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First published online March 30, 2006
Journal of Experimental Biology 209, 1487-1501 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02167

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Ontogeny of osmoregulation in embryos of intertidal crabs (Hemigrapsus sexdentatus and H. crenulatus, Grapsidae, Brachyura): putative involvement of the embryonic dorsal organ

Deepani Seneviratna and H. H. Taylor^*

School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch 8020, New Zealand

View larger version (49K): [in a new window]   Fig. 1. Representative stages in embryonic development of Hemigrapsus sexdentatus. (A) Stage 1, cleavage (morula shown). (B) Stage 2, gastrula. The clear zone is the embryo. (C) Stage 3, eyespot. Yolk is present as a single mass. (D) Stage 4. The yolk forms four lobes, the heart is beating, and chromatophores are forming. (E) Stage 5, pre-hatching. The yolk has now been reduced to two lobes. (F) Hatched zoea. Scale bars, 250 µm (A–E and F). The steps in development of H. crenulatus are similar although the eggs are smaller (see text).

View larger version (23K): [in a new window]   Fig. 2. Time course of changes in the internal osmolality of embryos (homogenised eggs) sampled from ovigerous crabs (A, Hemigrapsus sexdentatus; B, H. crenulatus) reared in dilutions of seawater: red, 100% (osmolality 1000 mmol kg–1); blue 50% (osmolality 500 mmol kg–1); green 25% (osmolality 250 mmol kg–1). The abscissa shows the estimated elapsed time from egg extrusion. The hyposaline exposure was commenced either during cleavage (1 day after extrusion, broken lines and open symbols) or after gastrulation (11 days and 8 days respectively after extrusion, solid lines and closed symbols). The final measurement was made just before hatching (for embryos in 100% seawater and those exposed to 50% seawater as gastrulae) or before abortion (25% seawater and those commenced during cleavage). Horizontal coloured lines show the osmolalities of the corresponding external seawaters. Values are means ± s.e.m., N=5–7 (A) and 3–5 (B).

View larger version (36K): [in a new window]   Fig. 3. Changes in the volume of eggs sampled from ovigerous crabs (A,B, Hemigrapsus sexdentatus; C,D, H. crenulatus) reared in dilutions of seawater (black bars, 100% seawater; grey bars, 50% seawater; white bars, 25% seawater). The hyposaline exposure was commenced either during stage 1 (cleavage, A,C) or at stage 2 (early gastrula, B,D). The abscissa shows the estimated elapsed times from egg extrusion. The final measurement was taken just before hatching (for embryos in 100% seawater and for embryos exposed to 50% seawater after gastrulation) or before abortion (25% seawater and those commenced during cleavage). Values are means ± s.e.m., N=10 (A) or 5 (B–D). Letters above the 100% seawater bars indicate means that are significantly different (one-way ANOVA, Tukey test). Asterisks above the 50% and 25% seawater bars indicate values that are significantly different from those in 100% seawater (two-way ANOVA, Tukey tests).

View larger version (22K): [in a new window]   Fig. 4. Representative efflux curves for labelled water at different developmental stages of Hemigrapsus crenulatus. Eggs were pooled from five ovigerous crabs, batch-labelled in tritiated seawater for 24 h, and washed in groups of 4–8 in unlabelled 100% seawater for varying times. (A) Egg radioactivity (expressed as nl exchangeable water at the radioactive concentration of the loading medium) is shown on a linear scale; (B) the natural logarithm of the radioactivity is plotted. Each point is the mean (± s.e.m. in the linear plot) of 5–8 groups of eggs sampled at each time. Trend lines were fitted by single or bi-exponential regression (see Materials and methods).

View larger version (22K): [in a new window]   Fig. 5. Representative efflux curves for 22Na at different developmental stages of Hemigrapsus crenulatus. Eggs were pooled from five ovigerous crabs, batch-labelled in 22Na-labelled seawater for 24 h, and washed in groups of 4–8 in unlabelled 100% seawater for varying times. (A) Egg radioactivity (expressed as nmol exchangeable sodium at the radioactive concentration of the loading medium) is shown on a linear scale; (B) natural logarithm of the radioactivity is plotted. Each point is the mean (± s.e.m. in the linear plot) of 5–8 groups of eggs sampled at each time. Trend lines were fitted by single or bi-exponential regression (see Materials and methods).

View larger version (89K): [in a new window]   Fig. 6. Silver staining of live embryos of Hemigrapsus sexdentatus and H. crenulatus. Eggs were washed in distilled water, stained briefly with AgNO3, washed again in distilled water and returned to seawater. (A–F) Appearance in vivo. In post-gastrula embryos a silver deposit is observed over the yolk (arrows). It is hypothesised that this patch corresponds to the embryonic dorsal organ and is responsible for water and salt excretion. Scale bars, 250 µm. (A) H. sexdentatus, stage 1, blastulae. Weak mottled silver staining over the whole embryo occasionally highlighting cell boundaries. (B) H. crenulatus, stage 2, gastrulae. (C) H. sexdentatus, stage 3, eyespots formed. (D) H. sexdentatus, stage 4–5, two yolk lobes. (E) H. crenulatus, stage 5, two yolk lobes, pre-hatching. (F) H. sexdentatus, Zoea. Some larvae hatched during staining. Free larvae did not stain but in those that hatched after staining, the silver deposit was cast off with the exuviae at the final embryonic moult (arrows). (G) Semi-thin epoxy section of AgNO3 stained embryo of H. sexdentatus at stage 4 showing the dorsal surface and the yolk. A black deposit of silver is present between the outer and inner membranes. Scale bar, 25 µm.

View larger version (104K): [in a new window]   Fig. 7. Scanning electron microscopy of embryos of Hemigrapsus sexdentatus and H. crenulatus fixed after AgNO3 treatment. AgCl deposits are indicated by arrows. (A) H. sexdentatus stage 4 with outer membrane intact. No silver deposit is visible. (B) H. sexdentatus stage 4 after partial removal of outer membrane. A thick amorphous deposit of AgCl adheres to the surface of the inner membrane. In C–F the outer membrane has been removed. (C) H. sexdentatus at stage 1, blastula. Spots of AgCl are distributed over the whole embryo, typically encircling small holes or craters. (D) H. crenulatus at stage 2, (E) H. sexdentatus at stage 3, and (F) H. crenulatus at stage 4. Scale bar, 25 µm (note that some shrinkage has occurred during processing).

View larger version (59K): [in a new window]   Fig. 8. X-ray microanalysis of the AgNO3-stained area on an embryo of Hemigrapsus sexdentatus at stage 4. (A) Secondary electron image. (B) Corresponding mapping of the energy peak for silver atoms showing its localisation over the deposit on the inner membrane. (C) Energy spectrogram showing Ag and Cl atoms are major constituents of the material in the patch (the Au peak is from the sputter coat).

View larger version (32K): [in a new window]   Fig. 9. A hypothetical model accounting for steady state water and salt turnover, hyper-osmoregulation and silver staining of post-gastrula embryos of Hemigrapsus. Active uptake of sodium and chloride takes place across the embryonic ectoderm into a hyperosmotic extracellular compartment by bounded embryonic and extra-embryonic ectoderm. Continuous osmotic entry of water into the extracellular compartment generates a small internal hydrostatic pressure causing both water and salts to leak out between the cells. In embryos vital stained with AgNO3, Cl– ions are precipitated as AgCl between the outer and inner membranes. After gastrulation this paracellular flow occurs primarily in the region of the embryonic dorsal organ, which therefore functions as a simple filtration-type excretory organ.