M. Loudiki

Isolation, characterization and quantification of microcystins (heptapeptides hepatotoxins) in Microcystis aeruginosa dominated bloom of Lalla Takerkoust lake–reservoir (Morocco)

This paper presents the first data on the identification, characterization and quantification of microcystins isolated from both an extract of a cyanobacteria natural bloom, collected from a eutrophic Moroccan reservoir (Lalla Takerkoust, Marrakesh) and an isolated strain cultivated under laboratory conditions. The isolation and purification of toxins was performed by reverse phase HPLC and then characterized by amino acid analysis and fast atom bombardment mass spectrometry (FAB-MS). Chemical characterization of the toxins from the bloom revealed variants of microcystins such as Mcyst-LR, Mcyst-RR, Mcyst-YR and [D-Asp3]Mcyst-LR. However, the Microcystis aeruginosa strain produced only Mcyst-RR. Using an ELISA assay the total microcystin contents of eight bloom samples collected from 1994 to 1997 ranged from 0.7 to 8.8 microg/mg of lyophilized material. The two isolated Microcystis strains contained higher amounts of microcystins (0.65 microg/ mg of dry weight) than the Pseudanabaena strains (0.021 microg/mg of dry weight). Our results show that the presence of cyanobacteria toxins in water used for drinking in a North African country may be regarded as an health hazard. These results are a contribution to the knowledge of the biogeography of toxic cyanobacteria and their toxins, namely in north African countries.

Contributed Article Dynamics and toxicity of Anabaena aphanizomenoides (Cyanobacteria) waterblooms in the shallow brackish Oued Mellah lake (Morocco)

Abstract The structure and abundance of phytoplankton communities were investigated during 1997 to 1999 in Oued Mellah, a shallow brackish and hypertrophic lake, with particular regard to Anabaena aphanizomenoides dynamics. Important events of algal blooms were observed mostly by the cyanobacteria Microcystis ichthyoblabe, Anabaena aphanizomenoides and Oscillatoria chlorina and by the ichthyotoxic haptophyceae Prymnesium parvum. Anabaena aphanizomenoides proliferated during late summer after the Microcystis ichthyoblabe blooms. The percentage of Anabaena aphanizomenoides of the phytoplankton biomass varied from 88 to 94 percent during bloom periods. Maximum biomass was 146 and 120 mg fresh weight l−1 during the 1997 and 1999 summer periods, respectively. The main environmental factors leading to the ecological success of A. aphanizomenoides were high temperature (25–28ˆC), high incident light intensities (1488–1912 μ E m−2 s−1, high nutrient deficiency (0 μ g P-PO−4 l−1; 0–0.18 mg N-NO3 l−1) and decrease of alkalinity (329–494 mg HCO3 − l−1). The toxicity of the Anabaena aphanizomenoides bloom was evaluated by bioassays and analyses. The lethal dose50 of the bloom sample tested in mice was 254 mg DW kg−1 body weight while toxicity (24 h LC−50) in the brine shrimp Artemia salina was 3.68 mg DW ml−1. The low microcystin content (3.28 μ g g DW −1) determined by ELISA was not consistent with the tested bioassays and is suggestive of the presence of other toxic compounds in the bloom extracts. Four toxic fractions were separated by HPLC-PDA and identified as microcystins according to their UV spectra. The production of microcystins by Anabaena aphanizomenoides bloom was confirmed by the analysis of the isolated strain which, in Z8 medium under controlled laboratory conditions, produced three variants of microcystins, two of them being similar to those produced by the natural bloom.

Effect of light and temperature on the population dynamics of two toxic bloom forming Cyanobacteria – Microcystis ichthyoblabe and Anabaena aphanizomenoides

The effect of light and temperature on the growth of Microcystis ichthyoblabe and Anabaena aphanizomenoides, isolated from the subtropical Oued Mellah lake, Morocco (33°30′N–07°20′W), were investigated in batch culture. Growth rates at 66 light–temperature combinations were determined and fitted with different mathematical models. The results show that the two Cyanobacteria grow at all light intensities and temperatures, except at 10 °C for A. aphanizomenoides, where the growth was strongly limited. The μmax of M. ichthyoblabe increased with temperature from 0.56 d−1 at 10 °C to 1.32 d−1 at 35 °C. At all tested temperatures, a relative photoinhibition within the studied range of irradiance was observed and the photosensitivity was thermodependent. For Anabaena, the obtained μmax ranged between 0.07 d−1 at 10 °C and 1.46 d−1 at 35 °C, and a weak photoinhibition was observed at 15 °C. The positive correlation between μmax and Iopt (r2≥0.93) indicates a close interaction between light and temperature on the cyanobacteria growth. The results obtained in this work suggest that the growth of these two species is possible under low light and low temperature.

Growth responses of Microcystis ichthyoblabe Kützing and Anabaena aphanizomenoides Forti (cyanobacteria) under different nitrogen and phosphorus conditions

Experiments in batch cultures under controlled sub-optimal light and temperature conditions were undertaken to determine the effect of nitrogen, phosphorus and N:P ratios on the growth of Microcystis ichthyoblabe Kütz. 1843 and Anabaena aphanizomenoides Forti 1912, two toxic cyanobacteria forming blooms from Oued Mellah lake. Phosphorus experiments show that densities of M. ichthyoblabe and A. aphanizomenoides increased differently in the various media. Under non-limiting phosphorus conditions (1000–6960 μg P l−1), 5–7 days of exponential growth was observed, while in P-free and in P-deficient media (0–500 μg l−1), the growth was limited. As with phosphorus experiments, cell growth of M. ichthyoblabe was substantially favoured under high nitrate concentrations (50–84 mg l−1), whereas cultures under N-free or N-deficient conditions (0–10 mg l−1) seemed to be limited. Nitrate-nitrogen at all tested concentrations was not limiting for the growth of A. aphanizomenoides cultures, which reached high density during an exponential growth of 8–9 days. Under low nitrate concentrations (0–5 mg l−1), an increased number of heterocysts was observed. There was a markedly diminished growth with the lower N:P ratio experiments (≤5) only for Microcystis and on the highest N:P ratio experiments (≥30) for both Microcystis and Anabaena aphanizomenoides.

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.