Experimental Design

Two separate experiments were conducted to specifically isolate the effects of environmental pH/Ω_ar manipulation during the first larval shell formation (0–48 h pf = prodissoconch I = “PD1 trial”) and the veliger shell development stage (2–21 days pf = prodissoconch II = “PD2 trial”). Both trials examined the acute effects on the larvae and carry-over effects when the juveniles returned to a control environment. The PD1 trial, therefore, included a 3-wk veliger assessment period, whereas the PD2 trial was extended to 10 days postsettlement (Fig. 1).

Seawater was either left unmodified (Ω_ar ∼2 = control treatments) or adjusted to an approximate Ω_ar of ∼0.5 and 1.0 by elevating pCO₂, or enriched with sodium carbonate solution to Ω_ar ∼3, 4, or 7. The latter treatment was chosen as an extreme, likely to result in suboptimal performance because of uncontrolled carbonate deposition (G. Waldbusser, personal communication). Endpoint modification was determined by continuous measurement of free pH (EcoSense EC1030A, YSI, Yellow Springs, OH; calibrated using Merck Hanna pH standards), with target values estimated by CO2calc (v4.0.9; Robbins & Kleypas 2011) using dissociation constants established by Mehrbach et al. (1973) and refitted by Dickson and Millero (1987), using a mean total alkalinity (A _T) of 2,274 μmol kg^–1 previously measured by Ericson (2010). Carbonate system parameters, including Ω_ar and total pH, were subsequently verified by taking 1 L water samples, fixed with 100 μL saturated mercuric chloride, for assessment of total dissolved inorganic carbon (DIC) and A _T (Currie Laboratory, University of Otago). Dissolved inorganic carbon was determined (±1 μmol kg^–1) using a coulometer (UIC model 5011) and SOMMA-style CO₂ extraction system (Dickson et al. 2007; SOP2), and A _T was assessed using closed-cell potentiometric titration (±2 μmol kg^–1; after Dickson et al. 2007; SOP3). Both assays were calibrated against certified reference material (A. Dickson, Scripps Institution of Oceanography).

Brood Stock and Spawning

Mussel collection and spawning for PD1 took place in November 2014 (austral spring) and in March 2015 (autumn) for PD2. Mature adult Perna canaliculus were carefully removed from aquaculture long-lines in Pelorus Sound (South Island, New Zealand) and transported in humid air (∼4 h at 8°C) to the Cawthron Aquaculture Park. Epifauna was removed by mechanical scrubbing, a freshwater rinse followed by 2 min immersion in 0.4% (v/v) sodium hypochlorite, and brief rehydration in seawater. Mussels were then emersed and held in humid air at 16°C for 12 h overnight. Spawning was induced by thermal shock: Mussels were immersed in 1-μm-filtered 34 PSU seawater at 22°C for 2 h and then cycled between 9°C and 22°C at 30 min intervals until spawning was apparent after 2.5–3 h.

Figure 1.

Experimental and sampling design for PD1 and 2 trials. “*” identifies net performance assessment points (population counts, visual assessment, and shell length measurement). “+” identifies sample points for oxidative stress profiling (DNA, lipid, and protein damage markers: 8-OHdG, LP, and protein carbonyls; antioxidant enzymes: SOD, CAT, GR, glutathione peroxidase, and glutathione S-transferase) and dry mass. “o” represents sampling for total ROS. Note expanded time scale (d pf = days postfertilization). V and pH refer to mean aragonite saturation state and total pH, respectively.

Spawning females were rinsed and isolated in 1 L containers with 0.5 L of 8°C seawater, where the negatively buoyant eggs were regularly aspirated and consolidated in a storage vial. Spawning males were wiped dry and “inverted” (siphons directed downward) over a 60-mL collection container in 16°C air, allowing the mussels to eject a concentrated mixture of sperm and residual mantle water. Eggs and sperm from the 30 most fecund individuals of either sex were stored at 4°C for up to 4 h before being pooled and counted. Eggs were added at 1,000 mL^–1 to 15 L nylon buckets containing 1-μm-filtered, 17°C seawater preincubated with 4 μM EDTA for 24 h (ethylenediamine tetra-acetic acid disodium salt, molecular grade; 5 mM stock prepared in distilled water buffered to pH 8.0), followed by sperm at a ratio of 500 per egg. Fertilization was confirmed by the appearance of polar bodies in greater than 80% of eggs within 30 min. Fertilized eggs were then transferred to 160 L conical nylon tanks containing identical seawater/ EDTA (PD2 trial) or a modified Ω_ar seawater environment (PD1 trial). Eggs were stocked at 50 mL^–1 and incubated for 2 days. Air temperature regulation maintained water temperature at 17 ± 1°C, and gentle aeration prevented stratification.

PD1 Trial: Effects of Ωar on Embryogenesis and Trochophore Development

Manipulation of Water Chemistry

Each Ω_ar treatment was established in triplicate 160-L tanks. Reduced Ω_ar treatments received CO₂-enriched aeration [using a WMA-5 CO₂ gas analyzer (PP Systems) driving a PLC system admixing CO₂/air via a 3/2-way valve (SMC Pneumatics, type S070)], which equilibrated water to Ω_ar ∼1; CO₂saturated seawater was also added at 3 L day^–1 to create the Ω_ar 0.5 treatment. A concentrated sodium carbonate solution was made by dissolving 37 g anhydrous Na₂CO₃ in distilled water at 35°C; 5, 14, and 50 mL of this solution were added slowly to the tank water to create Ω_ar 3, 4, and greater than 7, respectively. Small additional volumes were added as required to buffer a gradual pH decline. Temperature and free pH were measured in situ in each tank at 2–4 h intervals. Bottle samples were taken from each tank for A _T and DIC analysis immediately before the addition of eggs and following the removal of 2-day-old larvae (Table 1). Total dissolved calcium concentration was also measured in the incubation tank water, 48 h after the addition of eggs, by ICP-MS (Hill Laboratories, Hamilton, NZ; protocol: “Saline-ultratrace,” APHA 3125 B, 22nd ed. 2012).

TABLE 1.

Carbonate chemistry of 160 L incubation tanks used for the PD1 trial.

PD2: Manipulation of Larval Culture Water 2–21 days pf

Spawning and initial embryogenesis to 2 days pf followed the approach described for the PD1 controls mentioned earlier, using a single 160-L incubation tank. Veliger larvae were then stocked in each of six replicate 2.5-L rearing tanks (500,000 individuals tank^–1; see Ragg et al. (2010) for design details) which were assigned to one of six Ω_ar treatments designed to reproduce the carbonate environments created in PD1. The exception was the extreme “Ω_ar 7″ treatment, where a slightly reduced target of ∼6.5 was used to reduce the risk of spontaneous precipitation. Reduced Ω_ar environments were achieved by the continuous dilution of CO₂-saturated seawater in 100 L header tanks, whereas elevated Ω_ar required the addition of concentrated sodium carbonate solution (as described earlier). Axenic algal cultures were added to the modified water of each header tank to achieve a mean effluent concentration of 40 cells μL^–1 [1:2 cell ratio, Tisochrysis lutea: Chaetoceros calcitrans; Ragg et al. (2010)] and allowed to flow through each rearing tank at 80 mL min^–1. Water temperature was maintained at 20°C using air temperature control. Temperature and free pH were measured daily in each tank, and mercuric chloride-fixed water samples were taken on four occasions from each system, as described for PD1, to assess A _T and DIC entering and leaving each larval tank (Table 4).

PD1 Trial: Effects of Ω_ar on Embryogenesis and Trochophore Development

PD1 Biological Performance

Treatment had no significant effect on survival 43 h pf (F _5,12 = 2.093, P = 0.13; Fig. 2A) but substantially influenced development. Under control conditions (Ω_ar 1.9), 72.8% ± 2.9% of eggs developed into apparently normal veliger; this value rose to 82.6% ± 3.8% in the Ω_ar 4.5 treatment (Fig. 2A), but the increase was not found to be significant (Tukey's HSD, P > 0.05). Development was severely compromised in the extreme treatments, with most larvae displaying physical abnormality in the Ω_ar 0.8 and Ω_ar ∼7 treatments. Larvae reared under Ω_ar 0.5 conditions failed to reach the veliger stage (Fig. 2A). Underdeveloped control treatment larvae failed to develop to veliger, even when incubated for a further 15 h (P > 0.05; Fig. 2B and C). Further development was, however, apparent in the Ω_ar 2.9 treatment, with an initial veliger yield of 70.6% ± 2.8% (Fig. 2A) rising to 77.0% ± 4.2% after 68 h (Fig. 2C); this increase was also found to be nonsignificant (P > 0.05). By treating Ω_ar as a continuous variable, a quadratic linear model described the (arcsine transformed) 43-h pf veliger yield data very well (R² = 0.90; Table 2). Peak yield (zero gradient, determined by differential analysis) was predicted to occur at Ω_ar = 4.03. Larvae raised under Ω_ar ∼7 conditions appeared to destabilize and were progressively lost beyond 43 h pf (Fig. 2A–C). Correspondingly, individual dry mass was relatively constant across treatments 18 h pf, but subsequently failed to increase significantly by 48 h pf in the aggressive Ω_ar 0.5, 0.8, and ∼7 treatments (P < 0.05; Fig. 3).

Intracellular ROS concentrations, determined by D399 fluorescence, were statistically similar between treatments and the control (Ω_ar 1.9) in samples taken 24 h pf (Fig. 4); only Ω_ar 0.8 and Ω_ar ∼7 levels were significantly different from each other (F _{5, 12} = 3.685, P = 0.03). By 52 h pf, ROS levels had fallen in control and elevated Ω_ar treatments, but remained significantly elevated in low Ω_ar treatments (F _{5, 12} = 21.72, P < 0.001; Fig. 4). DNA and protein damage, quantified as 8-OHdG and protein carbonyl content, respectively, remained stable across treatments and life stage samples 18 and 48 h pf (Fig. 5A and C). The exception was a significant accumulation in the extreme Ω_ar 0.5 and ∼7 treatments after 48 h. Lipid hydroperoxide levels not only followed a similar pattern, but also notably reduced in the intermediate Ω_ar treatments between 18 and 48 h pf (effects of age F _{1, 24} = 13.21, P = 0.001; Fig. 5B). Defense enzymes showed the reciprocal pattern, with SOD, CAT, GR, glutathione peroxidase, and glutathione S-transferases all being significantly reduced below control treatment levels after 48 h (Fig. 5D–H). Superoxide dismutase, GR, and GPOX showed significant elevation above the control after 48 h in the Ω_ar 4.5 treatment (Fig. 5D, F and G).

Despite low or apparently zero veliger yields in extreme treatments (Fig. 2A–C), the large biomass available allowed sufficient veligers to be harvested from all incubation tanks for assessment of carry-over effects. Veligers originating from extreme Ω_ar treatments showed rapid attrition (Fig. 6). All individuals previously incubated in Ω_ar 0.5 water died within a week. Most larvae previously exposed to Ω_ar 0.8 or Ω_ar ∼7 also died within 3 days of stocking in veliger rearing tanks, with only 20%–30% of veligers surviving (Fig. 6). Control and moderately elevated treatments (Ω_ar 2.9 and 4.5) showed the steady mortality rate typical of commercial Perna canaliculus larval rearing (c.f. Ragg et al. 2010), with approximately half of the veligers surviving by 19 days pf (Fig. 6). Similar shell growth rates were observed across all surviving treatments (mean 10.01 ± 0.20 μm day^–1), so significant differences in the size of veligers entering the rearing system (3 days pf; F _5,11 = 83.08, P < 0.001) were perpetuated to the start of pediveliger harvesting (20 days pf; Fig. 7; F _4,9 = 13.4, P < 0.001). When pediveliger yield was expressed as a percentage of population initially added to the veliger tanks (counted 3 days pf; Fig. 8A), the compound effects of reduced growth and survival following Ω_ar 0.8 or ∼7 treatment were apparent, with a yield of 12%–14% compared with 38%–41% from intermediate treatments. As a result of large within-treatment variability however, differences were nonsignificant (P > 0.05). When standardized to original egg population (Fig. 8B), the acute effects experienced during the incubation treatment itself are also accommodated. No net yield occurred in the low Ω_ar treatments, and only 1.9% ± 0.6% of eggs yielded pediveligers in the Ω_ar ∼7 treatment (Fig. 8B). A net control yield of 29.4% ± 5.8% increased to 33.2% ± 3.7% in the Ω_ar 4.5 treatment (Fig. 8B), but the increase was not found to be significant (P > 0.5). A quadratic regression model fitted the pediveliger yield data well (Fig. 8A, Table 3; R² = 0.69), predicting maximum yield at Ω_ar = 3.98.

Figure 2.

Net survival and proportion of population resembling normal veligers as a percentage of initial number of fertilized eggs stocked in each tank, following (A) 43 h, (B) 51 h, and (C) 68 h of incubation in modified V_ar seawater.

TABLE 2.

Quadratic regression model fitted to initial veliger yield 43 h pf.

Figure 3.

Individual dry mass of larvae sampled at 18 h (narrow bars) and 48 h pf (wide bars). Inorganic (ash) component is represented by the clear area, and organic component by the shaded area. Mean dry mass that differs significantly from the corresponding (18 or 48 h) control is marked * (P < 0.05). Bars represent mean ± SEM, n = 3.

PD2 Trial: Effects of Ω_ar Veliger Development

PD2 Biological Performance

Seawater Ω_ar did not appear to significantly influence veliger survival, with all treatments showing steady mortality rates for the first 2 wk of life, plateauing to a final mean survival of 30.6% ± 2.1% of 2 days pf population surviving 20 days pf, when pediveliger harvesting commenced (F _5,30 = 0.439, P = 0.818; Fig. 9). Shell growth was not affected by Ω_ar elevation (Fig. 10), with mean rates of 5.9–6.6 μm day^–1; however, growth fell significantly in the reduced Ω_ar treatments, 4.9–5.0 μm day^–1 (F _5,28 = 6.666, P < 0.001). Net pediveliger yield, standardized by 2 days pf population stocked in the tanks, showed substantial within-treatment variability, with only the Ω_ar 0.7 yield being significantly lower than the control (F _5,30 = 3.243, P = 0.02; Fig. 11). A quadratic regression model failed to adequately describe the yield data (R² = 0.16), preventing the estimation of an optimum Ω_ar level. No statistical differences were detected between either inorganic (ash) or total dry mass of individual pediveligers produced in the various treatments (F _5,22 = 1.386, P = 0.268; Fig. 12). It should, however, be noted that some tanks failed to produce sufficient larvae for dry matter assessment, resulting in reduced replication and statistical power. Net survival following pediveliger transfer into an unmodified seawater setting system is shown in Figure 13. Juveniles developing from larvae raised in Ω_ar 0.7, but not Ω_ar 0.6, showed significantly reduced survival (F _5,30 = 4.345, P = 0.004; Fig. 13).

Figure 4.

Relative intracellular ROS levels in larvae incubated in a range of V_ar environments for 24 and 52 h, expressed as fluorescence of the D399' molecular probe standardized to larval dry mass. Mean ± SEM, n = 3. Signals significantly different to their corresponding control are marked * (P < 0.05).

Figure 5.

Indicators of oxidative damage and defense in larvae raised for 18 or 48 h in modified V_ar water. (A) 8-OHdG indicating DNA damage; (B) LP, standardized against larval dry mass; (C) protein carbonyls; (D) SOD; (E) CAT; (F) GR; (G) GPOX; and (H) glutathione S-transferase, standardized against protein mass. Mean ± SEM, n = 3. Signals significantly different from their corresponding control are marked * (P < 0.05).

Figure 6.

Relative survival of veligers raised in control conditions, following incubation in different V_ar treatments (mean ± SEM).

Water Chemistry Manipulation

Dosing with either CO₂-saturated seawater or concentrated sodium carbonate solution was effective in modulating carbonate chemistry to achieve a range of Ω_ar environments. In the large water tanks used for the PD1 incubation, unintended pH decline was also apparent. During an initial 24-h incubation without embryos, pH_T fell from 8.10 to 7.87 and then again to 7.71 following 48 h of incubation with embryos (Table 1). The addition of EDTA may be partially responsible for the initial decrease, but it is suspected that microbial and larval metabolism accounted for most of the subsequent decline. Modified Ω_ar treatments also tended to decline, but to a lesser extent than the control seawater; by contrast, Ω_ar 4.5 increased to 5.0 during embryo occupancy possibly because of a slight overcorrection during sodium carbonate adjustment. The continuously flowing rearing system used in the PD2 trial was more stable, with only the reduced Ω_ar treatments tending to rise significantly within the larval tanks (Table 4) possibly because of progressive equilibration with air bubbling through these tanks.

Effects of Ω _ar on Early Larval Stages (0–48 h pf)

Embryogenesis, trochophore development, and initial shell formation were sensitive to the Ω_ar environment. Development was retarded in the undersaturated Ω_ar 0.5 and 0.8 treatments; larvae either failed to form a shell or became malformed and apparently nonviable (Fig. 2). Most embryos exposed to the extreme Ω_ar ∼7 also tended to have malformed shells, showing signs of hypercalcification. These aggressive Ω_ar treatments showed little sign of mass gain after 18 h pf (Fig. 3) and began to destabilize by 68 h pf (Fig. 2C). More than 70% of fertilized eggs incubated in the control (Ω_ar 1.9) or moderately elevated Ω_ar (2.9–4.5) formed normal prodissoconch I shells, becoming apparently viable veligers (Fig. 2). The relative differences between these treatments were fairly subtle; the statistical design (n = 3) was, therefore, insufficiently powerful to identify potential significant differences. The mean yield of veligers, however, increased by 13% by 43 h pf if Ω_ar was increased to 4.5% and by 8% if veligers were reared in Ω_ar 2.9 for 51–68 h (Fig. 2). Larvae incubated in Ω_ar 2.9 were also 11% heavier than controls by 48 h pf (Fig. 3). Gray et al. (2017) demonstrated a similar correlation between Ω_ar and shell length 2 days pf in Mytilus californianus larvae.

Figure 7.

Mean shell length of veligers raised in control conditions, following incubation in different V_ar treatments. Bars represent mean ± SEM 1 day after stocking in the veliger rearing system (3 days pf) and immediately before pediveliger harvesting, 20 days pf. “*” represent values significantly different from their corresponding control treatment (Tukey's HSD, P < 0.05).

Mode of Action

Under control conditions, similar antioxidant enzyme levels (SOD and GPOX) have been described in the eggs (Gale et al. 2014) and 18 h and 43 h pf larvae of Perna canaliculus (Fig. 5A and G, present study), suggesting that antioxidant protection is reliant on maternal transfer. For clarity, a simple outline of the mitigation role played by these antioxidants in stress homeostasis is presented in Figure 14. The incubation environment subtly influenced antioxidant enzyme levels of embryos within the first day of life, with significant lowering of SOD, CAT, GR, glutathione peroxidase, and glutathione S-transferase in the most undersaturated Ω_ar treatment (18 h pf; Fig. 5D–H). The 18-h pf embryos did not, however, show signs of elevated oxidative damage to DNA, lipids, or proteins, indicated by 8-OHdG, lipid hydroperoxide, and protein carbonyl levels that resembled the control in all treatments (Fig. 5A–C). By the end of the second day (43–52 h pf) however, protective enzyme levels were markedly reduced under extreme Ω_ar 0.5 and ∼7 conditions (Fig. 5D–H) and indicators of oxidative damage were elevated (Fig. 5A–C), along with intracellular ROS concentration (Fig. 4). Tomanek et al. (2011) suggest that elevated pCO₂ may cause oxidative stress by direct increase of ROS via reaction with peroxynitrite, or indirectly by lowering intracellular pH and reducing the efficiency of the electron transport chain and generating ROS. An elevation of Ω_ar and pH may, therefore, mitigate these impacts. In the present study, reduced antioxidant enzyme activity appears to precede the elevation of ROS and detectable oxidative damage, suggesting that an impairment of antioxidant synthesis or function may exacerbate the direct elevation of ROS. Interestingly, a significant elevation in antioxidant enzymes GR and GPOX was detected following 43 h incubation in Ω_ar 4.5. The mechanisms supporting these increased levels remain to be elucidated, but may relate to energy sparing in the facilitated shell-growing environment. Calcifiers use macromolecules and membrane transporters to enhance shell formation rates beyond passive precipitation rates; however, both mechanisms are energetically costly (Waldbusser et al. 2013). In young Mytilus californianus veligers exposed to undersaturated Ω_ar conditions, the energetic burden was exacerbated by a delay in the onset of feeding (Gray et al. 2017); the reverse may, therefore, apply under elevated Ω_ar. Adult bivalves (Crassostrea virginica and Mercenaria mercenaria) also showed signs of oxidative stress following acute exposure to elevated pCO₂ (800 ppm); however, most parameters had returned to baseline by the end of a two-month exposure (Matoo et al. 2013), suggesting resilience may increase in postlarval stages. The bivalve C. virginica was, however, shown to upregulate antioxidant proteins, including SOD, when challenged with a more extreme pCO₂ of ∼3,500 ppm (Tomanek et al. 2011).

Figure 8.

Net survival to pediveliger stage following 2 days of incubation in modified V_ar water and subsequent rearing under control conditions. Pediveligers are isolated based on size (>∼215 μm; Ragg et al. 2010), by removal on a 178-μm mesh 20 and 22 days pf. Values are expressed as (A) percentage of veligers stocked in tanks (3 days pf) and (B) percentage of fertilized eggs initially added to each incubation tank. Mean ± SEM, n= 3. Net yields significantly different from their corresponding control are marked * (Tukey's HSD, P < 0.05).

TABLE 3.

Quadratic regression model fitted to final pediveliger yield.

Veligers removed from modified incubation environments and transferred to control seawater rearing conditions failed to compensate for perturbations experienced during the first 2 days of life. Larvae previously exposed to undersaturated (Ω_ar 0.5–0.8) or hyper-saturated conditions (Ω_ar ∼7) displayed elevated mortality; all Ω_ar 0.5 larvae died, whereas net survival to pediveliger was substantially reduced in Ω_ar 0.8 and ∼7 larvae (30%–50% of control levels; Figs. 6 and 8A). Young veligers exposed to these aggressive calcifying conditions were significantly smaller than controls; these differences were still apparent 3 wk later, at the start of metamorphosis (Fig. 7). Gray et al. (2017) developed models suggesting that pediveliger development would be delayed by over 4 days if pre-veliger stages were exposed to undersaturated water. The findings of the present study concur, but final pediveliger yields were reduced rather than delayed, suggesting that additional factors prevent prolongation of the veliger stage. When polynomial models were fitted to yield data, the predicted optimal Ω_ar environment for early incubation was estimated to be 4.03 based on initial veliger yield and an almost identical 3.98 based on net pediveliger yield (Tables 2 and 3). Overall, the data suggest that larval status at the start of the veliger prodissoconch II stage has a substantial influence on the net capacity to recruit to the juvenile population.

Effects of Ω_ar on Veliger Larvae (2–20 days pf)

Veliger larvae exposed to adjusted Ω_ar environments from 2 days pf showed surprising resilience. Survival trajectories appeared unaffected (Fig. 9), but shell length growth was reduced by about 25% in the undersaturated treatments (Fig. 10). Consequently, the net proportion of the initial (2 days pf) population successfully attaining pediveliger size was reduced in the low Ω_ar treatments, but only Ω_ar 0.7 declines were significant (Fig. 11). Surprisingly, the pediveligers produced in undersaturated water appeared to be heavier than those produced in higher Ω_ar treatments (Fig. 12). It is suggested that carbonate limitation influenced the deposition of the prodissoconch II shell, slowing longitudinal development, resulting in a deeper shell cavity and a higher mass:shell length ratio.

TABLE 4.

Carbonate chemistry of continuous flow 2.5-L veliger rearing tanks. Values represent mean ± SEM (n= 4), for water flow into and out of each tank.

Figure 9.

Relative survival of veligers raised in continuous flow tanks (200 mL^–1, 2.5 L) receiving V_ar-modified water. Values are expressed as the mean percentage of initial tank population (2 days pf) ± SEM (n= 6) and continue to first harvest of pediveligers, 20 days pf.

Unexplained variance may have masked some treatment effects in this trial. Overall batch performance was poorer than in PD1, with 15.3% ± 2.1% of 2 days pf larvae reaching the pediveliger stage, compared with 40.3% ± 10.2% in the PD1 trial, and tank-to-tank variability was frequently higher than between-treatment effects. High between-cohort variability and within-cohort tank effects have been previously observed in Perna canaliculus larval trials (Ragg et al. 2010), as well as in other bivalve larvae (e.g., Robert & Gerard 1999). As water quality was carefully controlled during trials, it is suspected that endogenous effects associated with the acquisition of brood stock in a different season (PD1: austral spring, PD2: autumn) may drive the batch differences; however, the reasons for tank-to-tank variability remain elusive.

Figure 10.

Veliger shell growth between 2 and 14 days pf in a range of V_ar environments. Bars represent mean daily shell length gain ± SEM. Growth rates significantly different from the control are marked * (Tukey's HSD, P < 0.05).

Figure 11.

Yield of pediveliger larvae expressed as a percentage of initial tank population 2 days pf; screening was repeated 20, 22, and 23 days pf. Values are expressed as mean ± SEM, n = 6. Total yields significantly different from the control are marked * (Tukey's HSD, P < 0.05).

Carry-over effects were apparent following the removal of pediveligers from the Ω_ar 0.7 treatment, with net survival through metamorphosis and juvenile settlement being significantly lower than controls (Fig. 13). Curiously, however, no such legacy effects were apparent following rearing in Ω_ar 0.6; again, it is suspected that unexplained variance may mask a treatment effect. Hettinger et al. (2013b) observed reduced juvenile growth as a carry-over effect following rearing of Olympia oyster larvae under low Ω_ar, suggesting that the measurement of short-term survival used in the present study may not be the most suitable parameter to quantify. The development of the scallop Argopecten irradians and clam Mercenaria mercenaria veligers was also significantly influenced by pCO₂, with effects most apparent in the postmetamorphosis juveniles (Talmage & Gobler 2010). The larvae of the slow-growing Olympia oyster Ostrea lurida raised under a broad range of Ω_ar conditions also showed growth rates that remained relatively independent of Ω_ar, but were prone to malformation if grown above Ω_ar 6 (Waldbusser et al. 2016 ).

Figure 12.

Dry mass of individual pediveliger larvae retained by a 178-μm screen 20 days pf following rearing in different V_ar environments. Inorganic (ash) component is represented by the clear area, and organic component by the shaded area. Mean ± SEM, n = 3–6.

Figure 13.

Proportion of 5,000 pediveligers, transferred from modified V_ar treatments into an unmodified environment, that successfully metamorphosed and survived to 13 days postmetamorphosis (mean ± SEM, n= 6).

Ecological Implications

The experimental conditions created were deliberately designed to allow elucidation of relative sensitivity to the pH/ carbonate environment, rather than to reproduce ecologically relevant conditions. Some ecological inference can, however, be made from the data presented here. The reduced Ω_ar treatments were created by elevating pCO₂ and, therefore, mimic the effects of ocean acidification. The conditions created were extreme (pH_T 7.09–7.47), exceeding likely medium-term projections for the southern oceans (IPCC 2014). The Greenshell mussel Perna canaliculus, however, inhabits the shallow sublittoral zone of New Zealand coasts, where local conditions may result in pH and carbonate saturation levels substantially lower than the oceanic mean (Law et al. 2018). Exposure of pre-veliger life stages to pelagic conditions where Ω_ar remains below 0.8 could result in a complete recruitment failure (Fig. 8). The parents used in this study, however, were naive, being raised in water that appears to remain above Ω_ar of 1.55 (Kim Currie, NZOAON, unpublished data); as both selection pressure and conferral of resilience represent possible mechanisms for the production of offspring with increased tolerance to high pCO₂ conditions (e.g., Parker et al. 2012, Munday et al. 2013), the current findings may not fully represent vulnerability in future populations. Veliger stages are less likely to be severely impacted by ocean acidification. In the presence of abundant food, veligers exposed to undersaturated water in the PD2 trial displayed performance that was broadly comparable with the controls. Importantly, development rates were unaffected by the Ω_ar environment (Fig. 11). In a natural, food-limited environment, the veliger stage may be protracted and the length of the free-living stage may, in turn, be correlated to reduced recruitment (Alfaro et al. 2010). For example, Gray et al. (2017) observed delayed onset of feeding and lower subsequent ingestion rates in Mytilus californianus larvae exposed to undersaturated Ω_ar, reducing the scope for growth. Waldbusser et al. (2013) also argue that with a completed prodissoconch shell allowing some degree of isolation from external chemistry, veligers with access to sufficient food to overcome energetic limitations would be far more resilient to varying Ω_ar than earlier life stages. Melzner et al. (2011) have demonstrated that abundant food supply can, for example, substantially mitigate nacre dissolution caused by elevated pCO₂. Hettinger et al. (2013a) found that compromised performance of Olympia oyster veligers under elevated pCO₂ was mitigated by the presence of abundant food. An exploration of the interacting effects of ocean acidification and food limitation would, therefore, be useful.

Although the effects of Ω_ar elevation are relatively subtle, they support an important hypothesis that progressive acidification from the start of the industrial revolution is already influencing the performance of marine species. For example, in a direct exploration of this hypothesis, Talmage and Gobler (2010) exposed scallop and clam larvae to simulated preindustrial seawater conditions (Ω_ar ∼3.4) and observed significant increases in the development rate and more robust shell structure. In the present study, net veliger and pediveliger yields conformed to a parabolic, quadratic relationship with Ω_ar. Below the apex of ∼Ω_ar 4, the response curve was exponential, with the magnitude of response predicted to increase rapidly as Ω_ar declines. The implication is that Perna canaliculus, and other bivalve larvae, exposed to present-day seawater are already displaying phenotypic responses as part of a continuum, rather than true resistance awaiting a “tipping point” before effects become apparent.

Figure 14.

Simple schema of selected processes associated with the development of oxidative stress in shellfish. Red arrows track the development of oxidative stress, whereas blue arrows represent antioxidant defense and return to normality. The antioxidant defense markers targeted in the present study are SOD, CAT, and GPOX.

Commercial Application

The importance of Ω_ar to commercial bivalve hatcheries operating in the upwelling regions of the Pacific United States has been carefully explored. An economic assessment even proposes a commercial “break-even Ω_ar” of 1.7 (Barton et al. 2015). In the present study, elevating Ω_ar of the pre-veliger incubation medium to 4.5 (PD1 trial) increased both immediate veliger and subsequent pediveliger yields by an average of 13% (Figs. 2A and 8). The relative economic effects of post-industrialization OA or artificial carbonate enrichment on Greenshell mussel aquaculture would be challenging to calculate. As a broad indication, hatchery production is forecast to supply 30% of Perna canaliculus commercial spat by 2020 (Anon. 2015); if the industry size and value remain stable at ∼250m USD exports p.a. (Jeffs et al. 2018), a 13% increase in the limited hatchery seed supply would correspond to ∼10m USD of increased sales.

Incubation at Ω_ar 4.5 also significantly elevated levels of the antioxidant enzymes GR and GPOX (Fig. 5F and G). Quadratic modeling suggested that optimum veliger and pediveliger yields would, in fact, be obtained following incubation in Ω_ar ∼4 seawater (Tables 2 and 3). It should be noted that the predicted optimum Ω_ar of 4 will be influenced by the true saturation state of the hyper-saturated treatment, which was assumed to be 7 for computational purposes, but could not be assessed directly because of precipitation within the sample. Waldbusser et al. (2015) described continuing increases in shell growth of young Crassostrea gigas veligers in saturation states as high as Ω_ar 6.5, suggesting more focused investigation of high Ω_ar environments would be informative. Importantly, Ω_ar and pH declined to potentially dangerous levels in the control incubation water in the present study (Ω_ar 1.28, pH 7.71). Comparable levels are described in the Pacific NW United States, where sporadic intake of Ω_ar undersaturated seawater into commercial bivalve hatcheries can occur (Barton et al. 2012). For both scenarios, carbonate enrichment represents a benign mechanism to buffer these changes. Manipulation of Ω_ar in the constantly flowing veliger rearing system is more involved, and the potential benefits of increasing Ω_ar are equivocal (PD2 trial). As carbonate enrichment of pre-veliger incubation water is inexpensive and technically straightforward, it is recommended that Perna canaliculus tanks be enriched to Ω_ar 4 as standard practice. It is also suggested that subsequent veliger culture should be left unmodified, until compelling evidence of potential benefits becomes available. The logistical constraints associated with trials using large-volume commercial tanks resulted in low replication and statistical power in the PD1 trial. It is, therefore, recommended that culture trials continue to contrast performance with unmodified seawater, to develop a robust comparative dataset.

The combined effects of carbonate and EDTA enrichment need further consideration. The presence of EDTA is considered essential in the commercial production of many bivalve veligers (FAO 2004), and recent findings suggest that increasing EDTA concentration above 4 μM will further improve veliger yield (Gale et al. 2016). Metal speciation and bioavailability will vary with both pH and the effects of a chelator, such as EDTA; bioavailability of both beneficial and potentially toxic metals should, therefore, be considered when designing an enriched medium.

Conclusions and Future Work

The pre-veliger larvae of Perna canaliculus were found to be sensitive to the aragonite saturation state of their incubation seawater. Embryos and young larvae incubated in Ω_ar undersaturated (#0.8) or hyper-saturated (∼7) water showed acute signs of oxidative stress and compromised development. Ongoing performance issues were apparent when the juveniles were returned to normal seawater, resulting in little or no net recruitment. Elevating Ω_ar to 4.5 increased antioxidant levels and supported a 13% increase in net recruitment. By contrast, veliger performance differences were dominated by unexplained variability, rather than Ω_ar. A valuable enhancement to the approach presented here would be to consider the ramifications of Ω_ar manipulation on the other organisms in the hatchery mesocosm, notably the dietary algae and the microbiome associated with the larvae themselves. Repeated commercial trials are also required to improve the robustness of the conclusions presented here and allow a cost–benefit analysis of carbonate enrichment. Young P. canaliculus are vulnerable to extreme ocean acidification conditions. Comprehensive trials are now required to characterize the integrated life-cycle responses of this keystone ecological and commercial species to realistic near-future seawater conditions.