Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
• Data Files
• Related Publications
• Parameters
• Instruments
• Project Information
• Funding

Refer to the Methods section of Park & Dam (2021).

Sample collection and culture:
The toxigenic dinoflagellate, Alexandrium catenella (strain BF-5, isolated from the Bay of Fundy, Canada) was grown in F/2 medium without silicate. Cultures were maintained in the exponential growth phase and all experiments were conducted in an environmental chamber kept at 18°C and illuminated with fluorescent lighting (~100 µM m⁻² s⁻¹) set to a 12 h:12 h light:dark photoperiod. The calanoid copepod Acartia hudsonica, historically co-occurring with toxic A. catenella, was collected from Casco Bay, Maine, U.S.A. (43°39′N, 74°47′W), a location in which blooms of A. catenella are common. Triplicate copepod cultures were maintained with a mixed diet of Rhodomonas sp., Tetraselmis sp., and Thalassiosira weissflogii.

Grazer-induced toxin production assay:
Alexandrium catenella cells were placed in 500 ml bottles (300 cells ml⁻¹) either without copepods (controls: constitutive toxin production) or with 20 adult female A. hudsonica (treatments) for a period of 96 h. Experiments were done in quadruplicate sets for both control and treatment, and carried out at 18 °C in a walk-in environmental chamber as described above. For the time series analysis, A. catenella cells were harvested at the conclusion of each exposure time (0, 4, 8, 24, 48, 72, and 96 h) after which cells were gently separated by wet-sieving onto a 63 μm mesh to remove copepods, nauplii, eggs, and fecal pellets. Two aliquots from each bottle were filtered onto 5 μm pore size polycarbonate membranes; one for toxin analysis and another for RNA extraction.

Toxin and gene expression analysis:
PST concentrations were determined by reverse-phase ion-pairing high performance liquid chromatography (HPLC) using the post-column oxidative fluorescence method. Gene expression analyses were conducted using reverse transcription quantitative PCR (RT-qPCR). From previous studies several pairs of primers were designed and tested for specificity and PCR efficiency by RT-qPCR. All quantitative PCRs (qPCRs) were performed on a StepOnePlus™ real-time PCR system (Applied Biosystems) and run in 10 μL reactions with Fast SYBR® Green Master Mix (Applied Biosystems).

Data Processing:
A Kruskal-Wallis one-way ANOVA on ranks was used and all pairwise comparisons among controls/treatments were assessed with the Student-Newman-Keul’s (SNK) post-hoc procedure. The effects of day, presence of grazer, and their interaction on cell concentration, toxin content, toxin profile, growth rate, and relative gene expression (four independent replicates per test and two samples for PST analysis) were tested by two-way ANOVA. Time-dependent changes in these variables were also tested separately by regression analysis for the control and treatments. Ingestion rates were tested with a Mann-Whitney U Test to compare the Frost equation to our modified equation. All statistical analyses were performed using SigmaPlot version 11.0 and SPSS version 26 software.

Description from NSF award abstract:
Species of the dinoflagellate genus Alexandrium occur around the globe, and some species, because of their toxin production, have been hypothesized to be keystone species. Alexandrium produces chemical compounds that appear to target different consumers. Neurotoxins such as PST target metazoan grazers. In preliminary experiments in their laboratory, the investigators also verified the presence of reactive oxygen species that target, at a minimum, protistan grazers. Such compounds reduce grazer fitness, and, at least in the case of PST, have been shown to have profound evolutionary effects on grazers. Grazer adaptation, in turn, can affect Alexandrium population dynamics. A common assumption is that production of toxic compounds in phytoplankton represents an adaptive defense. However, unequivocal experimental evidence in support of this hypothesis is scarce. This project will be a rigorous experimental test of the chemical defense hypothesis. The project's investigators will investigate a series of experimentally falsifiable hypotheses with both metazoan and protistan grazers challenged with Alexandrium. This project will provide novel understanding of, and insight into, the factors that determine grazer-induced toxin production, the relationship between degree of chemical defense and susceptibility to grazing, and the costs and tradeoffs of the purported mechanisms of chemical defense in Alexandrium. Verification or refutation of the chemical defense hypothesis is essential to conceptual models of the formation, control and persistence of toxic algal blooms, and chemically-mediated predator-prey interactions.