Species Of Freshwater Cyanobacteria Research Articles

Fate of Saxitoxins in Lake Water: Preliminary Testing of Degradation by Microbes and Sunlight

The cyanobacterial toxin saxitoxin (STX) is mainly associated with the blooms of marine dinoflagellates, but it is also produced by several species of freshwater cyanobacteria. So far, the degradation of STX has only been demonstrated by physicochemical treatments, but in this study, we demonstrated that natural factors, such as bacterioplankton and sunlight, had the capacity for degrading STX in the eutrophic Lake Arresø, Denmark. Natural lake bacterioplankton could reduce STX concentrations by 41–59%. A similar reduction was shown for four saxitoxin analogs. The exposure of the lake water to natural sunlight or simulated sunlight also reduced both intracellular and extracellular, dissolved STX. During 4–8 h exposure, natural sunlight reduced intracellular STX by 38–52% but increased extracellular, dissolved STX by 7–29%. Corresponding values for simulated sunlight were a reduction in intracellular STX by 16–45% and increased levels of extracellular STX by 10–33%. In particle-free lake water, the two types of sunlight reduced ambient, dissolved STX by 13–17%. The light exposure was observed to damage >94% of the sxtA gene involved in STX synthesis. This study demonstrated that lake water bacterioplankton and sunlight can modify STX by degradation and cell destruction, and that the biosynthesis of STX may be inhibited by exposure to sunlight.

Genome of a novel Sediminibacterium discovered in association with two species of freshwater cyanobacteria from streams in Southern California.

Here, we report the discovery of a novel Sediminibacterium sequenced from laboratory cultures of freshwater stream cyanobacteria from sites in Southern California, grown in BG11 medium. Our genome-wide analyses reveal a highly contiguous and complete genome (97% BUSCO) that is placed within sediminibacterial clades in phylogenomic analyses. Functional annotation indicates the presence of genes that could be involved in mutualistic/commensal relationship with associated cyanobacterial hosts.

Advances in metabolic engineering of cyanobacteria for production of biofuels

Current trends in fossil fuel use, having a direct impact on the global carbon cycle, could potentially be mitigated by adopting the use of biofuels with sustainable features. While bioethanol is currently being used as a major biofuel, especially for transportation worldwide, it is hygroscopic and contains less energy. Therefore, the prospect of biofuels has now shifted to higher alcohols, alkanes, and other molecules that have similar properties to existing gasoline and diesel fuels. Microalgae and cyanobacteria are aquatic photosynthetic microorganisms that can convert CO2 and water into carbon-rich lipids, a substrate for biodiesel production, more efficiently than the agricultural oleaginous crops, making them a superior option to traditional oil-seed crops. Cyanobacteria are gram-negative photosynthetic bacteria that have instrumental roles in global biological carbon sequestration, oxygen evolution, and nitrogen fixation. Furthermore, cyanobacteria have established genetic backgrounds, flexibility in genetic manipulation, and tolerance to the introduction of foreign genes. Metabolic engineering strategies in these photosynthetic hosts have resulted in the conception of cell factories capable of one-step synthesis of biofuel molecules from CO2, light, and water. Two freshwater cyanobacteria species, Synechocystis sp. PCC 6803 (Synechocystis 6803) and Synechococcus elongatus sp. PCC 7492 (Synechococcus 7492), and marine Synechococcus sp. PCC 7002 (Synechococcus 7002) are among the highly studied cyanobacteria for the production of biofuels. Here, we reviewed the recent progress in metabolic engineering and synthetic biology approaches in cyanobacteria for the synthesis of various biofuel molecules, ranging from alcohols to biodiesels and terpenes or terpenoids.

First Identification of 12β-Deoxygonyautoxin 5 (12α-Gonyautoxinol 5) in the Cyanobacterium Dolichospermum circinale (TA04) and 12β-Deoxysaxitoxin (12α-Saxitoxinol) in D. circinale (TA04) and the Dinoflagellate Alexandrium pacificum (Group IV) (120518KureAC).

Saxitoxin and its analogues, paralytic shellfish toxins (PSTs), are potent and specific voltage-gated sodium channel blockers. These toxins are produced by some species of freshwater cyanobacteria and marine dinoflagellates. We previously identified several biosynthetic intermediates of PSTs, as well as new analogues, from such organisms and proposed the biosynthetic and metabolic pathways of PSTs. In this study, 12β-deoxygonyautoxin 5 (12α-gonyautoxinol 5 = gonyautoxin 5-12(R)-ol) was identified in the freshwater cyanobacterium, Dolichospermum circinale (TA04), and 12β-deoxysaxitoxin (12α-saxitoxinol = saxitoxin-12(R)-ol) was identified in the same cyanobacterium and in the marine dinoflagellate Alexandrium pacificum (Group IV) (120518KureAC) for the first time from natural sources. The authentic standards of these compounds and 12α-deoxygonyautoxin 5 (12β-gonyautoxinol 5 = gonyautoxin 5-12(S)-ol) were prepared by chemical derivatization from the major PSTs, C1/C2, produced in D. circinale (TA04). These standards were used to identify the deoxy analogues by comparing the retention times and MS/MS spectra using high-resolution LC-MS/MS. Biosynthetic or metabolic pathways for these analogues have also been proposed based on their structures. The identification of these compounds supports the α-oriented stereoselective oxidation at C12 in the biosynthetic pathway towards PSTs.

Whole-genome characterization and comparative genomics of a novel freshwater cyanobacteria species: Pseudanabaena punensis

Cyanobacteria are emerging as a potential source of novel, beneficial bioactive compounds. However, some cyanobacteria species can harm water quality and public health through the production of toxins. Therefore, surveying the occurrence and generating genomic resources of cyanobacteria producing harmful compounds could help develop the control methods necessary to manage their growth and limit the release contaminants into the water bodies. Here, we describe a novel strain, Pseudanabaena punensis isolated from the open ends of pipelines supplying freshwater. This isolate was characterized morphologically, biochemically and by whole-genome sequence analysis. We also provide genomic information for P. punensis to help understand and highlight the features unique to this isolate. Morphological and genetic (analysis using 16S rRNA and rbcL genes) data were used to assign this novel strain to phylogenetic and taxonomic groups. The isolate was identified as a filamentous and non-heterocystous cyanobacteria. Based on morphological and 16S rRNA phylogeny, this isolate shares characteristics with the Pseudanabaenaceae family, but remains distinct from well-characterized species suggesting its polyphyletic assemblage. The whole-genome sequence analysis suggests greater genomic and phenotypic plasticity. Genome-wide sequence and comparative genomic analyses, comparing against several closely related species, revealed diverse and important genes associated with synthesizing bioactive compounds, multi-drug resistance pathway, heavy metal resistance, and virulence factors. This isolate also produces several important fatty acids with potential industrial applications. The observations described in this study emphasize both industrial applications and risks associated with the freshwater contamination, and therefore genomic resources provided in this study offer an opportunity for further investigations.

Stability Analyses by HPLC-MS of Guanitoxin Isolated from Sphaerospermopsis torques-reginae

Guanitoxin (GNT) is a natural organophosphate produced by some species of freshwater cyanobacteria, which inhibits the active site of acetylcholinesterase, preventing the hydrolysis of cholinesterases and consequently causing serious disturbances in the neuromuscular system. Despite having a chemical structure like synthetic organophosphates, there is still no analytical standard available for environmental and freshwater monitoring. Therefore, this study investigated the stability of GNT under different storage conditions, pH, and temperature. The toxin is produced by the cyanobacterium Sphaerospermopsis torques-reginae and monitored by liquid chromatography coupled to mass spectrometry (LC-MS) and LC-MS/MS for the identification and verification of its stability. The main degradation product formed is the hydroxy-amino-guanidinic derivative of the toxin. The results also indicate that GNT is stable in acidic medium (pH = 3.0), but can gradually degrade at room temperature (> 23 ºC) over a period of 96 h. Lyophilized biomass of S. torques-reginae containing GNT remained stable when stored in a refrigerator below 4 ºC. In addition, the extraction yield is higher when prepared from fresh S. torques-reginae cells than from lyophilized material. Thus, the results shown here contribute with valuable information for studies that aim at the isolation, identification, and monitoring of GNT in samples of raw water and cyanobacterial blooms.

Effects of cylindrospermopsin, its decomposition products, and anatoxin-a on human keratinocytes

Toxins produced by cyanobacteria (cyanotoxins) are among the most dangerous natural compounds. In recent years, there have been many published papers related to the toxic alkaloids cylindrospermopsin (CYN) and anatoxin-a (ANTX-a), which are synthesized by several freshwater species of cyanobacteria (i.e. Raphidiopsis raciborskii and Anabaena flos-aquae) and are some of the most common cyanotoxins in aquatic reservoirs. The harmful properties of CYN are wide and primarily include cytotoxicity. To date, several analogs and decomposition products of CYN have been described, which can potentially increase its toxic effects in living organisms. The mode of action of ANTX-a is different than that observed after CYN exposure and involves structures in the nervous system. One of the most frequent situations in which cyanotoxins are introduced into the human body is by skin contact with contaminated water, i.e., during water sports, fishing or agriculture. Unfortunately, to date, knowledge on the influence of CYN, its decomposition products, and ANTX-a on human skin is limited. In this paper, we investigated the impact of CYN, its decomposition products, and ANTX-a on the proliferation of human keratinocytes, which provide a protective barrier on the skin. Moreover, we described the cytotoxic effects developed in the selected cell type and estimated the ability of the keratinocytes to migrate under the influence of the studied cyanotoxins. The obtained results suggest that CYN and its decomposition products at concentrations corresponding to that determined for CYN in nature (1 μg·mL−1) are strong inhibitors of keratinocyte proliferation (70% inhibition within 24 h for pure CYN). The cytotoxic effects of CYN and the CYN decomposition products on keratinocytes was also significant, and the pure toxin (1 μg·mL−1) was estimated to be 35% after 24 h of exposure. Similarly, harmful effects caused by CYN and its byproducts were observed during keratinocyte migration, and the initial form of the toxin (1 μg·mL−1) showed 40% inhibition within 16 h. Different results were obtained for ANTX-a. The toxic effects of this compound on human keratinocytes estimated by the applied tests was observed only at the highest tested concentration (10 μg·mL−1) and after a long period of exposure. The results presented in this paper are, to the best of our knowledge, the first description of the influence of CYN, CYN decomposition products, and ANTX-a on human epidermal cells. Clearly, CYN and its decomposition products are serious threats not only when acting on internal organs but also during the skin contact stage. Further studies on cyanotoxins should focus on the determination of their decomposition products and ecotoxicology in natural aquatic environments.

Biosynthesis of Saxitoxin in Marine Dinoflagellates: An Omics Perspective.

Saxitoxin is an alkaloid neurotoxin originally isolated from the clam Saxidomus giganteus in 1957. This group of neurotoxins is produced by several species of freshwater cyanobacteria and marine dinoflagellates. The saxitoxin biosynthesis pathway was described for the first time in the 1980s and, since then, it was studied in more than seven cyanobacterial genera, comprising 26 genes that form a cluster ranging from 25.7 kb to 35 kb in sequence length. Due to the complexity of the genomic landscape, saxitoxin biosynthesis in dinoflagellates remains unknown. In order to reveal and understand the dynamics of the activity in such impressive unicellular organisms with a complex genome, a strategy that can carefully engage them in a systems view is necessary. Advances in omics technology (the collective tools of biological sciences) facilitated high-throughput studies of the genome, transcriptome, proteome, and metabolome of dinoflagellates. The omics approach was utilized to address saxitoxin-producing dinoflagellates in response to environmental stresses to improve understanding of dinoflagellates gene–environment interactions. Therefore, in this review, the progress in understanding dinoflagellate saxitoxin biosynthesis using an omics approach is emphasized. Further potential applications of metabolomics and genomics to unravel novel insights into saxitoxin biosynthesis in dinoflagellates are also reviewed.

Control of a toxic cyanobacterial bloom species, Microcystis aeruginosa, using the peptide HPA3NT3-A2.

Microcystis aeruginosa, a species of freshwater cyanobacteria, is known to be one of the dominant species causing cyanobacterial harmful algal blooms (CyanoHABs). M. aeruginosa blooms have the potential to produce neurotoxins and peptide hepatotoxins, such as microcystins and lipopolysaccharides (LPSs). Currently, technologies for CyanoHAB control do not provide any ultimate solution because of the secondary pollution associated with the control measures. In this study, we attempted to use the peptide HPA3NT3-A2, which has been reported to be nontoxic and has antimicrobial properties, for the development of an eco-friendly control against CyanoHABs. HPA3NT3-A2 displayed significant algicidal effects against M. aeruginosa cells. HPA3NT3-A2 induced cell aggregation and flotation (thereby facilitating harvest), inhibited cell growth through sedimentation, and eventually destroyed the cells. HPA3NT3-A2 had no algicidal effect on other microalgal species such as Haematococcus pluvialis and Chlorella vulgaris. Additionally, HPA3NT3-A2 was not toxic to Daphnia magna. The algicidal mechanism of HPA3NT3-A2 was intracellular penetration. The results of this study suggest the novel possibility of controlling CyanoHABs using HPA3NT3-A2.

Decomposition products of cylindrospermopsin – a cyanotoxin produced by Raphidiopsis raciborskii (Woloszynska)

Abstract Toxins produced by cyanobacteria (cyanotoxins) and released into water have become a serious problem worldwide due to the increasing morbidity and mortality of living organisms they have caused. The ability to synthesize the cytotoxic alkaloid cylindrospermopsin (CYN) has been demonstrated in several freshwater species of cyanobacteria. CYN is highly chemically stable under environmental factors and decomposes only under alkaline conditions, where it forms derivatives. The toxicity potential of the decomposition products formed at pH 10 combined with high temperature (100°C) or UV-B irradiation (36 μmol m−2 s−1) has been research based on the crustacean Thamnocephalus platyurus (Thamnotoxkit FTM) and bacteria Vibrio fischeri (Deltatox® II) bioassays. This paper is a continuation and completion of our previous experiments and the obtained results showed that the applied conditions contributed to the decomposition of the CYN molecule to non-toxic products and its structural modifications by separating the uracil ring or/and the sulfate group from the tricyclic guanidine moiety, leading to a reduction in its toxicity. To the best of our knowledge, this is the first report describing the toxicity of CYN decomposition products formed under alkaline conditions combined with boiling temperature or UV-B irradiation.

The Metabolome of a Cyanobacterial Bloom Visualized by MS/MS-Based Molecular Networking Reveals New Neurotoxic Smenamide Analogs (C, D, and E).

Members of the cyanobacterial genus Trichodesmium are well known for their substantial impact on nitrogen influx in ocean ecosystems and the enormous surface blooms they form in tropical and subtropical locations. However, the secondary metabolite composition of these complex environmental bloom events is not well known, nor the possibility of the production of potent toxins that have been observed in other bloom-forming marine and freshwater cyanobacteria species. In the present work, we aimed to characterize the metabolome of a Trichodesmium bloom utilizing MS/MS-based molecular networking. Furthermore, we integrated cytotoxicity assays in order to identify and ultimately isolate potential cyanotoxins from the bloom. These efforts led to the isolation and identification of several members of the smenamide family, including three new smenamide analogs (1–3) as well as the previously reported smenothiazole A-hybrid polyketide-peptide compounds. Two of these new smenamides possessed cytotoxicity to neuro-2A cells (1 and 3) and their presence elicits further questions as to their potential ecological roles. HPLC profiling and molecular networking of chromatography fractions from the bloom revealed an elaborate secondary metabolome, generating hypotheses with respect to the environmental role of these metabolites and the consistency of this chemical composition across genera, space and time.

Elucidation of Microcystin-LR Degrading Mechanism of Bacillus cereus

Microcystin-LR (MC-LR) is a cyclic peptide produced as a secondary metabolite of certain freshwater cyanobacteria species such as, Microcystis, Anabaena, and Oscillatoria. Biodegradation of MC-LR by heterotrophic bacteria have been accepted as a reliable and cost effective method to treat MC-LR contamination. To date, more than 30 different bacterial genera have been recorded as potential MCs degraders. However, limited information are available on MC-LR degradation mechanism of bacteria. Intracellular MC-LR degradation by enzymes encoded by mlr A,B,C and D genes is the only hypothesis accepted so far. Hence, the present study focused on the elucidation of MC-LR degradation mechanism of KJ954304 Bacillus cereus 12GK strain which was previously isolated and characterized by authors as an efficient MC-LR degrader. Overnight grown and starved B. cereus bacterial suspension (0.5 μl) was inoculated into 100 ml of filter sterile (0.2 μm) Beira lake water containing MC-LR at a final concentration of 5 μgml-1. Control sample was prepared without bacterial inoculation. All flasks were incubated at 28°C and shaken at 100 rpm for 3 days. Following three days of incubation 0.5 ml sub sample aliquot was removed from both experimental and control flasks and frozen at (-20°C). Then 45 ml of experimental sample was kept aside as initial experimental sample and remaining 45 ml of sample was filtered under sterile conditions using 0.2 μm filter to remove bacterial cells. Then the filtrate was placed immediately in ice to prevent denature of enzymes. Subsequently, MC-LR was spiked to both initial experimental samples and filtered samples at a final concentration of 5μgml-1, and incubated at 280C, 100 rpm for 3 days. 1ml aliquots were removed from initial experimental sample and filtrate sample for 0-3 days of incubation. These samples were frozen and processed for High Performance Liquid Chromatography (HPLC) analysis. A PCR analysis was carried out to detect the presence of MC-LR degrading mlr A,B,C and D genes in B. cereus. Amplifications were performed in 50 μL volumes, containing 1 mM of each primer. A GeneAmps 2400 PCR System was utilized for the amplifications. At the end of 3 days of experiment MC-LR concentration of the initial experimental sample with bacteria was 1.8 μgml-1 whereas the filtrate sample without bacteria had 4.8 μgml-1 of MC-LR. Furthermore, the PCR study confirmed the presence of mlr A,B,C and D genes in B. cereus. Thus, MC-LR degradation is performed as an intracellular degradation by B. cereus with the involvement of intracellular enzymes. Keywords: Microcystin-LR, Biodegradation, Bacillus cereus, Intracellular degradation, mlr genes

Automated enumeration and size distribution analysis of Microcystis aeruginosa via fluorescence imaging

Due to climate change, toxic cyanobacteria and algae blooms and the associated exposure risk to humans has become a global issue. As a result, routine monitoring to evaluate cell concentrations is increasingly required to ensure safe water supplies. Current methods for cyanobacteria and algae cells enumeration are time consuming and cost-intensive due to the need for manual labor, which prevents their widespread adoption for routine water monitoring.. Automated enumeration with computer-assisted image analysis has strong potential to become a viable solution for continuous routine monitoring; however, the design of such automated systems is challenging due to: a) poor contrast between the target cells and the background, b) presence of confounding cells and abiotic particles and b) image quality variability depending on factors such as the underlying microscopy system in use and the sample condition. In this study, we introduce a novel integrated imaging-based method for automated enumeration and size distribution of Microcystis aeruginosa, a species of freshwater cyanobacteria that can originate harmful blooms. The target cells were excited using a 546nm light source and the resulting fluorescent imaging signal was acquired. A probabilistic unsupervised classification approach was taken to detect Microcystis cells from the surrounding background based on the fluorescent signal. A Gaussian mixture model was learned from the fluorescent imaging signal. The detected Microcystis cells were then enumerated and statistics regarding their size distribution automatically computed. When compared to the manual enumeration data using an hemacytometer, the developed method achieved higher accuracy using much less time and resources, without cell staining. These preliminary results demonstrate the potential of the proposed method as a powerful and robust tool for water quality monitoring and safe water quality control when used alongside gold standard methods.

Effects of depuration on histopathological changes in tilapia (Oreochromis niloticus) after exposure to cylindrospermopsin.

Cylindrospermopsin (CYN) is a highly water-soluble cytotoxin produced by several species of freshwater cyanobacteria and it is considered the second most studied cyanotoxin worldwide. CYN acts as a potent protein and glutathione synthesis inhibitor, as well as inducing genotoxicity, oxidative stress and histopathological alterations. Studies concerning the depuration of cyanobacterial toxins in aquatic organisms, especially in fish, are of great interest for fish economy and public health, but are scarce in the case of CYN. This is the first study reporting the ability of depuration (3 - 7 days) in reversing or ameliorating the histopathological lesions induced in liver, kidney, heart, intestines, and gills of tilapia (Oreochromis niloticus) due to exposure by immersion to repeated doses of a CYN-containing culture of A. ovalisporum for 14 days. The main histopathological changes induced by CYN were glucogenic degeneration and loss of the normal hepatic cord-structure (liver), hyperemia, dilated Bowman's capsule and cellular tumefaction (kidney), myofibrolysis, hemorrhages and edema (heart), necrosis and partial loss of microvilli (gastrointestinal tract), and hyperemia and inflammatory cells infiltrates (gills). After 3 days of depuration, gills were totally recovered, while the liver, kidney, and gastrointestinal tract required 7 days, and longer depuration periods may be needed for a full recovery of the heart. In addition, the morphometric study indicated that depuration managed to reverse the affectation in the hepatocytes nuclear diameters and cross sections of the proximal and distal convoluted tubules induced in CYN-exposed fish. In general, these results validate depuration as an effective practice for detoxification of fish contaminated with CYN. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1318-1332, 2017.

Microphytoplankton variations during coral spawning at Los Roques, Southern Caribbean.

Phytoplankton drives primary productivity in marine pelagic systems. This is also true for the oligotrophic waters in coral reefs, where natural and anthropogenic sources of nutrients can alter pelagic trophic webs. In this study, microphytoplankton assemblages were characterized for the first time in relation to expected coral spawning dates in the Caribbean. A hierarchical experimental design was used to examine these assemblages in Los Roques archipelago, Venezuela, at various temporal and spatial scales for spawning events in both 2007 and 2008. At four reefs, superficial water samples were taken daily for 9 days after the full moon of August, including days before, during and after the expected days of coral spawning. Microphytoplankton assemblages comprised 100 microalgae taxa at up to 50 cells per mL (mean ± 8 SD) and showed temporal and spatial variations related to the coral spawning only in 2007. However, chlorophyll a concentrations increased during and after the spawning events in both years, and this was better matched with analyses of higher taxonomical groups (diatoms, cyanophytes and dinoflagellates), that also varied in relation to spawning times in 2007 and 2008, but asynchronously among reefs. Heterotrophic and mixotrophic dinoflagellates increased in abundance, correlating with a decrease of the diatom Cerataulina pelagica and an increase of the diatom Rhizosolenia imbricata. These variations occurred during and after the coral spawning event for some reefs in 2007. For the first time, a fresh-water cyanobacteria species of Anabaena was ephemerally found (only 3 days) in the archipelago, at reefs closest to human settlements. Variability among reefs in relation to spawning times indicated that reef-specific processes such as water residence time, re-mineralization rates, and benthic-pelagic coupling can be relevant to the observed patterns. These results suggest an important role of microheterotrophic grazers in re-mineralization of organic matter in coral reef waters and highlight the importance of assessing compositional changes of larger size fractions of the phytoplankton when evaluating primary productivity and nutrient fluxes.

Intensive DNA Replication and Metabolism during the Lag Phase in Cyanobacteria.

Unlike bacteria such as Escherichia coli and Bacillus subtilis, several species of freshwater cyanobacteria are known to contain multiple chromosomal copies per cell, at all stages of their cell cycle. We have characterized the replication of multi-copy chromosomes in the cyanobacterium Synechococcus elongatus PCC 7942 (hereafter Synechococcus 7942). In Synechococcus 7942, the replication of multi-copy chromosome is asynchronous, not only among cells but also among multi-copy chromosomes. This suggests that DNA replication is not tightly coupled to cell division in Synechococcus 7942. To address this hypothesis, we analysed the relationship between DNA replication and cell doubling at various growth phases of Synechococcus 7942 cell culture. Three distinct growth phases were characterised in Synechococcus 7942 batch culture: lag phase, exponential phase, and arithmetic (linear) phase. The chromosomal copy number was significantly higher during the lag phase than during the exponential and linear phases. Likewise, DNA replication activity was higher in the lag phase cells than in the exponential and linear phase cells, and the lag phase cells were more sensitive to nalidixic acid, a DNA gyrase inhibitor, than cells in other growth phases. To elucidate physiological differences in Synechococcus 7942 during the lag phase, we analysed the metabolome at each growth phase. In addition, we assessed the accumulation of central carbon metabolites, amino acids, and DNA precursors at each phase. The results of these analyses suggest that Synechococcus 7942 cells prepare for cell division during the lag phase by initiating intensive chromosomal DNA replication and accumulating metabolites necessary for the subsequent cell division and elongation steps that occur during the exponential growth and linear phases.

Cylindrospermopsin induces neurotoxicity in tilapia fish (Oreochromis niloticus) exposed to Aphanizomenon ovalisporum

Cylindrospermopsin (CYN) is a cytotoxic cyanotoxin produced by several species of freshwater cyanobacteria, such as Aphanizomenon ovalisporum. CYN is a tricyclic alkaloid known for its ability to inhibit both protein and glutathione synthesis, and the alteration of different oxidative stress biomarkers in mammals and vertebrates. Although the liver and kidney appear to be the main CYN targets for this toxin, it also affects other organs. In fish, there is no evidence about the neurotoxicity of CYN yet. In the present study, we aimed to study the potential neurotoxicity of CYN, based on the measure of Acetylcholinesterase (AChE) activity, lipid peroxidation (LPO) levels and histopathological studies in brain of tilapia (Oreochromis niloticus) subchronically exposed to repeated concentrations of 10μg CYN/L by immersion in an A.ovalisporum culture for 14 days. The results showed significant inhibition of AChE activity and increases in LPO levels, as well as relevant histopathological alterations in the brain of fish (O. niloticus) subchronically exposed to the toxin. Moreover, we also investigated the potential recovery of these parameters by subjecting the fish to two depuration periods (3 and 7 days) in clean uncontaminated water, showing a recovery of the biochemical parameters since 3 days of depuration, and being necessary 7 days to recover the histopathological changes. In order to support these results, CYN was detected and quantified by enzyme-linked immunosorbent assay (ELISA) in brain of all the exposed fish and the effects of the depuration periods were also observed. Based on these results, it was demonstrated for the first time the neurotoxicity of CYN and its presence in brain of tilapia fish subchronically exposed to CYN.

Immunohistochemical Approach to Study Cylindrospermopsin Distribution in Tilapia (Oreochromis niloticus) under Different Exposure Conditions

Cylindrospermopsin (CYN) is a cytotoxic cyanotoxin produced by several species of freshwater cyanobacteria (i.e., Aphanizomenon ovalisporum). CYN is a tricyclic alkaloid combined with a guanidine moiety. It is well known that CYN inhibits both protein and glutathione synthesis, and also induces genotoxicity and the alteration of different oxidative stress biomarkers. Although the liver and kidney appear to be the main target organs for this toxin based on previous studies, CYN also affects other organs. In the present study, we studied the distribution of CYN in fish (Oreochromis niloticus) under two different exposure scenarios using immunohistochemical (IHC) techniques. In the first method, fish were exposed acutely by intraperitoneal injection or by gavage to 200 µg pure CYN/Kg body weight (bw), and euthanized after 24 h or five days of exposure. In the second method, fish were exposed by immersion to lyophilized A. ovalisporum CYN-producing cells using two concentration levels (10 or 100 µg/L) for two different exposure times (7 or 14 days). The IHC was carried out in liver, kidney, intestine, and gills of fish. Results demonstrated a similar pattern of CYN distribution in both experimental methods. The organ that presented the most immunopositive results was the liver, followed by the kidney, intestine, and gills. Moreover, the immunolabeling signal intensified with increasing time in both assays, confirming the delayed toxicity of CYN, and also with the increment of the dose, as it is shown in the sub-chronic assay. Thus, IHC is shown to be a valuable technique to study CYN distribution in these organisms.

Evolution and Distribution of Saxitoxin Biosynthesis in Dinoflagellates

Numerous species of marine dinoflagellates synthesize the potent environmental neurotoxic alkaloid, saxitoxin, the agent of the human illness, paralytic shellfish poisoning. In addition, certain freshwater species of cyanobacteria also synthesize the same toxic compound, with the biosynthetic pathway and genes responsible being recently reported. Three theories have been postulated to explain the origin of saxitoxin in dinoflagellates: The production of saxitoxin by co-cultured bacteria rather than the dinoflagellates themselves, convergent evolution within both dinoflagellates and bacteria and horizontal gene transfer between dinoflagellates and bacteria. The discovery of cyanobacterial saxitoxin homologs in dinoflagellates has enabled us for the first time to evaluate these theories. Here, we review the distribution of saxitoxin within the dinoflagellates and our knowledge of its genetic basis to determine the likely evolutionary origins of this potent neurotoxin.

Toxic risk associated with sporadic occurrences of Microcystis aeruginosa blooms from tidal rivers in marine and estuarine ecosystems and its impact on Artemia franciscana nauplii populations

Microcystis aeruginosa is a species of freshwater cyanobacteria which can form harmful algal blooms in freshwater water bodies worldwide. However, in spite its sporadic occurrences for short periods of time in estuarine waters, their influence on zooplankton populations present in these ecosystems has not been extensively studied. In this work, Artemia franciscana was used as test organism model, studying mortality against several strains of M. aeruginosa with different degrees of toxigenicity, measuring whole-live cells and homogenate extracts. Results were compared with microcystin-LR equivalent content, measured by immunoassay. The results show that there were no significant differences between both exposure models (whole cells and extracts), and there are significant differences respect to the toxigenicity of cyanobacterial blooms depending of the M. aerugionosa strain involved in the process. Analysis of microcystin-LR equivalent concentration test immediately below the lowest significant concentration in all M. aerugionosa strains was used to determine the potential risk associated with the cell densities during a bloom. Comparison among the selected M. aerugionsa strains show that these factors have influence in the results obtained, and thus, several differences have been evidenced depending of the microcystin-LR equivalent production and the strain type involved.