Ammonia excretion and acid-base regulation in the American horseshoe crab, Limulus polyphemus.

Many studies have investigated ammonia excretion and acid-base regulation in aquatic arthropods, yet current knowledge of marine chelicerates is non-existent. In American horseshoe crabs (Limulus polyphemus), book gills bear physiologically distinct regions: dorsal and ventral half-lamellae, a central mitochondria-rich area (CMRA) and peripheral mitochondria-poor areas (PMPAs). In the present study, the CMRA and ventral half-lamella exhibited characteristics important for ammonia excretion and/or acid-base regulation, as supported by high expression levels of Rhesus-protein 1 (LpRh-1), cytoplasmic carbonic anhydrase (CA-2) and hyperpolarization-activated cyclic nucleotide-gated K+ channel (HCN) compared with the PMPA and dorsal half-lamella. The half-lamellae displayed remarkable differences; the ventral epithelium was ion-leaky whereas the dorsal counterpart possessed an exceptionally tight epithelium. LpRh-1 was more abundant than Rhesus-protein 2 (LpRh-2) in all investigated tissues, but LpRh-2 was more prevalent in the PMPA than in the CMRA. Ammonia influx associated with high ambient ammonia (HAA) treatment was counteracted by intact animals and complemented by upregulation of branchial CA-2, V-type H+-ATPase (HAT), HCN and LpRh-1 mRNA expression. The dorsal epithelium demonstrated characteristics of active ammonia excretion. However, an influx was observed across the ventral epithelium as a result of the tissue's high ion conductance, although the influx rate was not proportionately high considering the ∼3-fold inwardly directed ammonia gradient. These novel findings suggest a role for the coxal gland in excretion and in the maintenance of hemolymph ammonia regulation under HAA. Hypercapnic exposure induced compensatory respiratory acidosis and partial metabolic depression. Functional differences between the two halves of a branchial lamella may be physiologically beneficial in reducing the backflow of waste products into adjacent lamellae, especially in fluctuating environments where ammonia levels can increase.

Fluid absorption in the isolated midgut of adult female yellow fever mosquitoes (Aedes aegypti).

The transepithelial voltage (Vte) and the volume of isolated posterior midguts of adult female yellow fever mosquitoes (Aedes aegypti) were monitored. In all experiments, the initial Vte after filling the midgut was lumen negative, but subsequently became lumen positive at a rate of approximately 1 mV min(-1). Simultaneously, the midgut volume decreased, indicating spontaneous fluid absorption. When the midguts were filled and bathed with mosquito saline, the average rate of fluid absorption was 36.5±3.0 nl min(-1) (N=4, ±s.e.m.). In the presence of theophylline (10 mmol l(-1)), Vte reached significantly higher lumen-positive values, but the rate of fluid absorption was not affected (N=6). In the presence of NaCN (5 mmol l(-1)), Vte remained close to 0 mV (N=4) and fluid absorption was reduced (14.4±1.3 nl min(-1), N=3, ±s.e.m.). When midguts were filled with buffered NaCl (154 mmol l(-1) plus 1 mmol l(-1) HEPES) and bathed in mosquito saline with theophylline, fluid absorption was augmented (50.0±5.8 nl min(-1), N=12, ±s.e.m.). Concanamycin A (10 µmol l(-1)), ouabain (1 mmol l(-1)), and acetazolamide (1 mmol l(-1)) affected Vte in different ways, but all reduced fluid absorption by 60-70% of the value before addition of the drugs.

Osmoregulation and excretion.

The article discusses advances in osmoregulation and excretion with emphasis on how multicellular animals in different osmotic environments regulate their milieu intérieur. Mechanisms of energy transformations in animal osmoregulation are dealt with in biophysical terms with respect to water and ion exchange across biological membranes and coupling of ion and water fluxes across epithelia. The discussion of functions is based on a comparative approach analyzing mechanisms that have evolved in different taxonomic groups at biochemical, cellular and tissue levels and their integration in maintaining whole body water and ion homeostasis. The focus is on recent studies of adaptations and newly discovered mechanisms of acclimatization during transitions of animals between different osmotic environments. Special attention is paid to hypotheses about the diversity of cellular organization of osmoregulatory and excretory organs such as glomerular kidneys, antennal glands, Malpighian tubules and insect gut, gills, integument and intestine, with accounts on experimental approaches and methods applied in the studies. It is demonstrated how knowledge in these areas of comparative physiology has expanded considerably during the last two decades, bridging seminal classical works with studies based on new approaches at all levels of anatomical and functional organization. A number of as yet partially unanswered questions are emphasized, some of which are about how water and solute exchange mechanisms at lower levels are integrated for regulating whole body extracellular water volume and ion homeostasis of animals in their natural habitats. © 2014 American Physiological Society.

Multiple functions of the crustacean gill: osmotic/ionic regulation, acid-base balance, ammonia excretion, and bioaccumulation of toxic metals.

The crustacean gill is a multi-functional organ, and it is the site of a number of physiological processes, including ion transport, which is the basis for hemolymph osmoregulation; acid-base balance; and ammonia excretion. The gill is also the site by which many toxic metals are taken up by aquatic crustaceans, and thus it plays an important role in the toxicology of these species. This review provides a comprehensive overview of the ecology, physiology, biochemistry, and molecular biology of the mechanisms of osmotic and ionic regulation performed by the gill. The current concepts of the mechanisms of ion transport, the structural, biochemical, and molecular bases of systemic physiology, and the history of their development are discussed. The relationship between branchial ion transport and hemolymph acid-base regulation is also treated. In addition, the mechanisms of ammonia transport and excretion across the gill are discussed. And finally, the toxicology of heavy metal accumulation via the gill is reviewed in detail.

Signaling by intracellular Ca2+ and H+ in larval mosquito (Aedes aegypti) midgut epithelium in response to serosal serotonin and lumen pH.

The midgut of larval mosquitoes (Aedes aegypti) mediates a cycle of alkali secretion in the anterior segment (AMG) followed by partial reacidification in the posterior segment (PMG); both processes are serotonin-dependent. Here we report that intracellular Ca(2+)(Ca(i)(2+)) as indicated by Fura-2 fluorescence, is elevated in both tissues in response to serotonin, but the time courses differ characteristically in the two gut segments, and Ca(2+)-free solution abolishes the serotonin response in AMG, but not in PMG, whereas Thapsigargin, an inhibitor of endoplasmic Ca(2+) transport, abolished responsiveness to 5-HT in PMG. These results suggest the origins for the Ca(2+) signal differ between the two tissues. Quantitative real-time RT-PCR revealed expression of 5 putative 5-HT receptor types in AMG, including 5-HT(2)-like receptors which would be expected to initiate a Ca(2+) signal. None of these receptors were highly expressed in PMG. Cyclic AMP (cAMP) is a secretagogue for both tissues, but H89, an inhibitor of Protein Kinase A (PKA), is also a secretagogue, suggesting that the stimulatory effect of cAMP involves a non-PKA pathway. Cytochalasins B and D block the effect of 5-HT in AMG, suggesting a vesicle-fusion mechanism of activation of the basal V-ATPase in this tissue. Finally, in PMG, elevation of luminal pH increases (Ca(i)(2+)) and decreases intracellular pH as measured by BCECF fluorescence. These responses suggest that the rate of acid secretion by PMG might be responsive to local demand for luminal reacidification as well as to serosal serotonin.

Serotonin-induced high intracellular pH aids in alkali secretion in the anterior midgut of larval yellow fever mosquito Aedes aegypti L.

The anterior midgut of the larval yellow fever mosquito Aedes aegypti generates a luminal pH in excess of 10 in vivo and similar values are attained by isolated and perfused anterior midgut segments after stimulation with submicromolar serotonin. In the present study we investigated the mechanisms of strong luminal alkalinization using the intracellular fluorescent indicator BCECF-AM. Following stimulation with serotonin, we observed that intracellular pH (pH(i)) of the anterior midgut increased from a mean of 6.89 to a mean of 7.62, whereas pH(i) of the posterior midgut did not change in response to serotonin. Moreover, a further increase of pH(i) to 8.58 occurred when the pH of the luminal perfusate was raised to an in vivo-like value of 10.0. Luminal Zn(2+) (10 micromol l(-1)), an inhibitor of conductive proton pathways, did not inhibit the increase in pH(i), the transepithelial voltage, or the capacity of the isolated tissue to alkalinize the lumen. Finally, the transapical voltage did not significantly respond to luminal pH changes induced either by perfusion with pH 10 or by stopping the luminal perfusion with unbuffered solution which results in spontaneous luminal alkalinization. Together, these results seem to rule out the involvement of conductive pathways for proton absorption across the apical membrane and suggest that a serotonin-induced alkaline pH(i) plays an important role in the generation of an alkaline lumen.

The anterior midgut of larval yellow fever mosquitoes (Aedes aegypti): effects of amino acids, dicarboxylic acids, and glucose on the transepithelial voltage and strong luminal alkalinization.

Isolated anterior midguts of larval Aedes aegypti were bathed in aerated mosquito saline containing serotonin (0.2 micromol L(-1)) and perfused with NaCl (100 mmol L(-1)). The lumen negative transepithelial voltage (V(te)) was measured and luminal alkalinization was determined through the color change of luminal m-cresol purple from yellow to purple after luminal perfusion stops. Addition of 10 mmol L(-1) amino acids (arginine, glutamine, histidine or proline) or dicarboxylic acids (malate or succinate) to the luminal perfusate resulted in more negative V(te) values, whereas addition of glucose was without effect. In the presence of TRIS chloride as luminal perfusate, addition of nutrients did not change V(te). These results are consistent with Na(+)-dependent absorption of amino acids and dicarboxylic acids. Effects of serotonin withdrawal indicated that nutrient absorption is stimulated by this hormone. Strong luminal alkalinization was observed with mosquito saline containing serotonin on the hemolymph-side and 100 mmol L(-1) NaCl in the lumen, indicating that alkalinization does not depend on luminal nutrients. Omission of glucose or dicarboxylic acids from the hemolymph-side solution had no effect on luminal alkalinization, whereas omission of amino acids significantly decelerated it. Re-addition of amino acids restored alkalinization, suggesting the involvement of amino acid metabolism in luminal alkalinization.

Revisiting the cellular mechanisms of strong luminal alkalinization in the anterior midgut of larval mosquitoes.

Here we critically review two recent hypotheses about the mechanism of strong alkalinization by the anterior midgut of mosquito larvae and our tests of these hypotheses. We present experimental evidence against the major components of transport models proposed in these hypotheses. Measurements of the transapical and transbasal proton electrochemical gradients provide an indication of driving forces faced by and generated by the transport mechanisms of the tissue. These measurements confirmed that basal V-ATPase energizes alkalinization. Serotonin stimulates the V-ATPase, as indicated by the ensuing increase in proton-motive force across the basal membrane. Moreover, the neurohormone resulted in a surprisingly large increase in the intracellular pH. The results of inhibitor studies indicate that, contrary to previous proposals, carbonic anhydrase is apparently not involved in supplying acid-base equivalents to the respective transporters. Furthermore, any apical processes proposed to be involved in alkali secretion or acid absorption must be Cl(-) independent and insensitive to DIDS, amiloride, Zn(2+) and ouabain. These results argue against the involvement of putative apical Cl(-)/HCO (-)(3) exchangers, apical H(+) channels, apical cation/proton exchangers and the importance of the apical Na(+)/K(+) pump. The studies analyzed here thus provide both a limitation and direction for further studies of the mechanism of strong alkalinization in this system.

Strong alkalinization in the anterior midgut of larval yellow fever mosquitoes (Aedes aegypti): involvement of luminal Na+/K+-ATPase.

Recently, Na(+)/K(+)-ATPase has been detected in the luminal membrane of the anterior midgut of larval yellow fever mosquitoes (Aedes aegypti) with immunohistochemical techniques. In this study, the possible involvement of this ATPase in strong alkalinization was investigated on the level of whole larvae, isolated and perfused midgut preparations and on the molecular level of the Na(+)/K(+)-ATPase protein. Ouabain (5 mM) did not inhibit the capability of intact larval mosquitoes to alkalinize their anterior midgut. Also in isolated and perfused midgut preparations the perfusion of the lumen with ouabain (5 mM) did not result in a significant change of the transepithelial voltage or the capacity of luminal alkalinization. Na(+)/K(+)-ATPase activity was completely abolished when KCl was substituted with choline chloride, suggesting that the enzyme cannot act as an ATP-driven Na(+)/H(+)-exchanger. Altogether the results of the present investigation indicate that apical Na(+)/K(+)-ATPase is not of direct importance for strong luminal alkalinization in the anterior midgut of larval yellow fever mosquitoes.

A structure-function analysis of ion transport in crustacean gills and excretory organs.

Osmotic and ionic regulation in the Crustacea is mostly accomplished by the multifunctional gills, together with the excretory organs. In addition to their role in gas exchange, the gills constitute organs of active, transepithelial, ion transport, an activity of major importance that underlies many essential physiological functions like osmoregulation, calcium homeostasis, ammonium excretion and extracellular pH regulation. This review focuses on structure-function relationships in crustacean gills and excretory effectors, from the organ to molecular levels of organization. We address the diversity of structural architectures encountered in different crustacean gill types, and in constituent cell types, before examining the physiological mechanisms of Na(+), Cl(-), Ca(2+) and NH(4)(+) transport, and of acid-base equivalents, based on findings obtained over the last two decades employing advanced techniques. The antennal and maxillary glands constitute the principal crustacean excretory organs, which have received less attention in functional studies. We examine the diversity present in antennal and maxillary gland architecture, highlighting the structural similarities between both organ types, and we analyze the functions ascribed to each glandular segment. Emphasis is given to volume and osmoregulatory functions, capacity to produce dilute urine in freshwater crustaceans, and the effect of acclimation salinity on urine volume and composition. The microanatomy and diversity of function ascribed to gills and excretory organs are appraised from an evolutionary perspective, and suggestions made as to future avenues of investigation that may elucidate evolutionary and adaptive trends underpinning the invasion and exploitation of novel habitats.

Alkalinization in the isolated and perfused anterior midgut of the larval mosquito, Aedes aegypti.

In the present study, isolated midguts of larval Aedes aegypti L. (Diptera: Culicidae) were mounted on perfusion pipettes and bathed in high buffer mosquito saline. With low buffer perfusion saline, containing m-cresol purple, transepithelial voltage was monitored and luminal alkalinization became visible through color changes of m-cresol purple after perfusion stop. Lumen negative voltage and alkalinization depended on metabolic energy and were stimulated in the presence of serotonin (0.2 micromol l(-1)). In some experiments a pH microelectrode in the lumen recorded pH values up to 10 within minutes after perfusion stop. The V-ATPase inhibitor concanamycin (50 micromol l(-1)) on the hemolymph side almost abolished V(te) and inhibited luminal alkalinization. The carbonic anhydrase inhibitor, methazolamide (50 micromol l(-1)), on either the luminal or hemolymph-side, or the inhibitor of anion transport, DIDS (1 mmol l(-1)) on the luminal side, had no effect on V(te) or alkalinization. Cl(-) substitution in the lumen or on both sides of the tissue affected V(te), but the color change of m-cresol purple was unchanged from control conditions. Hemolymph-side Na(+) substitution or addition of the Na(+)/H(+) exchange inhibitor, amiloride (200 micromol l(-1)), reduced V(te) and luminal alkalinization. Luminal amiloride (200 micromol l(-1)) was without effects on V(te) or alkalinization. High K(+) (60 mmol l(-1)) in the lumen reduced V(te) without affecting alkalinization. These results indicate that strong luminal alkalinization in isolated and perfused anterior midgut of larval A. aegypti depends on basolateral V-ATPase, but is apparently independent of carbonic anhydrase, apical Cl(-)/HCO(3)(-) exchange or apical K(+)/2H(+) antiport.

The isolated anterior stomach of larval mosquitoes (Aedes aegypti): voltage-clamp measurements with a tubular epithelium.

The anterior stomach of larval Aedes aegypti was isolated and perfused via two pipettes. For transepithelial voltage (V(te)) measurement, the inflow pipette and the bath were connected via agar bridges to calomel electrodes. For voltage-clamping, the lumen of the tissue contained an Ag/AgCl wire held by the outflow pipette, and the preparation was placed in a bath within a spiral of Ag/AgCl wire. After equilibrating the tissue in mosquito saline on both sides, a V(te) of -8+/-1 mV was measured (+/-S.E.M., N=32). Current-voltage curves (+/-100 mV) demonstrated ohmic behaviour of the epithelium. Short-circuiting resulted in a current (I(sc)) of 103+/-16 microA cm(-2) and a mean transepithelial conductance (G(te)) of 11.8+/-1.3 mS cm(-2) (+/-S.E.M., N=32). A Yonath-Civan plot of G(te) of individual preparations over the corresponding I(sc) resulted in a straight line (r(2)=0.8422), indicating that the difference in I(sc) of individual preparations is mainly based on different transcellular conductances (G(c)). This analysis allowed to estimate the mean leak conductance (G(l) approximately 3.9 mS cm(-2)) and the mean transcellular electromotive force (E(c) approximately 13 mV). After administering 0.2 micromol L(-1) serotonin, I(sc) and G(te) significantly increased, to 457+/-49 microA cm(-2) and to 21.3+/-2.3 mS cm(-2) (+/-S.E.M., N=31, P<0.05), respectively. The Yonath-Civan plot after serotonin resulted again in a straight line (r(2)=0.8219), indicating a mean G(l) of about 1 mS cm(-2) and a mean E(c) of about 22 mV. Dinitrophenol (2.5 mmol L(-1)) almost abolished I(sc) and significantly reduced G(te) (N=6). Concanamycin A (100 micromol L(-1)) reduced I(sc) by more than 90% without significantly affecting G(te).

Adaptation to hypoosmotic challenge in brachyuran crabs: a microanatomical and electrophysiological characterization of the intestinal epithelia.

Besides its role in digestion and nutrient absorption, the crustacean gut participates in osmo/ionic regulation. We investigate microanatomy, ionic permeability and transepithelial electrophysiological parameters in the mid- and hindguts of three hyperosmoregulating crabs that inhabit estuarine waters (Chasmagnathus granulata), brackish mangrove swamp (Sesarma rectum) or freshwater (Dilocarcinus pagei). The abdominal hindguts are cuticle lined, the single-layered epithelia consisting of narrow, columnar cells exhibiting apically dense, unvesiculated cytoplasm. In the saltwater species, the thoracic midgut epithelium consists of tall, narrow, columnar cells displaying numerous, apical microvilli above dense apical cytoplasm. However, the corresponding gut segment in the hololimnetic species, D. pagei, consists of squat cells lacking apical microvilli, overlain by a heavy cuticle, constituting a thoracic or anterior hindgut. The midgut/thoracic hindgut epithelia in all three crabs, and abdominal (posterior) hindgut of D. pagei, exhibit similar, small, lumen-negative voltages when perfused symmetrically with hemolymph-like salines. The hindguts of the saltwater species show similar, small, lumen-positive voltages. Small short-circuit currents are detectable after voltage clamping. Washout and/or addition of luminal glucose or amino acids do not alter current or conductance, suggesting the absence of active, electrogenic nutrient absorption. Ion substitution did not disclose active, electrogenic absorption or secretion of Na+ and/or Cl-. The midguts of the saltwater species exhibit similar conductances, greater than in D. pagei, but no ion selectivity; hindgut conductance is low, the epithelia showing moderate anion selectivity. The thoracic (anterior) and abdominal (posterior) hindgut epithelia of D. pagei, the freshwater species, exhibit similar, low conductances, and are ion selective. These findings reveal that active, electrogenic, salt and nutrient transport is undetectably low or absent. The reduced transepithelial conductances and notable ion selectivities in the abdominal and thoracic hindguts of D. pagei may reduce passive salt losses in fresh water, contributing to osmotic and ionic regulation.

Ion-motive ATPases and active, transbranchial NaCl uptake in the red freshwater crab, Dilocarcinus pagei (Decapoda, Trichodactylidae).

The present investigation examined the microanatomy and mRNA expression and activity of ion-motive ATPases, in anterior and posterior gills of a South American, true freshwater crab, Dilocarcinus pagei. Like diadromous crabs, the anterior gills of this hololimnetic trichodactylid exhibit a highly attenuated (2-5 microm), symmetrical epithelium on both lamellar surfaces. In sharp contrast, the posterior gill lamellar epithelia are markedly asymmetrical. Their proximal side consists of thick (18-20 microm) cells, displaying features typical of a transporting epithelium, while the distal epithelium is thin (3-10 microm) and formed entirely by apical pillar cell flanges. Both anterior and posterior gills express Na+/K+- and V-ATPases. Phylogenetic analysis of partial cDNA sequences for the Na+/K+-ATPase alpha-subunit and V-ATPase B-subunit among various crab species confirmed the previous classification and grouping of D. pagei based on morphological criteria. Semi-quantitative RT-PCR clearly showed that mRNA for both ion pump subunits is more intensely expressed in posterior gills. Na+/K+-ATPase activity in the posterior gills was nearly fourfold that of anterior gills, while V-ATPase and F-ATPase activities did not differ. A negative short-circuit current (Isc) was measured using the distal side of split, posterior gill lamellae, mounted in a modified Ussing chamber and perfused symmetrically with identical hemolymph-like salines. Although hemolymph-side ouabain did not affect this current, concanamycin significantly reduced Isc without altering preparation conductance, suggesting V-ATPase-driven Cl- absorption on the distal side of the posterior gill lamellae, as known to occur in diadromous crabs adapted to freshwater. These findings suggest that active Na+ uptake predominates across the thick proximal epithelium, and Cl- uptake across the thin, distal epithelium of the posterior gill lamellae.

Active NaCl absorption across posterior gills of hyperosmoregulating Chasmagnathus granulatus.

Split lamellae of posterior gills of Chasmagnathus granulatus adapted to 2.5 per thousand salinity were mounted in a modified Ussing chamber. With NaCl-saline on both sides of the preparation a transepithelial voltage (V(te)) of 4.1+/-0.5 mV (outside positive) was measured. After voltage-clamping, the negative short-circuit current (I(sc)) amounted to -142+/-21 micro A cm(-2) at a conductance (G(te)) of 44+/-5 mS cm(-2). Substitution of either chloride (by nitrate) or sodium (by choline) on both sides of split gill lamellae significantly reduced I(sc) (by 70-80%) and G(te) (by 30-50%). External CsCl (but not BaCl(2) or furosemide) inhibited the negative I(sc) without affecting G(te). Addition of ouabain, BaCl(2) or diphenylamine-2-carboxylate to the internal bath inhibited I(sc) at unchanged G(te). Internal acetazolamide did not affect I(sc) or G(te) across split gill lamellae. Unidirectional Na(+) influx across isolated and perfused posterior gills, however, was reduced by internal acetazolamide by approximately 20% at constant V(te). The results suggest that posterior gills of hyperosmoregulating C. granulatus display a high conductance epithelium that actively absorbs NaCl in a coupled way by an electrogenic mechanism similar to that seen in the thick ascending limb of Henle's loop and, to a minor degree, by an electroneutral mechanism, presumably via apical Na(+)/H(+)- and Cl(-)/HCO(3)(-)-antiports.

Ion transport across posterior gills of hyperosmoregulating shore crabs (Carcinus maenas): amiloride blocks the cuticular Na(+) conductance and induces current-noise.

Split gill lamellae and gill cuticles of shore crabs (Carcinus maenas) adapted to 10 per thousand salinity were mounted in a modified Ussing-type chamber. With NaCl saline on both sides, split gill lamellae generated a short-circuit current (I(sc)) of -301+/-16 microA cm(-2) at a conductance (G(te)) of 40+/-2 mS cm(-2). The net influxes of Na(+) and Cl(-) were 8.3+/-2.6 and 18.2+/-2.7 micromol cm(-2) h(-1), respectively. External amiloride (100 micromol l(-1)) reduced G(te) to approximately 50 % of the original value at unchanged I(sc); Cl(-) fluxes remained unaffected, whereas Na(+) fluxes were markedly reduced by 70-80 %. The I(sc) in the presence of external amiloride was almost completely inhibited by internal ouabain. At a clamp voltage of 50 mV (outside-positive), a positive current was measured at unchanged G(te). Under these conditions, amiloride reduced the current and conductance at half-maximal concentrations of 3.6 and 2.0 micromol l(-1), respectively. At outside-positive voltages, but not under short-circuit conditions, external amiloride induced Lorentzian components in the power density spectra. The amiloride-dependent changes in the corner frequency (linear) and of the low-frequency plateau ('bell-shaped') were as expected for channel blockade by amiloride with pseudo-first-order kinetics. With an outside-positive clamp voltage of 50 mV across isolated cuticles, a positive cuticular current (I(cut)) of 25 188+/-3791 microA cm(-2) and a cuticular conductance (G(cut)) of 547+/-76 mS cm(-2) were measured. External amiloride reduced I(cut) and G(cut) at half-maximal concentrations of 0.7 and 0.6 micromol l(-1), respectively. Amiloride-induced current-noise analysis gave similar results to those observed with split gill lamellae. Ion-substitution experiments with isolated cuticles further support inhibition by external amiloride of the cuticular Na(+) conductance of shore crab gills and not amiloride-sensitive transporters (Na(+) channels or Na(+)/H(+) antiports) in the apical membrane.

Hyperosmoregulation in the red freshwater crab Dilocarcinus pagei (Brachyura, Trichodactylidae): structural and functional asymmetries of the posterior gills.

The osmotic and ionic status of the haemolymph and the structural and ion-transport characteristics of the posterior gills of Dilocarcinus pagei, a hololimnetic crab, were investigated. Haemolymph osmolality was 386 +/- 18 mosmol kg(-1), while [Na(+)] and [Cl(-)] were 190 +/- 13 and 206 +/- 12 mmol l(-1), respectively; [K(+)], [Ca(2+)] and [Mg(2+)] were 9.7 +/- 0.7, 10.2 +/- 0.5 and 2.8 +/- 0.4 mmol l(-1), respectively (means +/- S.E.M., N=12-17). The gill lamellae possess a central, osmiophilic area, which exhibits a marked structural asymmetry. The thick (18-20 microm) proximal epithelium is characterised by basal invaginations and a few apical vesicles, while the thin (3-10 microm) distal epithelium consists of apical pillar cell flanges populated by vesicles and membrane invaginations. Isolated gills, bathed and perfused with NaCl saline, spontaneously generate a negative transbranchial potential difference (V(te)), which stabilises at positive or negative values. Ouabain shifts V(te) to more positive values. When mounted in an Ussing chamber, distal split lamellae generate a negative, Cl(-)-dependent short-circuit current (I(sc)). Na(+) substitution leads to more negative values of I(sc). Internal ouabain is without effect, while diphenylamine-2-carboxylate and acetazolamide abolish I(sc). Proximal split lamellae show a positive, Na(+)-dependent I(sc), which decreases after internal application of ouabain. These data suggest that the thin epithelium actively absorbs Cl(-), while the thick epithelium actively absorbs Na(+).