Coral calcification responds to seawater acidification: a working hypothesis towards a physiological mechanism

The decrease in the saturation state of seawater, Ω, following seawater acidification, is believed to be the main factor leading to a decrease in the calcification of marine organisms. To provide a physiological explanation for this phenomenon, the effect of seawater acidification was studied on the calcification and photosynthesis of the scleractinian tropical coral Stylophora pistillata. Coral nubbins were incubated for 8 days at three different pH (7.6, 8.0, and 8.2). To differentiate between the effects of the various components of the carbonate chemistry (pH, CO₃²⁻, HCO₃⁻, CO₂, Ω), tanks were also maintained under similar pH, but with 2-mM HCO₃⁻ added to the seawater. The addition of 2-mM bicarbonate significantly increased the photosynthesis in S. pistillata, suggesting carbon-limited conditions. Conversely, photosynthesis was insensitive to changes in pH and pCO₂. Seawater acidification decreased coral calcification by ca. 0.1-mg CaCO₃ g⁻¹ d⁻¹ for a decrease of 0.1 pH units. This correlation suggested that seawater acidification affected coral calcification by decreasing the availability of the CO₃²⁻ substrate for calcification. However, the decrease in coral calcification could also be attributed either to a decrease in extra- or intracellular pH or to a change in the buffering capacity of the medium, impairing supply of CO₃²⁻ from HCO₃⁻.     

Coral reef calcifiers buffer their response to ocean acidification using both bicarbonate and carbonate

Central to evaluating the effects of ocean acidification (OA) on coral reefs is understanding how calcification is affected by the dissolution of CO₂ in sea water, which causes declines in carbonate ion concentration [CO₃²⁻] and increases in bicarbonate ion concentration [HCO₃⁻]. To address this topic, we manipulated [CO₃²⁻] and [HCO₃⁻] to test the effects on calcification of the coral Porites rus and the alga Hydrolithon onkodes, measured from the start to the end of a 15-day incubation, as well as in the day and night. [CO₃²⁻] played a significant role in light and dark calcification of P. rus, whereas [HCO₃⁻] mainly affected calcification in the light. Both [CO₃²⁻] and [HCO₃⁻] had a significant effect on the calcification of H. onkodes, but the strongest relationship was found with [CO₃²⁻]. Our results show that the negative effect of declining [CO₃²⁻] on the calcification of corals and algae can be partly mitigated by the use of HCO₃⁻ for calcification and perhaps photosynthesis. These results add empirical support to two conceptual models that can form a template for further research to account for the calcification response of corals and crustose coralline algae to OA.     

The metabolic responses and acid–base status after feeding, exhaustive exercise, and both feeding and exhaustive exercise in Chinese catfish (Silurus asotus Linnaeus)

Feeding and exhaustive exercise are known to elevate metabolism. However, acid–base status may be oppositely affected by the two processes. In this study, we first investigated the acid–base response of Chinese catfish to feeding (the meal size was about 8% of body mass) to test whether an alkaline tide (a metabolic alkalosis created by gastric HCl secretion after feeding) would occur. We then determined the combined effects of feeding and exhaustive exercise on excess post-exercise oxygen consumption and acid–base status to determine whether the alkaline tide induced by feeding protects against acid–base disturbance during exhaustive exercise and affects subsequent recovery. Arterial blood pH increased from 7.74 ± 0.02 before feeding to 7.88 ± 0.02 and plasma [HCO₃ ⁻]_pl increased from 5.42 ± 0.29 to 7.83 ± 0.37 mmol L⁻¹ 6 h after feeding, while feeding had no significant effect on P_\textCO2 P_{{{\text{CO}}_{2} }} . Exhaustive exercise led to a significant reduction in pH by 0.46 units and a reduction of [HCO₃ ⁻]_pl by ~3 mmol L⁻¹. Lactate concentrations in white muscle and plasma increased by 2.4 mmol L⁻¹ and 13.4 μmol g⁻¹, respectively. Fed fish had a higher pH and [HCO₃ ⁻]_pl than fasting fish at rest, and the reductions in pH (0.36 units) and [HCO₃ ⁻]_pl (~2 mmol L⁻¹) were thus lower after exhaustive exercise. However, the recovery of acid–base status and metabolites were similar in digesting and fasting fish. Overall, a significant alkaline tide was found in Chinese catfish after feeding. The alkaline tide elicited by feeding significantly prevented the decreases in pH and [HCO₃ ⁻]_pl immediately after exhaustive exercise, but recovery from exhaustive exercise was not affected by digestion.     

A nonlinear calcification response to CO2-induced ocean acidification by the coral Oculina arbuscula

Anthropogenic elevation of atmospheric pCO₂ is predicted to cause the pH of surface seawater to decline by 0.3–0.4 units by 2100 AD, causing a 50% reduction in seawater [CO₃ ²⁻] and undersaturation with respect to aragonite in high-latitude surface waters. We investigated the impact of CO₂-induced ocean acidification on the temperate scleractinian coral Oculina arbuscula by rearing colonies for 60 days in experimental seawaters bubbled with air-CO₂ gas mixtures of 409, 606, 903, and 2,856 ppm pCO₂, yielding average aragonite saturation states (Ω_A) of 2.6, 2.3, 1.6, and 0.8. Measurement of calcification (via buoyant weighing) and linear extension (relative to a ¹³⁷Ba/¹³⁸Ba spike) revealed that skeletal accretion was only minimally impaired by reductions in Ω_A from 2.6 to 1.6, although major reductions were observed at 0.8 (undersaturation). Notably, the corals continued accreting new skeletal material even in undersaturated conditions, although at reduced rates. Correlation between rates of linear extension and calcification suggests that reduced calcification under Ω_A = 0.8 resulted from reduced aragonite accretion, rather than from localized dissolution. Accretion of pure aragonite under each Ω_A discounts the possibility that these corals will begin producing calcite, a less soluble form of CaCO₃, as the oceans acidify. The corals’ nonlinear response to reduced Ω_A and their ability to accrete new skeletal material in undersaturated conditions suggest that they strongly control the biomineralization process. However, our data suggest that a threshold seawater [CO₃ ²⁻] exists, below which calcification within this species (and possibly others) becomes impaired. Indeed, the strong negative response of O. arbuscula to Ω_A = 0.8 indicates that their response to future pCO₂-induced ocean acidification could be both abrupt and severe once the critical Ω_A is reached.     

Anion uptake and acid-base and ionic effects during isolated and combined exposure to hypercapnia and nitrite in the freshwater crayfish, Astacus astacus

Nitrite influx into crayfish showed saturation kinetics, supporting a carrier-mediated uptake. Addition of 4,4′-diisothiocyanatostilbene-2,2′-disulfonate (DIDS: at 10⁻⁵, 10⁻⁴ and 10⁻³M) and bumetanide (at 10⁻⁵ M and 10⁻⁴ M) to the ambient water did not significantly affect nitrite influx. Rather than suggesting that neither Cl⁻/HCO₃ ⁻ exchange nor K⁺/Na⁺/2Cl⁻ cotransport were involved in the transport, this may reflect that the gill cuticle has a low permeability to the pharmacological agents, or that the sensitivity of the transport mechanism to the inhibitors is low. Nitrite accumulation in the haemolymph was significantly decreased during hypercapnic conditions compared to normocapnic conditions. This supports the idea that an acid–base regulatory decrease in Cl⁻(influx)/HCO₃ ⁻ (efflux) induced by hypercapnia should decrease NO₂ ⁻ uptake if NO₂ ⁻ and Cl⁻ share this uptake route. The respiratory acidosis caused by exposure to hypercapnia alone was partially compensated by HCO₃ ⁻ accumulation in the haemolymph. Combined exposure to hypercapnia and nitrite improved pH recovery, mainly by augmenting the [HCO₃ ⁻] increase, but also by decreasing haemolymph PCO₂. Exposure to nitrite in normocapnic water induced an initial increase in haemolymph [HCO₃ ⁻] and later also a decrease in PCO₂. Thus, the improved acid-base compensation during combined hypercapnia and nitrite exposure was an amplification of this nitriteinduced response. Haemolymph base excess rose much more than haemolymph [Ca], suggesting that transfer of acid-base equivalents between animal and water was more important than H⁺ buffering by exoskeletal CaCO₃ in mediating the increase in haemolymph [HCO₃ ⁻]. Accepted: 27 June 2000     

Acquisition of Ca2+ and HCO3 −/CO3 2− for shell formation in embryos of the common pond snail Lymnaea stagnalis

Embryos of the freshwater common pond snail Lymnaea stagnalis develop to hatch within 10 days under control conditions (22°C, Miami-Dade tap water) and this development is impaired by removal of ambient calcium. In contrast, embryos did not exhibit dependence upon an ambient HCO₃ ⁻/CO₃ ²⁻ source, developing and hatching in HCO₃ ⁻/CO₃ ²⁻-free water at rates comparable to controls. Post-metamorphic, shell-laying embryos exhibited a significant saturation-type calcium uptake as a function of increasing ambient calcium concentration. However, changes in ambient bicarbonate concentration did not influence calcium or apparent titratable alkalinity uptake. There was a distinct shift from no significant flux in pre-metamorphic embryos to net uptake of calcium in post-metamorphic stages as indicated by an increased uptake from the micro-environment surrounding the egg mass and increased net uptake in 24-h, whole egg mass flux measurements. Furthermore, HCO₃ ⁻/CO₃ ²⁻ acquisition as measured by titratable alkalinity flux is at least partially attributable to an endogenous carbonate source that is associated with acid extrusion. Thus, calcium requirements for embryonic shell formation are met via uptake but HCO₃ ⁻/CO₃ ²⁻, which is also necessary for shell formation is acquired in part from endogenous sources with no detectable correlation to ambient HCO₃ ⁻/CO₃ ²⁻ availability.     

Effects of Carbon Dioxide Enrichment and Nitrogen Addition on Inorganic Carbon Leaching in Subtropical Model Forest Ecosystems

Soil mineral weathering may serve as a sink for atmospheric carbon dioxide (CO₂). Increased weathering of soil minerals induced by elevated CO₂ concentration has been reported previously in temperate areas. However, this has not been well documented for the tropics and subtropics. We used model forest ecosystems in open-top chambers to study the effects of CO₂ enrichment alone and together with nitrogen (N) addition on inorganic carbon (C) losses in the leachates. Three years of exposure to an atmospheric CO₂ concentration of 700 ppm resulted in increased annual inorganic C export through leaching below the 70 cm soil profile. Compared to the control without any CO₂ and N treatments, net biocarbonate C (HCO₃ ⁻-C) loss increased by 42%, 74%, and 81% in the high CO₂ concentration treatment in 2006, 2007, and 2008, respectively. Increased inorganic C export following the exposure to the elevated CO₂ was related to both increased inorganic C concentrations in the leaching water and the greater amount of leaching water. Net annual inorganic C (HCO₃ ⁻-C and carbonate C: CO₃ ²⁻-C) loss via the leaching water in the high CO₂ concentration chambers reached 48.0, 49.5, and 114.0 kg ha⁻¹ y⁻¹ in 2006, 2007, and 2008, respectively, compared with 33.8, 28.4, and 62.8 kg ha⁻¹ y⁻¹ in the control chambers in the corresponding years. The N addition showed a negative effect on the mineral weathering. The decreased inorganic C concentration in the leaching water and the decreased leaching water amount induced by the high N treatment were the results of the adverse effect. Our results suggest that tropical forest soil systems may be able to compensate for a small part of the atmospheric CO₂ increase through the accelerated processing of CO₂ into HCO₃ ⁻-C during soil mineral weathering, which might be transported in part into ground water or oceans on geological timescales.     

Is the response of coral calcification to seawater acidification related to nutrient loading?

The effect of decreasing aragonite saturation state (Ω_Arag) of seawater (elevated pCO₂) on calcification rates of Acropora muricata was studied using nubbins prepared from parent colonies located at two sites of La Saline reef (La Réunion Island, western Indian Ocean): a back-reef site (BR) affected by nutrient-enriched groundwater discharge (mainly nitrate), and a reef flat site (RF) with low terrigenous inputs. Protein and chlorophyll a content of the nubbins, as well as zooxanthellae abundance, were lower at RF than BR. Nubbins were incubated at ~27°C over 2 h under sunlight, in filtered seawater manipulated to get differing initial pCO₂ (1,440–340 μatm), Ω_Arag (1.4–4.0), and dissolved inorganic carbon (DIC) concentrations (2,100–1,850 μmol kg⁻¹). Increasing DIC concentrations at constant total alkalinity (A_T) resulted in a decrease in Ω_Arag and an increase in pCO₂. A_T at the beginning of the incubations was kept at a natural level of 2,193 ± 6 μmol kg⁻¹ (mean ± SD). Net photosynthesis (NP) and calcification were calculated from changes in pH and A_T during the incubations. Calcification decrease in response to doubling pCO₂ relative to preindustrial level was 22% for RF nubbins. When normalized to surface area of the nubbins, (1) NP and calcification were higher at BR than RF, (2) NP increased in high pCO₂ treatments at BR compared to low pCO₂ treatments, and (3) calcification was not related to Ω_Arag at BR. When normalized to NP, calcification was linearly related to Ω_Arag at both sites, and the slopes of the relationships were not significantly different. The increase in NP at BR in the high pCO₂ treatments may have increased calcification and thus masked the negative effect of low Ω_Arag on calcification. Removing the effect of NP variations at BR showed that calcification declined in a similar manner with decreased Ω_Arag (increased pCO₂) whatever the nutrient loading.     

Carbonate precipitates and bicarbonate secretion in the intestine of sea bass, <Emphasis Type="Italic">Dicentrarchus labrax</Emphasis>

The aim of this paper was to study the chemical composition of the precipitates found in the intestine of Dicentrarchus labrax and the source of HCO₃ ⁻ secreted into the intestinal lumen. The chemical analysis was performed by employing the potentiometric double titration method and by means of an electron microscope coupled with a spectrometer and X-ray powder diffraction. The results obtained suggest the presence of very insoluble intestinal precipitates, presumably formed by a mixture of CaCO₃ and MgCO₃, with a higher quantity of the former with respect to the latter. HCO₃ ⁻ secretion rate was investigated with the aid of the pH stat method in isolated tissues mounted in Ussing chamber, where the transepithelial electrical parameters were also measured. When the serosal surface of the intestinal mucosa was bathed in HCO₃ ⁻-Ringer bubbled with 1% CO₂ in O₂ while the serosal surface was bathed in HCO₃ ⁻ free Ringer solution bubbled with pure O₂, bicarbonate secretion proceeded at an almost stable rate of 0.9 ± 0.05 μeq cm⁻² h⁻¹ for about 3 h while I _sc maintained a constant value of 38 ± 1.5 μA cm⁻². The carbonic anhydrase inhibitor ethoxyzolamide elicited a progressive reduction of HCO₃ ⁻ secretion that was about 75% of the initial value after 80 min. When serosal HCO₃ ⁻–CO₂ saline was substituted with Hepes–O₂ saline base secretion progressively declined reaching a value of about 20% of the initial value. It was also strongly inhibited when Na⁺ was substituted with the impermeant cation choline and when either DIDS or ouabain were added to the basolateral side. These results suggest that most of the bicarbonate secreted is of extracellular source and is probably transported across the basolateral membrane by both Na⁺ independent mechanism and Na⁺ dependent transporter, presumably a NaHCO₃ cotransport.     

Seasonal variations of pH, pCO2, pO2, HCO3- and Ca2+ in the haemolymph: implications on the calcification physiology in Anodonta cygnea

A study about the relationship between the physical–chemical parameters and the calcium carbonate balance between the haemolymph fluid and mantle calcareous structures was carried out in Anodonta cygnea. An intense peak of HCO₃ ⁻ and a highest pH in December–January months may be understood as a preparation period for creating alkaline conditions. An intense pH decrease from January to February in parallel with the HCO₃ ⁻ reduction seems to indicate the beginning process of carbonate precipitation. On the other hand, the following calcium and HCO₃ ⁻ increases in February–May associated with a continuous and gradual pH fall profile may infer two combined aspects: calcium and HCO₃ ⁻ absorption from external environment and a simultaneous intense calcium carbonate deposition in the haemolymph. So, the pCO₂ peak in this period reflects a subsequent result on equilibrium balance between HCO₃ ⁻ absorption and deposition. The only significant pO₂ increase in the next period, from February to June, is related with an energetic increase to support the metabolic activity favouring the posterior intense pCO₂ peaks. The extended time of CO₂ production in the haemolymph from May to November should induce an increased metabolic acidosis with subsequent intense formation of both HCO₃ ⁻ and Ca²⁺ ions in the same period. This seems to result from CaCO₃ deposits dissolution in the haemolymph, the most direct calcareous source. Additionally, the later increase of metabolic succinic acid during autumn may greatly potentiate this acidosis increasing the dissolution process. Consequently, the pH profile present two simultaneous alkaline peaks in July and October, probably due to a strong HCO₃ ⁻ release from the CaCO₃ dissolution. So, the present seasonal results indicate that in the freshwater bivalve A. cygnea, the low metabolism with higher pH from the early winter is the main cause which may favour a calcareous precipitation, while the high metabolism with lower pH from the early summer may function as an inductor of calcareous dissolution in the haemolymph.     

Coral reef calcification: carbonate,bicarbonate and proton flux under conditions of increasing ocean acidification

Data on calcification rate of coral and crustose coralline algae were used to test the proton flux model of calcification. There was a significant correlation between calcification (G) and the ratio of dissolved inorganic carbon (DIC) to proton concentration ([DIC] : [H⁺] ratio). The ratio is tightly correlated with [CO₃²⁻] and with aragonite saturation state (Ω_a). An argument is presented that correlation does not prove cause and effect, and that Ω_a and [CO₃²⁻] have no basic physiological meaning on coral reefs other than a correlation with [DIC] : [H⁺] ratio, which is the driver of G.     

Effects of different ionic compositions on survival and growth of <Emphasis Type="Italic">Physa acuta</Emphasis>

Most inland saline waters in southern Australia predominantly contain Na⁺ and Cl⁻ as major ions. The proportions of Ca²⁺, Mg²⁺, SO₄ ²⁻, HCO₃ ⁻ and CO₃ ²⁻ in these waters somewhat vary and might influence salinity tolerance of freshwater organisms. Here the salinity stress of five ionic compositions to the freshwater snail Physa acuta Draparnaud (Gastropoda: Physidae) was compared: commercial sea salt Ocean Nature (ON), synthetic Ocean Nature (ONS) and three saline water types that are common in southern Australia (ONS but without [1]: SO₄ ²⁻, HCO₃ ⁻ and CO₃ ²⁻, [2]: Ca²⁺, HCO₃ ⁻ and CO₃ ²⁻, [3]: Ca²⁺, Mg²⁺), Milli-Q water was used as a negative control. The 96-h LC₅₀ values for all treatments did not differ. However in prolonged sub-lethal exposures, results varied depending on the ionic composition. Growth was negative and shell strength reduced in treatments lacking Ca. Though the content of major cationic elements (Ca, Mg, Na and K) did not differ per unit dry weight of snail across the treatments, the total load of these elements per individual snail varied among treatments. Furthermore, at the sub-lethal salinities tested, 1 and 5 mS cm⁻¹, ionic compositions had more effect on the snail’s growth than salinity. The long-term effects on freshwater animals, especially taxa with calcium-based exoskeletons, from exposure to common saline water types with low calcium concentrations will likely be greater than from exposure to saline waters with an ionic composition similar to seawater.     

Climate change and ocean acidification effects on seagrasses and marine macroalgae

Although seagrasses and marine macroalgae (macro‐autotrophs) play critical ecological roles in reef, lagoon, coastal and open‐water ecosystems, their response to ocean acidification (OA) and climate change is not well understood. In this review, we examine marine macro‐autotroph biochemistry and physiology relevant to their response to elevated dissolved inorganic carbon [DIC], carbon dioxide [CO₂], and lower carbonate [CO₃^2?] and pH. We also explore the effects of increasing temperature under climate change and the interactions of elevated temperature and [CO₂]. Finally, recommendations are made for future research based on this synthesis. A literature review of >100 species revealed that marine macro‐autotroph photosynthesis is overwhelmingly C₃ (≥ 85%) with most species capable of utilizing HCO₃^?; however, most are not saturated at current ocean [DIC]. These results, and the presence of CO₂‐only users, lead us to conclude that photosynthetic and growth rates of marine macro‐autotrophs are likely to increase under elevated [CO₂] similar to terrestrial C₃ species. In the tropics, many species live close to their thermal limits and will have to up‐regulate stress‐response systems to tolerate sublethal temperature exposures with climate change, whereas elevated [CO₂] effects on thermal acclimation are unknown. Fundamental linkages between elevated [CO₂] and temperature on photorespiration, enzyme systems, carbohydrate production, and calcification dictate the need to consider these two parameters simultaneously. Relevant to calcifiers, elevated [CO₂] lowers net calcification and this effect is amplified by high temperature. Although the mechanisms are not clear, OA likely disrupts diffusion and transport systems of H⁺ and DIC. These fluxes control micro‐environments that promote calcification over dissolution and may be more important than CaCO₃ mineralogy in predicting macroalgal responses to OA. Calcareous macroalgae are highly vulnerable to OA, and it is likely that fleshy macroalgae will dominate in a higher CO₂ ocean; therefore, it is critical to elucidate the research gaps identified in this review.     

Framework of barrier reefs threatened by ocean acidification

To date, studies of ocean acidification (OA) on coral reefs have focused on organisms rather than communities, and the few community effects that have been addressed have focused on shallow back reef habitats. The effects of OA on outer barrier reefs, which are the most striking of coral reef habitats and are functionally and physically different from back reefs, are unknown. Using 5‐m long outdoor flumes to create treatment conditions, we constructed coral reef communities comprised of calcified algae, corals, and reef pavement that were assembled to match the community structure at 17 m depth on the outer barrier reef of Moorea, French Polynesia. Communities were maintained under ambient and 1200 μatm pCO₂ for 7 weeks, and net calcification rates were measured at different flow speeds. Community net calcification was significantly affected by OA, especially at night when net calcification was depressed ~78% compared to ambient pCO₂. Flow speed (2–14 cm s^?1) enhanced net calcification only at night under elevated pCO₂. Reef pavement also was affected by OA, with dissolution ~86% higher under elevated pCO₂ compared to ambient pCO₂. These results suggest that net accretion of outer barrier reef communities will decline under OA conditions predicted within the next 100 years, largely because of increased dissolution of reef pavement. Such extensive dissolution poses a threat to the carbonate foundation of barrier reef communities.     

Acid–base regulation in the plainfin midshipman (Porichthys notatus): an aglomerular marine teleost

The plainfin midshipman (Porichthys notatus) possesses an aglomerular kidney and like other marine teleosts, secretes base into the intestine to aid water absorption. Each of these features could potentially influence acid–base regulation during respiratory acidosis either by facilitating or constraining HCO₃ ⁻ accumulation, respectively. Thus, in the present study, we evaluated the capacity of P. notatus to regulate blood acid–base status during exposure to increasing levels of hypercapnia (nominally 1–5% CO₂). Fish exhibited a well-developed ability to increase plasma HCO₃ ⁻ levels with values of 39.8 ± 2.8 mmol l⁻¹ being achieved at the most severe stage of hypercapnic exposure (arterial blood PCO₂ = 21.9 ± 1.7 mmHg). Consequently, blood pH, while lowered by 0.15 units (pH = 7.63 ± 0.06) during the final step of hypercapnia, was regulated far above values predicted by chemical buffering (predicted pH = 7.0). The accumulation of plasma HCO₃ ⁻ during hypercapnia was associated with marked increases in branchial net acid excretion (J _NETH⁺) owing exclusively to increases in the titratable alkalinity component; total ammonia excretion was actually reduced during hypercapnia. The increase in J _NETH⁺ was accompanied by increases in branchial carbonic anhydrase (CA) enzymatic activity (2.8×) and CA protein levels (1.6×); branchial Na⁺/K⁺-ATPase activity was unaffected. Rectal fluids sampled from control fish contained on average HCO₃ ⁻ concentrations of 92.2 ± 4.8 mmol l⁻¹. At the highest level of hypercapnia, rectal fluid HCO₃ ⁻ levels were increased significantly to 141.8 ± 7.4 mmol l⁻¹ but returned to control levels during post-hypercapnia recovery (96.0 ± 13.2 mmol l⁻¹). Thus, the impressive accumulation of plasma HCO₃ ⁻ to compensate for hypercapnic acidosis occurred against a backdrop of increasing intestinal HCO₃ ⁻ excretion. Based on in vitro measurements of intestinal base secretion in Ussing chambers, it would appear that P. notatus did not respond by minimizing base loss during hypercapnia; the increases in base flux across the intestinal epithelium in response to alterations in serosal HCO₃ ⁻ concentration were similar in preparations obtained from control or hypercapnic fish. Fish returned to normocapnia developed profound metabolic alkalosis owing to unusually slow clearance of the accumulated plasma HCO₃ ⁻. The apparent inability of P. notatus to effectively excrete HCO₃ ⁻ following hypercapnia may reflect its aglomerular (i.e., non-filtering) kidney coupled with the normally low rates of urine production in marine teleosts.     

Mechanisms of inorganic carbon acquisition in Gracilaria gaditana nom. prov. (Rhodophyta)

The mechanisms for acquisition of dissolved inorganic carbon (DIC) in the red macroalga Gracilaria gaditana nom. prov. have been investigated. The capacity for HCO₃ ⁻ use by an extracellular carbonic anhydrase (CA; EC 4.2.1.1), and by an anion exchanger with similar properties to that of red blood cells (AE1), has been quantified. It was illustrated by comparing O₂ evolution rates with those theoretically supported by CO₂, as well as by photosynthesis-pH curves. Both external and internal CA, and a direct uptake were involved in HCO₃ ⁻ use, since photosynthesis and pH evolution were affected by acetazolamide, 6-ethoxyzolamide (inhibitors of external and total CA, respectively) and 4,4′-diisothiocyanatostilbene-2,2′-disulfonate, (DIDS; an inhibitor of HCO₃ ⁻ exchanger protein). The activity of the external CA was detected by a potentiometric method and by an alternative method based on the study of O₂ evolution after addition of CO₂ and acetazolamide. The latter method showed a residual photosynthetic rate due to direct HCO₃ ⁻ use. Inhibitors caused a reduction in the pH compensation points in pH-drift experiments. The CO₂ compensation points for photosynthesis increased when the inhibitors were applied, indicating a suppresion of the pathways involved in the carbon-concentrating mechanism. The net photosynthesis rates as a function of DIC concentration displayed a biphasic pattern that could be supported by the occurrence of the two mechanisms of HCO₃ ⁻ use. The potential contribution to HCO₃ ⁻ acquisition by the DIDS-sensitive mechanism was higher after culturing at a high pH. Our results suggest that the HCO₃ ⁻ use by Gracilaria gaditana is carried out by the two DIC uptake mechanisms. These operate simultaneously with different affinities for DIC, the indirect HCO₃ ⁻ use by an external CA activity being the main pathway. The presence of a carbon-concentrating mechanism confers eco-physiological advantages in a fluctuating ecosystem subjected daily to high pHs and low DIC concentrations. Received: 3 July 1998 / Accepted: 30 November 1998     

Carbonic anhydrase,a respiratory enzyme in the gills of the shore crabCarcinus maenas

This paper summarizes investigations on the enzyme carbonic anhydrase (CA) in the gills of the osmoregulating shore crabCarcinus maenas. Carbonic anhydrase, an enzyme catalyzing the reversible hydration of CO₂ to HCO₃ ⁻ and H⁺, is localized with highest activities in the posterior salt-transporting gills of the shore crab- and here CA activity is strongly dependent on salinity. Contrary to the earlier hypothesis established for the blue crabCallinectes sapidus that cytoplasmic branchial CA provides the counter ions HCO₃ ⁻ and H⁺ for apical exchange against Na⁺ and Cl⁻, the involvement of CA in NaCl uptake mechanisms can be excluded inCarcinus. Differential and density gradient centrifugations indicate that branchial CA is a predominantly membrane-associated protein. Branchial CA was greatly inhibited by the sulfonamide acetazolamide (AZ) K_i=2.4·10⁻⁸ mol/l). Using the preparation of the isolated perfused gill, application of 10⁻⁴ mol/l AZ resulted in an 80% decrease of CO₂/HCO₃ ⁻ excretion. Thus we conclude that CA is localized in plasma membranes, maintaining the CO₂ gradient by accelerating adjustment of the pH-dependent CO₂/HCO₃ ⁻ equilibrium.     

A Mathematical Model of the Pancreatic Ductal Epithelium

A mathematical model of the HCO⁻ ₃-secreting pancreatic ductal epithelium was developed using network thermodynamics. With a minimal set of assumptions, the model accurately reproduced the experimentally measured membrane potentials, voltage divider ratio, transepithelial resistance and short-circuit current of nonstimulated ducts that were microperfused and bathed with a CO₂/HCO⁻ ₃-free, HEPES-buffered solution, and also the intracellular pH of duct cells bathed in a CO₂/HCO⁻ ₃-buffered solution. The model also accurately simulated: (i) the effect of step changes in basolateral K⁺ concentration, and the effect of K⁺ channel blockers on basolateral membrane potential; (ii) the intracellular acidification caused by a Na⁺-free extracellular solution and the effect of amiloride on this acidification; and (iii) the intracellular alkalinization caused by a Cl⁻-free extracellular solution and the effect of DIDS on this alkalinization. In addition, the model predicted that the luminal Cl⁻ conductance plays a key role in controlling both the HCO⁻ ₃ secretory rate and intracellular pH during HCO⁻ ₃ secretion. We believe that the model will be helpful in the analysis of experimental data and improve our understanding of HCO⁻ ₃-transporting mechanisms in pancreatic duct cells. Received: 18 October 1995/Revised: 5 July 1996     

Photosynthetic CO2 affinity of the aquatic carnivorous plant Utricularia australis (Lentibulariaceae) and its investment in carnivory

Aquatic carnivorous plants usually grow in shallow dystrophic waters poor in inorganic N and P. Utricularia australis was chosen as a model plant for its prolific distribution and great ecological plasticity. The photosynthetic CO₂ compensation point and factors associated with investment in carnivory and capture of prey were measured in 17 U. australis micropopulations in Třeboň basin, Czech Republic, together with water chemistry factors at these sites differing greatly in their trophic level, water hardness, and prey availability. Apical shoot growth rate was estimated at some oligotrophic sites. The micropopulations differed greatly in the proportion of traps with animal prey (2.7–70%, mean 26%), trap proportion to total biomass (1.4–42%, mean 26%), mean trap biomass (0.7–63 μg trap⁻¹, mean 19 μg), and maximum trap size (1–3 mm, mean 2.0 mm). CO₂ compensation points ranged from 0.7 to 6.1 μM (mean 2.6 μM). A weak HCO₃ ⁻ use (compensation point 0.51 mM) was found in plants growing in alkaline water. Trap biomass proportion did not correlate significantly with prey capture and CO₂ compensation points with ambient [CO₂]. A very rapid apical growth (2.5–4.2 new nodes day⁻¹) occurred in sand pits. Thus, HCO₃ ⁻ use in U. australis can be induced by growing at very high pH. CO₂ compensation points resembled those known in other aquatic non-carnivorous plants. They did not reflect carnivory. In spite of very rapid apical shoot growth, the relative growth rate of U. australis can be zero in oligotrophic habitats without prey.     

Intracellular pH regulation and buffer capacity in CO2/HCO− 3-buffered media in cultured epithelial cells from rainbow trout gills

The influence of a CO₂/HCO⁻ ₃-buffered medium on intracellular pH regulation of gill pavement cells from freshwater rainbow trout was examined in monolayers grown in primary culture on glass coverslips; intracellular pH (pH_i) was monitored by continuous spectrofluorometric recording from cells loaded with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxy-fluoroscein. When cells in HEPES-buffered medium at normal pH=7.70 were transferred to normal CO₂/HCO⁻ ₃-buffered medium {P _CO2=3.71 mmHg, [HCO⁻ ₃]= 6.1 mmol l⁻¹, extracellular pH (pH_e)=7.70}, they exhibited a brief acidosis but subsequently regulated the same pHi (∼7.41) as in HEPES. Buffer capacity (β) increased by the expected amount (5.5–8.0 slykes) based on intracellular [HCO⁻ ₃], and was unaffected by most drugs and treatments. However, after transfer to high P _CO2=11.15 mmHg, [HCO⁻ ₃]= 18.2 mmol l⁻¹ at the same pH_e=7.70, the final regulated pHi was elevated (∼7.53). The rate of correction of alkalosis caused by washout of this high P _CO2, high-HCO⁻ ₃ medium was unaffected by removal of extracellular Cl⁻. Removal of extracellular Na⁺ lowered resting pH_i and greatly inhibited the rate of pH_i recovery from acidosis. Bafilomycin A₁ (3 μmol l⁻¹) had no effect on these responses. However amiloride (0.2 mmol l⁻¹) inhibited recovery from acidosis caused by washout of an ammonia prepulse, but did not affect resting pH_i, the latter differing from the response in HEPES where amiloride also lowered resting pH_i. Similarly 4-acetamido-4′- isothiocyanatostilbene-2,2′-disulfonic acid, sodium salt (0.1 mmol l⁻¹) did not affect resting pH_i but slowed the rate of recovery from acidosis, though to a lesser extent than amiloride. Removal of extracellular Cl⁻ also slowed the rate of recovery but greatly increased β by an unknown mechanism; when this was taken into account, H⁺ extrusion rate was unaffected. These results are consistent with the presence of Na⁺-(HCO⁻ ₃)_N co-transport and/or Na⁺-dependent HCO⁻ ₃/Cl⁻ exchange, in addition to Na⁺/H⁺ exchange, as mechanisms contributing to “housekeeping” pH_i regulation in gill cells in CO₂/HCO⁻ ₃ media, whereas only Na⁺/H⁺ exchange is seen in HEPES. Both Na⁺-independent Cl⁻/HCO⁻ ₃ exchange and V-type H⁺-ATPase mechanisms appear to be absent from these cells cultured in isotonic media. Accepted: 30 November 1999