Regulation of Fatty Acid Production and Release in Benthic Algae: Could Parallel Allelopathy Be Explained with Plant Defence Theories?

Many organisms produce chemical compounds, generally referred as secondary metabolites, to defend against predators and competitors (allelopathic compounds). Several hypotheses have been proposed to explain the interaction between environmental factors and secondary metabolites production. However, microalgae commonly use simple metabolites having a role in primary metabolism as allelopathic compounds. The aim of this study was to determine whether classical theories of plant chemical defences could be applied to microalgae producing allelochemicals derived from the primary metabolism. Our study was designed to investigate how growth phase, algal population density, nutrient limitation and carbon assimilation affect the production and release of allelopathic free fatty acids (FFAs) among other FFAs. The model species used was Uronema confervicolum, a benthic filamentous green alga that produces two allelopathic FFAs (linoleic and α-linolenic acids) inhibiting diatom growth. FFAs have been quantified in algal biomass and in culture medium. Our results were analysed according to two classical plant defence theories: the growth-differentiation balance hypothesis (GDBH) and the optimal defence theory (ODT), based on the metabolic capacities for defence production and on the need for defence, respectively. While a higher production of allelopathic compounds under increased light conditions supports the use of GDBH with this microalga, the observation of a negative feedback mechanism mostly supports ODT. Therefore, both theories were insufficient to explain all the observed effects of environmental factors on the production of these allelochemicals. This highlights the needs of new theories and models to better describe chemical interactions of microalgae.

Warming lowers critical thresholds for multiple stressor-induced shifts between aquatic primary producers.

In aquatic ecosystems, excessive nutrient loading is a global problem that can induce regime shifts from macrophyte- to phytoplankton-dominated states with severe consequences for ecosystem functions. Most agricultural landscapes are sites of nutrient and pesticide loading, which can interact with other stressors (e.g., warming) in additive, antagonistic, synergistic or reversed forms. The effects of multiple stressors on the resilience of macrophyte-dominated states and on critical thresholds for regime shifts are, however, unknown. We test the effects of individual and combined stressors of warming, nitrate, and various pesticides typically found in agricultural run-off (ARO) on the growth of macrophytes, periphyton, and phytoplankton in microcosms. We applied a one-level replicated design to test whether ARO induces a regime shift and a multifactorial dose-response design to model stressor thresholds and disentangle stressor interactions along a gradient. The individual stressors did not induce a regime shift, but the full ARO did. Nitrate and pesticides acted synergistically, inducing a shift with increasing phytoplankton biomass and decreasing macrophyte biomass. Warming amplified this effect and lowered critical thresholds for regime shifts. Shallow aquatic ecosystems in agricultural landscapes affected by global warming thus increasingly risk shifting to a turbid, phytoplankton-dominated state, and negatively impacting ecosystem service provisioning. Multiple stressor interactions must be considered when defining safe operating spaces for aquatic systems.

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.

Association of Diopsys® Short-duration Transient Visual Evoked Potential Latency with Visual Field Progression in Chronic Glaucoma.

AIM: To determine the association of Diopsys® NOVA-LX amplitude and latency abnormality scores with perimetric staging of chronic glaucoma, and to explore potential single-visit short-duration transient visual evoked potential (SD-tVEP) trend detection ability utilizing Humphrey 30-2 field progression data. MATERIALS AND METHODS: Setting: Glaucoma subspecialty clinic. Participants: Treated adult chronic glaucoma patients undergoing SD-tVEP evaluation. Main outcome measures: (1) Proportion of eyes designated as suspect or abnormal by the NOVA-LX multifactorial algorithm were determined as a function of glaucoma severity using the most recent Humphrey visual field analyzer (HVFA) 30-2 field. (2) Association between long-term HVFA-guided progression analysis (GPA) annual slopes and SD-tVEP abnormality was assessed to determine whether a single VEP test might help to identify eyes more prone to progressive visual field (VF) loss. RESULTS: One hundred and thirty-three eyes of 84 patients (mean age 68 years) were analyzed. The SD-tVEP abnormality increased proportionately with severity of VF loss under high-contrast (Hc) test conditions for both latency (p = 0.001) and amplitude (p < 0.01). The HVFA progression analysis printouts existed for 91 eyes (mean 12.3 fields per eye/range 5-18). Nearly three-quarters (72.5%) of eyes with mean annual HVFA progression >0.7 dB/year (n = 29) had single-visit VEP latency abnormalities. Fewer than half (46.7%) of the remainder (n = 62) showed latency abnormality. Mean progression for eyes with abnormal vs normal VEP latency was -0.87 ± 0.3 dB/year vs -0.32 ± 0.4 dB/year. CONCLUSION: Diopsys NOVA-LX Hc latency abnormality shows strong association with VF loss among a diverse population of clinical patients undergoing active treatment for chronic glaucoma, and appears likely to afford clinically useful trend-detecting test. CLINICAL SIGNIFICANCE: The SD-tVEP has the potential to serve as a single-visit clinical indicator to identify glaucoma patients at high risk for VF progression.How to cite this article: Trevino R, Sponsel WE, Majcher CE, Allen J, Rabin J. Association of Diopsys® Short-duration Transient Visual Evoked Potential Latency with Visual Field Progression in Chronic Glaucoma. J Curr Glaucoma Pract 2018;12(1):29-35.

Allelopathic interactions involving benthic phototrophic microorganisms.

As a way to prevent resource depletion by other species, many phototrophic aquatic microorganisms produce inhibitory compounds. This process, known as allelopathy, has been widely studied in planktonic environments, where it is recognized as being a driving force of planktonic communities. However, in benthic environments, biofilms provide very particular micro-environments. The present review focuses on allelopathic interactions involving benthic phototrophic prokaryotes and micro-eukaryotes ('microalgae'), which generally form biofilms, and includes any interaction involving benthic microalgae either as the emitter or as the target in both marine and freshwater habitats. To support our hypothesis on the importance of allelopathy in biofilms due to the particularities of biofilms, we show that (i) reported allelopathic species and compounds are diverse and numerous in the three major groups of benthic phototrophic microorganisms, (ii) allelopathic benthic species could affect community composition, (iii) allelopathy in biofilms is currently underestimated because of the lack of suitable methods. As benthic primary producers represent an important source of organic carbon in some streams and littoral areas, these interactions could impact the whole ecosystem in these areas, probably more than in areas dominated by planktonic communities.

Impairment of benthic diatom adhesion and photosynthetic activity by allelopathic compounds from a green alga: involvement of free fatty acids?

The role of chemical interactions in shaping microbial communities has raised increasing interest over the last decade. Many benthic microorganisms are known to develop chemical strategies to overcome competitors, but the real importance of chemical interactions within freshwater biofilm remains unknown. This study focused on the biological and chemical mechanisms of an interaction involving two benthic microorganisms, an allelopathic filamentous green alga, Uronema confervicolum, and a common diatom, Fistulifera saprophila. Our results showed that functions critical for benthic phototrophic microorganisms were inhibited by U. confervicolum extracts. Growth, cell motility, adhesion, and photosynthetic activity were impaired at extract concentrations ranging between 5 and 20 µg ml(-1). The adhesion inhibition was mediated by intracellular nitric oxide (NO) induction. A bioassay-guided fractionation of the extract with HPLC helped to identify two C18 fatty acids present in the growth-inhibiting fractions: linoleic (LA) and α-linolenic (LNA) acids. These compounds represented 77% of the total free fatty acids of U. confervicolum and were present in the culture medium (1.45 µg l(-1) in total). Both could inhibit the diatom growth at concentrations higher than 0.25 µg ml(-1), but had no effect on cell adhesion. The discrepancy between the effective concentrations of fatty acids and the concentration found in culture medium may be explained by the presence of high-concentration microenvironments. The compounds involved in adhesion inhibition remain to be identified. Though further experiments with complex biofilms are needed, our results suggest that U. confervicolum may participate to the control of biofilm composition by inhibiting diatom adhesion.