Aquaculture 479 (2017) 455–466

Contents lists available at ScienceDirect

Aquaculture

journal homepage: www.elsevier.com/locate/aquaculture

Physiological and histopathological impacts of increased carbon dioxide and MARK temperature on the cultured under upwelling influences in northern

⁎ Marco A. Lardiesa,b, , Samanta Beniteza,b, Sebastian Osoresa,b, Cristian A. Vargasb,c, Cristián Duarted, Karin B. Lohrmanne, Nelson A. Lagosf a Facultad de Artes Liberales, Universidad Adolfo Ibañez, Santiago, Chile b Center for the Study of Multiple-Drivers on Marine Socio-Ecological Systems (MUSELS), Universidad de Concepción, Concepción, Chile c Aquatic Ecosystem Functioning Lab (LAFE), Department of Aquatic Systems, Faculty of Environmental Sciences, Environmental Sciences Center EULA Chile, Universidad de Concepción, Chile d Facultad de Ecología y Recursos Naturales, Universidad Andrés Bello, Santiago, Chile e Facultad de Ciencias del Mar, Universidad Católica del Norte, Centro Innovación Acuícola Aquapacífico, Coquimbo, Chile f Centro de Investigación e Innovación para el Cambio Climático (CiiCC), Universidad Santo Tomás, Ejercito 146, Santiago, Chile

ARTICLE INFO ABSTRACT

Keywords: In addition to the increase in temperature occurring in the world's , new evidences suggest a tendency for fi acidi cation an increase in upwelling-favorable winds, bringing to surface cold and high pCO2 corrosive waters. Changing Histopathology temperature and pCO2 conditions may have significant implications for the shellfish farming socio-ecological Metabolism system. In order, to setup the basis for understand the impact of both environmental variables, in this study, we Food ingestion investigated the combined effects of changing temperature and pCO on the physiological rates and histo- Multiple stressor impacts 2 pathology of scallops Argopecten purpuratus farmed in Tongoy Bay, an area permanently influenced by coastal Aquaculture upwelling. Juvenile scallops were reared at two pCO2 levels (400 and 1000 μatm) and two temperatures (14 and 18 °C). After 18 d of experimental exposure, growth, metabolic and clearance rates increased significantly at

high temperature but independent of pCO2 level, indicating a positive effect of warming on the physiological processes associated with energy acquisition. However, ingestion rates of scallops showed a synergistic inter-

active effect when exposed to both stressors. Increased pCO2 also impacts the health of A. purpuratus through atrophy in the digestive gland. These results suggest that, the presence of trade-offs in energy allocation during

upwelling-induced stress (low temperature and high pCO2) can impact growth, metabolism, ingestion rates and

health status of scallops cultured in Tongoy Bay. But, less severe response to high pCO2 levels, suggest that natural variability in upwelling areas may promote acclimation and adaptation potential in this farmed scallops.

1. Introduction Doney 2009; Perry et al. 2010). Essentially, humans' adaptation capa- cities based on a better understanding of the combined impacts of Coastal oceans are arguably one of the most important ecosystems multiple global and local drivers on marine ecosystems services as food of the planet and impacted by human activities in the ocean. Global provision through fisheries and aquaculture activities will facilitate the environmental stressors, such as Ocean Warming (OW), Ocean success of our societies in establishing sustainable pathways for the Acidification (OA) and Deoxygenation (DO) are undergoing pro- future (Gattuso et al., 2015). nounced change in the world's coastal oceans today. There is growing In Chile, coastal shellfish aquaculture activities are focused on bi- concern with respect to the combined effects (thereby synergistically, valves (e.g. , , scallops), and gastropods (e.g. ). additively or antagonistically) of these environmental stressors on the From 1990, shellfish aquaculture has experienced an annual average coastal marine biota. During the last decades, there is growing interest growth rate of ~18%. Shellfish farming accounted with ca. about the need to understand the interplay between environmental and 265,000 tons in 2013, or 14% of total biomass and Chile is the third and socio-economic drivers in marine resource management (Cooley and second largest producer of scallops and mussels in the world,

⁎ Corresponding author. E-mail address: [email protected] (M.A. Lardies). http://dx.doi.org/10.1016/j.aquaculture.2017.06.008 Received 29 December 2016; Received in revised form 6 June 2017; Accepted 8 June 2017 Available online 10 June 2017 0044-8486/ © 2017 Elsevier B.V. All rights reserved. M.A. Lardies et al. Aquaculture 479 (2017) 455–466

Fig. 1. Location of the study site at the Northern Chile coast (a); Circle indicates the location of the farm inside Tongoy Bay, which is northward of the Punta Lengua de Vaca upwelling center (b). 72ºW

30ºS Coquimbo

30ºS

Tongoy Bay

10 km 71.4ºW

respectively (www.sernapesca.cl). Although the industry has thrived in northward of 37°S [in northern (18–30°S) and central Chile (30°–37°S)] the past decade, global drivers such as climate, market variability, and showing SST anomalies that varies between 4 and 7 °C, whereas in la- anthropogenic activities provide major challenges in sustaining the titudes above 37°S, SST varies between 2 °C to 4 °C (Lagos et al. 2008; future of the aquaculture sector in our coastal ocean. This is of parti- Aravena et al. 2014). The Chilean scallop farming industry in northern cular concern in the case of valuable commercially exploited species Chile is localized near to these upwelling centres and has been regularly such as the scallop Argopecten purpuratus. A recent crash in the scallop affected by events of larval and adult mortality, potentially driven by production has been recorded in Chile, where the number of scallop the effect of corrosive upwelling waters. The environmentally induced farms became closed due to market and environmental drivers, with phenotypic change, namely, phenotypic plasticity has been recognized concomitant reduction in national production from 20.000 tons in year as an important strategy for maximizing or maintaining fitness in an 2000 to 5000 tons in year 2013 (Lagos et al. 2016). organism that inhabits variable environments (Bouvet et al. 2005; Eastern Boundary Upwelling Ecosystems, located off the mid-lati- Pigliucci 2005; Schlichting and Pigliucci 1998; Via and Lande 1985). tude western coasts of the U.S., South America, Iberia and Africa, are These notions, suggest that may be expected that populations of A. predicted to be one of the most increasingly stressed marine ecosystems purpuratus that inhabit waters influenced by coastal upwelling, namely, under multiple-stressors (e.g. ocean acidification, warming and deox- subject to environmental heterogeneity in pH, temperature and oxygen ygenation; Gruber, 2011). Upwelling ecosystems account for only 1.9% concentration may be “naturally” acclimated to this environmental of the world's oceans, but provide an estimated 23% of global fisheries variation in temperature and pCO2 (i.e., phenotypic plasticity) probably (Kudela et al. 2005; Chavez et al. 2008), being the Humboldt Current as end product of local adaptation (see Lardies et al. 2014). System the most biologically productive ecosystem of world' coastal In this study we evaluate the combined effects of temperature and oceans (Thiel et al. 2007). These areas are characterized by strong pCO2 on scallops Argopecten purpuratus cultured under the influence of seasonal winds that promote the upwelling of cold and CO2 super- coastal upwelling. A. purpuratus represents an important species for saturated subsurface waters. Climatic projections suggest an increase in aquaculture, since it is the most cultivated mollusk species in the north the intensification of upwelling events due to the change in the in- of Chile (over 5000 tons in 2013) with its main production zone is lo- tensity and patterns of wind that favor the upwelling (Sydeman et al. calized in Tongoy Bay (von Brandt et al. 2006; SERNAPESCA 2013). A. 2014). In northern Chile, upwelling dynamics occur year around, with purpuratus is a relatively well studied species regarding the effect of foci located at major point or headlands of the coastline. In particular, a environmental variables on physiological processes (see Navarro and strong latitudinal gradient in SST anomalies located at 30°S is asso- Jaramillo, 1994, Navarro and González, 1998; Labarta et al. 1997; ciated with the coastal area of Punta Lengua de Vaca (PLV) (Figueroa Uriarte et al. 2004; Martínez et al. 1995, 2000; Fernández-Reiriz et al., and Moffat 2000). This coastal area at the PLV upwelling system might 2005; Soria et al., 2007, 2011; Ramajo et al. 2016a) and the impacts of result in under-saturated subsurface waters with respect to aragonite increased pCO2 and temperature on shell properties of this species (Ωarag < 1) and in lower pH in nearshore and bays areas (Torres et al. (Lagos et al. 2016). However, no studies have addressed how combined 2011), characteristics previously expected to occur in the open ocean effects of these climate stressors will affect the physiological rates and until the year 2100 in a scenario of ocean acidification (Orr et al. 2005; histopathology of this scallop species. Thus, in this study we evaluate Gruber et al. 2012). Coastal waters with low levels of carbonate sa- trough laboratory experiments the effect of cold temperatures and in- turation state are corrosives, challenging calcification of shells and creased pCO2 conditions, which commonly occur during upwelling skeletons of marine organisms (Fabry et al. 2008; Beniash et al. 2010; events influencing the study area (i.e. Tongoy Bay) upon growth rates Duarte et al. 2015). Furthermore, large upwelling areas are located and metabolic and feeding performance of A. purpuratus.

456 M.A. Lardies et al. Aquaculture 479 (2017) 455–466

2. Materials and methods 1000 ppm, we blended dry air with pure CO2 to each target con- centration using mass flow controllers (MFCs, www.aalborg.com) for

2.1. Study site and collection air and CO2; then this blend was bubbled into the corresponding con- tainer. Dry and clean air was generated by compressing atmospheric air Juvenile individuals of A. purpuratus (< 40 mm in size (LT)) were (117 psi) using an oil-free air compressor. The experimental containers collected from culture ropes (2 m of depth) in Tongoy Bay (30°12′S, were constantly bubbled with the corresponding CO2 concentration. 71°34′O) located in northern Chile (Fig. 1). Thus, the juvenile scallops During the experiment, total alkalinity (TA) of the water was monitored experienced during its early life stages the influence of Punta Lengua de every 4 days (two replicates) and pH, temperature, salinity of the water Vaca upwelling center which waters penetrate into the Tongoy Bay was monitored every day in each aquaria (i.e. replicate) and each seasonally (i.e. spring and summer) when winds coming from the south, header tank (Navarro et al. 2013). Seawater was changed every day, transport Equatorial subsurface waters (ESSW) with high salinity, high with the corresponding pCO2 levels from the seawater acidification nutrients content, low temperature, low pH, and low oxygen content to head-tank following the methodology proposed by Navarro et al. the same scallop farming area (Rutllant 1993; Uribe and Blanco 2001; (2013).pHNBS and total alkalinity (AT) was monitored on day 2, 6, 10, Lagos et al. 2016). 14 and 18 for carbonate systems estimates. pH samples were collected These experimental scallops are produced in hatchery from brood- in 50 mL syringes and immediately transferred to a 25 mL thermo- stock of the same Tongoy Bay. After collection, the scallops were statted cell at 25.0 ± 0.1 °C for standardization, with a pH meter transported in chilled conditions to the Calfuco Coastal Laboratory of Metrohm® using a glass combined double junction Ag/AgCl electrode the Universidad Austral de Chile (LCC, ca. 39°S, Valdivia) and placed in (Metrohm model 6.0258.600) calibrated with standard calibration plastic containers (30 cm in diameter and 40 cm in height). To accli- buffer Metrohm® 4 (6.2307.200), 7 (6.2307.210) y 9 (6.2307.220). pH matize them to experimental conditions, the scallops were kept for values are reported on the NBS scale. Samples for AT were poisoned 6 5 days and fed daily with microalgae Tetraselmis spp. (~65 × 10 cell/ with 50 μL of saturated HgCl2 solution and stored in 500 mL bor- ml) before the beginning of the experiments. In addition, during the day osilicate BOD bottles with ground-glass stoppers lightly coated with of the scallop collection, 17 juvenile individuals (hereafter natural Apiezon L® grease and kept in darkness at room temperature. Tem- control) were immediately preserved for histological procedures (see perature and salinity were monitored during incubations by using a below). salinometer Eco Sense EC 300, YSI. AT was determined using the open- cell titration method (Dickson et al. 2007), by using an automatic Al- 2.2. Seawater acidification system kalinity Tritrator Model AS-ALK2 Apollo SciTech. The AS-ALK2 system is equipped with a combination pH electrode (8102BNUWP, Thermo After the acclimation period, the scallops were randomly assigned to Scientific, USA) and temperature probe for temperature control (Star one of four treatments: (Angilletta et al., 2004) 14 °C and 400 μatm ATC probe, Thermo Scientific, USA) connected to a pH meter (Orion fi pCO2 (ambient condition), (Angilletta, 2009) 14 °C and 1000 μatm Star A211 pH meter, Thermo Scienti c, USA). All samples were ana- pCO2 (upwelling condition), (Aravena et al., 2014) 18 °C and 400 μatm lyzed at 25 °C ( ± 0.1 °C) with temperature regulation using a water- pCO2 (warming and upwelling relaxation), (Beniash et al., 2010)18°C bath (Lab Companion CW-05G). The accuracy was controlled against a fi and 1000 μatm pCO2 (ocean acidification and warming) (Table 1), for a certi ed reference material (CRM, supplied by Andrew Dickson, Scripps period 18 days which is larger time than two complete upwelling cycles Institution of Oceanography, San Diego, USA) and the AT repeatability − in northern Chile (see Strub et al. 1998; Garreaud et al. 2011). Each averaged 2–3 μmol kg 1. Temperature and salinity data were used to 2 − treatment was replicated five times and each replicate contained four calculate the rest of carbonate system parameters (e.g. pCO2,CO3 ) scallops (N = 80). The experimental were identified using bee and the saturation stage of Omega Aragonite (Ωarag). Analyses were tags. We used a semi-automatic system for long-term seawater carbo- performed using CO2SYS software for MS Excel (Pierrot et al. 2006) set nate chemistry manipulation (Torres et al. 2013; Duarte et al. 2014; with Mehrbach solubility constants (Mehrbach et al. 1973)refitted by Navarro et al. 2013; Manríquez et al., 2013; Vargas et al. 2013; Duarte Dickson and Millero (1987). The KHSO4 equilibrium constant de- et al. 2015). The experimental temperatures represent the average termined by Dickson (1990) was used for all calculations. surface value (14 °C) and alternatively the maximum surface value (18 °C) of the study area (Uribe and Blanco 2001; Pérez et al. 2012; 2.3. Physiological rates

Aravena et al. 2014). The pCO2 levels were defined considering the mean range reported for this coastal embayment by Torres & Ampuero 2.3.1. Growth rate (2009) and Vargas et al. (2017). The temperatures were adjusted and Growth rates of juvenile individuals of A. purpuratus were estimated maintained using external chillers. To obtain the different CO2 con- from changes in the Total Weight (TW) (to the nearest 0.01 mg) of the centrations, the experimental containers were adjusted following the scallops (Palmer 1982). For all measurements, each scallop was care- methodology described by Navarro et al. (2013). Briefly, for 400 ppm, fully removed from the water, immediately weighed under the water pure atmospheric air was bubbled into experimental containers; for (buoyant weight = BW), then gently blotted, and weighed in air (TW).

Table 1 − Average (+SE) conditions of carbonate system parameters during experiments conducted with juvenile scallops: pH (NBS scale), Total Alkalinity (TA in μmol kg 1), partial pressure of − 2 −1 CO2 (levels of pCO2 in seawater in μatm), carbonate ion concentration (CO3 in μmol kg ), saturation state of the water with respect to aragonite minerals (Ωarag).

Low (400 μatm) High (1000 μatm)

14 °C 18 °C 14 °C 18 °C

Temperature (°C) 14.13 ± 0.19 18.13 ± 0.11 14.08 ± 0.14 18.14 ± 0.13 Salinity (PSU) 34.48 ± 1.78 33.28 ± 1.97 35.86 ± 0.50 34.00 ± 0.68

pHNBS 8.058 ± 0.017 8.032 ± 0.041 7.754 ± 0.027 7.720 ± 0.021 Total alkalinity (TA) 1580.41 ± 206.40 1666.75 ± 117.55 1778.14 ± 127.82 1695.96 ± 133.49 − 2 CO3 86.54 ± 10.15 94.76 ± 3.92 53.76 ± 5.58 52.13 ± 5.07

pCO2 367.77 ± 62.17 435.96 ± 74.43 891.26 ± 77.76 969.48 ± 74.27

Ωaragonite 1.32 ± 0.15 1.48 ± 0.06 0.82 ± 0.09 0.81 ± 0.08

Ωcalcite 2.07 ± 0.24 2.29 ± 0.09 1.28 ± 0.13 1.25 ± 0.13

457 M.A. Lardies et al. Aquaculture 479 (2017) 455–466

Furthermore we estimated biomass increase (i.e. metabolically active 2.4. Histopathological measurements tissue) as the change in the relation between (TW–BW) during the time of the experiment. 2.4.1. Histopathological analysis Tissues for histology analysis were excised from scallops exposed to

the different treatments of pCO2 and temperature (N = 40) and from 2.3.2. Standard metabolic rate (SMR) those 17 scallops representing natural control conditions. The tissues Scallops were left in aquaria for 24 h with no food after exposition excised included gills, digestive gland and mantle. The tissue samples − 1 − 1 time. Then, we measured oxygen consumption (mg O2 ×h g ) were fixed for 24 h. in Davidson's fluid, transferred to 70% ethanol, and using a Presens Mini Oxy-4 respirometer (see Gaitán-Espitía et al. sent to the Histopathology Laboratory of the Universidad Católica del 2017). To quantify the SMR, animals of each replicate were used and Norte (Coquimbo, Chile). They were routine processed for histology, placed individually in respirometric chambers filled with 805 mL of 5 μm sections were cut, and stained with Harris Hematoxilyn and Eosin. seawater with the corresponding pCO2 levels and temperature of each The slides were analyzed using a Zeiss Axiostar plus microscope. treatment (see above). In addition 33 psu salinity and oxygen-saturated seawater through air bubbling were added before starting measuring. 2.4.2. Pathological conditions The measurements were performed at a controlled temperature of 14 °C The prevalence, percentage of individuals presenting each parasite and 18 °C using an automated temperature chiller. In each chamber, and condition was assessed for A. purpuratus from the four experimental dissolved oxygen was quantified every 15 s for 55 min. Sensors were treatments and the natural controls. For the two most prevalent in- previously calibrated in anoxic water, using a saturated solution of dicators observed (rickettsiales-like organisms, RLOs, and digestive

Na2SO3 and in water 100% saturated with oxygen using bubbled air. gland atrophy), also the intensity of infection was assessed. For the The same respirometric chambers were used as controls, but without RLOs this was accomplished counting all the inclusions of these para- animals inside, under the same experimental conditions (the control sites from the whole 5 μm thick section. The mean intensity of infection never had a decayment of the oxygen concentration higher than 3% of was calculated dividing the total number of parasites through the total the measurements). Each oxygen decayment due to background noise number of infected hosts. The intensity of digestive gland atrophy was was deducted from the individual measurements performed in the ex- assessed registering the percentage of this condition when present, and perimental chambers. The first and the last 5 min were discarded in the mean percentage was calculated. Prevalence, infection intensity and order to avoid possible disturbances when the scallops were placed in mean intensity of infection were calculated following Bush et al. respirometric chambers. Thus, oxygen estimations are the average of (1997). the remaining 45 min of measurements. Before and after each mea- surement, bouyant weight of scallops were recorded using an analytical 2.5. Statistical analyses balance (ADAM AFA-180LC with precision ± 0.001 mg). The average of both body mass measurements was used in the statistical analyses. To avoid pseudo-replication problems, the variables measured were Slopes were calculated by the decrease of oxygen in the chamber per averaged for the four scallops of each replicate. Two-way ANOVA were hour and normalized to fresh mass to get respiration rates in units of used to evaluate whether temperature and CO2 levels and the interac- −1 − 1 mg O2 L h . tion between factors affected growth rate (increase in TW and Biomass increase with time), ingestion and clearance rate (Zar, 1999). A two- way analysis of covariance (ANCOVA) was performed to determine the

2.3.3. Ingestion rate (IR) and clearance rate (CR) effects of temperature and pCO2 level on mass-specific metabolism, Immediately after completion of the experiment period (i.e. using body mass as covariate. When the analysis showed significant 18 days), we determined clearance and ingestion rates. We used interactions, a one-way ANOVA was carried out for each factor sepa- 1000 mL polycarbonate bottles previously cleaned with HCl. rately in each level from the other factor, followed by Tukey's a pos- Individuals belonging to each experimental group were individually teriori HSD test. When the analysis did not show significant interac- sorted into 1000 mL acid-washed polycarbonate bottles filled with fil- tions, multiple comparisons were carried out using Tukey's a posteriori tered seawater (1 μm, salinity of 33‰) and equilibrated with the re- HSD test on each factor that showed significant differences spective CO2 treatment. Three control bottles without A. purpuratus and (Underwood, 1997). Buoyant weight increases were expressed in g per three experimental bottles with one individual for each four treatment scallop per day. All analyses were carried out using Statistica version were incubated for approximately 2 h, and periodically rotated by hand 7.0. Assumptions of normality and homoscedasticity of the one-way to avoid particle sedimentation. Previous to the experiment juvenile ANOVA were evaluated using the Kolmogorov–Smirnov and Burtlett body mass (mb) was determined with an analytical balance tests, respectively (Sokal and Rohlf, 1995). Finally, we used an exact ( ± 0.01 mg) in order to standardize our clearance and ingestion rate binomial calculation to estimate the 2-tail probability that the pre- − − estimates in g 1 h 1. For these experiments, we added Tetraselmis spp. valence of conditions evaluated in the histological analysis of scallop − at a concentration of ~32,000 cell/mL 1, to supply food at saturation tissues was higher or lower than expected by chance (p = 0.5) in n level. Bottles were immersed in a container with flow-through seawater independent trials or scallops observations (Zar 1999). system used for maintaining temperature fluctuation during a given experiment within one degree. After incubation we took a sub-sample 3. Results of 200 mL from each bottle, which was filtered through a GF/F filter of 0.75 μm mesh-size. The filter was stored in an aluminum envelope at 3.1. Physiological rates −20 °C until analysis of Chlorophyll (Chl-a) concentration. Chl-a was dark extracted in acetone 95% before measurement on a TD 6700 3.1.1. Growth rate Turner Fluorometer (Strickland and Parsons 1968). Clearance (CR) and Growth rate of the scallops increased significantly in treatment with Ingestion (IR) rates were estimated through the measurement of Chl-a higher temperature compared with individuals maintained at low depletion and cell removal, according to Frost (1972) modified by temperature, 14 °C (Two way ANOVA, F1,19 = 24.40; P < 0.0000), Marín et al. (1986). For all experiments, there were no significant dif- but were not influenced by increased pCO2 levels within each tem- ferences in food availability among treatments. No mortality effects perature treatment (F1,19 = 0.97; P = 0.340) (Fig. 2a). Although, in- were found on juveniles scallops in any of the experimental treatments, creased growth rate was also recorded in those scallops reared at the which indicates that the projected increase of pCO2 levels and tem- treatment with low temperature and high pCO2 levels, representing a perature had chronic, but not lethal, effects on these organisms. typical ‘upwelling’ condition, these do not lead to a significant

458 M.A. Lardies et al. Aquaculture 479 (2017) 455–466

Fig. 2. Biological responses (mean ± S.E.) of Argopecten purpuratus reared at two temperatures and two nominal levels of CO2: (a) Growth; (b) Biomass increase; (c) Metabolic; (d)

Ingestion and (e) Clearance rates. Different letters (a,b) represents significant differences after a post hoc Tukey Test between pCO2 or temperature treatments.

interaction term (Temperature × pCO2;F1,19 = 3.89; P = 0.066) although there was an subtle increase from low to high temperatures − − − − (Fig. 2a). Otherwise, biomass, which represents metabolically active treatments (~175 ml g 1 h 1 to 206 ml g 1 h 1), respectively (Two- tissue, showed an significant increase in scallops exposed to low pCO2 way ANOVA, F1,17 = 0.003; P = 0.990) (Fig. 2c). treatments (Two way ANOVA; F1,19 = 3.17; P = 0.045), but were not The interaction between temperature and pCO2 levels affected sig- influenced by temperature treatments (F1,19 = 0.92; P = 0.991) nificantly the ingestion rates A. purpuratus juveniles (Two-way ANOVA, (Fig. 2b), and also leading to non-significant interaction between F1,17 = 6.72; P = 0.021) (Fig. 2d). The scallops exposed to the com- treatments (Fig. 2b). bination of high-pCO2/low temperature treatment (‘upwelling condi- tion’) presented significant higher ingestion rate than organisms ex- 3.1.2. Ingestion and clearance rates posed to the resto of treatments combinations (Fig. 2d). Non-significant The scallops A. purpuratus significantly increased their clearance differences were found between levels of temperature (14 °C and 18 °C), rates when exposed to the high temperature (18 °C), with a ~ 35% in- and neither for high and low pCO2 treatments in particles ingestion. crease recorded in individuals exposed to high temperature/low pCO2 Scallops maintained at low temperature and high pCO2 raised the in- combination (Two-way ANOVA, F1,17 = 7.68, P = 0.015) (Fig. 2c). gestion rate over 20% relative to low pCO2 conditions (high pCO2: −1 − 1 But, non-significant changes were observed in clearance rates in rela- 1.91 ± 0.23 mg Chla g h ; low pCO2 pH: 1.49 ± 0.27 − 1 − 1 tion to pCO2 levels for scallops at each temperature conditions, mg Chla g h )(Fig. 2d).

459 M.A. Lardies et al. Aquaculture 479 (2017) 455–466

3.1.3. Standard metabolic rate (SMR) upwelling conditions operating in Tongoy Bay, did not affect most Metabolic rate of A. purpuratus, measured as oxygen consumption physiological rates measured in juveniles of A. purpuratus, except for a increased significantly at the high temperature condition (Two-way significant increase in about of 40% of the ingestion rate at the high

ANCOVA, F1,17 = 7.29; P = 0.017) (Fig. 2e). Despite that oxygen pCO2 scenario. Nevertheless, physiological processes were sensitive to consumption in scallops was higher in both high pCO2 treatments, these changes in temperature, which induced a positive effect on the phy- differences were not significant (F1,17 = 3.35; P = 0.088). Further- siological processes associated with increased energy acquisition. more, a non-significant Temperature × pCO2 interaction was observed Specifically, our factorial experiment showed that the environmental for metabolic rate (Fig. 2e) (Two-way ANCOVA, F1,17 = 0.45; drivers studied, temperature and pCO2 operate in an additive way upon P = 0.515). Mass-specific metabolic rate in juveniles of A. purpuratus the physiological performance of A. purpuratus from Tongoy Bay. was lower in the low temperature/low-pCO2 level treatment than in low Nevertheless, the current study also suggests that under experimental temperature/high-pCO2 level combination (0.73 ± 0.19 and conditions the combination of low temperature across both pCO2 con- − 1 −1 0.97 ± 0.37 mg O2 h g , respectively). Metabolic rate of in- ditions may reduce the health of A. purpuratus. Histopathological as- dividuals exposed to high temperature and high pCO2 level reached the sessment of the soft tissues of juvenile scallops exposed to acidification highest values observed in the experiment (1.60 ± 0.40 and temperature treatments, showed damage, particularly in digestive −1 −1 mg O2 h g ), almost doubling oxygen consumption of scallops ex- gland, but without mortality. In addition, though not fully assessed, it posed to low temperature conditions (Fig. 2e). also suggests the role of high pCO2 on statocyst volume. Temperature profoundly affects growth and its underlying processes 3.2. Histopathological measurement in ectotherms (Angilletta 2009; Somero 2010), rising temperature leads to increasing rates of biochemical-physiological processes (e.g. meta- 3.2.1. Histopathological analysis bolic and heart rate) and life-history characteristics (Gillooly et al. The main affected tissue in juvenile scallops was the digestive gland, 2001; Willmer et al. 2005). Temperature increases the growth rate of showing degenerative alterations of the digestive tubules (Fig. 3a scallops, an expected pattern because temperature increases growth and normal tubule) and secondary (transport) tubules across treatments body size in marine (see Sicard et al., 1999; Woods et al. (Fig. 3b,c). This alteration was evidenced as a desquamation of the 2003; Angiletta et al. 2004). The increase of growth rate with tem- epithelial cells (Fig. 3b), flattening the epithelium, and thus in some perature increment has been demonstrated previously in A. purpuratus extreme cases leaving only the presence of the basal lamina (Fig. 3c). for populations from Chile and Perú (see Martínez and Pérez 2003; The secondary tubule atrophy, although present, showed a low in- Thébault et al. 2008; Soria et al. 2011; Lagos et al. 2016). Water tem- tensity across all treatments. Although statocysts had not specifically perature is the environmental factor most often cited as influencing been sampled for, they appeared located close to the pedal ganglia and bivalve reproduction (see Sastry 1968; Wolff 1998; Navarro et al. the digestive gland (Fig. 3g, right and left statocysts from natural 2016). Nevertheless, we detected a significant increase in biomass of control scallops). The left statocyst showed no alteration in the ex- the scallops (i.e., metabolically active tissue) raised under low pCO2 perimental scallops (Fig. 3h), however, the right statocyst showed ir- levels. Thus, juveniles scallops experiencing high pCO2 conditions, regular outlines, due to epithelial desquamation in the high pCO2 probably due to higher energetic cost of homeostasis, decrease the condition at both levels of temperature (Fig. 3i). tissue mass (see Lardies et al. 2014). Recently, we reported a reduction in shell length and growth in juveniles of A. pururatus from Tongoy Bay

3.2.2. Pathological conditions under high pCO2 treatments (Lagos et al. 2016). Reduced growth under The prevalence of parasites and pathological conditions is shown in acidified conditions has been reported also for other scallops species Table 2. The rickettsiales–like organisms (RLOs) were detected as ba- (e.g. Talmage and Gobler 2009; White et al. 2013). A similar pattern sophilic, ovoid-spherical inclusions in epithelial cells of the digestive was informed in the bivalve Mytilus galloprovincialis where high pCO2 tubules (Fig. 3a), residing in otherwise completely normal digestive significantly reduced growth rate at low temperature but this effect cells. RLOs showed low prevalence across the experimental treatments gradually decreased with warming (Kroeker et al. 2014). Negative and higher, but non-significant prevalence increment in scallops natural impacts on biomass under low pH could be explained because energy control conditions. These RLOs were most prevalent in the natural allocation is being diverted away to other key physiological processes control scallops (24%) and ranged from 0% to 10% in experimental such as calcification, acid–base balance, reproduction, and immune treatments (Table 2). The intensity of infection by RLOs ranged from 1 function (Wood et al. 2008; Findlay et al. 2009; Lagos et al. 2016). to 27 inclusions counted from one section, with a mean intensity of 8 in Nevertheless, previous studies report that juveniles of A. purpuratus the case of natural control scallops (Table 2). Another, unidentified maintained between 10 °C and 18 °C showed no effect in growth rate microorganism, showed increased and significant prevalence in the and their scope for growth (Gonzalez et al. 2002). The origin of ex- Lumina of digestive and secondary tubules, but also only in the natural perimental populations may explain this contradictory results, whereas control scallops (76%, Table 2); it had a strongly eosinophilic thick the juveniles scallops used in our experiment were obtained from cover resembling a resting cyst, sometimes with cells inside (Fig. 3d). Tongoy Bay (northern Chile, 30° 12′S), these authors collected scallops Hemocyte infiltration was also observed in gills (Fig. 3e) and digestive from southern Chile (41° 54′S), thus suggesting the role of a possible gland (Fig. 3f) mainly, and occasionally in gonad as well (Fig. 3f). This acclimatization and/or adaptation of local populations to environ- hemocyte infiltration in gills was increased (70%) but not significantly, mental factors experienced in their native habitats. Temperature re- at the high-pCO2/low temperature combination (Table 2). At low gimes vary widely along the Chilean coast and specifically in > 6 °C temperature and irrespective of pCO2 level, the 100% of individuals between these two localities (Thiel et al. 2007; Lagos et al. 2008). Thus, showed digestive tubule atrophy (Table 2). The approximate coverage geographical variation in the degree of plasticity and/or potential for percentage (intensity) of this condition ranged from 5 to 30% in the local adaptation for these traits could be present within A. purpuratus natural control scallops and varies across experimental treatments, species along the Chilean coast and partially may explain the differ- showing the highest increment in scallops raised under the low tem- ences in biological responses observed between local population of A. perature and high-pCO2 condition (39%; 20%–60%, Table 2). purpuratus. There are few studies on combined effects of temperature and pCO2 4. Discussion on metabolism and performance of marine invertebrates (Queirós et al., 2015). For instance, Navarro et al. (2016) found that the metabolic rate

Our results indicate that under rise in pCO2 from 400 to 1000 μatm was not affected by temperature but detected an increase in metabolism in a scenario of low temperature, which may resemble the influence of on the farmed mussels Mytilus chilensis when exposed to low pH level.

460 M.A. Lardies et al. Aquaculture 479 (2017) 455–466

Fig. 3. Histopathological effect of two temperatures and two nominal levels of pCO2 on tissues of Argopecten purpuratus (Stain H & E). A: Normal digestive tubules, composed of digestive cells (DC) and basophilic cells (arrowhead), L: lumen of digestive tubule. RLO inclusions present in digestive cells (arrows). B & C: Digestive gland atrophy, flat epithelium (arrowheads), desquamating cells (desq. cells), *: basal lamina. D: Unknown organism in lumen (L) of secondary tubule. Ep: epithelium of secondary tubule. E & F: Hemocytic infiltration (*), E: in gill within distended gill filaments (GF). F: in digestive gland (DG), and in male gonad (MG). G: Statocysts in natural control from Tongoy Bay, left statocyst (LS) and right statocyst (RS). In the interior the statoconia (*) can be observed, in the left statocyst (**) it forms a mass filling it completely, in the right statocyst it only occupies about 25% of the volume. Part of the pedal ganglion visible on the upper part of the photograph. H: perfectly normal looking left statocyst from the low pCO2/high temperature treatment. I: Right statocyst from the high pCO2/high temperature treatment, were desquamation of the epithelium is observed (arrows).

On the contrary, in our study temperature increased significantly the 2014; Navarro et al. 2016). Navarro et al. (2000) found that oxygen metabolic rate in scallops but did not vary across pCO2 level. This result uptake and scope for growth were not affected in A. purpuratus by suggests a major role for warmer temperatures, which could potentially temperatures in a range between 16 °C and 20 °C. However, these ex- offset the reduction in energy budget originated by ocean acidification perimental temperatures are higher to the average conditions (e.g., (see Byrne and Przeslawski 2013, Kroeker et al. 2013, Duarte et al. 12–14 °C, Figueroa and Moffat 2000; Lagos et al. 2016) dominating

461 M.A. Lardies et al. Aquaculture 479 (2017) 455–466

Table 2

Prevalence (P) of pathological conditions evaluated in scallops collected from field (natural control) and under the four experimental CO2/temperature combinations. Mean infection intensity (I) was also evaluated for Rickettsia-like organisms and digestive tubule atrophy. Significant prevalence evaluated using the 2-tail binomial probability test is shown in bold. The number of scallops used in each evaluation (n) is also showed.

Condition evaluated Natural control Low (400 μatm) High (1000 μatm)

14 °C 18 °C 14 °C 18 °C

(n = 17) (n = 10) (n = 10) (n = 10) (n = 10)

Rickettsia-Like organisms (P) 24 10 10 0 10 Rickettsia-Like organisms (I) 8 (1–27) 1 8 (8) 0 1 Unidentified microorganism (P) 76 0000 Gill hemocyte infiltration (P) 18 40 40 70 50 Digestive gland infiltration (P) 0 50 90 40 100 Digestive tubule atrophy (P) 24 100 80 100 80 Digestive tubule atrophy (I) 18% (5–30) 12% (5–30) 35% (10–40) 39% (20–60) 24% (10–40) Secondary tubule atrophy (P) 0 20 50 10 40 Distended gill filaments (P) 0 10 10 30 30 during upwelling processes influencing Tongoy Bay. Similar results to cope with acidic conditions (Ramajo et al. 2016b; Hendriks et al. were found in the scallop where the respiration rate was 2015). Metabolic up-regulation to face stressful environments is en- not affected in juveniles displaying tolerance to levels of pH ranging ergetically costly and may impose limits to the energy available to between 8.2 and 7.6 (Sanders et al. 2013). It is important to emphasize sustain other physiological activities like growth or ingestion rate, that in our experiment, as well as in the Sanders et al. (2013) study, generating trade-offs between phenotypic traits (Wood et al. 2008; food availability was not restricted. In addition to temperature, food Deigweiher et al. 2010; Lardies et al. 2014; Navarro et al. 2016). supply seems to play a major role modulating organismal responses of Studies analyzing the effect of high pCO2 on bivalve species have A. purpuratus by providing the energetic means to support the physio- shown negative effects on feeding activities, such as clearance and in- logical cost imposed by ocean acidification stress (Ramajo et al. 2016a). gestion rates, possibly due to deficiencies in the functioning of the di-

Our results can be discussed in the framework of natural pCO2 gestive and filtering systems (Fernández-Reiriz et al. 2011; Navarro variability in coastal upwelling areas influencing scallop farming and et al. 2013; Vargas et al. 2013; Vargas et al. 2015). Reduced feeding the potential for local adaptation in these regions (Vargas et al. 2017). under ocean acidification and warming conditions results in lower This can be rationalized because the scallops were exposed to a pH and survival, size, and calcification (Dupont & Thorndyke, 2009; temperature range that the animals experience in their natural en- Byrne & Przeslawski 2013). In contrast, in our study juvenile scallops vironment (see Torres and Ampuero 2009; Lagos et al. 2016; Vargas showed a significant increase of ingestion rate at high pCO2/low tem- et al. 2017). Coastal upwelling modulates the physical and chemical perature treatment (i.e., upwelling conditions) and clearance rate was properties of seawater over large areas of the coastal ocean. In relation increased as expected in higher temperatures without effect of pH. It is to the current study, the Pt. Lengua de Vaca upwelling center increases evident from our results and the other available studies that high pCO2 CO2-fluxes and promotes strong reductions in pH (pH ~ 7.6) in Tongoy increases juvenile feeding of scallop species. Our findings are consistent Bay and the surrounding area (Torres and Ampuero 2009; Torres et al. with results reported for other scallops such as Chlamys nobilis and 2011) and adds also a high variability in sea surface temperature (see Pecten maximus under low pH treatments (Wenguang & Maoxian, 2012; Aravena et al. 2014). That is, the moderate effect found on physiolo- Sanders et al., 2013) and in the juveniles of A. purpuratus under dif- gical performance of juvenile individuals of A. purpuratus from Tongoy ferent scenarios of pH and food availability (Ramajo et al. 2016a). Bay is likely due to the presence of phenotypic plasticity leading to Navarro et al. (2000) show that A. purpuratus can actively regulate increased tolerances which is characteristic of organisms that inhabit clearance rate and does not simply switch between feeding and non- heterogeneous environments (Via et al. 1995). Most upwelling zones feeding states. A different pattern have been reported in other mollusk are acidic compared to the coastal and open ocean (Hofmann et al. species for the Chilean coast, for instance in newly hatched larvae of the 2011). Furthermore, it has recently been demonstrated that most up- gastropod concholepas and juvenile stages of the welling zones are indeed characterized by natural variations in sea- Perumytilus purpuratus, whose clearance rates decreased between 15 up water chemistry, suggesting that in some particular habitats, resident to 70% under high pCO2 respect to control conditions (Vargas et al. organisms are already experiencing pH regimes projected for the year 2013; Vargas et al. 2015). On the other hand, the maintenance of 2100 (Hofmann et al. 2011; Vargas et al. 2017). This can be the case of scallop biomass across treatments (see Fig. 2b) may require high food the Tongoy Bay due to the presence of a semi-permanent upwelling ingestion at the same time that more energy needs to be canalized to the (Vargas et al. 2017). Our results evidenced that a less severe response to shell calcification process (see Lagos et al. 2016). In upwelling areas the high pCO2 levels of the scallops that are naturally exposed to higher pH exposure of the organisms to low temperatures is concomitant with and temperature variability, suggesting the acclimatization and adap- high-pCO2, which would depress the saturation state for carbonate tation potential for this species. This moderate effect of temperature below the level derived from high pCO2 alone (Feely et al. 2008). The and high pCO2 has also been reported in other marine invertebrates that previous is because the kinetics of dissolution/solubility of carbonate inhabit highly variable environments on terms of pH and temperature shells increases at low temperatures (Morse et al. 2007). These “up- (see Lardies et al. 2014; Collard et al. 2016; Vargas et al. 2017). We welling conditions” experienced by scallops in our experimental treat- observed an increase of scallop metabolism in high pCO2 at both tem- ments (i.e. low temperature and high pCO2) are already experienced by peratures. This up-regulation observed in metabolic rate of scallops scallop farmed in Tongoy Bay, hence an adaptation to local environ- under high pCO2 scenarios has also been observed in others bivalves mental conditions trough phenotypic plasticity may be part of the re- species exposed to low pH conditions, and may be a requirement for sponses expressed by this scallop population. It is important to maintain maintaining intracellular pH, and cellular homeostasis (e.g., Cummings a perspective about the several organismal responses as an integrated et al. 2011; Lardies et al. 2014; Ramajo et al. 2016a), which is con- phenotype because low pH regimes can significantly affect calcification sistent with the metabolic costs of mechanisms developed by calcifiers process in A. purpuratus, which becomes enhanced under low

462 M.A. Lardies et al. Aquaculture 479 (2017) 455–466 temperatures such as those occurring during an active upwelling phase 1977). In our study the right statocyst observed in scallop exposed to

(Lagos et al. 2016; Vargas et al. 2017). Thus, the presence of trade-offs the high pCO2 treatment suggests that the statoconia have been par- in energy allocation during stress induced by upwelling processes is tially lost due to increased pCO2 or acidification conditions. Observa- present at least in terms of calcification, growth rate and metabolism. tion on scallops farmed at Tongoy bay (natural control), evidenced a Therefore, studies that aim to improve our understanding about the right statocyst less developed and partially filled with statoconia, as has mechanisms that govern bivalve aquaculture species in terms of calci- been described for other pectinids (e.g., Von Buddenbrock 1915). In

fication, growth and physiological performance in upwelling zones are addition, this right statocyst of the high pCO2 treatment, regardless of urgently needed. the temperature, showed some desquamation of the epithelium. It is not For A. purpuratus the most striking histopathological effect was on clear if this loss of integrity of the right statocyst epithelium could the digestive gland integrity, which consisted in disintegrating and impair the gravity perception of the scallops, as only removal of the left sloughing of digestive cells. Many stressors can generate atrophied di- one seemed to cause harm in other pectinids (Buddenbrock 1915). gestive tubules, such as pollution (Syasina et al. 1997), food avail- Recently, Schalkhausser et al. (2013) demonstrate that Pecten maximus ability, saline and thermal stress (Carella et al. 2015). We measured reduce their clapping performance when exposed to low temperature that natural only a small proportion of scallops collected from the farm and increased pCO2 conditions suggesting potential link between the (natural control) showed reconstituting tubules morphology (Mathers impact on the escape behaviour of scallops and the reduced epithelial 1976, Henry et al. 1991, Mathers et al. 1979, Syasina et al. 1997), integrity of the statocyst observed in our study. which was atrophied (60%) in scallops subjected to the experimental There are several possible reasons to explain the moderate effect in conditions. This implies a reduction of digestive tubules available for A. purpuratus of ocean acidification and in scallops in general; firstly, accomplishing intracellular digestion, which may alter an adequate the main mineral component of the shell is entirely of calcite a less ingestion during the exposure time. Clearly, this pattern of tubules soluble form of carbonates (Gazeau et al. 2013) and also a secretion of a atrophy in scallops across treatments may be associated with differ- thicker periostracum has been reported involving higher levels of ences between the natural diet from Tongoy Bay and the artificial food polysaccharides under OA conditions as shell protection mechanism in used during the experiment. It has been reported for juvenile bivalve juveniles (Ramajo et al. 2016a). Secondly, seawater remained in all molluscs that food availability can outweigh ocean acidification, as treatments in our experiments above the calcite saturation state shown for Mytilus edulis (Thomsen et al. 2013), Pecten maximus (Sanders (Ωcalcite > 1). Lastly, Scanes et al. (2014) propose that swimming ca- et al. 2013) and Argopecten purpuratus (Ramajo et al., 2016a, b). This pacity of scallops which produce elevations of CO2 in the haemolymph latter study was undertaken in a population of juvenile scallops from and alterations in acid-base status and is another possible reason for the the same population as this study and consisted in a 30-day exposure to moderate effects of ocean acidification on scallops when compared to different pH and food concentrations (Ramajo et al. 2016a) similar to the negative physiological effects widely reported in oysters and mus- those used in our incubations. Thus, an explanation of this positive sels which are sessile mollusks. The previous is applicable for wild responses could be the existence of a greater proportion of healthy di- scallops in natural stocks; nevertheless, this mechanism is potentially gestive tubules, providing the capacity to digest the available food, and less plausible in conditions of suspended culture where density in the the amelioration of ocean acidification effects trough increased tem- lantern net is high (i.e., from 200 to 25 scallops per lantern level in perature. Tongoy Bay farm), which reduces the opportunities for swimming in Hemocytic infiltration is an inflammatory response to pathogens, juvenile scallops. However, the underlying mechanism by which high chemicals (such as pollutants) or physical cell injury (De Vico and pCO2 impacts moderately scallops physiology remains unclear for us. Carella 2012; Carella et al. 2015). High prevalences but non-significant Although our treatment of low temperature and high pCO2 or “up- gill hemocytic infiltration were observed in the experimental treat- welling conditions” did not evidence strong effect on the studied phy- ments, respect to the natural control group. Gills had highest pre- siological traits, it is still uncertain the potential effect of future ocean valence in the high temperature treatment (i.e., independent of pCO2 acidification upon biological responses measured. One of the main level). Mytilus edulis showed highest hemocytes infiltration when ex- problems in order to evaluate the effect of ocean acidification in marine posed to high temperature conditions, but was absent under high pCO2 organisms inhabiting these regions is the lack of information regarding treatments (Mackenzie et al. 2014). In addition, two parasites or sym- to potential scenarios of ocean acidification for eastern boundary re- bionts were found in this study, a prokaryote, rickettsiales-like or- gions. There is no consensus of how the effects of warming and ocean ganism (RLO) and an unidentified protistan. Rickettsiales-like organ- acidification will interact (e.g. additive, synergistic, or antagonistic) to isms (RLO) have been described as basophilic intracellular inclusion influence the physiology and performance and health of mollusks (e.g. bodies in the gills or digestive gland of most bivalve molluscs (Bower Findlay et al. 2009, Gooding et al. 2009, Comeau et al. 2010, Byrne et al. 1994), usually not causing any harm. They have also been de- 2011, Kroeker et al. 2013, Byrne and Przeslawski 2013; Duarte et al. scribed from digestive gland of adult cultivated A. purpuratus from 2014; Lagos et al. 2016; Navarro et al. 2016). Although high pCO2 Caldera, and the bays of Guanaqueros and Tongoy (Lohrmann 2009). In significantly reduced growth rate of scallops at 14 °C, this effect is not this study RLOs were most prevalent in the natural control group than significant at temperatures of 18 °C, showing how moderate warming in the experimental treatment, but with similar mean intensity. RLOs can ameliorate the effects of OA through temperature effects on both prevalence have though been linked to serious mortalities in some bi- physiology and histopathology. However, these scenarios of progressive valves such as in the scallop Pecten maximus (Legall et al. 1988), giant acidification and warming may be not fully applicable to highly pro- Hippopus hippopus (Norton et al. 1993) and clams Venerupis ductive upwelling ecosystems, where the projected intensification of rhomboides (Villalba et al. 1999). Although, RLOs have always been upwelling events due to the change in the intensity and patterns of wind detected at low prevalence and intensity of infection in A. purpuratus, (Sydeman et al. 2014) forecast more extreme upwelling events with with no host hemocyte responses, is suggested that a harmless parasite cold subsurface waters, which are supersatured with dissolved CO2 can become a pathogen under the influence of as climate stressors that during longer periods. Therefore, the consideration of this kind of cli- can enhance the virulence and/or the parasite pathogenicity (Burge matic scenarios in multistressor experiments is urgently needed for et al. 2014). shellfish aquaculture species cultivated in upwelling zones in such as, In scallops, statocysts are sacs formed by a ciliated epithelium with for instance North America, Iberia and Africa. a single statolith and a loose statoconia filling about 25% up to 100% of the sac, lie close to the pedal ganglion, are asymmetrical and function Acknowledgements as gravity receptor, which epithelium development, cell types and cilia allow to differentiate the left and right statocysts (Cragg and Nott We thank Pamela Tapia and Christian Tapia at Invertec–Ostimar

463 M.A. Lardies et al. Aquaculture 479 (2017) 455–466

Hatchery and fieldwork facilities. We extend our acknowledgment to acidification on the juvenile individuals of the mussel Mytilus chilensis. J. Sea Res. 85, 308–314. Jorge López, and Paulina Contreras for their valuable assistance during Duarte, C., Navarro, J., Acuña, K., Torres, R., Manríquez, P.H., Lardies, M.A., Vargas, the experiments and fieldwork. We thank two anonymous reviewers for C.A., Lagos, N.A., Aguilera, V., 2015. Intraspecific variability in the response of the insightful criticisms to the manuscript. This study was supported by the edible mussel Mytilus chilensis (Hupe) to ocean acidification. Estuar. Coasts 38, 590–598. Millennium Nucleus Center for the Study of Multiple drivers on Marine Dupont, S., Thorndyke, M.C., 2009. Impact of CO2-driven ocean acidification on in- Socio-Ecological Systems (MUSELS) funded by MINECON NC120086 vertebrates early life-history-what we know, what we need to know and what we can and FONDECYT grant nos. 1140938 and 1140092 to N.A.L. and M.A.L., do. Biogeosciences 6, 3109–3131. fi and 1130254 to CAV. CAV was also funded by the Millennium Institute Fabry, V.J., Seibel, B.A., Feely, R.A., Orr, J.C., 2008. Impacts of ocean acidi cation on marine fauna and ecosystem processes. ICES J. Mar. Sci. 65, 414–432. of Oceanography (IMO), MINECON IC120019 during the preparation of Feely, R.A., Sabine, C.L., Hernandez-Ayon, J.M., Ianson, D., Hales, B., 2008. Evidence for this manuscript. upwelling of corrosive ‘acidified’ water onto the continental shelf. Science 320, 1490–1492. Fernández-Reiriz, M., Navarro, J.M., Labarta, U., 2005. Enzymatic and feeding behaviour References of Argopecten purpuratus under variation in salinity and food supply. Comp. Biochem. Physiol. 141A, 153–163. Angilletta Jr., M.J., 2009. Thermal Adaptation: A Theoretical and Empirical Synthesis. Fernández-Reiriz, M.J., Range, P., Alvarez-Salgado, X.A., Labarta, U., 2011. Physiological Oxford University Press, Oxford. energetics of juvenile clams Ruditapes decussatus in a high CO2 coastal ocean. Mar. – Angilletta Jr., M.J., Oufieroa, C.E., Searsb, M.W., 2004. Thermal adaptation of maternal Ecol. Prog. Ser. 433, 97 105. ff fl and embryonic phenotypes in a geographically widespread ectotherm. Int. Congr. Figueroa, D., Mo at, C., 2000. On the in uence of topography in the induction of coastal – Ser. 1275, 258–266. upwelling along the Chilean coast. Geophys. Res. Lett. 27, 3905 3908. Aravena, G., Broitman, B., Stenseth, N.C., 2014. Twelve years of change in coastal up- Findlay, H.S., Wood, H.L., Kendall, M.A., Spicer, J.I., Twitchett, R.J., Widdicombe, S., fi welling along the central-northern coast of Chile: spatially heterogeneous responses 2009. Calci cation, a physiological process to be considered in the context of the – to climatic variability. PLoS One 9, e90276. whole organism. Biogeosciences 6, 2267 2284. ff Beniash, E., Ivanina, A., Lieb, N.S., Kurochkin, I., Sokolova, I.M., 2010. Elevated level of Frost, B.W., 1972. E ects of size and concentration of food particles on the feeding be- fi carbon dioxide affects metabolism and shell formation in oysters virginica. havior of the marine planktonic copepod Calanus paci cus. Limnol. Oceanogr. 17, – Mar. Ecol. Prog. Ser. 419, 95–108. 805 815. Bouvet, J.M., Vigneron, P., Saya, A., 2005. Phenotypic plasticity of growth trajectory and Gaitán-Espitia, J.D., Bacigalupe, L.D., Opitz, T., Lagos, N.A., Osores, S., Lardies, M.A., ontogenic allometry in response to density for Eucalyptus hybrid clones and families. 2017. Thermal physiological responses of an intertidal crab along a latitudinal cline. Ann. Bot. 96, 811–821. J. Theor. Biol. http://dx.doi.org/10.1016/j.jtherbio.2017.02.011. Bower, S., McGladdery, S., Price, I., 1994. Synopsis of infectious diseases and parasites of Garreaud, R., Rutllant, J.A., Muñoz, R.C., Rahn, D.A., Ramos, M., Figueroa, D., 2011. commercially exploited shellfish. Annu. Rev. Fish Dis. 4, 1–199. VOCALS-CUpEx: the Chilean upwelling experiment. In: Atmospheric Chemistry and – von Brand, E., Merino, G., Abarca, A., Stotz, W., 2006. Scallop fishery and aquaculture in Physics. VOCALS-REx Special Issue. 11. pp. 2015 2029. Chile. In: Parsons, J. (Ed.), Scallop, Biology, Ecology and Aquaculture: Developments Gattuso, J.P., Magnan, A., Billé, R., Cheung, W.W.L., Howes, E.L., Joos, F., Hoegh- ff in Aquaculture and Fisheries Sciences. Elsevier, The Netherlands, pp. 1293–1311 Guldberg, O., 2015. Contrasting futures for ocean and society from di erent an- (1460). thropogenic CO2 emissions scenarios. Science 349 (6243), aac4722. Buddenbrock, W., 1915. Die Statocyste von Pecten, ihre Histologie und Physiologie. Zool Gazeau, F., Parker, L.M., Comeau, S., Gattuso, J.P., O'Connor, W.A., Martin, S., Ross, P.M., fi Jb Abt Anat Physiol Bd 35, 302–358. 2013. Impacts of ocean acidi cation on marine shelled molluscs. Mar. Biol. 160 (8), – Burge, C.A., Eakin, C.M., Friedman, C.S., Froelich, B., Hershberger, P.K., Hofmann, E.E., 2207 2245. ff Petes, L.E., Prager, K.C., Weil, E., Willis, B.L., Ford, S.E., Harvell, C.D., 2014. Climate Gillooly, J.F., Brown, J.H., West, G.B., Savage, V.M., Charnov, E.L., 2001. E ects of size – Change Influences on Marine Infectious Diseases: Implications for Management and and temperature on metabolic rate. Science 293 (5538), 2248 2251. ff Society. In: Carlson, C.A., Giovannoni, S.J. (Eds.), Ann. Rev. Mar. Sci. Vol 6 (Book 6). González, M., López, D., Pérez, M., Castro, J.M., 2002. E ect of temperature on the scope Bush, A.O., Lafferty, K.D., Lotz, J.M., Shostak, A.W., 1997. Parasitology meets ecology on for growth in juvenile scallops Argopecten purpuratus (Lamark, 1819). Aquac. Int. 10, – its own terms: Margolis et al. revisited. J. Parasitol. 83 (4), 575–583. 339 349. Byrne, M., 2011. Impact of ocean warming and ocean acidification on marine in- Gooding, R.A., Harley, C.D.G., Tang, E., 2009. Elevated water temperature and carbon vertebrate life history stages: vulnerabilities and potential for persistence in a dioxide concentration increase the growth of a keystone echinoderm. Proc. Natl. – changing ocean. Oceanogr. Mar. Biol. Annu. Rev. 49, 1–42. Acad. Sci. U. S. A. 106, 9316 9321. Byrne, M., Przeslawski, R., 2013. Multistressor impacts of warming and acidification of Gruber, N., 2011. Warming up, turning sour, losing breath: ocean biogeochemistry under – the ocean on marine Invertebrates' life histories. Integr. Comp. Biol. 53, 582–596. global change. Philos. Transact. A Math. Phys. Eng. Sci. 369, 1980 1996. Carella, F., Feist, S.W., Bignell, J.P., De Vico, G., 2015. Comparative pathology in bi- Gruber, N., Hauri, C., Lachkar, Z., Loher, D., Fröhlicher, T.L., Plattner, G.K., 2012. Rapid fi valves: aetiological agents and disease processes. J. Invertebr. Pathol. 131, 107–120. progression of ocean acidi cation in the California current system. Science 337, – Chavez, F.P., Bertrand, A., Guevara, R., Soler, P., Csirke, J., 2008. The northern Humboldt 220 223. Current System: brief history, present status and a view towards the future. Prog. Hendriks, I.E., Duarte, C.M., Olsen, Y.S., Steckbauer, A., Ramajo, L., Moore, T.S., ... Oceanogr. 79, 95–105. McCulloch, M., 2015. Biological mechanisms supporting adaptation to ocean acid- fi – Collard, M., Rastrick, S.P.S., Calosi, P., Demolder, Y., Dille, J., Findlay, H.S., Hall-Spencer, i cation in coastal ecosystems. Estuar. Coast. Shelf Sci. 152, A1 A8. J.M., Milazzo, M., Moulin, L., Widdicombe, S., Dehairs, F., Dubois, P., 2016. The Henry, M., Boucaud-Camou, E., Lefort, Y., 1991. Functional micro-anatomy of the di- – impact of ocean acidification and warming on the skeletal mechanical properties of gestive gland of the scallop Pecten maximus (L.). Aquat. Living Resour. 4, 191 202. the sea urchin Paracentrotus lividus from laboratory and field observations. ICES J. Hofmann, G.E., Smith, J.E., Johnson, K.S., Send, U., Levin, L.A., Micheli, F., Paytan, A., Mar. Sci. 73 (3), 727–738. Price, N.N., Peterson, B., Takeshita, Y., Matson, P.G., Crook, E.D., Kroeker, K.J., Comeau, S., Gorsky, G., Alliouane, S., Gattuso, J.P., 2010. Larvae of the pteropod Gambi, M.C., Rivest, E.B., Frieder, C.A., Yu, P.C., Martz, T.R., 2011. High-frequency Cavolinia inflexa exposed to aragonite undersaturation are viable but shell-less. Mar. dynamics of ocean pH: a multi-ecosystem comparison. PLoS One 6 (12), e28983. Biol. 157, 2341–2345. Kroeker, K.J., Kordas, R.L., Crim, R., Hendriks, I.E., Ramajo, L., Singh, G.S., ... Gattuso, fi Cooley, S.R., Doney, S.C., 2009. Anticipating ocean acidification's economic consequences J.P., 2013. Impacts of ocean acidi cation on marine organisms: quantifying sensi- – for commercial fisheries. Environ. Res. Lett. 4 (2), 024007. tivities and interaction with warming. Glob. Chang. Biol. 19 (6), 1884 1896. Cragg, S.M., Nott, J.A., 1977. Ultrastructure of statocysts in pediveliger larvae of Pecten Kroeker, K.J., Gaylord, B., Hill, T.M., Hosfelt, J.D., Miller, S.H., Sanford, E., 2014. The fi maximus (l) (). J. Exp. Mar. Biol. Ecol. 27, 23–36. role of temperature in determining Species' vulnerability to ocean acidi cation: a Cummings, V., Hewitt, J., Van Rooyen, A., Currie, K., Beard, S., Thrush, S., Norkko, J., case study using Mytilus galloprovincialis. PLoS One 9 (7), e100353. Barr, N., Heath, P., Halliday, N.J., Sedcole, R., Gomez, A., McGraw, C., Metcalf, V., Kudela, R., Pitcher, G., Probyn, T., Figueiras, F., Moita, T., Trainer, V., 2005. Harmful – 2011. Ocean Acidification at High Latitudes: Potential Effects on Functioning of the algal blooms in coastal upwelling systems. Oceanography 18, 184 197. ff Antarctic Bivalve Laternula elliptica. PLoS ONE 6 (1) e16069. Labarta, U., Fernández-Reiríz, M.J., Babarro, J.M.F., 1997. Di erences in physiological De Vico, G., Carella, F., 2012. Morphological features of the inflammatory response in energetics between intertidal and raft cultivated mussels Mytilus galloprovincialis. – molluscs. Res. Vet. Sci. 93, 1109–1115. Mar. Ecol. Prog. Ser. 152, 167 173. Deigweiher, K., Hirse, T., Bock, C., Lucassen, M., Pörtner, H.O., 2010. Hypercapnia in- Lagos, N., Castilla, J.C., Broitman, B.R., 2008. Spatial environmental correlates of inter- duced shifts in gill energy budgets of Antarctic notothenioids. J. Comp. Physiol. tidal recruitment: a test using in the northern Chile. Ecol. Monogr. 78 (2), – 180B, 347–359. 245 261. Dickson, A.G., 1990. Standard potential of the reaction AgCl(s)+1/2 H2(g) = Ag(s) Lagos, N.A., Benítez, S., Duarte, C., Lardies, M.A., Broitman, B.R., Tapia, C., Tapia, P., ff fi +HCl(aq) and the standard acidity constant of the ion HSO4 − in synthetic sea water Widdicombe, S., Vargas, C.A., 2016. E ects of temperature and ocean acidi cation on from 273.15 to 318.15 K. J. Chem. Thermodyn. 22, 113–127. shell characteristics of Argopecten purpuratus: implications for scallop aquaculture in fl – Dickson, A.G., Millero, F.J., 1987. A comparison of the equilibrium constants for the an upwelling-in uenced area. Aquac. Environ. Interact. 8, 357 370. dissociation of carbonic acid in seawater media. Deep-Sea Res. 34 (10A), 1733–1743. Lardies, M.A., Arias, M.B., Poupin, M.J., Manríquez, P.H., Torres, R., Vargas, C.A., ff fi Dickson, A.G., Sabine, C.L., Christian, J.R. (Eds.), 2007. Guide to Best Practices for Ocean Navarro, J.M., Lagos, N.A., 2014. Di erential response to ocean acidi cation in physiological traits of Concholepas concholepas populations. J. Sea Res. 90, 127–134. CO2 Measurements. PICES Special Publication Vol. 3 North Pacific Marine Sciences Organization, Sidney. Legall, G., Chagot, D., Mialhe, E., Grizel, H., 1988. Branchial rickettsiales-like infection Duarte, C., Navarro, J.M., Acuña, K., Torres, R., Manríquez, P.H., Lardies, M.A., Vargas, associated whit a mass mortality of sea scallop Pecten maximus. Dis. Aquat. Org. 4, – C.A., Lagos, N.A., Aguilera, V., 2014. Combined effects of temperature and ocean 229 232.

464 M.A. Lardies et al. Aquaculture 479 (2017) 455–466

Lohrmann, K.B., 2009. How healthy are cultivated scallops (Argopecten purpuratus) from subtidal scallop Mimachlamys asperrima (Lamarck, 1819). PLoS One 9 (4), e93649. Chile? A histopathological survey. Rev. Biol. Mar. Oceanogr. 44, 35–47. Schalkhausser, B., Bock, C., Stemmer, K., Brey, T., Pörtner, H.O., Lannig, G., 2013. Impact Mackenzie, C.L., Lynch, S.A., Culloty, S.C., Malham, S.K., 2014. Future oceanic warming of ocean acidification on escape performance of the king scallop, Pecten maximus, and acidification alter immune response and disease status in a commercial shellfish from Norway. Mar. Biol. 160, 1995–2006. species, Mytilus edulis L. PLoS One 9 (6), e99712. Schlichting, C.D., Pigliucci, M., 1998. Phenotypic Evolution: A Reaction Norm Manríquez, P.H., Jara, M.E., Mardones, M.L., Navarro, J.M., Torres, R., Lardies, M.A., Perspective. Sinauer Associates, Sunderland, MA. Vargas, C.A., Duarte, C., Widdicombe, S., Salisbury, J., Lagos, N.A., 2013. Ocean SERNAPESCA (Servicio Nacional de Pesca y Acuicultura), 2013. Anuarios estadísticos del acidification disrupts prey responses to predator cues but not net prey shell growth in Servicio Nacional de Pesca y Acuicultura. www.sernapesca.cl. Concholepas concholepas (loco). PLoS One 8 (7), e68643. Sicard, M.T., Maeda-Martinez, A.N., Omart, P., Reynoso-Granados, T., Carvalho, L., 1999. Marin, V., Huntley, M.E., Frost, B., 1986. Measuring feeding rate of pelagic : Optimum temperature for growth in the catarina scallop (Argopecten ventricosus cir- analysis of experimental design and methods. Mar. Biol. 93 (1), 49–58. cularis, Sowerby II, 1842). J. Shellfish Res. 18, 385–392. Martínez, G., Pérez, H., 2003. Effect of different temperature regimes on reproductive Sokal, R.R., Rohlf, F.J., 1995. Biometry: The Principles and Practice of Statistics in conditioning in the scallop Argopecten purpuratus. Aquaculture 228, 153–167. Biological Research. Freeman, W.H., New York. Martínez, G., Caceres, L.A., Uribe, E., Díaz, M.A., 1995. Effects of different feeding re- Somero, G.N., 2010. The physiology of climate change: how potentials for acclimatization gimens on larval growth and the energy budget of juvenile Chilean scallops, and genetic adaptation will determine “winners” and “losers”. J. Exp. Biol. 213, Argopecten purpuratus Lamarck. Aquaculture 132 (3–4), 313–323. 912–920. Martínez, G., Brokordt, K., Aguilera, C., Soto, V., Guderley, H., 2000. Effect of diet and Soria, G., Merino, G., von Brand, E., 2007. Effect of increasing salinity on physiological temperatura upon muscle metabolic capacities and biochemical composition of response in juvenile scallops Argopecten purpuratus at two rearing temperatures. gonad and muscle in Argopecten purpuratus Lamarck, 1819. J. Exp. Biol. Ecol. 247, Aquaculture 270, 451–463. 29–49. Soria, G., Merino, G., Uribe, E., von Brand, E., 2011. Effect of increasing salinity on Mathers, N.F., 1976. Effects of tidal currents on rhythm of feeding and digestion in weight-specific filtration rate of juvenile scallop Argopecten purpuratus reared at two pecten-maximus l. J. Exp. Mar. Biol. Ecol. 24, 271–283. temperatures: is any effect related to ammonia buildup? J. Shellfish Res. 30 (2), Mathers, N.F., Smith, T., Colins, N., 1979. Monophasic and diphasic digestive cycles in 279–286. Venerupis decussata and Chlamys varia. J. Molluscan Stud. 45, 68–81. Strickland, J.D.H., Parsons, T.H., 1968. A Practical Handbook of Seawater Analysis, Mehrbach, C., Culberson, C.H., Hawley, J.E., Pytkowicz, R.N., 1973. Measurement of the Fisheries Research Board of Canada, Bulletin 167. Ottawa, 293 pp. apparent dissociation constants of carbonic acid in seawater at atmospheric pressure. Strub, P.T., Mesías, J.M., Montecino, V., Rutllant, J., Salinas, S., 1998. Coastal ocean Limnol. Oceanogr. 18, 897–907. circulation off western South America. In: Robinson, A.R., Brink, K.H. (Eds.), The Sea. Morse, J.W., Arvidson, R.S., Lüttge, A., 2007. Calcium carbonate formation and dis- John Wiley and Sons, New York, pp. 273–314. solution. Chem. Rev. 107, 342–381. Syasina, I.G., Vaschenko, M.A., Zhadan, P.M., 1997. Morphological alterations in the Navarro, J.M., González, C.M., 1998. Physiological responses of the Chilean scallop digestive diverticula of (Bivalvia: Pectinidae) from polluted Argopecten purpuratus to decreasing salinities. Aquaculture 167, 315–327. areas of Peter the Great Bay, Sea of Japan. Mar. Environ. Res. 44, 85–98. Navarro, J.M., Jaramillo, R., 1994. Evaluación de la oferta alimentaria natural disponible Sydeman, W.J., García-Reyes, M., Schoeman, D.S., Rykaczewski, R.R., Thompson, S.A., a organismos filtradores en la Bahía de Yaldad (43 –08; 73–44), sur de Chile. Rev. Black, B.A., Bograd, S.J., 2014. Climate change and wind intensification in coastal Biol. Mar. 29, 57–75. upwelling ecosystems. Science 345, 77–80. Navarro, J.M., Leiva, G.E., Martínez, G., Aguilera, C., 2000. Interactive effects of diet and Talmage, S., Gobler, C., 2009. The effects of elevated carbon dioxide concentrations on temperature on the scope for growth of the scallops Argopecten purpuratus during the metamorphosis, size, and survival of larval hard clams (Mercenaria mercenaria), reproductive conditioning. J. Exp. Mar. Biol. Ecol. 247, 67–83. bay scallops (), and eastern oysters (Crassostrea virginica). Limnol. Navarro, J.M., Torres, R., Acuña, K., Duarte, C., Manríquez, P.H., Lardies, M., Lagos, N.A., Oceanogr. 54, 2072–2080. Vargas, C., Aguilera, V., 2013. Impact of medium-term exposure to elevated pCO2 Thébault, J., Thouzeau, G., Chauvaud, L., Cantillánez, M., Avendaño, M., 2008. Growth of levels on the physiological energetics of the mussel Mytilus chilensis. Chemosphere 90, Argopecten purpuratus (: Bivalvia) on natural bank in northern Chile: 1242–1248. sclerochronological record and environmental controls. Aquat. Living Resour. 21, Navarro, J.M., Duarte, C., Manríquez, P.H., Lardies, M.A., Torres, R., Acuña, K., Vargas, 44–45. C.A., Lagos, N.A., 2016. Ocean warming and elevated carbon dioxide: multiple Thiel, M., Macaya, E.C., Acuña, E., Arntz, W.E., et al., 2007. The Humboldt current system stressor impacts on juvenile mussels from southern Chile. ICES J. Mar. Sci. 73, of northern and central Chile. Oceanographic processes, ecological interactions and 764–771. socioeconomic feedback. Oceanogr. Mar. Biol. Annu. Rev. 45, 195–344. Norton, J.H., Shepherd, M.A., Abdonnaguit, M.R., Lindsay, S., 1993. Mortalities in the Thomsen, J., Casties, I., Pansch, C., Kortzinger, A., Melzner, F., 2013. Food availability giant Hippopus hippopus associated with rickettsiales-like organisms. J. outweighs ocean acidification effects in juvenile Mytilus edulis: laboratory and field Invertebr. Pathol. 62, 207–209. experiments. Glob. Chang. Biol. 19, 1017–1027.

Orr, J.C., Fabry, V.J., Aumont, O., Bopp, L., Doney, S.C., Feely, R.A., Key, R.M., 2005. Torres, R., Ampuero, P., 2009. Strong CO2 outgassing from high nutrient low chlorophyll Anthropogenic ocean acidification over the twenty-first century and its impact on coastal waters off central Chile (30° S): the role of dissolved iron. Estuar. Coast. Shelf calcifying organisms. Nature 437 (7059), 681–686. Sci. 83, 126–132. Palmer, A.R., 1982. Growth in marine gastropods: a non-destructive technique for in- Torres, R., Pantoja, S., Harada, N., González, H.E., Daneri, G., Frangopulos, M., Fukasawa, dependently measuring shell and body weight. Malacologia 23, 63–73. M., 2011. Air-sea CO2 fluxes along the coast of Chile: From CO2 outgassing in central Pérez, E.P., Azócar, C., Araya, A., Astudillo, O., Ramos, M., 2012. Comparación del cre- northern upwelling waters to CO2 uptake in southern Patagonian fjords. J. Geophys. cimiento de Argopecten purpuratus entre cohortes obtenidas de captación de en Res. Oceans Atmos. 116 (C9). ambiente natural y de hatchery. Lat. Am. J. Aquat. Res. 40 (4), 1026–1038. Torres, R., Manriquez, P.H., Duarte, C., Navarro, J.M., Lagos, N.A., Vargas, C.A., Lardies, Perry, R.I., Ommer, R.E., Barange, M., Werner, F., 2010. The challenge of adapting M.A., 2013. Evaluation of a semi-automatic system for long-term seawater carbo- marine social–ecological systems to the additional stress of climate change. Curr. natechemistry manipulation. Rev. Chil. Hist. Nat. 86, 443–451. Opin. Environ. Sustain. 2, 356–363. Underwood, A.J., 1997. Experiments in Ecology: Their Logical Design and Interpretation Pierrot, D., Lewis, E., Wallace, D.W.R., 2006. MS Excel Program Developed for CO2 Using Analysis of Variance. Cambridge University Press, Cambridge. System Calculations, ORNL/CDIAC- 105. Carbon Dioxide Information Analysis Uriarte, I., Farías, A., Hernández, J., Schäfer, C., Sorgeloos, P., 2004. Reproductive con- Center. Oak Ridge National Laboratory, US Department of Energy, Oak Ridge, TN. ditioning of Chilean scallop (Argopecten purpuratus) and the Pacific (Crassostrea Pigliucci, M., 2005. Evolution of phenotypic plasticity: where are we going now? Trends gigas): effects of enriched diets. Aquaculture 230, 349–357. Ecol. Evol. 20, 481–486. Uribe, E., Blanco, J.L., 2001. Capacidad de los sistemas acuáticos para el sostenimiento del Queirós, A.M., Fernandes, J.A., Faulwetter, S., Nunes, J., Rastrick, S.P.S., Mieszkowska, cultivo de pectínidos: El caso de Argopecten purpuratus en la Bahía de Tongoy, Chile. N., Artioli, Y., Yool, A., Calosi, P., Arvanitidis, C., Findlay, H.S., Barange, M., Cheung, Los Moluscos Pectínidos de Iberoamérica: Ciencia y Acuicultura A. N. Maeda- W.W.L., Widdicombe, S., 2015. Scaling up experimental ocean acidification and Martínez (ed.) 2001. Cap. 12, 000. warming research: from individuals to the ecosystem. Glob. Chang. Biol. 21 (1), Vargas, C.A., de la Hoz, M., Aguilera, V., San Martín, V., Manríquez, P.H., Navarro, J.M.,

130–143. Torres, R., Lardies, N.A., Lagos, N.A., 2013. CO2-driven ocean acidification reduces Ramajo, L., Marba, N., Prado, L., Peron, S., Lardies, M.A., Rodriguez-Navarro, A.B., larval feeding efficiency and changes food selectivity in the mollusk Concholepas Vargas, C.A., Lagos, N.A., Duarte, C.M., 2016a. Biomineralization changes with food concholepas. J. Plankton Res. 33 (5), 1059–1068. supply confer juvenile scallops (Argopecten purpuratus) resistance to ocean acidifica- Vargas, C.A., Aguilera, V., Martín, V., Manríquez, P., Navarro, J., Duarte, C., Torres, R.,

tion. Glob. Chang. Biol. 22, 2025–2037. Lardies, M., Lagos, N., 2015. CO2-driven ocean acidification disrupts the filter feeding Ramajo, L., Prado, L., Rodriguez-Navarro, A.B., Lardies, M.A., Duarte, C.M., Lagos, N.A., behavior in Chilean gastropod and bivalve species from different geographic lo- 2016b. Plasticity and trade-offs in physiological traits of intertidal mussels subjected calities. Estuar. Coasts 38, 1163–1177. to freshwater-induced environmental variation. Mar. Ecol. Prog. Ser. 553, 93–109. Vargas, C.A., Lagos, N.A., Lardies, M.A., Duarte, C., Manríquez, P.H., Aguilera, V.H., Rutllant, J., 1993. Coastal Lows and Associated Southerly Winds Events in North-Central Broitman, B., Widdicombe, S., Dupont, S., 2017. Species specific responses to ocean Chile. Proceeding Confer. Southern Hemisphere Meteorology and Oceanography, acidification should account for local adaptation and adaptive plasticity. Nature Ecol. Australia (2 pp). Evol. 1, 00. Sanders, M.B., Bean, T.P., Hutchinson, T.H., Le Quesne, W.J., 2013. Juvenile king scallop, Via, S., Lande, R., 1985. Genotype-environment interaction and the evolution of pheno- Pecten maximus, is potentially tolerant to low levels of ocean acidification when food typic plasticity. Evolution 505–522. is unrestricted. PLoS One 8 (9), e74118. Via, S., Gomulkiewicz, R., De Jong, G., Scheiner, S.M., Schlichting, C.D., Van Tienderen, Sastry, A.N., 1968. The relationships among food, temperature, and gonad development P.H., 1995. Adaptive phenotypic plasticity: consensus and controversy. Trends Ecol. of the bay scallop Aequipecten irradians Lamarck. Physiol. Zool. 41, 44–53. Evol. 10 (5), 212–217.

Scanes, E., Parker, L.M., O'Connor, W.A., Ross, P.M., 2014. Mixed effects of elevated pCO2 Villalba, A., Carballal, M.J., Lopez, C., Cabada, A., Corral, L., Azevedo, C., 1999. on , larval and juvenile development and adult responses in the mobile Branchial rickettsia-like infection associated with clam Venerupis rhomboides

465 M.A. Lardies et al. Aquaculture 479 (2017) 455–466

mortality. Dis. Aquat. Org. 36, 53–60. Blackwell, Carlton. Von Buddenbrock, W., 1915. Die statocysten von Pecten, ihre histologie und physiologie. Wolff, M., 1998. Spawning and recruitment in the Peruvian scallop Argopecten Zool Jb.(abt. allg. Zool. Physiol.) 35, 301–356. Purpuratus. Mar. Ecol. Prog. Ser. 42 (1998), 213–217. Wenguang, L., Maoxian, H., 2012. Effects of ocean acidification on the metabolic rates of Wood, H.L., Spicer, J.I., Widdicombe, S., 2008. Ocean acidification may increase calci- three species of bivalve from southern coast of China. Chin. J. Oceanol. Limnol. 30 fication rates, but at a cost. Proc. R. Soc. Lond. B Biol. Sci. 275, 1767–1773. (2), 206–211. Woods, H.A., Makino, W., Cotner, J.B., Hobbie, S.E., Harrison, J.F., Acharya, K., Elser, White, M.M., McCorkle, D.C., Mullineaux, L.S., Cohen, A.L., 2013. Early exposure of bay J.J., 2003. Temperature and the chemical composition of poikilothermic organisms.

scallops (Argopecten irradians) to high CO2 causes a decrease in larval shell growth. Funct. Ecol. 17 (2), 237–245. PLoS One 8 (4), e61065. Zar, J.H., 1999. Biostatistical Analysis, 4th ed. Prentice Hall, Upper Saddle River. Willmer, P., Stone, G., Johnston, I., 2005. Environmental Physiology of Animals, 2nd edn.

466