Isolation of polyketides from Prymnesium parvum (Haptophyta) and their detection by liquid chromatography/mass spectrometry metabolic fingerprint analysis

Prymnesium parvum is a microalga that forms blooms coupled with the presence of potent exotoxins; however, no chemical standards are currently available for the toxins. Streamlined methods are presented for the separation and enrichment of polyketide toxins, prymnesin-1 (prym1) and prymnesin-2 (prym2). Prymnesins were separated by reversed-phase chromatography and detected by positive-mode electrospray ionization MS to generate a unique metabolic fingerprint. More than 10 ions were detected and mass assignments were in agreement with predicted isotopic distributions for the intact compounds and related fragments; ions occurred as multiply protonated species and with common salt adducts. The most prevalent ion was observed at 919.88 m/z, which represents the aglycone [prym_agly + 2H]²⁺ backbone structure common to both molecules. Expanded mass spectra for this and related ions were in excellent agreement (<0.5 ppm) with empirically derived spectra based on elemental composition and naturally occurring isotopes. These investigations have confirmed the isolation of polyketide prymnesins from whole cells, which heretofore has not been reproduced since their original characterization. Moreover, this study represents the first time these compounds have been verified in aqueous materials. These tools should allow the direct identification and analysis of polyketide prymnesins, which will greatly improve our understanding of these toxins in P. parvum.     

Increase in the production of allelopathic substances by Prymnesium parvum cells grown under N- or P-deficient conditions

The haptophyte Prymnesium parvum is known to produce a set of highly potent exotoxins, commonly called prymnesins. These toxins have been shown to have several biological activities, including ichthyotoxic, neurotoxic, cytotoxic, hepatotoxic and hemolytic activity towards a range of marine organisms. In addition, recent studies have shown that the toxicity of P. parvum is enhanced when the cells are grown under N- or P-deficient conditions. In this study, the influence of prymnesium toxins on the growth of other phytoplankton species was investigated by addition of cell-free filtrate of P. parvum cultures grown under nutrient-deficient (N or P) or non-deficient conditions. Addition of cell-free filtrate from P. parvum cultures grown under N or P limitation inhibited the growth of Thalassiosira weissflogii, Prorocentrum minimum and Rhodomonas cf. baltica. In contrast, a strain of Prymnesium patelliferum known to produce prymnesium toxins was not negatively affected under any conditions. Furthermore, addition of filtrates from nutrient-sufficient P. parvum cultures did not negatively influence the growth of any of the tested species. These findings suggest that prymnesium toxins may play an allelopathic role, and that the production of allelopathic substances is regulated by the availability of nutrients.     

The ecophysiology and bloom dynamics of Prymnesium spp.

Members of Prymnesium belong to the division Haptophyta, class Prymnesiophyceae, order Prymnesiales and family Prymnesiaceae. As most haptophytes, members of the genus Prymnesium are unicellular and planktonic. The most known of these species is the ichthyotoxic P. parvum, which may form nearly monospecific dense blooms in coastal and inland waters. This species possesses extraordinary plasticity concerning life survival strategies, and is specifically addressed in this review.Toxins produced by P. parvum have hemolytic properties, that not only kill fish but also co-existing plankton. These substances are allelopathic (when other algae are killed) and grazer deterrent (when grazers are killed). Allelopathy enables P. parvum to utilize inorganic nutrients present in the surrounding water without competition from other algal species; and by eliminating its grazers P. parvum reduces cell losses. The paralized microalgae and/or zooplankton, are therefter ingested by the P. parvum cells, a process called phagotrophy. P. parvum is also able of osmotrophy, i.e. utilization of dissolved organic matter. In this review, the cellular characteristics, life cycles, bloom formation, and factors affecting toxicity, allelopathy, phagotrophy, and osmotrophy of P. parvum are discussed.     

Identification of toxic fatty acid amides isolated from the harmful alga Prymnesium parvum carter

The golden alga Prymnesium parvum has been implicated in fish and aquatic animal kills globally for over a century. In addition to widespread ecological impacts through the loss of entire fish populations within lakes, an economic burden is also felt by state and local agencies due to year class losses of fish raised for stocking lakes as well as loss of fishing and recreational use of the affected water. Multiple compounds have been implicated in P. parvum toxicity, but the unequivocal identification and characterization of all P. parvum toxins remained to be accomplished. To unambiguously characterize these toxins, we analyzed laboratory-cultured cells exposed to limited nitrogen and phosphorus concentrations, uni-algal wild cells collected from an ichthytoxic bloom event at Lake Wichita, TX, and the water from both cultured and field-collected algae. A bioassay-guided fractionation process was employed to chemically isolate P. parvum toxins using both mammalian cells and larval fish. The results of these assays revealed that there was a distinct similarity in the toxic compounds characterized as seven primary fatty acid amides (myristamide, palmitamide, linoleamide, oleamide, elaidamide, stearamide, and erucamide) and one hydroxamic acid (linoleyl hydroxamic acid). These compounds display cytotoxic and ichthytoxic activity and have not yet been reported in P. parvum toxicity or in the toxicity of harmful algal species.     

The mechanism of the action of prymnesium toxin on membranes

The mechanism of the lytic action of prymnesin, a toxin produced by the alga, Prymnesium parvum, was studied using liposomes as a model membrane system. Prymnesin showed severe damage to liposomes containing cholesterol but did not affect those without cholesterol. The requirement of cholesterol for the susceptibility to prymnesin was much more strict than the reported requirement for the susceptibility to polyene antibiotics. The net charge on the membranes was shown not to be important for the reaction.     

The effects of aeration on growth and toxicity of Prymnesium parvum grown with and without algal prey

We investigated the effects of aeration on growth and toxicity of the haptophyte Prymnesium parvum in the presence and absence of the algal prey Rhodomonas salina. Batch monocultures of P-limited P. parvum and N and P sufficient R. salina and mixed cultures of the two microalgae were grown with no, low (20) and high (100) ml min⁻¹ aeration for 18 days. Cell growth of P. parvum and R. salina and cell toxicity of P. parvum were studied over the experimental period. The highest specific growth rates of P. parvum were found at low aeration rates. R. salina in monocultures showed typical growth patterns, while R. salina numbers declined rapidly in the mixed cultures. Of the initial cell densities, 98–100% of the R. salina cells were lysed or ingested within 24 h of mixing with P. parvum cells. The maxima P. parvum biomasses were significantly higher in the mixed cultures than in the monocultures. Cell toxicity of P. parvum increased significantly in response to aeration rates and the highest levels were found in the high aeration condition. Availability of prey and resupply of inorganic nutrients decreased P. parvum cell toxicity. Our study suggests that P. parvum is tolerant and is able to grow over a broad range of aeration and associated turbulence effects though low aeration represents an optimal condition for growth. As P. parvum toxicity was higher in the high aeration treatment we suggest that the higher concentrations of oxygen cause more toxins to be produced, as these are oxygen rich compounds. We suggest that oxygenation and turbulence of surface waters caused by mixing may be involved in promoting high toxic P. parvum blooms in shallow lakes and coastal waters.     

Prymnesium parvum invasion success into coastal bays of the Gulf of Mexico: Galveston Bay case study

The toxic haptophyte Prymnesium parvum regularly forms fish-killing blooms in inland brackish water bodies in the south-central USA. Along the Texas coast smaller blooms have occurred in isolated areas. There appears to be an increasing risk that harmful P. parvum blooms will propagate into open coastal waters with implementation of future water plans. These plans will include increased interbasin water transfers from the Brazos River, regularly impacted by P. parvum blooms, to the San Jacinto-Brazos Coastal Basin, which ultimately flows into Galveston Bay (GB). Persisting source populations of P. parvum in inland waters elevates this risk. Thus, there is a need for an increased understanding of how P. parvum might perform in coastal waters, such as those found in GB. Here, two in-field experiments were conducted to investigate the influence of various plankton size-fractions of GB water on inoculated P. parvum during fall and winter, periods when blooms are typically initiating and developing inland. Stationary- and log-growth phase P. parvum were used to represent high and low toxicity initial conditions. Results revealed that P. parvum could grow in GB waters and cause acute mortality to silverside minnows (Menidia beryllina). Depending on season and growth phase, however, P. parvum growth and toxicity varied in different size fractions. During the fall, P. parvum inoculated from stationary-, but not log-growth phase culture, was negatively affected by bacteria-sized particles. During the winter, bacteria and nanoplankton together had a negative effect on P. parvum inoculated from stationary- and, to a lesser degree, log-growth phase cultures. Intermediate- and large-sized grazers when combined with bacteria and nanoplankton had complex relationships with inoculated P. parvum, sometimes stimulating and sometimes suppressing population growth. Toxicity to fish occurred in almost all plankton size fractions. The inclusion of progressively larger sized plankton fractions resulted in trends of decreased toxicity in treatments inoculated with stationary-, but not log-growth phase P. parvum in the fall. In the winter, however, inclusion of larger sized plankton fractions resulted in trends of increased toxicity to fish in treatments inoculated with both stationary- and log-growth phase P. parvum. This study indicates that understanding P. parvum population dynamics in open waters of estuaries and bays will be challenging, as there appears to be complex relationships with naturally occurring components of the plankton. The observations that P. parvum is able to grow to high population density and produce fish-killing levels of toxins underscores the need for advanced risk assessment studies, especially in light of water use plans that will result in P. parvum invasions of greater size.     

Occurrence of toxic Prymnesium parvum blooms with high protease activity is related to fish mortality in Hungarian ponds

The unicellular alga Prymnesium parvum has been responsible for toxic incidents with severe ecological impacts in many parts of the world, and causes massive fish kills worldwide. Recently the haptophyte microalgae have caused water-bloom (4.3 × 10⁴ cells ml⁻¹) in 6 fish ponds with high conductivity in Hungary, and caused fish mortality with typical symptoms. Toxicity of P. parvum from water samples was quantified by the assay of the influence of its cell-free filtrates on haemolysis (346 ± 42.2) and in fish and daphnia toxicity tests. High amount of proteases in P. parvum containing waterbloom samples were detected with the help of activity gel electrophoresis. The proteases of investigated P. parvum samples (125–18 kDa) showed high gelatinolytic activity and some of them showed sensitivity to EDTA (inhibitors of metalloproteases) and to PMSF (inhibitors of serine proteases).     

Rapid Quantification of the Toxic Alga Prymnesium parvum in Natural Samples by Use of a Specific Monoclonal Antibody and Solid-Phase Cytometry

The increasing incidence of harmful algal blooms around the world and their associated health and economic effects require the development of methods to rapidly and accurately detect and enumerate the target species. Here we describe use of a solid-phase cytometer to detect and enumerate the toxic alga Prymnesium parvum in natural samples, using a specific monoclonal antibody and indirect immunofluorescence. The immunoglobulin G antibody 16E4 exhibited narrow specificity in that it recognized several P. parvum strains and a Prymnesium nemamethecum strain but it did not cross-react with P. parvum strains from Scandinavia or any other algal strains, including species of the closely related genus Chrysochromulina. Prymnesium sp. cells labeled with 16E4 were readily detected by the solid-phase cytometer because of the large fluorescence signal and the signal/noise ratio. Immunofluorescence detection and enumeration of cultured P. parvum cells preserved with different fixatives showed that the highest cell counts were obtained when cells were fixed with either glutaraldehyde or formaldehyde plus the cell protectant Pluronic F-68, whereas the use of formaldehyde alone resulted in significantly lower counts. Immunofluorescence labeling and analysis with the solid-phase cytometer of fixed natural samples from a bloom of P. parvum occurring in Lake Colorado in Texas gave cell counts that were close to those obtained by the traditional method of counting using light microscopy. These results show that a solid-phase cytometer can be used to rapidly enumerate natural P. parvum cells and that it could be used to detect other toxic algae, with an appropriate antibody or DNA probe.     

Phylogenetic Characterization of Virulence and Resistance Phenotypes of Pseudomonas syringae

Individual strains of the plant pathogenic bacterium Pseudomonas syringae vary in their ability to produce toxins, nucleate ice, and resist antimicrobial compounds. These phenotypes enhance virulence, but it is not clear whether they play a dominant role in specific pathogen-host interactions. To investigate the evolution of these virulence-associated phenotypes, we used functional assays to survey for the distribution of these phenotypes among a collection of 95 P. syringae strains. All of these strains were phylogenetically characterized via multilocus sequence typing (MLST). We surveyed for the production of coronatine, phaseolotoxin, syringomycin, and tabtoxin; for resistance to ampicillin, chloramphenicol, rifampin, streptomycin, tetracycline, kanamycin, and copper; and for the ability to nucleate ice at high temperatures via the ice-nucleating protein INA. We found that fewer than 50% of the strains produced toxins and significantly fewer strains than expected produced multiple toxins, leading to the speculation that there is a cost associated with the production of multiple toxins. None of these toxins was associated with host of isolation, and their distribution, relative to core genome phylogeny, indicated extensive horizontal genetic exchange. Most strains were resistant to ampicillin and copper and had the ability to nucleate ice, and yet very few strains were resistant to the other antibiotics. The distribution of the rare resistance phenotypes was also inconsistent with the clonal history of the species and did not associate with host of isolation. The present study provides a robust phylogenetic foundation for the study of these important virulence-associated phenotypes in P. syringae host colonization and pathogenesis.     

Stability of the intra- and extracellular toxins of Prymnesium parvum using a microalgal bioassay

Prymnesium parvum produces a variety of toxic compounds, which affect other algae, grazers and organisms at higher trophic levels. Here we provide the method for development of a sensitive algal bioassay using a microalgal target, Teleaulax acuta, to measure strain variability in P. parvum toxicity, as well as the temporal stability of both the intracellular and the extracellular lytic compounds of P. parvum. We show high strain variation in toxicities after 3 h incubation with LC₅₀s ranging from 24 to 223 × 10³ cells ml⁻¹. Most importantly we prove the necessity of testing physico-chemical properties of P. parvum toxins before attempting to isolate and characterize them. The extracellular toxin in the supernatant is highly unstable, and it loses significant lytic effects after 3 days despite storage at −20 °C and after only 24 h stored at 4 °C. However, when stored at −80 °C, lytic activity is more easily maintained. Reducing oxidation by storing the supernatant with no headspace in the vials significantly slowed loss of activity when stored at 4 °C. We show that the lytic activity of the intracellular toxins, when released by sonication, is not as high as the extracellular toxins, however the stability of the intracellular toxins when kept as a cell pellet at −20 °C is excellent, which proves this is a sufficient storage method for less than 3 months. Our results provide an ecologically appropriate algal bioassay to quantify lytic activity of P. parvum toxins and we have advanced our knowledge of how to handle and store the toxins from P. parvum so as to maintain biologically relevant toxicity.     

Production of BMAA and DAB by diatoms (Phaeodactylum tricornutum,Chaetoceros sp., Chaetoceros calcitrans and,Thalassiosira pseudonana) and bacteria isolated from a diatom culture

Microalgae have previously been reported to contain β-N-methylamino-l-alanine (BMAA), and the global presence of these primary producers has been associated with the widespread occurrence of BMAA in marine organisms. It has been repeatedly shown that filter-feeding bivalves accumulate phytoplankton species and their toxins. In this study, the concentrations of total soluble BMAA and DAB as a function of growth phase were observed for four non-axenic diatom species (i.e. Phaeodactylum tricornutum, Chaetoceros sp., Chaetoceros calcitrans and Thalassiosira pseudonana). These strains had previously been shown to contain BMAA using a highly selective HILIC-MS/MS method. BMAA cell quota appeared to be species-specific, however, highest BMAA concentrations were always obtained during the stationary growth phase, for all four species, suggesting that BMAA is a secondary metabolite. While DAB was detected in a bacterial culture isolated from a culture of P. tricornutum, the presence or absence of a bacterial population did not influence production of BMAA and DAB by P. tricornutum, i.e. no significant difference was noted for BMAA and DAB production between axenic and non-axenic cultures. The presence of DAB in bacteria had previously been shown, and raised the question as to whether DAB observed in many species of microalgae may arise from the non-axenic culture conditions or from the microalgae themselves.     

Hemolysis induced by Prymnesium parvum toxin effect of primaquine treatment

The effect of primaquine (1 mM) incubation on rabbit erythrocytes was studied at 25° using the hemolytic toxin (prymnesin) of the chrysomonad Prymnesium parvum. One notable effect is the alteration of rate of prymnesin-induced hemolysis. The hemolysis rate constant, k_ψ, showed a biphasic dependence on length of primaquine incubation: a gradual increase in k_ψ was observed (0–4 h), followed by a more pronounced increase (4–6 h). Incubation with primaquine (an antimalarial) is known to cause invaginations and loss of cell surface by subsequent internalization. No biphasic effect of primaquine incubation was noted in the tendency of prymnesin to be bound (using ³H-labeled toxin). Median cell volume, however, does show a biphasic relationship, and it appears that the biphasic effect depends upon changes in one out of two or more populations of cells.     

Fusion of chicken erythrocytes by phospholipase C (Clostridium perfringens): The requirement for hemolytic and hydrolytic factors for fusion

Phospholipase C (Clostridium perfringens) preparations are able to induce fusion of chicken erythrocytes only in the presence of low concentrations (0.6 mM) of Mn²⁺. Anti-phospholipase C serum inhibits hemolysis and fusion of the cells only when added during the first 20 min of the incubation time. Heated phospholipase C preparations which fail to hemolyze chicken erythrocytes are able to induce fusion only in the presence of another hemolytic reagent such as prymnesin (an hemolytic toxin from Prymnesium parvum).     

Nuclear Ribosomal DNA Variation and Pathogenic Specialization in Alternaria Fungi Known To Produce Host-Specific Toxins

A total of 99 strains of 11 Alternaria species, including 68 strains of seven fungi known to produce host-specific toxins, were subjected to analysis of restriction fragment length polymorphism (RFLP) in nuclear ribosomal DNA (rDNA). Total DNA was digested with XbaI, and the Southern blots were probed with a nuclear rDNA clone of Alternaria kikuchiana. The hybridization gave 17 different RFLPs from the 99 strains. On the basis of these RFLPs, populations of host-specific toxin-producing fungi could not be differentiated from one another nor from nonpathogenic A. alternata. Each population of the toxin-producing fungi carried rDNA variants. Nine different types, named A1 to A6 and B1 to B3, were detected among the toxin-producing fungi and nonpathogenic A. alternata. All of the populations contained the type A4 variant, and the other rDNA types were also shared by different toxin-producing fungi and A. alternata. In contrast, Alternaria species that are morphologically distinguishable from A. alternata could be differentiated from A. alternata on the basis of the rDNA RFLPs. Polymorphisms in rDNA digested with HaeIII and MspI were also evaluated in 61 Alternaria strains. These restriction enzymes produced 31 variations among all of the samples. The seven toxin-producing fungi and nonpathogenic A. alternata could not be resolved by phylogenetic analysis based on the RFLPs, although they could be differentiated from the other Alternaria species studied. These results provide support for the hypothesis that Alternaria fungi known to produce host-specific toxins are intraspecific variants of A. alternata specialized in pathogenicity.     

Fish kills related toPrymnesium parvum N. Carter (Haptophyta) in the People's Republic of China

Prymnesium parvum has been known to cause mass mortality of fish in PR of China since 1963. It usually occurs in brackish waters and inland high-mineral waters. The fish-breeding industry (mainly species of carp) in these regions of the PRC has been threatened by this microalga. Electron microscopic examination of isolates from Dalian and Tianjin revealed that the isolates wereP. parvum, based on specific scale patterns and two kinds of scales. The symptoms of the poisoned fish and the control of this toxic alga are also discussed. The addition of ammonium sulfate, copper sulfate, mud, reduced salinity and organic fertilizer to fish ponds has been partially successful in controlling blooms of this toxic alga. Adding 50–70 kg ha^–1 day^–1 manure (dry weight) to the fish pond to inhibitP. parvum from becoming the dominant species in the fish pond is recommended. A reduction in salinity to less than 2 is the easiest way to save freshwater fish from being poisoned byP. parvum. Use of ammonium sulfate is an efficient, economical and safer method to controlP. parvum than copper sulfate or mud.     

Incorporating molecular tools into routine HAB monitoring programs: Using qPCR to track invasive Prymnesium

Microscopy, a staple of monitoring programs for tracking the occurrence and abundance of harmful algal bloom (HAB) species, is time consuming and often characterized by high uncertainty. Alternate methods that allow rapid and accurate assessment of presence and abundance of HAB species are needed. For many HAB species, such as the toxigenic haptophyte, Prymnesium parvum, molecular methods including quantitative real-time PCR (qPCR) have been developed with the suggestion that they should be useful for monitoring programs. However, this suggestion rarely has been put into action. In this study, we modified a recently developed method for detecting P. parvum using qPCR and tested its efficacy as an alternative to microscopy for P. parvum detection and enumeration in a long-term monitoring program in a recently invaded subtropical US reservoir. Abundance estimates of P. parvum were similar for both methods, but we detected P. parvum at multiple sites using qPCR where it previously had gone undetected by microscopy. Using qPCR, we substantially reduced processing time, increased detection limit and reduced error in P. parvum abundance estimates compared to microscopy. Thus, qPCR is an effective tool for detecting and monitoring P. parvum, particularly at pre-bloom densities, and should likewise prove useful in monitoring programs for the other HAB species for which qPCR methods have been developed.     

Understanding the winning strategies used by the bloom-forming cyanobacterium Cylindrospermopsis raciborskii

The cyanobacterium Cylindrospermopsis raciborskii is a widespread species increasingly being recorded in freshwater systems around the world. It is of particular concern because strains in some geographic areas are capable of producing toxins with implications for human and animal health. Studies of this species have increased rapidly in the last two decades, especially in the southern hemisphere where toxic strains are prevalent. A clearer picture is emerging of the strategies adopted by this species to bloom and out-compete other species. This species has a high level of flexibility with respect to light and nutrients, with higher temperatures and carbon dioxide also promoting growth. There are two types of toxins produced by C. raciborskii: cylindrospermopsins (CYNs) and saxitoxins (STXs). The toxins CYNs are constitutively produced irrespective of environmental conditions and the ecological or physiological role is unclear, while STXs appear to serve as protection against high salinity and/or water hardness. It is also apparent that strains of this species can vary substantially in their physiological responses to environmental conditions, including CYNs production, and this may explain discrepancies in findings from studies in different geographical areas. The combination of a flexible strategy with respect to environmental conditions, and variability in strain response makes it a challenging species to manage. Our ability to improve bloom prediction will rely on a more detailed understanding of the complex physiology of this species.     

Absence of negative allelopathic effects of cylindrospermopsin and microcystin-LR on selected marine and freshwater phytoplankton species

Cyanobacterial toxins have been regarded by some researchers as allelopathic substances that could modulate the growth of competitors. Nevertheless, often the concentrations of toxins used are too high to be considered ecologically relevant. In this work we tested the hypothesis that microcystin-LR (MC-LR) and cylindrospermopsin (CYN) at ecologically relevant concentrations have no allelopathic effects on some species of phytoplankton. Extracts containing the toxins as well as pure MC-LR and CYN toxins were used to assess their effects on the growth rates of Nannochloropsis sp., Chlamydomonas reinhardtii, and Chlorella vulgaris. Cyanobacterial crude extracts induced more pronounced effects on growth rates than pure toxins. Microcystis aeruginosa and Aphanizomenon ovalisporum crude extracts containing MC-LR and CYN at 0.025–2.5 mg l^?1 stimulated growth rates of microalgae, whereas A. ovalisporum crude extracts containing 2.5 mg l^?1 of CYN strongly inhibited growth rates of microalgae after 4 and 7 days of exposure. MC-LR and CYN at environmentally occurring concentrations were unable to affect negatively the growth of microalgae, and therefore these molecules may play roles other than allelopathy in natural ecosystems.