Interactive comment on “ Physiological compensation for environmental acidification is limited in the deep-sea urchin Strongylocentrotus fragilis ”

Taylor et al. present the first long term acclimation study of a deep sea calcifying invertebrate to the combined impacts of ocean acidification and hypoxia. This is an important area of research that has been neglected so far. The study has been carried out well and gives valuable insight into the ecophysiology of the sea urchin S. fragilis. The discussion would benefit from a stronger focus on expected scenarios of change and habitat variability. The paper will be a very good contribution to the field following careful revision.


Introduction
Anthropogenic CO 2 is penetrating to the deep waters of the oceans through thermohaline circulation and other vertical mixing processes, and has now reached depths over 1000 m in the Eastern Pacific (Gruber and Sarmiento, 2002;Sabine et al., 2004), overlapping the Oxygen Minimum Zone (OMZ) and driving ocean acidification (OA).Increased CO 2 concentrations reduce even further the typically low pH of OMZs, which are known to be stressful environments for many taxa.As OA and climate-related Figures deoxygenation (Doney et al., 2009b;Keeling and Garcia, 2002;Stramma et al., 2011) intensifies in the future, animals in OMZs are expected to experience increased physiological stress, particularly for calcifying taxa (Dupont et al., 2010;Melzner et al., 2012;Portner et al., 2011).Deep-sea animals living in this dark, energy-poor environment have evolved with moderate to mild temporal variation in biochemical conditions, but very large spatial variation in biochemical conditions.In particular, taxa spanning a range of depths within and beyond an OMZ experience very large ranges of temperature, pH, oxygen, and food; not as individuals, but as populations.The high amount of genetic diversity presumably required for a population to persist throughout a bathymetric range including an OMZ suggests we should expect fairly high tolerance to at least the extent of conditions seen within its bathymetric range.We expect the apparent limitation in tolerance of OMZ taxa to global climate change is not a product of evolutionary constancy, but rather is a product of the limited energy (food and oxygen) available to cope with the stress of environmental change (Barry et al., 2011;Pane and Barry, 2007;Seibel and Walsh, 2003).
Environmental acidification can affect an individual marine animal's energy budget by disrupting homeostatic processes; parameters including growth rates, reproduction, and nutritional status may consequently be reduced (Dupont et al., 2012;Portner, 2012;Uthicke and Fabricius, 2012).The extent to which a marine animal can regulate extracellular fluid acid-base balance thus offers a reasonable predictor of the extent to which behavior, growth, and reproduction may be affected by increases in environmental acidity (Seibel and Walsh, 2003;Spicer et al., 2007).Proper function of enzymes, including those necessary for homeostatic processes, demands precise internal pH conditions; precise regulation of acid-base balance is typically far better honed in more phylogenetically derived animals (e.g., bony fishes as compared to aquatic invertebrates).Studies have shown major species-dependence in the acid-base regulatory capacity of sea urchins (Calosi et al., 2013).Shallow-living sea urchins Psammechinus miliaris and Echinus esculentus were shown to perfectly compensate the respiratory acidosis induced by emersion (Spicer et al., 1988) a taxa experiencing considerable environmental variability, is able to fully or partially compensate extracellular pH changes by accumulation of bicarbonate (Stumpp et al., 2012).However, taxa distributed around a CO 2 vent revealed very different capacities for accumulation of extracellular bicarbonate between sea urchins Arbacia lixula and Paracentrotus lividus (Calosi et al., 2013).To our knowledge, the ability of deep-sea urchin taxa to regulate extracellular acid-base balance is yet unknown.Ocean acidification typically does not happen as a solitary environmental change; it is often accompanied by warming temperatures and less frequently by hypoxia.Each of these parameters can act simultaneously on individual physiology; occasionally in a way that offsets negative effects of one stressor, but additive (or synergistic) negative effects are also observed.For example, concomitant low O 2 and high CO 2 interfere with aquatic respiration as O 2 becomes more difficult to obtain from the hypoxic water and CO 2 becomes more difficult to excrete to the water along a weaker concentration gradient.The scientific and popular literature is rich with examples of the effects of multiple climate stressors on aquatic animals (Crain et al., 2009;Howell, 2012;Najjar et al., 2010;Wahl, 2009); however, the OA literature has yet to be enriched by the addition of practical multiple stressor studies.
A high priority motivating research concerning the effects of OA on marine animal physiology is to understand more fully the potential impacts of OA on ecosystem services important to society.For most, the deep-sea is unseen and unheard, but society depends upon the continuing function and health of deep-sea ecosystems for many fisheries and more fundamentally for their role in key biogeochemical cycles (Armstrong et al., 2010;Danovaro et al., 2008;Dell'Anno and Danovaro, 2005;Introduction Conclusions References Tables Figures

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Full Jørgensen and Boetius, 2007;Suttle, 2007).Unfortunately, measuring and valuing deep-sea ecosystem functions and services is difficult, as our quantitative knowledge of these goods and services, and their interactions, is very limited (Armstrong et al., 2010).The deep-sea fragile urchin, Strongylocentrotus fragilis, is a member of the Strongylocentrotidae, a worldwide, and largely shallow water family, that inhabits the upper continental slope along the eastern North Pacific, ranging in depth from 200-1200 m (in seawater chemistry described in Fig. 1) off central California.It is a key member of the benthic megafaunal community on the upper slope, where it is an important detritivore.The bathymetric range of S. fragilis spans a fairly large gradient in temperature, oxygen, pH, and presumably food supply, each of which may affect its individual performance and population distribution.We hypothesize that the physiological performance of S. fragilis will vary considerably over the large range of environmental variability across its bathymetric range, and it will be most vulnerable to ocean acidification and hypoxia in the core of the OMZ.Two experiments have been used to test this hypothesis.The first addresses the ability of S. fragilis to regulate internal acid-base balance at near-and far-future levels of environmental CO 2 .The second set of experiments addresses the behavioral, nutritional, and growth consequences of similar levels of CO 2 on S. fragilis, and also examined whether this taxon is able to improve upon baseline rates of locomotion, feeding, and growth, on exposure to surface levels of O 2 rather than typical OMZ levels.Lobos, animals were kept in 5 • C seawater for 3-5 h until arrival at the Monterey Bay Aquarium Research Institute (MBARI).Urchins were then held in climate-controlled environmental chambers in the laboratory, in an approx.50 L acrylic tank receiving flowthrough chilled seawater pumped in from Monterey Bay (5.5 ± 0.5 34 ± 1 ppt salinity).

Experimental design
During both experiments, individual urchins were held in 1 L glass jars overflowing with seawater with the desired chemistry at a rate of approx.30 mL min −1 , for the duration of the experiment.Target conditions for seawater oxygen and pH were maintained throughout the study period by a PC-manipulated, Gas-Controlled Aquarium (GCA) system supplying seawater to the laboratory's climate-controlled environmental chamber.In brief, oxygen and pH for each seawater treatment holding tank were controlled using a combination of sensors (Aanderra oxygen optodes and Honeywell pH probes) and membrane contactors connected to recirculation pumps and gas sources.A Lab-VIEW software system integrated with mass flow controllers for oxygen, carbon dioxide, and nitrogen sources allows real-time, automated regulation of gas concentrations in each tank (Barry et al., 2008).Experimental seawater conditions were monitored throughout using a logging system for pH, oxygen, and temperature sensors in the seawater head tanks, and from samples collected directly from jars containing urchins (pH and Total CO 2 (C CO 2 ) measurements).During regular spot-sampling, treatment and control conditions within jars were checked using a Thermo Scientific Orion 5-Star handheld meter with an optode and temperature compensated pH probe.The Orion optode was calibrated using a twopoint method at temperature (approx.6 • C).The probe comes with a sleeve to provide a 100 % saturation calibration point; a sodium dithionate solution was used to generate a 0 % saturation point.An Aanderraa optode was also used to crosscheck the oxygen (% saturation, µM) measurements determined by the GCA system.During jar sampling, the water flow delivery line was removed and the probes and cables (bound Figures

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Full together) were lowered mid-way into the 1 L volume of seawater in the jars, easily avoiding the animal contained therein.Output was given 5 min to stabilize, after which values from each probe were recorded.
During animal sampling events, jar seawater and tap samples were also collected in glass, gas-tight Scintillation vials with negligible headspace and analyzed within one hour, using the same protocols by which concurrently sampled animal fluids were analyzed.For these samples, C CO 2 was measured by non-dispersive infrared analysis (LI-COR model 6262), as detailed by Friederich et al. (2002) In brief, samples were acidified (5 % phosphoric acid) and the stripped gas introduced into an infrared analyzer.A standard curve was created using sodium carbonate (dried for 4 h at 250 • C before making carbonate standards), and resultant values standardized to certified reference material (A.Dickson CRM, Scripps Institute of Oceanography, La Jolla, CA).Seawater pH was measured by spectrophotometry using the indicator dye m-cresol purple (Clayton and Byrne, 1993;SOP 6b of Dickson et al., 2007;Part 1 of Riebesell et al., 2010).

Experiment one: internal acid-base balance
Experiment one was used to measure the ability of S. fragilis to regulate internal acidbase balance under a range of environmental conditions, and evaluate the hypothesis that its acid-base regulatory capacity is weak.The impact of hypercapnia on urchin acid-base balance was examined over a 31 day period.Four treatments (30 individuals per treatment) were defined as pH (total scale) 8.0, 7.5, 7.1, and 6.7 at in situ O 2 (25 µM) and 5 • C. No animals were fed during the experimental period.

Blood buffering capacity
The non-bicarbonate buffering capacity (β) of extracellular fluids was determined for urchins collected at the same time, but not undergoing other experimentation.Samples gas-tight Hamilton syringes rinsed with a modified Crab Ringer comprised of NaCl (460 mM), KCl (10 mM), CaCl 2 (20 mM), MgCl 2 (9.5 mM), adjusted to pH 7.8 (Lang and Gainer, 1969;Pane and Barry, 2007).Fluid samples were then centrifuged aerobically at 4000 × g at 5 • C for 1 min to remove debris, added (200 µL) to round-bottom flasks for equilibration with humidified gas mixtures of CO 2 and nitrogen from pre-analyzed cylinders (Airgas), and allowed to equilibrate for 90 min in a shaking cooler at 5 • C.
Samples were then drawn into gas-tight syringes and pH (total scale) was measured using a microelectrode and in-line reference electrode (Microelectrodes) thermostatted to 5  Boutilier et al. (1984) and Truchot (1976).βs were derived from linear regression of pH-bicarbonate plots.

Blood chemistry
Five animals were terminally sampled from each pH treatment 5 days before treatment conditions were achieved; and on days 0 (when treatment conditions were achieved after a 5 day ramp to conditions), 1, 3, 7, and 31 of treatment.Extracellular fluids were attained while animals were gently held by hand at the top of their respective jars in the environmental chamber, aboral surface just below the water's surface.Approx.Introduction

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Full non-dispersive infrared analysis (LI-COR, as above) by diluting 300 µL extracellular fluids in 3300 µL Nanopure water in glass scintillation vials (by Hamilton syringe through a septum with negligible head-space).A 16-gauge needle was used to vent the septa, and another fed sample into the LI-COR.A standard curve was created using three sets of four standards prepared as above, in parallel, and measured before, during, and after extracellular fluid samples.Resultant values were standardized to certified reference material (A.Dickson CRM, Scripps Institute of Oceanography, La Jolla, CA).

Experiment two: behavior and nutritional status
The second of our two experimental groups of S. fragilis was used to investigate the impacts of hypercapnia and O 2 availability on locomotion, feeding, growth, and reproductive capacity during approx.4 months (140 d) exposure.Urchins collected from a depth of 660 m at Sponge Ridge were allowed two days to acclimate en masse in aquaria as described above.The health of 150 urchins (< 48 mm diameter) was evaluated prior to their inclusion in the experiment using a flip test by turning them aboral side down and monitoring the time required to 'right' themselves (oral side down).These 150 urchins were segregated haphazardly into 5 groups (30 individuals per group) and each individual placed in a 1 L jar.The jars (A-E; 1-30) were then connected to water supply lines of the Gas Controlled Aquaria (GCA) as described above.The five treatments were defined as pH (total scale) 7.9, 7.6, 7.2, and 6.7 at in situ O 2 (25 µM), and pH 7.9 at surface O 2 (220 µM).All treatments were applied at the in situ temperature of 5 • C. In each treatment, two feeding regimes were applied; odd numbered urchins were fed kelp to satiation, while even numbered urchins were fasted during the experimental period.Introduction

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Full Routinely over the course of the experiment, individuals from all treatments were rotated through the video station and evaluated using a flip test.

Feeding
Urchins in the feeding treatment groups (odd numbered jars in treatments A-E) were fed weighed stipes of the kelp Macrocystis pyrifera to satiation; kelp in jars was replaced every 4-12 days and weighed for calculations of feeding rates.Visual evidence of kelp consumption (Fig. 3b) was also recorded to document feeding frequency; this was used as a Y/N measure of feeding during the feeding period.The weight change of kelp upon cutting and exposure to seawater was quantified in parallel for each feeding period, and found to be negligible.A validated blotting technique was used to ensure consistency in attaining kelp weights on removal from treatment jars.

Photo sizing methods
All urchins included in this experiment were digitally photographed (Fig. 3a) in anticipation of sizing animals from images.Once urchins had been initially evaluated for their ability to right themselves and placed in their assigned jars, a digital 35 mm camera mounted on a tripod and operated via infrared remote was used to take photographs of 8322 Figures

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Full each animal against a solid black background.Two internal metric scales, one on the background and one on a wand allowing its placement along the major axis mid-line of the animal, were included in each image.Midway through the experiment this process was repeated.At the termination of the experiment (half of each treatment group at 30 and the other half at 140 days), digital images were again collected.As before overhead shots were taken of the aboral surface of the animal; additionally, images of the oral side were taken at this time.Animals were then removed from jars and measured to the nearest 0.1 mm using calipers; these measurements were used to validate image measurements.

Reproductive capacity
Gonadosomatic index (GSI) was measured as a proxy for reproductive capacity.Urchin whole body wet weight was measured in the urchins sacrificed after 140 days of treatment exposure; gonads were also extracted and weighed separately in order to calculate the terminal GSI (percentage of gonad to whole body wet weight) for each animal at this timepoint.

Statistical analyses
All data were analyzed using StatPlus (AnalystSoft) software.Each mean value is reported with its standard error of mean (mean ± SEM).One-way analysis of variance (ANOVA) was used to test the impact of the treatment conditions on parameters measured in experiment one, as these data met assumptions for normality and equal variance as determined by the Shapiro-Wilk test (Shapiro and Wilk, 1965) and the F Test for equal variance.In experiment two, most data did not meet assumptions of normality; the Mann-Whitney U test was thus used to determine statistical significance as detailed below.Introduction

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Full

Water chemistry
GCA logging and spot-sampling revealed that target treatment conditions for experiments one and two were maintained ±5 µM O 2 , 0.5 • C, and 0.05 pH units.

Experiment one: internal acid-base balance
In all treatments except pH 8.0, extracellular fluids mirrored closely the pH of treatment seawater.As shown in pH-bicarbonate (Davenport) diagrams (Fig. 4), a pattern of significant (ANOVA, F = 9.68, 30.46, and 30.55 for pH 7.5, 7.1, and 6.7, respectively; p < 0.0001 in all cases) hypercapnic-induced acidosis persists in S. fragilis.Within the first 24 h of all three hypercapnic exposures, extracellular fluid chemistry was titrated in an acidic direction, roughly along the non-bicarbonate buffering (β) line and consistent with CO 2 -derived respiratory acidosis (Fig. 4).Metabolic compensation of respiratory acidosis is typical of aquatic regulators and is characterized by large gains in bicarbonate concentration and movement in an alkalotic direction across P CO 2 isopleths; there was evidence of this type of compensation only in animals from the pH 7.5 treatment, and even in this case compensation was incomplete after 31 days (Fig. 4b, two-tailed t test, t crit = 2.45, p < 0.05).No mortality occurred in experiment one.

Mortality
In the pH 6.6 group, there was one mortality (3.3 %) before the first terminal samples were taken (day 30) and four before the final terminal samples were taken on day 140.In the pH 7.2 group, three mortalities occurred before the final sampling day.In the pH 7.6 group, there was one mortality before day 140.In the pH 7.9 group, no Figures

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Full mortality occurred.In the pH 7.9/high O 2 group, there were two mortalities before the final sampling day.

Locomotion
Time required for an overturned urchin to right itself (flip time) increased for the pH 6.6 treatment after 6 days (two-tailed t test, t crit = 1.99, p < 0.05), and remained increased for all timepoints after day 16 (two-tailed t test, t crit = 1.99, p < 0.0001), compared to pre-treatment flip times.Increased flip response time was also seen in S. fragilis exposed to pH 7.2 for 16 days (two-tailed t test, t crit = 1.99, p < 0.001).

Feeding
During the entire experimental period, urchins fed significantly less frequently (Fig. 6a) than the pH 7.9/high O 2 group when exposed to pH 6.6 (Mann-Whitney U test, U = 289, p < 0.001) and pH 7.2 (Mann-Whitney U test, U = 234, p = 0.002).Urchins at pH 6.6 consumed significantly less kelp than pre-treatment at most timepoints after 10 days of exposure to treatment conditions (Fig. 6b, denoted by "+").Feeding rates of urchins at pH 6.6 were also significantly less than those of urchins at pH 7.9/high O 2 at timepoints after 10 days of exposure (Fig. 6b, denoted by "*").Urchins exposed to pH 7.2 also fed less often than urchins at pH 7.9/high O 2 at timepoints 16 (Mann-Whitney U test, U = 48, p = 0.037) and 37 (Mann-Whitney U test, U = 42, p = 0.024) days into the experiment (Fig. 6b).The F Test for equal variances revealed significantly greater variation in feeding rates of animals at pH 7.6 during the first week of exposure (F = 40, p < 0.001), as compared with pre-treatment variance (Fig. 6b).

Growth
After 30 days of exposure to treatment conditions, changes in body width were significantly correlated (negatively) only with pH 6.6 fed animals (Fig. 7a; Mann-Whitney U test, U = 52, p = 0.005); change in body height was correlated (negatively) with pH 6.6 Introduction

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Reproduction
S. fragilis exposed to pH 6.6 and fed for 140 days showed significantly lower GSI (Mann-Whitney U test, U = 29, p < 0.05) when compared with animals from all other treatments (Fig. 8).Fasted animals from all treatments showed GSI values statistically equivalent to zero (Fig. 8, two-tailed, one-sample t tests, p > 0.05).

Discussion
S. fragilis has negligible ability to regulate acid-base balance during environmental acidification, substantiated by the low non-bicarbonate buffer capacity of its extracellular fluids (Fig. 4).At each reduced pH treatment condition S. fragilis showed an uncompensated respiratory acidosis, which increased in intensity as treatment pH Introduction

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Full decreased.This result is in accordance with findings for shallower living Strongylocentrotidae (Spicer et al., 2007(Spicer et al., , 2011;;Stumpp et al., 2012), and is not surprising considering the lack of available internal body compartments for ion exchange and thereby movement of H + for acid-base balance regulation (Portner, 1993).S. fragilis is largely devoid of internal structures, visibly only possessing a thin, relatively straight-thru gastrointestinal tract and variable gonadal mass (typically with GSI of approx.5 %; see Fig. 8) within otherwise non-compartmentalized extracellular fluids.The pH along the bathymetric range of S. fragilis drops to near pH 7.6 in the OMZ (Fig. 1).On exposure to environmental acidification of only 0.1 pH units below this in situ value, S. fragilis was still unable to fully compensate a respiratory acidosis after 30 days (Fig. 4).The consequences of prolonged systemic acidosis are addressed by experiment two, in which results show that among the parameters measured the effects of a presumably slight extracellular acidosis are negligible at pH 7.6.However, during further reduction of pH to a treatment 0.4 units less than the current OMZ low of pH 7.6, prolonged systemic acidosis (seen in Fig. 4) is correlated with significant adverse impacts-at least transiently-on locomotion and feeding (see treatment pH 7.2 of Figs. 5 and 6, respectively).
Adding to the complexity of the story, Dupont et al. (2012) show that although Strongylocentrotus drobacheiensis is impaired by elevated pCO 2 up to 4 months, by 16 months it has fully compensated and has acclimated in many ways.This result is in accordance with our measurements of transient adverse effects at treatment pH 7.2, but suggests that acclimation may be possible on much longer time scales (i.e.4-16 months) than we considered.We expect that S. fragilis exposed to pH 7.2 for upwards of 4 months may see continued improvements in metrics of physiological compensation of acidosis.
The results of experiment two suggest S. fragilis is vulnerable to adverse effects of OA via impaired locomotion, reduced feeding, decreased somatic growth and reduced reproductive capacity.Effects on locomotion (Fig. 5) and feeding (Fig. 6 ( Caldeira and Wickett, 2003;Doney et al., 2009a, b).However, at pH 6.6 the effects on locomotion and feeding are more catastrophic and persistent, and are accompanied by decreased somatic growth and a significant decline in reproductive capacity.Not surprising but perhaps most worrisome for the persistence of S. fragilis at its current bathymetric range is the adverse impact of fasting on reproductive capacity (Fig. 8).
Presumably, GSI is prioritized over any other process in the energy budget, such that the population persists, although very few studies have examined energy budget priorities in sea urchins.Those available show that reproductive growth in fact tends not to be prioritized (Stumpp et al., 2012) unless food quality (i.e., protein content) is high (Poorbagher et al., 2010).The ability to search for, consume, and assimilate prey items is thus of utmost concern for the persistence of S. fragilis; effective locomotion is an essential aspect of the detritivore lifestyle and may be compromised by OA.
Based on the expected impact of global climate change on ocean pH and oxygen, our results suggest that we should not expect a mass die-off and ultimate extinction of S. fragilis in the eastern North Pacific; rather, we expect to see a contraction of its bathymetric range.S fragilis will doubtfully have a problem at the shallow end of its bathymetric range (i.e., 200-300 m, see Fig. 1) in the future, but perhaps will suffer a decline in the heart of the OMZ (i.e., 400-1000 m).While a shoaling of the S. fragilis bathymetric range seems likely, we have only examined one taxon of a complex ecosystem (Barry et al., 2011;Loreau et al., 2001;Russell et al., 2012).It is possible that while S. fragilis may better tolerate the chemistry of the shallower range, competition in fact may be a more considerable limitation in the new depth range.The OMZ, while generally considered an energetically limited, arduous area of the ocean to inhabit, has seemingly few competing taxa.
The effect of global climate change on deep-sea ecosystems and the services they provide society is undoubtedly a complex issue in need of further investigation.While the impacts of OA on individual animal physiology and population dynamics are becoming better understood, consequent changes in taxa interactions and resultant community structure are important next considerations.Introduction

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Full  .S. fragilis feeding frequency (A) and rates (B), before and during exposure to pH 2 7.9, 7.6, 7.1, and 6.7 at in situ O2 levels, and pH 7.9 with 100% O2 saturation.fragilis Gonadosomatic Index (GSI) after 140 days of exposure to pH 7.9, 7.6, 7.2, and 6.6 at in situ O 2 levels, and pH 7.9 with 100 % O 2 saturation, following feeding and fasting regimes.GSI is shown as mean ± SEM.
Discussion Paper | Discussion Paper | Discussion Paper | . Similarly, Strongylocentrotus droebachiensis, Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | S. fragilis were captured by suction sampler from the Remotely Operated Vehicle (ROV) Ventana, operated from the R/V Point Lobos, from depths of approx.500 to 1000 m in Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | of extracellular fluids were drawn anaerobically through the oral membrane into ice-cold Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | The day following urchin segregation into jars, video recordings were made of a flip test for each animal.Down-looking video through the open-top jar was collected using simple web cameras in waterproof housings connected to a PC.With this setup, 8-10 individuals were monitored simultaneously.The time taken for each individual to right itself was noted, with recordings stopped if the individuals failed to turn over within two hours.
Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | ) are transient at pH 7.2, a level of OA feasible in the OMZ by the end of the century Discussion Paper | Discussion Paper | Discussion Paper |

Figure 1 . 4 Fig. 1 . 8 Fig. 4 .
Figure 1. S. fragilis lives at depths of 200-1200 m in Monterey Bay, CA, in seawater with 2 chemistry described in the shaded area of the plot.3 4
• C and coupled to an Accumet (Fisher Scientific) pH meter.pH was standardized with Radiometer Analytical precision buffers adjusted to the ionic strength of S. fragilis extracellular fluid (∼ 1050 mOsm).C CO 2 was measured as for seawater samples by non-dispersive infrared analysis (LI-COR).Extracellular fluid CO 2 tension (P aCO 2 ) and [HCO − 3 ], based on measured pH and CCO 2 , were calculated by rearrangement of the Henderson-Hasselbach equation with values for CO 2 solubility (αCO 2 ) and apparent pK (pK ) at the appropriate temperature taken from