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A morphological study on gills of a crab acclimated to fresh water
TISSUE & CELL 1983 15 (4) 58%5% @ 1983 Longman Group Ltd
JEAN-A.
BARRA, ANDRt
PEQUEUX”
and WILLY HUMBERT
A MORPHOLOGICAL STUDY ON GILLS OF A CRAB ACCLIMATED TO FRESH WATER Key words: tacea.
Gills,
ultrastructure,
fresh water,
salt-transporting
epithelium,
En’ocheir,
Crus-
ABSTRACT. The gills of the fully euryhaline Chinese crab En’ocheir sinens& were studied by light and electron microscopy. In these Phyllobranchiates, the gills consist of a double row of lamellae extending laterally from a central shaft. Haemolymph flow pattern inside the gill is described and the existence of a complex secondary vascularization inside the platelets is reported. It is shown that important differences exist between the ultrastructure of the three anterior and the three posterior pairs of large gills. The epithelium of the posterior gills is much thicker and possesses an extensive elaboration of the plasma membranes in the form of infoldings, crypts and interdigitations, along which are packed numerous mitochondria. The presence of such a complex membrane system opening to the extracellular space and closely associated with mitochondria is common to all salt-transporting tissues. This study corroborates the idea that the posterior pairs of gills of Eriocheir sinemis are the only ones implicated in active Na+ uptake when the crab lives in dilute aquatic environment. The epithelium of anterior gills is much thinner and the cells poor in intracellular organelles. It seems to be involved essentially in respiration. Thus this work clearly corroborates the existence already suggested by physiological approach of a functional difference between the different pairs of E. sinensis branchiae with respect to their participation in the respiration and in the regulation of the blood ions content. Common to both types of gills is the presence of a lamellar septum separating the haemolymph space into two compartments. The part played by that structure in determining the pattern of haemolymph flow, together with periodic bridges forming pillars across the haemolymph space, is emphasized.
Callinectes:
Copeland and Fitzjarrel, 1968; Copeland, 1968; Jueru: Bubel and Jones, 1974), nothing is known on the fine structure of the gills of the Chinese crab. From the physiological point of view, it has been established that the salt loss occurring when in dilute media is counterbalanced by an active absorption of Na+ and Cl- which probably takes place in the gill tissue (for review, see Gilles, 1975; Pequeux and Gilles, 1981). The purpose of this work was therefore to understand the morphology of both the anterior and posterior gills of E. sinensis in order to try to explain the physiological differences reported in ion movements, cellular ionic content, transepithelial potential and enzyme activity. This paper deals with light and electron microscopy observations made on the gills of Chinese crabs acclimated to fresh water.
Introduction
Gecarcinus:
Like many other euryhaline crustaceans, the Chinese crab Eriocheir sinensis has a migratory life cycle with a growing period of long duration in fresh water and a shorter breeding stay in salt water. In the laboratory it withstands direct acclimation either to fresh water or to sea water without any apparent damage. Despite work on the normal histology and fine structure of gill tissues from various crustacean species (Astucus:Bielawski, 1971;
Laboratoire de Zoologie et d’Embryologie experimentale, 12 rue de I’Universite, F 67MM Strasbourg, France. * Laboratory of Animal Physiology, University of Litge, 4020 Liege, Belgium. Received 19 November 1982. Revised 27 April 1983. 583
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Materials and Methods Animals Adult Chinese crabs E. sinensis were obtained from commercial fishermen in Bad-Zwischenahn, near Oldenburg, West Germany. Capture occurred in October when animals were still living in fresh pools, before their breeding migration towards salt water. In the laboratory they were kept at II-12°C in continuously renewed aerated tap water. Light microscopy Samples were fixed in aqueous Bouin fixative. Transverse sections (7 pm) were stained by classical staining methods (Trichrome of Prenant). Semithin sections (1-2 pm) were stained with toluidine blue. Transmission electron microscopy (TEM) Pieces of gills were fixed for 2 hr at room temperature in a solution of 4% glutaraldehyde buffered with 0.4 M cacodylate at pH 7.4. The final osmolarity of the fixative medium was 300 mOsmo1 kg-’ H20. A mixture of 4% glutaraldehyde and 2% osmium tetroxide buffered with 0.4 M cacodylate at pH 7.4 was also tested. The pieces were rinsed several times in 0.4 M cacodylate buffer with addition of 5% saccharose; they were then post-fixed for 1 hr at room temperature in 2% osmium tetroxide. Specimens were embedded in Araldite. Thin sections were stained with ethanolic uranyl-acetate and lead citrate (Reynolds, 1963) and examined with a Siemens Elmiskop 101 electron microscope. Silver nitrate treatment In a preliminary survey of the gill tissues, the patches of cells presumed to absorb salt were
PEQUEUX
AND
located by use of AgNOs following the method of Koch (1934, pers. comm.; see also Ewer and Hattingh, 1952). Living crabs first washed with distilled water were placed in a 0.05% solution of silver nitrate for 5 min (Ewer and Hattingh, 1952). After removal of the excess AgNOs by a second rinse in distilled water, the animals were killed and the branchiostegite was removed. Gills soaked in the fixative were then exposed to sunlight for 15 min until appearance and stabilization of a brown violet colour. Transverse sections of AgNOs stained gills were prepared for electron microscopy. X-ray microanalysis X-ray microanalyses of Ag+ and Cl- were performed by means of a microanalyser CAMECA MS46 operating under the following conditions: acceleration voltage, 15 kV; beam current, 50 nA; beam diameter, 1 pm, PET Crystal for Cl- and Ag+. Analyses were made on carbon-coated semithin sections placed on Mylar slides. In an attempt to corroborate the results obtained with the CAMECA MS46 analyser, ultrathin sections were also mounted on copper grids and analysed with CAMEBAX microanalyser under the following conditions: acceleration voltage, 50 kV; beam current. 100 nA. Silver lactate treatment Cl- ions were detected and located in ultrathin unstained sections prepared for TEM observation, after precipitation with silver lactate according to the method of Komnick (1962) and Komnick and Bierther (1969). 1% silver lactate (Fluka) was added to the fixative solution (2% 0~0~) in cacodylate buffer acidified with acetic acid
Table 1. Branchial formula of Eriocheir sinensis Maxillipeds
Cheliped
2
3
1
Pleurobranch Arthrobranch Podobranch 1 Epipodite 1 1 Total: 8 gills, 3 epipodites
2 1 1
_ 2 _
1
HUMBERT
Peraeopods 2
3
4
5 ~-
I -
1 _ -
_ -
_ _
CRAB GILLS ACCLIMATED
TO FRESH WATER
A ___
B ___ C _--
Fig. 1. Diagram of a cross-section of Chinese crab gill. The arrows indicate the main routes of the haemolymph flow within gill Iamellae. The left side of the diagram represents the platelet outer side covered with chitineous cuticle. The dotted curved lines represents the distribution of pilaster cells (PC). CA and DA respectively correspond to clear areas in the platelets near the efferent vessel (Ev) and dark areas located near the afferent vessel (Av). The right side of the diagram presents an internal view of the platelet showing the fenestrated lamellar septum (Ls) and arterioles related to the larger epibranchial and hypobranchial vessels (respectively Ep.v and Hy.v). The dotted lines A, B and C show the levels where cross-sections shown in Figs. 3-5 have been made. MC, marginal canal; Ne, nephrocytes; S, shaft; Lv, lamellar vessel.
Fig. 2. Schematic model of part of a branchial platelet in the so-called ‘dark area’, showing the fenestrated lamellar septum (Ls). Blood flow in the platelet is tentatively shown by solid arrows, with the larger arrow representing greater flow in the marginal canal (MC). C, cuticle; Be, branchial epithelium; Lv, lamellar vessel; PC, pillar cell.
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(pH 6.4). During dehydration, 0.1 N NHOs was added to the 20% acetone solution in order to eliminate non-specific precipitation. Pieces were embedded in Araldite. Ultrathin sections were observed without staining. ‘Cl- ions’ (AgCI) appear as electron-dense precipitates. Polysaccharide staining The periodic acid-thiocarbohydrazide silver proteinate method (PATAG) for polysaccharides as described by Thiery (1967, 1969) has been applied to branchial lamellae. Thin sections were pre-treated for 30 min with 1% hydrogen peroxide in order to remove osmium from tissues and avoid non-specific deposits. Sections were then treated with the following reagents: (a) 1% periodic acid for 30 min at room temperature, (b) 0.2% thiocarbohydrazide (TCH) (Merck) reagent for 60 min, (c) 1% silver proteinate solution for 30 min. Controls were treated with silver proteinate omitting the periodic acid oxidation or TCH treatment. Results General organization Eriocheir sinensis has eight pairs of gills in the branchial chamber. The branchial formula is given in Table 1. The gills are phyllobranchiae, composed of a double row of closely spaced lamellae extending anteriorly and posteriorly from a median shaft.
Fig. 3-5. Cross-sections different levels as indicated
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The cuticular surface of the gill lamellae shows two distinct areas characterized by different colour densities: a dorsal dark area and a ventral clear area (Fig. 1). The lamellar septum, essentially restricted to the dark area, exhibits a lot of apertures, allowing for complex movements of haemolymph between upper and lower compartments of the haemolymph space (Figs. 2-5). Histologically, the lamellae are composed of a single layer of cells lining the cuticle on each side of the leaflet. Both cell layers are connected periodically by pilaster cells which keep them separated and maintain a large central haemolymph space. Macroscopically. the insertion points of the pilaster cells in the cuticle can be seen by transparency ai the lamella surface where they appear as curved concentric lines (Fig. 1). An afferent and an efferent blood vessel line respectively the outer and the inner side of each gill. A lacunose median shaft built of support connective cells joins both blood vessels (Fig. 6). Facing the lamella septum, clusters of nephrocytes are located near the efferent vessel (Figs. 7, 8). Since the structure of small podobranches on maxillipeds 2 and 3 appears to be similar to the structure of the other gills. only the six pairs of larger gills have been studied in detail. Main circulatory routes The main circulatory
routes
of the same scrics of three lateral lamcllac in Fig. 1 by the dotted lines A. B and C.
Fig. 3. Semithin cross-section through the dark area. showing lamellar septum and the marginal vessel (MC). x 170.
of
pcrformcd
the connection
the
at three
hetwccn
the
Fig. 4. Cross-section through both the dark and clear arcas, showing the lamcllar septum to he essentially restricted to the dark area. Note the thinness of the median shaft (S). x 170. Fig, 5. Cross-section through the clear area bordering absence of lamcllar septum. x 170.
on
the efferent
blood vessel. Note the
Fig. 6. Cross-section of a paraffin embedded gill, showing the affcrcnt blood vessel (Ev). A small epibranchial vessel (Ep.v) can he seen inside the median shaft (S). Fig. 7. Cross-section of a paraffin embedded gill, showmg the effercnt blood vc~~el (Eb). A smaller hypobranchial vessel (Hy.v) can he seen among the abundant nephrocytes (NC). X220.
--
BARRA,
PEQUEUX
AND HUMBERT
Fig. 8. Longitudinal semithin section through the efferent blood vessel showmg lateral lamellae. At their proximal joining parts, nephrocytesclusters (Ne) hang down in the efferent vessel. Pillar cells (PC) of lateral lam&e can distinctly be seen. x140. Fig. 9. Cross-section through a lamellar vessel (Lv) located near the median septum. x 16,Mx).
Fig. 10. Cross-sectionof the branchml ‘respiratory’ epithelium. Cuticular surface at top, thick basal lamina (Bl) lining the haemolymph lacuna (L) below. Arrows indicate deep interdigitations of latero-basal lamina (see text tar other pomts). m. mltochondna; N, nucleus; C. cuticle. X34,CKl Fig. 11. Cross-section of the branchial ‘salt-transporting’ epithelium. Apical membranes exhibit extensive folds(F). Basal and latero-basal membranes show deep, complex and regular interdigitations (I) coming into close contact with mitocbondria. Note the abundance of mitochondria (m). See text for other points. C, cuticle: N, nucleus. x34,wO. Fig. 12. Unstained cross-sectionthrough the dark area of a gill lamella treated to reveal polysaccharides (PATAG method). Glycogen (g) appears as very abundant in the median lamellar septum. Ep, gill epithelium. xfLO0
BARRA,
haemolymph in the crab gills lamella are speculatively represented in the schematic diagram of Fig. 2. Haemolymph reaches the gill by the outer afferent vessel which communicates laterally with the marginal vessel of each gill lamella by means of oblong slits. Blood thus enters the haemolymph lacuna from the outer side of the platelet towards the inner part. The marginal canal which is lined by a thin epithelium, appears as a rapid circulatory route where the haemolymph does not meet any constraint. On the contrary, inside the lamella, blood flow is impeded by pilaster cells, by the lamellar septum and by a thicker epithelium. Blood reaches the efferent vessel either directly from the marginal vessel, or by percolating through the central part of the lamella. The efferent vessel wall is much more complex than that of the afferent vessel, its outer face being lined by a thin chitinous cuticle, while its inner side is limited by the nephrocytes. Its lateral walls correspond to the pilaster cells of the clear area. The positioning of these pillars
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undoubtedly determines the pattern of blood flow from the marginal vessel and the central haemolymph lacuna towards the efferent vessel (Fig. 7). The smaller vessels, called epibranchial and hypobranchial vessels, are located in the median shaft near the afferent and the efferent vessels respectively (Figs. 1, 6, 7). They will henceforth be identified by their topographical position in the gill. In phasecontrast microscopy the lamella of the gills shows a complex dichotomic network of thin. so-called lamellar vessels (Fig. 1) which are related to the epibranchial and hypobranchial vessels, as clearly shown by serial cross-sections. Each lamellar vessel wall is constituted of two endothelial ceils closely bonded to each other by means of deep interdigitations (Fig. 9). Facing the vessel lumen, the intima is coated with a thick glycocalyx. Generally the inner folds completely obliterate the central lumen although, the vessel diameter can be modified to allow haemocytes passage. The lamellar vessels very probably open on the
Fig. 13. Single lamclla isolated from an anterior located gill after in viva trcatmcnt with silver nitrate (see Materials and Methods). Only an extremely weak coloration can hc seen at the lamella surface. Fig. 14. Single lamella isolated from a posterior located gill after in viva treatment with silver nitrate. Note the very dense coloration essentially restricted to the ‘dark area’ (DA), and contrasting with the weak coloration of the ‘clear area’ (CA). Fig. 15. Unstained cross-section through the thin ‘respiratory’ epithelium of an an&&r gill treated in viva with silver nitrate (same gill as shown in Fig. 13). Dense clusters of silver chloride granules appear asregularly distributed in, and essentially restricted to, the cuticle. x26,OGil. Fig. 16. Unstained cross-section through the thick ‘salt transporting’ epithelium (dark area) of a posterior gill treated in viva with silver nitrate (same gill as shown in Fig. 14). Silver chloride granules appear as uniformly distributed in, and restricted to. the cuticle, and the extracellular compartment between the cuticle and the apical side of the epithelial cells. ~26,000. Fig. 17. Unstained cross-section through the ‘respiratory’ epithelium of an anren’or gill, after treatment in vitro with silver lactate (specific ‘staining’ method for Cl- as described in Materials and Methods). The clusters of electron-dense granules have been identified as containing Cl and Ag by X-ray microanalysis (see inset record). x 18,000. Fig. 18. Unstained cross-section through the ‘salt-transporting’ cpithelium of a posrerior gill, after treatment in virro with silver lactate. A large amount of AgCl containing granules appears as uniformly distributed in, and restricted to, the cuticle. and the extracellular space between the cuticle and the apical infoldings of the epithelial cells. x 15,600.
BARRA,
lamellar haemolymph lacuna but, up to now, this has not been observed on electron micrographs. Anterior gills
The three anterior pairs correspond to gills 1, 2, 3. During intermolt, the cuticle is from 0.7 to 1.0 pm thick in the central part of the lamella and about 2 pm thick near its insertion point on the median shaft. It is thicker (3-6 pm) in the wall of the marginal vessel. From the outermost layer inward, the cuticle consists of an epicuticle and an uncalcified endocuticle (Fig. 10). The epithelium of anterior gills is about 2-4 pm thick. At the base of the lamellae and at their apical side, however, epithelium thickness may reach up to 10 pm. Nuclei generally protrude within the haemolymph space. The cells’ apical side just beneath the cuticle is very lightly folded. The plasma membrane of the folds appears a little denser. The extracellular compartment under the cuticle is extremely reduced. The amount of intracellular organelles seems to be very limited. The hyaloplasma is well populated with mitochondria and glycogen granules, but, whereas glycogen granules are uniformly distributed in the cells, mitochondria are essentially restricted to the apical area. All the other organelles are poorly represented and the plasma membrane does not exhibit extensive basal infoldings. Periodically, three to four cells are associated to form a pillar. Posterior gills
The three posterior gills correspond to gills 4, 5, 6. Serial cross-sections through a posterior gill show that (a) the thickness of the gill epithelium decreases from the dark towards the clear area. (b) the lamellae are much thicker in the dark area, and (c) the median shaft is thinner in the central part of the gill (Figs. 3-5). The cuticle structure is quite similar to that described prevously. However, it is worth noticing that, in the dark area, its thickness can be as little as 0.3 pm. From the shaft towards the distal area of the lamella, the epithelium thickness decreases, reaching about 1 pm near the marginal vessel, while the epithelial cells in the dark area near the shaft are larger, up to 10 pm and more. Such a size difference has to be related to a sudden change in the structure
PEQUEUX
AND HUMBERT
of the cells in connection with the distribution of the clear and the dark areas. The large epithelial cells near the median shaft are characterized by a complex and well-developed network of large apical folds. These membrane folds may contain cytoplasm and produce a large extracellular compartment under the cuticle (Fig. 11). Basal folds generally come into close contact with mitochondria membranes. Mitochondria appear as the most abundant organelles in these epithelial cells of posterior gills. They may completely fill the cytoplasmic space. As reported above, structural changes occur from the shaft area towards the lamella apex: (a) the amount of mitochondria considerably decreases in the centre of the cells, (b) apical and latero-basal folds become shorter and shorter (the cell still may be considered however as typical ‘salttransporting cells’). These transformations are very progressive from the proximal to the distal part of the lamella where the epithelium appears to be very similar to the ‘respiratory epithelium’ of the anterior gills. In the clear area where the lamella septum is lacking it is 2 pm thick or less. The lamellar septum is a stratified fenestrated sheet which is 9-15 pm thick and is surrounded by a basal lamina. The septum is supported by pilaster cells. A large amount of glycogen has been identified histochemitally by the Patag reaction (Thiery, 1967, 1969) (Fig. 12). The ergastoplasm appears as long flattened and sinuous saccules. Mitochondria are very abundant and the Golgi apparatus reduced to three to four saccules. Neighbouring cells are bound by desmosomes. The early in vivo experiments of Koch (1934-54) have been repeated and the selective staining of posterior gills of the Chinese crab E. sinensis has been confirmed (Figs. 13, 14). Gills of the crabs treated by AgN03 were then fixed and prepared for electron microscopy observation. In vitro experiments with silver lactate according to the method of Komnick (1962, 1963) have also been performed and give similar results. Following in vivo treatment by AgNOs, the lamellae of anterior gills exhibit a uniform pale brown colour. It is worth noticing that the coloration is extremely light and not always evident macroscopically. The reaction remains negative in the clear area of the
CRAB GILLS ACCLIMATED
TO FRESH WATER
gills (Fig. 13). Photomicrographs (Fig. 15) show that light coloration is due to clusters of dense granules (AgCl precipitate) regularly distributed in and mainly restricted to the cuticle. No significant precipitate has been observed in the respiratory epithelium or in extracellular lacunae. Quite similar observations were made after in viva treatment with silver lactate (Fig. 17). At variance with these observations, the lamellae of posterior gills strongly react to AgNOa exposure. After treatment, the area adjoining the afferent vessel exhibits a strong brown colour while the clear area near the afferent vessel remains uncoloured (Fig. 14). AgCl precipitates are uniformly distributed in the cuticle and in the compartment under the cuticle near the apical folds of the salt-transporting cells (Fig. 16). Similar results were obtained with silver lactate. Granules were moreover observed in the epithelium itself (Fig. 18). As illustrated by the peak recordings of Fig. 17, electron probe X-ray microanalysis of semithin and ultrathin sections confirm the AgCl nature of electron-dense granules. Discussion Since the early works of Koch et al. (1954) on the Chinese crab Eriocherir sinensis, conclusive proof exists that the gills of euryhaline crabs absorb salts actively from dilute media. More recently Na+ transport processes at work in the branchial epithelium of the Chinese crab have been investigated more extensively using perfused preparations of iolated gills (Gilles, 1975; Pequeux and Gilles, 1978a, b, 1981). Among other things, these works clearly establish the existence of functional differences between the three anterior and the three posterior pairs of branchiae with respect to their participation in the blood osmoregulation that this species is able to achieve when in dilute media. Up to now, the structure of the Chinese crab gill has not however received much attention. The Chinese crab Eriocheir sinensis is a typical hyperosmoregulator which can maintain a pronounced blood hyperosmotic state when in diluted medium, and whose blood is isosmotic to the external medium when the animal is acclimated to sea water (Berger, 1931; Krogh, 1939; Schoffeniels and Gilles, 1970). In hyperosmoregulators, active Na+
uptake at the gill level is an essential mechanism in counterbalancing the salt loss in dilute media. In E. sinensis, it has been established that the active Na+ influx is mostly achieved by the three posterior pairs of gills (Pequeux and Gilles, 1981). The results of the present structural study are in complete agreement with that finding. A so-called ‘salt-transporting epithelium’ is reported to be localized and essentially restricted to the same three posterior pairs of gills. At variance with most marine or fresh water brachyuran decapods, which have nine pairs of gills, Eriocheir sinensk has only eight pairs (Peters and Panning, 1933). Several authors have therefore considered that the loss of one pair of branchiae by E. sinensis could perhaps be related to the well-known gill reduction trend reported in terrestrial or semi-terrestrial crab species (Gray, 1957; Bliss, 1968; Taylor and Greenaway, 1979). The way in which water flows over the gills, and haemolymph flows in vessels and lacunae is characteristic of the well-known counter-current system (Hughes et al., 1969; Nakao, 1974; see Dejours, 1975, for review) but differs from the more complex bidirectional system of crustacea having trichobranchiae (Fisher, 1972; Burggren et al., 1974). The epithelium of the anterior gills is much thinner (2-4 pm) and covered by a 1 pm thick cuticle supposed to be permeable to gas. The structure of the Chinese crab cuticle is very similar to the gill cuticle of other crustacean species (Copeland, 1967, 1968; Copeland and Fitzjarrel, 1968; Bielawski, 1971; Fisher, 1972; Starch and Welsch, 1975; Foster and Howse, 1978; Taylor and Greenaway, 1979; Kiimmel, 1981). In these epithelia the distance between the haemolymph and the external medium is only about 3-5 pm, more often less than 2 pm, and such a thinness of the epithelium doubtless favours gas passage from the outside towards the haemolymph. Therefore, it seems quite reasonable to consider that the main and essential function of that tissue must be respiration. The epithelium of the posterior gills is much thicker, up to 10 pm, while its cuticle tends to be thinner (O-3 pm). It is characterized by infoldings of the plasma membrane along which are packed numerous mitochon-
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dria. Such extensive elaboration of the plasma membranes in the form of tubules, crypts, infoldings and interdigitations opening to the extracellular space and in close association with mitochondria seems to be common to all the ‘salt-transporting tissues’ (Berridge and Oschman, 1972): in gills of other crustacean species (Copeland, 1963, 1964a; Copeland and Fitzjarrel, 1968; Foster and Howse, 1978), in the ‘salt gland’ of marine turtles (Ellis and Abel, 1965) and birds (Komnick, 1963), and in the anal papillae of insect larvae (Copeland, 1964b; Koch, 1938). Apparently, cells of the gill filaments of fish (Philpott and Copeland, 1963), of the elasmobranch rectal gland (Bulger, 1963a, 1965; Komnick and Wohlfarth-Bottermann, 1966), of the insect midgut (Berridge, 1970), or of the insect rectum (Gupta and Berridge, 1966; Oschman and Wall, 1969) also reveal a quite similar organization. Large interdigitating plasma membrane folds, highly populated with mitochondria, are also present in the striated duct cells of mammalian salivary glands (Tandler, 1963) as well as in epithelium of mammalian kidney tubules (Tisher et al., 1966). In light of this the results of the present study are in complete agreement with physiological data, since the posterior gills are the only ones implicated in active Na’ uptake (Pequeux and Gilles, 1981). As early as 1934, Koch, studying the staining of gills of different crustaceans with silver salts, suggested that those showing fixation of silver nitrate were involved in active Na+ uptake. This method and hypothesis were later used on several species to localize patches of cells presumed to absorb salt: Artemia (Copeland, 1967), Callinectes (Copeland and Fitzjarrel, 1968), Potamon (Ewer and Hattingh, 1952), Pacifastacus (Morse et al., 1970). To the best of our knowledge, these experiments were never followed by an ultrastructural study of the tissues treated. It is shown here that AgCl precipitates, as identified by microanalysis, are localized in the endocuticle of both gill types, either as isolated clusters in anterior gills or as a continuous layer in posterior gills. Treatment with silver lactate gives identical results, which are moreover in agreement with results obtained with other
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Arthropod species (Haase, 1975. for bibliography; Schmitz and Komnick, 1976; Humbert, 1978). Up to now the exact significance of these results remains unclear. Precipitates obviously do not allow the localization of Cl- ions in the living tissue, but more likely have to be considered as an indication of the gill area presumed to be more permeable to ions. As early as 1898, Kimus already reported the existence of an intralamellar septum so-called ‘tissu intermediaire ’ in Azellus, Cirolona, Idothea, Anilocra and Cymothoa which is described as variable in form and size. In Eriocheir, it has been shown to induce a ‘dark area’ in the gill tissue. The lamellar septum must be similar to what has been described in Cardina as ‘trabecular cells’ by Nakao (1974). and in several species of decapods as ‘reseau’ by Drach (1930). If the septum seems thus to be of rather general occurrence in crustacea, its function still remains unclear. In the light of our results we believe that, besides the fact that it divides the haemolymph into compartments, and must undoubtedly determine the pattern of haemolymph flow through the platelet, the septum appears as a storage tissue able to accumulate a large amount of glycogen. For more details on the structure of the various types of septa encountered in other species of crustacea, the reader is referred to Cuenot (1895), Morse et al. (1970), Fisher (1972) and Burggren et al (1974). One of the outstanding points of the present structural study is the description of a complex vascularization system inside the Chinese crab gill platelet itself. The existence of such a vascularization has already been reported in several species of brachyurans but, up to now, these reports remained quite scanty (Taylor and Greenaway, 1979, for bibliography). The intralamellar network in E. sinensis seem to be very similar to what has been reported in Holthuisana transversa. Their functional significance still remains unclear. According to Taylor and Greenaway (1979), such vascularization could be involved in oxygen supply to the gill tissue when the crab lives out of the water. The origin of epi- and hypobranchial vessels as well as their physiological significance in E. sinensis have yet to be studied more extensively.
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WATER
595
References Berger, A. 1931. Uber die Anpassung eines Silsswasser und eines Brackwasserkrebses an Medien van verschiedenen Salzgehalt. Pfriigers Arch. ges. Physiol., 228, 790-807. Berridge, M. J. 1970. A structural analysis of intestinal absorption. In Insecf U~rrasrrucrure (ed. A. C. Neville), pp. 135-151. Blackwell, Oxford. Berridge, M. J. and Oschman, J. L. 1972. Transporting Epithelia. Academic Press. Bielawski, J. 1971. Ultrastructure and ion transport in gill epithelium of the crayfish, Asfacur leptodoctylus Esch. Protoplasma, 73, 177-190. Bliss, D. E. 1968. Transition from water to land in decapod Crustaceans. Am. Zool., 8, 355-392. Bubel, A. and Jones, M. B. 1974. Fine structure of the gills of Jaera nordmanni (Rathke) (Crustacea, Isopoda). J. mar. biol. Ass. U.K., 54, 737-743. Bulger, R. E. 1963. Fine structure of the rectal (salt-secreting) gland of the spiny dogfish, Squalus acanrhias. Anat. Rec., 147, 95-127. Bulger, R. E. 1965. Electron microscopy of the stratified epithelium living the excretory canal of the dogfish rectal gland. Anot. Rec., 151, 589-607. Burggren, W. W., McMahon, B. R. and Costerton, J. W. 1974. Branchial water- and blood-flow patterns and the structure of the gill of the crayfish Procambaw clarkii. Can. .I. Zool., 52, 1511-1518. Copeland, D. E. 1963. Granular cells, possibly osmoregulatory in the gill of Callinecfes. Biol. Bull., 125, 376. Copeland, D. E. 1964a. Salt absorbing cells in gills of Crabs Collinecfes and Carcinus. Biol. Bull., 127, 367-368. Copeland, D. E. 1964b. A mitochondrial pump in the cells of the anal papillae of the mosquito larvae. J. CeNBiol., 23, 253-263. Copeland, D. E. 1967. A study of salt secreting cells in the brine shrimp (Artemia saline). Protoplasma, 63, 363-384. Copeland, D. E. 1968. Fine structure of salt and water uptake in the landcrab Gecarcinus later&. Am. Zool., 8, 417-432. Copeland, D. E. and Fitzjarrel, A. 1968. The salt absorbing cells in the gills of the Blue Crab (Callinectes sapidus Rathbun) with notes on modified mitochondria. Z. Zellforsch. mikrosk. Anat., 92, l-22. Cuenot, L. 1895. Etudes physiologiques sur les Crustaces Decapodes. Arch. Biol., Paris, 13, 245-303. Dejours, P. 1975. Principles of Comparative Respiratory Physiologic. North-Holland/American Elsevier. Drach, P. 1930. Etude sur le systeme branchial des Crustaces Dtcapodes. Archs. Anat. microsc. Paris, 26, 83-133. Ellis, K. and Abel, J. 1965. Intracellular channels in the salt-secreting glands of marine Turtles. Science, 144, 1340-1342. Ewer, D. W. and Hattingh, I. 1952. Absorption of silver on the gills of a freshwater crab. Nature, Lond., 169, 460. Fisher, J. M. 1972. Fine-structural observations on the gill filaments of the fresh-water crayfish. Astacus pallipes Lereboullet. Tissue & Cell, 4, 287-299. Foster, C. A. and Howse, H. D. 1978. A morphological study on gills of the brown shrimp. Pamzeus aztecus. Tissue & Cell, 10, 77-92. Gilles, R. 1975. Mechanisms of ion and osmoregulation. In Marine Ecology (ed. D. Kinne), Vol. 2, pp. 259-347. Wiley-Interscience, London, New York. Gray, 1. E. 1957. A comparative study of the gill area of Crabs. BioL Bull., 112, 34-42. Gupta, B. L. and Berridge, M. J. 1966. A coat of repeating subunits on the cytoplamic surface of the plasma membrane in the rectal papillae of the blowfly, Cailiphora erythrocephala (Meig.) studied in situ by electron microscopy. .I. Cell Biol., 29, 376-382. Haase, W. 1975. Ultrastruktur and Funktion der Carapaxfelder van Argulus folicaceus (L.) (Crustacea, Branchiura). Z. Morph. Tiere, 81, 161-189. Hughes, G. M., Knights, B. and Schammel, C. A. 1%9. The distribution of PO2 and hydrostatic pressure changes within the branchial chambers in relation to gill ventilation of the shore crab. Carciws maenas L. J. exp. Biol., 51,203-220. Humbert, W. 1978. Cytochemistry and X-ray microprobe analysis of the midgut of Tomocerus minor Lubbock (Insecta, Collembola) with special reference to the physiological significance of the mineral concretions. Cell Tics. Res., 187, 397-416. Kimus, J. 1898. Recherches sur les branchies des Crustaces. La CelluIe, 15, 297-404. Koch, H. J. 1934. Essai d’interpretation de la soi-disant ‘reduction vitale’ de sels d’Argent par certains organes d’Arthropodes. AnnIs Sot. scientifique Brwelles, B, 54, 346-361. Koch, H. J. 1938. The absorption of chloride ions by the anal papillae of Diptera larvae. J. exp. Biol., 15, 152-160. Koch, H. J. 1954. Cholinesterase and active transport of sodium chloride through the isolated gills of the Crab Eriocheir sinensis (M. Edw.). In Recent Developments in Cell Physiology (ed. J. A. Kitching), pp. 15-27. Butterworth, London. Koch, H. J., Evans, J. and Schicks, E. 1954. The active absorption of ions by the isolated gills of the Crab Eriocheir sine&s. Meded. K. Vlaam, Acad. K. Wet., 16, 1-16. Komnick, H. 1962. Elektronenmikroskopische Lokalisation van Na+ and Cl- in den Zellen und Geweben. Protoplasma, 55,414-418.
39
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Komnick, H. 1963. Elcctroncnmikroskopische Untersuchungen zur funktionelle Morphologic des lonentransportes in der Salzdriise van Lam argentutus. I. Bau und Feinstruktur der Salzdriise. Protoplosmu, 56, 274-314. Komnick, H. and Bierther, M. 1969. Zur histochemischen lonenlokalisation mit Hilfe der Elektronen mikroskopic unter besonderer Beriicksichtigung der chloridreaktion. Histochimie. 18, 337-362. Komnick, H. and Wohlfarth-Bottermann. K. E. 1966. Zur Cytologic der rectaldriisen van Knorpelfishen I. Die Fcinstruktur der Tubulusepithelzellen. Z. ZeNfor&. mikrosk. Anat., 74, 123- 144. Krogh, A. 1939. Osmotic Regulation in Aquatic Animals, 242 pp. Cambridge University Press. Kummel, G. 1981. Fine structural indications of an osmoregulatory function of the ‘gills’ in tcrrcstrlal Isopodcs (Crustacea, Oniscoidea). Cell Tin. Res., 214, 663-666. Morse, H. C., Harris, P. J. and Dornfcld. E. J. 1970. Pacifastacus leniusculus: fine structure of arthrobranch with reference to active ion uptake. Trans. Am. Microsc. Sot.. 89, 12-27. Nakao, T. 1974. Electron microscopic study of the open circulatory system of the shrimp Caridina japonica. I. Gill capillaries. J. Morph., 144, 361-380. Oschman. J. L. and Wall, B. S. 1969. The structure of the rectal pads of Periplaneta americana L. with regard to fluid transport. J. Morph., 127, 475-509. Pequeux, A. and Gillcs. R. lY78a. Osmoregulation of the euryhaline Chinese crab Eriocheir sinensb. Ionic transport across isolated perfused gills as related to the salinity of the environment. In Physiology and Behaviour of Marrne Organisms (eds. D. S. McLusky and A. J. Berry), pp. 105-111. Pcrgamon Press, Oxford, New York. in isolated perfused gills of the Chincsc crab Eriochcir Pequeux, A. and Gil&, R. lY78b. Na+iNH,+ co-transport sinensis acclimated to fresh water. Experentia. 34, 1593-1594. Pequeux, A. and Gilles. R. 1981. Na+ fluxes across isolated perfused gills of the Chinese crab Eriocheirsrnensrs. J. exp. Biol.. 92, 173-186. Peters. N. and Panning. A. 1933. Die chineaiche Wollhandrkrabbe (Errocheir sinensrs. Milne-Edwards) in Deutschland. Zool. Am.. 104, l-177. Philpott. C. W. and Copeland, D. E. 1963. Fme structure of chloride cells from three specica of Fundu/us. J. Cc/l Rio/., 18, 389-404. Reynolds. E. S. 1963. The USC of lead citrate at high pH as an electron opaque stain in electron microscopy. J Cell Bio[., 17, 208-212. Schmitz. M. and Komnick. H. 1976. Rectale chloridepithelicn und osmoregulatonschc Salzaufnahme durch den Enddarm van Zygopteren und Anisopteren Libellenlarven. J. Inrecr. Physiol.. 22, 875-883. Schoffcniels, E. and Gilles, R. 1970. Osmoregulation in aquatic arthropods. In Chemical Zoology (cds M. Florkin and B. T. Schccr) Vol. 5A. pp. 255-286. Academic Press, New York. Starch. V. and Welsch. U. 197.5. Uber Bau und Funktion der Kiemcn und Lungcn van Ocypode cerutophthuhnu (Decapoda: Crustacea). Mar. Biol., 29, 363-371. Tandler, B. 1963. Ultrastructurc of the human submaxillary gland. II. The base of the striated duct cells. J. Ultrastruct. Res.. 9, 65-75. Taylor, H. H. and Greenaway, P. lY7Y. The structure of the gills and lungs of the arid-zone crab Holthuisam (Austrothelphusa) transverse (Brachyura: Sundathelphusidac) including observations on arterial vessels within the gills. J. Zool. Lond.. 189, 359-384. Thi@ry, J. P. 1967. Mise en Cvidence dcs polysaccharidcs sur coupes fines cn microscopic Clcctroniquc. .I. Microscopic, 6, 987-101X. ThiCry, J. P. 1969. Role dc I’appareil de Golgi dam la synthese des mucopolysaccharides. Etude cytochimique I. Mlsc en @vidcncc dc mucopolysaccharides dam les vCsicules de transition entre l’ergastoplasme ct I’appareil de Golgi J. Microscopic. 8, 689-708. Tisher, C. C., Bulger, R. E. and Trump, B. F. 1966. Human renal ultrastructure I. Proximal tubule of healthy individuals. Lab. /mat., 15, 1357-1394.
JEAN-A.
BARRA, ANDRt
PEQUEUX”
and WILLY HUMBERT
A MORPHOLOGICAL STUDY ON GILLS OF A CRAB ACCLIMATED TO FRESH WATER Key words: tacea.
Gills,
ultrastructure,
fresh water,
salt-transporting
epithelium,
En’ocheir,
Crus-
ABSTRACT. The gills of the fully euryhaline Chinese crab En’ocheir sinens& were studied by light and electron microscopy. In these Phyllobranchiates, the gills consist of a double row of lamellae extending laterally from a central shaft. Haemolymph flow pattern inside the gill is described and the existence of a complex secondary vascularization inside the platelets is reported. It is shown that important differences exist between the ultrastructure of the three anterior and the three posterior pairs of large gills. The epithelium of the posterior gills is much thicker and possesses an extensive elaboration of the plasma membranes in the form of infoldings, crypts and interdigitations, along which are packed numerous mitochondria. The presence of such a complex membrane system opening to the extracellular space and closely associated with mitochondria is common to all salt-transporting tissues. This study corroborates the idea that the posterior pairs of gills of Eriocheir sinemis are the only ones implicated in active Na+ uptake when the crab lives in dilute aquatic environment. The epithelium of anterior gills is much thinner and the cells poor in intracellular organelles. It seems to be involved essentially in respiration. Thus this work clearly corroborates the existence already suggested by physiological approach of a functional difference between the different pairs of E. sinensis branchiae with respect to their participation in the respiration and in the regulation of the blood ions content. Common to both types of gills is the presence of a lamellar septum separating the haemolymph space into two compartments. The part played by that structure in determining the pattern of haemolymph flow, together with periodic bridges forming pillars across the haemolymph space, is emphasized.
Callinectes:
Copeland and Fitzjarrel, 1968; Copeland, 1968; Jueru: Bubel and Jones, 1974), nothing is known on the fine structure of the gills of the Chinese crab. From the physiological point of view, it has been established that the salt loss occurring when in dilute media is counterbalanced by an active absorption of Na+ and Cl- which probably takes place in the gill tissue (for review, see Gilles, 1975; Pequeux and Gilles, 1981). The purpose of this work was therefore to understand the morphology of both the anterior and posterior gills of E. sinensis in order to try to explain the physiological differences reported in ion movements, cellular ionic content, transepithelial potential and enzyme activity. This paper deals with light and electron microscopy observations made on the gills of Chinese crabs acclimated to fresh water.
Introduction
Gecarcinus:
Like many other euryhaline crustaceans, the Chinese crab Eriocheir sinensis has a migratory life cycle with a growing period of long duration in fresh water and a shorter breeding stay in salt water. In the laboratory it withstands direct acclimation either to fresh water or to sea water without any apparent damage. Despite work on the normal histology and fine structure of gill tissues from various crustacean species (Astucus:Bielawski, 1971;
Laboratoire de Zoologie et d’Embryologie experimentale, 12 rue de I’Universite, F 67MM Strasbourg, France. * Laboratory of Animal Physiology, University of Litge, 4020 Liege, Belgium. Received 19 November 1982. Revised 27 April 1983. 583
584
BARRA.
Materials and Methods Animals Adult Chinese crabs E. sinensis were obtained from commercial fishermen in Bad-Zwischenahn, near Oldenburg, West Germany. Capture occurred in October when animals were still living in fresh pools, before their breeding migration towards salt water. In the laboratory they were kept at II-12°C in continuously renewed aerated tap water. Light microscopy Samples were fixed in aqueous Bouin fixative. Transverse sections (7 pm) were stained by classical staining methods (Trichrome of Prenant). Semithin sections (1-2 pm) were stained with toluidine blue. Transmission electron microscopy (TEM) Pieces of gills were fixed for 2 hr at room temperature in a solution of 4% glutaraldehyde buffered with 0.4 M cacodylate at pH 7.4. The final osmolarity of the fixative medium was 300 mOsmo1 kg-’ H20. A mixture of 4% glutaraldehyde and 2% osmium tetroxide buffered with 0.4 M cacodylate at pH 7.4 was also tested. The pieces were rinsed several times in 0.4 M cacodylate buffer with addition of 5% saccharose; they were then post-fixed for 1 hr at room temperature in 2% osmium tetroxide. Specimens were embedded in Araldite. Thin sections were stained with ethanolic uranyl-acetate and lead citrate (Reynolds, 1963) and examined with a Siemens Elmiskop 101 electron microscope. Silver nitrate treatment In a preliminary survey of the gill tissues, the patches of cells presumed to absorb salt were
PEQUEUX
AND
located by use of AgNOs following the method of Koch (1934, pers. comm.; see also Ewer and Hattingh, 1952). Living crabs first washed with distilled water were placed in a 0.05% solution of silver nitrate for 5 min (Ewer and Hattingh, 1952). After removal of the excess AgNOs by a second rinse in distilled water, the animals were killed and the branchiostegite was removed. Gills soaked in the fixative were then exposed to sunlight for 15 min until appearance and stabilization of a brown violet colour. Transverse sections of AgNOs stained gills were prepared for electron microscopy. X-ray microanalysis X-ray microanalyses of Ag+ and Cl- were performed by means of a microanalyser CAMECA MS46 operating under the following conditions: acceleration voltage, 15 kV; beam current, 50 nA; beam diameter, 1 pm, PET Crystal for Cl- and Ag+. Analyses were made on carbon-coated semithin sections placed on Mylar slides. In an attempt to corroborate the results obtained with the CAMECA MS46 analyser, ultrathin sections were also mounted on copper grids and analysed with CAMEBAX microanalyser under the following conditions: acceleration voltage, 50 kV; beam current. 100 nA. Silver lactate treatment Cl- ions were detected and located in ultrathin unstained sections prepared for TEM observation, after precipitation with silver lactate according to the method of Komnick (1962) and Komnick and Bierther (1969). 1% silver lactate (Fluka) was added to the fixative solution (2% 0~0~) in cacodylate buffer acidified with acetic acid
Table 1. Branchial formula of Eriocheir sinensis Maxillipeds
Cheliped
2
3
1
Pleurobranch Arthrobranch Podobranch 1 Epipodite 1 1 Total: 8 gills, 3 epipodites
2 1 1
_ 2 _
1
HUMBERT
Peraeopods 2
3
4
5 ~-
I -
1 _ -
_ -
_ _
CRAB GILLS ACCLIMATED
TO FRESH WATER
A ___
B ___ C _--
Fig. 1. Diagram of a cross-section of Chinese crab gill. The arrows indicate the main routes of the haemolymph flow within gill Iamellae. The left side of the diagram represents the platelet outer side covered with chitineous cuticle. The dotted curved lines represents the distribution of pilaster cells (PC). CA and DA respectively correspond to clear areas in the platelets near the efferent vessel (Ev) and dark areas located near the afferent vessel (Av). The right side of the diagram presents an internal view of the platelet showing the fenestrated lamellar septum (Ls) and arterioles related to the larger epibranchial and hypobranchial vessels (respectively Ep.v and Hy.v). The dotted lines A, B and C show the levels where cross-sections shown in Figs. 3-5 have been made. MC, marginal canal; Ne, nephrocytes; S, shaft; Lv, lamellar vessel.
Fig. 2. Schematic model of part of a branchial platelet in the so-called ‘dark area’, showing the fenestrated lamellar septum (Ls). Blood flow in the platelet is tentatively shown by solid arrows, with the larger arrow representing greater flow in the marginal canal (MC). C, cuticle; Be, branchial epithelium; Lv, lamellar vessel; PC, pillar cell.
585
586
BARRA.
(pH 6.4). During dehydration, 0.1 N NHOs was added to the 20% acetone solution in order to eliminate non-specific precipitation. Pieces were embedded in Araldite. Ultrathin sections were observed without staining. ‘Cl- ions’ (AgCI) appear as electron-dense precipitates. Polysaccharide staining The periodic acid-thiocarbohydrazide silver proteinate method (PATAG) for polysaccharides as described by Thiery (1967, 1969) has been applied to branchial lamellae. Thin sections were pre-treated for 30 min with 1% hydrogen peroxide in order to remove osmium from tissues and avoid non-specific deposits. Sections were then treated with the following reagents: (a) 1% periodic acid for 30 min at room temperature, (b) 0.2% thiocarbohydrazide (TCH) (Merck) reagent for 60 min, (c) 1% silver proteinate solution for 30 min. Controls were treated with silver proteinate omitting the periodic acid oxidation or TCH treatment. Results General organization Eriocheir sinensis has eight pairs of gills in the branchial chamber. The branchial formula is given in Table 1. The gills are phyllobranchiae, composed of a double row of closely spaced lamellae extending anteriorly and posteriorly from a median shaft.
Fig. 3-5. Cross-sections different levels as indicated
PEQUEUX
AND
HUMBERT
The cuticular surface of the gill lamellae shows two distinct areas characterized by different colour densities: a dorsal dark area and a ventral clear area (Fig. 1). The lamellar septum, essentially restricted to the dark area, exhibits a lot of apertures, allowing for complex movements of haemolymph between upper and lower compartments of the haemolymph space (Figs. 2-5). Histologically, the lamellae are composed of a single layer of cells lining the cuticle on each side of the leaflet. Both cell layers are connected periodically by pilaster cells which keep them separated and maintain a large central haemolymph space. Macroscopically. the insertion points of the pilaster cells in the cuticle can be seen by transparency ai the lamella surface where they appear as curved concentric lines (Fig. 1). An afferent and an efferent blood vessel line respectively the outer and the inner side of each gill. A lacunose median shaft built of support connective cells joins both blood vessels (Fig. 6). Facing the lamella septum, clusters of nephrocytes are located near the efferent vessel (Figs. 7, 8). Since the structure of small podobranches on maxillipeds 2 and 3 appears to be similar to the structure of the other gills. only the six pairs of larger gills have been studied in detail. Main circulatory routes The main circulatory
routes
of the same scrics of three lateral lamcllac in Fig. 1 by the dotted lines A. B and C.
Fig. 3. Semithin cross-section through the dark area. showing lamellar septum and the marginal vessel (MC). x 170.
of
pcrformcd
the connection
the
at three
hetwccn
the
Fig. 4. Cross-section through both the dark and clear arcas, showing the lamcllar septum to he essentially restricted to the dark area. Note the thinness of the median shaft (S). x 170. Fig, 5. Cross-section through the clear area bordering absence of lamcllar septum. x 170.
on
the efferent
blood vessel. Note the
Fig. 6. Cross-section of a paraffin embedded gill, showing the affcrcnt blood vessel (Ev). A small epibranchial vessel (Ep.v) can he seen inside the median shaft (S). Fig. 7. Cross-section of a paraffin embedded gill, showmg the effercnt blood vc~~el (Eb). A smaller hypobranchial vessel (Hy.v) can he seen among the abundant nephrocytes (NC). X220.
--
BARRA,
PEQUEUX
AND HUMBERT
Fig. 8. Longitudinal semithin section through the efferent blood vessel showmg lateral lamellae. At their proximal joining parts, nephrocytesclusters (Ne) hang down in the efferent vessel. Pillar cells (PC) of lateral lam&e can distinctly be seen. x140. Fig. 9. Cross-section through a lamellar vessel (Lv) located near the median septum. x 16,Mx).
Fig. 10. Cross-sectionof the branchml ‘respiratory’ epithelium. Cuticular surface at top, thick basal lamina (Bl) lining the haemolymph lacuna (L) below. Arrows indicate deep interdigitations of latero-basal lamina (see text tar other pomts). m. mltochondna; N, nucleus; C. cuticle. X34,CKl Fig. 11. Cross-section of the branchial ‘salt-transporting’ epithelium. Apical membranes exhibit extensive folds(F). Basal and latero-basal membranes show deep, complex and regular interdigitations (I) coming into close contact with mitocbondria. Note the abundance of mitochondria (m). See text for other points. C, cuticle: N, nucleus. x34,wO. Fig. 12. Unstained cross-sectionthrough the dark area of a gill lamella treated to reveal polysaccharides (PATAG method). Glycogen (g) appears as very abundant in the median lamellar septum. Ep, gill epithelium. xfLO0
BARRA,
haemolymph in the crab gills lamella are speculatively represented in the schematic diagram of Fig. 2. Haemolymph reaches the gill by the outer afferent vessel which communicates laterally with the marginal vessel of each gill lamella by means of oblong slits. Blood thus enters the haemolymph lacuna from the outer side of the platelet towards the inner part. The marginal canal which is lined by a thin epithelium, appears as a rapid circulatory route where the haemolymph does not meet any constraint. On the contrary, inside the lamella, blood flow is impeded by pilaster cells, by the lamellar septum and by a thicker epithelium. Blood reaches the efferent vessel either directly from the marginal vessel, or by percolating through the central part of the lamella. The efferent vessel wall is much more complex than that of the afferent vessel, its outer face being lined by a thin chitinous cuticle, while its inner side is limited by the nephrocytes. Its lateral walls correspond to the pilaster cells of the clear area. The positioning of these pillars
PEQUEUX
AND
HUMBERT
undoubtedly determines the pattern of blood flow from the marginal vessel and the central haemolymph lacuna towards the efferent vessel (Fig. 7). The smaller vessels, called epibranchial and hypobranchial vessels, are located in the median shaft near the afferent and the efferent vessels respectively (Figs. 1, 6, 7). They will henceforth be identified by their topographical position in the gill. In phasecontrast microscopy the lamella of the gills shows a complex dichotomic network of thin. so-called lamellar vessels (Fig. 1) which are related to the epibranchial and hypobranchial vessels, as clearly shown by serial cross-sections. Each lamellar vessel wall is constituted of two endothelial ceils closely bonded to each other by means of deep interdigitations (Fig. 9). Facing the vessel lumen, the intima is coated with a thick glycocalyx. Generally the inner folds completely obliterate the central lumen although, the vessel diameter can be modified to allow haemocytes passage. The lamellar vessels very probably open on the
Fig. 13. Single lamclla isolated from an anterior located gill after in viva trcatmcnt with silver nitrate (see Materials and Methods). Only an extremely weak coloration can hc seen at the lamella surface. Fig. 14. Single lamella isolated from a posterior located gill after in viva treatment with silver nitrate. Note the very dense coloration essentially restricted to the ‘dark area’ (DA), and contrasting with the weak coloration of the ‘clear area’ (CA). Fig. 15. Unstained cross-section through the thin ‘respiratory’ epithelium of an an&&r gill treated in viva with silver nitrate (same gill as shown in Fig. 13). Dense clusters of silver chloride granules appear asregularly distributed in, and essentially restricted to, the cuticle. x26,OGil. Fig. 16. Unstained cross-section through the thick ‘salt transporting’ epithelium (dark area) of a posterior gill treated in viva with silver nitrate (same gill as shown in Fig. 14). Silver chloride granules appear as uniformly distributed in, and restricted to. the cuticle, and the extracellular compartment between the cuticle and the apical side of the epithelial cells. ~26,000. Fig. 17. Unstained cross-section through the ‘respiratory’ epithelium of an anren’or gill, after treatment in vitro with silver lactate (specific ‘staining’ method for Cl- as described in Materials and Methods). The clusters of electron-dense granules have been identified as containing Cl and Ag by X-ray microanalysis (see inset record). x 18,000. Fig. 18. Unstained cross-section through the ‘salt-transporting’ cpithelium of a posrerior gill, after treatment in virro with silver lactate. A large amount of AgCl containing granules appears as uniformly distributed in, and restricted to, the cuticle. and the extracellular space between the cuticle and the apical infoldings of the epithelial cells. x 15,600.
BARRA,
lamellar haemolymph lacuna but, up to now, this has not been observed on electron micrographs. Anterior gills
The three anterior pairs correspond to gills 1, 2, 3. During intermolt, the cuticle is from 0.7 to 1.0 pm thick in the central part of the lamella and about 2 pm thick near its insertion point on the median shaft. It is thicker (3-6 pm) in the wall of the marginal vessel. From the outermost layer inward, the cuticle consists of an epicuticle and an uncalcified endocuticle (Fig. 10). The epithelium of anterior gills is about 2-4 pm thick. At the base of the lamellae and at their apical side, however, epithelium thickness may reach up to 10 pm. Nuclei generally protrude within the haemolymph space. The cells’ apical side just beneath the cuticle is very lightly folded. The plasma membrane of the folds appears a little denser. The extracellular compartment under the cuticle is extremely reduced. The amount of intracellular organelles seems to be very limited. The hyaloplasma is well populated with mitochondria and glycogen granules, but, whereas glycogen granules are uniformly distributed in the cells, mitochondria are essentially restricted to the apical area. All the other organelles are poorly represented and the plasma membrane does not exhibit extensive basal infoldings. Periodically, three to four cells are associated to form a pillar. Posterior gills
The three posterior gills correspond to gills 4, 5, 6. Serial cross-sections through a posterior gill show that (a) the thickness of the gill epithelium decreases from the dark towards the clear area. (b) the lamellae are much thicker in the dark area, and (c) the median shaft is thinner in the central part of the gill (Figs. 3-5). The cuticle structure is quite similar to that described prevously. However, it is worth noticing that, in the dark area, its thickness can be as little as 0.3 pm. From the shaft towards the distal area of the lamella, the epithelium thickness decreases, reaching about 1 pm near the marginal vessel, while the epithelial cells in the dark area near the shaft are larger, up to 10 pm and more. Such a size difference has to be related to a sudden change in the structure
PEQUEUX
AND HUMBERT
of the cells in connection with the distribution of the clear and the dark areas. The large epithelial cells near the median shaft are characterized by a complex and well-developed network of large apical folds. These membrane folds may contain cytoplasm and produce a large extracellular compartment under the cuticle (Fig. 11). Basal folds generally come into close contact with mitochondria membranes. Mitochondria appear as the most abundant organelles in these epithelial cells of posterior gills. They may completely fill the cytoplasmic space. As reported above, structural changes occur from the shaft area towards the lamella apex: (a) the amount of mitochondria considerably decreases in the centre of the cells, (b) apical and latero-basal folds become shorter and shorter (the cell still may be considered however as typical ‘salttransporting cells’). These transformations are very progressive from the proximal to the distal part of the lamella where the epithelium appears to be very similar to the ‘respiratory epithelium’ of the anterior gills. In the clear area where the lamella septum is lacking it is 2 pm thick or less. The lamellar septum is a stratified fenestrated sheet which is 9-15 pm thick and is surrounded by a basal lamina. The septum is supported by pilaster cells. A large amount of glycogen has been identified histochemitally by the Patag reaction (Thiery, 1967, 1969) (Fig. 12). The ergastoplasm appears as long flattened and sinuous saccules. Mitochondria are very abundant and the Golgi apparatus reduced to three to four saccules. Neighbouring cells are bound by desmosomes. The early in vivo experiments of Koch (1934-54) have been repeated and the selective staining of posterior gills of the Chinese crab E. sinensis has been confirmed (Figs. 13, 14). Gills of the crabs treated by AgN03 were then fixed and prepared for electron microscopy observation. In vitro experiments with silver lactate according to the method of Komnick (1962, 1963) have also been performed and give similar results. Following in vivo treatment by AgNOs, the lamellae of anterior gills exhibit a uniform pale brown colour. It is worth noticing that the coloration is extremely light and not always evident macroscopically. The reaction remains negative in the clear area of the
CRAB GILLS ACCLIMATED
TO FRESH WATER
gills (Fig. 13). Photomicrographs (Fig. 15) show that light coloration is due to clusters of dense granules (AgCl precipitate) regularly distributed in and mainly restricted to the cuticle. No significant precipitate has been observed in the respiratory epithelium or in extracellular lacunae. Quite similar observations were made after in viva treatment with silver lactate (Fig. 17). At variance with these observations, the lamellae of posterior gills strongly react to AgNOa exposure. After treatment, the area adjoining the afferent vessel exhibits a strong brown colour while the clear area near the afferent vessel remains uncoloured (Fig. 14). AgCl precipitates are uniformly distributed in the cuticle and in the compartment under the cuticle near the apical folds of the salt-transporting cells (Fig. 16). Similar results were obtained with silver lactate. Granules were moreover observed in the epithelium itself (Fig. 18). As illustrated by the peak recordings of Fig. 17, electron probe X-ray microanalysis of semithin and ultrathin sections confirm the AgCl nature of electron-dense granules. Discussion Since the early works of Koch et al. (1954) on the Chinese crab Eriocherir sinensis, conclusive proof exists that the gills of euryhaline crabs absorb salts actively from dilute media. More recently Na+ transport processes at work in the branchial epithelium of the Chinese crab have been investigated more extensively using perfused preparations of iolated gills (Gilles, 1975; Pequeux and Gilles, 1978a, b, 1981). Among other things, these works clearly establish the existence of functional differences between the three anterior and the three posterior pairs of branchiae with respect to their participation in the blood osmoregulation that this species is able to achieve when in dilute media. Up to now, the structure of the Chinese crab gill has not however received much attention. The Chinese crab Eriocheir sinensis is a typical hyperosmoregulator which can maintain a pronounced blood hyperosmotic state when in diluted medium, and whose blood is isosmotic to the external medium when the animal is acclimated to sea water (Berger, 1931; Krogh, 1939; Schoffeniels and Gilles, 1970). In hyperosmoregulators, active Na+
uptake at the gill level is an essential mechanism in counterbalancing the salt loss in dilute media. In E. sinensis, it has been established that the active Na+ influx is mostly achieved by the three posterior pairs of gills (Pequeux and Gilles, 1981). The results of the present structural study are in complete agreement with that finding. A so-called ‘salt-transporting epithelium’ is reported to be localized and essentially restricted to the same three posterior pairs of gills. At variance with most marine or fresh water brachyuran decapods, which have nine pairs of gills, Eriocheir sinensk has only eight pairs (Peters and Panning, 1933). Several authors have therefore considered that the loss of one pair of branchiae by E. sinensis could perhaps be related to the well-known gill reduction trend reported in terrestrial or semi-terrestrial crab species (Gray, 1957; Bliss, 1968; Taylor and Greenaway, 1979). The way in which water flows over the gills, and haemolymph flows in vessels and lacunae is characteristic of the well-known counter-current system (Hughes et al., 1969; Nakao, 1974; see Dejours, 1975, for review) but differs from the more complex bidirectional system of crustacea having trichobranchiae (Fisher, 1972; Burggren et al., 1974). The epithelium of the anterior gills is much thinner (2-4 pm) and covered by a 1 pm thick cuticle supposed to be permeable to gas. The structure of the Chinese crab cuticle is very similar to the gill cuticle of other crustacean species (Copeland, 1967, 1968; Copeland and Fitzjarrel, 1968; Bielawski, 1971; Fisher, 1972; Starch and Welsch, 1975; Foster and Howse, 1978; Taylor and Greenaway, 1979; Kiimmel, 1981). In these epithelia the distance between the haemolymph and the external medium is only about 3-5 pm, more often less than 2 pm, and such a thinness of the epithelium doubtless favours gas passage from the outside towards the haemolymph. Therefore, it seems quite reasonable to consider that the main and essential function of that tissue must be respiration. The epithelium of the posterior gills is much thicker, up to 10 pm, while its cuticle tends to be thinner (O-3 pm). It is characterized by infoldings of the plasma membrane along which are packed numerous mitochon-
BARRA,
dria. Such extensive elaboration of the plasma membranes in the form of tubules, crypts, infoldings and interdigitations opening to the extracellular space and in close association with mitochondria seems to be common to all the ‘salt-transporting tissues’ (Berridge and Oschman, 1972): in gills of other crustacean species (Copeland, 1963, 1964a; Copeland and Fitzjarrel, 1968; Foster and Howse, 1978), in the ‘salt gland’ of marine turtles (Ellis and Abel, 1965) and birds (Komnick, 1963), and in the anal papillae of insect larvae (Copeland, 1964b; Koch, 1938). Apparently, cells of the gill filaments of fish (Philpott and Copeland, 1963), of the elasmobranch rectal gland (Bulger, 1963a, 1965; Komnick and Wohlfarth-Bottermann, 1966), of the insect midgut (Berridge, 1970), or of the insect rectum (Gupta and Berridge, 1966; Oschman and Wall, 1969) also reveal a quite similar organization. Large interdigitating plasma membrane folds, highly populated with mitochondria, are also present in the striated duct cells of mammalian salivary glands (Tandler, 1963) as well as in epithelium of mammalian kidney tubules (Tisher et al., 1966). In light of this the results of the present study are in complete agreement with physiological data, since the posterior gills are the only ones implicated in active Na’ uptake (Pequeux and Gilles, 1981). As early as 1934, Koch, studying the staining of gills of different crustaceans with silver salts, suggested that those showing fixation of silver nitrate were involved in active Na+ uptake. This method and hypothesis were later used on several species to localize patches of cells presumed to absorb salt: Artemia (Copeland, 1967), Callinectes (Copeland and Fitzjarrel, 1968), Potamon (Ewer and Hattingh, 1952), Pacifastacus (Morse et al., 1970). To the best of our knowledge, these experiments were never followed by an ultrastructural study of the tissues treated. It is shown here that AgCl precipitates, as identified by microanalysis, are localized in the endocuticle of both gill types, either as isolated clusters in anterior gills or as a continuous layer in posterior gills. Treatment with silver lactate gives identical results, which are moreover in agreement with results obtained with other
PEQUEUX
AND HUMBERT
Arthropod species (Haase, 1975. for bibliography; Schmitz and Komnick, 1976; Humbert, 1978). Up to now the exact significance of these results remains unclear. Precipitates obviously do not allow the localization of Cl- ions in the living tissue, but more likely have to be considered as an indication of the gill area presumed to be more permeable to ions. As early as 1898, Kimus already reported the existence of an intralamellar septum so-called ‘tissu intermediaire ’ in Azellus, Cirolona, Idothea, Anilocra and Cymothoa which is described as variable in form and size. In Eriocheir, it has been shown to induce a ‘dark area’ in the gill tissue. The lamellar septum must be similar to what has been described in Cardina as ‘trabecular cells’ by Nakao (1974). and in several species of decapods as ‘reseau’ by Drach (1930). If the septum seems thus to be of rather general occurrence in crustacea, its function still remains unclear. In the light of our results we believe that, besides the fact that it divides the haemolymph into compartments, and must undoubtedly determine the pattern of haemolymph flow through the platelet, the septum appears as a storage tissue able to accumulate a large amount of glycogen. For more details on the structure of the various types of septa encountered in other species of crustacea, the reader is referred to Cuenot (1895), Morse et al. (1970), Fisher (1972) and Burggren et al (1974). One of the outstanding points of the present structural study is the description of a complex vascularization system inside the Chinese crab gill platelet itself. The existence of such a vascularization has already been reported in several species of brachyurans but, up to now, these reports remained quite scanty (Taylor and Greenaway, 1979, for bibliography). The intralamellar network in E. sinensis seem to be very similar to what has been reported in Holthuisana transversa. Their functional significance still remains unclear. According to Taylor and Greenaway (1979), such vascularization could be involved in oxygen supply to the gill tissue when the crab lives out of the water. The origin of epi- and hypobranchial vessels as well as their physiological significance in E. sinensis have yet to be studied more extensively.
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ACCLIMATED
TO FRESH
WATER
595
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