Cécile Bernard

CYLINDROSPERMOPSIS RACIBORSKII (CYANOBACTERIA) INVASION AT MID-LATITUDES: SELECTION, WIDE PHYSIOLOGICAL TOLERANCE, ORGLOBALWARMING?1

The tropical bloom‐forming cyanobacterium Cylindrospermopsis raciborskii (Woloszynska) Seenaya et Subba Raju is causing increasing concern because of its potential toxicity and invasive behavior at mid‐latitudes. This species has recently been identified in several temperate areas and was first reported in France in 1994, but the mechanisms underlying this acclimation process remain to be elucidated. We performed a range of laboratory experiments in an attempt to identify the physiological characteristics that could account for this behavior. We investigated the three following hypotheses: 1) clones of C. raciborskii adapted to temperate climate have been selected as it advanced north, 2) C. raciborskii has high physiological tolerance that allows it to prosper in a wide range of conditions, and 3) changes inclimate (global warming) have favored the development of C. raciborskii in mid‐latitudes. Ten strains of C. raciborskii from Australia n=1), Brazil (2), France (2), Germany (1), Hungary (1), Mexico (1), and Senegal (2) were cultured at different temperatures and light intensities. The in vitro growth parameters (μ and Topt) were the same for the tropical and temperate strains. All 10 strains displayed positive net growth in a wide range of temperatures (from 20 to 35°C) and light intensities (from 30 to 400 μmol photons·m−2·s−1), with maximum growth rates at around 30°C and 80 μmol photons·m−2·s−1. This suggests that the colonization of mid‐latitudes by C. raciborskii may result from a combination of its ability to tolerate a rather wide range of climatic conditions and the global warming phenomenon, which provides this species with better environmental conditions for its growth.

FIRST EVIDENCE OF PALYTOXIN ANALOGUES FROM AN OSTREOPSIS MASCARENENSIS (DINOPHYCEAE) BENTHIC BLOOM IN SOUTHWESTERN INDIAN OCEAN1

Benthic dinoflagellates of the genus Ostreopsis Schmidt are common in tropical and subtropical water, and some species produce toxins potentially involved in human intoxication events. A benthic bloom of Ostreopsis mascarenensis Quod was observed near Rodrigues Island during a survey of benthic dinoflagellates in the southwestern Indian Ocean. The morphology of O. mascarenensis was studied by LM and SEM. Preliminary screening of a crude extract of an O. mascarenensis bloom revealed neurotoxicity in mice similar to that induced by palytoxin. After partition of the crude extract, the highest toxicity was retained in the butanol‐soluble fraction, which retained hemolytic activity suggestive of palytoxin analogues. Two new toxins, mascarenotoxin‐A and ‐B, were resolved from this fraction by HPLC coupled to a diode array detector. The closed mass spectrum profile and fragmentation pattern obtained by advanced nano–electrospray ionization quadrupole time‐of‐flight mass spectrometry between purified toxins and a reference palytoxin confirmed the mascarenotoxins as palytoxin analogues. These results were confirmed by tandem mass spectrometry with the identification of specific fragment ion m/z 327. An on‐line liquid chromatography protocol coupled to tandem mass spectrometry was developed for detection of these palytoxin analogues. The present study describes the first purification, chemical, and toxicological characterization of new palytoxin analogues isolated from a benthic bloom of O. mascarenensis. These results suggest that O. mascarenensis, which is largely distributed in the southwestern Indian Ocean, could be a source of palytoxin poisoning in this tropical area.

Genetic diversity of Cylindrospermopsis strains (cyanobacteria) isolated from four continents.

ABSTRACT The genetic diversity of Cylindrospermopsis strains (cyanobacteria) was examined using mainly the 16S-23S internally transcribed spacer (ITS1) sequences. Strains were grouped in three clusters: (i) America, (ii) Europe, and (iii) Africa and Australia. These results suggested a recent spread of Cylindrospermopsis across the American and European continents from restricted warm refuge areas instead of exchanges between continents. On the other hand, they also suggested a recent colonization of Australia by African strains.

Temporal Variations in the Dynamics of Potentially Microcystin-Producing Strains in a Bloom-Forming Planktothrix agardhii (Cyanobacterium) Population

ABSTRACT The concentration of microcystins (MCs) produced during blooms depends on variations in both the proportion of strains containing the genes involved in MC production and the MC cell quota (the ratio between the MC concentration and the density of cells with the mcyA genotype) for toxic strains. In order to assess the dynamics of MC-producing and non-MC-producing strains and to identify the impact of environmental factors on the relative proportions of these two subpopulations, we performed a 2-year survey of a perennial bloom of Planktothrix agardhii (cyanobacteria). Applying quantitative real-time PCR to the mcyA and phycocyanin genes, we found that the proportion of cells with the mcyA genotype varied considerably over time (ranging from 30 to 80% of the population). The changes in the proportion of cells with the mcyA genotype appeared to be inversely correlated to changes in the density of P. agardhii cells and also, to a lesser extent, to the availability of certain nutrients and the abundance of cladocerans. Among toxic cells, the MC cell quota varied throughout the survey. However, a negative correlation between the MC cell quota and the mcyA cell number during two short periods characterized by marked changes in the cyanobacterial biomass was found. Finally, only 54% of the variation in the MC concentrations measured in the lake can be explained by the dynamics of the density of cells with the MC producer genotype, suggesting that this measurement is not a satisfactory method for use in monitoring programs intended to predict the toxic risk associated with cyanobacterial proliferation.

Suitability of the Neuro-2a cell line for the detection of palytoxin and analogues (neurotoxic phycotoxins)

Palytoxin and related compounds are neurotoxic phycotoxins produced by benthic microalgae belonging to the genus Ostreopsis. For several years this family of phycotoxins has been posing a threat to human health since they can bioaccumulate in shellfish. With the aim of replacing current biological assays, such as the mouse or hemolytic assays, we investigated using the Neuro-2a neuroblastoma cell line to detect palytoxin and related compounds. Cell death induced by the effects of PlTX and analogues on Na+, K+-ATPase were measured using the 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) assay for mitochondrial reductase activity as a surrogate for cell number. The specificity of the Neuro-2a cell-based assay for palytoxin detection was confirmed by using ouabain, which also acts on Na+, K+-ATPase. Pre-treatment of the Neuro-2a cells with ouabain minimizes the effects of palytoxin. The specificity of the Neuro-2a assay was confirmed by the finding that cell death was not detected when Neuro-2a cells were exposed to other phycotoxins with unrelated cellular targets. When the Neuro-2a assay was used to detect palytoxin in mussel extracts spiked with levels of palytoxin around the proposed regulatory value of 250 microg palytoxin/kg shellfish, a good correlation was observed between the levels found and the expected values. We conclude by proposing an experimental design for functional assays using the Neuro-2a cell line for the specific detection of four neurotoxic phycotoxin families: saxitoxins, brevetoxins, ciguatoxins and palytoxins.

Localization of microcystin-LR in medaka fish tissues after cyanotoxin gavage.

Microcystins (MCs) are toxic monocyclic heptapeptides produced by many cyanobacteria. Over 70 MCs have been successfully isolated and identified, of which MC-LR is the most commonly occurring toxin. Microcystins, especially MC-LR, cause toxic effects in mammals, birds and fish and are a recognized potent cause of environmental stress and pose a potential health hazard in aquatic ecosystems when heavy blooms of cyanobacteria appear. They also constitute a public health threat to people via drinking water and food chains. The concentrations of MC-LR can be very low, even in fish displaying severely disrupted tissues, which makes it essential to devise selective and sensitive histochemical methods for identifying and localizing MC-LR in target organs, such as liver and intestine. The aim of the study reported here was to analyze the presence of MC-LR in contaminated fish tissues using immunohistochemical methods. The present experiment involving subacute exposure confirmed our initial hypothesis that subacute and acute exposure to microcystin contamination can exacerbate physiological stress, induce sustained pathological damage, and affect the immune response in exposed medaka fish.

Collapse of a Planktothrix agardhii perennial bloom and microcystin dynamics in response to reduced phosphate concentrations in a temperate lake

Planktothrix agardhii dynamics, microcystin concentration and limnological variables were monitored every 2 weeks for 2 years (2004-2006) in a shallow hypereutrophic artificial lake (BNV, Viry-Châtillon, France). Time-series analysis identified two components in the P. agardhii biomass dynamics: (1) a significant decreasing trend in P. agardhii biomass (65% of the overall variance) and (2) a residual component without significant seasonal periodicity. A path-analysis model was built to determine the main factors controlling the P. agardhii dynamics over the period studied. The model explained 66% of P. agardhii biomass changes. The decreasing trend in P. agardhii biomass was significantly related to a decrease in the PO4(3-) concentration resulting from an improved treatment of the incoming watershed surface water. The residual component was related to zooplankton dynamics (cyclopoid abundances), supporting the hypothesis of a top-down control of P. agardhii, but only when the biomass was low. Forty-nine percent of the variability in the microcystin (MC) concentration (min:<0.1 microg equivalent MC-LR L(-1); max: 7.4 microg equivalent MC-LR L(-1)) could be explained by changes in the P. agardhii biomass. The highest toxin content was observed when P. agardhii biomass was the lowest, which suggests changes in the proportion of microcystin-producing and -nonproducing subpopulations and/or the physiological status of cells.

Application of the neuroblastoma assay for paralytic shellfish poisons to neurotoxic freshwater cyanobacteria: interlaboratory calibration and comparison with other methods of analysis.

Paralytic shellfish poisons (PSPs) are produced by freshwater cyanobacteria and pose a threat to human and animal drinking-water supplies. The wide range of toxin analogues (and the likelihood that further analogues remain to be discovered) means that chromatographic methods are not always reliable indicators of toxicity. Although the mouse bioassay remains the method of choice in the seafood industry, its use is increasingly being questioned on ethical grounds. The cell-based Neuro-2A neuroblastoma toxicity assay is an alternative bioassay validated for testing shellfish extracts, so it was of interest to determine its applicability with the different suite of toxin analogues produced by cyanobacteria. Cyanobacterial bloom samples from Australia, Brazil, and France were assayed using the neuroblastoma assay, liquid chromatography-tandem mass spectrometry (LC-MS/MS), high-performance liquid chromatography with postcolumn derivatization and fluorescence detection, and the Jellett Rapid Test for PSP. To assess interlaboratory variability, the neuroblastoma assay was set up in laboratories in Paris (France) and Adelaide (Australia). Neuroblastoma and chromatographic methods gave comparable results except in the case of the neurotoxic Brazilian samples: LC-MS/MS did not detect the putative new PSPs contained in these samples. Inter- and intralaboratory variability of the neuroblastoma assay was typical of biological assays but no greater than that found for interassay variability between different chromatographic determinations. The batch of Jellett Rapid Tests for PSP used did not yield quantitative results. Overall, the neuroblastoma assay was useful as a screening assay for determination of toxicity caused by saxitoxin neurotoxins in freshwater cyanobacteria, having the advantage of being sensitive to unidentified toxins that currently cannot be quantified by chromatographic means.

THE CONTRIBUTION OF SUB-SAHARAN AFRICAN STRAINS TO THE PHYLOGENY OF CYANOBACTERIA: FOCUSING ON THE NOSTOCACEAE (NOSTOCALES, CYANOBACTERIA)1

To date, phylogenies have been based on known gene sequences accessible at GenBank, and the absence of many cyanobacterial lineages from collections and sequence databases has hampered their classification. Investigating new biotopes to isolate more genera and species is one way to enrich strain collections and subsequently enhance gene sequence databases. A polyphasic approach is another way of improving our understanding of the details of cyanobacterial classification. In this work, we have studied phylogenetic relationships in strains isolated from freshwater bodies in Senegal and Burkina Faso to complement existing morphological and genetic databases. By comparing 16S rDNA sequences of African strains to those of other cyanobacteria lineages, we placed them in the cyanobacterial phylogeny and confirmed their genus membership. We then focused on the Nostocaceae family by concatenated analysis of four genes (16S rDNA, hetR, nifH, and rpoC1 genes) to characterize relationships among Anabaena morphospecies, in particular, Anabaena sphaerica var. tenuis G. S. West. Using a polyphasic approach to the Nostocaceae family, we demonstrate that A. sphaerica var. tenuis is more closely related to Cylindrospermospsis/Raphidiopsis than to other planktonic Anabaena/Aphanizomenon. On the basis of phylogeny and morphological data, we propose that these three significantly different clusters should be assigned to three genera.

Design and application of a stratified sampling strategy to study the regional distribution of cyanobacteria (Ile-de-France, France)

This study describes the design and application of a stratified sampling strategy of waterbodies to assess and analyze the distribution of cyanobacteria at a regional scale (Ile-de-France, IDF). Ten groups of hydrographical zones were defined within the IDF on the basis of their anthropogenic and geomorphologic characteristics. Sampling effort (n=50) was then randomly allocated according to the number of waterbodies in each group. This sampling strategy was tested in August 2006, using a field probe to estimate total phytoplankton as well as cyanobacteria biomasses. The sampled waterbodies exhibited a wide range of phytoplankton (< 1-375 microg equiv.Chla L(-1)) and cyanobacteria biomasses (< 1-278 microg equiv.ChlaL(-1)). 72% of the waterbodies in the IDF were classified as eutrophic (42% hypereutrophic), and 24% of the sites studied were dominated by cyanobacteria. Waterbodies connected to hydrographical networks (n=26) showed significantly higher total (p<0.0001; 3.5 times greater) and cyanobacterial (p<0.001, 3.2 times greater) biomasses than the isolated ones (n=24). No significant overall relationship was found through contingency analysis between waterbody trophic status and global land use categories (urban, periurban, and rural) within their hydrographical zones. However, concerning the waterbodies linked to hydrographical networks, the percentage of land covered by forest appeared as a good indicator of phytoplankton and cyanobacterial biomasses. This observation may be a consequence of lower amounts of nutrients being discharged into waterbodies from highly forested hydrological zone than from urban and/or agricultural areas. Our results illustrate a successful means of selecting representative waterbodies to conduct a regional assessment of cyanobacteria distribution using accessible GIS analyses.