Ocean acidification represents a deleterious anthropogenic influence that can affect the vital functions of marine organisms1, with many studies reporting implications for growth, development and reproduction due to energetic trade-offs in important energy-demanding processes2,3,4. If organisms cannot maintain their energy budgets then their reproductive contribution to subsequent generations may be compromised, with implications for species’ abundances and distributions5. Predicting the fate of marine biodiversity in the face of climate change, including ocean acidification, will therefore require scaling up from impacts upon individuals’ physiology and fitness to population level demographic processes.

We investigated the effects of ocean acidification on the gastropod Hexaplex trunculus (L.), a widespread Mediterranean mollusc. Our study made use of CO 2 seeps off the coast of Isola Vulcano (Sicily, Italy), which provide a natural gradient of pH and a carbonate chemistry analogue for future oceans6, as well as the means to investigate naturally-assembled communities. As a slow-moving and predominantly direct developing organism H. trunculus has low dispersal capacity (relative to the spatial scale of the seep system), resulting in the potential for population genetic structuring at a fine geographical scale. This makes it an ideal model with which to investigate individual- to population-level responses to prolonged (multigenerational), exposure to elevated pCO 2 .

Three sites along the natural pH gradient were used (Fig. 1; full carbonate chemistry details are presented in Supplementary Table 1). All three sites experience stable ambient temperature and salinity, with the mean surface seawater pH progressively decreasing with proximity to the CO 2 seeps7. The Low pH site experienced elevated pCO 2 conditions (7.65 ± 0.01 pH T ), the Control site experienced ambient pH conditions (8.00 ± 0.01 pH T ) over the long-term, albeit with periods of short-term (hours/days) pH variability (likely associated with wind-driven currents7) and the Reference site experienced long-term stable, ambient pH conditions (8.07 ± 0.01 pH T ), unaffected by the seeps.

Figure 1 Map of the study area. Baia di Levante (Isola Vulcano, Sicily), showing sampling sites ‘Low pH’ (pH T 7.65 ± 0.01), ‘Control’ (pH T 8.00 ± 0.01) and ‘Reference’ (pH T 8.07 ± 0.01), with ‘x’ indicating the gas seeps. The map was generated using ArcGIS 9.3 software (http://www.esri.com/software/arcgis/). Full size image

Environmental factors modulate metabolic activity through alterations in resource demand and/or availability. Should insufficient resources be available, then stress-induced trade-offs between energy demanding processes can occur (e.g. growth, shell maintenance and reproduction)4. The capacity of an organism to maintain elevated metabolic rates (relative to their tissue mass4) may allow them to sustain positive life-history traits8 and determine organism fitness in future acidified oceans4.

Using reciprocal transplants of H. trunculus between two sites (Low pH and Control site) we tested how ocean acidification modulates metabolic activity using oxygen consumption rate ( ) as a proxy. Results demonstrate that metabolic rate was significantly increased by exposure to elevated pCO 2 (ANOVA, F 1,20 = 4.87, p = 0.039). The mean of individuals collected and caged at the Low pH site was significantly higher compared to individuals collected from and caged in control conditions (Fig. 2). This suggests that individuals of H. trunculus experience increased energetic demand under persistent exposure to ocean acidification, in part this is likely due to higher predicted costs of maintaining calcification and acid–base homeostasis9. Both nuclear and mtDNA genetic markers showed no evidence of restricted gene flow between populations at these two sites (see Supplementary Table 2 and 3) suggesting that differences in metabolic rate were a result of acclimatisation rather than being driven by genetic differences attributable to drift or population isolation. When H. trunculus individuals were reciprocally transplanted between Control and Low pH sites, metabolic rates did not significantly differ (Tukey HSD post-hoc, p > 0.05), with mean intermediate between the other two treatments (Fig. 2). These results suggest that individuals entering or leaving acidified areas may acclimate via physiological plasticity8 (Fig. 2).

Figure 2 Effect of exposure to different pCO 2 /pH conditions on the mean (± S.E.) oxygen consumption rate of H. trunculus that were either (i) collected in the Control site and re-transplanted in the Control site (Control-Control), (ii) transplanted from the Control site to the Low pH site (Control-Low pH), (iii) re-transplanted within Low pH site (Low pH-Low pH) and (iv) transplanted from Low pH into the Control Site (Low pH-Control). Mean is expressed as nmol O 2 h−1 mg−1 (WW) STPD. Significantly different treatments are indicated by different lower case letters above the column (Tukey HSD, p ≤ 0.05). Full size image

Acclimatisation can buffer populations against the immediate impacts of ocean acidification and even provide time for adaptation10. However, it can also result in stress-induced energetic trade-offs. Mean shell length (ANOVA, F 2,208 = 3.33, p = 0.034) and thickness (ANOVA, F 2,66 = 4.36, p = 0.017) in the acidified conditions were significantly lower (Fig. 3), but by contrast the mean (shell-free) body mass of individuals was significantly greater in the Low pH site (ANOVA, F 2,66 = 22.37, p < 0.001; Fig. 4). Regulation of pH at the site of calcification is energetically costly11 and associated increases in energy partitioning towards maintaining acid–base homeostasis may reduce net calcification rates9. Reduced shell thickness may also lessen any growth-limiting effect that the shell structure has on somatic tissue growth11, allowing for a larger body mass (assuming sufficient energy is available).

Figure 3 Shells of the investigated species. Samples of H. trunculus living at the Low pH site (a) showing shell dissolution and reduced shell size when compared to individuals from the Control (b) and Reference site (c). The individuals displayed are representative for both the mean shell length, as well as the overall shell condition and shape based on geometric morphometric analyses (Harvey, unpublished data). Full size image

Figure 4 Effect of exposure to different pCO 2 /pH conditions on the mean (±S.E.) dry shell-free body mass of male (open bars) and female (grey bars) individuals (length 44 ± 1.2 mm [S.E.]) from the Low pH, Control and Reference site. Full size image

When scaling from the individual to the population there is a need to consider whether responses are sex-specific, since any disproportionate effects on one or the other sex could subsequently affect population demographics. To our knowledge, no previous studies have provided evidence for skewed sex ratios associated with ocean acidification. However, it has been previously demonstrated that sex-specific effects can alter how an individual responds to, or is affected by ocean acidification. For instance, the metabolome of males and females of the blue mussel Mytilus edulis differed significantly when exposed to reduced pH12. Moreover, it has been suggested that selection pressures to ocean acidification may differ between sexes13. We found that significantly fewer females were present in the Low pH site (32.26%, χ2 1, 31 = 3.90, p = 0.048), while the sex ratio in the ambient pH sites did not significantly differ from equality (Control, 54.84% female; Reference, 45.83% female, χ2 both p > 0.50). It should be noted that there are increased levels of trace elements and heavy metals at our low pH site (due to the use of the CO 2 seep site14). While a previous study, at the same study sites and using the same species as the present work, found no spatial pattern in trace element bioaccumulation15 it is not possible to disentangle the effects of reduced pH from potential changes in sex ratios that can occur through the bioaccumulation of heavy metals and trace elements16,17. Therefore, while our findings imply that sex-specific responses to ocean acidification may occur with consequences for individual performance and fitness, as well as for population dynamics, some caution in their interpretation is advised.

Despite the absence of genetic differentiation between the sites’, the prominent feature of the genetic data was the large, significant F IS values reported for the Low pH site compared to non-significant values reported for both ambient pH sites (Low pH multi-locus F IS = 0.2036, p < 0.0001; Control multi-locus F IS = −0.0368, p = 0.2692; Reference multi-locus F IS = −0.0379, p = 0.9879). The F IS values suggest that for H. trunculus some aspect of the acidified environment results in greater departures from Hardy-Weinberg equilibrium outcrossing genotype proportions in the form of heterozygote deficits. Such site-specific heterozygote deficits could be generated by inbreeding, natural selection acting on the genetic markers, or spatial/temporal structure within samples known as the Wahlund effect18. As the significant multi-locus F IS values resulted from significant values at the majority of individual loci, locus-specific selection effects are unlikely. Inbreeding effects within a small population can also be discounted, as the Low pH site sample reported significantly reduced mean individual relatedness (mr) values compared to the Control (p = 0.03) and Reference (p = 0.048) sites (Low pH mr = −0.514; Control mr = 0.0077; Reference mr = 0.0066).

For a Wahlund effect to be observed, genetically distinct populations must occur within the geographical scale of the sampling. Due to the high gene flow indicated throughout the sampled area, any heterogeneity in allele frequencies is more likely to be generated by increased variance in reproductive success among individuals, rather than by spatial population structuring per se. Such variance in reproductive success among individuals has been reported for many highly fecund marine invertebrates and is often referred to as sweepstakes recruitment19. Within such a reproductive system many individuals do not contribute to recruitment in a given cycle, resulting in short-term genetic drift generating allele frequency differences among groups of recruits even though they are all derived from a single population. These differences among groups generate the observed heterozygote deficits and decreased mean relatedness within samples.

Variance in reproductive success among individuals may be driven by any deterministic and/or stochastic processes influencing fitness and ocean acidification has been shown to strongly influence recruitment processes20. Previous studies have demonstrated that climate derived stressors may result in a reduction in the proportion of the population that are reproductively active and an increase in reproductive failure (such as in the limpet Patella vulgata21). In our study significantly reduced numbers of females in the Low pH site represents an obvious mechanism that could increase variance in reproductive success among males, however altered energy allocations and/or other cryptic factors may also contribute.