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Flavobacteriaceae (strain S03) and Cytophaga sp. (strain 41-DBG2) are algicidal bacteria active against the brevetoxin (PbTx)-producing, red tide dinoflagellate, Karenia brevis. Little is known about the fate of PbTx associated with K. brevis cells following attack by such bacteria. The fate and distribution of PbTx in K. brevis cultures exposed to these algicidal strains were thus examined by receptor binding assay and liquid chromatography/mass spectrometry (LC/MS) in three size fractions (45, 0.22–5, o0.22mm) over a 2-week time course. In control cultures, brevetoxin concentrations in the 45mm particulate size fraction correlated with changes in cell density, whereas significant increases in dissolved (i.e., o0.22mm) toxin were observed in the later stages of culture growth. Exposure of K. brevis to either of the two algicidal bacteria tested caused cell lysis, coinciding with a rapid decline in the 45mm PbTX size fraction and a simultaneous release of dissolved toxin into the growth medium. Upon cell lysis, dissolved brevetoxin accounted for ca. 60% of total toxin and consisted of 51–82% open A-ring derivatives. Open A-ring PbTx-2 and PbTx-3 derivatives bound with lower affinity (approximately 22- and 57-fold, respectively) to voltage-gated sodium channels and were considerably less cytotoxic (86- and 142-fold, respectively) to N2A cells than their individual parent toxins (i.e., PbTx-2 and PbTx-3). These novel findings of changes in PbTx size-fractioned distribution and overall reduction in K. brevis toxicity following attack by algicidal bacteria improve our understanding of potential trophic transfer routes and the fate of PbTx during red tide events. Moreover, this information will be important to consider when evaluating the potential role of algicidal bacteria in harmful algal bloom (HAB) management strategies involving control of bloom populations. r 2007 Elsevier Ltd. All rights reserved.

Ciguatoxins (CTX) are a suite of cyclic polyether toxins produced by the marine dinoflagellate Gambierdiscus sp., are potent activators of voltage-gated sodium channels and a leading cause of human poisoning from food fish. This report characterizes the genomic and proteomic response in whole blood of adult male mice exposed i.p. to 264 ng/kg of the Pacific congener of CTX (P-CTX-1) at 1, 4 and 24h. Whole genome microarray expression data were filtered by tightness of fit between replicates, fold change (1.8) and p-value (10(-5)), resulting in 183 annotated genes used for trending analysis, K-means clustering and ontology classification. Genes involved with cytokine signaling, proteasome complex and ribosomal function were dominant. qPCR performed on 19 genes of interest had a correlation of 0.95 to array results by Pearson's correlation coefficient. Serum protein analysis showed small but significant changes in 6 of 60 proteins assayed: Ccl2, Ccl12, CD40, IL-10, leptin and M-CSF. In large part, the gene expression was consistent with a Th2 immune response with interesting similarities to expression seen in asthmatic models.

Brevetoxins (PbTxs) and ciguatoxins (CTXs) are two suites of dinoflagellate derived marine polyether neurotoxins that target the voltage gated sodium channel (VGSC). PbTxs are commonly responsible for massive fish kills and unusual mortalities in marine mammals. CTXs, more often noted for human intoxication, are suspected causes of fish and marine mammal intoxication, although this has never been reported in the field. VGSCs, present in the membrane of all excitable cells including those found in skeletal muscle, nervous and heart tissues, are found as isoforms with differential expression within species and tissues. To investigate the tissue and species susceptibility to these biotoxins, we determined the relative affinity of PbTx-2 and -3 and P-CTX-1 to native VGSCs in the brain, heart, and skeletal muscle of rat and the marine teleost fish Centropristis striata by competitive binding in the presence of [(3)H]PbTx-3. No differences between rat and fish were observed in the binding of PbTxs and CTX to either brain or skeletal muscle. However, [(3)H]PbTx-3 showed substantial lower affinity to rat heart tissue while in the fish it bound with the same affinity to heart than to brain or skeletal muscle. These new insights into PbTxs and CTXs binding in fish and mammalian excitable tissues indicate a species related resistance of heart VGSC in the rat; yet, with comparable sensitivity between the species for brain and skeletal muscle.

In Florida (USA), numerous cases of human ciguatera fish poisoning, as well as neurotoxic shellfish poisoning following consumption of local seafood products, have been reported. By using in parallel, the sodium channel receptor binding assay (RBA), and the ouabain/veratridine-dependent cytotoxicity assay (N2A assay), we established criteria to identify, detect, and quantify ciguatoxins in fish extracts, with a brevetoxin as internal standard. Results showed that the Caribbean ciguatoxin C-CTX-1 exhibited an 8-fold higher potency in the RBA than brevetoxins and, a 440 and 2300-fold higher potency in the N2A assay than PbTx-1 and PbTx-3, respectively. Moreover, a sensitivity comparison between assays revealed that the N2A assay was more sensitive (12-fold) for ciguatoxin analysis, whereas the RBA was more sensitive (3–24-fold) for brevetoxins analysis. Based on the relative potency between toxins and the opposite sensitivity of both assays we have used the RBA and the N2A assay to screen great barracuda ( Sphyraena barracuda ) collected from the Florida Keys for ciguatoxins and brevetoxins. Fish extract analysis showed a sodium channel-dependent activity consistent with the presence of ciguatoxins, and not brevetoxins. Among 40 barracudas analyzed, 60% contained ciguatoxin levels in their liver measurable by the N2A assay with the most toxic fish containing 2.1 ppb C-CTX-1 equivalents.