PARALYTIC SHELLFISH TOXINS ARE RESTRICTED TO FEW SPECIES AMONG AUSTRALIA'S TAXONOMIC DIVERSITY OF CULTURED MICROALGAE1

There are at least 40,000 species of microalgae in the aquatic environment. Fifteen species of marine dinoflagellates and freshwater cyanobacteria are known to produce paralytic shellfish toxins (PSTs) and represent a threat to human and/or livestock health. Although known toxic species are regularly monitored, the wider cross‐section of microalgae has not been systematically tested for PSTs. Advances in rapid screening techniques have resulted in the development of highly sensitive and specific methods to detect PSTs, including the sodium channel and saxiphilin binding assays. These assays were used in this study in 96‐well formats to screen 234 highly diverse isolates of Australian freshwater and marine microalgae for PSTs. The screening assays detected five toxic species, representing one freshwater cyanobacterium (Anabaena circinalis Rabenhorst) and four species of marine dinoflagellates (Alexandrium minutum Halim, A. catenella Balech, A. tamarense Balech, and Gymnodinium catenatum Graham). Liquid chromatography‐fluorescence detection was used to identify 14 saxitoxin analogues across the five species, and each species exhibited distinct toxin profiles. These results indicate that PST production is restricted to a narrow range of microalgal species found in Australian waters.     

Microalgae and cyanobacteria as a source of glycosidase inhibitors

Culture filtrates and organic solvent extracts of over 500 freshwater and marine eukaryotic microalgae and cyanobacteria were screened for the presence of glycosidase inhibitors. Rapid colorimetric assays were used to detect inhibitors of alpha-glucosidase, alpha-amylase and beta-galactosidase. Inhibitors were found from 38 species. The results suggest that microalgae and cyanobacteria have potential as a source of glycosidase inhibitors which may have clinical applications.     

Identification of teratogenic polymethoxy-1-alkenes from Cylindrospermopsis raciborskii,and taxonomically diverse freshwater cyanobacteria and green algae

Cylindrospermopsis raciborskii is among the most commonly recognized toxigenic cyanobacteria associated with harmful algal blooms (HAB) in freshwater systems, and specifically associated with multiple water-soluble toxins. Lipophilic metabolites from C. raciborskii, however, were previously shown to exert teratogenicity (i.e. inhibition of vertebrate development) in the zebrafish (Danio rerio) embryo model, specifically suggesting the presence of additional bioactive compounds unrelated to the currently known toxins. In the present study, a series of known teratogenic polymethoxy-1-alkenes (PMA) were identified, purified and chemically characterized from an otherwise well-characterized strain of toxigenic C. raciborskii. Although PMA have been previously identified in other cyanobacteria, this is the first time they have been identified from this recognized HAB species. Following their identification from C. raciborskii, the taxonomic distribution of the PMA was additionally investigated by chemical screening of a freshwater algal (i.e. cyanobacteria, green algal) culture collection. Screening suggests that these compounds are distributed among phylogenetically diverse taxa. Furthermore, parallel screening of the algal culture collection, using the zebrafish embryo model of teratogenicity, the presence of PMA was found to closely correlate with developmental toxicity of these diverse algal isolates. Taken together, the data suggest PMA contribute to the toxicity of C. raciborskii, as well as apparently several other taxonomically disparate cyanobacterial and green algal genera, and may, accordingly, contribute to the toxicity of diverse freshwater HAB.     

Cyanotoxins from Black Band Disease of Corals and from Other Coral Reef Environments

Many cyanobacteria produce cyanotoxins, which has been well documented from freshwater environments but not investigated to the same extent in marine environments. Cyanobacteria are an obligate component of the polymicrobial disease of corals known as black band disease (BBD). Cyanotoxins were previously shown to be present in field samples of BBD and in a limited number of BBD cyanobacterial cultures. These toxins were suggested as one of the mechanisms contributing to BBD-associated coral tissue lysis and death. In this work, we tested nine cyanobacterial isolates from BBD and additionally nine isolated from non-BBD marine sources for their ability to produce toxins. The presence of toxins was determined using cell extracts of laboratory grown cyanobacterial cultures using ELISA and the PP2A assay. Based on these tests, it was shown that cyanobacterial toxins belonging to the microcystin/nodularin group were produced by cyanobacteria originating from both BBD and non-BBD sources. Several environmental factors that can be encountered in the highly dynamic microenvironment of BBD were tested for their effect on both cyanobacterial growth yield and rate of toxin production using two of the BBD isolates of the genera Leptolyngbya and Geitlerinema. While toxin production was the highest under mixotrophic conditions (light and glucose) for the Leptolyngbya isolate, it was highest under photoautotrophic conditions for the Geitlerinema isolate. Our results show that toxin production among marine cyanobacteria is more widespread than previously documented, and we present data showing three marine cyanobacterial genera (Phormidium, Pseudanabaena, and Spirulina) are newly identified as cyanotoxin producers. We also show that cyanotoxin production by BBD cyanobacteria can be affected by environmental factors that are present in the microenvironment associated with this coral disease.     

Pharmacology and toxicology of pahayokolide A, a bioactive metabolite from a freshwater species of Lyngbya isolated from the Florida Everglades

The genus of filamentous cyanobacteria, Lyngbya, has been found to be a rich source of bioactive metabolites. However, identification of such compounds from Lyngbya has largely focused on a few marine representatives. Here, we report on the pharmacology and toxicology of pahayokolide A from a freshwater isolate, Lyngbya sp. strain 15-2, from the Florida Everglades. Specifically, we investigated inhibition of microbial representatives and mammalian cell lines, as well as toxicity of the compound to both invertebrate and vertebrate models. Pahayokolide A inhibited representatives of Bacillus, as well as the yeast, Saccharomyces cerevisiae. Interestingly, the compound also inhibited several representatives of green algae that were also isolated from the Everglades. Pahayokolide A was shown to inhibit a number of cancer cell lines over a range of concentrations (IC50 varied from 2.13 to 44.57 microM) depending on the cell-type. When tested against brine shrimp, pahayokolide was only marginally toxic at the highest concentrations tested (1 mg/mL). The compound was, however, acutely toxic to zebrafish embryos (LC50=2.15 microM). Possible biomedical and environmental health aspects of the pahayokolides remain to be investigated; however, the identification of bioactive metabolites such as these demonstrates the potential of the Florida Everglades as source of new toxins and drugs.     

Application of molecular genetic and microbiological techniques in ecology and biotechnology of cyanobacteria

The review discusses the advances and problems in biotechnology and ecology of cyanobacteria and considers the possibilities of molecular genetic and microbiological techniques in this field. Due to the ease of cultivation, high growth rate, availability of synchronous cultures, and existence of numerous molecular genetic and microbiological techniques for various cyanobacterial strains, cyanobacteria—prokaryotic organisms that are ancient relatives of the chloroplasts—are model organisms in the studies of photosynthesis, dinitrogen fixation, cell division, hydrogen production, and in a number of other areas of basic and applied science. These techniques make possible deeper understanding of the role of cyanobacteria in various ecosystems and utilization of their potential in numerous applied projects, including production of molecular hydrogen, phycobiliproteins, and cyanophycin; formation of nanoparticles; removal of heavy metals from the environment; substrate biodegradation; manufacture of products for medicine and food industry; and solution of the problem of cyanobacterial toxins in freshwater and marine environments.     

163 Identification of Euglenoids That Produce Ichthyotoxin(S) (Euglenophyta)

Diatoms, dinoflagellates, pelagiophytes, prymnesiophytes, and cyanobacteria are the only divisions of microalgae known to produce toxins. We now report toxin production by freshwater members of the genus Euglena. Fish mortalities (sheepshead minnows, catfish, striped bass, and tilapia) have been observed following exposure in the field to Euglena blooms and in the laboratory when exposed to unialgal isolates of two species of Euglena ( E. sanguinea Ehrenberg and E. granulata (Klebs) Lemm.). Three toxic fractions have been isolated from unialgal isolates of both species, and include both water soluble and lipophilic compounds having ichthyotoxic activity. The toxins are stable at −80°C for at least 60 days and are heat stable to 30°C. Erratic swimming behavior of fish suggests a neurological toxin. This is the first report of fish kills by any freshwater algal taxa from both field and laboratory studies.     

Biodiversity and application of microalgae

The algae are a polyphyletic, artificial assemblage of O₂-evolving, photosynthetic organisms (and secondarily nonphotosynthetic evolutionary descendants) that includes seaweeds (macroalgae) and a highly diverse group of microorganisms known as microalgae. Phycology, the study of algae, developed historically as a discipline focused on the morphological, physiological and ecological similarities of the subject organisms, including the prokaryotic bluegreen algae (cyanobacteria) and prochlorophytes. Eukaryotic algal groups represent at least five distinct evolutionary lineages, some of which include protists traditionally recognized as fungi and protozoa. Ubiquitous in marine, freshwater and terrestrial habitats and possessing broad biochemical diversity, the number of algal species has been estimated at between one and ten million, most of which are microalgae. The implied biochemical diversity is the basis for many biotechnological and industrial applications.     

Identification of a diagnostic marker to detect freshwater cyanophages of filamentous cyanobacteria

Cyanophages are viruses that infect the cyanobacteria, globally important photosynthetic microorganisms. Cyanophages are considered significant components of microbial communities, playing major roles in influencing host community diversity and primary productivity, terminating cyanobacterial water blooms, and influencing biogeochemical cycles. Cyanophages are ubiquitous in both marine and freshwater systems; however, the majority of molecular research has been biased toward the study of marine cyanophages. In this study, a diagnostic probe was developed to detect freshwater cyanophages in natural waters. Oligonucleotide PCR-based primers were designed to specifically amplify the major capsid protein gene from previously characterized freshwater cyanomyoviruses that are infectious to the filamentous, nitrogen-fixing cyanobacterial genera Anabaena and Nostoc. The primers were also successful in yielding PCR products from mixed virus communities concentrated from water samples collected from freshwater lakes in the United Kingdom. The probes are thought to provide a useful tool for the investigation of cyanophage diversity in freshwater environments.     

Freshwater cyanophages

Cyanophages are double-stranded DNA viruses that infect cyanobacteria, and they can be found in both freshwater and marine environments. They have a complex pattern of host ranges and play important roles in controlling cyanobacteria population. Unlike marine cyanophages, for which there have been a number of recent investigations, very little attention has been paid to freshwater cyanophages. This review summarizes the taxonomy and morphology, host range, distribution, seasonal dynamics, and complete genomes of freshwater cyanophages, as well as diagnostic markers that can be used to identify them.     

New horizons in culture and valorization of red microalgae

Research on marine microalgae has been abundantly published and patented these last years leading to the production and/or the characterization of some biomolecules such as pigments, proteins, enzymes, biofuels, polyunsaturated fatty acids, enzymes and hydrocolloids. This literature focusing on metabolic pathways, structural characterization of biomolecules, taxonomy, optimization of culture conditions, biorefinery and downstream process is often optimistic considering the valorization of these biocompounds. However, the accumulation of knowledge associated with the development of processes and technologies for biomass production and its treatment has sometimes led to success in the commercial arena. In the history of the microalgae market, red marine microalgae are well positioned particularly for applications in the field of high value pigment and hydrocolloid productions. This review aims to establish the state of the art of the diversity of red marine microalgae, the advances in characterization of their metabolites and the developments of bioprocesses to produce this biomass.     

Identification of a Diagnostic Marker To Detect Freshwater Cyanophages of Filamentous Cyanobacteria

Cyanophages are viruses that infect the cyanobacteria, globally important photosynthetic microorganisms. Cyanophages are considered significant components of microbial communities, playing major roles in influencing host community diversity and primary productivity, terminating cyanobacterial water blooms, and influencing biogeochemical cycles. Cyanophages are ubiquitous in both marine and freshwater systems; however, the majority of molecular research has been biased toward the study of marine cyanophages. In this study, a diagnostic probe was developed to detect freshwater cyanophages in natural waters. Oligonucleotide PCR-based primers were designed to specifically amplify the major capsid protein gene from previously characterized freshwater cyanomyoviruses that are infectious to the filamentous, nitrogen-fixing cyanobacterial genera Anabaena and Nostoc. The primers were also successful in yielding PCR products from mixed virus communities concentrated from water samples collected from freshwater lakes in the United Kingdom. The probes are thought to provide a useful tool for the investigation of cyanophage diversity in freshwater environments.     

Distribution and diversity of natural product genes in marine and freshwater cyanobacterial cultures and genomes

Natural products are a functionally diverse class of biochemically synthesized compounds, which include antibiotics, toxins, and siderophores. In this paper, we describe both the detection of natural product activities and the sequence identification of gene fragments from two molecular systems that have previously been implicated in natural product production, i.e., nonribosomal peptide synthetases (NRPSs) and modular polyketide synthases (PKSs), in diverse marine and freshwater cyanobacterial cultures. Using degenerate PCR and the sequencing of cloned products, we show that NRPSs and PKSs are common among the cyanobacteria tested. Our molecular data, when combined with genomic searches of finished and progressing cyanobacterial genomes, demonstrate that not all cyanobacteria contain NRPS and PKS genes and that the filamentous and heterocystous cyanobacteria are the richest sources of these genes and the most likely sources of novel natural products within the phylum. In addition to validating the use of degenerate primers for the identification of PKS and NRPS genes in cyanobacteria, this study also defines numerous gene fragments that will be useful as probes for future studies of the synthesis of natural products in cyanobacteria. Phylogenetic analyses of the cyanobacterial NRPS and PKS fragments sequenced in this study, as well as those from the cyanobacterial genome projects, demonstrate that there is remarkable diversity and likely novelty of these genes within the cyanobacteria. These results underscore the potential variety of novel products being produced by these ubiquitous organisms.     

Detection of saxitoxin-producing cyanobacteria and Anabaena circinalis in environmental water blooms by quantitative PCR

Saxitoxins (STXs) are carbamate alkaloid neurotoxins produced by marine "red tide" dinoflagellates and several species of freshwater filamentous cyanobacteria, including Anabaena circinalis, Aphanizomenon spp., Lyngbya wollei, and Cylindrospermopsis raciborskii. A specific quantitative PCR (qPCR) method based on SYBR green chemistry was developed to quantify saxitoxin-producing Anabaena circinalis cyanobacteria, which are major bloom-forming freshwater cyanobacteria. The aim of this study was to infer the potential toxigenicity of samples by determining the copy number of a unique and unusual polyketide synthase (PKS) sequence (sxtA) in the STX biosynthesis gene cluster identified in cyanobacteria. Our qPCR approach was applied to water samples collected from different Australian lakes, dams, and rivers. The STX concentration and cyanobacterial cell density of these blooms were also determined by high-pressure liquid chromatography (HPLC) and microscopic cell counting, respectively. STX concentrations correlated positively with STX gene copy numbers, indicating that the latter can be used as a measure of potential toxigenicity in Anabaena circinalis and possibly other cyanobacterial blooms. The qPCR method targeting STX genes can also be employed for both monitoring and ecophysiological studies of toxic Anabaena circinalis blooms and potentially several other STX-producing cyanobacteria.     

MICROALGAE AND CYANOBACTERIA: FOOD FOR THOUGHT1

In non‐Western civilizations, cyanobacteria have been part of the human diet for centuries. Today, microalgae and cyanobacteria are either produced in controlled cultivation processes or harvested from the natural habitats and marketed as food supplements around the world. Cyanobacteria produce a vast array of different biologically active compounds, some of which are expected to be used in drug development. The fact that some of the active components from cyanobacteria potentially have anticancer, antimicrobial, antiviral, anti‐inflammatory, and other effects is being used for marketing purposes. However, introduction of these products in the form of whole biomass for alimentary purposes raises concerns regarding the potential toxicity and long‐term effects on human health. Here, we review data on the use of cyanobacteria and microalgae in human nutrition and searched for available information on legislature that regulates the use of these products. We have found that, although the quality control of these products is most often self‐regulated by the manufacturers, different governmental agencies are introducing strict regulations for placing novel products, such as algae and cyanobacteria, on the market. The existing regulations require these products to be tested for the presence of toxins, such as microcystin; however, other, sometimes novel, toxins remain undetected, and their long‐term effects on human health remain unknown.     

Genetic engineering in marine cyanobacteria

Many species of microalgae producing useful materials have been isolated from marine environments. For their industrial application, widely applicable and stable gene expression is required. It is necessary to establish gene transfer methods as an essential first step in genetic manipulation. Although gene transfer techniques for cyanobacteria have been developed, only naturally transformable strains have been used. Here, we describe recent progress made in developing gene transfer methods for marine cyanobacteria. The following are covered: (1) transformation, (2) electroporation, (3) conjugation, (4) particle gun. A plasmid from the marine cyanobacterium, Synechococcus sp., whose copy number is dependent on salinity, was characterized. This plasmid is being used to develop a stable and controllable gene expression system.     

Blue green algal (cyanobacterial) toxins, surface drinking water, and liver cancer in Florida

The blue green algae or cyanobacteria represent a diverse group of organisms that produce potent natural toxins. There have been case reports of severe morbidity and mortality in domestic animals through drinking water contaminated by these toxins. Microcystins, in particular, have been associated with acute liver damage and possibly liver cancer in laboratory animals. Although, there has been little epidemiologic research on toxin effects in humans, a study by Yu (1995) found an association between primary liver cancer and surface water. Surface water drinking supplies are particularly vulnerable to the growth of these organisms; current US drinking water treatment practices do not monitor or actively treat for blue green algal toxins including the microcystins.After a monitoring survey in Florida found organisms and microcystins (among other cyanobacterial toxins) in surface water drinking sources, a pilot ecological study was performed using a Geographic Information System (GIS) to evaluate the risk of primary hepatocellular carcinoma (HCC) and proximity to a surface water treatment plant at cancer diagnosis. The study linked all HCC cancers diagnosed in Florida from 1981 to 1998 with environmental databases.A significantly increased risk for HCC with residence within the service area of a surface water treatment plant was found compared to persons living in areas contiguous to the surface water treatment plants. However, this increased risk was not seen in comparison to persons living in randomly selected ground water treatment service areas or compared to the Florida cumulative incidence rate for the study period, using various comparison and GIS methodologies. Furthermore, these findings must be interpreted in light of significant issues of latency, high population mobility, and the lack of individual exposure information. Nevertheless, the issue of acute and chronic human health effects associated with the consumption of surface waters possibly contaminated by blue green algal toxins merits further investigation.     

Metabolites from freshwater aquatic microalgae and fungi as potential natural pesticides

Microorganisms are recognized worldwide as the major source of secondary metabolites with mega diverse structures and promissory biological activities. However, as yet many of them remain little or under-explored like the microbiota from freshwater aquatic ecosystems. In the present review, we undertook a recompilation of metabolites reported with pesticidal properties from microalgae (cyanobacteria and green algae) and fungi, specifically from freshwater aquatic habitats.     

Harmful blooms of cyanobacteria (Oscillatoriaceae) and dinoflagellates (Gymnodiniaceae) in the Golfo de Nicoya, Costa Rica

Recently, the Pacific coast of Costa Rica has experienced an increase in both magnitude and frequency of harmful algae blooms (HAB). The lack of data regarding the dynamics of these events in the area, and the species of microalgae that produce them, are themes of great interest. The blooms have produced negative impacts on fishery resources and on human health in Costa Rica. In May 2002 a HAB left a large number of dead fish along the central Pacific coast. Water samples were collected using a phytoplankton net and fixed for subsequent processing by electron microscopy. In addition, a one liter sample of surface water was taken for later cell count. In the observed HAB, the dominating organisms found were the cyanobacteria Trichodesmiun erythraeum surrounded by high concentrations of Gram-bacteria and the dinoflagellate Cochlodinium cf. polykrikoides. T. erythraeum, is one of the most important N2 fixing cyanobacteria in marine waters that has been associated with HAB events in diverse parts of the world as well as with symptoms that produce contact dermatitis and other discomforts. C. cf. polykrikoides is a dinoflagellete associated with fish kills; although the type of associated toxins are unknown. In a national newspaper 17 cases of intoxication in humans were reported during this same period, which presented respiratory disorders and burning of the eyes. This is the first report in Costa Rica where a cyanobacteria and a dinoflagellate were observed together producing HAB.     

Analysis of the hli gene family in marine and freshwater cyanobacteria

Certain cyanobacteria thrive in natural habitats in which light intensities can reach 2000 micromol photon m(-2) s(-1) and nutrient levels are extremely low. Recently, a family of genes designated hli was demonstrated to be important for survival of cyanobacteria during exposure to high light. In this study we have identified members of the hli gene family in seven cyanobacterial genomes, including those of a marine cyanobacterium adapted to high-light growth in surface waters of the open ocean (Prochlorococcus sp. strain Med4), three marine cyanobacteria adapted to growth in moderate- or low-light (Prochlorococcus sp. strain MIT9313, Prochlorococcus marinus SS120, and Synechococcus WH8102), and three freshwater strains (the unicellular Synechocystis sp. strain PCC6803 and the filamentous species Nostoc punctiforme strain ATCC29133 and Anabaena sp. [Nostoc] strain PCC7120). The high-light-adapted Prochlorococcus Med4 has the smallest genome (1.7 Mb), yet it has more than twice as many hli genes as any of the other six cyanobacterial species, some of which appear to have arisen from recent duplication events. Based on cluster analysis, some groups of hli genes appear to be specific to either marine or freshwater cyanobacteria. This information is discussed with respect to the role of hli genes in the acclimation of cyanobacteria to high light, and the possible relationships among members of this diverse gene family.