Optical plankton analyser: a flow cytometer for plankton analysis, II: Specifications

An analysing flow cytometer, the optical plankton analyser (OPA), is presented. The instrument is designed for phytoplankton analysis, having a sensitivity comparable with commercially available flow cytometers, but a significantly extended particle size range. Particles of 500 microns in width and over 1,000 microns in length can be analysed. Sample flow rates of up to 55 microliters/s can be used. Also, the dynamic range of the instrument is significantly increased for particles larger than about 5 microns. The optics, hydraulics, and electronics of the instrument are described, including the best form for a low fluid shear cuvette. The new pulse quantification technique we call digital integration is presented. This technique is essential for the instrument to handle both short and very long particles with a large dynamic range. Test measurements demonstrating particle size range and dynamic range are presented. Dynamic ranges of 10,000 and 100,000 were typically observed, measuring field samples with Microcystis aeruginosa colonies, whereas one sample showed a dynamic range of 10(6). A simple method for interpretation of time of flight (TOF) data in terms of particle morphology is presented. The specifications of the instrument are given.     

Patchy agglomeration as a transition from monospecies to recurrent plankton blooms

We propose a model for explaining both red tides and recurring phytoplankton blooms. Three assumptions are made, namely the presence of toxin producing phytoplankton, the satiation phenomenon in zooplankton's feeding, modelled by a Holling type II response, and phytoplankton aggregation leading to formation of patches. The dynamics of the plankton population is shown to depend on the fraction of the phytoplankton population that aggregates to form colonies and on the number of the latter.     

Syringe pumped high speed flow cytometry of oceanic phytoplankton.

BACKGROUND: Nanophytoplankton (2-20 microm) are less numerous than picophytoplankton (<2 microm) in the oceans but their biomass and production are comparable and sometimes higher. The accuracy of cytometry-based enumeration of phytoplankton ultimately depends on cell abundance and sample flow rate. Commercial flow cytometers in which sheath and core streams are driven by air pressure cannot produce sufficiently high, stable sample flow rate. The present study demonstrates the applicability of a syringe pump for flow cytometric enumeration of oceanic nanophytoplankton on two meridional transects across the Atlantic Ocean. METHODS: Commercially available syringe pumps were used to deliver live phytoplankton samples into a flow cell of standard flow cytometers (FACSort, FACSCalibur, BD) with increased flow rate of > 1.0 ml min(-) (1) compared to the normal air pressure sample delivery of < 0.1 ml min(-) (1). An auxiliary application of syringe pump flow cytometry for calibrating 0.5 microm bead concentration standards is also discussed. RESULTS: The results demonstrated that flow cytometry of samples injected at rates above 0.1 ml min(-) (1) is achievable and worthwhile. Counts of phytoplankton in air and syringe pumped samples agreed closely. Syringe pumping of samples offered a broader range of flow rates up to 0.8-1.0 ml min(-) (1) without detrimental effect on flow cytometric enumeration of cells. The increased number of coincidences at high flow rates led to an approximate 10% decrease of Cyanobacteria counts when the acquisition rate approached 1,000 particles s(-) (1), but seemed to have a lesser effect on counting rarer phytoplankton. The syringe pump flow cytometry allowed enumeration of phytoplankton groups at concentrations of 5-100 cells ml(-) (1), cell concentrations equivalent to those of Cyanobacteria in the twilight deep ocean. CONCLUSION: The proposed syringe pump modification of a FACS instrument represents a significant improvement for accurate enumeration of the less abundant phytoplankton and so gives better estimations of phytoplankton distribution and standing stocks.     

From mice to elephants: overturning the ‘one size fits all’ paradigm in marine plankton food chains

It is widely believed that consumer control is a weak regulator of marine phytoplankton communities. It remains unclear, however, why this should be the case when marine consumers routinely regulate their prey at higher trophic levels. One possibility is that the weak consumer control of phytoplankton communities results from the inability of field researchers to effectively account for consumer–prey trophic relationships operating at the scale of the plankton. We explored this issue by reviewing studies of trophic control in marine plankton. Experimental studies indicate that size is a critical determinant of feeding relationships among plankton. In sharp contrast, of the 51 field studies reviewed, 78% did not distinguish among the sizes or species of phytoplankton and their consumers, but instead assumed a general bulk phytoplankton–zooplankton trophic connection. Such an approach neglects the possibility that several trophic connections may separate the smallest phytoplankton (0.2 μm) from the larger zooplankton (~ 1000 μm), a remarkable size differential exceeding that between a mouse (~10 cm) and an elephant (~2500 cm). The size‐based approach we propose integrates theory, experiments and field observations and has the potential to greatly enhance our understanding of the causes and consequences of recently documented restructuring of plankton communities.     

Flow cytometric characterization of marine microbes

The analysis of marine phytoplankton using flow cytometry has enabled the discovery of new taxa and has contributed new understanding to the dynamics and ecological contributions of phytoplankton to the global carbon cycle. Marine phytoplankton are uniquely suited to analysis by flow cytometry because of their size, pigment content, and ability to remain in suspension. Cytometric analysis of marine populations is not without challenges. Phytoplankton communities span a broad range of sizes. The smallest microbes are a few tenths of a micron, while the largest are a few tenths of a millimeter. The improvement of cytometric measurements of scattered laser light allows one to investigate marine microbes whose sizes span several orders of magnitude. To effectively leverage the advantages that marine microbes possess, cytometers have to be carefully engineered for marine use.     

The effects of abrupt topography on plankton dynamics

Plankton population dynamics in the upper layer of the ocean depends on upwelling processes that bring nutrients from deeper waters. In turn, these depend on the structure of the vertical velocity field. In coastal areas and in oceanic regions characterized by the presence of strong submarine topographic features, the variable bottom topography induces significant effects on vertical velocities and upwelling/downwelling patterns. As a consequence, large plankton and fish abundances are frequently observed above seamounts, canyons and steep continental shelves. In this work, the dynamics of an NPZ (nutrient-phytoplankton-zooplankton) system is numerically studied by coupling the ecosystem model with a quasi two-dimensional (2D) fluid model with topography. At variance with classical 2D approaches, this formulation allows for an explicit expression of the vertical motions produced when fluid columns are squeezed and stretched as they experience changes of depth. Thus, input or output of nutrients at the surface are associated with fluid motion over the bottom topography. We examine the dynamics of a cyclonic vortex over two basic topographies: a steep escarpment and a submarine mountain. We show that plankton abundance over the escarpment is modulated by the passing of topographic Rossby waves, generated by the vortex-topography interaction. In such configuration, advection effects driven by the flow over the escarpment are of limited relevance for the dynamics of biological fields. By contrast, we find that the flow resulting from the interaction of a vortex with a seamount is sufficiently strong and persistent to allow for a remarkable increase of nutrients, and a corresponding enhancement of phytoplankton and zooplankton concentrations. Over the seamount, advection effects associated with trapped flow perturbations around the summit play an essential role.     

Extracellular phosphatase activity of natural plankton studied with ELF97 phosphate: fluorescence quantification and labelling kinetics

ELF(R)97 phosphate (ELFP) is a phosphatase substrate which produces ELF(R)97 alcohol (ELFA), a fluorescent water-insoluble product, upon hydrolysis. We studied the kinetics of ELFA precipitation in freshwater samples at levels of total plankton and single phytoplankton cells, and tested the suitability of ELFP for measurement of surface-bound algal extracellular phosphatases. Samples from acidic Plesné Lake (pH approximately 5; high phosphatase activity) and eutrophic Rímov reservoir (pH approximately 7-10; moderate phosphatase activity) were incubated with ELFP for 5-300 min, fixed with HgCl2 and filtered through polycarbonate filters. Relative fluorescence of filter-retained ELFA precipitates was quantified with image analysis. Time-courses of ELFA formation exhibited lag periods followed by finite periods of linear increase. In Plesné Lake, lag-times were shorter (1-18 min) and rates of increase in ELFA fluorescence higher (by approximately 2 orders of magnitude) than in Rímov reservoir (lag-times 30-200 min). Similar patterns of ELFA formation kinetics were also observed in Plesné Lake samples in cuvette spectrofluorometer measurements (which failed in Rímov reservoir). Linear regression of seasonal data on rates of increase in ELFA fluorescence from image cytometry and spectrofluorometry (r2 = 0.65, n = 10) allowed for calibration of image cytometry in terms of amount of cell-associated ELFA. Preliminary measurements of extracellular phosphatase activities of several algae resulted in rates (10-2260 fmol cell-1 h-1) which are comparable to data reported in the literature for algal cultures.     

The effects of abrupt topography on plankton dynamics

Plankton population dynamics in the upper layer of the ocean depends on upwelling processes that bring nutrients from deeper waters. In turn, these depend on the structure of the vertical velocity field. In coastal areas and in oceanic regions characterized by the presence of strong submarine topographic features, the variable bottom topography induces significant effects on vertical velocities and upwelling/downwelling patterns. As a consequence, large plankton and fish abundances are frequently observed above seamounts, canyons and steep continental shelves. In this work, the dynamics of an NPZ (nutrient-phytoplankton-zooplankton) system is numerically studied by coupling the ecosystem model with a quasi two-dimensional (2D) fluid model with topography. At variance with classical 2D approaches, this formulation allows for an explicit expression of the vertical motions produced when fluid columns are squeezed and stretched as they experience changes of depth. Thus, input or output of nutrients at the surface are associated with fluid motion over the bottom topography. We examine the dynamics of a cyclonic vortex over two basic topographies: a steep escarpment and a submarine mountain. We show that plankton abundance over the escarpment is modulated by the passing of topographic Rossby waves, generated by the vortex-topography interaction. In such configuration, advection effects driven by the flow over the escarpment are of limited relevance for the dynamics of biological fields. By contrast, we find that the flow resulting from the interaction of a vortex with a seamount is sufficiently strong and persistent to allow for a remarkable increase of nutrients, and a corresponding enhancement of phytoplankton and zooplankton concentrations. Over the seamount, advection effects associated with trapped flow perturbations around the summit play an essential role.     

A field portable gas-exchange system for measuring carbon dioxide and water vapour exchange rates of leaves during fumigation with SO2

Abstract A field portable, steady-state gas-exchange system which measures both CO₂ and water vapour exchange of single intact leaves during fumigations with SO₂ is described. Within the leaf cuvette temperature, light, humidity and both CO₂ and SO₂ concentrations are controlled to preset levels. Gas flow and concentrations are controlled by mass flow controllers. Photosynthetic uptake of CO₂ can be determined either by differential depletion or null balance measurement. Water vapour exchange is measured differentially and transpiration and conductance to water vapour determined. Sulphur dioxide is measured directly within the cuvette exhaust gas line by UV-pulse fluorescence. The performance of this system under field conditions is described and the physiological measurements compared with those obtained with other systems.     

Simulated seasonal marine mining impacts on plankton

A computer simulated model is presented which predicts changes in phytoplankton populations from six-week mining operations conducted at different times of the year. Barely perceptable changes are predicted from the six-week May mining operation, while similar operations at other times of the year were predicted to have variable and sometimes long term impacts on the phytoplankton dynamics.     

Noninvasive monitoring of adrenocortical function in captive jaguars (Panthera onca)

Jaguars are threatened with extinction throughout their range. A sustainable captive population can serve as a hedge against extinction, but only if they are healthy and reproduce. Understanding how jaguars respond to stressors may help improve the captive environment and enhance their wellbeing. Thus, our objectives were to: (1) conduct an adrenocorticotrophic hormone (ACTH) challenge to validate a cortisol radioimmunoassay (RIA) for noninvasive monitoring of adrenocortical function in jaguars; (2) investigate the relationship between fecal corticoid (FCM) and androgen metabolite (FAM) concentrations in males during the ACTH challenge; and (3) establish a range of physiological concentrations of FCMs for the proposed protocol. Seven jaguars (3 M, 4 F) received 500 IU/animal of ACTH. Pre‐ and post‐ACTH fecal samples were assayed for corticoid (M and F) and androgen metabolites (M) by RIA. Concentrations of FCMs increased (P80.01) after ACTH injection (pre‐ACTH: 0.90 ± 0.12 µg/g dry feces; post‐ACTH: 2.55 ± 0.25 µg/g). Considering pre‐ and post‐ACTH samples, FCM concentrations were higher (P80.01) in males (2.15 ± 0.20 µg/g) than in females (1.30 ± 0.20 µg/g), but the magnitude of the response to ACTH was comparable (P>0.05) between genders. After ACTH injection, FAMs increased in two (of 3) males; in one male, FCMs and FAMs were positively correlated (0.60; P80.01). Excretion of FCMs was assessed in 16 jaguars (7 M, 9 F) and found to be highly variable (range, 80.11–1.56 µg/g). In conclusion, this study presents a cortisol RIA for monitoring adrenocortical function in jaguars noninvasively. Zoo Biol 31:426–441, 2012. © 2011 Wiley Periodicals, Inc.     

Water temperature and stratification depth independently shift cardinal events during plankton spring succession

In deep temperate lakes, the beginning of the growing season is triggered by thermal stratification, which alleviates light limitation of planktonic producers in the surface layer and prevents heat loss to deeper strata. The sequence of subsequent phenological events (phytoplankton spring bloom, grazer peak, clearwater phase) results in part from coupled phytoplankton–grazer interactions. Disentangling the separate, direct effects of correlated climatic drivers (stratification‐dependent underwater light climate vs. water temperature) from their indirect effects mediated through trophic feedbacks is impossible using observational field data, which challenges our understanding of global warming effects on seasonal plankton dynamics. We therefore manipulated water temperature and stratification depth independently in experimental field mesocosms containing ambient microplankton and inocula of the resident grazer Daphnia hyalina. Higher light availability in shallower surface layers accelerated primary production, warming accelerated consumption and growth of Daphnia, and both factors speeded up successional dynamics driven by trophic feedbacks. Specifically, phytoplankton peaked and decreased earlier and Daphnia populations increased and peaked earlier at both shallower stratification and higher temperature. The timing of ciliate dynamics was unrelated to both factors. Volumetric peak densities of phytoplankton, ciliates and Daphnia in the surface layer were also unaffected by temperature but declined with stratification depth in parallel with light availability. The latter relationship vanished, however, when population sizes were integrated over the entire water column. Overall our results suggest that, integrated over the entire water column of a deep lake, surface warming and shallower stratification independently speed up spring successional events, whereas the magnitudes of phytoplankton and zooplankton spring peaks are less sensitive to these factors. Therefore, accelerated dynamics under warming need not lead to a trophic mismatch (given similar grazer inocula at the time of stratification). We emphasize that entire water column dynamics must be studied to estimate global warming effects on lake ecosystems.     

A computerized plankton counter

A portable computerized plankton counting system is presented, which has the potential to enumerate 255 different species per sample. The software is used to count phytoplankton samples and produce summary data. The hardware allows the results to be reproduced in the form of a hardcopy, stored on a cassette tape, or transferred to a larger computer for analysis.     

Rapid and straightforward estimates of photosynthetic characteristics using a portable gas exchange system

Procedures are described for estimating photosynthetic characteristics using a portable infra-red gas analysis (IRGA) system. Once the effects of stomatal limitation on CO2 assimilation have been established, up to ten parameters of photosynthesis can be estimated for a single leaf within 2 h, including: photosynthetic efficiency and capacity on both photon and CO2 bases; compensation irradiances and CO2 compensation concentrations; and light and dark respiration rates. These measurements can be made in the laboratory, glasshouse or field with relative ease. Methods for obtaining near instantaneous ("snapshot") measurements of leaf photosynthesis are also described, using carefully pre-set conditions within the leaf cuvette. Representative results are shown for Phaseolus vulgaris L. Important aspects of the procedure's experimental design, assumptions made in the analysis, and limitations of this approach are analysed. This revised version was published online in June 2006 with corrections to the Cover Date.     

A low volume 3D-printed temperature-controllable cuvette for UV visible spectroscopy

We report the fabrication of a 3D-printed water-heated cuvette that fits into a standard UV visible spectrophotometer. Full 3D-printable designs are provided and 3D-printing conditions have been optimised to provide options to print the cuvette in either acrylonitrile butadiene styrene or polylactic acid polymers, extending the range of solvents that are compatible with the design. We demonstrate the efficacy of the cuvette by determining the critical micelle concentration of sodium dodecyl sulphate at 40 °C, the molar extinction coefficients of cobalt nitrate and dsDNA and by reproducing the thermochromic UV visible spectrum of a mixture of cobalt chloride, water and propan-2-ol.     

Rapid and straightforward estimates of photosynthetic characteristics using a portable gas exchange system

Procedures are described for estimating photosynthetic characteristics using a portable infra-red gas analysis (IRGA) system. Once the effects of stomatal limitation on CO2 assimilation have been established, up to ten parameters of photosynthesis can be estimated for a single leaf within 2 h, including: photosynthetic efficiency and capacity on both photon and CO2 bases; compensation irradiances and CO2 compensation concentrations; and light and dark respiration rates. These measurements can be made in the laboratory, glasshouse or field with relative ease. Methods for obtaining near instantaneous ("snapshot") measurements of leaf photosynthesis are also described, using carefully pre-set conditions within the leaf cuvette. Representative results are shown for Phaseolus vulgaris L. Important aspects of the procedure's experimental design, assumptions made in the analysis, and limitations of this approach are analysed.     

Design and first results of CytoBuoy: a wireless flow cytometer for in situ analysis of marine and fresh waters.

BACKGROUND:The high costs of microscopical determination and counting of phytoplankton often limit sampling frequencies below an acceptable level for the monitoring of dynamic ecosystems. Although having a limited discrimination power, flow cytometry allows the analysis of large numbers of samples to a level that is sufficient for many basic monitoring jobs. For this purpose, flow cytometers should not be restricted to research laboratories. We report here on the development of an in situ flow cytometer for autonomous operation inside a small moored buoy or on other platforms. METHODS AND RESULTS: Operational specifications served a wide range of applications in the aquatic field. Specific conditions had to be met with respect to the operation platform and autonomy. A small, battery-operated flow cytometer resulted, requiring no external sheath fluid supply. Because it was designed to operate in a buoy, we call it CytoBuoy. Sampling, analysis, and radio transmission of the data proceed automatically at user-defined intervals. A powerful feature is the acquisition and radio transmission of full detector pulse shapes of each particle. This provides valuable morphological information for particles larger than the 5-microm laser focus. CONCLUSIONS:CytoBuoy allows on-line in situ particle analysis, estimation of phytoplankton biomass, and discrimination between different phytoplankton groups. This will increase the applicability of flow cytometry in the field of environmental monitoring.     

Towards the development of a new generation of whole-cell bioreporters to sense iron bioavailability in oceanic systems—learning from the case of Synechococcus sp. PCC7002 iron bioreporter

Whole-cell bioreporters are genetically modified micro-organisms designed to sense bioavailable forms of nutrients or toxic compounds in aquatic systems. As they represent the most promising cost-efficient tools available for such purpose, engineering and use of bioreporters is rapidly growing in association with wide applicability. Bioreporters are urgently needed to determine phytoplankton iron (Fe) limitation, which has been reported in up to 30% of the ocean, with consequences affecting Earth's global carbon cycle and climate. This study presents a critical evaluation and optimization of the only Cyanobacteria bioreporter available to sense Fe limitation in marine systems (Synechococcus sp. PCC7002). The nonmonotonic biphasic dose–response curve between the bioreporters’ signal and Fe bioavailability impairs an appropriate data interpretation, highlighting the need for new carefully designed bioreporters. Here, limitations under low Fe concentrations were related to cellular energy stress, nonlinear expression of the targeted promoter and siderophore expression. Furthermore, we provide critical standard criteria for the development of new Fe bioreporters. Finally, based on gene expression data under a range of marine Fe concentrations, we propose novel sensor genes for the development of new Cyanobacteria Fe bioreporters for distinct marine regions.     

Species Richness and Invasion Vectors: Sampling Techniques and Biases

During a European Union Concerted Action study on species introductions, an intercalibration workshop on ship ballast water sampling techniques considered various phytoplankton and zooplankton sampling methods. For the first time, all the techniques presently in use worldwide were compared using a plankton tower as a model ballast tank spiked with the brine shrimp and oyster larvae while phytoplankton samples were taken simultaneously in the field (Helgoland Harbour, Germany). Three cone-shaped and 11 non-cone shaped plankton nets of different sizes and designs were employed. Net lengths varied from 50 to 300 cm, diameters 9.7–50 cm, and mesh sizes 10–100 μm. Three pumps, a Ruttner sampler, and a bucket previously used in ballast water sampling studies were also compared. This first assessment indicates that for sampling ballast water a wide range of techniques may be needed. Each method showed different results in efficiency and it is unlikely that any of the methods will sample all taxa. Although several methods proved to be valid elements of a hypothetical `tool box' of effective ship sampling techniques. The Ruttner water sampler and the pump P30 provide suitable means for the quantitative phytoplankton sampling, whereas other pumps prevailed during the qualitative trial. Pump P15 and cone-shaped nets were the best methods used for quantitative zooplankton sampling. It is recommended that a further exercise involving a wider range of taxa be examined in a larger series of mesocosms in conjunction with promising treatment measures for managing ballast water. This revised version was published online in July 2006 with corrections to the Cover Date.     

Phases in the development of riverine plankton: Examples from the rivers Rhine and Meuse

Observations on phyto and zooplankton in two hydrographically different rivers were compared in order to discriminate phases in plankton development. Along the longitudinal axis of the River Rhine a gradual increase in the development of phytoplankton was observed, which reached its maximumca. 100 km before the river flows into its artificial sedimentation area. The development of rotifer populations was slightly retarded as compared with that of phytoplankton and highest population densities were only reached in the sedimentation area. Crustaceans developed in significant numbers, not until the river water had entered the sedimentation area. Development of zooplankton coincided here with a strong decrease in the density of phytoplankton. A similar trend in plankton development was observed in the River Meuse, although in this river the highest densities of phyto and zooplankton already occurred in its middle reaches. The differences in the timing of plankton growth in the two rivers are probably caused by differences in flow regime between both rivers. The River Rhine, which is fed by rainwater and melting of glaciers in the Alps, has a relatively constant discharge. On the other hand, the low discharge of the rain-fed River Meuse combined with an increased residence time of the water as a consequence of large numbers of weirs, allows a full cycle of plankton development long before its discharge into the sea. This phenomenon was also reflected in the silicate cycle in the Meuse, where the consumption by planktonic diatoms and the regeneration of silicate of deposits seem to be important. In contrast, in the main branches of the River Rhine only the effects of silicate consumption were detectable.