Morphological, physiological and molecular responses of Nitzschia palea under cadmium stress.

The impact of cadmium on the diatom Nitzschia palea (Kützing) W. Smith 1856 was studied by examining the relation between valve deformities and response through biological processes and genetic expression. Cultures of N. palea were exposed to two Cd treatments (C1 = 2.4 ± 0.6 and C2 = 42.6 ± 4.2 µg Cd/L) along with a control (C0 = 0 µg Cd/L) for 28 days. Cadmium bioaccumulation, diatoms growth, photosynthetic efficiencies, valve deformities and genetic expression were investigated during the course of the experiment. Cadmium exposure had significant effects on bioaccumulation, growth, valve deformities and genetic expression. Maximal effects for all studied endpoints were recorded after 7 days of exposure for the C2 treatment, which corresponded to the sampling time and condition with maximum cadmium bioaccumulation. Abnormal raphe formations (deviation from its lateral position) were significantly more abundant in the C2 treatment compared to the control. Molecular responses were related to cadmium level based on the number of genes impacted, intensity of the response and the frequency of observations. The expression of genes involved in the regulation of mitochondrial metabolism, photosynthesis, oxidative stress and silica metabolism was affected by cadmium exposure.

Genomic Insights of Alnus-Infective Frankia Strains Reveal Unique Genetic Features and New Evidence on Their Host-Restricted Lifestyle.

The present study aimed to use comparative genomics to explore the relationships between Frankia and actinorhizal plants using a data set made of 33 Frankia genomes. The determinants of host specificity were first explored for "Alnus-infective strains" (i.e., Frankia strains belonging to Cluster Ia). Several genes were specifically found in these strains, including an agmatine deiminase which could possibly be involved in various functions as access to nitrogen sources, nodule organogenesis or plant defense. Within "Alnus-infective strains", Sp+ Frankia genomes were compared to Sp- genomes in order to elucidate the narrower host specificity of Sp+ strains (i.e., Sp+ strains being capable of in planta sporulation, unlike Sp- strains). A total of 88 protein families were lost in the Sp+ genomes. The lost genes were related to saprophytic life (transcriptional factors, transmembrane and secreted proteins), reinforcing the proposed status of Sp+ as obligatory symbiont. The Sp+ genomes were also characterized by a loss of genetic and functional paralogs, highlighting a reduction in functional redundancy (e.g., hup genes) or a possible loss of function related to a saprophytic lifestyle (e.g., genes involved in gas vesicle formation or recycling of nutrients).

Intra-population genomic diversity of the bloom-forming cyanobacterium, Aphanizomenon gracile, at low spatial scale.

Cyanobacteria are oxygenic photosynthetic bacteria that perform a substantial part of the global primary production. Some species are responsible for catastrophic environmental events, called blooms, which have become increasingly common in lakes and freshwater bodies as a consequence of global changes. Genotypic diversity is considered essential for marine cyanobacterial population, allowing it to cope with spatio-temporal environmental variations and to adapt to specific micro-niches in the ecosystem. This aspect is underestimated in the study of bloom development, however, and given little notice in studies of the ecology of harmful cyanobacteria. Here we compared the genomes of four strains of Aphanizomenon gracile, a species of filamentous toxinogenic cyanobacteria (Nostocales) found worldwide in fresh and brackish water. Millimeter-sized fascicles were isolated from a single water sample and have been maintained in culture since 2010. A comparative study revealed extensive heterogeneity in gene contents, despite similar genome size and high similarity indices. These variations were mainly associated with mobile genetic elements and biosynthetic gene clusters. For some of the latter, metabolomic analysis confirmed the production of related secondary metabolites, such as cyanotoxins and carotenoids, which are thought to play a fundamental role in the cyanobacterial fitness. Altogether, these results demonstrated that an A. gracile bloom could be a highly diverse population at low spatial scale and raised questions about potential exchanges of essential metabolites between individuals.

Use of polar organic chemical integrative samplers to assess the effects of chronic pesticide exposure on biofilms.

The responses of aquatic organisms to chronic exposure to environmental concentrations of toxicants, often found in mixtures, are poorly documented. Here passive sampler extracts were used in experimental contamination of laboratory channels, to investigate their effects on natural biofilm communities. A realistic mixture of pesticides extracted from Polar Organic Chemical Integrative Samplers was used to expose biofilms in laboratory channels to total pesticide concentrations averaging 0.5 ± 0.1 µg l⁻¹. The level of exposure was representative of field conditions in terms of relative proportions of the substances but the exposure concentration was not maintained (decreasing concentrations between contamination occasions). The impact on the structural as well as the functional characteristics of the autotrophic and heterotrophic components was determined, using biofilm grown in uncontaminated conditions (reference site) and in sites exposed to pesticides (contaminated site). The exposure imposed did not significantly modify the structure or functions of reference biofilms, nor did it modify tolerance as measured by mixture EC50 (EC50 mix). In contrast, the communities from the more contaminated downstream section lost tolerance following decreased dose exposure, but community composition remained fairly stable. Overall, these results indicate that low levels of contamination did not lead to strong changes in community structure, and 14-day changes in tolerance seemed to depend mainly on physiological adaptation, suggesting that other environmental factors or longer-lasting processes prevailed. This study reports the first attempt to use passive sampler extracts as a realistic composite contaminant for experimental exposure of biofilms, with promising perspectives in further ecotoxicology studies.

Pesticide toxicity towards microalgae increases with environmental mixture complexity.

Effect-directed analysis (EDA) aims at identifying the compound(s) responsible for toxicity in a complex environmental sample where several dozens of contaminants can be present. In this study, we used an environmental mixture extracted from the Polar Organic Chemical Integrative Sampler (POCIS) previously immersed downstream a landfill (River Ponteils, South West France), to perform an EDA approach using a microalgal bioassay based on the photosynthetic capacities of diatom (Nitzschia palea) cultures. Adverse effects on photosynthetic capacities were recorded when algae were exposed to the entire POCIS extract (> 85% inhibition at the highest concentration tested). This result was coherent with the detection of diuron and isoproturon, which were the 2 most concentrated herbicides in the extract. However, the EDA process did not allow pointing out the specific compound(s) responsible for the observed toxicity but rather suggested that multiple compounds were involved in the overall toxicity and caused mixture effects.

The success of the bloom-forming cyanobacteria Planktothrix: Genotypes variability supports variable responses to light and temperature stress.

Cyanobacterial blooms can modify the dynamic of aquatic ecosystems and have harmful consequences for human activities. Moreover, cyanobacteria can produce a variety of cyanotoxins, including microcystins, but little is known about the role of environmental factors on the prevalence of microcystin producers in the cyanobacterial bloom dynamics. This study aimed to better understand the success of Planktothrix in various environments by unveiling the variety of strategies governing cell responses to sudden changes in light intensity and temperature. The cellular responses (photosynthesis, photoprotection, heat shock response and metabolites synthesis) of four Planktothrix strains to high-light or high-temperature were studied, focusing on how distinct ecotypes (red- or green-pigmented) and microcystin production capability affect cyanobacteria's ability to cope with such abiotic stimuli. Our results showed that high-light and high-temperature impact different cellular processes and that Planktothrix responses are heterogeneous, specific to each strain and thus, to genotype. The ability of cyanobacteria to cope with sudden increase in light intensity and temperature was not related to red- or green-pigmented ecotype or microcystin production capability. According to our results, microcystin producers do not cope better to high-light or high-temperature and microcystin content does not increase in response to such stresses.

Dynamics of the Metabolome of Aliinostoc sp. PMC 882.14 in Response to Light and Temperature Variations.

Cyanobacteria are microorganisms able to adapt to a wide variety of environmental conditions and abiotic stresses. They produce a large number of metabolites that can participate in the dynamic adaptation of cyanobacteria to a range of different light, temperature, and nutrient conditions. Studying the metabolite profile is one way to understand how the physiological status of cells is related to their adaptive response. In this study, we sought to understand how the diversity and dynamics of the whole metabolome depended on the growth phase and various abiotic factors such as light intensity and temperature. The cyanobacterium, Aliinostoc sp. PMC 882.14, was selected for its large number of biosynthetic gene clusters. One group of cells was grown under normal conditions as a control, while other groups were grown under higher light or temperature. Metabolomes were analyzed by mass spectrometry (qTOF-MS/MS) combined with untargeted analysis to investigate metabolite dynamics, and significant variation was found between exponential and stationary phases, regardless of culture conditions. In the higher light group, the synthesis of several metabolites, including shinorine, was induced while other metabolites, such as microviridins, were synthesized under higher temperature conditions. Among highly regulated metabolites, we observed the presence of mycosporine-like amino acids (MAAs) and variants of somamides, microginins, and microviridins. This study demonstrated the importance of considering the physiological state of cyanobacteria for comparative global metabolomics and studies of the regulatory processes involved in production of specific metabolites. Our results also open up new perspectives on the use of organisms such as cyanobacteria for the targeted production of bioactive metabolites.

Light stress in green and red Planktothrix strains: The orange carotenoid protein and its related photoprotective mechanism.

Photosynthetic organisms need to sense and respond to fluctuating environmental conditions, to perform efficient photosynthesis and avoid the formation of harmful reactive oxygen species. Cyanobacteria have developed a photoprotective mechanism that decreases the energy arriving at the reaction centers by increasing thermal energy dissipation at the level of the phycobilisome, the extramembranal light-harvesting antenna. This mechanism is triggered by the photoactive orange carotenoid protein (OCP). In this study, we characterized OCP and the related photoprotective mechanism in non-stressed and light-stressed cells of three different strains of Planktothrix that can form impressive blooms. In addition to changing lake ecosystemic functions and biodiversity, Planktothrix blooms can have adverse effects on human and animal health as they produce toxins (e.g., microcystins). Three Planktothrix strains were selected: two green strains, PCC 10110 (microcystin producer) and PCC 7805 (non-microcystin producer), and one red strain, PCC 7821. The green strains colonize shallow lakes with higher light intensities while red strains proliferate in deep lakes. Our study allowed us to conclude that there is a correlation between the ecological niche in which these strains proliferate and the rates of induction and recovery of OCP-related photoprotection. However, differences in the resistance to prolonged high-light stress were correlated to a better replacement of damaged D1 protein and not to differences in OCP photoprotection. Finally, microcystins do not seem to be involved in photoprotection as was previously suggested.

Development of a new extraction method based on high-intensity ultra-sonication to study RNA regulation of the filamentous cyanobacteria Planktothrix.

Efficient RNA extraction methods are needed to study transcript regulation. Such methods must lyse the cell without degrading the genetic material. For cyanobacteria this can be particularly challenging because of the presence of the cyanobacteria cell envelope. The great breath of cyanobacterial shape and size (unicellular, colonial, or filamentous multicellular) created a variety of cell lysis methods. However, there is still a lack of reliable techniques for nucleic acid extraction for several types of cyanobacteria. Here we designed and tested 15 extraction methods using physical, thermic or chemical stress on the filamentous cyanobacteria Planktothrix agardhii. Techniques based on the use of beads, sonication, and heat shock appeared to be too soft to break the Planktothrix agardhii cell envelope, whereas techniques based on the use of detergents degraded the cell envelope but also the RNA. Two protocols allowed to successfully obtain good-quality RNA. The first protocol consisted to manually crush the frozen cell pellet with a pestle and the second was based on the use of high-intensity ultra-sonication. When comparing these two, the high-intensity ultra-sonication protocol was less laborious, faster and allowed to extract 3.5 times more RNA compared to the liquid nitrogen pestle protocol. The high-intensity ultra-sonication protocol was then tested on five Planktothrix strains, this protocol allowed to obtain >8.5 µg of RNA for approximatively 3.5 × 108 cells. The extracted RNA were characterized by 260/280 and 260/230 ratio > to 2, indicating that the samples were devoid of contaminant, and RNA Quality Number > to 7, meaning that the integrity of RNA was preserved with this extraction method. In conclusion, the method we developed based on high-intensity ultra-sonication proved its efficacy in the extraction of Planktothrix RNA and could be helpful for other types of samples.

Insights into the Diversity of Secondary Metabolites of Planktothrix Using a Biphasic Approach Combining Global Genomics and Metabolomics.

Cyanobacteria are an ancient lineage of slow-growing photosynthetic bacteria and a prolific source of natural products with diverse chemical structures and potent biological activities and toxicities. The chemical identification of these compounds remains a major bottleneck. Strategies that can prioritize the most prolific strains and novel compounds are of great interest. Here, we combine chemical analysis and genomics to investigate the chemodiversity of secondary metabolites based on their pattern of distribution within some cyanobacteria. Planktothrix being a cyanobacterial genus known to form blooms worldwide and to produce a broad spectrum of toxins and other bioactive compounds, we applied this combined approach on four closely related strains of Planktothrix. The chemical diversity of the metabolites produced by the four strains was evaluated using an untargeted metabolomics strategy with high-resolution LC-MS. Metabolite profiles were correlated with the potential of metabolite production identified by genomics for the different strains. Although, the Planktothrix strains present a global similarity in terms of a biosynthetic cluster gene for microcystin, aeruginosin, and prenylagaramide for example, we found remarkable strain-specific chemodiversity. Only few of the chemical features were common to the four studied strains. Additionally, the MS/MS data were analyzed using Global Natural Products Social Molecular Networking (GNPS) to identify molecular families of the same biosynthetic origin. In conclusion, we depict an efficient, integrative strategy for elucidating the chemical diversity of a given genus and link the data obtained from analytical chemistry to biosynthetic genes of cyanobacteria.

Improving Toxicity Assessment of Pesticide Mixtures: The Use of Polar Passive Sampling Devices Extracts in Microalgae Toxicity Tests.

Complexity of contaminants exposure needs to be taking in account for an appropriate evaluation of risks related to mixtures of pesticides released in the ecosystems. Toxicity assessment of such mixtures can be made through a variety of toxicity tests reflecting different level of biological complexity. This paper reviews the recent developments of passive sampling techniques for polar compounds, especially Polar Organic Chemical Integrative Samplers (POCIS) and Chemcatcher® and the principal assessment techniques using microalgae in laboratory experiments. The progresses permitted by the coupled use of such passive samplers and ecotoxicology testing as well as their limitations are presented. Case studies combining passive sampling devices (PSD) extracts and toxicity assessment toward microorganisms at different biological scales from single organisms to communities level are presented. These case studies, respectively, aimed (i) at characterizing the "toxic potential" of waters using dose-response curves, and (ii) at performing microcosm experiments with increased environmental realism in the toxicant exposure in term of cocktail composition and concentration. Finally perspectives and limitations of such approaches for future applications in the area of environmental risk assessment are discussed.

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.

Is the toxicity of pesticide mixtures on river biofilm accounted for solely by the major compounds identified?

Comparative effects of long-term exposure to Polar Organic Chemical Integrative Sampler (POCIS) extracts (PE) and to a reconstituted mixture based on the major compounds quantified in the PE were evaluated on river biofilm communities. The study aimed to characterize the effects of long-term and low-dose exposure to pesticides on natural biofilm communities and to evaluate if the effects due to PE exposure could be explained solely by the major compounds identified in the extracts. Biofilms from an uncontaminated site were exposed in artificial channels to realistic environmental concentrations using diluted PE, with the 12 major compounds quantified in the extracts (Mix) or with water not containing pesticides (Ctr). Significant differences between biofilms exposed to pesticides or not were observed with regard to diatom density, biomass (dry weight and ash-free dry mass), photosynthetic efficiency (ΦpsII) and antioxidant enzyme activities. After 14 days of exposure to the different treatments, the observed trend towards a decrease of mean diatom cell biovolumes in samples exposed to pesticides was related to the control biofilms' higher relative abundance of large species like Cocconeis placentula or Amphora copulata and lower relative abundance of small species like Eolimna minima compared to the contaminated ones. Principal component analyses clearly separated contaminated (PE and Mix) from non-contaminated (Ctr) biofilms; on the contrary, the analyses did not reveal separation between biofilms exposed to PE or to the 12 major compounds identified in the extract.

Environmental effects of realistic pesticide mixtures on natural biofilm communities with different exposure histories.

This study deals with the use of Polar Organic Chemical Integrative Sampler (POCIS) extracts to assess the impact of low-dose pesticide mixtures on natural biofilm communities originating from either a chronically contaminated or a reference field site. To investigate how natural biofilm communities, pre-exposed to pesticides in situ or not might respond to environmentally realistic changes in pesticide pressure, they were exposed to either clean water or to POCIS extracts (PE) in order to represent toxic pressure with a realistic pesticide mixture directly isolated from the field. The impacts of PE were assessed on structure, physiology and growth of biofilms. Initial levels of tolerance of phototrophic communities to PE were also estimated at day 0. PE exposure led to negative effects on diatom growth kinetics independently of in-field biofilm exposure history. In contrast, the impacts observed on dry weight, ash-free dry mass and algal fluorescence-related parameters followed different trends depending on biofilm origin. Exposure to PE induced changes in diatom assemblages for the biofilm originating from the reference field site with higher relative abundance of Eolimna minima and Nitzschia palea with PE exposure. Initial tolerance of phototrophic communities to PE was 8-fold higher for the biofilm originating from the chronically contaminated site compared to the reference field site. The use of POCIS extracts allowed us to highlight both chronic impacts of low doses of a mixture of pesticides on natural communities with regard to biofilm exposure history as well as initial levels of tolerance of phototrophic communities.

Development of q-PCR approaches to assess water quality: effects of cadmium on gene expression of the diatom Eolimna minima.

This study was undertaken to develop molecular tools to assess water quality using diatoms as the biological model. Molecular approaches were designed following the development of a rapid and easy RNA extraction method suited to diatoms and the sequencing of genes involved in mitochondrial and photosystem metabolism. Secondly the impact of cadmium was evaluated at the genetic level by q-PCR on 9 genes of interest after exposure of Eolimna minima diatom populations cultured in suspension under controlled laboratory conditions. Their growth kinetics and Cd bioaccumulation were followed. Population growth rates revealed the high impact of Cd at 100 µg/L with total inhibition of growth. These results are linked to the high bioaccumulation values calculated after 14 days of exposure, 57.0±6.3 µg Cd/g dw and 734.1±70 µg Cd/g dw for exposures of 10 and 100 µg Cd/L respectively. Genetic responses revealed the impact of Cd on the mitochondrial metabolism and the chloroplast photosystem of E. minima exposed to 10 and 100 µg Cd/L with induction of cox1, 12S, d1 and psaA after 7 days of exposure for the concentration of 100 µg Cd/L and of nad5, d1 and psaA after 14 days of exposure for both conditions. This is the first reported use of q-PCR for the assessment of toxic pollution on benthic river diatoms. The results obtained presage interesting perspectives, but the techniques developed need to be optimized before the design of new water quality diagnosis tools for use on natural biofilms.

Do inactivated microbial preparations improve life history traits of the copepod Acartia tonsa?

We have tested a microbial preparation with probiotic effects (PSI; Sorbial A/S DANISCO) on the calanoid copepod Acartia tonsa (Dana) development time and reproduction effectiveness in culture. The hypotheses were that PSI increases the productivity and quality of copepods in culture (increased egg production and hatching success, HS). This was carried out because the use of copepods as live prey in aquaculture could increase the number of fish successfully raised through their entire life cycle. However, the availability of copepods is limited by their difficulty to be effectively raised. Our results show that the addition of PSI to the algal food increased the individual size of the adult females and their egg production. The PSI, together with Rhodomonas salina, also increased the HS of the eggs produced by PSI-treated females. These effects were observed despite that the biochemical analysis of the PSI revealed that it is a nutritionally poor food lacking essential fatty acids, and hence it cannot be used alone to raise copepods but instead as a food additive. This is the first demonstration that the effectiveness of copepod culturing can be improved using microbial preparations as a food additive.