5.1. Strain origin and laboratory acclimation
The clonal strain of G. catenatum (IO13-04) used in the present study was established by reisolation of a single cell from a culture obtained by germination of a wild cyst from phytoplankton net samples collected in Cascais (Portugal) (38°41′37″ N 9°24′52.5″ W) in September 2018 during a bloom of G. catenatum. Once established, cultures were maintained in L1 medium [33] salinity 33 ppt, at 19.0 ± 1.0 °C, under 12 h:12 h light: dark cycle and photosynthetic photon flux density (PPFD) of ca. 40 μmol photons m−2s−1. Cultures were maintained in the algae culture collection at Lisbon University (ALISU).
To obtain the necessary inoculum for the initiation of the planned experiments, cultures were scaled-up at the facilities of Laboratório Marítimo da Guia (Cascais, Portugal). Here, cultures were acclimated to the control experimental conditions for two months in temperature-controlled (STC-3000 temperature controllers, hysteresis 0.3 °C) water baths at 18.5 °C (control temperature for Cascais, Portugal) with 10:14 L:D photoperiod under AquaRay GroBeam lamps (600 ultima, TMC, UK) lights.
5.2. Experimental design
Following acclimation, all the cultures were combined and re-distributed between experimental treatments, in 2L Schott flasks as follows: i) Control temperature (18.5 °C, n = 8 flasks), ii) MHW category I (19.9 °C, n = 8) and iii) MHW category IV (24.1 °C, n = 8). To simulate MHWs, a 30-year (1989-2019) dataset for seawater surface temperature from Cascais, Portugal, was obtained from NOAA [accessed on 15/01/2021]. With this dataset, it was determined that the average temperature in Cascais throughout the 30 years was 18.5 °C. In R, through the package ‘heatwaveR’ [33] - which uses the definition of MHWs according to [4] - the intensity and rates of onset and offset of MHWs was determined. Table 1 summarises the details pertaining to the simulation of MHWs. Here, Category I MHW was determined to have a peak intensity of 19.9 °C when climatology was 18.5 °C, whereas Category IV MHW was determined to have a peak intensity of 24.1 °C, with the same climatology.
Table 1. Exposure of Gymnodinium catenatum to simulated MHW categories I and IV.
|
Category I
|
Category IV
|
Peak intensity (°C)
|
19.9 °C
|
24.1 °C
|
Rate of onset (°C day-1)
|
0.13
|
0.55
|
Duration (days)
|
10
|
10
|
Rate of offset (°C day-1)
|
0.14
|
0.56
|
Total duration (days)
|
30
|
30
|
The duration of 30 days corresponds to the following phases: i) 10 days of onset temperature increasing slope, ii) 10 days in peak temperature and iii) 10 days in offset temperature decreasing slope. The duration was chosen to accompany the cultures’ growth curve (circa 10 days) and the definition of MHW: “(…) MHW needs to persist for at least five days” [3].
5.3. Culture maintenance and sample acquisition
During exposure to MHWs, daily additions of small amounts (50ml) of fresh L1 growth medium [34] were carried out to avoid sudden changes in water quality. Since the cultures were immersed in the circulating water-bath, temperature parameters were obtained from the water-bath and not directly from the cultures to avoid contamination. Every three days, samples were collected from four random replicates in each treatment for cell count, cell size and photobiological measurements. Every ten days, half of the culture volume (500ml) was collected for toxin analysis, corresponding to the amount of medium added in the 10-day period. Before sampling cultures were always carefully homogenised by gently turning the flasks. Cell counts were carried out in a Sedgewick-Rafter counting chamber, where the total number of single cells and the number of cells in chains was registered. Photographs (n = 100 per treatment) were taken with a stereomicroscope (Leica S APO coupled with MC 190 HD camera, Leica Microsystems) to carry out cell size measurements in ImageJ. Photobiological parameters (basal fluorescence (Fo), maximum fluorescence (Fm) and maximum quantum PSII yield (Fv/Fm) were obtained using a Water-PAM (Walz) in dark-acclimated (15 min.) cultures. From each replicate, the volume extracted was filtered onto 47mm Whatman GF/C with a nominal pore size of 1.2 mm, under a low vacuum and stored at -80 °C for posterior toxin quantification.
5.4. Toxin extraction and quantification
5.4.1 Extraction
Gymnodinium catenatum extraction was performed following [35] with slight modifications. Toxins were extracted in 4 mL of 0.05M acetic acid and sonicated for 1 min at 25 W, 50% pulse duty cycle (Vibracell, Sonic & Materials, Newtown, CT, USA) in an ice bath. The extract was then centrifuged (3000 g) for 10 min, and the supernatant was collected.
5.4.2 Solid-phase extraction (SPE) clean-up
The supernatant was cleaned by solid-phase extraction (SPE) following [36], with slight modifications. Briefly, 1 mL of the acetic acid extract was transferred to a polypropylene centrifuge tube, and 5 µL of NH4OH was added.
The SPE procedure was performed on an SPE Vacuum Manifold with amorphous graphitised polymer carbon Supelco ENVI-Carb 250 mg/3 mL cartridges (P/N:57088, Sigma–Aldrich, St. Louis, MO, USA). The ENVI-Carb cartridges were conditioned with 3 mL of acetonitrile/water/acetic acid (20:80:1 v/v/v), followed by 3 mL of water/NH4OH (1000:1 v/v), being both solutions eluted to waste. From the acetic acid extracts/NH4OH solution, 400 µL were loaded onto the conditioned cartridges and were washed with 700 µL of water, both eluting to waste.
The toxins were then eluted with 2 mL of acetonitrile/water/acetic acid (20:80:1 v/v/v), into a polypropylene test tube. The eluate was transferred to a polypropylene autosampler vial and diluted with acetonitrile before analysis.
5.4.3 LC-HRMS analysis
The samples were analysed by Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS). Chromatographic separation was carried out using an UltiMate 3000 UHPLC system coupled to an Orbitrap Elite mass spectrometer (Thermo Scientific, Germany) equipped with a Heated EletroSpray Ionization source (HESI-II). The PST analogues were separated using an ACQUITY Premier BEH Amide (2.1×100 mm, 1.7 μm, Waters, USA) at 35 °C. Samples were held in the autosampler at 4 °C. The mobile phase was composed of water with 0.1% formic acid and 10 mM ammonium formate (A) and acetonitrile with 0.1% formic acid and 2% 10 mM ammonium formate solution (B). The gradient (in v/v %) started with 5% of B and increased linearly to 95% in 11 min. This composition was maintained for 1 min and then returned to 5% of B in 1 min and maintained at this composition for 2 min before the next run [37]. The flow rate was 0.3 mL min-1 and the injection volume was 10 µL. Data were acquired under positive (ESI+) and negative (ESI-) polarity using the following ionization parameters: spray voltage, 3.8 kV; sheath gas, 40 arbitrary units; auxiliary gas, 10 arbitrary units; heater temperature, 300 °C; capillary temperature, 325 °C; S-Lenses RF level, 69.10%. The LC-HRMS acquisition was performed under multiple-reaction monitoring (MRM) mode. A minimum of two MRM transitions for each PST analogue were selected based on MS/MS spectra collision-induced dissociation (CID) acquired by infusing the individual standards into the mass spectrometer. The collision energy used was 35 arbitrary units. Positive mode (ESI+) transitions were used exclusively for STX, NEO, dcSTX, and dcNEO quantitation. Negative mode (ESI-) transitions were used for the remaining analogues (GTX1to 6, dcGTX2 and 3, and C1 to 4) quantitation. The MRM transitions used are summarized in Table S3. Quantitation was performed by running calibration curves, with five levels of different concentrations of PST analogues standards mixture, in triplicate. The correlation coefficient (R2) was determined using a linear regression model. Toxicity equivalency factors (TEF) used were taken from those recommended by European Food Safety Authority – EFSA [38].
5.5. Data analysis
To confirm the influence of the temperature treatments on cell parameters and toxin production, generalized linear models (GLM, “glm” function) were fitted to the response data [39]. Treatments were set as a factor with three levels (i.e., Control, MHW I, and MHW IV), replicates as a factor with four levels, and MHW stage as a factor with three levels. A GLM from the Poisson family (log link) was fitted to cell counts, the binomial family (i.e., using the logit link) was used to fit chain formation data (0/1). Lastly, linear models (i.e., using the identity link) were fitted to cell length, photosynthesis, and toxin concentrations. Type II Wald chi-square tests (function “Anova”) were performed on the models, to evaluate the effect of the different treatments on the response variables. The model assumptions of normality, homoscedasticity of residuals, as well as independence between data points and influential observations were checked. Post-hoc comparisons (function “emmeans” in the “emmeans” package) were performed on the temperature treatment and MHW stage whenever an influence of these variables was detected by the Wald chi-squared test. Significance levels were set at p < 0.05, and p-values were adjusted through Tukey corrections, in order to avoid type I errors. Statistical analyses were carried out in RStudio, PBC (version 2021.09.1, R Core Team, 2022).