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The assessment of genetic diversity of the mtDNA ND4 gene in benzimidazolr-resistant and 11 Teladorsagia circumcinta populations from France and Morocco is reported. The findings indicate that no strong, recurrent bottlenecks occur during the acquisition of BZ resistance by a worm population.

The effects of temperature, light intensity, and quality on the growth of the cyanobacteria Planktothrix agardhii (Gomont) Anag. et Komárek and P. rubescens (Gomont) Anag. et Komárek were assessed in batch cultures. The relative competitiveness of the green-pigmented P. agardhii and the red-pigmented P. rubescens was evaluated in separate and mixed cultures, under different light intensities and qualities (green, red, and white), and at two different temperatures, chosen as representative of the natural conditions favoring the respective blooms of each species. In monocultures, the P. rubescens strain appeared to be particularly well adapted to low intensities of green light and displayed a strong photoinhibition under high irradiance levels. The P. agardhii strain appeared less specialized with regard to light quality and also less sensitive to photoinhibition at higher irradiances. In competition experiments, temperature (15°C vs. 25°C) was the most important parameter in determining relative fitness of the species and competitive success. At 15°C, P. rubescens appeared to be much more competitive than P. agardhii, while P. agardhii was more competitive at 25°C. Under low irradiance, however, the pigmentation of these strains was of primary importance in determining the outcomes of the competition experiments. On the basis of our experimental results and on field observations, we propose that the successful growth leading to the proliferation of these two differently pigmented strains may largely depend on the combined conditions of light and temperature. The two strains, being genetically close relatives, could therefore be considered as two ecotypes that are adapted to different light and temperature environments. Competition experiments showed that the combination of these parameters largely controls the success of one strain in comparison to the other. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

With the aim of explaining the variations in microcystin (MC) concentrations during cyanobacterial blooms, we studied several Microcystis aeruginosa populations blooming in different freshwater ecosystems located in the same geographical area. As assessed by real-time PCR, it appeared that the potentially MC-producing cells (mcyB+) were predominant (70 to 100%) in all of these M. aeruginosa populations, with the exception of one population in which non-MC-producing cells always dominated. Apart from the population in the Grangent Reservoir, we found that the proportions of potentially MC-producing and non-MC-producing cells varied little over time, which was consistent with the fact that according to a previous study of the same populations, the intergenic transcribed spacer (ITS) genotype composition did not change (38). In the Grangent Reservoir, the MC-RR variant was the dominant microcystin variant throughout the bloom season, despite changes in the ITS composition and in the proportions of mcyB cells. Finally, the variations in total MC concentrations (0.3 to 15μg liter-1) and in the MC cellular quotas (0.01 to 3.4 pg cell-1) were high both between and within sites, and no correlation was found between the MC concentrations and the proportion of mcyB+ cells. All of these findings demonstrate that very different results can be found for the proportions of potentially MC-producing and non-MC-producing cells and MC concentrations, even in M. aeruginosa populations living in more or less connected ecosystems, demonstrating the importance of the effect of very local environmental conditions on these parameters and also the difficulty of predicting the potential toxicity of Microcystis blooms. [ABSTRACT FROM AUTHOR]

Stream and lake ecosystems in agricultural watersheds are exposed to fungicide inputs that can threaten the structure and functioning of aquatic microbial communities. This research analyzes the impact of the triazole fungicide tebuconazole (TBZ) on natural biofilm and plankton microbial communities from sites presenting different degrees of agricultural contamination. Biofilm and plankton communities from less-polluted (LP) and polluted (P) sites were exposed to nominal concentrations of 0 (control), 2 and 20 μg TBZ L(-1) in 3-week microcosm experiments. Descriptors of microbial community structure (bacterial density and chlorophyll-a concentration) and function (bacterial respiration and production and photosynthesis) were analyzed to chart the effects of TBZ and the kinetics of TBZ attenuation in water during the experiments. The results showed TBZ-induced effects on biofilm function (inhibition of substrate-induced respiration and photosynthetic activity), especially in LP-site communities, whereas plankton communities experienced a transitory stimulation of bacterial densities in communities from both LP and P sites. TBZ attenuation was stronger in biofilm (60-75%) than plankton (15-18%) experiments, probably due to greater adsorption on biofilms. The differences between biofilm and plankton responses to TBZ were likely explained by differences in community structure (presence of extracellular polymeric substances (EPS) matrix) and microbial composition. Biofilm communities also exhibited different sensitivity levels according to their in-field pre-exposure to fungicide, with P-site communities demonstrating adaptation capacities to TBZ. This study indicates that TBZ toxicity to non-targeted aquatic microbial communities essentially composed by microalgae and bacteria was moderate, and that its effects varied between stream and lake microbial communities., (© 2013.)

The impact of climate change and of other anthropogenic pressures on the structure and composition of phytoplankton communities of large European rivers remains poorly documented. Here we report the findings of a study of the changes in the phytoplankton community of the middle segment of the river Loire over the past 24 years. An attempt is made to distinguish between the impact of changes acting at the local scale and that of those acting more globally. A dramatic reduction in phytoplankton abundance was observed, particularly in the mid -1990s; this was concomitant with an increase in the relative proportion of cyanobacteria. At the same time, the phytoplankton community displayed increasing richness and diversity, and little change in its size structure. All these changes seem to be related to local changes, in particular to the reduction in phosphorus concentrations, as well as to changes in climate, throughout modifications in the river discharge and water temperature. Interestingly, herbicide contamination also appeared to be of particular importance in explaining the unexpected increase in the proportion of cyanobacteria in the phytoplankton community after the 1990s. These findings suggest that combinations of numerous anthropogenic pressures acting at different spatial and temporal scales have led to a mix of predictable and unpredictable changes occurring in the phytoplankton community of the river Loire, with probable consequences for the trophic networks in this river., (© 2013 Blackwell Publishing Ltd.)

The evolution of benzimidazoles (BZ) resistance in Teladorsagia circumcincta was investigated in a controlled trial with lambs, submitted to different treatment regimens. Four paddocks were seeded with a T. circumcincta strain constituted by 25% of BZ-resistant nematodes. Ten permanent lambs were allocated to each paddock, from April to November in order to renew the contamination of pasture. Monthly, three tracer lambs were allocated in each paddock. BZ-resistant nematode frequency was determined (PCR diagnosis). The faecal egg count reduction test (permanent lambs) and the number of nematodes in lambs were also determined (permanent and tracer lambs). Four different regimens of treatments were performed: control, levamisole (a non-BZ drug), fenbendazole (a BZ drug), and an alternation of levamisole and fenbendazole every second treatment. The same protocol was repeated on two consecutive grazing seasons, increasing the number of treatments (3 in first year and 5 in second year). The proportions of BZ-resistant nematodes did not change during all the study in both the control and the levamisole paddocks, supporting an equal global fitness of BZ-resistant and susceptible nematodes. Thus, no reversion of BZ resistance is to be expected. In the alternated drug group and in the BZ treated group, BZ-resistant nematodes increased from 25% to 47% and to 78%, respectively. BZ resistance increased proportionally to the selective pressure (number of BZ treatments). The drug alternation is not a good solution to delay importantly the evolution of resistance when more than 25% of nematodes are BZ-resistant. This study is the first evaluation of BZ-resistance evolution (using individual genotyping) in controlled conditions. It showed that when a monogenic anthelmintic resistance is established at 25% in a sexually reproducing nematode population, it seems to be impossible to prevent its increase even when using limited number of BZ treatments., ((c) 2010 Elsevier B.V. All rights reserved.)

Cylindrospermopsis raciborskii, a potentially toxic blooming cyanobacterium (blue-green alga), responsible for public health problems in Australia, was identified in France in 1994 in a shallow pond south of Paris. A program monitoring the occurrence of C. raciborskii in this pond was conducted from July 1998 to October 1999. The phytoplankton assemblages were studied, and limnological parameters (water temperature, dissolved oxygen, pH, conductivity, and dissolved inorganic nutrients) were measured. By multivariate analysis (principal component analysis), we showed that sufficiently high temperatures to allow the germination of akinetes, relatively low nutrient concentrations (soluble reactive phosphorus with a mean concentration of 1 microM and nitrate between 0 and 5 microM, except in February 1999 (21 microM)) and a characteristic high and constant sulfate concentration (8981+/-471 microM) seemed to be the main factors involved in the proliferation of C. raciborskii in the "Francs-Pêcheurs" (FP) pond. In the light of these findings and of bibliographic data, C. raciborskii would seem to be characterized by good adaptability, but also by low competitiveness with other phytoplanktonic species in the temperate study area.

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 beta-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 beta-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 beta-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.

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., (Copyright 2000 Academic Press.)

We have developed a new molecular tool for the diagnosis of the benzimidazole (BZ)-susceptibility or resistance in Teladorsagia circumcincta a nematode parasite of small ruminants. This tool is based on the use of the PCR and allows the genotyping of resistant (rr) or susceptible (rS and SS) adult worms or larvae. By using four primers in the same reaction mixture, worms can be genotyped in regard to the mutation on the residue 200 (phenylalanine to tyrosine) of the beta-tubulin which is implicated in BZ resistance. A very high proportion of homozygous SS (Phe/Phe) individuals characterized the BZ susceptible populations, whereas a variable proportion of homozygous rr (Tyr/Tyr) individuals characterized the BZ resistant populations. A positive correlation was observed between the LD50 estimated by egg hatch assay, and the proportion of mutant homozygous individuals rr (Tyr/Tyr). Our PCR method allows the rapid genotyping of numerous worms and permits the detection of the first resistant individuals in a worm population.

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.

Four species of lungworms of the Metastrongylus genus are found sympatrically in the lungs of the wild boars (Sus scrofa L.) on the Chambord game reserve (France): Metastrongylus asymmetricus, M. confusus, M. pudendotectus, and M. salmi. These species are difficult to identify using morphological characters alone, and epidemiological evidence raises doubt about the existence of 4 distinct species. Two molecular approaches (sequencing of ribosomal DNA Internal Transcribed Spacer 2 [ITS 2] and Random Amplified Polymorphic DNA assay [RAPD]) were used to evaluate the genetic similarities between the 4 taxa. The ITS 2 sequences of M. salmi and M. confusus were identical, whereas the sequences of the other species were distinct. On the other hand, RAPD analysis indicated unambiguously that the 4 species are genetically different. These results reveal the limitation of using only 1 molecular approach for taxonomic studies and indicate the need to examine different regions (ribosomal, mitochondrial DNA) in the genome of a species in complement with morphological and epidemiological data.