New insights into ion regulation of cephalopod molluscs: a role of epidermal ionocytes in acid-base regulation during embryogenesis

The constraints of an active life in a pelagic habitat led to numerous convergent morphological and physiological adaptations that enable cephalopod molluscs and teleost fishes to compete for similar resources. Here we show for the first time that such convergent developments are also found in the ontogenetic progression of ion regulatory tissues: as in teleost fish epidermal ionocytes scattered on skin and yolk sac of cephalopod embryos appear to be responsible for ionic and acid-base regulation before gill epithelia become functional. Ion and acid-base regulation is crucial in cephalopod embryos, as they are surrounded by a hypercapnic egg fluid with a pCO2 of 0.2-0.4 kPa. Epidermal ionocytes were characterized via immunohistochemistry, in situ hybridization and vital dye staining techniques. We found one group of cells that is recognized by Concavalin A and MitoTracker, which also expresses Na+/H+ exchangers (NHE) and Na+/K+-ATPase. Similar to findings obtained in teleosts these NHE3-rich cells take up sodium in exchange for protons, illustrating the energetic superiority of NHE based proton excretion in marine systems. In vivo electrophysiological techniques demonstrated that acid equivalents are secreted by the yolk and skin integument. Intriguingly, epidermal ionocytes of cephalopod embryos are ciliated as demonstrated by scanning electron microscopy suggesting a dual function of epithelial cells in water convection and ion regulation. These findings add significant knowledge to our mechanistic understanding of hypercapnia tolerance in marine organisms, as it demonstrates that marine taxa which were identified as powerful acid-base regulators during hypercapnic challenges already exhibit strong acid-base regulatory abilities during embryogenesis

Haemolymph constituents and osmolality as functions of moult stage, body weight, and feeding status in marron, Cherax cainii (Austin and Ryan, 2002) and yabbies, Cherax destructor (Clark, 1936)

The study investigates the change in osmolality and haemolymph constituents in marron Cherax cainii and yabbies Cherax destructor associated with moult stages, body weights and their feeding status. A total of 582 haemolymph samples from 5 moult stages (postmoult-AB, intermoult-C, and premoult stages – D0, D1, D2), two body weight classes (2–15 g and 61–75 g) and nutritional status were used for analysis of osmolality, protein, glucose, and ionic concentrations of potassium and chloride following the standard biochemical procedures. The haemolymph protein, glucose, potassium and chloride levels were highest at intermoult and early premoult stages, and lowest at postmoult in both crayfish species. Except protein, no significant differences were seen in analyzed parameters between various weight classes and two species. Haemolymph osmolality, protein and glucose were significantly higher in fed crayfish, whereas no variations in haemolymph potassium and chloride concentrations were observed between the fed and unfed crayfish. Maximum osmolality was recorded at 7–8 h after feeding in both crayfish species. The results showed that the biochemical changes in the haemolymph of marron and yabbies are related to moult stages, body weight and feeding and thus can be used as tools for determining suitable diets

Ontogeny of osmoregulation in the grapsid crab Armases miersii (Crustacea, Decapoda)

Osmoregulation was studied in the zoeal stages I to III, the megalopa, juvenile crab stages I and II, and in adults of the grapsid crab Armases miersii. The larvae hatch and develop in tropical supratidal rock pools, where ample variations of salinity occur. To cope with this harsh environment, the capacity for osmoregulation is well developed at hatching, and becomes further accentuated in the larvae and juveniles. All zoeal stages hyper-regulated at low salinity (5 to 26 PSU), but at higher salinity (33 to 44 PSU), they were hyper- osmoconformers. The type of osmoregulation changed in the megalopa stage to a hyper-hypo-regulation pattern. While the hyper-osmoregulatory capacity increased gradually throughout postembryonic development from hatching to adult, the hypo- osmoregulatory capacity increased from the megalopa to the adult. The ontogenic acquisition of osmoregulation was faster for hyper-than for hypo-regulation: 85 and 41% of the adult capacity, respectively, was acquired in the crab I stage. These findings confirm that important physiological changes occur at metamorphosis. A correlation is established between the osmoregulatory ability of each developmental stage and its salinity tolerance. The ecological implications and the adaptive and evolutionary significance of osmoregulation in early Life-history stages of A. miersii and other aquatic crustaceans are discussed

Salinity tolerance, osmoregulation, and immunolocalization of Na+/K+-ATPase in larval and early juvenile stages of the Chinese mitten crab, Eriocheir sinensis (Decapod, Grapsoidea)

The ontogeny of osmoregulation was studied in laboratory-reared early developmental stages of the Chinese mitten crab, Eriocheir sinensis, from the Elbe estuary (North Sea, Germany). At salinities ranging from 0.1644.3 , survival rate was quantified, hemolymph osmolality was measured, and osmoregulatory capacity was calculated as difference between osmolality of the hemolymph and the external medium. Zoea larvae hyper-regulated in dilute media, but osmoconformed in seawater and at higher salinities (≥32.2 ). Megalopae and stage I-II juveniles hyper-regulated at low salinities and hypo-regulated at ≥32.2 , with an ontogenetic increase in osmoregulatory capacity. Survival at ca. 10-32 was generally high (90100 %), while complete mortality occurred in all zoeal stages (except for zoea I) at 0.165.3 . By contrast, nearly 50 % of the megalopae and all juvenile crabs survived at such low salinities. The expression of Na+/K+-ATPase and the development of transporting epithelia were studied by means of immunofluorescence light microscopy (ILM) and transmission electron microscopy (TEM). In early (stage I-II) zoeae, fluorescence staining was observed along the inner epithelium of the branchiostegites, and epithelial cells showed typical features of ionocytes. In the megalopa and first juvenile crab, ionocytes and immunolabeled Na+/K+-ATPase were located in the filaments of the most posterior gills, while no immunodetection occurred in the anterior gills. Comparison of histological and physiological results shows a close relationship between the ontogeny of osmoregulation and the expression of Na+/K+-ATPase within the transporting epithelia of the branchial chamber. In conclusion, the adult pattern of osmoregulation develops in E. sinensis through two molts, (1) from a moderately hyper-iso-regulating zoeal phase to the moderately hyper-/hypo-regulating megalopa, (2) from the megalopa to a strongly euryhaline, hyper-/hypo-regulating first juvenile crab stage. The results of this study are consistent with an export strategy in this holo-euryhaline crab species

Ontogeny of osmoregulation, physiological plasticity, and larval export strategy in the grapsid crab Chasmagnathus granulata (Crustacea, Decapoda)

The grapsid crab Chasmagnathus granulata populates brackish-water lagoons and other estuarine environments. In its reproduction, this species follows a strategy of larval export, i.e. its larvae live under different salinity conditions from the juveniles and adults. In the present experimental investigation, ontogenetic changes in the capability for osmoregulation were studied in all 4 zoeal stages, the megalopa, the juvenile crab instars I, II and IV, and adults (all reared in seawater, 32). Moreover, we studied effects of embryonic and larval acclimation on osmoregulation. The zoea I larvae were slight hyper-regulators at low salinities (10 to 17) and hyper-osmoconformers at higher salinities. Stages II to IV zoe generally hyper-osmoconformers. At metamorphosis to the megalopa, the type of osmoregulation changed to hyper-hypo-regulation. The osmoregulatory capacities under both hypo- and hypersaline conditions increased strongly in the crab I and throughout later juvenile development. These patterns in osmoregulation match the ontogenetic changes that typically occur in the ecology of C. granulata: the zoea I hatches in brackish estuarine waters, where the juveniles and adults live, before it is exported to coastal marine zones. This initial larval stage is euryhaline and capable of hyper-osmoregulation at low salinities. The same capabilities were observed in the megalopa, which re-invades the brackish adult environment. This stage is known to settle in semiterrestrial habitats near the adult burrows, where both brackish and hypersaline conditions are likely to occur; this coincides with the first ontogenetic appearance of the hyper-hypo-osmoregulation pattern. The zoeal stages II, III and IV, in contrast, develop in the adjacent sea, where the salinity is higher and more stable. Correspondingly, these intermediate larval stages were found to be stenohaline osmoconformers. Preceding exposure of the eggs and larvae to a reduced salinity (20) enhanced the hyper-osmoregulatory capacity at low salinities (5 to 10) in all zoeal stages. This indicates an effect of non-genetic acclimation and, hence, phenotypic plasticity. This trait should have an adaptive value, as it increases the chance larval survival, at least in the initial larval stage, which is in the field exposed to highly variable, mostly reduced salinities

Osmoregulation, immunolocalization of Na+/K+-ATPase, and ultrastructure of branchial epithelia in the developing brown shrimp, Crangon crangon (Decapoda, Caridea)

Aspects of osmoregulation including salinity tolerance, osmoregulatory capacity, location of transporting epithelia, and the expression of the enzyme Na+/K+-ATPase were investigated in the developing brown shrimp, Crangon crangon (L.), from the North Sea. Early developmental stages and large juveniles were exposed to a wide range of salinities, for measurement of hemolymph osmolality and survival rates. In media ranging from 17.0 to 32.2 , salinity tolerance was generally high (survival rates: 70-100 %) in all developmental stages, but it decreased in media <10.2 . Zoeal stages and decapodids slightly hyper-regulated at 17.0 and osmoconformed in media ?25.5 . At 10.2 , these stages showed high mortality, and only juveniles survived at 5.3 . Juveniles hyper-regulated at 10.2 and 17.0 , osmoconformed at 25.5 , and hypo-regulated in media ?32.2 . Large juveniles hyper-regulated also at 5.3 . Expression of the Na+/K+-ATPase and ion-transporting cells were located through immunofluorescence microscopy and transmission electron microscopy. In zoeae I and VI, a strong immunoreactivity was observed in cells of the inner epithelia of the branchiostegites and in epithelial cells lining the pleurae. Their ultrastructure showed typical features of ion-transporting cells. In decapodids and juveniles, ionocytes and expression of Na+/K+-ATPase remained located in the branchiostegite epithelium, but they disappeared from the pleurae and appeared in the epipodites. In large juveniles, the cells of the gill shaft showed positive immunolabeling and ultrastructural features of ionocytes. In summary, the adult pattern of osmoregulation in C. crangon is accomplished after metamorphosis from a moderately hyper-osmoconforming decapodid to an effectively hyper-/hypo-regulating juvenile stage. Salinity tolerance and osmoregulatory capacity are closely correlated with the development of ion-transporting cells and the expression of Na+/K+-ATPase

Ontogeny of osmoregulatory structures and functions in the green crab, Carcinus maenas (Crustacea, Decapoda)

The ontogeny of osmoregulation, the development of branchial transporting epithelia and the expression of the enzyme Na+/K+-ATPase were studied in Carcinus maenas (L.) from the North Sea, Germany. Laboratory reared zoea larvae, megalopae and young crabs were exposed to a wide range of salinities, and hemolymph osmolality was measured after 24 h exposure time (72 h in juveniles). Zoea I larvae slightly hyper-regulated in dilute media (10.2 and 17.0 ) and osmoconformed at >17 . All later zoeal stages (II IV) osmoconformed in salinities from 10.2 to 44.3 . The megalopa hyper-regulated at salinities from 10.2 to 25.5 . Young crabs hyper-regulated at salinities from 5.3 to 25.5, showing an increase in the osmoregulatory capacity. The development of transporting epithelia and the expression of Na+/K+-ATPase were investigated by means of transmission electron microscopy and immunofluorescence microscopy. In the zoea IV, only a very light fluorescence staining was observed in gill buds. Epithelial cells were rather undifferentiated without showing features of ionocytes. Gills were found to be present in the megalopa, where Na+/K+-ATPase was located in basal filaments of the posterior gills. In crab I juveniles and adults, Na+/K+-ATPase was noted in the three most posterior pairs of gills, whereas anterior gills lacked the enzyme. Ionocytes could first be recognized in filaments of megalopal posterior gills, persisting through subsequent stages at the same location. Thus, the development of the gills and the expression of Na+/K+-ATPase are closely correlated with the ontogeny of osmoregulatory abilities. The morphological two-step metamorphosis of C.maenas can also be regarded as an osmo-physiological metamorphosis, (1) from the osmoconforming zoeal stages to the weakly regulating megalopa, and (2) to the effectively hyper-regulating juvenile and adult crabs

Osmoregulation and salinity tolerance in zoeae and juveniles of the snow crab

Osmoregulation and salinity tolerance were studied in zoeae 1 and instar IX juveniles of Chionoecetes opilio. In zoeae 1, the lower and upper lethal salinities for 50% of the animals (LS 50) at 14 °C were about 10 and 42‰ at 24 h, 18 and 41‰ at 48 h, 25 and 38‰ at 96 h. In juveniles, the approximate 48 h LS 50 s at 6 °C were 13.5 and 46‰. Both developmental stages are able to withstand relatively ample but brief variations of salinity. Their short-term euryhalinity is discussed in relation to the salinity of their habitat. Zoeae were hyper-osmoconformers. Juveniles were osmoconformers and isoionic to the external medium except for Mg++ which was hypo-regulated. Isosmotic and isoionic regulation in post-metamorphic stages are presumably an attribute of the family Majidae. The pattern of osmoregulation in zoeae 1 and juveniles relate C. opilio to the first of three groups of species previously characterized by their pattern of ontogeny of osmoregulation