CBA-10018; No of Pages 10 Comparative Biochemistry and Physiology, Part A xxx (2016) xxx–xxx

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Seawater acidification affects the physiological energetics and spawning capacity of the Manila clam Ruditapes philippinarum during gonadal maturation Xian Xu, Feng Yang ⁎, Liqiang Zhao ⁎,1, Xiwu Yan ⁎ Engineering Research Center of Shellfish Culture and Breeding in Liaoning Province, Dalian Ocean University, Dalian 116023, China

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Article history: Received 23 October 2015 Received in revised form 15 February 2016 Accepted 22 February 2016 Available online xxxx

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Ocean acidification is predicted to have widespread implications for marine bivalve mollusks. While our understanding of its impact on their physiological and behavioral responses is increasing, little is known about their reproductive responses under future scenarios of anthropogenic climate change. In this study, we examined the physiological energetics of the Manila clam Ruditapes philippinarum exposed to CO2-induced seawater acidification during gonadal maturation. Three recirculating systems filled with 600 L of seawater were manipulated to three pH levels (8.0, 7.7, and 7.4) corresponding to control and projected pH levels for 2100 and 2300. In each system, temperature was gradually increased ca. 0.3 °C per day from 10 to 20 °C for 30 days and maintained at 20 °C for the following 40 days. Irrespective of seawater pH levels, clearance rate (CR), respiration rate (RR), ammonia excretion rate (ER), and scope for growth (SFG) increased after a 30-day stepwise warming protocol. When seawater pH was reduced, CR, ratio of oxygen to nitrogen, and SFG significantly decreased concurrently, whereas ammonia ER increased. RR was virtually unaffected under acidified conditions. Neither temperature nor acidification showed a significant effect on food absorption efficiency. Our findings indicate that energy is allocated away from reproduction under reduced seawater pH, potentially resulting in an impaired or suppressed reproductive function. This interpretation is based on the fact that spawning was induced in only 56% of the clams grown at pH 7.4. Seawater acidification can therefore potentially impair the physiological energetics and spawning capacity of R. philippinarum. © 2016 Published by Elsevier Inc.

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Keywords: Ocean acidification Physiology Energy budget Manila clam

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Rapidly increasing anthropogenic CO2 emissions are resulting in ocean acidification and global warming. These environmental changes may have serious consequences for a variety of marine organisms. For example, elevated seawater pCO2 levels, and the associated reductions in pH, are predicted to affect essential physiological processes (e.g., acid-base regulation, oxygen transport, and metabolic rate), ultimately affecting growth and reproduction, and even survival in extreme cases (Pörtner et al., 2004; Orr et al., 2005; Doney et al., 2009; Kroeker et al., 2013). While our current understanding of the impact of future global climate change scenarios on overall individual organism performance is increasing, to date, relatively little is known about the potential for multigenerational adaptation of marine biota to such persistent environmental changes (Somero, 2010; Kelly and Hofmann, 2013). However, such knowledge is crucial to predict how marine ecosystems

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1. Introduction

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⁎ Corresponding authors. E-mail addresses: [email protected] (F. Yang), [email protected] (L. Zhao), [email protected] (X. Yan). 1 Present address: Institute of Geosciences, University of Mainz, Mainz 55128, Germany.

will be altered in the context of global climate change (Dupont and Pörtner, 2013). The potential for fecundity loss is one of the most serious consequences of future global climate change scenarios and may affect the long-term sustainability of individual populations (Parmesan, 2006; Pörtner, 2008; Berg et al., 2010). Despite species-specific differences, a reduction in fecundity is likely to reflect an increase in the energetic costs required to compensate for ocean acidification impacts. This interpretation is consistent with the observations of Stumpp et al. (2012), according to which increased energetic costs resulted in significantly reduced gonadal development in adult sea urchins (Strongylocentrotus droebachiensis) when exposed to 2840 μatm pCO2. According to Podolsky and Moran (2006), females under increased stress such as that predicted under climate change scenarios may respond through increasing maternal energetic investment-per-offspring (e.g., increasing egg size) in order to favor larval performance. However, to ensure survival, they must allocate a large amount of energy to accommodate these stressful challenges (Wingfield and Sapolsky, 2003), consequently limiting the energy available for reproductive processes (Petes et al., 2008). Reproduction is energetically expensive and is therefore inevitably compromised under stressful conditions. Bibby et al. (2008), for

http://dx.doi.org/10.1016/j.cbpa.2016.02.014 1095-6433/© 2016 Published by Elsevier Inc.

Please cite this article as: Xu, X., et al., Seawater acidification affects the physiological energetics and spawning capacity of the Manila clam Ruditapes philippinarum during gonadal maturation..., Comp. Biochem. Physiol., A (2016), http://dx.doi.org/10.1016/j.cbpa.2016.02.014

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2. Materials and methods

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2.1. Adult collection and maintenance

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Wild-type adult R. philippinarum specimens (32.2 ± 2 mm shell length) were collected from the intertidal shores of Liangshui Bay (39°04′14.41″ N, 122°01′47.70″ E), Liaodong Peninsula, Northeast China, in April 2014. Environmental conditions at the sample collection site were as follows: temperature 8.6 °C, salinity 32.4, and pH (total scale) 8.18. Clams were transported to an aquarium facility where they were allowed to acclimate to laboratory conditions for 2 weeks prior to experimentation. Clams were held in recirculating seawater that mimicked natural conditions. Temperature, salinity, pH, and light:dark cycles were maintained at constants of 9 °C, 32, 8.1, and 12 h:12 h, respectively. They were fed twice daily with an equal mixture of the microalgae Chaetoceros muelleri, Nitzschia closterium, Chlorella vulgaris, and Isochrysis galbana to fulfill their nutritional requirements for gonadal development. Furthermore, given that food availability may counteract the influence of ocean acidification on marine bivalves (Thomsen et al., 2012), a constant food supply (ca. 20,000 cells/mL) was maintained over the duration of the experiment. In the present study, histological analysis indicated that 63% of clams were in the early developmental stage prior to experimentation.

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2.2. Experimental setup

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To assess the influence of seawater pH, three separate, but identical, recirculating systems were established, as outlined in Fig. 1. Each system comprised three exposure chambers (i.e., three replicates), a filter chamber, a temperature-controlling chamber, and a gas mixing chamber, and each held a total volume of ca. 600 L seawater. In these experimental systems, elevated seawater pCO2, and the associated reduced pH, were maintained by vigorously bubbling pure CO2 gas into the gas mixing chamber and monitored using a digital pH controller. Gas flow was adjusted using a mass flow controller. At the same time, a constant rate of fresh air was supplied into the gas mixing chamber. After ca. 36 h of equilibration, the whole system reached a steady state. Two low pH conditions, 7.7 and 7.4, were successfully established, corresponding to the predicted levels for 2100 and 2300 (Caldeira and Wickett, 2003), respectively. The control system was bubbled with ambient air only. Temperature targets in each system were controlled in the temperature controlling chamber using a digital temperature regulator. Clams were randomized into three groups and assigned into either the control or experimental system at a density of 150 individuals per exposure chamber. During the subsequent 2 weeks, they were acclimated to the experimental conditions; temperature was kept at a constant level of 10 °C and pH was slowly adjusted to target levels (ca. 0.5 pH unit per day). Over the following 30 days (from May 1st to 30th, 2013), clams were subjected to increasing temperature regimes. In all

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evidence suggests that the feeding activity of many bivalves is positively related with temperature up to an optimum, beyond which it declines sharply (e.g., Beiras et al., 1995; Sobral and Widdows, 1997; GuzmánAgüero et al., 2013). The thermal optimum for R. philippinarum is 20 °C (Han et al., 2008). Therefore, in the present study, we adhered to a previously established protocol for artificial induction of gonadal maturation as follows: (1) progressive temperature increases from 10 to 20 °C within 30 days (ca. 0.3 °C per day) and (2) over the following 40 days maintaining a constant temperature of 20 °C. After 70 days of artificial ripening, clams were then induced to spawn by thermal shock. During the experiment, physiological energetics were measured in terms of key physiological parameters: energy acquisition (from ingestion and digestion), energy expenditure (from respiration and ammonia excretion), and energy budget (i.e., scope for growth). In addition, spawning rate was utilized as an estimation of clam reproductive response at the end of the experiment.

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example, demonstrated that exposure to elevated pCO2 between 1160 and 3316 μatm alters the reproductive condition of blue mussels (Mytilus edulis), forcing them to reabsorb their gametes as an energysaving or survival strategy. Impaired or suppressed reproduction can have serious detrimental consequences for recruitment and population dynamics and, in the most extreme cases, species persistence (Berg et al., 2010). Therefore, predicting the long-term severity of ocean acidification on the reproductive success of marine organisms is crucial for gaining a better understanding of the energetic trade-offs between stress resistance and reproduction (Petes et al., 2008; Sokolova et al., 2012). The Manila clam, Ruditapes philippinarum, is an ecologically and economically significant molluscan species, widespread in intertidal and shallow subtidal habitats. This species is extensively cultivated and forms the largest and oldest aquaculture industry in China, Japan, and Korea (Zhang and Yan, 2006; Uddin et al., 2012). For aquaculture purposes, this species has been introduced on the west coast of North America and in European countries (e.g., Portugal, Italy, France, and Spain) and has subsequently spread rapidly (Gosling, 2003). In all regions, R. philippinarum has proven readily adaptable to various coastal environments and its sustainability for aquaculture practices has been demonstrated (Drummond et al., 2006; Zhang and Yan, 2006; Dang et al., 2010). While the potential for significant ecological and economic impacts of future scenarios of anthropogenic environmental change on Mytilus species has been widely recognized (Michaelidis et al., 2005; Range et al., 2012; Navarro et al., 2013; Múgica et al., 2015; Wang et al., 2015), R. philippinarum has received relatively little attention. Recent studies have shown that sediment acidification due to CO2 leakage from sub-seabed storage greatly facilitates heavy metal bioaccumulation in R. philippinarum (e.g., Rodríguez-Romero et al., 2014; Basallote et al., 2015). In a study of its close relative, Ruditapes decussatus, Range et al. (2011) hypothesized that increased survival of clams exposed to elevated pCO2 of 3702 μatm may be associated with a delay in their reproductive processes. This hypothesis is supported by the observation of reduced energy acquisition (i.e., feeding activity) in R. decussatus exposed to the same pCO2 level of 3702 μatm (Fernández-Reiriz et al., 2011). However, despite its ecological and cultural significance, the reproductive performance of R. philippinarum in the context of global climate change, to the best of our knowledge, has not yet been investigated. Considering the available data from previous studies and the highly variable environments clams inhabit, it is conceivable that clam reproduction may respond plastically to increasing environmental stress, potentially allocating energy for reproduction to costly physiological defenses, that in extreme cases, translates to impaired or suppressed reproductive function. An improved understanding of the reproductive response of R. philippinarum under future climate change scenarios is therefore crucial for evaluating its population dynamics. The aim of the present study was to experimentally evaluate the potential effect of seawater acidification on the physiological energetics of R. philippinarum during gonadal maturation. For this purpose, reproductive control was a prerequisite. Out of all environmental parameters, temperature is considered the major forcing and timing factor in the regulation of marine bivalve gonadal development and spawning cycles (Gosling, 2003). In the Yellow Sea, for example, sea surface temperature (SST) ranges from 3.2 °C in February to 25.2 °C in August. Uddin et al. (2012) observed that the onset of gametogenesis in female Manila clams was initiated in February when the SST is 3.2 °C. Clams in the early developmental stage were dominant in March (7 °C) and April (12 °C). Ripe clams were first observed in May (17 °C) and the proportion of clams in the ripe stage rapidly increased to 56.5% in July (22 °C). The first spawning clams were observed in May and, in August and September, spawning activity reached its annual peak when SST ranged between 25.6 and 24.7 °C. A continuous energy supply is essential for the continuous ripening of gonads, i.e., clams need to actively filter more food. Accumulating

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Please cite this article as: Xu, X., et al., Seawater acidification affects the physiological energetics and spawning capacity of the Manila clam Ruditapes philippinarum during gonadal maturation..., Comp. Biochem. Physiol., A (2016), http://dx.doi.org/10.1016/j.cbpa.2016.02.014

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2.3. Physiological measurements

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Four major physiological parameters, including CR, absorption efficiency (AE), respiration rate (RR), and excretion rate (ER) were measured, and the ratio of oxygen to nitrogen (O:N) and scope for growth (SFG) were calculated accordingly. The recirculating system was terminated and the clams were allowed to depurate for 12 h prior to measurements. Six randomly selected individual clams from each exposure chamber (replicate) for each system were measured on days 10, 30, 44, and 70. After all measurements were completed, the clams tested were dissected, and all of the soft tissues and shells were then dried in an oven at 60 °C for 48 h to obtain their dry weight. Following this, the general condition index (CI) of each clam was calculated as follows (Lucas and Beninger, 1985):

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systems, temperature was simultaneously increased ca. 0.3 °C per day from 10 to 20 °C. From May 31th (on day 31), clams were maintained at 20 °C for 40 days. Throughout the duration of the experiment, constant low pH conditions were maintained. Temperature, salinity, and pH were recorded every day. Half of the water in each system was replenished every 4–5 days to maintain water quality. Changes in water chemistry between two renewals were monitored. Water samples for total alkalinity (TA) measurements were filtered through 0.45 μm membranes and immediately frozen at −20 °C until measurements. TA was measured using an alkalinity titrator. Temperature, salinity, pH, and TA were used to calculate the carbonate system parameters using the CO2SYS software program according to Pierrot et al. (2006). Clams were fed with concentrated microalgae once or twice daily based on clearance rates (CR) in order to minimize the potential impact of high phytoplankton biomass on seawater carbonate chemistry. Mortality in each system was recorded every day.

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CR ¼ ðVÞ  ð ln C1 – lnC2 Þ=ðTÞ where CR is the clearance rate (L h–1 ind–1), V is the volume (L) of fil- 252 tered seawater used in the aquarium (3 L), C1 and C2 are the algal cell concentrations (cells L–1) at the beginning and end of the measure- 253 ments, and T is the time elapsed (2 h). 254 2.3.2. Absorption efficiency AE is the measurement of the amount of organic matter absorbed by the clams from ingested food. After the CR experiments, feces produced by clams from each aquarium were collected using a pipette. Food and feces samples were filtered through pre-ashed (at 450 °C) and preweighed Whatmann® GF/C filter papers, and rinsed with distilled water. Following this, the filters were dried in an oven at 110 °C for 24 h, cooled in desiccators and weighed, combusted in a muffle furnace at 450 °C for 6 h, cooled, and then weighed again to obtain the dry weight and ash-free weight. AE was calculated following the ratio method of Conover (1966):

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AE ¼ ð F–EÞ=ð½1–E  EÞ  100

CI ¼ dry soft tissue massðgÞ=dry shell massðgÞ  100

where AE is the absorption efficiency (%), F is the ratio of ash-free 267 weight to dry weight for the algae, and E is the ratio of ash-free weight to dry weight for feces. 268

2.3.1. Clearance rate CR is the measurement of the volume of water cleared of suspended particles per unit of time. Clams were removed individually from each exposure chamber and placed in a 5 L aquarium filled with 3 L of filtered

2.3.3. Respiration rate RR, the rate of oxygen consumption by clams, was measured in a closed glass respirometer (500 mL) filled with oxygen-saturated natural seawater. To ensure that the clams had resumed respiration, the

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seawater. After 30 min acclimation, 60 × 106 cells mL–1 of I. galbana was added to the aquarium. In a preliminary experiment, this algal concentration determined the threshold concentration for the prevention of pseudo-faeces production. One identical aquarium without a clam was simultaneously used as the blank in order to account for the sedimentation of algae. In each aquarium, water samples were taken every 30 min over a period of 2 h. Algal cell concentration was determined using a haemocytometer. CR was calculated using the following equation, slightly modified from Coughlan (1969):

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Fig. 1. Overview of the exposure setup used in the present study.

Please cite this article as: Xu, X., et al., Seawater acidification affects the physiological energetics and spawning capacity of the Manila clam Ruditapes philippinarum during gonadal maturation..., Comp. Biochem. Physiol., A (2016), http://dx.doi.org/10.1016/j.cbpa.2016.02.014

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RR ¼ ðVÞ  ðC1 –C2 Þ=ðTÞ 282 283 284 285 286 287 288 289 290 291 292

where RR is the respiration rate (mg O2 h–1 ind–1), C1 and C2 are the oxygen levels (mg O2 L−1) at the beginning and end of the experiment, respectively, and T is the time elapsed (1 h). 2.3.4. Excretion rate ER was measured immediately after completion of the RR measurements. Individual clams were placed in a sealed beaker filled with 800 mL of filtered seawater to measure ammonia–nitrogen excretion. One additional sealed beaker without a clam served as the blank. Water samples (50 mL) were collected initially and at the end of 1 h, and analyzed for ammonia–nitrogen excretion according to the phenol–hypochlorite method (Solorzano, 1969). ER was calculated using the following equation:

3. Results

ER ¼ ðVÞ  ðC1 –C2 Þ=ðTÞ

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2.3.5. Oxygen to nitrogen ratio The measured clam RR and ER values were used to calculate the atomic ratio of oxygen utilized to nitrogen excreted (O:N) following the equation of Widdows (1985): O:N ¼

h i  –1 –1 mg O2 h =16Þ=ðmg NH4 ‐N h =14

2.3.6. Scope for growth SFG was calculated using the energy balance equation given by Widdows and Johnson (1988) after converting all physiological parameters into energy equivalents:

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Continuous monitoring of the recirculating systems revealed that temperature and pH targets were successfully reached and constantly maintained throughout the experiment (Fig. 2). Seawater carbonate chemistry parameters are shown in Table 1. TA ranged from 2097 to

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3.1. Seawater chemistry, mortality, and condition index

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where ER is the ammonia–nitrogen excretion rate (mg NH4–N h–1 ind–1), C1 and C2 are the ammonia levels (mg NH4–N L–1) at the beginning and end of the measurements, respectively, and T is the time elapsed (1 h).

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to the analysis, all data were checked for normal distribution (Shapiro– Wilk's test) and homogeneity of variances (Bartlett's test). Following this, within each sampling day, the potential effect of pH on CI was analyzed using one-way analysis of variance (ANOVA) with pH as a fixed factor, while pH effects on physiological parameters (e.g., CR, RR, ER, O:N, and SFG) were examined by means of one-way analysis of covariance (ANCOVA), with dry soft tissue mass of each clam as a covariate and pH as a factor. To evaluate the combined effects of temperature and pH, and their interaction, on physiological clam responses, data collected on days 10 and 30 were analyzed by means of two-way ANCOVA with dry soft tissue mass of each clam as a covariate, and pH and temperature as fixed factors. From day 30, when a constant temperature was maintained, the effects of pH and experimental duration (day), and their interaction, were evaluated by means of two-way ANCOVA with dry soft tissue mass of each clam as a covariate, and pH and experimental day (i.e., day 30, 44, and 70) as fixed factors. The effects of temperature and pH (and the effects of pH and experimental duration) on CI were examined by means of two-way ANOVA, with temperature and pH (pH and experimental duration) as fixed factors. At the end of the experiment, one-way ANOVA was used to test whether pH had a significant effect on spawning rate. Data were expressed as mean ± standard deviation (SD). A statistically significant difference was accepted when p b 0.05.

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experiment was initiated when their siphons had drawn out. The respirometer was then sealed off for 1 h. One respirometer without a clam was used as the blank, simultaneously. Water samples were taken from each respirometer at the beginning and end of the experiment, and the rate of oxygen reduction was measured using an oxygen meter (YSI 58) with a Clark type electrode. Oxygen consumption in each respirometer did not fall below 75% of saturation after measurements. RR was calculated using the following equation:

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where SFG is the scope for growth (J h–1 ind–1), A is the total absorbed energy (J h–1 ind–1), R is the energy lost in respiration (J h–1 ind–1), and U is the energy lost in excretion (J h–1 ind–1).

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    –1 –1  POM mg L–1  23:5 J mg–1  AR A ¼ CR L h ind   –1 –1  14:3 J mg–1 O2 R ¼ RR mg O2 h ind   –1 –1  25 J mg–1 NH4 ‐N U ¼ ER mg NH4 ‐N h ind SFG ¼ A–ðR þ UÞ

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2.3.7. Spawning rate On day 70, a total of 100 clams randomly selected from each exposure chamber for each system were induced to spawn by thermal shock (+4°°C; Zhang and Yan, 2006). The number of spawning individuals was recorded and the percentage of spawners was calculated. We considered only the overall percentage of spawning individuals within each system within 8 h.

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Statistical analyses were performed using the Statistical Package for Social Sciences for Windows (version 17.0; SPSS, Chicago, IL, USA). Prior

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Fig. 2. Continuous monitoring of seawater temperature and salinity (A), and pHT (B) in the recirculating systems throughout the 70 days of the experiment. Considering that temperature manipulations were made simultaneously in control and experimental systems and salinity was rather stable, the average of temperature and salinity in all three systems was shown in Fig. 2A.

Please cite this article as: Xu, X., et al., Seawater acidification affects the physiological energetics and spawning capacity of the Manila clam Ruditapes philippinarum during gonadal maturation..., Comp. Biochem. Physiol., A (2016), http://dx.doi.org/10.1016/j.cbpa.2016.02.014

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Table 1 Seawater carbonate chemistry throughout the experiment. Temperature, salinity, and pHT were measured every day, and total alkalinity (TA) every four or five days. Partial pressure of CO2 in seawater (pCO2), dissolved inorganic carbon (DIC), saturation state of calcite (Ωcal), and aragonite (Ωara) were calculated from the measured temperature, salinity, pHT, and TA. Temperature and salinity values are shown in Fig. 2.

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8.0 7.7 7.4

7.96 ± 0.04 7.70 ± 0.02 7.39 ± 0.03

2291 ± 92 2275 ± 68 2252 ± 85

505 ± 63 990 ± 95 2086 ± 120

2082 ± 116 2190 ± 74 2256 ± 59

3.58 ± 0.55 2.13 ± 0.42 1.09 ± 0.33

2.32 ± 0.33 1.38 ± 0.25 0.67 ± 0.19

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3.2.1. Clearance rate Fig. 5 illustrates the feeding behavior activity (i.e., CR) of the clams in response to seawater acidification and temperature elevation. Irrespective of temperature, the CR values of clams grown at elevated seawater pCO2, and therefore reduced pH, were significantly lower in comparison with those under ambient pCO2. However, a further reduction in pH from 7.7 to 7.4 did not significantly affect CR, except on day 70. With increasing temperature, CR values in control and CO2-exposed clams increased rapidly, as observed on days 10 and 30. When temperature was maintained at a constant from day 30, CR was relatively stable over time in both the control and moderate exposure systems. However, a significant reduction in CR by clams exposed to pH 7.4 was observed on day 70.

3.2.4. Excretion rate Clams exposed to low pH showed a significant increase in ER as the experiment progressed from day 30 (Fig. 8). In comparison with the control, ER increases of ca. 41.20 and 52.15% were observed in clams exposed at pH 7.7 and 7.4 between days 30 and 44, whereas at the end of the experiment (day 70), ca. 174% and 209% increases were observed. Temperature, irrespective of seawater pH, had a statistically significant effect on ER. ER increased with increasing temperature; the sensitivity of ER to pH reduction was clearly amplified at higher temperature. 3.2.5. Oxygen-to-nitrogen ratio The O:N ratio was highly variable during the experiment (Fig. 9). As the experiment progressed from day 30, the O:N ratio decreased significantly. Similar to ER, the influence of pH was significantly pronounced at 20 °C. In comparison with the control clams, reduced seawater pH significantly decreased the ratio of O:N. However, a significant difference between clams reared at pH 7.4 and 7.7 was only observed on day 70.

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3.2.6. Scope for growth Low pH conditions had serious detrimental effects on the SFG of clams by reducing the energy budget, as illustrated in Fig. 10. These effects were more pronounced on day 70, at the end of the experiment,

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3.2.2. Absorption efficiency As shown in Fig. 6, neither reduced seawater pH nor increased temperature had a significant effect on clam AE. However, AE values observed in clams exposed at ambient and moderate pCO2 levels increased from 66.73 and 71.94% on day 10 to 87.29 and 87.48% on day 70, respectively, while the AE of clams at pH 7.4 increased from 77.02 to 84.43%.

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3.2.3. Respiration rate The influence of seawater pH on the RR of clams was only pronounced at the first measurement, i.e., on day 10 (Fig. 7). Clams

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grown at pH 7.4 had a significantly higher RR compared with those grown at pH 8.0 and 7.7. However, moderately reduced pH (from 8.0 to 7.7) did not significantly affect RR. Temperature had a significant influence; at 20 °C from day 30, the CR was approximately two-fold higher than that at 15 °C on day 10. Fluctuations of RR across pH treatments were not statistically significant when temperature was kept constant until the end of the experiment.

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2315 μmol kg–1 and remained stable across treatments. Estimates of cumulative mortality were within 5% and did not differ significantly between the control and pH manipulated systems throughout the 70 days of the experiment (Fig. 3). Furthermore, all mortalities occurred progressively. Over the duration of the experiment, CI markedly increased (Fig. 4). Significant differences between the different pH treatments were observed at the end of the experiment (on day 70).

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Fig. 3. Cumulative mortality of Ruditapes philippinarum throughout the experiment. The initial clam number at each treatment was 450.

Fig. 4. Condition index measurements of Ruditapes philippinarum throughout the experiment. Measurements were carried out on days 10, 30, 44, and 70. Data were presented as mean ± SD (n = 6). Asterisks indicate a significant difference between different pH levels at each sampling time (p b 0.05). Data collected on days 10 and 30 were performed to evaluate the combined effects of temperature and pH on the condition index by means of two-way ANOVA. From day 30, the effects of pH and experimental duration (day) on the condition index were evaluated by means of twoway ANOVA.

Please cite this article as: Xu, X., et al., Seawater acidification affects the physiological energetics and spawning capacity of the Manila clam Ruditapes philippinarum during gonadal maturation..., Comp. Biochem. Physiol., A (2016), http://dx.doi.org/10.1016/j.cbpa.2016.02.014

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Over the duration of the whole experiment, brood stock cumulative mortality estimates were less than 5% in all three scenarios, implying that adult survival, especially during the stage of gonadal maturation, may be resilient to forthcoming ocean acidification. This is consistent with previous observations of long-term chronic exposure to elevated pCO2 in other bivalve species (e.g., 77-day exposure on Crassostrea virginica at 802 μatm [Dickinson et al., 2012]; 90-day exposure on M. edulis and Arctica islandica over a wide range of pCO2 from 768 to 1654 μatm [Hiebenthal et al., 2012]; 75-day exposure on R. decussatus at 3702 μatm [Range et al., 2011]; and 84-day exposure on Mytilus galloprovincialis at pCO2 up to 4203 μatm [Range et al., 2012]). CI is a promising indicator that tracks the gonadal development and spawning time of bivalve mollusks (Gosling, 2003). For example, the CI of R. philippinarum increases during gonadal maturation and decreases during spawning (Drummond et al., 2006; Dang et al., 2010). It is therefore reasonable to assume that clam gonadal development can be reflected in the CI variability in the present study. An elevation of CI was observed throughout the experiment, indicating gonad maturation. Furthermore, our findings showed that the detrimental effect of reduced pH on CI was significantly pronounced at the end of the experiment (i.e., day 70), suggesting that seawater acidification can potentially affect the reproductive capacity of R. philippinarum. Beninger and Lucas (1984) and Drummond et al. (2006) demonstrated that the variability of CI during gonadal maturation in R. philippinarum was mainly affected by energy investment and utilization strategy, i.e., continuous and adequate energy supply is required for gonad ripening. Therefore, the reproductive response of R. philippinarum to seawater acidification can most likely be attributed to an energetic limitation. Bivalve mollusks exposed to acidified seawater often allocate more energy resources to essential physiological processes, especially the maintenance of acid–base homeostasis (Pörtner, 2008; Melzner et al., 2009; Gazeau et al., 2013; Kroeker et al., 2013). Therefore, overall individual performance in an acidifying ocean is determined by the capacity

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3.2.7. Spawning rate A total of 300 clams in each system were induced to spawn on day 70. Of the clams tested, 285, 289, and 168 individual clams at pH levels 8.0, 7.7, and 7.4 were successfully induced to spawn within 8 h,

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the day the clams were induced to spawn. In comparison with control individuals, SFG values of clams grown at pH 7.7 and 7.4 were ca. 31.30% and 79.26% lower, potentially indicating a reduction of the energy utilized for spawning. Such estimations were partially consistent with the following spawning rate results.

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Fig. 7. Respiration rate measurements of Ruditapes philippinarum throughout the experiment. Measurements were carried out on days 10, 30, 44, and 70. Data were presented as mean ± SD (n = 6). Asterisks indicate a significant difference between different pH levels at each sampling time (p b 0.05). Data collected on days 10 and 30 were performed to evaluate the combined effects of temperature and pH on the respiration rate by means of two-way ANCOVA with dry soft tissue of each clam as a covariate and pH and temperature as fixed factors. From day 30, the effects of pH and experimental duration (day) on the respiration rate were evaluated by means of twoway ANCOVA with dry soft tissue of each clam as a covariate and pH and experimental day as fixed factors.

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Fig. 5. Clearance rate measurements of Ruditapes philippinarum throughout the experiment. Measurements were carried out on days 10, 30, 44, and 70. Data were presented as mean ± SD (n = 6). Asterisks indicate a significant difference between different pH levels at each sampling time (p b 0.05). Data collected on days 10 and 30 were performed to evaluate the combined effects of temperature and pH on the clearance rate by means of two-way ANCOVA with dry soft tissue of each clam as a covariate and pH and temperature as fixed factors. From day 30, the effects of pH and experimental duration (day) on the clearance rate were evaluated by means of two-way ANCOVA with dry soft tissue of each clam as a covariate and pH and experimental day as fixed factors.

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Fig. 6. Food absorption efficiency measurements of Ruditapes philippinarum throughout the experiment. Measurements were carried out on days 10, 30, 44, and 70. Data were presented as mean ± SD (n = 6). Asterisks indicate a significant difference between different pH levels at each sampling time (p b 0.05). Data collected on days 10 and 30 were performed to evaluate the combined effects of temperature and pH on the food absorption efficiency by means of two-way ANCOVA with dry soft tissue of each clam as a covariate and pH and temperature as fixed factors. From day 30, the effects of pH and experimental duration (day) on the food absorption efficiency were evaluated by means of two-way ANCOVA with dry soft tissue of each clam as a covariate and pH and experimental day as fixed factors.

Please cite this article as: Xu, X., et al., Seawater acidification affects the physiological energetics and spawning capacity of the Manila clam Ruditapes philippinarum during gonadal maturation..., Comp. Biochem. Physiol., A (2016), http://dx.doi.org/10.1016/j.cbpa.2016.02.014

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Fig. 8. Ammonia excretion rate measurements of Ruditapes philippinarum throughout the experiment. Measurements were carried out on days 10, 30, 44, and 70. Data were presented as mean ± SD (n = 6). Asterisks indicate a significant difference between different pH levels at each sampling time (p b 0.05). Data collected on days 10 and 30 were performed to evaluate the combined effects of temperature and pH on the ammonia excretion rate by means of two-way ANCOVA with dry soft tissue of each clam as a covariate and pH and temperature as fixed factors. From day 30, the effects of pH and experimental duration (day) on the ammonia excretion were evaluated by means of two-way ANCOVA with dry soft tissue of each clam as a covariate and pH and experimental day as fixed factors.

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relative of R. philippinarum) to three pCO2 conditions (730, 1813, and 3702 μatm). Evidently, seawater acidification will most dramatically affect the capacity of bivalves to obtain sufficient energy from food. Consequently, bivalves may be unable to adequately overcome any negative effects of reduced seawater pH on gonadal maturation by enhancing feeding activity (i.e., energy input). Our findings demonstrate that AE was virtually unaffected under acidified conditions, implying that the functioning of the digestive system remained in a steady state. Similarly, Wang et al. (2015) found that the AE of Mytilus coruscus was insensitive to seawater acidification. Indeed, as intertidal species, R. philippinarum and M. coruscus may experience strong fluctuations in food quality and quantity over a diel cycle. They are therefore likely to be capable of exhibiting great digestive plasticity, allowing them to maintain adequate energy acquisition (Stead et al., 2003). These adjustments are achieved primarily through controlling the gut volume and gut residence time of ingested material (Gosling, 2003). Furthermore, certain digestive enzymes are also insensitive to seawater acidification (Zhang et al., 2015). Therefore, the mechanisms by which ocean acidification affects the energy acquisition

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to adequately balance the increased metabolic demands with a sufficiently high energy supply. This is particularly applicable in gonadal maturation since this requires a considerable amount of energetic investment (Gosling, 2003). At the whole organism level, CR is generally considered the most sensitive parameter for the overall energy budget (Sobral and Widdows, 1997). Therefore, the CR variability could track the feeding activity, and consequently, the energy acquisition, of R. philippinarum during the present experiment. Irrespective of the duration of exposure, reductions in CR occurred concomitantly with elevated pCO2 and the associated reductions in seawater pH. This finding is consistent with the observations by FernándezReiriz et al. (2011) following the exposure of R. decussatus (a close

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Fig. 10. Scope for growth measurements of Ruditapes philippinarum throughout the experiment. Measurements were carried out on days 10, 30, 44, and 70. Data were presented as mean ± SD (n = 6). Asterisks indicate a significant difference between different pH levels at each sampling time (p b 0.05). Data collected on days 10 and 30 were performed to evaluate the combined effects of temperature and pH on the scope for growth by means of two-way ANCOVA with dry soft tissue of each clam as a covariate and pH and temperature as fixed factors. From day 30, the effects of pH and experimental duration (day) on the scope for growth were evaluated by means of twoway ANCOVA with dry soft tissue of each clam as a covariate and pH and experimental day as fixed factors.

Fig. 9. O:N measurements of Ruditapes philippinarum throughout the experiment. Measurements were carried out on days 10, 30, 44, and 70. Data were presented as mean ± SD (n = 6). Asterisks indicate a significant difference between different pH levels at each sampling time (p b 0.05). Data collected on days 10 and 30 were performed to evaluate the combined effects of temperature and pH on the O:N by means of two-way ANCOVA with dry soft tissue of each clam as a covariate and pH and temperature as fixed factors. From day 30, the effects of pH and experimental duration (day) on the O:N were evaluated by means of two-way ANCOVA with dry soft tissue of each clam as a covariate and pH and experimental day as fixed factors.

Fig. 11. Spawning rate measurements of Ruditapes philippinarum (n = 300) at the end of the experiment (on day 70) at three pH levels.

Please cite this article as: Xu, X., et al., Seawater acidification affects the physiological energetics and spawning capacity of the Manila clam Ruditapes philippinarum during gonadal maturation..., Comp. Biochem. Physiol., A (2016), http://dx.doi.org/10.1016/j.cbpa.2016.02.014

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M. galloprovincialis to elevated pCO2 of 3820 μatm leads to a significant reduction of extracellular pH from 7.55 to 7.36, whereas intracellular pH remains virtually unchanged and close to pre-exposure levels. During reproductive conditioning, the maintenance of intracellular acid–base balance in the gonads of marine invertebrates is of paramount importance for defending gametes in a quiescent state prior to spawning (Byrne et al., 2010). Continuous energy investments are required for these processes (Melzner et al., 2009), consequently resulting in the enhancement of protein catabolism. This interpretation supports the observations of Ip et al. (2006), according to which H+ in the extracellular fluid, where shell calcification occurs, can be transported in combination with NH3 in the form of NH+. Therefore, the removal of H+ by NH3 may facilitate shell deposition. Generally, bivalve mollusks rely on carbohydrates to fuel energy demands during reproductive conditioning (Gosling, 2003). Lipids have also been reported to play a major role in gamete development. However, under stress conditions, they tend to utilize more nutrient reserves to maintain basal metabolism and fuel additional stress responses. Proteins are considered as an emergency energy supply. In the present study, a reduction in O:N ratios in combination with an increase of ER suggested enhanced protein catabolism during exposure to elevated pCO2. Furthermore, a reduction in the O:N ratio is potentially associated with a decrease of the rate of protein synthesis or the use of substrate metabolism (Langenbuch and Pörtner, 2002). When combined, our results support the concept that energy balance is fundamental in environmental stress adaptation and tolerance in marine organisms (Pörtner and Farrell, 2008; Sokolova et al., 2012). Therefore, overall individual conditioning in bivalve mollusks depends on whether energy acquisition can keep pace with energy expenditure that may increase at elevated pCO2. In this respect, SFG is an ideal index for representing the energy budget at the whole organism level and therefore provides a robust estimation of the effects of climaterelated stressors on the fitness of an organism (Widdows and Johnson, 1988). SFG can respond strongly to elevated pCO2. Reduced seawater pH may shift the energy budget, inflating metabolic demands that initially reduce the SFG. This may eventually result in negative growth or, in extreme cases, mass mortality, when the compensatory mechanisms are overwhelmed (Pörtner et al., 2004; Pörtner and Farrell, 2008; Gazeau et al., 2013). In agreement with the majority of previous studies on bivalve mollusks (Fernández-Reiriz et al., 2012; Navarro et al., 2013; Wang et al., 2015), exposure of R. philippinarum to elevated pCO2 in the present study resulted in a significant SFG reduction. Until now, the influence of ocean acidification on the energy budget of bivalve mollusks during gonadal maturation has received relatively little attention. A hot topic in ocean acidification research is whether marine organisms are able to adapt to long-term multigenerational exposure, i.e., whether long-term chronic exposure of adults to elevated pCO2 can influence the fecundity and survival of their offspring (Parker et al., 2012, 2015). However, the responses of marine organisms, specifically bivalve mollusks, to seawater acidification during reproductive conditioning are unknown. This information is essential for accurately determining fecundity loss in transgenerational transmission. In our study, on day 70, the gonad cover area (unpublished data) of control and CO2-exposed clams exceeded 90%, indicating a ripe stage. Following this, more than 95% of clams exposed to ambient and moderate pCO2 levels were successfully induced to spawn with thermal shock (+4 °C), whereas 44% of individuals failed at pH 7.4. During reproductive conditioning, clams are likely to compensate the negative effects of seawater acidification at the expense of an energy budget shift; however, if left uncompensated, they may be unable to fulfill the energy demand of spawning, a process that requires a considerable amount of energy investment (Gosling, 2003). Spawning failure may have serious detrimental consequences for this species at the population level through a diminished larval supply and recruitment to adult populations. Overall, the results of the present study support the concept that an integral understanding of the ecophysiological responses of marine

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of bivalve mollusks may be related to the regulation of their feeding behavior (CR) rather than digestive performance (AE). The rate of oxygen consumption in bivalve mollusks is typically defined as an estimation of metabolic energy expenditure (Gosling, 2003). Previous studies have demonstrated that bivalves can trigger metabolic depression (i.e., reduction in respiration rate) to save energy when exposed to elevated pCO2 (e.g., M. galloprovincialis exposed at 3820 μatm [Michaelidis et al., 2005]; R. decussatus at 3702 μatm [Fernández-Reiriz et al., 2011]; Mytilus chilensis at 981 μatm [Navarro et al., 2013]; and Mytilus coruscus at 4485 μatm [Wang et al., 2015]). This interpretation is perhaps best exemplified by uncompensated extracellular pH following exposure to seawater acidification (Thomsen et al., 2010; Heinemann et al., 2012; Zittier et al., 2015). To counteract ongoing extracellular pH reductions and maintain an intracellular acid–base steady state, bivalves need to actively remove excess proton ions, specifically through active proton equivalent ion exchange (Fabry et al., 2008; Melzner et al., 2009). In these processes, Na+/K+-ATPases are considered the primary motor of ion exchange through the creation of ion and electrochemical gradients, subsequently utilized by secondary active ion transporters, as mentioned above (Melzner et al., 2009). Therefore, the compensation for intracellular acid–base imbalance caused by acidified seawater is believed to be an energy-demanding process, although the underlying mechanism has not yet been fully elucidated (Melzner et al., 2009; Gazeau et al., 2013). Support for the existence of these energetically demanding processes is derived from elevations in RR observed in CO2-exposed bivalves such as Crassostrea virginica at 3523 μatm (Beniash et al., 2010), Laternula elliptica at 735 μatm (Cummings et al., 2011), and Saccostrea glomerata at 856 μatm (Parker et al., 2012). Given these ambiguous results, interpreting the differential responses of respiration between studies is still a very challenging task. RR may show a variable response to elevated pCO2, even within the same species. For example, while exposure of M. gallorovincialis to elevated pCO2 of 3820 μatm caused a significant reduction in RR (Michaelidis et al., 2005), it remained unaffected up to 3790 μatm pCO2 (Fernández-Reiriz et al., 2012). In our study, we found no significant effect of pH on RR, suggesting that R. philippinarum has a higher tolerance to hypercapnia than previously thought. This is in accordance with the study by Thomsen and Melzner (2010) in which the RR of M. edulis remained virtually unaffected at moderate levels of acidification (2400 μatm pCO2). Similarly, RRs of Pinctada fucata and Perna viridis have been shown to be insensitive to extreme pH reductions of up to 0.7 units (Liu and He, 2012). Ammonia ER is a useful indicator of environmental stress on molluscan health. However, although ER is usually defined as a measure of the rate of ammonia excretion and therefore protein catabolism, it is affected by the nutritional and reproductive status of the animal (Griffiths and Griffiths, 1987). The latter is exemplified by the findings of the present study. By day 70, ER had increased rapidly in both control and CO2-exposed clams. This can most likely be attributed to the rapidly ripening gonads that incurs significant energetic costs. For example, glycogen has been identified as an important energy source during gonadal maturation (Taylor and Venn, 1979). High levels of lytic oocytes toward the end of pectinid gonadal maturation have been shown to be associated with a lack of glycogen (Le Pennec et al., 1998). It has been elucidated that a pH reduction in molluscan body fluids may cause a shift of metabolic conditions to partial anaerobiosis (De Zwaan et al., 1976). Protein degradation may then occur as a consequence. This interpretation is well supported by previous observations of CO2-induced increases of ER in various species of bivalve mollusks (e.g., Michaelidis et al., 2005; Thomsen and Melzner, 2010; Fernández-Reiriz et al., 2011, 2012; and Navarro et al., 2013), in addition to the present study's findings. Building on the work of Michaelidis et al. (2005), recent studies have demonstrated that the enhancement of protein metabolism contributes to the compensation of intracellular pH imbalance under ocean acidification (e.g., Fernández-Reiriz et al., 2011, 2012). According to Michaelidis et al. (2005), exposure of

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The findings of the present study demonstrate that seawater acidification can potentially affect the physiological energetics and spawning capacity of R. philippinarum during gonadal maturation. Reductions in seawater pH significantly decreased CR but increased ER, thereby compromising the energy budget and contributing to a reduction in the energy allocated for gonadal development and spawning activity. However, the oxygen uptake of the clams remained virtually unaffected under acidified conditions, indicating that this ecologically and economically important species may to some extent alleviate the negative effects of seawater acidification exposure. Further in-depth studies are required to demonstrate the effect of seawater acidification on the stages of gonadal maturation.

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We thank Xin Du for her help in conducting the experiments. Two anonymous reviewers kindly provided comments that helped to substantially improve the quality of this paper. This study has been made possible by the earmarked fund for Modern Agro-industry Technology Research System (CARS-48).

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Please cite this article as: Xu, X., et al., Seawater acidification affects the physiological energetics and spawning capacity of the Manila clam Ruditapes philippinarum during gonadal maturation..., Comp. Biochem. Physiol., A (2016), http://dx.doi.org/10.1016/j.cbpa.2016.02.014

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Seawater acidification affects the physiological energetics and spawning capacity of the Manila clam Ruditapes philippinarum during gonadal maturation.

Ocean acidification is predicted to have widespread implications for marine bivalve mollusks. While our understanding of its impact on their physiolog...
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