ABSTRACT Animals inhabiting the intertidal zone are exposed to abrupt changes in environmental conditions associated with the rise and fall of the tide. For convenience, the majority of laboratory studies on intertidal organisms have acclimated individuals to permanently submerged conditions in seawater tanks. In this study, green shore crabs, Carcinus maenas , were acclimated to either a simulated tidal regime of continuous emersion–immersion (‘tidal’) or to permanently submerged conditions (‘non-tidal’) to assess their physiological responses to subsequent emersion. Tidal crabs exhibited an endogenous rhythm of oxygen consumption during continuous submersion with lower oxygen consumption during periods of anticipated emersion, which was not detected in non-tidal crabs. During emersion, tidal crabs were able to buffer apparent changes in acid–base balance and exhibited no change in venous pH, whereas non-tidal crabs developed an acidosis associated with a rise in lactate levels. These results indicate that tidal crabs were better able to sustain aerobic metabolism and had lower metabolic costs during emersion than non-tidal crabs. It is likely that the elevated levels of haemocyanin exhibited by tidal crabs allowed them to maintain oxygen transport and buffer pH changes during emersion . This suggests that acclimation of C. maenas to submerged conditions results in a loss of important physiological mechanisms that enable it to tolerate emersion. The results of this study show that caution must be taken when acclimating intertidal organisms to submerged conditions in the laboratory, as it may abolish important physiological responses and adaptations that are critical to their performance when exposed to air.

ABSTRACT 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 P CO 2 . At 13°C, H. gammarus juveniles were able to fully compensate for acid–base disturbances caused by the exposure to elevated seawater P CO 2 at levels associated with ocean acidification and carbon dioxide capture and storage (CCS) leakage scenarios, via haemolymph [HCO 3 − ] regulation. However, metabolic rate remained constant and food consumption decreased under elevated P CO 2 , 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 P CO 2 at 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.

ABSTRACT The H + -ATPase-rich (HR) cells of zebrafish larvae are a sub-type of ion-transporting cell located on the yolk sac epithelium that are responsible for Na + uptake and H + extrusion. Current models of HR cell ion transport mechanisms in zebrafish larvae are well established, but little is known about the involvement of the various ion transport pathways in regulating intracellular acid–base status. Here, a ratiometric imaging technique was developed and validated to monitor intracellular pH (pHi) continuously in larval zebrafish HR cells in vivo . Gene knockdown or CRISPR/Cas9 knockout approaches were used to evaluate the roles of the two principal apical membrane acid excretory pathways, the Na + /H + exchanger (NHE3b; slc9a3.2 ) and the H + -ATPase ( atpv1aa ). Additionally, the role of HR cell cytosolic carbonic anhydrase (CAc) was investigated because of its presumed role in providing H + for Na + /H + exchange and H + -ATPase. The temporal pattern and extent of intracellular acidification during exposure of fish to 1% CO 2 and the extent of post-CO 2 alkalisation were altered markedly in fish experiencing knockdown/knockout of CAc, NHE3b or H + -ATPase. Although there were slight differences among the three knockdown/knockout experiments, the typical response was a greater degree of intracellular acidification during CO 2 exposure and a reduced capacity to restore pHi to baseline levels post-hypercapnia. The metabolic alkalosis and subsequent acidification associated with 20 mmol l −1 NH 4 Cl exposure and its washout were largely unaffected by gene knockdown. Overall, the results suggest markedly different mechanisms of intracellular acid–base regulation in zebrafish HR cells depending on the nature of the acid–base disturbance.

SUMMARY Experiments on Gulf toadfish Opsanus beta demonstrate how feeding impacts osmoregulation in the marine teleost intestine. A high Ca 2+ diet of pilchards Sardina pilchardus ([Ca 2+ ]=404.2 mmol kg -1 ) was compared to a low Ca 2+ diet of common squid Loligo forbesi ([Ca 2+ ]=1.3 mmol kg -1 ), as high[Ca 2+ ] has been shown to stimulate intestinal anion exchange. Gastrointestinal fluids and blood plasma were collected over a time course from pre-feeding to 216 h post feeding. Following food intake, monovalent ions were largely absorbed across the intestinal epithelium, leaving a fluid rich in divalent ions, which have a lower osmotic coefficient and effectively reduce osmotic pressure in the lumen to allow for enhanced fluid absorption. Concentrations of Cl - and HCO - 3 in fluid along the gastrointestinal tract of fish fed both diets, particularly 1 and 2 days post-feeding, demonstrate that apical Cl - /HCO - 3 exchange plays a vital role in postprandial Cl - and water absorption. Postprandial acid-base balance disturbance as indicated by plasma alkalinization was limited or absent,indicating compensation for gastric acid secretion in this teleost fish. Plasma osmolality peaked 12 h post-feeding in toadfish fed squid, but was not accompanied by a significant increase in inorganic ion concentrations. Transient fluid secretion by the gastrointestinal tract was evident from reduced luminal Mg 2+ and SO 2- 4 concentrations for 24-48 h post feeding. Discrepancy between the sum of inorganic osmolytes and measured osmotic pressure was attributed to organic osmolytes, which occurred at high concentrations in the stomach and anterior intestine for up to 24 h post feeding.

SUMMARY The objective of the present study was to examine the branchial distribution of the recently identified rainbow trout cytoplasmic carbonic anhydrase isoform (tCAc) and to investigate its role in the regulation of acid-base disturbances in rainbow trout ( Oncorhynchus mykiss ). In situ hybridization using an oligonucleotide probe specific to tCAc revealed tCAc mRNA expression in both pavement cells and mitochondria-rich cells (chloride cells). Similarly, using a homologous polyclonal antibody,tCAc immunoreactivity was localized to pavement cells and mitochondria-rich cells in the interlamellar region and along the lamellae of the gills. Exposure of rainbow trout to hypercarbia (∼0.8% CO 2 ) for 24 h resulted in significant increases in tCAc mRNA expression (∼20-fold;quantified by real-time PCR) and protein levels (∼1.3-fold; quantified by western analysis) but not enzyme activity (assessed on crude gill homogenates using the delta-pH CA assay). Inhibition of branchial CA activity in vivo using acetazolamide reduced branchial net acid excretion significantly by 20%. This effect was enhanced to a 36% reduction in branchial net acid excretion by subjecting the trout to hypercarbia (∼0.8%CO 2 ) for 10 h prior to acetazolamide injection, an exposure that significantly increased branchial net acid excretion. The results of the present study support the widely held premise that branchial intracellular CA activity (tCAc) plays a key role in regulating acid-base balance in freshwater teleost fish.

In fish, exhaustive exercise stress differs from steady-state aerobic exercise in causing (1) a depletion of glycogen, creatine phosphate (CP) and ATP reserves and an accumulation of lactate and metabolic acid (H + m ) in white muscle; (2) blood respiratory and metabolic acidoses {P CO 2 and H + m elevations, respectively); (3) marked ionic and fluid volume disturbances; and (4) a surge in plasma catecholamines. During recovery, the smaller fast component (20%) of excess postexercise oxygen consumption (EPOC) is explained by CP and ATP resynthesis and aerobic demands, but the larger slow component (80%) is considerably greater than the cost of lactate clearance and glycogen resynthesis. Ionic and H 2 O shifts may contribute significantly to EPOC; net fluxes are greatest between extracellular (ECF) and intracellular fluid (ICF) compartments, with smaller disturbances at the kidney (increased filtration, reabsorption and excretion) and gills (passive ion losses and H 2 O uptake). Modulation of branchial Na + and Cl − exchange is important in the temporary storage of H + m in the environment during recovery. Movements of lactate and H + from ICF to ECF are dissociated processes; the major portions of both are retained in the white muscle and are probably cleared by oxidation and/or glycogen resynthesis in situ. Elevated catecholamine levels are implicated in many of these responses and serve to protect metabolic processes against acid-base disturbances, but do not appear to contribute to EPOC directly. Catecholamines also cause an elevation in blood Pco 2 by a mechanism linked to the β-adrenergic activation of red blood cell Na + /H + exchange that protects O 2 transport. The compound blood addosis stimulates ventilation to meet the demands of EPOC.

Whole-animal net electrolyte fluxes (Ca 2+ , apparent H + , titratable acidic equivalents, ammonia, Na + , Cl − , K + , Mg 2+ , phosphate and sulphate) and respiratory gas exchange were monitored throughout the moulting cycle in juvenile freshwater crayfish Procambarus clarkii (Girard) at 21 °C. Intermoult crayfish were essentially in ion balance. As crayfish approached ecdysis (−3 days, where t=0 is the day when the cuticle is shed), there was a net efflux of Ca 2+ (−1000μmolkg −1 h −1 ) correlated with a corresponding uptake of acidic equivalents (or base output) of +2000μmolkg −1 h −1 . Following ecdysis, both fluxes switched vector; uptake of Ca 2- (+2000μmolkg − h − ) and basic equivalents (+4000μmolkg −1 h −1 ) were completed within 6 days. The moulting cycle also affected fluxes of electrolytes other than those involved in CaCO 3 resorption and deposition. Crayfish remained in Na + ; and C − ; balance from intermoult up to ecdysis. Following ecdysis, both were taken up actively at rates of around plus;500μanolkg −1 h − 1 for 3 days, presumably restoring the haemodilution that would have resulted from water loading. A premoult efflux of K + was partially offset by postmoult uptake. Meanwhile, crayfish experienced increased efflux of phosphate following ecdysis, probably because of increased integumentary permeability. Rates of O 2 uptake (MO 2 ) and CO 2 excretion (Mco 2 ) increased to peak values (double intermoult rates) immediately prior to ecdysis. While MO 2 recovered during postmoult, Mco 2 dropped precipitously, significantly reducing the gas exchange ratio. Since the Mco 2 deficit agreed well with the postmoult basic equivalent uptake, the latter is probably attributable to HCO 3 − uptake for calcification.

1. Under short-circuit conditions the outer mantle epithelium of Anodonta cygnea is known to produce an acidification of the solution bathing the shell side and an alkalinization of the solution bathing the haemolymph side. 2. At steady state, the rates of secretion of acid and base were numerically equal to the simultaneously measured short-circuit current, expressed in the same units. 3. The rates of acid and base secretion, and the short-circuit current, showed close similarity in the reductions caused by anoxia, diamox, DIDS (from haemolymph side), DNP and iodoacetamide. 4. The short-circuit current ( I sc ) was sensitive to the concentration of CO 2 , bicarbonate or protons in the solution on the shell side. 5. The short-circuit current was insensitive to vanadate or oligomycin, was slowly inhibited by DCCD added under anoxia to the shell side, and was almost completely inhibited within seconds by TBTO (shell side) which also caused a 40% reduction in transepithelial conductance. 6. It is suggested that I sc is due to a Cl − /HCO − exchange shunted by a Cl − recirculation across the basolateral membrane and to the operation of an electrogenic proton pump located in the apical membrane.

The mechanisms of adaptation that allow the teleost Silurus glanis to maintain its resting oxygen consumption constant when the O 2 partial pressure (P O 2 ) m the inspired water (PI O 2 ) varied between 40 and 3kPa were studied at 13 °C. Steadystate values of oxygen consumption, ventilatory and circulatory flow rates, P O 2 in the inspired and expired water, P O 2 and O 2 concentration in the arterial and venous blood, haematocrit and acid--base status in the arterial blood were determined after 1-day exposures at selected PI O 2 values. Whole-blood O 2 -binding characteristics were also determined. The key adaptation after 1 day of acclimation was maintenance of oxygen consumption by ventilatory adjustment with no change in blood flow rate or pH (no Bohr effect). At each PI O 2 value (i) the ventilatory adjustment was minimal as the O 2 extraction coefficient from water always remained around 80–90 % and (ii) Pa O 2 stayed constant at about 2kPa. Data are compared with previous results in crayfish and other teleosts. It is concluded that the principle of a constant O 2 status in the milieu intérieur -- independent of large changes in PI O 2 for a given state of activity -- should be valid in many crustaceans and teleosts.

Handling of electrolytes and acidic equivalents by the antennal gland was monitored in freshwater crayfish ( Pacifastacus leniusculus ) during control normoxia (P O 2 = 148mmHg; 1 mmHg = 133.3Pa), 72h of hyperoxia (P O 2 = 500mmHg) and 24 h of recovery. A preparation was developed for direct collection of urinary flow (UFR) which was confirmed using inulin clearance (C IN ). Renal effluxes of Na + , Cl − , Mg 2+ , K + , Ca 2+ , sulphate, phosphate, titratable and nontitratable acidity and CO 2 were monitored. These were used in conjunction with filtration rates to calculate net rates of reabsorption. UFR was elevated by around 50 % during hyperoxia. Contributing to this were an increase in C IN and a reduction in [IN] U : [IN] e . Excretion of Na + (E Na ) tended to increase initially whereas E Cl tended to decrease after 36 h. E K and E Mg showed a similar profile to that of E Na with increases averaging 60 %. The increases in excretion of Ca 2+ , phosphate and sulphate were more pronounced (threefold). In control crayfish, 65–95 % of filtered electrolytes were reabsorbed at the antennal gland except for ammonia which was secreted. Electrolyte reabsorption increased during hyperoxia; percentage increases varied from 70% (Na + , Cl − ) to 150% (sulphate, phosphate) with HCO 3 − showing a fourfold increase above control values. Ammonia secretion correspondingly increased. Control urine was acid with respect to the haemolymph and became increasingly acidified during initial hyperoxic exposure. Net renal proton excretion largely reflected (90%) ammonia excretion; both were approximately doubled during hyperoxia. Unlike the situation in mammals, ammonia appears to be more important than phosphate in buffering urine p H. Urinary parameters generally required 24 h for complete recovery when normoxia was reinstated. Renal net efflux and reabsorption rates were compared with branchial net and unidirectional influx rates from an earlier part of this study. Branchial and renal net effluxes of Na + and Cl − had similar vectors and magnitudes during the control period but tended to counter each other during hyperoxia. Renal reabsorption rates of these ions were threefold greater than branchial influx rates, confirming increased transporting capability. Both epithelia exhibited a net H + efflux during hyperoxia; the kidney contributed only 10% of the whole-animal response. Possible mechanisms of renal postfiltrational electrolyte and acidic equivalent processing are discussed.