Dark Production of Extracellular Superoxide by the Coral Porites astreoides and Representative Symbionts

Abstract

The reactive oxygen species (ROS) superoxide has been implicated in both beneficial and detrimental processes in coral biology, ranging from pathogenic disease resistance to coral bleaching. Despite the critical role of ROS in coral health, there is a distinct lack of ROS measurements and thus an incomplete understanding of underpinning ROS sources and production mechanisms within coral systems. Here, we quantified in situ extracellular superoxide concentrations at the surfaces of aquaria-hosted Porites astreoides during a diel cycle. High concentrations of superoxide (∼10's of nM) were present at coral surfaces, and these levels did not change significantly as a function of time of day. These results indicate that the coral holobiont produces extracellular superoxide in the dark, independent of photosynthesis. As a short-lived anion at physiological pH, superoxide has a limited ability to cross intact biological membranes. Further, removing surface mucus layers from the P. astreoides colonies did not impact external superoxide concentrations. We therefore attribute external superoxide derived from the coral holobiont under these conditions to the activity of the coral host epithelium, rather than mucus-derived epibionts or internal sources such as endosymbionts (e.g., Symbiodinium). However, endosymbionts likely contribute to internal ROS levels via extracellular superoxide production. Indeed, common coral symbionts, including multiple strains of Symbiodinium (clades A to D) and the bacterium Endozoicomonas montiporae LMG 24815, produced extracellular superoxide in the dark and at low light levels. Further, representative P. astreoides symbionts, Symbiodinium CCMP2456 (clade A) and E. montiporae, produced similar concentrations of superoxide alone and in combination with each other, in the dark and low light, and regardless of time of day. Overall, these results indicate that healthy, non-stressed P. astreoides and representative symbionts produce superoxide externally, which is decoupled from photosynthetic activity and circadian control. Corals may therefore produce extracellular superoxide constitutively, highlighting an unclear yet potentially beneficial role for superoxide in coral physiology and health.

Species-specific control of external superoxide levels by the coral holobiont during a natural bleaching event

“…On the basis of these results and the low diffusivity of superoxide, the most likely sources of external superoxide are coral epithelial cells and/or microbial epibionts residing on the coral's surface mucus layer, rather than Symbiodinium . Similarly, previous studies also showed superoxide production at the surface of aquaria-hosted pigmented colonies of P. astreoides and both bleached and pigmented colonies of S. pistillata in the absence of light, pointing to non-algal superoxide sources1840. Yet in the presence of light, superoxide production by S. pistillata was moderately elevated for pigmented colonies but not bleached corals in this previous study18.…”

“…2 and Supplementary Table 2) rules out abiotic photo-oxidation mechanisms as a major mode of superoxide generation. Similarly, a recent aquaria-based study also found dark production of extracellular superoxide by another Porites species, P. astreoides 40. Lastly, the fact that superoxide levels are species-specific (Fig.…”

“…18) and from ∼1 to 35 nM (ref. 40), respectively. Furthermore, up to ∼33 nM superoxide was detected in the deep chlorophyll maximum at the subtropical front east of New Zealand35.…”

“…Indeed, heterotrophic bacteria17 and corals44 have previously been shown to simultaneously produce and degrade extracellular ROS. In fact, corals have been shown to release antioxidants into their surroundings18404448, which also varies widely as a function of coral species44. This differential antioxidant release may explain why superoxide concentrations in the direct vicinity of M. capitata were lower than the surrounding seawater.…”

Marine Animal Microbiomes: Toward Understanding Host–Microbiome Interactions in a Changing Ocean

“…In addition to nutrition, the microbiome plays a role in coral health and stress. Temperature and light stress to corals can result in overproduction of reactive oxygen species (ROS), which can be detrimental to Symbiodinium and result in bleaching, but the associated bacteria have also recently been shown to contribute extracellular ROS Zhang et al, 2016), which could play a signaling role with the host or within the microbiome. Given the recent mass bleaching occurring on reefs (Hughes et al, 2017), corals will likely continue to be a useful and popular system for symbiosis and dysbiosis research.…”