The scaling of metabolic traits differs among larvae and juvenile colonies of scleractinian corals

Body size profoundly affects organism fitness and ecosystem dynamics through the scaling of physiological traits. This study tests for variation in metabolic scaling and its potential drivers among corals differing in life history strategies and taxonomic identity. Data were compiled from published sources and augmented with empirical measurements of corals in Moorea, French Polynesia. The data compilation revealed metabolic isometry in broadcasted larvae, but size-independent metabolism in brooded larvae; empirical measures of Pocillopora acuta larvae also supported size-independent metabolism in brooded coral larvae. In contrast, for juvenile colonies (i.e., 1–4 cm diameter), metabolic scaling was isometric for Pocillopora spp. and negatively allometric for Porites spp. The scaling of biomass with surface area was isometric for Pocillopora spp., but positively allometric for Porites spp., suggesting the surface area:biomass ratio mediates metabolic scaling in these corals. The scaling of tissue biomass and metabolism was not affected by light treatment (i.e., either natural photoperiods or constant darkness) in both juvenile taxa. However, biomass was reduced by 9–15% in the juvenile corals from the light treatments and this coincided with higher metabolic scaling exponents, thus supporting the causal role of biomass in driving variation in scaling. This study shows that metabolic scaling is plastic in the early life stages of corals, with intrinsic differences between life history strategy (i.e., brooded and broadcasted larvae) and taxa (i.e., Pocillopora spp. and Porites spp.), and acquired differences attributed to changes in area-normalized biomass.

Methods
Data were compiled from published sources and augmented with empirical measurements of corals in Moorea, French Polynesia. Data were processed through Excel and R software (R Development Core Team 2021, v1.4.1103) using the stats package (R Core Team, v4.0.3), and the car package (Fox and Weisberg, 2020, v3.0-10) used to test for the statistical assumptions of the procedures employed.

Metabolic scaling of larvae - data compilation

To investigate metabolic scaling of brooded and broadcasted larvae, data were compiled from peer-reviewed and grey literature identified using Web of Science, Google Scholar, and the published proceedings of the International Coral Reef Symposia which were available from 1972 to 2012 (accessed 11 May 2020). The keywords, “(scleractinia*) and (larva* or planula*) and (respirat* or metabolism or "metabolic rate" or metabolic)” were utilized, with the asterisks indicating different suffixes. The citation lists in the papers included in the data compilation were also searched for additional relevant sources. We searched for studies that reported oxygen consumption and dry biomass that were either standardized to or could be converted to, a per larva scale. Studies that reported proximal tissue composition (i.e., lipid or protein content) from which dry biomass could be calculated based on conversion factors (Table S1, see details in the supplementary materials of Bean and Edmunds 2024) were also included. Only data from studies in which larvae were kept under ambient seawater conditions (relative to the time and location of the study) with respect to temperature and pCO2 were retained. Data were extracted from sources using values reported in the manuscript or supplementary material, or by digitizing plots presented therein using the online tool, WebPlot Digitizer (Rohatgi, 2021). Some data points from one study were not retrievable due to the resolution of the graphics and were omitted from the analysis.

Metabolic scaling of larvae – empirical data

To empirically quantify the scaling of metabolism in brooded larvae, respiration was measured for larvae freshly released from Pocillopora acuta (Lamark 1816) in Moorea, French Polynesia. To obtain larvae from P. acuta, 6 adult colonies were collected from a site on the north shore (17°28'59.67"S, 149°48'49.91"W) at ~1 m depth. Colonies were collected on May 15th, 2021 (lunar day 4) to sample peak release of larvae that occurs around lunar days 8–12.

The colonies were taken to the Richard B. Gump South Pacific Research Station, where each was placed in a separate aquarium under shaded natural sunlight (~9 µmol photons m-2 s-1) where they were supplied with sand-filtered seawater pumped directly from Cook’s Bay at 8 m depth. Larvae were collected by overflowing seawater from these aquaria into a container fitted with a 110 µm mesh window. Following the overnight release of larvae, they were collected the next morning from a single parent colony that had released the greatest number of larvae. 

Larval respiration was measured for groups of similarly-sized larvae placed in 2 mL glass Autosampler vials (Biomed Scientific, part number: MGV2-9-CS-100Z), with the results expressed per larva. Respiration trials were conducted with 6 larvae vial-1 with each trial considered a statistical replicate. To obtain larvae of similar sizes, freshly released larvae were coarsely sorted into 3 size classes (i.e., small, medium, large) by visual inspection using a dissecting microscope (40x magnification). Approximately 40 each of the largest, smallest, and medium-sized larvae (relative to the larval sizes on each day of release) were grouped and dark acclimated for 100 minutes. Following dark acclimation, batches of 6 similarly-sized larvae were randomly selected from the three size groups and allocated to autosampler vials filled with filtered seawater (0.45 µm) with known oxygen saturation and covered with parafilm. At the conclusion of the respiration trial, larval size was measured by quantifying their protein content as described below. Throughout dark acclimation and respiration trials, larvae were maintained in filtered seawater (0.45 µm) at ambient seawater temperature (27.9°C) and salinity (34.7 ppt) relative to the time of collection of the maternal colonies. The temperature was regulated (± 0.1°C) using a water bath (Lauda Ecoline, model RE 104, Lauda-Königshofen, Germany), and measured with a certified thermometer (Fisher Scientific, model 15-077-8, Waltham, MA, USA, accuracy ± 0.1°C), and salinity were measured with a benchtop conductivity meter (ThermoFisher Scientific Orion Star A212, Waltham, MA, USA).

Oxygen saturation was measured using a NEOFOX-GT optical oxygen sensing system fitted with a 1.6-mm diameter FOSPOR-R probe (Ocean Insight, Orlando, FL, USA). The probe was two-point calibrated at 0% (sodium sulfite and 0.01 M sodium tetraborate) and 100% oxygen saturation (air-saturated 0.45 µm filtered seawater (FSW)). The respiration of the larvae in the vials was measured in darkness for ~0.5–1.5 hrs, over which oxygen saturation remained > 80% to prevent oxygen-dependent respiration. After the incubation period, the vials were carefully inverted to mix the seawater, and the oxygen probe was inserted through the parafilm to record the oxygen saturation. Oxygen saturation was converted to concentration using Unisense conversion tables and corrected with controls consisting of filtered seawater (0.45 µm). Following the respiration trials, larvae were frozen in seawater (-18°C) for determination of protein biomass.

The protein content of the larvae was measured using the Bio-Rad Protein Assay (Bio-Rad Laboratories, CA, USA). Larvae were thawed, sonicated (Sonic Dismembrator 500, Fisher Scientific, Waltham, MA, USA), heated in alkaline conditions using 1 M NaOH, and neutralized with 1.65 M HCl. Protein content (mg) was calculated using bovine serum albumin standards and converted to dry biomass (mg) assuming the larvae contain 17% protein

Metabolic scaling of juvenile corals

11 juvenile colonies of Pocillopora spp. (excluding P. acuta) and 10 of massive Porites spp. (i.e., not including branching Porites spp.) were collected from the back reef of Moorea (17°28’25.65”S, 149°48’46.53”W) at ~2 m depth on November 8th, 2020. Juvenile colonies were collected to vary in size from 1 to 4 cm in diameter so that the scaling exponent for respiration could be measured. Corals were not morphologically distinguishable by species at this early life stage and were identified by genus. Colonies were chipped from the reef using a small chisel and attached to a plastic base using underwater epoxy (Z-Spar Splash Zone A-788). The portions of the colony bases that were not covered in live coral tissue were cleaned of organisms and covered with the same underwater epoxy. The colonies were left for 21 hours in flowing seawater under a shaded roof to let the epoxy harden before respiration was measured.

The respiration rates of the juvenile corals were measured within 4 days of collection to minimize photo-acclimation to laboratory conditions and following 14–54 hours of darkness in flowing, sand-filtered water pumped from Cook’s Bay and maintained at an ambient temperature of ~27°C. Respiration trials were run over two days and were conducted by coral colony, alternating between taxa, with two seawater controls run daily. For each trial, a juvenile coral was placed inside a sealed 240 mL cylindrical chamber filled with 0.45 µm FSW and surrounded by a water jacket to regulate the temperature. All trials began at ~100% oxygen saturation and were conducted at ~27.3°C and ~34.3 ppt, which were the ambient conditions at the collection site when the analyses were completed (November 2020). A stir bar below the juveniles mixed the seawater during the trials, and juvenile corals were acclimated to chamber conditions for 10 minutes before sealing the chamber and measuring respiration through oxygen consumption recorded using a FOSPOR-R optical oxygen probe (Ocean Insight, Orlando, FL, USA). The probe was calibrated in the same manner as described above for larval respiration, and incubations lasted 20–60 minutes until a steady decline of oxygen saturation was recorded while ensuring that saturation remained > 80%.

Following respiration trials, the surface area and the dry biomass of the corals were measured. The surface area of the juveniles was estimated geometrically by measuring two perpendicular diameters (i.e., length and width) and height, and assuming the colonies were vertical cylinders (see details in the supplementary materials of Bean and Edmunds 2024). Biomass was quantified by fixing the juvenile corals in 10% formalin in seawater for 48 hours, then decalcified in 5% HCl in distilled water over 1–7 days. The tissue was rinsed of formalin residue with deionized (DI) water before being placed in HCl to avoid the formation of bis-chloromethyl ether, a carcinogen. The tissue tunic was rinsed in DI water before being dried to a constant weight at 60°C.

 Metabolic scaling between taxa in light and dark

To evaluate the role of biomass in mediating scaling relationships, the biomass of corals was indirectly manipulated by exposing corals to darkness (i.e., starvation) or natural photoperiods (i.e., 12:12 hr light:dark) of ambient light relative to the collection site as a control. Two tanks were maintained in darkness and two at a maximum of 967 ± 9 (± s.e.m.) μmol photons m−2 s−1 (measured with a Li-Cor 4π sensor fitted to a Li-Cor meter (LI-1400, Lincoln, NE, USA)) on a 12:12 hr light:dark photoperiod supplied through LED lights (75 W, Sol LED Module, Aqua-illumination, Ames, Iowa, USA). Thirty-two juvenile colonies of both Pocillopora spp. and massive Porites spp. were collected on May 20th, 2021, from the back reef of Moorea at ~2 m depth (17°28’25.65” S, 149°48’46.53” W). Colonies were acclimated to laboratory conditions for 4 days before experimental trials took place. Tanks (150 L) were held at 28.5°C and 34.7 ppt and supplied with sand-filtered seawater from Cook’s Bay at ~400 mL min-1 and each contained eight colonies of Pocillopora spp. and eight colonies of Porites spp. Colonies were held at treatment conditions for 9–12 days, depending on when they were used for respiration trials, and arranged daily in each tank to minimize position effects. Respiration was measured starting at 9 days because the colonies in the dark tanks showed signs of physiological stress (i.e., paling). Two juveniles were measured concurrently for respiration in separate chambers with one control completed in each chamber daily. Colony selection for respiration trials alternated between tanks within each treatment and between taxa, and with 14–18 corals processed each day, all corals were processed in four days. The respiration rate of each colony was measured using the methods described above for juvenile corals.

Following respiration trials, corals were frozen (-20 °C) and crushed with 2500 µL of 0.45 µm FSW to produce a slurry of skeleton and tissue. To maintain consistency of sampling, ~13 cm2 of each coral colony was crushed with the area chosen as the size of the smallest colony in the experiment. The homogenate was used for the analysis of ash-free dry weight (AFDW) using a muffle furnace (5 hours at 450°C) (Fisher Scientific, model: 650–126, Waltham, MA) and symbiont density using a hemocytometer (8 replicate counts/sample).