J Phycol. 35, 870-883 (1999) DETECTION OF THE TOXIC DINOFLAGELLATE AIEXANDRIUMFUNDYENSE (DINOPHYCEAE) WITH OLIGONUCLEOTIDE AND ANTIBODY PROBES: VARIABILITY IN LABELING INTENSITY WITH PHYSIOLOGICAL CONDITION1 2 Donald M. Anderson , David M. Kulis, Bruce A. Keafer Woods Hole Oceanographic Institution, Biology Department, Woods Hole, Massachusetts, 02513 and Elisa Berdalet Institut de Ciencies del Mar, P. Joan de Borbo s/n 08039, Barcelona, Spain The toxic dinoflagellate Alexandrium fundyense Bal­ rRNA levels, and the number or accessibility of cell ech was grown under temperature- and nutrient-lim­ surface proteins. Of the two probes tested, the ited conditions, and changes in labeling intensity on rRNA probe was the most variable, suggesting that intact cells were determined for two probe types: an in automated, whole-cell assays, it can be used only oligonucleotide probe targeting rRNA and a mono­ in a semiquantitative manner. For manual counts, clonal antibody (MAb) targeting a cell surface pro­ the human eye will likely accommodate the labeling tein. In nutrient-replete batch culture, labeling with differences. The MAb probe was less variable, and the rRNA probe was up to 400% brighter during thus should be amenable to both manual and auto­ exponential phase than during stationary phase, mated counts. whereas MAb labeling did not change significantly Key index words: Alexandrium; antibody; oligonucle­ with growth stage at the optimal growth tempera­ otide; physiology; probe; rRNA , ture. In cultures grown at suboptimal, low temper­ atures, there was a significant difference between Abbreviations: FITC, fluorescein isothiocyanate; labeling intensity in stationary versus exponential FLS, forward light scatter; FSB, fetal bovine serum; phase for both probe types, with exponential cells MAb, monoclonal antibody; PAb, polyclonal anti­ labeling brighter with the rRNA probe and slightly body; RFU, relative fluorescence units; SET, salt, weaker with the MAb. The decrease in rRNA probe EDTA, Tris buffer (750 mM NaCl, 5 mM EDTA, 100 labeling with increasing culture age was likely due mM Tris-HCl [pH 7 .8] buffer); SSC, side scatter 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 A common problem ,in phytoplankton field ecol­ at low, nonoptimal temperature labeled 35% and ogy occurs when the species of interest is only a mi­ 50% brighter than cells growing faster at warmer nor component of the mixed planktonic assem­ blage. Many potentially useful physiological mea­ temperatures. Some differences in labeling intensity 14 per cell disappeared when the data were normalized surements (e.g. chlorophyll, C fixation, C:N:P ra­ to surface area or volume, which indicated that the tios) are not feasible or are extremely difficult to number of target antigens or rRNA molecules was obtain for individual species because of the co-oc­ relatively constant per unit area or volume, respec­ currence of other organisms and detritus. Another tively. Slow growth accompanying phosphorus and common problem arises from the difficulty inherent nitrogen limitation resulted in up to a 400% de­ in identifYing species or strains, which are so mor­ crease in labeling intensity with the rRNA probe phologically similar that distinguishing characteris­ compared to nutrient-replete levels, whereas the tics are difficult to discern under the light micro­ MAb labeling intensity increased by a maximum of scope. Such fine levels of discrimination are not 60%. With both probes, labeling was more intense generally practical in monitoring programs or other under phosphorus limitation than under nitrogen studies that generate large numbers of samples for limitation, and for all conditions tested, labeling in­ cell enumeration. This situation is encountered fre­ tensity was from 600% to 3600% brighter with the quently in studies of harmful or toxic algae, where MAb than with the rRNA probe. Thus, it is clear that toxic and nontoxic varieties of species exist and significant levels of variability in labeling intensity sometimes even co-occur (e.g. Yentsch et al. 1978, can be expected with both probe types because of Smith et al. 1990). the influence of environmental conditions and A new technology that may well alleviate these growth stage on cellular biochemistry, cell size, problems utilizes molecular "probes" that are spe­ cific for certain species of phytoplankton (reviewed in Anderson 1995, Scholin and Anderson 1998). Ol­ 1 Received 11 September 1998. Accepted 23 April 1999. igonucleotide probes target particular genes or 2 Author for reprint requests; e-mail [email protected]. gene products inside cells using short, synthetic 870 VARIABILITY IN PROBE LABELING 871 DNA segments that bind selectively to sequences that rRNA varies systematically with growth rate; fast­ specific for a particular organism. For marine sys­ er growing cells have more RNA per cell than do tems, this technique has thus far been used primar­ cells growing at slower rates (e.g. DeLong et al. ily on prokaryotes (e.g. DeLong et al. 1989, Amann 1989). Efforts are underway to use the variation in et al. 1990, Distel et al. 1991), but oligonucleotide rRNA content, measured using oligonucleotide (rRNA) probes for toxic phytoplankton, such as probes, to obtain estimates of bacterial growth rate Pseudo-nitzschia and Alexandrium, are emerging (Lee et al. 1993, Lee and Kemp 1994). In that in­ (Scholin et al. 1994, Adachi et al 1996, Miller and stance, variability in labeling intensity is meaningful Scholin 1996, Anderson, unpubl.). An alternative to as an indicator of physiological state, but for other the rRNA probe approach uses antibodies raised applications, the variability in labeling may lead to against cell surface proteins. These probes are used ambiguous results. An example of the latter is in to label cells of interest by using indirect immuno­ flow cytometry, where the intensity of positive label­ fluorescence or related procedures for visualizing ing must be significantly higher than the back­ antibody-antigen binding (e.g. Campbell and Car­ ground fluorescence of control or unlabeled cells if penter 1987, Shapiro et al. 1989, Vrieling and An­ a particular species is to be identified and gated with derson 1996). For harmful or toxic phytoplankton, electronic thresholds (Vrieling et al. 1994, Anderson this work has progressed to the stage where poly­ 1995). Even if signal enhancement techniques are clonal and monoclonal antibodies (PAbs and MAbs) used for cells with lower antigen-expression levels, are available that identify target species in several flow cytometric gating will introduce estimate errors different algal classes. of the abundance of a target species in mixed algal Anderson et al. (1989) developed a PAb specific assemblages, mostly because of the natural disper­ for the "brown tide" chrysophyte Aureococcus ano­ sion of the parameter distributions for natural pop­ phagefferens that has been used along the northeast­ ulations of cells. ern U.S. coastline in order to characterize the dis­ In this study, the toxic dinoflagellate A. fundyense tribution of A. anophagefferens (Anderson et al. was grown under different nutrient and tempera­ 1993). Several PAbs were developed that recognize ture conditions, and changes in labeling intensity toxic and nontoxic varieties of the diatom Pseudo­ were determined for intact cells using two probe nitzschia pungens (Bates et al. 1993). Antibodies have types: an oligonucleotide probe targeting ribosomal also been raised against several species of toxic di­ RNA and a MAb targeting cell surface proteins. The noflagellates. A monoclonal antisen1m produced by fluorescently labeled probes were detected and Nagasaki et al. (1991) labeled nine strains of Gym­ quantified with flow cytometry. nodinium nagasakiense from Japan, and antisera pro­ duced against cell surface antigens of Gyrodinium au­ MATERIALS AND METHODS reolum from western Europe have been reported Cultures. All experiments utilized A. fundyense (strain GTCA2S) (Vrieling et al. 1994). Additionally, MAbs have been isolated from the western Gulf of Maine and rendered clonal by single cell isolation. Inoculum cultures were maintained for sev­ developed for Prorocentrum micans (Vrieling et al. eral transfers in mid to late exponential growth in modified f/2 1993) and for toxic Alexandrium species (Adachi et medium (Guillard and Ryther 1962) made with 0.2 [LM filtered al. 1993, Sako et al. 1993). Further promise for the Vineyard Sound seawater (31 psu). The f/2 medium was modified by adding Na Se0 and by reducing the concentration of application of antibodies in phytoplankton autecol­ 2 3 CuS04·5H20 to a final concentration of 10-s M each. Cultures ogy was demonstrated by the separation of toxic Al­ were grown at the experimental temperatures of so and 15° C on exandrium cells from mixed plankton assemblages a 14:10 h LD cycle (ca. 200 [Lmol photons·m-2-sec1 irradiance using immunomagnetic bead methodologies, which provided by cool white fluorescent bulbs). may eventually be utilized for species-specific physi­ Experimental. At the start of the experiment, nutrient-replete ological measurements (Aguilera et al. 1996). cultures were inoculated into six 2.8-L Fern bach flasks containing 1.3 L of temperature-equilibrated f/2 medium for each treat­ As work progresses on molecular-probe approach­ ment. Eight 1-L flasks were also inoculated, each with 400 mL of es to phytoplankton identification, enumeration, f/40 nitrate or phosphate (identical to f/2, but containing one­ and separation, one area that requires investigation twentieth the normal amount ofNaN03 or NaH2P04). The initial relates to the variability in labeling intensity caused cell density in the experimental flasks was calculated to be 200 cells· mL-l. Cell densities of the so and 15° C cultures were deter­ by changes in the physiological condition of the tar­ mined both microscopically, using a Sedgewick Rafter counting get species. Both antibody and rRNA probes are po­ chamber, and with a Coulter Counter (Coulter model ZF with P- tentially affected by environmental effects on the 12S size distribution analyzer).