Multiple simultaneous detection of Harmful Algal Blooms (HABs) through a high throughput bead array technology, with potential use in phytoplankton community analysis.

As an alternative to traditional, morphology-based methods, molecular techniques can provide detection of multiple species within the HAB community and, more widely, the phytoplankton community in a rapid, accurate and simultaneous qualitative analysis. These methods require detailed knowledge of the molecular diversity within taxa in order to design efficient specific primers and specific probes able to avoid cross-reaction with non-target sequences. Isolates from Florida coastal communities were sequence-analyzed and compared with the GenBank database. Almost 44% of the genotypes obtained did not match any sequence in GenBank, showing the existence of a large and still unexplored biodiversity among taxa. Based on these results and on the GenBank database, we designed 14 species-specific probes and 4 sets of specific primers. Multiple simultaneous detection was achieved with a bead array method based on the use of a flow cytometer and color-coded microspheres, which are conjugated to the developed probes. Following a parallel double PCR amplification, which employed universal primers in a singleplex reaction and a set of species-specific primers in multiplex, detection was performed in a cost effective and highly specific analysis. This multi-format assay, which required less than 4 h to complete from sample collection, can be expanded according to need. Up to 100 different species can be identified simultaneously in a single sample, which allows for additional use of this method in community analyses extended to all phytoplankton species. Our initial field trials, which were based on the 14 species-specific probes, showed the co-existence and dominance of two or more species of Karenia during toxic blooms in Florida waters.

Polyketide synthase genes from marine dinoflagellates.

Rapidly developing techniques for manipulating the pathways of polyketide biosynthesis at the genomic level have created the demand for new pathways with novel biosynthetic capability. Polyketides derived from dinoflagellates are among the most complex and unique structures identified thus far, yet no studies of the biosynthesis of dinoflagellate-derived polyketides at the genomic level have been reported. Nine strains representing 7 different species of dinoflagellates were screened for the presence of type I and type II polyketide synthases (PKSs) by polymerase chain reaction (PCR) and reverse transcriptase PCR. Seven of the 9 strains yielded products that were homologous with known and putative type I PKSs. Unexpectedly, a PKS gene was amplified from cultures of the dinoflagellate Gymnodinium catenatum, a saxitoxin producer, which is not known to produce a polyketide. In each case the presence of a PKS gene was correlated with the presence of bacteria in the cultures as identified by amplification of the bacterial 16S ribosomal RNA gene. However, amplification from polyadenylated RNA, the lack of PKS expression in light-deprived cultures, residual phylogenetic signals, resistance to methylation-sensitive restriction enzymes, and the lack of hybridization to bacterial isolates support a dinoflagellate origin for most of these genes.

Are Pfiesteria species toxicogenic? Evidence against production of ichthyotoxins by Pfiesteria shumwayae.

The estuarine genus Pfiesteria has received considerable attention since it was first identified and proposed to be the causative agent of fish kills along the mid-Atlantic coast in 1992. The presumption has been that the mechanism of fish death is by release of one or more toxins by the dinoflagellate. In this report, we challenge the notion that Pfiesteria species produce ichthyotoxins. Specifically, we show that (i) simple centrifugation, with and without ultrasonication, is sufficient to "detoxify" water of actively fish-killing cultures of Pfiesteria shumwayae, (ii) organic extracts of lyophilized cultures are not toxic to fish, (iii) degenerate primers that amplify PKS genes from several polyketide-producing dinoflagellates failed to yield a product with P. shumwayae DNA or cDNA, and (iv) degenerate primers for NRPS genes failed to amplify any NRPS genes but (unexpectedly) yielded a band (among several) that corresponded to known or putative PKSs and fatty acid synthases. We conclude that P. shumwayae is able to kill fish by means other than releasing a toxin into bulk water. Alternative explanations of the effects attributed to Pfiesteria are suggested.

Brevetoxin derivatives that inhibit toxin activity.

BACKGROUND: The brevetoxins are marine neurotoxins that interfere with the normal functions of the voltage-gated Na(+) channel. We have identified two brevetoxin derivatives that do not exhibit pharmacological properties typical of the brevetoxins and that function as brevetoxin antagonists. RESULTS: PbTx-3 and benzoyl-PbTx-3 elicited Na(+) channel openings during steady-state depolarizations; however, two PbTx-3 derivatives retained their ability to bind to the receptor, but did not elicit Na(+) channel openings. alpha-Naphthoyl-PbTx-3 acted as a PbTx-3 antagonist but did not affect Na(+) channels that were not exposed to PbTx-3. beta-Naphthoyl-PbTx-3 reduced openings of Na(+) channels that were not exposed to PbTx-3. CONCLUSIONS: Some modifications to the brevetoxin molecule do not alter either the binding properties or the activity of these toxins. Larger modifications to the K-ring sidechain do not interfere with binding but have profound effects on their pharmacological properties. This implies a critical function for the K-ring sidechain of the native toxin.

Polyketides from dinoflagellates: origins, pharmacology and biosynthesis.

Dinoflagellates, unicellular marine protists, produce some of the largest and most complex polyketides identified to date. The biological activities of these compounds are quite diverse. Compounds having potential therapeutic value as anti-cancer agents as well as deadly neurotoxins, whose production has resulted in severe public health hazards and economic hardships, are represented in this group of secondary metabolites. Stable isotope feeding experiments have firmly established the polyketide origins of representative compounds from each of the three structural classes, the polyether ladders, the macrocycles and the linear polyethers. Yet some unusual labeling patterns have been observed in each class. Pendant methyl groups are most often derived from C-2 of acetate and deletions of C-1 of acetate are common. Studies on the biosynthesis of dinoflagellate derived polyketides at the genomic level have not been reported, in part due to the peculiarities of the dinoflagellate nucleus and the lack of a dinoflagellate transformation system. Nevertheless, a fundamental understanding of the genetics of polyketide biosynthesis by dinoflagellates could be the catalyst for developing several fruitful avenues of research. Dinoflagellate derived polyketides are reviewed with special emphasis on pharmacology and biosynthesis.

Brevetoxin-6 (PbTx-6), a nonaromatic marine neurotoxin, is a ligand of the aryl hydrocarbon receptor.

Brevetoxins (PbTx) are a family of marine polyether toxins that exert their toxic action by activating voltage-sensitive sodium channels. Two forms of brevetoxin, PbTx-2 and -3, induce hepatic cytochrome P4501A1, measured as ethoxyresorufin O-deethylase (EROD) activity, in redfish and striped bass. P4501A1 induction is transcriptionally regulated through the binding of a ligand, typically a planar aromatic compound, to the aryl hydrocarbon receptor (AhR) and subsequent complex formation with the dioxin response element (DRE), an upstream regulatory region of the CYP1A1 gene. To determine if PbTx, a nonaromatic compound, induced EROD by this mechanism, two sets of experiments were performed. Initially, saturation binding assays with PbTx-2, -3, and -6 were carried out to determine if PbTx-2, -3, or -6 was an AhR ligand. Results showed that PbTx-6 inhibited specific binding of dioxin to the AhR, whereas PbTx-2 and -3 had no effect. Subsequently, gel retardation assays showed that PbTx-6 caused a concentration-dependent increase in AhR-DRE complex formation. The most abundant and neurotoxic forms of brevetoxin, PbTx-2 and -3, did not appear to be involved in this process. However, PbTx-6, the epoxide which is a likely biotransformation product, is at least one of the forms of PbTx involved in EROD induction.

The relationship of brevetoxin 'length' and A-ring functionality to binding and activity in neuronal sodium channels.

BACKGROUND: Brevetoxins are polyether ladder toxins that are ichthyotoxic at nanomolar concentrations. They bind to voltage-gated sodium channels, causing four distinct electrophysiological effects: (i) a shift of activation potential; (ii) occurrence of subconductance states; (iii) induction of longer mean open times of the channel; and (iv) inhibition of channel inactivation. We set out to determine whether these functions all require the same structural elements within the brevetoxin molecules. RESULTS: Several synthetically prepared structural analogs of brevetoxin B were examined in synaptosome receptor binding assays and by functional electrophysiological measurements. A truncated analog is not ichthyotoxic at micromolar concentrations, shows decreased receptor-binding affinity, and causes only a shift of activation potential without affecting mean open times or channel inactivation. An analog with the A-ring carbonyl removed binds to the receptor with nanomolar affinity, produces a shift of activation potential and inhibits inactivation, but does not induce longer mean open times. An analog in which the A-ring diol is reduced shows low binding affinity, yet populates five subconductance states. CONCLUSIONS: Our data are consistent with the hypothesis that binding to sodium channels requires an elongated cigar-shaped molecule, approximately 30 A long. The four electrophysiological effects of the brevetoxins are not produced by a single structural feature, however, since they can be decoupled by using modified ligands, which are shown here to be partial sodium channel agonists. We propose a detailed model for the binding of brevetoxins to the channel which explains the differences in the effects of the brevetoxin analogs. These studies also offer the potential for developing brevetoxin antagonists.

Modified immunoassays for polyether toxins: implications of biological matrixes, metabolic states, and epitope recognition.

Polyether marine toxins are responsible for the seafood intoxication phenomena known as neurotoxic shellfish poisoning (due to brevetoxins), ciguatera (due to ciguatoxin), and diarrheic shellfish poisoning (due to okadaic acid). Using traditional techniques of hapten (pure toxin) conjugation to protein to create complete antigen, animal immunization and antibody isolation, and specific antibody subpopulation purification, discriminating antibodies have been isolated that detect brevetoxins and ciguatoxin, but not okadaic acid, in a dose-dependent fashion. Using microorganic chemistry and purified toxins, a unique set of tools has been created for the study of polyether ladder toxin accumulation; depuration; and specific site localization in tissues, food sources, and clinical samples. Developed test protocols can detect toxin in dinoflagellate cells, in extracts from food sources, in seawater and culture media, and in human serum samples. Enzyme-linked immunosorbent assay protocols developed for eventual collaborative testing have been successful in limited applications within the laboratory (correlation coefficient of 0.92 excluding 2 outliers), and alternative formats are being developed to optimize the basic test for use in research laboratories, regulatory laboratories, and field inspections.

Radioimmunoassay for PbTx-2-type brevetoxins: epitope specificity of two anti-PbTx sera.

Antiserum against PbTx-2-type brevetoxins was produced by immunizing rabbits with a PbTx-2-bovine serum albumin (BSA) conjugate. This serum had a higher affinity, but lower titer, than our current goat serum. Using 4 natural brevetoxins and 6 synthetic derivatives as competitors in our brevetoxin radioimmunoassay, we determined the epitope specificity of both sera. Modification of the backbone structure at C-42 on the K-ring had little or no effect on the antigen-binding capability of either serum. Reduction of the double bond between C-2 and C-3 on the A-ring by reduction of the lactone decreased binding 500 to 750-fold. Epoxidation of the double bond between C-27 and C-28 on the H-ring did not affect binding, which suggested that the goat serum is specific for the A-ring region of the brevetoxin backbone. In contrast, modifying the A-ring had no effect on rabbit serum binding. However, epoxidation of the H-ring decreased binding 5 to 20-fold, which suggested that the rabbit antiserum is specific for the H-ring region of the molecule. These results suggest that assays utilizing only one antibody may not adequately detect toxin metabolites if molecules are altered in the critical region of antibody recognition.

Brevetoxin PbTx-2 immunology: differential epitope recognition by antibodies from two goats.

The epitopic regions of the brevetoxin PbTx-3 molecule, produced by the marine dinoflagellate Ptychodiscus brevis, have been identified by structural modification at three distinct regions of the toxin. These are: the A-ring lactone region of the molecule, the K-ring side-chain and the H-ring. The modified PbTx-3 derivatives were tested for their ability to bind brevetoxin goat antisera directed against the PbTx-3 molecule, by radioimmunoassay. The results showed that at least two major epitopes and one minor epitope are recognized: the A-ring lactone region of the molecule and the K-ring side-chain, and the H-ring. The results illustrate the variety of antibodies which may be produced, even within a species, and suggests that epitope characterization is important in the development of assays which are to be employed in seafood safety issues.

Is the A-ring lactone of brevetoxin PbTx-3 required for sodium channel orphan receptor binding and activity?

Brevetoxin PbTx-3 and non-toxic derivative 4 were investigated for their abilities to bind to the specific brevetoxin receptor site on rat brain synaptosomes and to modulate the normal function of voltage-gated sodium channels as determined by patch clamping of cultured neurons. Compounds 4 and 5 are produced from PbTx-3 by opening of the A-ring lactone to the saturated and unsaturated diols using sodium borohydride in ethanol. Natural PbTx-3 exhibited tighter binding to rat brain synaptosomes by at least 3 orders of magnitude as determined by competitive radioligand binding experiments, and was also more effective at activating voltage-gated channels. Patch clamping revealed the 3 orders of magnitude greater potency of PbTx-3 toxin over 5, although each produced delayed sodium channel opening and a pronounced delay in inactivation. Conformational modeling of the Brevetoxin B backbone indicates that the two molecules are identical except for the region of the A-Ring lactone. Thus, we conclude that the brevetoxin PbTx-3 backbone requires electrophilic functionality in the region of the lactone in PbTx-3, and that opening of the ring in 5 is sufficient to substantially reduce both binding and activity.

Binding of brevetoxins and ciguatoxin to the voltage-sensitive sodium channel and conformational analysis of brevetoxin B.

The marine toxins known generically as brevetoxins, as well as their structural relative ciguatoxin, are known as polyether ladder toxins, and bind uniquely to site 5 of the voltage-sensitive sodium channel. Rat brain synaptosome binding data show similarities in binding affinity for brevetoxins having the same structural (ladder) backbone, but different affinities between brevetoxins having different backbones. Ciguatoxin has a different backbone from the brevetoxins, but binds even more strongly to the same site. Could the flexibility of the backbone be related to their relative toxicities? As part of an effort to identify the common pharmacophore for the toxins, Monte Carlo methods were used to generate conformational models of the polyether ladder toxin brevetoxin B (PbTx-2) which shows significant flexibility at the juncture of the two 7-membered rings.

Methods for detecting brevetoxins in seawater, in biological matrices, and on excitable membranes.

Although the toxins differ in each of the principal polyether marine seafood intoxication syndromes, developing technology is of general applicability. The brevetoxins can be readily detected in seawater, in dinoflagellate cells, and in shellfish using standardized mouse bioassay, isocratic high performance liquid chromatography, radioimmunoassay and enzyme-linked immunoassay...each possessing distinct advantages and disadvantages. Clinical diagnosis, pharmacokinetics, and membrane-receptor studies on the contrary require specialized radioligand technology and immunofluorescent and immunodense imaging. The applicability of brevetoxins assays towards predictive, detection, and molecular mechanism of action of ciguatoxin is presented.