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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

^* Author for correspondence (e-mail: harry.taylor{at}canterbury.ac.nz)

Accepted 13 February 2006

	Summary

TOP Summary Introduction Materials and methods Results Discussion References

 
This study examined whether the existence of hyperosmotic internal fluids in embryos of euryhaline crabs (Hemigrapsus sexdentatus and H. crenulatus) in dilute seawater reflects osmotic isolation due to impermeability of the egg envelope, as proposed for other decapods, or active osmoregulation. When ovigerous crabs with eggs at gastrula stage were transferred from 100% seawater (osmolality 1000 mmol kg^–1) to 50% seawater, embryogenesis and hatching of zoea were completed normally, but were delayed. Hatching failed if the transfer to 50% seawater occurred before gastrulation, and embryogenesis was abnormal in 25% seawater. In 100% seawater, embryos at all stages were internally hyperosmotic by 150–250 mmol kg^–1. On transfer to 50% seawater, osmolality initially decreased but remained 200–350 mmol kg^–1 hyperosmotic to the medium for several weeks until hatching. High efflux rates of tritium-labelled water (t_1/2 16–75 min) and ²²Na (t_1/2 109–374 min) from H. crenulatus embryos were inconsistent with the osmotic isolation hypothesis. It is concluded that post-gastrula embryos were actively hyper-osmoregulating. The diffusional water permeability of the embryos decreased during development while the sodium efflux rate increased 10-fold. Very rapidly exchanging pools of water and sodium (t_1/2 a few seconds to minutes) probably corresponded to peri-embryonic fluid and implied that the egg envelope was a negligible barrier to diffusion of water and salts. Higher Na⁺/K⁺-ATPase activities in late embryos of H. crenulatus incubated in 50% seawater than in embryos incubated in full strength seawater were consistent with an acclimation response. An area of the embryonic surface located over the yolk in the region of the embryonic dorsal organ stained with AgNO₃. Staining appeared at gastrulation, persisted throughout development and was lost at hatching. Deposits of AgCl between the outer and inner membranes, identified by X-ray microanalysis, suggest that the dorsal organ was a site of chloride extrusion. A model for osmoregulation in post-gastrula embryos is proposed: osmotic uptake of water is balanced by excretion of water and salts via the dorsal organ and salt loss is balanced by active uptake over the general embryonic ectoderm.

Key words: crab, Hemigrapsus, osmoregulation, excretion, development, water permeability, sodium transport, embryonic dorsal organ, silver staining

	Introduction

TOP Summary Introduction Materials and methods Results Discussion References

 
Hemigrapsus sexdentatus (formerly H. edwardsii) and H. crenulatus are euryhaline intertidal crabs endemic to New Zealand. The adults of both species are strong hyperosmotic regulators, consistent with their exposure to rainfall at low tide and their frequent occurrence near to freshwater streams and, in the case of H. crenulatus, within estuaries (Hicks, 1973; Jones, 1976; Bedford and Leader, 1977; McLay, 1988). Ovigerous females brood their embryos (i.e. between cleavage and hatching as zoea) externally, within eggs attached ventrally to the abdominal pleopods. Thus, the adults and their embryos are potentially exposed to similar osmotic regimes. Studies of other hyper-regulating decapods, including homarid lobsters (Charmantier and Aiken, 1987; Charmantier et al., 1988; Charmantier and Charmantier-Daures, 1991; Charmantier, 1998), grapsid crabs (Charmantier et al., 2002), and astacid freshwater crayfish (Susanto and Charmantier, 2000; Susanto and Charmantier, 2001) have concluded that the capacity for osmoregulation is absent in embryonic stages. Although the embryonic and peri-embryonic fluids of Homarus and Astacus have been observed to be hyperosmotic to the external medium, this state is believed to be maintained by the low permeability of the membranes of the egg envelope to water and salts, thus protecting the embryos from osmotic and diffusive exchanges with the external medium (Charmantier and Charmantier-Daures, 2001).

We have shown previously that the embryos of both species, at all stages between gastrulation and hatching, survive acute transfer to seawater diluted by as much as 100-fold (10 mmol kg^–1 osmolality) for several days without significant mortality or obvious impairment of development (Taylor and Seneviratna, 2005). During this time, the internal osmolality of these eggs (i.e. the embryo, peri-embryonic fluid, and external membranes) was maintained hyperosmotic to the external medium, displaying the characteristics of strong osmoregulation. In contrast, cleavage stages osmoconformed within 6 h and experienced higher mortality in dilute seawater.

Interpretation of the osmotic behaviour of Hemigrapsus embryos in terms of the above osmoprotection hypothesis implies that the egg envelope becomes impermeable to water and solutes a few days after extrusion as has been proposed for two other grapsid crabs (Bas and Spivac, 2000). Such absolute impermeability ostensibly conflicts with the requirements for exchange of respiratory gases, ammonia and other excretory products during development, with the observed increases in water and electrolyte contents during embryogenesis in these crabs (Seneviratna, 2003) and other decapodan eggs (Pandian, 1970), and with qualitative observations on the solute permeability of homarid eggs (Yonge, 1937; Yonge, 1946). However, quantitative measurements of water and solute fluxes and permeabilities that are required to critically evaluate osmoprotection versus osmoregulation hypotheses, are unavailable for crustacean eggs.

Steady state hyperosmotic regulation requires both the active uptake of salts from the external medium and a route for the elimination of water gained osmotically. Gills, important in the osmoregulation of adult crabs (Mantel and Farmer, 1983; Pequeux, 1995), are absent from the embryonic stages of crabs and the renal (antennal) organs are not fully formed until larval stages (Anderson, 1973). In contrast to the proposed situation in decapods, embryonic osmoregulation has been demonstrated in euryhaline gammarid and talitrid amphipods, and is believed to involve the embryonic dorsal organ the ultrastructure of which is suggestive of ion transport and it stains characteristically with silver ions (Meschenmoser, 1989; Morritt and Spicer, 1995; Morritt and Spicer, 1996). Embryonic dorsal organs have also been reported in brachyuran embryos (Anderson, 1973; Fioroni, 1980) but have not been linked to an osmoregulatory function. Dorsal organs, also referred to as nuchal or neck organs, are present in other larval and adult crustaceans, notably in the nauplii of branchiopods. Their homology with embryonic dorsal organs is uncertain but they are believed to function as either ionoregulatory or sensory organs (Martin and Laverack, 1992; Aladin and Potts, 1995).

To examine the longer term osmotic homeostasis of Hemigrapsus embryos, and its significance for normal embryogenesis, we observed embryonic development and hatching success, and changes in the osmolality and volume of the eggs during chronic exposures of both species to dilute seawater, commencing either during the osmoconforming cleavage stages or after gastrulation, when we hypothesised that osmoregulation begins. To further investigate the basis of apparent hyper-osmoregulation we measured steady state fluxes of isotopically labelled water and sodium ions in H. crenulatus eggs. Demonstration of turnover times that are short in relation to the duration of the hyperosmotic state, would favour active rather than passive maintenance of this condition. We measured the activity of Na⁺/K⁺-ATPase in H. crenulatus chronically exposed to either normal or diluted seawater as a possible indicator of osmoregulatory acclimation. We describe, using light and electron microscopy, the selective staining with silver ions of a discrete patch on the surface of Hemigrapsus embryos and discuss its possible importance in embryonic osmoregulation.

	Materials and methods

TOP Summary Introduction Materials and methods Results Discussion References

 
Ovigerous crabs carrying early embryos were collected on the east coast of South Island, New Zealand at the start of their spawning periods. Hemigrapsus sexdentatus H. Milne Edwards 1837 [formerly H. edwardsii Hilgendorf 1882 (see McLay and Schubart, 2004)] were obtained from Glenafric Beach, Waipara in April and Hemigrapsus crenulatus (H. Milne Edwards 1837) from the Avon-Heathcote Estuary, Christchurch in August to September. The crabs were tagged for identification and transferred to recirculating seawater aquaria at 15°C under a 12 h:12 h light:dark cycle, provided with a gravel base and rocky refugia, fed on mussel pieces, and subjected to simulated tidal emersion:immersion on a 6.2 h:6.2 h cycle. The osmolality of the seawater was adjusted every few days to either 1000 mmol kg^–1 (nominally `100% seawater', salinity 35), 500 mmol kg<sup>–1</sup> (`50% seawater') or 250 mmol kg^–1 (`25% seawater'), and the water was changed completely every 2 weeks.

Effect of chronic exposure to dilute seawater on embryonic development
For observations of developmental stage and viability, ten ovigerous crabs of each species with embryos either at stage 1 or at stage 2 (see below) were placed into each of the three tidal aquaria (100%, 50% and 25% seawater) and observed until the embryos hatched or were aborted. Crabs were removed briefly, a small sample of the eggs was detached from the pleopods for microscopical examination, and the crab replaced. The developmental stage of embryos is reported on a five-step scheme (Taylor and Seneviratna, 2005) (Fig. 1). The approximate timings of these stages in 100% seawater at 15°C are for H. sexdentatus: (1) cleavage, 0–5 days; (2) gastrula, 6–28 days; (3) eyespot, 29–37 days; (4) four-lobe, 38–50 days; (5) two-lobe to hatching zoea, 51–62 days. For H. crenulatus the timings are: (1) cleavage, 0–2 days; (2) gastrula, 3–18 days; (3) eyespot, 19–27 days; (4) four-lobe, 28–36 days; (5) two-lobe to hatching zoea, 37–43 days.

View larger version (49K): [in this window] [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).

The Na⁺/K⁺-ATPase activity of embryos was measured separately using H. crenulatus only. Groups of ovigerous crabs were transferred at stage 2 to the 100% and 50% seawater tidal systems. Crabs in 100% seawater were sampled immediately and the remainder maintained at 15°C and removed at either stage 4 or stage 5. The Na⁺/K⁺-ATPase activity was measured on 0.4 g of homogenized eggs (less than five samples in each stage and salinity combination) as described previously (Taylor and Seneviratna, 2005) and expressed as pmol P_i embryo^–1 min^–1.

Morphological observations
Live embryos were examined using a stereomicroscope (Zeiss Stemi 2000c) and photographed using a digital camera (Sony DSC-S75) mounted on the microscope. Embryos were also aldehyde-fixed for preparation of semi-thin (1 µm) resin sections: 2 h under vacuum at room temperature, overnight at 4°C, in 3% glutaraldehyde, 1.2% paraformaldehyde, 0.05% tannic acid, 0.05% saponin, 0.1 mol l^–1 sodium cacodylate buffer in 50% seawater, pH 7.3, osmolality 1300 mmol kg^–1, post-fixed for 4 h at 4°C in 1% osmium tetroxide, 0.2 mol l^–1 sodium cacodylate, dehydrated in graded ethanol and propylene oxide series and embedded in epoxy resin (Spurr, 1969). Sections were stained with 1% Toluidine Blue in a 1% borax solution, observed using a Zeiss Axioskop 2 microscope, and images captured digitally (Zeiss Axiocam).

Silver staining
Eggs were detached from ovigerous crabs and rinsed in distilled water (changed twice) for about 30 s. They were then transferred to 5 g l^–1 AgNO₃ for 5 min, again rinsed in distilled water and exposed to sunlight for 10-15 min. Light microscope observations were made on unfixed silver-stained embryos in seawater and on epoxy sections.

Scanning electron microscopy and X-ray microanalysis
Silver-stained and unstained eggs with embryos at all developmental stages of both H. sexdentatus and H. crenulatus were examined using the scanning electron microscope (SEM). Eggs were fixed in 2.5% glutaraldehyde in 0.1 mol l^–1 sodium cacodylate buffer (pH 7.3) containing 0.3 mol l^–1 sucrose for 48 h at 4°C, washed in buffer overnight, dehydrated in a graded ethanol series, transferred via a graded ethanol/amyl acetate series to liquid CO₂, and critical-point dried. The eggs were mounted on aluminium stubs using carbon-impregnated double-sided adhesive tabs. In order to view the silver deposit, the outer egg membrane was carefully ruptured with the tip of a fine scalpel blade. Finally, the samples were sputter coated with 60 nm gold/palladium and observed and photographed in the SEM (Leica S440) at an accelerating voltage of 15 kV. Energy dispersive X-ray microanalysis was carried out on the silver-stained patch in the SEM {Link Pentafect detector, ISIS Software v3.34 [Oxford Instruments (UK) Ltd, High Wycombe, UK], 20 kV, 600 pA probe current} using the spot analysis mode to generate spectra (2 min) and X-ray dot mapping to show the distribution of silver within the patch area.

Water and sodium turnover in H. crenulatus eggs
Tritiated water fluxes
Eggs with embryos at each of the five stages were pooled from four to five ovigerous crabs and sub-sampled for measurement of the mean volume and mass of individual eggs. They were then equilibrated for 24 h in batches of 100 in 2 ml of seawater labelled with tritiated water (2 MBq ml^–1) in a covered solid watch-glass. Groups of four to eight eggs were removed with minimal fluid carry over and washed in 5 ml of unlabelled seawater. The seawater was gently stirred and changed periodically during the washing period. After 0.5, 1, 2, 4, 8, 10, 25, 50, 75, 90 and 115 min, eggs were pipetted onto a filter paper. As soon as adherent fluid had been absorbed (a few seconds), they were transferred with forceps to scintillation vials containing 1.0 ml of aqueous scintillation fluid (Ready-Solv MP, Beckman Instruments, Inc., Fullerton, CA, USA). For the zero time measurement, the eggs were transferred directly from the labelling medium to the filter paper. The activity of tritium was measured by liquid scintillation counting (Beckman 5800) and corrected for quenching. Five or more replicate groups of eggs were measured for each developmental stage, at each wash time. The radioactivity of the labelling medium was also measured. Mean egg volume was measured on sub-samples of the same batch. Measurements were made at room temperature (20°C).

Sodium efflux from H. crenulatus eggs was measured using a protocol similar to that used for tritium. After estimating their mean volume and mass, eggs were equilibrated for 24 h in ²²Na-labelled seawater (200 kBq ml^–1). Five or more replicates were performed for each developmental stage and wash time. Radioactivity was measured by liquid scintillation counting (Beckman 5800) without quench correction.

Water and sodium contents
The total exchangeable water and sodium contents (C nl or C nmol, respectively) of single eggs were estimated from their radioactivity at time zero (A₀; d.p.m. egg^–1).

where A_m is the radioactivity of the labelling seawater (d.p.m. nl^–1 or d.p.m. nmol^–1).

Compartmental analysis of tracer washout data
³H and ²²Na radioactivities of the eggs generally did not decline as first order exponentials. Thus, washout curves were analysed as bi-exponential decay curves of the form:

where A_t is the total radioactivity (expressed as either nl egg^–1 of ³H₂O or nmol egg^–1 of ²²Na, at the radioactivity concentration of the loading medium) remaining at time t (min), and P and Q represent radioactivities of `fast' and `slow' pools, declining with rate constants of a and b (min^–1), respectively. For initial analysis, an iterative non-linear curve fitting routine (Marquardt method) supplied with the FigP graphing package (Biosoft, Cambridge, UK) was used to determine P, Q, a and b. Mean data were weighted inversely by their standard errors. Where all four parameters were statistically significant (P<0.05 that they were non-zero), these values are reported, along with standard errors of the estimates and their corresponding half times (t_1/2=ln(0.5)/a or ln(0.5)/b). Where a rapidly exchanging pool was present but its rate constant (a) was too high in relation to the sampling intervals to be determined by this method, Q and b were determined by linear regression of ln(At) versus time when none of the fast pool remained (>2 min for ³H₂O, >25 min for ²²Na). The size of the fast pool (P) was then determined by subtraction of Q from the specific activity at time zero (C=P+Q).

Calculation of water and sodium efflux rates and permeabilities
These estimates are reported for the slow pool only (assumed to represent the embryo, see Discussion section) relative to the surface area of the egg envelope (A, cm²), assumed to be a sphere of diameter equal to the mean of the largest and smallest diameters. The diffusional permeability constant of water is:

The permeability constant for sodium influx was calculated assuming that the eggs were in steady state turnover in seawater. Thus, the permeability constant for sodium is:

where ([Na]_ext is the sodium concentration in seawater (0.49x10^–3 mol cm^–3). The mass-specific efflux of sodium,

where M is the mass of one egg, estimated by weighing a known number of eggs from the same batch.

Data analysis
Data are presented as means ± s.e.m. Changes in the volume of eggs with development time in the control series (100% seawater) were analysed using single factor analysis of variance (ANOVA). The effects of salinity (% seawater) and development time or stage on egg ATPase activity and volume were analysed using two-factor ANOVA. In the latter case, to avoid missing cells resulting from differential survival in 50% and 25% seawater of stage 1 embryos, separate ANOVAs were performed (see Results). Tukey HSD tests were employed for post-hoc comparison of means. Differences were considered statistically significant where P<0.05.

	Results

TOP Summary Introduction Materials and methods Results Discussion References

 
Development and hatching success in dilute seawater
Hyposaline exposure commencing before gastrulation
Ovigerous H. sexdentatus that were introduced to the 100% seawater tidal system with embryos at stage 1 (cleavage) had incubated essentially all of their eggs to stage 5 (two yolk lobes, pre-hatch) at 60 days and these all hatched to motile zoea after 62 days (cf. Fig. 1; see Materials and methods). When stage 1 ovigerous crabs were transferred to the 50% seawater system, cleavage proceeded normally but embryogenesis was progressively retarded from gastrulation onwards. Although there was a higher incidence of non-viable embryos, the majority reached stage 5 after about 70 days when development was arrested. In about 10% of the eggs, the envelope ruptured but the hatched larvae appeared incapable of swimming. In 25% seawater, development did not proceed beyond gastrulation and the crabs aborted their clutches of disintegrating embryos between 30 and 40 days. Similar effects were observed in H. crenulatus that were introduced into the tidal system at stage 1. In 100% seawater, embryogenesis occurred normally and all had successfully hatched at 37 days. In 50% seawater, development invariably proceeded to completion but was delayed. In this case, the majority of the eggs hatched around 43 days but, as for H. sexdentatus, the motility of the larvae was impaired. In 25% seawater, gastrulation of H. crenulatus was unsuccessful and the eggs were aborted between 18 and 30 days.

Hyposaline exposure commencing after gastrulation
Delaying the introduction to dilute seawater until after gastrulation markedly improved the viability of the embryos. Although retarded similarly to those commenced at cleavage, embryos of both species successfully hatched as active larvae in 50% seawater (by 70 days for H. sexdentatus and by 43 days for H. crenulatus). The yolk reserves in late hatching zoea were noticeably reduced (and white rather than yellow) compared with those from 100% seawater. In 25% seawater, embryogenesis continued apparently normally until about stage 3 (at 40 days and 24 days, respectively) but then ceased, and the eggs were subsequently aborted.

Osmoregulation
The mean osmolalities of the embryos (homogenised whole eggs) carried by H. sexdentatus and H. crenulatus maintained in the 100% seawater tidal systems were consistently hyperosmotic (150–250 mmol kg^–1) to that of the seawater (997–1005 mmol kg^–1) throughout development (Fig. 2). On transfer to 50% seawater (498–505 mmol kg^–1) and 25% seawater (248–260 mmol kg^–1), the osmolality of the eggs of both species decreased during the first few days but stabilised hyperosmotic to the external medium by several 100 mmol kg^–1. Embryonic osmolality decreased more slowly when the period of hyposaline exposure commenced after gastrulation (stage 2) compared with those started during cleavage (stage 1) and, in the case of H. crenulatus the plateaus were higher (Fig. 2). Stage 1 embryos of H. sexdentatus became almost iso-osmotic with 25% seawater after 11 days. The final upturn in osmolality of both species introduced to 25% seawater at stage 1 corresponded to a period of abnormal morphology, and possibly autolysis, just prior to their abortion. The general hyper-osmoticity exhibited by the embryos was statistically highly significant. Thus at each sample time, in all 12 treatment series shown in Fig. 2A,B, the mean value of osmolality, the lower 95% confidence limit, and indeed every replicate, was higher than the simultaneously measured osmolality of the external seawater.

View larger version (23K): [in this window] [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 this window] [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).

Exchangeable water content and water efflux rates of H. crenulatus eggs
Exchangeable water (EW) contents and efflux rates were determined using tritium-labelled seawater. The volume of EW per egg increased during development from about 6 nl at stage 1 (cleavage) to about 18 nl at stage 5 (pre-hatching). During stages 1 and 2 (cleavage to gastrula), the EW comprised a small component that turned over extremely rapidly and a larger more slowly exchanging pool (Fig. 4, Table 1). The fast pool was very small in the eyespot and four-lobe stages (stages 3 and 4) but increased at stage 5. In the two trials using stage 1 embryos, the efflux rate constants for the fast pool were 302 h^–1 and 116 h^–1 and were immeasurably high in stages 2 and 3, representing half times for exchange of less than 1 min). At stage 5 the fast pool efflux rate constants were more than an order of magnitude lower at 7–11 h^–1 (t_1/2=2.3–6.5 min; Table 1).

View larger version (22K): [in this window] [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 this table: [in this window] [in a new window]   Table 1. Exchangeable water contents and efflux rates for Hemigrapsus crenulatus eggs in 100% seawater (36 salinity) at 20°C

Efflux rate constants for the more slowly exchanging water compartment were in the range 0.5–2.6 h^–1 with half exchange times of 0.25 to 1.25 h. The efflux rate constant of the slow compartment decreased, and t_1/2 increased, by approximately a factor of three between cleavage and hatching due mainly to the increase in egg volume. The diffusive water permeability of the slow pool was variable but tended to decrease during development with a mean value for all stages of 0.91±0.09x10^–6 cm s^–1 (N=10).

Exchangeable sodium and sodium efflux rates of H. crenulatus eggs
Exchangeable sodium contents and sodium efflux rates of H. crenulatus eggs into 100% seawater were measured using ²²Na as a tracer. The exchangeable sodium content of individual eggs increased from about 1 nmol to 4 nmol between cleavage and hatching (Table 2). Sodium efflux was resolved into a rapidly exchanging pool and a slowly exchanging pool at all stages (Fig. 5, Table 2). Significant estimates were obtained for the fast pool rate constants only at stages 1 and 5, with t_1/2 values of a few seconds and a few minutes, respectively. As for water, there was a large (>30-fold) decrease in the sodium efflux from the fast pool in stage 5 eggs. The slow pool data indicated that the embryos of H. crenulatus were relatively permeable to sodium at all stages, with efflux rate constants of 0.11–0.68 h^–1 and half times for sodium turnover of 1–6 h. In contrast to water fluxes, and the fast sodium pool, the mass-specific sodium efflux from the slow pool increased about 10-fold between cleavage and hatching (Table 2) and was associated with a corresponding increase in the permeability constant (from 0.14x10^–7 to 1.89x10^–7 cm s^–1).

View this table: [in this window] [in a new window]   Table 2. Exchangeable sodium contents and turnover rates for eggs of Hemigrapsus crenulatus at different developmental stages in 100% seawater (36 salinity) at 20°C

View larger version (22K): [in this window] [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).

Effect of dilute seawater on Na⁺/K⁺-ATPase activity of H. crenulatus embryos
The mean initial Na⁺/K⁺-ATPase activity of embryos in 100% seawater at stage 2 was 0.50±0.17 pmol P_i embryo^–1 min^–1 (N=10). Their activity increased sixfold to 3.25±0.31 pmol P_i embryo^–1 min^–1 (N=5) at stage 4 and by a further factor of four to 12.89±0.39 pmol P_i embryo^–1 min^–1 (N=5) at stage 5. At stage 4, the mean activity of embryos from the 50% seawater system was 6.61±0.60 pmol P_i embryo^–1 min^–1 (N=10), double that in 100% seawater (P<0.01). The value for embryos at stage 5 in 50% seawater was 11.79±0.84 pmol P_i embryo^–1 min^–1 (N=5) and not significantly different from that in 100% seawater (two-factor ANOVA, significant effect of stage F_1,21=131, P<0.0001, salinity not significant F_1,21=3.0, significant interaction F_1,21=29.3, P=0.002; Tukey tests).

Silver staining of embryos
Optical microscopy following the silver nitrate staining procedure revealed an oval brownish patch on the surface of embryos of H. sexdentatus and H. crenulatus at all stages of development except during cleavage (stage 1). The patch was irregular in outline, generally with a more densely stained rim, represented 5–8% of the surface area of the egg, and was always positioned over the yolk at the opposite pole from the embryo (Fig. 6). A discrete area of staining was not present in cleavage and blastula stages but smaller spots of variable size and intensity were distributed over the whole surface of the egg and in some cases cell outlines were emphasised (Fig. 6A).

View larger version (89K): [in this window] [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 this window] [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 this window] [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).

	Discussion

TOP Summary Introduction Materials and methods Results Discussion References

Water and sodium contents
The exchangeable water contents of H. crenulatus eggs in normal seawater increased from about 6 nl to 18 nl, between cleavage and hatching. Slightly larger measured volumes of the eggs (Table 1, Fig. 3) presumably reflect the volume occupied by yolk and other solid material. Exchangeable sodium contents (increasing from about 1–4 nmol) were comparable with analyses by atomic absorption spectroscopy (Seneviratna, 2003). Water and sodium in the eggs therefore appear to have fully equilibrated with the tracers in the 24 h loading period. Each pool was resolved into an extremely rapidly exchanging component that varied in size during development and a more slowly exchanging component. The rapidly exchanging fractions are necessarily located superficially and presumably correspond to peri-embryonic fluid situated between the embryo and the outer egg envelope. The outer membrane of the egg therefore appears to be a negligible barrier to water and ion movements.

Turnover of water and salts
The more slowly exchanging pools are believed to correspond to the embryo itself. Half times for efflux of the slow pools of water (0.25–1.25 h) and sodium ions (1–6 h) were much shorter than the periods during which post-gastrula stages of H. crenulatus eggs remained hyperosmotic to full-strength and dilute seawater. This implies that post-gastrula embryos actively hyper-osmoregulate with the effluxes of water and sodium balanced by equal influx rates. Although isotopically measured water fluxes primarily represent diffusive exchange (Potts and Parry, 1964; Rasmussen and Andersen, 1996) under the anisosmotic conditions observed here, net uptake of water by osmosis (and excretion by another route) would have taken place continuously. Additionally, at least one primary active transport step is required to drive the steady state flux of sodium through the embryo.

Water permeability of the embryo
The rate-limiting permeability barrier for turnover of the embryonic (slow) water pool probably resides within the ectoderm or the associated inner membrane. In Carcinus maenas gastrulae the inner membrane is chitinous (Cheung, 1966). It is secreted by the embryo and appears to be equivalent to the embryonic cuticle, later becoming a multiple layer as a result of embryonic moults (Goudeau and Lachaise, 1983). Diffusional water permeabilities for a range of cells and aquatic organisms, determined using isotopically labelled water, have been compiled by several authors (Potts and Parry, 1964; Stein, 1967; Prosser, 1973; Taylor, 1989; Rasmussen and Andersen, 1996). Normalised to the surface of H. crenulatus eggs the diffusional permeability constant of the slow pool (10^–6 cm s^–1; Table 1) was many orders of magnitudes lower than reported for internal cells such as erythrocytes and squid axons (10^–2–10^–4 cm s^–1) (Stein, 1967) and also lower than the permeabilities of most adult euryhaline and freshwater crustaceans (10^–4–10^–5 cm s^–1) (Potts and Parry, 1964; Taylor, 1989). The present values are comparable with animals generally regarded as rather impermeable e.g. the larvae of the freshwater insect Sialis lutaria (Shaw, 1955), goldfish (Potts and Parry, 1964) and shed fish eggs (Prescott and Zeuthen, 1953; Potts and Eddy, 1973). However, such comparisons obscure the importance of surface/volume ratio. When the water permeabilities of adult crustaceans are expressed in terms of their hourly exchange fractions (Taylor, 1989; Rasmussen and Andersen, 1996), the rate constants of H. crenulatus embryos (1–3 h^–1; Table 1) are positioned in the middle of the range. Clearly, the relatively low water permeability of H. crenulatus embryos does not eliminate the requirement to osmoregulate, although it is certainly an adaptation that reduces the metabolic cost of osmoregulation. The rate of water turnover decreased somewhat during development, implying a corresponding decrease in water permeability and further energetic saving, perhaps resulting from multiplication of the inner membrane.

Sodium turnover
Mass-specific sodium efflux rates from the embryonic pool of H. crenulatus increased tenfold between cleavage (6.6 mmol kg^–1 h^–1) and hatching (63.4 mmol kg^–1 h^–1; Table 2), indicating increasing active ion transport, and were associated with increased Na⁺/K⁺-ATPase activity of embryos (present data) (Taylor and Seneviratna, 2005). Sodium efflux rates for a range of cells and animals were documented by Potts and Parry (Potts and Parry, 1964). Turnover rates observed in H. crenulatus embryos were higher than in most freshwater animals (e.g. Asellus 0.4 mmol kg^–1 h^–1; Eriocheir 2 mmol kg^–1 h^–1), spanned the range exhibited by marine animals and euryhaline osmoregulators (e.g. Carcinus maenas 25 mmol kg^–1 h^–1 in seawater, 14 mmol kg^–1 h^–1 in 40% seawater; Nereis diversicolor 10.9 mmol kg^–1 h^–1 in seawater, 5.5 mmol kg^–1 h^–1 in freshwater), and even exceeded steady state turnover of many internal tissues (e.g. frog muscle 8.7 mmol kg^–1 h^–1; Sepia axons 25 mmol kg^–1 h^–1; rat diaphragm 117 mmol kg^–1 h^–1). It is concluded that the outer layers of H. crenulatus embryos do not provide isolation from ionic exchanges with the external seawater and the requirement to osmoregulate.

Tolerance of dilute seawater by developing embryos
Early ovigerous females of H. edwardsii have been observed in rock pools with salinity <5 near freshwater streams and ovigerous H. crenulatus may be collected from estuarine channels where river and seawaters mix. The previously demonstrated tolerance of post-gastrula embryos to acute changes in osmolality over a very wide range is therefore adaptive to conditions that may be encountered in their habitat (Taylor and Seneviratna, 2005). Prior to gastrulation, cleavage and blastula stages of H. sexdentatus and H. crenulatus were osmoconformers and were less tolerant of dilution. Similarly, embryos of the estuarine grapsids Chasmagnathus granulata and Cyrtograpsus angulatus acquired greater tolerance of extreme salinities (3–44) 2 or 3 days following egg extrusion (Bas and Spivak, 2000). These authors attributed the increased tolerance to a reduction in egg permeability but in H. crenulatus water permeability did not change appreciably at this time. A plausible alternative interpretation is that the increased survival was associated with gastrulation and the acquisition of the capacity to hyper-osmoregulate.

In the longer term trials reported here the embryos tolerated a narrower range of salinity. Normal embryogenesis and hatching of H. sexdentatus and H. crenulatus occurred in 100% and 50% seawater (36 to 18 salinity) but not in 25% seawater (9 salinity). Although adult crabs are capable of indefinite survival below 10% seawater (Hicks, 1973; Bedford and Leader, 1977) (H.H.T., unpublished observations), ovigerous crabs presumably must remain within regions of the shore or estuary where the average salinity is higher. Similarly, complete development of C. granulata and C. angulatus only occurred between salinity 12 and 40 (Bas and Spivak, 2000), and in the estuarine ocypodid Macrophthalmus hirtipes, between salinity 18 and 35 (Jones and Simons, 1982). Interestingly, although exposure of embryos to 25% and 50% seawater before gastrulation was not obviously detrimental in the short term (present study) (Taylor and Seneviratna, 2005) when exposure was prolonged, development and hatching were subsequently impaired. Possibly, the osmotic stress disrupted critical morphogenetic events around cleavage and gastrulation and this contributed to their failure to develop normally. In the light of the greater vulnerability of pregastrula embryos of intertidal grapsids to osmotic shock, it is important to investigate whether ovigerous crabs exhibit behavioural or other mechanisms to protect the egg clutch during this short period.

The progressive increase in the volume of the Hemigrapsus spp. eggs incubated in seawater (Fig. 3) accords with observations on other Brachyura (Wear, 1974; Valdes et al., 1991). Little additional swelling occurred in 50% and 25% seawater, implying that egg volume was regulated, i.e. the embryos possess mechanisms for excreting water gained osmotically. Interestingly, swelling was observed in the eggs that were introduced to dilute seawater during cleavage and failed to gastrulate normally. This also suggests that egg volume depends on embryonic physiological processes rather than the properties of the envelope.

Acclimation to dilute seawater
A link between activities of the Na⁺/K⁺-ATPase in branchial and other epithelia and the osmoregulatory capacity of larval and adult crustaceans is well-established and many authors have demonstrated that this primary active transporter is up-regulated in response to lowered external salt concentrations (Holliday, 1985; Wheatley and Henry, 1987; Thuet et al., 1988; Holliday et al., 1990; Bouaricha et al., 1991; Bouaricha et al., 1994; Charmantier et al., 2001; Towle et al., 2001). The present observation of doubled Na⁺/K⁺-ATPase activity in H. crenulatus embryos at stage 4 that had been chronically exposed to 50% seawater, taken together with observations of increased Na⁺/K⁺-ATPase activity during acute (24 h) hyposaline exposures (Taylor and Seneviratna, 2005) suggest that the capability to mount an osmoregulatory acclimation response is present even in the earliest embryonic stages.

Sites of uptake and excretion of water and salts in embryos
Silver staining has been used to identify putative osmoregulatory epithelia in a number of insects (Krogh, 1939) and crustaceans (Koch, 1934; Ewer and Hatlingh, 1952; Talbot et al., 1972; Barra et al., 1983; Felder et al., 1986; Dickson and Dillaman, 1991; Kikuchi and Matsumasa, 1993; Lindhjem et al., 2000; Haond et al., 2001). The appearance of the silver-staining patch on the surface of the embryo at gastrulation coincided with the acquisition of hyper-osmoregulatory capacity, tempting the suggestion that the patch is concerned with ion transport. Silver-stained areas often correspond to epithelia involved in active ion uptake although the mechanism of staining is uncertain. Ag⁺ may bind to Na⁺ transporters because of similarities between these two ions (Koch, 1934; Krogh, 1939). Conversely, epithelia concerned with salt extrusion may stain with silver ions by precipitating AgCl at sites of Cl^– efflux, e.g. the neck organ of hypo-regulating Artemia salina nauplii (Conte et al., 1972) and the apical pit of marine teleosts (Philpott, 1965). Identification of considerable deposits of AgCl within the silver stained-patch on H. sexdentatus and H. crenulatus eggs implicates this region in chloride extrusion. Furthermore, the location of AgCl between the outer and inner membranes of the egg indicates that at this location the inner membrane is permeable to Cl^– ions and the outer membrane to Ag⁺ ions.

The essential features of all multicellular hyperosmoregulators are, firstly, a site on the body surface for the active uptake of salts from the external medium into a hyperosmotic extracellular compartment and secondly, a mechanism for the delivery of extracellular fluid and salts (i.e. urine) to the exterior, to compensate for the osmotic water uptake. In adult decapod crustaceans these requirements typically are met by ion uptake into the haemolymph by branchial ionocytes and ultrafiltration via the antennal organs (Mantel and Farmer, 1983; Pequeux, 1995). The same general scheme has been applied even to very simple animals such as a freshwater coelenterate (Marshall, 1969) or embryos of freshwater molluscs (Beadle, 1969a; Beadle, 1969b; Beadle and Beadle, 1969; Taylor, 1977) in which extracellular fluid is expelled from the enteron and the blastocoel, respectively. What are the corresponding structures in hyper-regulating crab embryos?

In post-gastrula decapod embryos the extracellular space is delimited by the embryonic and the extra-embryonic ectoderm, which extend over and enclose the yolk (Anderson, 1973). In decapods (e.g. Palaemonetes, Leander, Crangon, Homarus, Astacus, Palinurus, Galathea, Eupagurus, and another grapsid Leptograpsus) the embryonic dorsal organ is a thickening of the extraembryonic ectoderm in the dorsal midline at the opposite pole to the developing embryo (Anderson, 1973; Fioroni, 1980). It is believed to be concerned with the histolysis of the extraembryonic ectoderm during the formation of the body wall (Anderson, 1973). Precipitation of AgCl in this region indicates that the embryonic dorsal organs of H. sexdentatus and H. crenulatus allow the passage of electrolytes to the exterior. We propose that continuous osmotic entry of water into the extracellular compartment generates a small internal hydrostatic pressure that drives the paracellular exit of extracellular fluid; i.e. the embryonic dorsal organ serves as a simple filtration-type excretory organ. Whether cells in this region have podocyte-like specialisations for ultrafiltration or simply form a discontinuous barrier permitting leakage, and whether there are mechanisms for reclaiming useful molecules, are topics for future investigation. A hypothetical scheme for osmoregulation in Hemigrapsus embryos is presented in Fig. 9. The site of salt uptake is unknown but is perhaps a property of the general ectoderm. The dorsal organ persists until hatching, by which time the antennal organs are formed and are presumably functional (Anderson, 1973).

View larger version (32K): [in this window] [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.

Water and salt balance during cleavage
Pregastrula stages are hyper-osmoconformers maintaining a small positive osmolality difference (150 mmol l^–1) between the medium and the water (Taylor and Seneviratna, 2005). Given their relatively high permeability to water, there is a requirement for water excretion in cleavage stages also. Possibly, the mottled deposits of AgCl observed in blastulae indicate salt release at multiple loci by temporary rupture of cell junctions between blastomeres as observed in pulmonate embryos (Taylor, 1977).

Conclusions
In summary, the present investigations have provided further evidence for the development of osmoregulatory function from the earliest embryonic stages of crabs. The generality of these conclusions for decapods should now be investigated. Previous studies on embryos of homarid lobsters and freshwater crayfish emphasised the osmoprotective role and apparently low permeability of the egg envelopes. Although the permeabilities of crab embryos in the present study were low compared with larger aquatic animals, they were certainly too high to provide effective osmotic isolation from the external medium. It was suggested (Taylor and Seneviratna, 2005) that the envelopes of homarid and astacid embryos may be more permeable to salts and/or water than previously supposed. These embryos also possess embryonic dorsal organs (Fioroni, 1980; Martin and Laverack, 1992). Comparative studies on the development, ultrastructure and fate of embryonic dorsal organs among crustaceans, immunocytochemical investigations of the appearance and localisation of membrane transporters, and their transcriptional control during embryogenesis, clearly would help to elucidate the specific adaptations required to achieve water and salt balance of these critical life stages.

	Acknowledgments

 
This work was carried out during the tenure of a PhD scholarship awarded to D.S. by the New Zealand Ministry of Foreign Affairs and Trade under the NZODA program. We are grateful to Jan McKenzie and Gavin Robinson for skilled technical assistance.

	References

TOP Summary Introduction Materials and methods Results Discussion References

 

Aladin, N. V. and Potts, W. T. W. (1995). Osmoregulatory capacity of the Cladocera. J. Comp. Physiol. 164B,671 -683.

Anderson, D. T. (1973). Embryology and Phylogeny of Annelids and Arthropods. Oxford: Pergamon Press.

Barra, J., Pequeux, A. and Humbert, W. (1983). A morphological study on gills of a crab acclimated to fresh water. Tissue Cell 15,583 -596.[CrossRef][Medline]

Bas, C. C. and Spivak, E. D. (2000). Effect of salinity on embryos of two southwestern Atlantic estuarine grapsid crab species cultured in vitro. J. Crust. Biol. 20,647 -656.

Beadle, L. C. (1969a). Salt and water regulation in the embryos of freshwater pulmonate molluscs. I. The embryonic environment of Biomphalaria sudanica and Lymnea stagnalis. J. Exp. Biol. 50,473 -479.[Abstract/Free Full Text]

Beadle, L. C. (1969b). Salt and water regulation in the embryos of freshwater pulmonate molluscs. III. Regulation of water during the development of Biomphalaria sudanica. J. Exp. Biol. 50,491 -499.[Abstract/Free Full Text]

Beadle, L. C. and Beadle, S. F. (1969). Salt and water regulation in the embryos of freshwater pulmonate molluscs. II. Sodium uptake during the developement of Biomphalaria sudanica. J. Exp. Biol. 50,481 -489.[Abstract/Free Full Text]

Bedford, J. J. and Leader, J. P. (1977). The composition of the haemolymph and muscle tissue of the shore crab, Hemigrapsus edwardsii, exposed to different salinities. Comp. Biochem. Physiol. 57A,341 -345.[CrossRef]

Bouaricha, N., Thuet, P., Charmantier, G., Charmantier-Daures, M. and Trilles, J.-P. (1991). Na⁺-K⁺ ATPase and carbonic anhydrase activities in larvae, postlarvae and adults of the shrimp Penaeus japonicus (Decapoda, Penaeidea). Comp. Biochem. Physiol. 100A,433 -437.[Medline]

Bouaricha, N., Charmantier-Daures, M., Thuet, P., Trilles, J.-P. and Charmantier, G. (1994). Ontogeny of osmoregulatory structures in the shrimp Penaeus japonicus (Crustacea, Decapoda). Biol. Bull. 186,29 -40.[Abstract]

Charmantier, G. (1998). Ontogeny of osmoregulation in crustaceans: a review. Invertebr. Reprod. Dev. 33,177 -190.

Charmantier, G. and Aiken, D. E. (1987). Osmotic regulation in late embryos and prelarvae of the American lobster Homarus americanus H. Milne-Edwards Crustacea, Decapoda. J. Exp. Mar. Biol. Ecol. 109,101 -108.[CrossRef]

Charmantier, G. and Charmantier-Daures, M. (1991). Ontogeny of osmoregulation and salinity tolerance in Cancer irroratus elements of comparison with C. borealis.Biol. Bull. 180,125 -134.[Abstract]

Charmantier, G. and Charmantier-Daures, M. (2001). Ontogeny of osmoregulation in crustaceans: the embryonic phase. Am. Zool. 41,1078 -1089.

Charmantier, G., Charmantier-Daures, M., Bouaricha, N., Thuet, P., Aiken, D. E. and Trilles, J. P. (1988). Ontogeny of osmoregulation and salinity tolerance in two decapod crustaceans: Homarus americanus and Penaeus japonicus. Biol. Bull. 175,102 -110.[Abstract/Free Full Text]

Charmantier, G., Haond, C., Lignot, J. and Charmantier-Daures, M. (2001). Ecophysiological adaptation to salinity throughout a life cycle: a review in homarid lobsters. J. Exp. Biol. 204,967 -977.[Abstract]

Charmantier, G., Gimenez, L., Charmantier-Daures, M. and Anger, K. (2002). Ontogeny of osmoregulation, physiological plasticity and larval export strategy in the grapsid crab Chasmagnathus granulata. Mar. Ecol. Prog. Ser. 229,185 -194.

Cheung, T. S. (1966). The development of egg-membranes and egg attachment in the shore crab, Carcinus maenas, and some related decapods. J. Mar. Biol. Assoc. UK 46,373 -400.

Conte, F. P., Hootman, S. R. and Harris, P. J. (1972). Neck organ of Artemia salina Nauplii. J. Comp. Physiol. 80,239 -246.[CrossRef]

Dickson, J. S. and Dillaman, R. M. (1991). Distribution and characterization of ion transporting and respiratory filaments in the gills of Procambarus clarkii. Biol. Bull. 180,154 -166.[Abstract]

Ewer, D. W. and Hatlingh, I. (1952). Absorption of silver on the gills of a freshwater crab. Nature 169, 460.[Medline]

Ewing, R. D., Peterson, G. L. and Conte, F. P. (1974). Larval salt gland of Artemia salina nauplii. J. Comp. Physiol. 88,217 -234.[CrossRef]

Felder, J. M., Felder, D. L. and Hand, S. C. (1986). Ontogeny of osmoregulation in the estuarine ghost shrimp Callianassa jamaicense var. louisianensis Schmitt (Decapoda, Thalassinidea). J. Exp. Mar. Biol. Ecol. 99, 91-105.[CrossRef]

Fioroni, V. P. (1980). The dorsal organ of arthropods with special reference to crustacea malacostraca – a comparative embryological survey. Zool. Jb. Anat. 104,425 -465.

Goudeau, M. and Lachaise, F. (1983). Structure of the egg funicululus and deposition of embryonic envelopes in a crab. Tissue Cell 15,47 -62.[CrossRef][Medline]

Haond, C., Charmantier, G., Flik, G. and Wendelaar Bonga, S. E. (2001). Identification of respiratory and ion-transporting epithelia in the phyllosoma larvae of the slipper lobster Scyllarus arctus. Cell Tissue Res. 305,445 -455.[Medline]

Hicks, G. R. F. (1973). Combined effects of temperature and salinity on Hemigrapsus edwardsi (Hilgendorf) and H. crenulatus (Milne Edwards) from Wellingon Harbour, New Zealand. J. Exp. Mar. Biol. Ecol. 13, 1-14.

Holliday, C. W. (1985). Salinity-induced changes in gill Na⁺,K⁺-ATPase activity in the Mud Fiddler Crab, Uca pugnax. J. Exp. Zool. 233,199 -208.[CrossRef]

Holliday, C. W., Roye, D. B. and Roer, R. D. (1990). Salinity-induced changes in branchial Na⁺/K⁺–ATPase activity and transepithelial potential difference in the brine shrimp Artemia salina. J. Exp. Biol. 151,279 -296.[Abstract/Free Full Text]

Hootman, S. R. and Conte, F. P. (1975). Functional morphology of the neck organ in Artemia salina nauplii. J. Morphol. 145,371 -386.[CrossRef]

Hootman, S. R., Harris, P. J. and Conte, F. P.</