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Monitoring natural phytoplankton communities: A comparison between traditional methods and pulse-shape recording flow cytometry
Abstract
The phytoplankton community can vary within hours (physiology) to years (climatic and anthropogenic responses), and monitoring at different timescales is relevant for understanding community functioning and assessing changes. However, standard techniques used in monitoring programmes are time-consuming and/or expensive, limiting sampling frequency. The use of faster methods, such as flow cytometry, has become more frequent in phytoplankton studies, although comparisons between this technique and traditional ones are still scarce. This study aimed to assess if natural phytoplankton communities analysed with pulse-shape recording flow cytometry (PFCM) and classical techniques (chl a extracts and microscopy) provide comparable results. Monthly samples (March to September 2015) from 4 stations in Roskilde Fjord (Denmark) were analysed with PFCM and classical techniques. Results showed a highly significant correlation between total red fluorescence and chl a, and comparable cell counts from PFCM and microscopy for cell sizes >5 µm, but not for sizes <5 µm. We propose an empirical algorithm to obtain cell volumes from the integrated forward scatter signal from PFCM, making it possible to estimate carbon biomass with PFCM, applying the same conversion factors as for microscopy. Biomasses obtained with PFCM, estimated from live cells, were higher than microscopy for natural samples. We conclude that PFCM results are comparable to classical techniques, yet the data from PFCM had poor taxonomic resolution without support of other techniques. With the faster analysis capacity of PFCM, post-processing of data and analysis of high-resolution time series may be made easier.
... There are several challenges related to quantifying the scale of depletion. Phytoplankton assemblages are heterogeneous in space and time, requiring monitoring at several spatiotemporal scales with the capacity to accurately characterize community composition (Zingone et al., 2015;Haraguchi et al., 2017). The particle depletion effect is not straightforward, as capture efficiency is variable for particles with different dimensions and can be low or negligent with particles below 4 µm diameter . ...
... A CytoSense™ pulse shape flow cytometer (CytoBuoy b.v. Woerden, NL) was used to count and size phytoplankton (Haraguchi et al., 2017) with 400 -800 µL aliquots from each sample. The flow cytometer was equipped with a 488 nm excitation laser, red (650−700 nm), orange (600−650 nm), and yellow (550 nm) fluorescence emission sensors in addition to side-and forward-scattering sensors. ...
... Gating was performed in a sequential fashion until particles unassigned in a cluster constituted ~5% or less of the total particle count. Particle bio-volume was estimated following Haraguchi et al. (2017). Briefly, the integrated forward scatter signal was calibrated against a range of commercial beads of known diameter/volume using a model II regression. ...
The ecological footprint of modern industrialized society now encompasses the entire biosphere. Interrelated themes of global warming, biodiversity loss, ocean acidification, and eutrophication are defining features of the Anthropocene. The nutritive enrichment of our coastal seas and estuaries has been widely recognized as a core environmental issue that has taken shape in national and international regulation. Despite the improvements in nutrient load reductions to some coastal waters, ensuing ecological rebounding or otherwise expected improvements have not been realized. In light of internal loading and the manifold negative interactions, there is increasing recognition that multifaceted intervention in coastal ecology is required to rectify the deterioration of coastal ecosystems. One such intervention is leveraging the intense particle filtration capacity of marine bivalves, where in the western Baltic Sea, the blue mussel (Mytilus edulis) cultivated for the primary purpose of mitigating eutrophication can extract large quantities of nutrients from coastal waters while providing additional ecosystem services.
Several studies have examined bivalve farming practices and their quantitative effects on the environment in relation to seston immobilization and nutrient dynamics. While sharing some features of conventional shellfish production, mitigation mussel cultivation shifts cultivation objectives from product quality to total nutrient content at minimal costs. Accordingly, modified or new modes of production require optimization procedures in terms of cultivation methods and spatial prioritization. Eutrophic environments are highly dynamic and the interactions of marine mitigation mechanisms with the environment require careful investigation to assess the scale of ecosystem services rendered. Suspended mussel farms function as large-scale reactors for organic particles, transforming a portion into somatic mass, another into particulate organic wastes deposited on the sea floor, and the remaining as dissolved metabolic wastes. In an organic particle-rich environment, this large-scale filtration mechanism functions to clarify the water column and locally enhance nutrient dynamics, to a degree contingent upon the environmental contexts.
To characterize mitigation production, important ecosystem services and impacts, this PhD thesis encompasses three years of field and model studies across Denmark and the western Baltic Sea. Experimental results from commercial-scale field trials demonstrated that increasing substrate density in the water column or utilization of cultivation technologies with high substrate surface area and nominal requirements for buoyancy maintenance dramatically increase prior estimates of nutrient extractive potential of mitigation mussel farming. Improved extractive potentials could then be used to evaluate mitigation potentials over the western Baltic Sea by development of a spatial model. Spatial modeling results exhibited several regions where mitigation farms can be prioritized in order to meet water quality goals, while also identifying regions where mussel production may be limited by food depletion. Therefore, a farm-scale model was developed to evaluate the mechanisms behind food depletion in detail, by exploring scenarios with multiple environmental interactions representative of the varying gradations of conditions across the western Baltic Sea. From model results and field observations of food depletion, relatively minor variability in environmental conditions or farm configuration drove highly differential intensity and extent of depletion signals. To interpret the complex real-world structure of depletion in a large-scale mitigation mussel farm, field studies were conducted alongside collection and analysis of satellite remote sensing data. Spatial patterns of seston depletion are primarily influenced by hydrodynamic regimes, yet indications of basin-scale feedbacks were suggestive with high biomass loads. Considering the potential feedbacks of particle immobilization on nutrient dynamics, further field study was conducted to assess the effects of particle deposition at a mitigation mussel farm on biogeochemical processes. Impacts of mitigation farms related to intensive particle immobilization were generally localized within farm areas, and quickly obscured by variability in the existing highly-productive systems.
This thesis and the papers herein establish parameters for optimizing this mitigation instrument and present novel means to evaluate the potential for mitigation in different environmental conditions, including ecological impacts.
... to analyse phytoplankton. This technique provides phytoplankton counts (cell sizes 1-1000 µm) comparable to those obtained with traditional microscopy, although with more reliable counts for cells <5 µm (Haraguchi et al. 2017). Additionally, it also provides information on cell size and morphology due to its capacity to store the optical profile for each particle, recorded as they travel through the flow cell. ...
... Taxonomical information was obtained for some of the clusters based on their optical characteristics, pictures taken by the equipment and cross-referenced with microscopy. Carbon biomass was obtained by converting total FWS to volume (Haraguchi et al. 2017) and then converting volume to biomass using a generic protist volume-to-carbon conversion formula (Menden-Deuer and Lessard 2000). In order to assess phytoplankton physiological state, we estimated the carbon-to-chlorophyll ratio (C:Chla) of each cluster of the phytoplankton community for all samples separately. ...
... In order to assess phytoplankton physiological state, we estimated the carbon-to-chlorophyll ratio (C:Chla) of each cluster of the phytoplankton community for all samples separately. For this, total carbon biomass was divided by total Chla, which was estimated by converting the total red fluorescence to Chla concentration using an empirical formula proposed by Haraguchi et al. (2017), for the same location where the inoculum were taken. It needs to be emphasized that the carbon and the Chla estimated by CytoSense differs from the carbon and Chla estimated by microscopy and organic solvent extraction method respectively. ...
Dissolved organic matter (DOM) is an important component of nutrient cycling, but the role of different organisms controlling the processing of autochthonous DOM remains poorly understood. Aiming to characterize phytoplankton-derived DOM and the effects of complex pelagic communities on its dynamics, we incubated natural plankton communities from a temperate mesohaline estuary under controlled conditions for 18 days. The incubations were carried out in contrasting seasons (spring and autumn) and changes in the planktonic community (phytoplankton, bacteria and microzooplankton), nutrients and DOM were assessed. Our results highlight the complexity of DOM production and fate in natural planktonic communities. Small changes in DOM composition were observed in the experiments relative to the orders-of-magnitude variations experienced in the phytoplankton assembly. We argue that the tight coupling between microbial processing and DOM production by phytoplankton and grazers stabilizes variations in quantity and characteristics of autochthonous DOM, resulting in apparently homogeneous semi-labile DOM pool throughout the experiments. However, seasonal differences in the production and processing of DOM were observed, reflecting differences in the nutrient regimes and initial DOM characteristics in each experiment, but also likely influenced by changes in the successional status of the pelagic community. Acknowledging that characteristics of the DOM derived from phytoplankton growth can vary broadly, heterotrophic processing and successional status of the community are synergistically important factors for shaping those characteristics, and thus affecting the seasonal signature of the semi-labile autochthonous DOM pool.
... Similar to other flow cytometers, the suspended particles (sample) are injected into a particle-free carrying fluid (sheath). The laminar flow of the moving sheath fluid aligns the cells in single file sample stream that intersects a laser (488 nm) (Haraguchi et al. 2017). To match the density and refractive index of the sheath fluid as closely as possible to the samples, prior to analysis, the sheath fluid was replaced with a 3% NaCl solution (w/v) made with Milli-Q water and filtered through a 0.2 μm filter. ...
... Automated analyses of plankton traits complexity, such as cell wall granularity (e.g., coccoliths in coccolithophores), ornaments (processes, setae, and heavily silicified cell walls in diatoms), and internal vacuoles (also commonly found in large diatoms; Woods and Villareal 2008). The FWS total signal has been suggested as a suitable descriptor for particle volume (Haraguchi et al. 2017), thus, photo-physiological information for the community, such as the amount of Chl a per cell volume (Chl a/Vol), either as a single cell or chain, was analyzed by FR total/FWS total. Other fluorescence signatures, such as FO total/FR total related to their size (FWS total), provide information about the pigment composition in the phytoplankton community, allowing the differentiation of cyanobacteria from other picophytoplankton groups, given that they contain high concentrations of phycobiliproteins (e.g., phycoerythrin [PE]), which fluoresce in the orange part of the spectrum, per cell size (FWS total) ( Table 1). ...
... The FWS total signal has been suggested as a better descriptor for particle (cells and colonies/chains) volume than SWS (Haraguchi et al. 2017), so in this study, volume (Vol) is taken as FWS total. To estimate plankton group biovolume, Vol was converted to μm 3 following Haraguchi et al. (2017), where biovolume (μm 3 ) = 4.24 × 10 −6 Vol 1.88 . ...
Plankton are an extremely diverse and polyphyletic group, exhibiting a large range in morphological and physiological traits. Here, we apply automated optical techniques, provided by the pulse‐shape recording automated flow cytometer—CytoSense—to investigate trait variability of phytoplankton and plastidic ciliates in Arctic and Atlantic waters of the subpolar North Atlantic. We used the bio‐optical descriptors derived from the CytoSense (light scattering [forward and sideward] and fluorescence [red, yellow/green and orange from chlorophyll a, degraded pigments, and phycobiliproteins, respectively]) and translated them into functional traits to demonstrate ecological trait variability along an environmental gradient. Cell size was the master trait varying in this study, with large photosynthetic microplankton (> 20 μm in cell diameter), including diatoms as single cells and chains, as well as plastidic ciliates found in Arctic waters, while small‐sized phytoplankton groups, such as the picoeukaryotes (< 4 μm) and the cyanobacteria Synechococcus were dominant in Atlantic waters. Morphological traits, such as chain/colony formation and structural complexity (i.e., cellular processes, setae, and internal vacuoles), appear to favor buoyancy in highly illuminated and stratified Arctic waters. In Atlantic waters, small cell size and spherical cell shape, in addition to photo‐physiological traits, such as high internal pigmentation, offer chromatic adaptation for survival in the low nutrient and dynamic mixing waters of the Atlantic Ocean. The use of automated techniques that quantify ecological traits holds exciting new opportunities to unravel linkages between the structure and function of plankton communities and marine ecosystems.
... Using these technologies in high frequency monitoring of plankton has demonstrated that short-term events can be easily missed with sampling frequencies typically employed for monitoring (Thyssen et al., 2008;Campbell et al., 2013;Dugenne et al., 2014). Furthermore, in-flow systems allow analysis of live samples, avoiding loss and shrinkage of cells due to fixation (Jakobsen and Carstensen, 2011;Haraguchi et al., 2017). Thus, the use of in-flow systems can improve our knowledge on the coupled dynamics of phytoplankton and ciliates, by allowing a large number of samples to be analyzed in relatively short time. ...
... We employed a pulse-shape recording flow cytometer (PFCM) (CytoSense, Cytobuoy, NL) to analyse phytoplankton. This technique is suitable for rapid analysis of the phytoplankton size spectra, providing cell counts comparable to those obtained with traditional microscopy and more reliable information for picoplankton (Haraguchi et al., 2017). Additionally, it also provides information on cell size and morphology due to its capacity to store the optical profile for each particle, recorded as they travel through the flow cell. ...
... Taxonomical information was obtained for some of the clusters based on their optical characteristics and photos taken by the equipment, which were cross-referenced with qualitative information obtained from live samples examined by light microscopy. Carbon biomass was obtained by converting total FWS to volume by applying the empirical formula in Haraguchi et al. (2017) and then converting volume to biomass using a generic protist volume-to-carbon conversion formula (Menden-Deuer and Lessard, 2000). Note that for some characteristic and/or abundant groups (e.g., chains, pico-eukaryotes, Teleaulax spp.) group-specific clusters were identified based on cells characteristics (size, shape, fluorescence). ...
Phytoplankton plays a key role as primary producers and mediating biogeochemical cycles in the water column. The understanding of the temporal dynamic of primary grazers channeling energy and carbon from primary producers is important for evaluating aquatic ecosystems functioning. This study investigates the coupling between phytoplankton and ciliates from live samples collected with approximately daily frequency during an almost 2-year cycle. The study site is a nutrient-rich temperate estuary, Roskilde Fjord (Denmark). Our aim is to evaluate the importance of protist grazers, especially ciliates, as predators on phytoplankton and to evaluate differences among multiple nutritional strategies through different seasons. The phytoplankton community, was mostly dominated by small organisms (<20 μm) with few observations of diatoms. In most of observations, heterotrophic dinoflagellates biomass was smaller than biomass of ciliates (<10%), indicating that ciliates are the main component of microzooplankton. Except for the spring 2016, the ciliate community closely followed the phytoplankton community, showing a tight coupling between the primary producers and grazers during all seasons. This somehow contradicts the general assumption that ciliate dominance is restricted to periods of nutrient limitation dominated by the microbial food web and suggests a year-round key role of ciliates as consumers of phytoplankton biomass. Biomasses of ciliates increased during spring and were highest during summer. Relative importance of mixotrophs were high due to occurrence of Mesodinium rubrum blooms as well as other mixotrophic ciliates in late spring/early summer. M. rubrum biomass had the opposite pattern of the cryptophyte prey Teleaulax spp., and the coupling between the two populations was very strong in late spring. Ciliates that grazed on selected phytoplankton, had a smaller potential grazing impact regarding their biomasses, likely due to food limitation; conversely ciliates that feed on diverse prey items were less constrained by food limitation, and their seasonality appear to be driven by other factors. These findings suggest that the ciliate community structure and dynamics is important in structuring the phytoplankton community on short and seasonal scale.
... These techniques generate data comparable to those obtained by manual light microscopy, but in a high-throughput way. Still, some differences can be detected due to the differences in manual vs automatic classification, sample preservation vs in situ observations, and between sampled seawater volumes Jakobsen & Carstensen, 2011;Álvarez et al., 2014;Schmid et al., 2016;Haraguchi et al., 2017;Detmer et al., 2019;Hrycik et al., 2019;Kraft et al., 2021). ...
A major challenge in characterizing plankton communities is the collection, identification and quantification of samples in a time-efficient way. The classical manual microscopy counts are gradually being replaced by high throughput imaging and nucleic acid sequencing. DNA sequencing allows deep taxonomic resolution (including cryptic species) as well as high detection power (detecting rare species), while RNA provides insights on function and potential activity. However, these methods are affected by database limitations, PCR bias, and copy number variability across taxa. Recent developments in high-throughput imaging applied in situ or on collected samples (high-throughput microscopy, Underwater Vision Profiler, FlowCam, ZooScan, etc) has enabled a rapid enumeration of morphologically-distinguished plankton populations, estimates of biovolume/biomass, and provides additional valuable phenotypic information. Although machine learning classifiers generate encouraging results to classify marine plankton images in a time efficient way, there is still a need for large training datasets of manually annotated images. Here we provide workflow examples that couple nucleic acid sequencing with high-throughput imaging for a more complete and robust analysis of microbial communities. We also describe the publicly available and collaborative web application EcoTaxa, which offers tools for the rapid validation of plankton by specialists with the help of automatic recognition algorithms. Finally, we describe how the field is moving with citizen science programs, unmanned autonomous platforms with in situ sensors, and sequencing and digitalization of historical plankton samples.
... A commercial option that epitomizes this latter category is the CytoSense (Cytobuoy, Inc.), which, like the Imaging Flow Cytobot (McLane Labs), also records the shapes of cytometric signals from cells larger than the laser beam and chains of cells. The CytoSense relies principally on characterization of shapes of scatter and fluorescence signals for detecting and discriminating phytoplankton into optical categories (which do not necessarily correlate well to phylogenetic categories) (Haraguchi et al., 2017). Images can also be collected on subsets of cells classified first by the pulse-shape parameters in 2-dimensional gates. ...
Harmful algal blooms (HABs) are a major concern for Chilean scientists and governmental authorities, due to their deleterious effects on the aquaculture industry, resulting in complex sanitary, economic, and social problems. Increasingly, intense and frequent HABs events have distressed the aquaculture industry in recent years, especially for the occurrence of previously unreported toxic microalgae species. In the context of HAB monitoring programs, we review recent major advances in new approaches that have enabled deeper understanding of HAB species diversity in Chile. In particular, molecular approaches have revealed exceptional cryptic diversity, and chemotaxonomic approaches exposed the presence of new phycotoxins belonging to still undetected HAB species using conventional light microscopy. New advances in the detection of ichthyotoxins through the use of a fish cell line assay promises an early incorporation of this technique in routine monitoring of community composition. Emerging imaging flow cytometry is progressively pushing monitoring to more automated capabilities, and citizen science is gradually involving local communities in the complex phenomenon of harmful algal blooms.
... These methods analyse samples and produce data at a fraction of the time compared to microscopical analyses and could increase the temporal frequency of data. However, using conventional monitoring methods, i.e. light microscopy provides better taxonomical precision for nanoplankton and microplankton compared to for example, flow-cytometry (Haraguchi et al., 2017), and imaging-based techniques require an investment of time to build training sets of images of focal biota to be used by the software in taxonomic identification before they can be routinely taken into use. In due time, we expect the novel methods to contribute valuably to conventional microscopy-based plankton monitoring, and thus enable more extensive temporal monitoring data collection. ...
Heterotrophic protists are essential components of the marine ecosystem, yet they are often excluded from monitoring programmes. With limited resources, monitoring strategies need to be optimised considering both scientific knowledge and available resources. In doing so, it is crucial to understand how sampling frequency affects the value of the data. We analysed 11 years of weekly heterotrophic protist time-series data from Station L4 in the Western English Channel to explore how different sampling intervals impact data quality. In the L4 dataset, comprising 55 protist taxa, the reduction of sampling frequency from weekly to four times a year at specific seasons decreased the number of taxa encountered by 38% for ciliates and 29% for heterotrophic dinoflagellates while the mean annual biomass or its mean variation were not affected. Furthermore, when samples were taken only four times a year, biomass peaks of the ten most important taxa were often missed. The primary motivator for this study was furthering the development of the heterotrophic protist monitoring in temperate and subarctic marine areas, e.g. the Baltic Sea. Based on our findings, we give recommendations on sampling frequency to optimise the value of heterotrophic protist monitoring.
... De plus les abondances du groupe Phaeocystis étaient élevées au niveau au large de Brest alors que ce groupe n'a pas été compté dans les analyses microscopiques à cette saison. Malheureusement, aucune donnée pigmentaire n'a été acquise à l'automne (campagne CGFS 2018.Les différences observées entre ces différentes techniques d'analyse ont déjà été mises en évidence dans des études qui combinaient analyse pigmentaire, cytométrie en flux et fluoroprobe en Manche-Mer du Nord(Bonato, 2015) ; analyse pigmentaire, cytométrie en flux et LISST en Méditerranée(Leroux et al., 2017), de la cytométrie en flux automatisée et la microscopie(Haraguchi et al., 2017) et toutes techniques confondues (projet INTERREG IVA « 2 Mers » DYMAPHY, ...
... The CellCognize pipeline represents a promising tool to achieve this, as it provides the ability to detect and quantify focal strains, can detect different growth phases of focal strains even within a diverse microbiota background, can track the growth and enrichment of populations within a community, and can be deployed to estimate biomass. We acknowledge that biomass calculations should be further improved: they are very sensitive to the existing estimates of the mass of individual standards, which could be improved using recent techniques 36,37 . Nevertheless, the alternative 14 C-methods carry their own disadvantages, tending to overestimate substrate usage as a result of unspecific sorption to cells. ...
The study of complex microbial communities typically entails high-throughput sequencing and downstream bioinformatics analyses. Here we expand and accelerate microbiota analysis by enabling cell type diversity quantification from multidimensional flow cytometry data using a supervised machine learning algorithm of standard cell type recognition (CellCognize). As a proof-of-concept, we trained neural networks with 32 microbial cell and bead standards. The resulting classifiers were extensively validated in silico on known microbiota, showing on average 80% prediction accuracy. Furthermore, the classifiers could detect shifts in microbial communities of unknown composition upon chemical amendment, comparable to results from 16S-rRNA-amplicon analysis. CellCognize was also able to quantify population growth and estimate total community biomass productivity, providing estimates similar to those from ¹⁴C-substrate incorporation. CellCognize complements current sequencing-based methods by enabling rapid routine cell diversity analysis. The pipeline is suitable to optimize cell recognition for recurring microbiota types, such as in human health or engineered systems.
... Although small photoautotrophs significantly dominated abundance (Fig. 8) in both WEC and CEC during the cruise, the contribution of nanoeukaryotes and microphytoplankton to total red fluorescence, which is an estimation of chlorophyll a fluorescence (Haraguchi et al., 2017), was important (supplementary table A1, Fig. 8). The relative contribution of the Coccolithophore-like cells to the total red fluorescence was almost seven times higher in C1 than in any other community. ...
... The technology, include pulse-shape recording flow cytometry that has shown promising results. This technology has demonstrated a potential in the analysis of phytoplankton community composition because of its rapid analysis time, that it can estimate particle volumes and because it has a counting precision comparable to microscopic counting (Haraguchi et al. 2017). PFCM data has already demonstrated that the technology provides reliable data with a time frequency as low as minutes. ...
... Techniques such as image analysis and laser diffraction techniques (e.g. flow cytometers) are increasingly used for phyto- plankton identification and size structure analysis ( Haraguchi et al. 2017). Several sophisticated instru- ments have been developed to analyze phytoplank- ton communities in situ. ...
Changes in phytoplankton composition reveal relevant information about the re- sponse of aquatic systems to environmental drivers. Here, we propose the combined use of particle size measurements and pigment signatures to analyze the changes in the composition of phyto- plankton communities at a coastal location. Canonical correlation analysis (CCA) was applied to separate phytoplankton signals from non-algal components of particulate matter in concurrent measurements of particle size distribution and pigment concentrations (chlorophylls a, b and c). Using this method, we were able to identify phytoplankton community structure variations at size and functional levels associated with the water column during the spring to summer transition at a Mediterranean coastal site. Some taxa with characteristic size spectrum signatures such as Pseudo- nitzschia sp., which produced an intense (up to 5 × 105 cells l−1), but ephemeral bloom, could also be identified. The general patterns of phytoplankton succession obtained using this methodology were corroborated by light microscopy and flow cytometry identification. Phytoplankton biovolume comparisons between both methods were highly consistent (r = 0.75, p < 0.01). We consider that combined pigment and size structure analysis using CCA is a useful tool to determine reorganiza- tion patterns of phytoplankton via changes in species composition.
... These methods generate data at a fraction of the time and is a better option for picoplankton (< 2 µm), which is difficult to identify by microscopy without using dyes and immersion oil. However, microscopy still produces better taxonomical resolution for nano-and microplankton compared with flow-cytometry (Haraguchi et al. 2017). Genetic sequencing is a good tool for obtaining taxonomic data, but has limitations in terms of quantification of biomass and detection of relevant ecological data e.g. ...
Finnish plankton monitoring is divided into phytoplankton and mesozooplankton sampling.
Using the phytoplankton protocol, we included all organisms identified in samples
from the Baltic Sea during spring (n = 125). The plankton was converted to carbon, and
including all microscopy derived carbon (MDC), increased the carbon content by 22%, on
average, compared with only phytoplankton. Particulate organic carbon (POC) and chlorophyll
a (Chl a) were also measured, and the general relationship between MDC and POC
was: slope = 1.04, intercept = 240 μg POC l–1, R2 = 0.66; for phytoplankton to Chl a: 0.037
g Chl a (g MDC)–1, R2 = 0.68. Our results demonstrate that a variable fraction of the plankton
biomass is not recorded in the monitoring programs. Most of the unaccounted biomass
was ciliates, which constituted 14.1% ± 3.7% (mean ± maximum error) of the plankton
biomass. Based on the results we recommend including microzooplankton in the existing
phytoplankton monitoring program.
Across the Southern Ocean, large (≥20 μm) diatoms are generally assumed to be the primary vector for carbon export, although this assumption derives mainly from summertime observations. Here, we investigated carbon production and export potential during the Atlantic Southern Ocean's spring bloom from size-fractionated measurements of net primary production (NPP), nitrogen (nitrate, ammonium, urea) and iron (labile inorganic iron, organically complexed iron) uptake, and a high-resolution characterization of phytoplankton community composition. The nanoplankton-sized (2.7 to 20 μm) diatom, Chaetoceros spp., dominated the biomass, NPP, and nitrate uptake across the basin (40°S to 56°S), which we attribute to their low iron requirement, rapid response to increased light, and ability to escape grazing when aggregated into chains. We estimate that the spring Chaetoceros bloom accounted for >25% of annual export production across the Atlantic Southern Ocean, a finding consistent with recent observations from other regions highlighting the central role of the phytoplankton "middle class" in carbon export.
To broaden our understanding of pelagic ecosystem responses to environmental change, it is essential that we improve the spatiotemporal resolution of in situ monitoring of phytoplankton communities. A key challenge for existing methods is in classifying and quantifying cells within the nanophytoplankton size range (2–20 μ m). This is particularly difficult when there are similarities in morphology, making visual differentiation difficult for both trained taxonomists and machine learning‐based approaches. Here we present a rapid fluoro‐electrochemical technique for classifying nanophytoplankton, and using a library of 52 diverse strains of nanophytoplankton we assess the accuracy of this technique based on two measurements at the individual level: charge required to reduce per cell chlorophyll a fluorescence by 50% and cell radius. We demonstrate a high degree of accuracy overall (92%) in categorizing cells belonging to widely recognized key functional groups; however, this is reduced when we consider the broader diversity of “nano‐phytoflagellates'.” Notably, we observe that some groups, for example, calcifying Isochrysidales, have much greater resilience to electrochemically driven oxidative conditions relative to others of a similar size, making them more easily categorized by the technique. The findings of this study present a promising step forward in advancing our toolkit for monitoring phytoplankton communities. We highlight that, for improved categorization accuracy, future iterations of the method can be enhanced by measuring additional predictor variables with minimal adjustments to the set‐up. In doing so, we foresee this technique being highly applicable, and potentially invaluable, for in situ classification and enumeration of the nanophytoplankton size fraction.
The recent development of biological sensors has extended marine plankton studies from conducting laboratory bench work to in vivo and real-time observations. Flow cytometry (FCM) has shed new light on marine microorganisms since the 1980s through its single-cell approach and robust detection of the smallest cells. FCM records valuable optical properties of light scattering and fluorescence from cells passing in a single file in front of a narrow-collimated light source, recording tens of thousands of cells within a few minutes. Depending on the instrument settings, the sampling strategy, and the automation level, it resolves the spatial and temporal distribution of microbial marine prokaryotes and eukaryotes. Cells are usually classified and grouped on cytograms by experts and are still lacking standards, reducing data sharing capacities. Therefore, the need to make FCM data sets FAIR (Findability, Accessibility, Interoperability, and Reusability of digital assets) is becoming critical. In this paper, we present a consensus vocabulary for the 13 most common marine microbial groups observed with FCM using blue and red-light excitation. The authors designed a common layout on two-dimensional log-transformed cytograms reinforced by a decision tree that facilitates the characterization of groups. The proposed vocabulary aims at standardising data analysis and definitions, to promote harmonisation and comparison of data between users and instruments. This represents a much-needed step towards FAIRification of flow cytometric data collected in various marine environments.
Plankton communities form the basis of aquatic ecosystems and elucidating their role in increasingly important environmental issues is a persistent research question. Recent technological advances in automated microscopic imaging, together with cloud platforms for high-performance computing, have created possibilities for collecting and processing detailed high-frequency data on planktonic communities, opening new horizons for testing core hypotheses in aquatic ecosystems. Analyzing continuous streams of big data calls for development and deployment of novel computer vision and machine learning systems. The implementation of these analysis systems is not always straightforward with regards to operationality, and issues regarding data flows, computing and data treatment need to be considered. We created a data pipeline for automated near-real-time classification of phytoplankton during remote deployment of imaging flow cytometer (Imaging FlowCytobot, IFCB). Convolutional neural network (CNN) is used to classify continuous imaging data with probability thresholds used to filter out images not belonging to our existing classes. The automated data flow and classification system were used to monitor dominating species of filamentous cyanobacteria on the coast of Finland during summer 2021. We demonstrate that good phytoplankton recognition can be achieved with transfer learning utilizing a relatively shallow, publicly available, pre-trained CNN model and fine-tuning it with community-specific phytoplankton images (overall F1-score of 0.95 for test set of our labeled image data complemented with a 50% unclassifiable image portion). This enables both fast training and low computing resource requirements for model deployment making it easy to modify and applicable in wide range of situations. The system performed well when used to classify a natural phytoplankton community over different seasons (overall F1-score 0.82 for our evaluation data set). Furthermore, we address the key challenges of image classification for varying planktonic communities and analyze the practical implications of confused classes. We published our labeled image data set of Baltic Sea phytoplankton community for the training of image recognition models (~63000 images in 50 classes) to accelerate implementation of imaging systems for other brackish and freshwater communities. Our evaluation data set, 59 fully annotated samples of natural communities throughout an annual cycle, is also available for model testing purposes (~150000 images).
The climate-driven changes in temperature, in combination with high inputs of nutrients through anthropogenic activities, significantly affect phytoplankton communities in shallow lakes. This study aimed to assess the effect of nutrients on the community composition, size distribution, and diversity of phytoplankton at three contrasting temperature regimes in phosphorus (P)–enriched mesocosms and with different nitrogen (N) availability imitating eutrophic environments. We applied imaging flow cytometry (IFC) to evaluate complex phytoplankton communities changes, particularly size of planktonic cells, biomass, and phytoplankton composition. We found that N enrichment led to the shift in the dominance from the bloom-forming cyanobacteria to the mixed-type blooming by cyanobacteria and green algae. Moreover, the N enrichment stimulated phytoplankton size increase in the high-temperature regime and led to phytoplankton size decrease in lower temperatures. A combination of high temperature and N enrichment resulted in the lowest phytoplankton diversity. Together these findings demonstrate that the net effect of N and P pollution on phytoplankton communities depends on the temperature conditions. These implications are important for forecasting future climate change impacts on the world’s shallow lake ecosystems.
Carbon-to-chlorophyll a ratios (C:Chl a; weight : weight) were analyzed for 7578 coastal seawater samples collected from Danish waters from 1990 to 2014. The aim was to identify the seasonal and spatial dynamics relative to nutrient richness and to study the effect of reduced nitrogen loadings over time. C:Chl a values were lowest during winter, about 15 across all stations. During the spring, C:Chl a increased to summer values between 20 and 96, depending on the annual mean of total nitrogen concentration. An inter-annual sinusoidal model with monthly time steps described the seasonal C:Chl a pattern well. The amplitudes of the model varied inversely with the annual mean of total nitrogen. Data also showed that a reduction in nitrogen loadings to the area by ~ 40% over the past 24 yr, resulted in a statistically significant increase in mean annual C:Chl a values of 0.8±0.2 yr.-1. The patterns derived from this large data set can be used to predict C:Chl a values for temperate coastal phytoplankton. Use of the empirical relationships derived from the data set improves predictions of C:Chl a values and thereby, e.g., carbon based food-web calculations and carbon based ecosystem models, which often are validated using chlorophyll measurements.
Taxonomic data on phytoplankton composition is important for ecological studies, however, such information is not easy to gather. Imaging devices and image classification software have been developed in the past decades for rapid phytoplankton assessment. Taxonomic resolution output of classification software are primarily limited by the quality of images produced by these imaging instruments. FlowCAM has been utilized in several studies for this endeavor. However, the phytoplankton categories that the instrument is currently able to discriminate are still few compared to the outputs of microscopy. This study aimed to produce high resolution FlowCAM images of fixed phytoplankton samples from natural environments without compromising sample analysis time. It was also aimed to optimize the capability of FlowCAM's VisualSpreadsheet software to automatically classify phytoplankton images. The use of FOV300 flow cell and 10X objective combination has proven to be effective in producing good quality images at a faster rate. The modified hardware configuration resulted to FlowCAM counts that were comparable to that of the standard microscopy method. FlowCAM was able to automatically classify images of dominant phytoplankton groups in the two key sardine fishery areas in the Philippines with relatively high accuracy values. These phytoplankton groups are represented by genera with complex morphological structures (e.g., setae) such as Chaetoceros and Bacteriastrum as well as those genera with simple shapes such as Pseudo-nitzschia (thin-elongate) and Coscinodiscus (spherical).
Phytoplankton observation in the ocean can be a challenge in oceanography.
Accurate estimations of its biomass and dynamics will help to understand
ocean ecosystems and refine global climate models. Relevant data sets of
phytoplankton defined at a functional level and on a sub-meso- and daily scale are thus required. In order to achieve this, an automated,
high-frequency, dedicated scanning flow cytometer (SFC, Cytobuoy b.v., the Netherlands) has been
developed to cover the entire size range of phytoplankton cells whilst
simultaneously taking pictures of the largest of them. This cytometer was
directly connected to the water inlet of a PocketFerryBox during a cruise
in the North Sea, 08–12 May 2011 (DYMAPHY project, INTERREG IV A "2 Seas"),
in order to identify the phytoplankton community structure of near surface
waters (6 m) with a high spatial resolution basis (2.2 ± 1.8 km). Ten
groups of cells, distinguished on the basis of their optical pulse shapes,
were described (abundance, size estimate, red fluorescence per unit volume).
Abundances varied depending on the hydrological status of the traversed
waters, reflecting different stages of the North Sea blooming period.
Comparisons between several techniques analysing chlorophyll a and the
scanning flow cytometer, using the integrated red fluorescence emitted by
each counted cell, showed significant correlations. For the first time, the
community structure observed from the automated flow cytometry data set was
compared with PHYSAT reflectance anomalies over a daily scale. The number of
matchups observed between the SFC automated high-frequency in situ sampling and
remote sensing was found to be more than 2 times better than when using
traditional water sampling strategies. Significant differences in the
phytoplankton community structure within the 2 days for which matchups
were available suggest that it is possible to label PHYSAT anomalies using
automated flow cytometry to resolve not only dominant groups but also community
structure.
A fundamental understanding of the interaction between physical and biological factors that regulate plankton species composition requires, first of all, detailed and sustained observations. Only now is it becoming possible to acquire these types of observations, as we develop and deploy instruments that can continuously monitor individual organisms in the ocean. Our research group can measure and count the smallest phytoplankton cells using a submersible flow cytometer (FlowCytobot), in which optical properties of individual suspended cells are recorded as they pass through a focused laser beam. However, FlowCytobot cannot efficiently sample or identify the much larger cells (10 to >100 μm) that often dominate the plankton in coastal waters. Because these larger cells often have recognizable morphologies, we have developed a second submersible flow cytometer, with imaging capability and increased water sampling rate (typically, 5 mL seawater analyzed every 20 min), to characterize these nano- and microplankton. Like the original, Imaging FlowCytobot can operate unattended for months at a time; it obtains power from and communicates with a shore laboratory, so we can monitor results and modify sampling procedures when needed. Imaging FlowCytobot was successfully tested for 2 months in Woods Hole Harbor and is presently deployed alongside FlowCytobot at the Martha's Vineyard Coastal Observatory. These combined approaches will allow continuous long-term observations of plankton community structure over a wide range of cell sizes and types, and help to elucidate the processes and interactions that control the life cycles of individual species. © 2007, by the American Society of Limnology and Oceanography, Inc.
A brief overview is given of current applications of flow cytometry (FCM) in marine phytoplankton research. This paper presents a selection of highlights and various technical and analytical problems we encountered during the past 10 years. In particular, the conversion of the relative values obtained in terms of size and fluorescence applying FCM to quantitative estimates of cell size, pigment concentration, genome size etc., is addressed. The introduction of DNA -cell-cycle analysis made easily assessable by flow cytometry has been of great impor tance, allowing in situ measurement of species specific growth rates. Key questions in ecology such as factors determining the wax and wane of phytoplankton bloom can now be better answered in terms of species specific growth and mortality. Finally, flow cytometry provides detailed information of the physiological status of the individual algal cells. New staining methods enable us to distinguish between viable and non-viable cells and so will help us to elucidate the importance of 'automortality' in aquatic ecosystems.
Instruments for in vivo identification and quantification of marine organisms are becoming more common, and the interpretation of data from these instruments is still evolving. In the present study, we compare the sizing performance of 3 instruments: (1) a black and white (B/W) FlowCAM II; (2) a BeckmanCoulter Multisizer III (MIII); and (3) an inverted microscope. We applied 3 different particle sizing algorithms available from the FlowCAM to suspensions of 5 different particle morphotypes (4 different phytoplankton species and a spherical NIST calibration bead). The FlowCAM generated size distributions similar to those reported by the MIII for the spherical calibration beads. However, differences in reported sizes emerged among FlowCAM algorithms as well as among instruments when applied to morphologically more complex particles, such as diatom chains. There was an immediate and substantial loss of cell counts when live cells were fixed in Lugol’s solution, but only minor differences in cell size distributions among the different FlowCAM algorithms. The difference in sizing performance of the FlowCAM algorithms affects biovolume estimates of the natural plankton samples analysed. Species diversity was apparently higher in samples analysed by microscopy than with the FlowCAM, but the cell size distribution from the microscope was extremely narrow compared to FlowCAM and MIII. The present study demonstrates that the particle sizing algorithm has severe impact on the characteristics of the particle size distribution and on the total community biomass estimate.
Phytoplankton is a key component in marine ecosystems. It is responsible for most of the marine primary production, particularly in eutrophic lagoons, where it frequently blooms. Because they are very sensitive to their environment, the dynamics of these microbial communities has to be observed over different time scales, however, assessment of short term variability is often out of reach of traditional monitoring methods. To overcome these limitations, we set up a Cytosense automated flow cytometer (Cytobuoy b.v), designed for high frequency monitoring of phytoplankton composition, abundance, cell size, and pigment content, in one of the largest Mediterranean lagoons, the Berre lagoon (South-Eastern France). During October 2011, it recorded the cell optical properties of 12 groups of pico-, nano- and microphytoplankton. Daily variations in the cluster optical properties were consistent with individual changes observed using microscopic imaging, during the cell cycle. We therefore used an adaptation of the size-structured matrix population model, developed by Sosik et al. (2003) to process the single cell analysis of the clusters and estimate the division rates of 2 dinoflagellate populations before, during and after a strong wind event. The increase in the estimated in situ daily cluster growth rates suggest that physiological changes in the cells can prevail over the response of abundance.
Most of phytoplankton influence is barely understood at the sub meso scale and daily scale because of the lack of means to simultaneously assess phytoplankton functionality, dynamics and community structure. For a few years now, it has been possible to address this objective with an automated in situ high frequency sampling strategy. In order to study the influence of environmental short-term events (nutrients, wind speed, precipitation, solar radiation, temperature, and salinity) on the onset of the phytoplankton bloom in the oligotrophic Bay of Villefranche-sur-Mer (NW Mediterranean Sea), a fully remotely controlled automated flow cytometer (CytoSense) was deployed on a solar-powered platform (EOL buoy, CNRS-Mobilis). The CytoSense carried out single-cell analyses on particles (1-800 μm in width, up to several mm in length), recording optical pulse shapes when analyzing several cm(3). Samples were taken every 2 h in the surface waters during 2 months. Up to 6 phytoplankton clusters were resolved based on their optical properties (PicoFLO, Picoeukaryotes, Nanophytoplankton, Microphytoplankton, HighSWS, HighFLO). Three main abundance pulses involving the 6 phytoplankton groups monitored indicated that the spring bloom not only depends on light and water column stability, but also on short-term events such as wind events and precipitation followed by nutrient pulses. Wind and precipitation were also determinant in the collapse of the clusters' abundances. These events occurred within a couple of days, and phytoplankton abundance reacted within days. The third abundance pulse could be considered as the spring bloom commonly observed in the area. The high frequency data-set made it possible to study the phytoplankton cell cycle based on daily cycles of forward scatter and abundance. The combination of daily cell cycle, abundance trends and environmental pulses will open the way to the study of phytoplankton short-term reactivity to environmental conditions.
Plankton counting and analysis is essential in ecological study, yet scant literature exists as to the reliability of those counts and the consistency of the experts who make the counts. To assess how variable expert taxonomic identifications are, a set of six archived mesozooplankton samples from a series of Longhurst Hardy Plankton Recorder net hauls were counted by expert zooplankton analysts located at six marine laboratories. Sample identifications were repeated on two separate days with over 700 target specimens counted and identified on each day across the samples. Twenty percent of the analysts returned counts that varied by more than 10%. Thirty-three percent of analysts exhibited low identification consistencies, returning Intraclass Correlation Coefficient scores of less than 0.80. Statistical analyses of these data suggest that over 83% of the observed categorical count variance can be attributed to inconsistencies within analysts. We suggest this is the root cause of variation in expert specimen labelling consistency.
Present automated systems for counting and measuring marine plankton include flow cytometers and in situ plankton video recorders. Neither of these approaches are optimal for the microplankton cells which range in size from 20 to 200 mu m and can be fewer than 10(4) l(-1). We describe here an instrument designed for rapid counting, imaging and measuring of individual cells and particles in the microplankton size range from cultures and natural populations. It uses a unique optical element to extend the depth of focus of the imaging lens, allowing a sample stream flow rate of 1 ml min(-1) The instrument stores a digital image of each particle along with real time fluorescence and size measurements. An interactive cytogram links a dot-plot of the size and fluorescence data to the stored cell images, allowing rapid characterization of populations. We have tested the system on live phytoplankton cultures and bead standards, proving the system counting and sizing accuracy and precision. The system provides images and size distributions for cultures or natural marine samples. It has been used successfully at sea to continuously monitor particles while underway. It may prove useful in studies of plankton community structure, ocean optics and monitoring for harmful algal species.
Short-term variations of phytoplankton communities are poorly documented. To overcome these limitations and make observations
on a short-time (hours) scale, we moored a submersible flow cytometer (CytoBuoy b.v.) in the Bay of Marseille. The CytoSub
monitored phytoplankton every 30 min at a fixed site (2 m depth) during summer 2005. The data treatment, conducted on the
basis of pulse-shape analysis, resolved seven clusters. Daily sampling of nutrients and continuous information on salinity,
temperature and wind speed allowed distinction between diel cycles and the impact of environmental factors on phytoplankton
communities. Autocorrelation of the time series showed a significant periodicity of ∼24 h for most of the clusters during
undisturbed meteorological conditions. Two clusters had regular daily abundance variations in the range 0–>103 cells cm−3. Two strong wind events revealed similar cluster succession patterns occurring over several days after the wind events. These
results provided by the high frequency in situ analysis suggest that the flow cytometry resolved clusters, showing independent
behaviour and distinct environment-correlated variations, which may be considered as functional groups. We point out its potential
for global oceanic observing systems for which such systems could provide real biological information.
Measurements of the spectral scattering and attenuation properties of coccolithophores (Emiliania huxleyi; clone 88E) and their associated coccoliths were made for three growth phases as well as for acidified cultures. These measurements allow a clean separation and determination of the optical effects of the various components. The beam attenuation cross sections (m2 particle-1) were found to be 8.4E-12, 2.6E-10, and 4.9E-11 for coccoliths, plated cells, and naked cells, respectively, at 440 nm. The spectral dependence of these factors followed a power law dependence, with a wavelength exponent of -1.9, 0.42, and -0.52 for the coccoliths, plated cells, and naked cells. The volume scattering functions for all appeared similar; however, the backscattering cross sections (m2 particle-1) at 456 nm were 1.4E-13, 6.7E-12, and 9.9E-13, respectively. The wavelength dependence of this parameter also followed a power law and was -1.4, -1.2, and -1.0. Overall, these results show that optical properties of a coccolithophore bloom are sensitive to the coccolith:cell ratio and can vary between and within blooms.
Any technique developed to enumerate plankton must take into account the size structure of the plankton community. Automatic sampling devices must be capable of analysing a minimum number of cells of the largest size to cover the whole size range intended to be sampled effectively. The Flow Cytometer And Microscope (FlowCAM ®) has been used in the last decade to estimate the size structure of the plankton community. Few attempts, however, have been made to compare FlowCAM measurements with the results provided by traditional microscopy methods for size-structure estimations. FlowCAM can operate in three working modes: autoimage, fluorescence triggered and side-scatter triggered. Autoimage and fluorescence triggered cannot only count accurately a mono-specific suspension of cells, but they are also useful to estimate the size structure of natural samples. The side-scatter-triggered mode is not effective to estimate the size structure of natural samples, although it can count a sparse mono-specific solution accurately. The analysis of natural samples with FlowCAM requires a planned pre-processing of the samples to adjust the density of triggering particles (concentrating or diluting the sample) and to pre-filtrate the sample to avoid cell clumping or obstruction of the flow chamber. The size structure obtained with FlowCAM and with microscopy counts on preserved samples are comparable. Sample preservation, however, alters the size structure of the sample, which suggests that results based on preserved samples must be taken with caution. Automatic sampling devices like FlowCAM could provide a more precise analysis of plankton communities, increasing the resolution of surveys and avoiding the effects of preservation and sample storage. © The Author 2011. Published by Oxford University Press. All rights reserved.
Cellular carbon and nitrogen content and cell volume of nutritionally and morphologically diverse dinoflagellate species were measured to determine carbon to volume (C:vol) and nitrogen to volume (N:vol) relationships. Cellular C and N content ranged from 48 to 3.0 x 104 pgC cell-1 and 11 to 2,656 pgN cell-1 for cells ranging in volume from 180 to 2.8 X 105 μm3. C and N density in dinoflagellates decreased significantly with increasing cell volume. C:N ratios ranged from 3.44 to 6.45. C:vol and N:vol in dinoflagellates are significantly related as expressed by the equations pgC cell-1 = 0.760 X volume0.819 and pgN cell-1 = 0.118 X volume0.849. Previously published data were combined to compare C:vol relationships in different phylogenetic protist groups, including chlorophytes, chrysophytes, prasinophytes, and prymnesiophytes. Our analysis indicated differences between the C:vol relationships available for ciliates. A new C:vol relationship for diatoms was established (pgC cell-1 = 0.288 X volume0.811). Dinoflagellates are significantly more C dense than diatoms. Except for diatoms, we found few significant differences between C: vol relationships of different phylogenetic groups. Consequently, one C: vol relationship for taxonomically diverse protist plankton excluding diatoms was determined (pgC cell-1 = 0.216 x volume0.939). In the combined data set, carbon density was not constant but decreased significantly with increasing cell volume. Using constant C:vol conversion factors for plankton over large size ranges will cause systematic errors in biomass estimates.
The relationship between chlorophyll a and phytoplankton biomass (organic carbon content) is highly variable as is the yield of in vivo fluorescence per unit chlorophyll. Thus, vertical profiles of chlorophyll or in vivo fluorescence must be interpreted with caution if their ecological significance is to be established. Although the variability of carbon-to-chlorophyll ratios and fluorescence yield is large, much of it can be anticipated, corrected for, and usefully interpreted. Vertical profiles from different regions of the sea are presented; each has a deep chlorophyll maximum, but the probable mechanisms of their formation and maintenance differ widely. Most vertical distributions of chlorophyll can be explained by the interaction between hydrography and growth, behavior, or physiological adaptation of phytoplankton with no special consideration of grazing by herbivores, even though vertical distributions of epizooplankton are not uniform. The interaction between vertical profiles of zooplankton and chlorophyll will be better understood when the relationships between chlorophyll and phytoplankton biomass in those profiles is determined.Key words: chlorophyll a, fluorescence, phytoplankton, vertical structure
This paper offers a synoptic account of studies on the phytoplankton communities in the deep southern subalpine lakes (DSL)
Garda, Iseo, Como, Lugano and Maggiore. The main cause of the degradation of the water quality in the DSL is eutrophication.
The euphotic layers of these lakes are trophically different, ranging from the oligo-mesotrophy of lakes Maggiore and Garda
to the meso-eutrophy of lakes Iseo and Lugano. The trophic status as estimated by using total phosphorus and chlorophyll a has provided consistent results in agreement with the models proposed by OECD (1982. Eutrophication of Waters. Monitoring, Assessment and Control, OECD, Paris). Though related with chlorophyll a and TP, the Secchi disk depths have significantly underestimated the trophic status of the DSL. Two trophic indices using
the algal orders (PTIorders) and species (PTIspecies) were drawn up on the basis of the distribution of phytoplankton along a trophic gradient defined by the application of multivariate
methods; the scores emerging from these indices were used to make a definitive ecological classification of water bodies on
a scale from 1 to 5, in accordance with the Water Framework Directive. A third index (PTIOE) was computed as the ratio between the annual mean values of the cumulative biovolumes of two groups of algal orders with
opposite trophic characteristics. The three PTI indices were highly correlated, providing a consistent classification of the
water bodies. The indices proposed in this work were specifically adopted for use in the DSL. However, the criteria for their
implementation constitute a robust and impartial tool for assessing similar indices in other lake typologies and for evaluating
the degree of specificity of the trophic indicator values assigned to the single phytoplankton orders and species.
During the last several decades, harmful algal bloom (HAB) events have been observed in more locations than ever before throughout
the United States. Scientists have identified a larger number of algal species involved in HABs, more toxins have been uncovered,
and more fisheries resources have been affected. Whether this apparent increase in HAB events is a real phenomenon or is the
result of increased sampling and monitoring is a topic of intense discussions within the scientific community. We also have
an inchoate understanding of the reasons for the apparent increase, particularly concerning the role of anthropogenic nutrient
loadings as a causal factor. Whatever the reasons, virtually all coastal regions of the U.S. are now regarded as potentially
subject to a wide variety and increased frequency of HABs. It is important to begin to understand the scale of the economic
costs to society of such natural hazards. It is a common, but not yet widespread, practice for resource managers and scientists
in many localities to develop rough estimates of the economic effects of HAB events in terms of lost sales in the relevant
product or factor markets, expenditures for medical treatments, environmental monitoring and management budgets, or other
types of costs. These estimates may be invoked in policy debates, often without concern about how they were developed. Although
such estimates are not necessarily good measures of the true costs of HABs to society, they may help to measure the scale
of losses and be suggestive of their distribution across political jurisdictions or industry sectors. With adequate interpretation,
our thinking about appropriate policy responses may be guided by these estimates. Here we compile disparate estimates of the
economic effects of HABs for events in the U.S. where such effects were measured during 1987–1992. We consider effects of
four basic types: public health, commercial fisheries, recreation and tourism, and monitoring and management. We discuss many
of the issues surrounding the nature of these estimates, their relevance as measures of the social costs of natural hazards,
and their potential for comparability and aggregation into a national estimate.
Altered freshwater inflows have affected circulation, salinity, and water quality patterns of Florida Bay, in turn altering the structure and function of this estuary. Changes in water quality and salinity and associated loss of dense turtle grass and other submerged aquatic vegetation (SAV) in Florida Bay have created a condition in the bay where sediments and nutrients have been regularly disturbed, frequently causing large and dense phytoplankton blooms. These algal and cyanobacterial blooms in turn often cause further loss of more recently established SAV, exacerbating the conditions causing the blooms. Chlorophyll a (CHLA) was selected as an indicator of water quality because it is an indicator of phytoplankton biomass, with concentrations reflecting the integrated effect of many of the water quality factors that may be altered by restoration activities. Overall, we assessed the CHLA indicator as being (1) relevant and reflecting the state of the Florida Bay ecosystem, (2) sensitive to ecosystem drivers (stressors, especially nutrient loading), (3) feasible to monitor, and (4) scientifically defensible. Distinct zones within the bay were defined according to statistical and consensual information. Threshold levels of CHLA for each zone were defined using historical data and scientific consensus. A presentation template of condition of the bay using these thresholds is shown as an example of an outreach product.
Microalgal biovolume is commonly calculated to assess the relative abundance (as biomass or carbon) of co-occurring algae varying in shape and/or size. However, a standardized set of equations for biovolume calculations from microscopically measured linear dimensions that includes the entire range of microalgal shapes is not available yet. In comparison with automated methods, the use of microscopical measurements allows high taxonomic resolution, up to the species level, and has fewer sources of error. We present a set of geometric shapes and mathematical equations for calculating biovolumes of >850 pelagic and benthic marine and freshwater microalgal genera. The equations are designed to minimize the effort of microscopic measurement. The similarities and differences between our proposal for standardization and previously published proposals are discussed and recommendations for quality standards given.
Links between biodiversity and ecosystem function provide compelling reasons for conserving maximal numbers of species in ecosystems. Here we describe a previously unrecognized effect of biodiversity on ecosystem predictability, where predictability is inversely related to temporal and spatial variation in ecosystem properties. By manipulating biodiversity in aquatic microbial communities, we show that one process, ecosystem respiration, becomes more predictable as biodiversity increases. Analysis of similar patterns extracted from other studies indicates that biodiversity also enhances predictability in terrestrial ecosystems. Biodiversity can also affect average levels of ecosystem performance, but the extent to which different species make unique or redundant contributions to ecosystem processes remains controversial. Nonlinear effects of biodiversity on the decomposition of particulate organic matter and resistance of communities to invasion indicate that different species have redundant functions in our system. The consequences of biodiversity are also not restricted to early successional situations as described in previous studies, because strong effects persist even after ecosystems develop for periods corresponding to 40-80 generations of dominant organisms.
Phytoplankton biovolume can be measured or calculated through the calculation of similar geometric models. A set of geometric models is suggested for calculating cell biovolume and surface area for 284 phytoplankton genera in China Sea waters. Thirty-one geometric shapes have been assigned to estimate the biovolume and surface area of phytoplankton cells. Reductions of error and microscopic effort are also discussed. The model has been verified by its application in the China Seas regions. The software to make these calculations is available at http://www.ouc.edu.cn/csmxy/sunjun/biovolume.htm 10.1093/plankt/fbg096
All the important questions in counting phytoplankton are reviewed. The methods described refer primarily to fresh water but are applicable to marine phytoplankton as well. No attempt has been made to review the whole of the voluminous literature on counting technique or describe its development. The main aim is to describe the counting-chamber method. The numerous difficulties encountered in quantitative plankton research are discussed and ways of avoiding them are described together with improvements of technique that save time. Among the equipment described are the filling-chamber (Füllkammer) and the combination-chamber (Verbundkammer). A mixture of potassium iodide in iodine and sodium acetate is used for the preservation of phytoplankton. A new sort of fractionated counting designed to overcome a certain degree of unavoidably uneven distribution of the plankton sediments is described.
We utilized an extensive data set (1977–2013) from a water quality monitoring program to investigate the recovery of a Danish estuary following large reductions in total phosphorus (TP) and total nitrogen (TN) loading. Monthly rates of net transport and biogeochemical transformation of dissolved inorganic nitrogen (DIN) and phosphorus (DIP) were computed in two basins of the estuary using a box model approach, and oxygen-based rates of net ecosystem production (NEP) were determined. Since 1990, nutrient loading was reduced by 58 % for nitrogen and 80 % for phosphorus, causing significant decreases in DIN (60 %) and DIP (85 %) concentrations. Reductions in nutrient loadings and concentrations reduced annual chlorophyll levels by 50 % in the inner estuary and improved Secchi depth by approximately 1 m during the same period, particularly in the summer period. In the outer, deeper region of the estuary trends in water quality was less evident. Improvements in the inner estuary were strongly coupled to declines in DIN. Thresholds of DIN and DIP concentrations limiting phytoplankton growth indicated that both regions of the estuary were nitrogen limited. NEP rates indicated the development of more net autotrophic conditions over time that were likely associated with higher benthic primary production stimulated by improved light conditions. Box model computations revealed a modest reduction in summer net production of DIP over time, despite the persistence of elevated fluxes for several years after external loads were reduced. Since the mid-1990s, nutrient loading and transformation were stable while nutrient concentrations continued to decline and water quality improved in the inner estuary. The oligotrophication trajectory involved an initial fast transformation and modest retention of nutrients followed by a gradual decline in the rate of improvement towards a new stable condition.
We introduce a new design for the optical cuvette and a nem optical lay-out for the Scanning Flow Cytometer (SFC) that permits measurement of the angular dependency of the scattered Light from individual moving particles, The improved optical scheme of the SFC allows measurement of the angular scattering pattern of individual particles at polar angles from 10 degrees to 120 degrees with integration at azimuthal angles from 0 degrees to 360 degrees and with angular resolution of better than 0.5 degrees. The performance of the SFC is demonstrated using certified polystyrene particles as reference material, The aim of this work is to develop a flow cytometer, which, by recording the entire light scattering pattern of individual biological particles, would provide more information about the particle structure than the ordinary wide angle, forward and side scattering concepts. (C) 1998 Wiley-Liss, Inc.
Changes in oceanic primary production, linked to changes in the network of global biogeochemical cycles, have profoundly influenced
the geochemistry of Earth for over 3 billion years. In the contemporary ocean, photosynthetic carbon fixation by marine phytoplankton
leads to formation of ∼45 gigatons of organic carbon per annum, of which 16 gigatons are exported to the ocean interior. Changes
in the magnitude of total and export production can strongly influence atmospheric CO2 levels (and hence climate) on geological time scales, as well as set upper bounds for sustainable fisheries harvest. The
two fluxes are critically dependent on geophysical processes that determine mixed-layer depth, nutrient fluxes to and within
the ocean, and food-web structure. Because the average turnover time of phytoplankton carbon in the ocean is on the order
of a week or less, total and export production are extremely sensitive to external forcing and consequently are seldom in
steady state. Elucidating the biogeochemical controls and feedbacks on primary production is essential to understanding how
oceanic biota responded to and affected natural climatic variability in the geological past, and will respond to anthropogenically
influenced changes in coming decades. One of the most crucial feedbacks results from changes in radiative forcing on the hydrological
cycle, which influences the aeolian iron flux and, in turn, affects nitrogen fixation and primary production in the oceans.
Layers and patches of phytoplankton at sub-meter scales in the vertical dimension and kilometer scales in horizontal dimensions are common features in the coastal ocean. These heterogeneous distributions of cells are fundamental to their population dynamics and the function of pelagic ecosystems. To better understand biological processes at these small scales, methods were developed to assess phytoplankton community composition and physiological characteristics based on high-resolution, in situ optical measurements. Scanning flow cytometry of discrete samples was used to determine the effects of phytoplankton and non-algal particle abundance, size, and pigment content on the spectral shape and relative magnitude of particulate attenuation, absorption, scatter, and backscatter coefficients. The slope of particulate attenuation varied with phytoplankton size and morphology, the slope of particulate absorption and the ratio of scatter to absorption varied primarily with cellular pigment content, and the backscatter ratio varied primarily with the relative abundance of non-algal particles. Determination of particle and phytoplankton characteristics from optical measurements over small spatial and temporal scales was tested with 2 independent high-resolution data sets collected from an in situ autonomous profiling system. Comparison of these high-resolution optical data with flow cytometric sample analyses generally agreed with the previously determined relationships but suggest that complex morpho-logy of large colonial diatoms may result in higher than expected particulate attenuation slopes. High-resolution data revealed variations in community size structure and physiology that would be difficult to visualize with discrete samples or measures of total chlorophyll concentration.
Measurements of the spectral scattering and attenuation properties of coccolithophores (Emiliania huxleyi; clone 88E) and their associated coccoliths were made for three growth phases as well as for acidified cultures. These measurements allow a clean separation and determination of the optical effects of the various components. The beam attenuation cross sections (m² particle⁻¹) were found to be 8.4E-12, 2.6E-10, and 4.9E-11 for coccoliths, plated cells, and naked cells, respectively, at 440 nm. The spectral dependence of these factors followed a power law dependence, with a wavelength exponent of −1.9, 0.42, and −0.52 for the coccoliths, plated cells, and naked cells. The volume scattering functions for all appeared similar; however, the backscattering cross sections (m² particle⁻¹) at 456 nm were 1.4E-13, 6.7E-12, and 9.9E-13, respectively. The wavelength dependence of this parameter also followed a power law and was −1.4, −1.2, and −1.0. Overall, these results show that optical properties of a coccolithophore bloom are sensitive to the coccolith:cell ratio and can vary between and within blooms.
Understanding of how coastal plankton communities are regulated has traditionally been limited by undersampling, but cabled observatories now provide opportunities to deploy submersible sensors that have high power and data transmission requirements. We have developed an in situ instrument to carry out high-resolution, long term monitoring of phytoplankton and microzooplankton in the size range 10 to100 micrometers, to be deployed at cabled research facilities such as the Martha's Vineyard Coastal Observatory (MVCO). The new instrument is designed to complement FlowCytobot, a submersible flow cytometer currently deployed at MVCO that uses fluorescence and light scattering signals from a laser beam to characterize the smallest phytoplankton cells (less than 10 micrometers). Imaging FlowCytobot uses a combination of flow cytometric and video technology to capture images of organisms for identification and to measure chlorophyll fluorescence associated with each image. Images will be classified using neural net software, while the measurements of chlorophyll fluorescence will allow us to discriminate heterotrophic from phototrophic cells. The new instrument, like the original FlowCytobot is autonomous but remotely programmable. It utilizes a computer controlled syringe pump and distribution valve that allows periodic anti-fouling treatment and analysis of standard beads. Samples are analyzed continuously (0.25 to 2.5 ml per min) and data is sent over a fiber optic link to a remote computer for analysis. Preliminary results indicate that we can detect cells as small as 5 micrometers and discriminate several taxa of diatoms and dinoflagellates.
This article is essentially a review of the temporal and spatial scales of variability in both marine and freshwater planktonic environments and the algal responses to those scales. I assert that there are problems with our present understanding of these scales and the use of inappropriate assumptions concerning the occurrence of steady-state conditions. In a nonsteady-state environment the concepts of limiting nutrients must be changed, and the extrapolation from culture to field conditions is fraught with problems. In this paper I review the evidence for the existence and importance of small-scale, high frequency and large-scale, low frequency variation in the planktonic environment and show that such variation fundamentally affects our understanding of existing processes. Methodology and models must also reflect the true scales of variability which exist. I show that there are, at present, problems with our understanding of planktonic processes which greatly affect our ability to manage water quality. New concepts and models are urgently needed. Finally I propose a new model of community structure and process in variable environments which accounts for the correct 'algal' scales of perturbation and response and allows certain predictions to be made. It is possible to reconcile certain problems and controversies in the literature by the use of such a model. An enhanced ability to manage planktonic systems should result from an improved understanding of the true scales of variability which exist.Key words: lakes, oceans, phytoplankton, communities, nutrients, models, management, eutrophication, fluctuations, scales
The fluorescence excitation spectrum is sometimes used as a proxy for the action spectrum of photosynthesis in phytoplankton. The main assumption behind this approximation is that the shapes of absorption and fluorescence excitation spectra are similar except for the absorption by photoprotective pigments, which do not contribute to the fluorescence spectrum. In this study, we compare the shapes of the absorption and fluorescence spectra in three species of phytoplankton grown at different irradiances: two diatoms (Thalassiosira weissflogii and Chaetoceros sp.) and a cyanophyte (Synechococcus sp.). The contribution to absorption by photoprotective pigments was estimated for each experiment. Results showed that the differences between the shapes of absorption and fluorescence spectra were similar to the estimated absorption by photoprotective pigments only in the case of T. weissflogii. In Synechococcus sp., and to a lesser degree in Chaetoceros sp., the differences between the two types of spectra were larger than the absorption by photoprotective pigments. In the case of Synechococcus sp., the difference between these spectra was apparently due mainly to the extreme imbalance of chlorophyll a distribution between the two photosystems. Chaetoceros sp. seemed to be an intermediate case: a small part of the chlorophyll a of the cell appeared to be exclusively associated with photosystem I and therefore did not contribute to fluorescence. Fluorescence and absorption values were normalized to their values at 545 nm, and the ratio of normalized absorption to normalized fluorescence was computed for the blue (439 nm) and red (676 nm) peaks in the spectra. The results showed that these peak ratios can be used to distinguish between the effects of photoprotective pigments and the arrangement of the photosynthetic apparatus on differences between fluorescence and absorption spectra.
The relationships between photoadaptation, photoacclimation, cell size and the optical characteristics of the phytoplankton community were studied in 2 areas of the Atlantic Ocean. Pigment composition, absorption and fluorescence excitation spectra were analyzed for samples collected during 2 spring cruises: one in the Labrador Sea and the other in the Central North Atlantic. Photoadaptation (i.e. evolutionary adaptation of different species leading to acquisition of different pigment composition) was evident in the distribution of the main phytoplankton pigments in the area. Photoacclimation (i.e. temporary changes in pigment concentrations in a given species) was also noticeable in the changes in pigment composition and optical characteristics of phytoplankton with changes in depth. Size fractionation of samples from the depth of the chlorophyll a (chl a) maximum showed that, on average, chl a concentration and the values of absorption and fluorescence were dominated by the > 2 mum fraction of phytoplankton. Spectral variations in absorption and fluorescence excitation were, however, similar for the small and for the large size fractions. A distinct regional distribution of algal groups was observed, in which prokaryotic picophytoplankton (size class < 2 mum) dominated in oligotrophic subtropical regions and larger cells (size class > 2 mum) dominated in coastal areas and at higher latitudes. Some significant relationships were observed between the relative abundances of pigments characteristic of certain algal groups and optical properties of the sample. For example, the ratio of absorption at 440 nm to that at 676 nm was positively correlated with the ratio of zeaxanthin to chl a, indicating high abundance of picophytoplankton, especially the cyanobacteria Synechococcus and Prochlorococcus. The ratio of absorption at 555 nm to that at 623 nm was positively correlated with the abundance of phycoerythrin-containing Synechococcus. These results show that useful information about phytoplankton group composition and photoacclimation state can be retrieved from optical properties at a regional scale.
We describe a database of the single-particle optical properties of marine microbial particles. This database includes representatives from five classes of particles: viruses (VIR), heterotrophic bacteria (BAC), cyanobacteria (CYA), small nanoplanktonic diatoms (DIA), and nanoplanktonic chlorophytes (CHLO). The optical properties of VIR, whose mean size is 0.07 μm, were determined from Mie scattering calculations using reasonable approximations about the size distribution and refractive index of vital particles. The database for BAC, CYA, DIA, and CHLO was created from laboratory measurements of microbial cultures and modeling of particle optics. BAC are represented by a mixed natural population of bacterial species (~ 0.55 μm in size), CYA by Synechococcus (clone WH 8103, ~ 1 μm), DIA by Thalassiosira pseudonana (~ 4 μm), and CHLO by Dunaliella tertiolecta (~ 7.5 μm). The database includes the single-particle optical properties that are useful in radiative transfer modeling: the absorption and scattering cross sections and scattering phase functions. Additionally, the database includes the attenuation cross sections, optical efficiency factors, single-scattering albedos, and backscattering properties. For phytoplankton species, chlorophyll- and carbon-specific optical coefficients are also available. The optical quantities are generally determined at 1-nm intervals in the spectral region from 350 to 750 nm. The scattering phase function is determined at 5-nm intervals in wavelength and 1°intervals in scattering angle. The size distribution and refractive index of the particles are also included. This database, when combined with radiative transfer modeling, provides a powerful approach to advancing our understanding of oceanic optics.
The size of 30 small (2-60 pm) phytoplankton species was examined with a microscope and a Coulter Counter before and after fixation. Acid Lugol's iodine caused cells to shrink immediately. The shrinkage effect was constant for concentrations of l-10% Lugol's iodine (in seawater). For optically measured cells fixed in 2% Lugol's iodine, volume of live cells = 1.33 x (volume of fixed cells). Coulter Counter and optically measured volumes did not agree. For live cells, optical cell volume = 1.24-2.04 x (Coulter Counter determined volume); this difference is likely due to inaccurate volume measurements of non- spherical cells by the Coulter Counter and by inaccurate microscopy resulting from optical distortions (errors of ~0.5 pm in cell dimensions). Cell quota estimates were presented following the relation y = a.x?, where x = optically measured cell volume (pm3), y = any cell constituent (pg cell-'), and a and b are constants. The constants a and b were 0.109 and 0.99 1 for carbon, 0.0172 and 1.023 for nitrogen, 0.043 and 1.058 for protein, and 0.00428 and 0.9 17 for Chl a. Our relation of carbon to volume differs from other literature values, in which there is no consensus. Our data can be used to determine carbon, nitrogen, protein, and Chl a estimates from field material that has been fixed with Lugol's iodine, observed live, optically measured, or Coulter Counter measured; however, the variability in published data suggests that any of these estimates will have a large potential error.
Monitoring programs for harmful algal blooms (HABs) typically rely on time-consuming manual methods for identification and enumeration of phytoplankton, which make it difficult to obtain results with sufficient temporal resolution for early warning. Continuous automated imaging-in-flow by the Imaging FlowCytobot (IFCB) deployed at Port Aransas, TX has provided early warnings of six HAB events. Here we describe the progress in automating this early warning system for blooms of Karenia brevis. In 2009, manual inspection of IFCB images in mid-August 2009 provided early warning for a Karenia bloom that developed in mid-September. Images from 2009 were used to develop an automated classifier that was employed in 2011. Successful implementation of automated file downloading, processing and image classification allowed results to be available within 4 h after collection and to be sent to state agency representatives by email for early warning of HABs. No human illness (neurotoxic shellfish poisoning) has resulted from these events. In contrast to the common assumption that Karenia blooms are near monospecific, post-bloom analysis of the time series revealed that Karenia cells comprised at most 60-75 % of the total microplankton.
The latitudinal distributions of picoeukaryote phytoplankton (PEUK), coccolithophores (COCCO), cryptophytes (CRYPTO) and other nanoeukaryote phytoplankton (NEUK) were studied in the Atlantic Ocean between 49°N and 46°S in September–October 2003 and April–June 2004 by flow cytometry. Phytoplankton abundance and carbon (C) biomass varied considerably with latitude and down through the water column. Abundance and C biomass of all eukaryotic groups studied were highest in North and South Atlantic temperate waters and in the Mauritanian Upwelling off the west coast of Africa, where the total C biomass of eukaryotic phytoplankton smaller than 10 μm reached almost 150 mg C m−3. Phytoplankton in the Equatorial Upwelling region was concentrated well below the surface at 50–80 m, with total C biomass in this layer being approximately 4 times that in the mixed layer. The North and South Atlantic Gyres supported much lower eukaryotic phytoplankton C biomass, with total eukaryote C biomass only reaching 2–3 mg C m−3, peaking below 100 m. Of the four eukaryote groups studied, the PEUK were the most abundant, reaching densities of up to 40,000 cells cm−3. They often contributed between 25% and 60% of total C biomass, particularly in the deep chlorophyll maxima of the different oceanic regions and also in the South Atlantic temperate waters, both in austral spring and autumn. NEUK also contributed significantly to C biomass. They generally dominated in the mixed layer, where they contributed 65–85% of total C biomass in the subtropical gyres and in North Atlantic temperate waters. CRYPTO and COCCO were generally less abundant. CRYPTO attained highest abundance in the Southern Temperate waters of over 500 cells cm−3 on both cruises. COCCO were often undetectable but on the European continental shelf abundance reached up to 2600 cells cm−3 during AMT 14. The C biomass standing stock of eukaryotic phytoplankton (<10 μm) for the Atlantic Ocean as a whole was estimated to be 80 million tonnes C during AMT 13, approximately one-third of total phytoplankton C biomass in the Atlantic Ocean.
Imaging FlowCytobot (IFCB) combines video and flow cytometric technology to capture images of nano- and microplankton (∼10 to >100 μm) and to measure the chlorophyll fluorescence associated with each image. The images are of sufficient resolution to identify many organisms to genus or even species level. IFCB has provided >200 million images since its installation at the entrance to the Mission-Aransas estuary (Port Aransas, TX, USA) in September 2007. In early February 2008, Dinophysis cells (1–5 · mL−1) were detected by manual inspection of images; by late February, abundance estimates exceeded 200 cells · mL−1. Manual microscopy of water samples from the site confirmed that D. cf. ovum F. Schütt was the dominant species, with cell concentrations similar to those calculated from IFCB data, and toxin analyses showed that okadaic acid was present, which led to closing of shellfish harvesting. Analysis of the time series using automated image classification (extraction of image features and supervised machine learning algorithms) revealed a dynamic phytoplankton community composition. Before the Dinophysis bloom, Myrionecta rubra (a prey item of Dinophysis) was observed, and another potentially toxic dinoflagellate, Prorocentrum, was observed after the bloom. Dinophysis cell-division rates, as estimated from the frequency of dividing cells, were the highest at the beginning of the bloom. Considered on a daily basis, cell concentration increased roughly exponentially up to the bloom peak, but closer inspection revealed that the increases generally occurred when the direction of water flow was into the estuary, suggesting the source of the bloom was offshore.
The cellular fluorescence of chlorophyll a in natural phytoplankton was measured during vertical profiling in marine coastal waters. The ratio of in situ fluorescence to chlorophyll a concentration, which was considered as an index of cellular fluorescence, varied over a wide range, with large changes occurring both within the water column and between profiling sites. The variations were caused in part by an inhibition in the fluorescence of cells exposed to intense sunlight. The inhibition, which occurred at irradiances exceeding 0.15 langley (ly)/min, led to diel fluctuations in the fluorescence of those phytoplankton near the sea surface. The remaining variations were independent of changes in temperature, but were unexplained. Both light-dependent and light-independent variations in cellular fluorescence will affect the accuracy of the continuous, fluorometric measurement of in vivo chlorophyll.
Based on an 18-year data base (1984–2002), seasonal (spring, summer) phytoplankton relationships to specific environmental determinants were identified within different salinity regions of Chesapeake Bay. Growth conditions in these areas were identified as either less favorable (Impaired) or favorable (Least Impaired) for phytoplankton development. Diatoms represented the greatest cellular abundance and biomass during spring in different salinity regions and water quality conditions. In contrast, the dominant summer floral biomass was produced by a combination of diatoms, chlorophytes, and cyanobacteria in tidal freshwater and oligohaline waters, with diatoms and dinoflagellates representing the major algal biomass in mesohaline and polyhaline regions. Chlorophyte and cyanobacteria abundance and biomass decreased with the increasing salinities of the mesohaline and polyhaline regions, in contrast to increased biomass and abundance by dinoflagellates and diatoms. The common background taxa and an additional biomass source throughout these seasons were cryptophytes. Increased summer cyanobacteria abundance and biomass in the Impaired water of the tidal fresh and oligohaline regions were associated with reduced light availability and higher nutrient concentrations. The summer diatoms and dinoflagellates had increased mean cell sizes in the Least Impaired mesohaline and polyhaline waters compared to their populations in Impaired regions. This relationship was enhanced by increased abundance of neritic diatoms and dinoflagellates entering the Bay from Atlantic coastal waters. The data suggested a general reduction of existing nutrient levels and improved light availability in the Impaired waters would still continue the dominance of diatom flora over any additional cyanobacteria development.