Vol. 66: 199–209, 2012 AQUATIC MICROBIAL ECOLOGY Published online May 31 doi: 10.3354/ame01570 Aquat Microb Ecol OPENPEN ACCESSCCESS Reactive oxygen species are linked to the toxicity of the dinoflagellate Alexandrium spp. to protists Hayley S. Flores1,3, Gary H. Wikfors2, Hans G. Dam1,* 1Department of Marine Sciences, University of Connecticut, 1080 Shennecossett Road, Groton, Connecticut 06340, USA 2Northeast Fisheries Science Center, National Marine Fisheries Service, 212 Rogers Avenue, Milford, Connecticut 06460, USA 3Present address: Algenol Biofuels, 16121 Lee Road, Ft. Myers, Florida 33912, USA ABSTRACT: Short-term experiments were conducted to examine the response of the ciliate Tia- rina fusus and the heterotrophic dinoflagellate Polykrikos kofoidii to 3 strains in the Alexandrium tamarense species complex, each with a different paralytic shellfish toxin (PST) content. Both pro- tist species fed on all 3 Alexandrium strains, but significant mortality occurred within 24 h of initial exposure to high densities of each dinoflagellate isolate. Protist mortality was not related, how- ever, to the PST content of the Alexandrium strains, indicating a different mechanism of toxicity. Exposure of T. fusus to cell-free culture filtrates or cell extracts did not cause significant ciliate mortality. In contrast, significant mortality occurred when ciliates were separated physically from a live Alexandrium sp. culture by a 10 µm nylon mesh, suggesting that the toxicity is dependent upon the viability of the Alexandrium spp. cells but does not require physical contact or ingestion. Addition of antioxidant compounds significantly increased the survival of both protist species when exposed to Alexandrium, suggesting that reactive oxygen species and/or the secondary compounds produced by ROS-induced lipid peroxidation are involved in the toxicity of Alexan- drium spp. to ciliates and heterotrophic dinoflagellates. This mechanism of toxicity is previously unknown for Alexandrium spp. and may play an important role in bloom dynamics and toxin transfer within the food web. KEY WORDS: Alexandrium · Ciliate · Harmful algae · Heterotrophic dinoflagellate · Polykrikos kofoidii · Reactive oxygen species · Tiarina fusus Resale or republication not permitted without written consent of the publisher INTRODUCTION Durbin et al. 2002). Given the potential economic and human health risks associated with blooms of toxic Harmful algal blooms (HABs) in marine ecosys- Alexandrium spp., it is important to understand the tems are increasing worldwide, presenting a scientif- mechanisms controlling the population dynamics of ically complex and economically significant chal- these harmful dinoflagellates. lenge to the management of coastal waters (Smayda Grazing is thought to be an important biological 1990, Hallegraeff 1993). Among HAB species, dino - factor influencing the formation and termination of flagellates in the genus Alexandrium are among the HABs (Buskey et al. 1997, Turner & Tester 1997, most ecologically important because some species Colin & Dam 2007, Smayda 2008). Microzooplank- produce neurotoxins referred to as paralytic shellfish ton, particularly ciliates and heterotrophic dinofla- toxins (PSTs). PSTs can be accumulated by filter- gellates, are often the most active grazers of phyto- feeding shellfish and other grazers and transferred to plankton, consuming 60 to 70% of daily planktonic humans and other animals, leading to severe illness primary production (Sherr & Sherr 2002, Calbet & and possibly death (White 1980, Shum way 1990, Landry 2004, Calbet 2008). Although high abun- *Corresponding author. Email: [email protected] © Inter-Research 2012 · www.int-res.com 200 Aquat Microb Ecol 66: 199–209, 2012 dances of ciliates and heterotrophic dinoflagellates understand the interactions between heterotrophic have been observed during blooms of Alexandrium protists and Alexandrium spp. and to evaluate the spp. (Needler 1949, Prakash 1963, Watras et al. role of these grazers in the formation and termina- 1985, Carreto et al. 1986), the interactions of these tion of blooms. protists with Alexandrium spp. are not well under- In the present study, we investigated the effects of stood. Several heterotrophic protist species have 3 isolates in the Alexandrium tamarense species been reported to ingest Alexandrium spp. with no complex on the survival of the ciliate Tiarina fusus apparent adverse effects (Stoecker et al. 1981, and the heterotrophic dinoflagellate Polykrikos Hansen 1992, Matsuoka et al. 2000, Kamiyama et al. kofoidii. Further, we tested a hypothesis that ROS are 2005), but other protists exhibit altered swimming linked to the toxicity of Alexandrium spp. to protists behavior, reduced ingestion, growth inhibition, or by examining the effect of free-radical-scavenging mortality (Hansen 1989, Hansen et al. 1992, Till- enzymes on the survival of T. fusus and P. kofodii mann & John 2002, Fistarol et al. 2004, Fulco 2007, exposed to Alexandrium. Tillmann et al. 2007, 2008). The disparate results from previous research cannot be attributed to dif- ferences in the PST content of the algal isolates MATERIALS AND METHODS (Tillmann & John 2002, Tillmann et al. 2008, 2009). Instead, it appears that uncharacterized metabolites Experimental cultures produced by Alexandrium spp. are re sponsible for the toxicity of these dinoflagellates to protists (Till- Three strains in the Alexandrium tamarense spe- mann & John 2002, Fistarol et al. 2004). These cies complex (Table 1; hereafter referred to as harmful compounds are often referred to as ‘allelo- Alexandrium spp.) and one strain each of the dino - chemicals,’ i.e. secondary metabolites that in hibit flagellates Lingulodinium polyedra and Scrippsiella the growth of competing organisms (Legrand et al. trochoidea were maintained in f/2 medium without 2003, Granéli & Hansen 2006). In marine microbial silicate (Guillard 1975) at 18°C on a 12:12 h light: dark ecology, the category of allelochemicals can include cycle. The cultures were transferred biweekly to compounds that incapacitate or deter grazers (Cem- fresh medium and were in exponential growth for all bella 2003, Granéli & Hansen 2006). experiments. The cultures were not axenic, but asep- The bioactive, allelochemical compounds pro- tic techniques were used to minimize additional duced by Alexandrium spp. affect the structure and microbial contamination. Prior to experimentation, function of cell membranes, causing immobilization Alexandrium spp. strains were examined for the pro- of co-occurring protist cells, followed by cell duction of PSTs. Triplicate samples were extracted swelling and lysis (Hansen 1989, Tillmann & John according to Anderson et al. (1994) and analyzed 2002). The specific mode of action, however, is cur- using high performance liquid chromatography for rently unknown. Emura et al. (2004) speculated saxitoxin (STX), neosaxitoxin (NEO), and gonyautox- that a protein-like toxin is responsible for the lytic ins I-IV (GTX1-4) using the methods of Oshima et al. activity of Alexandrium spp. Further research by (1989). Toxin standards were obtained from the Ma et al. (2009) has suggested that the allelochem- National Research Council, Marine Analytical icals may be amphipathic compounds that form Chemistry Standards Program, Halifax, Nova Scotia, large aggregates or macromolecular complexes. Canada. Based upon the analysis, the 3 Alexandrium Elevated concentrations of reactive oxygen species sp. isolates were designated ‘High PST’ (NB-05), (ROS) can disrupt a variety of cellular processes, ‘Low PST’ (CB-307), and ‘No PST’ (CCMP115) including cell membrane integrity (Halliwell & Gut- (Table 1). teridge 1985). Although these com- pounds have been linked to the toxi- Table 1. Alexandrium spp. strains, source location, isolation date, and toxin city of other HAB species to aquatic content. STX eq: saxitoxin equivalent. –: no measurable toxin content organisms (Yang et al. 1995, Ishi- matsu et al. 1997, Kim et al. 1999, Strain name Source location Isolation date Average toxin content Tang & Gobler 2009), their possible (pg STX eq. cell−1) role in the toxicity of Alexandrium spp. to grazers has not been exam- NB-05 Bay of Fundy, NB 2001 22.25 CB-307 Casco Bay, ME 2001 11.98 ined. Determination of the mecha- CCMP115 Tamar Estuary, UK 1957 – nism of toxicity is needed to better Flores et al.: Toxicity of Alexandrium spp. to protists 201 The ciliate Tiarina fusus was isolated from Long Is- tion, 5 ml (in triplicate) was added to each well of 12 land Sound off Avery Point, CT, in June 2008. Ciliate well polystyrene tissue-culture plates. Groups of 15 cultures were maintained in 25 cm2 polystyrene tis- ciliates or heterotrophic dinoflagellates were added sue-culture flasks containing 20 ml of f/2 medium, to to each experimental well, and treatments were incu- which the dinoflagellate Lingulodinium polyedra was bated for 24 h at 18°C on a 12:12 h light:dark cycle. added as a food source. The heterotrophic dinoflagel- Following incubation, acidic Lugol’s solution (2% late Polykrikos kofoidii was isolated from Northport final concentration) was added to each well, and Bay, located on the north shore of Long Island, NY, intact T. fusus or P. kofoidii cells were enumerated by during a bloom of Alexandrium spp. in May 2009. P. light microscopy. Controls consisted of the dinofla- kofoidii cultures were maintained in 6 well poly- gellate Lingulodinium polyedra (63 to 1000 cells styrene tissue-culture plates and were fed a mixture ml−1) and 0.2