Concurrent bioimaging of microalgal photophysiology and oxidative stress.

The production of reactive oxygen species (ROS) is an unavoidable consequence of oxygenic photosynthesis and represents a major cause of oxidative stress in phototrophs, having detrimental effects on the photosynthetic apparatus, limiting cell growth, and productivity. Several methods have been developed for the quantification of cellular ROS, however, most are invasive, requiring the destruction of the sample. Here, we present a new methodology that allows the concurrent quantification of ROS and photosynthetic activity, using the fluorochrome dichlorofluorescein (DCF) and in vivo chlorophyll a fluorescence, respectively. Both types of fluorescence were measured using an imaging Pulse Amplitude Modulation (PAM) fluorometer, modified by adding a UVA-excitation light source (385 nm) and a green bandpass emission filter (530 nm) to enable the sequential capture of red chlorophyll fluorescence and green DCF fluorescence in the same sample. The method was established on Phaeodactylum tricornutum Bohlin, an important marine model diatom species, by determining protocol conditions that permitted the detection of ROS without impacting photosynthetic activity. The utility of the method was validated by quantifying the effects of two herbicides (DCMU and methyl viologen) on the photosynthetic activity and ROS production in P. tricornutum and of light acclimation state in Navicula cf. recens Lange-Bertalot, a common benthic diatom. The developed method is rapid and non-destructive, allowing for the high-throughput screening of multiple samples over time.

Machine learning techniques to characterize functional traits of plankton from image data.

Plankton imaging systems supported by automated classification and analysis have improved ecologists' ability to observe aquatic ecosystems. Today, we are on the cusp of reliably tracking plankton populations with a suite of lab-based and in situ tools, collecting imaging data at unprecedentedly fine spatial and temporal scales. But these data have potential well beyond examining the abundances of different taxa; the individual images themselves contain a wealth of information on functional traits. Here, we outline traits that could be measured from image data, suggest machine learning and computer vision approaches to extract functional trait information from the images, and discuss promising avenues for novel studies. The approaches we discuss are data agnostic and are broadly applicable to imagery of other aquatic or terrestrial organisms.

Multiple-stressor exposure of aquatic food webs: Nitrate and warming modulate the effect of pesticides.

Shallow lakes provide essential ecological and environmental services but are exposed to multiple stressors, including agricultural runoff (ARO) and climate warming, which may act on different target receptors disrupting their normal functioning. We performed a microcosm experiment to determine the individual and combined effects of three stressors-pesticides, nitrate and climate warming-on two trophic levels representative of communities found in shallow lakes. We used three submerged macrophyte species (Myriophyllum spicatum, Potamogeton perfoliatus, Elodea nuttallii), eight benthic or pelagic microalgal species and three primary consumer species (Daphnia magna, Lymnaea stagnalis, Dreissena polymorpha) with different feeding preferences for benthic and pelagic primary producers. Eight different treatments consisted of a control, only nitrate, a pesticide cocktail, and a combination of nitrate and pesticides representing ARO, each replicated at ambient temperature and +3.5°C, mimicking climate warming. Pesticides negatively affected all functional groups except phytoplankton, which increased. Warming and nitrate modified these effects. Strong but opposite pesticide and warming effects on Myriophyllum drove the response of the total macrophyte biomass. Nitrate significantly suppressed Myriophyllum final biomass, but not overall macrophyte and microalgal biomass. Nitrate and pesticides in combination caused a macrophyte decline, and the system tipped towards phytoplankton dominance. Strong synergistic or even reversed stressor interaction effects were observed for macrophytes or periphyton. We emphasize the need for more complex community- and ecosystem-level studies incorporating multiple stressor scenarios to define safe operating spaces.

Disentangling the direct and indirect effects of agricultural runoff on freshwater ecosystems subject to global warming: A microcosm study.

Aquatic ecosystems are exposed to multiple stressors such as agricultural run-off (ARO) and climate-change related increase of temperature. We aimed to determine how ARO and the frequency of its input can affect shallow lake ecosystems through direct and indirect effects on primary producers and primary consumers, and whether warming can mitigate or reinforce the impact of ARO. We performed a set of microcosm experiments simulating ARO using a cocktail of three organic pesticides (terbuthylazine, tebuconazole, pirimicarb), copper and nitrate. Two experiments were performed to determine the direct effect of ARO on primary producers (submerged macrophytes, periphyton and phytoplankton) and on the grazing snail Lymnaea stagnalis, respectively. Three different ARO concentrations added as single doses or as multiple pulses at two different temperatures (22°C and 26°C) were applied. In a third experiment, primary producers and consumers were exposed together to allow trophic interactions. When functional groups were exposed alone, ARO had a direct positive effect on phytoplankton and a strong negative effect on L. stagnalis. When exposed together, primary producer responses were contrasting, as the negative effect of ARO on grazers led to an indirect positive effect on periphyton. Periphyton in turn exerted a strong control on phytoplankton, leading to an indirect negative effect of ARO on phytoplankton. Macrophytes showed little response to the stressors. Multiple pulse exposure increased the effect of ARO on L. stagnalis and periphyton when compared with the same quantity of ARO added as a single dose. The increase in temperature had only limited effects. Our results highlight the importance of indirect effects of stressors, here mediated by grazers and periphyton, and the frequency of the ARO input in aquatic ecosystems.

Probing the Adhesion of the Common Freshwater Diatom Nitzschia palea at Nanoscale.

Freshwater biofilms play an essential ecological role, but they also adversely affect human activities through undesirable biofouling of artificial submerged structures. They form complex aggregates of microorganisms that colonize any type of substratum. In phototrophic biofilms, diatoms dominate in biomass and produce copious amount of extracellular polymeric substances (EPSs), making them efficient early colonizers. Therefore, a better understanding of diatoms adhesive properties is essential to develop new anti-biofouling strategies. In this context, we used atomic force microscopy (AFM) to decipher the topography and adhesive mechanisms of the common freshwater diatom Nitzschia palea. Images taken in physiological conditions revealed typical ultrastructural features with a few nanometers resolution. Using single-cell force spectroscopy, we showed that N. palea strongly adheres to hydrophobic surfaces as compared to hydrophilic ones. Chemical force spectroscopy with hydrophobic tips further confirmed that the adhesion is governed by surface-associated hydrophobic EPS distributed in clusters at the frustule surface, and mostly composed of (glyco)-lipids as revealed by Raman spectroscopy. Collectively, our results demonstrate that AFM-based nanoscopy, combined with Raman spectroscopy, is a powerful tool to provide new insights into the adhesion mechanisms of diatoms.

Modeling the impact of high temperatures on microalgal viability and photosynthetic activity.

BACKGROUND: Culture collapse due to high temperatures can significantly impact the profitability of outdoor algal cultivation systems. The objective of this study was to model for the first time the impact of high temperatures on algal activity and viability. RESULTS: Viability measurements on Dunaliella salina cultures were based on cytometry with two fluorescent markers (erythrosine and fluorescein di-acetate), and photosynthetic activity was measured by Pulse Amplitude Modulation (PAM) fluorometry. Kinetic studies revealed that viability and activity losses during exposure to high temperatures could be described by a Weibull model. Both mortality and activity were shown to be functions of the thermal dose received by the algae, defined as the product of duration of exposure to high temperatures and an exponential function of temperature. Simulations at five climatic locations revealed that culture collapse due to high temperatures could impact productivity of D. salina in non-temperature-controlled outdoor photobioreactors by 35 and 40% in arid and Mediterranean climates, respectively. CONCLUSIONS: The model developed in this study can be used to forecast the impact of high temperatures on algal biofuel productivity. When coupled with models predicting the temperature of outdoor cultivation systems, this model can also be used to select the best combination of location, system geometry, and algal species to minimize the risks of culture collapse and therefore maximize biofuel productivity.

Herbicide toxicity on river biofilms assessed by pulse amplitude modulated (PAM) fluorometry.

The use of Rapid light curves (RLCs) as a toxicity endpoint for river biofilms was examined in this study and compared to "classical fluorescence parameters" i.e. minimal fluorescence (F0), optimal and effective quantum yields of photosystem II (Fv/Fm and ФPSII). Measurements were performed after exposure to five concentrations of diuron (from 0.3 to 33.4µgL(-1)), its main degradation product (DCPMU) (from 1.0 to 1014µgL(-1)) and norflurazon (from 0.6 to 585µgL(-1)) with the lowest exposure concentrations corresponding to levels regularly encountered in chronically contaminated sites. Biofilm responses were evaluated after 1, 5, 7 and 14 days of exposure to the different toxicants. Overall, the responses of both "classical fluorescence parameters" and RLC endpoints were highly time dependent and related to the mode of action of the different compounds. Interestingly, parameters calculated from RLCs (α, ETRmax and Ik) were useful early markers of pesticide exposure since they revealed significant effects of all the tested toxicants from the first day of exposure. In comparison, classical fluorescence endpoints (F0 and Fv/Fm) measured at day 1 were only affected in the DCPMU treatment. Our results demonstrated the interest of RLCs as early markers of toxicant exposure particularly when working with toxicants with less specific mode of action than PSII inhibitors.

Response of intertidal benthic microalgal biofilms to a coupled light-temperature stress: evidence for latitudinal adaptation along the Atlantic coast of Southern Europe.

Although estuarine microphytobenthos (MPB) is frequently exposed to excessive light and temperature conditions, little is known on their interactive effects on MPB primary productivity. Laboratory and in situ experiments were combined to investigate the short-term joint effects of high light (HL) and high temperature (37 °C versus 27 °C) on the operating efficiency of photoprotective processes [vertical migration versus non-photochemical quenching (NPQ)] exhibited by natural benthic diatom communities from two intertidal flats in France (FR) and Portugal (PT). A clear latitudinal pattern was observed, with PT biofilms being more resistant to HL stress, regardless the effect of temperature, and displaying a lower relative contribution of vertical migration to photoprotection and a stronger NPQ in situ. However, higher temperature leads to comparable effects, with photoinhibition increasing to about three times (i.e. from 3% to 10% and from 8% to 22% in PT and FR sites respectively). By using a number of methodological novelties in MPB research (lipid peroxidation quantification, Lhcx proteins immunodetection), this study brings a physiological basis to the previously reported depression of MPB photosynthetic productivity in summer. They emphasize the joint role of temperature and light in limiting, at least transiently (i.e. during emersion), MPB photosynthetic activity in situ.

In vivo estimation of pigment composition and optical absorption cross-section by spectroradiometry in four aquatic photosynthetic micro-organisms.

The objective of the present study was to estimate in vivo pigment composition and to retrieve absorption cross-section values, a(∗), of photosynthetic micro-organisms using a non-invasive technique of reflectance spectrometry. To test the methodology, organisms from different taxonomical groups and different pigment composition were used (Spirulina platensis a Cyanophyta, Porphyridium cruentum a Rhodophyta, Dunaliella tertiolecta a Chlorophyta and Entomoneis paludosa a Bacillariophyta) and photoacclimated to two different irradiance levels: 25 µmol photonm(-2)s(-1) (Low Light, LL) and 500 µmol photonm(-2)s(-1) (High Light, HL). Second derivative spectra from reflectance were used to identify pigment in vivo absorption bands that were linked to specific pigments detected by high performance liquid chromatography. Whereas some absorption bands such as those induced by Chlorophyll (Chl) a (416, 440, 625 and around 675 nm) were ubiquous, others were taxonomically specific (e.g. 636 nm for Chl c in E. paludosa) and/or photo-physiological dependent (e.g. 489 nm for zeaxanthin in the HL-acclimated S. platensis). The optical absorption cross-section, a(∗), was retrieved from reflectance data using a radiative transfer model previously developed for microphytobenthos. Despite the cellular Chl a decrease observed from LL to HL (up to 88% for S. platensis), the a(∗) increased, except for P. cruentum. This was attributed to a 'package effect' and to a greater absorption by photoprotective carotenoids that did not contribute to the energy transfer to the core Chl a.

A method for the rapid generation of nonsequential light-response curves of chlorophyll fluorescence.

Light-response curves (LCs) of chlorophyll fluorescence are widely used in plant physiology. Most commonly, LCs are generated sequentially, exposing the same sample to a sequence of distinct actinic light intensities. These measurements are not independent, as the response to each new light level is affected by the light exposure history experienced during previous steps of the LC, an issue particularly relevant in the case of the popular rapid light curves. In this work, we demonstrate the proof of concept of a new method for the rapid generation of LCs from nonsequential, temporally independent fluorescence measurements. The method is based on the combined use of sample illumination with digitally controlled, spatially separated beams of actinic light and a fluorescence imaging system. It allows the generation of a whole LC, including a large number of actinic light steps and adequate replication, within the time required for a single measurement (and therefore named "single-pulse light curve"). This method is illustrated for the generation of LCs of photosystem II quantum yield, relative electron transport rate, and nonphotochemical quenching on intact plant leaves exhibiting distinct light responses. This approach makes it also possible to easily characterize the integrated dynamic light response of a sample by combining the measurement of LCs (actinic light intensity is varied while measuring time is fixed) with induction/relaxation kinetics (actinic light intensity is fixed and the response is followed over time), describing both how the response to light varies with time and how the response kinetics varies with light intensity.

Short term recovery of periphyton photosynthesis after pulse exposition to the photosystem II inhibitors atrazine and isoproturon.

Aquatic organisms are exposed to fluctuating concentrations of herbicides which contaminate rivers following their use for agricultural or domestic purposes. The development of sensitive bioanalytical tests enabling us not only to detect the effects of those pollutants but to take into account this pattern of exposure should improve the ecological relevance of river toxicity assessment. In this respect, the use of chlorophyll fluorescence measurements is a convenient way to probe the effect of photosystem II (PSII) inhibitors on primary producers. This study was devoted to validate the combined use of two fluorescence parameters, the effective and the optimal quantum yields of PSII photochemistry (Φ(PSII) and F(v)/F(m)), as reliable biomarkers of initial isoproturon (IPU) or atrazine (ATZ) toxicity to natural periphyton in a pulse exposition scenario. Φ(PSII) and F(v)/F(m) were regularly estimated during a 7 h-exposure to each pollutant (0-100 µM) and also later after being transferred in herbicide-free water (up to 36 h). Our results showed that IPU was more toxic than ATZ, but with effects reversible within 12 h. Moreover, these two similarly acting herbicides (i.e. same target site) presented contrasted short term recovery patterns, regarding the previous exposure duration.

Light modulated toxicity of isoproturon toward natural stream periphyton photosynthesis: a comparison between constant and dynamic light conditions.

This study tested if a variation in light intensity, in comparison to constant light required in well-designed toxicity test, could have measurable consequences on the sensitivity of phototrophic biofilms (periphyton) to isoproturon. Two independent experiments were carried out to investigate the combined effects of light and isoproturon on the photochemical behavior of intact natural biofilms by measurements of chlorophyll fluorescence and pigment composition. Experiment 1 consisted of exposing biofilms to series of isoproturon concentrations (0-2 mg L(-1)) for 7 h under constant light at different irradiance levels (25-300 micromol m(-2) s(-1)). In experiment 2, biofilms were exposed using more environmentally realistic conditions to three selected concentrations of isoproturon (2, 6 and 20 microg L(-1)) during a 7-h-simulated daily light cycle. Our results demonstrated that light, considered here as a direct physical stressor, slightly modulated the acute toxicity of isoproturon on these diatom dominated communities. This was attributed to the fact that these two factors act specifically on the photosynthetic activity. Furthermore, it was shown that a dynamic light regime increased periphyton sensitivity to isoproturon by challenging its photoprotective mechanisms such as the xanthophyll cycle, therefore implying that traditional ecotoxicological bioassays lead to underestimate the effect of isoproturon.

STREAM PERIPHYTON PHOTOACCLIMATION RESPONSE IN FIELD CONDITIONS: EFFECT OF COMMUNITY DEVELOPMENT AND SEASONAL CHANGES(1).

The photochemical behavior of intact stream periphyton communities in France was evaluated in response to the time course of natural light. Intact biofilms grown on glass substrata were collected at three development stages in July and November, and structural parameters of the biofilms were investigated (diatom density and taxonomy). At each season, physiological parameters based on pigment analysis (HPLC) and pulse-amplitude-modulated (PAM) chl fluorescence technique were estimated periodically during a day from dawn to zenith. Regardless of the community studied, the optimal quantum yield of PSII (Fv /Fm ), the effective PSII efficiency (ΦPSII ), the nonphotochemical quenching (NPQ), and the relative electron transport rate (rETR) exhibited clear dynamic patterns over the morning. Moreover, microalgae responded to the light increase by developing the photoprotective xanthophyll cycle. The analysis of P-I parameters and pigment profiles suggests that July communities were adapted to higher light environments in comparison with November ones, which could be partly explained by a shift in the taxonomic composition. Finally, differences between development stages were significant only in July. In particular, photoinhibition was less pronounced in mature assemblages, indicating that self-shading (in relation to algal biomass) could have influenced photosynthesis in older communities.