patch as above. Meantime, detailed sampling will document the distributions of Alexandrium cells and nutrients. A number of physiological and behavioral measurements will be made within the patch, and for most, comparisons will be made to Alexandrium cells from outside the patch and/or outside the coastal current, when possible.

Life History Stages (PI: Anderson) The Alexandrium population will be monitored to determine the percentage of planozygotes (Anderson et al. 1983). To obtain sufficient biomass, net tows will be collected or water will be pumped through a "dino-box" used by the Anderson lab to concentrate the 20-80 _m size fraction.

Vertical Migration (PIs: Anderson,, Cullen, Loder): Samples will be taken at one meter intervals within the population every two hours over 36 hours to determine migration behavior. The physical structure of the water column, light penetration and nutrient distributions will be characterized as well. Information on vertical movements is critical to understanding of the mechanisms underlying population accumulation in fronts or other physical features. It will also provide guidance and input to the model, which has detailed vertical resolution of the water column structure, but thus far no swimming behavior for Alexandrium.

In situ Growth Rates (PI: Anderson) During the vertical migration study and at other times, samples will be collected to measure in situ growth rates (Cheng and Carpenter 1991). We will employ our proven method, using species-specific antibody probes to label Alexandrium cells and DNA-specific fluors to stain nuclei so that DNA content can be determined through the diel cycle using epifluorescence microscopy and image analysis.

Grazing Rates and Food Web Transfer of Toxin (PIs: Doucette, Turner). The zooplankton community in the patch will be characterized and then time-series measurements made of toxin in different plankton size fractions. Grazing rates on Alexandrium will be determined for dominant zooplankters using the natural phytoplankton assemblage.

Physiological Condition: (PIs: Anderson, Cullen). Fluorescein diacetate (FDA) will be used to distinguish dead from metabolically active cells (Selvin et al. 1989). Gentien (pers. comm.) has found that up to 30% of cells can be non-viable during blooms. This simple method will be used to see whether the percentage of viable Alexandrium cells varies significantly through time, with depth, or with location, thereby influencing net transport of viable cells.

Several other techniques rely on a new method developed in the Anderson laboratory to separate Alexandrium cells from the field assemblage, permitting species-specific analyses. The method relies on our Alexandrium antibody, which is coupled to magnetic beads, allowing cells and beads to be pulled from solution with a magnet. Alexandrium cells can be collected from a mixed assemblage with 95-99% purity (Aguillera et al., 1996). Once sufficient cells are concentrated using the plankton net or the "dino-box" they will be processed with the immunomagnetic method and collected cells analyzed for chlorophyll content, fluorescence +/- DCMU, toxicity, carbohydrate, lipid, protein, and toxicity to explore how these parameters change with time and bloom stage. The suite of assays will be chosen based on their efficacy and how much sample is required. Other assays are under development, including measurements of C:N ratios, ¹⁴C-uptake, and an assay for Fe limitation. P. Glibert and F. Lipshultz have both expressed interest in working with us to see if the magnetic bead approach can be used to measure species-specific N uptake, enzyme activity, and internal inorganic N pools. These latter measurements are dependent on methods development, and are mentioned here only to indicate our thinking. Given rapid development of these and other physiological "indicators" in oceanography, new procedures will be added to our program by the time the process studies begin.

Mixotrophic Nutrition (PI: Anderson). There is now reason to believe that Alexandrium can ingest particulate matter (e.g., Jacobson and Anderson 1996). If mixotrophy is confirmed in Alexandrium, there are obvious implications with respect to linkages between blooms and dissolved nutrients. Samples will be collected at different depths and times and examined for the conspicuous food vacuoles described in Jacobson and Anderson (1996). Cells will also be examined with epifluorescence, as Stoecker (pers. comm.) has had great success documenting mixotophy in Prorocentrum by detecting orange fluorescing cryptophytes inside the dinoflagellate.

The EMCC Region (PIs: Townsend, Loder, Pettigrew, Anderson, Keller):

Rationale: Alexandrium cells from the western Bay of Fundy are likely advected along the coast of Maine via the EMCC (e.g. Fig. 3), but we are not at all certain of the significance of in situ growth of Alexandrium in eastern Maine. The extent of lateral exchange between the EMCC and nearshore waters may lie at the heart of the PSP incidents in the region. Process studies will thus focus on physical exchanges between: (a) the western Bay of Fundy and the EMCC (i.e. a "source region" study; and, (b) the EMCC and nearshore waters of eastern coastal Maine. The details of the experimental plan will no doubt be refined in response to the results from Years 1 and 3 though the rationale will most likely be altered little.

Approach: Our experimental approach will focus on finer spatial scales, in both the horizontal and vertical than in Years 1 and 3. We will combine small-scale hydrographic surveys of critical features known to be sites for cell accumulation or delivery. Water properties, Alexandrium cells, and nutrients will be determined with higher resolution in the vertical and horizontal in order to elucidate Alexandrium dynamics in relation to physical processes. Accumulation of cells and transport to nearshore waters and to deep water clam beds will be the focus. Measures of water column structure and movement will be determined with shipboard ADCP measurements, surface and "deep" drogued drifters, and high-resolution shallow water broadband Doppler moorings. GPS drifters will be used for small scale studies. Moorings will be deployed at locations chosen to characterize the circulation in the nearshore bloom areas and the transverse circulation patterns. ARGOS surface drifters will be released in Fundy to assess the seeding role of populations there. Because there has been only a single (1980) survey cruise in this region of the Gulf, it is difficult at this time to state specifically what we will find in Years 1 & 3, and exactly how the details of the process study will take shape.