Hyperosmoregulation in the red freshwater crab Dilocarcinus pagei (Brachyura, Trichodactylidae): structural and functional asymmetries of the posterior gills
Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes 3900, Ribeirão Preto 14040-901, São Paulo, Brasil
*e-mail: onkenh{at}ffclrp.usp.br
Accepted 24 October 2001
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Summary |
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Key words: Crustacea, red freshwater crab, Dilocarcinus pagei, haemolymph, osmolality, ion concentration, gill, transbranchial potential difference, split gill lamellae, Ussing chamber, short-circuit current.
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Introduction |
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Unlike the freshwater Macrura, the freshwater crabs do not appear to have evolved the ability to produce dilute urine. Their particularly low rate of iso-osmotic urine production seems not to depend solely on the low water permeability of the body surfaces since reabsorption of iso-osmotic fluid by the antennal gland also reduces urine volume. This strategy, apparently typical of freshwater crabs, may be a water-conserving adaptation to amphibious life (Greenaway, 1981; Harris, 1975
; Morris and Van Aardt, 1998
). However, since a reduced flow of iso-osmotic urine also conserves salt, this same adaptation reduces dependence on the mechanisms of active NaCl absorption from the freshwater medium that counterbalance diffusive losses.
Hyperosmoregulating Crustacea compensate for passive salt loss in dilute media by actively absorbing NaCl across their gill epithelia (for reviews, see Péqueux et al., 1988; Péqueux, 1995
). In diadromous crabs from marine and brackish waters, these organs, which play vital roles in gas exchange, in osmotic and ionic regulation, in pH regulation and in the excretion of N2 compounds, have been investigated from the whole gill to the molecular level, employing a wide variety of techniques (for a review, see Taylor and Taylor, 1992
). In contrast, investigations of the gills of the hololimnetic or true freshwater crabs have been limited, and gill ultrastructure has been examined only in Potamon niloticus (Maina, 1990
). No structural differences regarding the gills of other hyperosmoregulating crabs are evident. Freshwater crabs absorb salt against considerable ionic gradients: the external sodium concentration at which half-maximal uptake occurs is less than 0.2 mmol l1, which is clearly lower than for brackish-water animals. Consistent with the reduced passive salt loss typical of freshwater crabs, the maximal rate of sodium uptake in whole crabs (<2 µmol g1 h1) is also lower than that for brackish-water animals (see Morris and Van Aardt, 1998
; Potts and Parry, 1964
).
Dilocarcinus pagei Stimpson is a hololimnetic, trichodactylid crab endemic to the Amazon and Paraguai/Paraná river basins of South America (Magalhães, 1991). Virtually nothing is known of its osmotic and ion-regulatory capabilities. In the present investigation, we evaluated the haemolymph osmotic and ionic status and analysed the microanatomy and ion-transport characteristics of the posterior gills in this species. Our findings reveal novel structural and physiological asymmetries that underlie the ion-transport capabilities of these gills, disclosing adaptations that may have contributed to the successful invasion of the freshwater biotope by the trichodactylid crabs.
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Materials and methods |
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Before killing the crabs, a haemolymph sample of approximately 3 ml was collected via the arthrodial membrane of the posterior-most pereiopod using an insulin syringe coupled to a 28-6 gauge needle. The samples were stored in individual vials at 25°C until ion concentration analysis.
To obtain the gills, the crabs were quickly killed by destroying the dorsal brain and the ventral ganglion with a large pair of scissors. The carapace was removed, and the gills were excised at their bases with a pair of fine scissors and removed with tweezers. The gills were used immediately for the structural analysis and physiological experiments.
Haemolymph osmolality and ionic composition
Haemolymph osmolality was measured in 10 µl samples using a Wescor 5500 vapour pressure micro-osmometer. Na+, K+, Ca2+ and Mg2+ concentrations were measured by atomic absorption spectroscopy (GBC 933AA spectrophotometer) in 1020 µl samples diluted 1:1501:5000 times in distilled water. Cl concentration was measured in 10 µl haemolymph samples by microtitration against mercuric nitrate using s-diphenylcarbazone as the indicator (Santos and McNamara, 1996).
Microscopic studies
After dissection on ice, the gills were immediately perfused via the afferent vessel over a 23 min period with 1 ml of ice-cold primary fixative containing (in mmol l1): paraformaldehyde, 200; glutaraldehyde, 250; Na+, 100; K+, 10; Ca2+, 13; Mg2+, 2 (as chlorides); buffered in 100 mmol l1 sodium cacodylate at pH 7.5. Medial portions of selected gills consisting of approximately five lamellae each were then fixed on ice in fresh primary fixative for 1.5 h. After rinsing in buffered saline alone (3x5 min), the gill lamellae were post-fixed in 1 % osmium tetroxide in buffered saline for 1 h, dehydrated in an ethanol/propylene oxide series and embedded in Araldite 502 resin. Thick (0.5 µm) sections were prepared using a Porter-Blum Sorvall MT-2B ultramicrotome and stained with 1 % Methylene and Toluidine Blue in 1 % aqueous borax.
Measurement of transbranchial voltage
To perfuse an isolated gill, the afferent vessel was connected by a fine polyethylene catheter (0.61.2 mm outer diameter) to a perfusion system, and the gills were flushed in a Petri dish under a binocular microscope for 12 min with saline containing (in mmol l1): NaCl, 200; NaHCO3, 2; KCl, 5; CaCl2, 10; glucose, 5; Hepes, 5; at pH 7.6 (Tris). A second catheter was inserted into the efferent vessel, and both catheters were then fixed in position with a small Lucite clamp covered with smooth neoprene to avoid gill damage and to isolate the gill interior from the bathing medium. The gill was bathed in a beaker containing approximately 50 ml of aerated saline and was perfused by gravity flow, the perfusate being collected in a second beaker. In 22 experiments, the mean rate of perfusion was 21±4 ml h1. Perfusion rate was verified every 15 min for constancy.
To measure the transepithelial voltage (Vte) generated by the perfused gills, two calomel electrodes were connected via agar bridges (3 % agar in 3 mol l1 KCl) to the beakers containing the bath and perfusate. Vte was measured using a digital multimeter (model 8050A, Fluke, USA), the reference electrode being placed in the perfusate (internal side), and was recorded every minute for periods of up to 8 h.
Measurements with whole gill lamellae, split gill lamellae and isolated cuticles
Gill lamellae were isolated from a medial portion of the whole gills. Split gill lamellae were obtained by mechanically separating the two halves of a single gill lamella using two pairs of ultra-fine tweezers (see Schwarz and Graszynsky, 1989; Onken and Riestenpatt, 1998
). To obtain isolated cuticles, the epithelium was carefully removed from a split lamella preparation with a blunt micro-scraper. All manipulations and mounting of the preparations in a modified Ussing chamber were performed under a stereomicroscope.
A surface area of 0.01 cm2 was exposed to the chamber compartments (approximately 50 µl volume) bathing the external and internal surfaces of the split gill lamella. Both chamber compartments were continuously perfused with aerated saline by gravity flow (approximately 2 ml min1). When Na+-free saline was used, NaCl was substituted by choline chloride and NaHCO3 by KHCO3. In Cl-free saline, NaNO3, KNO3 and calcium gluconate served as Cl substitutes.
For voltage measurements, calomel electrodes were connected via agar bridges (3 % agar in 3 mol l1 KCl) to both sides of the preparation, the distance from the bridge tip to the tissue being less than 1 mm. The reference electrode was placed in the internal bath. Silver wires coated with AgCl served as electrodes to apply current for short-circuiting (i.e. measurement of the short-circuit current, Isc) through an automatic clamping device (model VCC 600; Physiologic Instruments, USA). The conductance of the preparations (Gte) was calculated from imposed voltage pulses (V) and the resulting current deflections (
I). The data were recorded continuously on a chart recorder (type 3229 I/85; Linseis, Germany).
Chemical reagents
All reagents were of analytical grade. Unless mentioned otherwise, all substances were purchased from Labsynth (Diadema, São Paulo, Brazil). Choline chloride, calcium gluconate and acetazolamide were from Sigma, and KNO3 and diphenylamine-2-carboxylate (free acid) were obtained from Fluka. Ouabain was from Serva, and agar agar, Hepes and Tris were purchased from Roth (Karlsruhe, Germany).
Statistical analyses
All data are given as the mean ± standard error of the mean (N). Differences between mean values were compared using Students t-test (P=0.05).
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Results |
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Posterior gill 7 was examined by light microscopy to provide a detailed analysis of the lamellar microanatomy. Near the central shaft, the lamellae are approximately 30 µm thick and contain a tenuous (45 µm thick), continuous, intralamellar septum that separates the two thin epithelia underlying the cuticle on either side of the lamella (Fig. 1E). Distally from the central axis, the septum becomes finer and discontinuous, disappearing approximately 80 µm from the gill shaft. At this point, the lamellae thicken to approximately 45 µm, and the opposing epithelial layers become conspicuously asymmetrical: on the proximal side, which faces the gill insertion point, the lamellar epithelium is notably thicker (1820 µm) than on the distal side (310 µm), which faces the gill tip (Fig. 1E). The dense proximal epithelium (Fig. 1F) is characterised by basal invaginations and a few apical vesicles. The thin distal epithelium (Fig. 1F) is characterised by the extensive apical expansions of the frequent pillar cells in the form of thin flanges, populated by vesicles and invaginations of the apical membrane. The pillar cell bodies extend across the haemolymph space, abutting on the thick proximal epithelium (Fig. 1F).
Electrical potential differences of isolated and perfused posterior gills
Posterior gills, perfused and bathed with NaCl saline, spontaneously generated a negative transepithelial voltage (Vte) of 16±4 mV (N=22), which stabilised after 30 min at 11±2 mV in 17 preparations, and at positive values (+5±3 mV; N=5) in five other gills. The addition of ouabain (2 mmol l1) to the perfusate significantly reduced the transepithelial voltage from 13±3 to +1±2 mV (N=8; P<0.05). The time courses of the voltage changes in individual gills during ouabain perfusion reveal interesting differences (see Fig. 2). In gills exhibiting a negative Vte, the voltage was reduced to values near 0 mV (Fig. 2A) or the polarity reversed and the gill produced a positive Vte (Fig. 2B). In gills showing a positive Vte under control conditions, ouabain perfusion increased Vte to more positive values (Fig. 2C).
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When Cl was substituted by nitrate on both sides of the preparation, Isc decreased significantly from 58±18 to 8±3 µA cm2 (N=5) (Fig. 3). Simultaneously, Gte was reduced from 4.05±0.36 to 1.65±0.34 mS cm2. When Cl was restored, a rapid current overshoot resulted, Isc then stabilising at approximately 70 % of the original value. In two experiments, Cl was initially substituted by nitrate only in the external bath. This almost abolished the negative Isc and markedly reduced Gte. Subsequent replacement of Cl in the internal bathing medium had no further effect on Isc or Gte.
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In five experiments, diphenylamine-2-carboxylate (DPC), a Cl channel blocker (Di Stefano et al., 1985), was added to the medium representing the haemolymph side (Fig. 3). At 1 mmol l1, DPC reduced Isc from 28±7 to 2±2 µA cm2. During DPC washout, Isc recovered to over 80 % of the control value. Gte was not significantly affected by perfusion of DPC over the internal surface. Dimethylsulphoxide alone, the primary solvent for DPC, had no effect on Isc or Gte (Fig. 3). In five experiments, ouabain (2 mmol l1), a specific inhibitor of the Na+/K+-ATPase (Skou, 1965
), was added to the perfusate bathing the haemolymph side of the epithelium for 1020 min (Fig. 3). Ouabain had no effect on the negative Isc or on Gte.
Acetazolamide (0.2 mmol l1), a carbonic anhydrase inhibitor (Maren, 1967), was added to the internal bathing medium (Fig. 3). Acetazolamide caused a rapid decrease in Isc from 23±7 to 2±2 µA cm2 (N=5). Gte was unaffected. The reduction in Isc was only slightly reversible, and the current recovered very slowly during washout.
Split gill lamellae: proximal side
Proximal split lamellae were perfused on both sides with NaCl saline in a modified Ussing chamber. This epithelium spontaneously generated a negative voltage (Vte) of 5±2 mV (N=7). Clamping Vte to 0 mV gave a positive short-circuit current (Isc) of +41±12 µA cm2. The conductance (Gte) was 18±5 mS cm2. These values reveal that the proximal split lamellae consist of the proximal epithelium and cuticle.
Substitution of Na+ by choline on both sides of the preparations abolished the positive Isc of +26±5 (2±1 µA cm2, N=5) (Fig. 4). Gte was reduced simultaneously from 18±5 to 2.9±1.0 mS cm2. The effect of Na+ substitution was completely reversible.
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Whole gill lamellae and cuticle
To evaluate the quality of the split lamella preparations, the electrical resistances of whole gill lamellae and isolated cuticles were measured. Fig. 5 shows these respective resistances together with those of the distal and proximal split lamella preparations and their sum.
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Discussion |
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Microanatomy of the posterior gills
While most Brachyura possess nine pairs of phyllobranchiate gills, the red freshwater crab has only eight (see Fig. 1A,B), although the African freshwater crab Potamon niloticus has seven pairs (Maina, 1990). Regions similar to the well-defined, central, dark area in the posterior gill lamellae of Dilocarcinus pagei (see Fig. 1D) have been found after silver nitrate staining of the posterior gills of other hyperosmoregulating crabs such as Callinectes sapidus (Copeland and Fitzjarrell, 1968
), Carcinus maenas (Compère et al., 1989
) and Eriocheir sinensis (Barra et al., 1983
). Such areas correspond to a thick epithelium of approximately 10 µm in height showing features typical of transporting cells, including extensive apical and basal membrane infoldings and an elevated mitochondrial density. In Carcinus maenas gills, this area increases when the crabs are adapted to a dilute medium (Compère et al., 1989
).
A strikingly thick epithelium is also present in the dense region of the posterior gills of Dilocarcinus pagei. However, while the lamellar epithelium in marine and brackish water crabs is symmetrically thickened on both lamellar surfaces, and punctuated by occasional pillar cells (Taylor and Taylor, 1992), only the proximal lamellar epithelium is thickened in Dilocarcinus pagei. The distal lamellar surface consists of a thin epithelium composed mainly of apical pillar cell flanges. The posterior gill lamellae of Dilocarcinus pagei thus exhibit a remarkable epithelial asymmetry (see Fig. 1E,F). The only other histological study on freshwater crab gills revealed that the lamellar epithelia in Potamon niloticus gills are symmetrical and that the central, dense region is apparently lacking. The squamous, 6 µm thick, epithelial cells exhibit an extensive system of apical leaflets and basal membrane invaginations associated with mitochondria, which is typical of a structure with transport function (Maina, 1990
).
Asymmetrical epithelia resembling those of Dilocarcinus pagei occur in the book gill lamellae of the euryhaline horseshoe crab Limulus polyphemus (Henry et al., 1996). The ventral epithelium exhibits a thick (510 µm), central dense area that displays features characteristic of a transporting epithelium, such as numerous mitochondria associated with basal membrane invaginations and elevated Na+/K+-ATPase and carbonic anhydrase activities. However, in Dilocarcinus pagei lamellae, both epithelia are much thicker (1020 µm) than in Limulus polyphemus lamellae, in which the pillar cell flanges do not constitute the thin epithelium. The irregular pillar cell arrangement in Dilocarcinus pagei, where thin apical flanges (see Fig. 1E,F) constitute the distal epithelium, is very similar to that of the lamellar epithelium of freshwater palaemonid shrimps, which is involved in both gas exchange and ion absorption (Taylor and Taylor, 1992
; Freire and McNamara, 1995
; McNamara and Lima, 1995
; McNamara and Torres, 2000
).
Electrophysiological characteristics of posterior gills
Transbranchial voltage (Vte) has been measured in a variety of isolated, perfused, whole crustacean gills (see Péqueux et al., 1988). Interestingly, the posterior gills of Dilocarcinus pagei generate Vte values of different polarity, which are not due to gill position or seasonal variation. Vte values of opposite polarity were found on consecutive days and when using gills from the same insertion position (gills 68). A novel mechanism of integration of the two different epithelia in the lamellae of individual gills may provide a plausible explanation for these variable Vte polarities in the different gills. This hypothesis requires future investigation. In all cases, however, ouabain altered Vte to more positive values, indicating inhibition of active, electrogenic Na+ absorption generated by the Na+/K+-ATPase (Fig. 2). The maximum Vte values under control conditions for each polarity (23 and +16 mV, respectively) suggest at least a moderately tight epithelium, as expected for freshwater animals maintaining large ionic gradients.
Split gill lamellae have been used successfully in an Ussing chamber to characterise active NaCl absorption across the gills of hyperosmoregulating Chinese crabs, Eriocheir sinensis, and shore crabs, Carcinus maenas (for a review, see Onken and Riestenpatt, 1998). In Dilocarcinus pagei, the Cl-dependence of the negative Isc and of the transepithelial conductance (Gte) of distal split lamellae (see Fig. 3) indicates that the thin epithelium generates active, electrogenic Cl absorption. Negative Isc and Gte were not reduced when ouabain was added to the internal bathing medium (see Fig. 3), demonstrating that Cl absorption across this epithelium does not depend on a functioning Na+/K+-ATPase. Substitution of Na+ by choline even increased the negative Isc. Although difficult to interpret at present, this increase does indicate that active Cl absorption is independent of Na+. Internal addition of the Cl channel blocker diphenylamine-2-carboxylate (Di Stefano et al., 1985
) almost abolished the negative Isc without affecting Gte (see Fig. 3). Thus, active Cl absorption seems to proceed via basolateral Cl channels, as observed in many other Cl-absorbing epithelia (see Greger and Kunzelmann, 1990
). The lack of effect of diphenylamine-2-carboxylate on Gte may simply reflect the presence of other electrogenic pathways in the basolateral membrane, the conductance of which, in many epithelia, is determined mainly by K+ channels. The internal addition of acetazolamide, a carbonic anhydrase inhibitor (Maren, 1967
), reduced the negative Isc, indicating the involvement of carbonic anhydrase in active Cl absorption. These results show striking similarities with active Cl absorption by Eriocheir sinensis split gill lamellae (Onken et al., 1991
), in which an apical V-type H+-pump is thought to drive electrogenic and Na+-independent Cl absorption via apical Cl/HCO3 antiports and basolateral Cl channels (Onken and Putzenlechner, 1996
). This same transport mechanism seems likely for the thin epithelium of the posterior gill lamellae in Dilocarcinus pagei.
The reduction in the positive Isc across proximal split lamellae after the addition of ouabain to the internal bathing medium or after substitution of Na+ by choline (see Fig. 4) indicates that the thick epithelium generates active, electrogenic Na+ absorption. As in Eriocheir sinensis split gill lamellae (Zeiske et al., 1992), Na+ absorption may proceed via apical Na+ channels. Strictly, however, since an apical transport pathway requires confirmation, Na+ absorption may also proceed via an electrogenic 2Na+/1H+ antiporter, which has been found in crustacean gill (Shetlar and Towle, 1989
) and other transport epithelia (Kimura et al., 1994
). In preliminary experiments, external amiloride caused only a minor decrease in current, even at high concentrations (1 mmol l1). Apparently, this Na+ channel blocker does not permeate the cuticle and cannot be used to distinguish between channels and antiporters. Substitution of Na+ by choline not only abolished the positive Isc, but also markedly reduced the conductance to values below 2 mS cm2 (see Fig. 4), indicating that the preparation exhibits a marked selectivity for Na+. Such selectivity may be due to the nature of the transporter in the apical membrane and/or to ion-selective paracellular junctions. The cuticle may also contribute, since ion-selectivity by isolated crustacean cuticles has been observed (Lignon and Péqueux, 1990
).
A comparison of the resistances of whole gill lamellae with the sum of those of the distal and proximal split lamellae (Fig. 5) reveals a difference of approximately 20 %. In particular, the resistance of the thick, proximal epithelium seems to be low. Such preparations generated negative voltages, but these were not as large as those observed with isolated, perfused gills. These data suggest that the proximal split lamellae may suffer some damage during splitting. However, the basic electrophysiological parameters for the distal split lamellae (see Results) demonstrate that these preparations are mainly unaffected by the mechanical splitting process.
The electrophysiological characteristics of the posterior gills of Dilocarcinus pagei show clear similarities with those of Eriocheir sinensis, a crab that spends most of its life in fresh water. Unlike Carcinus maenas (see Riestenpatt et al., 1996), which migrates between sea water and brackish water, both freshwater-inhabiting species generate active, independent, electrogenic absorption of Na+ and Cl. Both apparently possess a tight gill epithelium, which is able to generate high voltages and to maintain large ionic gradients, as expected for freshwater animals. The principal difference in active, transbranchial NaCl absorption between Dilocarcinus pagei and Eriocheir sinensis lies in the intralamellar distribution of transport functions. The posterior gills of the red freshwater crab actively absorb Na+ and Cl across opposite sides of the lamellae, while the active absorption of Na+ and Cl across Chinese crab gills is effected on both sides.
Transport mechanisms have been proposed for the amphibian skin, fish gills and Chinese crab gills in which two ATPases (an apical H+ pump and a basolateral Na+/K+-ATPase) drive Na+ absorption via apical Na+ channels and Cl absorption via apical Cl/HCO3 antiports (Goss et al., 1992; Larsen, 1988
; Onken and Riestenpatt, 1998
). Although the intraepithelial organisation of the transporters involved appears to be different in these three examples, the model seems to be characteristic of freshwater animals. Absorption of NaCl across the gills of the red freshwater crab apparently conforms to the same principle, although it is manifest in a structurally different manner. It will be a rewarding task to examine this hypothesis and to reveal details of the transport characteristics of Dilocarcinus pagei gills in future studies.
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Acknowledgments |
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