Dataset: Chemical Defenses-1: Flores et al. 2012

Refer to the Methods section of Flores et al. (2012).

Culture and culturing conditions:
Three strains in the Alexandrium tamarense species complex (hereafter referred to as Alexandrium spp.) and one strain each of the dinoflagellates Lingulodinium polyedra and Scrippsiella trochoidea were maintained in f/2 medium without silicate (Guillard 1975) at 18°C on a 12:12 h light-dark cycle. Cultures were transferred biweekly to fresh medium and were in exponential growth for all experiments. The ciliate, Tiarina fusus, was isolated from Long Island Sound off Avery Point, Connecticut in June 2008. Ciliate cultures were maintained in 25-cm² polystyrene tissue-culture flasks containing 20 ml of f/2 medium, to which the dinoflagellate, Lingulodinium polyedra, was added as a food source. The heterotrophic dinoflagellate, Polykrikos kofoidii, was isolated from Northport Bay, located on the north shore of Long Island, NY, during a bloom of Alexandrium spp. in May 2009. P. kofoidii cultures were maintained in 6-well, polystyrene tissue-culture plates and were fed a mixture of L. polyedra and Scrippsiella trochoidea. All heterotrophic protist cultures were incubated at 18°C with a 12:12h light-dark cycle and were transferred weekly or biweekly into fresh medium containing prey.

Interactions between Alexandrium spp. and heterotrophic protists:
Observational experiments were conducted to examine qualitatively the effects of each Alexandrium spp. strain upon Tiarina fusus and Polykrikos kofoidii. Groups of 25 T. fusus or P. kofoidii cells were transferred by micropipette into individual wells of 12-well, polystyrene tissue-culture plates containing 2 ml of 0.2-μm filtered seawater (FSW). Both heterotrophic protist species were starved for 24 h prior to experimentation to ensure digestion of any recently-ingested Lingulodinium polyedra or Scrippsiella trochoidea cells from the stock cultures. Following starvation, aliquots of each Alexandrium spp. culture were added to the wells containing T. fusus or P. kofoidii. For each Alexandrium spp. strain, cell densities of 200 and 2,000 cells ml⁻¹ were tested. Controls consisted of FSW and L. polyedra (200 or 2,000 cells ml⁻¹). The behavior of individual T. fusus and P. kofoidii cells was observed under a stereomicroscope at 15-min intervals for 2 h.

Culture filtrates and extracts:
Experiments were conducted with Tiarina fusus to examine the effects of cell-free Alexandrium spp. culture filtrates and extracts upon ciliate survival. Alexandrium spp. cultures were diluted with f/2 medium to a density of 1,000 cells ml⁻¹. An aliquot (20 ml) of each Alexandrium spp. culture was filtered gently through a 0.2-μm syringe filter, resulting in a filtrate free of both Alexandrium spp. cells and bacteria. To examine the possible effects of bacteria present in the Alexandrium spp. cultures on T. fusus survival, additional aliquots (20 ml) from each Alexandrium spp. culture were filtered through 5-μm syringe filters, allowing bacteria, but not Alexandrium spp. cells, to pass through the filter. Cell extracts from Alexandrium spp. cultures were prepared by sonicating Alexandrium spp. culture aliquots (20 ml), on ice, with a Fisher model 100 sonic dismembrator until cells were completely disrupted (as confirmed by microscopy). Following sonication, the extracted samples were filtered through a 0.2-μm syringe filter to remove cell debris. Filtrates and extracts were added (5 ml; in triplicate) to individual wells of a 12-well, polystyrene tissue-culture plate. Controls consisted of intact Alexandrium spp. cultures and FSW. Fifteen ciliates were added to each experimental well, and treatments were incubated and enumerated as described above.

Physical separation from live Alexandrium spp. culture:
In order to determine if the observed mortality of Tiarina fusus exposed to Alexandrium spp. is a result of physical contact with and/or ingestion of the dinoflagellate. Groups of 15 T. fusus cells were placed into individual wells of 12-well, polystyrene tissue-culture plates containing FSW (2.5 ml). A culture plate insert with a10-μm nylon mesh bottom was added to each experimental well. Aliquots (1.5 ml) of each Alexandrium spp. culture were added to each culture insert, resulting in a final concentration of dissolved compounds in the treatment equivalent to a ~1,000 cells ml⁻¹ Alexandrium spp. culture. The 10-μm mesh separating the Alexandrium spp. culture from T. fusus cells prevented physical contact between the species while permitting exchange of dissolved compounds. Controls consisted of Alexandrium spp. cultures (1,000 cells ml⁻¹) in direct contact with T.fusus, and FSW. All experimental treatments and controls were conducted in triplicate and were incubated and enumerated as described in the above experiments.

Mitigation of toxicity:
Experiment was conducted to examine the effects of scavengers of reactive oxygen species on Tiarina fusus and Polykrikos kofoidii survival when exposed to Alexandrium spp. The antioxidant enzymes peroxidase (MP Biomedicals, #191370), catalase (MP Biomedicals, #100429), and superoxide dismutase (MP Biomedicals, # 190117) were prepared as aqueous solutions according to manufacturer specifications. All solutions were used within 1 h of preparation or were frozen immediately (-20°C) and thawed just before use. An additional treatment testing the protease, trypsin, was included to examine the possibility that protein or protein-like compounds are responsible for the toxicity of Alexandrium spp. to protists. Alexandrium spp. cultures were diluted with f/2 medium to a density of 1,000 cells ml⁻¹. Each Alexandrium spp. culture was subdivided, and peroxidase (1.25 μg ml⁻¹), catalase (2 U ml⁻¹), superoxide dismutase (5 U ml⁻¹), or trypsin (500 μg ml⁻¹) was added. Aliquots (5 ml, in triplicate) of each culture were added to individual wells of 12-well, polystyrene tissue-culture plates. A group of 15 T. fusus or P. kofoidii cells was added to each experimental well, and treatments were incubated and enumerated as described above. Controls consisted of ciliates and heterotrophic dinoflagellates exposed to Alexandrium spp. cultures without the addition of the enzymes and also FSW with the addition of each enzyme.