Azadinium Poporum (Dinophyceae) in the Southeast Pacific: Morphology, Molecular Phylogeny, and Azaspiracid Profile Characterization
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Journal of Plankton Research academic.oup.com/plankt J. Plankton Res. (2017) 39(2): 350–367. First published online January 20, 2017 doi:10.1093/plankt/fbw099 Downloaded from https://academic.oup.com/plankt/article/39/2/350/2929416 by guest on 12 October 2020 Identification of Azadinium poporum (Dinophyceae) in the Southeast Pacific: morphology, molecular phylogeny, and azaspiracid profile characterization URBAN TILLMANN1*, NICOLE TREFAULT2, BERND KROCK1, GÉNESIS PARADA-POZO2, RODRIGO DE LA IGLESIA3 AND MÓNICA VÁSQUEZ3 ALFRED WEGENER INSTITUTE, HELMHOLTZ CENTRE FOR POLAR AND MARINE RESEARCH, AM HANDELSHAFEN , D- BREMERHAVEN, GERMANY, CENTER FOR GENOMICS AND BIOINFORMATICS, FACULTY OF SCIENCES, UNIVERSIDAD MAYOR, CAMINO LA PIRÁMIDE , HUECHURABA, SANTIAGO, CHILE AND DEPARTMENT OF MOLECULAR GENETICS AND MICROBIOLOGY, PONTIFICIA UNIVERSIDAD CATÓLICA DE CHILE, AV. LIBERTADOR BERNARDO O´HIGGINS , SANTIAGO, CHILE *CORRESPONDING AUTHOR: [email protected] Received November 7, 2016; editorial decision December 21, 2016; accepted December 24, 2016 Corresponding Editor: John Dolan Azaspiracids (AZA), a group of lipophilic phycotoxins, are produced by some species of the marine dinophycean genus Azadinium. AZA have recently been detected in shellfish from the Southeast Pacific, however, AZA-producing species have not been recorded yet from the area. This study is the first record of the genus Azadinium and of the species Azadinium poporum from the Pacific side of South America. Three strains of A. poporum from Chañaral (Northern Chile) comply to the type description of A. poporum by the presence of multiple pyrenoids, in thecal plate details, and in the position of the ventral pore located on the left side of the pore plate. Molecular phylogeny, based on internal transcribed spacer and large subunit ribosomal DNA sequences, revealed that Chilean strains fall in the same ribotype clade as European and strains from New Zealand. Analyses of AZA profiles using LC–MS/MS showed an identical profile for all three strains with the presence of AZA-11 and two phosphorylated AZA. This is the first confirmation of the presence of AZA producing Azadinium in the Chilean coastal area and underlines the risk of AZA shellfish and concomitant human contamination episodes in the Southeast Pacific region. KEYWORDS: Azadinium; azaspiracids; Southeast Pacific; Chile available online at academic.oup.com/plankt © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected] U. TILLMANN ET AL. j AZADINIUM POPORUM FROM THE SOUTHEAST PACIFIC INTRODUCTION poisoning (Twiner et al., 2014). This group of compounds fi fl has been reported in shell sh from numerous geograph- The Humboldt Current owing along the west coast of ical sites, such as the Atlantic coasts of various European South America is one of the major ocean current systems countries including Denmark, France, Ireland, Norway, of the world where cold, nutrient-rich upwelling water Portugal, Spain, Sweden and the United Kingdom drives high rates of primary and secondary productivity. (James et al., 2002; Braña Magdalena et al., 2003; Amzil Consequently, the water masses off Chile are among the et al.,2008; Vale et al., 2008). AZA are known from the most productive marine areas on a global scale (Alheit Pacific coast of the United States (Trainer et al., 2013) et al. fi and Bernal, 1993; Daneri ,2000) and marine sheries and Mexico (García-Mendoza et al., 2014), from the are a key component of the Chilean economy (Gelcich Atlantic coasts of NW Africa (Taleb et al., 2006) and et al.,2010). During strong upwelling periods, occurring Canada (M. Quilliam, personal communication in Downloaded from https://academic.oup.com/plankt/article/39/2/350/2929416 by guest on 12 October 2020 typically in winter and spring, diatoms dominate the sys- Twiner et al., 2008), and from the Asian Pacific off tem. However, dinophytes and other smaller sized flagel- China (Yao et al., 2010) and Japan (Ueoka et al., 2009). lates may dominate during weakened upwelling periods With respect to South America, AZA have been found (typically in summer and autumn) and El Niño periods on both the Atlantic and Pacific coasts in Brazil when nutrient availability is reduced (Iriarte and Gonzá- (Massucatto et al., 2014), Argentina (Turner and Goya, et al. lez, 2004; Ochoa ,2010). This is of particular import- 2015) and Chile (Álvarez et al.,2010; López-Rivera et al., ance as many members of the Dinophyceae or other 2010), respectively. planktonic flagellates are known for the production of AZA are produced by some species of the family potent toxins and other bioactive compounds. This phyto- Amphidomataceae. This was discovered in 2009 driven planktonic class may form Harmful Algal Blooms (HAB) by the targeted search for the planktonic source of AZA causing human health problems, massive killing of fish, (Krock et al., 2009b) with the first described species birds or mammals and/or other ecosystem disruptions Azadinium spinosum Elbrächter et Tillmann (Tillmann (Smayda, 1997; Hallegraeff, 2003, 2014). et al., 2009). Among the eleven species of the genus Records of phycotoxins and potentially toxic plankton Azadinium (Tillmann and Akselman, 2016), three have species as well as reports of HAB for the Humboldt been found to produce AZA (i.e. Azadinium spinosum, Current system off Chile indicate the presence of almost Azadinium poporum Tillmann et Elbrächter, Azadinium dex- all known phycotoxins. Pectenotoxins and okadaic acid fi teroporum Percopo et Zingone) (Krock et al., 2012; including dinophysistoxins in plankton, lter feeders, Percopo et al., 2013). AZA have also been found in the Dinophysis and in cells of local spp. are common (Blanco related species Amphidoma languida Tillmann, Salas et et al., 2007; Krock et al., 2009a; Trefault et al., 2011). fi Elbrächter (Tillmann et al., 2012). Moreover, yessotoxins are reported both in shell sh Just as for records of AZA in shellfish there is increas- (Yasumoto and Takizawa, 1997) and in plankton sam- ing evidence that species of Azadinium and Amphidoma ples (Krock et al., 2009a), and blooms of the producing have a wide geographical distribution in both coastal Lingulodinium polyedra species have been also recorded and open ocean areas of both the North and South et al. fi (Álvarez , 2011). Spirolides are found in shell sh in Atlantic and Pacific(Tillmann et al., 2014b). For the northern Chile (Álvarez et al., 2010), and gymnodimines fi Atlantic side of South America, Azadinium now is retro- were recently detected in Chile for the rst time spectively known to have been present almost two dec- et al. (Trefault , 2011). In Northern Chile off Bahía ades ago and at least three bloom episodes of a diverse Inglesa, blooms of Pseudo-nitzschia australis and detection community of Azadinium species occurred in 1990, 1991 of domoic acid above regulatory levels in scallops have and 1998 (Akselman and Negri, 2012; Akselman et al., et al. been reported (Suárez-Isla , 2002). Paralytic shell- 2014; Tillmann and Akselman, 2016). In addition, the fish poisoning (PSP) is a major problem in southern presence of an AZA producing Azadinium, an AZA-2 pro- et al. Chile (Lagos, 1998; Guzmán , 2002). Nevertheless ducing A. poporum in the Argentinean coastal area, was it is present in the Humboldt Current system, and recently confirmed (Tillmann et al. 2016). A. poporum pro- blooms of Alexandrium as well as PSP toxins in shellfish ducing the same AZA compound, but differing signifi- were detected in several aquaculture sites in northern cantly in ribosomal sequence data from the Argentinean et al. Chile (Álvarez , 2009). population, was recorded in the Gulf of Mexico (Luo The group of azaspiracids (AZA) were recently also et al., 2016). In the Pacific Ocean, Azadinium has been et al. found in the area (Álvarez ,2010; López-Rivera recorded in Korea, China and New Zealand (Potvin et al. et al. , 2010; Trefault ,2011). AZA are the most et al., 2012; Gu et al., 2013; Smith et al., 2016). However, recently identified group of lipophilic marine biotoxins, on the Pacific side of South America there has been only which are associated with human incidents of shellfish JOURNAL OF PLANKTON RESEARCH j VOLUME j NUMBER j PAGES – j one record of a species determined as A. spinosum on the of Azadinium and to obtain and grow isolates of Azadinium Pacific coast of Mexico (Hernández-Becerril et al., 2012). from Chile for a thorough morphological, molecular and Despite the known presence of AZA in Chile (Álvarez toxinological characterisation. et al.,2010; López-Rivera et al., 2010), the causative spe- cies in Chilean coastal waters thus have not been fi detected or identi ed yet. This is important because it is METHOD quite likely that the diversity of the Amphidomataceae is not yet fully explored and thus new toxigenic species Field campaign might be present. Knowledge on the species present is In 2014, a sampling campaign was setup to study effects an indispensable prerequisite for any monitoring and/or of heavy metal contamination on plankton in the early warning system to be successfully implemented. Chañaral area, a site heavily affected by copper depos- Downloaded from https://academic.oup.com/plankt/article/39/2/350/2929416 by guest on 12 October 2020 This is especially true for small and inconspicuous species ition from the local mining industry. As a side aspect of fi like Azadinium whose routine detection and identi cation this campaign, plankton samples were taken for deter- require molecular detection tools, which are only avail- mining microalgal toxins and for detecting and charac- able for known species. Large areas of the Chilean coast terising potentially toxic species with the special aim for fi are dif cult to access for routine plankton sampling and AZA and Azadinium. thus detailed knowledge on the species composition of the small plankton fraction is rare. The aim of the pre- sent study was thus to specifically search for the presence Sampling Seawater samples were taken from the Chañaral area at three stations (Fig.