Differential sensitivity of larvae to ocean acidification in two interacting mollusc species

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Differential sensitivity of larvae to ocean acidification in two interacting mollusc species Camilla Campanatia, Sam Dupontb, Gray A. Williamsa, Vengatesen Thiyagarajana,∗ a

The Swire Institute of Marine Science and School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong, China Department of Biological and Environmental Sciences, University of Gothenburg, The Sven Lovén Centre for Marine Infrastructure, Kristineberg, Fiskebäckskil, 45178, Sweden b

A R T I C LE I N FO

A B S T R A C T

Keywords: Saccostrea cucullata Reishia clavigera Larvae Ocean acidification Predator Prey

Anthropogenically-induced ocean acidification (OA) scenarios of decreased pH and altered carbonate chemistry are threatening the fitness of coastal species and hence near-shore ecosystems' biodiversity. Differential tolerances to OA between species at different trophic levels, for example, may alter species interactions and impact community stability. Here we evaluate the effect of OA on the larval stages of the rock oyster, Saccostrea cucullata, a dominant Indo-Pacific ecosystem engineer, and its key predator, the whelk, Reishia clavigera. pH as low as 7.4 had no significant effect on mortality, abnormality or growth of oyster larvae, whereas whelk larvae exposed to pH 7.4 experienced increased mortality (up to ∼30%), abnormalities (up to 60%) and ∼3 times higher metabolic rates compared to controls. Although these impacts' long-term consequences are yet to be investigated, greater vulnerability of whelk larvae to OA could impact predation rates on intertidal rocky shores, and have implications for subsequent community dynamics.

1. Introduction Oceans are absorbing a fourth of the atmospheric carbon dioxide emitted through human activities, which is continuously rising (Raven et al., 2005; IPCC et al., 2014). As a consequence, through the process of “ocean acidification” (OA), seawater pH is decreasing and carbonate chemistry is shifting towards an undersaturation state in respect to calcium carbonate minerals (Orr et al., 2005; Raven et al., 2005; Feely et al., 2009). OA, therefore, threatens to reduce the survival, growth and shell calcification of marine molluscs, which include important commercial species and key components for coastal/rocky shore ecosystems (Salisbury et al., 2008; Beck et al., 2009; Gazeau et al., 2013; Waldbusser et al., 2013). Alteration of the seawater carbonate system can impair shell deposition and maintenance in molluscs (Gazeau et al., 2007; Hofmann et al., 2010; Roleda et al., 2012; Waldbusser and Salisbury, 2014). Many species exposed to acidified conditions show decreased shell growth (e.g. Berge et al., 2006; Talmage and Gobler, 2011) and/or thinner shells (e.g. Beniash et al., 2010; Welladsen et al., 2010; Gaylord et al., 2011), with sometimes shell dissolution outpacing carbonate deposition (Bibby et al., 2007; Nienhuis et al., 2010). Sensitivity to OA, however, can vary between species, populations and developmental stages (Parker et al., 2010, 2011; Byrne, 2012; Kroeker et al., 2010, 2013). Bottlenecks may be defined by certain species' life

∗

stages, which are particularly vulnerable to OA (Kurihara, 2008; Dupont et al., 2010; Hofmann et al., 2010; Byrne, 2012). Many molluscs, for example, show extreme sensitivity to OA at the larval stage (Gazeau et al., 2013; Parker et al., 2013; Przeslawski et al., 2015), with larvae having smaller and deformed shells or being unable to create a shell when exposed to lower pH (e.g. Parker et al., 2009; Watson et al., 2009; Byrne et al., 2011; Kimura et al., 2011; Ko et al., 2014). The implications of these effects will vary with life stages and between individual species, but their impacts will eventually depend on the ecological role of each species. For example any OA impact on the survival and/or development of a given species could affect its abundance within an ecosystem and could, thus, indirectly, alter the species' interspecific interactions (Kroeker et al., 2014). In a predator and prey system, differential species sensitivity to OA scenarios at their early-life stages could, for example, have important consequences on their population dynamics, and could eventually alter subsequent interactions (Palmer et al., 1996; Wieters et al., 2008; Sanford et al., 2014). This might, in turn, change the benthic community structure and cause a cascade effect, disrupting trophic webs (Sanford, 1999; Zarnetske et al., 2012; Kroeker et al., 2014). Sheltered rocky shores in the Indo-Pacific are dominated by extensive beds of the rock oyster, Saccostrea cucullata, which is an important ecosystem engineer, providing habitat for numerous species

Corresponding author. E-mail address: [email protected] (V. Thiyagarajan).

https://doi.org/10.1016/j.marenvres.2018.08.005 Received 27 May 2018; Received in revised form 3 August 2018; Accepted 5 August 2018 0141-1136/ © 2018 Published by Elsevier Ltd.

Please cite this article as: Campanati, C., Marine Environmental Research (2018), https://doi.org/10.1016/j.marenvres.2018.08.005

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2.1.2. Whelk egg capsules Whelk capsules were collected from natural deposition on rocks and tiles inside the tank where the adult whelks were acclimated (50 l). Newly laid egg capsules were transferred into 3 l tanks filled with new 0.22-μm FSW, constantly aerated, held at ∼28 °C. The water was changed every other day and, after ∼20 days of intracapsular development, hatched larvae were collected through a 260-μm mesh. The larvae were washed several times with natural FSW (T = 28 °C, Salinity = 32) to separate them from capsules with infected and/or dead embryos (Tong, 1988). Hatched larvae were subsequently resuspended in a known volume of fresh FSW and sub-counts were conducted in triplicate to estimate larval concentration. Within 24 h from hatching, the larvae were randomly distributed among 16 culture tanks (volume = 1 l) at the final density of ∼2 larvae ml−1. In order to measure the initial larval size before exposure to the treatments, for both oyster and whelk larvae, three samples of 1 ml from the hatched larvae, concentrated in known volume of FSW, were fixed in 10% buffered formalin prior to measurement of morphological structures.

(Chiu, 1997; Gutiérrez et al., 2003). The rock oyster is also the prey of the whelk, Reishia clavigera, which can cause high mortality of the oyster (Chiu, 1997; Chow, 2004), hence influencing local biodiversity (e.g. Silliman et al., 2011; St. Pierre and Kovalenko, 2014; McAfee et al., 2016). These two species, therefore, inhabit the same physical habitat, but play very different interacting roles; the outcome of which may be influenced by differential responses of the species to environmental conditions, such as OA. The aim of this work was to evaluate the sensitivity to low pH of the larval stages of these two interacting species and to determine thresholds of pH levels suitable for the survival and development of these larval phases. In this context, many studies still investigate the sensitivities to OA by comparing the organismal responses between ambient pH and only one specific pH treatment. However, approaches that test the organisms' physiological responses against a wider range of pH, representing the seawater chemistry's natural variability at the specific habitat, have proved to be more insightful methods to better predict the impacts of OA (e.g. ‘physiological tipping points’; Dorey et al., 2013; Ventura et al., 2016; Vargas et al., 2017). We hypothesized that within the pH range naturally experienced at the collection site, both oyster and whelk larvae would survive and develop normally, but at lower pH levels (< pH 7.6) both calcifying species would be impacted in their larval phase. To test this hypothesis, newly hatched planktotrophic larvae of S. cucullata and R. clavigera were exposed for 12 days to four different pH levels, covering present and future natural conditions (ranging from 8.1 to 7.4), and several endpoints (larval survival, growth, metabolic rate and shell dissolutioninduced abnormalities) were scored. The comparison of these two species responses to OA at, presumably, their most vulnerable life stages is an important step to understand the future outcome of this predator-prey interaction and subsequent community dynamics.

2.2. Experimental design In order to investigate the responses of the larvae to OA, the effects of four pH levels (Nominal pH: 8.1, 7.8, 7.6, 7.4) were tested, each replicated 4 times (Σn = 4 pH levels x 4 replicates = 16 culture tanks). The levels of tested pH were selected to present natural fluctuation and projected pH ranges for the year 2100 at the collection site. Future scenarios were calculated from a reduction of 0.42 pH units from present (Site SM1; HKEPD, 2014; scenario RCP 8.5; IPCC et al., 2014; Fig.S1). The pH 7.8 represents the minimum value experienced in the present day in the summer, as well as the mean pH value expected to occur by 2100. The extreme low-pH value expected by 2100 is pH 7.4 (Fig.S1). The pH was manipulated by mixing ambient air and CO2 through gas flow meters (Cole-Parmer Inc.) whereas only ambient air was bubbled in the highest pH treatment (after Lane et al., 2013). Seawater temperature (∼28 °C) was maintained by placing the 16 randomly distributed tanks in temperature -controlled water baths with circulation pumps. In each culture tank pH, temperature and salinity (∼30–32) were measured on a daily basis using a Mettler-Toledo (SG2) probe waterproof salinity meter (Model 837–1, Gain Express Holding Ltd., HK). The pH probe was calibrated using NIST buffers (pH = 4.01, 7.00 and 9.21; Mettler Toledo, Gmbh Analytical CH8603 Schwerzenbach, Switzerland). Samples of seawater from each culture tank were collected every week, poisoned with 10 μl of a 250 mM mercuric chloride solution for later total alkalinity (TA) measures using an Apollo Scitech titrator (T50, Mettler Toledo). Total dissolved inorganic carbon (DIC μmol kg−1), pCO2 levels (μatm), bicarbonate and carbonate ions concentration ([HCO3−], [CO3−]; μmol kg−1] as well as calcite and aragonite saturation states (Ωcal, Ωara) were calculated using the CO2SYS program (Pierrot et al., 2006) from total alkalinity and pH (NBS) with the dissociation constants of Mehrbach (1973) as refitted by Dickson and Millero (1987).

2. Material and methods 2.1. Adult collection, spawning and egg capsules maintenance Adult oysters and whelks were collected at low tide in Tai Tam bay, Hong Kong (22°14′N, 114°13′ E), in 2015, during their reproductive period (summer months: May–August; Tong, 1988; Chiu, 1997; Sea surface temperature ∼ 28 °C; Salinity = 31). Animals were transferred to the aquarium at the Swire Institute of Marine Science where they were maintained in two separate open flow-through systems (volume = 50 l) supplied with ambient, sand-filtered natural seawater and fed ad libitum. The oysters were fed with a mixture of Isochrysis galbana and Chaetoceros gracilis, (LB 2307, 2658, UTEX culture collection, Austin, USA), whereas the whelks were fed with the flesh of Saccostrea cucullata. 2.1.1. Oyster spawning Oyster eggs and sperm were obtained from twenty male and twenty females, strip-spawned in 0.22-μm filtered seawater (FSW; 27.6 °C, salinity = 31.3, pHNBS = 8.1) as in Ko et al. (2014). Briefly, gametes were removed from mature gonads of multiple parents and suspended in FSW. The eggs were concentrated in a known volume of seawater and subcounts of 50 μl enabled the determination of eggs number. Fertilization was then facilitated by gently mixing a dense sperm solution with the concentrated eggs (about 5 ml for 1 million eggs; Ko et al., 2014). After ∼1 h, when > 50% of the eggs were fertilized, embryos were separated from the excess sperm using 20-μm nylon mesh and the remaining suspension was divided into three 50 l tanks, filled with natural FSW, constantly aerated, at the density of ∼13 embryos ml−1. According to our preliminary data, this density was sufficient to not affect hatching size or subsequent development and ensured sufficient larval density for the experiments. After 24 h, hatched oyster larvae were collected through a 30 μm nylon mesh, washed several times with new FSW and randomly assigned to 16 culture/experimental tanks (5 l) at the density of ∼3 larvae ml−1.

2.3. Larval culture and measurement of survival, development and growth 2.3.1. Oyster larvae The seawater in the oysters' culture tanks was changed every two days. At this time, the larvae were sieved through meshes of appropriate size and concentrated in 200 ml of adjusted-pH FSW for culture sampling. The respective treatments pH were re-established before reintroducing the larvae into the acclimation tanks. Larvae were fed with an increasing concentration of microalgae, from 5 × 103 cells ml−1 of Isochrysis galbana to 2 × 104 cells ml−1 of a mixed diet (1: 1 I. galbana and Chaetoceros gracilis) by day 10 of development. On days 3, 5, 8, 10, 12, 1 ml sub-samples from the concentrated cultures were collected to estimate larval density and morphology. Depending on the larval density in the culture, morphometrics of the shell (the length and width 2

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(μm) and the shell area (μm2)) were measured on the left valve of 10–20 individuals per tank per sampling day. No measurements were possible for the oxygen consumption of oyster larvae due to their relatively small size compared to the whelk larvae and the low culture density at the end of the experiment (see below).

as the regression coefficient of the significant logarithmic relationship between mortality (% of initial density) and days of development (Fig. S3; Tables S2, S3). The slopes of the relationships from each replicate tank were then tested against the measured pH (averaged within tank), using linear regression, to test for significant relationships between larval mortality rate and pH of acclimation. Further fitting with the generalised least square (GLS, gls fuction in the nlme package) method for the whelk larval mortality rate, was conducted, using the fixed variance structure (varFixed), to test for differences between pH treatments. Variation in growth rates for both oyster and whelk larvae were analysed from the slopes derived from the size increase over time of one shell dimension (shell area for the oyster larvae and shell length for the whelk larvae). The relationship between oyster larval shell area (μm2) and days of development was linearized by log-log transformation and the resulting coefficients of regression from each replicate tank (Table S4) were tested against the treatment pH using One-way Kruskal-Wallis test due to failure to meet the required ANOVA assumptions. Whelk growth rate was obtained from the slopes of the linear regression between shell length (μm) and days of development (Table S5) in each replicate tank and compared using One-way ANOVA against pH treatment. Allometries of shell morphometrics (shell length vs. shell width) were also compared among pH treatment groups. For each replicate tank, estimated coefficients of the allometric relationships were used to investigate differences amongst pH treatments using a Kruskal-Wallis test (pH treatment as factor). Notably, abnormal larvae (defined in section 2.3) were not included in the analyses of growth and allometric relationships. In order to test the differences in whelk larval metabolic rates among different pH treatments, a linear mixed model was used, with pH as a fixed factor and replicate tank as random (lme function in nlme package in R). Whenever pH was found to have a significant effect on the larval respiration, post-hoc analyses (multcomp package in R) followed to discriminate on the treatment differences.

2.3.2. Whelk larvae Reishia clavigera larvae were fed daily with 4 × 104 cell ml−1 of the diatom, Chaetoceros gracilis (Romero et al., 2004). Water was changed daily. The larvae were sieved through 200-250 μm-meshes and the 16 culture tanks were momentary concentrated in plastic vessels, from where triplicate subsamples were used to measure larval density on days 3, 5, 7 and 9. At the same time points, shell length and width (μm) were measured from ∼10 larvae per tank. For both the larval cultures, samples were fixed with 10% buffered formalin and larvae were photographed under a LED trinocular microscope (Kaves SL-700) connected to a camera (CANON, EOS 700D). Photographs were then examined using Image J (Schneider et al., 2012). As described in Li et al. (2013), abnormalities in the whelk larvae were assessed as a reduction of body size (abnormal soft body features) or as a reduced shell size compared to the body size, which impaired the complete withdrawal of the velum. Abnormalities in the oyster larvae were investigated after Ventura et al. (2016) and included phenotypes as indented margin, convex hinge, and cupped shells. 2.4. Whelk larval metabolic rates As previous trial experiments showed that Reishia clavigera larvae during their planktonic stages exhibited maximum oxygen uptake during the 10th day of development (Campanati, unpublished data), O2 consumption was measured between day 9 and 10 of development. Larvae were sieved from each culture tank during normal water changes and concentrated in glass beakers (200 ml). For each replicate, 50 to 150 larvae were manually transferred into triplicate glass vials (7 ml) filled with autoclaved natural FSW at the respective treatment pH and fitted with optical oxygen sensors (PSt3, Presens, Precision Sensing GmbH). Three vials per pH treatment, filled up with pre-adjusted FSW served as blanks. All the vials were sealed air-tight and placed on racks immersed in a thermostatically-regulated water bath in the dark. Oxygen concentration inside the vials was constantly measured for 24 h using a non-invasive Fibox4 system (PreSens, Precision Sensing GmbH). Larval oxygen uptake (MO2 = μmol O2 h−1 ind−1) as calculated from the difference in oxygen concentration throughout the time of incubation (linear slope). The value was corrected (blank values) and standardised by the number of larvae in the vessel. Oxygen in the vials did not drop below 70% saturation during the experiment.

3. Results 3.1. Seawater chemistry The nominal pH targeted for the experiments were reached in both the oyster and whelk cultures and were similar and consistent in time (delta pH from ∼0.35 to 0.39 to ∼0.72–0.76 units; Table 1; Table S2). The oyster culture system (5 L) was, however, subject to higher fluctuations of pH over time compared to the smaller whelk culture system (1 L) (Fig. S2), possibly due to the fact that in the latter the water was changed everyday rather than every other day as in the oyster experiment. In both experiments aragonite was undersaturated with respect to seawater (Ω < 1) at the lowest pH treatment (nominal pH 7.4; Table 1). Replicate tanks were included in the analyses as they improved the statistical mixed model, stabilising the model residual variance. The replicate tank effect accounted for ∼22 and ∼60% of total variance in models assessing differences in total alkalinity between treatments, in the oyster and whelk larval experiments respectively (Table S1). For the other seawater parameters % of the total variance associated to tanks was relatively low (ranging from 0.1 to 7.2%) (Table S1).

2.4.1. Data analyses Data were checked for normality and heteroscedasticity through visual inspection of the models' residuals, coupled with Shapiro-Wilk and Levene's tests before parametric tests such as ANOVA. All statistical analyses and graphics were performed in the R environment (R Core Team, 2015) with alpha set at 0.05. Differences in seawater chemistry parameters were tested among pH treatments with linear mixed models using a random intercept model with the lme function (nlme package in R; Lindstrom and Bates, 1990) with time as a repeated measure fixed factor and replicate tanks as a random factor. Models were compared by the Akaike Information Criterion (AIC). From the validated model with best fit, reported statistics for the factors of interest were obtained by nested models comparisons through log likelihood ratio test (anova function) following the procedures in Zuur et al. (2009) and described in Sunday et al. (2011). Percentage of total variance associated with fixed and random factors were obtained from the marginal and conditional effects, as proposed by Nakagawa and Schielzeth (2013), using the piecewise SEM package (Lefcheck, 2015). Larval mortality rate was estimated for each replicate

3.2. Larval mortality Larval mortality rate was estimated for each replicate as the regression coefficient of the significant logarithmic relationship between mortality (% of initial density) and days of development (Fig. S3; Table S2, S3). Only the replicate tanks with significant relationships (p < 0.05) between the larval density and time were used in the analyses (Table S2, S3). The mortality rate was not correlated with pH for the oyster larvae (Linear regression: F1,13 = 3.416; R2 = 0.21; p = 0.09; Fig. 1) but it was for the whelk larvae (Linear regression: F1,12 = 13.26; R2 = 0.52; p = 0.003; Fig. 1, Fig. S3; Table S3). The 3

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Table 1 Seawater carbonate chemistry parameters. pH (NBS), temperature (°C), salinity and total alkalinity (TA; μequiv kg−1) were used to calculate pCO2 (μatm), HCO3−1 (mmol kgSW−1), CO3−2 (mmol kgSW−1) and saturation state of calcite and aragonite (Ωca and Ωar). Mean ( ± SD) of the replicate culture tanks (n = 4) are presented for the seawater parameters. Calculated seawater parameters were obtained using the CO2SYS program (Pierrot et al., 2006) from total alkalinity and pH (NBS) with the dissociation constants of Mehrbach (1973) as refitted by Dickson and Millero (1987). Species

Saccostrea cucullata

Measured Nominal pH

pH

T(°C)

Salinity (ppt)

TA (μequiv kgSW-1)

pCO2 (μatm)

HCO3-1 (mmol kgSW-1)

CO3-2 (mmol kgSW-1)

Ω Ca

Ω Ar

8.1

8.17 ± 0.00 7.78 ± 0.01 7.61 ± 0.01 7.41 ± 0.01

28.42 ± 0.05 28.35 ± 0.08 28.36 ± 0.06 28.36 ± 0.02

30.47 ± 0.11 30.51 ± 0.02 30.52 ± 0.10 30.46 ± 0.07

2156 ± 54.34

404.35 ± 9.46

1678.81 ± 43.66

199.4 ± 5.59

3.31 ± 0.09

2176.13 ± 26.87 2160.74 ± 22.56 2165.33 ± 14.61

1148.45 ± 36.46 1843.30 ± 23.31 3057.98 ± 9.56

1945.76 ± 22.55

94.83 ± 3.46

2008.58 ± 20.40

62.60 ± 1.23

2066.78 ± 13.44

40.60 ± 0.58

5.03 ± 0.15 2.39 ± 0.09 1.58 ± 0.03 1.02 ± 0.01

8.14 ± 0.15 7.79 ± 0.03 7.61 ± 0.04 7.42 ± 0.04

27.85 ± 0.22 27.82 ± 0.22 27.84 ± 0.23 27.84 ± 0.25

31.86 ± 0.74 32.05 ± 0.75 32.12 ± 0.78 32.07 ± 0.77

2202 ± 73.94

436.81 ± 16.46 1162.49 ± 32.79 1725.87 ± 20.84 2762.04 ± 282.21

1728.92 ± 47.39

193.83 ± 13.44

1962.80 ± 36.54

93.53 ± 1.31

2016.16 ± 22.68

66.57 ± 1.43

2086.94 ± 24.16

44.85 ± 4.66

7.8 7.6 7.4

Reishia clavigera

Calculated

8.1 7.8 7.6 7.4

2190.67 ± 38.70 2178.41 ± 25.36 2196 ± 25.23

average mortality rate in the oyster larval culture was ∼72% log (day)−1. In the case of the whelk larvae, by the 10th day of development, ∼66% mortality was observed in control pH condition, ∼62% in pH 7.8, ∼52% in pH 7.6, and ∼93% in pH 7.4. The mortality rate was significantly higher in whelk larvae developed at pH 7.4 as compared to those raised at pH 7.8 (gls model fit by REML: b = −24.615; t(14) = 3.752; p = 0.004) and pH 8.1 (gls model fit by REML: b = −25.927; t (14) = -3.914; p = 0.003). No difference was, however, detected between pH 7.4 and pH 7.6 treatments, possibly as a result of the large variation within the pH 7.6 treatment condition (Fig. 1; gls model fit by REML: b = −5.705; t(14) = -0.713; p = 0.492).

4.83 ± 0.33 2.33 ± 0.03 1.66 ± 0.04 1.12 ± 0.12

1.58 ± 0.06 1.04 ± 0.02 0.67 ± 0.01 3.19 ± 0.22 1.54 ± 0.02 1.10 ± 0.02 0.74 ± 0.08

growth of the whelk larvae was obtained from the linear regression coefficients relating shell length and days of development (Fig. 2 and Tablel S6). There was a significant increase in growth rate for snail larvae developed at pH 7.6 compared to the other treatments (One-way ANOVA: F3,12 = 5.74; p < 0.05). The shell length and width allometric relationship for the whelk larvae was similar under different pH conditions (Fig. 3B and Table S8; KW: χ2 = 5.91, d.f. = 3, p > 0.05). At the lowest pH treatment (pH 7.4), various abnormalities were observed in the whelk larvae (Fig S5). At day 9, up to 62% malformed larvae were found at pH 7.4, compared to 7% in pH 7.6 larvae, and none in pH 8.1 and pH 7.8. No abnormalities were detected in any oyster larvae treatment. Scanning electron microscopy (SEM) of larval shell surfaces revealed signs of dissolution in the embryonic portion of the shell for both oyster and whelk larvae exposed to low pH conditions (Fig. S7, S8). The larval portion of the shell, however, only greatly impacted (with pitting and cracks) in the whelk larvae (Fig. S7, S9).

3.3. Larval growth Shell growth of the oyster larvae (obtained from the linear relationship coefficients between log-log area and days of development) did not differ among pH treatments (Fig. 2 and Table S4; KW: χ2 = 2.93, d.f. = 3, p > 0.05). Allometries of the oyster larval shell dimensions (shell length and width) were also not affected by pH (Fig. 3A and Table S7; KW: χ2 = 3.57, d.f. = 3, p > 0.05). Shell

3.4. Snail larval metabolic rates Whelk larval metabolic rates, as measured by oxygen uptake (μmol

Fig. 1. Mortality rate of oyster and whelk larvae exposed to different pH treatments (Nominal pH). Median and confidence interval (median ± 1.57 × IQR/√n) are shown, with n = 4 replicate tanks in all treatments but n = 3 in the oyster larvae cultured at pH 7.4 and n = 2 in the whelk larvae cultured at 7.6 pH treatment. 4

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Fig. 2. Shell growth rate of oyster (left) and whelk (right) larvae developed under different pH treatments (Nominal pH). Median and confidence interval (median ± 1.57 × IQR/√n) of four replicate tanks are shown. The accretions of the respective shell dimensions over time are reported in Fig S4.

O2 h−1ind−1), were affected by pH (linear mixed model with random intercept: χ2(1) = 11.24; p < 0.001). Particularly, the oxygen consumption of larvae developed at pH 7.4 significantly differed from the other treatments, being ∼3 times higher (Fig. 4). The replicate tank was included as random factor in the linear mixed model as it brought a significant improvement to the statistical model (i.e. lower AIC; homogeneous variances; Log-likelihood ratio test: χ2(1) = 6.56; p = 0.01). However, the replicate tank had little influence on the results: the variance for the random factor was estimated as d2 = 4.75 × e−5, with a residual variance estimated as σ2 = 4.12 × e−5.

of the Sydney rock oyster, S. glomerata, raised under decreased pH conditions (Watson et al., 2009; Parker et al., 2012). Reduced pH (−0.2 and −0.5 pH units) also decreased the larval size and delayed metamorphosis in the Eastern oyster, Crassostrea virginica, and its survival was significantly reduced after 10 days of exposure to pH 7.5 (∼-0.5 pH units from control; Talmage and Gobler, 2009). From an interspecific comparison between the early life stages of the Pacific oyster, Crassostrea gigas, and the Sydney rock oyster, Saccostrea glomerata, it emerged that C. gigas, which is characterized by a relatively faster growth and a broader biogeographic distribution, was less impacted by OA (Parker et al., 2010). Although acidified waters negatively affected both species, the impact on the fertilization success and larval development was more pronounced in S. glomerata larvae (Parker et al., 2010). Moreover, selectively bred S. glomerata oysters, which grew fast and had high resistance to diseases, produced offspring which were less impacted by acidified waters compared to wild oyster populations (Parker et al., 2011). From this study, S. cucullata larvae appear more tolerant to OA than other oyster species. Only the embryonic portion of the shell (prodissoconch I) was altered in the oyster larvae exposed to pH 7.4 (Ωara < 1), which might derive from periostracum maintenance and dissolution rather than calcification impairments. The oyster larvae high tolerance to pH down to pH 7.4 can explain its large geographical distribution. Possibly native from India, S. cucullata has a broad distribution, as it extends from the South African coasts to the Pacific waters of Guam, occurring along the Chinese and Philippines coasts, and it stretches across latitudes from Japan to Australia and New

4. Discussion 4.1. Oyster larvae response to ocean acidification The larvae of the oyster, Saccostrea cucullata, survived and exhibited normal development at the same rate throughout the range of pH tested. They appear more tolerant to increased acidification as compared to the Australian congeneric, S. glomerata, in which survival in the first week of development was significantly reduced at pH 7.8 (pCO2 ∼500 μatm; Watson et al., 2009). Almost 100% of abnormal D-shape larvae were found in cultures of S. glomerata exposed to a reduction of 0.4 pH units from control conditions (pCO2∼1000 μatm; Parker et al., 2009). In the present study, no change in morphology was observed in the shells of the oyster larvae developed under low pH, while in contrast the shell length and width were significantly smaller in the larvae

Fig. 3. Allometries of shell morphometrics (shell length vs. shell width) in oyster and whelk larvae developed under different pH treatments. Linear relationships between the shell dimensions for all the four replicate tanks in each treatment are shown. Black round shaped points fitted with the continuous line represent control conditions (pH = 8.1); Squared points and long-dashed lines represent larvae developed at pH = 7.8; White round shaped points and dashed line larvae at pH = 7.6; Triangles and dotted line represent larvae at pH = 7.4. The regression coefficient, intercept, pvalue, R2, F-value and df are given for each replicate culture tank in Table S5 and S6.

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whelk larvae under pH 7.6. This could be beneficial for whelk larvae and could possibly compensate for the higher rates of mortality. On the other hand, faster larval growth in this treatment could be the result of selective mortality of slow growing cohorts at this pH level (Rumrill, 1990). In a study that separated the genetic and physiological components of differential growth rates in oyster larvae, Pace et al. (2006) found that most of the differences in growth rates between fast-and slow-growing larvae could be explained by different feeding rates and differential energy allocation of the individuals. It is possible that, as the larval density decreased, due to high mortality in some treatments (e.g. pH 7.4 and 7.6), then the surviving larvae would have reduced competition for food and so grown faster. In our study, however, microalgae in each culture tank were provided ad libitum, and, thus, it is unlikely there was any competition between larvae for food, which might have affected growth. However, it is possible that whelk larvae at this pH level could have increased their feeding rates (e.g. Pace et al., 2006), as shown in other studies (e.g. Melzner et al., 2011; Campanati et al., 2015; Ramajo et al., 2016). Another explanation is that larvae developed at pH 7.6 could have had substantial differences in energy allocation at the cellular level, without apparent changes in oxygen consumption rates (e.g. Pan et al., 2015). As commonly found for larvae of other molluscan larvae (e.g. Zhang et al., 2014; Waldbusser et al., 2015), we showed that whelk larvae had higher metabolic rates when raised at pH 7.4, which deviated from natural pH range at the sampling site by ∼0.2 units. Increased metabolic rates under elevated pCO2/low pH levels have been hypothesized to help restore the acid-base balance in the extracellular medium (Pörtner, 2012; Gibson et al., 2012). The increase in metabolic rates could, therefore, reflect increased energy costs to maintain shell size and growth rate at the lowest pH (Day et al., 2000). Subtle increases in the metabolism of planktotrophic larvae involved in shell calcification could also reduce the energy reserves critical for larval survival (Miller et al., 2009). At the lowest pH conditions (pH 7.4), the increased metabolic rate of R. clavigera larvae was, in fact, accompanied by a significantly increased mortality rate and at day 9 of development, up to 60% of the whelk larvae at pH 7.4 were malformed. Shell and body abnormalities have been often found in larval stages of molluscs exposed to low pH/elevated pCO2 conditions (Kurihara et al., 2007; Barros et al., 2013; Li et al., 2013; Ventura et al., 2016). Malformations in abalone larvae ranged from 20% in Haliotis discus hannai exposed to pCO2∼1650 μatm (ΔpH ∼ −0.45 units from control; Kimura et al., 2011) to 40% in Haliotis kamtschatkana exposed to pCO2∼800 μatm (ΔpH ∼ −0.2 units from control; Crim et al., 2011). These morphological abnormalities may affect the larval swimming behaviour and increase their vulnerability to predation in the plankton (e.g. Chan, 2012; Zhang et al., 2014; Chan et al., 2015). Furthermore, micro protuberances in gastropod veligers shells (Fig. S6, S7, S8) result from more remote biomineralization (Hickman, 1999), which may partly explain the drastic difference observed in this study in the shell surface between larvae developed in seawater conditions above aragonitic saturation state (Ωara > 1) compared to those in Ωara < 1. At pH 7.4, both the embryonic and larval portion of the veliger shell (protoconch I and II) showed fewer micro protuberances. Although the link between these shell features and their functional traits is still unknown, the impact of acidified conditions on the whelk veliger shells could have further mechanical consequences on the shell structure (Hickman, 1999).

Fig. 4. Metabolic rate of 9-day old whelk larvae exposed to different pH conditions (Nominal pH). Values (mean ± SD) represent the mean of n = 4 replicate culture tanks. Each culture tank was comprehensive of 3 glass vials with 50–150 larvae each. Asterisks represent significant differences (linear mixed model and post hoc test, p < 0.001).

Zealand (Chiu, 1997 and ref therein). More recently putative S. cucullata species appeared to be introduced also in the Mediterranean Sea (Galil and Zenetos, 2002), Hawaii (Coles et al., 1999) and along the Caribbean coasts (Lohan et al., 2015).

4.2. Whelk larvae response to ocean acidification Increased mortality, shell abnormality and respiration rates were observed in the whelk, R. clavigera, larvae developed under low pH conditions. This is consistent with observations in the larval stages of other gastropods (Gazeau et al., 2013). For example, larvae of the muricid, Concholepas concholepas, reared under pH 7.5 (pCO2 levels ∼1000 μatm) since their embryonic development within the egg capsules, suffered up to 30% mortality in the first 96 h post-hatching (Manríquez et al., 2014). This low-pH treatment also increased the time to hatch and induced a lower hatching success, although geographic variability and developmental plasticity were also observed (Manríquez et al., 2014). Reduced survival due to acidified conditions (pCO2∼1260 μatm) was also found in scavenger gastropod larvae over a period of 3 days exposure (Zhang et al., 2014). A 20% reduction in embryos viability was observed in Littorina obtusata, exposed to seawater pH 0.5 units lower than control conditions (Ellis et al., 2009), whereas a decrease in 0.2 and 0.5 pH units reduced the survival of the endangered abalone (Haliotis kamtschatkana) larvae by 43% as compared to control conditions (Crim et al., 2011). As energy is oftentimes devoted to sustain maintenance costs under stress, rates of larval development and larval size have been affected by acidified conditions in many shelled molluscs (Gazeau et al., 2013). For example, the protoconch growth rate in veligers of Crepipatella dilatata, which developed under lower pH environments, experienced within the brood chamber, was significantly reduced (Montory et al., 2009). Shell morphology and size were also negatively impacted by elevated pCO2 in Haliotis kamtschatkana larvae developed at ΔpH of −0.2 and −0.5 pH units from control conditions (Crim et al., 2011). In this study we observed a subtle increase in the shell growth of

4.3. Comparison between oysters and whelk responses to ocean acidification Several recent articles highlight the key role of species' niche and local adaptation to sensitivity to OA (e.g. Dorey et al., 2013; Thor and Dupont, 2015; Ventura et al., 2016). Re-evaluating the literature on the impact of decreased pH on several species and populations along the coast of Chile, Vargas et al. (2017) showed that species/population sensitivity to pH was dependent on present natural variability. As a 6

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consequence, a population tipping point can be hypothesized to be close to the extremes of the present range of natural variability. In the present study, we hypothesized that negative effects of decreased pH should appear at pH lower than 7.6 for both species as they reproduce at the same time and then experience the same natural variability. However, this hypothesis was only valid for the whelk larvae, as oyster larvae appeared to withstand low pH better without any apparent plastic adjustment in their shell morphology. There are several plausible explanations for this difference between species. Firstly, studies suggest that gastropod larvae might be more vulnerable to future ocean changes than bivalve veligers (Byrne et al., 2011). Secondly, the two species have different modes of embryonic development: The rock oyster is a broadcast spawner, with embryonic development occurring in the water column in a relative short time (∼22 h; Sukumar and Joseph, 1988) whereas the whelk females lay eggs into capsules (∼400 eggs per capsule; Tong, 1988), where the embryos develop for longer time (∼20 days at 28 °C; pers. obs.). The protective benefits of the gastropod egg capsules where the embryos develop seem to be less than previously assumed (Rawlings, 1999; Montory et al., 2009; Noisette et al., 2014). Longer developmental time and reduced viability were found, for example, in encapsulated embryos of the intertidal snail Littorina obtusata exposed to pH 7.6 (Ellis et al., 2009). Snail larvae hatched under OA conditions showed impacts on shell morphology (Ellis et al., 2009), with sometimes-higher frequency of abnormalities (Noisette et al., 2014). To avoid confounding factors associated with the time in the egg capsules and to only test for the larval vulnerabilities, we exposed the organisms to decreased pH conditions from the day of larval hatching. Finally, the tested oyster species have a wider distribution range compared to the whelk (see Guo et al., 2015), suggesting a general higher tolerance to stress that may limit the potential for local adaptation (Lenz et al., 2011). Oyster larvae, however, experienced greater mortality compared to the whelk larvae. As the species used in this study were not broadly used as model species in laboratory experiments and standard larval culture protocols are not available (e.g. Sukumar and Joseph, 1988), we could not exclude the fact that this result might derive from a better capacity of the whelk larvae to withstand laboratory conditions as compared to the oyster larvae. Alternatively, a potential explanation would be related to the differences in growth rates and sizes, as well as evolutionary and life-history strategies (i.e. different embryonic development) in the two species.

The authors would like to thank Khan Cheung and Fiona Chong for their help during animal collection. We are grateful to two anonymous reviewers who offered valuable comments on the previous version of the manuscript. This work was primarily supported by a grant from the Research Grant Council (RGC) of Hong Kong SAR, China (HKSAR-RGC, No. 17303517) and partially supported by a seed grant from the State Key Laboratory of Marine Pollution (SKLMP). Appendix A. Supplementary data Supplementary data related to this chapter can be found at https:// doi.org/10.1016/j.marenvres.2018.08.005. References Arnold, W.S., 2008. Application of larval release for restocking and stock enhancement of coastal marine bivalve populations. Rev. Fish. Sci. 16 (1–3), 65–71. Barros, P., Sobral, P., Range, P., Chícharo, L., Matias, D., 2013. Effects of sea-Water acidification on fertilization and larval development of the oyster Crassostrea Gigas. J. Exp. Mar. Biol. Ecol. 440, 200–206. 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5. Conclusions The survival and responses of key coastal foundation species are critical for the persistence of biodiversity in near-shore ecosystems (Harley et al., 2006; Gaylord et al., 2011; Harley, 2011; Cheng et al., 2015). By comparing the larval responses of Saccostrea cucullata and its predator, Reishia clavigera, we found that the prey larvae were more resistant to lower pH conditions as compared to those of the predator. Unless the whelk population would rely on other/external sources of larval pool, the OA-induced increased mortality, shell abnormalities and energetic demand, as well as altered growth rates, found in the larval stage, could affect its recruitment on the shore: A bottleneck at this early stage has, in fact, the potential to compromise the persistence of the predator population and, thus, alter the balance of the whole ecosystem (Schneider et al., 2003; Arnold, 2008). This hypothesis needs to be tested and longer term impacts on both species (e.g. transgenerational effects; Parker et al., 2009, 2012; Lane et al., 2015) and their interaction should be further evaluated. Author Contributions. C.C. designed and carried out experimentations, V.T. provided experimental resources, C.C. lead the co-writing of the manuscript with S.D., G.A.W. and V.T. Declaration of interests: none.

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