Dinophysis Species Associated with Diarrhetic Shellfish Poisoning Episodes in North Patagonian Gulfs (Chubut, Argentina) Author(s): Leiéen Gracia Villalobos, Norma Santinelli, Viviana Sastre, Bernd Krock and José Luis Esteves Source: Journal of Shellfish Research, 34(3):1141-1149. Published By: National Shellfisheries Association DOI: http://dx.doi.org/10.2983/035.034.0339 URL: http://www.bioone.org/doi/full/10.2983/035.034.0339 BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use. 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DINOPHYSIS SPECIES ASSOCIATED WITH DIARRHETIC SHELLFISH POISONING EPISODES IN NORTH PATAGONIAN GULFS (CHUBUT, ARGENTINA) LEILEN GRACIA VILLALOBOS,1* NORMA SANTINELLI,2 VIVIANA SASTRE,2 BERND KROCK3 AND JOSE LUIS ESTEVES1 1Centro Nacional Patagonico (CONICET), Boulevard Brown 2915, 9120 Puerto Madryn, Chubut, Argentina; 2Facultad de Ciencias Naturales, Universidad Nacional de la Patagonia ‘‘San Juan Bosco’’, Julio Argentino Roca 115 1° piso, 9100 Trelew, Chubut, Argentina; 3Alfred Wegener Institut-Helmholtz Zentrum fur€ Polar- und Meeresforschung, Chemische Okologie,€ Am Handelshafen 12, 27570 Bremerhaven, Germany ABSTRACT The marine dinoflagellate genus Dinophysis Ehrenberg is globally distributed in coastal and oceanic waters and can produce lipophilic toxins. These toxins can accumulate in filter-feeding shellfish and cause diarrhetic shellfish poisoning (DSP). Between 2009 and 2011 the two most frequent and abundant Dinophysis species found in North Patagonian gulfs were Dinophysis tripos Gourret and Dinophysis acuminata Claparede and Lachmann, and in 2015 D. tripos was the only toxic species found in moderate to high relative abundances when mouse bioassay results for DSP were positive. The positive results from mouse bioassay for DSP agree with moderate to high relative abundances of D. tripos and it was the only potentially toxic Dinophysis species found in the samples. The toxin profiles consisted mainly of pectentoxin-2 (PTX-2) followed by PTX-11 and PTX-2 seco acid. The toxin profiles of the samples could be associated with D. tripos, because the maximum proportion of D. acuminata did not exceed 1.3% of total Dinophysis cells in any of the samples. The results suggest that shellfish accumulated high levels of PTX when D. tripos was at a high abundance. To our knowledge, this is the first record of positive results in the mouse bioassay for DSP related to D. tripos and with closures of shellfish harvesting in North Patagonian gulfs. KEY WORDS: Dinophysis tripos, Dinophysis acuminata, diarrhetic shellfish poisoning, mouse bioassay, liquid chromatography- tandem mass spectrometry, North Patagonian gulfs INTRODUCTION their potential threat to human health is currently under debate (Miles et al. 2004, Reguera et al. 2012). Yessotoxins are disulfated Diarrhetic shellfish poisoning (DSP) is a specific type of food polycyclic polyether toxins and are toxic to mice only by in- poisoning and a severe gastrointestinal illness caused by the traperitoneal injection (Aune et al. 2002, Tubaro et al. 2003). ingestion of filter-feeding bivalves contaminated with a specific Okadaic acid and DTX are mainly produced by planktonic suite of marine toxins (Yasumoto et al. 1984, Domınguez et al. dinoflagellates of the genus Dinophysis and Prorocentrum lima 2010). Diarrhetic shellfish poisoning is globally reported with Ehrenberg (Dodge) (Gayoso et al. 2002, Maranda et al. 2007). recurring cases in Europe, South America, and Southeast Asia To date, 12 species of Dinophysis have been reported to contain (Hallegraeff 1993, Van Dolah 2000, Reguera et al. 2012). The lipophilic toxins (Yasumoto et al. 1985, Reguera & Pizarro most characteristic symptoms of DSP intoxication are diarrhea, 2008, Rodrıguez et al. 2012). Of these species, seven (Dinophysis nausea, vomiting, abdominal pain, and chill. No human acuminata, Dinophysis acuta Ehrenberg, Dinophysis caudata fatalities attributed to DSP have been reported; however, some of the DSP toxins may promote stomach tumors and thus cause Saville-Kent, Dinophysis fortii Pavillard, Dinophysis miles € chronic problems in shellfish consumers (Suganuma et al. 1988, Cleve, Dinophysis ovum Schutt, Dinophysis sacculus Stein) have Larsen & Moestrup 1992).Three chemically different lipophilic been associated with DSP events (Reguera et al. 2012). In New groups of toxins have been historically associated with DSP: Zealand, Chile, and along the Atlantic coasts of Europe, okadaic acid (OA) and dinophysistoxins (DTX), pectenotoxins Dinophysis acuminata and D. acuta have been reported as the (PTX), and yessotoxins (Domınguez et al. 2010). Both OA and main causative agents of lipophilic shellfish toxin events in the DTX are acid polyethers that inhibit protein phosphatases, shellfish above regulatory levels (van Egmond et al. 1993, Raho and are the only toxins of the old ÔÔDSP complexÕÕ with et al. 2008, Reguera & Pizarro 2008). Previously, many diarrheogenic effects (Reguera et al. 2012). Okadaic acid and questions about the ecology, behavior, toxin content, and its congeners DTX-1 and DTX-2 are the main toxins respon- genetic diversity of Dinophysis populations remained unan- sible for the DSP syndrome (Yasumoto et al. 1985); however, swered. This is partly due to the fact that researchers were PTX and yessotoxins have been considered as DSP toxins unable to successfully establish laboratory cultures of Dinoph- mainly because they are extracted together with OA and DTX ysis species for many years. This obstacle was overcome for DSP testing (Yasumoto et al. 1985, Murata et al. 1987, when Park et al. (2006) successfully cultured an isolate of Domınguez et al. 2010). The PTX are polyether-lactones, some D. acuminata by providing the ciliate prey Mesodinium rubrum of which are hepatotoxic to mice by intraperitoneal injection. (Lohmann) Hamburger and Buddenbrock which in turn was The PTX-2 and its shellfish-mediated derivative, PTX-2 seco acid allowed to feed on the cryptophyte Teleaulax sp. As a result (PTX-2sa), are not toxic to mice when administered orally and of this culturing achievement, D. fortii (Nagai et al. 2008), D. caudata (Nishitani et al. 2008a), D. acuta (Jaen et al. 2009) *Corresponding author. E-mail: [email protected] and Dinophysis infundibulus Schiller (Nishitani et al. 2008b) have DOI: 10.2983/035.034.0339 also been successfully cultured. Later, Rodrıguez et al. (2012) 1141 1142 GRACIA VILLALOBOS ET AL. described morphological, toxicological and genetic characteris- detected the toxigenic Dinophysis species Dinophysis acuminata tics of D. tripos from cultures fed with the ciliate Mesodinium and Dinophysis caudata and lipophilic shellfish toxins in two rubrum. This was the first report of the presence of PTX-2 in D. bivalve species from the coast of Buenos Aires Province. This tripos and of the establishment of cultures of this species. was the first record of detection of an outbreak of DSP The first record of potential DSP producers along the associated with the presence of Dinophysis species in Argentina. Patagonian coast dates back to 1980. Gil et al. (1989) reported Most recently, another PTX producing species, Dinophysis the occurrence of Dinophysis acuminata and Dinophysis fortii in tripos, was reported for the Argentine Sea (Fabro et al. 2015). the San Jose Gulf, being the first report of DSP producers from Previous work was further supplemented by the survey carried Chubut Province, northern Patagonia, Argentina. One year after out by Sar et al. (2012), which focused on the detection of DSP D. acuminata was reported from the Chubut River Estuary toxins in shellfish samples. Their results showed that shellfish (Sastre et al. 1990) and D. acuminata and Prorocentrum lima were were contaminated with a DSP toxin profile composed of OA, recorded in phytoplankton samples from the San Jose Gulf and DTX-1 and DTX-3. Besides this report, however, little nothing Nuevo Gulf (Santinelli et al. 1994, Santinelli et al. 2002). Later the is known about the linkage of positive DSP mouse bioassays occurrence of D. acuminata in Nuevo Gulf was also recorded in and the original phytoplankton toxin profiles in this area. For December 1993 and November 1994 (Santinelli 2008). this reason the major aim of this study was to investigate the Even though DSP producing species in this region had been toxin profile of toxigenic phytoplankton present in shellfish observed before, it was not before 1999 that the first recognized harvesting areas during harvest closure due to DSP in the DSP outbreak reported by Gayoso and Ciocco (2001) and Chubut Province in the years 2009 to 2011. Gayoso et al. (2002). At a celebration, more