Variation in symbiont density is linked to changes in constitutive immunity in the facultatively symbiotic coral, Astrangia poculata

Scleractinian corals are essential ecosystem engineers, forming the basis of coral reef ecosystems. However, these organisms are in decline globally, in part due to rising disease prevalence. Most corals are dependent on symbiotic interactions with single-celled algae from the family Symbiodiniaceae to meet their nutritional needs, however suppression of host immunity may be essential to this relationship. To explore immunological consequences of algal symbioses in scleractinian corals, we investigated constitutive immune activity in the facultatively symbiotic coral, Astrangia poculata. We compared immune metrics (melanin synthesis, antioxidant production, and antibacterial activity) between coral colonies of varying symbiont density. Symbiont density was positively correlated to both antioxidant activity and melanin concentration. Our results suggest that the relationship between algal symbiosis and host immunity may be more complex than originally hypothesized and highlight the need for nuanced approaches when considering these relationships.MetaDataA21.csv- raw data used in the publication including data on symbiont density and immune activity FinalStats_A21.R- R code used to process data files and generate statistics reported in the manuscriptFunding provided by: National Institute of General Medical SciencesCrossref Funder Registry ID: http://dx.doi.org/10.13039/100000057Award Number: P20GM103430Funding provided by: Texas State UniversityCrossref Funder Registry ID: http://dx.doi.org/10.13039/100008882Award Number:Sample collection Astrangia poculata colonies were collected from Fort Wetherill in Jamestown, Rhode Island in April 2021 (41°28′40″ N, 71°21′34″ W) at a depth of 10-15 meters, via SCUBA. Colonies were visually assessed and sorted into either high or low symbiont density groups (termed "brown" or "white" colonies respectively); 10 colonies of each type were collected. Visual assessment of colony color is a reliable method for distinguishing corals with high symbiont density (>106 cells cm-2) from those with low symbiont density (104-106 cells cm-2). It should be noted that we use the terms "brown" and "white" as colonies grouped in the white category are rarely completely aposymbiotic. Following collection, the colonies were returned to Roger Williams University (Bristol, RI) where they were maintained for several weeks in closed, recirculating systems containing locally sourced seawater and fed three times weekly with frozen copepod feed. This period allowed corals to acclimatize to common garden conditions, reducing the effect of environmental variation on our measured variables. Samples were then flash frozen in liquid nitrogen and shipped to Texas State University for analyses. Protein extraction Tissue was removed from colonies with extraction buffer (TRIS with DTT, pH 7.8) using protocols outlined by Fuess et al. 2016. First, tissue was removed and isolated from a fixed surface area (2.14 cm2) on the flattest portion of the coral for Symbiodiniaceae density calculation. Then, tissue from the remaining fragment was removed and isolated into a separate aliquot. Both aliquots of tissue extracts were homogenized using a Fisherbrand Homogenizer 150 prior to downstream processing. The Symbiodiniaceae aliquot was processed using a series of consecutive centrifugation and wash steps. The homogenate was centrifuged at 2000 RPM for 3 minutes and the supernatant was removed. The resultant pellet was resuspended in 500µL of deionized water, and the product was centrifuged again using the same procedure. This step was repeated, and the sample was preserved in 500µL of 0.01% SDS in deionized water, stored at 4C. The host aliquot was processed to obtain subsamples for protein activity assays and melanin concentration estimation. Following homogenization, 1 mL of the host aliquot was flash frozen, and stored at 20oC for melanin concentration estimation. The remainder of the host aliquot was centrifuged for 5 minutes at 3500 RPM using an Eppendorf Centrifuge 5425 R. The resulting supernatant (protein enriched cell-free extract) was flash frozen in liquid nitrogen and stored at –80oC for downstream assays. Symbiont density Symbiodiniaceae density was estimated using a standard hemocytometer and Nikon Eclipse E600 microscope. Symbiodiniaceae counts were repeated in triplicate and averaged to calculate symbiont density/tissue area. Biochemical Immune Assays Biochemical immune assays were conducted following established protocols for scleractinian corals, with minor modifications necessary to adapt the procedures for A. poculata. Constitutive immunity was measured using assays estimating activity of the prophenoloxidase cascade (total phenoloxidase activity and melanin concentration), antioxidant activity (catalase and peroxidase) and antibacterial activity. All assays were standardized by either protein concentration or dry tissue weight, as appropriate. Assays were run in duplicates on 96 well plates using a Cytation 1 cell imaging multi-mode reader with Gen5 software (BioTek)