Immunolocalization of NA +,K + -ATPase in the Branchial Cavity During the Early Development of the European Lobster Homarus Gammarus (Crustacea,Decapoda)

Abstract

We examined the ontogeny of the osmoregulatory sites of the branchial cavity in embryonic and early postembryonic stages of the European lobster Homarus gammarus through transmission electron microscopy, immunofluorescence microscopy, and immunogold electron microscopy using a monoclonal antibody IgGα₅ raised against the avian α-subunit of the Na⁺,K⁺-ATPase. In mid-late embryos, Na⁺,K⁺-ATPase was located along the pleurites and within the epipodite buds. In late embryos just before hatching, the enzyme was confined to the epipodite epithelia. After hatching, slight differentiations of ionocytes occured in the epipodites of larval stages. Na⁺,K⁺-ATPase was also located in the ionocytes of the epipodites of larvae exposed to seawater (35.0‰) and to dilute seawater (22.1 ‰). After metamorphosis, the inner-side branchiostegite epithelium appeared as an additional site of enzyme location in postlarvae held in dilute seawater. Within the ionocytes, Na⁺,K⁺-ATP-ase was mostly located along the basolateral infoldings. These observations are discussed in relation to the physiological shift from osmoconforming larvae to slightly hyper-regulating (in dilute seawater) postmetamorphic stages. The acquisition of the ability to hyper-osmo-regulate probably originates from the differentiation, on the epipodites and mainly along the branchiostegites, of ionocytes that are the site of ion pumping as evidenced by the location of Na⁺,K⁺-ATPase. (J Histochem Cytochem 49:1013–1023, 2001)

Keywords

osmoregulation ionocytes ontogeny metamorphosis crustacean lobster

Despite the wealth of information available concerning crustacean osmoregulation in adult stages (reviewed in Mantel and Farmer 1983; Péqueux 1995), detailed data related to changes in osmoregulation throughout the postembryonic development are still scanty (reviewed in Charmantier 1998). However, ontogeny of osmoregulation has been clearly demonstrated as a major adaptive process to environmental salinity, because the establishment of a species in a given habitat depends on the ability of each of its developing stages to adapt to the environment. Three alternative ontogenetic patterns have been described: (a) osmoregulation is weak and varies only little with the progress of the developmental stages; (b) the adult type of efficient osmoregulation is established in the first postembryonic stage; (c) whereas larval stages are osmoconformers, later developmental stages, usually after metamorphosis, have an efficient osmoregulatory ability (Charmantier 1998).

In homarid lobsters, the third ontogenetic pattern of osmoregulation prevails. Embryos do not osmoregulate (Charmantier and Aiken 1987), planktonic larval stages are osmoconformers, and benthic postmetamorphic stages are hyper-isoregulators (Charmantier et al. 1988a, b; Thuet et al. 1988), as are the adults (Dall 1970; Charmantier et al. 1984a). Metamorphosis also marks the setup of neuroendocrine control of hydromineral metabolism in Homarus americanus (Charmantier et al. 1984b; Charmantier–Daures et al. 1994). Similarly, in H. gammarus, the adult type of osmoregulation acquired at the metamorphic molt is correlated with a change in sodium regulation at low salinity (from isoionic to hyperionic regulation), an increase in osmoregulatory capacity, and increased activity of the sodium-potassium adenosinetriphosphatase (Na⁺,K⁺-ATPase) and of the carbonic anhydrase activity (Thuet et al. 1988).

Na⁺,K⁺-ATPase, a heterodimeric integral membrane protein composed of a catalytic α-subunit (M_r ≈ 100 kD) and a smaller glycosylated β-subunit (M_r ≈ 40–60 kD), enables ion transport, either directly through movements of ions across epithelial membranes (e.g., by pumping Na⁺ from the surrounding medium for hyper-regulation at low salinity) or indirectly through the generation of ionic electrochemical gradients (reviewed in Mantel and Farmer 1983; Siebers et al. 1985; Péqueux and Gilles 1988; Lucu 1990; Taylor and Taylor 1992; Towle 1993; Péqueux 1995). This enzyme is therefore of special interest for the study of the ontogeny of osmoregulation. In particular, its precise cellular localization can further ascertain functional and morphological identification of ion transport.

High levels of Na⁺,K⁺-ATPase activity have been recorded in branchial and extrabranchial osmoregulatory tissues of many decapods, with increased activity after transfer from seawater to dilute seawater (Towle et al. 1976; Siebers et al. 1982; Péqueux and Gilles 1988; Thuet et al. 1988; Bouaricha et al. 1991; Dickson et al. 1991; Piller et al. 1995; Lima et al. 1997). Na⁺,K⁺-ATPase has been ultracytochemically localized through the use of ouabain-sensitive p-nitrophe-nylphosphatase (p-NPPase) in the basolateral membranes of gill lamellae of strongly osmoregulating crabs such as Callinectes sapidus and Carcinus maenas (Towle and Kays 1986). Similar observations have also been reported in the gills of the crab Eriocheir sinensis (Péqueux et al. cited in Taylor and Taylor 1992) and in the infolding membranes of the hindgut epithelium of the terrestrial isopod Armadillo officinalis (Warburg and Rosenberg 1989). Na⁺,K⁺-ATPase has also been localized through immunostaining techniques in the basolateral membranes of the epidermal cells and salt gland organ of developing brine shrimp Artemia sp. (Sun et al. 1991) and in the posterior and anterior calcium-transporting sternal epithelium of the terrestrial isopod Porcellio scaber (Ziegler 1997). We also recently localized Na⁺,K⁺-ATPase by immunostaining in the basolateral membranes of epipodite and inner-side branchiostegite epithelia of 1-year-old juvenile European lobster H. gammarus (Lignot et al. 1999). In this species, a lack of specific reactivity has been reported in the branchial filaments. This is in agreement with previous ultrastructural descriptions of the osmoregulatory epithelia located in the branchial cavity of the European lobster (Haond et al. 1998) and suggests the involvement of extrabranchial organs of H. gammarus in hyper-osmoregulation at low salinity.

The aim of the present study was therefore to examine the localization of Na⁺,K⁺-ATPase during the embryonic and postembryonic development of H. gammarus (L., 1758) after exposure to seawater and dilute seawater. This study is aimed at further ascertaining the ontogenetic involvement in osmoregulation of ion-transporting epithelia within the branchial cavities. They were therefore studied in embryonic, larval, and postlarval lobsters by scanning and transmission electron microscopy, immunofluorescence light microscopy, and immunogold electron microscopy using a monoclonal antibody IgGα₅ raised against the avian α-subunit of the Na⁺,K⁺-ATPase.

Materials and Methods

Animals

Adult berried females of H. gammarus caught off the coast of Brittany and obtained from a shellfish retailer (La Pêcherie; Montpellier, France) were maintained at the Montpellier laboratory in individual compartments containing aerated and recirculated (Eheim systems) natural seawater (35.0 ± 1.3‰, 20 ± 1C) and were fed three times a week with mussels. A 12-hr light:12-hr dark photoperiod was maintained. The rate of embryonic development was monitored by measuring the size of the eyes and calculating the eye index (EI) according to the method of Perkins (1972) adapted to the European species of lobster (Helluy and Beltz 1991; Charmantier and Mounet-Guillaume 1992). After hatching, larval Stages I–III were maintained in planktonkreisels (Hughes et al. 1974) containing 40 liters of aerated and recirculated natural seawater. They were fed three times a day with frozen Artemia sp. To avoid increased cannibalism occurring after metamorphosis, late larval Stages III were transferred to individual compartments containing aerated and recirculated natural seawater. Larval and postlarval (IV) stages were held either in seawater (SW: 1030 mOsm.kg^–1, ≈ 35.0‰) or acclimated to dilute seawater (DSW: 650 mOsm.kg^–1, = 22.1‰) over a 24-hr period by progressive addition of dechlorinated tapwater. Before sampling, DSW-acclimated individuals were held in that medium for another 24-hr period. Larvae and postlarvae were sampled in the middle of each developmental stage, thus corresponding to intermolt Stage C (Drach 1939). Other H. gammarus females caught off Helgoland Island, North Sea, Germany, were used to provide mid-late and late embryos (EI = 520 and 607 μm).

Scanning Electron Microscopy

Dehydrated larvae and postlarvae from SW were critical point-dried with liquid CO₂ in a Baltec CPD 030 critical-point dryer. After being mounted on stubs, the samples were coated with silver in a Baltec SOD 050 ion coater and examined with a Jeol JSM-6300f scanning electron microscope operated at 15 kV.

Transmission Electron Microscopy

Larvae and postlarvae from SW and DSW were fixed on ice for 1–2 hr with 2.5% glutaraldehyde in 0.1 M Na-cacodylate buffer, pH 7.5, fitted to 1030 mOsm.kg^–1 (animals held in SW) and 650 mOsm.kg^–1 (DSW-acclimated animals) by NaCl to avoid osmotic shocks. After rinsing in diluted sodium cacodylate buffer (pH 7.2), samples were fully dehydrated in a graded ethanol series and infiltrated with LR White, which was polymerized for 24 hr at 60C. Ultrathin sections were cut on a Reichert TM60 ultramicrotome and collected on Formvar-coated nickel grids. Sections were stained with uranyl acetate (30 min) and lead citrate (3 min) and observed with a JEOL 1200 EX2 transmission electron microscope.

Na⁺,K⁺-ATPase Antibody and Western Blotting

Mouse monoclonal antibody IgGα₅ was raised against the α-subunit of the chicken Na⁺,K⁺-ATPase (Takeyasu et al. 1988). This antibody, kindly provided by D. M. Fambrough (Baltimore, MD), specifically reacts with its counterpart in different crustacean species (Porcellio scaber, Ziegler 1997; Carcinus maenas, Lucu and Flik 1999; H. gammarus, Lignot et al. 1999). Epipodites of adults maintained in DSW were quickly removed and homogenized in ice-cold sample buffer (2 mM DTT, 0.5% bromophenol blue, 30% glycerol, 20 mM Tris-HCl, pH 6.8) to cleave disulfide bonds, boiled for 2 min, sonicated, and centrifuged at 16,000 × g for 5 min. This treatment was critical to extract a single immunoreactive species from the membrane preparation. The supernatant was then subjected to SDS-PAGE on 5% gels, and the proteins electrotransferred onto nitrocellulose sheets. After blocking of the membranes for 30 min with TBST buffer (20 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 0.05% Tween-20) containing 1% skim milk (to block nonspecific binding sites), the proteins were incubated for 1 hr in primary antibody diluted at 1 μg/ml in TBST and reacted for 1 hr with the horseradish peroxidase labeled anti-mouse secondary antibody (Jackson Immunoresearch; Avondale, PA) diluted 1:3000 in TBST. Bound antibodies were visualized by incubating the blots in an ECL chemiluminescent detection system (Amersham; Poole, UK) for 1 min, followed by exposure to autoradiographic film for 5–20 sec.

Immunofluorescence Light Microscopy

Embryos, larvae, and postlarvae cephalothorax were fixed for 24 hr in Bouin's fixative. Specimens were fully dehydrated and embedded in paraffin. Sagittal sections (3 μm) were cut on a Leitz Wetzlar microtome and collected on poly-l-lysine-coated slides. The technique for the immunocytochemical demonstration of Na⁺,K⁺-ATPase in epithelial cells was similar to that of Lignot et al. (1999). Sections were preincubated for 10 min in 0.01 mM Tween-20, 150 mM NaCl in 10mM phosphate buffer, pH 7.3, and then treated with 50 mM NH₄Cl in PBS, pH 7.3, for 5 min. The sections were washed in PBS and incubated for 10 min with a blocking solution (BS) containing 1% bovine serum albumin (BSA) and 0.1% gelatin in PBS. Droplets (10 μl) of primary antibody diluted in BS to 20 μg.ml^–1 were placed on the sections and incubated for 2 hr at room temperature in a wet chamber. After being washed in BS (six times for 5 min), the sections were incubated for 2 hr in droplets of secondary antibody [FITC-conjugated goat anti-mouse IgGH&L (Jackson Immunoresearch)]. After extensive washes in BS (three times for 5 min) and in PBS (three times for 5 min), sections were mounted with anti-bleaching mounting medium (Sigma; Poole, UK). Sections were then examined with a fluorescent microscope (Leitz Diaplan coupled to a Ploemopak 1-Lambda lamp) equipped with the appropriate filter set (450–490-nm bandpass excitation filter) and a phase-contrast device. The procedure was similar for the control sections, which were incubated without the primary antibody.

Immunogold Electron Microscopy and Quantitative Analysis of Gold Particles

A postembedding immunostaining technique on LR White sections was applied, as previously described (Lignot et al. 1999). Larval and postlarval stages acclimated to DSW were fixed for 1–2 hr with 0.5% glutaraldehyde in 0.1 M Nacacodylate buffer, pH 7.5, adjusted to DSW osmolality, fully dehydrated, infiltrated with LR White, and polymerized for 18 hr at 50C. Ultrathin sections were cut on a Reichert TM60 ultramicrotome and collected on Formvar-coated nickel grids. Selected grids were placed on 5% gelatin containing 50 mM glycine. The grids were successively preincubated on droplets of 50 mM glycine in PBS (once for 5 min) and 1% BSA-PBS (3 × PBS). The grids were then transferred to droplets of IgGα₅ antibody diluted to 20 μg.ml^–1 in BSA-PBS and incubated for 2 hr at room temperature in a wet chamber. The sections were washed in BSA-PBS (six times for 5 min) and incubated for 1 hr in droplets of 10-nm gold-conjugated goat anti-mouse IgG (Jackson Immunoresearch). After washing in BSA-PBS (three times for 5 min) and PBS (three times for 5 min), sections were stained with uranyl acetate (30 min) and lead citrate (3 min) and examined with a JEOL 1200 EX2 electron microscope. For controls, the procedure was similar but the grids were incubated without the primary antibody.

For the quantitative immunocytochemical study of the distribution of Na⁺,K⁺-ATPase, all the reactions were carried out close to the saturation level of the antibody (20 μg.ml^–1), as previously determined by Lignot et al. (1999). The relative labeling density D was calculated by dividing the number of particles by the length of infolding membrane analyzed. A 1-4-μm length of the considered infolding domain was analyzed.

Statistical Analysis

Data are presented as means ± SE. Statistical comparisons of experimental data were performed by one-way and two-way analyses of variance (ANOVA) and Fisher's multiple-range least significant difference (LSD) post-hoc test by using the software Statview 4.02 (Abacus Concepts; Tucson, AZ) (Sokalf and Rohlf 1981). The level of statistical significance was set at p < 0.05.

Results

General Morphology and Ultrastructure

Lobster branchial cavities, already well formed after hatching, are located on both sides of the cephalothorax and are sheltered by the branchiostegites (taken off in Figure 1 presenting the branchial chamber in Stage IV). Each branchial cavity contains 20 tricho-branchiate gills and seven epipodites. These organs have already been described in the adult stage (Haond et al. 1998). Briefly, each gill (podobranch or arthrobranch) connected to the pleurite possesses short filaments attached to a central axis. Epipodites are lamellar organs associated with the podobranchs (except on the first maxilliped) and are well formed in larval and postlarval stages (Figure 1). These epipodites are lined on both sides by an epithelium made of columnar cells and thickening throughout the larval development (5– 12 μm) (Figures 2A-2C). The branchiostegites possess an outer limiting epithelium secreting a thick and strongly calcified outer cuticle, a connective tissue, and an inner-side epithelium bordered by a basal lamina on the connective tissue side and a thin cuticle on the side limiting the branchial cavity (Figures 2D and 2E). Depending on the organ (epipodite or branchiostegite), tissue differentiation occurs at different times in development. In larval and postlarval stages, the epipodite epithelia present cells with many mitochondria associated with a basolateral infolding system lined by a basal lamina (Figure 2A). From Stage I to Stage IV, these epipodite ionocytes differentiate progressively. They become thicker (from 2–5 to 8–12 μm) and their infolding system depth increases (from 2 to 8 μm) (Figures 2A-2C). Within the cells of the branchiostegite inner-side epithelium, no ionocyte-like differentiation has been observed in early larvae (Figure 2D); these cells, 1–4 μm thick, contain a few mitochondria but no infoldings. In Stage III, the infolding system is present in some cells of the inner-side epithelium (Figure 2E). In postlarval Stage IV, however, typical ion-transporting features are present along the inner-side epithelium of the branchiostegites (Figure 2F). These ionocytes, 2–5 μm thick, host many mitochondria associated with a well-developed basolateral infolding system lined by a basal lamina (Figure 2F).

Western Blotting of Na⁺,K⁺-ATPase

Immunological specificity of the purified antibody against the α-subunit of the chicken Na⁺,K⁺-ATPase was tested by immunoblotting analysis of the solubilized epipodite and branchiostegite epithelia. The IgGα₅ antibody bound specifically to one band (Figure 3). The apparent molecular weight of this polypeptide in the epipodites was approximately 90 kD. We then used this antibody in all immunocytochemical experiments.

Immunofluorescence Light Microscopy

Bouin fixation and paraffin-embedding procedures yielded good antigenicity as observed with the fluorescent micrographs (Figures 4A, 4C, 4E, 5A, 5C, 5E, and 5G) and good structural preservation as observed with phase-contrast pictures (Figures 4B, 4D, 4F, 5B, 5D, 5F, and 5H). Immunofluorescence microscopy also showed consistent results in embryonic and post-embryonic stages and the two treatment groups. Controls run at all stages and, in particular, in Stage IV showed no specific binding within gill, branchiostegite, and epipodite epithelia (Figure 4E). In mid–late embryos (EI = 520 μm), fluorescent staining was observed in the epipodite buds and along the pleurites. In late embryos (EI = 607 μm), the immunoreactivity concerned only the now well-formed epipodites. In both embryonic stages, gills and branchiostegites showed no immunoreactivity (Figures 4A and 4C).

In larval Stages I to III held either in SW or DSW, enzyme labeling was observed in the epipodite epithelial layers (Figures 5A and 5E). The inner-side epithelium of the branchiostegites presented almost no specific immunofluorescence (Figures 5A and 5E) and the pleurites and gill filaments showed no immunoreactivity (Figures 5A and 5E).

Figure 1

Homarus gammarus. View of the left branchial chamber of a postlarval Stage IV. The branchiostegite has been cut to reveal the six podobranchs and the epipodites connected to them. The arthrobranchs are not visible as they lie beneath the podobranch. E, epipodite; F, filament; G, gill; S, Scaphognatite. Bar = 1 mm.

Figure 2

Homarus gammarus. Transmission electron micrographs of epipodite and branchiostegite epithelia of larval and postlarval stages maintained in DSW. (A) Epipodite epithelium of a larval Stage I. Bar = 0.6 μm. (B) Epipodite epithelium of a larval Stage III. Bar = 1.4 μm. (C) epipodite epithelium of a postlarval Stage IV. Bar = 1.2 μm. (D) Inner-side branchiostegite epithelia of a larval Stage I. Bar = 0.3 μm. (E) Inner-side branchiostegite epithelium of a larval Stage III. Bar = 0.7 μm. (F) Inner-side branchiostegite epithelium of a postlarval stage IV. Bar = 2 μm. BL, basal lamina; CU, cuticle; I, infolding system; M, mitochondrion; N, nucleus.

In postlarval Stage IV held in either SW or DSW, copious staining was observed in the epipodites (Figures 5C and 5G). Along the inner-side epithelial layer of the branchiostegites, no enzyme labeling was detected after exposure to SW (Figure 5C), but strong immunoreactivity was observed after exposure to DSW (Figure 5G). Gill epithelia showed no fluorescent staining (Figures 5C and 5G).

Immunogold Electron Microscopy and Quantitative Analysis of Gold Particles

In larval Stages I to III held in DSW, gold particles were exclusively localized in the ionocytes of the epipodites (Figure 6A). In Stage IV, gold labeling was observed in the ionocytes located in the epipodites, and along the inner-side of the branchiostegites in DSW (Figure 6B). In both the epipodite and branchiostegite ionocytes, gold labeling was well confined to the mitochondria-rich basolateral infolding systems (Figures 6A and 6B). Gold particles were present along the basal lamina and mostly on the cytoplasmic side of the infolding membranes. Almost no gold labeling was observed along the apical microvilli of the epithelial cells.

Figure 3

Western blotting analysis of homogenized epipodites of H. gammarus. The mouse monoclonal antibody IgGα₅ raised against the α-subunit of chicken Na⁺,K⁺-ATPase recognized a 90.4-kD protein as the prominent molecular species. A, branchiostegite; B, epipodite.

The variation in the average number (n) of gold particles per micrometer of membrane in the infolding systems is shown in Figure 7. In animals held in SW, no difference was observed between n in the epipodite cells of larval Stage III and postlarval Stage IV and a small amount of gold particles were observed in the branchiostegite cells of postlarval Stage IV only. After exposure to DSW, no significant increase in the number of gold particles was observed in the epipodite cells of Stage III. In the branchiostegite cells of this stage, n was at a very low level. In Stage IV exposed to DSW, however, n significantly increased in the ionocytes of epipodites and branchiostegites and was respectively twice and four times higher than in Stage IV maintained in SW.

Discussion

Depending on the organ of the branchial cavity, the tissue differentiation towards ion transport appears at different times throughout the postembryonic development of the European lobster H. gammarus (Haond and Charmantier, unpublished; and this study). In larval stages, the epipodite cells present characteristic features of ionocytes such as apical microvilli, basolateral infoldings, and many mitochondria. They progressively differentiate from Stages I to III. In individuals maintained at low salinity, the inner-side branchiostegite epithelia also possess ionocytes (Haond and Charmantier, unpublished), but tissue differentiation appears mainly after metamorphosis. The involvement of these extrabranchial organs in ion transport has already been observed in adult lobsters (Haond et al. 1998). When adults are exposed to low salinity, profound ultrastructural changes also occur in the epipodite and inner-side branchiostegite epithelia, i.e., vacuolization, increased thickness of the ionocytes, and development of the basolateral infoldings and apical microvilli (Haond et al. 1998). The present immunocytochemical study furthermore indicates that Na⁺,K⁺-ATPase is located in embryos along the pleurites and in the epipodite buds and is then confined to the epipodite epithelia in larval stages maintained either in seawater or at low salinity. After metamorphosis, the enzyme is still located in the epipodites and also along the inner-side branchiostegite epithelia of individuals held at low salinity. Within these ion-transporting organs, the enzyme is mostly located along the basolateral infolding system of the ionocytes.

A few other species of crustaceans, in which the ontogeny of osmoregulation and of the activity of Na⁺, K⁺-ATPase has been studied, are available for comparative purposes. In non-decapod crustaceans, early differentiation of osmoregulatory epithelia has been observed along the dorsal organ in the early nauplius stage of Artemia salina (Conte et al. 1972; Hootman and Conte 1975), in the neck organ of newborn Daphnia magna (Aladin and Potts 1995), and in the branchiostegites and pleurae of the zoea and mysis stages of the shrimp Callianassa jamaicense (Felder et al. 1986). In decapods, ionocytes have been found in young Penaeus aztacus (Talbot et al. 1972) and in Penaeus japonicus (Bouaricha et al. 1994), with differentiated osmoregulatory structures appearing in the branchiostegites and pleurae of the zoae and mysis stages (Bouaricha et al. 1994). After metamorphosis, these osmoregulatory structures progressively disappear from the branchiostegites and pleurae, and ionocytes appear in gills and mainly in epipodites as these organs develop (Bouaricha et al. 1994). In the European lobster, this study shows that osmoregulatory structures are also acquired very early during its development, because Na⁺,K⁺-ATPase is already present in the mid-late and late embryonic stages.

The lack of positive staining in the gill filaments, indicating a very low level of Na⁺,K⁺-ATPase, that we report in all the studied embryonic and postembryonic stages of H. gammarus is in agreement with recent ultrastructural and immunocytochemical studies conducted in juvenile and adult H. gammarus (Haond et al. 1998; Lignot et al. 1999). In contrast, brachyuran crab species such as Callinectes sapidus (Neufeld et al. 1980), Carcinus maenas (Towle and Kays 1986), Leptograpsus variegatus (Morris and Greenaway 1992), and Eriocheir sinensis (Péqueux et al. cited in Taylor and Taylor 1992) have large amounts of Na⁺,K⁺-ATPase in their gills. The thin and poorly differentiated epithelial layer of the lobster branchial filaments is considered to be most probably involved mainly in gas exchange (and also perhaps in other metabolic pathways such as ammonia excretion) (Haond et al. 1998). In addition, the pleurites, covered by a very thick cuticle, displayed no fluorescent staining throughout the studied postembryonic stages.

Figure 4

Homarus gammarus. Immunolocalization of Na⁺,K⁺-ATPase on thin sections. (A,B) Branchial chamber of a mid-late embryo (EI = 520 μm). (C,D) Branchial chamber of a late embryo (EI = 607 μm); (E,F) Control branchial chamber of a postlarval Stage IV held in DSW. (A,C,D) Fluorescent micrographs; (B,D,F) corresponding phase-contrast pictures. BST, branchiostegite; CU, cuticle; EP, epipodite; GF, gill filaments; HL, hemolymph lacuna; IEP, inner-side limiting epithelium; N, nucleus; OEP, outer-side limiting epithelium; PC, pillar cells; PL, pleura. Bars = 40 μm.

The strong fluorescent staining indicating the presence of Na⁺,K⁺-ATPase in the epipodites of all the studied embryonic and postembryonic stages of H. gammarus highlights the early setup of ion transport in embryos and confirms the prominent role of these organs in osmoregulation throughout development (Haond et al. 1998; Lignot et al. 1999). This has been recently confirmed through Na⁺,K⁺-ATPase titrations in adults of H. gammarus (Lucu and Devescovi 1999). The enzyme activities recorded in the epipodites of lobsters held in seawater and at low salinity were respectively 40% and 30% higher than those found in the gills. In the adult peneid shrimp Penaeus japonicus, Na⁺,K⁺-ATPase activity was also studied in the various organs of the branchial chamber and was determined as low in the branchiostegites, high in the gills, and very high in the epipodites (Bouaricha et al. 1991). Well-differentiated osmoregulatory epithelia have also been observed in epipodites of the crayfish Astacus pallipes and Astacus leptodactylus (Dunel-Erb et al. 1982, 1997) and in the peneid shrimp Penaeus japonicus (Bouaricha et al. 1994).

Figure 5

Homarus gammarus. Immunolocalization of the Na⁺,K⁺-ATPase on thin sections. (A,B) Branchial chamber of a larval Stage I held in SW. (C,D) Branchial chamber of a postlarval Stage IV held in SW. (E,F) Branchial chamber of a larval Stage I maintained in DSW. (G,H) Branchial chamber of a postlarval Stage IV maintained in DSW. (A,C,E,G) Fluorescent micrographs; (B,D,F,H) corresponding phase-contrast pictures. Abbreviations as in Figure 4. Bars = 40 μm.

Figure 6

Homarus gammarus. Immunogold localization of Na⁺,K⁺-ATPase on ultrathin sections. (A) Basolateral infoldings of the epipodite epithelium close to the basal lamina of a DSW-acclimated larval Stage I. (B) Basolateral region of the branchiostegite inner-side epithelium of a DSW-acclimated postlarval Stage IV. AS, apical side; BL, basal lamina; BS, basal side; CP, cytoplasm; GP, gold particles; I, invagination of the basal membrane. Bars = 0.2 μm.

In postlarval stages acclimated to low salinity, the development of well-formed ionocytes along the inner-side epithelial layer of the branchiostegites and the copious presence of Na⁺,K⁺-ATPase at this location reinforce previous findings pointing to the involvement of branchiostegites in osmoregulation (Haond et al. 1998; Lignot et al. 1999). Ionocytes located along the internal sheet of the branchiostegites have also been observed in the peneid shrimp Penaeus aztecus (Talbot et al. 1972) and Penaeus japonicus (Bouaricha et al. 1994) and in the estuarine tanaid Sinelobus stanfordi (Kikuchi and Matsumasa 1993). The strong immunostaining of branchiostegites in Stage IV of H. gammarus acclimated to low salinity emphasizes the role of metamorphosis in osmoregulatory processes. Postlarval stages acquire the capacity to slightly hyper-regulate in dilute medium right after metamorphosis (Charmantier et al. 1988b; Thuet et al. 1988). We therefore hypothesize that the change in osmotic and ionic metabolism acquired at metamorphosis results from the occurence of differentiated ionocytes on the branchiostegites, in addition to the increased differentiation of ionocytes already present on the epipodites.

Figure 7

Homarus gammarus. Labeling density of gold particles on basolateral infolding surfaces of the epipodite and inner-side branchiostegite epithelial cells in the branchial cavity of larval Stage III and postlarval Stage IV held in SW and DSW. Number of gold particles per micrometer of basolateral infolding measured from 8–18 segments. Error bars = mean ± SD; n = 6–15 infoldings. Letters on top of the error bars indicate significant differences between the considered areas (p < 0.05).

In homarid lobsters, the setup of neuroendocrine control of the osmotic and ionic regulation and an increased carbonic anhydrase activity have also been observed at metamorphosis (Charmantier et al. 1984b; Thuet et al. 1988; Charmantier-Daures et al. 1994). These cumulative physiological changes are part of metamorphosis and result in a higher tolerance to salinity in postlarvae compared to larval stages. The establishment of osmoregulatory structures and the appearance of Na⁺,K⁺-ATPase in ion-transporting tissues of epipodites and branchiostegites are therefore strongly correlated with the ontogeny of osmoregulatory physiological processes and are correlated with the ecology of H. gammarus.

In conclusion, a progressive partitioning of respiratory (and perhaps excretory) and ion regulatory functions occurs within the branchial chamber throughout lobster development. Respiration is effected by the gills and osmoregulation is performed by osmoregulatory structures located in the epipodite epithelia in all postembryonic stages and along the branchiostegites in postmetamorphic stages.

The ontogeny of osmoregulation in H. gammarus is therefore based on extrabranchial organs according to a two-step process: (a) an early embryonic expression of Na⁺,K⁺-ATPase in epipodite epithelia and (b) a further tissue differentiation at metamorphosis involving enhanced Na⁺,K⁺-ATPase activity along with activated and/or “de novo” synthesis of Na⁺,K⁺-ATPase in the branchiostegites. These are important cellular and molecular changes that enable lobsters to set a moderately efficient osmoregulatory ability after metamorphosis, i.e., at the time of ecophysiological transition from a planktonic to a benthic habitat.

Footnotes

Acknowledgments

We wish to thank Dr D.M. Fambrough for his generous gift of the anti-Na⁺,K⁺-ATPase antibody, Dr I. Ulrich for providing lobsters, Dr M. Charmantier-Daures for critical reading of the manuscript, and Ms E. Grousset, F. Justy, V. Richard, and Mr J. McKay for their technical help.

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Immunolocalization of NA +,K + -ATPase in the Branchial Cavity During the Early Development of the European Lobster Homarus Gammarus (Crustacea,Decapoda)

Abstract

Keywords

Materials and Methods

Animals

Scanning Electron Microscopy

Transmission Electron Microscopy

Na+,K+-ATPase Antibody and Western Blotting

Immunofluorescence Light Microscopy

Immunogold Electron Microscopy and Quantitative Analysis of Gold Particles

Statistical Analysis

Results

General Morphology and Ultrastructure

Western Blotting of Na+,K+-ATPase

Immunofluorescence Light Microscopy

Immunogold Electron Microscopy and Quantitative Analysis of Gold Particles

Discussion

Footnotes

Acknowledgments

References

Na⁺,K⁺-ATPase Antibody and Western Blotting

Western Blotting of Na⁺,K⁺-ATPase