Youness Ouahid

Physiological changes in Triticum durum, Zea mays, Pisum sativum and Lens esculenta cultivars, caused by irrigation with water contaminated with microcystins: a laboratory experimental approach.

The aim of the present study was to investigate the effect of exposure to a microcystin (MC)-containing extract from a cyanobacteria bloom on growth, development, mineral nutrient accumulation, and photosynthetic activity of Triticum durum, Zea mays, Pisum sativum and Lens esculenta cultivars. The MCs in the extract, identified by HPLC and/or mass spectrometry (MS) were: MC-RR, -LR, -YR, -(H4)YR, -WR, and -FR. Plant growth and development was tested along 30 exposure days. After this period, MC-extract caused a clear reduction in plant growth and productivity, as well as deleterious effects on development and Photosystem II activity, measured by Fv/Fm fluorescence. However, the chlorophyll (a + b) content hardly varied, and the accumulation of Na+, K+, Ca2+, P and N was enhanced. All the effects observed were plant species, MC concentration, and exposure-time dependent. Relative accumulation of each MC variant greatly varied among plant species and plant organ. The data obtained supports the idea that the use of surface water containing MCs for crop irrigation can affect both plant yield and quality, and secondly, that MC accumulation in edible plants might pose a potential risk for human and animal health, if the MC intake exceeded the recommended tolerable limits.

Identification of microcystins from three collection strains of Microcystis aeruginosa

Microcystins (MCs) are toxic cyclic heptapeptides produced by various cyanobacteria genera, especially Microcystis. We identified 10 out of 12 MCs produced by three Microcystis aeruginosa strains from cyanobacteria collections, UTEX 2666, UTEX 2670 and UAM 1303, by using two analytical methods: Matrix-assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF/MS) and HPLC Photodiode Array Detector coupled to a hybrid Quadrupole Time of Flight Mass Spectrometry (HPLC-PDA-QTOF/MS). MALDI-TOF/MS failed to detect non-polar MCs, such as MC-LY and MC-LW. HPLC-QTOF/MS permitted the accurate identification of most MCs present in methanolic extracts. Besides, three new MCs, namely: [D-Glu(OCH3)6, D-Asp3] MC-LAba, MC-YL and MC-YM were detected by HPLC-QTOF/MS.

Effect of different microcystin profiles on toxin bioaccumulation in common carp (Cyprinus carpio) larvae via Artemia nauplii

In this study, a 12-day growth trial was conducted to compare the effect of the variation in microcystin (MC) composition in two Microcystis aeruginosa bloom samples on the growth performance and MC accumulation/transfer in the common carp (Cyprinus carpio L.) larvae. Fish were fed Artemia salina nauplii that had been preexposed to extracts from two M. aeruginosa natural blooms with different microcystins (MCs) profiles. Bloom A had MC-LR as major toxin (74.05%) while bloom B had a diversity of MC (MC-RR; MC-(H4)YR; MC-YR; MC-LR; MC-FR; MC-WR) with no dominance of MC-LR. Newly-hatched Artemia nauplii were exposed separately to the two M. aeruginosa extracts A and B (100 microg L(-1)EqMC-LR) for 2h. The MC concentration in the nauplii was 73.60+/-7.88ngEqMC-LRg(-1)FW (n=4, mean+/-SE) for bloom A and 87.04+/-10.31ngEqMC-LRg(-1)FW for bloom B. These contaminated nauplii were given at the same ration to different groups (A and B) of fish larvae. Larval weight and length from day 9 were significantly different between groups A and B, and in both cases lower than that of a control group fed non-exposed nauplii. MCs accumulation by larvae, inversely correlated with the growth performance, was also significantly different between groups A and B (37.43+/-2.61 and 54.55+/-3.01ngEqMC-LRg(-1) FW, respectively, at the end of the experimental period). These results indicate that MC profile of a bloom may have differential effects on toxin accumulation/transfer and toxicity.

Detection of potentially producing cylindrospermopsin and microcystin strains in mixed populations of cyanobacteria by simultaneous amplification of cylindrospermopsin and microcystin gene regions.

Cyanobacterial blooms are frequently formed by heterogeneous populations of toxin-producing and non-producing strains. Microcystins (MC) and cylindrospermopsin (CYN) are the most representative cyanobacterial toxins. We have developed a multiplex PCR assay that allows simultaneous detection of MC(+) and/or CYN(+) strains in mixed populations of cyanobacteria. Various primer sets were designed using mcy and aoa gene sequences related with MC and CYN synthesis respectively, to amplify at the same time aoa and mcy sequences. Purified DNA, cultured cell mixtures and field samples with MC and CYN producing strains were used as DNA template. The results show: (i) the expected amplicons were only observed with toxic strains; (ii) cells were suitable as a source of purified DNA for the multiplex PCR; (iii) the assay could detect simultaneously 3 aoa and 3 mcy gene regions with mixed CYN(+) and MC(+) cyanobacteria cells. The method could be applied to environmental samples, allowing in a rapid, economical and easy way to detect simultaneously the presence of CYN(+) and MC(+) cyanobacteria in sestonic fractions of water samples.

Compensatory Growth Induced in Zebrafish Larvae after Pre-Exposure to a Microcystis aeruginosa Natural Bloom Extract Containing Microcystins

Early life stage tests with zebrafish (Danio rerio) were used to detect toxic effects of compounds from a Microcystis aeruginosa natural bloom extract on their embryolarval development. We carried out the exposure of developing stages of fish to complex cyanobacterial blooms containing hepatotoxic molecules - microcystins. Fish embryo tests performed with the bloom extract containing 3 mg·L−1 Eq microcystin-LR showed that after 24 h of exposure all fish embryos died. The same tests performed with other diluted extracts (containing 0.3, 0.1 and 0.03 mg·L−1 Eq microcystin-LR) were shown to have an influence on zebrafish development and a large number of embryos showed malformation signs (edema, bent and curving tail). After hatching the larvae were transferred to a medium without toxins to follow the larval development under the new conditions. The specific growth of the pre-exposed larvae was significantly more important than that of the control larvae. This may represent a compensatory growth used to reduce the difference in size with the control fish noted after hatching.

Typing of toxinogenic Microcystis from environmental samples by multiplex PCR

Microcystin (MC)-producing Microcystis strains from environmental samples were assessed by the simultaneous amplification of up to five DNA sequences, corresponding to specific regions of six mcy genes (mcyA, mcyB, mcyC, mcyD, mcyE and mcyG), codifying for key motifs of the non-ribosomal peptide synthetase and polyketide synthase of the microcystin synthetase complex. Six primer pairs with the same melting temperature, one of them of new design, were used. A crucial point for the good performance of the new multiplex PCR test was the concentration of each primer pair. In the test, cell suspensions from laboratory cultures, field colonies and blooms were directly used as DNA source. The results of the multiplex PCR were consistent with the toxinogenic character of the samples, as checked by high performance liquid chromatography and/or matrix-assisted laser desorption ionisation time-of-flight mass spectrometry. As a whole, the newly developed test could be used for a reliable, rapid and low-cost screening of potential MC-producing Microcystis in field samples, even scattered colonies.

Effects of the microcystin profile of a cyanobacterial bloom on growth and toxin accumulation in common carp Cyprinus carpio larvae

A 12 day growth trial was conducted to compare the effect of the variation in microcystins (MC) composition of two bloom samples of Microcystis aeruginosa on the growth performance and microcystin accumulation in common carp Cyprinus carpio larvae. Two M. aeruginosa natural bloom samples with different MC profiles were collected and larvae were exposed to cyanobacterial cells through their diet. Three diets, a basal control diet and two diets prepared from the basal diet plus the same toxins content (60 ng MC g(-1) diet) of each cyanobacterial bloom, were given at the same ration level to three groups of larvae during the experimental period. Larval mass and standard length from day 9 were significantly different between cyanobacterial treatments and in both cases lower than that of the control. The MC accumulation by larvae, inversely correlated with the growth performance, was also significantly different between cyanobacterial treatments (26.96 v. 17.32 ng g(-1) at the end of the experimental period). These results indicate that MC variants profile may have effects on the toxin uptake and toxicity. To date, this is the first laboratory study to show that fish accumulate MC depending on the toxin profile of the cyanobacterial bloom.

A single microcystin in a toxic Microcystis bloom from the river Río de la Plata (Argentina)

Microcystis is one of the most common bloom-forming cyanobacteria genera in diverse ecosystems. More than 80% of its strains are toxic, mainly due to their ability to produce metabolites known as microcystins (MC). Here we report on a M. aeruginosa bloom that appeared in the summer of 2001 at a site of the Río de la Plata, within the city of Buenos Aires. The symptoms in mice indicated that the bloom was hepatotoxic. LC-PDA analysis revealed a similar high concentration (0.9–1 mg g–1 d w) of just one MC in the bloom biomass during the 3-month study period. During this period most of the MC (ca. 98 %) was found intracellularly, and the content remained almost the same. The molecular mass of the bloom MC was 1036 Da, as deduced from LC-ESI-MS data. Fragment ion analysis by LC-IT-MS-MS allowed identifying 6 out of the 7 amino acids, as well as their position in the molecule. The molecular mass of the unidentified amino acid residue was 155 Da. According to the data obtained, the MC under study was MC-XR, X representing the unidentified amino acid. This is the first report both on the characterisation of MCs in an urban site of the Río de la Plata waters, and on an Argentinean bloom exhibiting only one MC variant.

The first detection of potentially toxic Microcystis strains in two Middle Atlas Mountains natural lakes (Morocco)

Aguelmam Azizgza (LAZ) and Dayet Afourgah (DAF) are two Moroccan natural lakes located in a humid hydrographic basin of the Middle Atlas Mountains. Both are considered important reservoirs of plant and animal biodiversity. In addition, they are extensively used for recreational and fishing activities and as a water source for irrigation of agricultural crops. Recurrent cyanobacteria scum episodes in the two water bodies have been reported, Microcystis being the main genus in the scums. Here, we report on the toxic potential of three Microcystisaeruginosa strains isolated from those lakes: Mic LAZ and Mic B7 from LAZ and Mic DAF isolated from DAF. The toxic potential was checked by their microcystin (MC) content and the presence of mcy genes involved in MC synthesis. The identification and quantification of MC variants were performed by high-performance liquid chromatography-photo-diode array. The detection of mcy genes was achieved by whole-cell multiplex PCR that allowed the simultaneous amplification of DNA sequences corresponding to specific mcy regions. MC content of cultured cells, as MC-LR equivalents per gram cell biomass, was slightly higher in Mic LAZ (ca. 860) than in Mic B7 (ca. 700) and Mic DAF (ca. 690). Four MC variants were identified in the three isolates: MC-WR, MC-RR, MC-DM-WR, and MC-YR. The presence of toxic Microcystis strains in the two studied lakes may be regarded as an environmental and health hazard, especially during periods of bloom proliferation. It would be recommended the use of two complementary techniques, as those utilized herein (HPLC and mcy detection) to alert on highly probable toxicity of such lakes.