Surf smelt accelerate usage of endogenous energy reserves under climate change.

Surf smelt (Hypomesus pretiosus) are ecologically critical forage fish in the North Pacific ecosystem. As obligate beach spawners, surf smelt embryos are exposed to wide-ranging marine and terrestrial environmental conditions. Despite this fact, very few studies have assessed surf smelt tolerance to climate stressors. The purpose of this study was to examine the interactive effects of climate co-stressors ocean warming and acidification on the energy demands of embryonic and larval surf smelt. Surf smelt embryos and larvae were collected from spawning beaches and placed into treatment basins under three temperature treatments (13°C, 15°C, and 18°C) and two pCO2 treatments (i.e. ocean acidification) of approximately 900 and 1900 μatm. Increased temperature significantly decreased yolk size in surf smelt embryos and larvae. Embryo yolk sacs in high temperature treatments were on average 7.3% smaller than embryo yolk sacs from ambient temperature water. Larval yolk and oil globules mirrored this trend. Larval yolk sacs in the high temperature treatment were 45.8% smaller and oil globules 31.9% smaller compared to larvae in ambient temperature. There was also a significant positive effect of acidification on embryo yolk size, indicating embryos used less maternally-provisioned energy under acidification scenarios. There was no significant effect of either temperature or acidification on embryo heartrates. These results indicate that near-future climate change scenarios may impact the energy demands of developing surf smelt, leading to potential effects on surf smelt fitness and contributing to variability in adult recruitment.

Introduction

Surf smelt (Hypomesus pretiosus; Girard 1854 [1]) are small, schooling forage fish that provide a critical link in the marine food web by facilitating energy transfer from primary producers to higher trophic levels, including numerous species of sea birds, marine mammals, and other commercially important fish [2-5]. Surf smelt range extends from Prince William Sound, Alaska, to southern California [6], and they interact closely with the nearshore environment, especially during spawning events and juvenile development. Like other forage fish (e.g. herring, sand lance), surf smelt require unaltered shoreline to thrive [7, 8], making them indicators of overall marine system health. Any reductions to their population abundance can have cascading effects on marine ecosystem productivity and functioning [4, 5]. Despite their ecological importance, they are an understudied forage fish in the North Pacific ecosystem.

Surf smelt spawn on gravel beaches during high tides, utilizing the upper third of a beach’s tidal range, typically at tidal heights of seven feet or above [4, 7, 9]. Female surf smelt deposit eggs which adhere to gravel substrate [4, 8]. Once deposited and fertilized, eggs and their developing embryos are subjected to daily exposure in both air and seawater throughout their maturation. Consequently, embryos experience a dynamic range of environmental conditions over diel cycles [8, 10]. During this critical time, surf smelt embryos develop by utilizing maternally provided (endogenous) energy in the form of yolk and lipid droplets for approximately 14 to 21 days depending on ambient temperature and sediment humidity [4, 10]. When embryos are fully developed, hatching is triggered by physical disturbance, primarily resulting from tidal activity and wave action [4, 7, 10]. Once hatched, the planktonic surf smelt larvae continue to utilize yolk reserves and lipids contained within an oil globule for energy and development [4] until exogenous feeding begins. Any accelerated usage of stored energy in forage fish could negatively affect performance and survival, and ultimately, adult recruitment [11].

One mechanism that may force accelerated usage of endogenous energy reserves in surf smelt embryos and larvae is energy demand for compensatory processes like maintaining physiological homeostasis under temperature stress [12, 13]. For an obligate beach spawner like surf smelt, this is reason for concern. Shoreline development is increasingly removing nearshore vegetation and the shading it provides for developing surf smelt embryos, causing a significant increase in beach gravel temperature and concurrent decreases in gravel humidity [7]. A survey of modified and natural beaches in the Salish Sea found that temperatures on modified beaches were, on average, 4.7°C higher than temperatures of natural beaches [7]. Research on the effects of temperature on surf smelt have predominantly focused on the effects of air temperature and desiccation stress on mortality and hatching in the field [7, 10]. For example, increased air temperature was linked to increased developmental rates in surf smelt embryos [7], indirectly suggesting increased yolk utilization.

The effects of temperature on larval fish fitness are often amplified under coincident environmental stressors [e.g. 14–16]. One potential concomitant stressor is ocean acidification (OA). OA is a suite of chemical changes in marine carbonate parameters (e.g. pH, pCO2, [CO32]) that are driven on a global scale by the rise in total seawater carbon (CT) through dissolution of atmospheric CO2 into seawater. Exposure of embryonic and larval fish to both temperature stress and OA conditions may drive reallocation of energy reserves. OA is shown to have wide-ranging effects on marine organisms across multiple taxonomic groups [17], including on related species of forage fish [18-20]. Temperate cold-water ecosystems like the northeast Pacific Ocean are vulnerable to OA due to the ability of cold water to absorb atmospheric gasses more efficiently than warm water [21]. Additionally, seasonal upwelling and strong tidal mixing introduce deep waters enriched in CO2 into surface waters [22, 23], acting to exacerbate global OA processes and signals. In the Salish Sea’s urbanized Puget Sound estuary, where surf smelt spawning is widespread [8], observed minimum pH in surface waters varied between 7.4 and 7.7 [22], indicating pCO2 values on the order of 1000–2000 μatm. Global ocean pH values are predicted to reach about 7.7 at the end of the century under a business as usual scenario (RCP8.5) [24]. Due to the intertidal spawning behavior of surf smelt, which in some locations across the northeast Pacific Ocean occurs year-round [4], embryos are not continuously submerged during embryogenesis. Thus, the high daily and seasonal variability of pCO2 and pH exposure the embryos experience may select for embryo phenotypes that are robust to variable and at times high pCO2. However, the pelagic lecithotrophic larvae 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 conditions. 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 elevated 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 Center (SPMC) in Washington State, U.S.A. This water was dispensed into a header tank that gravity fed seawater into six mixing basins. In each mixing basin, submersible powerhead pumps (Marineland® Maxi-jet 900) circulated water using magnetically driven impellers. Three mixing basins continuously received a slow feed of pure CO2 gas from an 8 channel Masterflex® 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 amendment 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 Aqueon® submersible heaters attached to Inkbird® temperature regulators. The temperature regulators engaged the heaters anytime the temperature deviated below 0.3°C of the set temperature. Treatment temperatures were chosen to represent 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 temperature 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.

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 inundation surf smelt embryos experience in nature. Nonetheless, our embryonic results are informative 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 HOBO® 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. Spectrophotometric 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 extensive probe specific cross-comparison with pH values on the total scale derived from spectrophotometric 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 metabolism 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 temperature 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 exception 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 experiment to avoid repeated measurements. Photos were later analyzed using the software ImageJ [30] to determine the egg and total yolk area. These measurements were used to calculate the ratio of yolk area to total egg area. This ratio was used to account for initial differences in egg size.

Surf smelt embryo heart rate measurements were taken as another measure of energy demand. Due to heart rate being affected by fish developmental stage [31], heart rate measurements were taken within treatments at a specific, identifiable developmental stage: when the eye spots of the developing embryos darken [26]. When embryos were removed from glass bowls to be photographed for yolk measurements, if the eye spots had darkened since the previous day, the embryos were also video recorded for 10 seconds. Heart beats were later counted in each video.

Energy consumption of surf smelt larvae was assessed through a 3-day experiment, during which 2 larvae were haphazardly selected each day from each bowl within each treatment basin. Larvae were anesthetized using tricaine following the methods of Massee et al. [32]. This method limits larval movement and allows still images of the larvae to be taken. Leica software and a stereomicroscope were used to photograph the anesthetized larvae. After being photographed, larvae were removed from the experiment to avoid repeated measurements. Photographs were later analyzed using ImageJ [30] to determine yolk area and oil globule area.

Statistical analysis

Larval yolk sac, oil globule size, and embryo heart rates were analyzed using linear mixed effect models with day, pCO2, temperature, and their interactions as fixed factors, and experimental basin as a random factor. The ratio of yolk size to embryo size was analyzed using a generalized linear mixed effect model (family = binomial, link = logistic) with day, pCO2, temperature, and their interactions as fixed factors, and experimental basin as a random factor. For each response variable measured, models were built with and without the interaction terms based on a combination of p-value significance (α = 0.05) and AIC comparisons to choose the most appropriate model. Interaction terms that were not significant and did not improve the model fit were left out. Random slope or intercept terms were applied to experimental basin depending on model fit determined by AIC comparisons. Data from elevated and ambient pCO2 treatments are configured categorically in figures for clarity, but pCO2 was treated as a continuous variable in statistical models. All statistics were completed using R [33].

Ethics approval

All experimental procedures were carried out in accordance with the recommendations and approval of the Animal Care and Use Committee at Western Washington University (protocol approval 19–001), and embryos were collected in collaboration with the Washington State Department of Fish and Wildlife.

Results

Treatment conditions

Direct injection of pure CO2 into the elevated pCO2 mixing tanks followed by equilibration of both ambient and elevated pCO2 treatments to three temperatures resulted in two distinct pCO2/pH conditions, modified somewhat by temperature (Table 1 embryo and larval yolk and oil globule experiments) (S1 Table heartbeat experiment). The average pH variability was similar between ambient and high pCO2 treatments at comparable temperatures (± 0.03 pH units) (Table 1). The CO2 amendment in the elevated pCO2 treatment resulted in an average increase in 42 μmol kg/SW relative to the ambient treatment (Table 1). Because pCO2 is derived from temperature, pH, and CT, the variability in all three measurements results in relatively high variability in pCO2 estimates. The ambient pCO2 tanks had an average standard deviation of ± 50.58 μatm pCO2, while the elevated pCO2 tanks had considerably higher variability, with an average standard deviation of ± 275.89 μatm (Table 1). Differences in mean pCO2 between temperature levels within pCO2 treatments and were generally less than 150 μatm, considerably smaller than the approximately 800 μatm difference between the ambient and elevated pCO2 treatments (Table 1). Therefore, data are presented in the elevated pCO2 and ambient pCO2 treatments in figures for embryo and larval characteristics. Given the need to use electrode pH measurements, we acknowledge the limitation of these estimates of pCO2.

Table 1

Treatment conditions.

		In-Situ Measurements		Discrete Samples
Experiment	Conditions	pH	Temperature	pCO2	C T	Salinity
	(pCO2 +°C)	(NBS Scale)	(°C)	(μatm)	(μmol kg*SW-1)
Embryo Yolk	ambient +13	7.88 ± 0.01 (36)	13.77 ± 0.24 (36)	793. 85 ± 28.59 (15)	2015.06 ± 10.44 (15)	30.8 ± 0.6 (15)
	ambient +15	7.87 ± 0.03 (42)	14.89 ± 0.03 (42)	749.37 ± 141.69 (15)	2013.21 ± 16.53 (15)	30.7 ± 0.5 (15)
	ambient +18	7.83 ± 0.02 (39)	17.94 ± 0.23 (39)	872.25 ± 133.73 (15)	2010.92 ± 14.86 (15)	30.6 ± 0.6 (15)
	elevated+13	7.56 ± 0.09 (39)	13.44 ± 0.24 (39)	1695.41 ± 337.05 (15)	2049.61 ± 11.56 (15)	31.1 ± 0.9 (15)
	elevated+15	7.56 ± 0.06 (39)	15.02 ± 0.03 (39)	1617.05 ± 324.02 (15)	2050.43 ± 15.9 (15)	30.9 ± 0.9 (15)
	elevated+18	7.53± 0.03 (39)	17.93 ± 0.23 (39)	1695.41 ± 368.98 (15)	2047.64 ± 17.34 (15)	30.6 ± 0.6 (15)
Larvae Yolk and Oil Globule	ambient +13	7.85 ± 0.01 (9)	13.22 ± 0.35 (9)	828.75 ± 25.25 (6)	2032.91 ± 11.06 (6)	31.0 ± 0.6 (6)
	ambient +14	7.83 ± 0.01 (9)	14.15 ± 0.08 (9)	888.25 ± 25.49 (6)	2028.65 ± 12.21 (6)	30.8 ± 0.4 (6)
	ambient +18	7.79 ± 0.01 (9)	17.42 ± 0.15 (9)	992.22 ± 39.38 (6)	2022.67 ± 14.04 (6)	30.6 ± 0.6 (6)
	elevated+13	7.54 ± 0.07 (9)	13.43 ± 0.43 (9)	1759.74 ± 316.37 (6)	2067.31 ± 16.84 (6)	31.2 ± 0.7 (6)
	elevated+14	7.51 ± 0.06 (9)	14.15 ± 0.23 (9)	1787.97 ± 247.35 (6)	2077.95 ± 17.39 (6)	30.9 ± 0.6 (6)
	elevated+18	7.51 ± 0.06 (9)	17.32 ± 0.11 (9)	1907.61 ± 309.72 (6)	2065.77 ± 16.79 (6)	31.0 ± 0.6 (6)

Average seawater temperature, pH, pCO2, and CT for two experiments. Data for the embryo heartrate experiment are presented in S1 Table. Data are shown as time-averaged means ± 1 SD of (n) measurements. pH and temperature were measured daily, while CT was measured 3 times per week. pCO2 was derived from temperature, pH, and CT measurements.

Embryo yolk usage and heartrate

The proportion of embryo yolk size relative to total egg size (Y:E) decreased throughout the duration of the experiment in all treatments with day having a significant negative effect on Y:E (Table 2; Fig 1). Temperature also had a significant, negative effect on Y:E throughout the 13-day experiment (Table 2; Fig 1). The average Y:E decreased by 21.6% after 13 days of incubation at 13°C compared to a 28.2% decrease at 18°C. This equates to Y:E being 7.3% smaller at 18°C compared to 13°C at the final time point. There was also a significant, positive effect of elevated pCO2 on Y:E (Table 2). Embryo heart rates were not significantly affected by temperature or pCO2 stressors (Table 3). Surf smelt embryo heart rates in ambient temperature treatments were similar between pCO2 treatments. As temperature increased, the average heartrate increased between 13°C and 18°C at elevated pCO2 and a decreased at ambient pCO2 but variability was high, and these patterns were not significant (S1 Fig).

Table 2

Generalized linear model results for Y:E.

	Value	Std. Error	z-value	p-value
Intercept	6.5614	0.5062	12.960	<2.00e-16
Day	-0.49	0.0181	-27.927	< 2.00e-16
pCO2	0.4460	0.1024	4.356	1.32e-05
Temperature	-0.2321	0.0303	-7.654	1.94e-14

Summary of results of a generalized linear mixed effect model (family = binomial, link = logistic) examining the effect of day, temperature, and pCO2 on embryo yolk sac relative to total egg size.

Fig 1

Embryo yolk usage.

Proportion of yolk area to total egg area (Y:E) of surf smelt embryos (n = 216/day) that were submerged in treatment water for 13 days. Each point is an average measurement representative of the temperature treatment, pCO2 treatment (points offset for visualization), and day with standard error shown as whiskers. Day, pCO2 and Temperature were significant factors predicting embryo yolk usage.

Table 3

Linear mixed effect model results.

		Value	Std. Error	df	t-value	p-value
Embryo heart rate	Intercept	11.8181	0.9540	65	12.3879	<0.0000
	Temperature	-0.0252	0.0230	26	-1.0925	0.2846
	pCO 2	0.0008	0.0005	65	1.8712	0.0657
Larval yolk sac size	Intercept	0.3389	0.0270	383	12.5504	<0.0000
	Day	-0.0283	0.0019	383	-14.3675	<0.0000
	pCO 2	0.000004	<0.0000	16	0.6234	0.5418
	Temperature	-0.0111	0.0017	383	-6.4106	<0.0000
Larval oil globule size	Intercept	0.0823	0.0071	383	11.5417	<0.0000
	Day	-0.0075	0.0007	383	-10.8844	<0.0000
	pCO 2	-0.000001	<0.0000	16	-0.8062	0.4319
	Temperature	-0.0018	0.0005	383	-4.1379	<0.0000

Summary of linear mixed effect models examining the effect of day, temperature, and pCO2 treatment level on multiple parameter measurements.

Larvae yolk and oil globule usage

Larval yolk size decreased over time in all treatments with day having a significant negative effect on larval yolk size (Table 3; Fig 2). Temperature also had a significant, negative effect on larval yolk size throughout the duration of the experiment (Table 3; Fig 2) and for every 1°C increase in temperature, the average larval yolk size decreased by 0.011 mm2 (Table 2). The average size of larval yolk sacs decreased by 27.1% at 13°C and 49.8% at 18°C over the duration of the experiment. Larval yolk size was not significantly affected by pCO2 (Table 3; Fig 2).

Fig 2

Larvae yolk usage.

Yolk sac area of surf smelt larvae (n = 144/ day) that were submerged in treatment water for 3 days. Data are averages representative of the temperature treatment, pCO2 treatment (points offset for visualization), and day with standard error shown as whiskers. Day, and Temperature were significant factors predicting larvae yolk usage.

Surf smelt oil globule size decreased over time in all treatments with day having a significant negative effect on oil globule size (Table 3; Fig 3). Temperature had a significant negative effect on the size of larval oil globules and for every 1°C increase in temperature, the average oil globule area decreased by 0.0018 mm2 (Table 3; Fig 3). Over the three-day incubation, oil globule size decreased by 25.7% at 13°C compared to 43.2% at 18°C. On average, oil globules at 18°C were 31.9% smaller than oil globules at 13°C on the final day of data collection (Fig 3). pCO2 had no significant effect on oil globule size (Table 3).

Fig 3

Larvae oil globule usage.

Oil globule area of surf smelt larvae (n = 144/day) that were submerged in treatment water for 3 days. Each point is an average measurement representative of the temperature treatment, pCO2 treatment (points offset for visualization), and day with standard error shown as whiskers. Day, and Temperature were significant factors predicting larvae oil globule usage.

Discussion

Increased energy usage due to elevated metabolism across fish life histories can result in trade-offs in growth, performance, and reproduction [34-36]. We found that elevated temperature was a primary abiotic driver affecting early life history stages of surf smelt, accelerating yolk usage in both surf smelt embryos and larvae, and oil globule usage in larvae. Our results showing increased usage of endogenous energy in response to increasing temperatures agree with findings from numerous other fish species [e.g. 37–43], including the forage fish Engraulis japonica [38], Clupea pallasi [40] and Clupea harengus [41], Brevoortia tyrannus [42], and Sardinops sagax [43]. However, our results also suggest that surf smelt may be more tolerant to temperature variance than other fish species. Heming and Buddington [37] summarized the findings of 23 species of fish and showed that Q10 values for yolk absorption in fish over a temperature span of 1–30°C averaged 2.9. In this study we observed Q10 values of 1.58 and 2.09 for surf smelt embryos and larvae, respectively. In the broadcast spawning Pacific sardine, Lasker [42] found a Q10 of 4 for yolk absorption between environmental temperature range of 15 to 21°C. The fact that our observed Q10 values are slightly below the averages summarized in Heming and Buddington [37] and well-below those reported in Lasker [43] suggests that surf smelt, while not immune to temperature effects, are less sensitive to temperature stress compared to other fish species. More robust tolerance to elevated temperature compared to other fish species may be an adaptation to withstand the wide-ranging temperatures surf smelt embryos experience during their high intertidal incubation, which can last between 10 and 21 days [4].

Elevated temperature was associated with a significant decrease in oil globule size in addition to decreased larval yolk area in this study, indicating ongoing taxation on endogenous energy in the larval stage. While fish embryos use free amino acids found in their yolk as their primary energy source prior to hatching, they switch to using fatty acids from their oil globule post hatching [44]. Ehrlich and Muszynski [45] proposed that in addition to being a source of energy for metabolism, the lipids that make up the oil globules in surf smelt likely aid in larval buoyancy. This was supported by findings in Blaxter and Ehrlich [46], who observed that Atlantic herring and European plaice were oriented head down in the water column post yolk absorption. These larvae were unable to feed even when offered food due to their heads sinking when attempting to follow prey. The rapid use of the oil globule in surf smelt at higher temperatures could result in compromised fitness when larvae enter the plankton, and buoyancy effects likely reduce exogenous energy acquisition through decreased swimming performance and prey capture efficiency. Swimming is arguably the largest sink for energy in fish larva [46], and an essential behavior for acquiring prey and for locating abiotic conditions that maximize fitness and survival. Thus, any environmental factor that accelerates the use of this endogenous energy source prior to the onset of feeding may affect larval survival and, ultimately, adult recruitment.

In the Salish Sea’s Puget Sound, elevated temperatures can be driven by human activities. Regionally, approximately one third of the coastline has been modified by humans [47]. Human activities and development alter surf smelt spawning grounds by reducing nearshore vegetation and the shading it provides, causing a significant increase in beach gravel temperature and concurrent decreases in gravel humidity [7]. A survey of modified and natural beaches in the Puget Sound found that modified beaches had an average substrate temperature of 18.8°C, which was 4.7°C higher than the average temperature of natural beaches, and a temperature known to cause increased surf smelt embryo mortality [7]. The observed temperature increase caused by shoreline modification was represented by the high temperature treatment (18°C) in this study. It is likely that contemporaneous shoreline modifications and subsequent temperature effects are realigning surf smelt energy budgets and, potentially, the timing of hatching [7, 10]. Ongoing shoreline development coupled with the large proportion of already developed beaches in the Salish Sea poses an immediate threat as surf smelt rely on a relatively small proportion of beaches for spawning each year, and disturbance to any of them could have population-level implications [8]. Additionally, climate models predict that Salish Sea seawater temperatures will increase by approximately 1.2°C by the year 2040 [48]. This temperature increase was captured in this study as the 15°C treatment and was found to reduce yolk size by 22% compared to ambient seawater. Thus, findings here suggest that global climate change will accelerate surf smelt endogenous energy usage in the northeast Pacific Ocean.

We observed little direct effects of elevated pCO2 on early life history stages of surf smelt, which is in agreement with recent findings on another Salish Sea forage fish, Clupea pallasii [40]. Embryo heartrate, larval yolk size, and oil globule size were not directly affected by variation in pCO2, despite the very high concentrations used in this study. Only surf smelt embryos were directly affected by elevated pCO2, with a significant positive effect on Y:E. A review of the effects of OA on marine fish metabolic rates found that 21% of studies reported a consistent reduction in metabolic rate in response to OA [49]. The positive effect of elevated pCO2 on surf smelt embryos and the absence of a pCO2 treatment effect on the surf smelt larval metrics may reflect the unique spawning behavior and habitat use of surf smelt, and an overall robustness to OA sensitivity. In comparison to open-ocean species, marine fish that spawn in the intertidal or near-shore habitat possess broad tolerance to environmental variability due to the high frequency and magnitude of environmental change [7, 40, 50, 51]. With respect to habitat-dependent CO2 sensitivity, Baumann [52] called this the Ocean Variability Hypothesis. The surf smelt embryos used here were collected from a shallow embayment that experiences highly variable and ephemeral pH, ranging from ≤ 6.5 to > 8.5, with the majority of observations between 7.15 to 8.45 [53]. Thus, the lack of a strong OA effect on surf smelt, even on early life histories, can be expected.

While this study shows that the utilization of endogenous energy reserves in early life-history stages of surf smelt are affected by elevated temperature and, to a lesser degree, pCO2, it remains unknown whether these effects will be detrimental to later life history stages, or in annual population abundance and recruitment. Despite this limitation, this study highlights the importance of including surf smelt in ecological and climate change research given the evidence provided here that surf smelt early life stages are affected by stressors associated with near-present climate change and under conditions associated with current nearshore urban development. Given their keystone role in marine ecosystems, reductions to forage fish fitness and population abundance have the potential to cause ecosystem-wide effects. For example, ecosystem models show that Puget Sound seabird populations are negatively affected by reduction in forage fish biomass [5, 54] and piscivorous fish and marine mammals may share similar vulnerability to changes in forage fish abundance [4, 5]. Finally, surf smelt remain one of the truly understudied forage fish in the north Pacific ecosystem, and further investigation of their potential role in ecosystem response and resilience to global change is warranted.

Experimental conditions–embryo heartrate experiment.

Average seawater temperature, pH, pCO2, and CT for the embryo heartrate experiment. Data are shown as time-averaged means ± 1 SD of (n) measurements. pH and temperature were measured daily, while CT was measured 3 times per week. pCO2 was derived from temperature, pH, and CT measurements.

(PDF)

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Embryo heartrate.

The number of heart beats from embryos (n = 96) per ten seconds from each treatment. Whiskers extend from the upper and lower quartiles to 1.5 times the interquartile range. Data outside of this range are shown as points. Neither temperature nor pCO2 were significant predictors of heartrate.

(TIF)

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8 Nov 2021

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Reviewer #1: Review for “Surf smelt accelerate usage of endogenous energy reserves under climate change”

This is an interesting manuscript in which the short-term effects of high temperature and high pCO2 exposure on energy reserves of embryos and larvae of surf smelt are described. I have several questions regarding the experimental design and a few remarks for the results section. The discussion is much too long and often discusses topics that are only distantly related to what was actually measured in this study. The authors describe what other studies found but often miss to connect it to their own findings.

Abstract

Line 12-14: One can also argue that because surf smelt are beach spawners, they are exceptionally robust to environmental stress

Line 18:19: Why are you specific with the temperature used but not the pCO2 level? At least give an approximation what “ambient” and what “elevated” means.

Line 26: “CT” has not been explained before use

Line 27: “may impact”. It is more informative if you give a direction. Is it negative or positive impact?

Introduction

Line 107-109: This sentence is repetitive to the previous sentence

Methodology

Line 130: Refer to Table 1.

Line 135-136: Figure 1 is not needed. If you want to show it somewhere put it in the supplementary material

Line 142: Were the embryos shock exposed to the treatment conditions or was there a gradual increase/decrease in water temperature and pCO2?

Line 145-150: Why did you not use the embryos from your embryo experiment to be further studied as larvae? There might be cascading effects from embryo to larva that you missed because of your experimental design which I see as a drawback for your results

Line 172-174: I don’t understand what is meant. Were the temperature treatments not run at the same time?

Line 185-188: What was done to maintain treatment conditions during measurements of heart rates? How long after removal of the embryos from their tanks did it take to start recording the heart rate videos?

Results

Line 206-227: I find this a very weird way of showing the treatment conditions. It gives the impression that you did not exactly know what the treatment conditions were. E.g. line 208: “temperatures remained consistent throughout the duration of the experiment”. If your experiment was planned to use stable temperatures, this is a given. I consider this unnecessary to take up so much space in your manuscript. Referring to Table 1 in your methods section would simply be enough.

Line 211-214: Why is the treatment then called 15C instead of 16C if the water temperature was 16C and not 15C?

Line 213: “summer warming elevated the seawater temperature in the ambient temperature treatment” by how much? For how long?

Line 235-237: Figure 2 does not show this result. I suggest to use a glmer to account for the ratio which cannot be above 1 or below 0. Also I would prefer showing the error as 95% intervals and offsetting the CT data for clarity of the error bars. Maybe this would then visually support the result that pCO2 had an effect. Also you describe that data from the 13C treatment are missing in line 173 (which I commented on before) but why is there a data point for day 13 for the ambient pCO2 treatment but not for the elevated pCO2 treatment?

Line 254: In Figure 4 it looks like larvae used for the 18C treatment started off with smaller yolk areas already. How can this be and was the difference in yolk area size at the start accounted for when comparing the yolk area at the final day?

Discussion

Line 277-285: Can you draw some conclusion from all these Q10 values? The Q10 that you calculated is smaller than the average 2.9 calculated by Heming and Buddington. If you are using this reference, it indicates that surf smelt are less temperature dependent than most fish species studied (in case this is the point which you want to make by referring to Heming and Buddington). Why are you stating this reference? What is the point that you want to make with it? This is especially true for the last sentence in this paragraph (line 283-285).

Line 286-292: Similar to the previous paragraph. Which point do you want to make with the review of Villalobos et al 2020? We do not know the temperature performance curve or at least the temperatures at which C. pallasi occurs in nature.

Line 310 to 322: Although there is nothing wrong with this paragraph I question why it is included. You mention that you did not measure developmental rate and you don’t know if a faster development due to higher temperature is present in surf smelt, so why do you then include this paragraph in your manuscript?

Line 337: Which temperatures are considered “temperature stress” in surf smelt larvae?

Line 338-342: What do these sentences add to your story?

Line 354-363: Again, why is this paragraph included? This paragraph is about acid-base regulation which was not part of what you have measured. You are discussing topics that you did not measure in your study.

Line 370-374: Did your embryos and larvae come from adults that were exposed to high pCO2? Relate the studies that you refer to to your own study.

Line 383-388: Also have a look at Leo et al 2018. They used herring from Norway and found higher susceptibility of embryos and larvae to high pCO2 than Sswat and Franke. But also here, the point that you want to make by referring to these herring studies is not really clear.

Line 388-390: I don’t think that you can draw this conclusion by studying surf smelt for only 3 days.

Line 415-424: While this all is correct I think it is too far off from what you did in your study and does not need to be included here.

Line 425-465: These three paragraphs are great and if they are filled out with your main points from your results they would make a great discussion making most of the previous discussion (line 264-424) redundant

Reviewer #2: General comments:

The manuscript (PONE-D-21-27467) titled: “Surf smelt accelerate usage of endogenous energy reserves under climate change” aimed to determine if energy reserves of surf smelt embryos and larvae are affected by single and interactive effect of ocean warming and ocean acidification. To address this aim, before reaching 24 hours old, surf smelt were acclimated to a combination of three temperatures 13, 15, and 18°C and two treatment of total carbon (CT). Yolk sac and globule size, embryo and larval size, and heart rates were then compared between the 6 treatment groups (3 temperature x 2 CT). The main conclusion of the study is that temperature but not CT affect energy reserves in surf smelt early stages and that the energy demand increase with the combination of the two stressors.

Overall the manuscript is clear and well written. Over the past decade a number of studies addressed the impact of future warming and pCO2 levels on fish metabolism but only recently, studies have begun to address how ocean acidification and warming affect the metabolism of fish embryos. While the importance of studying surf smelt is well justify, reasons for investigating warming and CT are unclear is this manuscript and make partly sense only once reading the “Ecological Implications” section at the end of the discussion. Similarly, the choice for the experimental conditions lacks in justification and supporting statement. The following are additional suggestions for improvement.

Major changes:

Lines 59-77: The discussion state (lines 427-430) that human activities have induce a reduction in vegetation in surf smelt spawning grounds leading to an increase in temperature due to the lack of shading. A few sentences in the introduction would be useful to justify the importance of investigating the effect of temperature stress on surf smelt.

Lines 78-84: Similarly here, a justification for studying the effect of ocean acidification is missing. It appears only in the discussion lines 444-446. I think it would be benefic to reshape the introduction for better understand the basis of the study.

Lines 95-97: Is it possible to provide the range of pCO2 encounter in the northeast Pacific Ocean systems? What are the values predicted by Feely et al. (2020) for the Puget Sound ecosystem and how do they compare with predictions from climate model forced with IPCC?

Lines 125-127: How much the temperature fluctuates along the Salish Sea shoreline? It would be good to add this information to provide context on the choice of acclimation temperatures (13, 15, 18°C). The justification for using 15 and 18°C is lacking. Presumably these represent future climate conditions encounter in shallow Salish Sea waters during the summer but according to which climate model and how far in the future?

Line 204: Is there a possibility to mention/display mortality and hatching success? Were there any difference between treatments?

Lines 211-214: What was the temperature of the water at time of collection? For how long was the temperature changed from 15°C to 16°C? Why choose 16°C, did the ambient condition only increase of 1°C (13 to 14°C) during the summer? Then would using a delta be beneficial: Δ2°C from the “ambient” condition. Thus, if temperature of the ambient condition increased to 14°C during the summer the medium condition was always 2°C above. Was the 18°C condition always maintain at 18°C?

Lines 223-224: Only the average standard deviation is displayed, it would be a nice reminder of the condition and for better flow to have the value presented as: mean ±SD. In addition, it is highlighted here that variability in pCO2 was higher for the high pCO2 condition. It would be interesting to mention it in the discussion. How higher variability (compare to more stable treatment) impacted the results? What is usually observed in the wild?

Lines 299-300: Tradeoffs is a hypothesis that may explained the results but results can also be explained by the multiple performances –multiple optima (MPMO) (Clark et al. 2013), stating that different physiological activities have potentially different thermal optima. While this theory have been developed in regards to performance traits of developing larvae it may be applicable in embryos.

Lines 355-356: In accordance with the discussion here, a recent study (Dahlke et al. 2020), demonstrated that early-life stages without functional gills appear to be better equipped in terms of ion homeostasis than previously thought.

Lines 375-381: It would be interesting to link further the behavior/biological cycle of surf smelt with their capacity to withstand elevated CT. The author might want to look into the “Ocean variability hypothesis” which connect the duration and distance of the stay inshore of migratory fish with their resilience to CO2 (Baumann, 2019).

Lines 426-447: The effort to discuss the ecological implications of the results is to be applauded, however, I believe that an important part of this section should be moved to the introduction to justify why this study focus on the two stressors: temperature and pCO2.

Minor changes

Line 39: What is implied by “clean, cold water and unaltered shoreline”? It would be beneficial to provide thermal range for surf smelt and maybe and index of water quality.

Line 58: A reference is missing to support this statement.

Line 173: It is not clear why ambient temperature treatment only lasted for 10 days.

Line 183: A reference is missing to support this statement.

Results: Were there any basins effects? While the details of statistic test are provided in the table, it would be engaging to have the p-value in the text.

Lines 229-237: How the combination of the stressors affected yolk usage?

Line 354-355: “despite the very high concentration”, again we have no indication of the concentration find in the wild. What is it normally observed in their habitat? Why is it considered “very high”?

Figures: Indication of significant differences using symbols (*) or letters would be benefic.

References:

Clark TD, Sandblom E, Jutfelt F. Aerobic scope measurements of fishes in an era of climate change: respirometry, relevance and recommendations. J Exp Biol. 2013;216(15):2771–2782.

Dahlke FT, Leo E, Mark FC, Pörtner HO, Bickmeyer U, Frickenhaus S, Storch D. Effects of ocean acidification increase embryonic sensitivity to thermal extremes in Atlantic cod, Gadus morhua. Glob Chang Biol. 2017 Apr;23(4):1499-1510.

Baumann, H. (2019). Experimental assessments of marine species sensitivities to ocean acidification and co-stressors: How far have we come? 1. Canadian Journal of Zoology, 97.

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Submitted filename: Review for.docx

Click here for additional data file.

20 Jan 2022

Editor Comments:

In addition to points raised by the reviewers, one confusing aspect of this manuscript is highlighted in the last paragraph of the introduction. It seems clear that the primary hypotheses of this research were related to increased PCO2, however for some reason, increased PCO2 in the introduction changed to total dissolved carbon in the results and discussion sections. Where are the results for the hypotheses that increased PCO2 has effects on these biological factors? It is clearly stated that "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.", yet, none of these results are shown or discussed. The MS (lines 154-160) does a fine job justifying the use of the pH electrode, so why weren't they used for PCO2? At the end of the methods section "CT was the only carbonate parameter used in statistical analyses." is added without any explanation. OA and CT are not synonymous. This confusion in the manuscript needs to be resolved by including the results of the relevant statistical analyses and revising the manuscript accordingly. Sure spectrophotometer pH measurements may be good to the second decimal place and the pH probe might only be good to one decimal place, but you demonstrated the accuracy as noted above. The weak link in your chemical analyses looks to be the refractometer (only one decimal place). I can imagine several ways to approach this (changing/adding figures, results of statistical analyses, clearly stating results of all analyses, etc. in the MS and/or supplemental data).

We appreciate your feedback on the clarity of our approach to which carbonate system parameters are used in the analysis and reporting of the study. We recognize that pCO2 is the primary parameter used in most OA discussions, and there are good physical and experimental reasons for this. Many systems (including the atmosphere/ocean system writ large) do rely on gas exchange, and so pCO2 is the primary parameter to encapsulate OA. However, the design of our system is such that total carbon is the carbonate parameter we are most directly manipulating as CO2 gas is quantitatively dissolved on introduction to the mixing tanks. Therefore, we were careful to state our broad hypothesis in terms of OA and our more specific ones in terms of CT:

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…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 CT (i.e. acidification). It was also predicted that the highest energy usage would be observed under simultaneous increased temperature and elevated CT due to the additive effect of these climate stressors on compensatory homeostatic processes.

We also chose total carbon as the primary carbonate variable in our statistical analysis because pCO2 is highly temperature dependent. The use of total carbon rather than pCO2 maintains the least amount of correlation between potential variables which would make it more difficult to disentangle the effects of carbonate system changes versus temperature changes.

Clearly, we can do better in making a case for total carbon as the most appropriate measure of OA in this instance. In order to address your concerns in this area, added some text in the methods section with recognition that pCO2 is the parameter most commonly used to represent the suite of changes associated with ocean acidification and making our choice to use total carbon in the analysis here more clear. This explanation emphasizes the design of the system and the co-variation of pCO2 and temperature, which is not desirable when we want to understand the effects of both temperature and OA related changes. We have reviewed the introduction and discussion to be sure we are careful about where the use of OA, pCO2 and total carbon are appropriate. We could also bring more attention to the pCO2 values that we did calculate in the results and discussion if you recommend that we do so.

Additional revisions we could make include adding data about the correlations that exist between pCO2, total carbon and temperature in our data, to make the point that total carbon does a good job of representing OA, and is highly correlated with pCO2, but that it is less correlated with temperature, which is desirable in this situation for the statistical analysis. This might be more appropriate in supplementary information if it is needed. While salinity does not have a strong effect on pCO2 with the kind of variability observed in this study, there are high quality monitors on the seawater system and we could retrieve those data if necessary. However, the variability in the pH numbers will swamp any minor salinity effects, so we suggest this step may not be necessary.

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These documents were reviewed and the format was adjusted accordingly.

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We added the specific name of the Bay that samples were collected as well as coordinates, and this can be referenced with any available regional map.

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This project was conducted in collaboration with the Washington State Department of Fish and Wildlife (WDFW) forage fish team. Embryos were collected under their auspices and, as such, we did not need our own WDFW collection permit.

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This was reviewed and ensured to match.

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Please note that funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

We moved this section to the ‘Funding’ section. We hope this rectifies the issue.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

The project described in this publication was supported by the Department of the Interior Northwest Climate Adaptation Science Center (NW CASC- https://nwcasc.uw.edu/) through a Cooperative Agreement (G17AC000218) from the United States Geological Survey (USGS). Its contents are solely the responsibility of the authors and do not necessarily represent the views of the NW CASC or the USGS. This manuscript is submitted for publication with the understanding that the United States Government is authorized to reproduce and distribute reprints for Governmental purposes. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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The data has been published in the SENOE repository, citation:

Russell Megan (2018). Surf Smelt Embryo and Larvae Data. SEANOE. https://doi.org/10.17882/85830

Our cover letter has been updated to reflect this change.

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This section has been moved to the Methods section of the manuscript

Reviewer #1 Comments/responses

Reviewer #1: Review for “Surf smelt accelerate usage of endogenous energy reserves under climate change”

This is an interesting manuscript in which the short-term effects of high temperature and high pCO2 exposure on energy reserves of embryos and larvae of surf smelt are described. I have several questions regarding the experimental design and a few remarks for the results section. The discussion is much too long and often discusses topics that are only distantly related to what was actually measured in this study. The authors describe what other studies found but often miss to connect it to their own findings.

We appreciate this comment. This manuscript describes the M.S. thesis work of the lead author. We recognize that the rather long discussion in the original manuscript reflects this style. In the revision we cut the discussion down from seven pages to just over three pages, and have removed all components that we believe only peripherally supported our findings. We believe this change in response to your suggestion makes for a much stronger discussion.

Abstract

Line 12-14: One can also argue that because surf smelt are beach spawners, they are exceptionally robust to environmental stress

Excellent point. We changed this sentence to reflect that as obligate beach spawners, they are exposed to both marine and terrestrial stressors. We also highlighted this argument in the discussion.

Line 18:19: Why are you specific with the temperature used but not the pCO2 level? At least give an approximation what “ambient” and what “elevated” means.

We amended this to show specificity with total carbon and pCO2.

Line 26: “CT” has not been explained before use

Thank you for letting us know. It is now defined on line 19.

Line 27: “may impact”. It is more informative if you give a direction. Is it negative or positive impact?

We amended this sentence to reflect simply the direction of change.

Introduction

Line 107-109: This sentence is repetitive to the previous sentence

We meant for the first sentence to reference these stressors as isolated, and in the following sentence as occurring simultaneously. We amended the sentence to make this more clear.

Methodology

Line 130: Refer to Table 1.

Thank you for this suggestion. We added this reference.

Line 135-136: Figure 1 is not needed. If you want to show it somewhere put it in the supplementary material

Excellent suggestion. We removed this figure from the manuscript.

Line 142: Were the embryos shock exposed to the treatment conditions or was there a gradual increase/decrease in water temperature and pCO2?

The embryos were not slowly acclimated to these conditions.

Line 145-150: Why did you not use the embryos from your embryo experiment to be further studied as larvae? There might be cascading effects from embryo to larva that you missed because of your experimental design which I see as a drawback for your results

We agree that this is a shortcoming, but did not have the manpower to include this design.

Line 172-174: I don’t understand what is meant. Were the temperature treatments not run at the same time?

They were run at the same time, but the ambient treatment was terminated after 10 days because the natural warming of the seawater brought the temperature close to the medium temperature treatment. As such, we lost temperature separation between treatments at 10 days. We amended this section for clarity.

Line 185-188: What was done to maintain treatment conditions during measurements of heart rates? How long after removal of the embryos from their tanks did it take to start recording the heart rate videos?

The embryos were moved individually in their treatment water and placed immediately under the scope. In preliminary experiments we did test to make sure that temperature did not increase or decrease during the short 10-30 seconds of filming.

Results

Line 206-227: I find this a very weird way of showing the treatment conditions. It gives the impression that you did not exactly know what the treatment conditions were. E.g. line 208: “temperatures remained consistent throughout the duration of the experiment”. If your experiment was planned to use stable temperatures, this is a given. I consider this unnecessary to take up so much space in your manuscript. Referring to Table 1 in your methods section would simply be enough.

We agree, and have cut much of this section.

Line 211-214: Why is the treatment then called 15C instead of 16C if the water temperature was 16C and not 15C?

This sentence is a mistake, and thank you for catching it. They were adjusted from 14C to 15C. This section is part of the passage that is now omitted.

Line 213: “summer warming elevated the seawater temperature in the ambient temperature treatment” by how much? For how long?

This fact was addressed in your earlier comment about lines 172-174, and was the reason that the ambient temperature treatment was stopped at 10 days.

Line 235-237: Figure 2 does not show this result. I suggest to use a glmer to account for the ratio which cannot be above 1 or below 0. Also I would prefer showing the error as 95% intervals and offsetting the CT data for clarity of the error bars. Maybe this would then visually support the result that pCO2 had an effect. Also you describe that data from the 13C treatment are missing in line 173 (which I commented on before) but why is there a data point for day 13 for the ambient pCO2 treatment but not for the elevated pCO2 treatment?

We analyzed the data now with a generalized linear model (family= binomial, link=logistic) to account for the ratio and give the model stronger power. Results have been modified to reflect this model change. We modified the figure to offset the data points and displayed error as standard error to allow for greater clarity in the figure. The data were binned based on temperature. There was an experimental basin in the ambient pCO2 treatment that remained in the 13C temperature bin, but there was not one for the elevated PCO2 treatment.

Line 254: In Figure 4 it looks like larvae used for the 18C treatment started off with smaller yolk areas already. How can this be and was the difference in yolk area size at the start accounted for when comparing the yolk area at the final day?

The data start in Figure 4 on day one. That is, larvae were in treatment water for 24 hours prior to the first measurement. Thus, the 18C treatments had already shown evidence of accelerated yolk usage. Unfortunately, we do not have time zero measurements. The reduction in yolk size expressed in lines 252-253 does take into account the original size in each treatment. The average size at the end does not (lines 253-254) and, because of this, has been removed. We are grateful you caught that error.

Discussion

Line 277-285: Can you draw some conclusion from all these Q10 values? The Q10 that you calculated is smaller than the average 2.9 calculated by Heming and Buddington. If you are using this reference, it indicates that surf smelt are less temperature dependent than most fish species studied (in case this is the point which you want to make by referring to Heming and Buddington). Why are you stating this reference? What is the point that you want to make with it? This is especially true for the last sentence in this paragraph (line 283-285).

We appreciate this comment, and now view it as a missed opportunity to elaborate on an earlier point that you made (…One can also argue that because surf smelt are beach spawners, they are exceptionally robust to environmental stress). We amended this section to include a short discussion of the relevance of our lower observed Q10 values.

Line 286-292: Similar to the previous paragraph. Which point do you want to make with the review of Villalobos et al 2020? We do not know the temperature performance curve or at least the temperatures at which C. pallasi occurs in nature.

Upon reflection, we see your point and agree that this adds little to no value to the discussion of our results. We have omitted this section.

Line 310 to 322: Although there is nothing wrong with this paragraph I question why it is included. You mention that you did not measure developmental rate and you don’t know if a faster development due to higher temperature is present in surf smelt, so why do you then include this paragraph in your manuscript?

This section has been removed from the manuscript.

Line 337: Which temperatures are considered “temperature stress” in surf smelt larvae?

Your point is well taken, and we amended this sentence.

Line 338-342: What do these sentences add to your story?

We amended this section, but believe accelerated use of the oil globule will, by way of its influence on fish buoyancy, negatively affect the ability of larval fish to capture prey. Thus, we are connecting our results to some ecological implications.

Line 354-363: Again, why is this paragraph included? This paragraph is about acid-base regulation which was not part of what you have measured. You are discussing topics that you did not measure in your study.

This section has been removed from the manuscript.

Line 370-374: Did your embryos and larvae come from adults that were exposed to high pCO2? Relate the studies that you refer to your own study.

This section has been removed from the manuscript.

Line 383-388: Also have a look at Leo et al 2018. They used herring from Norway and found higher susceptibility of embryos and larvae to high pCO2 than Sswat and Franke. But also here, the point that you want to make by referring to these herring studies is not really clear.

This section has been removed from the manuscript.

Line 388-390: I don’t think that you can draw this conclusion by studying surf smelt for only 3 days.

This section has been removed from the manuscript.

Line 415-424: While this all is correct I think it is too far off from what you did in your study and does not need to be included here.

This section has been removed from the manuscript.

Line 425-465: These three paragraphs are great and if they are filled out with your main points from your results they would make a great discussion making most of the previous discussion (line 264-424) redundant

As previously stated, we restructured our discussion based on this comment. In doing so, it is considerably shorter and we believe more directly aligns with our findings.

Reviewer #2 Comments/responses

The manuscript (PONE-D-21-27467) titled: “Surf smelt accelerate usage of endogenous energy reserves under climate change” aimed to determine if energy reserves of surf smelt embryos and larvae are affected by single and interactive effect of ocean warming and ocean acidification. To address this aim, before reaching 24 hours old, surf smelt were acclimated to a combination of three temperatures 13, 15, and 18°C and two treatment of total carbon (CT). Yolk sac and globule size, embryo and larval size, and heart rates were then compared between the 6 treatment groups (3 temperature x 2 CT). The main conclusion of the study is that temperature but not CT affect energy reserves in surf smelt early stages and that the energy demand increase with the combination of the two stressors.

Overall the manuscript is clear and well written. Over the past decade a number of studies addressed the impact of future warming and pCO2 levels on fish metabolism but only recently, studies have begun to address how ocean acidification and warming affect the metabolism of fish embryos.

While the importance of studying surf smelt is well justify, reasons for investigating warming and CT are unclear is this manuscript and make partly sense only once reading the “Ecological Implications” section at the end of the discussion. Similarly, the choice for the experimental conditions lacks in justification and supporting statement. The following are additional suggestions for improvement.

We have made considerable effort to address this concern throughout the manuscript.

Major changes:

Lines 59-77: The discussion state (lines 427-430) that human activities have induce a reduction in vegetation in surf smelt spawning grounds leading to an increase in temperature due to the lack of shading. A few sentences in the introduction would be useful to justify the importance of investigating the effect of temperature stress on surf smelt.

We appreciate your comment and have added a section highlighting this in the introduction.

Lines 78-84: Similarly here, a justification for studying the effect of ocean acidification is missing. It appears only in the discussion lines 444-446. I think it would be benefic to reshape the introduction for better understand the basis of the study.

We have added a section in the introduction justifying the OA component of this study.

Lines 95-97: Is it possible to provide the range of pCO2 encounter in the northeast Pacific Ocean systems? What are the values predicted by Feely et al. (2020) for the Puget Sound ecosystem and how do they compare with predictions from climate model forced with IPCC?

We have added a section to the introduction addressing this concern.

Lines 125-127: How much the temperature fluctuates along the Salish Sea shoreline? It would be good to add this information to provide context on the choice of acclimation temperatures (13, 15, 18°C). The justification for using 15 and 18°C is lacking. Presumably these represent future climate conditions encounter in shallow Salish Sea waters during the summer but according to which climate model and how far in the future?

We have added considerable justification for using these temperatures.

Line 204: Is there a possibility to mention/display mortality and hatching success? Were there any difference between treatments?

We did not measure this as we were focusing on energy utilization and were limited by person hours.

Lines 211-214: What was the temperature of the water at time of collection?

13℃

For how long was the temperature changed from 15°C to 16°C? Why choose 16°C, did the ambient condition only increase of 1°C (13 to 14°C) during the summer? Then would using a delta be beneficial: Δ2°C from the “ambient” condition. Thus, if temperature of the ambient condition increased to 14°C during the summer the medium condition was always 2°C above. Was the 18°C condition always maintain at 18°C?

This passage, based on comments from another reviewer, has been cut. The original passage had an error. Namely, the moderate treatment was not moved up to 16℃, but from 14℃ to 15℃. We are referring all readers to table 1.

Lines 223-224: Only the average standard deviation is displayed, it would be a nice reminder of the condition and for better flow to have the value presented as: mean ±SD. In addition, it is highlighted here that variability in pCO2 was higher for the high pCO2 condition. It would be interesting to mention it in the discussion. How higher variability (compare to more stable treatment) impacted the results? What is usually observed in the wild?

We agree that presenting standard deviations without the means is a little awkward and makes them less meaningful. Because pCO2 is temperature sensitive, we do not want to repeat a large number of mean values (18 in total) already presented in the data table and referenced.

Regarding the effect of variability, this pattern is very common in OA studies and is not usually included in the analysis of results. These animals are exposed to high variability in their natural spawning habitat, so large physiological effects associated with these difference in variability seem unlikely.

Lines 299-300: Tradeoffs is a hypothesis that may explained the results but results can also be explained by the multiple performances –multiple optima (MPMO) (Clark et al. 2013), stating that different physiological activities have potentially different thermal optima. While this theory have been developed in regards to performance traits of developing larvae it may be applicable in embryos.

Another reviewer was very critical of the length of our original discussion, and the weak connection to our own data. As such, and as you will see, we drastically reduced the length of our original discussion. In doing so, this section was cut from the manuscript.

Lines 355-356: In accordance with the discussion here, a recent study (Dahlke et al. 2020), demonstrated that early-life stages without functional gills appear to be better equipped in terms of ion homeostasis than previously thought.

This section has been removed from the manuscript.

Lines 375-381: It would be interesting to link further the behavior/biological cycle of surf smelt with their capacity to withstand elevated CT. The author might want to look into the “Ocean variability hypothesis” which connect the duration and distance of the stay inshore of migratory fish with their resilience to CO2 (Baumann, 2019).

This is an excellent suggestion. We added to this discussion, including pH variability at the site of collection and how this relates to the OVH Hypothesis.

Lines 426-447: The effort to discuss the ecological implications of the results is to be applauded, however, I believe that an important part of this section should be moved to the introduction to justify why this study focus on the two stressors: temperature and pCO2.

We added justification to the introduction and re-framed our discussion around these ecological implications.

Minor changes

Line 39: What is implied by “clean, cold water and unaltered shoreline”? It would be beneficial to provide thermal range for surf smelt and maybe and index of water quality.

We do not have or can find a thermal range for surf smelt, nor an index of water quality required for spawning. We removed the text associated with ‘clean’ and ‘cold’ seawater. We did, however, leave the in reference to ‘unaltered’ as this relates to our discussion of modified beaches, and the references we cite support that statement.

Line 58: A reference is missing to support this statement.

Thank you for alerting us to this omission. We have added a recent reference to support this statement.

Line 173: It is not clear why ambient temperature treatment only lasted for 10 days.

We amended this section to add clarity.

Line 183: A reference is missing to support this statement.

A reference was added for this statement (Thorarensen et al. 1996).

Results: Were there any basins effects? While the details of statistic test are provided in the table, it would be engaging to have the p-value in the text.

Basins were included as a random variable and none were observed. We understand your point about adding p values in the text, but we made the decision to keep them out and refer the reader to the tables. This way the text is more readable.

Lines 229-237: How the combination of the stressors affected yolk usage?

Stressor interactions were investigated when determining the best fit model. All interactions not shown were not significant and including them lowered the overall fit of the model based on AIC score comparisons.

Line 354-355: “despite the very high concentration”, again we have no indication of the concentration find in the wild. What is it normally observed in their habitat? Why is it considered “very high”?

As stated previously, we added significant text in the introduction informing the reader as to OA concentrations surf smelt experience in the study region.

Figures: Indication of significant differences using symbols (*) or letters would be beneficial.

Our figures are not a display of our models, but of the raw data that was used to create these models. Because we looked at overall trends over time rather than looking at individual time points, it would not make sense to annotate significant differences on our figures at specific time points.

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

All of our figures were uploaded to PACE and checked.

Submitted filename: Response to Reviewers.docx

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10 Feb 2022

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #3: (No Response)

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Reviewer #3: Partly

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Reviewer #3: I Don't Know

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Reviewer #3: Yes

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Reviewer #3: This paper poses that beach spawning forage fish, smelt, may be affected by increasing temperature and seawater CO2 and possible interactions between these variables. The question is interesting, but tricky. I’ve provided some additional things for the authors to consider below. The accelerated use of yolk at high temperatures is clear but not unexpected. The effects of CO2 and interactions with temperature are minimal/none. The paper is generally well-written.

See attachment for further comments and figures.

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Reviewer #3: No

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Submitted filename: Russell et al smelt CO2 temp PLoS One review.pdf

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25 Mar 2022

Response to Reviewers

Editors Comments

Thank you for addressing concerns of the two first reviewers. Your response to my previous comments about confusing total carbon and pCO2 was not adequate, and I solicited another reviewer's opinion. You will need to re-write the manuscript with regards to pCO2 instead of total carbon in order for it to be acceptable (cf. comment #6 of Reviewer #3). Please look over the comments of reviewer #3 carefully. You should find them useful in your data analyses and discussion. Considering those points should further improve your manuscript.

We have rewritten the manuscript focusing on pCO2 rather than total carbon treatments, including re-running the statistics using pCO2 as a continuous variable. We hope this change and modification to the subsequent manuscript fulfills this request.

Reviewer Comments

1. The eggs are found buried in sediment on the beach and its unclear how much time is spent immersed in seawater. At lower tides, the eggs are in a moist environment that may heat up due to sun exposure. I imagine the CO2 levels in the sediment increase at this point relative to that found in seawater. No measurements of pH or CO2 in the sand were made. If CO2 is higher in seawater than in the emmersed sand, then one would expect high CO2 to cooccur with lower (ambient) temperatures. However, the only interaction between CO2 and temperature found was at high temperature and high CO2 (but see point 2).

We understand that our experimental design does not replicate the dynamic nature of embryo exposure, and we acknowledge this in lines 147-151.

2. Heart rate was highly variable but not significantly different across temperatures. The high Temp/high CO2 treatment was significantly lower but the error bars are enormous and completely overlap with the other treatments. So that result is not compelling. The text states that heart rate increased at high temp/ambient CO2, but that is not significant and so should be changed (no effect).

When re-running the stats with pCO2 as a continuous variable, there was no significant effect of temperature or pCO2 on heartrate. The results and discussion have been modified to reflect this. The table showing experimental conditions for the heartrate experiment and the figure showing the data have been moved to supplemental materials.

3. The text makes the case that the reduced heart rate may have resulted from CO2/pH effects on oxygen binding in the blood. However, this would only be important if the embryos were near maximum metabolic rate for the available oxygen (i.e. near the Pcrit for whatever their oxygen consumption rate was). Aerobic scope (difference between max and rest metabolic rates) is largely unknown for eggs. However, Pcrits for fish eggs below 50% saturation have been reported. In that case, any reduction in oxygen transport would not translate into a reduced metabolic rate under the oxic experimental conditions. Something to ponder.

This possible mechanism was suggested in a source, see below. However, your points are valid and due to the unknown nature of this mechanism and wanting to avoid speculation, we have removed this sentence.

Esbaugh A (2018) Physiological implications of ocean acidification for marine fish: emerging patterns and new insights. J Comp Physiol B 188:1-13. doi:10.1007/s00360-017-1105-6

4. Heart rate would be expected to increase with metabolic rate, which nearly always increases with temperature. An increased metabolic rate is consistent with greater consumption of yolk and fat stores at high temperature. Its unclear why heart rate wouldn’t increase at high temperature except that its so variable as to be undetectable.

We agree and have largely removed all discussion of heart rate due, as stated above, to the fact that when we re-analyzed our data using pCO2 as a continuous variable, we did not observe a significant effect of temp or pCO2 on heart rate. And as you say, with variable data and our small sample size, it is unlikely to yield observable significant result.

5. There is a small, but likely significant, “experiment” effect on the CO2 levels achieved (see figure below). The embryo heart rate experiment had the highest PCO2 levels achieved of any experiment. Its not possible to say whether than influenced the outcome relative to the slightly lower values in the other two experiments.

We understand this could be an effect but as you said, it is not possible to say whether this influenced the outcome.

6. As pointed out by the editor, the paper discusses PCO2 in the introduction and frames hypotheses about the effects of PCO2 on biology. However, the authors instead perform statistical analyses on total carbon. As stated on line 124: “While pCO2 is the parameter most commonly used to represent the suite of changes associated with OA, and is the parameter actually manipulated in many systems, CT is the control variable in this system.” It is further stated that PCO2 is temperature sensitive and this is why CT was used instead. However, both variables are temperature sensitive. In fact, what changes with temperature is the solubility of CO2. If a given amount of CO2 is dissolved in seawater, both total carbon and carbon dioxide pressure will change and this change will be different at different temperatures because of changing solubility with temperature. Cold water will hold more of the CO2 (thus higher CT) and the dissolved CO2 will exert less pressure. This is seen clearly in a graphical representation of the data (below), especially within the “ambient” treatment. So ultimately it doesn’t matter which is stated throughout the text…the treatment has high total CO2 and high PCO2 relative to the ambient control. However, because the range of values is different for each variable, the statistical analyses could be influenced. For example, if the PCO2 doubles, the pH will decrease by 0.3 units and total CO2 increases by only a small fraction because of the large amount of CO2 already dissolved in seawater. Thus analyzing the interacting effects of CT and temperature may provide less clear results than analyzing the interacting effects of PCO2 and temperature…even though they are two sides of the same coin. Because CO2 was added at a particular rate, rather than a particular amount or to achieve a particular PCO2, pH or TC, the various carbonate parameters all change with temperature in interrelated ways and the different experiments have different values.

We agree with your summation and have re-analyzed the data using pCO2 as a continuous variable to look at pCO2 interactions. The only change to our results was the loss of statistical significance in the heartrate experiment. As a result, the manuscript has been modified to reflect these changes.

Submitted filename: Response to Reviewers.docx

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18 Apr 2022

1 Jun 2022

Response to Reviewers

1) The manuscript is still confusing pCO2 and CT. For example, in the abstract the sentence "two total carbon (CT) treatments" makes it seem like the treatments were made in a fashion other than manipulating pCO2. In fact, pCO2 was used to manipulate the system, and that should be make clear throughout the manuscript. Everyone knows that when you change pCO2, CT also changes. That sentence in the abstract should be re-written to accurately reflect the experiments.

The CT portion of the sentence was removed from the abstract.

2) Line 117: 20-lb

This edit was completed.

3) lines 133 and 134: see comment number 1 above.

CT was changed to PCO2 when describing the manipulated variable.

4) Lines 146 and 156: 200-mL

This edit was completed.

5) Lines 147-151: This sentence is awkward. Maybe break it up into 2 or three sentences?

This sentence was broken into two sentences.

6) Line 152: This sentence is awkward. Maybe explicitly state this is a different experiment to clarify this for the reader.

This sentence was split and states that larval experiment was started.

7) Line 153: 12-L

This edit was completed.

8) Line 167: 20-mL

This edit was completed.

9) Line 182: I suggest changing to ... 'into a 6-well plate labeled with its corresponding treatment.'

This edit was completed.

10) Please give citation for ImageJ

A citation for ImageJ was added.

11) Lines 189-194. This paragraph is a bit difficult to follow. I suggest re-writing it.

This paragraph was edited for clarity.

12) Lines 202-205 can be deleted as they appear on lines 220-222. Add the WA DFW info to the approval statement, too.

This edit was completed.

13) In the statistical analysis section of the methods, please state which stats software was used.

Added in a sentence stating R software was used and added in a citation for R.

14) Lines 225-239 and anywhere else: Please see comment number one above.

This edit was completed.

15) Table 3: Please correct the p for pCO2. It now reads 0.0.5418

This edit was completed.

16) A recent paper by Murray and Klinger (J Exp Biol (2022) 225 (5): jeb243501) is applicable to your study and could be a good addition to your discussion.

This paper was read and added to our discussion when relevant.

Submitted filename: Response to Reviewers.docx

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13 Jun 2022

Surf smelt accelerate usage of endogenous energy reserves under climate change

PONE-D-21-27467R3

Dear Dr. Russell,

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Additional Editor Comments (optional):

Reviewers' comments:

16 Jun 2022

PONE-D-21-27467R3

Surf smelt accelerate usage of endogenous energy reserves under climate change

Dear Dr. Russell:

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