Developing techniques to investigate superoxide dynamics associated with the coral holobiont

Reactive oxygen species (ROS), including superoxide, are produced by all aerobic organisms. In adult corals, intracellular ROS have been implicated in coral bleaching, while more recent studies have pointed to potential benefits of extracellular ROS in coral health. To enable real-time in situ superoxide measurements, we designed and developed the first diver-operated submersible chemiluminescent sensor (DISCO). Here, DISCO is optimized to measure extracellular superoxide associated with coral, enabling measurements to be collected non-invasively, in high resolution, and in a matter of minutes. During field surveys with DISCO in Jardines de la Reina, Cuba, extracellular superoxide levels were found to range widely as a function of coral species. In particular, Porites porites and Porites astreoides were associated with extremely high concentrations of extracellular superoxide ranging from 17 – 173 nM, levels that are orders of magnitude higher than surrounding seawater. Similarly high concentrations of extracellular superoxide were observed in association with aquaria hosted P. astreoides swimming larvae and settled polyps. The individual-normalized extracellular superoxide concentrations associated with swimming larvae averaged 115 pM, exhibiting a density dependent relationship. Extracellular superoxide concentrations associated with settled larvae decreased from 776 pM after 24 hours of settlement to 161 pM after 209 days of settlement, showing higher and more variable concentrations within the first few days of settlement. Through refinement of targeted in-gel assay techniques, the enzymes responsible for extracellular superoxide production by P. astreoides larvae are being purified, identified and characterized. Identifying such enzymes will allow for tracking protein derived superoxide production within more complex systems, such as adult coral holobionts exposed to differing environmental conditions. These recent superoxide sensing advances with DISCO coupled with methodological advances in identifying underlying biochemical mechanisms will enable a better understanding of ROS dynamics associated with corals from polyp to reef scale across varying environmental and geographic conditions. Increasing the accessibility of ROS measurements and developing techniques to track protein expression involved in superoxide production will serve as a foundation for and promote future studies in investigating the link between ROS production, coral health, and coral species variations.