Results of a Kruskal-Wallis nonparametric one factor ANOVA for differences in CTX toxicity among <i>Gambierdiscus</i> and <i>Fukuyoa</i> species.

<p><i>Gambierdiscus excentricus</i> and <i>G</i>. <i>silvae</i> were excluded from the analysis because only a single clone was examined. Abbreviations: n = sample size, M = median toxicity (fg CTX3C eq. cell^-1), <i>H</i> = Kruskal-Wallis test statistic, <i>df</i> = degrees of freedom. Brackets denote result of the Dunn’s follow up test. The statistic is designed to estimate median toxicities to determine if the species partitioned into distinct groups.</p

CTX-like and MTX-like activity in HPLC fractionated <i>Gambierdiscus</i> extracts from Caribbean/Atlantic isolates. Dichloromethane (DCM) and methanol (MeOH) extracts of Belize <i>G</i>. <i>carolinianus</i> (Dive 1 Gam 1), North Carolina <i>G</i>. <i>carolinianus</i> (Kenny 6), St Maartens <i>Gambierdiscus ribotype II</i> (St. Maartens Gam 6) and Puerto Rico <i>Gambierdiscus ribotype II</i> (Mixed PR Gam 3) were fractionated by HPLC with MS detection (black trace).

<p>Fractions were tested for activity using the FLIPR^TETRA assay. CTX-like (red trace) and MTX-like (blue trace) activity (%) is given relative to the most active MTX and CTX-containing fraction tested in this study.</p

CTX-like and MTX-like activity in HPLC fractionated <i>Gambierdiscus</i> and <i>Fukuyoa</i> extracts. Dichloromethane (DCM) and methanol (MeOH) extracts of Caribbean <i>F</i>. <i>ruetzleri</i> (Belize, Gam1), <i>G</i>. <i>belizeanus</i> (St. Barthelemy Island, CCMP 399), <i>G</i>. <i>carpenteri</i> (Belize, GT4) and <i>G</i>. <i>caribaeus</i> (Belize, Gam 19) isolates fractionated by HPLC with UV detection (black trace).

<p>Fractions were tested for activity using the FLIPR^TETRA assay. CTX-like (red trace) and MTX-like (blue trace) activity was expressed as percent activity (%) relative to the most active MTX and CTX-containing fraction tested in this study.</p

Cell viability assay. Cell death measured in Neuro2a cells treated with veratridine (10 μM), ouabain (100 μM) and varying concentrations of Pacific ciguatoxins.

<p>The pEC₅₀ values of P-CTX-1, -2 and -3 were 11.17 ± 0.03, 11.09 ± 0.04 and 11.31 ± 0.04, respectively. Data are presented as value ± standard error (n = 3).</p

The species, strain designations, isolate locations, replicate growth rates and toxicities of the <i>Gambierdiscus</i> and <i>Fukuyoa</i> strains examined in this study.

<p>The citations in the species column indicate where the species was described. The reference(s) under the strain designation indicate other publications where the strain has been studied. Many of the strains analyzed for CTX-like activity in this study were also assayed for maitotoxicity in separate investigations [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185776#pone.0185776.ref032" target="_blank">32</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185776#pone.0185776.ref033" target="_blank">33</a>]. The strain growth rates (± standard deviation) were determined from triplicate, independent cultures started for each isolate. Mean species growth rates and average toxicities were determined by averaging all replicate culture data for a given species. Toxicity was normalized both as femtograms (fg) CTX3C equivalents [eq.] cell^-1 and per biovolume attograms (ag) CTX3C eq. μm^-3. Numbers in parentheses in the data cells of the last three columns = coefficient of variation. Correlation coefficients (R²) for the time versus cell number relationships used to the calculate growth rates for each of the cultures exceeded 0.98.</p

CTX-like and MTX-like activity in HPLC fractionated <i>Gambierdiscus</i> extracts of Pacific isolates. Dichloromethane (DCM) and methanol (MeOH) extracts of <i>G</i>. <i>australes</i> (W B Gam 3), Hawaii <i>G</i>. <i>caribaeus</i> (Pat HI Jar 2 Gam 2), <i>G</i>. <i>carpenteri</i> (Pat HI Jar 7 Gam 11), and <i>G</i>. <i>carolinianus</i> (Pat HI Jar 3 Gam) were fractionated by HPLC with MS UV detection (black trace).

<p>Fractions were tested for activity using the FLIPR^TETRA assay. CTX-like (red trace) and MTX-like (blue trace) activity was expressed as percent activity (%) relative to the most active MTX and CTX-containing fraction tested in this study.</p

Representative plots showing the long-term steady state growth of the <i>Gambierdiscus</i> and <i>Fukuyoa</i> isolates achieved in this study.

<p>Exponential growth was achieved by acclimating cells to optimal temperature, light and nutrient conditions and maintained in exponential growth phase by periodic dilution with nutrient rich media.</p

Toxin production rates.

<p>This figure shows the estimated toxin production (fg CTX3C eq. cell^-1 d^-1) rate for each species.</p

Synergistic effects of P-CTX-1, 2, 3 on veratridine responses in SH-SY5Y cells.

<p>(A) Representative veratridine-induced responses in SH-SY5Y cells pre-incubated for 300 s with 0.5 nM P-CTX-1. Blue trace, response in the absence of veratridine; green trace, response elicited by 1 μM veratridine; purple trace, response elicited by 3 μM veratridine; red trace, response elicited by 5 μM veratridine. (B-D) P-CTX-1, P-CTX-2 and P-CTX-3 (30 fM–100 nM) were added 300 s prior to addition of 1 μM (green), 3 μM (purple) or 5 μM (red) veratridine. The blue data in each panel show the response to P-CTX-1, 2 and 3 in the absence of veratridine. (B) Concentration-dependent potentiation of veratridine responses by P-CTX-1 (30 fM–100 nM). (C) Concentration-dependent potentiation of veratridine responses by P-CTX-2 (30 fM–100 nM). (D) Concentration-dependent potentiation of veratridine responses by P-CTX-3 (30 fM–100 nM). Data are presented as mean ± SEM from at least n = 3 wells and are representative of three independent experiments.</p

Fluorescence response of SH-SY5Y cells to MTX-like or CTX-like activity.

<p>Fluorescent responses to addition of HPLC fractionated samples was measured in SH-SY5Y cells loaded with Calcium-4-NW dye using a two-addition protocol. The presence of MTX-like activity (blue trace, <i>Fukuyoa ruetzleri</i> methanol fraction) caused a rapid increase in fluorescence that was maintained for the 600 s time course. In contrast, addition of samples containing CTX-like activity (red trace, <i>Gambierdiscus carolinianus</i> DCM fraction) caused no measurable increase in fluorescence until addition of veratridine (5 μM) after 300 s due to potentiation of veratridine-induced effects on endogenous Na_V channels by CTX-like activity. The resultant Ca₂₊ transient is proportional to the toxicity of CTX-like compounds present. The black trace indicates the buffer control response on the first addition as well as the veratridine response (5 μM) on second addition in the absence of pre-treatment with CTX.</p