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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
scavenging gastropod, Nassarius festivus (Powys, 1835), are
affected by salinity and temperature but not by ocean acidification
Haoyu Zhang 1, Paul K. S. Shin 1,2, and Siu Gin Cheung 1,2*
1
Department of Biology and Chemistry, City University of Hong Kong, Hong Kong, China
State Key Laboratory in Marine Pollution, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, China
2
*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
For over 800 000 years, carbon dioxide has been relatively stable in
the atmosphere at 172– 300 matm by volume concentration (Luthi
et al., 2008). The level reached in 2000 (395 matm) is predicted
to rise to 1000 matm by 2100 (Collins et al., 2013). During the
period 2000– 2008, approximately one quarter of anthropogenic
carbon dioxide was dissolved in the ocean (Le Quéré et al., 2009),
and increasing CO2 availability is causing a global decrease in pH
of seawater, a phenomenon known as ocean acidification.
Effects of ocean acidification have been extensively reported
among marine organisms including bacteria, plants, and animals
[reviewed by Caldeira and Wickett (2003)]. The unsaturated state
of calcium carbonate caused by excess H+ and lower Ca2+ availability in acidifying seawater makes calcifying invertebrates potential
victims of changes to ocean chemistry. Among such organisms,
corals are considered to be one of the most vulnerable groups
(Bramanti et al., 2013; Reyes-Nivia et al., 2013). For molluscs,
Abduraji and Danilo (2015) found that the pH-driven survival
rate of Haliotis asinina was reduced from 86.3 to 47.2% at pH
7.99, and 18.3% at pH 7.62 and 7.42, respectively, after 20 d of exposure. Acidified seawater also restrained pteropods from maintaining shells made up of aragonite (Honjo et al., 2000). Dissolution of
the shell at the growing edge of the aperture was observed in the
pteropod Clio pyramidata within 48 h of exposure to 788 matm
pCO2 (Orr et al., 2005).
Physiological responses of ocean acidification are species-specific
with differential responses being observed in closely related species.
For example, the Mediterranean mussel Mytilus galloprovincialis
showed a reduced metabolic rate and slower growth when exposed
to pH 7.3 for 3 months (Michaelidis et al., 2005). In contrast, no
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Physiological responses and scope for growth in a marine scavenging gastropod
physiological disturbance was observed in the blue mussel Mytilus
edulis at pH 7.14 for 60 d (Thomsen and Melzner, 2010). Some
species are robust or show positive responses to ocean acidification
[reviewed by Andersson et al. (2011)]. The brittlestar Amphiura filiformis showed an increase in metabolism and calcification with a substantial cost (muscle wastage) upon exposure to acidified seawater
(pH 7.7) for 40 d (Wood et al., 2008). Neutral responses in metabolic
rates have been observed in three echinoderms, Asterias rubens,
Ophiothrix fragilis, and A. filiformis after 1 week of exposure to pH
7.5 (Carey et al., 2014). High metabolic rates commonly found in
crustaceans facilitate the control of extracellular pH through active
ion transport (Whiteley, 2011), hence reducing the impact of ocean
acidification. For instance, after 10 d of incubation, there were no
net changes in survival or overall development of larvae of the barnacle Amphibalanus improvisus raised at pH 7.6 compared with the
control pH of 8.0 (Pansch et al., 2013). This may be partly due to
the absence of calcified structures in barnacle larvae that are developed only when they settle and metamorphose into the juvenile stage.
Shifts in energy allocation upon exposure to ocean acidification
may reduce fitness and produce low functional capacities, hence increasing sensitivity to environmental stressors such as temperature,
food supply, and salinity (Zittier et al., 2013; Carey et al., 2014).
According to Pörtner (2008), ocean acidification enhances sensitivity to thermal stress, resulting in a narrowing of the thermal tolerance window and aerobic capacity. For example, the brown crab
Cancer pagurus reduced its upper thermal limit of aerobic scope
by 58C under hypercapnia (pH 7.06) exposure for 16 h (Metzger
et al., 2007). The scope for performance of the Arctic spider crab
Hyas araneus was reduced at the limits of thermal tolerance (48C)
and exacerbated by an elevated CO2 level of 3000 matm (Zittier
et al., 2013). Synergistic effects have also been observed between
ocean acidification and low salinity. Combined exposure to hypercapnia and low salinity negatively affected mortality, tissue growth,
energy storage, and mechanical properties of shells of juvenile
oysters Crassostrea virginica (Dickinson et al., 2012). In addition,
the larval mortality of a subtidal scavenging gastropod Nassarius
conoidalis was enhanced by high pCO2 level (1250 matm) at low salinity (10 psu) but not at normal salinity (30 psu; Zhang et al., 2014).
Acclimation occurs when organisms adjust physiologically to
changes in the environment, allowing them to maintain performance
relatively independently of the changes. Such adjustment occurs
over a short period and depends on lifespan (Barry et al., 2011).
Acclimation to temperature and salinity is commonly found in
marine animals. For instance, intertidal barnacles Elminus modestus
and Balanus balanoides can tolerate salinities as low as 14–17 psu following experimental or natural acclimation (Foster, 1970). A range of
homeostatic responses which serve to offset the passive effects of
reduced temperature have been shown to allow teleost fish to adapt
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
was observed after 6 months and resulted in an enhancement of calcification (Form and Riebesell, 2012). Physiological responses of the
subtidal scavenging gastropod, N. conoidalis, were sensitive to ocean
acidification under acute exposure for 3 d, but complete acclimation
was observed after incubation for 1 month (Zhang et al., 2015).
Scope for growth (SfG) is an integrated index reflecting energy
allocation strategies in living organisms and has been shown to be
a useful indicator of physiological stress (Bayne and Newell, 1983;
Liu et al., 2011). For instance, a reduced SfG has been observed
in juveniles of the sea star Asterias rubens upon exposure to
815
1120 matm pCO2 for 39 weeks with no acclimation observed
(Appelhans et al., 2014). In addition, SfG measurements of the sea
urchin Strongylocentrotus purpuratus raised under high pCO2
(129 Pa, 1271 matm) indicate that an average of 39 –45% of the
available energy was spent in somatic growth, while control larvae
could allocate between 78 and 80% of the available energy to
growth processes.
As one of the most dominant and competitive scavengers on
sandy shores in Hong Kong, Nassarius festivus plays an important
role in matter cycling and energy flow, and serves as an important
cleaner in removing carrion (Briton and Morton, 1992). Previous
studies on physiological energetics have shown that this species is tolerant of environmental stresses, including low salinity and hypoxia
(Cheung and Lam, 1995; Chan et al., 2008). The interactive effects
of ocean acidification, high temperature, and low salinity increased
the mortality of the veliger larvae (Zhang et al., 2014). The maintenance cost, as shown by the respiration rate, also increased with
temperature and pCO2 level. In the present study, N. festivus adults
were exposed to the combined effect of ocean acidification, temperature, and salinity for 31 d. The acute responses to the combined stresses and physiological adjustments, if any, following prolonged
exposure to the stresses were investigated. To understand if there
are any life-stage differences in sensitivities to multiple stressors
that could create a bottleneck in population performance, results
were compared with those obtained for the larvae in a previous
study (Zhang et al., 2014). Reduction in population performance
could eventually lessen the role of N. festivus in removing carrion
on sandy shores, hence resulting in a deterioration of environmental
quality.
Based on climate models in the IPCC Fifth Assessment Report
(AR5), The Hong Kong Observatory has predicted the temperature
and rainfall changes in Hong Kong in the 21st century (http://www
.hko.gov.hk/climate_change/proj_hk_rainfall_e.htm). Under the
high greenhouse gas concentration scenario (RCP8.5) proposed in
this report, temperature is expected to rise by 1.5 –3 and 3 – 68C in
the mid-21st century (2051 –2060) and late 21st century (2091 –
2100), respectively, when compared with the 1986–2005 average
of 23.38C. The number of extremely wet years is expected to increase
from 3 in 1885– 2005 to about 12 in 2006–2100. The annual rainfall
in the late 21st century is expected to rise by 180 mm when compared with the 1986–2005 average. Such predictions indicate that
Hong Kong is facing an increase in temperature and rainfall due
to climate change and by inference, salinity, and temperature stresses on coastal marine organisms would be both more frequent and
more abrupt. The results of this study can thus aid in predicting
the performance of an important beach cleaner under the combined
effects of ocean acidification temperature change and salinity
stresses.
Methods
Study organisms
Nassarius festivus (shell length: 13 + 2 mm) were collected from
Starfish Bay, a sandy beach located in the northeast of Hong Kong
(22.481308N, 114.244118E). As the experiment lasted for a month
and all the replicates could not be completed at the same time due
to logistical problems, individuals from each replicate were collected
from the field before each experiment and were acclimated to laboratory conditions (248C, 30 psu, 12 h light–12 h dark) for 2 weeks
before experimentation. The experiment was conducted with three
replicates in two periods (August 2012 and April 2013). The
816
H. Zhang et al.
experimental period were chosen to avoid the reproductive season
(between November and February) as this could affect the physiology
of the experimental animals. A preliminary experiment was conducted to compare physiological responses of individuals collected
in August and April and no significant differences were observed.
Individuals were fed with the short-necked clam Ruditapes philippinarum (a predominant food source of N. festivus at the collection
site) for 2 h to satiation once every 3–4 d, a feeding frequency
similar to that observed in their natural environment (Chan et al.,
2008). Seawater was changed immediately after feeding to avoid the
accumulation of unconsumed food and metabolic wastes.
Experimental set-up
Combined effects of pCO2, temperature, and salinity on the physiological responses of N. festivus were investigated using a full-factorial experimental design with three pCO2 levels (380, 950, and 1250 matm),
two temperatures (15 and 308C), and two salinities (10 and 30 psu).
The pCO2 concentration of 380 matm (LC) was the estimated
current global level, whereas 950 matm (MC) and 1250 matm (HC)
were the predicted levels in the years 2100 and 2300, respectively,
according to the IPCC (Intergovernmental Panel on Climate
Change, PCR8.5 scenario, Collins et al., 2013). The average winter
and summer temperatures in Hong Kong are 15 + 0.48C (LT) and
30 + 0.38C (HT), respectively (http://epic.epd.gov.hk/EPICRIVER/
marine/history/result/). A water bath at 158C was maintained by a
chiller and another water bath at 308C by a thermostatically controlled
heater. A salinity of 10 psu represents the lowest salinity N. festivus
experiences in summer during heavy rainfall at the study site, while
30 psu is the normal salinity in Hong Kong waters (Morton and
Morton, 1983). In this experiment, the lowest pH level N. festivus
were exposed to was 7.25, which was beyond the range experienced
in the field. According to the Hong Kong Environmental Protection
Department (2015), the pH of the sampling site varied between
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 pressures were prepared by mixing air and industrial CO2 gas (purity
of 99.5%, Hong Kong Oxygen and Acetylene Co., Ltd). The flow rate
of gases was regulated by digital flowmeters (GCR-B9SA-BA15,
Vogtlin, Sweden), and air and CO2 were mixed in sealed bottles
containing water, then dried in conical flasks with silica gel balls. The
gas mixture was then delivered to individual experimental chambers
using plastic tubing and valves (Zhang et al., 2015). The control
group was supplied only with ambient air. As N. festivus specimens
were collected at an ambient temperature of 248C and transferred
to experimental temperatures of either 15 or 308C, a control group
(248C, 30 psu) was set up to investigate changes in physiological
responses, if any, after they were transferred to a new temperature.
A carbon dioxide online analyser (LI-260, Li-Cor Company,
Switzerland) was used to monitor the real-time pCO2 levels.
Temperature, pH (NBS scale), pCO2, and salinity were recorded
daily using a thermometer, pH meter (HI9124, Hanna, USA),
carbon dioxide online analyser (LI-260, Li-Cor Company), and a refractometer (HI-211ATC, HT, China), respectively. The software
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
an alkalinity titrator (HANNA, HI 84431, Germany); the variation
between the calculated and measured total alkalinity was between
2.5 and 5%. Environmental parameters of the 12 treatments are
summarized in Table 1.
In each replicate, 20 individuals were maintained in each glass
bottle containing 1000 ml of 0.45 mm filtered natural seawater
collected from a pier 9 km away from the study site; five replicates
were prepared for each treatment. Replicates of 20 individuals
were used because N. festivus is small, and the amount of food consumed and faeces produced by an individual could not be accurately
determined. This inevitably would sacrifice individual variability.
Seawater was changed daily to minimize the effect of microbial
activity and to avoid the metabolism of the experimental animals
adversely affecting seawater chemistry. In our preliminary experiment, pH was measured at the beginning and after 24 h in the
experimental containers; the change in pH was only 0.02 –0.03
NBS unit. This practice was continued in the main experiment.
The exposure period was 31 d. All physiological parameters were
measured once between Days 1 and 3 and between Days 29 and 31
to investigate the acute (first 3 d of exposure) and short-term responses
and physiological adjustments, if any, to the combined stresses.
Ingestion rate
Ingestion rate (I ) was measured on Days 1 and 29. The tissue wet
weight of the clam R. philippinarum was measured to the nearest
0.0001 g. Individuals in each replicate were fed with excess clam
tissue for 2 h to ensure that they were satiated as a previous study
had shown that N. festivus required less than an hour to complete
a meal (Cheung, 1994). Dry weight of clam tissue, Wdry, was calculated using linear regression equations established in preliminary
Table 1. Environmental parameters of the 12 treatments (mean + SD).
LT –LC–LS
LT –LC–HS
LT –MC–LS
LT –MC–HS
LT –HC–LS
LT –HC–HS
HT– LC–LS
HT– LC–HS
HT– MC–LS
HT– MC–HS
HT– HC–LS
HT– HC–HS
Temperature (88 C)
15.2 + 0.2
15.1 + 0.3
15.4 + 0.1
15.2 + 0.2
15.3 + 0.1
15.5 + 0.3
29.1 + 0.7
29.4 + 0.4
29.2 + 0.6
29.4 + 0.3
29.3 + 0.4
29.2 + 0.6
Salinity (psu)
10.1 + 0.4
30.0 + 0.7
10.1 + 0.3
30.3 + 0.2
10.3 + 0.4
29.8 + 0.4
10.7 + 0.8
31.0 + 0.7
10.8 + 0.5
30.3 + 0.6
11.0 + 0.4
30.5 + 0.5
L, low; M, medium; H, high; T, temperature; C, pCO2 levels; S, salinity.
pCO2 (matm)
392 + 46
392 + 46
934 + 83
934 + 83
1260 + 76
1260 + 76
392 + 46
392 + 46
934 + 83
934 + 83
1260 + 76
1260 + 76
pH
7.80 + 0.18
8.05 + 0.09
7.50 + 0.07
7.75 + 0.02
7.34 + 0.09
7.52 + 0.04
7.84 + 0.10
8.18 + 0.08
7.56 + 0.05
7.70 + 0.07
7.37 + 0.06
7.60 + 0.06
At (mg l21)
90.3 + 15.4
199.3 + 11.9
97.7 + 0.6
201.1 + 19.4
97.3 + 5.5
197.9 + 6.1
87.3 + 17.2
199.0 + 10.8
102.8 + 4.2
198.1 + 8.8
96.9 + 2.1
216.4 + 22.0
VCa
0.58
4.62
0.33
2.14
0.21
0.98
1.20
7.73
0.75
3.18
0.41
2.30
VAr
0.33
2.93
0.19
1.36
0.12
0.62
0.71
5.11
0.44
2.10
0.24
1.52
817
Physiological responses and scope for growth in a marine scavenging gastropod
experiments at two different salinities:
Wdry−30psu = 0.2288 × Wfresh − 0.1369(g)
(n = 35, r 2 = 0.9528, p , 0.01),
Wdry−10psu = 0.2243 × Wfresh − 0.0730(g)
(n = 35, r 2 = 0.9591, p , 0.01),
where Wdry-30psu and Wdry-10psu are the tissue dry weight at a salinity
of 30 and 10 psu, respectively, and Wfresh is the tissue wet weight.
After feeding, the unconsumed tissue was oven-dried at 1058C
for 24 h to constant weight then weighed. Dry weight of tissue
consumed was the difference between initial tissue dry weight and
unconsumed tissue dry weight.
The calorific value of the dry body tissue of R. philippinarum
was 20.46 + 0.36 (1 SD) kJ g21 (Cheung, 1994). Ingestion rate
(I, J h21 ind21) was calculated by multiplying tissue dry weight consumed by the energy value of R. philippinarum.
I = 20.46 × 1000 ×
Wdry
.
3.5 × 24 × 20
Absorption efficiency
The absorption efficiency (A) was determined using the following
equation:
A=
F−E
× 100%,
[(1 − E) × F]
where F is the ash-free dry weight : dry weight ratio of clam tissue and
E the ash-free dry weight : dry weight ratio of faeces (Conover, 1966).
F was determined by drying the tissue of 30 clams separately at 1058C
to obtain dry tissue weight. Dry tissue was then ashed at 5008C for 3 h
to obtain the ash-free dry weight. F was estimated at 89.4%. To determine E, faeces were collected on Days 2 and 30 by filtering the seawater
through a dry preweighed glass filter paper of 0.45 mm (Whatman
GF/C 47 mm). Filter papers were rinsed with isotonic ammonium
formate (3%) to remove salts and dried to constant weight at
1058C for 24 h to obtain the dry weight, then ashed at 5008C for
3 h to get the ash-free dry weight.
Absorption rate
Absorption rate (Ab, J h21 ind21) was calculated using I (J h21 ind21)
and A as follows:
Ab = I ×
A
.
100
Energy expended on respiration
Energy expended on respiration (R) was determined on Days 3 and
31. As the individuals were small, 20 individuals in each replicate
were divided into four groups with five individuals each and respiration rate was determined for each group of five individuals by incubating them for 1 h in a sealed syringe containing 50 ml of seawater
from the corresponding treatment. The respiration rate obtained
was divided by five to obtain the rate per individual. The mean respiration rate was obtained for each replicate by averaging the values
obtained from the four groups. Precautions were taken to prevent
air bubbles being trapped in the syringe. One syringe without
gastropods served as the control for each treatment. The initial
and final dissolved oxygen (DO) levels in each syringe were monitored by a DO meter (TauTheta SOO-100) and respiration rate estimated by the software (TTI O2 1.08). The initial DO levels were ca.
6.0 mg l21 (21 kPa), and the final DO levels were not less than
3.0 mg l21 to prevent reduction in the metabolic rate due to low
DO content. The respiration rate (mg h21 ind21) was converted
to energy expended (J h21 ind21) using a conversion factor of
14.14 J mg21 O2 (Elliott and Davison, 1975). The mean values
and SDs obtained from the five replicates were used in subsequent
statistical analyses.
Energy expended on ammonia excretion
Energy expended on ammonia excretion rate (U ) was determined
immediately after respiration rate measurement. For each replicate,
five gastropods were assigned to each group and four groups were
prepared for each replicate. Each group was incubated for 1 h in a
well-sealed syringe with 50 ml of seawater from the corresponding
treatment. One syringe without gastropods was prepared for each
treatment and served as a control. Ammonia content in the samples
and blanks was determined using a Flow Injection Analyser (Lachat
QuikChem 8500).
The ammonia excretion rate was converted into an energy equivalent using a conversion factor of 0.025 J mg21 NH4-N (Elliott and
Davison, 1975). For each replicate, a mean value was calculated by
averaging the values obtained from the four groups. The mean
values and SDs obtained from the five replicates were used in subsequent statistical analyses.
Scope for growth
SfG (J h21 ind21) was calculated using the following equation
(Winberg, 1960):
SfG = Ab − (R + U),
where Ab was the absorption rate, R the energy expended on respiration, and U the energy expended on ammonia excretion. SfG was
calculated on Days 3 and 31 using data collected on Day 1, Day 2
and Day 29, Day 30, respectively.
Mortality
Cumulative mortality was recorded daily throughout the experiment. Individuals were defined as dead if they retracted their
siphon and could not extend their body out of the shell in
ambient seawater after 10 min in addition to the smell of decomposing soft tissue.
Data analysis
As the amount of food consumed and amount of faeces produced by
each gastropod were very small, ingestion rate, absorption rate and
efficiency, and SfG were determined for each replicate by incubating
20 individuals in the same experimental chamber. The data were
analysed by three-way ANOVA. When there was an interaction
among the three factors, the effects of temperature, salinity, and
pCO2 were analysed separately at each level of the other factors by
one-way ANOVA followed by multiple comparison Tukey test. As
the gastropods were maintained at 248C before the experiment
and transferred to either 15 or 308C, a control group at 248C, and
380 matm pCO2 was set up. Physiological responses of the control
group were compared by one-way ANOVA with the groups
exposed to either 158C and 380 matm pCO2 or 308C and
818
380 matm. Normality and equal variance of the data were checked by
the Shapiro –Wilk test and Levene’s test, respectively. All the analyses were performed using SPSS 20.0.
Results
Mortality
Most individuals (≥80%) survived the 31 d of exposure for all the
treatment groups. No mortality was observed for the control
group and some high salinity groups (i.e. LT–MC–HS and HT–
LC–HS). However, significantly higher cumulative mortality was
found under low salinity (d.f. ¼ 1, F ¼ 36.37, p , 0.001). On the
other hand, pCO2 and temperature did not have any effect on cumulative mortality (Figure 1).
H. Zhang et al.
Ingestion rate
Ingestion rate was reduced following exposure to low salinity (Day 1:
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
between temperature and pCO2 was observed on Day 1 as analysed
by three-way ANOVA (d.f. ¼ 2, F ¼ 4.20, p , 0.05). However,
when the effect of temperature was compared at each pCO2 level
and the effect of pCO2 at each temperature, no significant differences
were found. Ingestion rates at 15 and 308C were not significantly different 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).
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
Figure 1. Cumulative mortality of N. festivus upon exposure to
different combinations of temperature, salinity, and pCO2 level for 31 d.
Figure 2. Combined effect of temperature, salinity, and pCO2 on the
ingestion rate (I ) of N. festivus on Days 1 and 29.
Absorption rate (Ab) was reduced substantially (Figure 4) under low
salinity on Day 2 (d.f. ¼ 1, F ¼ 163.37, p , 0.001) and the effect was
Figure 3. Combined effect of temperature, salinity, and pCO2 on the
absorption efficiency (A) of N. festivus on Days 2 and 30.
Physiological responses and scope for growth in a marine scavenging gastropod
819
Figure 4. Combined effect of temperature, salinity, and pCO2 on the
absorption rate (Ab) of N. festivus on Days 2 and 30.
Figure 5. Combined effect of temperature, salinity, and pCO2 on the
respiration rate (R) of N. 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,
p , 0.001). Reduction in Ab was also observed at the lower temperature on both Day 2 (d.f. ¼ 1, F ¼ 6.65, p , 0.05) and Day 30 (d.f. ¼
1, F ¼ 10.71, p , 0.005). The effect of pCO2, however, was statistically
indistinguishable (d.f. ¼ 2, F ¼ 0.26, p ¼ 0.771). No interaction
between the three factors was observed throughout the experiment
(Day 2: temperature × pCO2: d.f. ¼ 2, F ¼ 3.08, p ¼ 0.055;
temperature × salinity: d.f. ¼ 1, F ¼ 3.87, p ¼ 0.055; pCO2 × salinity: d.f. ¼ 2, F ¼ 0.10, p ¼ 0.904; temperature × salinity × pCO2:
d.f. ¼ 2, F ¼ 1.62, p ¼ 0.208; Day 30: temperature × pCO2: d.f. ¼ 2,
F ¼ 0.29, p ¼ 0.750; temperature × salinity: d.f. ¼ 1, F ¼ 1.50, p ¼
0.227; pCO2 × salinity: d.f. ¼ 2, F ¼ 0.31, p ¼ 0.738; temperature ×
salinity × pCO2: d.f. ¼ 2, F ¼ 0.70, p ¼ 0.501).
Energy expended on ammonia excretion
Energy expended on respiration
Energy expended on respiration (R) did not change under elevated
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)
or salinities (d.f. ¼ 1, F ¼ 86.88, p , 0.001) and the effects persisted
until the end of the experiment (Figure 5). No interaction between
the three factors was observed throughout the experiment (Day 2:
temperature × pCO2: d.f. ¼ 2, F ¼ 0.55, p ¼ 0.579; temperature ×
salinity: d.f. ¼ 1, F ¼ 1.34, p ¼ 0.253; pCO2 × salinity: d.f. ¼ 2,
F ¼ 0.21, p ¼ 0.808; temperature × salinity × pCO2: d.f. ¼ 2, F ¼
1.62, p ¼ 0.208; Day 30: temperature × pCO2: d.f. ¼ 2, F ¼ 0.16,
p ¼ 0.85; temperature × salinity: d.f. ¼ 1, F ¼ 1.24, p ¼ 0.270;
pCO2 × salinity: d.f. ¼ 2, F ¼ 1.68, p ¼ 0.198; temperature ×
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 (Day 3:
p ¼ 0.087; Day 31, p ¼ 0.410), but was significantly higher than
that at 158C on both days (Day 3: p , 0.001; Day 31, p , 0.005).
Ammonia excretion (U ) was reduced significantly at low salinity or
temperature on both Days 3 and 31 (Figure 6). On Day 3, the interaction between temperature and pCO2 was significant (d.f. ¼ 2, F ¼
3.36, p , 0.05). No statistical difference in U was found between
three pCO2 levels at both 15 and 308C, but the rate was significantly
higher at 308C than at 158C for all the pCO2 levels (Table 2).
Significant interaction between the three factors was also found
on Day 31 (d.f. ¼ 2, F ¼ 3.95, p , 0.05; Table 3). The effect of temperature was significant at all combinations of pCO2 and salinity.
The effect of salinity, however, was only significant at 380 and
1250 matm pCO2 at 158C, and 950 and 1250 matm pCO2 at 308C.
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
0.163), whereas U at 308C was significantly higher on Day 3 (p ,
0.050) but not on Day 31 (p ¼ 0.804).
Scope for growth
SfG was reduced significantly at low salinity on Day 3 (d.f. ¼ 1, F ¼
121.94, p , 0.001) and Day 31 (d.f. ¼ 1, F ¼ 16.52, p , 0.001;
Figure 7). The interactive effect between temperature and pCO2
(d.f. ¼ 2, F ¼ 3.33, p , 0.05) and that between temperature and
salinity (d.f. ¼ 1, F ¼ 4.49, p ¼ 0.05) were significant on Day
3. Multiple comparison tests showed that SfG reduced at low salinity
for both temperatures (Table 4). SfG at 248C was not significantly
different from that at 15 and 308C on Day 3 (d.f. ¼ 1, F ¼ 0.70,
p ¼ 0.520) and Day 31 (d.f. ¼ 1, F ¼ 2.12, p ¼ 0.171).
Discussion
The physiological responses of N. festivus were positively correlated
with temperature and salinity. In contrast, the effect of pCO2 was
820
H. Zhang et al.
Figure 6. Combined effect of temperature, salinity, and pCO2 on the
excretion rate (U) of N. festivus on Days 3 and 31.
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 acidification 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
exist in a fluctuating environment (Maderira et al., 2014). This phenomenon 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.
158C
308C
380 matm
950 matm
1250 matm
d.f.
2
2
1
1
1
MS
9.233E20.006
3.293E20.005
0.000
0.000
0.000
F
0.488
1.515
28.875
5.608
5.678
p
0.619
0.238
0.000
0.029
0.028
Tukey test
380 matm
380 matma
158Ca
158Ca
158Ca
950 matm
950 matm
308Cb
308Cb
308Cb
1250 matm
1250 matm
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.
10 psu-158C
10 psu-308C
30 psu-158C
30 psu-308C
158C-380 matm
158C-950 matm
158C-1250 matm
308C-380 matm
308C-950 matm
308C-1250 matm
10 psu-380 matm
10 psu-950 matm
10 psu-1250 matm
30 psu-380 matm
30 psu-950 matm
30 psu-1250 matm
d.f.
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
MS
1.047E20.005
1.287E20.005
3.467E20.006
2.427E20.005
7.290E20.005
1.690E20.005
4.000E20.005
1.000E20.007
6.250E20.005
7.840E20.005
0.000
0.000
0.000
0.000
0.000
0.000
F
1.880
3.899
0.819
1.742
9.785
2.965
25.806
0.020
7.812
6.149
73.923
19.761
78.400
15.728
26.955
20.747
p
0.195
0.050
0.464
0.217
0.014
0.123
0.001
0.892
0.023
0.038
0.000
0.002
0.000
0.004
0.001
0.002
Tukey test
380 matm
380 matma
380 matm
380 matm
10 psua
10 psu
10 psua
10 psu
10 psua
10 psua
158Ca
158Ca
158Ca
158Ca
158Ca
158Ca
950 matm
950 matmb
950 matm
950 matm
30 psub
30 psu
30 psub
30 psu
30 psub
30 psub
308Cb
308Cb
308Cb
308Cb
308Cb
308Cb
1250 matm
1250 matmab
1250 matm
1250 matm
Values in bold indicate the differences were statistically significant. Values in the same row with different letter designations indicate that they are statistically
different.
821
Physiological responses and scope for growth in a marine scavenging gastropod
Paracentrotus lividus living in contrasting environments, with intertidal populations being more tolerant of a decrease in pH than
subtidal populations (Moulin et al., 2011). Tolerance of an organism
to elevated pCO2 may be due to its ability to compensate for
CO2-induced changes in extracellular pH (Wittmann and Pörtner,
2013). Our previous study on the subtidal gastropod N. conoidalis,
a congeneric counterpart of the intertidal N. festivus, demonstrated
metabolic depression when exposed to ocean acidification (Zhang
et al., 2015). This is a common phenomenon of uncompensated
changes in extracellular pH and intracellular pH, but was not
observed for N. festivus (Zhang et al., 2015).
Extracellular acid –base regulation during short-term hypercapnia has been shown in the Dungeness crab, Cancer magister, which
inhabits fluctuating shallow waters, but was absent in the relatively
stable habitat of the deep-sea Tanner crab Chionoecetes tanneri
(Pane and Barry, 2007). Compensation for hypercapnic acidosis
Figure 7. Combined effect of temperature, salinity, and pCO2 on the
SfG of N. festivus on Days 3 and 31.
in body fluids and calcification compartments during exposure to
ocean acidification involves pH and ion regulation across the epithelia of gills, gut, and kidneys and is driven by energy-consuming ion
pumps (Wittmann and Pörtner, 2013). The associated energetic
costs may shift the energy budget of the organism (Pörtner, 2008).
Although ocean acidification did not affect physiological responses
and SfG in N. festivus, an increase in the cost of acid–base regulation,
if any, may alter the energy allocation strategy (e.g. reduction in reproductive output), and this deserves further investigations.
Aweak interactive effect between temperature and pCO2 on physiological responses (ingestion, excretion and SfG) in N. festivus was
recorded in the early phase of the present experiment (Days 1–3).
Enhancement of temperature effects under low pH has been
shown in various marine species. For example, production of the
heat shock protein HSP70 in the crab Pachygrapsus marmoratus
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 (+2 to 48C)
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 (Pörtner
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 (Pörtner 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 ionosmoregulatory 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
reduced growth, elevated mortality, and impaired shell maintenance 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
F
p
(a) Temperature effect at each pCO2 level and pCO2 effect at each temperature
158C
2
0.104
0.378
0.689
308C
2
0.380
0.633
0.539
380 matm
1
0.605
1.463
0.242
950 matm
1
0.025
0.053
0.821
1250 matm
1
0.256
0.601
0.448
(b) Temperature effect at each salinity and salinity effect at each temperature
158C
1
5.270
62.804
0.000
308C
1
11.463
58.079
0.000
10 psu
1
0.248
1.986
0.170
30 psu
1
0.350
2.241
0.146
Tukey test
380 matm
380 matm
158C
158C
158C
950 matm
950 matm
308C
308C
308C
10 psua
10 psua
158C
158C
30 psub
30 psub
308C
308C
Values in the same row with different letter designations indicate that they are statistically significant (p , 0.05).
1250 matm
1250 matm
822
owing to strongly elevated basal energy demand. Low salinity also
modulated responses to elevated pCO2 through negatively affecting
the mechanical properties of the shell (Dickinson et al., 2013).
Low salinity had a major effect on the survival and physiological
energetics of N. festivus in the present study. Unlike M. mercenaria,
both energy intake and expenditure in N. festivus was reduced at low
salinity, possibly owing to the experimental salinity approaching the
lethal limit as the lowest salinity for N. festivus to survive indefinitely
has been estimated at 11.5 psu (Morton, 1990). This may also help
explain the absence of interactive effects between salinity and pCO2,
as the effect of low salinity possibly overshadowed that of pCO2.
pCO2 levels enhanced the effects of temperature/salinity on
physiological responses only in the first few days of the present experiment, but the pCO2 effect was absent after 1 month. This may
indicate rapid adjustment of physiological responses to pCO2, possibly through regulation of extra- and/or intracellular pH. In the
cold-water coral L. pertusa, short-term (1 week) high CO2 exposure
under pH 7.77 resulted in a decline of calcification by 26 –29% and a
net dissolution of calcium carbonate. Acclimation to acidified conditions, however, has been observed following long-term (6
months) experiments, leading to even slightly enhanced rates of calcification (Form and Riebesell, 2012). Although a short-term exposure (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
body conditions and result in negative effects on growth and reproduction. For example, reduction in female fecundity has been
observed in the sea urchin Strongylocentrotus droebachiensis following 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 exposure, but the rate was significantly reduced when the exposure
period was extended to 314 d. An experiment for an extended
period of several months could clarify whether the neutral effects
of ocean acidification on the physiological responses of N. festivus
observed in the present study were results of complete acclimation.
Physiological energetics reveals the energy allocation strategy of
individuals. However, many studies have shown that effects of ocean
acidification can be cumulative and carryover to successive life
stages as well as across generations (transgenerational effect).
Significant transgenerational responses are expected when environmental changes such as ocean acidification persist (Sunday et al.,
2011). For example, adult sea urchins pre-exposed for 4 months
to 1200 matm pCO2 had a direct negative impact on subsequent
larval settlement success, with five to nine times fewer offspring
reaching the juvenile stage (Dupont et al., 2013). After the calanoid
copepod Pseudocalanus acuspes was grown for two generations
under 1550 matm pCO2, there was an apparent alleviation of
effects on fecundity and metabolic stress as a result of transgenerational factors (Thor and Dupont, 2015).
Carryover effects of ocean acidification have also been reported
in the Olympia oyster (Ostrea lurida), where juveniles reared as
larvae under reduced pH exhibited a 41% decrease in shell growth
rate. This effect was persistent regardless of the pH level the
oysters experienced as juveniles, indicating a strong carryover
effect from the larval phase (Hettinger et al. 2012). In addition,
the carryover effect of larvae may reduce adult fitness, including
brain development (Trokovic et al., 2011) and reproduction
(Araki and Blouin, 2009). Our previous study on early life stages
of N. festivus has demonstrated that larvae hatched under an elevated
high pCO2 level (1250 matm) were smaller and the juveniles grew
H. Zhang et al.
slower (unpublished data). Smaller newly hatched larvae may experience a slower growth and take a longer time to metamorphose
into juveniles which, themselves, may be smaller. This highlights
the importance of tests for transgenerational and carryover effects
in future research programmes.
Under the combined effect of temperature, salinity, and ocean
acidification, mortality and the maintenance cost of the larvae of
N. festivus increased (Zhang et al., 2014). Larvae hatched under an
elevated pCO2 were smaller and the juveniles grew slower (unpublished data). Younger life stages, therefore, are more sensitive to
these stresses than adults. An extensive review of studies on sea
urchins has shown that larvae and juveniles are much more sensitive
to ocean acidification than adults and gametes (Dupont and
Thorndyke, 2013). Similar observations have been reported for
marine molluscs (Parker et al., 2013), possibly due to shell structure,
which is composed of more soluble forms of calcium carbonate, i.e.
amorphous calcium carbonate and aragonite (Wicks and Roberts,
2012) and the lack of the ability to maintain acid–base status
(Melzner et al., 2009b). Although neutral effects have been observed
for the energy budget of N. festivus adults upon exposure to multiple
stressors, population performance may be impacted through observed
effects on younger life stages.
Acknowledgements
We thank two anonymous reviewers for their constructive comments on the manuscript and Bruce Richardson for improving
the English. Our work was fully supported by a strategic research
grant (grant no. 7004027) of the City University of Hong Kong.
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