PHYTOPLANKTON COMMUNITY COMPOSITION AND GENE EXPRESSION OF FUNCTIONAL GENES INVOLVED IN CARBON AND NITROGEN ASSIMILATION1

A functional gene microarray was developed and used to investigate phytoplankton community composition and gene expression in the English Channel. Genes encoding the CO₂‐fixation enzyme RUBISCO (rbcL) and the nitrate assimilation enzyme nitrate reductase (NR) representing several major groups of phytoplankton were included as oligonucleotide probes on the “phytoarray.” Five major groups of eukaryotic phytoplankton that possess the Type 1D rbcL gene were detected, both in terms of presence (DNA) and activity (rbcL gene expression). Changes in relative signal intensity among the Type 1D rbcL probes indicated a shift from diatom dominance in the spring bloom to dominance by haptophytes and flagellates later in the summer. Because of the limitations of a smaller database, NR probes detected fewer groups, but due to the greater diversity among known NR sequences, NR probes provided higher phylogenetic resolution than did rbcL probes and identified two uncultivated diatom phylotypes as the most abundant (DNA) and active (NR gene expression) in field samples. Unidentified chlorophytes and the diatom Phaeodactylum tricornutum Bohlin were detected at both the DNA and cDNA (gene expression) levels. The reproducibility of the array was evaluated in several ways, and future directions for further improvement of probe development and sensitivity are outlined. The phytoarray provides a relatively high‐resolution, high‐throughput approach to assessing phytoplankton community composition in marine environments.     

CHARACTERIZATION OF DIATOM (BACILLARIOPHYCEAE) NITRATE REDUCTASE GENES AND THEIR DETECTION IN MARINE PHYTOPLANKTON COMMUNITIES1

The complete assimilatory nitrate reductase (NR) gene from the pennate diatom Phaeodactylum triconutum Bohlin was sequenced from cDNA and compared with NR sequences from fungi, green algae, vascular plants, and the recently sequenced genome of the centric diatom Thalassiosira pseudonana Hasle and Heimdal CCMP1335. In all the major eukaryotic nitrate reductase (Euk‐NR) functional domains, diatom NR gene sequences are generally 50%–60% identical to plant and alga sequences at the amino acid level. In the less conserved N‐terminal, hinge 1, and hinge 2 regions, homology to other NR sequences is weak, generally<30%. Two PCR primer sets capable of amplifying Euk‐NR from plants, algae, and diatoms were designed. One primer set was used to amplify a 750‐base pair (bp) NR fragment from the cDNA of five additional diatom strains. The PCR amplicon spans part of the well‐conserved dimer interface region, the more variable hinge 1 region, and part of the conserved cytochrome b heme binding region. The second primer set, targeted to the dimer region, was used to amplify an approximately 400‐bp fragment of the NR gene from DNA samples collected in Monterey Bay, California and in central New Jersey inner continental shelf (LEO‐15 site) waters. Only diatom‐like NR sequences were recovered from Monterey Bay samples, whereas LEO‐15 samples yielded NR sequences from a range of photosynthetic eukaryotes. The prospect of using DNA‐ and RNA‐based methods to target the NR genes of diatoms specifically is a promising approach for future physiological and ecological experiments.     

THE ELEMENTAL COMPOSITION OF SOME MARINE PHYTOPLANKTON1

We analyzed the cellular content of C, N, P, S, K, Mg, Ca, Sr, Fe, Mn, Zn, Cu, Co, Cd, and Mo in 15 marine eukaryotic phytoplankton species in culture representing the major marine phyla. All the organisms were grown under identical culture conditions, in a medium designed to allow rapid growth while minimizing precipitation of iron hydroxide. The cellular concentrations of all metals, phosphorus, and sulfur were determined by high‐resolution inductively coupled plasma mass spectrometry (HR‐ICPMS) and those of carbon and nitrogen by a carbon hydrogen nitrogen analyzer. Accuracy of the HR‐ICPMS method was validated by comparison with data obtained with ⁵⁵Fe radioactive tracer and by a planktonic reference material. The cellular quotas (normalized to P) of trace metals and major cations in the biomass varied by a factor of about 20 among species (except for Cd, which varied over two orders of magnitude) compared with factors of 5 to 10 for major nutrients. Green algae had generally higher C, N, Fe, Zn, and Cu quotas and lower S, K, Ca, Sr, Mn, Co, and Cd quotas than coccolithophores and diatoms. Co and Cd quotas were also lower in diatoms than in coccolithophores. Although trace element quotas are influenced by a variety of growth conditions, a comparison of our results with published data suggests that the measured compositions reflect chiefly the intrinsic (i.e. genetically encoded) trace element physiology of the individual species. Published field data on the composition of the planktonic biomass fall within the range of laboratory values and are generally close to the approximate extended Redfield formula given by the average stoichiometry of our model species (excluding the hard parts): While clearly this elemental stoichiometry varies between species and, potentially, in response to changes in the chemistry of seawater, it provides a basis for examining how phytoplankton influence the relative distributions of the ensemble of major and trace elements in the ocean.     

THE INFLUENCE OF FLUCTUATING LIGHT ON DIVERSITY AND SPECIES NUMBER OF NUTRIENT‐LIMITED PHYTOPLANKTON1

The influence of fluctuating light on diversity and species number of a natural phytoplankton assemblage competing for nutrients was investigated for 48 days under semicontinuous culture conditions. Light conditions were either changed periodically from high (65 μmol photons·m^?2·s^?1) to low intensity (15 μmol photons·m^?2·s^?1) at intervals of 1, 3, 6, and 12 days or fixed at constant light conditions of intermediate intensity (40 μmol photons·m^?2·s^?1). Fluctuating light at intervals of 1–12 days significantly affected phytoplankton diversity. The development of phytoplankton communities differed in treatments with different light regimes. In treatments with long light intervals, species abundance oscillated with the light phases. Differences in the temporal development of phytoplankton communities resulted in hump‐shaped relations between the interval length of the light phases and both species number and diversity index and can be explained by the intermediate disturbance hypothesis. Fluctuating light tends to sustain phytoplankton diversity under nutrient limitation if the light regime changes in the order of several days. This indicates that temporal changes in weather regime are important in preventing competitive exclusion of phytoplankton species in nature.     

INTRASPECIFIC GENETIC DIVERSITY IN THE MARINE COCCOLITHOPHORE EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE): THE USE OF MICROSATELLITE ANALYSIS IN MARINE PHYTOPLANKTON POPULATION STUDIES1

Using primer pairs for seven previously described microsatellite loci and three newly characterized microsatellite loci from the coccolithophore Emiliania huxleyi (Lohm.) Hay and Mohler, we assessed genetic variation within this species. Analysis of microsatellite length variants (alleles) was conducted for 85 E. huxleyi isolates representative of different ocean basins. These results revealed high intraspecific genetic variability within the E. huxleyi species concept. Pairwise comparison of a 1992 Coastal Fjord group (FJ92) (n=41) and a North East Atlantic (NEA) group (n=21), using F_ST as an indicator of genetic differentiation, revealed moderate genetic differentiation (F_ST=0.09894; P=0; significance level=0.05). Gene flow between the FJ92 and NEA groups was estimated to be low, which is in agreement with the moderate levels of genetic differentiation revealed by the microsatellite data. A genetic assignment method that uses genotype likelihoods to draw inference about the groups to which individuals belong was tested. Using FJ92 and NEA as reference groups, we observed that all the E. huxleyi groups tested against the two reference groups were unrelated to them. On a global biogeographical scale, E. huxleyi populations appear to be highly genetically diverse. Our findings raise the question of whether such a high degree of intraspecific genetic diversity in coccolithophores translates into variability in ecological function.     

THE UPTAKE OF IODATE BY MARINE PHYTOPLANKTON1

Several studies have suggested that phytoplankton play a role in the iodine cycle. Using a short-term incubation technique for determining the uptake of iodate by phytoplankton, cultures of Thalassiosira oceanica Hasle, Skeletonema costatum (Greville) Cleve, Emiliania huxleyi (Lohmann) Hay and Mohler, and Dunaliella tertiolecta Butcher were found to be capable of assimilating iodate at rates ranging from 0.003 to 0.24 nmol IO₃^?·μg chlorophyll a^?1·h^?1. The kinetics for the uptake of iodate can be modeled, and the similarity between the model and experimental results suggests that there is a steady state between iodate uptake and release of dissolved iodine from the cells, presumably in the form of iodide. Two experiments were conducted in the Sand Shoal Inlet of the Cobb Bay estuary (37°15′N, 75°50′W). The uptake of iodate was 0.26 and 0.08 nmol IO₃^?·μg chlorophyll a^?1·h^?1 during high and low tide, respectively. Using field estimates based on measured levels of iodate in the estuary, we estimate that phytoplankton can take up as much as 3% of the ambient pool of iodate on a daily basis and the entire pool in about 1 month. Thus, phytoplankton can be a significant component of the global iodine cycle by mediating changes in the speciation of iodine in the marine environment.     

EFFECTS OF CADMIUM TOXICITY ON GROWTH AND ELEMENTAL COMPOSITION OF MARINE PHYTOPLANKTON

Some classes of marine phytoplankton are believed to be more tolerant of high concentrations of trace metals than others, but the results of experimental tests of this hypothesis are ambiguous. Eleven species of phytoplankton representing five classes were grown in Aquil medium containing Cd concentrations between 10⁻⁸ and 10⁻⁵ M ([Cd²⁺]= 10^−9.85 to 10^−6.84 M), and growth rates and intracellular concentrations of Cd, C, N, and S were measured. The mean Cd²⁺ concentration (pCd⁵⁰) that reduced the growth rate of each species to 50% of its maximum varied by 2.5 orders of magnitude, from 10^−6.23 for Emiliania huxleyi to 10^−8.79 for Synechococcus sp. Taxonomic trends in Cd resistance were not apparent in these data. Cadmium quotas (mol Cd·L⁻¹ cell volume) were lowest in species of Bacillariophyceae (ANOVA, P < 0.001), suggesting that they might regulate Cd transport differently than other taxa. Cellular S:C molar ratios increased in four of seven phytoplankton grown at high pCd (7.37–6.84) compared to low Cd ion concentrations (no added Cd), a result of increases in S·L⁻¹ cell volume. Nitrogen:carbon molar ratios were also higher in Cd-exposed phytoplankton, as changes in N and S were highly correlated ( r = 0.98, P < 0.0001). In two species that were examined, S:C ratios increased as a linear function of increasing Cd concentration. The results demonstrate large variability in Cd resistance among phytoplankton that is primarily a function of interspecific differences in Cd detoxification.     

INFLUENCE OF IRRADIANCE ON THE FATTY ACID COMPOSITION OF PHYTOPLANKTON1

Eight species of marine phytoplankton commonly used in aquaculture were grown under a range of photon flux densities (PEDs) and analyzed for their fatty acid (FA) composition. Fatty and composition changed considerably at different PFDs although no consistent correlation between the relative proportion of a single FA and μ or chl a · cell^?1 was apparent. Within an individual species the percentage of certain fatty acids covaried with PFDs, growth rate and/or chl a · cell^?1. The light conditions which produced the greatest proportion of the essential fatty acids was species specific. Eicosapentaenoic acid. 20:5ω3 increased from 6.1% to 15.5% of the total fatty acids of Chaetoceros simplex Ostenfield grown at PFDs which decreased from 225 μE · m^?2· s^?1 to 6 μE · m^?2· s^?1, respectively. Most species had their greatest proportion of 20: 5ω3 at low levels of irradiance. Conversely, docosahexaenoic acid, 22:6ω3, decreased from 9.7% to 3.6% of the total fatty acids in Pavlova lutheri Droop as PFD decreased. The percentage of 22:6ω3 generally decreased with decreasing irradiances. In all diatoms the percentage of 16:0 was significantly correlated with PFD, and in three of five diatoms, with growth rate (μ). Results suggest that fatty acid composition is a highly dynamic component of cellular physiology, which responds significantly to variation in PFD.     

PHYSIOLOGICAL ACCLIMATION OF MARINE PHYTOPLANKTON TO DIFFERENT NITROGEN SOURCES1

We examined the energetic dependency of the biochemical and physiological responses of Thalassiosira pseudonana Hasle and Heimdal. Chaetoceros gracilis Schütt, Dunaliella tertiolecta Butcher, and Gymnodinium sanguineum Hirasaka to NH₄⁺, NO₃^?, and urea by growing them at subsaturating and saturating photon flux (PF). At subsaturating PF, when energy was limiting, NO₃^? and NH₄⁺ grown cells had similar growth rates and C and X quotas. Therefore, NO₃^? grown cells used up to 48% more energy than NH₄⁺ grown cells to assimilate carbon and nitrogen. Based on our measurements of pigments, chlorophyll-a-specific in vivo absorption cross-section, and fluorescence-chlorophyll a^?1, we suggest that NO₃^?, grown cells do not compensate for the greater energy requirements of NO₃^? reduction by trapping more light energy. At saturating PF, when energy is not limiting, the utilization of NO₃^?, compared to NH₄⁺ resulted in lower growth rates and N quotas in Thalassiosira pseudonana and lower N quotas in Chaetoceros gracilis, suggesting enzymatic rather than energetic limitations to growth. The utilization of urea compared to Nh₄⁺ resulted in lower growth rates in Chaetoceros gracilis and Gymnodinium sanguineum (saturating PF) and in lower N quotas in all species tested at both subsaturating and saturating PF. The high C:N ratios observed in all urea-grown species suggest that nitrogen assimilation may be limited by urea uptake or deamination and that symptoms of N limitation in microalgae may be induced by the nature of the N source in addition to the N supply rate. Our results provide new eridence that the maximum growth rates of microalgae may be limited by enzymatic processes associated with the assimilation of NO₃^?, or urea.     

PHYTOPLANKTON DIVERSITY OF SHALLOW TIDAL LAKES: INFLUENCE OF PERIODIC SALINITY CHANGES ON DIVERSITY AND SPECIES NUMBER OF A NATURAL ASSEMBLAGE1

The influence of periodic salinity changes was investigated for 42 days under semicontinuous culture conditions with phosphorus limitation using phytoplankton assemblages from Lake Waihola, a tidally influenced shallow lake. To simulate tidal effects on the phytoplankton community, salinity in the cultures was increased in pulses at different intervals (3.5, 7, and 14 days), and these cultures were compared with those that experienced constant freshwater conditions. Salinity pulses significantly affected competition and succession with a major loss in diversity during the first days of the experiment due to the initial pulse that caused a transition from freshwater to brackish conditions in the cultures. After this initial phase, diversity index (H') and species number (S_corr) decreased less rapidly. The loss in H' and S_corr over time was highest under constant freshwater conditions and lowest in the treatment with an interval of 3.5 days between salinity pulses. At the end of the experiment, the combination of initial loss in H' and S_corr and the time course of H' and S_corr resulted in a U‐shaped relation between the interval length of salinity pulses and both H' and S_corrtemp1.txttemp1.txt. Our results indicate that salinity pulses at intervals of a few days tend to promote phytoplankton diversity. If saline intrusions in coastal freshwater systems occur only at spring tides, this will lead to decreases in diversity and species richness.     

海水养虾池浮游动物对浮游植物牧食力的研究

利用常规显微镜直接计数法评估了海水养虾池浮游动物对浮游藻类的牧食力。测得虾池浮游动物对水柱浮游植物的总滤水率为８０．３８ｍｌ／（Ｌ·ｈ）,每个浮游动物的滤水率平均为１１．１３μｌ／（ｉｎｄ·ｈ）,总牧食率为１０．５０μｇ／（Ｌ·ｈ）,即１．２５ｎｇＣ／（ｉｎｄ·ｈ）。浮游动物对水柱原位主要浮游藻类的选食,以隐藻和舟形藻为最多,其次是扁藻和小环藻,６ｈ内对上述４种藻类的选食率分别为９２．６８％、７２．４６％、３６．２２％和３２．５６％,其相应的选择指数依次为０．７３３１、０．６７１７、０．４３４５和０．３９１３。对水柱总体而言,６ｈ内有３５．７２％的主要浮游藻类被选食了。     

A MODEL APPROACH FOR SIZE-SELECTIVE COMPETITION OF MARINE PHYTOPLANKTON FOR FLUCTUATING NITRATE AND AMMONIUM1

Phytoplankton size-selective competition for fluctuating nutrients was studied with the use of a numerical model, which describes nitrate and ammonium uptake, nitrate reduction to ammonium, and growth as a function of cell she under fluctuating nitrogen limitation. The only size-dependent parameter in the model was the cell nutrient quota. Related to this, the cell surface area per biomass was negatively correlated to cell volume, and the vacuole volume per biomass ratio was positively correlated to cell volume. Simulations showed an inverse correlation between the maximum specific growth rate and cell size under steady-state conditions. With nitrate as the limiting nitrogen source, nitrogen quotas were always higher than with ammonium at the same specific growth rate. Net passive transport of ammonium due to unspecific diffusion of ammonia across the plasma membrane decreased the affinity for ammonium, whereas the affinity for nitrate was not influenced. Transient state-specific ammonium uptake was not dependent on cell size. However, small algae always have the highest specific growth rate in ammonium-controlled systems according to our model. Transient state nitrate uptake rate was positively correlated to cell size because larger algae have a higher vacuole volume per biomass, in which nitrate can be stored. Despite their lower maximum growth rate, larger algae became dominant during simulations under fluctuating nitrate supply when amplitude of and the period between nitrate pulses were high enough. Results from model simulations were qualitatively validated by earlier observations that large diatoms become dominant under fluctuating conditions when nitrate is the main nitrogen source.     

EFFECTS OF ENVIRONMENTAL VARIATION ON SINKING RATES OF MARINE PHYTOPLANKTON1

The effects of environmental variables, particularly irradiance, on the sinking rates of phytoplankton were investigated using cultures of Chaetoceros gracilis Schütt and C. flexuosum Mangin in laboratory experiments; these data were compared with results from assemblages in the open ocean and marginal ice zone of the Greenland Sea. In culture experiments both the irradiance under which the diatom was grown and culture growth rate were positively correlated with sinking rates. Sinking rates (ψ) in the Greenland Sea were smallest when determined from chlorophyll (mean ψ_chl= 0.14 m · d^?1) and biogenic silica (ψ_si= 0.14 m · d^?1) and greatest when determined from particulate carbon (ψ_c= 0.55 m · d^?1) and nitrogen (ψ_N= 0.64 m · d^?1). Field measurements indicated that variations in sinking may be associated with changes in irradiance and nitrate concentrations. Because these factors do not directly affect water density, they must be inducing physiological changes in the cell which affect buoyancy. Although a direct response to a single environmental variable was not always evident, sinking rates were positively correlated with growth rates in the marginal ice zone, further indicating a connection to physiological processes. Estimats of carbon flux at stations with vertically mixed euphotic zones indicated that approximately 30% of the daily primary production sank from the euphotic zone in the form of small particulates. Calculated carbon flux tended to increase with primary productivity.     

CELL-ASSOCIATED PROTEOLYTIC ENZYMES FROM MARINE PHYTOPLANKTON1

Despite their central importance in cell metabolism, little is known about proteases in marine phytoplankton. We surveyed caseinolytic and leucine aminopeptidase (LAP) activities in log-phase cultures of the chlorophyte Dunaliella tertiolecta Butcher, the diatom Thalassiosira weissflogii (Gru.) Fryxell et Hasle, the chrysophyte Isochrysis galbana Parke, the coccolithophorid Emiliania huxleyi (Lohm.) Hay et Mohler, and the cyanobacterium Synechococcus sp. (WH 5701). LAP activity was very low at pH < 6 and peaked between pH 7.5 and 8.5 in all species, whereas caseinolytic activity in most species showed only minor peaks in the pH 4–5 range and broad maxima above pH 8. Thus, acidic vacuolar proteases apparently represented only a small fraction of total protease activity. Attempts to classify proteases using selective inhibitors were inconclusive. Neither the serine/cysteine protease inhibitor leupeptin nor the aspartic protease inhibitor pepstatin. A inhibited caseinolytic or LAP activity in any species. The metalloprotease inhibitor EDTA was only effective against LAP activity in some species, causing average decreases of 30–50%, whereas the cysteine/serine protease inhibitor phenyl methyl sulfonylfluoride achieved at best a 30–60% decrease in caseinolytic activity. Caseinolytic activities were remarkably stable. At pH 7.5 and 25°C, extracts of D. tertiolecta, E. huxleyi, and Synechococcus showed no changes in activity after 24 h, whereas activity declined by less than 50% in the other species. Incubation of cell extracts for 1 h at 25°C in pH 7.5 buffer did not alter patterns of cell proteins, suggesting that endogenous proteases did not effectively degrade endogenous proteins. Casein zymograms were used to identify >200-and <20-kDa proteases in homogenates of log-phase T. weissflogii; only the smaller protease was found in D. tertiolecta. Antibodies to the ATPase subunit (C) of the conserved, chloroplastic Clp protease from Pisum cross-reacted with proteins in Synechococcus, D. tertiolecta, and I. galbana, but no cross-reactions were found for any species with antibodies against the ClpP subunit from either E. coli or Nicotiana. Our results show that phytoplankton contain a diverse complement of proteases with novel characteristics.     

一些海洋浮游植物量子产值的研究

现场实验以及用硅藻、金藻和绿藻所做的实验表明,在光饱和深度以下,随着深度的增加,浮游植物的量子产懂具有不变、增加和下降3种垂直变化趋势。偏离理论模式的后两种结果可能是由浮游植物的光强适应(Light-shade adaptation)等原因引起。还探讨了量子产值作为初级生产力模型和光利用效率模型中的参数的问题。     

SORPTION OF PLUTONIUM-237 BY TWO SPECIES OF MARINE PHYTOPLANKTON11

The adsorption of plutonium-237, added from an acid solution, by two species of marine phytoplankton, Monochrysis lutheri Droop, a flagellated chrysomonad, and Phaeodactylum tricornutum Bohlin, a diatom, is governed by a passive mechanism. This is clearly indicated by the observation that the rapid rates of sorption were not significantly different for live and heat-killed cells incubated at 24°C and 0°C. The Q₁₀ values for the chrysomonad and the diatom of 0.95 and 1.18, respectively, are indicative of passive processes. The average ratio of (activity on algae/activity in filtrate) per unit volume at 24 h, the time when maximum levels of algal radioactivity were attained, of (32 ± 4) × 10³ was not significantly different for the diatom or the chrysomonad. On a per cell basis, maximum levels of radioactivity were attained within 2 h. Less than 20% of the adsorbed ²³⁷Pu desorbed after 24 h.     

RELATIONSHIPS BETWEEN NITRATE REDUCTASE ACTIVITY AND RATES OF GROWTH AND NITRATE INCORPORATION UNDER STEADY-STATE LIGHT OR NITRATE LIMITATION IN THE MARINE DIATOM THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE)1

Although activity of the enzyme nitrate reductase (NR) can potentially be used to predict the rate of nitrate incorporation in field assemblages of marine phytoplankton, application of this index has met with little success because the relationship between the two rates is not well established under steady-state conditions. To provide a basis for using NR activity measurements, the relationships among NR activity, growth rate, cell composition, and nitrate incorporation rate were examined in cultures of Thalassiosira pseudonana (Hustedt)Hasle and Heimdal, growing a) under steady-state light limitation, b) during transitions between low and high irradiance (15 or 90 μmol quanta.m^?2.s^?1), and c) under steady-state nitrate limitation. Using a modified assay for NR involving additions of bovine serum albumin to stabilize enzyme activity, NR activity in light-limited cultures was positively and quantitatively related to calculated rates of nitrate incorporation, even in cultures that were apparently starved of selenium. During transitions in irradiance, growth rates acclimated to new conditions within 1 day; through the transition, the relationship between NR activity and nitrate incorporation rate remained quantitative. In nitrate-limited chemostat cultures, NR activity was positively correlated with growth rate and with nitrate incorporation rates, but the relationship was not quantitative. NR activity exceeded nitrate incorporation rates at lower growth rates (<25% of nutrient-replete growth rates), but chemostats operating at such low dilution rates may not represent ecologically relevant conditions for marine diatoms. The strong relationship between NR activity and nitrate incorporation provides support for the idea that NR is rate-limiting for nitrate incorporation or is closely coupled to the rate-limiting step. In an effort to determine a suitable variable for scaling NR activity, relationships between different cell components and growth rate were examined. These relationships differed depending on the limiting factor. For example, under light limitation, cell volume and cell carbon content increased significantly with increased growth rate, while under nitrate limitation cell volume and carbon content decreased as growth rates increased. Despite the differences found between cell composition and growth rate under light and nitrate limitation, the relationships between NR activity scaled to different compositional variables and growth rate did not differ between the limitations. In field situations where cell numbers are not easily determined, scaling NR activity to particulate nitrogen content may be the best alternative. These results establish a strong basis for pursuing NR activity measurements as indices of nitrate incorporation in the field.     

AN EMPIRICAL PROTOCOL FOR WHOLE-CELL IMMUNOFLUORESCENCE OF MARINE PHYTOPLANKTON1

An immunofluorescence protocol for intracellular antigens of phytoplankton was developed empirically. Paraformaldehyde (4%) was used to fix samples for 6 h at 4° C; fixed samples can be stored in cold (–20°C) methanol for at least 1 month. We used Triton X-100, Nonidet P-40, and dimethyl sulfoxide to permeabilize the cells for antibody penetration across the cell wall and plasma membrane. After immunolabeling at room temperature, the samples were mounted on slides and could be stored at 4° C for up to 6 months without significant decay of the fluorescence. The staining was examined with an epifluorescence microscope; semiquantitative (percentage of cells with positive staining) and quantitative (staining intensity measured with an imaging system) analyses were performed. Reproducible staining was achieved for nuclear, chloroplastic, cytoplasmic, and cell surface antigens in species of Chlorophyceae, Prymnesiophyceae, Bacillariophyceae, and Dinophyceae including some field-collected samples. This protocol is advantageous to previously published protocols in that it allows relatively simple and long-term storage of fixed samples and allows staining of multiple antigens for the same sample. Although improvement on cell permeabilization is required for some species, the present protocol could prove useful for physiological or ecological studies when frequent sampling is required and immediate processing of the samples is not possible.