Aquaculture 576 (2023) 739891 Contents lists available at ScienceDirect Aquaculture journal homepage: www.elsevier.com/locate/aquaculture Experimental determination of differential seasonal response in seed of the Manila clam, Ruditapes philippinarum, in context of climate change Eve Galimany a, *, Arturo Lucas a, b, Francesc Maynou a, Montserrat Solé a, Carles Pelejero a, c, Montserrat Ramón a a Institut de Ciències del Mar, ICM-CSIC, Barcelona, Spain b Institut de Ciència i Tecnologia Ambienals (ICTA-UAB), Universitat Autònoma de Barcelona, 08193, Cerdanyola del Vallès, Barcelona, Spain c ICREA, Institució Catalana de Recerca i Estudis Avançats, Barcelona, Catalonia, Spain A R T I C L E I N F O A B S T R A C T Keywords: Marine bivalves are found as key components of coastal habitats and have several important roles, such as Bivalve serving as a food source for human beings and aquatic organisms. In fact, as the world’s population continues to Temperature grow, bivalve aquaculture is expected to increase in importance as a means of addressing demands for animal pH protein; however, its growth may be possibly compromised by unfavourable climatic conditions. Thus, we Physiology assessed the effects of increased water temperature and acidification on the seeds of a bivalve of commercial Biochemistry importance, the Manila clam, Ruditapes philippinarum, in order to understand how this species may be affected by climate change at its early life stages. We examined the expected response of clams by experimentally mimicking seasonal conditions that could be forecasted to occur at the end of the twenty-first century. Different physio­ logical responses were measured including growth rates, clearance rate, burrowing time and different biochemical biomarkers of metabolic stress. The results showed that growth decreased in acidic experimental conditions in spring, with a weak interaction with temperature. Clearance rate was negatively affected by a lower pH in spring and summer but, under extreme summer conditions, the effect of pH was overridden by the negative impact of a higher temperature. Burrowing rates were reduced by half under warm temperature conditions in spring and summer. In contrast, biochemical biomarkers were only significantly depicted under climate change conditions in autumn. Overall, this study demonstrates the need to consider seasonality when evaluating the potential effects of climate change on clam aquaculture in order to forecast consequences for biological production. 1. Introduction annum around the globe (van der Schatte Olivier et al., 2020). However, the ability to derive such products from bivalve aquaculture may be Marine bivalves are found as key components of coastal habitats and compromised by unfavourable environmental conditions occurring as a have many important roles with regard to maintaining a healthy result of increases in water temperature and acidification of ocean wa­ ecosystem including functions related to nutrient coupling and habitat- ters. It has been estimated that the detrimental costs of ocean acidifi­ forming capacity, enhancing coastal resilience. Additionally, bivalves cation on bivalves could reach over $100 billion by 2100; this value is yield a wide array of natural products that are based on the use of meat projected based on the assumption that there will be an increasing de­ as well as shells (Smaal et al., 2019). All of these functions have mand for cultured molluscs (Narita et al., 2012). worldwide economic impacts. For example, added-value goods obtained During the twenty-first century, oceans around the world are pre­ from bivalves are estimated to have an annual market value of $23.9 dicted to increase in water temperature and acidification with the pro­ billion globally, including not only meat destined for consumption, but gression of land-based human activities (Bindoff et al., 2019). The trends also ornamental pearls and shells, and components of poultry grit (van already observed over the past few decades will be strengthened by der Schatte Olivier et al., 2020). On the other hand, non-food bivalve future changes; these trends include a global sea surface temperature aquaculture products are worth $6.47 billion ($2.95–9.99 billion) per increase of ~0.9 ◦ C since the beginning of the 20th century (Arias et al., * Corresponding author. E-mail address: galimany@icm.csic.es (E. Galimany). https://doi.org/10.1016/j.aquaculture.2023.739891 Received 3 March 2023; Received in revised form 11 July 2023; Accepted 12 July 2023 Available online 17 July 2023 0044-8486/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by- nc/4.0/). E. Galimany et al. Aquaculture 576 (2023) 739891 2021) and a pH decrease of 0.017–0.027 units per decade across a range 2. Materials and methods of a 15 years-time series of pH observations (Bindoff et al., 2019). However, water temperature increases and pH decreases will reach 2.1. Experimental animals different values in each area. For example, the Mediterranean region, which represents a hot spot of climate change, temperature is projected Approximately 1000 seed-size Ruditapes philippinarum clams of 6–19 to highly rise and precipitation considerably decrease by the end of the mm total length (TL) and 6–30 mg dry weight (DW) were purchased 21st century (Calvo et al., 2011; Durrieu de Madron et al., 2011; Paeth from Satmar, the Marennes nurseries, France, before each seasonal et al., 2017). All these environmental changes could hamper the phys­ experiment. Clams were maintained in an open seawater system at the iological processes of bivalves, which may be related to shell calcifica­ Aquaria and Experimental Chambers (ZAE) of the Institute of Marine tion, growth, and immunological responses, also making them more Sciences (ICM-CSIC). They were acclimated to each seasonal in situ vulnerable to parasites and diseases (Fernández Robledo et al., 2018; seawater temperature for one week before the start of each experiment Fitzer et al., 2018; Matozzo and Marin, 2011; Tan and Zheng, 2020). The and fed daily a mixture (1:1 in volume) of the phytoplankton species metabolic processes of bivalves may be further compromised by the Tetraselmis sp. and Isochrysis galbana at a concentration of 4 × 103 cells combination of high temperature and low pH with other stressors such mL− 1, similar to other bivalve studies (Peteiro et al., 2018; Velasco, as marine pollution (Maynou et al., 2021). 2007). The phytoplankton species were cultured at an 18:6 h day:night The Manila clam, Ruditapes philippinarum (Adams & Reeve, 1850), is cycle at 23 ◦ C using standard protocols and F/2 media (Creswell, 2010). a commercial bivalve species reported to be affected by climate change. Mortality was scarce throughout the experiments, as detailed in This species is native to the Indo-Pacific area but it was brought to Table S1. Europe for commercial purposes in the 1970s; today it is found all over the world (Coelho et al., 2021). It is a bivalve species with high com­ 2.2. Experimental design mercial value and it is by far the most common cultured clam species in the world, with a total catch >4200 t in 2020 (FAO, 2022). The experiments were performed in a controlled temperature R. philippinarum may be affected by climate change in different ways. For chamber at the ZAE. Four experiments lasting four weeks each over example, ammonia excretion rate increased when seawater pH was different seasons were undertaken, in which clams were exposed to a experimentally reduced from 8.0 to 7.7, whereas clearance rate and the combination of two temperatures, ambient in each season (corre­ ratio of oxygen consumption to nitrogen excretion significantly sponding to the coastal mean sea surface temperature in a farming decreased (Xu et al., 2016). Similarly, R. philippinarum reduced the aquaculture area of the Ebre Delta, Western Mediterranean: spring clearance, ingestion and respiration rates and increased the ammonia 14.5 ◦ C, summer 20 ◦ C, autumn 18.8 ◦ C and winter 14.4 ◦ C) and ambient excretion rate at pH 7.7 and 7.4 (Fernández-Reiriz et al., 2011). How­ in each season +3 ◦ C, and three different pH levels, 8.1, 7.7, and 7.3 ever, other studies have reported contradicting results. While Velez et al. (Fig. 1, Table 1). The warming scenario attempted to mimic the expected (2016) reported that clams were able to maintain their physiological conditions for sea surface waters under the intensive-emission Repre­ status and biochemical performance at pH 7.8, Range et al. (2011) re­ sentative Concentration Pathway (RCP) 8.5 scenario by 2100 (Bopp ported no differences among pH treatments in net calcification or clam et al., 2013; Van Vuuren et al., 2011). The three pH levels were chosen as growth and weight for R. decussatus. These contradicting findings sug­ representatives of surface global oceans at present, at the end of 2100 gest that other factors, such as seasonality, may have affected the and at the end of 2300, respectively, also under RCP 8.5 (Bopp et al., physiology of the clams. In this sense, R. philippinarum biochemical 2013; Hartin et al., 2016). We note, however, that pH values between performance did not change as a result of low contamination levels but 7.7 and 7.3 may be temporarily reached earlier in coastal areas and as a result of seasonality (Costa et al., 2020a). shallow tide estuaries, which are typical environments for bivalve The projected temperature increase in the context of global climate cultivation that exhibit much larger pH variability than the open sea change scenarios will be an additional stressing factor to bivalves. When (Lowe et al., 2019). exposed to temperatures 3 ◦ C to 5 ◦ C higher than in ambient tempera­ A total of 20 to 50 seed clams were distributed in each 20-L glass tures, clams may suffer from thermal stress and reduce scope for growth aquarium, with three replicates per treatment, and they were fed a (Han et al., 2008; Zhang et al., 2019); however, physiological and phytoplankton mixture of 1:1 Tetraselmis sp. and Isochrysis galbana (in metabolic adaptative responses may lessen the impact in some species volume). A peristaltic pump continuously introduced the phytoplankton (Velez et al., 2017). When no interactions with other deleterious factors, into the head tank at 3 L h− 1 to reach a concentration of 4 × 103 cells like pH or emerging contaminants, are present, moderate temperature mL− 1. The clams were slowly acclimated to the targeted experimental increases may lead to enhanced growth and reproduction rates in clams conditions over 13–15 days by lowering the pH 0.05 units daily. Heaters and other bivalves, according to the results from mathematical models were used to increase the temperature 1 ◦ C every 4–5 days until the applied to physiological traits (Costa et al., 2020a; Maynou et al., 2021; desired final experimental conditions were achieved. Then, the seed Steeves et al., 2018). On larval stages of bivalves, the negative impact of clams were exposed for four weeks to ambient seasonal temperatures increased temperature may be even more acute than that on juveniles (control) and + 3 ◦ C (treatment) over the four seasons (Table 1). and adults, but evidence from experimental research is still limited HOBO data loggers were used to keep track of the temperature in (Prado et al., 2016). each treatment, which was controlled and manipulated by means of two The main goal of this study was to understand the potential influence temperature baths in which the experimental aquaria were placed, with of seasonality on several biological processes related to juvenile clam two electronic Pt100 regulators (Delta Ohm HD9022) connected to 300 (R. philippinarum) performance under forecasted climate change condi­ W heaters in the +3 ◦ C treatment (Fig. 1). Seawater pH was adjusted by tions. For this, the growth, clearance rate, burrowing activity, energy bubbling CO2 into two overhead tanks that delivered seawater at each of metabolism and detoxification defences of juvenile clams were the two acidic pH levels to all aquaria (Fig. 1). This CO2 delivery was compared throughout the seasons of the year under actual conditions performed in a controlled way by means of an automated system with and those that could be influenced by global and climate change, such as glass pH electrodes (Ecotrode Plus 6.0262.100, Metrohm) connected to pH decrease and temperature increase. two pH controllers (Consort R362), which opened and closed the sole­ noid valves of the CO2 line when needed. To avoid drifts in the pH measurements, glass electrodes were calibrated on a daily basis with TRIS buffer, following standard procedures (SOP6a of Dickson et al., 2007). For further details on the experimental setup for pH and tem­ perature manipulation, see Movilla et al. (2016) and Movilla et al. 2 E. Galimany et al. Aquaculture 576 (2023) 739891 Fig. 1. Diagram of the experimental setup. An open circulating water system was installed in a thermostatic room at the Experimental Aquarium Zone (ZAE) of the ICM-CSIC. Seawater with a concentration of 4 × 103 phytoplankton cells mL− 1 mixed in the header tank was continuously supplied to 20-L aquaria containing clams. This water was then circulated to three other head tanks where the pH was adjusted using a system of glass electrodes connected to a pH controller, which auto­ matically opened and closed the solenoid electrovalves of the CO2 line when needed. Water in all aquaria overflowed in the temperature baths. The ambient temperature bath was adjusted to room temperature, and the warm (ambient +3 ◦ C) bath was controlled with an electronic regulator. Note that this set up was designed to study the response of another species in addition to the clams in the experiment presented here, so only half of the aquaria were used in the experiments with Ruditapes philippinarum. (2012). In addition to the pH control using glass electrodes, approxi­ 2.3. Growth mately once a week, seawater samples from each treatment were taken to the laboratory for total alkalinity analyses by potentiometric titration All clams were measured (TL) with a Vernier calliper and weighed (± (Perez and Fraga, 1987; Pérez et al., 2000) and determination of pH by 0.001 g) before the seasonal experiments to ensure that sizes were spectrophotometry (Clayton and Byrne, 1993), which provides more equally distributed among the replicated aquaria in each treatment. At precise pH measurements than glass electrodes. At the same time, the the end of the 4-week experiments, all clams, that is, between 20 and 50 temperature and salinity of each treatment aquarium were also moni­ clams in each aquarium x 3 replicates per treatment x 6 treatments × 4 tored using a YSI-30 M probe and used to calculate the rest of the pa­ seasons, were measured and weighed again to compare the increase in rameters of the carbonate system in seawater using the CO2SYS Excel mean size among treatments. add-in (Pierrot et al., 2006) with dissociation constants for carbonate determined by Mehrbach et al. (1973) and refit by Dickson and Millero (1987). The physicochemical parameters of both treatments during the 2.4. Clearance rate experiment are shown in Table 1. To minimize potential pseudor­ eplication issues, weekly shifts in the tubes and the three head tanks The clearance rate, i.e., the volume of water cleared by clams per unit delivering seawater from each of the pH treatments were performed, so of time (Lh− 1) was estimated by quantifying the decrease in algal cell all aquaria received sequential seawater from all tanks throughout the density from the experimental water. Three clams, one from each experiment. replicate aquaria corresponding to the same combination of temperature For these four experiments, the following variables were measured: and pH, were placed at the bottom of 50-mL test tubes, which were growth, clearance rate and burrowing behaviour as physiological vari­ previously perforated with three holes on the base and three longitu­ ables and some selected biochemical biomarker determinations related dinal openings all around the tube, allowing water to pass through to oxidative stress and metabolism. All clams were measured at the (Fig. S1). The clams in the tube were immediately placed in a 250-mL beginning and end of the experiments. Then, each clam was only used to glass bottle with 200 mL of experimental water from the aquaria. The obtain measurements for one of the remaining variables, i.e., clearance test tubes with clams were maintained elevated and stable in the glass rate, burrowing or biomarker determinations. bottle using tin wire to allow the placement of a 2-cm magnet under­ neath the test tube. The bottles were then placed on a multi-position magnetic stirring plate to ensure resuspension of particles. Five bottles containing three clams each were used for each treatment, i.e., the combination of each of the two temperatures with each of the three pH 3 E. Galimany et al. Aquaculture 576 (2023) 739891 Table 1 Parameters of the seawater carbonate system in aquaria for each treatment corresponding to spring (A), summer (B), autumn (C) and winter (D). Spring treatment Measured parameters Calculated parameters Temperature pH T pHT Alk pCO2 DIC [CO2]aq [HCO−3 ] [CO2− 3 ] ΩC ΩA A Ambient (natural 8.1 14.5 ± 8.042 ± 2550 ± 1 441 ± 27 2299 ± 16.4 ± 2101 ± 16 182 ± 7 4.24 ± 2.73 ± cycle) 0.4 0.022 11 0.9 0.15 0.10 7.7 14.5 ± 7.722 ± 2547 ± 1 1020 ± 91 2444 ± 38.0 ± 2310 ± 19 96 ± 7 2.24 ± 1.44 ± 0.4 0.036 14 3.3 0.17 0.11 7.3 14.5 ± 7.393 ± 2548 ± 3 2315 ± 2563 ± 86 ± 12 2429 ± 14 48 ± 6 1.12 ± 0.72 ± 0.4 0.059 350 21 0.13 0.09 Warm (+3 C)◦ 8.1 17.9 ± 8.015 ± 2547 ± 1 473 ± 6 2282 ± 2 15.9 ± 2074 ± 4 192 ± 2 4.48 ± 2.90 ± 0.5 0.004 0.1 0.04 0.03 7.7 17.9 ± 7.736 ± 2557 ± 1 1000 ± 2427 ± 33.9 ± 2281 ± 29 112 ± 12 2.62 ± 1.70 ± 0.5 0.044 110 21 4.2 0.28 0.18 7.3 17.8 ± 7.352 ± 2564 ± 2 2624 ± 2579 ± 88.3 ± 13 2441 ± 15 50 ± 6 1.16 ± 0.75 ± 0.7 0.063 430 22 0.14 0.09 B Ambient (natural 8.1 20.2 ± 8.034 ± 2523 ± 441 ± 10 2229 ± 14.1 ± 2004 ± 20 210 ± 5 4.92 ± 3.21 ± cycle) 0.5 0.007 10 16 0.5 0.12 0.08 7.7 20.0 ± 7.728 ± 2542 ± 1 1014 ± 70 2404 ± 32.2 ± 2255 ± 14 117 ± 6 2.73 ± 1.78 ± 0.5 0.028 10 2.0 0.14 0.09 7.3 20.0 ± 7.373 ± 2533 ± 6 2431 ± 33 2531 ± 7 77.2 ± 2399 ± 7 55 ± 1 1.28 ± 0.84 ± 0.5 0.006 1.8 0.03 0.02 Warm (+3 ◦ C) 8.1 22.8 ± 7.996 ± 2526 ± 492 ± 11 2230 ± 14.5 ± 2004 ± 23 212 ± 5 4.97 ± 3.27 ± 0.6 0.006 13 19 0.5 0.12 0.08 7.7 23.1 ± 7.650 ± 2534 ± 6 1236 ± 18 2412 ± 9 36.2 ± 2266 ± 11 110 ± 3 2.56 ± 1.69 ± 0.5 0.005 1.0 0.07 0.05 7.3 23.0 ± 7.348 ± 2551 ± 2642 ± 2545 ± 77.6 ± 2409 ± 30 58 ± 3 1.36 ± 0.89 ± 0.5 0.018 24 130 32 5.0 0.07 0.05 C Ambient (natural 8.1 18.7 ± 8.072 ± 2562 ± 5 406 ± 1 2254 ± 3 13.4 ± 2021 ± 8 220 ± 5 5.13 ± 3.33 ± cycle) 0.7 0.001 0.3 0.13 0.09 7.7 18.8 ± 7.770 ± 2547 ± 7 907 ± 37 2398 ± 9 29.8 ± 2247 ± 8 122 ± 1 2.84 ± 1.85 ± 0.8 0.014 0.6 0.01 0.01 7.3 18.8 ± 7.403 ± 2544 ± 2257 ± 80 2536 ± 2 74.3 ± 2405 ± 2 56 ± 4 1.32 ± 0.86 ± 0.9 0.018 12 4.3 0.10 0.06 Warm (+3 ◦ C) 8.1 22.2 ± 8.019 ± 2561 ± 469 ± 14 2253 ± 14.0 ± 2019 ± 18 221 ± 3 5.17 ± 3.39 ± 0.1 0.007 24 21 0.2 0.08 0.06 7.7 21.7 ± 7.731 ± 2562 ± 1015 ± 32 2411 ± 30.8 ± 2256 ± 10 125 ± 1 2.92 ± 1.91 ± 0.6 0.001 11 11 0.5 0.01 0.01 7.3 21.9 ± 7.373 ± 2556 ± 6 2478 ± 2546 ± 2 74.9 ± 2412 ± 1 59 ± 3 1.38 ± 0.90 ± 0.3 0.018 101 3.6 0.07 0.05 D Ambient (natural 8.1 14.4 ± 8.055 ± 2570 ± 428 ± 4 2311 ± 8 16.0 ± 2108 ± 7 187 ± 2 4.37 ± 2.81 ± cycle) 0.2 0.004 11 0.1 0.04 0.03 7.7 14.4 ± 7.686 ± 2579 ± 1129 ± 92 2489 ± 2 42.1 ± 2357 ± 5 90 ± 6 2.10 ± 1.35 ± 0.2 0.034 10 3.2 0.15 0.09 7.3 14.4 ± 7.353 ± 2576 ± 2530 ± 2606 ± 94.4 ± 2468 ± 16 44 ± 1 1.02 ± 0.65 ± 0.2 0.015 14 100 19 3.8 0.03 0.02 Warm (+3 ◦ C) 8.1 17.8 ± 8.010 ± 2559 ± 4 482 ± 5 2296 ± 6 16.3 ± 2089 ± 6 190 ± 1 4.45 ± 2.88 ± 0.3 0.004 0.1 0.02 0.01 7.7 17.8 ± 7.638 ± 2565 ± 1280 ± 2475 ± 5 43.1 ± 2341 ± 3 91 ± 6 2.13 ± 1.38 ± 0.4 0.037 17 110 3.2 0.15 0.09 7.3 17.8 ± 7.314 ± 2558 ± 2805 ± 93 2587 ± 94.8 ± 2447 ± 15 45 ± 1 1.05 ± 0.68 ± 0.4 0.012 12 17 3.4 0.03 0.02 T (temperature, ◦ C), pHT (pH, in total scale), Alk (total alkalinity, μmol/kg-SW) and salinity (which was always 38.0) were used to calculate pCO2 (partial pressure of CO2, μatm), DIC (dissolved inorganic carbon, μmol/kg-SW); [CO2]aq (CO2 concentration in seawater, μmol/kg-SW), [HCO3− ] (bicarbonate ion concentration, μmol/ kg-SW), [CO2− 3 ] (carbonate ion concentration, μmol/kg-SW), ΩC (calcite saturation state) and ΩA (aragonite saturation state), using the CO2SYS Excel add-in (Pierrot et al., 2006) with dissociation constants for carbonate determined by Mehrbach et al. (1973) and refit by Dickson and Millero (1987). All parameters are expressed as the mean ± SE. values. Before the beginning of the experiments, 2 mL of 1:1 Tetraselmis analysis. At the end of the experiment, bivalves were dissected, and their sp.:Isochrysis galbana was added to each glass bottle to obtain a final soft tissue was dried at 60 ◦ C for 48 h to determine individual dry tissue concentration of 4 × 103 cells mL− 1. One extra bottle was left as a weight. To calculate the phytoplankton remaining in the bottles, water control to detect changes in the water particle concentration. Immedi­ samples were run on a BD Biosciences Flow Cytometer (San Jose, Cali­ ately after phytoplankton addition, samples (1.8 mL) were pipetted fornia) for 2 min on the fast flow rate setting (35 μl min− 1). The every 5 min, starting at time 0 and continuing for 30 min, after which phytoplankton species were identified and quantified using the con­ time samples were taken every 10 min until 1 h had passed. All collected trasting side scatter vs. green fluorescence plots produced by the flow samples were preserved in 1% formalin (final concentration) until cytometer. Then, the clearance rate was determined using the formula 4 E. Galimany et al. Aquaculture 576 (2023) 739891 (Riisgård, 2001): The kinetic assay was performed in a 50 mM phosphate buffer (pH = 7.4) containing 1 mM (final concentration) of the respective substrates CR = (V/nt ) ln (C0 /Ct ) and 25 μL of sample (S10). The formation of 4-nitrophenolate was where C0 and Ct are the algal concentrations (cells mL− 1) at time monitored spectrophotometrically at 405 nm at 25 ◦ C for 5 min. An 0 and time t, respectively, V is the volume of water, and nt is the number extinction coefficient of 18 mM− 1 cm− 1 was used to express the hy­ of individual clams (three per bottle). All CR values were standardized to drolysis of these nitrophenyl esters (Hosokawa and Satoh, 2005). 1 g of dried clam flesh using the following equation: 2.6.2. Antioxidant defences CRs = CR x (1/We )b Glutathione reductase (GR; EC 1.8.1.10) activity was measured using where CRs is the standardized clearance rate, CR is the experimen­ 1 mM glutathione disulphide (GSSG), 0.5 mM NADPH and 20 μL of tally determined clearance rate, We is the average dry body mass sample. The absorbance decrease was measured using a microplate measured for the clams in each bottle, and b is a predetermined constant reader for 3 min at 340 nm (ε = 6.22 mM− 1 cm− 1) following the protocol feeding rate. We used a value of b = 0.8 determined by Riisgård (1988) of Carlberg and Mannervik (1985). for the clam Mercenaria mercenaria, as no estimates for R. philippinarum Glutathione S-transferase (GST; EC 4.4.1.20) activity was quantified were found in the literature. using a reaction mixture containing 1 mM 1-chloro-2,4-dinitrobenzene For the clearance rate parameters, an additional fifth experimental (CDNB), 1 mM reduced glutathione (GSH) and 25 μL of sample. Mea­ setup was included to assess the influence of extreme summer condi­ surements were carried out for 3 min at 340 nm (ε = 9.6 mM− 1 cm− 1) tions, mimicking temperatures (> 28 ◦ C) that have actually been following the protocol of Habig et al. (1974). recorded in the area (Fernández-Tejedor et al., 2022; Galimany et al., 2011). This is a suspected temperature where the physiology of bivalves 2.6.3. Metabolic activity might be greatly compromised (Galimany et al., 2011). Thus, this rele­ Electron transport system (ETS) activity was measured following the vant physiological parameter was studied under the temperatures of method of King and Packard (1975) using p-iodonitrotetrazolium chlo­ 25 ◦ C (as ambient) and 28 ◦ C (extreme summer) for the three different ride as a substrate (0.6 mM). The absorbance was read at 490 nm for 5 pH levels (8.1, 7.7, and 7.3) as for the other experiments. min. The amount of formazan formed was calculated using ε = 15.9 mM− 1 cm− 1. 2.5. Burrowing behaviour 2.6.4. Protein determination The total protein content was determined by the Bradford method To assess the burrowing activity, aquaria were filled with 500 g of (Bradford, 1976) adapted to microplate readers using the Bradford dry sediment (depth 3–4 cm) collected from a clam bed. The sediment Bio-Rad Protein Assay reagent and bovine serum albumin (BSA) as a was previously calcinated to eliminate all organic matter. When the standard (0.05–1 mg mL− 1). Absorbance was read at 595 nm. sediment settled and no resuspension was observed in the aquaria (approximately 24 h later), burrowing bioassays were conducted by 2.7. Statistical analysis placing 15 clams of similar shell lengths over the sediment at each aquarium. Three replicate aquaria were used for each combination of Values of growth (log transformed), standard clearance rate and temperature and pH. Clam behaviour was continuously recorded by an biomarker determinations were analysed seasonally using a generalized observer per aquarium, and the burrowing time was determined as the linear model (GLM) against the environmental variables temperature, time that elapsed from the start of digging until the time the clam fully pH, and the interaction temperature × pH. Burrowing behaviour was disappeared from the sediment surface. analysed statistically with a Kaplan–Meier survival model for censored data, where individuals were assigned a value of 0 while on the surface 2.6. Biomarker determinations or 1 when burrowed. The survival curves determined under each experimental condition were tested for differences with the G-rho sta­ Biochemical determinations were conducted with the whole soft tistic. Analyses of the burrowing data were carried out with the R library tissue of two sets of three clams for each aquarium for a total of 6 in­ “survival” (Therneau, 2016). dividuals. Soft tissue was immediately and individually frozen at − 80 ◦ C until analyses. Before biomarker determinations, soft tissue was ho­ 3. Results mogenized at a 1:5 ratio (weight:volume) using a 50 mM phosphate buffer at pH 7.4 containing 1 mM ethylenediaminetetraacetic acid 3.1. Growth (EDTA). Homogenization was carried out using an electrically driven Polytron® homogenizer. All analytical determinations were carried out The growth of clams showed different trends throughout the studied on the supernatant resulting from a 10,000 g centrifugation for 30 min at period. The initial average (± SE) length and wet weight of the shell of 4 ◦ C, corresponding to the post-mitochondrial fraction (S10) and con­ clams per season were 8.94 ± 0.72 mm and 0.17 ± 0.04 g in spring, taining most of the cytosol soluble enzymes. 14.14 ± 1.29 mm and 0.61 ± 0.16 g in summer, 13.54 ± 0.85 mm and All assays were carried out in triplicate at 25 ◦ C in 96-well plates 0.48 ± 0.10 g in autumn, and 13.74 ± 0.72 mm and 0.50 ± 0.08 g in using a TECAN Infinite M200 microplate reader, and blanks (sample- winter, respectively. free) accompanied the sample batches to correct for non-enzymatic Clam length and weight at the end of the experiment did not differ reactivity of the substrates. Some purified proteins were included in among treatments in summer, autumn, and winter (Table S1). The the readings as methodological controls (Solé et al., 2020). All enzy­ average (± SE) lengths and wet weights of the shells were 14.11 ± 0.07 matic activities are expressed in nmol min− 1 mg− 1 of protein. mm and 0.63 ± 0.01 g, 13.56 ± 0.05 mm and 0.51 ± 0.01 g, and 14.52 ± 0.08 mm and 0.59 ± 0.01 g in summer, autumn, and winter, respec­ 2.6.1. Esterase activities tively. However, in spring, both final length and weight were signifi­ Carboxylesterase (CE; EC 3.1.1.1) activity was measured using the cantly higher in the experimental tanks at pH 8.1 than at pH 7.7 and 7.3 commercial colorimetric substrates p-nitrophenyl acetate (pNPA) and p- (lengths: 9.77 ± 0.06 mm, 9.57 ± 0.06 mm and 9.47 ± 0.06 mm, nitrophenyl butyrate (pNPB). The hydrolysis rate of pNPA and pNPB respectively, p < 0.0001; weights: 0.222 ± 0.013 g, 0.209 ± 0.012 g, was determined by a continuous spectrophotometric enzyme assay 0.205 ± 0.012 g, respectively, p < 0.0001) (Fig. 2). There was also a following Hosokawa and Satoh (2005). significant effect of the temperature × pH interaction (p = 0.053 for 5 E. Galimany et al. Aquaculture 576 (2023) 739891 Fig. 2. Average length and weight of clams at the end of each seasonal experiment plotted by temperature and pH separately. Letters denote significant differences (p < 0.05). The experimental ambient temperature conditions were as follows: spring 15 ◦ C, summer 23 ◦ C, autumn 18 ◦ C, and winter 14 ◦ C, with the “warm” treatment consisting of increasing the ambient temperature by 3 ◦ C. length; p = 0.025 for weight) in spring, indicating that growth at pH 7.7 the summer season, there was also an interaction between temperature was greater at ambient temperature than under warmer water and pH (F2,24 = 3.640, p = 0.044); a main effects test revealed that conditions. clearance rates at pH 8.1 and warm treatment were higher than those at the same temperature but in more acidic water. No differences among treatments were recorded for clearance rates in autumn and winter (p > 3.2. Clearance rate 0.05). Nonetheless, when clams were exposed to extreme summer temperatures (i.e., 24 ◦ C and 27 ◦ C), clearance rates were lower at the Clearance rates varied from 0.4 to 1.9 L h− 1 throughout all the ex­ highest temperature (F1,24 = 7.485, p = 0.012), while no differences periments, as detailed in Table 2. When comparing clearance rates be­ were found among pH values or for the interaction of both parameters tween the two different temperatures tested in each season, no (Fig. 4). significant differences were found; thus, clearance rates did not vary according to temperature throughout the four seasons. However, there 3.3. Burrowing behaviour were significant differences between pH levels in spring (F2,24 = 3.839, p = 0.036) and summer (F2,24 = 6.711, p = 0.006), showing higher Burrowing activity was always faster under ambient temperature in clearance rates at pH 8.1 and lower clearance rates at pH 7.3 (Fig. 3). For spring and summer. The time needed for 50% of the clams to burrow (Table 3) ranged between 11.30 and 16.20 min in spring and 8.67 and Table 2 11.75 min in summer under ambient temperature. In both seasons, Average standard clearance rates (± SE) for clams during each season, as increasing 3 ◦ C significantly slowed the time to 50% of the clams to observed under extreme temperature conditions and experimental conditions, e. burrow (17.90–21.50 min in spring and 12.67–21.67 min in summer), g., two water temperatures and three pH values. Ambient temperature and pH with G-rho values of 21 (1 d.f.) and 24 (1 d.f.) in spring and summer, 8.1 are considered to be current (control) conditions. respectively, with p < 0.001. In both seasons, pH was not significant (G- pH 8.1 pH 7.7 pH 7.3 rho with p > 0.05). The burrowing performance showed no significant Ambient Warm Ambient Warm Ambient Warm differences among treatments in autumn and winter (p > 0.05), neither 1.80 ± 1.91 ± 1.28 ± 1.35 ± 1.09 ± 1.27 ± for temperature nor pH (Table 3). Spring 0.43 0.36 0.17 0.16 0.10 0.13 1.50 ± 1.63 ± 1.50 ± 0.89 ± 1.13 ± 0.98 ± 3.4. Biomarker determinations Summer 0.13 0.19 0.08 0.09 0.18 0.12 1.05 ± 1.46 ± 1.99 ± 1.74 ± 1.09 ± 1.41 ± Autumn 0.10 0.19 0.60 0.38 0.17 0.27 All biomarker determinations showed no significant differences 0.96 ± 0.70 ± 0.64 ± 0.40 ± 0.43 ± 0.49 ± among treatments in spring, summer, or winter (p > 0.05) (Supple­ Winter 0.30 0.10 0.06 0.07 0.16 0.16 mental Figs. S2, S3, S4). However, in autumn, CE activities were Extreme 1.55 ± 1.39 ± 1.89 ± 0.96 ± 1.47 ± 0.86 ± different among pH values for pNPA-CE measures (F2,30 = 4.445, p = summer 0.20 0.29 0.35 0.17 0.21 0.27 0.020) and between temperatures when using pNPB as substrate (F1,30 6 E. Galimany et al. Aquaculture 576 (2023) 739891 Fig. 3. Seasonal clearance rates (average CR ± SD) estimated at each different experimental pH value (horizontal axis), pooling temperature treatment data. A) spring; B) summer; C) autumn; D) winter. Lowercase letters denote significant differences (p < 0.05). Table 3 Time to complete burrowing of 50% of the clam population (in minutes, with 95% confidence intervals) under experimental conditions. Season Temperature pH 50% 95% CI 8.1 11.30 (09.00–16.50) ambient 7.7 16.10 (14.20–20.70) 7.3 16.20 (11.30–18.30) Spring 8.1 20.60 (16.70–28.70) warm 7.7 21.50 (17.20–29.20) 7.3 17.90 (16.00–20.70) 8.1 11.75 (07.83–17.70) ambient 7.7 8.67 (07.00–13.80) 7.3 10.67 (08.50–16.80) Summer 8.1 19.50 (16.33–24.70) warm 7.7 21.67 (18.83–25.60) 7.3 12.67 (12.00–21.00) 8.1 17.00 (11.50–27.84) ambient 7.7 12.90 (11.20–26.00) 7.3 16.20 (11.50–23.80) Autumn 8.1 19.30 (15.00–29.20) warm 7.7 11.70 (09.50–28.80) 7.3 20.30 (12.20–28.30) 8.1 15.10 (10.80–21.50) ambient 7.7 26.80 (21.50–33.50) 7.3 15.80 (13.00–22.20) Winter 8.1 17.80 (16.00–28.20) warm 7.7 16.80 (14.00–23.80) 7.3 17.20 (14.30–29.20) = 9.526, p = 0.004). The warmer and more acidic treatments displayed higher enzymatic values (Fig. 5). Both antioxidant defences, GR and GST activities, were also higher in autumn at low pH (F2,30 = 7.940, p = 0.002 and F2,30 = 4.059, p = 0.028, respectively) (Fig. 5). In contrast, the metabolic activity, i.e., ETS, indicative of mitochondrial activity, dis­ Fig. 4. Clearance rates (average CR ± SD) estimated under extreme summer played the opposite response. conditions for both parameters measured, temperature (A) and pH (B). A good Pearson correlation coefficient was found among biomarker Lowercase letters denote significant differences between the ambient and responses considering the totality of the data (n = 143). That is, the warm treatments. antioxidant GR was positively correlated with GST (r = 0.560; p < 0.001), pNPA-CE (r = 0.336; p < 0.001) and pNPB-CE (r = 0.277; p = 0.001). Likewise, GST was correlated with CE measures with pNPA (r = 7 E. Galimany et al. Aquaculture 576 (2023) 739891 Fig. 5. Enzymatic activities (in nmol min− 1 mg− 1 prot) of whole clam (R. philippinarum) soft tissue during the autumn experiment as a function of water pH. A) Carboxylesterase phase I measures with the substrate pNPA, B) conjugative phase II glutathione S-transferase (GST) activity, C) antioxidant enzyme glutathione reductase (GR) activity and D) mitochondrial electron transport system (ETS) as an energy metabolism activity marker. The horizontal axis indicates pH treatments. 0.456; p < 0.001) and pNPB (r = 0.397; p < 0.001). In turn, pNPA-CE similar to what was seen in our study during spring and summer. Even and pNPB-CE were highly correlated (r = 0.796, p < 0.001). In though the temperatures were similar, other seasons (summer and contrast, ETS activity was negatively correlated with most parameters: autumn) did not follow this pattern. This may be because spring and GR (r = − 0.718; p < 0.001) and GST (r = − 0.204; p = 0.15) as well as summer are particularly vulnerable periods for clams, which exhibit with pNPA-CE (r = 0.252; p = 0.002) and pNPB-CE (r = 0.232; p = higher growth rates and adult animals may develop their gonads (Ojea 0.005). et al., 2004). The experimental clams may have been ripe despite their small size, as previously observed (Range et al., 2011). In 4. Discussion R. philippinarum, temperature may also cause variation in clearance rates, which typically rise with increasing temperatures (Han et al., The expansion of aquaculture is needed to feed the growing world’s 2008; Kang et al., 2016). However, the 3 ◦ C temperature difference population, but the sector is currently concerned about sustainability between the ambient and warm treatments used in our experiments and the effects of climate change on aquaculture production (Maulu might not be enough to allow for the detection of changes in this et al., 2021). In recent decades, many efforts have been made to un­ physiological parameter. The clearance rates of clams from the ambient derstand the effects of climate change on aquatic populations, but sea­ temperature and pH 8.1 treatment were higher in spring, slightly lower sonality is not included in most of the studies (Kwiatkowski and Orr, in summer, and they were the lowest in winter, the coldest season. This 2018). The life cycle of most marine organisms in temperate climates is pattern may be associated with the temperature gradient, given that strongly influenced by seasonality, which affect the reproductive cycle clearance, filtration and absorption rates for R. philippinarum increase and the availability of the trophic resources (Jung et al., 2019). More­ with temperature up to the threshold maximum (Bo et al., 2000). Clams over, seasonality has been determined to influence clam may adapt physiologically to low pH within limited bounds; for (R. philippinarum) performance under physical and chemical exposures example, despite temperature increases of up to 20 ◦ C and pH values as (Costa et al., 2020a). In this context, our study provides a novel low as 7.4 may alter clearance rates, food absorption efficiency may comprehensive experimental approach aimed at investigating the remain constant (Xu et al., 2016). The clams may, however, be physi­ response of this clam species to forecasted temperature and pH changes ologically compromised by temperatures above their thermal optimum, brought on by climate change. By focusing on clam seeds reared under which are estimated to be between 20 ◦ C and 22 ◦ C (Bo et al., 2000; Han different seasonal conditions, we were able to assess the extent of the et al., 2008). In our experiments, under extreme summer conditions changes seen throughout the year and pinpoint the most sensitive (28 ◦ C), the effect of temperature was found to override any influence of period. Changing environmental parameters have the potential to the pH decrease, when food absorption may have been altered. greatly influence physiological responses associated with feeding The ecological relevance of the effects of climate change on bivalve behaviour in bivalves. Growth rates, which are of key importance in burrowing activities have not been studied much. Burrowing provides aquaculture operations, were only significantly decreased in the spring protection from both predators and adverse environmental and tempo­ experiment, due to decreases in pH, while a 3 ◦ C change in temperature ral conditions, improving survivorship (Takeuchi et al., 2015; Zaklan was found to be not relevant. Likewise, clearance rates decreased in and Ydenberg, 1997). In the current study, burrowing was always faster clams exposed to pH 7.4 (Fernández-Reiriz et al., 2011; Xu et al., 2016) under controlled conditions than it was in warmer temperatures. Then, 8 E. Galimany et al. Aquaculture 576 (2023) 739891 clams would probably be more vulnerable to predation under predicted timing and success of larval settlement (Ghezzo et al., 2018; Maulu et al., climate conditions of increasing temperatures during those seasons 2021). However, each life cycle of bivalves occurs in a different envi­ (spring and summer). Although R. philippinarum is known to burrow ronment (planktonic larva and benthic adults) and has different physi­ quicker under higher temperature regimes (Takeuchi et al., 2015), clams ological requirements (Byrne et al., 2020). Given that the responses to may require more energy to manage the higher metabolic needs, or the increased temperatures and acidification seem to be season-specific, it is overall energy available for muscle movement may be reduced, resulting important to investigate the effects of climate change throughout the in longer burrowing times and impaired burrowing (Peng et al., 2017). course of the year, as we did in our study. Thus, year-round compre­ Larger clams require longer time to burrow because this activity is hensive studies are required as a basis to develop management strategies positively correlated with body mass (Takeuchi et al., 2015); thus, as in the near future for climate change mitigation to enhance clam was also observed for the clam Mulinia edulis (Abarca et al., 2019), aquaculture production, including the development of integrated multi- burrowing is a feature that may be adversely impacted by climate trophic aquaculture (IMTA) as well as genetic breeding techniques to change and compromise seasonal clam production. In our experiments, increase the resilience of bivalves to climate change (Byrne et al., 2020; the were no effects of pH on burrowing activity. However, an overall Tan and Zheng, 2020). protective role of sediments against acidification was reported in another study using the same clam species and at the same pH levels, in 5. Conclusions which comparisons were done between cultures with and without sed­ iments (Yuan et al., 2020). This contrasts with former studies that re­ Different climate change-related factors can negatively affect the ported a tendency of bivalves to burrow less in more acidic waters biology of Manila clams. This study indicates that clam seed seems to be considering that sediment pH tends to be lower than that of the nearby more affected by the anthropogenic-derived pH decrease than by the seawater due to microbial activity in the sediment (Clements and Hunt, 3 ◦ C increase in temperature. That is, low pH water values decreased 2017). A similar pattern was found in our experiments during spring for clearance rates and altered biomarker responses in a seasonally depen­ ambient temperatures, while it was not statistically significant, longer dent manner. However, high temperatures only increased the burrowing burrowing times were recorded under acidified treatments. time with spring and summer being the most vulnerable seasons. It is important to note that the biochemical responses of the clams Additionally, an extreme summer scenario was experimentally added, in were only detectable in the autumn specimens, which also happened to which the thermal optimum range of the clams was exceeded. In this be the season in which no physiological changes in energy use (growth case, clearance rates decreased at the highest temperature as opposed to and clearance rate) or burrowing activity were found. The biomarkers responding to acidification, as observed throughout the year, but within related to detoxification capacity, such as CE (phase I), GST (phase II) the clams’ optimal range. Considering that the physiological and bio­ and GR (antioxidant defence) activities, gradually increased under the logical responses of seed clams are influenced by seasonality, it is most unfavourable conditions (lower pH and higher temperature), with essential to develop strategies to improve aquaculture production antioxidant enzyme GR being the most sensitive to pH fluctuations. A globally while taking this variable into account. “preparation for stress” biochemical challenge was corroborated with the lowest ETS values, pinpointing to a metabolic shift towards an CRediT authorship contribution statement anaerobic pathway that is energetically less efficient. Therefore, the clams may reduce growth under longer-term exposures. Seasonal dif­ Eve Galimany: Investigation, Formal analysis, Writing – original ferences in ETS activity were found in adult clams (Costa et al., 2020a). draft. Arturo Lucas: Writing – review & editing. Francesc Maynou: Despite that ETS activity is typically reduced in adverse conditions, the Funding acquisition, Formal analysis, Writing – review & editing. same clam species were not affected when both physical and chemical Montserrat Solé: Methodology, Writing – review & editing. Carles stressors were present (Costa et al., 2020b). The fact that these Pelejero: Writing – review & editing. Montserrat Ramón: Conceptu­ biochemical changes from the autumn experiment did not result in a alization, Investigation, Writing – review & editing. reduction of the clams’ physiological performance (lower filtration rates or reduced growth) highlights their efficiency to overcome adverse Declaration of Competing Interest conditions under moderate physical stress. When adult clams were exposed to warm temperatures, low pH and additional chemicals, The authors declare that they have no known competing financial biochemical responses led to physiological changes such as reduced interests or personal relationships that could have appeared to influence respiration rates, which became significant for diclofenac (Costa et al., the work reported in this paper. 2020b) but not for triclosan (Costa et al., 2020c). In the present work, the fact that CE measures using pNPA substrate were more responsive to Data availability pH reductions but when using pNPB substrate they were more sensitive to temperature variations, may support a different isoenzyme compo­ Data will be made available on request. sition in clams, responding in a stressor-dependent manner. This occurred in spite of the high overlap in substrate specificity described for Acknowledgements this enzyme family in bivalves (Solé and Sanchez-Hernandez, 2018) and the significant correlation between both substrate measures. This research is part of the CERES project (H2020, EU 678193). We Overall, our experimental results indicate that the clams were more are grateful to the staff of the Aquaria and Experimental Chambers impacted by a pH decrease than by a 3 ◦ C temperature increase over the (ZAE) of the Institute of Marine Sciences (ICM-CSIC) and the institu­ tested pH and temperature ranges. In fact, bivalves have often been tional support of the ‘Severo Ochoa Centre of Excellence’ accreditation found to be more sensitive to acidification than other calcifying organ­ (CEX2019-000928-S). We also thank the following graduate students for isms, which have been observed to decrease survival, calcification, their help with the experimental setup and sample processing: Pere growth and general development (Kroeker et al., 2013). The construc­ Forteza Joy, Alizée Desrousseaux, Mireia Sierra, Alicia Paniego Gómez, tion and stability of bivalve shells are hampered by the decrease in and Ainoa Cubero Miró. carbonate ion concentration in seawater brought on by ocean acidifi­ cation, which has biological implications for the organisms (Matozzo Appendix A. Supplementary data and Marin, 2011; Range et al., 2011). More broadly, there will be a variety of effects of climate change on aquaculture. For example, seed Supplementary data to this article can be found online at https://doi. availability and farming practice may negatively be affected by the org/10.1016/j.aquaculture.2023.739891. 9 E. Galimany et al. 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