Surf smelt accelerate usage of endogenous energy reserves under climate change

remain nearshore, where pCO2 concentrations in northeast Pacific Ocean ecosystems can be extraordinarily high, and beyond those even predicted for global averages at the end of this century [22]. As such, surf smelt larvae under OA may consume yolk and the oil globule at high and, if an interaction between temperature and pCO2 exists, at exacerbated rates. The purpose of this study was to investigate the combined effects of elevated seawater temperature and OA on the energy demands of surf smelt embryos and larvae. This was accomplished through measuring yolk exhaustion and heart rates in embryos, and yolk and oil globule exhaustion in surf smelt larvae, under three temperatures and two pCO2 condi- tions. We hypothesized that energy demand, measured as embryo heart rate and yolk sac/oil globule exhaustion, for both embryos and larvae would increase under elevated seawater temperature and pCO2 (i.e. acidification) as isolated stressors. We also predicted that the highest energy usage would be observed under simultaneous increased temperature and ele- vated pCO2 due to the additive effect of these climate stressors on compensatory homeostatic processes. Methodology Experimental set-up All experimental seawater was collected from the Salish Sea at the Shannon Point Marine Cen- ter (SPMC) in Washington State, U.S.A. This water was dispensed into a header tank that grav- ity fed seawater into six mixing basins. In each mixing basin, submersible powerhead pumps (Marineland1 Maxi-jet 900) circulated water using magnetically driven impellers. Three mix-

ing basins continuously received a slow feed of pure CO2 gas from an 8 channel Masterflex1 L/S Digital Drive peristaltic pump attached to a 20-lb food grade CO2 gas cylinder. This flow of pure CO2 (19 mL/min), tuned to achieve the desired pCO2 given the flux of water into the tanks, was fed into the intakes of the powerhead pumps and completely dissolved while passing through the impeller chamber and associated turbulence [25]. This method of CO2 amend- ment is suitable for creating large volumes of water with elevated pCO2, as it does not depend on slower gas exchange processes. These pure CO2 additions generated two pCO2 treatments, ambient and elevated. Each mixing basin delivered water to three treatment basins held at the different temperatures (ambient seawater ~13˚C, 15˚C, and 18˚C). The tanks were heated using 100W Aqueon1 submersible heaters attached to Inkbird1 temperature regulators. The temperature regulators engaged the heaters anytime the tempera- ture deviated below 0.3˚C of the set temperature. Treatment temperatures were chosen to rep- resent ambient seawater temperatures during the summer surf smelt spawning period in the Salish Sea (13˚C), predicted seawater temperatures in the Salish Sea by the year 2040 (15˚C; [16]), and the current average sediment temperature (18˚C) observed at Salish Sea surf smelt spawning beaches that have been modified by human activity [7]. The two pCO2 conditions generated in the mixing tanks were modified slightly in the treatment basins as a result of tem-

perature driven differences in solubility and in pCO2, resulting in a range of OA scenarios. pCO2 across temperature treatments in ambient basins was about 750–1000 μ atm and about 1700–2200 μatm in elevated basins, with a pHNBS range of approximately 7.42–7.88. No CO2 additions were made to the ambient seawater treatments, which were representative of the ambient seawater at the SPMC. CO2 additions for the elevated treatment were chosen to result in pCO2 similar to those currently observed in the Salish Sea during naturally occurring CO2- enrichment [22], and elevated future levels that may result when global anthropogenic OA combines with natural regional acidification events. Treatment basins were covered with 5/8” acrylic sheets to limit gas exchange during experiments. PLOS ONE Surf smelt response to climate co-stressors PLOS ONE | https://doi.org/10.1371/journal.pone.0270491 June 27, 2022 3 / 14