Oxygen consumption measurements were undertaken to estimate photosynthetic rates. Oxygen saturation (O2 mg/liter) was measured using an SDR SensorDish Reader (PreSens, Regensburg, Germany) with light-emitting diode lights for noninvasive optical fluorescence oxygen sensing and a sealed 24-chamber glass microplate with 0.2-ml wells (Loligo Systems, Copenhagen, Denmark). Individuals of B. solaris were starved in sterile seawater and incubated at 16°C on a 14-hour light/10-hour dark cycle for 7 days and acclimatized to room temperature for 3 hours before the experiments.

Three wells each contained 25 worms in sterile seawater medium. The number of replicates was limited by the number of specimens available, as B. solaris is not culturable and specimens need to be collected in the field. A positive control consisted of three replicate wells containing a culture of the marine chlorophyte T. tetrathele (~500,000 cells/ml). Three replicate wells with sterile seawater were used as a negative control. Remaining wells contained deionized water.

Wells were overfilled, checked for air bubbles, and then sealed with transparent PCR film (Thermo Fisher Scientific, Waltham, MA, USA). The microplate was placed on the SDR reader and incubated in the dark for a 10-min acclimatization period before illumination. A 90-min light treatment in light-saturated conditions (light intensity, 120 μmol quanta m−2 s−1) was provided by a NanoTech T5 Reflector light (SunBlaster, Langley, Canada), followed by a 90-min dark treatment in which an opaque black box was placed over the wells. Oxygen measurements were made every 15 s during the treatments. The experiment was conducted at room temperature, with the microplate placed in a water bath to maintain constant temperature. Specimens were observed at the end of the experiment to check whether they were still alive.

For analysis, 20-min optimal measurement intervals were chosen from each 90-min measurement period based on having the least temperature fluctuation (with temperature ranges of 0.05°C and 0.04°C for light and dark treatments, respectively) to eliminate any confounding effect of temperature on oxygen saturation. The rate of change of oxygen saturation (O2 mg/liter per hour) over the treatment intervals was calculated for each replicate using a linear regression. The average of the negative control slopes was subtracted from the experimental wells to correct for background microbial respiration. Gross photosynthesis for each replicate was estimated as gross photosynthetic rate = net photosynthesis (oxygen evolution) + respiration. Mean and SE were calculated and visualized on a bar plot. All data analyses were conducted in RStudio v1.0.143 (RStudio Inc., Boston, MA, USA).

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