DOI: 10.5061/dryad.ffbg79d3x
title: <em>Astrangia poculata</em> heat stress pH response

description: Heat stress can disrupt acid-base homeostasis in reef-building corals and
 other tropical cnidarians, often leading to cellular acidosis that can
 undermine organismal function. Temperate cnidarians experience a high
 degree of seasonal temperature variability, leading us to hypothesize that
 temperate taxa have more thermally robust pH homeostasis than their
 tropical relatives. To test this, we investigated how elevated temperature
 affects intracellular pH and calcification in the temperate coral
 Astrangia poculata. Clonal pairs were exposed to elevated (30°C) or
 control (22°C) temperatures for 17 days. Despite causing damage to host
 tissues and symbiont cells, elevated temperature did not affect
 intracellular pH or inhibit calcification in A. poculata. These responses
 contrast with those of tropical cnidarians, which experience cellular
 acidification and decreased growth during heat stress. A. poculata
 therefore appears to have thermally resilient cellular acid-base
 homeostasis mechanisms, possibly due to adaptation to large seasonal
 temperature variations. However, we also observed tissue damage and lower
 egg densities in heat-treated individuals, suggesting that increasingly
 severe marine heatwaves can still threaten temperate coral fitness. These
 results provide insight into corals’ nuanced adaptive capacity across
 latitudes and biological scales.
description: (a)  Animal populations Adult
 <em>Astrangia poculata</em> corals were obtained from
 Narragansett Bay, RI, USA (41.49231 N, -71.41883 W) in August 2022 and
 transported in seawater first to the University of Rhode Island (Kingston,
 RI, USA) and then to XXXXXXXXXXXX (XXXXXXXXXXXX, USA)
 where they were maintained in a tank of artificial seawater
 (31–33 ppt; Instant Ocean Reef Crystals, Spectrum Brands, Blacksburg, VA,
 USA) at room temperature (~18°C) for 5 months prior to the experiment.
 Corals received ~200 µmol m-2 sec-1 photosynthetically active radiation as
 measured at colony depth below the surface using a LI-192 Underwater
 Quantum Sensor (LI-COR Biosciences, Lincoln, NE, USA) from NICREW
 HyperReef Dimmable Aquarium Lights (Shenzhen NiCai Technology Co.,
 Shenzhen, China) on the white + actinic setting, on a 12h:12h light:dark
 schedule. These light conditions were chosen to be well below the known
 photoinhibition point for this species [Jacques et al. 1983] and remained
 consistent through the acclimation and experimental periods. In late
 January 2023, 35 colonies ≥10 cm in diameter with relatively homogeneous
 coloration within each colony were selected, assigned colony numbers, and
 divided in half with a chisel (Fig. 1A-B). These genetically identical
 pairs (ramets) were affixed to tagged ceramic plugs (Frag Station Coral
 Frag Disks, The Alternative Reef, Green Bay, WI, USA) using cyanoacrylate
 reef glue on the face(s) of the colony lacking live tissue (CorAffix, Two
 Little Fishies, Miami Gardens, FL, USA). A total of N = 70 coral fragments
 (35 total colony pairs) were then placed into a tank that was gradually
 acclimated to 22°C by increasing the temperature from 18°C at a rate of
 ~0.5°C per day using an APEX Temperature Probe and 832 Power Strip energy
 bar (APEX, Neptune Systems, San Jose, CA, USA) powering a 50 W aquarium
 heater (Aqueon, Aqueon Products, Franklin, WI, USA). The temperature was
 monitored using a HOBO temperature logger (UA-001-64 64K Pendant, Onset
 Computer Corporation, Bourne, MA, USA) that recorded the temperature every
 15 minutes. Corals were held at 22°C for 12 days prior to the experiment.
 As the optimal respiration temperature for Rhode Island <em>A.
 poculata</em> is between 22-26°C [Aichelman et al. 2019 JEB], 22°C
 was chosen as a control temperature based on the Narragansett Bay, RI
 summer mean monthly maximum (NOAA) to represent normal non-heatwave heat
 exposure. Corals were fed 48-hour-old Artemia (1 full hatchery for the
 population; Brine Shrimp Direct, Ogden, UT, USA) weekly until the start of
 the experiment. 50-75% water changes were performed 4 hours after feeding,
 with the last pre-experimental water change five days before temperature
 treatments began. Water pH was maintained between 8.15-8.22 for the
 duration of the experiment and monitored using APEX pH probes.
 (b)  Experimental treatment A total of 6
 plastic tubs (7.57 L each) were randomized into one of two treatments,
 22°C (control) and 30°C (heated) for a total of 3 tubs per treatment (Fig.
 1C). Each tub was fitted with an egg crate, an air stone, a HOBO
 temperature logger, APEX pH and temperature probes, and an Aqueon 50 W
 aquarium heater. The corals were randomized into each tub, with one colony
 ramet placed into a control tub and its corresponding ramet placed into a
 heated tub. Control tubs were held at 22°C (22.53 ± 0.02°C; range =
 18.14°C-24.16°C) (Fig. 2A). Heated tubs increased by ~0.5°C every 12 hours
 (1°C per day) until the 30°C target was reached, after which temperatures
 were maintained at 30°C (30.11 ± 0.01°C; range = 27.21°C-31.88°C) for
 another 10 days (Fig. 2A) and recorded every 15 minutes using the HOBO
 temperature logger in the bottom of each tub. Water level was maintained
 over the course of the experiment with top-offs of deionized water to
 compensate for evaporation. Corals received a complete water change of
 each tub and a gentle removal of algae from each ramet on day 7 of the
 18-day experiment. After the water change, corals were fed 48-hour-old
 Artemia as described above. Each day for the first 17 days of treatment,
 between 12:00–14:30 (6-8.5 hours into the 12-hour light cycle), tubs were
 photographed, and every individual ramet was evaluated for mortality
 (scored as a binary), as well as polyp extension and ramet pigmentation.
 Aquarium lights were covered with black fabric during photography to
 prevent glare on the water surface, and photographs were taken using an
 iPhone 11 held ~40 cm above colonies. Both extension and color were scored
 qualitatively on a per-ramet basis in increments of 20% from 0%-100%.
 Color was assessed based on the coloration of live polyps per colony using
 a color standard made of white waterproof paper marked with red, green,
 and black electrical tape with color scale as follows: 0 = fully white, 20
 = mostly pale, 40 = mix of brown and pale, 60 = most polyps brown, 80 =
 all polyps brown, 100 = all polyps dark brown. The extension was estimated
 based on the percentage of live polyps per colony extended from the
 skeleton. (c)  Sampling Sampling was
 spread over the final two days of temperature treatment (days 17 and 18)
 so that in vivo intracellular pH measurements could be taken concurrently
 with physiological and histological sampling. Each sampling day, a
 haphazardly selected subset of approximately half of the corals (N = 17-18
 ramet pairs day-1) from each treatment were removed for sampling. A subset
 of 4-6 ramet pairs day-1 (10 pairs total) was further broken into 3
 pieces: 1) cell isolation for in vivo measurements of intracellular pH
 (see below), 2) freezing for later physiological assessments, and 3)
 preservation in 4% paraformaldehyde for histology. Nubbins sampled for
 physiological assessments, along with all other unbroken ramets, were
 placed in labeled sample bags (Whirl-Pak, Pleasant Prairie, WI, USA) and
 immediately stored at -80°C until processing. (d) 
 Physiological assessments Frozen ramets (N = 70) were
 thawed on ice and airbrushed (Vivohome 60 Hz air Compressor, City of
 Industry, CA, USA) using 10 mL chilled phosphate-buffered saline (PBS) as
 described [Innis et al. 2021]. As much filamentous algae as possible was
 excluded from the resulting tissue slurry. Tissue slurry was centrifuged
 at 7000 x g for 5 minutes at 4°C to separate host contents from algal
 cells. Host tissue supernatant was removed and stored at -80°C for protein
 quantification. Pelleted symbiont cells were immediately resuspended in
 PBS, homogenized at 25,000 rpm for 10 sec (Fisherbrand 850 Homogenizer,
 Thermo Fisher Scientific, Waltham, MA, USA), and kept on ice. Symbiont
 density was measured from these homogenates using existing protocols
 [Innis et al. 2021, Veal et al. 2010]. Briefly, each homogenized sample
 was diluted at a ratio of 1:3, 1:9, and 1:27 in 0.1% w/v sodium dodecyl
 sulfate (SDS) in PBS. Each dilution was loaded in triplicate into a
 round-bottom 96-well plate, and cell concentrations were determined using
 a Luminex Millipore Guava flow-cytometer (Guava Incyte Software, Austin,
 TX, USA). Relative chlorophyll fluorescence per symbiont was also measured
 on a per-ramet basis from the same flow cytometry run and was calculated
 as the average red autofluorescence of all events designated as symbionts
 based on existing gates [Innis et al. 2010]. Host protein was measured
 from the host fraction using the Bradford method in triplicate in a
 96-well plate using a spectrophotometer (BioTek PowerWave XS2, Agilent,
 Santa Clara, CA, USA). Host protein and symbiont densities were normalized
 to colony skeletal surface area, which was determined using the single wax
 dipping method on airbrushed skeletons as described [Veal et al. 2010].
 Care was taken to avoid dipping portions of each skeleton covered by glue
 or the ceramic base. As standards, eleven unique wooden dowels of known
 surface area spanning expected fragment sizes (1.5-114.0
 cm<sup>2</sup>) were also wax dipped to generate a linear fit
 relating surface area to dipped wax weight (R2=0.99).
 (e)  Calcification The net change in dry
 skeletal weight over the course of the experiment was determined using the
 buoyant weight method. Buoyant weight protocols were derived from previous
 studies that used this technique to measure calcification in <em>A.
 poculata</em> [Holcomb et al. 2012, Holcomb et al. 2010]. Briefly,
 each coral was placed in a glass beaker filled with seawater that remained
 submerged in a water bath to maintain room temperature (18.3-20.5°C) and
 suspended from a lab balance (Model AX223, OHAUS, Parsippany, NJ, USA). A
 DeWalt metal drill bit (Black & Decker, Towson, MD, USA) was
 weighed at the start of measurements as a standard, and reweighed if the
 temperature changed by at least 0.1°C to account for changes in seawater
 density at different temperatures. Seawater density at each temperature
 was calculated based on the buoyant weight, dry weight, and density of the
 metal standard. The density of the standard was calculated from its dry
 weight and volume, which was measured by submerging the standard in water
 and measuring displacement. The initial buoyant weight of all individuals
 (N = 70) was taken before placing each in their experimental tubs. After
 the experimental period (day 18), each coral was cleaned to remove any
 algal growth on its plug and re-weighed using the same procedure. Change
 in skeletal mass was calculated as described in [Davies 1989].
 (f)  Intracellular pH (pHi) measurements
 Intracellular pH (pHi) was measured for 10 colony pairs (N = 10
 fragments per temperature) using an existing confocal imaging method
 [Innis et al. 2021, Allen-Waller & Barott 2023, Allen-Waller et
 al. 2024, Venn et al. 2009]. After a 15-minute dark-acclimation period,
 cells were incubated with 10 µM of SNARF-1AM (Thermo Fisher Scientific) in
 filtered artificial seawater (FASW) for 20 minutes. FASW was sourced from
 the ASW used for coral husbandry (pH 8.15-8.22). Cells were then spun down
 to remove supernatant containing excess dye and resuspended in FASW. Cells
 were imaged on a confocal microscope (Leica SP8 DMi8, Leica Camera,
 Wetzlar, Germany) at 63x magnification (HC PL Apochromat C52 Oil
 objective, numerical aperture = 1.4). Cells were excited using 514 nm
 light from an argon laser (10% emission, 1.5% power, 458/514/561 nm
 beamsplitter). SNARF-1 fluorescent emission was simultaneously acquired at
 a frame rate of 400 Hz (frame rate = 0.77/s, pixel dwell time = 3.16 µs)
 in two channels (585 ± 15 and 640 ± 15 nm) using HyD detectors (gain = 75,
 pinhole = 1.00 airy unit). For each sample, 15-40 images were collected,
 about half of which pertained to each cell type (non-symbiocytes and
 symbiocytes). Images were analyzed using ImageJ, and coral cytoplasmic
 yellow:red fluorescence ratios were converted to pHi values using a
 standard curve generated using cells from non-experimental <em>A.
 poculata</em> incubated in solutions of known pH and osmolarity with
 30 µM nigericin [Allen-Waller et al. 2024, Venn et al. 2009] (Fig. S1).
 Cell quality was checked to ensure only live cells were measured as
 described previously [Allen-Waller et al. 2024]. Each colony measurement
 per cell type represents an average from N ≥ 8 cells.
 (g)  Histology Paraformaldehyde-fixed samples
 were decalcified in 0.5 M EDTA + 0.5% paraformaldehyde in calcium-free S22
 buffer (450 mM NaCl, 10 mM KCl, 58 mM MgCl<sub>2</sub>, 10 mM
 CaCl<sub>2</sub>, 100 mM HEPES, pH 7.8), changed daily until
 the skeleton was gone. Decalcified tissue was then held in 70% ethanol at
 4°C until samples were dehydrated for histology. Samples were dehydrated
 using a series of incubations (3 x 20 minutes each at 90% and then 100%
 ethanol), clarified in xylene substitute (SafeClear, Fisher HealthCare,
 Fisher Scientific), and sent to Pacific Pathology (San Diego, CA, USA) for
 paraffin wax embedding, slicing, and staining with hematoxylin and eosin.
 The resulting slides were treatment-blinded and assessed for tissue health
 by scoring (1) symbiont quality (roundness, cell integrity), (2) tissue
 layer definition/structural integrity, (3) cellular integrity (lack of
 necrosis/granulation), and (4) epidermal clarity. Each variable was
 qualitatively assessed on a scale of 1-5, adapting existing methods
 developed for tropical corals [Kruse et al. 2025]. A score of 5 was
 assigned to healthier tissue (e.g. clear definition between tissue layers,
 no necrosis, well-defined epidermis) while a score of 1 corresponded to
 more tissue damage (e.g. disordered tissue layers, granular/necrotic
 cells, degraded/no epidermis). Epidermal thickness was measured in ImageJ
 [Abramoff et al. 2025] wherever possible by drawing segments across the
 epidermis at regular intervals, wherever the epidermis was clearly defined
 (3-27 measurements per slide, depending on epidermal integrity of the
 ramet). Segment lengths were converted from pixel units to mm using a line
 drawn along a stage micrometer (0.01mm Microscope Camera Calibration
 Slide, OMAX Microscope), and average thickness per ramet was calculated
 using all measurements from each ramet. Egg production was also assessed
 as a binary by the presence/absence of eggs in all slides. Total eggs per
 area were calculated by counting the number of eggs per slide image and
 dividing it by the area of the tissue slice within the image. Sperm or
 spermatocytes were not seen in any individuals and were therefore not
 quantified. Slice area was measured by freehand outlining in ImageJ to
 obtain pixel area, which was then converted to mm<sup>2</sup>
 using the micrometer. (h)  Statistical
 methods All data were analyzed in RStudio version
 2022.07.2 (https://www.rstudio.com/) and plots were generated using the package ggplot2 [Wickham 2016]. Daily average treatment temperatures were compared between treatments using a linear model with treatment and date as fixed effects. To calculate the effect of temperature on qualitative metrics (colony polyp extension and pigmentation) over time, linear mixed effects models were constructed using the lme4 package [Bates et al. 2015] with temperature treatment and date fixed effects, and colony and tub as random intercepts. Post hoc pairwise comparisons by date were analyzed by Tukey’s HSD using the emmeans package [Lenth & Lenth 2017]. To calculate the impact of temperature treatment on each physiological metric, the value of each measured physiological parameter (symbiont cell density, chlorophyll fluorescence per symbiont, epidermal thickness, total daily calcification rate, nonsymbiocyte intracellular pH, and symbiocyte intracellular pH) for each 22°C-treated ramet was subtracted from the 30°C-treated ramet of the same colony. To test the effect of initial colony symbiont density on colonies’ temperature response, each resulting set of ∆ values was regressed against 22°C-treated ramets’ symbiont cell densities. Effects of temperature on final symbiont cell density, chlorophyll, colony color, and histological metrics were further tested via linear mixed effects models with temperature treatment as a fixed effect and colony as a random intercept. Q-Q and residual plots were checked to ensure each model met normality and homogeneity of variance assumptions. To test how colony bleaching response impacted organismal physiological metrics, colonies were divided between those that lost symbionts (∆ symbiont density &lt; 0) and those that gained symbionts (∆ symbiont density &gt; 0). Linear models for each subpopulation were constructed, testing the fixed effect of temperature treatment on every other physiological metric.
description: # *Astrangia poculata* heat stress pH response Dryad
 DOI: https://doi.org/10.5061/dryad.ffbg79d3x This repository contains data
 from an experiment testing the influence of heat treatment and symbiosis
 on intracellular pH homeostasis, growth, and host histology in the
 temperate coral *Astrangia poculata*.  ## Description of data and file
 structure The file **"ApocTempData.csv"** contains temperature
 data from HOBO loggers throughout experimental manipulations. Temperature
 is recorded in °C and °F at 15-minute intervals for the entire 18-day
 experiment. Columns: Treatment (Ambient vs. High), Tub (i.e. experimental
 container), Date, Time, DateTime (concatenation of Date and Time columns),
 Temperature (°F), and Temperature (°C). The file
 **"ApocDailyObservations.csv"** contains color and extension
 data from observations over the course of the experiment. Columns: Day,
 Colony (i.e. genet), Treatment (Ambient vs. High), Tub (i.e. experimental
 container), Extension, and Color. Extension and Color were qualitatively
 assigned % values in increments of 20%. The file
 **"ApocHeatStress.csv"** contains all data on *A. poculata*
 physiology, histology, and cell pH after temperature treatments. Columns:
 Treatment, Colony (i.e. genet), Tub (i.e. experimental container),
 Symbs.total (total cells per animal), SA.cm2 (surface area in cm^2^),
 Symbs.cm2 (symbiont cells per cm^2^), rel.chl (relative chlorophyll
 autofluorescence per cell; unitless), Nonsymb.pHi (nonsymbiocyte
 intracellular pH), Symb.pHi (symbiocyte intracellular pH), prot.ug.cm2
 (host protein in micrograms per cm^2^), Calcif.g.cm2.day (calcification in
 grams per cm^2^ per day). The following columns are histological health
 scores scored 1-5 (with 5 being the healthiest): Symbiont.Health,
 Epidermis.Integrity, Tissue.Integrity, and Necrosis.Granularity. The
 column Gametes is a binary score of whether coral eggs were seen in any of
 that ramet's histology slides. Egg.Size is the average egg size of
 all eggs seen in that ramet in square inches (as taken raw from the image
 file). Egg.Count is the total number of eggs seen in the ramet's
 histology slides. Total.Egg.Area is the summed area of all eggs seen in
 mm^2^. Eggs.mm2 is the number of eggs seen in the ramet's histology
 slides per total area of tissue in square millimeters assessed for that
 ramet. Epi.mm is the average thickness of the host epidermis in
 millimeters as measured based on all of that ramet's assessed tissue
 (see Methods). **"ApocRawBuoyantWeights.csv"** contains all raw
 buoyant weight measurements of ramets from the experiment as well as the
 seawater temperature at which those measurements were taken, standard
 buoyant and dry weights in g, calculated seawater density in g/L, and
 calculated calcification in grams per cm^2^ per day. Abbreviations: SW =
 seawater, SA = surface area. "Standard" refers to a metal drill
 bit (see Methods). **"ApocStdAllMeasurements.csv"** contains
 yellow:red fluorescence ratios of all individual cells measured for the
 *Astrangia poculata* intracellular pH standard described in this study.
 Columns: curve (i.e. first or second biological replicate curve),
 solution.pH (measured pH of the solutions in which those cells were
 calibrated), avg.yr.ratio (average ratio of yellow:red fluorescence in the
 1-2 measurements that were taken per cell).
 **"ApocHeatStressAnalysis.R"** contains code to reproduce the
 analysis of these data, including statistical tests and graphs, described
 in the manuscript "The temperate coral *Astrangia poculata* maintains
 acid-base homeostasis through heat stress." ## Other storage
 information This dataset and code are also available on Zenodo:
 [https://zenodo.org/records/17408148](https://zenodo.org/records/17408148)
 and on Github:
 [https://github.com/allenwaller/Astrangia.Heat.pHi](https://github.com/allenwaller/Astrangia.Heat.pHi).