Electrical parameters of the cuticle of the posterior gill lamella of the shore crab Carcinus maenas, were measured. When the cuticle was perfused with crab saline (CS) inside and sea water (SW) outside, ionic replacements or dilutions of the bathing solutions produced changes in potential that showed a selective permeability to cations with respect to anions. Similar measurements made with the cuticle bathed in single salt solutions gave the following permeability sequence: NH4+ > Rb+ > Cs+ > K+ > Na+ ≈ Ca2+ > Li+ > Tns+ > Mg2+ > HCO3−> CH3COC− > cl−>so42−.
From conductance measurements, the permeability of the cuticle to Ca2+ and Na+ was about 10−3cms−1. K+ and NH4+ were about five times more permeant, while Mg2+ permeability was 5 × 10−5 cm s−1. The permeability of the cuticle to anions 1 ay between 10−6 and 10−5cms−1. In addition, the cuticle showed an asymmetrical behaviour. These results suggest that the low anionic cuticular permeability can interfere with ionic exchanges across the whole gill.
Osmoregulation and ionoregulation in decapod crustaceans have been related to water and salt exchanges across the surface of the gut, the antennal gland and the gills. The latter is the main site of passive loss and active uptake of salts in hyperregulators (Mantel & Farmer, 1983). Isolated decapod gills have therefore been used with the aim of analysing Na+, NH4+, H+, Cl− and HCO3− transport mechanisms. In relation to these studies, diffusional transgill potentials have been measured in the crayfish Austropotamobius (Croghan, Curra & Lockwood, 1965) and in several crab species: Maja (King & Schoffeniels, 1969), Eriocheir (Pequeux & Gilles, 1981; Gilles & Pequeux, 1985), Callinectes (Mantel, 1967; Smith & Linton, 1971) and Carcinus (King & Schoffeniels, 1969; Siebers et al. 1985; Pequeux, Wanson & Gilles, 1984; Lucu & Siebers, 1986). From these measurements it has been suggested that the gills of crayfish and shore crabs are more permeable to Na+ than to Cl−.
Any possible action of the cuticle as a barrier (Mantel & Farmer, 1983) has generally been ignored in the studies quoted above on decapod gills. At least two factors have to be considered to relate the transbranchial ion transport to the cuticle properties: first, the cuticular ionic selectivity or relative ionic permeability across the cuticle, and second, the ionic cuticular permeability relative to that of the underlying epithelium. These two factors should be considered in conjunction with the properties of the subcuticular spaces that are limited by the apical infoldings of the gill epithelial membrane. Such spaces were first described by Copeland & Fitzjarrell (1968) in Callinectes, and can be observed in most hyperregulators, including Carcinus (Gilles & Pequeux, 1985; Towle & Kays, 1986).
Avenet & Lignon (1985) observed a high ionic permeability of the gill cuticle to all ionic species (10 −3cms−1) in the osmoconforming lobsters, Homarus and Nephrops. Such a high permeability of the cuticle is unlikely to affect the salt exchanges across the gills. However, in hyperregulating freshwater crayfish, Avenet & Lignon (1985) and Lignon & Lenoir (1985) showed a high ionic selectivity of the isolated gill cuticle. Besides, the permeability of the crayfish gill cuticle computed from conductance measurements was found to range from 10−7 to 10−3cms−1 depending upon the ionic species. These authors pointed out that, in crayfish, the cuticle could impede the transbranchial flux of some ionic species while allowing almost free passage of other ionic species. In their investigations on Callinectes gills, Smith & Linton (1971) also measured the diffusional transcuticular potential and stated that the preferential permeability of the gills to Na+ ‘is primarily a function of the cuticle’. Unfortunately, these authors did not make any estimate of the cuticular permeability so their statement is partly speculative. In contrast, Webb (1940) claimed that the permeability of Carcinus gill cuticle ‘is such that it does not affect the salt and water exchanges of the animal’. However, this claim was based on indirect evidence since Webb measured the salt permeabilities of the cuticle of the lobster foregut and presumed that the gill cuticle of Carcinus is more permeable since it is thinner.
In the present investigation, it is shown that the gill cuticle of Carcinus maenas has a marked selectivity for cations over anions. The cationic permeability computed from conductance measurements is relatively high while the anionic permeability is moderate. These results are discussed in relation to the overall permeability of the crab gill.
MATERIALS AND METHODS
The experiments were performed on large specimens of Carcinus maenas at intermoult stage at the Marine Biological Station in Roscoff (Brittany, France). The animals were kept in large tanks of running sea water (SW) prior to the experiment.
The isolated posterior gills were perfused with crab saline (CS) containing Evans Blue and, after a wash with pure CS, a lamella was isolated and cut along its edge with fine scissors. The two sides of the lamella cuticle were gently pulled apart from each other. Any patch of epithelium (selectively absorbing the dye) still adhering to the cuticle was peeled off mechanically.
The cuticle was sandwiched vertically between two identical half-chambers as described by Avenet & Lignon (1985). The volume of the half-chamber facing the exposed area of the cuticle (3 ·14 mm2) was 20 mm3. The cuticle was superfused on both sides at a rate of 10 –20 ml min−1 at room temperature (19 –20 °C). Salines flowing by gravity from storage vessels could be changed using multiway taps connected to the bottom of the half-chambers. Excess saline was removed by suction at the top of the chambers.
Experimental fluids were filtered sea water (SW) from the Station, artificial sea water (ASW) and crab saline (CS) made up with distilled water and pm analysis grade salts. ASW composition was close to that of SW, consisting of (in mmol 1−1), Na+, 479; K+, 10; Ca2+, 10; Mg2+, 55 ·5; Cl −, 559; SO42 −, 29; HCO3−, 2 ·5; pH8 ·1. CS had the following composition (in mmoll −1): Na+, 465; K+, 10; Ca2+, 10; Mg2+, 8; Cl −, 503; SO42 −, 4. The pH was adjusted to 7 ·5 with Hepes (2mmoll −1) and Tris.
In another experimental series, the cuticle was superfused with single salt solutions. The reference solution was usually a 500 mmol I −1 NaCl solution, having an ionic strength close to that of SW and CS. The reference concentrations for CaCl2, MgCl2 and Na2SO4 were 200mmoll−1 and 100mmoll−1 for MgSO4. CaSO4 was used at 10 mmol 1−1 (imposed by the solubility product). For comparative purposes, MgSO4 was also used at 10 mmol 1−1. All these solutions were buffered at pH 7 ·0 with the above-quoted buffer. The partial pressure of CO2 in the gas mixture used to equilibrate the NaHCO3 solutions was adjusted so as to hold the pH at 8 ·0. At this pH, 97 % of the total CO2 would be in the form of HCO3− so that Na+ and HCO3− concentrations can be considered as virtually equal.
Single ion activity coefficients were calculated from the salt activity coefficient given in the tables of Parson (1959) according to the convention of Bates (Meier el al. 1982).
Potential and conductance measurements
Electrical measurements were performed as described by Avenet & Lignon (1985). The potential (Voi) was measured with KCI (3 mol l −1)-agar bridges whose tips were held close to the cuticle. Voi was measured with respect to the inside. The junctional potentials were calculated using the Henderson equation.
Current-voltage (l/V) curves were obtained using constant-current pulses of varying amplitude fed from a floating current source to the cuticle through two silver plates. The current Io was taken as positive when flowing inwards. The series resistances (Rs: resistance of the fluid layers separating the tips of the bridges from the cuticle) were also calculated from I/V curves established at the end of the experiment after the cuticle had been disrupted. Means are given with their standard errors.
Potential, conductance and permeability relationships
Ionic flux across a membrane is proportional to the concentration, to the electrochemical gradient and to a permeability coefficient related to the transport properties of the membrane. Simple explicit solutions of the Poisson—Nernst—Planck equations are obtained if the Poisson equation is approximated either by the electroneutrality equation or by the constant-field equation and if boundary conditions are correctly chosen. At zero total transmembrane current (I = 0), the potential (VO1) is related to the permeability ratio between different ionic species αij= Pi/Pj) and to the ionic activities (aj). Flux rate constants (kj) and conductances (Gj) from which absolute values of the permeabilities (Pj) can be derived are generally functions of Pj, aj and Voi.
Potential in crab saline and artificial sea water
When the cuticle was superfused with CS on both sides, the transcuticular potential (VOI) was equal to zero, as expected from a cell-free system. The potential was negligibly small when SW bathed the outside and CS the inside. The effect of some ionic replacements performed in CS at constant ionic strength are illustrated in Fig. 1. Total replacement of Na+ by K+ or L1+ resulted in changes of VOI by up to 30mV and indicate the following permeability sequence: K+>Na+>L1+. Ca2+ replacement by Na+ was almost ineffective. Cl− replacement by CH3COO − induced a 4 mV change in Voi, showing a larger acetate permeability. The effect of the total replacement of Cl− by SO42− would support a larger permeability to SCL2 − but in this case [Na+]i was also reduced so that the negative value of Voi could also indicate a Na+ selectivity of the cuticle. Ionic substitutions performed in SW (ASW) had similar effects (Fig. 1), but the changes in Voi were then of the opposite sign and (slightly larger. These results show that the crab gill cuticle is permeable to monovalent cations.
The small changes in VO1 observed could result either from a low selectivity among monovalent cations or from a substantial permeability to anions. To distinguish between these two possibilities, saline solutions were diluted either inside or outside. When CS was diluted, VOI was negative (e.g. —15 mV for a dilution to half concentration; Fig. 2). Conversely, when SW was diluted, VO1 was positive. As shown in Fig. 2, Voi increased, respectively, to +16, +49 and +86 mV upon SW dilutions by a factor of 2, 10 or 100. These values of Voi are close to the monovalent cation equilibrium potentials, showing a high selectivity of the cuticle to cations over anions.
Potential in single salt solutions
Single salt solutions were used to distinguish the effects of each ionic species. Replacing either CS or SW by a pure NaCl solution (500 mmol 1−1) of similar ionic strength did not change VOI by more than 0 ·5 mV. Upon dilution of this pure NaCl solution either inside or outside, changes in VOI were recorded (Fig. 3) that were similar to those obtained with CS and SW dilutions (Fig. 2), clearly showing a selectivity of the cuticle for Na+ over Cl−.
During dilution of NaCl upon either side, Voi was close to the equilibrium potential for Na+ (ENa+). The initial slopes of the experimental curves (fitted by eye) were 57 ·4 ±0 ·6 and 57 ·2 ± 0 ·9 mV/decade, respectively, for an external and an internal dilution (N=5). These curves were virtually linear over the first two decades but the slopes decreased at higher dilutions so that Voi tended to a maximum value (140 ± 15 mV for an outside dilution). This reduction of the slope of the curve Voi/log (aNaCi) always occurred at higher NaCl activity when the dilution was performed inside. The maximum absolute value of VO1 was also found to be 20 –50 mV less than for an outside dilution. Similar results were obtained upon dilution of a 500 mmol 1−1 KCl or LiCl solution. Absolute values of Voi were slightly larger with KCl than with NaCl and slightly smaller with LiCl. It can thus be concluded that Na+, K+ and L1+ are much more permeant than Cl− across the crab gill cuticle.
Similar dilution experiments were performed with 500 mmol 1−1 solutions of Na+ salts containing Cl−, CH3COO− or HCO3−. NaCl and NaCH3COO dilution performed at pH 7 ·0 and 8 ·0 did not show any significant differences. NaHCO3 dilutions were performed at pH 8 ·0. In every case the cuticle showed a marked Na+ selectivity. However, the slopes of the curves Voi/log(aNa+) increased with the following salt sequence: NaHCO3, NaCH3COO, NaCl, indicating a larger impermeability of the cuticle to Cl− than to CH3COO− and HCO3−. For an outside dilution, typical values of the initial slope and Voi maximum for NaCl, NaCH3COO and NaHCO3 were 57 ·5, 57 ·0 and 55 ·5 mV/decade and 150, 120 and 115 mV, respectively.
Dilutions of salts containing divalent ions were also performed to assess the selectivity of the cuticle to Ca2+, Mg2+ and SO42−. The slope of Voi/log (aNa+) was close to 58 mV/decade upon diluting Na2SC4 and indicates a much higher permeability of the cuticle to Na+ than to SO42−. Voi was positive when CaCl2 was diluted outside. The curve relating VOI to log (aCa2+) was linear over two decades and had a slope of 28 ± 1 mV/decade (N = 3). Voi was slightly negative (—10 mV at most) when MgC12 was similarly diluted outside. This indicates that Mg2+ and Cl− have similar permeabilities while the cuticle permeability to Ca2+ is much higher. The dilution of both CaSO4 and MgSO4 (from 10 mmol 1−1 down to 0 ·1 mmol 1−1) outside induced the development of a positive potential, indicating a higher permeability of the cuticle to cations. The slopes of the curves Voi/log (aM2+) were 28 ± 1 mV/decade for CaSO4 (N = 4) and 18 ± 4mV/decade for MgSO4 (N = 5), respectively. Dilutions down to 1/100 were performed from solutions containing either 10 or 100 mmol 1−1 MgSO4 and gave similar initial slopes. However, the slope of the Voi/log (aMgsch) curves was reduced when a 100 mmol 1−1 MgSO4 solution was further diluted to 0 ·1 mmol I −1. VOI values of similar amplitude but of opposite sign were obtained when the dilutions were performed inside. These results indicate that SO42− is the less permeant ion across the crab cuticle and that Mg2+ is the less permeant cation while Ca2+ has a much higher permeability.
Permeability ratios between cations were assessed from Voi measurements in biionic conditions (total replacement of one cation by another one) (Table 1). When 500 mmol 1−1 NaCl was perfused inside and replaced outside, the potential sequence varied according to the sequence NH4C1, RbCl, CsCl, KCl, NaCl, LiCl, CaC12, TrisCi, MgC12 for each experiment (as given for the mean in Table 1). The same monovalent cation sequence was also obtained using SO42− instead of Cl− as a common ion. The values of Voi were then slightly larger. Perfusing the cuticle at 10 mmol 1−1 with CaSO4/MgSO4 (outside/inside) showed that Ca2+ is more permeant than Mg2+. Similar information was obtained when MgC12 and CaC12 were used (200mmol 1−1).
A similar ionic sequence was obtained when replacements of NaCl were performed inside while maintaining a 500 mmol 1−1 NaCl solution outside. The polarity of VO1 was reversed and the absolute values of Voi were usually slightly smaller. Exchanging MgC12 and CaC12 also resulted in a small drop (3 –4 mV) in the absolute value of VOI. In every case the time course of the change in VOI was slower when the ionic replacements were performed inside as compared with the effect of an external replacement.
Substitutions of Cl − in a 500 mmol 1−1 NaCl solution were also performed. In agreement with the much larger permeability of the cuticle to Na+, the changes in VO1 thus observed were small. These substitutions nevertheless confirm the following permeability sequence: HCO3− > CH3COO −> CI −.
The area-specific conductance (G) of the cuticle to various salts was estimated from the slope of the 1/V curve obtained when both sides of the cuticle were perfused with identical solutions. Both the total resistance (Rt) and the series resistance (Rs) were measured for each salt and for each concentration. As illustrated on Fig. 4A, the l/V curves were linear showing the ohmic behaviour of the cuticle in these conditions. G was deduced from the difference between Rt and Rs.
The conductance of the cuticle perfused with 500 mmol 1−1 NaCl was GNaCl = 3 ·4±0 ·7Scm −2 (N=5) and was not significantly different from the conductance obtained with CS and SW perfusions. Under these conditions, Rt and Rs were found to be of the same order of magnitude, thus increasing the relative error made for GNaCl More precise measurements were obtained when the cuticle was perfused with the relatively impermeant salt MgSO4. In this case Rt was much larger than Rs. was 5 ·2 ± 2 ·7 mScm −2 (N = 5) when the cuticle was perfused with 100mmoll−1 MgSO4 (activity = 15 mmol 1−1). dropped to 3 ·5±0 ·6 mS cm −2(N = 3) when [MgSO4] was 10 mmol 1−1 (activity = 4mmol 1−1). Replacing Mg2+ by Ca2+ (CaSO4 = 10 mmol 1−1) evoked a large increase of the cuticle areaspecific conductance to 84 ·6 ± 38 ·5 mScm −2 (N= 3) in agreement with the fact that Ca2+ is much more permeant than Mg2+.
GNa+ and GCl− were also obtained by perfusing both sides of the cuticle with MgSO4 (100mmoll−1) and progressively replacing either Mg2+ by Na + or SO42− by Cl− up to 20 mmol 1−1 (Fig. 4). R, was then kept constant and the change in G coule be directly related to GNa+ and GCl− It can be seen that the slope of the l/V curve was almost unchanged when a small amount of SO42− was replaced by Cl−. In contrast, the slope of the l/V curve increased markedly when a small amount of Mg2+ was replaced by Na+, indicating a large cuticular GNa+ (Fig. 4A). The change of the slope was linearly related to [Na+] (Fig. 4B). This observation allows us to calculate a molar area-specific conductance of the crab gill cuticle to Na+ (gNa+). This was found to be 2 ·05 ± 0 ·53 mS cm −2 (mmol l−1)−1 (N=5) on the concentration scale and 2 ·96 ± 0 ·76 mS cm −2 (mmol 1−1)−1 on the activity scale. The increase in G was noticeably smaller when SO42− was replaced by Cl− than when Mg2+ was replaced by Na+ : 50 ± 5 (N= 3) times smaller.
The experimental results in crab saline or sea water show a high selectivity of the crab gill cuticle for cations over anions normally present in SW and in the haemolymph. This was confirmed in single salt experiments that were performed to obtain precise values of the permeability ratios and of the conductance to each ionic species: monovalent cations and Ca2+ are much more permeant than other ionic species. The similarity of the results obtained in both conditions shows that the main properties of the crab gill cuticle are not modified if some ions are excluded from the perfusion medium. This allows for the use of permeability values, determined in single salt experiments, to compute the ionic fluxes in more complex solutions such as SW and CS. These permeability values should then be incorporated into a model that accounts for both the selectivity and the asymmetrical behaviour of the crab gill cuticle.
PNa+/Panion ratios were deduced from the fitting of the Voi/log (aNaX) curves obtained by dilution of Na+ salts containing Cl−, SO42−, CH3COO− or HCO3−. The experimental Voi/log (aNaCl) curves cannot be fitted by a model using the electroneutrality condition (equation 2b) that predicts a linear relationship. The curvilinear characteristic of the experimental Voi/log(aX) relationships such as those illustrated on Fig. 3 supports of the use of the constant-field condition (equation 2a). A PNa+/PCl− ratio of 260 ± 120 (N = 5) gave a relatively good fit (within a few mV) of the Voi/log (aNaCl)o curves. This ratio was reduced by a factor of 2 –3 for NaCH3COO and NaHCO3. The fit of the Voj/log (aNaCl)i curves was not as good but supported a PNa+/PCl− ratio of about 120. A PMS2+/PSO42− ratio of 7 was computed from the Voi values recorded in diluting MgSO4. PCa+/PSO42− was found to be greater than 100. The slope of the Voi/log (aCa2+) curves (28 mV/decade) also indicated a large PCa2+/PCl− value upon dilution of CaC12.
Since the ratios Pcation/PCl− and Pcation/Pso42− are large, equation 3a was used to calculate the permeability ratios between cations of like valencies from the Voi values recorded under bi-ionic conditions. The values of the computed ratios are shown in Stable 1 and give the following two sequences: NH4+ (6·7)>Rb+ (4·9) > Cs+ (4·5) > K+ (3·8) > Na+ (1) > L1+ (0·32) > Tris+ (0·032) and Ca2+ (12) > Mg2+ (1). The values of the permeability of the divalent cations relative to that of Na+ were estimated using equation 3b and gave the following sequence: Na+ (l)>Ca2+ (0·45)>Mg2+ (0·043).
The absolute values of the permeabilities were calculated, using equation 4, from the conductance measurements performed at low current intensities when both sides of the cuticle were perfused with identical solutions. The permeability to Na+ computed in this way was 7·1 ± l·8×l0− 4cms−1 (N= 5; activity scale) using gNa+ values obtained by addition of Na+ to a MgSO4 solution. A similar PNa+ value was deduced from the value of GNaCl. PCl− was, of course, 50 times lower (l·5× 10−5 ems−1) as ger was less than gNa+. PCl− can also be calculated in a different way from the individual pairs of gNa+ values and PNa+/PCl− ratios (as determined above) giving a lower PCl− value of 2·9 ± 1·9× 10− 6cms−1(N = 5). The large change in G when MgSO4 was replaced by CaSO4 can be entirely attributed to Ca2+. PCa2+ was then calculated as 1·3 ± 0·6×l0− 3cms−1 (N= 3). , is the sum of GMg2+ and . Both permeabilities and conductances were assumed to be proportional to compute PMg2+ (5 × 10− 5cms−1) and (5 ·7 × 10− 6cms−1).
Equivalent two-layer model of the cuticle
Permeabilities can be calculated with a limited number of assumptions from conductance measurements performed under symmetrical conditions. However, the permeabilities determined above should be incorporated in a model that accounts for the properties of the cuticle to predict transcuticular fluxes. Apart from its high selectivity to cations, the crab gill cuticle exhibits a functional asymmetry with regard to the time course and absolute values of VOI recorded when the salines are modified on either sides of the cuticle. This is not predicted by the classical model of Goldman (1943) that makes use of a single homogeneous membrane. The cuticle should, therefore, be divided into at least two functional layers. The larger potential obtained when the salts are diluted outside is best explained by a model such as that developed by Avenet & Lignon (1985) for the Cl−-selective gill lamina cuticle of the crayfish.
The model described above thus gives a relatively good account of the properties of the crab gill cuticle as far as Na+ and Cl− permeabilities are concerned. The outer selective layer could be tentatively identified as the epicuticle normally facing the SW. Such a conclusion that the impermeability and/or selectivity of the cuticle could be related to the epicuticle has also been stated by Jahn (1936) for the grasshopper egg membrane, by Yonge (1936) for the foregut of the lobster and by Avenet & Lignon (1985) for the crayfish gill lamina cuticle.
Physiological role of the cuticle
Our results on Carcinus maenas gill cuticle confirm and extend those of Smith & Linton (1971) on Callinectes sapidus in that the gill cuticle shows a marked selectivity for cations over anions. The larger Voi values obtained with Carcinus cuticle are likely to result from technical differences: Smith & Linton only diluted the external saline without perfusing the inside, thus creating an artificial unstirred layer that added to the effect of the endocuticle in reducing the cuticle selectivity. Besides, from our conductance measurements, the permeability of Carcinus cuticle to Ca2+ and Na+ is about 10− 3cms−1. K+ and NH4+ are about five times more permeant, while Mg2+ permeability is 5 ×l0− 5cms−1. The cuticle permeability to anions is noticeably lower and lies between 10− 6 and 10−5 cms−1.
It is now of interest to know how far the cuticle could interfere with Na+ and Cl− losses and uptake that occur when shore crabs are transferred to media of different salinities. Since Na+ and Cl− are found in equivalent amounts in the blood, they should be considered as ultimately being cotransported even though this overall cotransport can to some extent be dissociated when Na+/NH4+ and C1− /HCO3− (Pressley, Graves & Krall, 1981; Henry & Cameron, 1983; Lee & Pritchard, 1985) exchange mechanisms are switched on. The cuticle should not impede Na+/NH4+ and Na+/Na+ exchanges across the gills since its permeability to NH4+ and Na+ is high. However, this is not so for a HCO3− /Cl− exchange, since both Cl− and HCO3− have permeabilities lower than 10−5 cms−1. In this regard the cuticle could create a functional subcuticular compartment that should be taken into account when considering such an exchange across the gills. This conclusion could also hold for a Na+-Cl− cotransport (either loose or tight) since the salt flux will be limited by the slowly moving Cl− flux across the cuticle to satisfy the zero current condition across it.
These observations are, of course, to be considered in relation to the medium in which the gills are bathed. In full strength SW, it is most likely that the subcuticular ionic composition is close to that of SW. The transcuticular potential will then be zero and the cuticle as a whole will merely act as a single diffusion layer whose equivalent aqueous thickness will be given by the ratio of the cuticle permeability to the diffusion coefficient in water. This equivalent thickness is about 100 μm for cationic exchanges but about 10 mm for exchanges involving anions (either cotransported or counter-transported). For gills perfused with diluted SW, the subcuticular compartment could equilibrate with the external medium. The transepithelial ion leaks would thus be from the haemolymph to the subcuticular compartment. Recycling of these ions by the underlying epithelium could then occur from this compartment, favouring an apparent impermeability of the whole system in the steady state not only to ions but also to water. Alternatively, transbranchial ionic exchanges (either leak or uptake) could occur. In this case, a transcuticular electrochemical gradient will be established that will depend upon the relative permeabilities of the cuticle and of the epithelium and upon the pumping activity of the epithelium.
With respect to the latter alternative, the ionic permeabilities of the isolated cuticle, as determined in the present work, can be compared to the permeabilities of the whole gill (cuticle plus epithelium) deduced from the work of Lucu & Siebers (1986). According to these authors, the unidirectional Na+ flux across the isolated gill of Carcinus maenas perfused with 500 mmol 1−1 NaCl on both sides is 4000 μmol 1−1 h −1 and VO1 is then zero. They also calculated a PCl−/PNa+ ratio of 0 ·34 for the whole gill. Taking into account a gill surface area of about 50cm2g−1 fresh mass, the overall gill PNa+ and Per would be 5 ×10 −5 and 1 ·5 ×10 − 5cms −1, respectively. The gill PNA+ is much smaller than the gill cuticle PNA+. However, the overall gill Per is close to the gill cuticle PCl−. This, therefore, strongly suggests that the effect of the gill cuticle has to be taken into account in the study of transgill ion transport. Along these lines it is worth mentioning that the isolated cuticle and the whole gills both show a Na+ selectivity in Callinectes and that for a similar concentration gradient the transgill and the transcuticular potentials are close to each other (Smith & Linton, 1971). Published values of the transbranchial potential in Carcinus maenas (King & Schoffeniels, 1969; Pequeux, Wanson & Gilles, 1984, Siebers et al. 1985; Lucu & Siebers, 1986) also clearly show a Na+ selectivity, as we have observed for the isolated cuticle. However, it could be that both the cuticle and the epithelium show a Na+ selectivity and more extensive conductance measurements should be made before going further.
Our measurements, therefore, show that the inference made by Webb (1940) should be discarded : the gill cuticle of Carcinus maenas can no longer be considered as freely permeable to all ionic species. As mentioned above, a Na+-Cl− cotransport will be limited by the cuticle permeability to the slowest moving ion to satisfy the zero current condition across it. This permeability is 10 −3cms−1 in the lobster gill cuticle. It is less than 10 −6cms−1 in the freshwater crayfish (Avenet & Lignon, 1985). It lies between these values for Carcinus maenas gill cuticle. Since the lobster does not osmoregulate while Carcinus maenas has a limited power of regulation and the crayfish has an even higher power of regulation, it is tempting to relate the NaCl permeability of the cuticle to the power of regulation of decapods. A similar observation has been made for the carapace permeability of decapod crustaceans to salt and water: the carapace permeability has been related to the habitat of the animals (Gross, 1957; Herreid, 1969).
The author wishes to thank Professor P. Lasserre, director of the Marine Biological Station in Roscoff, and members of the staff, particularly Drs M. Moreau and J. P. Vilain, for their hospitality and kind material assistance. Mr Corbethau’s skill in designing the perfusion chamber is fully acknowledged.