Morphology, Phylogeny and Azaspiracid Profile of Azadinium Poporum (Dinophyceae) from the China Sea

See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/236668617 Morphology, phylogeny and azaspiracid profile of Azadinium poporum (Dinophyceae) from the China Sea Article in Harmful Algae · January 2013 DOI: 10.1016/j.hal.2012.11.009 CITATIONS READS 32 298 5 authors, including: Haifeng Gu Zhaohe Luo Third Institute of Oceanography China Third Institute of Oceanography China 48 PUBLICATIONS 420 CITATIONS 17 PUBLICATIONS 86 CITATIONS SEE PROFILE SEE PROFILE Urban Tillmann Alfred Wegener Institute Helmholtz Centre fo… 130 PUBLICATIONS 2,598 CITATIONS SEE PROFILE Some of the authors of this publication are also working on these related projects: Placing Marine Mixotrophs in Context View project All content following this page was uploaded by Haifeng Gu on 05 February 2017. The user has requested enhancement of the downloaded file. Harmful Algae 21–22 (2013) 64–75 Contents lists available at SciVerse ScienceDirect Harmful Algae jo urnal homepage: www.elsevier.com/locate/hal Morphology, phylogeny and azaspiracid profile of Azadinium poporum (Dinophyceae) from the China Sea a, a b c b, Haifeng Gu *, Zhaohe Luo , Bernd Krock , Mattias Witt , Urban Tillmann * a Third Institute of Oceanography, SOA, Xiamen 361005, China b Alfred Wegener Institute for Polar and Marine Research, Am Handelshafen 12, D-27570 Bremerhaven, Germany c Bruker Daltonik GmbH, Fahrenheitstr. 4, 28359 Bremen, Germany A R T I C L E I N F O A B S T R A C T Article history: Azadinium poporum is a small dinoflagellate from the family Amphidomataceae which is known for the Received 28 July 2012 production potential of azaspiracid toxins. A. poporum has been recorded from European and Korean Received in revised form 16 October 2012 waters. Here we present the first report of its occurrence along the coast of China. Morphology of Chinese Accepted 28 November 2012 A. poporum is similar to those from Europe and Korea. Several stalked pyrenoids surrounded by a starch sheath were revealed with light microscopy and confirmed by transmission electron microscopy. Among Keywords: 25 strains from the China Sea we identified two distinct ribotypes (referred to as ribotypes B and C). ITS Azadinium sequences of strains within the same ribotype are identical, whereas ribotype B and C differ from each Azadinium poporum other at 11 positions (98.3% similarity). A. poporum ribotypes B and C type differ from European strains Azaspiracids China (referred to as ribotype A) at 16 and 15 positions (97.5% and 97.7% similarity). The ITS region pairwise Genetic differentiation distance within A. poporum ranged from 0.017 to 0.022. Among all three ribotypes, no hemi- compensatory based changes were found within helix III of ITS indicating that they are conspecific. Azaspiracid profiles were analyzed for six strains and turned out to be unexpectedly diverse. Whereas no AZAs could be detected for one strain, another strain was found to contain a m/z 348 fragment type AZA previously found in a Korean Isolate and traces of two other unknown AZAs of higher masses. A third strain produced a novel AZA with a molecular mass of 871 Da. Three strains were found to contain considerable amounts of toxic AZA-2 as the sole AZA, a finding that might elegantly explain the detection of AZA-2 in sponges in the Sea of Japan and which underline the risk potential of A. poporum blooms with subsequent shellfish intoxication episodes for the Asian Pacific. ß 2012 Elsevier B.V. All rights reserved. 1. Introduction been described as new (Tillmann et al., 2012), and this species is the closest relative of Azadinium based on both molecular and The recently erected dinoflagellate genus Azadinium Elbra¨chter morphological data. Amphidoma and Azadinium are now grouped & Tillmann mainly attracts attention for its production of in the family Amphidomataceae, which forms an independent azaspiracids, a recently discovered group of lipophilic phycotoxins lineage among other monophyletic major groups of the dino- causing human intoxication via mussel consumption. With an phytes. Species of the genus Azadinium have so far been reported epithecal affinity to the Peridiniales and a hypothecal affinity to the from the North Sea (Tillmann et al., 2009, 2010, 2011), the French Gonyaulacales (Tillmann et al., 2009), the systematic position of and Irish coast of the eastern Atlantic (Salas et al., 2011; Ne´zan the genus within the dinoflagellates is not yet clarified. Three et al., 2012), the Argentinean coast (Akselman and Negri, 2012) and species have so far been described, i.e. Azadinium spinosum the Korean coast (Potvin et al., 2011). Nevertheless, the presence of Elbra¨chter & Tillmann, Azadinium obesum Tillmann & Elbra¨chter AZAs appeared to be distributed much more widely, reported in and Azadinium poporum Tillmann & Elbra¨chter (Tillmann et al., Northern Africa, northern Europe, Chile, USA and China (James 2009, 2010, 2011). Recently, Amphidoma caudata Halldal has been et al., 2002; Magdalena et al., 2003; Taleb et al., 2006; Klontz et al., transferred to Azadinium based on both morphology and molecular 2009; Lopez-Rivera et al., 2010; Yao et al., 2010). The discrepancy phylogeny (Ne´zan et al., 2012). Moreover, Amphidoma languida has between the distribution of Azadinium and AZAs suggest that A. spinosum might have a wider distribution, or strains of other Azadinium species could produce AZAs. Initially, A. spinosum was the only species for which AZAs were reported. For A. spinosum * Corresponding authors. strain 3D9, the toxin profile consisted of AZA-1, AZA-2 and an E-mail addresses: [email protected] (H. Gu), [email protected] (U. Tillmann). isomer of AZA-2 (Krock et al., 2009), which was later identified as 1568-9883/$ – see front matter ß 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.hal.2012.11.009 H. Gu et al. / Harmful Algae 21–22 (2013) 64–75 65 AZA-1 methyl ester and found to be an extraction artifact (Jauffrais coordinates and sample dates of the sites, see Table 1). The et al., 2012). AZA-1 and -2 production was subsequently confirmed sediment samples were stored in the dark at 4 8C until further for A. spinosum strains from Denmark (Tillmann et al., 2011) and treatment. Approximately 2 g of wet sediment were mixed with Ireland (Salas et al., 2011), indicating that production and profile of 20 mL of filtered seawater and sonicated for 2 min (100 W) to known AZAs is a stable characteristic of the species A. spinosum. dislodge detrital particles. The watery slurry was incubated Other related species/strains of Amphidomataceae have been directly in series of small containers in f/2-Si medium (Guillard À2 À1 reported not to contain any of the known azaspiracids. However, and Ryther, 1962) at 20 8C, 90 mE m s under a 12:12 h AZA production within Amphidomataceae probably is much more light:dark cycle (hereafter called ‘‘standard culture conditions’’). complex and diverse; recent evidences indicate the presence of Azadinium cells are characterized by swimming at low speed, new AZAs with a modified substitution pattern in A. poporum and interrupted by short, high-speed ‘jumps’ in various directions A. languida (Krock et al., 2012). North Sea strains and the Korean (Tillmann et al., 2009). Cells exhibiting such a characteristic isolate of A. poporum were found to produce AZAs with a swimming behavior were isolated by means of drawn-out Pasteur characteristic m/z 348-fragment, however, in two different pipettes and established into clonal cultures. Only one strain was variants with different masses in the North Sea strains and the established from one container to guarantee they represent true Korean strain, respectively (Krock et al., 2012). clonal strains. Strains were maintained under standard culture There is increasing evidence that AZAs are present in the Asian conditions. Pacific. In 2009, AZA-2 was isolated from a marine sponge collected off Amami-Oshima Island (southern Japan) (Ueoka et al., 2009). 2.2. Light microscopy (LM) Moreover, AZA1 was detected in shellfish collected in several areas of China (Yao et al., 2010), which encouraged us to search for Live cells were examined under a Zeiss Axio Imager microscope Azadinium species along the coast of China. By incubating sediment (Carl Zeiss, Go¨ ttingen, Germany) equipped with both differential samples we succeeded in obtaining 25 strains of A. poporum; their interference illumination and epifluorescence. Light micrographs morphology were examined in detail, and their partial large were obtained using a Zeiss Axiocam HRc digital camera. subunit (LSU) and internal transcribed spacer regions (ITS1, ITS2 Approximately 1 mL of live, healthy culture in mid exponential and 5.8S rDNA) sequences were compared with those of strains growth phase was transferred to a 1.5 mL microcentrifuge tube, 0 from Europe and Korea. Six of these strains were grown in larger and DAPI (4 ,6-diamidino-2-phenylindole dihydrochloride) stain quantities and analyzed for the presence of AZAs. (Sigma–Aldrich, St. Louis, USA) was added at a final concentration À1 of 10 mg mL . The cells were then incubated in the dark at room 2. Materials and methods temperature for 30 min. The cells were viewed and photographed through a Zeiss Filterset (emission: BP 365-445; beamsplitter: FT 2.1. Sample collection and treatment 395). Cells in mid exponential growth phase were fixed with 5% Lugol’s solution and cell size was measured at 400Â magnifica- Sediment samples were collected in the Bohai Sea, East China tion. Fifty cells were measured for each strain of G60, G64, G66, Sea and South China Sea using a grab sampler (Fig. 1, geographical and G68. Fig. 1. Locations of sampling stations. 66 H. Gu et al. / Harmful Algae 21–22 (2013) 64–75 Table 1 Strains of Azadinium poporum examined in the present study, including collection data and locations.

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