Physiological Responses and Scope for Growth in a Marine Scavenging

ICES Journal of Marine Science

ICES Journal of Marine Science (2016), 73(3), 814–824. doi:10.1093/icesjms/fsv208

Contribution to Special Issue: ‘Towards a Broader Perspective on Ocean Acidiﬁcation Research’ Original Article Physiological responses and scope for growth in a marine Downloaded from https://academic.oup.com/icesjms/article/73/3/814/2458919 by guest on 29 September 2021 scavenging gastropod, Nassarius festivus (Powys, 1835), are affected by salinity and temperature but not by ocean acidiﬁcation

Haoyu Zhang1, Paul K. S. Shin1,2, and Siu Gin Cheung1,2* 1Department of Biology and Chemistry, City University of Hong Kong, Hong Kong, China 2State Key Laboratory in Marine Pollution, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, China *Corresponding author: tel: + 852 34427749; fax: + 852 34420522; e-mail: [email protected] Zhang, H., Shin, P. K. S., and Cheung, S. G. Physiological responses and scope for growth in a marine scavenging gastropod, Nassarius festivus (Powys, 1835), are affected by salinity and temperature but not by ocean acidiﬁcation. – ICES Journal of Marine Science, 73: 814–824. Received 4 June 2015; revised 17 September 2015; accepted 19 October 2015; advance access publication 11 November 2015.

In the past few years, there has been a dramatic increase in the number of studies revealing negative or positive effects of ocean acidification on marine organisms including corals, echinoderms, copepods, molluscs, and fish. However, scavenging gastropods have received little attention despite being major players in energy flow, removing carrion, and recycling materials in marine benthic communities. The present study investigated the physiological responses (ingestion, absorption rate and efficiency, respiration, and excretion) and scope for growth (SfG) of an intertidal scavenging gastropod, Nassarius festivus, to the combined effects of ocean acidification (pCO2 levels: 380, 950, and 1250 matm), salinity (10 and 30 psu), and temperature (15 and 308C) for 31 d. Low salinity (10 psu) reduced ingestion, absorption rate, respiration, excretion, and SfG of N. festivus throughout the exposure period. Low temperature (158C) had a similar effect on these parameters, except for SfG at the end of the exposure period (31 d). However, elevated pCO2 levels had no effects in isolation on all physiological parameters and only weak interactions with temperature and/or salinity for excretion and SfG. In conclusion, elevated pCO2 will not affect the energy budget of adult N. festivus at the pCO2 level predicted to occur by the Intergovernmental Panel on Climate Change (IPCC) in the year 2300. Keywords: Nassarius festivus, ocean acidification, physiological energetics, salinity, scope for growth, temperature.

Introduction corals are considered to be one of the most vulnerable groups For over 800 000 years, carbon dioxide has been relatively stable in (Bramanti et al., 2013; Reyes-Nivia et al., 2013). For molluscs, the atmosphere at 172–300 matm by volume concentration (Luthi Abduraji and Danilo (2015) found that the pH-driven survival et al., 2008). The level reached in 2000 (395 matm) is predicted rate of Haliotis asinina was reduced from 86.3 to 47.2% at pH to rise to 1000 matm by 2100 (Collins et al., 2013). During the 7.99, and 18.3% at pH 7.62 and 7.42, respectively, after 20 d of ex- period 2000–2008, approximately one quarter of anthropogenic posure. Acidified seawateralso restrained pteropods from maintain- carbon dioxide was dissolved in the ocean (Le Que´re´ et al., 2009), ing shells made up of aragonite (Honjo et al., 2000). Dissolution of and increasing CO2 availability is causing a global decrease in pH the shell at the growing edge of the aperture was observed in the of seawater, a phenomenon known as ocean acidification. pteropod Clio pyramidata within 48 h of exposure to 788 matm Effects of ocean acidification have been extensively reported pCO2 (Orr et al., 2005). among marine organisms including bacteria, plants, and animals Physiological responses of ocean acidification are species-specific [reviewed by Caldeira and Wickett (2003)]. The unsaturated state with differential responses being observed in closely related species. + + of calcium carbonate caused by excess H and lower Ca2 availabil- For example, the Mediterranean mussel Mytilus galloprovincialis ity in acidifying seawater makes calcifying invertebrates potential showed a reduced metabolic rate and slower growth when exposed victims of changes to ocean chemistry. Among such organisms, to pH 7.3 for 3 months (Michaelidis et al., 2005). In contrast, no

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physiological disturbance was observed in the blue mussel Mytilus 1120 matm pCO2 for 39 weeks with no acclimation observed edulis at pH 7.14 for 60 d (Thomsen and Melzner, 2010). Some (Appelhans et al., 2014). In addition, SfG measurements of the sea species are robust or show positive responses to ocean acidification urchin Strongylocentrotus purpuratus raised under high pCO2 [reviewed by Andersson et al.(2011)]. The brittlestar Amphiura fili- (129 Pa, 1271 matm) indicate that an average of 39–45% of the formis showed an increase in metabolism and calcificationwith a sub- available energy was spent in somatic growth, while control larvae stantial cost (muscle wastage) upon exposure to acidified seawater could allocate between 78 and 80% of the available energy to (pH7.7) for40 d(Wood et al., 2008). Neutral responses in metabolic growth processes. rates have been observed in three echinoderms, Asterias rubens, As one of the most dominant and competitive scavengers on Ophiothrix fragilis,andA. filiformis after 1 week of exposure to pH sandy shores in Hong Kong, Nassarius festivus plays an important 7.5 (Carey et al., 2014). High metabolic rates commonly found in role in matter cycling and energy flow, and serves as an important crustaceans facilitate the control of extracellular pH through active cleaner in removing carrion (Briton and Morton, 1992). Previous ion transport (Whiteley, 2011), hence reducing the impact of ocean studies on physiological energetics have shown that this species is tol- acidification. For instance, after 10 d of incubation, there were no erant of environmental stresses, including low salinity and hypoxia Downloaded from https://academic.oup.com/icesjms/article/73/3/814/2458919 by guest on 29 September 2021 net changes in survival or overall development of larvae of the bar- (Cheung and Lam, 1995; Chan et al., 2008). The interactive effects nacle Amphibalanus improvisus raised at pH 7.6 compared with the of ocean acidification, high temperature, and low salinity increased control pH of 8.0 (Pansch et al., 2013). This may be partly due to the mortality of the veliger larvae (Zhang et al., 2014). The mainten- the absence of calcified structures in barnacle larvae that are devel- ance cost, as shown by the respiration rate, also increased with oped only when they settle and metamorphose into the juvenile stage. temperature and pCO2 level. In the present study, N. festivus adults Shifts in energy allocation upon exposure to ocean acidification were exposed to the combined effect of ocean acidification, tempera- may reduce fitness and produce low functional capacities, hence in- ture, and salinity for 31 d. The acute responses to the combined stres- creasing sensitivity to environmental stressors such as temperature, ses and physiological adjustments, if any, following prolonged food supply, and salinity (Zittier et al., 2013; Carey et al., 2014). exposure to the stresses were investigated. To understand if there According to Po¨rtner (2008), ocean acidification enhances sensitiv- are any life-stage differences in sensitivities to multiple stressors ity to thermal stress, resulting in a narrowing of the thermal toler- that could create a bottleneck in population performance, results ance window and aerobic capacity. For example, the brown crab were compared with those obtained for the larvae in a previous Cancer pagurus reduced its upper thermal limit of aerobic scope study (Zhang et al., 2014). Reduction in population performance by 58C under hypercapnia (pH 7.06) exposure for 16 h (Metzger could eventually lessen the role of N. festivus in removing carrion et al., 2007). The scope for performance of the Arctic spider crab on sandy shores, hence resulting in a deterioration of environmental Hyas araneus was reduced at the limits of thermal tolerance (48C) quality. and exacerbated by an elevated CO2 level of 3000 matm (Zittier Based on climate models in the IPCC Fifth Assessment Report et al., 2013). Synergistic effects have also been observed between (AR5), The Hong Kong Observatory has predicted the temperature ocean acidification and low salinity. Combined exposure to hyper- and rainfall changes in Hong Kong in the 21st century (http://www capnia and low salinity negatively affected mortality, tissue growth, .hko.gov.hk/climate_change/proj_hk_rainfall_e.htm). Under the energy storage, and mechanical properties of shells of juvenile high greenhouse gas concentration scenario (RCP8.5) proposed in oysters Crassostrea virginica (Dickinson et al., 2012). In addition, this report, temperature is expected to rise by 1.5–3 and 3–68Cin the larval mortality of a subtidal scavenging gastropod Nassarius the mid-21st century (2051–2060) and late 21st century (2091– conoidalis was enhanced by high pCO2 level (1250 matm) at low sal- 2100), respectively, when compared with the 1986–2005 average inity (10 psu) but not at normal salinity (30 psu; Zhang et al., 2014). of 23.38C. The number of extremely wet years is expected to increase Acclimation occurs when organisms adjust physiologically to from 3 in 1885–2005 to about 12 in 2006–2100. The annual rainfall changes in the environment, allowing them to maintain performance in the late 21st century is expected to rise by 180 mm when com- relatively independently of the changes. Such adjustment occurs pared with the 1986–2005 average. Such predictions indicate that over a short period and depends on lifespan (Barry et al., 2011). Hong Kong is facing an increase in temperature and rainfall due Acclimation to temperature and salinity is commonly found in to climate change and by inference, salinity, and temperature stres- marine animals. For instance, intertidal barnacles Elminus modestus ses on coastal marine organisms would be both more frequent and and Balanus balanoides can tolerate salinities as low as 14–17 psu fol- more abrupt. The results of this study can thus aid in predicting lowing experimental or natural acclimation (Foster, 1970). A range of the performance of an important beach cleaner under the combined homeostatic responses which serve to offset the passive effects of effects of ocean acidification temperature change and salinity reduced temperature have been shown to allow teleost fish to adapt stresses. to lower temperatures (Johnston and Dunn, 1987). In the cold-water coral Lophelia pertusa, short-term (1 week) exposure to pH 7.77 resulted in the dissolution of calcium carbonate, but acclimation Methods was observed after 6 months and resulted in an enhancement of cal- Study organisms cification (Form and Riebesell, 2012). Physiological responses of the Nassarius festivus (shell length: 13 + 2 mm) were collected from subtidal scavenging gastropod, N. conoidalis, were sensitive to ocean Starfish Bay, a sandy beach located in the northeast of Hong Kong acidification under acute exposure for 3 d, but complete acclimation (22.481308N, 114.244118E). As the experiment lasted for a month was observed after incubation for 1 month (Zhang et al., 2015). and all the replicates could not be completed at the same time due Scope for growth (SfG) is an integrated index reflecting energy to logistical problems, individuals from each replicate were collected allocation strategies in living organisms and has been shown to be from the field before each experiment and were acclimated to labora- a useful indicator of physiological stress (Bayne and Newell, 1983; tory conditions (248C, 30 psu, 12 h light–12 h dark) for 2 weeks Liu et al., 2011). For instance, a reduced SfG has been observed before experimentation. The experiment was conducted with three in juveniles of the sea star Asterias rubens upon exposure to replicates in two periods (August 2012 and April 2013). The 816 H. Zhang et al. experimental period were chosen to avoid the reproductive season were collected at an ambient temperature of 248C and transferred (between November and February) as this could affect the physiology to experimental temperatures of either 15 or 308C, a control group of the experimental animals. A preliminary experiment was con- (248C, 30 psu) was set up to investigate changes in physiological ducted to compare physiological responses of individuals collected responses, if any, after they were transferred to a new temperature. in August and April and no significant differences were observed. A carbon dioxide online analyser (LI-260, Li-Cor Company, Individuals were fed with the short-necked clam Ruditapes philippi- Switzerland) was used to monitor the real-time pCO2 levels. narum (a predominant food source of N. festivus at the collection Temperature, pH (NBS scale), pCO2, and salinity were recorded site) for 2 h to satiation once every 3–4 d, a feeding frequency daily using a thermometer, pH meter (HI9124, Hanna, USA), similar to that observed in their natural environment (Chan et al., carbon dioxide online analyser (LI-260, Li-Cor Company), and a re- 2008). Seawater was changed immediately after feeding to avoid the fractometer (HI-211ATC, HT, China), respectively. The software accumulation of unconsumed food and metabolic wastes. CO2SYS was used to calculate the saturation state of calcite (VCa) and aragonite (VAr), total alkalinity (At), and the relationship between these factors. Total alkalinity was also checked weekly using Downloaded from https://academic.oup.com/icesjms/article/73/3/814/2458919 by guest on 29 September 2021 Experimental set-up an alkalinity titrator (HANNA, HI 84431, Germany); the variation Combined effects of pCO2, temperature, and salinity on the physio- between the calculated and measured total alkalinity was between logical responses ofN. festivuswere investigated using afull-factorial ex- 2.5and5%.Environmentalparametersofthe12treatmentsare perimental design with three pCO2 levels (380, 950, and 1250 matm), summarized in Table 1. two temperatures (15 and 308C), and two salinities (10 and 30 psu). In each replicate, 20 individuals were maintained in each glass The pCO2 concentration of 380 matm (LC) was the estimated bottle containing 1000 ml of 0.45 mm filtered natural seawater current global level, whereas 950 matm (MC) and 1250 matm (HC) collected from a pier 9 km away from the study site; five replicates were the predicted levels in the years 2100 and 2300, respectively, were prepared for each treatment. Replicates of 20 individuals according to the IPCC (Intergovernmental Panel on Climate were used because N. festivus is small, and the amount of food con- Change, PCR8.5 scenario, Collins et al., 2013). The average winter sumed and faeces produced by an individual could not be accurately and summer temperatures in Hong Kong are 15 +0.48C(LT)and determined. This inevitably would sacrifice individual variability. 30 + 0.38C (HT), respectively (http://epic.epd.gov.hk/EPICRIVER/ Seawater was changed daily to minimize the effect of microbial marine/history/result/). A water bath at 158C was maintained by a activity and to avoid the metabolism of the experimental animals chiller and another water bath at 308C by a thermostatically controlled adversely affecting seawater chemistry. In our preliminary experi- heater. A salinity of 10 psu represents the lowest salinity N. festivus ment, pH was measured at the beginning and after 24 h in the experiences in summer during heavy rainfall at the study site, while experimental containers; the change in pH was only 0.02–0.03 30 psu is the normal salinity in Hong Kong waters (Morton and NBS unit. This practice was continued in the main experiment. Morton, 1983). In this experiment, the lowest pH level N. festivus The exposure period was 31 d. All physiological parameters were were exposed to was 7.25, which was beyond the range experienced measured once between Days 1 and 3 and between Days 29 and 31 in the field. According to the Hong Kong Environmental Protection to investigate the acute (first 3 d of exposure) and short-term responses Department (2015), the pH of the sampling site varied between and physiological adjustments, if any, to the combined stresses. 7.75 and 9.00, and pH values below 7.50 were recorded only three times during the last 17 years (1986–2013). Various CO2 partial pres- sures were prepared by mixing air and industrial CO2 gas (purity Ingestion rate of 99.5%, Hong Kong Oxygen and Acetylene Co., Ltd). The flow rate Ingestion rate (I) was measured on Days 1 and 29. The tissue wet of gases was regulated by digital flowmeters (GCR-B9SA-BA15, weight of the clam R. philippinarum was measured to the nearest Vogtlin, Sweden), and air and CO2 were mixed in sealed bottles 0.0001 g. Individuals in each replicate were fed with excess clam containing water, then dried in conical flasks with silica gel balls. The tissue for 2 h to ensure that they were satiated as a previous study gas mixture was then delivered to individual experimental chambers had shown that N. festivus required less than an hour to complete using plastic tubing and valves (Zhang et al., 2015). The control a meal (Cheung, 1994). Dry weight of clam tissue, Wdry, was calcu- groupwassuppliedonlywithambientair.AsN. festivus specimens lated using linear regression equations established in preliminary

Table 1. Environmental parameters of the 12 treatments (mean + SD).

21 Temperature (8C) Salinity (psu) pCO2 (matm) pH At (mg l ) VCa VAr LT–LC–LS 15.2 + 0.2 10.1 + 0.4 392 + 46 7.80 + 0.18 90.3 + 15.4 0.58 0.33 LT–LC–HS 15.1 + 0.3 30.0 + 0.7 392 + 46 8.05 + 0.09 199.3 + 11.9 4.62 2.93 LT–MC–LS 15.4 + 0.1 10.1 + 0.3 934 + 83 7.50 + 0.07 97.7 + 0.6 0.33 0.19 LT–MC–HS 15.2 + 0.2 30.3 + 0.2 934 + 83 7.75 + 0.02 201.1 + 19.4 2.14 1.36 LT–HC–LS 15.3 + 0.1 10.3 + 0.4 1260 + 76 7.34 + 0.09 97.3 + 5.5 0.21 0.12 LT–HC–HS 15.5 + 0.3 29.8 + 0.4 1260 + 76 7.52 + 0.04 197.9 + 6.1 0.98 0.62 HT–LC–LS 29.1 + 0.7 10.7 + 0.8 392 + 46 7.84 + 0.10 87.3 + 17.2 1.20 0.71 HT–LC–HS 29.4 + 0.4 31.0 + 0.7 392 + 46 8.18 + 0.08 199.0 + 10.8 7.73 5.11 HT–MC–LS 29.2 + 0.6 10.8 + 0.5 934 + 83 7.56 + 0.05 102.8 + 4.2 0.75 0.44 HT–MC–HS 29.4 + 0.3 30.3 + 0.6 934 + 83 7.70 + 0.07 198.1 + 8.8 3.18 2.10 HT–HC–LS 29.3 + 0.4 11.0 + 0.4 1260 + 76 7.37 + 0.06 96.9 + 2.1 0.41 0.24 HT–HC–HS 29.2 + 0.6 30.5 + 0.5 1260 + 76 7.60 + 0.06 216.4 + 22.0 2.30 1.52

L, low; M, medium; H, high; T, temperature; C, pCO2 levels; S, salinity. Physiological responses and scope for growth in a marine scavenging gastropod 817 experiments at two different salinities: gastropods served as the control for each treatment. The initial and final dissolved oxygen (DO) levels in each syringe were moni- Wdry−30psu = 0.2288 × Wfresh − 0.1369(g) tored by a DO meter (TauTheta SOO-100) and respiration rate estimated by the software (TTI O2 1.08). The initial DO levels were ca. (n = 35, r2 = 0.9528, p , 0.01), 6.0 mg l21 (21 kPa), and the final DO levels were not less than W − = 0.2243 × W − 0.0730(g) dry 10psu fresh 3.0 mg l21 to prevent reduction in the metabolic rate due to low 2 (n = 35, r = 0.9591, p , 0.01), DO content. The respiration rate (mg h21 ind21) was converted to energy expended (J h21 ind21) using a conversion factor of 21 where Wdry-30psu and Wdry-10psu are the tissue dry weight at a salinity 14.14 J mg O2 (Elliott and Davison, 1975). The mean values of 30 and 10 psu, respectively, and Wfresh is the tissue wet weight. and SDs obtained from the five replicates were used in subsequent After feeding, the unconsumed tissue was oven-dried at 1058C statistical analyses. for 24 h to constant weight then weighed. Dry weight of tissue consumed was the difference between initial tissue dry weight and Energy expended on ammonia excretion Downloaded from https://academic.oup.com/icesjms/article/73/3/814/2458919 by guest on 29 September 2021 unconsumed tissue dry weight. Energy expended on ammonia excretion rate (U) was determined The calorific value of the dry body tissue of R. philippinarum immediately after respiration rate measurement. For each replicate, 21 was 20.46 + 0.36 (1 SD) kJ g (Cheung, 1994). Ingestion rate five gastropods were assigned to each group and four groups were 21 21 (I,Jh ind ) wascalculated by multiplying tissue dry weight con- prepared for each replicate. Each group was incubated for 1 h in a sumed by the energy value of R. philippinarum. well-sealed syringe with 50 ml of seawater from the corresponding treatment. One syringe without gastropods was prepared for each W I = 20.46 × 1000 × dry . treatment and served as a control. Ammonia content in the samples 3.5 × 24 × 20 and blanks was determined using a Flow Injection Analyser (Lachat QuikChem 8500). The ammonia excretion rate was converted into an energy equiva- 21 Absorption efficiency lent using a conversion factor of 0.025 J mg NH4-N (Elliott and The absorption efficiency (A) was determined using the following Davison, 1975). For each replicate, a mean value was calculated by equation: averaging the values obtained from the four groups. The mean values and SDs obtained from the five replicates were used in subse- F − E quent statistical analyses. A = × 100%, [(1 − E)×F] Scope for growth where F is the ash-free dry weight : dry weight ratio of clam tissue and SfG (J h21 ind21) was calculated using the following equation E the ash-free dry weight : dry weight ratio of faeces (Conover, 1966). (Winberg, 1960): F was determined by drying the tissue of 30 clams separately at 1058C to obtain dry tissue weight. Dry tissue was then ashed at 5008Cfor3h SfG = Ab −(R + U), to obtain the ash-free dry weight. F was estimated at 89.4%. To deter- mineE,faeces werecollected onDays2and 30by filteringtheseawater where Ab was the absorption rate, R the energy expended on respir- through a dry preweighed glass filter paper of 0.45 mm(Whatman ation, and U the energy expended on ammonia excretion. SfG was GF/C 47 mm). Filter papers were rinsed with isotonic ammonium calculated on Days 3 and 31 using data collected on Day 1, Day 2 formate (3%) to remove salts and dried to constant weight at and Day 29, Day 30, respectively. 1058C for 24 h to obtain the dry weight, then ashed at 5008Cfor 3 h to get the ash-free dry weight. Mortality Cumulative mortality was recorded daily throughout the experi- Absorption rate ment. Individuals were defined as dead if they retracted their Absorption rate (Ab, J h21 ind21) was calculated using I (J h21 ind21) siphon and could not extend their body out of the shell in and A as follows: ambient seawaterafter 10 min in addition to the smell of decompos- ing soft tissue. A Ab = I × . 100 Data analysis As the amount of food consumed and amount of faeces produced by each gastropod were very small, ingestion rate, absorption rate and Energy expended on respiration efficiency, and SfG were determined for each replicate by incubating Energy expended on respiration (R) was determined on Days 3 and 20 individuals in the same experimental chamber. The data were 31. As the individuals were small, 20 individuals in each replicate analysed by three-way ANOVA. When there was an interaction were divided into four groups with five individuals each and respir- among the three factors, the effects of temperature, salinity, and ation rate was determined for each group of five individuals by incu- pCO2 were analysed separately at each level of the other factors by bating them for 1 h in a sealed syringe containing 50 ml of seawater one-way ANOVA followed by multiple comparison Tukey test. As from the corresponding treatment. The respiration rate obtained the gastropods were maintained at 248C before the experiment was divided by five to obtain the rate per individual. The mean res- and transferred to either 15 or 308C, a control group at 248C, and piration rate was obtained for each replicate by averaging the values 380 matm pCO2 was set up. Physiological responses of the control obtained from the four groups. Precautions were taken to prevent group were compared by one-way ANOVA with the groups air bubbles being trapped in the syringe. One syringe without exposed to either 158C and 380 matm pCO2 or 308C and 818 H. Zhang et al.

380 matm. Normality and equal variance of the datawere checked by Ingestion rate the Shapiro–Wilk test and Levene’s test, respectively. All the ana- Ingestion ratewasreduced following exposureto low salinity (Day 1: lyses were performed using SPSS 20.0. d.f. ¼ 1, F ¼ 181.71, p , 0.001; Day 29: d.f. ¼ 1, F ¼ 10.22, p , 0.005) or low temperature (Day 1: d.f. ¼ 1, F ¼ 5.20, p , 0.05; Day 29: d.f. ¼ 1, F ¼ 33.61, p , 0.001; Figure 2). Interaction Results between temperature and pCO2 was observed on Day 1 as analysed Mortality by three-way ANOVA (d.f. ¼ 2, F ¼ 4.20, p , 0.05). However, Most individuals (≥80%) survived the 31 d of exposure for all the when the effect of temperature was compared at each pCO2 level treatment groups. No mortality was observed for the control andthe effect of pCO2 at eachtemperature,nosignificant differences group and some high salinity groups (i.e. LT–MC–HS and HT– were found. Ingestion rates at 15 and 308C were not significantly dif- LC–HS). However, significantly higher cumulative mortality was ferent from the control at 248C, but the rate at 158C was significantly lower than at 308C (d.f. ¼ 1, F ¼ 4.62, p , 0.05). found under low salinity (d.f. ¼ 1, F ¼ 36.37, p , 0.001). On the Downloaded from https://academic.oup.com/icesjms/article/73/3/814/2458919 by guest on 29 September 2021 other hand, pCO2 and temperature did not have anyeffect on cumulative mortality (Figure 1). Absorption efficiency Absorption efficiency (A) varied between 71 and 96% and was not affected significantly by temperature (d.f. ¼ 1, F ¼ 0.298, p ¼ 0.588), salinity (d.f. ¼ 1, F ¼ 3.47, p ¼ 0.068), pCO2 (d.f. ¼ 2, F ¼ 0.06, p ¼ 0.939), or the interactions between these factors on Day 2 (temperature × pCO2: d.f. ¼ 2, F ¼ 0.06, p ¼ 0.940; temperature × salinity: d.f. ¼ 1, F ¼ 1.57, p ¼ 0.217; pCO2 × salinity: d.f. ¼ 2, F ¼ 0.13, p ¼ 0.876; temperature × pCO2 × salinity: d.f. ¼ 2, F ¼ 0.03, p ¼ 0.975). On Day 30, salinity reduced A significantly (d.f. ¼ 1, F ¼ 9.08, p , 0.005) (Figure 3). One-way ANOVA showed that A values at 15, 24, and 308C were not significantly different (Day 2: d.f. ¼ 1, F ¼ 0.38, p ¼ 0.694; Day 30: d.f. ¼ 1, F ¼ 2.49, p ¼ 0.133).

Absorption rate Absorption rate (Ab) was reduced substantially (Figure 4) under low Figure 1. Cumulative mortality of N. festivus upon exposure to salinity on Day 2 (d.f. ¼ 1, F ¼ 163.37, p , 0.001) and the effect was different combinations of temperature, salinity,and pCO2 levelfor 31 d.

Figure 2. Combined effect of temperature, salinity, and pCO2 on the Figure 3. Combined effect of temperature, salinity, and pCO2 on the ingestion rate (I)ofN. festivus on Days 1 and 29. absorption efﬁciency (A)ofN. festivus on Days 2 and 30. Physiological responses and scope for growth in a marine scavenging gastropod 819 Downloaded from https://academic.oup.com/icesjms/article/73/3/814/2458919 by guest on 29 September 2021

Figure 5. Combined effect of temperature, salinity, and CO on the Figure 4. Combined effect of temperature, salinity, and pCO2 on the p 2 absorption rate (Ab) of N. festivus on Days 2 and 30. respiration rate (R)ofN. festivus on Days 3 and 31. also seen at the end of the experiment on Day 30 (d.f. ¼ 1, F ¼ 6.84, Energy expended on ammonia excretion p , 0.001).Reduction in Ab wasalso observed at the lowertempera- Ammonia excretion (U) was reduced significantly at low salinity or ture on both Day 2 (d.f. ¼ 1, F ¼ 6.65, p , 0.05) and Day 30 (d.f. ¼ temperature on both Days 3 and 31 (Figure 6). On Day 3, the inter- 1, F ¼ 10.71, p , 0.005). The effect of pCO2, however, was statistically action between temperature and pCO2 was significant (d.f. ¼ 2, F ¼ indistinguishable (d.f. ¼ 2, F ¼ 0.26, p ¼ 0.771). No interaction 3.36, p , 0.05). No statistical difference in U was found between between the three factors was observed throughout the experiment three pCO2 levels at both 15 and 308C, but the rate was significantly × (Day 2: temperature pCO2:d.f.¼ 2, F ¼ 3.08, p ¼ 0.055; higher at 308C than at 158C for all the pCO2 levels (Table 2). temperature × salinity: d.f. ¼ 1, F ¼ 3.87, p ¼ 0.055; pCO2 × salin- Significant interaction between the three factors was also found ity: d.f. ¼ 2, F ¼ 0.10, p ¼ 0.904; temperature × salinity × pCO2: on Day 31 (d.f. ¼ 2, F ¼ 3.95, p , 0.05; Table 3). The effect of tem- × d.f. ¼ 2, F ¼ 1.62, p ¼ 0.208; Day 30: temperature pCO2:d.f.¼ 2, perature was significant at all combinations of pCO2 and salinity. F ¼ 0.29, p ¼ 0.750; temperature × salinity: d.f. ¼ 1, F ¼ 1.50, p ¼ The effect of salinity, however, was only significant at 380 and × × 0.227; pCO2 salinity: d.f. ¼ 2, F ¼ 0.31, p ¼ 0.738; temperature 1250 matm pCO2 at 158C, and 950 and 1250 matm pCO2 at 308C. × salinity pCO2:d.f.¼ 2, F ¼ 0.70, p ¼ 0.501). Differences in U among the three pCO2 levels were significant at 10 psu and 308C only. U at 158C were not statistically different from that at 248C on both days (Day 3: p ¼ 0.367; Day 31: p ¼ Energy expended on respiration 0.163), whereas U at 308C was significantly higher on Day 3 (p , Energy expended on respiration (R) did not change under elevated 0.050) but not on Day 31 (p ¼ 0.804). pCO2 levels (d.f. ¼ 2, F ¼ 0.24, p ¼ 0.791), but increased significantly at elevated temperatures (d.f. ¼ 1, F ¼ 216.56, p , 0.001) Scope for growth or salinities (d.f. ¼ 1, F ¼ 86.88, p , 0.001) and the effects persisted SfG was reduced significantly at low salinity on Day 3 (d.f. ¼ 1, F ¼ until the end of the experiment (Figure 5). No interaction between 121.94, p , 0.001) and Day 31 (d.f. ¼ 1, F ¼ 16.52, p , 0.001; the three factors was observed throughout the experiment (Day 2: Figure 7). The interactive effect between temperature and pCO2 temperature × pCO2:d.f.¼ 2, F ¼ 0.55, p ¼ 0.579; temperature × (d.f. ¼ 2, F ¼ 3.33, p , 0.05) and that between temperature and salinity: d.f. ¼ 1, F ¼ 1.34, p ¼ 0.253; pCO2 × salinity: d.f. ¼ 2, salinity (d.f. ¼ 1, F ¼ 4.49, p ¼ 0.05) were significant on Day F ¼ 0.21, p ¼ 0.808; temperature × salinity × pCO2:d.f.¼ 2, F ¼ 3. Multiple comparison tests showed that SfG reduced at low salinity 1.62, p ¼ 0.208; Day 30: temperature × pCO2:d.f.¼ 2, F ¼ 0.16, for both temperatures (Table 4). SfG at 248C was not significantly p ¼ 0.85; temperature × salinity: d.f. ¼ 1, F ¼ 1.24, p ¼ 0.270; different from that at 15 and 308C on Day 3 (d.f. ¼ 1, F ¼ 0.70, pCO2 × salinity: d.f. ¼ 2, F ¼ 1.68, p ¼ 0.198; temperature × p ¼ 0.520) and Day 31 (d.f. ¼ 1, F ¼ 2.12, p ¼ 0.171). salinity × pCO2: d.f. ¼ 2, F ¼ 3.02, p ¼ 0.058). At 380 matm pCO2, R at 248C was not significantly different from that at 308C(Day3: Discussion p ¼ 0.087; Day 31, p ¼ 0.410), but was significantly higher than The physiological responses of N. festivus were positively correlated that at 158C on both days (Day 3: p , 0.001; Day 31, p , 0.005). with temperature and salinity. In contrast, the effect of pCO2 was 820 H. Zhang et al.

insignificant and its combined effects with temperature and/or salinity were also weak and only occurred in the early phase of the experiment. Responses to future ocean acidification have been extensively studied in the past few years with negative effects observed in the most species tested, including molluscs (Kroeker et al., 2010; Parker et al., 2013), and neutral or positive effects appearing to differ among species and life stages (Dupont et al., 2013). For instance, the sea urchin Echinometra sp. showed no significant differences in somatic and gonadal growth under pCO2 1433 matm after 11 months exposure (Hazan et al., 2014). Sterechinus neumayeri also showed acclimation after 8 months exposure to low pH (20.5 U; Suckling, et al., 2015). Cross et al. (2015) reported no ocean acidifi- Downloaded from https://academic.oup.com/icesjms/article/73/3/814/2458919 by guest on 29 September 2021 cation effects on shell growth and repair in the New Zealand brachiopod Calloria inconspicua after exposure to pH 7.62 for 12 weeks. Nevertheless, increased rates of calcification in low pH waters have been observed for a few taxa including crustaceans (Ries et al., 2009; Kroeker et al., 2010), ophiuroids (Wood et al., 2008), and pisces (Melzner et al., 2009a; Hurst et al., 2013). The sea star, Asterias rubens, and the brittlestars, O. fragilis and A. filiformis, showed neutral responses in metabolic rate after being exposed to warming (208C) and ocean acidification (pH 7.5) for 1 week (Carey et al., 2014). In the present study, N. festivus showed high resilience to ocean acidification as no physiological effects were observed. Generally, intertidal organisms are more tolerant of variations in environmental variables, such as pH, temperature, and salinity, as they naturally Figure 6. Combined effect of temperature, salinity, and CO on the p 2 exist in a fluctuating environment (Maderira et al., 2014). This phe- excretion rate (U)ofN. festivus on Days 3 and 31. nomenon has been observed for the larvae of the sea urchin

Table 2. One-way ANOVA and the multiple comparison Tukey test for the temperature effect at each pCO2 level and the effect of pCO2 at each temperature on energy expended on excretion on Day 3. d.f. MS FpTukey test 158C 2 9.233E20.006 0.488 0.619 380 matm 950 matm 1250 matm 308C 2 3.293E20.005 1.515 0.238 380 matma 950 matm 1250 matm 380 matm 1 0.000 28.875 0.000 158Ca 308Cb 950 matm 1 0.000 5.608 0.029 158Ca 308Cb 1250 matm 1 0.000 5.678 0.028 158Ca 308Cb Values in bold are statistically signiﬁcant. Values in the same row with different letter designations indicate that they are statistically different.

Table 3. One-way ANOVA and the multiple comparison Tukey test of energy expended on excretion on Day 31. d.f. MS FpTukey test 10 psu-158C 2 1.047E20.005 1.880 0.195 380 matm 950 matm 1250 matm 10 psu-308C 2 1.287E20.005 3.899 0.050 380 matma 950 matmb 1250 matmab 30 psu-158C 2 3.467E20.006 0.819 0.464 380 matm 950 matm 1250 matm 30 psu-308C 2 2.427E20.005 1.742 0.217 380 matm 950 matm 1250 matm 158C-380 matm 1 7.290E20.005 9.785 0.014 10 psua 30 psub 158C-950 matm 1 1.690E20.005 2.965 0.123 10 psu 30 psu 158C-1250 matm 1 4.000E20.005 25.806 0.001 10 psua 30 psub 308C-380 matm 1 1.000E20.007 0.020 0.892 10 psu 30 psu 308C-950 matm 1 6.250E20.005 7.812 0.023 10 psua 30 psub 308C-1250 matm 1 7.840E20.005 6.149 0.038 10 psua 30 psub 10 psu-380 matm 1 0.000 73.923 0.000 158Ca 308Cb 10 psu-950 matm 1 0.000 19.761 0.002 158Ca 308Cb 10 psu-1250 matm 1 0.000 78.400 0.000 158Ca 308Cb 30 psu-380 matm 1 0.000 15.728 0.004 158Ca 308Cb 30 psu-950 matm 1 0.000 26.955 0.001 158Ca 308Cb 30 psu-1250 matm 1 0.000 20.747 0.002 158Ca 308Cb Values in bold indicate the differences were statistically signiﬁcant. Values in the same row with different letter designations indicate that they are statistically different. Physiological responses and scope for growth in a marine scavenging gastropod 821

Paracentrotus lividus living in contrasting environments, with inter- in body fluids and calcification compartments during exposure to tidal populations being more tolerant of a decrease in pH than ocean acidification involves pH andion regulation acrossthe epithe- subtidal populations (Moulin et al., 2011). Tolerance of an organism lia of gills, gut, and kidneys and is driven by energy-consuming ion to elevated pCO2 may be due to its ability to compensate for pumps (Wittmann and Po¨rtner, 2013). The associated energetic CO2-induced changes in extracellular pH (Wittmann and Po¨rtner, costs may shift the energy budget of the organism (Po¨rtner, 2008). 2013). Our previous study on the subtidal gastropod N. conoidalis, Although ocean acidification did not affect physiological responses a congeneric counterpart of the intertidal N. festivus, demonstrated and SfG inN. festivus, an increase in the cost of acid–base regulation, metabolic depression when exposed to ocean acidification (Zhang if any, may alter the energy allocation strategy (e.g. reduction in re- et al., 2015). This is a common phenomenon of uncompensated productive output), and this deserves further investigations. changes in extracellular pH and intracellular pH, but was not Aweak interactive effect between temperature and pCO2 on physio- observed for N. festivus (Zhang et al., 2015). logical responses (ingestion, excretion and SfG) in N. festivus was Extracellular acid–base regulation during short-term hypercap- recorded in the early phase of the present experiment (Days 1–3). nia has been shown in the Dungeness crab, Cancer magister, which Enhancement of temperature effects under low pH has been Downloaded from https://academic.oup.com/icesjms/article/73/3/814/2458919 by guest on 29 September 2021 inhabits fluctuating shallow waters, but was absent in the relatively shown in various marine species. For example, production of the stable habitat of the deep-sea Tanner crab Chionoecetes tanneri heat shock protein HSP70 in the crab Pachygrapsus marmoratus (Pane and Barry, 2007). Compensation for hypercapnic acidosis was significantly reduced by the combined effects of temperature and pH, but not by thermal stress alone (Maderira et al., 2014). Spine development in the sea urchin Heliocidaris erythrogramma was negatively affected by an increase in temperature (+2to48C) and extreme acidification (pH 7.4), with a complex interaction between the stressors (Wolfe et al., 2013). In our previous study, reduction in physiological responses (ingestion, absorption, respiration, and excretion) upon exposure to pCO2 was enhanced at high temperature (308C) in the benthic gastropod N. conoidalis (Zhang et al., 2015). Most organisms have an optimal temperature range within which physiological performance is maximized (Po¨rtner et al., 2005). However, acidification may intensify the sensitivity of the organisms to temperature change, resulting in a synergistic effect of elevated temperature and CO2-induced ocean acidification on energy metabolism that narrows the thermal tolerance window of marine ectotherms (Po¨rtner and Farrell, 2008). Reduced animal performance upon exposure to salinity outside of their natural range, regardless of hyper- or hyposalinity, is widely recognized (Newell, 1976; Chaparro et al., 2014). Hyposalinity may cause hypo-osmotic stress-induced physiological and ion- osmoregulatory responses in marine animals (Sinha et al., 2015) and enhances the effect of ocean acidification on acid–base regulation (Zhang et al., 2014). For instance, when both pH and salinity were reduced simultaneously (pH 7.6, salinity 26.2 psu), the interaction between the two stresses affected the predatory gastropod Limacina retroversa negatively both in terms of survival rate and an ability to swim upwards (Manno et al., 2012). Low salinity

Figure 7. Combined effect of temperature, salinity, and pCO2 on the reduced growth, elevated mortality, and impaired shell mainten- SfG of N. festivus on Days 3 and 31. ance in juveniles of the hard-shell clam, Mercenaria mercenaria,

Table 4. One-way ANOVA and the multiple comparison Tukey test for the (a) temperature effect at each pCO2 level and pCO2 effect at each temperature and (b) temperature effect at each salinity and salinity effect at each temperature on SfG on Day 3. d.f. MS FpTukey test

(a) Temperature effect at each pCO2 level and pCO2 effect at each temperature 158C 2 0.104 0.378 0.689 380 matm 950 matm 1250 matm 308C 2 0.380 0.633 0.539 380 matm 950 matm 1250 matm 380 matm 1 0.605 1.463 0.242 158C308C 950 matm 1 0.025 0.053 0.821 158C308C 1250 matm 1 0.256 0.601 0.448 158C308C (b) Temperature effect at each salinity and salinity effect at each temperature 158C 1 5.270 62.804 0.000 10 psua 30 psub 308C 1 11.463 58.079 0.000 10 psua 30 psub 10 psu 1 0.248 1.986 0.170 158C308C 30 psu 1 0.350 2.241 0.146 158C308C Values in the same row with different letter designations indicate that they are statistically significant (p , 0.05). 822 H. Zhang et al. owing to strongly elevated basal energy demand. Low salinity also slower (unpublished data). Smaller newly hatched larvae may ex- modulated responses to elevated pCO2 through negatively affecting perience a slower growth and take a longer time to metamorphose the mechanical properties of the shell (Dickinson et al., 2013). into juveniles which, themselves, may be smaller. This highlights Low salinity had a major effect on the survival and physiological the importance of tests for transgenerational and carryover effects energetics of N. festivus in the present study. Unlike M. mercenaria, in future research programmes. both energy intake and expenditure in N. festivus was reduced at low Under the combined effect of temperature, salinity, and ocean salinity, possibly owing to the experimental salinity approaching the acidification, mortality and the maintenance cost of the larvae of lethal limit as the lowest salinity for N. festivus to survive indefinitely N. festivus increased (Zhang et al., 2014). Larvae hatched under an has been estimated at 11.5 psu (Morton, 1990). This may also help elevated pCO2 were smaller and the juveniles grew slower (unpub- explain the absence of interactive effects between salinity and pCO2, lished data). Younger life stages, therefore, are more sensitive to as the effect of low salinity possibly overshadowed that of pCO2. these stresses than adults. An extensive review of studies on sea pCO2 levels enhanced the effects of temperature/salinity on urchins has shown that larvae and juveniles are much more sensitive physiological responses only in the first few days of the present ex- to ocean acidification than adults and gametes (Dupont and Downloaded from https://academic.oup.com/icesjms/article/73/3/814/2458919 by guest on 29 September 2021 periment, but the pCO2 effect was absent after 1 month. This may Thorndyke, 2013). Similar observations have been reported for indicate rapid adjustment of physiological responses to pCO2, pos- marine molluscs (Parker et al., 2013), possibly due to shell structure, sibly through regulation of extra- and/or intracellular pH. In the which is composed of more soluble forms of calcium carbonate, i.e. cold-water coral L. pertusa, short-term (1 week) high CO2 exposure amorphous calcium carbonate and aragonite (Wicks and Roberts, under pH 7.77 resulted in a decline of calcification by 26–29% and a 2012) and the lack of the ability to maintain acid–base status net dissolution of calcium carbonate. Acclimation to acidified con- (Melzner et al., 2009b). Although neutral effects have been observed ditions, however, has been observed following long-term (6 for the energy budget of N. festivus adults upon exposure to multiple months) experiments, leading to even slightly enhanced rates of cal- stressors,populationperformance maybeimpactedthrough observed cification (Form and Riebesell, 2012). Although a short-term expos- effects on younger life stages. ure (1 month) to ocean acidification had no effect on the physiological responses in N. festivus, reduction in physiological performance may eventually lead to a gradual deterioration of Acknowledgements body conditions and result in negative effects on growth and repro- We thank two anonymous reviewers for their constructive com- duction. For example, reduction in female fecundity has been ments on the manuscript and Bruce Richardson for improving observed in the sea urchin Strongylocentrotus droebachiensis follow- the English. Our work was fully supported by a strategic research grant (grant no. 7004027) of the City University of Hong Kong. ing exposure to pCO2 for 4 months (Dupont et al., 2013). Movilla et al. (2014) found that the calcification rate of the coral Desmophyllum dianthus was not reduced by pH 7.81 after 49 d of ex- References posure, but the rate was significantly reduced when the exposure Abduraji, T. S., and Danilo, D. T. 2015. Effects of reduced pH on the period was extended to 314 d. An experiment for an extended growth and survival of postlarvae of the donkey’s ear abalone, period of several months could clarify whether the neutral effects Haliotis asinina (L.). Aquaculture International, 23: 141–153. of ocean acidification on the physiological responses of N. festivus Andersson, A. J., Mackenzie, F. T., and Gattuso, J-P. 2011. 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