Physiological and behavioral diagnostics of nitrogen limitation for the toxic dinoflagellate Alexandrium fundyense

Abstract

One challenge in phytoplankton ecology is to measure species-specific physiological responses to changes in environmental conditions. Of particular importance in this regard are harmful algal bloom (RAB) species such as the toxic dinoflagellate Alexandrium fundyense which typically inhabit coastal regions where they are not usually dominant. Within the Gulf of Maine, environmental factors, specifically nitrogen, are likely to be a controlling factor for A. fundyense blooms. Therefore, the ability to ascertain the nutritional status of this species in field assemblages in critical to understanding its bloom dynamics. The aim of this thesis was to identify physiological and behavioral indicators or diagnostics of A. fundyense from the Gulf of Maine, and to evaluate these on natural populations in the Casco Bay region. Using a species-specific monoclonal antibody, two methods for identifying and separating A. fundyense from natural field assemblages were developed. The first used a species-specific antibody and flow cytometry to successfully detect and separate A. fundyense from co-occurring organisms, including other dinoflagellates of equivalent size. In particular the fluorescence associated with the antibody labeling was not sufficient of itself for species discrimination - natural red chlorophyll autofluorescence was also needed as a second parameter for identifying and sorting A. fundyense. A second antibody method was then investigated using immunomagnetic beads to successfully separate live A. fundyense from spiked field samples. The separated cells were then used to obtain accurate chlorophyll, protein and biomass estimates. CHN values were only accurate if the unbound magnetic beads were sieved from the sample prior to analysis. This is probably needed for carbohydrate analysis as well. Since A. fundyense usually inhabits coastal areas that are frequently limited by nitrogen, behavioral adaptations and intracellular responses to nitrogen availability are a primary consideration. It was therefore necessary to identify diagnostic indicators and behavioral adaptations of A. fundyense to nitrogen stress. Using laboratory water columns, nitrogen (N)-starved batch cultures, and N-limited, semi-continuous cultures, indicators of different N-nutritional states were identified. It was determined that low N concentrations in the surface of a mesocosm did not induce a Casco Bay A. fundyense isolate to vertically migrate to deep nutrient pools. Prolonged N-stress caused dramatic changes intracellular biochemistry, specifically chlorophyll a, carbohydrate, and protein content, as well as C:N, toxin content and composition. Ratios of different toxin derivatives were identified that increased with increasing N-stress and appear to be sensitive and robust indicators of N-status. Once indicators were developed for N-stress, variability in toxin content and composition were examined in the coastal waters of Casco Bay, Maine during an A. fundyense bloom in the spring of 1998. Over the course of the field season, toxin compositional changes did occur that were generally consistent with increasing levels of N-stress as the bloom progressed and N levels decreased. As observed in N-limited culture, large increases in some toxin ratios (e.g., GTX1,4:STX and NEO:STX) were observed during the latter portion of the field season, coinciding with low N:P ratios and undetectable levels of dissolved inorganic nitrogen. Overall, the toxin compositional trends are quite remarkable and suggest that this approach may provide valuable species-specific physiological information without the need for elaborate cell separation schemes such as flow cytometry or immunomagnetic bead sorting. Further laboratory studies are needed to better characterize the toxin response of A. fundyense isolates to environmental stresses before this suite of toxin indicators can be considered robust.