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.     

Cyanotoxins: bioaccumulation and effects on aquatic animals

Cyanobacteria are photosynthetic prokaryotes with wide geographic distribution that can produce secondary metabolites named cyanotoxins. These toxins can be classified into three main types according to their mechanism of action in vertebrates: hepatotoxins, dermatotoxins and neurotoxins. Many studies on the effects of cyanobacteria and their toxins over a wide range of aquatic organisms, including invertebrates and vertebrates, have reported acute effects (e.g., reduction in survivorship, feeding inhibition, paralysis), chronic effects (e.g., reduction in growth and fecundity), biochemical alterations (e.g., activity of phosphatases, GST, AChE, proteases), and behavioral alterations. Research has also focused on the potential for bioaccumulation and transferring of these toxins through the food chain. Although the herbivorous zooplankton is hypothesized as the main target of cyanotoxins, there is not unquestionable evidence of the deleterious effects of cyanobacteria and their toxins on these organisms. Also, the low toxin burden in secondary consumers points towards biodilution of microcystins in the food web as the predominant process. In this broad review we discuss important issues on bioaccumulation and the effects of cyanotoxins, with emphasis on microcystins, as well as drawbacks and future needs in this field of research.     

Indole Alkaloids of the Stigonematales (Cyanophyta): Chemical Diversity,Biosynthesis and Biological Activity

The cyanobacteria are well recognized as producers of a wide array of bioactive metabolites including toxins, and potential drug candidates. However, a limited number of taxa are generally considered with respect to both of these aspects. That said, the order Stigonematales, although largely overlooked in this regard, has become increasingly recognized as a source of bioactive metabolites relevant to both human and environmental health. In particular, the hapalindoles and related indole alkaloids (i.e., ambiguines, fischerindoles, welwitindolinones) from the order, represent a diverse, and phylogenetically characteristic, class of secondary metabolites with biological activity suggestive of potential as both environmental toxins, and promising drug discovery leads. The present review gives an overview of the chemical diversity of biologically active metabolites from the Stigonematales—and particularly the so-called hapalindole-type alkaloids—including their biosynthetic origins, and their pharmacologically and toxicologically relevant bioactivities. Taken together, the current evidence suggests that these alkaloids, and the associated cyanobacterial taxa from the order, warrant future consideration as both potentially harmful (i.e., “toxic”) algae, and as promising leads for drug discovery.     

Interpreting the Possible Ecological Role(s) of Cyanotoxins: Compounds for Competitive Advantage and/or Physiological Aide?

To date, most research on freshwater cyanotoxin(s) has focused on understanding the dynamics of toxin production and decomposition, as well as evaluating the environmental conditions that trigger toxin production, all with the objective of informing management strategies and options for risk reduction. Comparatively few research studies have considered how this information can be used to understand the broader ecological role of cyanotoxin(s), and the possible applications of this knowledge to the management of toxic blooms. This paper explores the ecological, toxicological, and genetic evidence for cyanotoxin production in natural environments. The possible evolutionary advantages of toxin production are grouped into two main themes: That of “competitive advantage” or “physiological aide”. The first grouping illustrates how compounds produced by cyanobacteria may have originated from the need for a cellular defence mechanism, in response to grazing pressure and/or resource competition. The second grouping considers the contribution that secondary metabolites make to improved cellular physiology, through benefits to homeostasis, photosynthetic efficiencies, and accelerated growth rates. The discussion also includes other factors in the debate about possible evolutionary roles for toxins, such as different modes of exposures and effects on non-target (i.e., non-competitive) species. The paper demonstrates that complex and multiple factors are at play in driving evolutionary processes in aquatic environments. This information may provide a fresh perspective on managing toxic blooms, including the need to use a “systems approach” to understand how physico-chemical conditions, as well biological stressors, interact to trigger toxin production.     

BMAA Inhibits Nitrogen Fixation in the Cyanobacterium Nostoc sp. PCC 7120

Cyanobacteria produce a range of secondary metabolites, one being the neurotoxic non-protein amino acid β-N-methylamino-L-alanine (BMAA), proposed to be a causative agent of human neurodegeneration. As for most cyanotoxins, the function of BMAA in cyanobacteria is unknown. Here, we examined the effects of BMAA on the physiology of the filamentous nitrogen-fixing cyanobacterium Nostoc sp. PCC 7120. Our data show that exogenously applied BMAA rapidly inhibits nitrogenase activity (acetylene reduction assay), even at micromolar concentrations, and that the inhibition was considerably more severe than that induced by combined nitrogen sources and most other amino acids. BMAA also caused growth arrest and massive cellular glycogen accumulation, as observed by electron microscopy. With nitrogen fixation being a process highly sensitive to oxygen species we propose that the BMAA effects found here may be related to the production of reactive oxygen species, as reported for other organisms.     

Phylogeny and Biogeography of Cyanobacteria and Their Produced Toxins

Phylogeny is an evolutionary reconstruction of the past relationships of DNA or protein sequences and it can further be used as a tool to assess population structuring, genetic diversity and biogeographic patterns. In the microbial world, the concept that everything is everywhere is widely accepted. However, it is much debated whether microbes are easily dispersed globally or whether they, like many macro-organisms, have historical biogeographies. Biogeography can be defined as the science that documents the spatial and temporal distribution of a given taxa in the environment at local, regional and continental scales. Speciation, extinction and dispersal are proposed to explain the generation of biogeographic patterns. Cyanobacteria are a diverse group of microorganisms that inhabit a wide range of ecological niches and are well known for their toxic secondary metabolite production. Knowledge of the evolution and dispersal of these microorganisms is still limited, and further research to understand such topics is imperative. Here, we provide a compilation of the most relevant information regarding these issues to better understand the present state of the art as a platform for future studies, and we highlight examples of both phylogenetic and biogeographic studies in non-symbiotic cyanobacteria and cyanotoxins.     

Floridean Starch and Floridoside Metabolic Pathways of Neoporphyra haitanensis and Their Regulatory Mechanism under Continuous Darkness

Floridean starch and floridoside are the main storage carbohydrates of red algae. However, their complete metabolic pathways and the origin, function, and regulatory mechanism of their pathway genes have not been fully elucidated. In this study, we identified their metabolic pathway genes and analyzed the changes in related gene expression and metabolite content in Neoporphyra haitanensis under continuous dark conditions. Our results showed that genes from different sources, including eukaryotic hosts, cyanobacteria, and bacteria, were combined to construct floridean starch and floridoside metabolic pathways in N. haitanensis. Moreover, compared with those in the control, under continuous dark conditions, floridean starch biosynthesis genes and some degradation genes were significantly upregulated with no significant change in floridean starch content, whereas floridoside degradation genes were significantly upregulated with a significant decrease in floridoside content. This implies that floridean starch content is maintained but floridoside is consumed in N. haitanensis under dark conditions. This study elucidates the “floridean starch–floridoside” metabolic network and its gene origins in N. haitanensis for the first time.     

Current Knowledge on Microviridin from Cyanobacteria

Cyanobacteria are a rich source of secondary metabolites with a vast biotechnological potential. These compounds have intrigued the scientific community due their uniqueness and diversity, which is guaranteed by a rich enzymatic apparatus. The ribosomally synthesized and post-translationally modified peptides (RiPPs) are among the most promising metabolite groups derived from cyanobacteria. They are interested in numerous biological and ecological processes, many of which are entirely unknown. Microviridins are among the most recognized class of ribosomal peptides formed by cyanobacteria. These oligopeptides are potent inhibitors of protease; thus, they can be used for drug development and the control of mosquitoes. They also play a key ecological role in the defense of cyanobacteria against microcrustaceans. The purpose of this review is to systematically identify the key characteristics of microviridins, including its chemical structure and biosynthesis, as well as its biotechnological and ecological significance.     

Can Some Marine-Derived Fungal Metabolites Become Actual Anticancer Agents?

Marine fungi are known to produce structurally unique secondary metabolites, and more than 1000 marine fungal-derived metabolites have already been reported. Despite the absence of marine fungal-derived metabolites in the current clinical pipeline, dozens of them have been classified as potential chemotherapy candidates because of their anticancer activity. Over the last decade, several comprehensive reviews have covered the potential anticancer activity of marine fungal-derived metabolites. However, these reviews consider the term “cytotoxicity” to be synonymous with “anticancer agent”, which is not actually true. Indeed, a cytotoxic compound is by definition a poisonous compound. To become a potential anticancer agent, a cytotoxic compound must at least display (i) selectivity between normal and cancer cells (ii) activity against multidrug-resistant (MDR) cancer cells; and (iii) a preferentially non-apoptotic cell death mechanism, as it is now well known that a high proportion of cancer cells that resist chemotherapy are in fact apoptosis-resistant cancer cells against which pro-apoptotic drugs have more than limited efficacy. The present review thus focuses on the cytotoxic marine fungal-derived metabolites whose ability to kill cancer cells has been reported in the literature. Particular attention is paid to the compounds that kill cancer cells through non-apoptotic cell death mechanisms.     

Chemoecological Screening Reveals High Bioactivity in Diverse Culturable Portuguese Marine Cyanobacteria

Marine cyanobacteria, notably those from tropical regions, are a rich source of bioactive secondary metabolites. Tropical marine cyanobacteria often grow to high densities in the environment, allowing direct isolation of many secondary metabolites from field-collected material. However, in temperate environments culturing is usually required to produce enough biomass for investigations of their chemical constituents. In this work, we cultured a selection of novel and diverse cyanobacteria isolated from the Portuguese coast, and tested their organic extracts in a series of ecologically-relevant bioassays. The majority of the extracts showed activity in at least one of the bioassays, all of which were run in very small scale. Phylogenetically related isolates exhibited different activity profiles, highlighting the value of microdiversity for bioprospection studies. Furthermore, LC-MS analyses of selected active extracts suggested the presence of previously unidentified secondary metabolites. Overall, the screening strategy employed here, in which previously untapped cyanobacterial diversity was combined with multiple bioassays, proved to be a successful strategy and allowed the selection of several strains for further investigations based on their bioactivity profiles.     

Activation of the Silent Secondary Metabolite Production by Introducing Neomycin-Resistance in a Marine-Derived Penicillium purpurogenum G59

Introduction of neomycin-resistance into a marine-derived, wild-type Penicillium purpurogenum G59 resulted in activation of silent biosynthetic pathways for the secondary metabolite production. Upon treatment of G59 spores with neomycin and dimethyl sulfoxide (DMSO), a total of 56 mutants were obtained by single colony isolation. The acquired resistance of mutants to neomycin was testified by the resistance test. In contrast to the G59 strain, the EtOAc extracts of 28 mutants inhibited the human cancer K562 cells, indicating that the 28 mutants have acquired the capability to produce bioactive metabolites. HPLC-photodiode array detector (PDAD)-UV and HPLC-electron spray ionization (ESI)-MS analyses further indicated that diverse secondary metabolites have been newly produced in the bioactive mutant extracts. Followed isolation and characterization demonstrated that five bioactive secondary metabolites, curvularin (1), citrinin (2), penicitrinone A (3), erythro-23-O-methylneocyclocitrinol (4) and 22E-7α-methoxy-5α,6α-epoxyergosta-8(14),22-dien-3β-ol (5), were newly produced by a mutant, 4-30, compared to the G59 strain. All 1–5 were also not yet found in the secondary metabolites of other wild type P. purpurogenum strains. Compounds 1–5 inhibited human cancer K562, HL-60, HeLa and BGC-823 cells to varying extents. Both present bioassays and chemical investigations demonstrated that the introduction of neomycin-resistance into the marine-derived fungal G59 strain could activate silent secondary metabolite production. The present work not only extended the previous DMSO-mediated method for introducing drug-resistance in fungi both in DMSO concentrations and antibiotics, but also additionally exemplified effectiveness of this method for activating silent fungal secondary metabolites. This method could be applied to other fungal isolates to elicit their metabolic potentials to investigate secondary metabolites from silent biosynthetic pathways.     

Bioactive peptides and depsipeptides with anticancer potential: sources from marine animals

Biologically active compounds with different modes of action, such as, antiproliferative, antioxidant, antimicrotubule, have been isolated from marine sources, specifically algae and cyanobacteria. Recently research has been focused on peptides from marine animal sources, since they have been found as secondary metabolites from sponges, ascidians, tunicates, and mollusks. The structural characteristics of these peptides include various unusual amino acid residues which may be responsible for their bioactivity. Moreover, protein hydrolysates formed by the enzymatic digestion of aquatic and marine by-products are an important source of bioactive peptides. Purified peptides from these sources have been shown to have antioxidant activity and cytotoxic effect on several human cancer cell lines such as HeLa, AGS, and DLD-1. These characteristics imply that the use of peptides from marine sources has potential for the prevention and treatment of cancer, and that they might also be useful as molecular models in anticancer drug research. This review focuses on the latest studies and critical research in this field, and evidences the immense potential of marine animals as bioactive peptide sources.     

Symbioses of Cyanobacteria in Marine Environments: Ecological Insights and Biotechnological Perspectives

Cyanobacteria are a diversified phylum of nitrogen-fixing, photo-oxygenic bacteria able to colonize a wide array of environments. In addition to their fundamental role as diazotrophs, they produce a plethora of bioactive molecules, often as secondary metabolites, exhibiting various biological and ecological functions to be further investigated. Among all the identified species, cyanobacteria are capable to embrace symbiotic relationships in marine environments with organisms such as protozoans, macroalgae, seagrasses, and sponges, up to ascidians and other invertebrates. These symbioses have been demonstrated to dramatically change the cyanobacteria physiology, inducing the production of usually unexpressed bioactive molecules. Indeed, metabolic changes in cyanobacteria engaged in a symbiotic relationship are triggered by an exchange of infochemicals and activate silenced pathways. Drug discovery studies demonstrated that those molecules have interesting biotechnological perspectives. In this review, we explore the cyanobacterial symbioses in marine environments, considering them not only as diazotrophs but taking into consideration exchanges of infochemicals as well and emphasizing both the chemical ecology of relationship and the candidate biotechnological value for pharmaceutical and nutraceutical applications.     

Mycosporine-like amino acids and marine toxins--the common and the different

Marine microorganisms harbor a multitude of secondary metabolites. Among these are toxins of different chemical classes as well as the UV-protective mycosporine-like amino acids (MAAs). The latter form a group of water-soluble, low molecular-weight (generally < 400) compounds composed of either an aminocyclohexenone or an aminocyclohexenimine ring, carrying amino acid or amino alcohol substituents. So far there has been no report of toxicity in MAAs but nevertheless there are some features they have in common with marine toxins. Among the organisms producing MAAs are cyanobacteria, dinoflagellates and diatoms that also synthesize toxins. As in cyclic peptide toxins found in cyanobacteria, amino acids are the main building blocks of MAAs. Both, MAAs and some marine toxins are transferred to other organisms e.g. via the food chains, and chemical modifications can take place in secondary consumers. In contrast to algal toxins, the physiological role of MAAs is clearly the protection from harmful UV radiation by physical screening. However, other roles, e.g. as osmolytes and antioxidants, are also considered. In this paper the common characteristics of MAAs and marine toxins are discussed as well as the differences.     

Isolation and Synthesis of Laxaphycin B-Type Peptides: A Case Study and Clues to Their Biosynthesis

The laxaphyci’s B family constitutes a group of five related cyclic lipopeptides isolated from diverse cyanobacteria from all around the world. This group shares a typical structure of 12 amino acids from the l and d series, some of them hydroxylated at the beta position, and all containing a rare beta-amino decanoic acid. Nevertheless, they can be differentiated due to slight variations in the composition of their amino acids, but the configuration of their alpha carbon remains conserved. Here, we provide the synthesis and characterization of new laxaphycin B-type peptides. In doing so we discuss how the synthesis of laxaphycin B and analogues was developed. We also isolate minor acyclic laxaphycins B, which are considered clues to their biosynthesis.     

Structural Characterization of New Peptide Variants Produced by Cyanobacteria from the Brazilian Atlantic Coastal Forest Using Liquid Chromatography Coupled to Quadrupole Time-of-Flight Tandem Mass Spectrometry

Cyanobacteria from underexplored and extreme habitats are attracting increasing attention in the search for new bioactive substances. However, cyanobacterial communities from tropical and subtropical regions are still largely unknown, especially with respect to metabolite production. Among the structurally diverse secondary metabolites produced by these organisms, peptides are by far the most frequently described structures. In this work, liquid chromatography/electrospray ionization coupled to high resolution quadrupole time-of-flight tandem mass spectrometry with positive ion detection was applied to study the peptide profile of a group of cyanobacteria isolated from the Southeastern Brazilian coastal forest. A total of 38 peptides belonging to three different families (anabaenopeptins, aeruginosins, and cyanopeptolins) were detected in the extracts. Of the 38 peptides, 37 were detected here for the first time. New structural features were proposed based on mass accuracy data and isotopic patterns derived from full scan and MS/MS spectra. Interestingly, of the 40 surveyed strains only nine were confirmed to be peptide producers; all of these strains belonged to the order Nostocales (three Nostoc sp., two Desmonostoc sp. and four Brasilonema sp.).     

Algae-Derived Anti-Inflammatory Compounds against Particulate Matters-Induced Respiratory Diseases: A Systematic Review

Air pollution has recently become a subject of increasing concern in many parts of the world. The World Health Organization (WHO) estimated that nearly 4.2 million early deaths are due to exposure to fine particles in polluted air, which causes multiple respiratory diseases. Algae, as a natural product, can be an alternative treatment due to potential biofunctional properties and advantages. This systematic review aims to summarize and evaluate the evidence of metabolites derived from algae as potential anti-inflammatory agents against respiratory disorders induced by atmospheric particulate matter (PM). Databases such as Scopus, Web of Science, and PubMed were systematically searched for relevant published full articles from 2016 to 2020. The main key search terms were limited to “algae”, “anti-inflammation”, and “air pollutant”. The search activity resulted in the retrieval of a total of 36 publications. Nine publications are eligible for inclusion in this systematic review. A total of four brown algae (Ecklonia cava, Ishige okamurae, Sargassum binderi and Sargassum horneri) with phytosterol, polysaccharides and polyphenols were reported in the nine studies. The review sheds light on the pathways of particulate matter travelling into respiratory systems and causing inflammation, and on the mechanisms of actions of algae in inhibiting inflammation. Limitations and future directions are also discussed. More research is needed to investigate the potential of algae as anti-inflammatory agents against PM in in vivo and in vitro experimental models, as well as clinically.     

Activation of Dormant Secondary Metabolite Production by Introducing Neomycin Resistance into the Deep-Sea Fungus,Aspergillus versicolor ZBY-3

A new ultrasound-mediated approach has been developed to introduce neomycin-resistance to activate silent pathways for secondary metabolite production in a bio-inactive, deep-sea fungus, Aspergillus versicolor ZBY-3. Upon treatment of the ZBY-3 spores with a high concentration of neomycin by proper ultrasound irradiation, a total of 30 mutants were obtained by single colony isolation. The acquired resistance of the mutants to neomycin was confirmed by a resistance test. In contrast to the ZBY-3 strain, the EtOAc extracts of 22 of the 30 mutants inhibited the human cancer K562 cells, indicating that these mutants acquired a capability to produce antitumor metabolites. HPLC-photodiode array detector (PDAD)-UV and HPLC-electron spray ionization (ESI)-MS analyses of the EtOAc extracts of seven bioactive mutants and the ZBY-3 strain indicated that diverse secondary metabolites have been newly produced in the mutant extracts in contrast to the ZBY-3 extract. The followed isolation and characterization demonstrated that six metabolites, cyclo(d-Pro-d-Phe) (1), cyclo(d-Tyr-d-Pro) (2), phenethyl 5-oxo-l-prolinate (3), cyclo(l-Ile-l-Pro) (4), cyclo(l-Leu-l-Pro) (5) and 3β,5α,9α-trihydroxy-(22E,24R)-ergosta-7,22-dien-6-one (6), were newly produced by the mutant u2n2h3-3 compared to the parent ZBY-3 strain. Compound 3 was a new compound; 2 was isolated from a natural source for the first time, and all of these compounds were also not yet found in the metabolites of other A. versicolor strains. Compounds 1–6 inhibited the K562 cells, with inhibition rates of 54.6% (1), 72.9% (2), 23.5% (3), 29.6% (4), 30.9% (5) and 51.1% (6) at 100 μg/mL, and inhibited also other human cancer HL-60, BGC-823 and HeLa cells, to some extent. The present study demonstrated the effectiveness of the ultrasound-mediated approach to activate silent metabolite production in fungi by introducing acquired resistance to aminoglycosides and its potential for discovering new compounds from silent fungal metabolic pathways. This approach could be applied to elicit the metabolic potentials of other fungal isolates to discover new compounds from cryptic secondary metabolites.