Detection of the toxic dinoflagellate Alexandrium fundyense (Dinophyceae) with oligonucleotide and antibody probes: Variability in labeling intensitywith physiological condition

The toxic dinonagellate Alexandrium fundyense Balech was grown under temper ature- and nutrient-limited conditions, and changes in labeling intensity o n intact cells were determined for two probe types: an oligonucleotide prob e targeting rRNA and a monoclonal antibody (MAb) targeting a cell surface p rotein. 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 temperatu res, there was a significant difference between labeling intensity in stati onary versus exponential phase for both probe types, with exponential cells labeling brighter with the rRNA probe and slightly weaker with the MAb. Th e decrease in rRNA probe labeling with increasing culture age was likely du e to lower abundance of the target nucleic acid, as extracted RNA varied in a similar manner. With the IMAI, and the rRNA probes, slower growing cultu res at low, nonoptimal temperature labeled 35% and 50% brighter than cells growing faster at warmer temperatures. Some differences in labeling intensi ty per cell disappeared when the data were normalized to surface area or vo lume, 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% de crease in labeling intensity with the rRNA probe compared to nutrient-reple te levels, whereas the MAb labeling intensity increased by a maximum of 60% . With both probes, labeling was more intense under phosphorus limitation t han under nitrogen limitation, and for all conditions tested, labeling inte nsity 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 inte nsity can be expected with both probe types because of the influence of env ironmental 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 tha t in automated, whole-cell assays, it can be used only in a semiquantitativ e manner. For manual counts, the human eye will likely accommodate the labe ling differences. The MAb probe was less variable, and thus should be amena ble to both manual and automated counts.