Ontogeny and osmoregulatory function of the urinary system in the Persian sturgeon, Acipenser persicus (Borodin, 1897).

The structure of the kidney and the localization of Na(+), K(+)-ATPase (NKA) immunopositive cells were examined throughout the postembryonic development of the Persian sturgeon, Acipenser persicus, from newly hatched prelarvae (10mm) to 20 days post hatch (20 DPH) larvae (31mm). Investigations were conducted through histology and immunohistochemistry by using the light and immunofluorescence microscopy. The pronephros was observed in newly hatched prelarvae. The cells lining the distal pronephric tubules and their collecting ducts showed laterally expressed NKA immunofluorescence that later extended throughout the whole cytoplasm. Mesonephrogenous placodes and pre-glomeruli were distinguished at 2 DPH along the collecting ducts posteriorly. Their tubules were formed and present in kidney mesenchyma, differentiated into neck, proximal, distal and collecting segments at 7 DPH when NKA immunopositive cells were observed. Their distal and collecting tubules showed an increasing immunofluorescence throughout their cytoplasm while the glomeruli remained unstained. From D 9 to D 17, the epithelial layer of pronephric collecting duct changed along the mesonephros to form ureters. Ureters, possessing isolated strong NKA immunopositive cells, appeared as two sac-like structures hanging under the trunk kidney. Since NKA immunopositive cells were not observed on the tegument or along the digestive tract of newly hatched prelarva, and also the gills are not formed yet, the pronephros is the only osmoregulatory organ until 4 DPH. At the larval stage, the pronephros and mesonephros are functional osmoregulatory organs and actively reabsorb necessary ions from the filtrate.

Ultrastructure and osmoregulatory function of the kidney in larvae of the Persian sturgeon Acipenser persicus.

The localization of Na(+) , K(+) -ATPase (NKA) and the ultrastructural features of kidney were examined in larvae of the Persian sturgeon Acipenser persicus (L 31-41 mm total length and 182·3-417·3 mg). Investigations were conducted through light and electron microscopy and through immunofluorescence for NKA detection. The kidney nephrons consisted of a large glomerulus and tubules (neck, proximal, distal and collecting), which connected to the ureters. Posteriorly, ureters extended and joined together into a thin-walled ureter terminal sac. Ultrastructurally, the glomerular cells (podocytes) possessed distinctive pedicels that extended to the basal membrane. The proximal tubule (PT) showed two different cells. The cells lining the anterior part of PT possessed apical tall microvilli (c. 2·7 µm), a sub-apical tubular system, a basal nucleus and dense granules. Posteriorly in the cells, the sub-apical tubular system and granules were absent and round mitochondria associated with basolateral infoldings were found; the apical microvilli were reduced. Distal and collecting tubular cells showed the typical features of osmoregulatory cells, i.e. well-developed basolateral infoldings associated with numerous mitochondria. No immunofluorescence of NKA was detected in the glomeruli. A weak immunostaining was observed at the basolateral side of the cells lining the neck and PT. A strong immunostaining of NKA was observed in the entire cells of the distal tubules, collecting tubules and in some isolated cells of the ureters. In all immunostained cells, the basolateral region showed a much higher fluorescence and nuclei were immunonegative. In conclusion, the epithelial cells of kidney tubules had morphological and enzymatic features of ionocytes, particularly in the distal and collecting tubules. Thus, the kidney of A. persicus larvae possesses active ion exchange capabilities and, beside its implication in excretion, participates in osmoregulation.

Early development of the digestive tract (pharynx and gut) in the embryos and pre-larvae of the European sea bass Dicentrarchus labrax.

The European sea bass Dicentrarchus labrax is a marine teleost important in Mediterranean aquaculture. The development of the entire digestive tract of D. labrax, including the pharynx, was investigated from early embryonic development to day 5 post hatching (dph), when the mouth opens. The digestive tract is initialized at stage 12 somites independently from two distinct infoldings of the endodermal sheet. In the pharyngeal region, the anterior infolding forms the pharynx and the first gill slits at stage 25 somites. The other three gill arches and slits are formed between 1 and 5 dph. Posteriorly, in the gut tube region, a posterior infolding forms the foregut, midgut and hindgut. The anus opens before hatching, at stage 28 somites. Associated organs (liver, pancreas and gall bladder) are all discernable from 3 dph. Some aspects of the development of the two independent initial infoldings seem original compared with data in the literature. These results are discussed and compared with embryonic and post-embryonic development patterns in other teleosts.

Physiological and behavioural responses of Gammarus pulex (Crustacea: Amphipoda) exposed to cadmium.

The aim of this study was to investigate the effects of cadmium on physiological and behavioural responses in Gammarus pulex. In a first experiment, cadmium LC50s for different times were evaluated in 264 h experiment under continuous mode of exposure (LC50(96 h)=82.1 microgL(-1), LC50(120 h)=37.1 microgL(-1), LC50(168 h)=21.6 microgL(-1), LC50(264 h)=10.5 microgL(-1)). In a second experiment, the physiological and behavioural responses of the amphipod exposed to cadmium (0, 7.5 and 15 microgL(-1)) were investigated under laboratory conditions. The mortality and the whole body cadmium concentration of organisms exposed to cadmium were significantly higher than in controls. Concerning physiological responses, cadmium exposure exerted a significant decrease on osmolality and haemolymph Ca(2+) concentration, but not on haemolymph Na(+) and Cl(-) concentrations, whereas the Na(+)/K(+)-ATPase activity was significantly increased. Behavioural responses, such as feeding rate, locomotor and ventilatory activities, were significantly reduced in Cd exposed organisms. Mechanism of cadmium action and consequent energetic reallocation in favour of maintenance functions (i.e., osmoregulation) are discussed. The results of this study indicate that osmolality and locomotor activity in G. pulex could be effective ecophysiological/behavioural markers to monitor freshwater ecosystem and to assess the health of organisms.

Physiological and behavioural responses of Gammarus pulex exposed to acid stress.

Physiological and behavioural responses of the acid-sensitive amphipod Gammarus pulex exposed to a wide range of acid conditions (pH 4.1, 5.1, 6.0) under laboratory conditions were investigated. An exposure of 38 h to acid conditions caused significant decreases in survival rate, osmolality, haemolymph Na+ concentration, ventilatory and locomotor activity compared to organisms exposed to a circumneutral medium (pH 7.9). We highlighted the interest of using individual response distribution, since drastic effects can be detected in organisms exposed to pH 6.0, in particular for osmolality: The response can be divided into two groups, unimpacted and impacted organisms. Moreover this representation permitted to evaluate the health level of individual organisms through the determination of threshold values. Significant correlations between mean pH and mean physiological/behavioural responses were observed. The relationships between individual responses permitted not only to compare endpoints, but also to show that affected organisms were impacted by ionoregulation failure, hypoventilation and low locomotor activity. The energetic reallocation in favour of maintenance functions, such as osmoregulation, is discussed. The results of this study indicate that the values of haemolymph Na+ concentration, osmolality and locomotor activity in G. pulex could be effective ecophysiological/behavioural markers to monitor freshwater ecosystems and to assess the health of organisms or populations.

Expression of Na(+)/K(+)-ATPase alpha-subunit mRNA during embryonic development of the crayfish Astacus leptodactylus.

Astacus leptodactylus is a decapod crustacean fully adapted to freshwater where it spends its entire life cycle after hatching under huge osmoconcentration differences between the hemolymph and surrounding freshwater. We investigated the expression of mRNA encoding one ion transport-related protein, Na(+)/K(+)-ATPase alpha-subunit, and one putative housekeeping gene, beta-actin, during crayfish ontogenesis using quantitative real-time PCR. A 216-amino acid part of the open reading frame region of the cDNA coding for the Na(+)/K(+)-ATPase alpha-subunit was sequenced from total embryo, juvenile and adult gill tissues. The predicted amino acid sequence showed a high percentage similarity to those of other invertebrates (up to 95%) and vertebrates (up to 69%). beta-actin expression exhibited modest changes through embryonic development and early post-embryonic stage. The Na(+)/K(+)-ATPase alpha-subunit gene was expressed in all studied stages from metanauplius to juvenile. Two peaks of expression were observed: one in young embryos at 25% of embryonic development (EI=100 mum), and one in embryos just before hatching (at EI=420 mum), continuing in the freshly hatched juveniles. The Na(+)/K(+)-ATPase expression profile during embryonic development is time-correlated with the occurrence of other features, including ontogenesis of excretory antennal glands and differentiation of gill ionocytes linked to hyperosmoregulation processes and therefore involved in freshwater adaptation.

Ontogeny of the antennal glands in the crayfish Astacus leptodactylus (Crustacea, Decapoda): anatomical and cell differentiation.

The ontogeny of the antennal glands was studied during the embryonic and post-embryonic development of Astacus leptodactylus. The future glands arising from undifferentiated columnar cells were detectable at the metanauplius stage EI 150 microm (EI: eye index; approximately 440 microm at hatching). The tubule and labyrinth differentiated in embryos at EI 190 microm, and the bladder and coelomosac at EI 250 microm. At EI 350 microm, the tubule lengthened and divided into proximal and distal sub-regions. In later stages, the gland retained the same morpho-anatomy but the differentiation and size of each part increased. The cells of the coelomosac displayed the cytological features of podocytes in late embryonic development at EI 440 microm. Only small apical microvilli and a few mitochondria were observable in the labyrinth cells at EI 250 microm; by EI 440 microm, these cells presented well-shaped apical microvilli, formed bodies, basal infoldings and mitochondria. In the cells of the tubules and bladder, mitochondria and basal infoldings occurred at EI 440 microm and EI 250 microm, respectively. The differentiation of the tubules and bladder cells suggested that they were involved in active transport at EI 440 microm. Following hatching, the differentiation of the cells and the size of the glands increased. The ontogeny of the antennal glands thus starts in early embryos, the specific cellular functional features being differentiated in the various parts of the glands by EI 440 microm. The antennal glands are probably functional just before hatching, i.e., before the juveniles are confronted with the low osmolality of freshwater.

Ontogeny of the antennal glands in the crayfish Astacus leptodactylus (Crustacea, Decapoda): immunolocalization of Na+,K+-ATPase.

The involvement of the antennal urinary glands in the ontogeny of osmoregulatory functions was investigated during the development of Astacus leptodactylus by measurements of hemolymph and urine osmolality in juvenile and adult crayfish and by the immunodetection of the enzyme Na+,K+-ATPase. In stage II juveniles, 1-year-old juveniles, and adults, all of which were maintained in freshwater, urine was significantly hypotonic to hemolymph. In adults, chloride and sodium concentrations were much lower in urine than in hemolymph. During embryonic development, Na+,K+-ATPase was detected by immunocytochemistry in ionocytes lining the tubule and the bladder, at an eye index (EI) of 220-250 microm, and in the labyrinth, at EI 350 microm. In all regions, immunofluorescence was mainly located at the basolateral side of the cells. No immunofluorescence was detected at any stage in the coelomosac. In late embryonic stages (EI 410-440 microm), in stage I juveniles, and in adults, strong positive immunofluorescence was found from the labyrinth up to and including the bladder. These results show that, as early as hatching, juvenile crayfish are able to produce dilute urine hypotonic to hemolymph. This ability originates from the presence of Na+,K+-ATPase in ion-transporting cells located in the labyrinth, the tubule, and the bladder of the antennal glands and constitutes one of the main adaptations of crayfish to freshwater.

Hydromineral regulation in the hydrothermal vent crab Bythograea thermydron.

This study investigates the salinity tolerance and the pattern of osmotic and ionic regulation of Bythograea thermydron Williams, 1980, a brachyuran crab endemic to the deep-sea hydrothermal vent habitat. Salinities of 33 per thousand-35 per thousand were measured in the seawater surrounding the captured specimens. B. thermydron is a marine stenohaline osmoconformer, which tolerates salinities ranging between about 31 per thousand and 42 per thousand. The time of osmotic adaptation after a sudden decrease in external salinity is about 15-24 h, which is relatively short for a brachyuran crab. In the range of tolerable salinities, it exhibits an iso-osmotic regulation, which is not affected by changes in hydrostatic pressure, and an iso-ionic regulation for Na(+) and Cl(-). The hemolymph Ca(2+) concentration is slightly hyper-regulated, K(+) concentration is slightly hyper-hypo-regulated, and Mg(2+) concentration is strongly hypo-regulated. These findings probably reflect a high permeability of the teguments to water and ions. In addition to limited information about salinity around hydrothermal vents, these results lead to the hypothesis that B. thermydron lives in a habitat of stable seawater salinity. The osmoconformity of this species is briefly discussed in relation to its potential phylogeny.

Identification of respiratory and ion-transporting epithelia in the phyllosoma larvae of the slipper lobster Scyllarus arctus.

Phyllosoma larvae of the Palinura lack a branchial cavity and gills. In the phyllosoma, gas and ion exchanges that occur at the level of the gill in the adult must occur in other parts of the body or through the entire body. The objective of this study was to localize epithelia bordering the body of the phyllosoma larvae that had features comparable to those of the gill epithelia of adult decapods. The first phyllosoma instar of the small Mediterranean slipper lobster Scyllarus arctus was studied. First, we used a silver nitrate staining method to identify parts of the body with high ionic permeability. Confocal laser scanning microscopy with a fluorescent vital stain for mitochondria, dimethylaminostyrylmethylpyridiniumiodine (DASPMI), was then used to localize cells with a high density of mitochondria. Next, an ultrastructural study of selected epithelia was carried out. A thick (5 microns) mitochondria-rich epithelium covers the ventral side of the cephalic shield; its cells are characterized by the presence of well-developed apical infoldings adjacent to the cuticle. This part of the body has a high ionic permeability as indicated by a positive silver nitrate staining. The ventral mitochondria-rich epithelium might be involved in active ion transport. The rest of the body, particularly the dorsal side of the shield and the appendages, shows a lower ionic permeability (no positive silver nitrate staining) and is limited by a thin (1 micron) epithelium with low numbers of mitochondria. This epithelium exhibits features of a typical respiratory epithelium.

Immunolocalization of NA(+),K(+)-ATPase in the branchial cavity during the early development of the European lobster Homarus gammarus (Crustacea, Decapoda).

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 IgGalpha(5) raised against the avian alpha-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.%o) and to dilute seawater (22.1 %o). 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.

Crayfish freshwater adaptation starts in eggs: ontogeny of osmoregulation in embryos of Astacus leptodactylus.

Osmoregulation was studied throughout the embryonic development of Astacus leptodactylus. Egg-carrying females were held in freshwater (FW) and in three dilute seawater media (200, 400, 600 mosm kg(-1), 6.8, 13.6, 20.4 per thousand salinity). In FW, changes in peri-embryonic fluid (PEF) and (when available) embryonic hemolymph osmolality were followed from newly-laid eggs to hatching (for an embryonic eye index, EI, of 430-450 microm) and in first-stage juveniles. The PEF and/or hemolymph osmolality remained stable at about 360-380 mosm kg(-1) from early to late (EI 410 microm) embryos; it decreased prior to hatching (EI 420 microm) and in newly-hatched juveniles, down to 290 mosm kg(-1). Artificial opening and removal of the egg membranes, followed by direct exposure to FW, demonstrated that the ability to hyper-osmoregulate, and consequently to survive, in FW appears in embryos with EI > or = 410 microm, i.e., only a few hours or days before hatching. Following a transfer to the dilute seawater media, the PEF/hemolymph osmolality increased slowly over 18-20 days and became isosmotic with the external media at 13.6 and 20.4 per thousand. The embryos died at EI 380-395 microm in these media, and only at 6.8 per thousand was the development completed until successful hatch. These results demonstrate that (1) the embryos become able to osmoregulate in FW shortly before hatching, (2) the embryos are osmo-protected in the eggs during their development, (3) embryonic development and hatching are possible up to a salinity of 7 per thousand. These results are discussed in relation to freshwater adaptation of crayfish.

Ecophysiological adaptation to salinity throughout a life cycle: a review in homarid lobsters.

Adaptations to salinity are reviewed throughout development in both species of the genus Homarus. Some populations of homarid lobsters are known to inhabit coastal and estuarine areas where salinity fluctuates. Salinity tolerance varies during development, with 50 % lethal salinities (LS(50)) ranging from approximately 15-17 in larvae to approximately 12 in postlarvae and 10 in adults. Larval and adult lobsters can avoid low-salinity areas using behavioural strategies. When exposed to low salinity, the capacity to osmoregulate varies with development. Embryos are osmoconformers and are osmotically protected by the egg membranes. Larvae are also osmoconformers, and the pattern of osmoregulation changes at metamorphosis to hyper-regulation, which is retained throughout the later stages up to the adult stage. Exposure to low salinity increases the activity of Na(+)/K(+)-ATPase in postlarvae and later stages. The level of osmoregulation evaluated through the osmoregulatory capacity (the difference between haemolymph and medium osmolalities) is negatively affected by low temperature (2 degrees C). The variations in haemolymph osmolality resulting from osmoconforming or partial osmoregulation are compensated by intracellular iso-osmotic regulation. Neuroendocrine control of osmoregulation appears in postlarvae and seems to involve the crustacean hyperglycaemic hormone. In adult lobsters, the gills appear to have a respiratory function only, and extracellular osmoregulation is effected by the epipodites, with the addition of the branchiostegites at low salinity. These organs are present at hatching. Transmission electron microscopy and immunolocalization of Na(+)/K(+)-ATPase reveal that the epipodites become functional in larvae and that the branchiostegites become functional in postlarvae. An integrated series of events links the appearance of osmoregulatory tissues, the increase in Na(+)/K(+)-ATPase activity, the occurrence in postlarvae of hyper-regulation at low salinity and the increase in salinity tolerance. Further ecological and physiological studies are proposed for a better understanding of the adaptive significance of the ontogeny of osmoregulation in lobsters.

Involvement of crustacean hyperglycemic hormone in the control of gill ion transport in the crab Pachygrapsus marmoratus.

Total extracts of sinus glands (SG) of the euryhaline grapsid crab Pachygrapsus marmoratus contain peptidic factor(s) that stimulate osmoregulatory processes in isolated and perfused posterior gills from crabs acclimated to dilute seawater. This study investigated the nature of the active factor(s). Separation of P. marmoratus SG peptides by reverse-phase HPLC, followed by a direct enzyme-linked immunosorbent assay using an anti-Carcinus maenas crustacean hyperglycemic hormone (CHH) antiserum, identified a major immunoreactive chromatographic peak. A glucose quantification bioassay demonstrated a strong and specific hyperglycemic activity following injection of the immunoreactive peak, therefore defined as the CHH of P. marmoratus. Isolated posterior gills were then perfused with HPLC fractions using a dose of 4 SG equivalents/assay. The CHH fraction consistently and significantly increased the transepithelial potential difference and Na(+) influx by about 50%. The effect was rapid and reversible. Another substance of unknown nature (eluted earlier than CHH in the HPLC gradient) caused a small increase in Na(+) influx (14%) but had no effect on the transepithelial potential difference. No other peptidic product from the SG had significant effect on the measured osmoregulatory parameters. These results indicate that CHH, in addition to its hyperglycemic activity, is also implicated in the control of branchial ionic transport. This neuropeptide may thus constitute a major factor involved in the control of osmoregulation in decapod crustaceans.

Ontogeny of osmoregulation and salinity tolerance in a mangrove crab, Sesarma curacaoense (Decapoda: Grapsidae).

The grapsid crab Sesarma curacaoense is believed to represent the closest saltwater relative to the ancestor which gave rise to an adaptive radiation of endemic freshwater and terrestrial species on the island of Jamaica. Living in mangrove swamps with variable salinity conditions and showing semiterrestrial behaviour, S. curacaoense exhibits ecological adaptations to non-marine conditions. In laboratory experiments, we studied the salinity tolerance during development from hatching to the end of the first juvenile stage. Successful development through metamorphosis occurred in the full salinity range tested (15-32 per thousand), although mortality was significantly enhanced and development delayed at 15 per thousand. In another series of experiments, we studied the ontogeny of the capability for osmoregulation, which is considered as the physiological basis of osmotic stress tolerance. Our results show that S. curacaoense is from hatching a fairly strong hyperosmoregulator in dilute media. This capability increased gradually from hatching throughout the larval and juvenile development. In seawater (32 per thousand) and at an enhanced salt concentration (44 per thousand), the zoeal stages remained hyperosmoconformers. The capability for hypoosmoregulation in concentrated media appeared first in the megalopa stage and increased thereafter. Adult crabs were observed to be strong hyper-hypo-osmoregulators in a salinity range from at least 1 per thousand to 44 per thousand. The unusually early appearance of strong regulatory capabilities, particularly in dilute media, is interpreted as a physiological preadaptation that should have facilitated the evolutionary process of adaptive radiation in non-marine environments on Jamaica.

Ontogeny of osmoregulation in the crayfish Astacus leptodactylus.

Osmoregulation was studied during the postembryonic development of Astacus leptodactylus Eschscholtz 1823 in juvenile stages 1-8 and in adults. Juveniles hatch and later stages develop in freshwater or in moderately saline waters. The time of acclimation from freshwater to a saline medium increased from early juveniles to adults. At all stages, it was longer than in comparable stages of marine crustaceans, reflecting the high impermeability of the teguments to water and ions. All stages were able to hyperisoosmoregulate. In freshwater, the ability to hyperosmoregulate was established at hatching and increased during development. The hemolymph osmolality increased from 286 mosm kg-1 in stage 1 juveniles to 419 mosm kg-1 in adults. All stages also hyperregulated at low salinities (7 per thousand and 13 per thousand salinity) and were osmoconformers at higher salinities up to 21 per thousand salinity. The lowest isosmotic salinity tended to increase with the developmental stages. The ability to osmoregulate at hatch and throughout postembryonic development is probably a key physiological adaptation in this and other freshwater crayfish.

Ontogeny of intracellular isosmotic regulation in the european lobster Homarus gammarus (L.).

Intracellular free amino acids were measured in the abdominal muscle of the three larval instars, postlarvae, and juveniles of the lobster Homarus gammarus, acclimated to seawater (35 per thousand) and to a dilute medium (22 per thousand), to study intracellular isosmotic regulation throughout the development of this species. Transfer to low salinity was followed by a highly significant drop of free amino acids level in all developmental stages. The main regulated amino acids were glycine, proline, and alanine. The level of regulation of total free amino acids changed at metamorphosis: the decrease in total free amino acids at low salinity was 46% in the three larval instars, but it was only 29% in postlarvae and 20% in juveniles. These results suggest that free amino acids, mainly glycine, proline, and alanine, are involved in intracellular isosmotic regulation in the lobster, with different levels of involvement in pre- and postmetamorphic stages. The ontogenetic changes in intracellular isosmotic regulation are discussed in relation to the changes in extracellular regulation (osmoregulation) in the lobster.

Immunolocalization of Na+,K(+)-ATPase in the organs of the branchial cavity of the European lobster Homarus gammarus (Crustacea, Decapoda).

The localization of Na+,K(+)-ATPase in epithelia of the organs of the branchial cavity of Homarus gammarus exposed to seawater and dilute seawater was examined by immunofluorescence microscopy and immunogold electron microscopy with a monoclonal antibody IgG alpha 5 raised against the avian alpha-subunit of the Na-,K(+)-ATPase. In juveniles held in seawater, fluorescent staining was observed only in the epithelial cells of epipodites. In juveniles held in dilute seawater, heavier immunoreactivity was observed in the epithelial cells of epipodites, and positive immunostaining was also observed along the inner-side epithelial layer of the branchiostegites. No fluorescent staining was observed in the gill epithelia. At the ultrastructural level, the Na+,K(+)-ATPase was localized in the basolateral infolding systems of the epipodite and inner-side branchiostegite epithelia of juveniles held in dilute seawater, mostly along the basal lamina. The expression of Na+,K(+)-ATPase therefore differs within tissues of the branchial cavity and according to the external salinity. These and previous ultrastructural observations suggest that the epipodites, and to a lesser extent the inner-side epithelium of the branchiostegites, are involved in the slight hyper-regulation displayed by lobsters at low salinity. Enhanced Na+,K(+)-ATPase activity and de novo synthesis of Na+,K(+)-ATPase within the epipodite and branchiostegite epithelia may be key points enabling lobsters to adapt to low salinity environments.

Confocal laser scanning and electron microscopical studies on osmoregulatory epithelia in the branchial cavity of the lobster homarus gammarus

The adult lobster Homarus gammarus is a weak hyper-regulator at low salinity. The objective of this study was to locate the ion-transporting tissues in the branchial chamber of this species, using electron microscopy and confocal laser scanning microscopy with a fluorescent vital stain for mitochondria, DASPMI, which is widely used to locate mitochondria-rich cells in ion-transporting epithelia of fish. A thick mitochondria-rich epithelium is present on the inner side of the branchiostegite and over the entire surface of the epipodites. Ultrastructural observations confirm that this tissue has features typical of an ion-transporting epithelium. When the lobster is transferred to low salinity, these epithelia undergo marked ultrastructural changes, such as an increase in thickness related to the development of basolateral infoldings, the appearance of numerous vesicles and an increase in height of the apical microvilli. In the gills, the branchial filaments are lined by a thin and poorly differentiated epithelium, containing numerous mitochondria; no significant ultrastructural changes were observed in the gills of animals acclimated to low salinity. In summary, in H. gammarus, no evidence of osmoregulatory structures was found in the gills. Differentiated ion-transporting epithelia are present in the branchial cavity, on the inner side of the branchiostegite and on the epipodites; these organs are probably involved in osmoregulation.

Stimulation of osmoregulating processes in the perfused gill of the crab Pachygrapsus marmoratus (Crustacea, Decapoda) by a sinus gland peptide.

Isolated posterior gills of the hyper-hyporegulating crab Pachygrapsus marmoratus were perfused with extracts of homologous sinus glands. Sinus gland extracts stimulated the influx of Na+ ions and increased the transepithelial potential difference in the gills in a dose-dependent and reversible fashion. The bioactivity of extracts prepared from crabs that had been acclimated to 10/1000 salinity for at least 1 week was not significantly different from that of extracts prepared from seawater (36/1000 salinity) crabs. The perfusion experiments with both extracts containing two sinus glands significantly increased Na+ influx by about 150% and transepithelial potential difference by about 45%. Sinus gland extracts also increased the Na+/K(+)-ATPase activity by 54% in incubated posterior gills. The bioactivity of extracts was reduced by pronase and trypsin, but not by heating for 10 min at 100 degrees. The molecular weight of the responsible factor(s) was > 5000 Da. Thus, the sinus gland of P. marmoratus is concluded to be involved in the neuroendocrine control of osmoregulation and to contain a peptide(s) that directly influences brachial function.