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International audience; Specific interactions have been highlighted betweencyanobacteria and chemotrophic bacteria within thecyanosphere, suggesting that nutrients recycling couldbe optimized by cyanobacteria/bacteria exchanges. Inorder to determine the respective metabolic roles ofthe cyanobacterial and bacterial consortia (micro-biome), a day–night metatranscriptomic analysis wasperformed onDolichospermumsp. (N2-fixer) andMicro-cystissp. (non N2-fixer) natural blooms occurringsuccessively within a Frenchperi-urban lake. The taxo-nomical and functional analysis of the metatran-scriptoms have highlighted specific association ofbacteria within the cyanosphere, driven by the cyano-bacteria identity, without strongly modifying the func-tional composition of the microbiomes, suggestingfunctional redundancy within the cyanosphere. More-over, the functional composition of these active com-munities was driven by the living mode. During the twosuccessive bloom events, it appeared that NH4+(newlyfixed and/or allochthonous) was preferentially trans-formed into amino acids for the both the microbiomeand the cyanobacteria, while phosphate metabolismwas enhanced, suggesting that due to a high cellulargrowth, P limitation might take place within the cyano-sphere consortium.

International audience; utrophication of Lake Victoria led to changes in its phytoplankton communities. However, different levels of eutrophication exist in the open lake and the bays, and between embayments. This study utilized spatial and temporal sampling of Napoleon Gulf and Murchison Bay, exhibiting different trophic conditions. Over one year, we investigated phytoplankton biomass, richness, diversity and dissimilarity, and related the dynamics of the dominant species to the limnological and climatic conditions. The results confirmed that Napoleon Gulf and Murchison Bay showed large differences in eutrophication status, with lower nutrient concentrations in Napoleon Gulf than in Murchison Bay, where a strong gradient was observed from inshore to offshore areas. These nutrient dynamics resulted in a 4 to 10 fold higher chlorophyll-a in Murchison Bay than in Napoleon Gulf. From the embayments, 135 phytoplankton taxa were recorded with no significant differences in alpha diversity. However, high dissimilarity in community structure was observed in beta diversity, mostly due to a turnover among the dominant toxigenic species. Thus, from a similar species pool, there was a shift in the dominant toxigenic cyanobacteria from Microcystis flos-aquae and M. aeruginosa in Murchison Bay, Dolichospermum circinale and Planktolyngbya circumcreta in Napoleon Gulf to D. circinale in the offshore stations. These cyanobacteria are toxigenic taxa with known health hazards. Using partial least square models, we showed that both climatic variables (e.g. wind, solar radiation) and levels of inorganic dissolved nutrients (e.g. SRP, NO3-, and NH4+) are the main drivers of differences and dominance in cyanobacteria communities in northern Lake Victoria.

International audience; Africa is experiencing high annual population growth in its major river basins. This growth has resulted in significant land use change and pollution pressures on the freshwater ecosystems. Among them, the Lake Victoria basin, with more than 42 million people, is a unique and vital resource that provides food and drinking water in East Africa. However, Lake Victoria (LV) has experienced a progressive eutrophication and substantial changes in the fish community leading to recurrent proliferation of water hyacinth and cyanobacteria. Based on an extensive literature review, we show that cyanobacterial biomasses and microcystin concentrations are higher in the bays and gulfs (B&Gs) than in the open lake (OL), with Microcystis and Dolichospermum as the dominant genera. These differences between the B&Gs and the OL are due to differences in their hydrological conditions and in the origins, type and quantities of nutrients. Using data from the literature, we describe the multiple ways in which the human population growth in the LV watershed is connected to the increasing occurrence of cyanobacterial blooms in the OL and B&Gs. We also described the consequences of cyanobacterial blooms on food resources and fishing and on direct water use and water supply of local populations, with their potential consequences on the human health. Finally, we discuss the actions that have been taken for the protection of LV. Although many projects have been implemented in the past years in order to improve the management of waste waters or to reduce deforestation and erosion, the huge challenge of the reduction of cyanobacterial blooms in LV by the control of eutrophication seems far from being achieved.

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International audience; High-throughput sequencing has given new insights into aquatic fungal community ecology over the last 10 years. Based on 18S ribosomal RNA gene sequences publicly available, we investigated fungal richness and taxonomic composition among 25 lakes and four rivers. We used a single pipeline to process the reads from raw data to the taxonomic affiliation. In addition, we studied, for a subset of lakes, the active fraction of fungi through the 18S rRNA transcripts level. These results revealed a high diversity of fungi that can be captured by 18S rRNA primers. The most OTU-rich groups were Dikarya (47%), represented by putative filamentous fungi more diverse and abundant in freshwater habitats than previous studies have suggested, followed by Cryptomycota (17.6%) and Chytridiomycota (15.4%). The active fraction of the community showed the same dominant groups as those observed at the 18S rRNA genes level. On average 13.25% of the fungal OTUs were active. The small number of OTUs shared among aquatic ecosystems may result from the low abundances of those microorganisms and/or they constitute allochthonous fungi coming from other habitats (e.g., sediment or catchment areas). The richness estimates suggest that fungi have been overlooked and undersampled in freshwater ecosystems, especially rivers, though they play key roles in ecosystem functioning as saprophytes and parasites.

International audience; Although they are widespread, diverse and involved in biogeochemical cycles, microbial eukaryotes attract less attention than their prokaryotic counterparts in environmental microbiology. In this study, we used publicly available 18S barcoding data to define biases that may limit such analyses and to gain an overview of the planktonic microbial eukaryotic diversity in freshwater ecosystems. The richness of the microbial eukaryotes was estimated to 100 798 operational taxonomic units (OTUs) delineating 1267 clusters or phylogenetic units (PUs, i.e. monophyletic groups of OTUs that are phylogenetically close). By summing the richness found in aquatic environments, we can predict the microbial eukaryotic richness to be around 200 000- 250 000 species. The molecular diversity of protists in freshwater environments is generally higher than that of the morphospecies and cultivated species catalogued in public databases. Amoebozoa, Viridiplantae, Ichthyosporea, and Cryptophyta are the most phylogenetically diverse taxa, and characterisation of these groups is still needed. A network analysis showed that Fungi, Stramenopiles and Viridiplantae play central role in lake ecosystems. Finally, this work provides guidance for compiling metabarcoding data and identifies missing data that should be obtained to increase our knowledge on microbial eukaryote diversity.

To overcome the limitations associated with studying the interactions between bacterial communities (BCs) and cyanobacteria in natural environments, we compared the structural and functional diversities of the BCs associated with fifteen non-axenic cyanobacterial strains in culture and two natural BCs sampled during cyanobacterial blooms. No significant differences in richness and diversity were found between the natural and cultivated BCs, although some of the cyanobacterial strains had been isolated 11 years earlier. Moreover, these BCs shared some similar characteristics, such as a very low abundance of Actinobacteria, but they display significant differences at the OTU level. Overall, our findings suggest that BCs associated with cyanobacteria in culture are good models to better understand the interactions between heterotrophic bacteria and cyanobacteria. Additionally, BCs associated with heterocystous cyanobacterial strains cultivated in Z8X culture medium without nitrate (Aphanizomenon-Dolichospermum) demonstrated significant differences compared to BCs associated with non-heterocystous strains cultivated in Z8 culture medium (Planktothrix-Microcystis) in terms of their composition and their ability to utilize different carbon sources, suggesting the potential influence of cyanobacterial metabolism and/or culture media on associated BCs. Finally, half of the dominant operational taxonomic units (OTUs) in these BCs were specifically associated with cyanobacteria or other phytoplankton, whereas the remaining OTUs were generally associated with ecosystems containing high Organic Matter (OM) content, such as sludge or intestines.

Glycogen is mainly found as the principal storage form of glucose in cells. Many bacteria are able to synthesize large amounts of glycogen under unfavorable life conditions. By combining infrared spectroscopy, single molecule force spectroscopy (SMFS) and immuno-staining technique, we evidenced that planktonic P. fluorescens (Pf) cells are also able to produce glycogen as an extracellular polymeric substance. For this purpose, Pf suspensions were examined at 3 and 21 h of growth in nutritive medium (LB, 0.5 g/L). The conformation of the extracellular glycogen, revealed through its infrared spectral signature, has been investigated by SMFS measurements using Freely Jointed Chain model. The analysis of force versus distance curves showed over growth time that the increase of glycogen production was accompanied by an increase in glycogen contour lengths and ramifications. These results demonstrated that the production of extracellular bacterial glycogen can occur even if the cells are not subjected to unfavorable life conditions.

The factors and processes driving cyanobacterial blooms in eutrophic freshwater ecosystems have been extensively studied in the past decade. A growing number of these studies concern the direct or indirect interactions between cyanobacteria and heterotrophic bacteria. The presence of bacteria that are directly attached or immediately adjacent to cyano-bacterial cells suggests that intense nutrient exchanges occur between these microorganisms. In order to determine if there is a specific association between cyanobac-teria and bacteria, we compared the bacterial community composition during two cyanobac-teria blooms of Anabaena (filamentous and N 2-fixing) and Microcystis (colonial and non-N 2 fixing) that occurred successively within the same lake. Using high-throughput sequencing, we revealed a clear distinction between associated and free-living communities and between cyanobacterial genera. The interactions between cyanobacteria and bacteria appeared to be based on dissolved organic matter degradation and on N recycling, both for N 2-fixing and non N 2-fixing cyanobacteria. Thus, the genus and potentially the species of cyanobacteria and its metabolic capacities appeared to select for the bacterial community in the phycosphere.

Ecosystem functioning depends on multiple interactions among physical, chemical and biological factors. Indeed, ecosystem processes (e.g., productivity and nutrient recycling) result directly from the diversity of functional traits in biotic communities, which in turn are determined by species composition and diversity. Species diversity results both from biotic introductions and from environmental pressures. As a result, changes in biodiversity in response to environmental selection pressures tend to have a direct impact on ecosystem processes. The relationships between biodiversity and ecosystem functioning have been the focus of numerous research studies for several years. Based on these studies, it appears that a low level of biodiversity allows an ecosystem to function effectively under constant conditions, but that greater biodiversity is called for in fluctuating environments. In this paper, we outline the concepts underlying the relationships between biodiversity and ecosystem functioning, and illustrate these concepts with examples from aquatic ecosystems. The importance of spatial scale will also be considered.

The resistance of gastro-intestinal nematodes of small ruminants (sheep and goat) to benzimidazole anthelmintic drugs seems to be linked primarily to a single mutation in the isotype 1 β-tubulin gene. This study was carried out to investigate the origin and diversity of benzimidazole-resistance alleles in trichostrongylid nematodes. We sequenced a 550 bp fragment of the isotype 1 β-tubulin gene from several benzimidazole-resistant Teladorsagia circumcincta populations isolated from dairy goat farms in the central and south-western France. We also sequenced the same β-tubulin fragment from Trichostrongylus colubriformis and Haemonchus contortus populations in south-western France. We found eight benzimidazole-resistance alleles in all T . circumcincta populations studied, six in H . contortus populations, and only one in T . colubriformis populations. In most cases, only one benzimidazole-resistance allele was present in T . circumcincta and H . contortus populations, but two alleles were found in a fewer number of them. Some T . circumcincta populations shared the same benzimidazole-resistance allele whereas some others had a specific benzimidazole-resistance allele. Similar findings were obtained for H . contortus . As no parasites are introduced once the flock of dairy goat farms has been constituted, these data indicate for the three studied species that rare pre-existing benzimidazole-resistance alleles already present before the isolation of populations had been selected. On the other hand, the fact that some benzimidazole-resistance alleles were specific to one population of T . circumcincta or H . contortus , seems to be in agreement with the hypothesis of the selection of spontaneous mutations. Thus, the origin of benzimidazole-resistance alleles in trichostrongylid nematodes seems to involve primarily the selection of rare alleles and possibly of spontaneous mutations.

[Departement_IRSTEA]Eaux [TR1_IRSTEA]BELCA; International audience; The pollution of lakes and rivers by pesticides is a growing problem worldwide. However, the impacts of these substances on microbial communities are still poorly understood, partly because next-generation sequencing (NGS) has rarely been used in an ecotoxicology context to study bacterial communities despite its interest for accessing rare taxa. Microcosm experiments were carried out to evaluate the effects of tebuconazole (TBZ) on the structure and composition of bacterial communities from two types of freshwater ecosystem (lakes and rivers) with differing histories of pollutant contamination (pristine vs. previously exposed sites). Pyrosequencing revealed that bacterial diversity was higher in the river than in the lakes and in previously exposed sites than in pristine sites. Lakes and river stations shared very few OTUs, and differences at the phylum level were identified between these ecosystems (i.e. the relative importance of Actinobacteria and Gammaproteobacteria). Despite differences between these ecosystems and their contamination history, no significant effect of TBZ on bacterial community structure or composition was observed. Compared to functional parameters that displayed variable responses, we demonstrated that a combination of classical methods and NGS is necessary to investigate the ecotoxicological responses of microbial communities to pollutants.

Publication online; International audience; Metatranscriptome analysis relates to the transcriptome of microbial communities directly sampled in the environment. Accessing the mRNA pool in natural bacterial communities presents some technical challenges such as the RNA extraction, rRNA depletion, and the choice of the high-throughput sequencing technique. The lack of technical details in scientific articles is a major problem to correctly obtained mRNA from a microbial community and thus the corresponding sequencing data. In our study, we present the methodological procedure that was developed in order to access to the metatranscriptome of the microbial communities during two cyanobacterial blooms successively occurring in a freshwater eutrophic lake. Each procedure step was detailed and discussed with regard to the choices and difficulties encountered and to the recent literature. Finally, the two major limits for metatranscriptomic approaches targeting bacterial communities from natural environments were (i) the removal of rRNA in order to increase the putative mRNA reads number after sequencing, and (ii) for most of the bacterial communities living in natural environments, the lack of reference genomes in databases that leads to the non-assignation of numerous reads. Once these challenges overcome, we managed to access putative mRNA of dominant species, i.e. cyanobacteria (from 6 to 72 % of mRNA assigned), and of the surrounding bacteria (from 1 to 5 % of mRNA assigned).

The use of microsatellites as highly polymorphic DNA markers for the typing of isolates of Aspergillus fumigatus was investigated. Four CA repeats were selected by screening an A. fumigatus DNA library with a (CA) 10 oligonucleotide. Primers flanking these CA repeats were designed to amplify each locus. One primer of each pair was labeled with a fluorophore, and the PCR products were analyzed with an automatic sequencer and the GeneScan software. For each primer set and for a given isolate, one band was detected and was assigned to an allele because A. fumigatus is haploid. With 50 clinical isolates, 50 environmental isolates, and 2 reference strains we obtained 12, 11, 10, and 23 different alleles for the four CA microsatellites, respectively (discriminatory power, 0.994). The results were identical by whatever DNA extraction technique was used. Interestingly, no clustering between environmental and clinical isolates was observed, suggesting that every isolate is potentially pathogenic. Microsatellite markers appear suitable for use in large epidemiological studies of invasive aspergillosis.

International audience; The cyanobacterium Microcystis aeruginosa is known to proliferate in freshwater ecosystems and to produce microcystins. It is now well established that much of the variability of bloom toxicity is due to differences in the relative proportions of microcystin-producing and non-microcystin-producing cells in cyanobacterial populations. In an attempt to elucidate changes in their relative proportions during cyanobacterial blooms, we compared the fitness of the microcystin-producing M. aeruginosa PCC 7806 strain (WT) to that of its non-microcystin-producing mutant (MT). We investigated the effects of two light intensities and of limiting and non-limiting nitrate concentrations on the growth of these strains in monoculture and co-culture experiments. We also monitored various physiological parameters, and microcystin production by the WT strain. In monoculture experiments, no significant difference was found between the growth rates or physiological characteristics of the two strains during the exponential growth phase. In contrast, the MT strain was found to dominate the WT strain in co-culture experiments under favorable growth conditions. Moreover, we also found an increase in the growth rate of the MT strain and in the cellular MC content of the WT strain. Our findings suggest that differences in the fitness of these two strains under optimum growth conditions were attributable to the cost to microcystin-producing cells of producing microcystins, and to the putative existence of cooperation processes involving direct interactions between these strains.

International audience; During cyanobacterial blooms, processes influencing the population dynamics of blooming species remain partially unexplained. To provide new information, we performed a high-frequency monitoring – every 2 days at six sampling points – of a Microcystis aeruginosa population blooming in a shallow lake. At each sampling date, there was no spatial heterogeneity in the ITS genotypic composition of the population and in the proportion of potentially microcystin-producing (mcyB+) cells, whereas high variations were recorded in cell abundances. In contrast, when looking at the temporal evolution of these parameters, the ITS genotypic composition of the population and in a lesser extent the percentage of mcyB+ cells displayed high variations during the growth phase of the bloom, but not during the plateau phase or the subsequent decline. This suggests that during the development of the bloom, there was no directional selection leading to the dominance of a restricted number of genotypes and that a balancing selection process permitted the maintenance of a high genetic diversity in the Microcystis population. Finally, no relationship was found between these variations occurring in the Microcystis population and those recorded for several environmental parameters, suggesting that many factors and processes interacting together might be involved in these variations.

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Editorial; International audience; Editorial for a special issue

Publisher Summary Cyanobacteria (or blue-green algae) are photosynthetic prokaryotes, which like the higher plants contain pigments that allow them to perform oxygenic photosynthesis. The morphology of cyanobacteria varies widely, and they include spherical, ovoid, and cylindrical unicellular species, as well as multicellular colonial and filamentous forms. In aquatic ecosystems, cyanobacteria can grow in the water column or live attached to rocks, sand, or plants. The parameters that can limit their growth are light, nutrients particularly, water temperature, and water column stability. The two main families of cyanotoxins that could potentially be used for chemical warfare are the hepatotoxins and the neurotoxins. Three families of toxins mainly target the liver: the Microcystins, which include more than 80 variants, the Nodularins, and the Cylindrospermopsins. Very few data are available about possible treatment protocols for use following cyanotoxin poisoning. Thus, in the case of the Microcystins, Rifampin seems to be an effective chemoprotectant and antidote against MCLR toxicity. Other drugs have also demonstrated protective effects, such as cyclosporin-A and silyramin. No terrorist attack involving cyanotoxins has been reported to date. However, cyanobacteria and their toxins cannot be discounted as a potential threat, because large quantities of toxins sometimes exist under natural conditions in aquatic ecosystems, and in most cases, it would be quite easy to extract them.

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.

International audience; The factors that control the production of microcystins (hepatotoxins) during cyanobacterial blooms, and the function of these metabolites remain largely unknown. In an attempt to provide answers to these questions, we compared the fitness of microcystin (MC)-producing and non-MC-producing Planktothrix agardhii strains under various experimental conditions. More specifically, we investigated the effects of temperature, light intensity and nitrate concentrations on several MC-producing and non-MC-producing strains in monoculture and competition experiments. In the monoculture experiments, no significant difference in cell growth rates was found for any of the environmental conditions tested. On the other hand, at the end of the competition experiments, we found that when the environmental conditions limited cell growth, MC-producing strains were clearly winning out over the non-MC-producing ones. This suggested that, under growth-limiting conditions, the benefits of producing MC outweigh the cost. Moreover, the reverse was found under non-growth-limiting conditions, suggesting that under environmental conditions that favour cyanobacterial growth, the cost of MC production must outweigh its benefits. These findings suggest that environmental factors may have an indirect effect on the MC production rate, and on the selection of MC-producing and non-MC-producing strains, via their direct impact on both the cell growth rate and the cell densities in the cultures. Several hypotheses have been advanced concerning the possible function of MCs, but none of them seems to be supported by our data.

Background The colonial cyanobacterium Microcystis proliferates in a wide range of freshwater ecosystems and is exposed to changing environmental factors during its life cycle. Microcystis blooms are often toxic, potentially fatal to animals and humans, and may cause environmental problems. There has been little investigation of the genomics of these cyanobacteria. Results Deciphering the 5,172,804 bp sequence of Microcystis aeruginosa PCC 7806 has revealed the high plasticity of its genome: 11.7% DNA repeats containing more than 1,000 bases, 6.8% putative transposases and 21 putative restriction enzymes. Compared to the genomes of other cyanobacterial lineages, strain PCC 7806 contains a large number of atypical genes that may have been acquired by lateral transfers. Metabolic pathways, such as fermentation and a methionine salvage pathway, have been identified, as have genes for programmed cell death that may be related to the rapid disappearance of Microcystis blooms in nature. Analysis of the PCC 7806 genome also reveals striking novel biosynthetic features that might help to elucidate the ecological impact of secondary metabolites and lead to the discovery of novel metabolites for new biotechnological applications. M. aeruginosa and other large cyanobacterial genomes exhibit a rapid loss of synteny in contrast to other microbial genomes. Conclusion Microcystis aeruginosa PCC 7806 appears to have adopted an evolutionary strategy relying on unusual genome plasticity to adapt to eutrophic freshwater ecosystems, a property shared by another strain of M. aeruginosa (NIES-843). Comparisons of the genomes of PCC 7806 and other cyanobacterial strains indicate that a similar strategy may have also been used by the marine strain Crocosphaera watsonii WH8501 to adapt to other ecological niches, such as oligotrophic open oceans.

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We investigated the environmental impact of diffuse pesticide pollution on natural river biofilms in the River Morcille, France, during 2 seasons in pristine and contaminated stations. Prokaryotic and eukaryotic community compositions were assessed by denaturing gradient gel electrophoresis, while microalgal community composition was determined by high pressure liquid chromatography pigment analysis. The sensitivity of microphytobenthos to the herbicide Diuron was investigated in the laboratory by short-term photosynthesis inhibition assays. Spatial changes in prokaryotic and eukaryotic species compositions were found in spring and in winter. Community structures were significantly different between contaminated areas and those that were uncontami- nated or less contaminated. Associated changes in biodiversity were not found. Community tolerance towards Diuron (based on EC50 values for photosynthesis) was significantly lower in upstream than in downstream photoautotrophic organisms. Pesticide concentration increased along the downstream gradient. These results strongly suggest contamination-driven changes in biofilm community struc- ture and in the tolerance of the photoautotrophic community, confirming the Pollution-Induced Community Tolerance (PICT) hypothesis.

We compared the genetic diversity of the 16S-23S spacer of the rRNA gene (ITS1) in benthic and pelagic colonies of the Microcystis genus isolated from two different sampling stations with different depths and at two different sampling times (winter and summer) in the French storage reservoir of Grangent. In all, 66 ITS1 sequences were found in the different clone libraries. The nucleotide diversity of all the sampled isolates were in the same range (average number = 0.022) regardless of their origin, showing that several clones are involved in the summer bloom event and contribute to the high biomass production. Phylogenetic study and analysis of molecular variance (AMOVA) revealed no obvious genetic differentiation between the benthic and pelagic isolates. This finding confirms that the Microcystis genus in this lake is characterized by having both a benthic phase in winter and spring allowing this organism to survive in unfavorable environmental conditions, and a pelagic phase in summer and autumn when environmental conditions allow them to grow in the water column. Finally, comparing these sequences with those available in the GenBank database showed that some highly conserved genotypes are found throughout the world.

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.

For the past 20 years, the increased development and routine application of molecular-based techniques has made it possible to carry out detailed evaluations of the biodiversity of aquatic microbial communities. It also offers great opportunities for finding out how this parameter responds to various environmental stresses. Most of these approaches involve an initial PCR amplification of a target, which is generally located within the ribosomal operon. The amplification is achieved by means of primers that are specific to the organisms of interest. The second step involves detecting sequence variations in the PCR fragments either by a cloning/sequencing analysis, which provides a complete characterization of the fragments, or by an electrophoretic analysis, which provides a visual separation of the mixture of fragments according to sequence polymorphism (denaturing or temperature gradient gel electrophoresis, single strand conformation polymorphism) or length polymorphism (terminal-restriction fragment length polymorphism, automated ribosomal intergenic spacer analysis). Other non-PCR-based methods are also commonly used, such as fluorescence in-situ hybridization and DNA re-association analysis. Depending on the technique used, the information gained can be quite different. Moreover, some of these analyses may be rather onerous in terms of time and money, and so not always suitable for screening large numbers of samples. The most widely used techniques are discussed in this paper to illustrate the principles, advantages and shortcomings of each of them. Finally, we will conclude by evaluating the techniques and discussing some emerging molecular techniques, such as real-time PCR and the microarray technique.

Between 1999 and 2002, a routine survey of water quality in the Lac du Bourget was performed to study the dynamics and microcystin (MC) production of Planktothrix rubescens. Using liquid chromatography coupled to diode array detection and mass spectrometry, we found that two main variants ([D-Asp3] and [D-Asp3, Dhb7] microcystin-RR) were produced. The proportion of these two variants was not influenced by the depth or season of sampling. Expressed in microcystin-LR equivalents, high microcystin concentrations were recorded from August to December each year, reaching values of up to 6.7 microg L-1. A significant correlation was found between the microcystin cell content and the cell densities of P. rubescens. Cellular quotas of microcystins ranged from 0.1 to 0.3 pg cell-1. Simultaneously, laboratory experiments were performed on a strain of P. rubescens isolated from the lake to assess the potential impact of various P-PO4 (3-) concentrations on intra- and extracellular microcystin production. Unlike natural populations, this strain only produced [D-Asp3] MC-RR. The intracellular microcystin content was similarly correlated to the cell density, but the cellular quota was slightly higher (0.3-0.7 pg cell-1) than in the natural population. Again, as in the natural population, a linear relationship was found between growth rate and microcystin production rate. These findings support the hypothesis that environmental factors, such as phosphate concentrations, have no direct impact on microcystin production by P. rubescens, but act indirectly by affecting growth rate.

International audience; Toxigenic cyanobacteria are photosynthetic prokaryotes that are most often recognized in marine and freshwater systems, such as lakes, ponds, rivers, and estuaries. When environmental conditions (such as light, nutrients, water column stability, etc.) are suitable for their growth, cyanobacteria may proliferate and form toxic blooms in the upper, sunlit layers. The biology and ecology of cyanobacteria have been extensively studied throughout the world during the last two decades, but we still know little about the factors and processes involved in regulating toxin production for many cyanobacterial species. In this minireview, we discuss these microorganisms, and more especially the toxins they produce, as a potential and important health risk for wild and domestic animals.

The genetic diversity of the mtDNA ND4 gene in 11 Teladorsagia circumcincta populations from France and Morocco was assessed by sequencing. Some of these nematode populations were resistant to benzimidazole (BZ) anthelmintics, while others were susceptible. The nucleotide diversity in all populations studied was very high, probably due to a high mutation rate in nematodes, but there was no significant difference between them. This suggests that no strong, recurrent bottlenecks occur during the acquisition of BZ resistance by a worm population. The conservation of genetic variations during the acquisition of BZ resistance is probably due to the fact that anthelmintic treatments do not kill all the susceptible adult worms and to the presence of numerous free-living larvae that are not submitted to this anthelmintic pressure. There was no genetic subdivision between worm populations on a small geographical scale (less than 200 km), but significant F S T s were found on a larger geographical scale. This kind of subdivision cannot be explained by different genetic flows between populations because all these populations were isolated from each other. This subdivision is probably due to the breeding management practices and the large size of these worm populations, which limit genetic drift.

We compared the species composition in phytobenthic communities at different sampling sites in a small French river presenting polluted and unpolluted areas. For each sampling point, the total DNA was extracted and used to construct an 18S rRNA gene clone library after PCR amplification of a ca 400 bp fragment. Phytobenthic community composition was estimated by random sequencing of several clones per library. Most of the sequences corresponded to the Bacillariophyceae and Chlorophyceae groups. By combining phylogenetic and correspondence analyses, we showed that our molecular approach is able to estimate and compare the species composition at different sampling sites in order to assess the environmental impact of xenobiotics on phytobenthic communities. Changes in species composition of these communities were found, but no evident decrease in the diversity. We discuss the significance of these changes with regard to the existing level of pollution and their impact on the functionality of the ecosystem. Our findings suggest that it is now possible to use faster molecular methods (DGGE, ARISA.) to test large numbers of samples in the context of ecotoxicological studies, and thus to assess the impact of pollution in an aquatic ecosystem.

This experiment was designed to determine the effects of under-dosing on the frequency of benzimidazole resistant allele in the nematode Teladorsagia circumcincta. Fenbendazole (FBZ) was tested at 1/32, 1/16, 1/8 and 1/4 of the recommended dose for sheep (5 mg/kg body weight). The fraction of the susceptible homozygote (SS), susceptible heterozygote (RS) and resistant homozygote (RR) genotypes were compared among FBZ dose groups to evaluate differences between SS and RS genotype selective advantage. Almost all SS genotype worms were eliminated by 1/4 of the FBZ recommended dose, whereas a significant fraction of the RS genotype worms survived treatment. The selective advantage was 4·5 times higher for the RS genotype. This selective advantage was determined at 1/4 of the manufacturer's recommended dose of FBZ. This value should be taken as an indictor of the selective advantage of RS over the SS genotype when lambs are under-dosed. A computer simulation was used to study the putative spread of anthelmintic resistance over a range of RS selective advantages (2, 4·5 and 10-fold), with two average sizes of individual host worm population (20 or 2000 worms/host) and two initial R allele frequencies (0·1% or 1%). In all situations, the lowest selective advantage of the RS genotype over the SS genotype was sufficient to promote the spread of resistance in susceptible populations. When the RS genotype had no selective advantage over the SS genotype, genetic drift almost always led to the loss of the R allele, except in the largest populations (average size = 2000 worms).

Molecular techniques are of growing importance in the study of anthelmintic resistance in trichostrongylid worm populations. A knowledge of the genetic determinants of benzimidazole (BZ) resistance has made it possible to construct a molecular tool for genotyping individual worms, in respect of mutation of the β-tubulin gene responsible for BZ resistance. This tool offers new possibilities in the diagnosis of BZ resistance, and also in the study of anthelmintic use and other breeding management factors that can affect the selection of BZ-resistant alleles in worm populations. New molecular methods have also made it possible to study the origin and diversity of BZ-resistant alleles in trichostrongylid populations. The results demonstrate the value of a multidisciplinary approach to the study of anthelmintic resistance, combining molecular, ecological and epidemiological techniques.

This report describes a molecular method for determining in a first step the generic composition of a nematode community and in a second step, the resistance of each species to benzimidazole (BZ). We first established a polymerase chain reaction (PCR) linked to a restriction fragment length polymorphism strategy using the isotype 1 beta-tubulin gene. This method overcame the limitations of morphological identification of larval stages of trichostrongylid nematode species. Geographically distant isolates from the three main gastrointestinal species in temperate zones, Teladorsagia circumcincta, Haemonchus contortus, and Trichostrongylus colubriformis, were distinguished using this method. We then used an allele-specific PCR (AS-PCR) to detect mutations of residue 200 of the beta-tubulin, which is implicated in BZ resistance. The sequences of several samples confirmed the BZ-resistance genotype determined by AS-PCR. The ability to process large numbers of samples simultaneously makes this PCR-based strategy particularly suitable for epidemiological studies. It may also be useful for monitoring the emergence of resistant alleles in nematode communities.

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SUMMARYThe 4 species of ruminants (dromedary, zebu cattle, sheep and goat) in arid areas of Mauritania harboured Haemonchus spp. as the most frequent internal parasite. This was a rare situation where the 3 putative species, H. longistipes (dromedary), H. placet (zebu cattle) and H. contortus (sheep and goat) occurred sympatrically. The study was undertaken on hosts slaughtered at the Nouakchott abattoir, on the basis of monthly collection of worms. The environment was very unfavourable to H. placei and unfavourable to H. contortus, as intensity of infection remained low throughout the year, whereas infection in the dromedary was 10 to 20-fold higher. The survival strategies during the long, dry season were different: the surviving stages were either 4th-stage larvae in digesta (dromedaries), 4th-stage larvae either in digesta or mucosae (cattle), or 4th-stage larvae in mucosae and few adults (sheep and goats). The prolificacy of female worms, indicative of the potential to contaminate pastures, was similar for all Haemonchus spp. in the rainy season. H. longistipes behave differently during the pre-rainy season as no increase of prolificacy could be demonstrated as observed in the other species. Traits of vulvar morphology are considered as markers of ecological adaptation and were studied. The knobbed and smooth female morphs (in equal proportions) were the most frequent in H. longistipes, the knobbed morph out-numbered the other morphs in H. placei, and all 3 morphs were present in sheep and goats with the linguiform form being predominant. Genetic characterization of the 3 species was performed by means of Random Amplified Polymorphic DNA (RAPD). Three groups were obtained from analysis of these data: 1 group with individuals of H. contortus, 1 group with individuals of H. placei, and 1 group with individuals of H. longistipes. This indicated that, although the 3 species were valid, H. contortus and H. placei were more similar. Intraspecific variability was 2-fold higher in H. contortus than in the 2 other species. The ecological, morphological and genetical studies showed that H. longistipes, H. placei and H. contortus could be arranged in increasing order of variability.

A genetic polymorphism study was performed in coccidia from the rabbit. A comparative analysis of the RAPD (random amplified polymorphic DNA)-generated fingerprints, using 11 arbitrary primers, was carried out (1) in nine Eimeria species (E. intestinalis, E. magna, E. piriformis, E. flavescens, E. vejdovskyi, E. coecicola, E. perforans, E. exigua, and E. media) and (2) in two strains of E. intestinalis and four strains of E. media originating from different geographic areas. For each of these four strains of E. media, three lines deriving from the multiplication of a single oocyst were compared. All the primers tested yielded about ten amplified fragments. The profiles obtained differed considerably according to the species; thus, it was not possible to establish a phylogeny. On the other hand, species-specific fingerprints were observed, showing that RAPD assays might be useful for diagnosis. In E. media, analysis of the RAPD products showed weak differences between each of the four strains but nevertheless allowed differentiation of the lines deriving from the multiplication of one oocyst. Similar results were obtained with three methods of analysis: correspondence analysis, the hierarchical unweighted pair-group method with arithmetic averages (UP-GMA), and parsimony analysis. RAPD proved to be a useful technique for these intraspecific studies.

The aim of this work was to evaluate random amplified polymorphic DNA (RAPD) as a source of markers for species identification and phylogenetic analysis of ruminant trichostrongylid nematodes. As these nematodes are often polymorphic, species identification may be difficult. We tested eight species and several of their morphs: Haemonchus contortus (three vulvar morphotypes: flap, smooth, and knobbed), Teladorsagia circumcincta, Ashworthius gagarini, Spiculopteragia boehmi, Ostertagia leptospicularis (and its morph Ostertagia kolchida), Cooperia oncophora (and its morph C. surnabada), Trichostrongylus colubriformis, and T. vitrinus. With five chosen 10-mer primers, genetic variations were assessed among individuals of each species or morphotype. In trichostrongylid nematodes, the identification of species is possible by means of RAPD on adult or larva DNA extracts, although the variability observed within species was very important for most species studied. The use of RAPD in phylogenetics studies is conversely questionable for this superfamily of parasitic nematodes. The interspecific distances were always larger than the intraspecific ones and did not vary much (between 0.8 and 0.9); they would not be of much use in the construction of a phylogenetic tree, at least for the species and the primers involved in this study.

The genetic diversity of two freshwater cyanobacteria, Planktothrix rubescens and P. agardhii, was studied by sequencing a 519 bp fragment of the 16S rRNA gene, a 550bp fragment of the psaAB locus, a 517bp fragment of the rbcLX locus and a 550bp fragment of the 16S-23S ITS. Most of the 19 isolates were obtained from five alpine lakes, except for four purchased from culture collections. There was no polymorphism between the P. rubescens/agardhii strains for the 16S rRNA gene (partial sequencing) and only a few polymorphic sites were found in the other sequences. These informative sites had a mosaic structure in the Planktothrix strains, suggesting the occurrence of recombination between them. In contrast, the population structure during a bloom appears to be clonal. The existence of recombinations between the strains of P. rubescens and P. agardhii and the 16S sequencing data suggest that they are conspecific.