Optimization of variable fluorescence measurements of phytoplankton communities with cyanobacteria

Excitation–emission fluorescence matrices of phytoplankton communities were simulated from laboratory-grown algae and cyanobacteria cultures, to define the optical configurations of theoretical fluorometers that either minimize or maximize the representation of these phytoplankton groups in community variable fluorescence measurements. Excitation sources that match the photosystem II (PSII) action spectrum of cyanobacteria do not necessarily lead to equal representation of cyanobacteria in community fluorescence. In communities with an equal share of algae and cyanobacteria, inducible PSII fluorescence in algae can be retrieved from community fluorescence under blue excitation (450–470 nm) with high accuracy (R ² = 1.00). The highest correlation between community and cyanobacterial variable fluorescence is obtained under orange-red excitation in the 590–650 nm range (R ² = 0.54). Gaussian band decomposition reveals that in the presence of cyanobacteria, the emission detection slit must be narrow (up to 10 nm) and centred on PSII chlorophyll-a emission (~683 nm) to avoid severe dampening of the signal by weakly variable phycobilisomal fluorescence and non-variable photosystem I fluorescence. When these optimizations of the optical configuration of the fluorometer are followed, both cyanobacterial and algal cultures in nutrient replete exponential growth exhibit values of the maximum quantum yield of charge separation in PSII in the range of 0.65–0.7.     

The effect of elevated CO2 on growth and competition in experimental phytoplankton communities

We report an experiment designed to identify the effect of elevated CO₂ on species of phytoplankton in a simple laboratory system. Major taxa of phytoplankton differ in their ability to take up CO₂, which might lead to predictable changes in the growth rate of species and thereby shifts in the composition of phytoplankton communities in response to rising CO₂. Six species of phytoplankton belonging to three major taxa (cyanobacteria, diatoms and chlorophytes) were cultured in atmospheres whose CO₂ concentration was gradually increased from ambient levels to 1000 parts per million over about 100 generations and then maintained for a further 200 generations at elevated CO₂. The experimental design allowed us to trace a predictive sequence, from physiological features to the growth response of species to elevated CO₂ in pure culture, from the growth response in pure culture to competitive ability in pairwise mixtures and from pairwise competitive ability to shifts in the relative abundance of species in the full community of all six species. CO₂ altered the dynamics of growth in a fashion consistent with known differences among major taxa in their ability to take up and use CO₂. This pure‐culture response was partly successful in predicting the outcome of competition in pairwise mixtures, especially the enhanced competitive ability of chlorophytes relative to cyanobacteria, although generally statistical support was weak. The competitive response in pairwise mixtures was a good predictor of changes in competitive ability in the full community. Hence, there is a potential for forging a logical chain of inferences for predicting how phytoplankton communities will respond to elevated CO₂. Clearly further extensive experiments will be required to validate this approach in the greater complexity found in diverse communities and environments of natural systems.     

The effect of elevated atmospheric carbon dioxide levels on soil bacterial communities

The effect of elevated carbon dioxide levels on total bacterial communities was studied in a series of controlled and replicated model terrestrial ecosystems over a period of 38 weeks. The bacterial community was profiled using Denaturing Gradient Gel Electrophoresis (DGGE) analysis of bacterial 16S rRNA gene fragments amplified by the Polymerase Chain Reaction from DNA extracted directly from soil. Bacterial community DGGE profiles provided three major findings: (i) there was a high degree of profile similarity after ≈ 12 weeks (one plant generation); (ii) whilst overall DGGE profile was maintained over the 38 weeks (three plant generations), the banding patterns became more diverse with time; (iii) DGGE data provided no evidence for a shift in bacterial community structure resulting from exposure of the ecosystem to an increased atmospheric CO₂ level.     

The effect of water column mixing on phytoplankton succession, diversity and similarity

The lake number was used to describe the mixing condition forthree consecutive years (1992–1994) in Lake Tegel, Berlin,and compared to the successions of diatoms, dinoflagellatesand cyanobacteria, the main phytoplankton groups in the lake,as well as to diversity and similarity indices. Using both diversityand similarity indices in juxtaposition provides an indicationof the growth type of prevailing species (r- or K-strategists)and the degree of competition in the aqua-ecological system.A genera] pattern of these indices can be recognized as threephases: (i) high diversity—during spring, summer and autumn,interrupted by (ii) phases of low diversity during the latespring clear-water phase as the number of spring species plummeted,and (iii) during the late summer, climax populations of K-strategists.On a smaller time scale, similarity and diversity proved toreact sensitively to disturbances at frequencies intermediatein relation to the generation times of the phytoplankton. Thissupports the ‘intermediate disturbance hypothesis’,as proposed for phytoplankton by Padisak et al. [(eds) IntermediateDisturbance Hypothesis in Phytoplankton Ecology. Kluwer Academic,1993]. Diversity may remain quite high even for extended periodsduring summer climax situations, in conjunction with a highdegree of similarity, if deeper mixing of the epilimnion occursat time intervals of 2–3 weeks, as during the summer of1993. This enables the prevalence of ‘ruderal’ species,together with some motile K-strategists who actively seek optimaldepths for photosynthesis. During such summer situations describedby frequent occurrences of lower lake numbers, the epilimnionof Lake Tegel is mixed deeply enough to support ruderals, butnot too vigorously to counteract competitive advantages of motilespecies. Thus, vertical niche separation enhances diversity.     

The dependence of phytoplankton fluorescence on the thermal stress factor

The results of a study of the temperature dependence of laser-induced fluorescence for four marine microalgae cultures that belonging to different taxonomic groups are described. The fluorescent properties of microalgae that were exposed to temperature stress up to irreversible changes in their cells have been examined. A comparative analysis has been performed and the models for the dependence of laser-induced fluorescence intensity on the temperature for the microalgal cell pigments have been constructed. The microalgae differ in both their dependence of fluorescence intensity on the thermal stress factor and the stress temperature value, which suggests that these characteristics are species specific.     

The effect of variable liposome brightness on quantifying lipid-protein interactions using fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) has been increasingly used to study the binding of fluorescently-labeled peptides and proteins to phospholipid vesicles. In this work, we present a new method to analyze partition data obtained by this technique based on the assumption that the number of fluorescently-labeled protein molecules bound per liposome follows a Poisson distribution. To not overestimate the recovered partition coefficients, we first show that the variation in liposome brightness caused by this statistical distribution must be considered explicitly in data analysis when the parameter used to establish the partition curves is the fractional instead of the absolute amplitudes associated with the slowest diffusing particles in the system (lipid vesicles), a choice frequently made in FCS partition studies. We further extend the theoretical model describing the membrane partition of a fluorescently-labeled protein by considering the presence of a trace amount of free fluorescent dye (non-binding component) in the system. We show that this situation can account for an apparent maximal binding level lower than 100% in the experimental partitioning curves obtained for Alexa 488 fluorescently-labeled lysozyme and liposomes prepared with variable anionic phospholipid content. The extreme sensitivity of the FCS technique allowed uncoupling lysozyme partition from the protein-induced liposome aggregation, confirming that lysozyme binding to negatively charged liposomes is dominantly driven by electrostatic interactions.     

The effect of low water levels on the water quality of Lake Biwa

Because of a lack of precipitation, water levels in Lake Biwa, Japan, were extremely low between the beginning of September 1984 and the end of February 1985. Approximately 13 million people depend upon the lake as a source of drinking water and for industrial use, and the severe water shortage became a serious concern for downstream communities. Also, there was concern that deterioration of water quality caused by rotting macrophytes and the release of nutrients from vegetation and nearshore sediments might create additional problems.In this paper, the release of nutrients from vegetation and sediments is examined under conditions which simulate both calm and turbulent water motions in the nearshore, and the magnitude of nutrient loadings are estimated in relation to the specific effects of low lake level.Sample stations were established around the south shore of Lake Biwa. Sampling was undertaken at the time of low water and during the rising water levels. Sediment samples were particle sized into 7 groups (<2000 µm). Other measured values ranged as follows: BOD (0.5–1.3), COD (1.2–3.5), TP (0.019–0.037), SRP (0.013–0.030), SOP (0.005–0.007), TN (0.45–0.90), NO₂-N (0.004–0.007), NO₃-N (0.04–0.08) and NH₄-N (0.026–0.053), all as mgL^-1. The sample data suggest that, overall, there was little impact on lake water quality as a result of low water levels. However, remedial actions may have had an important and beneficial impact on nearshore water quality in the southern basin of Lake Biwa.     

Effect of elevated levels of natural radioactivity on the fecundity of male mice

Male mice of the BALB/c strain were placed on the floor of a hut built at a site of Southwest France where the dose rate of gamma radiation amounts to about 10 mrad/h. Controls were kept near the radioactive site under comparable conditions. The doses of gamma rays received during a seven month period ranged from 0.1275 rad for the controls to 45 rad and 63 rad for the exposed animals. Due, possibly, to an effect on the libido exposure to 45 rad increased the fertility whereas the animals given 63 rad produced less offspring than the controls.     

Allelopathic effect of the aquatic macrophyte, Stratiotes aloides, on natural phytoplankton

1. A survey of different Dutch Stratiotes stands showed that the density of phytoplankton (except cyanobacteria) was always higher outside S. aloides than between the rosettes of S. aloides. Analyses of water samples revealed that nutrient limitation was unlikely to have caused the lower phytoplankton biomass in the vicinity of S. aloides. 2. An in situ incubation experiment in the Danube Delta, Romania, indicated allelopathic activity against phytoplankton in S. aloides stands. The growth rate of natural phytoplankton populations exposed to water from S. aloides stands was significantly lower than that of populations that had not been in contact with S. aloides exudates. 3. A laboratory microcosm experiment showed a significantly lower phytoplankton biomass in treatments with S. aloides exudates. Nutrient concentrations and the light intensity were high enough that the lower phytoplankton biomass could not be explained by nutrient or light limitation.     

The effect of abscisic acid on chlorophyll fluorescence in lichens under extreme water regimes

Chlorophyll fluorescence measurements were used to test whether externally applied abscisic acid (ABA) has an effect on the desiccation tolerance of lichens and their sensitivity to constant water saturation. A surplus of ABA did not decrease the time required to regain full photosynthetic capacity after prolonged dry periods. However, an effect of ABA could be observed when lichens were permanently hydrated at moderately high temperatures. Lichens suffered less from constant saturation if ABA was added to the incubation medium. These results suggest a positive effect of ABA on membrane function.     

The effect of tumour progression on lymphocyte responses to lectins,measured by fluorescence polarization techniques

The process of fluorochromasia involves the hydrolysis by cells of fluorescein diacetate resulting in an intracellular accumulation of fluorescein. The polarization of the fluorescence of the fluorescein appears to depend on the intracellular fluorescein concentration, the distribution of fluorescein within the cell and the viscosity of the cell cytoplasm.The parameters of fluorochromasia were studied with thymocytes from normal BALB/c mice and from mice bearing an intraperitoneal NK/LY/R lymphoma. During the course of tumour proliferation, the response toT-cell mitogens increased whereas the response to other lectins,e.g. wheat germ agglutinin, decreased or remained unaltered. These changes were consistent with the corresponding increase in immunocompetent cells within the thymus, observed by microelectrophoresis. Thus this sensitive technique provides a useful quantitative assessment of the lectin-lymphoid cell interaction.     

The effect of dynamic exercise on resting cold thermoregulatory responses measured during water immersion

The purpose of this study was to evaluate the effect of exercise on the subsequent post-exercise thresholds for vasoconstriction and shivering measured during water immersion. On 2 separate days, seven subjects (six males and one female) were immersed in water (37.5 degrees C) that was subsequently cooled at a constant rate of approximately 6.5 degrees C x h(-1) until the thresholds for vasoconstriction and shivering were clearly established. Water temperature was then increased to 37.5 degrees C. Subjects remained immersed for approximately 20 min, after which they exited the water, were towel-dried and sat in room air (22 degrees C) until both esophageal temperature and mean skin temperature (Tsk) returned to near-baseline values. Subjects then either performed 15 min of cycle ergometry (at 65% maximal oxygen consumption) followed by 30 min of recovery (Exercise), or remained seated with no exercise for 45 min (Control). Subjects were then cooled again. The core temperature thresholds for both vasoconstriction and shivering increased significantly by 0.2 degrees C Post-Exercise (P < 0.05). Because the Tsk at the onset of vasoconstriction and shivering was different during Pre- and Post-Exercise Cooling, we compensated mathematically for changes in skin temperatures using the established linear cutaneous contribution of skin to the control of vasoconstriction and shivering (20%). The calculated core temperature threshold (at a designated skin temperature of 32.0 degrees C) for vasoconstriction increased significantly from 37.1 (0.3) degrees C to 37.5 ( 0.3) degrees C post-exercise (P < 0.05). Likewise, the shivering threshold increased from 36.2 (0.3) degrees C to 36.5 (0.3) degrees C post-exercise (P < 0.05). In contrast to the post-exercise increase in cold thermal response thresholds, sequential measurements demonstrated a time-dependent similarity in the Pre- and Post-Control thresholds for vasoconstriction and shivering. These data indicate that exercise has a prolonged effect on the post-exercise thresholds for both cold thermoregulatory responses.     

Ultraviolet-B effects on photosystem II efficiency of natural phytoplankton communities from Antarctica

The impact of UVB on the Antarctic phytoplankton photosystem II repair cycle, involving the rapidly cycled D1 protein, was studied during summer 2002. On sunny and overcast days, phytoplankton (from 1-m depth) were exposed to natural light (+UVB) and Mylar-screened (–UVB) conditions. Half of the samples from each treatment were inoculated with lincomycin, an inhibitor of synthesis of chloroplast-encoded proteins including the D1 protein. Blocking D1 repair caused significant F_v/F_m depressions on sunny days but had not effect on the overcast day. Most of the F_v/F_m depression was caused by PAR and UVA with a non-significant contribution from UVB. In the presence of D1 repair, suppressing UVB had no effect on F_v/F_m when the samples originated from a weakly stratified water column with no defined upper mixed layer (UML) while it alleviated F_v/F_m depression when the phytoplankton samples originated from within an UML deeper than the depth of UVB penetration. These results suggest that UVB had more effect on the D1 repair process than on the damage process itself but that phytoplankton sensitivity to surface UVB exposure was influenced by their previous light history, partly determined by the vertical structure of the water column.     

The effect of acid water on the loss of divalent cations and primary amines from natural membranes

Losses of Ca2+, Mg2+ and primary amines into waters between pH = 5 and 3 from eyed Chinook salmon eggs, Oncorhynchus tshawytscha , and gills excised from the freshwater, bivalve mollusc Anodonta californiensis were measured and compared to effluxes into distilled water. Sulfuric, nitric and hydrochloric acids were used. Even at pH = 5 losses of Ca2+ and Mg2+ from both biological systems occur at short times, minutes, and can exceed those found in water of higher pH (non-acid waters). Increasing acidity increases short term primary amine loss from both systems. For both divalent cation and amino acid losses gills of A. californiensis are more sensitive to acidity than eggs of O. tshawytscha .     

Pigments and species composition of natural phytoplankton populations: effect on the absorption spectra

Data from three cruises (Arabesque 1 and 2 cruises in the ArabianSea and the Vancouver Island cruise) were examined to assessthe importance of species composition and accessory pigmentsin modifying specific absorption coefficients. The three cruisesdiffered widely in their phytoplankton assemblages with smallcells dominating the Arabesque 2 cruise and large diatoms theVancouver Island cruise. Absorption spectra from each cruisewere decomposed into 13 Gaussian bands representing absorptionby the major chlorophylls and accessory pigments. The maximumspecific peak height     

Interpretation of in vivo fluorescence and cell division rates of natural phytoplankton using a cell cycle model

A natural population of phytoplankton was collected from theMenai Straits and incubated in a continuous culture system undernatural illumination. In vivo fluorescence data were used toderive cell cycle parameters, and the division rates of cellsin the population were analysed using a transition-point modelof the cell cycle. The results indicated that for modellingpurposes the mixed population could be regarded as being composedof a single species having properties equivalent to the averagefor the population. Fluorescence measurements were subsequcntlyrepeated on samples of the in situ population at the collectionsite, and the cell cycle model used to interpret the growthrate of the phytoplankton in the natural environment.     

The vertical distribution of phytoplankton in stratified water columns

What determines the vertical distribution of phytoplankton in different aquatic environments remains an open question. To address this question, we develop a model to explore how phytoplankton respond through growth and movement to opposing resource gradients and different mixing conditions. We assume stratification creates a well-mixed surface layer on top of a poorly mixed deep layer and nutrients are supplied from multiple depth-dependent sources. Intraspecific competition leads to a unique strategic equilibrium for phytoplankton, which allows us to classify the distinct vertical distributions that can exist. Biomass can occur as a benthic layer (BL), a deep chlorophyll maximum (DCM), or in the mixed layer (ML), or as a combination of BL+ML or DCM+ML. The ML biomass can be limited by nutrients, light, or both. We predict how the vertical distribution, relative resource limitation, and biomass of phytoplankton will change across environmental gradients. We parameterized our model to represent potentially light and phosphorus limited freshwater lakes, but the model is applicable to a broad range of vertically stratified systems. Increasing nutrient input from the sediments or to the mixed layer increases light limitation, shifts phytoplankton towards the surface, and increases total biomass. Increasing background light attenuation increases light limitation, shifts the phytoplankton towards the surface, and generally decreases total biomass. Increasing mixed layer depth increases, decreases, or has no effect on light limitation and total biomass. Our model is able to replicate the diverse vertical distributions observed in nature and explain what underlying mechanisms drive these distributions.     

The pH-independent effect of aluminum on cultures of phytoplankton from an acidic Wisconsin lake

Negative correlations between aluminum and planktonic algal abundance have been reported in acidic lakes. Natural assemblages of phytoplankton from a low-pH, low-Al lake (Franklin Lake, WI) were grown in semi-continuous cultures consisting of four treatments at pH 5.7 with 0.0, 50, 100, and 200 μg Al L⁻¹ and one treatment at pH 4.7 with no Al added. Asterionella ralfsii var. americana (a common diatom plankter in acidic lakes) grew well at both pH 4.7 and 5.7 when no Al was added but declined in all other treatments and so may be useful as an indicator of acidic, low monomeric-Al conditions. Other common plankters that showed this pattern included: Arthrodesmus indentatus, Ar. octocornus, Ar. quiriferus, Staurastrum arachne var. curvatum, S. longipes var. contractum, and S. pentacerum. Common taxa showing no toxic effects of Al were Dinobryon bavaricum, Peridinium limbatum, Stenokalyx monilifera, Elaktothrix sp. and Oedogonium sp. We hypothesize that metal toxicity as a pulse at spring snowmelt could dramatically change algal succession in moderately acidic lakes. The experimental results agreed well with field observations. These types of experiments are useful for predicting the responses of natural phytoplankton communities to increases in Al concentration.     

Influence of phosphate and disinfection on the composition of biofilms produced from drinking water, as measured by fluorescence in situ hybridization

Biofilms were grown in annular reactors supplied with drinking water enriched with 235 microg C/L. Changes in the biofilms with ageing, disinfection, and phosphate treatment were monitored using fluorescence in situ hybridization. EUB338, BET42a, GAM42a, and ALF1b probes were used to target most bacteria and the alpha (alpha), beta (beta), and gamma (gamma) subclasses of Proteobacteria, respectively. The stability of biofilm composition was checked after the onset of colonization between T = 42 days and T = 113 days. From 56.0% to 75.9% of the cells detected through total direct counts with DAPI (4'-6-diamidino-2-phenylindole) were also detected with the EUB338 probe, which targets the 16S rRNA of most bacteria. Among these cells, 16.9%-24.7% were targeted with the BET42a probe, 1.8%-18.3% with the ALF1b probe, and <2.5% with the GAM42a probe. Phosphate treatment induced a significant enhancement to the proportion of gamma-Proteobacteria (detected with the GAM42a probe), a group that contains many health-related bacteria. Disinfection with monochloramine for 1 month or chlorine for 3 days induced a reduction in the percentage of DAPI-stained cells that hybridized with the EUB338 probe (as expressed by percentages of EUB338 counts/DAPI) and with any of the ALF1b, BET42a, and GAM42a probes. The percentage of cells detected by any of the three probes (ALF1b+BET42a+GAM42a) tended to decrease, and reached in total less than 30% of the EUB338-hybridized cells. Disinfection with chlorine for 7 days induced a reverse shift; an increase in the percentage of EUB338 counts targeted by any of these three probes was noted, which reached up to 87%. However, it should be noted that the global bacterial densities (heterotrophic plate counts and total direct counts) tended to decrease over the duration of the experiment. Therefore, those bacteria that could be considered to resist 7 days of chlorination constituted a small part of the initial biofilm community, up to the point at which the other bacterial groups were destroyed by chlorination. The results suggest that there were variations in the kinetics of inactivation by disinfectant, depending on the bacterial populations involved.