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 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.

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.

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.

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.

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.

Involvement of eyestalk factors in the neuroendocrine control of osmoregulation in adult American lobster Homarus americanus.

Osmoregulatory capacity (OC) decreased by approximately 50% after eyestalk ablation in adult Homarus americanus when in dilute media. OC was used to assay sinus gland extracts. Injection of total extracts and of some HPLC-separated fractions of sinus glands into destalked lobsters increased OC. One of the described crustacean hyperglycemic hormone isoforms influences osmoregulation. Another fraction of the sinus gland extracts modifies osmoregulation but its nature remains unknown. Variations in OC were examined in response to ecdysterone, Phe-Met-Arg-Phe-NH2, and atrial natriuretic factor but effects were minimal.

Ontogeny of Osmoregulatory Structures in the Shrimp Penaeus japonicus (Crustacea, Decapoda).

The ontogeny of differentiated osmoregulatory epithelia in the branchial chamber (gills, branchiostegite, pleura, epipodite) was studied by transmission electron microscopy throughout the postembryonic development of Penaeus japonicus. These epithelia are characterized by typical cytological features, including apical microvilli and numerous basal infoldings associated with mitochondria. Differentiated osmoregulatory structures are not observed in the early larval stages: nauplii and zoea 1. In the next larval stages, zoeas and mysis, gills and epipodites are developed as buds only, but osmoregulatory epithelia are observed in the branchiostegites and pleurae. The differentiated structures of the branchiostegites and pleurae are still present in postlarvae but disappear in juveniles and adults. Gills and epipodites develop progressively in the postlarval stages, with early differentiation of osmoregulatory epithelia in the epipodites. In juveniles and adults, the gill epithelium is poorly differentiated; in contrast, abundant differentiated osmoregulatory structures are observed in the epipodites. Ontogenetical comparisons of these observations with previous studies in the same species reveal strong correlations between the development of osmoregulatory epithelia, the ability to osmoregulate, the activity of Na+-K+ ATPase, and salinity tolerance during the postembryonic development of Penaeus japonicus.

Ontogeny of Osmoregulation and Salinity Tolerance in Cancer irroratus; Elements of Comparison with C. borealis (Crustacea, Decapoda).

Osmoregulation and salinity tolerance were studied in zoeae, megalopae, first crab stage (osmoregulation only), and adults of Cancer irroratus, and in zoeae and adults of C. borealis. In C. irroratus, salinity tolerance was moderate in zoeae, decreased in late zoeae 5, was at a minimum in megalopae, and increased in adults. The lower and upper lethal salinities for 50% of the animals (48 h LS 50) at 15°C were about 13-17‰/42-50‰ in zoeae, 24‰/37‰ in megalopae, and 8.5‰/65‰ in adults. In C. borealis, the corresponding values of LS 50s were 16-20‰/46-50‰ in zoeae and 12‰/65‰ in adults. In both species, zoeae were hyper-osmoconformers; adults were isosmotic in high salinities and slightly hyper-regulators in low salinities. In C. irroratus, the change from larval to adult type of regulation occurred from megalopa (hyper-osmoconformer) to first crab stage (hyper-regulator in dilute media), i.e., after the completion of metamorphosis. Osmoregulation and salinity tolerance appear correlated and are modified at metamorphosis. These results are discussed with an emphasis on the effects of metamorphosis on osmoregulation of developing decapods.

[Human somatotropin stimulates the growth of young American lobsters, Homarus americanus (Crustacea, Decapoda)]. / La somatotropine humaine stimule la croissance de jeunes homards américains, Homarus americanus (Crustacea, Decapoda).

Larval and postlarval lobsters, injected with human somatotropin (STH, or growth hormone), grew at a more rapid rate than untreated control animals over succeeding molts. Their mean carapace length was significantly longer than that of controls and they were heavier, although the moisture content of treated and control animals was similar. Injected STH increased the growth rate of individual animals by 10 to 20%. This is the first evidence for a growth enhancing effect of human somatotropin on a Crustacean.

Larval development and metamorphosis of the American lobster Homarus americanus (Crustacea, Decapoda): effect of eyestalk ablation and juvenile hormone injection.

Removal of eyestalks of Homarus americanus on different days and molting stages during larval development revealed that eyestalk tissue is involved in the larval and postlarval molting rhythm and in preparation for metamorphosis as early as the end of Stage II. Eyestalk removal in stages II and III reduced the duration of larval and postlarval stages. Eyestalk removal up to the end of Stage II delayed the completion of metamorphosis by one or two molts and caused additional development stages (designated IVa, IV', and V'). In this study, the critical stage for eyestalk ablation to delay metamorphosis occurred at the end of molt stage D1 of larval Stage II (the seventh day of development at 20 degrees). Injection of juvenile hormone before the critical stage resulted in a few intermediate stage IV' animals. This study demonstrates the involvement of eyestalk neuroendocrine tissue in the control of metamorphosis and investigates a possible involvement of juvenile hormone.

Neuroendocrine control of hydromineral regulation in the American lobster Homarus americanus H. Milne-Edwards, 1837 (Crustacea, Decapoda). 2. Larval and postlarval stages.

Sodium regulation in Homarus americanus changes from isoionic in Stage III to slightly hyperionic in Stage IV, and this is paralleled by improved survival of Stage IV lobsters in dilute media. Bilateral eyestalk ablation converts Stage IV and V lobsters to isoionic sodium regulation, but implantation of Stage IV or V eyestalk neuroendocrine tissue restores their normal hyperionic regulation. These results indicate that sodium regulation is controlled from Stage IV by eyestalk neuroendocrine factors. It is suggested that these changes between stages are part of a true metamorphosis that occurs between the last larval stage (III) and the first postlarval stage (IV).

Neuroendocrine control of hydromineral regulation in the American lobster Homarus americanus H. Milne-Edwards 1837 (Crustacea, Decapoda). 1. Juveniles.

Juvenile lobsters survive well in salinities above 10.2% (300 mOsm/kg), and their osmotic and ionic (Cl-, Na+, Ca2+) regulation in dilute sea water is slightly hyperosmotic, similar to that of adults. Approximately a month after eyestalk ablation, osmotic and ionic (Cl-, Na+) regulation becomes isosmotic, water content increases, and survival rate in dilute sea water declines, but these changes can be partially reversed by implantation of eyestalk neuroendocrine tissue. Regulation of Ca2+, in contrast, is only slightly affected by eyestalk ablation. These results indicate that osmotic regulation and regulation of hemolymph Na+ and Cl- concentrations are at least partly controlled by eyestalk neuroendocrine factors in this species.

[Eyestalk removal and intensive regeneration: joint effects on the molting cycle of Pachygrapsus marmoratus (Crustacea, Decapoda), variability of the critical regeneration stage]. / Epédonculation et régénération intensive: effets conjoints sur le cycle de mue de Pachygrapsus marmoratus (Crustacé, Décapode), variabilité de l'étape critique de régénération

When eyestalk-removal and pereiopod-removal, followed by intensive regeneration, are realized together on the same animal, their accelerative effect on the moulting rate is lower than the effect of only one of these two processes. The critical stage of pereiopod regeneration varies between the moulting stages C4 to D1 according to the physiological state of the Crab.