Surf smelt accelerate usage of endogenous energy reserves under climate change

Collection Surf smelt eggs containing embryos and their attached sediment were collected from Fidalgo Bay, a shallow embayment located in the Salish Sea’s northern Puget Sound in Washington State, U.S.A (48.4833816,-122.5868376). Embryos were transported to the SPMC where they were examined via stereomicroscopy to determine age. Experiments began if the embryos were determined to be < 24 hours old. Age was determined using the guidelines of Moulton and Penttila [26]. After aging, embryos were dispensed into experimental treatment basins. Approximately 50 embryos were added to each of 72, 200-mL glass bowls. These bowls were divided evenly into 18 treatment basins (4 bowls per basin). Our experimental design resulted in embryos being continuously bathed by seawater and does not replicate the sporadic inunda- tion surf smelt embryos experience in nature. Nonetheless, our embryonic results are informa- tive of the physiological responses of surf smelt embryos in response to abiotic stress and can be used to inform other studies. If the collected embryos were near hatch, then a larval experiment was started. For these experiments, the embryos and attached sediment were placed into 12-L bins where they were gently oscillated for 1-minute increments to simulate wave disturbance and initiate hatching. Hatched larvae were collected, and the bins were oscillated again until no additional hatch was observed. Approximately 15 freshly hatched larvae were distributed into each of 72, 200-mL glass bowls which were divided evenly into 18 treatment basins (4 bowls per basin).

Water chemistry measurements Temperature measurements from each basin were taken every 30 minutes by Onset HOBO1 data loggers to verify that temperature remained consistent throughout the experiments. Daily pH measurements from treatment water within basins were taken using a hand-held Orion Star A329 pH conductivity meter, calibrated with NBS-buffers prior to use each day. Spectro- photometric pH measurements were also taken, but the data were compromised and could not be used. While it is clear that pH measured with a glass electrode is not preferred, an exten- sive probe specific cross-comparison with pH values on the total scale derived from spectro- photometric pH methods (pHT) was carried out, yielding a strong linear relationship relating pHNBS to pHT (r2 0.997). Seawater samples stored in 20-mL plastic scintillation vials were also collected three times per week throughout the experiment for CT analyses. Water samples for CT analysis were poisoned with 20 μL mercuric chloride (HgCl2) to arrest microbial metabo- lism and stored for later analysis. Samples were analyzed for CT using an Apollo SciTech AS-C3. Sample salinity was measured using a refractometer, and salinity values and tempera- ture were used to convert CT measurements from μmol/L to μmol/kg. Measurements of CT were calibrated against a standard curve created from reference material (CRM, Batch 179, Dickson, Scripps Institute of Oceanography). CO2SYS [27] was used to calculate all remaining components of the carbonate system including pCO2, using K1 and K2 equilibrium constants

refit by Millero et al. [28] and the sulfate dissociation constant by Dickson [29]. Energy measurements Surf smelt embryo yolk usage was measured daily over a 13-day experiment, with the excep- tion of the ambient temperature treatment (~13˚C). This treatment ended after 10 days due to natural warming of the ambient seawater which made it redundant with the 15˚C treatment. Each day, 3 embryos from each of the bowls within each treatment basin were haphazardly selected and placed into 6-well plates labeled with its corresponding treatments. Embryos were photographed using a Leica M125 stereoscope attached to a Leica MC170 camera networked to Leica Suite software. After being photographed, embryos were removed from the PLOS ONE Surf smelt response to climate co-stressors PLOS ONE | https://doi.org/10.1371/journal.pone.0270491 June 27, 2022 4 / 14