Ramsy Agha

Phylogeography of cylindrospermopsin and paralytic shellfish toxin-producing Nostocales cyanobacteria from Mediterranean Europe (Spain)

ABSTRACT Planktonic Nostocales cyanobacteria represent a challenge for microbiological research because of the wide range of cyanotoxins that they synthesize and their invasive behavior, which is presumably enhanced by global warming. To gain insight into the phylogeography of potentially toxic Nostocales from Mediterranean Europe, 31 strains of Anabaena (Anabaena crassa, A. lemmermannii, A. mendotae, and A. planctonica), Aphanizomenon (Aphanizomenon gracile, A. ovalisporum), and Cylindrospermopsis raciborskii were isolated from 14 freshwater bodies in Spain and polyphasically analyzed for their phylogeography, cyanotoxin production, and the presence of cyanotoxin biosynthesis genes. The potent cytotoxin cylindrospermopsin (CYN) was produced by all 6 Aphanizomenon ovalisporum strains at high levels (5.7 to 9.1 μg CYN mg−1 [dry weight]) with low variation between strains (1.5 to 3.9-fold) and a marked extracellular release (19 to 41% dissolved CYN) during exponential growth. Paralytic shellfish poisoning (PSP) neurotoxins (saxitoxin, neosaxitoxin, and decarbamoylsaxitoxin) were detected in 2 Aphanizomenon gracile strains, both containing the sxtA gene. This gene was also amplified in non-PSP toxin-producing Aphanizomenon gracile and Aphanizomenon ovalisporum. Phylogenetic analyses supported the species identification and confirmed the high similarity of Spanish Anabaena and Aphanizomenon strains with other European strains. In contrast, Cylindrospermopsis raciborskii from Spain grouped together with American strains and was clearly separate from the rest of the European strains, raising questions about the current assumptions of the phylogeography and spreading routes of C. raciborskii. The present study confirms that the nostocalean genus Aphanizomenon is a major source of CYN and PSP toxins in Europe and demonstrates the presence of the sxtA gene in CYN-producing Aphanizomenon ovalisporum.

Multi-scale strategies for the monitoring of freshwater cyanobacteria: reducing the sources of uncertainty.

Cyanobacterial blooms are a frequent phenomenon in eutrophic freshwaters worldwide and are considered potential hazards to ecosystems and human health. Monitoring strategies based on conventional sampling often fail to cover the marked spatial and temporal variations in cyanobacterial distribution and fluctuating toxin concentrations inherent to cyanobacterial blooms. To deal with these problems, we employed a multi-scale approach for the study of a massive Microcystis bloom in Tajo River (Spain) utilizing 1) remote sensing techniques, 2) conventional water sampling and 3) analysis of chemotypical subpopulations. Tajo River at the study area is influenced by high temperatures waters diverted upstream from a nuclear power plant, the presence of a dam downstream and a high nutrient load, which provide optimal conditions for massive cyanobacterial proliferation. MERIS imagery revealed high Chl-a concentrations that rarely fell below 20 μg L(-1) and moderate spatiotemporal variations throughout the study period (March-November 2009). Although the phytoplanktonic community was generally dominated by Microcystis, sampling points highly differed in cyanobacterial abundance and community composition. Microcystin (MC) concentrations were highly heterogeneous, varying up to 3 orders of magnitude among sampling points, exceeding in some cases WHO guideline values for drinking and also for recreational waters. The analysis of single colonies by MALDI-TOF MS revealed differences in the proportion of MC-producing colonies among points. The proportion of toxic colonies showed a highly significant linear correlation with total MC: biovolume ratio (r(2) = 0.9; p < 0.001), evidencing that the variability in toxin concentrations can be efficiently addressed by simple analysis of subpopulations. We propose implementing a multi-scale monitoring strategy that allows covering the spatiotemporal heterogeneities in both cyanobacterial distribution (remote sensing) and MC concentrations (subpopulation analysis) and thereby reduce the main sources of uncertainty in the assessment of the risks associated to bloom events.

Diversity of toxin and non-toxin containing cyanobacterial mats of meltwater ponds on the Antarctic Peninsula: a pyrosequencing approach

Abstract Despite their pivotal role as primary producers, there is little information as to the diversity and physiology of cyanobacteria in the meltwater ecosystems of polar regions. Thirty cyanobacterial mats from Adelaide Island, Antarctica were investigated using 16S rRNA gene pyrosequencing and automated ribosomal intergenic spacer analysis, and screened for cyanobacterial toxins using molecular and chemical approaches. A total of 274 operational taxonomic units (OTUs) were detected. The richness ranged between 8 and 33 cyanobacterial OTUs per sample, reflecting a high mat diversity. Leptolyngbya and Phormidium (c. 55% and 37% of the OTUs per mat) were dominant. Cyanobacterial community composition was similar between mats, particularly those obtained from closely adjacent locations. The cyanotoxin microcystin was detected in 26 of 27 mats (10–300 ng g-1 organic mass), while cylindrospermopsin, detected for the first time in Antarctica, was present in 21 of 30 mats (2–156 ng g-1 organic mass). The latter was confirmed via liquid chromatography-mass spectrometry and by the presence of the cyrAB and cyrJ genes. This study demonstrates the usefulness of pyrosequencing for characterizing diverse cyanobacterial communities, and confirms that cyanobacteria from extreme environments produce a similar range of cyanotoxins as their temperate counterparts.

Oligopeptides as Biomarkers of Cyanobacterial Subpopulations. Toward an Understanding of Their Biological Role

Cyanobacterial oligopeptides comprise a wide range of bioactive and/or toxic compounds. While current research is strongly focused on exploring new oligopeptide variants and their bioactive properties, the biological role of these compounds remains elusive. Oligopeptides production abilities show a remarkably patchy distribution among conspecific strains. This observation has prompted alternative approaches to unveil their adaptive value, based on the use of cellular oligopeptide compositions as biomarkers of intraspecific subpopulations or chemotypes in freshwater cyanobacteria. Studies addressing the diversity, distribution, and dynamics of chemotypes in natural systems have provided important insights into the structure and ecology of cyanobacterial populations and the adaptive value of oligopeptides. This review presents an overview of the fundamentals of this emerging approach and its most relevant findings, and discusses our current understanding of the role of oligopeptides in the ecology of cyanobacteria.

First detection of cyanobacterial PSP (paralytic shellfish poisoning) toxins in Spanish freshwaters

Presence of the paralytic shellfish toxins (PSTs) saxitoxin, neosaxitoxin, decarbamoyl saxitoxin and gonyautoxin-5 was analyzed by mass spectrometry in 41 Spanish freshwaters and 13 strains of potential PST-producing planktonic cyanobacteria. Toxins were detected in five waterbodies, but were absent from the isolated strains. PST containing samples belonged to the same geographical region (South Western Spain) and were most likely related to the presence of the genus Aphanizomenon.

Effects of harmful cyanobacteria on the freshwater pathogenic free-living amoeba Acanthamoeba castellanii

Grazing is a major regulating factor in cyanobacterial population dynamics and, subsequently, considerable effort has been spent on investigating the effects of cyanotoxins on major metazoan grazers. However, protozoan grazers such as free-living amoebae can also feed efficiently on cyanobacteria, while simultaneously posing a major threat for public health as parasites of humans and potential reservoirs of opportunistic pathogens. In this study, we conducted several experiments in which the freshwater amoeba Acanthamoeba castellanii was exposed to pure microcystin-LR (MC-LR) and six cyanobacterial strains, three MC-producing strains (MC-LR, MC-RR, MC-YR, MC-WR, [Dha7] MC-RR) and three strains containing other oligopeptides such as anabaenopeptins and cyanopeptolins. Although the exposure to high concentrations of pure MC-LR yielded no effects on amoeba, all MC-producing strains inflicted high mortality rates on amoeba populations, suggesting that toxic effects must be mediated through the ingestion of toxic cells. Interestingly, an anabaenopeptin-producing strain caused the greatest inhibition of amoeba growth, indicating that toxic bioactive compounds other than MCs are of great importance for amoebae grazers. Confocal scanning microscopy revealed different alterations in amoeba cytoskeleton integrity and as such, the observed declines in amoeba densities could have indeed been caused via a cascade of cellular events primarily triggered by oligopeptides with protein-phosphatase inhibition capabilities such as MCs or anabaenopeptins. Moreover, inducible-defense mechanisms such as the egestion of toxic, MC-producing cyanobacterial cells and the increase of resting stages (encystation) in amoebae co-cultivated with all cyanobacterial strains were observed in our experiments. Consequently, cyanobacterial strains showed different susceptibilities to amoeba grazing which were possibly influenced by the potentiality of their toxic secondary metabolites. Hence, this study shows the importance of cyanobacterial toxicity against amoeba grazing and, that cyanobacteria may contain a wide range of chemical compounds capable of negatively affect free-living, herbivorous amoebae. Moreover, this is of high importance for understanding the interactions and population dynamics of such organisms in aquatic ecosystems.

Limited Stability of Microcystins in Oligopeptide Compositions of Microcystis aeruginosa (Cyanobacteria): Implications in the Definition of Chemotypes

The occurrence of diverse oligopeptides in cyanobacteria, including the cyanotoxins microcystins, has been recently used to classify individual clones into sub-specific oligopeptide chemotypes, whose composition and dynamics modulate microcystin concentrations in cyanobacterial blooms. Cyanobacterial chemotyping allows the study of the ecology of chemotypical subpopulations, which have been shown to possess dissimilar ecological traits. However, the stability of chemotypes under changing abiotic conditions is usually assumed and has not been assessed in detail. We monitored oligopeptide patterns of three strains of Microcystis aeruginosa under different nutrient and light conditions. MALDI-TOF MS revealed alterations in the microcystins signatures under N and P poor conditions and high light intensities (150 and 400 μmol photons m−2s−1). Variations in the general oligopeptide composition were caused by a gradual disappearance of microcystins with low relative intensity signals from the fingerprint. The extent of such variations seems to be closely related to physiological stress caused by treatments. Under identical clonal compositions, alterations in the oligopeptide fingerprint may be misinterpreted as apparent shifts in chemotype succession. We discuss the nature of such variations, as well as the consequent implications in the use of cyanobacterial chemotyping in studies at the subpopulation level and propose new guidance for the definition of chemotypes as a consistent subpopulation marker.