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.

Resilience of aggregated microbial communities subjected to drought--small-scale studies.

The response of microbial communities to disturbance is a major concern for microbial ecologists since potential modifications in their composition and functioning may affect ecosystems to a larger extent. Microbial ecosystems may be resistant (not affected) or may present engineering (return to initial state) or ecological resilience. In the latter case, when the disturbance is released, the ecosystem evolves towards a new equilibrium state. The aim of this study was to determine if variations in the magnitude of a disturbance could induce either engineering or ecological resilience. We used phototrophic biofilms grown in mesocosms as a model of microbial ecosystem and increasing drought duration (1-8 weeks) as a range of disturbances. Biofilm composition (algal and prokaryotic), photosynthetic activity (PhytoPAM), and potential functional diversity (Biolog) were determined at the end of dry phase and after a 2-week rewetting phase in individual aquaria. We only observed an ecological resilience of the biofilm, with a resistance of phototrophic component for the weakest disturbance. After rewetting, the biofilm could fulfill the same functions, but its species composition was highly modified. We observed a shift from cyanobacteria dominance towards diatom dominance. The disturbance caused a transition towards a new steady state of the biofilm. We also observed a positive effect of stress duration on biofilm productivity after resilience.

Ecotoxicity of Heteroaggregates of Polystyrene Nanospheres in Chironomidae and Amphibian.

Due to their various properties as polymeric materials, plastics have been produced, used and ultimately discharged into the environment. Although some studies have shown their negative impacts on the marine environment, the effects of plastics on freshwater organisms are still poorly studied, while they could be widely in contact with this pollution. The current work aimed to better elucidate the impact and the toxicity mechanisms of two kinds of commercial functionalized nanoplastics, i.e., carboxylated polystyrene microspheres of, respectively, 350 and 50 nm (PS350 and PS50), and heteroaggregated PS50 with humic acid with an apparent size of 350 nm (PSHA), all used at environmental concentrations (0.1 to 100 µg L-1). For this purpose, two relevant biological and aquatic models-amphibian larvae, Xenopus laevis, and dipters, Chironomus riparius-were used under normalized exposure conditions. The acute, chronic, and genetic toxicity parameters were examined and discussed with regard to the fundamental characterization in media exposures and, especially, the aggregation state of the nanoplastics. The size of PS350 and PSHA remained similar in the Xenopus and Chironomus exposure media. Inversely, PS50 aggregated in both exposition media and finally appeared to be micrometric during the exposition tests. Interestingly, this work highlighted that PS350 has no significant effect on the tested species, while PS50 is the most prone to alter the growth of Xenopus but not of Chironomus. Finally, PSHA induced a significant genotoxicity in Xenopus.

Effects of 2E,4E-decadienal on motility and aggregation of diatoms and on biofilm formation.

Phototrophic biofilm formation and dynamics result from the interaction between several parameters, including chemical interactions. Some of the secondary metabolites released by microalgae can influence the composition of benthic communities. We determined the effects of decadienal (DD), a polyunsaturated aldehyde produced by diatoms, on a benthic diatom, Fistulifera saprophila. At 5 µg ml(-1), DD reduced cell motility by 88% and cell adhesion to the substrate by 91%. The effects occurred in less than 30 min. Using a fluorescent probe, we showed that DD could induce nitric oxide (NO) accumulation in F. saprophila cells. Cells exposed to a NO donor presented reduced adhesion and motility, which suggests the involvement of this cellular messenger in the mode of action of DD. Short-term experiments in microcosms showed that the presence of DD on a substrate strongly inhibited biofilm formation. Moreover, when the biofilm was bispecific, DD modified the proportion of the two species present. This indicates that the presence of DD-producing diatoms in a biofilm may favor the presence of certain microalgae at the expense of others. In addition to the effects on adhesion and motility, DD induced the formation of aggregates of F. saprophila cells. Aggregation was independent of NO production. Complementary experiments were performed with two other benthic diatoms, Nitzschia palea and Mayamea atomus. They showed that the effects of DD on adhesion and aggregation were species-dependent.

Heteroaggregates of Polystyrene Nanospheres and Organic Matter: Preparation, Characterization and Evaluation of Their Toxicity to Algae in Environmentally Relevant Conditions.

The environmental fate and behavior of nanoplastics (NPs) and their toxicity against aquatic organisms are under current investigation. In this work, relevant physicochemical characterizations were provided to analyze the ecotoxicological risk of NPs in the aquatic compartment. For this purpose, heteroaggregates of 50 nm polystyrene nanospheres and natural organic matter were prepared and characterized. The kinetic of aggregation was assimilated to a reaction-limited colloid aggregation mode and led to the formation of heteroaggregates in the range of 100-500 nm. Toxicities of these heteroaggregates and polystyrene nanospheres (50 and 350 nm) were assessed for a large range of concentrations using four benthic and one planktonic algal species, in regards to particle states in the media. Heteroaggregates and nanospheres were shown to be stable in the exposure media during the ecotoxity tests. The algal species exhibited very low sensitivity (growth and photosynthetic activity), with the noteworthy exception of the planktonic alga, whose growth increased by more than 150% with the heteroaggregates at 1 µg L-1. Despite the lack of a strong direct effect of the NPs, they may still impair the functioning of aquatic ecosystems by destabilizing the competitive interactions between species. Moreover, further work should assess the toxicity of NPs associated with other substances (adsorbed pollutants or additives) that could enhance the NP effects.

Chemical interactions in diatoms: role of polyunsaturated aldehydes and precursors.

Chemicals produced by aquatic organisms, and especially micro-organisms, have received increasing attention in the last decade for their role in shaping interactions and communities. Several cases emphasize the fact that chemical signals or defence may modulate interspecific interactions. Notably, it has been shown that diatoms, unicellular algae and key primary producers in aquatic ecosystems produce a wide range of bioactive metabolites. Among these compounds, polyunsaturated short-chain aldehydes in vitro strongly impair the reproduction of various potential grazers. In the field, the relationship between aldehyde production and reproductive failure in copepods remains unclear. Recent studies have suggested that these putative defence compounds may also be involved in intercellular communication and in interactions with competitors. Potential effects of the aldehyde precursors on various organisms have also been described. This review presents an overview of various results obtained in the last decade that could help us to understand the role of polyunsaturated aldehydes and their precursors in the ecology of diatoms. It is focused on the dichotomy between freshwater and marine environments. Indeed, most of the results on anti-proliferative aldehydes concern marine planktonic diatoms, whereas they are also known to be produced by benthic and freshwater species.

Interactive Impacts of Silver and Phosphorus on Autotrophic Biofilm Elemental and Biochemical Quality for a Macroinvertebrate Consumer.

Autotrophic biofilms are complex and fundamental biological compartments of many aquatic ecosystems. In particular, these biofilms represent a major resource for many invertebrate consumers and the first ecological barrier against toxic metals. To date, very few studies have investigated the indirect effects of stressors on upper trophic levels through alterations of the quality of biofilms for their consumers. In a laboratory study, we investigated the single and combined effects of phosphorus (P) availability and silver, a re-emerging contaminant, on the elemental [carbon (C):nitrogen (N):P ratios] and biochemical (fatty acid profiles) compositions of a diatom-dominated biofilm initially collected in a shallow lake. We hypothesized that (1) P and silver, through the replacement of diatoms by more tolerant primary producer species, reduce the biochemical quality of biofilms for their consumers while (2) P enhances biofilm elemental quality and (3) silver contamination of biofilm has negative effects on consumers life history traits. The quality of biofilms for consumers was assessed for a common crustacean species, Gammarus fossarum, by measuring organisms' survival and growth rates during a 42-days feeding experiment. Results mainly showed that species replacement induced by both stressors affected biofilm fatty acid compositions, and that P immobilization permitted to achieve low C:P biofilms, whatever the level of silver contamination. Gammarids growth and survival rates were not significantly impacted by the ingestion of silver-contaminated resource. On the contrary, we found a significant positive relationship between the biofilm P-content and gammarids growth. This study underlines the large indirect consequences stressors could play on the quality of microbial biomass for consumers, and, in turn, on the whole food web.

Colony induction and growth inhibition in Desmodesmus quadrispina (Chlorococcales) by allelochemicals released from the filamentous alga Uronema confervicolum (Ulotrichales).

In biofilms, the competition between microorganisms for light, nutrients and space is extreme. Moreover, planktonic algae can be considered as competitors insofar as they decrease the available light for the benthic algae. One of the strategies employed by microorganisms to eliminate competitors is the release of inhibiting compounds, a process known as allelopathy. Here we demonstrate that a benthic/epiphytic alga, Uronema confervicolum, produces allelopathic compounds that induce oxidative stress and growth inhibition in the planktonic Desmodesmus quadrispina. Some of these compounds can also trigger the formation of colony in D. quadrispina. As colonies have higher sedimentation rates than unicells, their induction by U. confervicolum might decrease shading. This study is the first report of colony induction in the context of alga-alga interaction. Our results also suggest the implication of mitogen-activated protein (MAP) kinases in the transduction of the signal leading to the formation of reactive oxygen species in the cells. A comparison with allelochemicals from another planktonic green alga, Monoraphidium aff. dybowski, emphasizes the specificity of colony induction by U. confervicolum, in contrast with oxidative stress which is induced by several compounds. The reciprocal production of inhibiting compounds by D. quadrispina makes this interaction an interesting example of co-evolution between two microorganisms belonging to different compartments of the ecosystem.

Nutrient effects on the genetic and functional diversity of aquatic bacterial communities.

Studies on the effects of biodiversity on ecosystem functioning have generally revealed a positive asymptotic relationship between biodiversity and single functions, suggesting species redundancy with respect to these functions. However, most research was performed on specific processes and did not consider ecosystem 'multifunctionality'. There is also little information on the relationship between genetic and functional diversity. To analyze this relationship, we performed a microcosm experiment on a complex lake assemblage of decomposers, in the presence of the green alga Scenedesmus obliquus, which acted as carbon source for decomposers. By manipulating nutrient enrichment and the N : P input ratio, we observed that the structures of particle-associated and free bacterial assemblages were highly predictable in response to stoichiometric constraints. For a given treatment, the taxonomic compositions of free and particle-associated bacterial communities appeared close to each other only when phosphorus was not depleted. A coinertia analysis revealed a clear coupling between the genetic diversity of the microbial community, assessed using PCR-denatured gradient gel electrophoresis, and its potential functional diversity, studied with Biolog Ecoplates. This suggests that an ecologically relevant fraction of bacterial communities is characterized by lower level of redundancy than frequently thought, highlighting the necessity of exploring further the role of biodiversity in multifunctionality within ecosystems.

A long-term copper exposure on freshwater ecosystem using lotic mesocosms: Primary producer community responses.

Copper is commonly used as an algicide and plant herbicide in the aquatic environment. Despite of its frequent application in rivers, ponds and lakes, few studies have been performed at the ecosystem level on primary producers' communities. A long-term lotic mesocosms study was carried on, in 20m long channels, under environmentally realistic concentrations of copper (0, 5, 25 and 75 microgL(-1)) delivered continuously for 18 months. Results showed significant effects at the 25 and 75 microgL(-1) copper treatment levels on the phytoplankton, periphyton and macrophyte community structure. Effects on biomass and abundance were dissimilar depending on the studied community. Phytoplankton showed no abundance variation with treatment, whereas periphyton had higher biomass and macrophytes had lower coverage at 25 and 75 microgL(-1). Taxa richness was significantly affected for all primary producers communities at 25 and 75 microgL(-1). Some taxa seemed to be sensitive to copper, i.e. Cocconeis spp., Epithemia sp., Gomphonema spp., Lemna minor, Nasturtium officinale and filamentous macroalgae. Other taxa appeared to be tolerant or facilitated by indirect effects, i.e. Leptolyngbya sp., Mougeotia sp., Nitzschia palea, Pseudanabaena sp. and Ulothrix sp. and therefore increased in the 25 and 75 microgL(-1) treatments. Important indirect factors influenced the community responses, such as trophic interactions like decrease of grazing pressure, competition for light and nutrients and temperature favouring blue-green algae development. After 18 months of copper exposure, the no observed effect concentration at community level (NOEC(community)) for primary producers was set up at 5 or 4mugL(-1), if we consider the average effective concentration, with a lowest observed effect concentration (LOEC(community)) at 25 or 20 microgL(-1) (AEC).

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.

Assessment of self-organizing maps to analyze sole-carbon source utilization profiles.

The use of community-level physiological profiles obtained with Biolog microplates is widely employed to consider the functional diversity of bacterial communities. Biolog produces a great amount of data which analysis has been the subject of many studies. In most cases, after some transformations, these data were investigated with classical multivariate analyses. Here we provided an alternative to this method, that is the use of an artificial intelligence technique, the Self-Organizing Maps (SOM, unsupervised neural network). We used data from a microcosm study of algae-associated bacterial communities placed in various nutritive conditions. Analyses were carried out on the net absorbances at two incubation times for each substrates and on the chemical guild categorization of the total bacterial activity. Compared to Principal Components Analysis and cluster analysis, SOM appeared as a valuable tool for community classification, and to establish clear relationships between clusters of bacterial communities and sole-carbon sources utilization. Specifically, SOM offered a clear bidimensional projection of a relatively large volume of data and were easier to interpret than plots commonly obtained with multivariate analyses. They would be recommended to pattern the temporal evolution of communities' functional diversity.

Analysis of bacterial diversity in river biofilms using 16S rDNA PCR-DGGE: methodological settings and fingerprints interpretation.

Reliability of bacterial diversity assessment using polymerase chain reaction (PCR) denaturing gradient gel electrophoresis (DGGE) analysis of 16S rDNA fragments was evaluated for a particular complex microbial assemblage: river epilithic biofilm. By comparing 3 routine protocols on replicates of one river biofilm sample, we found that common DNA extraction procedures gave comparable diversity (from 28.0 to 30.7 bands detected) and community composition (> 75% of homology) despite differences in the total amount of extracted DNA (from 0.9 to 4.2 microg). Therefore methodological improvements only concerned electrophoretic separation of DNA fragments (range of denaturing gradient from 35% to 70% and migration time=18h) and standardisation of DNA amounts used (PCR-template=50 ng, gel loading=700 ng). Using such a standardised methodology we found a good reproducibility of all steps of the procedure. When an Escherichia coli strain was introduced as a contaminant in a biofilm sample, we were able to recover ribotypes from the strain. As concerns fields sampling, a satisfactory repeatability of banding patterns from neighbouring pebbles (sampling point) allowed discriminating between the biofilm intrasite variability (various points from a cross-profile). These trials confirmed that PCR-DGGE is suitable to assess a reliable genetic fingerprint of epilithic biofilms in the river. Phylogenetic analysis of 40 partial sequences of 16S rDNA from DGGE gels of two sets of river biofilms samples proved evidences for the retrieval of DNA fragments related to phototroph Eukarya. However, in both cases plastidial 16S rDNA represented less than 25% of the analysed operational taxonomic units. Taking into account that Cyanobacteria, as members of the Bacteria, were also detected, sequence analysis of relevant bands from the pattern is required to target "bacteria", i.e. the functional group of prokaryotic microorganisms to which one commonly refers as a key component in sustaining the nutrient turnover.

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.

Effects of toxic extracts and purified borbotoxins from Prorocentrum borbonicum (Dinophyceae) on vertebrate neuromuscular junctions.

Benthic dinoflagellates of the genus Prorocentrum are common in tropical and subtropical water and several species produce phycotoxins potentially involved in human toxic outbreaks. The toxic dinoflagellate Prorocentrum borbonicum collected at La Réunion Island (France) was cultured in laboratory. A crude extract of the organism displayed significant toxicity in mice characterized by progressive limb paralysis, severe dyspnea, and death, and the toxicity was retained, after partition, in the extract's butanol-soluble fraction (BSF). Electrophysiological experiments characterizing the fraction's effect on isolated vertebrate neuromuscular preparations revealed that it depolarizes the muscle membrane and reduces the driving force for endplate potentials (EPPs) evoked by nerve stimulation, blocking directly- and indirectly-elicited muscle twitches. The depolarization induced by P. borbonicum BSF was not due to Na(+) influx through voltage-dependent Na(+) channels, since tetrodotoxin neither prevented nor suppressed the depolarization. However, ouabain, a specific ligand of the Na/K ATPase, reduced the depolarization. These results suggest the presence of palytoxin-like compounds in the fraction. HPLC-MS and MS/MS analysis showed the presence of several toxins having identical UV absorbance, among which two new isomeric toxins, borbotoxin-A and -B, of molecular mass of 1037.6 Da were isolated. The purified borbotoxin-A, had no effect on the resting membrane potential of muscle fibers and did not affect directly-elicited muscle twitches. However, the toxin reduced nerve-evoked muscle twitches, in a dose-dependent manner, reduced EPPs' amplitudes and completely blocked miniature endplate potentials. These observations suggest that the main action of borbotoxin-A is to block post-synaptic nicotinic ACh receptors.

Impairment of benthic diatom adhesion and photosynthetic activity by 2E,4E-decadienal.

Within biofilms, microorganisms are exposed to a wide range of chemicals released by phototrophic organisms. Those chemicals are likely to influence the dynamics and functioning of biofilms. 2E,4E-decadienal (DD) is a polyunsaturated aldehyde produced by diatoms which is known to induce adverse effects in many aquatic organisms. It has been shown to inhibit the adhesion and motility of one benthic diatom. The aim of this article was to determine if the effects of DD on diatom adhesion were widespread and if it could affect biofilm formation and functioning. The adhesion of 5 of 10 benthic diatom strains was strongly inhibited at 2.5 µg ml(-1) DD. This indicates a high variability in diatom sensitivity to DD. Several experiments in microcosms showed that the presence of DD diffusing from a substrate decreased biofilm formation. This effect was dose-dependent and persisted for 72 h, though the molecule is highly volatile. Using a PHYTO-PAM fluorometer, we also showed that the effective quantum efficiency of charge separation of PSII of biofilms exposed to DD was negatively affected. This indicates a decrease in the efficiency of the photochemical processes. All these results suggest that the presence of DD-producing strains may have a significant impact on the composition and physiology of biofilms.