Mixed Effects of Elevated pCO2 on Fertilisation, Larval and Juvenile Development and Adult Responses in the Mobile Subtidal Scallop Mimachlamys asperrima (Lamarck, 1819)

Elliot Scanes; Laura M. Parker; Wayne A. O’Connor; Pauline M. Ross

doi:10.1371/journal.pone.0093649

Abstract

Ocean acidification is predicted to have severe consequences for calcifying marine organisms especially molluscs. Recent studies, however, have found that molluscs in marine environments with naturally elevated or fluctuating CO₂ or with an active, high metabolic rate lifestyle may have a capacity to acclimate and be resilient to exposures of elevated environmental pCO₂. The aim of this study was to determine the effects of near future concentrations of elevated pCO₂ on the larval and adult stages of the mobile doughboy scallop, Mimachlamys asperrima from a subtidal and stable physio-chemical environment. It was found that fertilisation and the shell length of early larval stages of M. asperrima decreased as pCO₂ increased, however, there were less pronounced effects of elevated pCO₂ on the shell length of later larval stages, with high pCO₂ enhancing growth in some instances. Byssal attachment and condition index of adult M. asperrima decreased with elevated pCO₂, while in contrast there was no effect on standard metabolic rate or pH_e. The responses of larval and adult M. asperrima to elevated pCO₂ measured in this study were more moderate than responses previously reported for intertidal oysters and mussels. Even this more moderate set of responses are still likely to reduce the abundance of M. asperrima and potentially other scallop species in the world’s oceans at predicted future pCO₂ levels.

Citation: Scanes E, Parker LM, O’Connor WA, Ross PM (2014) Mixed Effects of Elevated pCO₂ on Fertilisation, Larval and Juvenile Development and Adult Responses in the Mobile Subtidal Scallop Mimachlamys asperrima (Lamarck, 1819). PLoS ONE 9(4): e93649. https://doi.org/10.1371/journal.pone.0093649

Editor: Vengatesen Thiyagarajan (Rajan), University of Hong Kong, Hong Kong

Received: September 30, 2013; Accepted: March 8, 2014; Published: April 14, 2014

Copyright: © 2014 Scanes et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was funded via a grant from the Australian National Climate Change Adaption Research Facility (www.nccarf.edu.au) Marine Adaption Network for Marine Biodiversity and Resources. This work would not have been possible without their generous support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: Pauline Ross is a PLOS ONE Editorial Board member. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Introduction

Ocean acidification has been called the “other CO₂ problem”. Anthropogenic emissions of CO₂ released into the atmosphere are being absorbed by the world’s oceans causing them to acidify [1], [2]. Compared with pre-industrial levels, the mean pH of the surface oceans has declined by more than 0.1 units [2]–[4] and assuming median emission scenarios, the Intergovernmental Panel on Climate Change [5], [6] has predicted pH will fall a further 0.3–0.5 units (pH 7.8–7.6) by 2100 and another 0.7–0.77 units (pH 7.4–7.43) by 2300 [1], [2], [4].

All evidence suggests that the decline in pH of the oceans will impact on marine ecosystems and organisms, and one of the most vulnerable phylum will be the calcifying molluscs [7]–[13]. Already along the northwest coast of the USA, oyster hatcheries have experienced mass mortalities from upwelling of deep acidified seawater onto the coasts [14]. Studies on adult molluscs including the oysters Crassostrea gigas, Crassostrea virginica, Saccostrea glomerata, [13], [15], [16] and mussels Mytilus edulis [17], [19], [20] have found decreased calcification and growth in response to elevated pCO₂ [19], [20], perhaps because of the lowered saturation state of carbonate (CO₃²⁻) and alterations to the organism’s acid-base status, which they attempt to compensate for by increasing their standard metabolic rate [21]–[23]. Studies on the responses of early life-history stages of molluscs to elevated pCO₂ have found them to be even more sensitive than adult molluscs [9], [24]. Reduced fertilisation success, decreased larval size and development and increased larval abnormality and mortality have been found in the oysters C. gigas, C. virginica, S. glomerata, [11]–[13], [25]–[27] and mussels, Mytilus galloprovincialis, Mytilus edulis [18], [28]. Molluscs are not the only phylum affected by elevated pCO₂, with negative responses also recorded for echinoderms, including the sea urchins Hemicentrotus pulcherrimus, Enchinometra mathaei [29], [30], Paracetrotus lividus [31], Tripneustes gratilla, Pseudechinus huttoni, Evechinus chloroticus, Sterechinus neumayeri [32], Heliocidaris erythrogramma [33]–[35] and the brittle star Ophiothrix fragilis [7].

Against this backdrop, recent studies have found that both echinoderms and molluscs have a capacity to acclimate to exposure of elevated pCO₂ and fluctuations in pH [13], [36], [37]. Miller et al. [36] found that the larvae of the oyster Crassostrea ariakensis were not as adversely affected by the chronic exposure to high pCO₂ as some other mollusc species. They hypothesised [36] that the increased resilience of the C. ariakensis to elevated pCO₂ may be due to the exposure of this species to high levels of respiratory CO₂ in the benthos [38]. Thomsen et al. [37] also found communities dominated by calcifying organisms, mostly mussels, thrived in naturally CO₂ enriched waters of the Western Baltic Sea. When placed under artificially elevated pCO₂ in the laboratory, this long term exposure to naturally elevated pCO₂ increased the ability of Mytilus spp. to calcify. Similarly, Moulin et al. [31] found that echinoderms, Paracentrotus lividus, exposed to naturally fluctuating pH coped better with elevated pCO₂ than those from a stable pH environment. It was found that fertilisation and larval development of P. lividus increased under elevated pCO₂ when the parents were sourced from rock pools characterised by fluctuations in pH compared to adults that were sourced from rock pools where pH was more stable. Also, Parker et al. [13] found exposing adult estuarine oysters (S. glomerata) to elevated pCO₂ during reproductive conditioning increased the resilience of their larvae. Larvae spawned from adults exposed to elevated pCO₂ were larger and developed faster, but displayed similar survival compared with larvae spawned from adults exposed to ambient pCO₂. In contrast, the subtidal echinoderms, Strongylocentrotus droebachiensis were less resilient when exposed to elevated pCO₂ in the laboratory and less able to acclimatise over successive generations [39].

If long term fluctuating or stable exposure to elevated pCO₂ increases the capacity of an organism to acclimate [31], [32], [37] then molluscs living in more stable pH environments, as found in some subtidal habitats, may be more susceptible to future elevated pCO₂. Subtidal environments experience more stable physio-chemical properties than shallow subtidal or intertidal habitats where physio-chemical properties vary on a daily and seasonal basis [40].

Previous exposure to elevated pCO₂ alone is unlikely to reliably predict the response of an organism to elevated pCO₂. Mode of life and the capacity for ion regulation and maintenance of extracellular pH has been linked with responses of molluscs to elevated pCO₂ [41]–[43]. The more tolerant taxa are characterised by high metabolic rates and high levels of mobility and activity [43]. For example, cephalopods, which experience high levels of haemolymph CO₂ during bouts of physical exertion are able to increase calcification during exposure to elevated pCO₂ [42], [44]. In contrast, bivalve and gastropod molluscs with a hypo-metabolic [13], [16], [43] mode of life and a decreased capacity to regulate ions and acid-base balance, typically show greater sensitivity than cephalopods to elevated pCO₂. Some bivalves, such as the scallops (Pectinidae), are capable of swimming and during these swimming periods have elevated metabolism and oxygen consumption, often with 2 to 5 fold fluctuations in O₂/CO₂ gas exchange [45]. These short bouts of exercise, which result in an elevation of internal CO₂ and subsequent acidification of the haemolymph, may provide scallops with a slightly increased capacity to cope with future external elevations in environmental pCO₂ [43]. There is a strong link between the taxa that are tolerant to elevated pCO₂ and/or organisms which experience metabolic fluctuations due to physical activity and higher metabolic rates [43]. Scallops are not as physiologically advanced as teleost fish or cephalopods [41]–[44], however, compared to other more sessile bivalves, their capacity for locomotion and metabolic fluctuations is pronounced [13], [16], [43], [44]. It may be that physiological fluctuations of internal pCO₂ induced by physical activity, have enhanced the internal buffering systems of scallops, and in turn, this may enable them to better withstand external reductions of environmental pH.

To date, studies on the response of scallops to elevated CO₂ have found reductions in calcification and larval growth in the scallop Argopecten irradians after 7 [46]and 36 days [47] of exposure. Also, larval development decreased, and abnormality increased when the scallop Pecten maximus was exposed to elevated pCO₂ for 7 days [40]. In one of the first investigations on the physiological responses of scallops to elevated pCO₂, Schalkhausser et al. [47] found that although there was no change in the standard metabolic rate of P. maximus when exposed to elevated pCO₂, its ability to clap the upper and lower valves was reduced [48].

The aim of this study was to measure the responses of the scallop M. asperrima to elevated environmental pCO₂. It was hypothesised that a subtidal species of scallop experiencing a stable physio-chemical environment and capable of swimming may have greater capacity to cope with elevated pCO₂ compared to sessile molluscs such as oysters and mussels. Such increased capacity may be found in scallops that experience bouts of swimming which produce elevations of CO₂ in the haemolymph and alterations in acid-base status. To test this hypothesis, the effects on both the larval and adult stages of the doughboy scallop Mimachlamys asperrima, were measured in response to elevated pCO₂.

Materials and Methods

Broodstock Collection

M. asperrima is a commercially exploited subtidal bivalve endemic to south eastern Australia. Individuals grow to a shell length of 100 mm and remain a single sex throughout life [49]. M. asperrima is usually found in depths of 20–100 m. M. asperrima will attach to hard substrate using byssal threads, however, these threads can be easily dropped and the individual will swim away if threatened [49].

M. asperrima broodstock were collected by SCUBA from a depth of 20 m, approximately 500 m west of Honeymoon Bay (35.055434°S, 150.763794°E), on the eastern shore of Jervis Bay, NSW, Australia. As the collection site fell within Jervis Bay Marine Park, the relevant collection permit was acquired from NSW Department of Primary Industries. Following collection, scallops were placed in moist towels and hessian, packed in plastic tubs and transported to Port Stephens Fisheries Institute (PSFI). All seawater used in experimentation was collected from Little Beach (33.2°S, 152.11667°E), Nelson Bay, NSW, Australia and was filtered through 1 µm nominal filters (filtered sea water, FSW) prior to being used.

Monitoring of CO₂

In all early life experiments three elevated concentrations of pCO₂ (600 µatm, 750 µatm, and 1000 µatm) (Table 1) and one ambient concentration (current atmospheric concentration of CO₂: 390 µatm) were used being based on projections of the Intergovernmental Panel on Climate Change (IPCC) [5], [6] and the same as Parker et al. [12], [26]. These pCO₂ levels correlated to a mean ambient pH_NBS of (mean ± SE = 8.20±0.02) and a mean elevated pH_NBS levels of (mean ± SE = 7.89±0.02, 7.81±0.02, 7.69±0.03), respectively. The pH of the seawater was manipulated to the chosen pCO₂ concentration via direct bubbling of food grade CO₂ in FSW while being mixed thoroughly. To determine the pH level corresponding to the pCO₂ levels, total alkalinity (TA) was quantified at each water change (every two days) for all experiments using triplicate Gran-titration (Table 1, n = 3) [50], [51]. Following the titration, the TA and chosen pCO₂ levels were entered into a CO₂ system calculation program [52], using the dissociation constants of Mehrbach et al. [53], (Table 1) and the pH level corresponding with the desired pCO₂ level was calculated. When the desired pH was reached the container (tightly capped jar or bucket depending on the experiment) was capped shut and there was very little change in pH over a 12 h period [12], [13], [26]. The pH was monitored using a pH electrode (WTW 3400i) calibrated daily using NBS buffers [54]. The pH of each replicate was checked three times daily and adjusted with additional direct bubbling of food grade CO₂ where necessary.

Download:

Table 1. Mean recorded temperature, pH_NBS, salinity and target pCO₂ for each ontogenetic stage.

https://doi.org/10.1371/journal.pone.0093649.t001

Fertilisation and Early Development

To determine the effect of acute exposure to elevated pCO₂ on fertilisation after 2 h and early development after 24 h, spawning of adult M. asperrima was induced by injection of 0.05 ml 1×10⁻² M serotonin solution into ten females and five male scallops through multiple injections into the gonad and one injection into the adductor muscle [55]–[57]. Serotonin solution was made by dissolving creatine sulphate complex (C₁₄H₂₁N₅O₆S H₂O; Merck, Damnstdat, Germany) in filtered sea water [55]–[57]. Following injections, each scallop was isolated in a 400 mL container of FSW and observed.

Eggs from five females were collected from the containers, mixed in equal proportions and stocked in 5 L glass jars at a density of 16 eggs mL⁻¹. For each pCO₂ level (390 µatm, pH 8.2; 600 µatm, pH 7.89; 750 µatm, pH 7.81; and 1000 µatm, pH 7.69), 20 L containers of FSW were adjusted to the appropriate pH corresponding to the three levels of elevated pCO₂ by direct bubbling of CO₂. Once equilibrated this water was distributed among replicate 5 L jars, which were then tightly capped shut. There were 4 replicate jars per pCO₂ level. Sperm was collected and pooled in equal volumes from five males. To determine the correct sperm quantity, a 1 mL subsample of egg solution was taken. The sperm solution was then added incrementally to these eggs in known volumes and observed microscopically (Leica 100x). When enough sperm was added for there to be one active sperm visible per egg the volume of sperm was recorded. The volume of sperm to provide the equivalent concentration as the subsample was then added to all replicate jars. Two hours after sperm addition each replicate jar was thoroughly mixed and a 10 mL sample was taken. To each sample, 1 mL of 10% formalin was added to prevent further embryo development. Each sample was then passed through a 20 µm mesh screen and the contents were resuspended to a 1 mL sample. The percentage fertilisation for each sample was then determined under a compound microscope (Leica 100x) on a Sedgewick-Rafter counting slide. The first 30 eggs encountered were examined for signs of fertilisation. Fertilisation was defined as when a cleavage plane through the egg could be distinguished [12]. Data were expressed as a percentage fertilised per replicate. Twenty four hours after sperm addition, another 10 mL sample was taken from each replicate jar and filtered using the method above. The first 30 embryos encountered within each sample were examined and were deemed to be either fully developed trochophores or “other”. Trochophores were defined as ciliated and motile [58], [59] and “other” was primarily composed of embryos which had arrested development and unfertilised eggs. This was then expressed as percentage trochophore at 24 h (Table 2).

Download:

Table 2. Summary of experimental design for each ontogenetic stage.

https://doi.org/10.1371/journal.pone.0093649.t002

Larval Development

To determine the effect of elevated pCO₂ on larval development, adult M. asperrima were induced to spawn using the technique of temperature shock [57], [60], [61]. This technique was used because it yields a larger number of gametes, and serotonin injection does not provide the numbers of eggs and sperm required for this experiment. The temperature shock technique involved placing ten males and ten female scallops haphazardly selected from the broodstock population in a bath of FSW at 24°C (4°C warmer than the ambient temperature of 20°C). Following spawning, gametes were collected separately to avoid contamination and returned to individual containers of FSW at ambient temperature (20°C). Collected eggs from at least 20 females (15 million) were added to a 20 L bucket. Sperm was collected from at least 10 males and added to eggs at a concentration of 825 mL⁻¹ (1∶1.1 egg to sperm ratio, sperm concentration determined using a haemocytometer under a light microscope; Leica 100x) and mixed thoroughly, once sufficient fertilisation was observed microscopically (90% of eggs showing cleavage, about 1 h post sperm addition Leica 100x). Gametes were stocked in a 1000 L polyethylene tank filled with FSW (390 µatm; pH 8.20; 20°C; salinity 34.5 psu). They were then reared as a single batch using the methods of O’Connor et al. [55] and transferred to experimental conditions at 3 stages; D-veliger (4d), umbonate veliger (10 d), and pediveliger (16 d). Larvae were fed twice daily on a known algal diet consisting of equal parts Tahitian Isochrysis aff. galbana, Pavlova lutheri and Chaetoceros calcitrans. Algal concentrations ranged according to larval size, an equivalent of 3500 T. Isochrysis cells larva⁻¹ day⁻¹ was fed at the D-veliger stage. This incrementally increased to 20,000 T. Isochrysis cells larva⁻¹ day⁻¹ at the pediveliger stage [62], [63].

Once larvae reached the beginning of each ontogenetic stage (D-veliger, 4 d; umbonate veliger, 10 d; and pediveliger, 16 d), they were sieved through a 45, 90 and 130 µm mesh respectively. Larvae retained were then counted and added to 10 L replicate buckets in each experiment. The larval density at the beginning of each experiment was 5 mL⁻¹ for D-veligers, and 2.5 mL⁻¹ for umbonate and pediveliger stages. Larvae were placed in replicate 10 L buckets filled with 8 L of FSW and were maintained at a constant 20°C. Buckets were capped shut at all times. Each bucket had a small hole in the lid through which air was very gently pumped in by air hose to promote some water movement. There were four replicate buckets for D-veliger experiments and three replicate buckets for the umbonate, and pediveliger experiments at each pCO₂ level (390 µatm, pH 8.2; 600 µatm, pH 7.89; 750 µatm, pH 7.81; and 1000 µatm, pH 7.69;Table 1, 2). The pH of each container was measured 3 times daily, and if necessary, the pH was adjusted with additional CO₂. Every two days the seawater in each replicate bucket was changed. New replicate buckets were cleaned using Virkon S solution (Antec Corp, North Bend, WA, USA) rinsed with new FSW, and allowed to air dry. They were then filled with FSW of the same temperature and adjusted to the appropriate pH level. Each experimental bucket was sieved through 45 µm mesh screen and larvae were captured. The larvae were then transferred to their new replicate bucket set at the correct pCO₂ level and capped shut. For each larval stage, after four days of exposure the contents of each replicate bucket were stirred until homogenised and 500 mL of water was collected. Fifty mL of 10% buffered formalin was added to each collected sample. The first 30 larvae encountered were measured for shell length along their antero-posterior axis. This was done on a Sedgewick-Rafter slide under a compound microscope (Leica 100x) using an ocular micrometer. This collection and measurement was then repeated at six days of exposure.

Settlement and Exposure of Spat

To collect spat, settlement surfaces (balls of plastic netting) were suspended in the 1000 L main culture tank (originally stocked with larvae) 20 days after fertilisation. Spat were collected by rinsing these settlement surfaces with seawater over a mesh screen [55]. All spat removed from the screen were counted by eye and separated into 12 groups of 50. Spat were then added to a small purpose built “spat screen”. There were 50 spat per spat screen. Each spat screen was circular and 100 mm in diameter with raised plastic sides and a 130 µm mesh base, that prevented the spat from passing through. During experimental conditions, spat were fed twice daily on a known algal diet consisting of equal parts Tahitian Isochrysis aff. galbana, Pavlova lutheri and Chaetoceros calcitrans, at a concentration equivalent to 20,000 T. Isochrysis cells larva⁻¹ day⁻¹. To determine the effect of elevated pCO₂ on the growth of spat, spat were exposed to the experimental levels of pCO₂ (µatm, pH 8.2; 600 µatm, pH 7.89; 750 µatm, pH 7.81; and 1000 µatm, pH 7.69 Table 1, 2) for 6 days. The bucket set up and pH manipulation remained the same as per larval experiments. There were three 10 L replicate buckets at each pCO₂ level, one “spat screen” each containing 50 spat was added to each replicate bucket. Every two days the culture seawater was changed, at the time of each water change, each spat screen was individually removed from the experimental bucket and rinsed gently with FSW set at the experimental pCO₂ level to remove any bacteria or algae that had settled on the screen. The “spat screen” was then gently placed into the new replicate bucket of corresponding pCO₂ adjusted FSW (Table 2). The pH of each replicate bucket was monitored 3 times per day and adjusted where necessary. After 6 days exposure, the shell length (antero-posterior measurement) of 30 randomly selected spat were measured from each replicate bucket using a Sedgewick-Rafter slide under a compound microscope (Leica 100x) with an ocular micrometer.

Physiological Measurements on Adults

To determine the effect of elevated pCO₂ on adult physiology, 60 male and 60 female broodstock individuals were randomly selected. From this pool, 30 males and 30 females were randomly selected and assigned to the elevated pCO₂ and ambient pCO₂ treatment. The ambient level was the current atmospheric pCO₂ level of 390 µatm, mean pH_NBS (±SE) 8.20 (±0.02). The elevated level used was that predicted for the year 2100, 750 µatm, mean pH_NBS (±SE) 7.81 (±0.03) [5], [6]. The correct pH level for the elevated pCO₂ treatment was recalculated every water change (every 2 days) following triplicate gran titration. Following the titration, the total alkalinity (TA) and selected pCO₂ level were entered into a CO₂ system calculation program [52], using the dissociation constants of Mehrbach et al. [53] to determine the correspong pH value for the desired pCO₂ level. There were 3 replicate 200 L tanks for each the two pCO₂ levels, each tank was stocked with 20 scallops (10 male, 10 female). Scallops were housed mid water in large mesh bags for 5 weeks and water temperature was maintained at 20°C, each tank was filled with FSW. To ensure that there were no a-priori differences in sizes of scallops among groups, shell lengths (antero-posterior measurement) and whole weight of 15 randomly selected individuals from each of the ambient and elevated treatments were measured and analysed using ANOVA (Shell length: F_{(1, 28)} = 0.68 P = >0.05, Whole weight: F_{(1, 28)} = 0.29 P = >0.05).

The elevated level of pCO₂ was maintained using pH negative feedback systems (Aqua Medic, Aqacenta Pty Ltd, Kingsgrove, NSW, Australia; accuracy ± 0.01 pH units). Food grade CO₂ was bubbled directly into the tanks containing adult individuals via a CO₂ reactor to ensure proper mixing and in turn, reduce pH. A pH probe connected to a controlling computer was placed within each tank (probes were calibrated weekly using NBS buffers). When the desired pH level was reached, the delivery of CO₂ was automatically stopped by a computer signal to a solenoid valve. Each tank set to elevated pCO₂ was controlled by its own independent pH controlling system. The pH values of each tank were monitored daily, and the pH electrode of each controlling system was checked against another calibrated pH probe (NBS buffers, WTW 3400i) daily. Every second day of the five weeks of exposure, each tank received a complete water change. New FSW was pumped into identical tanks at least 24 h prior to the scheduled change, the CO₂ monitoring systems were also transferred to the new tanks prior to the water change. This allowed the new FSW to be at the same temperature, and CO₂ level as the previous FSW. It also allowed for scallops to be out of the water only briefly during the water change. Following the water change, each old tank was cleaned vigorously using Virkon S solution (Antec Corp, North Bend, WA, USA), and then rinsed with fresh water and allowed to air dry prior to re-filling. At each water change, dead scallops (if any) were removed, recorded and replaced with a scallop of the same sex where possible; replacement scallops were isolated in each tank from the original stocking population to prevent them being mistaken as part of the original stocked scallops. At every water change the number of scallops byssally attached to the mesh bag or another scallop’s shell in each replicate from the original stocked scallops (not replacements) was also recorded.

In weeks 2, 4 and 5 of experimental conditions, a male and female scallop from the original stocked scallops of each tank was selected at random for adult measurements. These measurements included Standard Metabolic Rate (SMR), and Haemolymph pH (pH_e) and O₂ (p_eO₂ hPa) levels. After 5 weeks exposure, each scallop from the original stocking (not replacements) was removed by slicing their byssal threads with a scalpel. The number of threads severed for each scallop was recorded. Adults were fed each day an algal mixture consisting of 50% Chaetoceros muelleri and 50% T. Isochrysis at a concentration equivalent to 2×10⁹ T. Isochrysis cells scallop⁻¹ d⁻¹ [62].

Standard Metabolic Rate (SMR)

SMR was determined using the closed respiratory system method of Parker et al. [13]. At each sampling time (weeks 2, 4 and 5), two scallops (one male, one female) were randomly selected from each tank for measurements (total 12; 6 from 390 µatm, 6 from 750 µatm). Scallops were placed in individual 500 mL airtight chambers filled with FSW at the correct pCO₂ level. Each chamber was fitted with a fibre-optic O₂ probe (PreSens dipping probe DP-PSt3, AS1 Ltd, Palmerston North, New Zealand). When scallops were placed in the chambers there were no air spaces which may interfere with the O₂ readings. The probes were calibrated using a two-point calibration (0% and 100%) and all measurements were done at the experimental temperature of 20°C. The time taken to reduce the percentage oxygen saturation of seawater in the chamber from 100% to 80% was recorded. Time was only recorded when scallops were actively respiring (time that oxygen levels were decreasing). Prior to these SMR measurements, feeding had ceased for 24 h to remove any variability associated with digestive metabolism. Following the measurements, scallops were removed from the chambers and the dry tissue mass (tissue removed and dried in an oven at 80°C for 72 h) was measured. SMR was calculated for each scallop using the following equation (1):

Equation 1. SMR calculation.

Where: SMR is oxygen consumption normalised to 1g of dry tissue mass (mg O₂ g⁻¹ dry tissue mass h⁻¹), V_r is the volume of the respiratory chamber minus the volume of the scallop (L), ΔC_WO₂ is the change in water oxygen concentration measured (mg O₂L⁻¹), Δt is the measuring time (h), bw is the dry tissue mass (g) [13].

Condition Index

The condition index of scallops was determined at weeks 2, 4 and 5, from the individuals used for SMR measurements (total 12; 6 from 390 µatm, 6 from 750 µatm). Condition Index was defined as a ratio of tissue weight to shell weight (as a proxy for shell internal volume). Scallop tissue and shells were dried in an oven for 72 h at 80°C, and weighed separately. Condition index was calculated for each individual as in Equation 2.

Equation 2. Condition Index calculation.

Where: CI = condition index, bw is the dry tissue mass (g), sw is the dry shell mass (g).

Haemolymph pH (pH_e) and O₂ (p_eO₂)

To determine haemolymph pH (pH_e) and O₂ (p_eO₂ hPa) approximately 0.5 mL of haemolymph fluid was extracted from the scallop’s adductor muscle using a 1 mL needled syringe. This was done on the scallops after they had their SMR analysed (total 12; 6 from 390 µatm, 6 from 750 µatm). While each scallop was held open, a 1 mL needled syringe was placed in the adductor muscle and haemolymph slowly extracted. This was done to prevent any air bubbles from passing into the haemolymph sample. The haemolymph was immediately placed into a 2 mL centrifuge vial where it was analysed for O₂ content. To measure O₂ content, a fibre-optic probe (PreSens dipping probe DP-PSt3, AS1 Ltd, Palmerston North, New Zealand) was placed in the haemolymph and O₂ levels recorded (p_eO₂ hPa). Following this measurement, haemolymph pH was measured using a fine pH electrode (Metrohm 826 pH mobile). The pH electrode was calibrated for each use with NBS buffers.

Data Analysis

To determine differences among pCO₂ treatments for shell length of D-veliger and umbonate veliger larvae, as well as physiological measurements of adults (SMR, condition index, and haemolymph pH and O₂), data were analysed using a three factor analysis of variance. “Time” and “pCO₂” were fixed factors and “Tank” (for adult measurements) or “Container” (for larval experiments) was nested in pCO₂. To determine any differences in shell length of pediveligers and spat among pCO₂ treatments data were analysed using a two factor analysis of variance where “pCO₂” was fixed and orthogonal and “Container” was nested in pCO₂. In all analysis with “Tank/Container” as a factor, if the “Tank/Container” factor was not significant (using α >0.25 to guard against increased possibility of Type 1 statistical error [64]), replicates were pooled for this factor and the analysis redone without “Tank/Container” as a factor. To determine any differences in means of percentage fertilisation, development to trochophore, and the number of byssal threads per scallop were analysed using a one factor analysis of variance with pCO₂ as the fixed factor. Spat growth from beginning (t₀) to six days treatment, and adult exposure starting shell length and whole weight were analysed using a one factor analysis of variance where shell length or whole weight was the fixed factor. The program GMAV 5 for Windows was used for all analyses of variance, heterogeneity of variances was assessed prior to analysis using Cochran’s test, but no significant heterogeneities among variances were detected [64]. Differences in the means were detected using a series of Student Newman Kuels (SNK test) on each parameter [65]. Planned comparisons were made to assess differences between extremes of treatment gradients. Linear regression analysis was used to determine the significance of the slope for both pCO₂ treatments when assessing the percentage of individuals byssally attached over time. For the linear regression analysis pCO₂ treatment was the dependent variable and days exposed was the independent variable.

Results

Fertilisation and Early Development

There was a significant effect of elevated pCO₂ on fertilisation success, and percentage development to trochophore larvae (Figure 1; Table 3). As the pCO₂ concentration increased, fertilisation decreased. Post-hoc analysis using SNK tests found the percentage of fertilisation in the ambient treatment (mean 85%) was significantly greater than all other treatments. The percentage fertilisation at 600 µatm (mean 75%) was significantly greater than the 750 and 1000 µatm treatments which were not significantly different from each other (Figure 1). Similarly, as pCO₂ concentration increased the percentage of embryos reaching the trochophore stage significantly decreased, with only about 50% of embryos developing to trochophore at 1000 µatm. Post-hoc analysis using SNK tests found there was no significant difference in the percentage of trochophores between ambient (mean 85%) and 600 µatm, (mean 75%) treatments. There was, however, a significantly lower percentage of embryos to reach the trochophore stage at 750 µatm (mean 65%), which was significantly greater than 1000 µatm treatment (mean 50%) (Figure 1, Table 3).

Download:

Figure 1. The effect of elevated pCO₂ on the fertilisation and early development of M. asperrima gametes.

Mean percentage of fertilisation and development to trochophore of M. asperrima among ambient and elevated pCO₂ levels (390 µatm, 600 µatm, 750 µatm, 1000 µatm) ±MSE, n = 4. Letters above columns represent results of SNK tests. Different letters represent significant differences detected among CO₂ treatments, corresponding letters represent no significant differences among treatments.

https://doi.org/10.1371/journal.pone.0093649.g001

Download:

Table 3. Summary of analysis of variance on M. asperrima early life history exposure to elevated pCO₂.

https://doi.org/10.1371/journal.pone.0093649.t003

Larval Development in Acute Exposure Experiments

In general, as pCO₂ increased, the size of D-veliger larvae decreased (Figure 2; Table 3). After four days, the shell length of D-veligers in ambient and 600 µatm conditions were significantly greater than the 750 and 1000 µatm treatments (Figure 2; Table 3). After six days of exposure the shell length of D-veligers in ambient conditions (mean ± SE = 124±1.7 µm n = 4 and in the 600 µatm (mean ± SE = 125±1.1 µm n = 4) treatment was significantly greater than D-veligers in the 750 µatm (mean ± SE = 121 µm±0.6 n = 4) and 1000 µatm (mean ± SE = 116 µm±1.9 n = 4) treatments, which were not significantly different from each other. There was no overall significant effect of elevated pCO₂ on the shell growth of umbonate veligers, however, post hoc analysis found that after 4 d exposure umbonate veligers in the 750 µatm treatment were significantly smaller than in the ambient and 1000 µatm treatments (Figure 3A; Table 3). Following 6 d exposure there were no significant differences in shell length of umbonates among treatments (Figure 3A; Table 3). There was a significant effect of elevated pCO₂ on the shell length of pediveligers, but in contrast to the trend for decreased shell length with increased pCO₂, following six days exposure, shell length was significantly greater in the 600 µatm treatment (Figure 3B; Table 3). During the pediveliger experiment, after 4 d many of the larvae in the control treatments died and the experiment was halted, explaining the absence of pediveliger data on day 6. The mean shell length of spat was greatest in the control treatment (mean ± SE = 1031 µm±10.4 n = 3), and the 1000 µatm treatment had the smallest mean spat shell length (mean ± SE = 997 µm±12.8 n = 3) this was, however, not a significant difference (Figure 3C; Table 3).

Download:

Figure 2. The effects of elevated pCO₂ on M. asperrima D-veliger shell length.

Mean shell length of M. asperrima D-veliger larvae following 4 and 6 days treatment at ambient and elevated pCO₂ levels (390 µatm, 600 µatm, 750 µatm, 1000 µatm) ±MSE, n = 4. Letters above columns represent results of SNK tests, different letters represent significant differences detected, corresponding letters represent no significant differences detected among those treatments. Roman numerals i – ii represent differences among CO₂ treatments after 4 days, Greek letters α – β represent differences among CO₂ treatments after 6 days and Latin letters a –c represent SNK results of overall mean larval shell length disregarding sampling times.

https://doi.org/10.1371/journal.pone.0093649.g002

Download:

Figure 3. The effects of elevated pCO₂ on M. asperrima Umbonate veliger, pediveliger and spat shell length.

Mean shell length (antero-posterior axis) following exposure to Ambient, and elevated (600, 750, 1000 µatm) levels of pCO₂. A) Umbonate larvae shell length following 4 (White) and 6 (grey) days exposure. B) Pediveliger larvae shell length following 4 days exposure. C) Spat shell length following 6 days exposure. ±MSE, n = 3. Letters above columns represent results of SNK tests, different letters represent significant differences detected, corresponding letters represent no significant differences detected among those treatments. Letters a - c represent differences in shell length among CO₂ treatments for umbonate veligers (A) and pediveligers (B) following 4 d exposure. No significant differences in shell length were detected among treatments for umbonate veligers (A) and spat (C) following 6 d exposure.

https://doi.org/10.1371/journal.pone.0093649.g003

Adults

Overall there was a significant increase in the SMR of M. asperrima between two and four weeks of the experiment (Figure 4, Table 4), but there was no significant difference in SMR in response to elevated pCO₂ levels at any sampling time (Table 4). There was, however, a significant effect of elevated pCO₂ on the condition index. The combined mean of the condition index for all sampling times was greater in the ambient (390 µatm, mean ± SE = 16.2±0.5 n = 3) than the elevated pCO₂ treatment (750 µatm, mean ± SE = 14.1±0.1 n = 3) (Figure 5, Table 4). Condition index was not, however, significantly different between treatments at week 4. There were also no significant differences in pH_e (Figure 6) and p_eO₂ (hPa) at elevated compared to ambient pCO₂ at either sampling time (Table 4), although there were differences over time in p_eO₂ (Table 4).

Download:

Figure 4. The mean of the standard metabolic rate (SMR) of M. asperrima in response to ambient and elevated pCO₂.

Mean SMR after exposure to ambient and elevated pCO₂ (390 µatm, 750 µatm), for 2, 4 and 5 weeks ±MSE, n = 3. Letters above columns represent results of SNK tests, different letters represent significant differences detected among sampling times disregarding CO₂ treatment.

https://doi.org/10.1371/journal.pone.0093649.g004

Download:

Figure 5. The mean condition index of M. asperrima individuals in response to ambient and elevated pCO₂.

Mean condition index after exposure to ambient and elevated pCO₂ (390 µatm, 750 µatm), for 2, 4 and 5 weeks ±MSE, n = 3. Symbols above columns represent results of SNK tests, Different Latin a - b, Greek α - α, and Roman i - ii symbols represent differences within each sampling time between pCO₂ treatments.

https://doi.org/10.1371/journal.pone.0093649.g005

Download:

Figure 6. The mean haemolymph pH (pH_e) of M. asperrima in response to ambient and elevated pCO₂.

Mean haemolymph pH after exposure to ambient and elevated pCO₂ (390 µatm, 750 µatm), after exposure for 2, 4 and 5 weeks ±MSE, n = 3.

https://doi.org/10.1371/journal.pone.0093649.g006

Download:

Table 4. Summary of analysis of variance on M. asperrima adult physiological responses to elevated pCO₂.

https://doi.org/10.1371/journal.pone.0093649.t004

While there was no significant difference in the amount of byssal threads per scallop between ambient and elevated pCO₂ treatments after 5 weeks of exposure (Table 4), there were fewer byssal threads produced by scallops in the elevated (750 µatm, mean ± SE = 2.6±0.4 n = 3) compared to ambient (390 µatm, mean ± SE = 1.5±0.2 n = 3) treatments with some differences among tanks as shown by the “Tank” x “pCO₂” interaction (Table 4). The percentage of scallops byssally attached over 5 weeks of exposure was dependent on the pCO₂ treatment. The percentage of scallops byssally attached in the ambient treatment increased significantly over time, with a strong correlation (R² = 0.6094, F_{1, 14} = 21.8, P = <0.001; Figure 7), but remained constant and not significant in the elevated (750 µatm) treatment (R² = 0.0946, F_{1, 14} = 1.4, P = >0.05; Figure 7). After four days of exposure in the elevated treatment, 65% of scallops were attached, and after five weeks of exposure 66% of scallops were attached. In contrast, after four days in the ambient treatment 57% of scallops were attached and 81% of scallops were attached at the end of the five weeks of exposure (Figure 7).

Download:

Figure 7. Mean Number of M. asperrima individuals byssally attached over experimental period.

Mean byssal attachment is represented every 2 days over the 5 weeks of ambient (390 µatm) and elevated (750 µatm) treatments. Line represents linear regression with linear equation and R² value n = 3.

https://doi.org/10.1371/journal.pone.0093649.g007

Discussion

Fertilisation and Larvae

This study found that exposure to elevated pCO₂ significantly affected the early life-history of M. asperrima. The fertilisation, development to trochophore stage and shell length of D-veliger larvae all decreased at elevated pCO₂. The shell length of umbonate and pediveliger larvae and newly metamorphosed spat, however, were less sensitive to elevated CO₂, with no significant reduction in shell length following acute exposure to elevated pCO₂, and in some instances, positive effects of elevated pCO₂ on shell growth. For many broadcast spawning marine organisms such as M. asperrima, fertilisation occurs in the water column and is directly affected by the environmental conditions [66]. Fertilisation is the first step in embryonic development, and is of particular importance to the reproductive success and population survival of marine free spawning invertebrates [67]. In this study, fertilisation of M. asperrima decreased with elevated pCO₂. At the highest pCO₂ level of 1000 µatm, the percentage fertilisation was reduced by 30%. Fertilisation of other marine organisms in response to elevated pCO₂ has been shown to be quite robust [68], [69], although variable. Some studies on northern hemisphere bivalve populations have reported no effect of elevated pCO₂ on the fertilisation of C. gigas [11], [70] and M.s galloprovincialis [28] at 2000 µatm pCO₂. In contrast, when experiments were conducted on southern hemisphere populations, elevated pCO₂ was found to reduce fertilisation in C. gigas and S. glomerata [12], [26]. The robustness of fertilisation has been questioned for other marine groups which are also broadcast spawners. For example, fertilisation was reduced during exposure to elevated pCO₂ in the sea urchins, Hemicentrotus pulcherrimus, Echinometra mathaei [30], [71], and Heliocidaris erythrogramma [35]. However, in another study, when H. erythrogramma was collected from a similar location, Byrne [66] found that there was no difference in the percentage fertilisation under elevated pCO₂ compared to ambient levels. Such contrasting results, from the same species sometimes from the same location, call into question the methodologies used to measure fertilisation [9], [26]. Byrne [72] has suggested a more consistent and integrated approach to facilitate accurate comparisons of fertilisation needs to be taken in future investigations.

Studies have questioned the effect of timing of exposure to elevated pCO₂ on development, suggesting it is not until the trochophore stage and shell mineralisation (calcification) that the effects of elevated pCO₂ will be observed [8], [11], [70]. Similar to this study, Parker et al. [12], [26] found there was a reduction in fertilisation of the oysters S. glomerata and C. gigas in response to elevated pCO₂ just 2 h after sperm addition, which is well before the beginning of the trochophore stage. In a natural setting, mortality of marine invertebrates during their early development can often exceed 90% [73]. Exposure to stressors early in life can also elicit the “developmental domino effect” where the physiological performance of the earlier stages has severe implications on the success of the following ontogenetic stages [72]. A decrease in fertilisation and early development, as observed in this study, could have severe flow on effects to the number of settlers and affect settlement and recruitment [9].

The shell length of D-veliger larvae of M. asperrima decreased with increased pCO₂ after four and six days of exposure. The effects of increased pCO₂ on the size of D-veliger larvae has been observed for other bivalve species such as M. edulis [18] and M. galloprovincialis [28] the clam Mercenaria mercenaria [47] and gastropods such as the abalone H. coccoriadata [33]. Reductions in the size of larvae in response to elevated pCO₂ has also been found in the sea urchins H. pulcherrimus, E. mathaei [30] T. gratilla, Evechinus chloroticus, and Sterechinus neumayeri [32], this is likely due to a decrease in larval calcification. Most studies report stunted larval development and decreased growth once larvae reach the calcifying stage [8], [11], [28]. Reductions in the calcification of adult marine organisms in response to elevated pCO₂ are also widely documented [20], [74]–[78]. It has also been suggested by Stumpp et al. [79] that reductions in larval growth at elevated pCO₂ can be attributed to a reduction of energy. Stumpp et al. [79] found decreased scope for growth in the larvae of the sea urchin S. purpuratus at elevated pCO₂. In an attempt to combat the physiological effect of elevated the sea urchin larvae diverted energy away from somatic growth to homeostatic processes. The reduced energy available for growth meant that the sea urchin larvae at elevated pCO₂ could not grow as fast as those under ambient pCO₂ conditions.

The results of this study are consistent with others, finding a significant reduction in the percentage fertilisation [12], [26] and decreased size of D-veliger at elevated pCO₂ [8], [11], [12], [26], [27], [36], [47]. The size of umbonate, pediveliger larvae and spat of M. asperrima were not, however, reduced at elevated pCO₂, and at some stages growth may have been enhanced under elevated pCO₂. This is in contrast to studies which have reported reduced shell length of umbonate, pediveliger and spat stages of the oysters S. glomerata and C. gigas, in response to elevated pCO₂ [12], [26], [27]. Similar to M. asperrima, larvae of the bay scallop Argopecten irradians were only affected by elevated pCO₂ (≈2000 µatm) on the first day of exposure [46] and similarly, White et al. [46] found that shell growth of larvae of A. irradians were reduced only up until day 7. Further, Talmage and Gobler [47] found larvae of A. irradians were only vulnerable in the very early life history stage and significantly less affected by elevated pCO₂ compared to the clam Mercenaria mercenaria.

Adults

It is known that as pCO₂ increases, dissolved CO₂ readily diffuses across the epithelial surfaces of marine molluscs and other marine organisms, where it equilibrates across the organism’s body spaces [23], [80] causing intra and extracellular acidification [23], [81]. If the organism cannot compensate, this acid-base balance disturbance can have detrimental consequences, including changes in energy metabolism [15]–[17], [37], [82], reductions in thermal tolerance and aerobic scope [83], compromises in immune responses [84], protein degradation [17], [85], decreases in somatic growth [15], [79], and in some extreme cases, increased mortality [86]–[89]. In an attempt to ameliorate these effects, organisms often try to increase their ion exchange to maintain acid-base balance. This requires energy, which can come at a cost to other physiological functions, or energy production (metabolic rate) is increased [15], [21]–[23], [43].

During the chronic five week exposure, adult M. asperrima showed no significant change in their standard metabolic rate (SMR), extracellular pH (pH_e) and extracellular O₂ (p_eO₂) in response to elevated pCO₂. There were, however, significant effects on the condition and byssal attachment of adult M. asperrima over the duration of the exposure. The present study, like most other studies to date, measured the rate of respiration (SMR) of the whole organism. SMR of the whole organism provides an estimation of the overall sum of energy consuming processes, making it possible that small changes in a specific metabolic system may be overlooked. The absence of an alteration of the SMR (either an increase or decrease) of adult M. asperrima to elevated pCO₂ in this study suggests that there was either (i) no change in metabolic functions in response to elevated pCO₂ or (ii) a shift in the energy budget and metabolic functions, where a metabolic decrease in one tissue was compensated by a metabolic increase in another, giving no net change in SMR. The decline in condition index and byssal attachment suggests that latter (ii) had occurred. Schalkhausser et al. [48] found that the great scallop Pecten maximus also had no change in overall resting SMR when exposed to elevated pCO₂, although it experienced a decrease in shell clapping power suggesting an energy trade off to maintain to maintain acid-base balance [48].

In this investigation, while there was a significant linear increase over time in the percentage of adult M. asperrima attached by byssal threads in the ambient treatment, there was no relationship between the percentage of adult M. asperrima attached in the elevated treatment. There was also a trend for the mean number of byssal threads secreted per animal to be greater in the ambient compared to the elevated treatment. Byssal attachment of scallops is known to be an energy demanding process, ranging between 4–14% of all somatic production [90], which is affected by environmental factors (mainly abiotic) and an indicator of physiological stress [91]–[96]. The relationship between stressful abiotic factors reducing byssal attachment such as increasing temperature and decreasing salinity, has been found for the Atlantic sea scallop Placopecten magellanicus [91], the great scallop P. maximus [92], M. asperrima [93], the queen scallop Chlamys opercularis [94], and the lions paw scallop Nodipecten nodosus [96]. Byssal attachment is also known to mirror the pattern of scallop growth and survival [93]. In this study, the decreased byssal attachment by M. asperrima suggests that elevated pCO₂ is acting in a similar way to other abiotic factors such as salinity and temperature to cause a decrease in byssal attachment. The observed byssal attachment reduction is most likely the result of one or both of two mechanisms; a behavioural response and/or a metabolic response. Most bivalves close their valves in response to an environmental stressor such as salinity, scallops however, cannot seal their shell [97]. As a result species including M. asperrima, A. irradians, and C. opercularis attempt to escape via swimming. Byssal attachment obviously hinders the swimming response, so detachment allows the individual to swim away from the irritant, or to be washed away via currents [97]. The reduced percentage of scallops byssally attached may also be due to a metabolic trade-off to favour increased homeostatic functions.

The mode of life of M. asperrima may increase the capacity of this bivalve to cope with elevated pCO₂ compared to more sessile species such as oysters and mussels. Water respiring aquatic organisms rely almost exclusively on ion exchange mechanisms to compensate for external CO₂ induced disturbances in acid-base balance [21], [22], [43]. Melzner et al. [43] argues that organisms with a more active mode of life such as teleost fish, brachyurans, and cephalopods have a greater ability to regulate acid-base balance primarily due to their more advanced non-carbonate buffering system. It is presumed that these more advanced buffering mechanisms have developed to mitigate the acidifying effect of excess CO₂ produced during exercise [43], [44]. During the rapid contractions of the adductor muscle associated with swimming, the heart rate of scallops can increase 2–3 fold, rising stroke volume and resulting in a total 5 fold increase in cardiac output [45]. This increased aerobic metabolism results in a spiking of CO₂ in the haemolymph which must be dealt with. The scallop is the only bivalve which can facilitate locomotion on a relatively large scale, via this swimming response [97]. It may be possible, that scallops too have developed increased acid-base regulation to cope with elevated CO₂ generated during their swimming response. An increased capacity to cope with elevated pCO₂ within the circulatory system may explain why no change in SMR or pH_e was observed at rest in this investigation, yet such a response has been observed in more sessile bivalves [13], [16], [17], [48].

Overall, when adult M. asperrima were exposed to elevated pCO₂ (750 µtm) there was no effect on SMR and pH_e, but a significant reduction in the condition index and byssal attachment. The increased ion-exchange capacity of the scallops could possibly be more energy demanding resulting in a trade-off decreasing somatic growth [16], [17], [23], [79], as measured by a reduction in condition index. Decreased condition index under elevated pCO₂ may also be due to reduction in protein turnover, resulting in lowered protein synthesis in some marine organisms [98]. Although sacrificing metabolic processes for the sake of homeostatic processes may be successful in the short term, it is likely that there are long term consequences for individuals and populations. Diverting energy away from and reducing somatic growth will potentially slow the reproductive development of scallops, which will in turn, affect reproductive output. If the response of M. asperrima is contrasted to studies on other Australian bivalves, Parker et al. [13] found S. glomerata significantly increased SMR, significantly decreased pH_e with no effect on condition index when exposed to elevated pCO₂ of 750 µatm.

Previous investigations have confirmed the ability of intertidal organisms to adapt and respond to elevated pCO₂ with increased resilience if they are previously exposed to a low pH or fluctuating environment [13], [31], [32], [37]. These results prompted the hypothesis that those organisms from stable pH environments such as the subtidal, would be more vulnerable to elevated pCO₂. This study does not support this hypothesis. Although M. asperrima were negatively affected across many life history stages by elevated CO₂, M. asperrima (and most other scallop species previously investigated [46], [47], [48]) were not affected to the extent that has been reported for intertidal bivalves such as M. edulis, M. galloprovincialis [18], [28] S. glomerata and C. gigas [12], [13], [16], [26]. Benthic respiration as suggested by Miller et al. [36] or similar environmental processes may allow M. asperrima and other scallops to develop a capacity to cope with an elevated pCO₂ environment, although measurement of benthic respiration and CO₂ flux monitoring is required to confirm this.

This study has found that fertilisation and early larval development of the economically and ecologically significant M. asperrima decreased with elevated pCO₂. Later larval stages were not as affected by elevated pCO₂ as early larval stages and some stages may have experienced enhanced growth when exposed to elevated pCO₂. It appears that M. asperrima larvae were less sensitive to elevated pCO₂ than the larval stages of the estuarine oysters S. glomerata and C. gigas [12], [13], [16], [26]. After a five week exposure to elevated pCO₂ adult M. asperrima were also in poorer condition and had decreased byssal attachment. The results of this study suggest that species responses to elevated pCO₂ are complex, and when attempting to predict and explain their response, the physiochemical characteristics of the environment, mode of life and responses across life history stages need to be considered. Even with the more moderate set of responses measured in this study the effects of ocean acidification are still likely to reduce the abundance of M. asperrima and other scallop species in the world’s oceans.

Acknowledgments

We wish to acknowledge the tremendous support of New South Wales Department of Primary Industries and NSW Marine Parks Authority who joined with the University of Western Sydney to complete this study. We especially thank Nathan Knott, Kyle Johnston, Andrew Parnell, Brandt Archer, Justin Kelly, Lyne Foulkes and Steve O’Connor. We thank the two anonymous reviewers and editor of this paper for their comments which improved the manuscript. We also need to thank the School of Science and Health at the University of Western Sydney and the support of all our colleagues.

Author Contributions

Conceived and designed the experiments: LP WO PR. Performed the experiments: ES. Analyzed the data: ES PR. Contributed reagents/materials/analysis tools: WO. Wrote the paper: ES PR LP.

References

1. Caldeira K, Wickett ME (2005) Ocean model predictions of chemistry changes from carbon dioxide emissions to the atmosphere and ocean. J Geophys Res 110: C09S04.
- View Article
- Google Scholar
2. Caldeira K, Wickett ME (2003) Anthropogenic carbon and ocean pH. Nature 425: 365–365.
- View Article
- Google Scholar
3. Hoegh-Guldberg O, Mumby P, Hooten A, Steneck R, Greenfield P, et al. (2007) Coral reefs under rapid climate change and ocean acidification. Science 318: 1737.
- View Article
- Google Scholar
4. Raven J, Caldeira K, Elderfield H, Hoegh-Guldberg O, Liss P, et al.. (2005) Ocean acidification due to increasing atmospheric carbon dioxide: Policy Document 12/05. London: The Royal Society.
5. Houghton JT, Ding Y, Griggs DJ, Noguer M, Van Der Linden PJ, et al.. (2001) Climate change 2001: the scientific basis. Cambridge: Cambridge University Press.
6. Solomon S (2007) Climate change 2007-the physical science basis: Working group I contribution to the fourth assessment report of the IPCC. Cambridge: Cambridge University Press.
7. Dupont S, Havenhand J, Thorndyke W, Peck L, Thorndyke M (2008) Near-future level of CO₂-driven ocean acidification radically affects larval survival and development in the brittlestar Ophiothrix fragilis. Mar Ecol Prog Ser 373: 285–294.
- View Article
- Google Scholar
8. Kurihara H (2008) Effects of CO₂-driven ocean acidification on the early developmental stages of invertebrates. Mar Ecol Prog Ser 373: 275–284.
- View Article
- Google Scholar
9. Ross PM, Parker L, O’Connor WA, Bailey EA (2011) The impact of ocean acidification on reproduction, early development and settlement of marine organisms. Water 3: 1005–1030.
- View Article
- Google Scholar
10. Doney SC, Fabry VJ, Feely RA, Kleypas JA (2009) Ocean acidification: the other CO₂ problem. Mar Sci 1: 169–192.
- View Article
- Google Scholar
11. Kurihara H, Kato S, Ishimatsu A (2007) Effects of increased seawater pCO₂ on early development of the oyster Crassostrea gigas. Aquat Biol 1: 91–98.
- View Article
- Google Scholar
12. Parker LM, Ross PM, O’Connor WA (2010) Comparing the effect of elevated pCO₂ and temperature on the fertilization and early development of two species of oysters. Mar biol 157: 2435–2452.
- View Article
- Google Scholar
13. Parker LM, Ross PM, O'Connor WA, Borysko L, Raftos DA, et al. (2013) Adult exposure influences offspring response to ocean acidification in oysters. Glob Chang Biol 18: 82–92.
- View Article
- Google Scholar
14. Feely RA, Sabine CL, Hernandez-Ayon JM, Ianson D, Hales B (2008) Evidence for upwelling of corrosive" acidified" water onto the continental shelf. Science 320: 1490–1492.
- View Article
- Google Scholar
15. Beniash E, Ivanina A, Lieb NS, Kurochkin I, Sokolova IM (2010) Elevated level of carbon dioxide affects metabolism and shell formation in oysters Crassostrea virginica. Mar Ecol Prog Ser 419: 95–108.
- View Article
- Google Scholar
16. Lannig G, Eilers S, Pörtner HO, Sokolova IM, Bock C (2010) Impact of ocean acidification on energy metabolism of oyster, Crassostrea gigas—changes in metabolic pathways and thermal response. Mar drugs 8: 2318–2339.
- View Article
- Google Scholar
17. Michaelidis B, Ouzounis C, Paleras A, Pörtner HO (2005) Effects of long-term moderate hypercapnia on acid-base balance and growth rate in marine mussels Mytilus galloprovincialis. Mar Ecol Prog Ser 293: 109–118.
- View Article
- Google Scholar
18. Gazeau F, Gattuso J, Dawber C, Pronker A, Peene F, et al. (2010) Effect of ocean acidification on the early life stages of the blue mussel (Mytilus edulis). Biogeosci Discuss 7: 2061–2050.
- View Article
- Google Scholar
19. Gazeau F, Quiblier C, Jansen JM, Gattuso JP, Middelburg JJ, et al. (2007) Impact of elevated CO₂ on shellfish calcification. Geophys Res Lett 34: L07603.
- View Article
- Google Scholar
20. Ries JB, Cohen AL, McCorkle DC (2009) Marine calcifiers exhibit mixed responses to CO₂-induced ocean acidification. Geology 37: 1131.
- View Article
- Google Scholar
21. Pörtner HO (2008) Ecosystem effects of ocean acidification in times of ocean warming: a physiologist's view. Mar Ecol Prog Ser 373: 203–217.
- View Article
- Google Scholar
22. Pörtner HO, Farrell AP (2008) Physiology and climate change. Science 322: 690–692.
- View Article
- Google Scholar
23. Pörtner HO, Langenbuch M, Reipschläger A (2004) Biological impact of elevated ocean CO₂ concentrations: lessons from animal physiology and earth history. J Oceanogr 60: 705–718.
- View Article
- Google Scholar
24. Parker LM, Ross PM, O'Connor WA, Pörtner HO, Scanes E, et al. (2013) Predicting the response of molluscs to the impact of ocean acidification. Biology 2: 651–692.
- View Article
- Google Scholar
25. Parker L, Ross PM, O’Connor WA (2011) Populations of the Sydney rock oyster, Saccostrea glomerata, vary in response to ocean acidification. Mar biol 158: 689–697.
- View Article
- Google Scholar
26. Parker LM, Ross PM, O'Connor WA (2009) The effect of ocean acidification and temperature on the fertilization and embryonic development of the Sydney rock oyster Saccostrea glomerata (Gould 1850). Glob Chang Biol 15: 2123–2136.
- View Article
- Google Scholar
27. Watson SA, Southgate PC, Tyler PA, Peck LS (2009) Early larval development of the Sydney rock oyster Saccostrea glomerata under near-future predictions of CO₂-driven ocean acidification. J Shellfish Res 28: 431–437.
- View Article
- Google Scholar
28. Kurihara H, Asai T, Kato S, Ishimatsu A (2009) Effects of elevated pCO₂ on early development in the mussel Mytilus galloprovincialis. Aquat Biol 4: 225–233.
- View Article
- Google Scholar
29. Ericson JA, Lamare MD, Morley SA, Barker MF (2010) The response of two ecologically important Antarctic invertebrates (Sterechinus neumayeri and Parborlasia corrugatus) to reduced seawater pH: effects on fertilisation and embryonic development. Mar biol 157: 2689–2752.
- View Article
- Google Scholar
30. Kurihara H, Shimode S, Shirayama Y (2004) Sub-lethal effects of elevated concentration of CO₂ on planktonic copepods and sea urchins. J Oceanog 60: 743–750.
- View Article
- Google Scholar
31. Moulin L, Catarino AI, Claessens T, Dubois P (2011) Effects of seawater acidification on early development of the intertidal sea urchin Paracentrotus lividus (Lamarck 1816). Mar Poll Bull 62: 48–54.
- View Article
- Google Scholar
32. Clark D, Lamare M, Barker M (2009) Response of sea urchin pluteus larvae (Echinodermata: Echinoidea) to reduced seawater pH: a comparison among a tropical, temperate, and a polar species. Mar biol 156: 1125–1137.
- View Article
- Google Scholar
33. Byrne M, Ho M, Wong E, Soars NA, Selvakumaraswamy P, et al. (2011) Unshelled abalone and corrupted urchins: development of marine calcifiers in a changing ocean. Proc Biol Sci 278: 2376.
- View Article
- Google Scholar
34. Byrne M, Soars N, Selvakumaraswamy P, Dworjanyn SA, Davis AR (2010) Sea urchin fertilization in a warm, acidified and high pCO₂ ocean across a range of sperm densities. Mar Environ Res 69: 234–239.
- View Article
- Google Scholar
35. Havenhand JN, Buttler FR, Thorndyke MC, Williamson JE (2008) Near-future levels of ocean acidification reduce fertilization success in a sea urchin. Curr Biol 18: R651–R652.
- View Article
- Google Scholar
36. Miller AW, Reynolds AC, Sobrino C, Riedel GF (2009) Shellfish face uncertain future in high CO₂ world: influence of acidification on oyster larvae calcification and growth in estuaries. PLoS One 4: e5661.
- View Article
- Google Scholar
37. Thomsen J, Melzner F (2010) Moderate seawater acidification does not elicit long-term metabolic depression in the blue mussel Mytilus edulis. Mar Biol 157: 2667–2676.
- View Article
- Google Scholar
38. Cai WJ, Pomeroy LR, Moran MA, Wang Y (1999) Oxygen and carbon dioxide mass balance for the estuarine-intertidal marsh complex of five rivers in the southeastern U. S. Limnol and Oceanogra. 44: 639–649.
- View Article
- Google Scholar
39. Dupont S, Dorey N, Stumpp M, Melzner F, Thorndyke M (2012) Long-term and trans-life-cycle effects of exposure to ocean acidification in the green sea urchin Strongylocentrotus droebachiensis. Mar Biol 160: 1835–1843.
- View Article
- Google Scholar
40. Andersen S, Grefsrud E, Harboe T (2013) Effect of increased pCO₂ on early shell development in great scallop (Pecten maximus Lamarck) larvae. Biogeosci discuss 10: 3281–3310.
- View Article
- Google Scholar
41. Gutowska M, Pörtner HO, Melzner F (2008) Growth and calcification in the cephalopod Sepia officinalis under elevated seawater pCO₂. Mar Ecol Prog Ser 373: 303–309.
- View Article
- Google Scholar
42. Gutowska MA, Melzner F, Langenbuch M, Bock C, Claireaux G, et al. (2010) Acid–base regulatory ability of the cephalopod (Sepia officinalis) in response to environmental hypercapnia. J Comp Phys Biol 180: 323–335.
- View Article
- Google Scholar
43. Melzner F, Gutowska M, Langenbuch M, Dupont S, Lucassen M, et al. (2009) Physiological basis for high CO₂ tolerance in marine ectothermic animals: pre-adaptation through lifestyle and ontogeny? Biogeosci 6: 2313–2331.
- View Article
- Google Scholar
44. Portner H, Webber D, Boutilier R, O'dor R (1991) Acid-base regulation in exercising squid (Illex illecebrosus, Loligo pealei). Am J Physiol Regul Interg Comp Physiol 261: R239–R246.
- View Article
- Google Scholar
45. Thompson R, Livingstone D, De Zwaan A (1980) Physiological and biochemical aspects of the valve snap and valve closure responses in the giant scallop Placopecten magellanicus. J Comp Physiol 137: 97–104.
- View Article
- Google Scholar
46. White MM, McCorkle DC, Mullineaux LS, Cohen AL (2013) Early exposure of bay scallops (Argopecten irradians) to high CO₂ causes a decrease in larval shell growth. PloS one 8: e61065.
- View Article
- Google Scholar
47. Talmage SC, Gobler CJ (2010) Effects of past, present, and future ocean carbon dioxide concentrations on the growth and survival of larval shellfish. Proc Natl Acad Sci 107: 17246–17251.
- View Article
- Google Scholar
48. Schalkhausser B, Bock C, Stemmer K, Brey T, Pörtner H-O, et al. (2012) Impact of ocean acidification on escape performance of the king scallop, Pecten maximus, from Norway. Mar Biol 160: 1995–2006.
- View Article
- Google Scholar
49. Lamprell K, Whitehead T (1992) Bivalves of Australia. Sydney, Balogh Scientific Books.
50. Butler JN (1982) Carbon dioxide equilibria and their applications. Boston, Addison-Wesley.
51. Gran G (1952) Determination of the equivalence point in potentiometric titrations. Part II. Analyst 77: 661–671.
- View Article
- Google Scholar
52. Lewis E, Wallace DWR (1998) Program developed for CO₂ system calculations. Oak Ridge, Tennessee: Oak Ridge national laboratory, US Department of Energy.
53. Mehrbach C, Culberson C, Hawley J, Pytkowicz R (1973) Measurement of the apparent dissociation constants of carbonic acid in seawater at atmospheric pressure. Limnol Oceanogr 18: 897–907.
- View Article
- Google Scholar
54. Perez FF, Fraga F (1987) The pH measurements in seawater on the NBS scale. Mar Chem 21: 315–327.
- View Article
- Google Scholar
55. O'Connor W, Heasman M, Frazer A, Taylor J (1994) Hatchery rearing the Doughboy scallop, Chlamys (Mimachlamys) asperrimus (Lamarck). Mem Queensl Mus 36: 357–360.
- View Article
- Google Scholar
56. Krassoi R, Anderson I, Everett D (1997) Larval abnormalities in doughboy scallops Chlamys(mimachlamys) asperrima L. in response to test conditions and six reference toxicants. Aust J Ecotox 3: 65–74.
- View Article
- Google Scholar
57. O'Connor WA, Heasman MP (1995) Spawning induction and fertilisation in the doughboy scallop Chlamys (Mimachlamys) asperrima. Aquaculture 136: 117–129.
- View Article
- Google Scholar
58. Cragg SM, Crisp DJ (1991) The Biology of Scallop Larvae. In: Shumway SE, editor. Scallops: Biology, Ecology and Aquaculture. Amsterdam: Elsevier.
59. Morton B, Prezant RS, Wilson B (1998) Class Bivalvia. In: Beesley PL, Ross, G. J B., Wells, A., editor. Mollusca: The Southern Synthesis Fauna of Australia Vol 5 . Melbourne: CSIRO Publishing.
60. Loosanoff VL, Davis HC (1963) Rearing of bivalve mollusks. Adv Mar Biol 1: 1–136.
- View Article
- Google Scholar
61. Rose R, Dix T (1984) Larval and juvenile development of the doughboy scallop, Chlamys (Chlamys) asperrimus (Lamarck)(Mollusca: Pectinidae). Mar Freshw Res 35: 315–323.
- View Article
- Google Scholar
62. O'connor W, Heasman M, O'connor S (2000) Algal diets for broodstock maintenance of the doughboy scallop Mimachlamys asperrima (Lamarck). Aquacul Res 31: 627–635.
- View Article
- Google Scholar
63. O'Connor WA (1997) Reproductive Biology and Hatchery Culture of the Doughboy Scallop Mimachlamys asperrima (Lamarck, 1819). Sydney: University of Technology, Sydney.
64. Underwood AJ (1996) Experiments in ecology: their logical design and interpretation using analysis of variance: Cambridge: Cambridge University Press.
65. Sokal R, Rohlf F (1995) Biometry (3rd edn). New York: WH Freman and company.
66. Byrne M, Ho M, Selvakumaraswamy P, Nguyen HD, Dworjanyn SA, et al. (2009) Temperature, but not pH, compromises sea urchin fertilization and early development under near-future climate change scenarios. Proc Biol Sci 276: 1883–1888.
- View Article
- Google Scholar
67. Marshall DJ, Styan CA, Keough MJ (2002) Sperm environment affects offspring quality in broadcast spawning marine invertebrates. Ecol lett 5: 173–176.
- View Article
- Google Scholar
68. Dupont S, Lundve B, Thorndyke M (2010) Near future ocean acidification increases growth rate of the lecithotrophic larvae and juveniles of the sea star Crossaster papposus. J Exp Zool B Mol Dev Evol 314: 382–389.
- View Article
- Google Scholar
69. Kroeker KJ, Kordas RL, Crim RN, Singh GG (2010) Meta-analysis reveals negative yet variable effects of ocean acidification on marine organisms. Ecol Lett 13: 1419–1434.
- View Article
- Google Scholar
70. Havenhand J, Schlegel P (2009) Near-future levels of ocean acidification do not affect sperm motility and fertilization kinetics in the oyster Crassostrea gigas. Biogeosci 6: 3009–3015.
- View Article
- Google Scholar
71. Kurihara H, Shirayama Y (2004) Effects of increased atmospheric CO₂ on sea urchin early development. Mar Ecol Prog Ser 274: 161–169.
- View Article
- Google Scholar
72. Byrne M (2012) Global change ecotoxicology: identification of early life history bottlenecks in marine invertebrates, variable species responses and variable experimental approaches. Mar Env Res 76: 3–16.
- View Article
- Google Scholar
73. Gosselin LA, Qian PY (1997) Juvenile mortality in benthic marine invertebrates. Mar Ecol Prog Ser 146: 265–282.
- View Article
- Google Scholar
74. Bijma J, Spero H, Lea D (1999) Reassessing foraminiferal stable isotope geochemistry: Impact of the oceanic carbonate system (experimental results). Use of proxies in paleoceanography: Springer. pp. 489–512.
75. Gao K, Aruga Y, Asada K, Ishihara T, Akano T, et al. (1993) Calcification in the articulated coralline alga Corallina pilulifera, with special reference to the effect of elevated CO₂ concentration. Mar Biolo 117: 129–132.
- View Article
- Google Scholar
76. Orr JC, Fabry VJ, Aumont O, Bopp L, Doney SC, et al. (2005) Anthropogenic ocean acidification over the twenty-first century and its impact on calcifying organisms. Nature 437: 681–686.
- View Article
- Google Scholar
77. Riebesell U, Zondervan I, Rost BÈ, Tortell PD, Zeebe RE, et al. (2000) Reduced calcification of marine plankton in response to increased atmospheric CO₂. Nature 407: 364–367.
- View Article
- Google Scholar
78. Reynaud S, Leclercq N, Romaine-Lioud S, Ferrier-Pagés C, Jaubert J, et al. (2003) Interacting effects of CO₂ partial pressure and temperature on photosynthesis and calcification in a scleractinian coral. Glob Chang Biol 9: 1660–1668.
- View Article
- Google Scholar
79. Stumpp M, Wren J, Melzner F, Thorndyke M, Dupont S (2011) CO₂ induced seawater acidification impacts sea urchin larval development I: Elevated metabolic rates decrease scope for growth and induce developmental delay. Comp Biochem Physiol A Mol Integr Physiol 160: 331–340.
- View Article
- Google Scholar
80. Reipschläger A, PÖRtner H (1996) Metabolic depression during environmental stress: the role of extracellular versus intracellular pH in Sipunculus nudus. J Exp Biol 199: 1801–1807.
- View Article
- Google Scholar
81. Fabry VJ, Seibel BA, Feely RA, Orr JC (2008) Impacts of ocean acidification on marine fauna and ecosystem processes. ICES J Mar Sci 65: 414–432.
- View Article
- Google Scholar
82. Cummings V, Hewitt J, Van Rooyen A, Currie K, Beard S, et al. (2011) Ocean acidification at high latitudes: potential effects on functioning of the Antarctic bivalve Laternula elliptica. PloS one 6: e16069.
- View Article
- Google Scholar
83. Nilsson GE, Crawley N, Lunde IG, Munday PL (2008) Elevated temperature reduces the respiratory scope of coral reef fishes. Glob Chang Biol 15: 1405–1412.
- View Article
- Google Scholar
84. Bibby R, Widdicombe S, Parry H, Spicer J, Pipe R (2008) Effect of ocean acidification on the immune response of the blue mussel, Mytilus edulis. Aquat Biol 2: 67–74.
- View Article
- Google Scholar
85. Neuberger-Cywiak L, Achituv Y, Garcia E (2007) Effects of sublethal Zn⁺ and Cd⁺ concentrations on filtration rate, absorption efficiency and scope for growth in Donax trunculus (Bivalvia; Donacidae). Bull Environ Contam Toxicol 79: 622–627.
- View Article
- Google Scholar
86. Berge JA, Bjerkeng B, Pettersen O, Schaanning MT, Oxnevad S (2006) Effects of increased sea water concentrations of CO₂ on growth of the bivalve Mytilus edulis L. Chemosphere. 62: 681–687.
- View Article
- Google Scholar
87. Range P, Chícharo M, Ben-Hamadou R, Piló D, Matias D, et al. (2011) Calcification, growth and mortality of juvenile clams Ruditapes decussatus under increased pCO₂ and reduced pH: Variable responses to ocean acidification at local scales? J Exp Mar Biol Ecol 396: 177–184.
- View Article
- Google Scholar
88. Shirayama Y, Thornton H (2005) Effect of increased atmospheric CO₂ on shallow water marine benthos. J Geophys Res 110: C09S08.
- View Article
- Google Scholar
89. Lischka S, Büdenbender J, Boxhammer T, Riebesell U (2010) Impact of ocean acidification and elevated temperatures on early juveniles of the polar shelled pteropod Limacina helicina: mortality, shell degradation, and shell growth. Biogeosci discuss 7: 8177–8214.
- View Article
- Google Scholar
90. Vahl O (1981) Energy transformations by the Iceland scallop, Chlamys islandica (OF Müller), from 70° N The age-specific energy budget and net growth efficiency. J Exp Mar Biol Ecol 53: 281–296.
- View Article
- Google Scholar
91. Caddy J (1972) Progressive loss of byssus attachment with size in the sea scallop, Placopecten magellanicus (Gmelin). J Exp Mar Biol Ecol 9: 179–190.
- View Article
- Google Scholar
92. Christophersen G, Strand Ø (2003) Effect of reduced salinity on the great scallop (Pecten maximus) spat at two rearing temperatures. Aquaculture 215: 79–92.
- View Article
- Google Scholar
93. O'Connor WA, Heasman MP (1998) Ontogenetic changes in salinity and temperature tolerance in the doughboy scallop, Mimachlamys asperrima. J Shellfish Res 17: 89–96.
- View Article
- Google Scholar
94. Paul JD (1980) Upper temperature tolerance and the effects of temperature on byssus attachment in the queen scallop, Chlamys opercularis (L.). J Exp Mar Biol Ecol 46: 41–50.
- View Article
- Google Scholar
95. Roberts D (1973) Some Sub-lethal effects of pesticides on the behaviour and physiology of bivalved molluscs: University of Liverpool (PhD Thesis).
96. Rupp GS, Parsons GJ (2004) Effects of salinity and temperature on the survival and byssal attachment of the lion's paw scallop Nodipecten nodosus at its southern distribution limit. J Exp Mar Biol Ecol 309: 173–198.
- View Article
- Google Scholar
97. Brand AR (1991) Scallop Ecology: Distributions and Behaviour. In: Shumway SE, editor. Scallops: Biology, Ecology and Aquaculture. Amsterdam: Elsevier.
98. Langenbuch M, Bock C, Leibfritz D, Pörtner H-O (2006) Effects of environmental hypercapnia on animal physiology: A ₁₃C NMR study of protein synthesis rates in the marine invertebrate Sipunculus nudus. Comp Biochem Physiol A Mol Integr Physiol 144: 479–484.
- View Article
- Google Scholar

[ref1] 1. Caldeira K, Wickett ME (2005) Ocean model predictions of chemistry changes from carbon dioxide emissions to the atmosphere and ocean. J Geophys Res 110: C09S04.
View Article
Google Scholar

[2] View Article

[3] Google Scholar

[ref2] 2. Caldeira K, Wickett ME (2003) Anthropogenic carbon and ocean pH. Nature 425: 365–365.
View Article
Google Scholar

[5] View Article

[6] Google Scholar

[ref3] 3. Hoegh-Guldberg O, Mumby P, Hooten A, Steneck R, Greenfield P, et al. (2007) Coral reefs under rapid climate change and ocean acidification. Science 318: 1737.
View Article
Google Scholar

[8] View Article

[9] Google Scholar

[ref4] 4. Raven J, Caldeira K, Elderfield H, Hoegh-Guldberg O, Liss P, et al.. (2005) Ocean acidification due to increasing atmospheric carbon dioxide: Policy Document 12/05. London: The Royal Society.

[ref5] 5. Houghton JT, Ding Y, Griggs DJ, Noguer M, Van Der Linden PJ, et al.. (2001) Climate change 2001: the scientific basis. Cambridge: Cambridge University Press.

[ref6] 6. Solomon S (2007) Climate change 2007-the physical science basis: Working group I contribution to the fourth assessment report of the IPCC. Cambridge: Cambridge University Press.

[ref7] 7. Dupont S, Havenhand J, Thorndyke W, Peck L, Thorndyke M (2008) Near-future level of CO₂-driven ocean acidification radically affects larval survival and development in the brittlestar Ophiothrix fragilis. Mar Ecol Prog Ser 373: 285–294.
View Article
Google Scholar

[14] View Article

[15] Google Scholar

[ref8] 8. Kurihara H (2008) Effects of CO₂-driven ocean acidification on the early developmental stages of invertebrates. Mar Ecol Prog Ser 373: 275–284.
View Article
Google Scholar

[17] View Article

[18] Google Scholar

[ref9] 9. Ross PM, Parker L, O’Connor WA, Bailey EA (2011) The impact of ocean acidification on reproduction, early development and settlement of marine organisms. Water 3: 1005–1030.
View Article
Google Scholar

[20] View Article

[21] Google Scholar

[ref10] 10. Doney SC, Fabry VJ, Feely RA, Kleypas JA (2009) Ocean acidification: the other CO₂ problem. Mar Sci 1: 169–192.
View Article
Google Scholar

[23] View Article

[24] Google Scholar

[ref11] 11. Kurihara H, Kato S, Ishimatsu A (2007) Effects of increased seawater pCO₂ on early development of the oyster Crassostrea gigas. Aquat Biol 1: 91–98.
View Article
Google Scholar

[26] View Article

[27] Google Scholar

[ref12] 12. Parker LM, Ross PM, O’Connor WA (2010) Comparing the effect of elevated pCO₂ and temperature on the fertilization and early development of two species of oysters. Mar biol 157: 2435–2452.
View Article
Google Scholar

[29] View Article

[30] Google Scholar

[ref13] 13. Parker LM, Ross PM, O'Connor WA, Borysko L, Raftos DA, et al. (2013) Adult exposure influences offspring response to ocean acidification in oysters. Glob Chang Biol 18: 82–92.
View Article
Google Scholar

[32] View Article

[33] Google Scholar

[ref14] 14. Feely RA, Sabine CL, Hernandez-Ayon JM, Ianson D, Hales B (2008) Evidence for upwelling of corrosive" acidified" water onto the continental shelf. Science 320: 1490–1492.
View Article
Google Scholar

[35] View Article

[36] Google Scholar

[ref15] 15. Beniash E, Ivanina A, Lieb NS, Kurochkin I, Sokolova IM (2010) Elevated level of carbon dioxide affects metabolism and shell formation in oysters Crassostrea virginica. Mar Ecol Prog Ser 419: 95–108.
View Article
Google Scholar

[38] View Article

[39] Google Scholar

[ref16] 16. Lannig G, Eilers S, Pörtner HO, Sokolova IM, Bock C (2010) Impact of ocean acidification on energy metabolism of oyster, Crassostrea gigas—changes in metabolic pathways and thermal response. Mar drugs 8: 2318–2339.
View Article
Google Scholar

[41] View Article

[42] Google Scholar

[ref17] 17. Michaelidis B, Ouzounis C, Paleras A, Pörtner HO (2005) Effects of long-term moderate hypercapnia on acid-base balance and growth rate in marine mussels Mytilus galloprovincialis. Mar Ecol Prog Ser 293: 109–118.
View Article
Google Scholar

[44] View Article

[45] Google Scholar

[ref18] 18. Gazeau F, Gattuso J, Dawber C, Pronker A, Peene F, et al. (2010) Effect of ocean acidification on the early life stages of the blue mussel (Mytilus edulis). Biogeosci Discuss 7: 2061–2050.
View Article
Google Scholar

[47] View Article

[48] Google Scholar

[ref19] 19. Gazeau F, Quiblier C, Jansen JM, Gattuso JP, Middelburg JJ, et al. (2007) Impact of elevated CO₂ on shellfish calcification. Geophys Res Lett 34: L07603.
View Article
Google Scholar

[50] View Article

[51] Google Scholar

[ref20] 20. Ries JB, Cohen AL, McCorkle DC (2009) Marine calcifiers exhibit mixed responses to CO₂-induced ocean acidification. Geology 37: 1131.
View Article
Google Scholar

[53] View Article

[54] Google Scholar

[ref21] 21. Pörtner HO (2008) Ecosystem effects of ocean acidification in times of ocean warming: a physiologist's view. Mar Ecol Prog Ser 373: 203–217.
View Article
Google Scholar

[56] View Article

[57] Google Scholar

[ref22] 22. Pörtner HO, Farrell AP (2008) Physiology and climate change. Science 322: 690–692.
View Article
Google Scholar

[59] View Article

[60] Google Scholar

[ref23] 23. Pörtner HO, Langenbuch M, Reipschläger A (2004) Biological impact of elevated ocean CO₂ concentrations: lessons from animal physiology and earth history. J Oceanogr 60: 705–718.
View Article
Google Scholar

[62] View Article

[63] Google Scholar

[ref24] 24. Parker LM, Ross PM, O'Connor WA, Pörtner HO, Scanes E, et al. (2013) Predicting the response of molluscs to the impact of ocean acidification. Biology 2: 651–692.
View Article
Google Scholar

[65] View Article

[66] Google Scholar

[ref25] 25. Parker L, Ross PM, O’Connor WA (2011) Populations of the Sydney rock oyster, Saccostrea glomerata, vary in response to ocean acidification. Mar biol 158: 689–697.
View Article
Google Scholar

[68] View Article

[69] Google Scholar

[ref26] 26. Parker LM, Ross PM, O'Connor WA (2009) The effect of ocean acidification and temperature on the fertilization and embryonic development of the Sydney rock oyster Saccostrea glomerata (Gould 1850). Glob Chang Biol 15: 2123–2136.
View Article
Google Scholar

[71] View Article

[72] Google Scholar

[ref27] 27. Watson SA, Southgate PC, Tyler PA, Peck LS (2009) Early larval development of the Sydney rock oyster Saccostrea glomerata under near-future predictions of CO₂-driven ocean acidification. J Shellfish Res 28: 431–437.
View Article
Google Scholar

[74] View Article

[75] Google Scholar

[ref28] 28. Kurihara H, Asai T, Kato S, Ishimatsu A (2009) Effects of elevated pCO₂ on early development in the mussel Mytilus galloprovincialis. Aquat Biol 4: 225–233.
View Article
Google Scholar

[77] View Article

[78] Google Scholar

[ref29] 29. Ericson JA, Lamare MD, Morley SA, Barker MF (2010) The response of two ecologically important Antarctic invertebrates (Sterechinus neumayeri and Parborlasia corrugatus) to reduced seawater pH: effects on fertilisation and embryonic development. Mar biol 157: 2689–2752.
View Article
Google Scholar

[80] View Article

[81] Google Scholar

[ref30] 30. Kurihara H, Shimode S, Shirayama Y (2004) Sub-lethal effects of elevated concentration of CO₂ on planktonic copepods and sea urchins. J Oceanog 60: 743–750.
View Article
Google Scholar

[83] View Article

[84] Google Scholar

[ref31] 31. Moulin L, Catarino AI, Claessens T, Dubois P (2011) Effects of seawater acidification on early development of the intertidal sea urchin Paracentrotus lividus (Lamarck 1816). Mar Poll Bull 62: 48–54.
View Article
Google Scholar

[86] View Article

[87] Google Scholar

[ref32] 32. Clark D, Lamare M, Barker M (2009) Response of sea urchin pluteus larvae (Echinodermata: Echinoidea) to reduced seawater pH: a comparison among a tropical, temperate, and a polar species. Mar biol 156: 1125–1137.
View Article
Google Scholar

[89] View Article

[90] Google Scholar

[ref33] 33. Byrne M, Ho M, Wong E, Soars NA, Selvakumaraswamy P, et al. (2011) Unshelled abalone and corrupted urchins: development of marine calcifiers in a changing ocean. Proc Biol Sci 278: 2376.
View Article
Google Scholar

[92] View Article

[93] Google Scholar

[ref34] 34. Byrne M, Soars N, Selvakumaraswamy P, Dworjanyn SA, Davis AR (2010) Sea urchin fertilization in a warm, acidified and high pCO₂ ocean across a range of sperm densities. Mar Environ Res 69: 234–239.
View Article
Google Scholar

[95] View Article

[96] Google Scholar

[ref35] 35. Havenhand JN, Buttler FR, Thorndyke MC, Williamson JE (2008) Near-future levels of ocean acidification reduce fertilization success in a sea urchin. Curr Biol 18: R651–R652.
View Article
Google Scholar

[98] View Article

[99] Google Scholar

[ref36] 36. Miller AW, Reynolds AC, Sobrino C, Riedel GF (2009) Shellfish face uncertain future in high CO₂ world: influence of acidification on oyster larvae calcification and growth in estuaries. PLoS One 4: e5661.
View Article
Google Scholar

[101] View Article

[102] Google Scholar

[ref37] 37. Thomsen J, Melzner F (2010) Moderate seawater acidification does not elicit long-term metabolic depression in the blue mussel Mytilus edulis. Mar Biol 157: 2667–2676.
View Article
Google Scholar

[104] View Article

[105] Google Scholar

[ref38] 38. Cai WJ, Pomeroy LR, Moran MA, Wang Y (1999) Oxygen and carbon dioxide mass balance for the estuarine-intertidal marsh complex of five rivers in the southeastern U. S. Limnol and Oceanogra. 44: 639–649.
View Article
Google Scholar

[107] View Article

[108] Google Scholar

[ref39] 39. Dupont S, Dorey N, Stumpp M, Melzner F, Thorndyke M (2012) Long-term and trans-life-cycle effects of exposure to ocean acidification in the green sea urchin Strongylocentrotus droebachiensis. Mar Biol 160: 1835–1843.
View Article
Google Scholar

[110] View Article

[111] Google Scholar

[ref40] 40. Andersen S, Grefsrud E, Harboe T (2013) Effect of increased pCO₂ on early shell development in great scallop (Pecten maximus Lamarck) larvae. Biogeosci discuss 10: 3281–3310.
View Article
Google Scholar

[113] View Article

[114] Google Scholar

[ref41] 41. Gutowska M, Pörtner HO, Melzner F (2008) Growth and calcification in the cephalopod Sepia officinalis under elevated seawater pCO₂. Mar Ecol Prog Ser 373: 303–309.
View Article
Google Scholar

[116] View Article

[117] Google Scholar

[ref42] 42. Gutowska MA, Melzner F, Langenbuch M, Bock C, Claireaux G, et al. (2010) Acid–base regulatory ability of the cephalopod (Sepia officinalis) in response to environmental hypercapnia. J Comp Phys Biol 180: 323–335.
View Article
Google Scholar

[119] View Article

[120] Google Scholar

[ref43] 43. Melzner F, Gutowska M, Langenbuch M, Dupont S, Lucassen M, et al. (2009) Physiological basis for high CO₂ tolerance in marine ectothermic animals: pre-adaptation through lifestyle and ontogeny? Biogeosci 6: 2313–2331.
View Article
Google Scholar

[122] View Article

[123] Google Scholar

[ref44] 44. Portner H, Webber D, Boutilier R, O'dor R (1991) Acid-base regulation in exercising squid (Illex illecebrosus, Loligo pealei). Am J Physiol Regul Interg Comp Physiol 261: R239–R246.
View Article
Google Scholar

[125] View Article

[126] Google Scholar

[ref45] 45. Thompson R, Livingstone D, De Zwaan A (1980) Physiological and biochemical aspects of the valve snap and valve closure responses in the giant scallop Placopecten magellanicus. J Comp Physiol 137: 97–104.
View Article
Google Scholar

[128] View Article

[129] Google Scholar

[ref46] 46. White MM, McCorkle DC, Mullineaux LS, Cohen AL (2013) Early exposure of bay scallops (Argopecten irradians) to high CO₂ causes a decrease in larval shell growth. PloS one 8: e61065.
View Article
Google Scholar

[131] View Article

[132] Google Scholar

[ref47] 47. Talmage SC, Gobler CJ (2010) Effects of past, present, and future ocean carbon dioxide concentrations on the growth and survival of larval shellfish. Proc Natl Acad Sci 107: 17246–17251.
View Article
Google Scholar

[134] View Article

[135] Google Scholar

[ref48] 48. Schalkhausser B, Bock C, Stemmer K, Brey T, Pörtner H-O, et al. (2012) Impact of ocean acidification on escape performance of the king scallop, Pecten maximus, from Norway. Mar Biol 160: 1995–2006.
View Article
Google Scholar

[137] View Article

[138] Google Scholar

[ref49] 49. Lamprell K, Whitehead T (1992) Bivalves of Australia. Sydney, Balogh Scientific Books.

[ref50] 50. Butler JN (1982) Carbon dioxide equilibria and their applications. Boston, Addison-Wesley.

[ref51] 51. Gran G (1952) Determination of the equivalence point in potentiometric titrations. Part II. Analyst 77: 661–671.
View Article
Google Scholar

[142] View Article

[143] Google Scholar

[ref52] 52. Lewis E, Wallace DWR (1998) Program developed for CO₂ system calculations. Oak Ridge, Tennessee: Oak Ridge national laboratory, US Department of Energy.

[ref53] 53. Mehrbach C, Culberson C, Hawley J, Pytkowicz R (1973) Measurement of the apparent dissociation constants of carbonic acid in seawater at atmospheric pressure. Limnol Oceanogr 18: 897–907.
View Article
Google Scholar

[146] View Article

[147] Google Scholar

[ref54] 54. Perez FF, Fraga F (1987) The pH measurements in seawater on the NBS scale. Mar Chem 21: 315–327.
View Article
Google Scholar

[149] View Article

[150] Google Scholar

[ref55] 55. O'Connor W, Heasman M, Frazer A, Taylor J (1994) Hatchery rearing the Doughboy scallop, Chlamys (Mimachlamys) asperrimus (Lamarck). Mem Queensl Mus 36: 357–360.
View Article
Google Scholar

[152] View Article

[153] Google Scholar

[ref56] 56. Krassoi R, Anderson I, Everett D (1997) Larval abnormalities in doughboy scallops Chlamys(mimachlamys) asperrima L. in response to test conditions and six reference toxicants. Aust J Ecotox 3: 65–74.
View Article
Google Scholar

[155] View Article

[156] Google Scholar

[ref57] 57. O'Connor WA, Heasman MP (1995) Spawning induction and fertilisation in the doughboy scallop Chlamys (Mimachlamys) asperrima. Aquaculture 136: 117–129.
View Article
Google Scholar

[158] View Article

[159] Google Scholar

[ref58] 58. Cragg SM, Crisp DJ (1991) The Biology of Scallop Larvae. In: Shumway SE, editor. Scallops: Biology, Ecology and Aquaculture. Amsterdam: Elsevier.

[ref59] 59. Morton B, Prezant RS, Wilson B (1998) Class Bivalvia. In: Beesley PL, Ross, G. J B., Wells, A., editor. Mollusca: The Southern Synthesis Fauna of Australia Vol 5 . Melbourne: CSIRO Publishing.

[ref60] 60. Loosanoff VL, Davis HC (1963) Rearing of bivalve mollusks. Adv Mar Biol 1: 1–136.
View Article
Google Scholar

[163] View Article

[164] Google Scholar

[ref61] 61. Rose R, Dix T (1984) Larval and juvenile development of the doughboy scallop, Chlamys (Chlamys) asperrimus (Lamarck)(Mollusca: Pectinidae). Mar Freshw Res 35: 315–323.
View Article
Google Scholar

[166] View Article

[167] Google Scholar

[ref62] 62. O'connor W, Heasman M, O'connor S (2000) Algal diets for broodstock maintenance of the doughboy scallop Mimachlamys asperrima (Lamarck). Aquacul Res 31: 627–635.
View Article
Google Scholar

[169] View Article

[170] Google Scholar

[ref63] 63. O'Connor WA (1997) Reproductive Biology and Hatchery Culture of the Doughboy Scallop Mimachlamys asperrima (Lamarck, 1819). Sydney: University of Technology, Sydney.

[ref64] 64. Underwood AJ (1996) Experiments in ecology: their logical design and interpretation using analysis of variance: Cambridge: Cambridge University Press.

[ref65] 65. Sokal R, Rohlf F (1995) Biometry (3rd edn). New York: WH Freman and company.

[ref66] 66. Byrne M, Ho M, Selvakumaraswamy P, Nguyen HD, Dworjanyn SA, et al. (2009) Temperature, but not pH, compromises sea urchin fertilization and early development under near-future climate change scenarios. Proc Biol Sci 276: 1883–1888.
View Article
Google Scholar

[175] View Article

[176] Google Scholar

[ref67] 67. Marshall DJ, Styan CA, Keough MJ (2002) Sperm environment affects offspring quality in broadcast spawning marine invertebrates. Ecol lett 5: 173–176.
View Article
Google Scholar

[178] View Article

[179] Google Scholar

[ref68] 68. Dupont S, Lundve B, Thorndyke M (2010) Near future ocean acidification increases growth rate of the lecithotrophic larvae and juveniles of the sea star Crossaster papposus. J Exp Zool B Mol Dev Evol 314: 382–389.
View Article
Google Scholar

[181] View Article

[182] Google Scholar

[ref69] 69. Kroeker KJ, Kordas RL, Crim RN, Singh GG (2010) Meta-analysis reveals negative yet variable effects of ocean acidification on marine organisms. Ecol Lett 13: 1419–1434.
View Article
Google Scholar

[184] View Article

[185] Google Scholar

[ref70] 70. Havenhand J, Schlegel P (2009) Near-future levels of ocean acidification do not affect sperm motility and fertilization kinetics in the oyster Crassostrea gigas. Biogeosci 6: 3009–3015.
View Article
Google Scholar

[187] View Article

[188] Google Scholar

[ref71] 71. Kurihara H, Shirayama Y (2004) Effects of increased atmospheric CO₂ on sea urchin early development. Mar Ecol Prog Ser 274: 161–169.
View Article
Google Scholar

[190] View Article

[191] Google Scholar

[ref72] 72. Byrne M (2012) Global change ecotoxicology: identification of early life history bottlenecks in marine invertebrates, variable species responses and variable experimental approaches. Mar Env Res 76: 3–16.
View Article
Google Scholar

[193] View Article

[194] Google Scholar

[ref73] 73. Gosselin LA, Qian PY (1997) Juvenile mortality in benthic marine invertebrates. Mar Ecol Prog Ser 146: 265–282.
View Article
Google Scholar

[196] View Article

[197] Google Scholar

[ref74] 74. Bijma J, Spero H, Lea D (1999) Reassessing foraminiferal stable isotope geochemistry: Impact of the oceanic carbonate system (experimental results). Use of proxies in paleoceanography: Springer. pp. 489–512.

[ref75] 75. Gao K, Aruga Y, Asada K, Ishihara T, Akano T, et al. (1993) Calcification in the articulated coralline alga Corallina pilulifera, with special reference to the effect of elevated CO₂ concentration. Mar Biolo 117: 129–132.
View Article
Google Scholar

[200] View Article

[201] Google Scholar

[ref76] 76. Orr JC, Fabry VJ, Aumont O, Bopp L, Doney SC, et al. (2005) Anthropogenic ocean acidification over the twenty-first century and its impact on calcifying organisms. Nature 437: 681–686.
View Article
Google Scholar

[203] View Article

[204] Google Scholar

[ref77] 77. Riebesell U, Zondervan I, Rost BÈ, Tortell PD, Zeebe RE, et al. (2000) Reduced calcification of marine plankton in response to increased atmospheric CO₂. Nature 407: 364–367.
View Article
Google Scholar

[206] View Article

[207] Google Scholar

[ref78] 78. Reynaud S, Leclercq N, Romaine-Lioud S, Ferrier-Pagés C, Jaubert J, et al. (2003) Interacting effects of CO₂ partial pressure and temperature on photosynthesis and calcification in a scleractinian coral. Glob Chang Biol 9: 1660–1668.
View Article
Google Scholar

[209] View Article

[210] Google Scholar

[ref79] 79. Stumpp M, Wren J, Melzner F, Thorndyke M, Dupont S (2011) CO₂ induced seawater acidification impacts sea urchin larval development I: Elevated metabolic rates decrease scope for growth and induce developmental delay. Comp Biochem Physiol A Mol Integr Physiol 160: 331–340.
View Article
Google Scholar

[212] View Article

[213] Google Scholar

[ref80] 80. Reipschläger A, PÖRtner H (1996) Metabolic depression during environmental stress: the role of extracellular versus intracellular pH in Sipunculus nudus. J Exp Biol 199: 1801–1807.
View Article
Google Scholar

[215] View Article

[216] Google Scholar

[ref81] 81. Fabry VJ, Seibel BA, Feely RA, Orr JC (2008) Impacts of ocean acidification on marine fauna and ecosystem processes. ICES J Mar Sci 65: 414–432.
View Article
Google Scholar

[218] View Article

[219] Google Scholar

[ref82] 82. Cummings V, Hewitt J, Van Rooyen A, Currie K, Beard S, et al. (2011) Ocean acidification at high latitudes: potential effects on functioning of the Antarctic bivalve Laternula elliptica. PloS one 6: e16069.
View Article
Google Scholar

[221] View Article

[222] Google Scholar

[ref83] 83. Nilsson GE, Crawley N, Lunde IG, Munday PL (2008) Elevated temperature reduces the respiratory scope of coral reef fishes. Glob Chang Biol 15: 1405–1412.
View Article
Google Scholar

[224] View Article

[225] Google Scholar

[ref84] 84. Bibby R, Widdicombe S, Parry H, Spicer J, Pipe R (2008) Effect of ocean acidification on the immune response of the blue mussel, Mytilus edulis. Aquat Biol 2: 67–74.
View Article
Google Scholar

[227] View Article

[228] Google Scholar

[ref85] 85. Neuberger-Cywiak L, Achituv Y, Garcia E (2007) Effects of sublethal Zn⁺ and Cd⁺ concentrations on filtration rate, absorption efficiency and scope for growth in Donax trunculus (Bivalvia; Donacidae). Bull Environ Contam Toxicol 79: 622–627.
View Article
Google Scholar

[230] View Article

[231] Google Scholar

[ref86] 86. Berge JA, Bjerkeng B, Pettersen O, Schaanning MT, Oxnevad S (2006) Effects of increased sea water concentrations of CO₂ on growth of the bivalve Mytilus edulis L. Chemosphere. 62: 681–687.
View Article
Google Scholar

[233] View Article

[234] Google Scholar

[ref87] 87. Range P, Chícharo M, Ben-Hamadou R, Piló D, Matias D, et al. (2011) Calcification, growth and mortality of juvenile clams Ruditapes decussatus under increased pCO₂ and reduced pH: Variable responses to ocean acidification at local scales? J Exp Mar Biol Ecol 396: 177–184.
View Article
Google Scholar

[236] View Article

[237] Google Scholar

[ref88] 88. Shirayama Y, Thornton H (2005) Effect of increased atmospheric CO₂ on shallow water marine benthos. J Geophys Res 110: C09S08.
View Article
Google Scholar

[239] View Article

[240] Google Scholar

[ref89] 89. Lischka S, Büdenbender J, Boxhammer T, Riebesell U (2010) Impact of ocean acidification and elevated temperatures on early juveniles of the polar shelled pteropod Limacina helicina: mortality, shell degradation, and shell growth. Biogeosci discuss 7: 8177–8214.
View Article
Google Scholar

[242] View Article

[243] Google Scholar

[ref90] 90. Vahl O (1981) Energy transformations by the Iceland scallop, Chlamys islandica (OF Müller), from 70° N The age-specific energy budget and net growth efficiency. J Exp Mar Biol Ecol 53: 281–296.
View Article
Google Scholar

[245] View Article

[246] Google Scholar

[ref91] 91. Caddy J (1972) Progressive loss of byssus attachment with size in the sea scallop, Placopecten magellanicus (Gmelin). J Exp Mar Biol Ecol 9: 179–190.
View Article
Google Scholar

[248] View Article

[249] Google Scholar

[ref92] 92. Christophersen G, Strand Ø (2003) Effect of reduced salinity on the great scallop (Pecten maximus) spat at two rearing temperatures. Aquaculture 215: 79–92.
View Article
Google Scholar

[251] View Article

[252] Google Scholar

[ref93] 93. O'Connor WA, Heasman MP (1998) Ontogenetic changes in salinity and temperature tolerance in the doughboy scallop, Mimachlamys asperrima. J Shellfish Res 17: 89–96.
View Article
Google Scholar

[254] View Article

[255] Google Scholar

[ref94] 94. Paul JD (1980) Upper temperature tolerance and the effects of temperature on byssus attachment in the queen scallop, Chlamys opercularis (L.). J Exp Mar Biol Ecol 46: 41–50.
View Article
Google Scholar

[257] View Article

[258] Google Scholar

[ref95] 95. Roberts D (1973) Some Sub-lethal effects of pesticides on the behaviour and physiology of bivalved molluscs: University of Liverpool (PhD Thesis).

[ref96] 96. Rupp GS, Parsons GJ (2004) Effects of salinity and temperature on the survival and byssal attachment of the lion's paw scallop Nodipecten nodosus at its southern distribution limit. J Exp Mar Biol Ecol 309: 173–198.
View Article
Google Scholar

[261] View Article

[262] Google Scholar

[ref97] 97. Brand AR (1991) Scallop Ecology: Distributions and Behaviour. In: Shumway SE, editor. Scallops: Biology, Ecology and Aquaculture. Amsterdam: Elsevier.

[ref98] 98. Langenbuch M, Bock C, Leibfritz D, Pörtner H-O (2006) Effects of environmental hypercapnia on animal physiology: A ₁₃C NMR study of protein synthesis rates in the marine invertebrate Sipunculus nudus. Comp Biochem Physiol A Mol Integr Physiol 144: 479–484.
View Article
Google Scholar

[265] View Article

[266] Google Scholar

Mixed Effects of Elevated pCO₂ on Fertilisation, Larval and Juvenile Development and Adult Responses in the Mobile Subtidal Scallop Mimachlamys asperrima (Lamarck, 1819)

Mixed Effects of Elevated pCO₂ on Fertilisation, Larval and Juvenile Development and Adult Responses in the Mobile Subtidal Scallop Mimachlamys asperrima (Lamarck, 1819)

Figures

Abstract

Introduction

Materials and Methods

Broodstock Collection

Monitoring of CO₂

Fertilisation and Early Development

Larval Development

Settlement and Exposure of Spat

Physiological Measurements on Adults

Standard Metabolic Rate (SMR)

Equation 1. SMR calculation.

Condition Index

Equation 2. Condition Index calculation.

Haemolymph pH (pH_e) and O₂ (p_eO₂)

Data Analysis

Results

Fertilisation and Early Development

Larval Development in Acute Exposure Experiments

Adults

Discussion

Fertilisation and Larvae

Adults

Acknowledgments

Author Contributions

References

Figures

Abstract

Introduction

Materials and Methods

Broodstock Collection

Monitoring of CO2

Fertilisation and Early Development

Larval Development

Settlement and Exposure of Spat

Physiological Measurements on Adults

Standard Metabolic Rate (SMR)

Equation 1. SMR calculation.

Condition Index

Equation 2. Condition Index calculation.

Haemolymph pH (pHe) and O2 (peO2)

Data Analysis

Results

Fertilisation and Early Development

Larval Development in Acute Exposure Experiments

Adults

Discussion

Fertilisation and Larvae

Adults

Acknowledgments

Author Contributions

References

Monitoring of CO₂

Haemolymph pH (pH_e) and O₂ (p_eO₂)