Science of the Total Environment 514 (2015) 261–272

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Physiological energetics of the thick shell mussel Mytilus coruscus exposed to seawater acidification and thermal stress Youji Wang a,b,c,1, Lisha Li a,b,1, Menghong Hu a,b,c,⁎, Weiqun Lu a,b,c,⁎ a b c

College of Fisheries and Life Science, Shanghai Ocean University, 999 Huchenghuan Road, Shanghai 201306, China Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai 201306, China Shanghai University Knowledge Service Platform, Shanghai Ocean University Aquatic Animal Breeding Center (ZF1206), Shanghai 201306, China

H I G H L I G H T S • Combined effects of pH and temperature on the mussel are investigated. • Thermal stress impairs the energy budget of mussels. • The mussels are tolerant to seawater acidification based on scope for growth.

a r t i c l e

i n f o

Article history: Received 1 November 2014 Received in revised form 11 January 2015 Accepted 27 January 2015 Available online xxxx Editor: C.E.W. Steinberg Keywords: Acidification CO2 Temperature Mussel Physiology Energy budget

a b s t r a c t Anthropogenic CO2 emissions have caused seawater temperature elevation and ocean acidification. In view of both phenomena are occurring simultaneously, their combined effects on marine species must be experimentally evaluated. The purpose of this study was to estimate the combined effects of seawater acidification and temperature increase on the energy budget of the thick shell mussel Mytilus coruscus. Juvenile mussels were exposed to six combined treatments with three pH levels (8.1, 7.7 and 7.3) × two temperatures (25 °C and 30 °C) for 14 d. We found that clearance rates (CRs), food absorption efficiencies (AEs), respiration rates (RRs), ammonium excretion rates (ER), scope for growth (SFG) and O:N ratios were significantly reduced by elevated temperature sometimes during the whole experiments. Low pH showed significant negative effects on RR and ER, and significantly increased O:N ratios, but showed almost no effects on CR, AE and SFG of M. coruscus. Nevertheless, their interactive effects were observed in RR, ER and O:N ratios. PCA revealed positive relationships among most physiological indicators, especially between SFG and CR under normal temperatures compared to high temperatures. PCA also showed that the high RR was closely correlated to an increasing ER with increasing pH levels. These results suggest that physiological energetics of juvenile M. coruscus are able to acclimate to CO2 acidification with a little physiological effect, but not increased temperatures. Therefore, the negative effects of a temperature increase could potentially impact the ecophysiological responses of M. coruscus and have significant ecological consequences, mainly in those habitats where this species is dominant in terms of abundance and biomass. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The combustion of fossil fuels by human is increasing carbon dioxide (CO2) at a rapid rate with an increase of 100 ppm from the industrial revolution and today stands at 390 ppm (Caldeira and Wickett, 2003; IPCC, 2007). The dissolution of CO2 in the oceans has dramatically altered the inorganic carbon chemistry of seawater by reducing the satuand the pH (Feely et al., 2004). It is now unequivocally ration of CO2− 3 accepted that an increase in atmospheric CO2 is causing global climatic ⁎ Corresponding authors. E-mail addresses: [email protected] (Y. Wang), [email protected] (M. Hu), [email protected] (W. Lu). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.scitotenv.2015.01.092 0048-9697/© 2015 Elsevier B.V. All rights reserved.

changes, with noticeable increases in global mean temperatures and ocean acidification (OA) (Caldeira and Wickett, 2003; IPCC, 2007). The global average surface temperature has increased by approximately 0.7 °C during the last century (Hansen et al., 2006) and a continued rising by between 1.8 °C and 4 °C by the end of the 21st century is predicted (IPCC, 2007). Temperature is a key factor that can influence animal physiological responses (Pörtner and Knust, 2007; Sara et al., 2011) and is, therefore, one of the most important factors determining the fundamental niche of a species (Hofmann and Todgham, 2010; Pörtner, 2010; Ezgeta-Balić et al., 2011). Beyond the borders of the thermal window marine invertebrates form internal (systemic) hypoxia (hypoxemia) with significant reductions in aerobic capacity, metabolic rate and scope for growth (Pörtner, 2002a,b; Pörtner and Knust, 2007). Marine bivalves, representing a reliable model to: (i), investigate

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adaptations to highly fluctuating environmental variance (Davenport and Davenport, 2005) and (ii), forecast the distribution and abundance of coastal biodiversity in the context of climate change (Helmuth et al., 2010), are particularly sensitive to external temperature changes, thus the effects of temperature on energy budgets were frequently studied (Wilbur and Hilbish, 1989; Helmuth et al., 2010; Sara et al., 2008, 2011). The oceans have dissolved up to 50% of CO2, leading to decreases in both pH and carbonate ions (e.g., Feely et al., 2004; Orr et al., 2005). The pH of the ocean surface is already nearly 0.1 units lower than the values from preindustrial epoch (Orr et al., 2005) and it is predicted to decrease by 0.4 units by the end of this century and nearly 0.8 units within the next 300 years (Caldeira and Wickett, 2003, 2005; Orr et al., 2005). A growing body of evidence indicates that OA impacts physiology, growth and reproduction in many marine species (e.g., Caldeira and Wickett, 2003; Pörtner et al., 2004; Michaelidis et al., 2005; Orr et al., 2005; Berge et al., 2006; Fabry et al., 2008; Kurihara, 2008; Pörtner, 2008; Doney et al., 2009; Melzner et al., 2009; Miller et al., 2009; Parker et al., 2009; Kroeker et al., 2010; Fernández-Reiriz et al., 2011; Gazeau et al., 2013). A rise in pCO2 levels can induce changes in the extracellular acid–base balance that can produce metabolic disturbances, adversely affecting relevant processes, such as calcification, metabolism, growth and fitness (Fabry et al., 2008; Melzner et al., 2009). Many marine calcifying organisms have exhibited negative responses to high pCO2, such as disorder in metabolic rates (Thomsen and Melzner, 2010), reduction of food uptake (Dupont and Thorndyke, 2010; Fernández-Reiriz et al., 2011) and alteration in calcification and development (Ross et al., 2011). However, adverse effects are not necessarily universal, with different species demonstrating different sensitivities to OA (Gutowska et al., 2008; Kroeker et al., 2010; Hendriks et al., 2010; Sanders et al., 2013). These previous studies showed important variability in the responses, among species, populations, and life stages (reviewed by Doney et al., 2009; Hendriks et al., 2010; Kroeker et al., 2010; Hofmann et al., 2010; Gazeau et al., 2013). Our current understanding of the effect of future OA on the physiological and ecological fitness of marine organisms is incomplete. Despite meriting considerable research effort in recent years, the biological impacts of OA have been mostly considered in isolation (Byrne, 2011). Interactive effects of OA and temperature are still poorly understood (Pörtner, 2008; Lannig et al., 2010; Fang et al., 2014). Although scarce, some recent studies have shown that the effects of CO2 could be modified by high temperatures (Parker et al., 2009). Duarte et al. (2014) investigated the combined effects of OA and temperature increase on Mytilus chilensis and found that mussels are able to overcome increased temperatures, but not increments of CO2 levels. As global warming and OA are occurring concomitantly and the responses of the organisms exposed to these environmental stressors are widely variable, the combined effects must be evaluated extensively in bivalves. Consequently, more studies are necessary to better understand how the interaction between temperature and OA affects both the individual organisms and the whole ecosystem. Measurements of the different physiological rates of bivalves (clearance, ingestion, absorption, respiration, excretion) can be integrated to determine the net energy balance (difference between the energy absorbed from the ingested food and the energy lost in respiration and excretion), which is commonly referred to the “Scope for Growth” (SFG) (Bayne and Widdows, 1978; Widdows and Hawkins, 1989; Smaal and Widdows, 1994; Fernández-Reiriz et al., 2012). This physiological index, besides being a good predictor of the growth rate (Bayne et al., 1979), is also a good indicator of the condition and fitness of organisms (Widdows, 1985) and a precise and sensitive index of the environmental conditions (Widdows, 1985; Albentosa et al., 2012). Being one of the main approaches to model bivalve growth, SFG has been successfully used in a range of bivalve species exposed to varying environmental conditions (Albentosa et al., 2012; Fernández-Reiriz et al., 2012; Duarte et al., 2014). Environmental temperature is a primary controlling factor in a host of physiological processes, including feeding and growth,

that are extremely relevant to many ecosystem level functions (Fry, 1971). Bivalve molluscs have a limited capacity for acid–base regulation due to the lack of developed ion-exchange and nonbicarbonate mechanisms (Melzner et al., 2009). The extracellular alterations caused by exposure to elevated pCO2 are likely to affect processes such as energy partitioning and metabolism (Pörtner, 1987; Melzner et al., 2009). The feeding physiology and energetic balance in bivalves are affected by various environmental factors, such as pH and temperature (Newell et al., 1977; Beiras et al., 1995; Fernández-Reiriz et al., 2011, 2012; Guzmán-Agüero et al., 2013; Duarte et al., 2014). The SFG approach provides an instantaneous estimate of the energy status of a given animal and also insights into the physiological components underlying observed growth rate changes (Smaal and Widdows, 1994; Han et al., 2008). Despite its relevance, SFG has rarely been used in the evaluation of the combined effects of OA and temperature on the physiology of marine organisms. Therefore, it is necessary to conduct specific studies to delineate the SFG of ecologically or commercially relevant species under OA and thermal stress. The potential for significant ecological and economic impacts of OA on bivalves has been explicitly recognized (Fabry et al., 2008; Fernández-Reiriz et al., 2011). However, there has been little investigation on the influence of temperature and OA on the physiology of Mytilus coruscus. M. coruscus is an important economic shellfish, widely distributed in the eastern coast of China, and especially cultured as an important aquaculture species along the coastal waters of Zhejiang province (Chang and Wu, 2007; Liao et al., 2013). During the last few decades, due to the overexploitation of wild resources of this species, it has been difficult to meet the market demands, particularly within the Yangtze River Delta area (Chang and Wu, 2007). As a common calcifier inhabiting coastal ecosystem, M. coruscus attaches to hard substrates in subtidal zones and forms extensive subtidal beds that play an important ecological role, affecting the community structure of the associated macrofauna. Therefore, this species is a key organism for studying the biological impacts of OA and warming, with great significance in ecology and aquaculture. Despite its importance, less information is available on the ecophysiology of M. coruscus apart from a few reports on its larval metamorphosis and settlement (Yang et al., 2014a,b). As an important aquaculture species in coast area of east China, it is necessary to study the feeding physiology of M. coruscus under global change scenario. The aim of this study was to investigate the combined effects of OA and temperature lasting for 14 d on the physiological processes in juvenile thick shell mussels M. coruscus. The physiological responses of the mussels were measured in terms of key physiological parameters, including clearance rate (CR), absorption efficiency (AE), respiration rate (RR), ammonia excretion rate (ER), oxygen to nitrogen (O:N) ratio and scope for growth (SFG). Such data are crucial for the management of populations of commercially exploited bivalves, and potentially useful as predictive indicators of changes in coastal biodiversity of East China Sea caused by OA and temperature elevation. 2. Materials and methods 2.1. Biological material and acclimation procedure Samples of juvenile mussels M. coruscus (27 ± 2 mm shell length, 68.0 ± 4.5 mg dry tissue weight) were collected from a mussel raft at Shengsi island of Zhejiang Province (30° 33′ 00.945″ N, 121° 49′ 59.757″ E), China during September 2013 (water temperature: 24.5 °C; salinity: 25.0‰; and pH: 8.11). Before being used for ecophysiological experimentation, they were held in fiber-glass tanks (500 l) equipped with a filtering system and air supply in the Shanghai Ocean University Shellfish Laboratory. The conditions mimicked the Shengsi island environment encountered in September: temperature, salinity, pH and oxygen content were kept constant at 25 °C, 25‰, 8.1 and 7–8 mg O2 l− 1. Mussels were fed daily twice with the microalgae

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algae Chlorella spp. (25,000 cells ml−1, approximate 3% of the mussel dry weight). One week before experimentation, the bivalves were progressively acclimated to the elevated temperature by increasing approximate 1 °C everyday from 25 °C.

calculated using the thermodynamic solubility product of calcite and aragonite following the procedures described by Morse et al. (1980).

2.2. Experimental design

2.5.1. Clearance rate measurements Clearance rate (CR), or the volume of water cleared of suspended particles per hour, was measured with all mussels pooled in each experimental tank. When the CR was determined, the flow system was stopped and the water in the tank was static. Before measuring the CR, the mussels were starved for 12 h to empty their guts. The evacuation time was previously determined by feeding mussels with microalgae for 1 h and the time when no more feces were produced was recorded. After acclimation for 30 min, to allow the mussels to open and resume pumping, the microalgae was added to achieve an initial concentration of 2.5 × 104 cells ml−1, the highest concentration at which no pseudo-feces were produced in a preliminary experiment. Two identical aquaria, without mussels, were used as the control. To measure the change in algal concentration in the experimental aquarium, an initial 20 ml sample was taken from the center of each aquarium with a syringe at the start of the experiment, and then 20 ml aliquots were sampled from each aquarium at 30 min intervals over a period of 120 min, and the decline in cell concentration was monitored using a Coulter Counter (Multisizer 3, Beckman, Irvine, USA) equipped with a tube of 60 μm aperture. Thus, the CR measurements were assured of not being affected by large decline of cell concentration (less than 30%). Control tanks, without mussels, showed no significant decline in cell concentration over the experimental period. CR was calculated by the following formula of Coughlan (1969):

To examine the combined effect of pH and temperature, the mussels were randomly assigned to six treatments, using three levels of pH (7.3, 7.7, 8.1) and two levels of temperature (25 °C, 30 °C). The experimental temperatures represent the summer average surface value (25 °C) of the study area and alternatively 5 °C above this average as a thermal condition. The carbonate chemistry of seawater was manipulated by bubbling pure CO2 gas, to achieve pH reductions of the magnitude estimated from the IPCC SRES-A2 emission scenarios for the year 2100 and 2300 (Caldeira and Wickett, 2003, 2005). Each treatment consisted of three flow-through tanks as three replicates, with 30 mussels per replicate, which were maintained in a 30 l aquarium and fed with the microalgae Chlorella spp. as above. The seawater was half renewed every two days during the experiment. The experiment lasted for 14 d, and measurements on feeding and metabolic activity of M. coruscus were conducted on the 1st, 4th, 7th and 14th days during the exposure period. 2.3. Acidification exposure system The experimental setup consisted of computer, pH controller (DAQM; 4 Channel; Loligo® Systems Inc., Tjele, Denmark), pH probe, air and CO2 supplies, valve, water pump, filter, temperature regulator, water tank, head tank and experimental tank. Low pH conditions in the seawater were controlled using two pCO2/pH feedback STAT systems connected with four WTW pH 3310 meters and SenTix 41 pH electrodes (Loligo Systems Inc., Tjele, Denmark), respectively, and these systems were operated by CapCTRL software (Loligo Systems Inc., Tjele, Denmark). The gas flux from the pure CO2 cylinder to the header tank was controlled through the pH-stat system by opening or closing a solenoid valve when the pH readings in the tank deviated from the predetermined set-points by ± 0.1 pH units. Such automated system can control four independent tanks with different pH/pCO2. To prevent excessive fluctuations in pCO2 and over exposure, the CO2 gas rates were manually set to the lowest possible levels that still allowed the desired pCO2 level to be obtained. Periodic measurements of pH were also taken to verify using a portable pH meter (pH-201, MSITECH (AsiaPacific) Pte. Ltd., Singapore) every day. A flow-through system with a header tank design was used to minimize any interference from the metabolic waste products of the mussels, and very little between-tank variation in pH/pCO2 was observed, as detected using the second pH meter/electrode. Water temperature was maintained at 25 °C and 30 °C using temperature regulators and the tanks were covered with acrylic plates to reduce or prevent external disturbance. 2.4. Monitoring of the physico-chemical variables of seawater Temperature and pH of seawater were continuously monitored during the experiment using pCO2/pH feedback STAT systems. Salinity was measured using a salinity refractometer (Hand Refractometer S/Mill-E, Atago, Itabashi-Ku, Tokyo, Japan). Total alkalinity (TA) was determined by Gran titration with a total alkalinity titrator system (AS-ALK2, Apollo SciTech Inc., Bogart, USA). The samples were titrated automatically with HCl (~0.25 M HCl in a solution of 0.45 M NaCl) past the endpoint of 4.5 (Range et al., 2011). Dissolved inorganic carbon (DIC) and partial pressure of CO2 (pCO2) in seawater were calculated from the temperature, pH and TA using the carbonic acid dissociation constants (Millero et al., 2006) and the CO2 solubility coefficient (Weiss, 1974). The CaCO3 saturation state for calcite (Ωcal) and aragonite (Ωara) were

2.5. Physiological measurements

CR ¼ V  ð ln C0 – ln Ct Þ=Nt where CR is the clearance rate (l h−1), V is the volume of test seawater in the tanks (l), C0 is the initial algal concentration (cells ml−1), Ct is the algal concentration at time t (cells ml−1), N is the number of mussels in the tanks, and t is the time elapsed (h). All physiological parameters were standardized to unit dry weight (see Section 2.5.5). 2.5.2. Absorption efficiency Absorption efficiency (AE) was measured by the method of Conover (1966), which represents the efficiency with which organic material is absorbed from the ingested food. It assumes that an animal can digest and absorb the organic component of the food but not the inorganic fraction. It was calculated for each replicate by collecting the feces after the CR measurements were completed. The organic content of the microalgae was calculated by filtering 1 h of filtered seawater with 2.5 × 104 cell ml− 1 algae using ashed and pre-weighed 40 mm glass fiber filters (Whatman® GF/C). These filter papers were rinsed using distilled water (Smaal and Widdows, 1994), dried in an oven (110 °C) for 24 h, weighed, ashed in a muffle furnace (450 °C for 6 h) and reweighed. Blank GF/C filters, also washed, ashed and pre-weighed were used with each batch of filters for corrections of weight change due to any daily variations of humidity. Filters were cooled in desiccators before weighing. Feces were collected by a pipette from the experimental aquarium 12 h after the CR measurements, and the organic content of the feces was determined using the same method as described above. AE was calculated according to Conover (1966) as follows: AE ¼ ð F–EÞ=½ð1–EÞ  F where AE is the absorption efficiency (%), F is the ratio of organic dry weight: dry weight in the food (0.85 for the diets in this study), and E is the ratio of organic dry weight: dry weight in the feces. Ingestion rate (IR) was calculated by multiplying CR by POM (particulate organic matter, mg l− 1) concentration (Hawkins et al., 1998),

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i.e., the amount of ingested food per hour. The POM concentration was converted to joules using a conversion factor of 23 J mg−1 for Chlorella spp. (Widdows and Johnson, 1988; Widdows et al., 1990). 2.5.3. Respiration rate measurements Because of the small size of the mussels, 15 mussels from each tank were randomly divided into three replicate groups, with five individuals each. The respiration rate (RR) was measured individually on 5 mussels in closed glass respirometers of (800 ml) containing air-saturated seawater from corresponding treatment tanks, which were stirred by a magnetic stirrer bar beneath a perforated glass plate supporting the mussel. Water tanks with temperature regulator were used to achieve corresponding temperatures where the mussels acclimated. To ensure the mussels had resumed respiration in the chambers, the experiment was started 20 min later when the valves of each mussel had opened. The respirometers were then sealed off for 60 min. Oxygen concentration in each chamber was not allowed to drop below 30% of saturation throughout the experiment. Two chambers without animals were used as the control. The rate of oxygen decline within the respirometer chamber was determined using an oxygen meter (model YSI 58). The initial and final oxygen concentrations in each chamber were measured. The respiration rate (RR) was then calculated using the following equation: RR ¼ ½Ct0 −Ct1 Þ  V=Nt where RR is the respiration rate (mg O2 h−1), Ct0 and Ct1 are the initial and final oxygen concentrations in the water (mg O2 l−1), respectively, V (l) is the volume of the water in the respirometer, N is the number of mussels in the chamber, and t (h) is the incubation time. Values for RR were transformed to J/h using a conversion factor of 13.98 J mg O−1 2 (Wong and Cheung, 2003).

then converted to energy equivalents (J h−1 g−1) in order to calculate the SFG, which represented the difference between the energy absorbed from the food and the energy loss via metabolic energy expenditure. SFG was calculated using the energy balance equation given by Smaal and Widdows (1994): SFG ¼ Ab–ðR þ UÞ where SFG is scope for growth (J h− 1 g− 1), Ab is the total absorbed energy (J h−1 g−1), R is the energy lost in respiration (J h−1 g−1), and U is the energy lost in ammonia excretion (J h−1 g−1). Absorption rate (Ab) is calculated as IR (J h−1) × AE (%). 2.6. Statistical analyses Prior to the analysis, normality of the data was evaluated by using the Shapiro–Wilk's W test and homogeneity of variances was checked by Levene's test using the statistical software SPSS 16.0. Two-way repeated measures ANOVA was used to evaluate whether temperature and pH and the interaction between factors affected physiological parameters. When the analysis showed significant interactions, a oneway ANOVA or t test was carried out for each factor separately in each level from the other factor. Multiple comparisons between different pH treatments were carried out using Tukey's HSD test. Finally, multivariate analysis was conducted by employing Principal Component Analysis (PCA) using XLSTAT®2014. A biplot was constructed showing both the measured variables and the observations, i.e. four sampling times of each experimental condition. The results are expressed as the means ± SD of the data. 3. Results 3.1. Monitoring of pH and temperatures in the experimental tanks

2.5.4. Ammonia excretion rate After RR measurement, excretion rates (ERs) of the same group of mussels were measured. Water samples were collected from each chamber and frozen to −20 °C until analysis. The concentration of ammonia excreted was determined with a spectrophotometer according to the phenol–hypochlorite method (Solorzano, 1969). Two containers without mussels were used as the control. ER was calculated from the difference in ammonia concentration between the chambers with and without animals using the equation: U ¼ ðCtest –Ccontrol Þ  ðV=1000Þ=Nt

Fig. 1 showed the trends of temperature and pH during the experiment. On average, the planned differences in pH and temperature between six treatments were achieved (Table 1). During the 14 d of exposure, stable trends were apparent in the reduced pH and elevated temperature treatments (Fig. 1). Salinity values consistently were maintained at 24–26‰, and dissolved oxygen in the exposure tanks consistently exceeded 6 mg l− 1 during the experiment. TA ranged from 2139 to 2383 μmol kg−1. The pH-stat system was generally effective in maintaining 3 clearly distinct levels of pH during the experimental periods. The carbonate chemistry of seawater in the six treatments was summarized in Table 1.

where U is the rate of ammonia excretion (mg NH4–N h−1), Ctest is the ammonia concentration (mg l−1) in the sample, Ccontrol is the ammonia concentration (mg l−1) in the control, V is the volume (ml) of seawater in which the animal is incubated, N is the number of mussels in the chamber and t is the incubation time (h). Values for excretion rate were transformed to J/h using the conversion factor: 1 mg NH4– N h−1 = 25 J h−1 (Elliott and Davison, 1975). The ratio of oxygen consumption to ammonia–nitrogen excretion in atomic equivalents (O:N) was calculated to determine the proportion of protein relative to carbohydrate and lipid catabolized for energy metabolism (Widdows, 1985). 2.5.5. SFG After the experiment the mussel tissues were removed from their shells and dried at 90 °C for 24 h to obtain their dry tissue weight. The CR (l h−1), RR (mg O2 h−1) and ER (mg NH4–N h−1) were converted to mass specific rates for a ‘standard mussel’ of 1 g dry weight using the following equation: Ys = (Ws/We)b × Ye, where Ys is the physiological rate for an animal of standard weight, Ws is the standard weight (1 g), We is the observed weight of the animal (g), is Ye the uncorrected (measured) physiological rate, and b is the weight exponent for the physiological rate function (b = 0.67). Each physiological rate was

Fig. 1. Daily values (mean ± SE) for pH and temperature of seawater during the 14 d of exposure.

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Table 1 Seawater carbonate chemistry variables (mean ± SE, n = 4) during the experimental period. Temperature, salinity and total alkalinity (TA) were measured on each sampling date. pH was measured continuously during the experiment. Dissolved inorganic carbon (DIC), partial pressure of CO2 in seawater (pCO2) and saturation state for calcite (Ωcal) and aragonite (Ωara) were calculated from the measured temperature, salinity, pH and TA. Treatments pH ∗ T

T (°C)

S

pH

TA (μmol kg−1)

DIC (μmol kg−1)

pCO2 (μatm)

Ωcal

Ωara

8.1 ∗ 25 7.7 ∗ 25 7.3 ∗ 25 8.1 ∗ 30 7.7 ∗ 30 7.3 ∗ 30

25.0 ± 0.2 24.9 ± 0.5 24.9 ± 0.3 29.9 ± 0.5 29.9 ± 0.5 30.0 ± 1.1

25.0 ± 0.4 24.9 ± 0.6 24.9 ± 0.9 25.0 ± 0.5 24.6 ± 0.4 25.0 ± 0.6

8.12 ± 0.01 7.70 ± 0.02 7.30 ± 0.02 8.14 ± 0.02 7.71 ± 0.02 7.29 ± 0.03

2365 ± 54 2383 ± 5 2356 ± 46 2241 ± 45 2249 ± 51 2139 ± 60

2211 ± 43 2196 ± 40 2114 ± 56 2332 ± 32 2391 ± 19 2352 ± 44

647 ± 13 2047 ± 78 4081 ± 229 715 ± 21 2091 ± 75 4485 ± 203

7.79 ± 0.15 4.33 ± 0.18 1.76 ± 0.07 7.50 ± 0.36 4.4 ± 0.18 1.78 ± 0.08

5.04 ± 0.16 2.72 ± 0.11 1.24 ± 0.08 5.11 ± 0.11 2.85 ± 0.16 1.32 ± 0.13

of pH and temperature were observed at day 1 and day 4 (Table 2; Fig. 3A). At day 1, ER was significantly lower at pH 7.7 and 7.3 than pH 8.1 at two temperatures, and significantly lower at 30 °C than 25 °C under the normal pH condition. At day 4, ER was significantly lower at pH 7.7 and 7.3 compared to pH 8.1 at 25 °C, and was significantly lower at pH 7.3 than pH 7.7 and 8.1 at 30 °C. At day 7 and 14, ammonia excretion rates were significantly different among three pH levels at two temperatures, with lowest values at pH 7.3. Respiration rates were significantly affected by pH, temperature and their interactions during the whole experiment (Table 2; Fig. 3B). Temperature only showed a significant effect under normal pH, and RR was lower at 30 °C than 25 °C during the whole experiment. At day 1, RR significantly decreased with pH reduction, and no significant difference was observed between pH 7.7 and 8.1 at 30 °C. At day 4, RR showed a similar pattern to day 1, but no significant difference was observed between pH 7.3 and 7.7 at 30 °C. At days 7 and 14, pH showed no significant effect at high temperature, and respiration rates were significantly reduced by low pH conditions. O:N ratios were significantly affected by pH during the whole experiment, and were only affected by temperature and interaction of both

3.2. Physiological measurements Clearance rates ranged from 0.4 to 1.6 l h−1 g−1, and were only significantly reduced by low pH at temperature 25 °C at day 14; temperature showed a significant effect on CR at day 7 and day 14, with higher values at temperature 25 °C than 30 °C when the pH was maintained at 7.7 and 8.1, respectively (Table 2; Fig. 2A). Absorption efficiencies were comparatively low (20–45%) throughout the experiment. ANOVA followed by a Tukey test of significance showed no significant differences in AE between the three pH levels tested. However, temperature only showed a significant effect at the beginning of the experiment when the pH was reduced to 7.3, and any other effects of temperature were not found at the rest sampling times (Table 2; Fig. 2B). Fecal organic weight ratio was significantly higher under pH 7.7 than normal pH at temperature 30 °C at day 4, and was significantly reduced by high temperature under pH 7.7 and 8.1 at day 14 (Table 2; Fig. 2C). Ammonia excretion rates were significantly different among three pH treatments during the whole experiment, and were only significantly affected by temperature at day 1. Moreover, interactive effects

Table 2 Summary of two-way ANOVA results on effects of pH and temperature (T) on clearance rate (CR), absorption efficiency (AE), fecal organic weight ration (E), respiration rate (RR), excretion rate (ER), O:N ratio, and scope for growth SFG). pH: 7.3, 7.7 and 8.1; temperature: 25 and 30 °C. Source

1d

4d

7d

14 d

CR

4d

7d

14 d

E

RR

T

pH

T ∗ pH

T

pH

T ∗ pH

T

pH

T ∗ pH

T

pH

T ∗ pH

df

1

2

2

1

2

2

1

2

2

1

2

2

MS F P MS F P MS F P MS F P

0.067 2.719 0.125 0.112 3.554 0.084 0.198 4.731 0.05 2.288 33.727 b0.001

0.011 0.449 0.648 0.096 3.045 0.085 0.04 0.962 0.41 0.013 0.186 0.833

0.044 1.778 0.211 0.027 0.855 0.45 0.108 2.588 0.116 0.219 3.228 0.076

97.189 6.29 0.028 33.11 2.221 0.162 17.579 1.476 0.248 66.727 3.775 0.076

10.649 0.689 0.521 0.887 0.059 0.943 15.869 1.332 0.3 32.594 1.844 0.2

16.418 1.063 0.376 9.123 0.612 0.558 10.248 0.86 0.447 17.117 0.968 0.408

0.003 0.869 0.370 b0.001 0.052 0.824 b0.001 0.346 0.567 0.114 37.408 b0.001

0.001 0.139 0.872 0.016 5.170 0.024 0.001 0.803 0.471 0.008 2.517 0.122

0.002 0.451 0.648 0.005 1.780 0.210 0.002 1.559 0.250 0.003 2.693 0.108

b0.001 20.142 0.001 b0.001 22.177 0.001 b0.001 29.272 b0.001 b0.001 29.188 b0.001

b0.001 61.677 b0.001 b0.001 36.598 b0.001 b0.001 14.294 0.001 b0.001 28.312 b0.001

b0.001 4.641 0.032 b0.001 4.544 0.034 b0.001 5.344 0.022 b0.001 12.206 0.001

Source

1d

AE

ER

O:N

SFG

T

pH

T ∗ pH

T

pH

T ∗ pH

T

pH

T ∗ pH

df

1

2

2

1

2

2

1

2

2

MS F P MS F P MS F P MS F P

4.815 10.322 0.007 0.653 2.262 0.158 11.627 4.086 0.066 3.717 3.557 0.084

36.105 77.399 b0.001 37.17 128.725 b0.001 132.576 46.589 b0.001 210.208 201.208 b0.001

2.002 4.291 0.039 12.173 42.158 b0.001 6.748 2.371 0.136 1.212 1.159 0.346

b0.001 b0.001 1.00 18.673 19.531 0.001 0.871 0.671 0.429 0.412 0.329 0.577

27.098 9.547 0.003 23.214 24.281 b0.001 79.237 61.053 b0.001 85.728 68.476 b0.001

1.165 0.411 0.672 21.003 21.969 b0.001 0.1 0.077 0.926 0.552 0.441 0.653

6.971 6.733 0.023 6.885 4.618 0.053 20.396 5.156 0.042 92.723 55.143 b0.001

0.592 0.571 0.579 3.924 2.632 0.113 5.24 1.325 0.302 0.856 0.509 0.614

2.09 2.018 0.176 0.179 0.12 0.888 7.68 1.942 0.186 4.212 2.505 0.123

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Fig. 2. A, Clearance rate (CR) and B, absorption efficiency (AE) of M. coruscus exposed to different combinations of pH and temperature for 14 d. The means sharing the asterisk between two temperatures at each fixed pH are significantly different at each sampling time (P b 0.05).

factors at day 4 (Table 2; Fig. 3C). At day 1, O:N ratio at pH 8.1 was significantly lower than that at pH 7.7 and 7.3 when the temperature was at 30 °C. At day 4, O:N ratio at pH 8.1 was significantly lower than that at pH 7.7 and 7.3 at 25 °C, and it was significantly lower at pH 8.1 and 7.7 than that at pH 7.3 when the temperature was higher. At days 7 and 14, all pH effects showed a similar trend, with significant high values under the lowest pH condition. The SFG values were positive for all treatments, and were not affected by pH (Table 2; Fig. 4). However, temperature showed a significant

Fig. 3. A, Ammonia excretion rate (ER), B, respiration rate (RR) and C, O:N ratio of M. coruscus exposed to different combinations of pH and temperature for 14 d. The means denoted by different superscripts at each fixed temperature are significantly different among three pH levels at each sampling time (P b 0.05). The means sharing the asterisk between two temperatures at each fixed pH are significantly different at each sampling time (P b 0.05).

effect at some pH conditions. At day 1, when the pH was reduced to 7.3, SFG was significantly lower at 30 °C than 25 °C. At day 7, SFG was significantly lower at 30 °C than 25 °C under normal pH condition. At day 14, high temperature significantly reduced the SFG when the pH was at 8.1 and 7.7, respectively. Principal component analysis (PCA) (Fig. 5) showed that 78.90% of total variance was explained by the two principal components. PC1 expressed 52.83% of total variance, the most significant response

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Fig. 4. Scope for growth (SFG) of M. coruscus exposed to different combinations of pH and temperature for 14 d. The means sharing the asterisk between two temperatures at each fixed pH are significantly different at each sampling time (P b 0.05).

referred to the separation between normal temperatures and high temperatures, where the high induction of most physiological functions, especially CR led to higher SFG levels under normal temperatures. PC2 representing 26.07% of total variance, separated low pH with normal pH exposed mussels, on the later ones RR and ER significantly increased. This clearly demonstrated that the decreasing SFG with increasing temperature observed in this study could be explained by decreased physiological activities. 4. Discussion Generally, in bivalves, increases in CR occurring concomitantly with increases in temperature up to an optimum temperature are often reported. However, with increases in temperature above the optimum, the CR has been shown to decrease rapidly (Newell et al., 1977; Winter, 1978; Buxton et al., 1981). Previous observations showed that CR increases when acclimation temperatures rise up to 15 °C–17 °C, and significantly reduced when temperature was beyond 25 °C in Mytilus species (Schulte, 1975; Gonzalez and Yevich, 1976; Bayne

Fig. 5. Biplot originating from principal component analysis integrating all measured variables (CR, AE, RR, ER, O:N, SFG, E) for four times (days: 1, 4, 7 and 14) at six different treatments (■–25 °C × pH 7.3, □–30 °C × pH 7.3, ▲–25 °C × pH 7.7, △ − 30 °C × pH 7.7, ♦–25 °C × pH 8.1, ◊–30 °C × pH 8.1). Both the loadings of the variables (●) and the scores of the experimental conditions were shown.

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et al., 1976). In the present study, mean CR values in M. coruscus were around 0.4–1.6 l h−1, which were very close to CR values given in the literature for Paphia rhomboids (Savina and Pouvreau, 2004), Ruditapes philippinarum (Goulletquer et al., 1989; Nakamura, 2001) and Tapes decussatus (Savina and Pouvreau, 2004), but comparatively lower than R. decussatus (Sobral and Widdows, 1997), Mytilus edulis (Widdows and Johnson, 1988) and Brachidontes pharaonis (Sara et al., 2008). Intertidal species living in highly variable habitats should seize an immediate advantage from every profitable environmental situation allowing them to acquire energy to be allocated to growth and reproduction. In contrast, M. coruscus showed a low rate of food acquisition which would equate more reasonably with a species living under more constant conditions. This hypothesis is supported by a study of Charles and Newell (1997) who reported that ribbed mussel Geukensia demissa under subtidal conditions showed lower feeding rates than intertidal conspecific. In East China Sea, M. coruscus live in a subtidal habitat where the temperature ranges from 10 to 25 °C (Zhang et al., 2009). During the beginning of our experiment, the results showed that the CR of M. coruscus was independent of temperature in the range 25–30 °C, which was similar to Pinctada margaritifera (Chavez-Villalba et al., 2013). Such feeding behavior was previously reported for P. margaritifera and Pinctada maxima between 23 °C and 32 °C (Yukihira et al., 2000), and Modiolus barbatus between 20 °C and 28 °C (Ezgeta-Balić et al., 2011). Working on R. philippinarum, Goulletquer et al. (1989) found that the filtration of R. philippinarum was nearly constant between 12 °C and 20 °C. However, there was an evidence that temperature significantly influenced CR with higher values at 25 °C than 30 °C, especially under pH 7.7 and 8.1 at day 7 and day 14, suggesting a food energy intake dependence on temperature. These results were very similar to Resgalla et al. (2007), who observed that under chronic conditions, Perna perna presented the capacity to compensate the CR between 15 °C and 30 °C, but with a tendency for this rate to increase up to 25 °C and reduction to 30 °C. The reduction in CR at 30 °C might be a result of the mussel's valve closure in response to warming as 30 °C may be an upper limit of optimum. As shown in a previous study, Mytilus galloprovincialis kept their valves closed for longer at 24 °C compared with 10–17 °C (Anestis et al., 2010). However, in the present study the CR was not examined over such a wide temperature range, but M. coruscus is likely to follow a similar normal-shaped curve as other bivalves with a decline in CR at higher and lower temperature extremes. Short term studies with various species of bivalves have reported differences in the sensitivity of the CR to altered pH. Fernández-Reiriz et al. (2011) found a reduction of the CR by the clam Ruditapes decussates under low pH conditions. Liu and He (2012) reported reductions in the CR of both the clam Chlamys nobilis and the mussel Perna viridis under pH conditions similar to this study. Conversely, the CR of the pearl oyster Pinctada fucata held under identical conditions was reported to increase. In contrast, CR of M. galloprovincialis remained unaffected by pH reductions of 0.6 units (Fernández-Reiriz et al., 2012). Adopting a similar approach, Sanders et al. (2013) found no significant effect of reduced pH of 7.7 on the filtration ability of juvenile king scallop Pecten maximus. In the present study, pH did not affect the CR of M. coruscus during the first week, probably due to the short experimental period, but low pH significantly reduced the CR at 25 °C at day 14. A longterm of 70 d experiment, established that the OA did not produce an immediate effect on the feeding activity of M. chilensis, but after 35 d of exposure a significant reduction in CR was observed (Navarro et al., 2013), which was similar to the present study. Thus, medium and long-term experiments avoid misinterpretations due to the initial acclimatization ability of a species to high levels of seawater acidification. The effects of seawater acidification on CR were also studied on three coastal bivalve species in different regions: the mussel M. galloprovincialis and the clams Chamelea gallina and R. decussates (Range et al., 2014). Reduced clearances were observed for R. decussatus under reduced pH. Clearance rates of M. galloprovincialis were significantly reduced by acidification in Italy, but not in Portugal, indicating large variations in

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the sensitivities of bivalves to climatic changes, among different species and between local populations of the same species. In this study, AE was relatively low, ranging from 25 to 45%, probably resulting from high fecal organic ratios. Low assimilation efficiencies are generally attributed to the indigestible cell wall possessed by some microalgae. In this study, it was probably attributed to the fact that Chlorella spp. can be less efficiently digested which was also inefficiently assimilated by oysters (17.4%, Babinchak and Ukeles, 1979) and mussels (40–50%, Wang et al., 2005). In contrast, AE of M. chilensis fed with Isochrysis galbana ranged from 50 to 60% (Navarro et al., 2013). Peirson (1983) reported high AE (83.3%) in Argopecten irradiansconcentricus for Dunaliella tertiolecta, algae which is known to foster poor growth due to its lack of essential polyunsaturated fatty acids. Therefore, high AE is not necessarily indicative of bivalve growth or of the nutritional value of microalgae (Han et al., 2008). AE was found to be relatively independent of temperature (Laing et al., 1987; Wilbur and Hilbish, 1989; Albentosa et al., 1994). The AE of M. edulis was independent of temperature, but even for this species, this relation can vary from one author to another (Widdows, 1978; Griffiths and Griffiths, 1987). AE of M. barbatus was significantly lower at temperature 28 °C than that at other temperatures (Ezgeta-Balić et al., 2011). According to Bayne and Newell (1983), AE depends on the length of time the food remains in the digestive tract and the rate of intake. In other words, there is a balance between CR and AE. In the present study, no significant difference was observed between two temperatures, except at the initial of the experiment under pH 7.3, which basically was in accordance with the above description. Little is known about the impact of increasing pCO2 levels on AE in marine organisms. Values measured in M. coruscus at the three pH levels were lower than those described by various authors for marine bivalves (Bayne and Newell, 1983; Fernández-Reiriz et al., 2005; Navarro et al., 2013), and did not show significantly difference among three pH levels, indicating the stable function of the digestive systems under conditions of seawater acidification. In M. chilensis, AE was significantly reduced by low pH conditions (Navarro et al., 2013). However, juvenile M. galloprovincialis showed a significant increment in AE caused by seawater acidification (Fernández-Reiriz et al., 2012). This pattern may be related to the optimization of certain digestive enzymes (amylase, glucosidase and peptidase) under conditions of reduced pH (Wojtowicz, 1972; Areekijseree et al., 2004), which could facilitate nutrient absorption. Thus, based on the existed data, the AE responses of bivalves under different pH conditions are species-specific. RR is a temperature-dependent process in mussels with evidence of some degree of acclimation to temperature change (Bayne et al., 1976; Ezgeta-Balić et al., 2011). At high temperatures, beyond the temperature-specific maximum respiration rate, there is a marked decline in oxygen consumption which is caused, in part, by oxygen limitation and a reduction in ventilation rate. This results in a rapid decline in respiration rates and an increase utilization of anaerobic metabolic pathways (Pörtner, 2002a; Tang et al., 2005; Jansen et al., 2007). The RR values obtained in the present study were around 0.025 mg O2 h−1, which is lower than other species, such as R. philippinarum (Bodoy et al., 1986; Goulletquer et al., 1989) and T. decussatus (Bodoy et al., 1986; Savina and Pouvreau, 2004). The RR of M. coruscus followed the temperature dependent relationship, although measured over a relatively narrow range between 25 and 30 °C in this study. This pattern was also common in Donax vittatus (Ansell, 1973) and Mytilus edulis (Sukhotin et al., 2003). In this study, the respiration rates of M. coruscus were lower at 30 °C than at 25 °C, suggesting the metabolic rate was depressed by thermal stress. Shumway (1982) pointed out that RR usually increases with rising temperature, up to a maximum or optimum limit beyond which it rapidly decreases. However, in the present study we were unable to investigate the response of M. coruscus to a wider range of temperatures, beyond a relatively high maximum of 30 °C. This prevents us from assessing the upper temperature limit on the physiological responses of this species and we therefore have to

infer from field observations and other species. Jansen et al. (2007) reported that the amplitude of the respiratory response of Mytilus spp. declined when temperature was beyond 24 °C in the field. It has been proposed that this modulation of metabolic thermal sensitivity is a protection mechanism that prevents excessive metabolic rates at high ambient temperatures. As pointed elsewhere, metabolic depression may be suitable to balance the temperature induced rise in energy demand. However, this would occur at the expense of reduced aerobic scope for activity (Pörtner, 2002a). Energy savings may thus contribute to passively alleviate thermal stress. Since M. coruscus normally survive at water temperature over 10–25 °C in East China Sea, the respiration of this species is unlikely to decline markedly over the range 25 to 30 °C compared with other bivalves (Sara et al., 2000, 2008). Thermal acclimation is not a universal feature of bivalve metabolism, the ability of bivalves to alter metabolism following a temperature change is species specific (Beiras et al., 1995). More particularly, the oxygen consumption in M. coruscus remains strongly dependent on the ambient temperature at normal pH level during the whole experiment, suggesting a limited ability to acclimate to temperature change (Newell et al., 1977; Shumway and Koehn, 1982). However, when the mussels were exposed high CO2, the difference between temperatures was minimal. Therefore, the marked thermal dependence of the respiration rates found in this work on juvenile M. coruscus is modulated by pH. Mechanisms like oxygen supply and respiration can respond strongly to alterations in surrounding CO2 conditions (Pörtner, 2008; Gazeau et al., 2013). Several studies on different species of marine invertebrates have described metabolic depression under high seawater pCO2 levels, suggesting that the uncompensated extracellular pH might be the cause for this reduction (Pörtner et al., 2004; Michaelidis et al., 2005). Some bivalve species that are pre-adapted to life in the intertidal and regularly experience associated oscillations in body fluid pCO2 between tidal cycles may more readily exploit metabolic depression to save energy in response to OA. Such a response was seen in the intertidal mussel M. galloprovincialis (Michaelidis et al., 2005) as well as in the clams R. decussatus (Fernández-Reiriz et al., 2011) and C. nobilis (Liu and He, 2012) following exposure to elevated CO2. Michaelidis et al. (2005), working on the mussel M. galloprovincialis, suggested that a reduction in RR is associated with seawater acidification. Reduced metabolic rates were also found in the clam R. decussates (Fernández-Reiriz et al., 2011; Range et al., 2014) and the mussel M. chilensis (Navarro et al., 2013) under reduced pH. Liu and He (2012) found that pH 7.4 significantly reduced the RR of C. nobilis. These results are in agreement with the present study, where the oxygen uptake was significantly depressed at pH 7.7 and 7.3. However, some authors (Wood et al., 2008; Fernández-Reiriz et al., 2012; Sanders et al., 2013) found that at moderate levels of acidification, metabolic rates remained unchanged or even increased. Three experiments concluded on an increase in bivalve metabolic rates during exposure to elevated CO2 (Lannig et al., 2010; Thomsen and Melzner, 2010; Parker et al., 2012), suggesting an ability to at least partially compensate for the increased energy costs of acidosis (Wicks and Roberts, 2012). With differing feeding regimes, acclimation duration, methodology and test species it is difficult to identify the reason for differing metabolic responses between studies. The ER has been used as an indicator of stress of the organism (Fernández-Reiriz et al., 2011, 2012), and it increases with temperature due to the metabolic energy demand in M. edulis (Anestis et al., 2010; Guzmán-Agüero et al., 2013). The rate of production of ammonia provides a measure of the protein catabolism rate, which can vary with the nutritional and reproductive status of the animal (Griffiths and Griffiths, 1987). Previous reports characterized the effects of temperature on bivalve excretion rates, and excretion rates were shown to increase with increasing temperatures (Albentosa et al., 1994; Anestis et al., 2010; Guzmán-Agüero et al., 2013). We found that ammonia excretion of mussel showed no significant difference between two temperatures except at the initial of the experiment under normal pH, indicating that M. coruscus could regulate catabolism of amino acids at 30 °C.

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According to De Zwaan et al. (1976), a pH decrease in the internal fluids caused a shift of mussel metabolism to partial anaerobiosis, with a consequent degradation of proteins. Michaelidis et al. (2005) suggested that short-term incubation of M. galloprovincialis under acidified conditions resulted in an increased excretion of ammonia, indicating net degradation of proteins. Thomsen and Melzner (2010) observed that ammonia excretion in M. edulis rose with increasing pCO2. This increase in ammonia excretion under reduced pH conditions can be interpreted as an intracellular pH regulatory mechanism. In fact, according to Boron (2004), greater excretion and protein degradation may support the production of HCO− 3 and, consequently, promote pH regulation. The values of ammonia excretion of M. coruscus were lower at the two higher pCO2 levels, which were similar to M. chilensis (Navarro et al., 2013). Liu and He (2012) reported that the excretion rates were significantly lower at pH 7.4 than pH 7.8 for three bivalve species. These results did not agree with some authors who explained the increase in ammonia excretion by the animals exposed to high pCO2 levels as a result of the enhancement of protein metabolism which contributed to intracellular pH regulation (Michaelidis et al., 2005; Fernández-Reiriz et al., 2005, 2011, 2012; Thomsen and Melzner, 2010; Range et al., 2014). In bivalves, most ammonia excreted is a product of the catabolism of amino acids from the metabolic reserve as from diet (Saucedo et al., 2004; Mao et al., 2006). In this study, since the initial food concentrations were identical for all treatments, a decrease in excretion rate may indicate a drastic reduction in the catabolism of amino acids. The mussels reduced their ER at low pH conditions probably because they are unable to compensate fully for disturbances in their acid–base balance when exposed to seawater acidification and this can lead to metabolic depression (Liu and He, 2012), which is, in many cases, an adaptive strategy for survival under stressful conditions (Michaelidis et al., 2005). The O:N ratio provides information on substrate catabolism, which is compared as an indicator of the nutritional conditions of the organisms. According to Fernández-Reiriz et al. (2011), a prevalence of catabolism of carbohydrates and lipids results in values higher than 30, while a protein catabolism (conditions of alimentary deficiency) results in values of less than 30. Accordingly, a high rate of protein catabolism compared to lipids or carbohydrates is expressed by a low ratio, which is generally indicative of a stressed condition (Anestis et al., 2010). Widdows (1985) demonstrated that index values below 30 lie on the stress threshold for M. edulis. During this experiment, all conditions of maintenance showed a prevalence of protein catabolism probably due to the low respiration rates and food absorption rates. For the temperature tests, the values for the O:N ratio were not significantly different between two temperatures except at day 4 at pH 7.7. However, for pH effect, higher values were recorded at pH 7.3 than at the other pH levels. In this case, the O:N ratio presented false results and lost its power to be used as an indicator of stress since a greater amount of protein was being metabolized at the normal pH conditions. SFG is a useful index for estimating effects of environmental stressors on the overall performance of bivalves (Navarro et al., 2013), and generally varies as a function of temperature (Brown et al., 2004). Ecophysiological information arising from the present experiment supports the view that temperature represents an important driver able to influence the energy budget and growth in bivalves (Ezgeta-Balić et al., 2011). The SFG values determined in this study were all positive, and comparable with literature values reported for M. edulis and M. galloprovincialis (Smaal and Widdows, 1994; Anestis et al., 2010). Whereas the SFG of M. barbatus were positive at temperature 20 °C and 26 °C, and negative at 28 °C (Ezgeta-Balić et al., 2011). Anestis et al. (2010) studied the response of M. galloprovincialis to increasing seawater temperature, and found that the SFG values became negative at temperatures higher than 24 °C, probably associated with a significant reduction in the clearance rate. In addition, the SFG for Mytilus californianus was highest at 17–22 °C and declined at 26 °C (Bayne et al., 1976). This is consistent with the fact that temperate Mytilus spp. are well known for their ability to tolerate low temperatures

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whereas very high temperatures can have serious detrimental effects on clearance rate and energy gain (Seed and Suchanek, 1992). In the present study, generally, SFG values were higher at 25 °C at each pH level during the whole experiment, and especially at day 7 and day 14, significant reductions of SFG at 30 °C were observed compared to 25 °C. Temperature increasing from 20 to 32 °C resulted in a marked reduction of SFG in the clam R. decussates (Sobral and Widdows, 1997). In oyster Crassostrea corteziensis, SFG increased with increasing temperature from 23 to 29 °C, and then decreased at 32 °C (Guzmán-Agüero et al., 2013). These reductions match with the lowering of the energy gain (i.e. clearance rate or food acquisition), which reflect the disruption of temperature adaptation mechanisms and inhibition of feeding activity at elevated temperatures as also found for species of Crassostrea, Ostrea, Ruditapes and Mytilus spp. (Winter, 1978; Widdows, 1978; Sobral and Widdows, 1997; Smaal and Widdows, 1994). The results of the SFG suggest that M. coruscus is able to grow at 30 °C in this work, but high temperatures (above 30 °C) are thus stressful to the mussels. Long-term increased pCO2 can significantly reduce the growth of bivalves such as M. edulis and Crassostrea virginica (Michaelidis et al., 2005; Berge et al., 2006; Beniash et al., 2010). Degradation of proteins and reduced energy input were reported in M. edulis, M. galloprovincialis and R. decussates following OA (Thomsen and Melzner, 2010; Fernández-Reiriz et al., 2011, 2012). Navarro et al. (2013) evaluated the impact elevated pCO2 on the physiological processes of juvenile M. chilensis, indicating that high pCO2 levels in the seawater have a negative effect on the health of M. chilensis. Duarte et al. (2014) found that in M. chilensis the calcium deposition and total weight were negatively affected by increased CO2, suggesting M. chilensis is not able to overcome increments of CO2. Some authors found metabolic depression under relatively high seawater pCO2 and suggested that uncompensated extracellular pH might be the trigger for these reductions (Pörtner et al., 2004; Michaelidis et al., 2005). However, in the present study, the growth of M. coruscus was not negatively affected by OA, since the present study revealed that the elevated pCO2 levels showed no significantly negative impact on the physiological energetics of juvenile M. coruscus from feeding, absorption and ultimately the energy available to growth. Similar to our study, using moderate levels of acidification, SFG remained unchanged or even increased under acidification stress in M. galloprovincialis (Fernández-Reiriz et al., 2012). Juvenile king scallop, P. maximus, is potentially tolerant to low levels of OA when food is unrestricted (Sanders et al., 2013). Miller et al. (2009) suggested that the biological responses to acidification, especially for calcifying biota, will be species-specific and much more variable and complex than previously reported. Some marine bivalves inhabiting the coastal region experience changes in body fluid pCO2 levels during periods of emersion and may be pre-adapted to utilize metabolic depression as a response to elevated pCO2 (Michaelidis et al., 2005; Thomsen and Melzner, 2010; Gazeau et al., 2013). Such pre-adaption to fluctuations could provide a mechanism for coping with changes in pCO2 levels due to ocean acidification. These results suggest that the predicted rise in seawater pCO2 levels will not affect the physiology and aquaculture of some bivalve species. There are few studies considering the impacts of OA in synergy with other environmental stressors (Gazeau et al., 2013). Studies on the interactive effect of OA and temperature on marine animals have led to two apparently contradictory views (Gazeau et al., 2013). First, the effects of OA are exacerbated in the presence of elevated temperature (Gazeau et al., 2013). Second, the effects of ocean acidification are ameliorated in the presence of elevated temperature (Brennand et al., 2010). For instance, higher temperature mitigated the impacts of reduced pH on the calcification of juvenile C. virginica (Waldbusser et al., 2011). This contradiction is apparent because the effect of warming depends on the control temperature used and where it is placed in the thermal window and on the performance curve of a species (Pörtner and Farrell, 2008). The first view starts from a temperature close to the upper optimum, warming causes a decrease in performance, with CO2

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exacerbating the effects of warming. The second view starts from a temperature below the optimum, and warming then improves performance and resistance to CO2. In the shelled mollusk species studied to date, most of the observations fall within the first views (Gazeau et al., 2013), in line with the hypothesis that CO2 causes a narrowing of thermal windows (Pörtner and Farrell, 2008). However, a recent review by Hendriks et al. (2010) proposed that marine biota may be more resistant to acidification than expected. In our study, although some interactive effects were observed in some physiological parameters, but OA did not show an additive or antagonistic effect to temperature on the feeding and physiological energetics of M. coruscus, which means that in a certain range of temperature pH may not have an interaction with other environmental factors for some species. A significant effect of pH on the CR at day 14 at 25 °C was found, but this effect was not observed when temperature was elevated (Fig. 2A), indicating that high temperature could reduce pH effect on this physiological function. At day 1, ER was significantly reduced by high temperature under normal pH condition, but showed no significant difference between two temperatures when the pH was reduce to 7.3 and 7.7, indicating the low pH eliminates the temperature effect. Similar patterns were also noted in four RR measurements (Fig. 3). All these results illustrated that temperature and pH interacted on some physiological functions in this species, although pH did not affect the scope for growth of the mussel. Interestingly, some physiological activities increased with time, especially CR and SFG in control treatments. pH 8.1 and temperature 25 °C were the optimal laboratory conditions for mussel culture, and the mussels displayed filtration activities and growth along with acclimation time better and better under such conditions. However, even exposed to stress conditions, the mussel still presented a positive growth, indicating a tolerance to acidification and thermal stress in this species. The PCA included all the data during the whole experiment. Thus, to see how the mussels react to environmental stress, such as the elevation of temperature and pH reduction in this work, and to see if the pH reduction was associated with a different physiological response under a temperature increase, a profile of active physiological functions with high AE, high CR and high SFG was established (Fig. 5). For the integration of physiological responses, PCA was applied to differentiate combined treatments as a function of principal components. The two dominant components strongly corresponded to CR, RR, ER, AE and SFG (PC1) and O:N and E (PC2). PCA distinguished control from exposed treatments since most low pH and high temperature treatments were grouped together by PC1 reflecting similar physiological responses. The ANOVA testing the relationship between PCA Component 1 and the temperature increase showed that the characteristics of physiology associated with the thermal stress were lower CR, AE and SFG coupled with lower RR and ER. The physiological function profile of CO 2 exposed mussels was similar to the physiological response of mussels' exposure to temperature stress. The present study has shown that juvenile M. coruscus exposed to reduced seawater pH are able to survive, although some physiological parameters are affected. In general, seawater acidification had a little effect on the feeding and digestive behavior of the juvenile bivalves as the CR, AE and SFG were almost not affected in acidified treatments. In contrast, temperature showed a significant effect on the feeding and SFG of juvenile mussels. Thus, a slight shift of temperature extremes towards the higher side would restrict slightly optimal performance of M. coruscus, pushing it closer to its ecophysiological limits. Although the present experiment has limitations in terms of the narrow temperature and local population, it provides a valuable insight into the physiological energetics of a commercially and ecologically important bivalve species. Future studies should consider long-term effects of wide temperature range and different geographic populations associated with OA for this species.

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Physiological energetics of the thick shell mussel Mytilus coruscus exposed to seawater acidification and thermal stress.

Anthropogenic CO₂ emissions have caused seawater temperature elevation and ocean acidification. In view of both phenomena are occurring simultaneously...
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