Extracellular Acid-base Balance Research Articles

The effects of elevated temperature and PCO2 on the energetics and haemolymph pH homeostasis of juveniles of the European lobster, Homarus gammarus.

Regulation of extracellular acid-base balance, while maintaining energy metabolism, is recognised as an important aspect when defining an organism's sensitivity to environmental changes. This study investigated the haemolymph buffering capacity and energy metabolism (oxygen consumption, haemolymph [l-lactate] and [protein]) in early benthic juveniles (carapace length <40 mm) of the European lobster, Homarus gammarus, exposed to elevated temperature and PCO2 At 13°C, H. gammarus juveniles were able to fully compensate for acid-base disturbances caused by the exposure to elevated seawater PCO2 at levels associated with ocean acidification and carbon dioxide capture and storage (CCS) leakage scenarios, via haemolymph [HCO3-] regulation. However, metabolic rate remained constant and food consumption decreased under elevated PCO2 , indicating reduced energy availability. Juveniles at 17°C showed no ability to actively compensate haemolymph pH, resulting in decreased haemolymph pH particularly under CCS conditions. Early benthic juvenile lobsters at 17°C were not able to increase energy intake to offset increased energy demand and therefore appear to be unable to respond to acid-base disturbances due to increased PCO2at elevated temperature. Analysis of haemolymph metabolites suggests that, even under control conditions, juveniles were energetically limited. They exhibited high haemolymph [l-lactate], indicating recourse to anaerobic metabolism. Low haemolymph [protein] was linked to minimal non-bicarbonate buffering and reduced oxygen transport capacity. We discuss these results in the context of potential impacts of ongoing ocean change and CCS leakage scenarios on the development of juvenile H. gammarus and future lobster populations and stocks.

Are control of extracellular acid-base balance and regulation of skeleton genes linked to resistance to ocean acidification in adult sea urchins?

Are control of extracellular acid-base balance and regulation of skeleton genes linked to resistance to ocean acidification in adult sea urchins?

Extracellular Acid-Base Balance and Ion Transport Between Body Fluid Compartments.

Clinical assessment of acid-base disorders depends on measurements made in the blood, part of the extracellular compartment. Yet much of the metabolic importance of these disorders concerns intracellular events. Intracellular and interstitial compartment acid-base balance is complex and heterogeneous. This review considers the determinants of the extracellular fluid pH related to the ion transport processes at the interface of cells and the interstitial fluid, and between epithelial cells lining the transcellular contents of the gastrointestinal and urinary tracts that open to the external environment. The generation of acid-base disorders and the associated disruption of electrolyte balance are considered in the context of these membrane transporters. This review suggests a process of internal and external balance for pH regulation, similar to that of potassium. The role of secretory gastrointestinal epithelia and renal epithelia with respect to normal pH homeostasis and clinical disorders are considered. Electroneutrality of electrolytes in the ECF is discussed in the context of reciprocal changes in Cl- or non Cl- anions and [Formula: see text].

Bicarbonate Transport During Enamel Maturation.

Amelogenesis (tooth enamel formation) is a biomineralization process consisting primarily of two stages (secretory stage and maturation stage) with unique features. During the secretory stage, the inner epithelium of the enamel organ (i.e., the ameloblast cells) synthesizes and secretes enamel matrix proteins (EMPs) into the enamel space. The protein-rich enamel matrix forms a highly organized architecture in a pH-neutral microenvironment. As amelogenesis transitions to maturation stage, EMPs are degraded and internalized by ameloblasts through endosomal-lysosomal pathways. Enamel crystallite formation is initiated early in the secretory stage, however, during maturation stage the more rapid deposition of calcium and phosphate into the enamel space results in a rapid expansion of crystallite length and mineral volume. During maturation-stage amelogenesis, the pH value of enamel varies considerably from slightly above neutral to acidic. Extracellular acid-base balance during enamel maturation is tightly controlled by ameloblast-mediated regulatory networks, which include significant synthesis and movement of bicarbonate ions from both the enamel papillary layer cells and ameloblasts. In this review we summarize the carbonic anhydrases and the carbonate transporters/exchangers involved in pH regulation in maturation-stage amelogenesis. Proteins that have been shown to be instrumental in this process include CA2, CA6, CFTR, AE2, NBCe1, SLC26A1/SAT1, SLC26A3/DRA, SLC26A4/PDS, SLC26A6/PAT1, and SLC26A7/SUT2. In addition, we discuss the association of miRNA regulation with bicarbonate transport in tooth enamel formation.

Ocean acidification affects parameters of immune response and extracellular pH in tropical sea urchins Lytechinus variegatus and Echinometra luccunter

The rising concentration of atmospheric CO2 by anthropogenic activities is changing the chemistry of the oceans, resulting in a decreased pH. Several studies have shown that the decrease in pH can affect calcification rates and reproduction of marine invertebrates, but little attention has been drawn to their immune response. Thus this study evaluated in two adult tropical sea urchin species, Lytechinus variegatus and Echinometra lucunter, the effects of ocean acidification over a period of 24h and 5days, on parameters of the immune response, the extracellular acid base balance, and the ability to recover these parameters. For this reason, the phagocytic capacity (PC), the phagocytic index (PI), the capacity of cell adhesion, cell spreading, cell spreading area of phagocytic amebocytes in vitro, and the coelomic fluid pH were analyzed in animals exposed to a pH of 8.0 (control group), 7.6 and 7.3. Experimental pH’s were predicted by IPCC for the future of the two species. Furthermore, a recovery test was conducted to verify whether animals have the ability to restore these physiological parameters after being re-exposed to control conditions. Both species presented a significant decrease in PC, in the pH of coelomic fluid and in the cell spreading area. Besides that, Echinometra lucunter showed a significant decrease in cell spreading and significant differences in coelomocyte proportions. The recovery test showed that the PC of both species increased, also being below the control values. Even so, they were still significantly higher than those exposed to acidified seawater, indicating that with the re-establishment of the pH value the phagocytic capacity of cells tends to restore control conditions. These results demonstrate that the immune system and the coelomic fluid pH of these animals can be affected by ocean acidification. However, the effects of a short-term exposure can be reversible if the natural values ​​are re-established. Thus, the effects of ocean acidification could lead to consequences for pathogen resistance and survival of these sea urchin species.

Branchial NH 4 + ‐dependent Acid‐base Transport Mechanisms and Energy Metabolism of Squid ( Sepioteuthis lessoniana ) Affected by Seawater Acidification

CO2 induced pH fluctuation in the extracellular compartments are a unifying physiological phenomenon that all animals are confronted with. Cephalopods have evolved acid-base regulatory abilities to accommodate temporal pH fluctuations via highly pH sensitive hemocyanins. Interestingly, it has been suggested that metabolic energetic limitations may be a critical factor when exposed to acidified seawater. Here we show the effects of short-term (few hours) to medium-term (up to 168 h) seawater acidification on pelagic squids, Sepioteuthis lessoniana. Routine metabolic rates, NH4+ secretion and extracellular acid-base balance were monitored during exposure to control (pH 8.1) and acidified conditions of pH 7.7 and 7.3 along a period of 168h. Metabolic rates were significantly depressed by 40% after 168 hours in pH 7.3 conditions exposure. The extracellular pH is fully restored within 20 h and is maintained along the period of 168 h, and the accompanied acid-base transporter transcripts were up-regulated including V-type H+-ATPase (VHA), Rhesus protein (RhP), Na/HCO3- cotransporter (NBC) and cystolic carbonic anhydrase (CAc). Immunohistochemical analyses demonstrated co-localization of Na+/K+-ATPase (NKA), VHA in basolateral and Na+/H+-exchanger 3 (NHE3), RhP in apical membranes of the branchial epithelium. Protein levels of branchial VHA and RhP were increased in response to acidified conditions. The present work demonstrated the NH4+ based proton secretion via RhP may contribute to a well-developed acid-base regulatory machinery in branchial epithelia of cephalopods.

Branchial NH4+-dependent acid–base transport mechanisms and energy metabolism of squid (Sepioteuthis lessoniana) affected by seawater acidification

Cephalopods have evolved strong acid–base regulatory abilities to cope with CO2 induced pH fluctuations in their extracellular compartments to protect gas transport via highly pH sensitive hemocyanins. To date, the mechanistic basis of branchial acid–base regulation in cephalopods is still poorly understood, and associated energetic limitations may represent a critical factor in high power squids during prolonged exposure to seawater acidification. The present work used adult squid Sepioteuthis lessoniana to investigate the effects of short-term (few hours) to medium-term (up to 168 h) seawater acidification on pelagic squids. Routine metabolic rates, NH4+ excretion, extracellular acid–base balance were monitored during exposure to control (pH 8.1) and acidified conditions of pH 7.7 and 7.3 along a period of 168 h. Metabolic rates were significantly depressed by 40% after exposure to pH 7.3 conditions for 168 h. Animals fully restored extracellular pH accompanied by an increase in blood HCO3− levels within 20 hours. This compensation reaction was accompanied by increased transcript abundance of branchial acid–base transporters including V-type H+-ATPase (VHA), Rhesus protein (RhP), Na+/HCO3− cotransporter (NBC) and cytosolic carbonic anhydrase (CAc). Immunocytochemistry demonstrated the sub-cellular localization of Na+/K+-ATPase (NKA), VHA in basolateral and Na+/H+-exchanger 3 (NHE3) and RhP in apical membranes of the ion-transporting branchial epithelium. Branchial VHA and RhP responded with increased mRNA and protein levels in response to acidified conditions indicating the importance of active NH4+ transport to mediate acid–base balance in cephalopods. The present work demonstrated that cephalopods have a well developed branchial acid–base regulatory machinery. However, pelagic squids that evolved a lifestyle at the edge of energetic limits are probably more sensitive to prolonged exposure to acidified conditions compared to their more sluggish relatives including cuttlefish and octopods.

Euechinoidea and Cidaroidea respond differently to ocean acidification

The impact of the chemical changes in the ocean waters due to the increasing atmospheric CO2 depends on the ability of an organism to control extracellular pH. Among sea urchins, this seems specific to the Euechinoidea, sea urchins except Cidaroidea. However, Cidaroidea survived two ocean acidification periods: the Permian–Trias and the Cretaceous–Tertiary crises. We investigated the response of these two sea urchin groups to reduced seawater pH with the tropical cidaroid Eucidaris tribuloides, the sympatric euechinoid Tripneustes ventricosus and the temperate euechinoid Paracentrotus lividus. Both euechinoid showed a compensation of the coelomic fluid pH due to increased buffer capacity. This was linked to an increased concentration of DIC in the coelomic fluid and thus of bicarbonate ions (most probably originating from the surrounding seawater as isotopic signature of the carbon – δ13C – was similar). On the other hand, the cidaroid showed no changes within the coelomic fluid. Moreover, the δ13C of the coelomic fluid did not match that of the seawater and was not significantly different between the urchins from the different treatments. Feeding rate was not affected in any species. While euechinoids are able to regulate their extracellular acid–base balance, many questions are still unanswered on the costs of this capacity. On the contrary, cidaroids do not seem affected by a reduced seawater pH. Further investigations need to be undertaken to cover more species and physiological and metabolic parameters in order to determine if energy trade-offs occur and how this mechanism of compensation is distributed among sea urchins.

Branchial NH

BackgroundCephalopods have evolved strong acid–base regulatory abilities to cope with CO2 induced pH fluctuations in their extracellular compartments to protect gas transport via highly pH sensitive hemocyanins. To date, the mechanistic basis of branchial acid–base regulation in cephalopods is still poorly understood, and associated energetic limitations may represent a critical factor in high power squids during prolonged exposure to seawater acidification.ResultsThe present work used adult squid Sepioteuthis lessoniana to investigate the effects of short-term (few hours) to medium-term (up to 168 h) seawater acidification on pelagic squids. Routine metabolic rates, NH4+ excretion, extracellular acid–base balance were monitored during exposure to control (pH 8.1) and acidified conditions of pH 7.7 and 7.3 along a period of 168 h. Metabolic rates were significantly depressed by 40% after exposure to pH 7.3 conditions for 168 h. Animals fully restored extracellular pH accompanied by an increase in blood HCO3− levels within 20 hours. This compensation reaction was accompanied by increased transcript abundance of branchial acid–base transporters including V-type H+-ATPase (VHA), Rhesus protein (RhP), Na+/HCO3− cotransporter (NBC) and cytosolic carbonic anhydrase (CAc). Immunocytochemistry demonstrated the sub-cellular localization of Na+/K+-ATPase (NKA), VHA in basolateral and Na+/H+-exchanger 3 (NHE3) and RhP in apical membranes of the ion-transporting branchial epithelium. Branchial VHA and RhP responded with increased mRNA and protein levels in response to acidified conditions indicating the importance of active NH4+ transport to mediate acid–base balance in cephalopods.ConclusionThe present work demonstrated that cephalopods have a well developed branchial acid–base regulatory machinery. However, pelagic squids that evolved a lifestyle at the edge of energetic limits are probably more sensitive to prolonged exposure to acidified conditions compared to their more sluggish relatives including cuttlefish and octopods.

Maintenance of coelomic fluid pH in sea urchins exposed to elevated CO2: the role of body cavity epithelia and stereom dissolution

Experimental ocean acidification leads to a shift in resource allocation and to an increased [HCO3 −] within the perivisceral coelomic fluid (PCF) in the Baltic green sea urchin Strongylocentrotus droebachiensis. We investigated putative mechanisms of this pH compensation reaction by evaluating epithelial barrier function and the magnitude of skeleton (stereom) dissolution. In addition, we measured ossicle growth and skeletal stability. Ussing chamber measurements revealed that the intestine formed a barrier for HCO3 − and was selective for cation diffusion. In contrast, the peritoneal epithelium was leaky and only formed a barrier for macromolecules. The ossicles of 6 week high CO2-acclimatised sea urchins revealed minor carbonate dissolution, reduced growth but unchanged stability. On the other hand, spines dissolved more severely and were more fragile following acclimatisation to high CO2. Our results indicate that epithelia lining the PCF space contribute to its acid–base regulation. The intestine prevents HCO3 − diffusion and thus buffer leakage. In contrast, the leaky peritoneal epithelium allows buffer generation via carbonate dissolution from the surrounding skeletal ossicles. Long-term extracellular acid–base balance must be mediated by active processes, as sea urchins can maintain relatively high extracellular [HCO3 −]. The intestinal epithelia are good candidate tissues for this active net import of HCO3 − into the PCF. Spines appear to be more vulnerable to ocean acidification which might significantly impact resistance to predation pressure and thus influence fitness of this keystone species.

Controversies surrounding the role of CFTR in airway bicarbonate secretion

Bicarbonate (HCO3−) serves many functions in the human body. It is an important biological buffer required for maintaining intracellular and extracellular acid–base balance, and plays a key role in CO2 transport by blood. Less well known is that HCO3− is secreted onto the mucosal surface of most epithelia where it is involved in a myriad of vital processes including sperm capacitation, innate defence, mucin expansion and mucosal protection. An important challenge in epithelial biology has been to understand the molecular coordination of transepithelial HCO3− secretion and how this simple anion impacts on such a wide variety of physiological processes. Through studies on the inherited disease cystic fibrosis (CF), it has become clear that the plasma membrane protein encoded by the CF gene, cystic fibrosis transmembrane conductance regulator (CFTR), is essential for epithelial HCO3− secretion, but only recently have we begun to fully appreciate the complex role that CFTR plays in this process.

Relationship between CO2-driven changes in extracellular acid–base balance and cellular immune response in two polar echinoderm species

Anthropogenic CO2 emissions are acidifying the world's oceans. A growing body of evidence demonstrates that ocean acidification can impact survival, growth, development and physiology of marine invertebrates. However, little is known on the impact of elevated pCO2 on immune-response. Here we investigate the impact of short-term (5–7days) exposure to elevated pCO2 (1275μatm compared to 350μatm in the control) on extracellular pH (pHe) and cellular immune response in two polar echinoderm species, the green sea urchin Strongylocentrotus droebachiensis and the seastar Leptasterias polaris. Both species experienced extracellular acidosis following short term exposure to elevated pCO2. While this acidosis remained uncompensated within 7days for L. polaris, pHe was fully compensated after 5days for S. droebachiensis. For both species, coelomic fluid acidosis was associated with an increase in total coelomocyte number and a reduction in vibratile cells in S. droebachiensis. A relationship between pHe and phagocyte numbers was observed in S. droebachiensis suggesting a direct link between pHe and cellular immune-response. Further studies would require the coordinated effort of ecologists and immunologists to understand the role of elevated pCO2 on the host–pathogen interactions that are involved in the stability of ecosystems.

Acute extracellular acid–base disturbance in the burrowing sea urchin Brissopsis lyrifera during exposure to a simulated CO2 release

We tested the hypothesis that as infaunal organisms are regularly exposed to elevated CO2, burrowing sea urchins will demonstrate a lower sensitivity to massive CO2 release than has previously been recorded for epifaunal organisms. Infaunal urchins Brissopsis lyrifera were exposed to CO2 acidified sea water (nominal pH 7.8 (control), 7.3, 6.5 and 5.9; T=10°C, S=34) for 12h and aspects of their extracellular acid–base balance measured every 2h. In common with epifaunal urchins B. lyrifera exhibited an uncompensated respiratory acidosis in its extracellular fluid, but was more sensitive to CO2 acidification than epifaunal urchins. The lower extracellular pH of B. lyrifera may indicate a higher metabolism than epifaunal urchins and this could explain the heightened sensitivity of this species to elevated CO2. Thus, the results of this present study do not support our original hypothesis. Instead we suggest an alternative hypothesis that as infaunal organisms are exposed naturally to high levels of CO2, they may already be closer to the limits of their physiological performance. Thus any further CO2 increase could compromise their function. As a result of this sensitivity, infaunal urchins may be more at risk from an accidental release of CO2 from geological sub-seabed storage sites, or from the deliberate injection of CO2 into deep water masses, than their epifaunal counterparts.

Regulation of gas exchange and haemolymph pH in the cockroach Nauphoeta cinerea

Ventilatory control of internal CO(2) plays an important role in regulating extracellular acid-base balance in terrestrial animals. While this phenomenon is well understood among vertebrates, the role that respiration plays in the acid-base balance of insects is in need of much further study. To measure changes in insect haemolymph pH, we implanted micro pH optodes into the haemocoel of cockroaches (Nauphoeta cinerea). They were then exposed to normoxic, hypoxic, hyperoxic and hypercapnic atmospheres while their haemolymph pH, VCO(2) and abdominal ventilation frequency were measured simultaneously. Intratracheal O(2) levels were also measured in separate experiments. It was found that cockroaches breathing continuously control their ventilation to defend a haemolymph pH of 7.3, except under conditions where hypoxia (<10% O(2)) induces hyperventilation, or where ambient hypercapnia is in excess of haemolymph (>1% CO(2)). In contrast, intratracheal O(2) levels fluctuated widely, but on average remained above 15% in normoxic (21% O(2)) atmospheres. Decapitation caused the cockroaches to display discontinuous gas exchange cycles (DGCs). The alternating periods of ventilation and apnoea during DGCs caused haemolymph pH to fluctuate by 0.11 units. Exposure to hypoxia caused haemolymph pH to increase and initiated brief bouts of spiracular opening prior to the active ventilation phase. The spontaneous occurrence of DGCs in decapitated cockroaches indicates that central pattern generators in the thoracic and abdominal ganglia generate the periodic gas exchange pattern in the absence of control from the cephalic ganglion. This pattern continues to maintain gas exchange, but with less precision.

Impact of CO2-acidified seawater on the extracellular acid–base balance of the northern sea urchin Strongylocentrotus dröebachiensis

We investigated the effect of five day exposure to CO2-acidified sea water treatments (pHNBS=7.89 [control], 7.44, 7.16 and 6.78, T=9.5°C) on the extracellular acid–base balance of the northern sea urchin Strongylocentrotus dröebachiensis. In each case there was an uncompensated respiratory acidosis which increased in intensity with decreasing environmental pH. This was very similar to results for another sea urchin species, Psammechinus miliaris (8d exposure, T=15°C). However, there were some important differences in the response to low seawater pH between the two urchin species S. dröebachiensis and P. miliaris. At the lowest pH tested (6.78) there was a metabolic component to this acidosis recorded (correlated with a significant increase in l-lactate) in S. dröebachiensis but not P. miliaris. The acidosis was accompanied by a very small, but significant increase in coelomic fluid calcium. Also the water used in our study was (controlling for pH) markedly undersaturated with respect to carbonate compared with that used in the Psammechinus study, highlighting the need for the environmental context to be assessed in future comparative studies.

Short-term exposure to hypercapnia does not compromise feeding, acid–base balance or respiration ofPatella vulgatabut surprisingly is accompanied by radula damage

The effect of short-term (5 days) exposure to CO2-acidified seawater (year 2100 predicted values, ocean pH = 7.6) on key aspects of the function of the intertidal common limpetPatella vulgata(Gastropoda: Patellidae) was investigated. Changes in extracellular acid–base balance were almost completely compensated by an increase in bicarbonate ions. A concomitant increase in haemolymph Ca2+and visible shell dissolution implicated passive shell dissolution as the bicarbonate source. Analysis of the radula using SEM revealed that individuals from the hypercapnic treatment showed an increase in the number of damaged teeth and the extent to which such teeth were damaged compared with controls. As radula teeth are composed mainly of chitin, acid dissolution seems unlikely, and so the proximate cause of damage is unknown. There was no hypercapnia-related change in metabolism (O2uptake) or feeding rate, also discounting the possibility that teeth damage was a result of a CO2-related increase in grazing. We conclude that although the limpet appears to have the physiological capacity to maintain its extracellular acid–base balance, metabolism and feeding rate over a 5 days exposure to acidified seawater, radular damage somehow incurred during this time could still compromise feeding in the longer term, in turn decreasing the top-down ecosystem control thatP. vulgataexerts over rocky shore environments.

Physiological responses of Daphnia pulex to acid stress

BackgroundAcidity exerts a determining influence on the composition and diversity of freshwater faunas. While the physiological implications of freshwater acidification have been intensively studied in teleost fish and crayfish, much less is known about the acid-stress physiology of ecologically important groups such as cladoceran zooplankton. This study analyzed the extracellular acid-base state and CO2 partial pressure (PCO2), circulation and ventilation, as well as the respiration rate of Daphnia pulex acclimated to acidic (pH 5.5 and 6.0) and circumneutral (pH 7.8) conditions.ResultsD. pulex had a remarkably high extracellular pH of 8.33 and extracellular PCO2 of 0.56 kPa under normal ambient conditions (pH 7.8 and normocapnia). The hemolymph had a high bicarbonate concentration of 20.9 mM and a total buffer value of 51.5 meq L-1 pH-1. Bicarbonate covered 93% of the total buffer value. Acidic conditions induced a slight acidosis (ΔpH = 0.16–0.23), a 30–65% bicarbonate loss, and elevated systemic activities (tachycardia, hyperventilation, hypermetabolism). pH 6.0 animals partly compensated the bicarbonate loss by increasing the non-bicarbonate buffer value from 2.0 to 5.1 meq L-1 pH-1. The extracellular PCO2 of pH 5.5 animals was significantly reduced to 0.33 kPa, and these animals showed the highest tolerance to a short-term exposure to severe acid stress.ConclusionChronic exposure to acidic conditions had a pervasive impact on Daphnia's physiology including acid-base balance, extracellular PCO2, circulation and ventilation, and energy metabolism. Compensatory changes in extracellular non-bicarbonate buffering capacity and the improved tolerance to severe acid stress indicated the activation of defense mechanisms which may result from gene-expression mediated adjustments in hemolymph buffer proteins and in epithelial properties. Mechanistic analyses of the interdependence between extracellular acid-base balance and CO2 transport raised the question of whether a carbonic anhydrase (CA) is involved in the catalysis of the reaction, which led to the discovery of 31 CA-genes in the genome of D. pulex.

Influence of CO2-related seawater acidification on extracellular acid–base balance in the velvet swimming crab Necora puber

We investigated the effect of different levels of hypercapnia-induced acidification (pH = 7.96, 7.31, 6.74 and 6.05) on the extracellular acid base balance of a shallow-water crustacean, the velvet swimming crab Necora puber over a period of 16 days. Any extracellular acidosis incurred was completely compensated by an increase in bicarbonate. Bicarbonate was partly, but not wholly, supplied by dissolution of the exoskeleton. This compensation was sustained for 16 days under all experimental treatments with two exceptions. First there was some evidence of extracellular acidosis in crabs after 16 days at pH = 6.74. Second at the lowest environmental pH (6.05) there was a marked uncompensated acidosis after 24 h. Necora puber appears less sensitive to low pH than many other species examined acutely. However, local acidification as a result of ocean CO2 dispersal or leakage from geological sequestration is likely to compromise even this species.

Effects of water salinity on acid-base balance in decapod crustaceans.

Extracellular acid-base balance in decapod crustaceans is influenced by water salinity, although the nature of this relationship is unclear. In euryhaline crabs, a decrease in salinity results in a metabolic alkalosis in the haemolymph and an increase in salinity results in a metabolic acidosis. Alterations in acid-base status by external changes in salinity are thought to be secondary to the adjustments required for ionic and osmotic regulation. In the present study, acid-base adjustments in the haemolymph of Eriocheir sinensis after transfer to 30 % sea water accompanied alterations in muscle pH and [HCO(3)(-)], as an initial acidosis coincided with an alkalosis in the leg muscle. By 48 h transfer, haemolymph pH increased as muscle pH and HCO(3)(-) declined. Haemolymph [Cl(-)] decreased significantly 3 h after transfer to a new steady state but haemolymph [Na(+)] and muscle [Na(+)] and [Cl(-)] remained unchanged. Muscle free amino acid concentration increased twofold 6 h after transfer, followed by a 2.5-fold increase in the haemolymph after 24 h. In contrast, 30 % sea water had no effect on haemolymph acid-base adjustments in the osmoconforming crab, Necora puber, which lacks ion and osmo-regulatory mechansims. Collectively these observations support the view that salinity-induced alterations in acid-base status are caused by adjustments consistent with cell volume regulation.

Ion Transport and Acid–Base Balance in Freshwater Bivalves

Blood acid&shy;base and ionic balance in freshwater bivalves is affected by the relative activities of epithelial Na+ and Cl- transporters. In the unionid Carunculina texasensis, the Na+/H+ exchanger is the predominant epithelial transporter that affects acid&shy;base state, while Cl-/HCO3- exchange is of lesser importance. In the corbiculid Corbicula fluminea, Cl- and Na+ transport are both significant components affecting acid&shy;base state. Serotonin (5-hydroxytryptamine, 5-HT) stimulates Na+ and Cl- transport in both species. In C. texasensis, the effect of exogenous serotonin is four times greater on Na+/H+ exchange than on Cl-/HCO3- transport, resulting in an increase in acid secretion and a rise in blood pH. In a Na+-free environment, serotonin had no effect on blood acid&shy;base state in C. texasensis. In C. fluminea, the acid&shy;base consequences of serotonin stimulation of Na+/H+ exchange were offset by similar increases in Cl-/HCO3- exchange and by alterations in blood PCO(sum) in medium containing Na+. In Na+-free medium, stimulation of the Cl- transporter with 5-HT resulted in a decrease in blood pH. The differences between these two species are related to the reliance of C. fluminea on Cl- as the major anion in the blood, requiring high levels of epithelial Cl- transport. In C. texasensis, the anionic component of the blood consists of both Cl- and HCO3- and these ions are interchangeable over a wide concentration range. Extracellular acid&shy;base balance in freshwater bivalves is governed, in part, by epithelial ion transporters.