John P. Berry

Cyanobacterial Toxins as Allelochemicals with Potential Applications as Algaecides, Herbicides and Insecticides

Cyanobacteria (“blue-green algae”) from marine and freshwater habitats are known to produce a diverse array of toxic or otherwise bioactive metabolites. However, the functional role of the vast majority of these compounds, particularly in terms of the physiology and ecology of the cyanobacteria that produce them, remains largely unknown. A limited number of studies have suggested that some of the compounds may have ecological roles as allelochemicals, specifically including compounds that may inhibit competing sympatric macrophytes, algae and microbes. These allelochemicals may also play a role in defense against potential predators and grazers, particularly aquatic invertebrates and their larvae. This review will discuss the existing evidence for the allelochemical roles of cyanobacterial toxins, as well as the potential for development and application of these compounds as algaecides, herbicides and insecticides, and specifically present relevant results from investigations into toxins of cyanobacteria from the Florida Everglades and associated waterways.

Toxicity of cylindrospermopsin, and other apparent metabolites from Cylindrospermopsis raciborskii and Aphanizomenon ovalisporum, to the zebrafish (Danio rerio) embryo.

Cyanobacteria produce a diverse array of toxic or otherwise bioactive compounds that pose growing threats to human and environmental health. We utilized the zebrafish (Danio rerio) embryo, as a model of vertebrate development, to investigate the inhibition of development pathways (i.e. developmental toxicity) by the cyanobacterial toxin, cylindrospermopsin (CYN), as well as extracts from various isolates of Cylindrospermopsis raciborskii and Aphanizomenon ovalisporum. CYN was toxic only when injected directly into embryos, but not by direct immersion at doses up to 50mug/ml. Despite the dose dependency of toxicity observed following injection of CYN, no consistent patterns of developmental defects were observed, suggesting that toxic effects of CYN may not target specific developmental pathways. In contrast, direct immersion of embryos in all of the extracts resulted in both increased mortality and reproducible, consistent, developmental dysfunctions. Interestingly, there was no correlation of developmental toxicity observed for these extracts with the presence of CYN or with previously reported toxicity for these strains. These results suggest that CYN is lethal to zebrafish embryos, but apparently inhibits no specific developmental pathways, whereas other apparent metabolites from C. raciborskii and A. ovalisporum seem to reproducibly inhibit development in the zebrafish model. Continued investigation of these apparent, unknown metabolites is needed.

Allelopathic activity among Cyanobacteria and microalgae isolated from Florida freshwater habitats.

We evaluated allelopathic interactions between strains of Cyanobacteria and green algae isolated from south and central Florida. Allelopathy, including inhibition or stimulation of growth, was assessed by cocultivation of each of the isolated strains, as well as by evaluation of extracts prepared from the isolates. All of the strains of Cyanobacteria, and four of the six isolates of green algae, showed some allelopathic activity (i.e. inhibition or stimulation of the growth of other strains). Of these, the most pronounced activity was observed for the cyanobacterial isolate Fischerella sp. strain 52-1. In the cocultivation experiments this cyanobacterium inhibited the growth of all tested green algae and Cyanobacteria. The crude lipophilic extracts from Fischerella sp. strain 52-1 isolated from both the biomass and the culture liquid inhibited photosynthesis of the green alga Chlamydomonas sp. in a concentration- and time-dependent manner and caused extensive loss of ultrastructural cell organization. Preliminary chemical characterization of compounds extracted from Fischerella sp. strain 52-1 indicated the presence of indole alkaloids, and further characterization has confirmed that these compounds belong to the hapalindoles previously isolated from other species of Fischerella and related genera. Further chemical characterization of these compounds, and further investigation of their apparent role in allelopathy is ongoing.

Evaluation of the human health threat associated with the hepatotoxin microcystin in the muscle and liver tissues of yellow perch (Perca flavescens)

During the summer of 2006, the western basin of Lake Erie experienced a bloom of the toxigenic cyanobacte- rium Microcystis aeruginosa. Across 11 sites, intracellular, particulate-bound microcystin levels in the seston increased to levels that exceeded World Health Organization guidelines for drinking water exposure (1 mg toxinL -1 ). In contrast, toxin concentrations in yellow perch (Perca flavescens) muscle tissue (n = 68) declined from June to August, were negatively related to algal toxin levels, and never exceeded a conservative chronic exposure concentration estimated using proposed United States Environmental Protection Agency (US EPA) guidelines. Microcystin concentrations in yellow perch liver exceeded US EPA chronic exposure guidelines, were on average 125 times higher than muscle toxin concentrations per unit dry weight, and varied little throughout the summer. With current guidelines, humans do not appear to be at risk when consuming the muscle tissue of Lake Erie yellow perch collected during large-scale cyanobacterial blooms. How- ever, this study highlights the need for a better understanding of the trophic transfer of cyanobacterial toxins through aquatic food webs in diverse ecosystems with an emphasis on understanding if these compounds could accumulate suffi- ciently to affect human health.

Apparent bioaccumulation of cylindrospermopsin and paralytic shellfish toxins by finfish in Lake Catemaco (Veracruz, Mexico).

Compared to the well-characterized health threats associated with contamination of fish and shellfish by algal toxins in marine fisheries, the toxicological relevance of the bioaccumulation of toxins from cyanobacteria (blue-green algae), as the primary toxigenic algae in freshwater systems, remains relatively unknown. Lake Catemaco (Veracruz, Mexico) is a small, tropical lake system specifically characterized by a year-round dominance of the known toxigenic cyanobacterial genus, Cylindrospermopsis, and by low, but detectable, levels of both a cyanobacterial hepatotoxin, cylindrospermopsin (CYN), and paralytic shellfish toxins (PSTs). In the present study, we evaluated, using enzyme-linked immunoassay (ELISA), levels of both toxins in several species of finfish caught and consumed locally in the region to investigate the bioaccumulation of, and possible health threats associated with, these toxins as potential foodborne contaminants. ELISA detected levels of both CYN and PSTs in fish tissues from the lake. Levels were generally low (≤1 ng g−1 tissue); however, calculated bioaccumulation factors (BAFs) indicate that toxin levels exceed the rather low levels in the water column and, consequently, indicated bioaccumulation (BAF>1). A reasonable correlation was observed between measured bioaccumulation of CYN and PSTs, possibly indicating a mutual source of both toxins, and most likely cells of Cylindrospermopsis, the dominant cyanobacteria in the lake, and a known producer of both metabolites. The potential roles of trophic transport in the system, as well as possible implications for human health with regards to bioaccumulation, are discussed.

Bioaccumulation of microcystins by fish associated with a persistent cyanobacterial bloom in Lago de Patzcuaro (Michoacan, Mexico)

Lago de Patzcuaro is a historically important freshwater fishery in Mexico. The lake is presently characterized by a persistent bloom of cyanobacteria, specifically dominated by recognized producers of toxic microcystins (MCYSTs). We evaluated MCYSTs in sestonic and dissolved fractions of the water column, as well as representative fish species (silversides, Chirostoma spp.; Goodea sp.; and carp, Cyprinus carpio) obtained from local markets and small commercial catches during the bloom. Samples were evaluated primarily by enzyme-linked immunosorbent assay (ELISA), and secondarily by protein phosphatase (PPase) inhibition assay and liquid chromatography-mass spectrometry (LC-MS). Sestonic MCYST concentration (0.02-0.36 µg/L) generally correlated inversely with distance from the bloom, supporting the bloom as the source of the toxin. Several MCYST variants, including MC-LR, -LA and -LY, as well as didemethyl variants, were identified by LC-MS/MS analysis. All three species of fish bioaccumulated MCYSTs in relevant tissues, and toxin content correlated with trophic level, with highest and lowest levels measured in phytoplanktivorous and zooplanktivorous representatives, respectively. Detection of MCYST in silversides and Goodea sp. is particularly relevant because both are consumed in their entirety, including viscera (e.g., liver) known to primarily accumulate MCYST. These results indicate that Lago de Patzcuaro is indeed characterized by a toxigenic bloom, and that commercially important fish species from the lake accumulate toxic MCYST in tissues relevant to human consumption. As such, this system may represent an ideal model of the trophic transfer of MCYSTs and its relevance to human and environmental health.

Cyanobacterial Microcystis aeruginosa Lipopolysaccharide Elicits Release of Superoxide Anion, Thromboxane B2, Cytokines, Chemokines, and Matrix Metalloproteinase-9 by Rat Microglia

Microcystis aeruginosa (M. aeruginosa) is a cosmopolitan Gram-negative cyanobacterium that may contaminate freshwater by releasing toxins, such as lipopolysaccharide (LPS) during aquatic blooms, affecting environmental and human health. The putative toxic effects of cyanobacterial LPS on brain microglia, a glial cell type that constitutes the main leukocyte-dependent source of reactive oxygen species in the central nervous system, are presently unknown. We tested the hypothesis that in vitro concentration- and time-dependent exposure to M. aeruginosa LPS strain UTCC 299 would activate rat microglia and the concomitant generation of superoxide anion (O₂⁻). After a 17-h exposure of microglia to M.aeruginosa LPS, the following concentration-dependent responses were observed: 0.1-100 ng/ml M. aeruginosa LPS enhanced O₂⁻ generation, with limited inflammatory mediator generation; 1000-10,000 ng/ml M. aeruginosa LPS caused thromboxane B₂ (TXB₂), matrix metalloproteinase-9 (MMP-9), and macrophage inflammatory protein-2 (MIP-2/CXCL2) release, concurrent with maximal O₂⁻ generation; 100,000 ng/mL M. aeruginosa LPS deactivated O₂⁻ production but maintained elevated levels of TXB₂, MMP-9, tumor necrosis factor-α (TNF-α), interleukin 1-α (IL-1α), and interleukin-6 (IL-6), macrophage inflammatory protein 1α (MIP-1α/CCL3), and MIP-2/CXCL2, with concomitant lactic dehydrogenase release. Although M. aeruginosa LPS was consistently less potent than Escherichia coli LPS, with the exception of O₂⁻, TXB₂, and MCP-1/CCL2 generation, it was more efficacious because higher levels of MMP-9, TNF-α, IL-1α, IL-6, MIP-1α/CCL3, and MIP-2/CXCL2 were produced. Our in vitro studies suggest that one or more of the inflammatory mediators released during M. aeruginosa LPS stimulation of microglia may play a critical role in the subsequent ability of microglia to generate O₂⁻. To our knowledge, this is the first experimental evidence that LPS isolated from a M. aeruginosa strain, can activate brain microglia in vitro, as well as the release of O₂⁻, and other inflammatory mediators hypothesized to be involved in neuroinflammation and neurodegeneration.

Detection of total microcystin in fish tissues based on lemieux oxidation and recovery of 2-methyl-3-methoxy-4-phenylbutanoic acid (MMPB) by solid-phase microextraction gas chromatography-mass spectrometry (SPME-GC/MS)

Microcystins (MCs) are widespread cyanobacterial toxins in freshwater systems, and have been linked to both acute and chronic health effects. A growing number of studies suggest that MC can bioaccumulate in food webs. Although, several methods (i.e. ELISA, LC-MS) have been developed for analysis of MC in water, extraction (for subsequent analysis) of the toxin from biological matrices (i.e. animal tissues) is impeded owing to covalent binding of toxins and active sites of their cellular targets, i.e. protein phosphatases. As an alternative approach, chromatographic methods for analysis of a unique marker, 2-methyl-3-methoxy-4-phenylbutanoic acid (MMPB), the product of the Lemieux oxidation of MCs, have been previously developed, and shown to measure total (bound and unbound) MC. Application, however, has been limited by poor recovery of the analyte. An improved recovery method is proposed – specifically the use of solid-phase microextraction (SPME). The MMPB analogue, 4-phenylbutanoic acid (4PB), and oxidized MC, were used to develop methods, and we specifically investigated several SPME fibres, and post-oxidation steps. Specifically, a method employing post-oxidation methyl esterification, followed by headspace SPME recovery of MMPB, was developed, and subsequently applied to analysis of environmental samples (i.e. fish tissues) previously shown to contain MCs. The method shows high linearity for both water and tissues spiked with MC, and an improved limit of quantitation of approximately 140 ng g−1. Evaluation of field samples by SPME-GC/MS detected considerably higher levels of MC, than detected by conventional methods (i.e. ELISA), and it is proposed that this technique reveals MC (particularly in the bound form) that is not detected by these methods. These results indicate that the developed method provides improved detection capability for MC in biological matrices, and will enhance our ability to understand bioaccumulation in freshwater food webs, as well as monitor exposure.

Effects of Cyanobacterial Lipopolysaccharides from Microcystis on Glutathione-Based Detoxification Pathways in the Zebrafish (Danio rerio) Embryo

Cyanobacteria (“blue-green algae”) are recognized producers of a diverse array of toxic secondary metabolites. Of these, the lipopolysaccharides (LPS), produced by all cyanobacteria, remain to be well investigated. In the current study, we specifically employed the zebrafish (Danio rerio) embryo to investigate the effects of LPS from geographically diverse strains of the widespread cyanobacterial genus, Microcystis, on several detoxifying enzymes/pathways, including glutathione-S-transferase (GST), glutathione peroxidase (GPx)/glutathione reductase (GR), superoxide dismutase (SOD), and catalase (CAT), and compared observed effects to those of heterotrophic bacterial (i.e., E. coli) LPS. In agreement with previous studies, cyanobacterial LPS significantly reduced GST in embryos exposed to LPS in all treatments. In contrast, GPx moderately increased in embryos exposed to LPS, with no effect on reciprocal GR activity. Interestingly, total glutathione levels were elevated in embryos exposed to Microcystis LPS, but the relative levels of reduced and oxidized glutathione (i.e., GSH/GSSG) were, likewise, elevated suggesting that oxidative stress is not involved in the observed effects as typical of heterotrophic bacterial LPS in mammalian systems. In further support of this, no effect was observed with respect to CAT or SOD activity. These findings demonstrate that Microcystis LPS affects glutathione-based detoxification pathways in the zebrafish embryo, and more generally, that this model is well suited for investigating the apparent toxicophore of cyanobacterial LPS, including possible differences in structure-activity relationships between heterotrophic and cyanobacterial LPS, and teleost fish versus mammalian systems.

Cyanobacterial LPS potentiates cadmium toxicity in zebrafish (Danio rerio) embryos.

Cyanobacteria are prevalent in the freshwater environment, reaching critical mass in harmful algal blooms. These organisms produce a variety of toxins including endotoxins such as lipopolysaccharides (LPS), which have been previously shown to decrease glutathione‐S‐transferase (GST) activity in zebrafish (Danio rerio) embryos. GST plays a vital role in detoxification response during oxidative stress and provides a first line of defense after toxic heavy metal insult, before increased metallothionein expression. Although some attention has focused on cyanobacterial LPS, little research has focused on effects of concurrent exposures with other toxicants. Because cyanobacterial LPS can alter detoxification enzymes including GST, we hypothesized that cyanobacterial LPS could potentiate metal toxicity. This study investigated the effects of LPS from two cyanobacterial species, Lyngbya spp. and Microcystis aeruginosa, on cadmium toxicity in zebrafish embryos. Forty‐eight‐hour CdCl2 LC50 values showed that coexposure of cadmium and Lyngbya LPS or Microcystis LPS resulted in significantly increased cadmium toxicity in comparison with cadmium alone. However, increased cadmium toxicity was not due to decreased GST activity as initially hypothesized. In concurrent Microcystis LPS‐cadmium exposures, GST activity was significantly increased in comparison with control embryos at all time points and cadmium concentrations sampled. Concurrent Lyngbya LPS‐cadmium exposures also resulted in increased GST activity at most exposure concentrations. These results indicate that regardless of mechanism, cyanobacterial LPS can potentiate the toxic effects of heavy metals. This represents a significant risk for aquatic organisms exposed to combinations of LPS and metals in the environment.