Award: EF-1315944

A portion of the CO2 released into the atmosphere by human activities dissolves in the ocean where it reacts with water to produce carbonic acid. This results in a reduction of the pH of seawater and a host of other chemical changes known as ocean acidification. In corals, ocean acidification reduces the formation rate of the calcium carbonate skeleton, and consequently growth. The detrimental effects of ocean acidification are compounded by numerous stressors harming coral reefs including high temperature-induced bleaching, overfishing, and nutrient pollution. This project sought to develop and apply new methods to determine the flows of inorganic carbon (CO2, HCO3-, CO32-) that supply inorganic carbon for new skeletal growth and photosynthesis in corals. Changes in these flows are likely a critical component of the response of corals to ocean acidification. The long-term goal of this research is to identify the mechanism by which ocean acidification reduces rates of skeletal growth. Work was primarily conducted in three areas: 1) modifying and applying membrane inlet mass spectrometry (MIMS) approaches that have been used to study inorganic carbon flows in microalgae to corals, 2) applying advanced microelectrode profiling methods to directly measure relevant chemicals in corals (H+, CO32-, Ca2+), and 3) developing numerical models that integrate these results to produce a unified view of inorganic carbon flows that support new skeletal growth and photosynthesis in corals. In microalgae, powerful MIMS methods have been developed that allow selected inorganic carbon flows and carbonic anhydrase activities to be measured directly. Carbonic anhydrase is an enzyme, pervasive in inorganic carbon delivery pathways, that catalyzes the hydration of CO2 and dehydration of HCO3-. We modified and extended these methods to corals, developing new models to analyze data on macroscopic organisms such as corals. We showed that corals possess an external, surface-associated carbonic anhydrase that converts HCO3- to CO2 for use in photosynthesis. This pathway supplies about half of the CO2 for net photosynthesis. Using these newly developed methods, internal carbonic anhydrase activity was measured and found to be quite high in coral tissues. Current conceptual models of inorganic carbon processing in corals posit multiple, critical roles for these internal carbonic anhydrases. Our work showed that there is sufficient carbonic anhydrase activity in coral tissues to fulfill these proposed roles. Finally, we showed that inorganic carbon is scarce for algal symbionts of corals, through measurements of inorganic carbon affinities of corals and isolated symbiotic algae. This finding is surprising given the abundance of inorganic carbon in seawater, and we hypothesize that coral hosts limit the inorganic carbon supply to their symbionts to control photosynthesis. Directly determining the concentration of inorganic carbon and related chemical forms within corals is extremely difficult. Microelectrodes are one tool that can provide such data, but their use requires much experimentation and care. We used microelectrodes sensitive to CO32-, H+, and Ca2+ to continuously measure these chemicals in corals as the microelectrode was passed through coral tissue. With this approach, we were able to directly measure the CO32- concentration in the calcifying fluid of corals for the first time. Coral skeletons are made of calcium carbonate (CaCO3), which is precipitated from the calcifying fluid. Consequently, the CO32- concentration in the calcifying fluid partially controls the rate of new skeleton formation and may be modified by ocean acidification. Our measurements indicated that corals achieve rapid rates of calcification by raising pH to very high levels, while keeping total inorganic carbon concentrations in the calcifying fluid low. The microelectrode profiles produced an abundance of novel data, for further study. The final focus of our work was integrative modeling to provide a more detailed understanding of inorganic carbon flows and probe ways in which these flows may change under ocean acidification. We developed a simple but powerful model of inorganic carbon processing in corals that represents the major biological compartments (diffusive boundary layer, oral tissue, coelenteron, aboral tissue, calcifying fluid) and fluxes (photosynthesis, calcification, bicarbonate transport, CO2/HCO3- interconversion). The model was constrained with data from the literature and new information obtained as part of this project. The model is capable of reproducing observed rates of photosynthesis, calcification, and their isotopic compositions showing progress in understanding the magnitude of and interdependence between these processes. Several graduate students participated in this project, including one student who obtained their PhD and one who obtained their masters working on this research. A new teaching module on ocean acidification was developed. A mini-symposium on symbiosis was held each year drawing participants from throughout the southeast. In summary, this project advanced our understanding of inorganic carbon supply pathways in corals and provided new approaches to studying this and related processes. This research thus helps us to better understand how ocean acidification affects corals. Last Modified: 10/09/2017 Submitted by: Brian Hopkinson