Evolutionary change during experimental ocean acidification

Rising atmospheric carbon dioxide (CO₂) conditions are driving unprecedented changes in seawater chemistry, resulting in reduced pH and carbonate ion concentrations in the Earth’s oceans. This ocean acidification has negative but variable impacts on individual performance in many marine species. However, little is known about the adaptive capacity of species to respond to an acidified ocean, and, as a result, predictions regarding future ecosystem responses remain incomplete. Here we demonstrate that ocean acidification generates striking patterns of genome-wide selection in purple sea urchins (Strongylocentrotus purpuratus) cultured under different CO₂ levels. We examined genetic change at 19,493 loci in larvae from seven adult populations cultured under realistic future CO₂ levels. Although larval development and morphology showed little response to elevated CO₂, we found substantial allelic change in 40 functional classes of proteins involving hundreds of loci. Pronounced genetic changes, including excess amino acid replacements, were detected in all populations and occurred in genes for biomineralization, lipid metabolism, and ion homeostasis—gene classes that build skeletons and interact in pH regulation. Such genetic change represents a neglected and important impact of ocean acidification that may influence populations that show few outward signs of response to acidification. Our results demonstrate the capacity for rapid evolution in the face of ocean acidification and show that standing genetic variation could be a reservoir of resilience to climate change in this coastal upwelling ecosystem. However, effective response to strong natural selection demands large population sizes and may be limited in species impacted by other environmental stressors.

Genes related to ion-transport and energy production are upregulated in response to CO2-driven pH decrease in corals: new insights from transcriptome analysis

Since the preindustrial era, the average surface ocean pH has declined by 0.1 pH units and is predicted to decline by an additional 0.3 units by the year 2100. Although subtle, this decreasing pH has profound effects on the seawater saturation state of carbonate minerals and is thus predicted to impact on calcifying organisms. Among these are the scleractinian corals, which are the main builders of tropical coral reefs. Several recent studies have evaluated the physiological impact of low pH, particularly in relation to coral growth and calcification. However, very few studies have focused on the impact of low pH at the global molecular level. In this context we investigated global transcriptomic modifications in a scleractinian coral (Pocillopora damicornis) exposed to pH 7.4 compared to pH 8.1 during a 3-week period. The RNAseq approach shows that 16% of our transcriptome was affected by the treatment with 6% of upregulations and 10% of downregulations. A more detailed analysis suggests that the downregulations are less coordinated than the upregulations and allowed the identification of several biological functions of interest. In order to better understand the links between these functions and the pH, transcript abundance of 48 candidate genes was quantified by q-RT-PCR (corals exposed at pH 7.2 and 7.8 for 3 weeks). The combined results of these two approaches suggest that pH≥7.4 induces an upregulation of genes coding for proteins involved in calcium and carbonate transport, conversion of CO₂ into HCO₃⁻ and organic matrix that may sustain calcification. Concomitantly, genes coding for heterotrophic and autotrophic related proteins are upregulated. This can reflect that low pH may increase the coral energy requirements, leading to an increase of energetic metabolism with the mobilization of energy reserves. In addition, the uncoordinated downregulations measured can reflect a general trade-off mechanism that may enable energy reallocation.

Environmental salinity modulates the effects of elevated CO2 levels on juvenile hard shell clams, Mercenaria mercenaria

Ocean acidification due to increasing atmospheric CO₂ concentrations results in a decrease in seawater pH and shifts in the carbonate chemistry that can negatively affect marine organisms. Marine bivalves such as the hard shell clams Mercenaria mercenaria serve as ecosystem engineers in estuaries and coastal zones of the western Atlantic and, as for many marine calcifiers, are sensitive to the impacts of ocean acidification. In estuaries, the effects of ocean acidification can be exacerbated by low buffering capacity of brackish waters, acidic inputs from freshwaters and land, and/or the negative effects of salinity on organisms’ physiology. We determined the interactive effects of 21 weeks of exposure to different levels of CO₂ (~395, 800 and 1500 µatm corresponding to pH of 8.2, 8.1 and 7.7 respectively) and salinity (32 vs. 16) on biomineralization, shell properties and energy metabolism of juveniles of the hard shell clam M. mercenaria. Low salinity had profound effects on survival, energy metabolism and biomineralization of hard shell clams and modulated their responses to elevated P_CO2. Negative effects of low salinity in juvenile clams were mostly due to the strongly elevated basal energy demand indicating energy deficiency that led to reduced growth, elevated mortality and impaired shell maintenance (evidenced by the extensive damage to the periostracum). The effects of elevated P_CO2 on physiology and biomineralization of hard shell clams were more complex. Elevated P_CO2 (~800-1500 µatm) had no significant effects on standard metabolic rates (indicative of the basal energy demand), but affected growth and shell mechanical properties in juvenile clams. Moderate hypercapnia (~800 µatm P_CO2) increased shell and tissue growth and reduced mortality of juvenile clams in high salinity exposures; however, these effects were abolished under the low salinity conditions or at high P_CO2 (~1500 µatm). Mechanical properties of the shell (measured as microhardness and fracture toughness of the shells) were negatively affected by elevated CO₂ alone or in combination with low salinity, which may have important implications for protection against predators or environmental stressors. Our data indicate that environmental salinity can strongly modulate responses to ocean acidification in hard shell clams and thus should be taken into account when predicting the effects of ocean acidification on estuarine bivalves.

Temperature and CO2 additively regulate physiology, morphology and genomic responses of larval sea urchins, Strongylocentrotus purpuratus

Ocean warming and ocean acidification, both consequences of anthropogenic production of CO₂, will combine to influence the physiological performance of many species in the marine environment. In this study, we used an integrative approach to forecast the impact of future ocean conditions on larval purple sea urchins (Strongylocentrotus purpuratus) from the northeast Pacific Ocean. In laboratory experiments that simulated ocean warming and ocean acidification, we examined larval development, skeletal growth, metabolism and patterns of gene expression using an orthogonal comparison of two temperature (13°C and 18°C) and pCO₂ (400 and 1100 μatm) conditions. Simultaneous exposure to increased temperature and pCO₂ significantly reduced larval metabolism and triggered a widespread downregulation of histone encoding genes. pCO₂ but not temperature impaired skeletal growth and reduced the expression of a major spicule matrix protein, suggesting that skeletal growth will not be further inhibited by ocean warming. Importantly, shifts in skeletal growth were not associated with developmental delay. Collectively, our results indicate that global change variables will have additive effects that exceed thresholds for optimized physiological performance in this keystone marine species.

Impact of ocean acidification on metabolism and energetics during early life stages of the intertidal porcelain crab Petrolisthes cinctipes

Absorption of elevated atmospheric CO₂ is causing surface ocean pH to decline, a process known as ocean acidification (OA). To date, few studies have assessed the physiological impacts of OA on early life-history stages of intertidal organisms, which transition from habitats with fluctuating pH (intertidal zone) to relatively stable (pelagic zone) pH environments. We used the intertidal crab Petrolisthes cinctipes to determine whether metabolic responses to year 2300 predictions for OA vary among early developmental stages and to examine whether the effects were more pronounced in larval stages developing in the open ocean. Oxygen consumption rate, total protein, dry mass, total lipids and C/N were determined in late-stage embryos, zoea I larvae and newly settled juveniles reared in ambient pH (7.93±0.06) or low pH (7.58±0.06). After short-term exposure to low pH, embryos displayed 11% and 6% lower metabolism and dry mass, respectively, which may have an associated bioenergetic cost of delayed development to hatching. However, metabolic responses appeared to vary among broods, suggesting significant parental effects among the offspring of six females, possibly a consequence of maternal state during egg deposition and genetic differences among broods. Larval and juvenile metabolism were not affected by acute exposure to elevated CO₂. Larvae contained 7% less nitrogen and C/N was 6% higher in individuals reared at pH 7.58 for 6 days, representing a possible switch from lipid to protein metabolism under low pH; the metabolic switch appears to fully cover the energetic cost of responding to elevated CO₂. Juvenile dry mass was unaffected after 33 days exposure to low pH seawater. Increased tolerance to low pH in zoea I larvae and juvenile stages may be a consequence of enhanced acid–base regulatory mechanisms, allowing greater compensation of extracellular pH changes and thus preventing decreases in metabolism after exposure to elevated P_CO2. The observed variation in responses of P. cinctipes to decreased pH in the present study suggests the potential for this species to adapt to future declines in near-shore pH.

Effects of ocean acidification on early life-history stages of the intertidal porcelain crab Petrolisthes cinctipes

Intertidal zone organisms naturally experience daily fluctuations in pH, presently reaching values beyond what is predicted for open ocean surface waters from ocean acidification (OA) by the year 2100, and thus present an opportunity to study the pH sensitivity of organisms that are presumably adapted to an acidified environment. The intertidal zone porcelain crab, Petrolisthes cinctipes, was used to study physiological responses to low pH in embryonic, larval and newly recruited juvenile life-history stages. In these crabs, embryonic development occurs in the pH-variable intertidal zone (pH 6.9–9.5), larvae mature in the more stable pelagic environment (pH 7.9–8.2), and juvenile crabs settle back into the pH-variable intertidal zone. We examined survival, cardiac performance, energetics and morphology in embryonic, larval and juvenile crabs exposed to two pH conditions (pH 7.9 and 7.6). Embryos and larvae were split by brood between the pH treatments for 9 days to examine brood-specific responses to low pH. Hatching success did not differ between pH conditions, but ranged from 30% to 95% among broods. Larval survival was not affected by acidification, but juvenile survival was reduced by ~30% after longer (40 days) exposure to low pH. Embryonic and larval heart rates were 37% and 20% lower at low pH, and there was a brood-specific response in embryos. Embryos did not increase in volume under acidified conditions, compared with a 15% increase in ambient conditions. We conclude that sustained exposure to low pH could be detrimental to P. cinctipes embryos and larvae despite the fact that embryos are regularly exposed to naturally fluctuating hypercapnic water in the intertidal zone. Importantly, our results indicate that early life-history stage responses to OA may be brood specific through as yet undetermined mechanisms.

Effects of in situ CO2 enrichment on the structural and chemical characteristics of the seagrass Thalassia testudinum

Seagrasses commonly display carbon-limited photosynthetic rates. Thus, increases in atmospheric pCO₂, and consequentially oceanic CO_2(aq) concentrations, may prove beneficial. While addressed in mesocosms, these hypotheses have not been tested in the field with manipulative experimentation. This study examines the effects of in situ CO_2(aq) enrichment on the structural and chemical characteristics of the tropical seagrass, Thalassia testudinum. CO_2(aq) availability was manipulated for 6 months in clear, open-top chambers within a shallow seagrass meadow in the Florida Keys (USA), reproducing forecasts for the year 2100. Structural characteristics (leaf area, leaf growth, shoot mass, and shoot density) were unresponsive to CO_2(aq) enrichment. However, leaf nitrogen and phosphorus content declined on average by 11 and 21 %, respectively. Belowground, non-structural carbohydrates increased by 29 %. These results indicate that increased CO_2(aq) availability may primarily alter the chemical composition of seagrasses, influencing both the nutrient status and resilience of these systems.

Calcification by juvenile corals under heterotrophy and elevated CO2

Ocean acidification (OA) threatens the existence of coral reefs by slowing the rate of calcium carbonate (CaCO₃) production of framework-building corals thus reducing the amount of CaCO₃ the reef can produce to counteract natural dissolution. Some evidence exists to suggest that elevated levels of dissolved inorganic nutrients can reduce the impact of OA on coral calcification. Here, we investigated the potential for enhanced energetic status of juvenile corals, achieved via heterotrophic feeding, to modulate the negative impact of OA on calcification. Larvae of the common Atlantic golf ball coral, Favia fragum, were collected and reared for 3 weeks under ambient (421 μatm) or significantly elevated (1,311 μatm) CO₂ conditions. The metamorphosed, zooxanthellate spat were either fed brine shrimp (i.e., received nutrition from photosynthesis plus heterotrophy) or not fed (i.e., primarily autotrophic). Regardless of CO₂ condition, the skeletons of fed corals exhibited accelerated development of septal cycles and were larger than those of unfed corals. At each CO₂ level, fed corals accreted more CaCO₃ than unfed corals, and fed corals reared under 1,311 μatm CO₂ accreted as much CaCO₃ as unfed corals reared under ambient CO₂. However, feeding did not alter the sensitivity of calcification to increased CO₂; ∆ calcification/∆Ω was comparable for fed and unfed corals. Our results suggest that calcification rates of nutritionally replete juvenile corals will decline as OA intensifies over the course of this century. Critically, however, such corals could maintain higher rates of skeletal growth and CaCO₃ production under OA than those in nutritionally limited environments.

Metabolic shifts in the Antarctic fish Notothenia rossii in response to rising temperature and PCO2

Introduction

Ongoing ocean warming and acidification increasingly affect marine ecosystems, in particular around the Antarctic Peninsula. Yet little is known about the capability of Antarctic notothenioid fish to cope with rising temperature in acidifying seawater. While the whole animal level is expected to be more sensitive towards hypercapnia and temperature, the basis of thermal tolerance is set at the cellular level, with a putative key role for mitochondria. This study therefore investigates the physiological responses of the Antarctic Notothenia rossii after long-term acclimation to increased temperatures (7°C) and elevated PCO₂ (0.2 kPa CO₂) at different levels of physiological organisation.

Results

For an integrated picture, we analysed the acclimation capacities of N. rossii by measuring routine metabolic rate (RMR), mitochondrial capacities (state III respiration) as well as intra- and extracellular acid–base status during acute thermal challenges and after long-term acclimation to changing temperature and hypercapnia. RMR was partially compensated during warm- acclimation (decreased below the rate observed after acute warming), while elevated PCO₂ had no effect on cold or warm acclimated RMR. Mitochondrial state III respiration was unaffected by temperature acclimation but depressed in cold and warm hypercapnia-acclimated fish. In both cold- and warm-exposed N. rossii, hypercapnia acclimation resulted in a shift of extracellular pH (pH_e) towards more alkaline values. A similar overcompensation was visible in muscle intracellular pH (pH_i). pH_i in liver displayed a slight acidosis after warm normo- or hypercapnia acclimation, nevertheless, long-term exposure to higher PCO₂ was compensated for by intracellular bicarbonate accumulation.

Conclusion

The partial warm compensation in whole animal metabolic rate indicates beginning limitations in tissue oxygen supply after warm-acclimation of N. rossii. Compensatory mechanisms of the reduced mitochondrial capacities under chronic hypercapnia may include a new metabolic equilibrium to meet the elevated energy demand for acid–base regulation. New set points of acid–base regulation under hypercapnia, visible at the systemic and intracellular level, indicate that N. rossii can at least in part acclimate to ocean warming and acidification. It remains open whether the reduced capacities of mitochondrial energy metabolism are adaptive or would impair population fitness over longer timescales under chronically elevated temperature and PCO₂.

Elevated CO2 alters larval proteome and its phosphorylation status in the commercial oyster, Crassostrea hongkongensis

Ocean acidification (OA) is beginning to have noticeable negative impact on calcification rate, shell structure and physiological energy budgeting of several marine organisms; these alter the growth of many economically important shellfish including oysters. Early life stages of oysters may be particularly vulnerable to OA-driven low pH conditions because their shell is made up of the highly soluble form of calcium carbonate (CaCO₃) mineral, aragonite. Our long-term CO₂ perturbation experiment showed that larval shell growth rate of the oyster species Crassostrea hongkongensis was significantly reduced at pH < 7.9 compared to the control (8.2). To gain new insights into the underlying mechanisms of low-pH-induced delays in larval growth, we have examined the effect of pH on the protein expression pattern, including protein phosphorylation status at the pediveliger larval stage. Using two-dimensional electrophoresis and mass spectrometry, we demonstrated that the larval proteome was significantly altered by the two low pH treatments (7.9 and 7.6) compared to the control pH (8.2). Generally, the number of expressed proteins and their phosphorylation level decreased with low pH. Proteins involved in larval energy metabolism and calcification appeared to be down-regulated in response to low pH, whereas cell motility and production of cytoskeletal proteins were increased. This study on larval growth coupled with proteome change is the first step toward the search for novel Protein Expression Signatures indicative of low pH, which may help in understanding the mechanisms involved in low pH tolerance.