ARTICLE |
Correspondence to: Guy Charmantier, Université de Montpellier II, CP 092, Place Eugène Bataillon, F-34095 Montpellier Cedex 05, France. E-mail: charmantier@univ-montp2.fr
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Summary |
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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 IgG5 raised against the avian
-subunit of the Na+,K+-ATPase. In midlate 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 (35.0ç) and to dilute seawater (22.1ç). 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+-ATPase 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-osmoregulate 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 49:10131023, 2001)
Key Words: Na+,K+-ATPase, osmoregulation, ionocytes, ontogeny, metamorphosis, crustacean, lobster, Homarus gammarus
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Introduction |
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DESPITE the wealth of information available concerning crustacean osmoregulation in adult stages (reviewed in
In homarid lobsters, the third ontogenetic pattern of osmoregulation prevails. Embryos do not osmoregulate (
Na+,K+-ATPase, a heterodimeric integral membrane protein composed of a catalytic -subunit (Mr
100 kD) and a smaller glycosylated ß-subunit (Mr
4060 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
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 (
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 IgG5 raised against the avian
-subunit of the Na+,K+-ATPase.
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Materials and Methods |
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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 ± 1.3ç, 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 35.0ç) or acclimated to dilute seawater (DSW: 650 mOsm.kg-1,
22.1ç) 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 (
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 12 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 IgG5 was raised against the
-subunit of the chicken Na+,K+-ATPase (
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
Immunogold Electron Microscopy and Quantitative Analysis of Gold Particles
A postembedding immunostaining technique on LR White sections was applied, as previously described (5 antibody diluted to 20 µg.ml-1 in BSAPBS and incubated for 2 hr at room temperature in a wet chamber. The sections were washed in BSAPBS (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 BSAPBS (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
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) (
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Results |
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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 Fig 1 presenting the branchial chamber in Stage IV). Each branchial cavity contains 20 trichobranchiate gills and seven epipodites. These organs have already been described in the adult stage (
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Western Blotting of Na+,K+-ATPase
Immunological specificity of the purified antibody against the -subunit of the chicken Na+,K+-ATPase was tested by immunoblotting analysis of the solubilized epipodite and branchiostegite epithelia. The IgG
5 antibody bound specifically to one band (Fig 3). The apparent molecular weight of this polypeptide in the epipodites was approximately 90 kD. We then used this antibody in all immunocytochemical experiments.
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Immunofluorescence Light Microscopy
Bouin fixation and paraffin-embedding procedures yielded good antigenicity as observed with the fluorescent micrographs (Fig 4A, Fig 4C, Fig 4E, Fig 5A, Fig 5C, Fig 5E, and Fig 5G) and good structural preservation as observed with phase-contrast pictures (Fig 4B, Fig 4D, Fig 4F, Fig 5B, Fig 5D, Fig 5F, and Fig 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 (Fig 4E). In midlate 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 (Fig 4A and Fig 4C).
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In larval Stages I to III held either in SW or DSW, enzyme labeling was observed in the epipodite epithelial layers (Fig 5A and Fig 5E). The inner-side epithelium of the branchiostegites presented almost no specific immunofluorescence (Fig 5A and Fig 5E) and the pleurites and gill filaments showed no immunoreactivity (Fig 5A and Fig 5E).
In postlarval Stage IV held in either SW or DSW, copious staining was observed in the epipodites (Fig 5C and Fig 5G). Along the inner-side epithelial layer of the branchiostegites, no enzyme labeling was detected after exposure to SW (Fig 5C), but strong immunoreactivity was observed after exposure to DSW (Fig 5G). Gill epithelia showed no fluorescent staining (Fig 5C and Fig 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 (Fig 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 (Fig 6B). In both the epipodite and branchiostegite ionocytes, gold labeling was well confined to the mitochondria-rich basolateral infolding systems (Fig 6A and Fig 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.
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The variation in the average number (n) of gold particles per micrometer of membrane in the infolding systems is shown in Fig 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.
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Discussion |
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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 (
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 (
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 (
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 (
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 (
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 (
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.
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Acknowledgments |
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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. CharmantierDaures for critical reading of the manuscript, and Ms E. Grousset, F. Justy, V. Richard, and Mr J. McKay for their technical help.
Received for publication May 25, 2000; accepted March 14, 2001.
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