Morphological and molecular characterization of the cyanobiont-bearing dinoflagellate Sinophysis canaliculata from the Canary Islands (eastern central Atlantic)

Abstract

Event Abstract Back to Event Morphological and molecular characterization of the cyanobiont-bearing dinoflagellate Sinophysis canaliculata from the Canary Islands (eastern central Atlantic) María García Portela1*, Pilar Riobó Agulla2 and Francisco Rodríguez Hernández1 1 Instituto Español de Oceanografía, Centro Oceanográfico de Vigo, Spain 2 Instituto de Investigaciones Marinas, CSIC (UA Microalgas Nocivas CSIC‐IEO), Spain In the course of a recent sampling of benthic dinoflagellate communities in the Canary Islands (late summer 2015) we observed the widespread occurrence of Sinophysis on microalgae samples (Fig. 1).As far as we know, this benthic dinoflagellate genus has not been reported previously in these islands. The morphological traits of Sinophysis cells corresponded with S. canalicuta but were pink coloured, a feature that has not been described for any species in that genus. Epifluorescence microscopy revealed multiple orange autofluorescence bodies inside S. canaliculata resembling the cyanobacterial-like endosymbionts reported by Escalera et al. (2011) in Japanese waters. For these reasons, we decided to elaborate the present study to describe the morphological and molecular characteristics of S.canaliculata and its cyanobacterial endosymbionts in the Canary Islands. Images and measurements of S.canaliculata cells were made with an Axiocam HRc digital camera (Zeiss, Germany) in bright field with a light microscopy Leica DM LA (Leica Microsystems GmbH, Wetzlar, Germany) at 630x magnification. Cyanobionts autoflorescence was observed in a Leica DM LA epifluorescence microscope with blue excitation. Fixed single cells were deposited through micromanipulation on polycarbonate membrane filters of 5.0 µm (ISOPORETM, Merck Millipore, Massachusetts, USA). Filters where stuck on the stubs and were coated with gold with a K5550X sputter coater (Emitech Ltd., Ashford, Kent, UK) and observed with a Phillips XL30 scanning electron microscope (FEI Company, Hillsboro, OR, USA). Primers Euk-A/Euk-B (Medlin et al., 1988) were used to amplify 18S host nuclear SSUrRNA. For 16S rRNA gene amplification of the endosymbionts and a Merismopedia-like colony, primers CYA106F/CYA781 (Nübel et al., 1997) were used.Purified DNA was sent to LIGHTrun sequencing services (GATC Biotech AG, Germany). Phylogenetic relationships were determined using the maximum likelihood (ML) method (MEGA 6) and the Bayesian inference method (BI) with a general time reversible model (Mr.Bayes v3.1; Huelsenbeck and Ronquist, 2001).The phylogenetic tree was represented using the ML results, with bootstrap values from the ML method (n=1000 replicates) and posterior probabilities from the BI method. Presence of lipophilic toxins in a Sinophysis sample (400 cells) was evaluated by LC-HRMS following Gersen et al.(2009). A methanolic extract from Fal 50 strain of Alexandrium ostenfeldii containing SPX-C and a toxin Standard mixture containing 10 ng/mL of OA, DTX2, DTX1, PTX2, AZA1, AZA2 and AZA3 were also analyzed. Chromathographyc separation was achieved at 400 µL min-1 on Gemini NX C18 (100 x 2 mm) 3 µm column mantained at 40 ºC using a Dionex Ultimate 3000 LC system (Thermo Fisher Scientific, San Jose, California). Mass spectrometry detection was performed with an Exactive mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with an Orbitrap mass analyzer and a heated electrospray source (H–ESI II). Mass acquisition was achieved in positive ion mode without and with all ion fragmentation (AIF) with a high-energy collisional dissociation (HCD) of 35 eV. The mass range was m/z 100–2000 in full-scan mode and m/z 500–900 in AIF mode. Live S.canaliculata cells had an average length (antero-posterior axis) of 43.44 (±2.77) µm and width of 43.23 (±2.50) µm. In vivo cells had a pale pink colour (Fig. 2) and one or two semicentral dark brown bodies with green UV autofluoresce (Fig. 3 B, E).Under blue epifluorescence, internal orange coloured globules were more concentrated near the cingulum of the cell and most of them were kidney-shaped or semi-oval (Fig. 3 C, F), and sometimes appeared grouped in pairs. Kidney-shaped endosymbionts displayed an average length of 5.82 (±0.62) µm and width of 3.83 (±0.24) µm. According to SEM images, pores were randomly located inside and among the areolae (Fig. 4 A). The number of pores and areolae on the right side (Fig. 5 A, B) were 5.53 (± 0.94) and 0.95 (± 0.18) per µm2 respectively, and on the left side, were 7.14 (± 2.25) and 1.25 (± 0.52) per µm2, respectively. The areolae and pores diameter were 1.02 (± 0.22) µm and 0.22 (± 0.05) µm, respectively. The narrow thecal cut (Fig. 4 B) located on the left side of the cell (Fig. 5 C), had a length of 7.2 (± 0.98) µm. The 18S rRNA based phylogeny including the sequence from a single cell of S.canaliculata (Acc. No. KX139004) from Tajao (Tenerife Island) is shown in Fig. 6. ML and BI topologies were very similar with slight differences regarding ML bootstrap and BI posterior probability values in S. verruculosa (JQ996377) and S. ebriola (JN587292) branches. S. canaliculata and S.microcephalus were grouped together in a subclade. Two incongruities were observed in the branch location of some sequences belonging to S. grandis (JN587291) and S. ebriola (JN587292). 16S sequences from two S. canaliculata endosymbionts (endosymbiont I, Acc. No. KX139005 and endosymbiont II, Acc. No. KX139006) appeared in a subclade associated to a robust cluster in which a variety of cyanobacterium and bacterium uncultured species were included (Fig. 7). The only sequences with any taxonomic information within that cluster were a Synechocystis sp. (Acc. No. KM020010; Friedl et al., unpublished), and a Chroococcales cyanobacterium (Acc. No. EF160009, EF160007). A Merismopedia-like colony was sequenced (Acc. No. KX139003) as it represented the only organism morphologically similar to the endosymbionts in Sinophysis. Nevertheless, pairwise comparisons among Merismopedia-like colony 16S rRNA sequence and those of the endosymbionts in S. canaliculata showed only 0.89 of similarity. Finally, according to LC-HMRS results, lipophilic toxins were not detected in extracts of S. canaliculata. In conclusion, the presence of S.canaliculata has been reported for the first time in the Canary Islands. Cells in the field were pink coloured and always contained orange autofluorescence endosymbionts. The consortium between S.canaliculata and its endosymbiont seems to be of temporal nature. Cyanobionts in S.canaliculata from Canary Islands (this study) matched those earlier reportedin Japan (Escalera et al., 2011), suggesting that this consortium would be widely distributed and species-specific. Its temporary nature is also likely given the rapid loose of colour of S. canaliculata in the laboratory and the inability of culturing them. Regarding toxins, new analyses on higher biomass would be desirable to confirm the absence of lipophilic toxins and to screen other compounds from its cyanobionts. Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Acknowledgements We thank to Isabel Ramilo for helping with Sinophysis cells identification in different sampling sites. This study was funded by CICAN project (CGL2013-40671-R. MINECO, Spain). The experimental part of this study was carried out at the Instituto Español de Oceanografía (IEO) in Vigo. References Escalera, L., Reguera, B., Takishita, K., Yoshimatsu, S., Koike, K. & Koike, K. 2011. Cyanobacterial endosymbionts in the benthic dinoflagellate Sinophysis canaliculata (Dinophysiales, Dinophyceae). Protist, 162:304-314. Friedl,T., Hepperle, D., Marrero-Callico, A., Jahn, R., Kusber,W. H. & Hallmann, C. Unpublished. Documentation of molecular phylogenetic features of terrestrial algae: Phylogenetic analyses of green algal and cyanobacterial reference strains within the AlgaTerra information system. Gerssen, A., Mulder, P. P., McElhinney, M. A. & de Boer, J. 2009. Liquid chromatography–tandem mass spectrometry method for the detection of marine lipophilic toxins under alkaline conditions. J. Chromatogr. A, 1216:1421-1430. Huelsenbeck, J. P. & Ronquist, F. 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics, 17:754-755. Medlin, L., Elwood, H. J., Stickel, S. & Sogin, M. L. 1988. The characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding regions. Gene, 71:491-499. Nübel, U., Garcia-Pichel, F. & Muyzer, G. 1997. PCR primers to amplify 16S rRNA genes from cyanobacteria. Appl. Environ. Microb. 63:3327-3332. Keywords: Canary Islands, GAF, endosymbiont, phylogenetics, SEM, Sinophysis Conference: XIX Iberian Symposium on Marine Biology Studies, Porto, Portugal, 5 Sep - 9 Sep, 2016. Presentation Type: Oral Presentation Topic: 1. ECOLOGY, BIODIVERSITY AND VULNERABLE ECOSYSTEMS Citation: García Portela M, Riobó Agulla P and Rodríguez Hernández F (2016). Morphological and molecular characterization of the cyanobiont-bearing dinoflagellate Sinophysis canaliculata from the Canary Islands (eastern central Atlantic). Front. Mar. Sci. Conference Abstract: XIX Iberian Symposium on Marine Biology Studies. doi: 10.3389/conf.FMARS.2016.05.00048 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 28 Apr 2016; Published Online: 02 Sep 2016. * Correspondence: PhD. María García Portela, Instituto Español de Oceanografía, Centro Oceanográfico de Vigo, Vigo, Pontevedra, 36390, Spain, maria.garcia@vi.ieo.es Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. 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