Cyanobacterial diversity held in microbial biological resource centers as a biotechnological asset: the case study of the newly established LEGE culture collection.

Cyanobacteria are a well-known source of bioproducts which renders culturable strains a valuable resource for biotechnology purposes. We describe here the establishment of a cyanobacterial culture collection (CC) and present the first version of the strain catalog and its online database (http://lege.ciimar.up.pt/). The LEGE CC holds 386 strains, mainly collected in coastal (48%), estuarine (11%), and fresh (34%) water bodies, for the most part from Portugal (84%). By following the most recent taxonomic classification, LEGE CC strains were classified into at least 46 genera from six orders (41% belong to the Synechococcales), several of them are unique among the phylogenetic diversity of the cyanobacteria. For all strains, primary data were obtained and secondary data were surveyed and reviewed, which can be reached through the strain sheets either in the catalog or in the online database. An overview on the notable biodiversity of LEGE CC strains is showcased, including a searchable phylogenetic tree and images for all strains. With this work, 80% of the LEGE CC strains have now their 16S rRNA gene sequences deposited in GenBank. Also, based in primary data, it is demonstrated that several LEGE CC strains are a promising source of extracellular polymeric substances (EPS). Through a review of previously published data, it is exposed that LEGE CC strains have the potential or actual capacity to produce a variety of biotechnologically interesting compounds, including common cyanotoxins or unprecedented bioactive molecules. Phylogenetic diversity of LEGE CC strains does not entirely reflect chemodiversity. Further bioprospecting should, therefore, account for strain specificity of the valuable cyanobacterial holdings of LEGE CC.

Toxic effects of pure anatoxin-a on biomarkers of rainbow trout, Oncorhynchus mykiss.

Anatoxin-a is a neurotoxin produced by various bloom-forming cyanobacteria. Although it shows widespread occurrence and is highly toxic to rodents, its mechanisms of action and biotransformation, and effects in fish species are still poorly understood. The main aim of this study was, thus, to investigate sub-lethal effects of anatoxin-a on selected biochemical markers in rainbow trout fry in order to get information about the mechanisms of toxicity and biotransformation of this toxin in fish. Trout fry were administered sub-lethal doses of anatoxin-a (0.08-0.31 µg g⁻¹) intraperitoneally. Livers and muscle tissue were collected 72 h later for quantification of key enzyme activities as biochemical markers. Enzymes assessed in muscle tissues were related to cholinergic transmission (acetylcholinesterase [AChE]), energy metabolism (lactate dehydrogenase [LDH] and NADP⁺-dependent isocitrate dehydrogenase [IDH]). Enzymes assessed in the liver were involved in biotransformation (ethoxyresorufin-O-deethylase [EROD] and glutathione S-transferases [GST]). The results indicated a significant increasing trend for AChE activity with the dose of anatoxin-a, possibly representing an attempt to cope with overstimulation of muscle activity by the toxin, which competes with acetylcholine for nicotinic receptors binding. Anatoxin-a was also found to significantly induce the activities of liver EROD and GST, indicating the involvement of phase I and II biotransformation in its detoxification. Likewise, lactate dehydrogenase activity recorded in fry muscle increased significantly with the dose of anatoxin-a, suggesting an induction of the anaerobic pathway of energy production to deal with toxic stress induced by the exposure. Altogether, the results suggest that under continued exposure in the wild fish may experience motor difficulties, possibly becoming vulnerable to predators, and be at increased metabolic demand to cope with energetic requirements imposed by anatoxin-a biotransformation mechanisms.

Production of anatoxin-a by cyanobacterial strains isolated from Portuguese fresh water systems.

The occurrence of anatoxin-a in several freshwater systems in Portugal and its production by Portuguese cyanobacterial strains, after cultivation in laboratory, were studied. Surface water samples from 9 water bodies, for recreational and human consumption usage, were surveyed for anatoxin-a presence and for obtaining cultures of pure cyanobacterial strains. Anatoxin-a analysis was performed by high performance liquid chromatography (HPLC) with fluorescence detection (FLD) followed by Mass Spectrometry (MS) confirmation. No anatoxin-a was detected in all the natural water samples (limit of detection (LOD) = 25 ng l(-1)) but among the 22 isolated cyanobacterial strains, 13 could produce anatoxin-a in laboratory conditions (LOD = 3 ng g(-1) dw). This proportion of anatoxin-a producing strains (59.1%) in laboratory is discussed considering the hypothesis that anatoxin-a is a more frequent metabolite in cyanobacteria than it was thought before and making its occurrence in Portuguese freshwaters almost certain. Therefore, health and ecological risks caused by anatoxin-a in Portugal, should be seriously considered.

First detection of anatoxin-a in human and animal dietary supplements containing cyanobacteria.

Anatoxin-a is a potent neurotoxin produced by several species of cyanobacteria. This alkaloid may cause fatal intoxication to exposed organisms and this has raised concerns over the increasing popularity of food supplements containing cyanobacteria. These are being marketed with alleged health properties for animal and human consumption. These supplements most commonly contain the genera Spirulina (Arthrospira) and Aphanizomenon and their consumption represent a potential route for anatoxin-a exposure in cases where adequate quality control is not undertaken. In this work, several dietary supplements containing cyanobacteria from different commercial suppliers were evaluated for the presence of anatoxin-a by high performance liquid chromatography with fluorescence detection. Additionally, the presence of the previously derivatized anatoxin-a was confirmed by using Gas chromatography-mass spectrometry. A total of 39 samples were analysed in the study. Results showed that three of the samples (7.7%) contained anatoxin-a, at concentrations ranging from 2.50 to 33 microg g(-1). Quality control of cyanobacterial food supplements is required to avoid potential health effects in humans and animals.

Uptake and depuration of anatoxin-a by the mussel Mytilus galloprovincialis (Lamarck, 1819) under laboratory conditions.

Cyanobacterial blooms tend to be more common in warm and nutrient-enriched waters and are increasing in many aquatic water bodies due to eutrophication. The aim of this work is to study the accumulation and depuration of anatoxin-a by Mytilus galloprovincialis a widespread distributed mussel living in estuarine and coastal waters and recognized worldwide as a bioindicator (e.g. Mussel Watch programs). Research on the distribution and biological effects of anatoxin-a in M. galloprovincialis is important. Nevertheless, the risk of human intoxication due to the consumption of contaminated bivalves should also be considered. A toxic bloom was simulated in an aquarium with 5 x 10(5) cell ml(-1) of Anabaena sp. (ANA 37), an anatoxin-a producing strain. Mussels were exposed to Anabaena for 15 days and then 15 days of depuration followed. Three or more animals were sampled every 24h for total toxin quantification and distribution in soft tissues (edible parts). Water samples were also taken every 24h in order to calculate total dissolved and particulate anatoxin-a concentrations. Anatoxin-a was quantified by HPLC with fluorescence detection. No deaths occurred during accumulation and depuration periods. One day after the beginning of depuration, the toxin could not be detected in the animals. Anatoxin-a is distributed in the digestive tract, muscles and foot and is probably actively detoxified.

Toxicology and detection methods of the alkaloid neurotoxin produced by cyanobacteria, anatoxin-a.

Freshwater resources are under stress due to naturally occurring conditions and human impacts. One of the consequences is the proliferation of cyanobacteria, microphytoplankton organisms that are capable to produce toxins called cyanotoxins. Anatoxin-a is one of the main cyanotoxins. It is a very potent neurotoxin that was already responsible for some animal fatalities. In this review we endeavor to divulgate much of the internationally published information about toxicology, occurrence and detection methods of anatoxin-a. Cyanobacteria generalities, anatoxin-a occurrence and production as well as anatoxin-a toxicology and its methods of detection are the aspects focused in this review. Remediation of anatoxin-a occurrence will be addressed with a public health perspective. Final remarks call the attention for some important gaps in the knowledge about this neurotoxin and its implication to public health. Alterations of aquatic ecosystems caused by anatoxin-a is also addressed. Although anatoxin-a is not the more frequent cyanotoxin worldwide, it has to be regarded as a health risk that can be fatal to terrestrial and aquatic organisms because of its high toxicity.

Analysis of anatoxin-a in biological samples using liquid chromatography with fluorescence detection after solid phase extraction and solid phase microextraction.

Anatoxin-a is a naturally occurring, potent neurotoxin produced by some species of cyanobacteria in freshwaters. This toxin, which is a potential health hazard, especially to animals, has been determined in different biological matrices such as several cyanobacterial cultures and water samples and carps and mussels tissue using a sensitive High Performance Liquid Chromatography with Fluorescence detection method. Sonication was the technique selected for the extraction of intracellular anatoxin-a and solid phase extraction using weak cation exchange was used for the concentration and purification of the samples. 4-Fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was used to convert anatoxin into a highly fluorescent derivative. Recovery experiments were performed for each type of matrix used in this work, and adequate values were obtained (71-87%). Limits of detection for anatoxin-a were estimated to be in the ng/L and ng/g level for water and cyanobacterial samples, respectively. The results obtained were also compared with those obtained after using solid phase microextraction, as an alternative for the extraction and purification of the samples. Advantages and disadvantages regarding to the efficiency for impurities removal, simplicity and rapidity and the potential for concentration enhancement of using both methodologies have been also discussed.

Acute effects of an anatoxin-a producing cyanobacterium on juvenile fish-Cyprinus carpio L.

The worldwide increase of eutrophication in fresh water bodies has caused cyanobacterial blooms to be more frequent. Anatoxin-a is a potent neurotoxin known to be produced by several genera of cyanobacteria including Anabaena. In this work, we exposed juvenile carps to freeze-dried cells of a toxic strain of the cyanobacterium Anabaena sp. during a 4-day period. Two different cell density--10(5) and 10(7) cell ml(-1)--were assayed. Lethality and anatoxin-a concentration in the whole fish were determined. In the higher cell density, all fish died between 26 and 29 h after exposure to toxin, whereas in the 10(5) cell ml(-1) density no deaths were observed. Levels of anatoxin-a in the whole fish ranged between 0.031 microg g(-1)d.w. at the 10(5) cell ml(-1) concentration and 0.768 microg g(-1)d.w. at 10(7) cell ml(-1). Minor uptake of anatoxin-a occurred.