DOI: 10.5061/dryad.1ns1rn8xb
title: Variation in symbiont density is linked to changes in constitutive immunity in the facultatively symbiotic coral, Astrangia poculata

description: 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.
description: 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
 (&gt;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).
description: 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 manuscript