Concurrent anthropogenic global climate change and ocean acidification is expected to have a negative impact on calcifying marine organisms. While knowledge of biological responses of organisms to oceanic stress has emerged from single species experiments, these do not capture ecologically relevant scenarios where the potential for multi-organism physiological interactions is assessed. Marine algae provide an interesting case study, as their photosynthetic activity elevates pH in the surrounding microenvironment, potentially buffering more acidic conditions for associated epiphytes. We present findings that indicate increased tolerance of an important epiphytic foraminifera, Marginopora vertebralis, to the effects of increased temperature (±3 °C) and pCO₂ (~1000 µatm) when associated with its common algal host, Laurencia intricata. Specimens of M. vertebralis were incubated for 15 days in flow-through aquaria simulating current and end-of-century temperature and pH conditions. Physiological measures of growth (change in wet weight), calcification (measured change in total alkalinity in closed bottles), photochemical efficiency (Fv/Fm), total chlorophyll, photosynthesis (oxygen flux), and respiration, were determined. When incubated in isolation, M. vertebralis exhibited reduced growth in end-of-century projections of ocean acidification conditions, while calcification rates were lowest in the high-temperature, low-pH treatment. Interestingly, association with L. intricata ameliorated these stress effects with the growth and calcification rates of M. vertebralis being similar to those observed in ambient conditions. Total chlorophyll levels in M. vertebralis decreased when in association with L. intricata, while maximum photochemical efficiency increased in ambient conditions. Net production estimates remained similar between M. vertebralis in isolation and in association with L. intricata, although both production and respiration rates of M. vertebralis were significantly higher when associated with L. intricata. These results indicate that the association with L. intricata increases the resilience of M. vertebralis to stress, providing one of the first examples of physiological buffering by a marine alga that can ameliorate the negative effects of changing ocean conditions.

Collection and acclimation

Specimens of Marginopora vertebralis (as identified by Renema 2018) and Laurencia intricata were collected from Coconut Beach (1-3 m depth), Lizard Island (014°40’08”S, 145°27’34”E) on the Great Barrier Reef, Australia, in October 2015 (Fig. 1). Samples were immediately transported back to Lizard Island Research Station and placed into flow-through ambient seawater conditions and light for 5 days to acclimate to laboratory conditions. Specimens of L. intricata were then separated into ~1 g (wet weight) replicates, and all visible epiphytes (M. vertebralis and other LBFs such as Calcarina hispida, Amphistegina lobifera, and Baculogypsina sphaerulata) removed. The M. vertebralis were separated into experimental replicates in which 6 M. vertebralis (~0.5 g wet weight, all approximately similar size of ~5 mm diameter) were placed into 60 mL jars with 40 mL of seawater, similar to densities found in situ (Doo, pers. obs.). The experimental treatment groups of M. vertebralis only, and L. intricata with M. vertebralis were established prior to the start of the experiment, and acclimated 3 days before the initiation of the experiment. Specimens were incubated in polypropylene jars with a hole cut from the side to prevent overflow over the top. A 462 µm plankton mesh was glued to the side of the jar to allow for overflow of water through the mesh, while maintaining flow-through conditions, and resulted in a total of ~40 mL water in each container. Light was provided using LED cool white lights (LED Type 3528) to an intensity of ~100 µmol photos m^-2 s^-1 for the duration of the experiment (2 weeks) in a 12hr:12hr, day-night cycle. A flow-through dripper tap system was used for experimental water delivery (~40 mL min^-1). Experimental conditions were gradually reached over a three-day period, with an increase of 1°C, and decrease of 0.1 pH unit each day prior to the start of the experiment when all replicates were incubated in ambient temperature (~25.5°C and ~pH_Total 7.95 conditions). Experimental water was collected from Lizard Island lagoon and filtered with a 5 µm filter bag, and delivered into 60L header tanks, from which all treatment groups were supplied incubation water.

Two temperatures (ambient [26°C] and high [29°C]), and two pH’s (ambient [8.0] and low [7.7] pH_Total units) fully orthogonal treatment groups were used. To determine the effect of symbiosis of L. intricata and M. vertebralis, 10 replicates each of M. vertebralis only, and L. intricata with M. vertebralis were incubated in each of the pH/temperature treatment groups for a total of 80 replicates.

Incubation parameters and seawater chemistry

The seawater pH and temperature conditions were controlled using a Neptune Apex system dosing pure CO₂ to regulate pH. In the experiment, a total of 4 sumps were used, one for each manipulated seawater condition (see above). This water was pumped into individual jars (see above), maintaining independence between replicates. Total alkalinity, pH, and temperature of the header tanks were measured on a daily basis, from randomly selected drippers. Total alkalinity samples were filtered with a 0.22 µm filter prior to analysis to eliminate possible contamination of calcium carbonate in the sample and measured using open-cell potentiometric titrations (DOE 1994). Seawater pH was monitored using m-cresol spectrophotometric measurements on an Ocean Optics USB4000+ spectrophotometer, and pH_Total calculated based using standard protocols (DOE 1994). These were referenced to seawater Certified Reference Material (CRM), Batch 161, prepared by A. Dickson in the Scripps School of Oceanography. Temperature and salinity measurements were collected with Vernier TMP-BTA and CON-BTA probes, respectively. Seawater parameters remained stable throughout the experimental incubation (Table 1).

Growth measurements

The wet weight of M. vertebralis across individual replicates were pooled within replicate jars and measured prior to the start of the experiment, and at the termination using a Mettler-Toledo ML240 balance to 10^-4g resolution. Measurements were then converted into a percentage daily change in weight. In treatments of both L. intricata and M. vertebralis, only the pooled M. vertebralis from each replicate were weighed at the initiation and termination of the experiment. 

Instantaneous calcification measurements

After a 2-week incubation, alkalinity anomaly measurements were made using close bottle experiments as a proxy of instantaneous calcification. Organisms were carefully sealed in ~20mL glass scintillation vials with their chosen treatment group water, and in the case of the algal associated groups, with the algal hosts. The sealed vials were immersed in the appropriate flow-through water system to maintain treatment temperature. Treatment groups were incubated for 8h in light conditions.

Analyses of water samples for total alkalinity were as above, and calcification (G) was calculated using Eqn. 1: TA is total alkalinity (µmol kg^-1), p is seawater density, V is chamber volume (mL), w.w. is wet weight (mg), and T is incubation time (h). All calculated values were normalized to final wet weight.

Photochemical efficiency measurements

At the termination of the experiment, maximum photochemical efficiency (Fv/Fm) data were measured using WALZ DIVING-PAM underwater fluorometer (similar to Schmidt et al. 2014a). Measurements were recorded 4 hours after sunset in dark conditions, and individual M. vertebralis, and averaged across pseudoreplicate M. vertebralis within individual treatments.

Total chlorophyll measurements

Following measurement of wet weight, samples were immediately frozen (-20°C) in dark conditions and stored for chlorophyll analyses. Samples were placed in 15 mL polypropylene plastic tubes with 10mL of 90% acetone, and subsequently mechanically ground with a hard metal rod. Samples were then incubated in 4°C overnight in the dark. Absorbance measurements were then taken from the supernatant using an Ocean Optics usb4000+ spectrophotometer, and wavelengths of 630nm, 647nm, 664nm, and 691nm were recorded. Total chlorophyll was calculated based on universal equations developed by (Ritchie 2008). In treatments of both L. intricata and M. vertebralis, M. vertebralis were pooled within the replicate sample jar, and measured separately. All measurements were normalized to final wet weight of the corresponding M. vertebralis replicate.

Oxygen flux measurements

Oxygen flux measurements were made with a Presens Oxy-10 mini 10-channel optical sensor. At the end of the 15-day incubation period, oxygen flux measurements were taken in 30 mL glass scintillation vials that were gently stirred. Replicate samples (including L. intricata in association treatments) were gently placed in the glass jars with corresponding pH and temperature conditions, and allowed to acclimate for at least 5 minutes before measurements were recorded. For oxygen production measurements, light conditions in replicates were ~100 µmol photons m^-2 s^-1 during measurements (similar to incubation levels) and measured for a total of ~30 min in light conditions first. Subsequently, respiration was measured in dark conditions for ~30 min, allowing for 5 min of acclimation, and rate of oxygen consumption measured after the acclimation period. All analyses were performed using standard protocols for LBFs outlined in (Uthicke and Fabricius 2012).

As the association treatment of M. vertebralis was incubated with L. intricata, an additional set of experiments was performed to separate the effect of LBF from macroalgae by independently measuring oxygen flux rates of algae in isolation. A total of 10 replicates were measured using similar incubations protocols described above, and the average of the four pH and temperature treatment groups were subtracted from the L. intricata with M. vertebralis replicates to obtain oxygen flux measurements of M. vertebralis in association treatment groups (Table 2).

Statistical analyses

For growth rate, instantaneous calcification, maximum photochemical efficiency (Fv/Fm), total chlorophyll, and oxygen flux measurement data, a three-way ANOVA was performed using pH (amb, -0.3 pH units), temperature (amb, and +3°C), and association (no association—treatments of M. vertebralis only, and with association--treatments of M. vertebralis and L. intricata) as fixed factors. Assumptions of ANOVA (homogeneity of variance and normality) were tested and met. All analyses were performed in R Tukey HSD test analyses conducted with the agricolae package.

Metadata are provided on the first tab of the excel file.