Cell growth and toxin production of Gambierdiscus spp. strains from the Macaronesian Region
Creators: name: Pisapia, Francesco
name: Marrero Alemán
name: Soler Onís, Emilio
name: Fernández Zabala, Juan
name: Acosta Arbelo, Félix Antonio
name: Bravo García, Jimena
name: Portillo Hahnefeld, Eduardo
name: Clemente Janeiro de Assunção, Patricia Alexandra
Publisher: name: Zenodo
Year: 2019
Subjects: Ciguatera
Gambierdiscus
Macaronesia

description: <strong>Introduction</strong> Ciguatera poisoning is a food-borne illness mostly caused by consumption of fish contaminated with polyether toxins known as ciguatoxins (CTXs). Originally known as a tropical disease, Ciguatera is being increasingly reported from areas previously not considered endemic, namely in the North-Eastern Atlantic. The epi-benthic dinoflagellates <em>Gambierdiscus</em> and <em>Fukuyoa</em> are considered to be the causative agents of Ciguatera. Furthermore, these genera have recently been shown to have an increasing number of species, also discovered in areas where it had not been observed before, namely the Canary Islands. The study presented here is part of the MIMAR project (MAC/4.6D/066) aiming at the characterization of hitherto unidentified CTXs from the Macaronesian region. A total of 54 strains of the genus <em>Gambierdiscus</em> were recently isolated from field samples collected from Macaronesia (species identification is ongoing). Five strains from the Canary Islands were used in this study for evaluation of growth rates and CTX-activity. <strong>Materials and methods</strong> Five strains of <em>Gambierdiscus</em> spp. from the Canary Islands were cultured using 75 mL of culture medium in 75 cm<sup>2</sup> culture flasks under the same culture conditions. Culture medium consisted of natural Atlantic seawater (salinity adjusted to 32), filtered at 0.2 µm and enriched with f/5 nutrients with the exception of silica. Cultures were maintained in a thermostated room at 25 °C under full spectrum lights with an incident photon flux density of 80 µmol photons m<sup>-2</sup> s<sup>-1</sup> and a daily light-dark cycle of 12 h:12 h light:dark. The light source was placed above the culture flasks. Flasks were randomly placed and the position was changed once a day in order to ensure a homogeneous exposure to light. Culture experiments were conducted using a semi-continuous batch method, as previously described by Pisapia et al. (2017) and Litaker et al. (2017). Two brands of the culture flasks (VWR<sup>®</sup> and Corning<sup>®</sup>) and four different treatments of the bottom surface, i.e. non-treated (NT), treated (T), cellbind–treated (CB) and ultra-low–attachment treated (ULA), were evaluated for their influence on the growth of the strains. Cell concentration (cells mL<sup>-1</sup>) was estimated once per week using a 1 mL Sedgewick-Rafter counting chamber under a light microscope. Maximum growth rate (µ<sub>max</sub>, d<sup>-1</sup>) was the slope calculated by the linear regression of the natural logarithm of the cell concentration versus time, after correcting for serial culture dilutions. Maximum growth rate (µ<sub>max</sub>, divisions day<sup>-1</sup>) was then calculated as follows: µ<sub>max</sub> (divisions day<sup>-1</sup>) = µ<sub>max</sub> (d<sup>-1</sup>) ln(2)<sup>-1</sup>. <em>Gambierdiscus</em> spp. cells were harvested in log phase growth and toxins were extracted using MeOH (x2) and MeOH:H<sub>2</sub>O (1:1, v/v) (x2). Crude extracts were adjusted to MeOH:H<sub>2</sub>O (3:2, v/v) and partitioned twice with dichloromethane (DCM). The lipophilic CTXs were partitioned into the DCM soluble fraction (DSF). Triplicate DSF samples were used for screening their CTX-activity using the O/V neuro-2a (n2a) cytotoxicity assay. The assay was carried out in 96-well flat-bottom Falcon<sup>®</sup> tissue culture plates with vacuum gas plasma treatment for cell adhesion. Pacific CTX1B was provided by R.J. Lewis (Queensland University, Australia) and used as reference standard material. For each DSF sample, four 10-fold serial dilutions were tested in three separate experiments and three replicate wells for each dilution were run for each experiment. <strong>Results</strong> Maximum growth rates (µ<sub>max</sub>, d<sup>-1</sup>) of <em>Gambierdiscus</em> spp. under ITC laboratory conditions ranged from 0.128 to 0.240 divisions day<sup>-1</sup>. The fastest growth was observed for G141 in all the culture flasks tested, reaching the highest µ<sub>max</sub> of 0.240 divisions day<sup>-1</sup> (R<sup>2</sup>=0.99, n=9 over 53 days) when cultured in VWR<sup>®</sup> NT flasks. G85 showed intermediate growth rate values, ranging from 0,156 (Corning<sup>®</sup> ULA, R<sup>2</sup>=0.96, n=10 over 52 days) to 0,183 (Corning<sup>®</sup> NT, R<sup>2</sup>=0.98, n=10 over 52 days) divisions day<sup>-1</sup>, depending on the bottom surface treatment. The slowest growing strain was G92, especially when cultured in Corning<sup>®</sup> NT flasks (0.088 d<sup>-1</sup>, R<sup>2</sup>=0.93, n=9 over 87 days). Estimation of µ<sub>max</sub> of G45 and G100 is currently ongoing. A first attempt in assessing CTX production of the strains using the n2a assay revealed that the strains distributed into three ranges of CTX-activity: (1) G100 showed the lowest CTX-activity, with a half maximal effective concentration (EC<sub>50</sub>) which fell into the range of 5000-50000 cells eq mL<sup>-1</sup> in well; (2) G141 and G85 showed low-intermediate CTX-activity, with EC<sub>50</sub>s of 500-5000 cells eq mL<sup>-1</sup> in well; and (3) G92 and G45 showed the highest CTX-activity, with EC<sub>50</sub>s of 50-500 cells eq mL<sup>-1</sup> in well. <strong>Discussion and Conclusions</strong> This study showed that <em>Gambierdiscus</em> spp. strains behaved as slow-growers under ITC laboratory conditions, with µ<sub>max</sub> &lt; 0.25 divisions day<sup>-1</sup>, which appeared similarly low or somewhat lower than those reported in other studies. All the different culture flasks employed for the culture experiments were suitable for cell growth, although some slight differences were observed in µ<sub>max</sub>. Still, more data points are needed to statistically determine the impact of the different brands and bottom surfaces tested. Preliminary results of the n2a assay suggest that G45 and G92 are the most CTX-producing strains. More serial dilution points with a narrower dilution factor are needed to determine sigmoidal dose-response curves, EC<sub>50</sub> values and, consequently, the quantitation of the amount of CTX eq per cell. Further studies will focus on culture scale-up and bioguided fractionation of the most toxic strains in combination with high resolution mass spectrometry to pinpoint known and/or previously undescribed toxins. <strong>References</strong> Litaker, R.W., Holland, W.C., Hardison, D.R., Pisapia, F., Hess, P., Kibler, S.R., Tester, P.A., 2017. Ciguatoxicity of <em>Gambierdiscus</em> and <em>Fukuyoa</em> species from the Caribbean and Gulf of Mexico. PLoS One 12. https://doi.org/10.1371/journal.pone.0185776 Pisapia, F., Holland, W.C., Hardison, D.R., Litaker, R.W., Fraga, S., Nishimura, T., Adachi, M., Nguyen-Ngoc, L., Séchet, V., Amzil, Z., Herrenknecht, C., Hess, P., 2017. Toxicity screening of 13 <em>Gambierdiscus</em> strains using neuro-2a and erythrocyte lysis bioassays. Harmful Algae 63. https://doi.org/10.1016/j.hal.2017.02.005