Phytoplankton light stress – dinoflagellate grazing experiments

General information

Emiliania huxleyi strains were grown in f/50 without added Si, except for CCMP1516 which was grown in f/2 for experiments D and I and in f/50 otherwise. All other phytoplankton were grown in f/2 medium without added Si. Most strains (designated CCMP) were obtained from the National Center for Marine Algae and Microbiota except Heterocapsa rotundata, which was from the Norwegian Culture Collection of Algae (NORCCA). Heterotrophic dinoflagellates Amphidinium longum and Oxyrrhis marina were isolated from marine waters of the Salish Sea, grown in ciliate medium (Gifford 1985), and maintained on a mixture of phytoflagellate species. All cultures of any type were grown at a salinity of 30 and a temperature of 15°C. Phytoplankton were grown at a range of low to moderate irradiances, depending on experiment on a 12L:12D cycle. Heterotrophic dinoflagellates were grown at 10-20 µmol photons m-2 s-1 on a 12L:12D cycle. Before use in experiments, dinoflagellate predators were fed only Rhodomonas sp. 755 (A. longum) or Dunaliella tertiolecta (O. marina) and allowed to consume these prey until they were nearly gone from the culture.

Cells were exposed to experimental light treatments outdoors in a shallow tank filled with flowing seawater supplied from nearby coastal waters. Temperature during experiments was monitored at regular intervals with a thermometer mounted in an unscreened incubation bottle, and ranged from 14-15°C except for Exp. A, where it averaged 17°C. Light (incident photosynthetically active radiation, or PAR) was measured with a Li-Cor 2π sensor, and logged at 5-min intervals so that total experiment light dose (mol photons m-2) could be computed for specific incubation periods. Control treatments were incubated in 60-ml polycarbonate bottles screened with sufficient neutral density screening to approximate growth irradiances. Higher light exposures were achieved using fewer (or no) layers of neutral density screening, depending on experiment. Except for Exp. E, which used polycarbonate bottles only, all high light treatments used 60-ml Teflon bottles, which are transparent to UV wavelengths. In some experiments high light treatments included both Teflon (UV-transparent) and polycarbonate (UV-opaque) bottles, to isolate the effects of UV on protist responses. Bottles were incubated at ~10 cm depth in the outdoor tank.

Experiments A-F exposed only the phytoplankton prey to the light stress treatments (‘Single_factor_grazing (prey-only)’ data set https://www.bco-dmo.org/dataset/779043). Cultures were divided into incubation bottles (n=3-5 depending on experiment) and placed in the outdoor tank for 60-120 min. Photosynthetic efficiency (Fv/Fm) was monitored before cells were taken outside (t=0) and, after gentle mixing, at 30-min intervals during the incubations (‘FvFm’ data set https://www.bco-dmo.org/dataset/779033). After outdoor exposure, phytoplankton were returned to the laboratory and a subsample from each replicate was added to a corresponding 30-ml polycarbonate bottle containing heterotrophic dinoflagellate predator A. longum to initiate predation experiments. The remainder of the phytoplankton culture volume was placed in an incubator at the culture growth irradiance level, and Fv/Fm monitored at regular intervals during this recovery period.

Prey concentrations for predation experiments ranged from 5.0 x 103 cells ml-1 for dinoflagellate Heterocapsa rotundata to 5.0 x 104 cells ml-1 for the various E. huxleyi strains. Prey biomass densities were equivalent for all prey types, at ~500 µg C liter-1. Carbon per cell for each phytoplankton species was estimated from measured cell volumes and published C:volume conversion factors (Menden-Deuer & Lessard 2000). A. longum concentrations were ~1-2 x 103 cells ml-1, and O. marina concentration (Exp. I only, see below) was 260 cells ml-1. For ‘prey only exposure’ experiments, predation tests were conducted for 50 min in a laboratory incubator at 15°C and ~50 µmol photons m-2 s-1. For ‘prey and predator exposure’ experiments, predation tests were conducted for 40-60 min under either control or high light outdoor illumination conditions. Predation tests were terminated by adding cells to cold 10% glutaraldehyde and DAPI stain (final concentrations 0.5% and 0.1 µg ml-1, respectively). After fixation overnight in 4°C and darkness, samples were filtered (3 or 5 µm pore-size polycarbonate filters), mounted on slides, and frozen for later examination by epifluorescence microscopy. UV excitation was used to locate and identify dinoflagellate predators from the DAPI-induced fluorescence of their nuclei. Ingested prey were detected using blue light excitation, from the orange (cryptophyte) or red (all other prey) autofluorescence of the prey pigments inside the predator food vacuoles Because A. longum uses a peduncle to feed on cryptophytes, rather than phagocytizing intact cells, the number of ingested prey per predator cannot be quantified for this predator – prey combination. Therefore for all predator and prey types, each micrograzer cell was scored as ‘feeding’ or ‘not feeding’. At least 250 micrograzers per slide were scored; predation intensity was calculated as fraction of the population feeding (= # micrograzers with ingested prey / total # micrograzers scored).

Experiments G, H, and I used a matrix design in which predators and prey were exposed to experimental irradiances separately, then combined in various ways and predation measured in outdoor irradiance conditions (‘Prey and predator exposure’ data set https://www.bco-dmo.org/dataset/779050). Cultures of predators and prey were incubated in separate bottles for the first 1-1.2 h of exposure time. After that, appropriate volumes of prey with various exposure histories were introduced into predator bottles with various exposure histories, and those predation tests incubated for an additional 40-60 min at the original predator irradiance level. Fv/Fm was monitored throughout (‘Photosynthetic efficiency’ data set https://www.bco-dmo.org/dataset/779033), first in the original phytoplankton-only bottles and then in the remaining phytoplankton volume after predation tests were initiated, and finally through a recovery period in the laboratory as described above. At the end of the predation test period, samples were fixed and slides prepared as described above.

For more information see Strom et al. (2020).