Ontogeny of Osmoregulation in Crustaceans: The Embryonic Phase 1

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1 AMER. ZOOL., 41: (2001) Ontogeny of Osmoregulation in Crustaceans: The Embryonic Phase 1 GUY CHARMANTIER 2 AND MIREILLE CHARMANTIER-DAURES Laboratoire d Ecophysiologie des Invertébrés, EA 3009 Adaptation Ecophysiologique au cours de l Ontogenèse, Université Montpellier II, Place Eugène Bataillon, Montpellier cedex 05, France SYNOPSIS. Following a brief overview of the patterns of ontogeny of osmoregulation in postembryonic stages, this review concentrates on the ontogeny of osmoregulation during the embryonic development of crustaceans, particularly in those species living under variable or extreme salinity conditions and whose hatchlings osmoregulate at hatch. Two situations are considered, internal development of the embryos in closed incubating, brood or marsupial pouches, and external development in eggs exposed to the external medium. In both cases, embryos are osmoprotected from the external salinity level and variation, either by the female pouches or by the egg envelopes. The mechanisms of osmoprotection are discussed. During embryonic life, temporary or definitive osmoregulatory organs develop, with ion transporting cells and enzymes such as Na -K ATPase, permitting the embryos and then the hatchlings to osmoregulate and tolerate the external salinity. INTRODUCTION Salinity is one of the main environmental factors that wield a selective pressure on aquatic organisms. Its level and variations have an impact on the composition and osmolality of the body fluids of animals, among which some groups or species possess physiological abilities of osmoregulation. Species that are able to efficiently osmoregulate are generally euryhaline. Osmoregulation may thus be viewed as an adaptive function enabling animals to occupy habitats with high, low and/or variable salinity, where a wide salinity tolerance is required for survival. Crustaceans display several patterns of osmoregulation, extensively studied in adult stages (reviews in Mantel and Farmer, 1983; Péqueux, 1995). Since natural selection acts on all stages of development (Bartholomew, 1987; Burggren, 1992), a complete overview of the adaptive strategy of a given species must be based on studies conducted on each stage of the life cycle, from adults to reproductive cells, embryos and larvae, postlarvae or juveniles (Charmantier and Wolcott, 2001). 1 From the Symposium Ontogenetic Strategies of Invertebrates in Aquatic Environments presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 3 7 January 2001, at Chicago, Illinois. 2 charmantier@univ-montp2.fr The ontogeny of osmoregulation has been studied in several species of crustaceans since the early work of Kalber and Costlow (1966). A previous review on the subject (Charmantier, 1998) showed that ontogenetic studies of osmoregulation have been conducted mainly in larvae and postlarvae, i.e., in postembryonic stages. The present overview will focus on the ontogeny of osmoregulation during the embryonic phase with a brief summary regarding the postembryonic stages. Three patterns of ontogeny of osmoregulation have been established from the data available in several species (Charmantier, 1998; Fig. 1). In one group of species (pattern 1), osmoregulation varies little with developmental stage; the adults of these species, mostly marine and stenohaline, are weak regulators or osmoconformers. In another group (pattern 2), the adult type of osmoregulation is established as early as the first post-embryonic stage; adults are euryhaline and generally live in environments where salinity is high, low, or variable; this group thus includes freshwater species. In the third group of species (pattern 3), larvae osmoconform or are slightly able to osmoregulate; metamorphosis marks the appearance of the adult type of osmoregulation; adults are mesohaline or euryhaline and live in environments of more or less variable salinity. 1078

2 OSMOREGULATION IN CRUSTACEAN EMBRYOS 1079 FIG. 1. Illustrated schematic patterns of post-embryonic ontogeny of osmoregulation in crustaceans. 1: Little variation in osmoregulation with developmental stage; adults osmoconform. 2 A, B: Adult type of osmoregulation established in first post-embryonic stage ( larvae and postlarvae correspond to early and late juveniles in cases of direct development); adults osmoregulate. 3 A, B: Shift from larval to adult type of osmoregulation during the post-embryonic development, usually at metamorphosis; larvae osmoconform or slightly osmoregulate; adults osmoregulate. In pattern 1, overlapping graphs corresponding to different stages have been separated for clarity of presentation. The physiological basis of these patterns and their ecological consequences have also been reviewed (Charmantier, 1998). As in adults, the occurrence of osmoregulation in young postembryonic stages is based on efficient ionic regulation (mainly of Na and Cl ) and on increased levels and/or presence of enzymes involved in ion transport (mainly Na -K ATPase, and other enzymes such as carbonic anhydrase). Osmoregulatory epithelia may appear at different times of development and at different locations, usually in organs of the branchial chamber. These events appear integrated during development, at least in those species in which sufficient data have been collected. Anatomical changes (development of osmoregulatory epithelia) lead to physiological modifications (increased Na -K ATPase activity and/or presence, linked to an increase in osmoregulatory capacity) that permit an augmented salinity tolerance allowing ecological adaptation to environments of variable and/or extreme salinity. In species or stages incapable of extracellular osmoregulation, i.e., in osmoconforming organisms, a limited degree of tolerance to salinity variation may originate from cell volume regulation through intracellular isosmotic regulation. This process has been extensively studied in adult crustaceans (reviews in Gilles and Péqueux, 1983; Kirschner, 1991). From the limited information available in early post-embryonic stages, intracellular free amino acids play, as in adults, a major function in the maintenance of the osmotic equilibrium between the cells and hemolymph (Haond et al., 1999). Depending on the pattern of ontogeny of osmoregulation, the ability to osmoregulate may occur at different stages of development. It never appears during the entire lifetime of species displaying pattern 1. In contrast, the time of occurrence of osmoregulation is well defined in pattern 3-species, often during the massive tissue reorganization at metamorphosis. In both cases, young larvae are totally or almost completely devoid of the ability to osmoregulate. By contrast, in pattern 2, the first post-embryonic stage is already able to osmoregulate at hatch. We may thus hypothesize that the ability to osmoregulate occurs earlier in development, at some time during embryogenesis. This hypothesis is the central issue discussed in the present review, along with the ontogeny of osmoregulatory mechanisms during the embryonic life. Studies on osmoregulation during embryonic development have been reported in a limited number of crustacean species (Table 1). They can be divided into two groups based on the model of separation of the embryonic cell mass from the external medium. Embryonic development is either internal, with embryos located inside the

3 1080 G. CHARMANTIER AND M. CHARMANTIER-DAURES List of studies conducted on the ontogeny of osmoregulation during the embryonic phase in crusta- TABLE 1. ceans. Internal development: Embryos or eggs isolated from external medium in the female body External development: Eggs exposed to external medium Branchiopoda Cladocera: several species Malacostraca Amphipoda: Orchestia gammarellus Isopoda: Sphaeroma serratum Branchiopoda Cladocera: several species Anostraca: Artemia sp. Malacostraca Amphipoda: Gammarus duebeni Isopoda: Cyathura polita Decapoda: Callianassa jamaicense Homarus americanus Astacus leptodactylus body of the female which shields them from the medium, or external, with embryos developing in eggs directly exposed to the external medium and with egg envelopes as the sole barrier between embryo and water. These two cases will be considered separately, starting with internal development. Aladin and Potts, 1995 Morritt and Spicer, 1996a, b, c, 1999 Charmantier and Charmantier- Daures, 1994 Aladin and Potts, 1995 Conte, 1984 Morritt and Spicer, 1995, 1996a Kelley and Burbanck, 1976 Felder et al., 1986 Charmantier and Aiken, 1987 Susanto and Charmantier, 2001 ONTOGENY OF OSMOREGULATION IN EMBRYOS: INTERNAL DEVELOPMENT Osmoregulation in Cladocera has been comprehensively reviewed by Aladin and Potts (1995). Within this order, incubation of eggs is effected in brood chambers formed by the carapace, except in laying resting eggs (Krogh, 1939). Brood chambers are closed in several species, some of them inhabiting fresh or slightly saline waters and most of them living in marine and saline continental waters. In both cases, the embryonic fluid osmolality is isosmotic to the brood chamber osmolality which is itself close to the hemolymph osmolality, the latter being either hypo-osmotic to the medium in hyper-saline waters or strongly hyper-regulated in freshwater (Aladin and Potts, 1995). This mechanism offers osmotic protection to the embryos until late in their development. In addition, nutrients destined for the embryos are secreted into the brood chamber liquid. Shortly before hatching, the brood chamber opens, which results in the exposure of its content to the ambient salinity. The emerging larvae are then able to osmoregulate with osmoregulatory organs, including the neck organ (in some cases later replaced by epipodites), which have developed during the embryonic life (Aladin and Potts, 1995). Sphaeromid isopods spend their entire life cycle in shallow coastal marine or lagoon waters, where salinity is variable. In Sphaeroma serratum, after spawning, eggs are transferred into ventral incubating pouches that open to the exterior by narrow slits (review in Charmantier and Charmantier-Daures, 1994). The developing embryos are bathed in a thick incubating pouch fluid (Leichmann, 1891), which may function in nourishing the brood (Monod, 1926; Saudray, 1954; Green, 1965; Hoese and Janssen, 1989). If extracted from the incubating pouches and exposed to low salinities, early and late embryos are, respectively, osmoconformers and slight hyper-osmoregulators. In the female, the embryos are isosmotic or slightly hyper-osmotic to the incubating pouch fluid osmolality, itself isosmotic to the hemolymph whose osmolality is strongly hyper-regulated at low salinity. The incubating pouches thus provide an osmotic protection to the brood of Sphaeroma serratum (Charmantier and Charmantier-Daures, 1994), as Klapow (1970) had theoretically postulated in the isopod Excirolana chiltoni. By the time they leave the pouches, the young sphaeromid juveniles have acquired a limited but efficient ability to hyper-osmoregulate, which increases in subsequent stages.

4 OSMOREGULATION IN CRUSTACEAN EMBRYOS 1081 Amphipods also provide different models for the study of the ontogeny of osmoregulation and an extensive series of works has been conducted on several of their species, either on naturally-developing broods or on artificially-cultured eggs and embryos (Morritt and Spicer, 1996a). Among these species, the semi-terrestrial beachflea Orchestia gammarellus lives in supralittoral strandline habitats where major changes in salinity can follow inundation by sea water or estuarine water, or rainfall, evaporation, etc. Adults are able to hyper-hypo-osmoregulate (Moore and Francis, 1985), i.e., to hyper-regulate at low salinity and to hyporegulate in sea water and at high salinity. The eggs are laid in a semi-closed marsupium limited by the ventral surface of the thoracic segments and the oostegites. While recently laid eggs cannot be cultured in salinities below 25% sea water (Morritt and Spicer, 1996a), females can raise broods at salinities down to 10% sea water (Morritt and Stevenson, 1993). This is most probably achieved through the control of the marsupial fluid osmolality by the female, which directs urine (isosmotic to hemolymph) from the antennary gland into the ventral chamber (Moore et al., 1993; Spicer and Taylor, 1994; Morritt and Spicer, 1996b). The marsupial fluid osmolality is thus maintained hyper-osmotic to the external medium at low salinity. In addition, the hyper-hypo-osmoregulatory ability develops early in the embryos of Orchestia gammarellus (Morritt and Spicer, 1996c, 1998). The embryos are able to maintain the concentration of the peri-embryonic fluid within narrow limits over the concentration range encountered in the marsupium. However the ability to hyper-hypoosmoregulate becomes temporarily much weaker in immediate post-hatch hatchlings. This physiological change might represent a switch in osmoregulatory sites, from a system associating the vitelline membrane and the embryonic dorsal organ (Turquin, 1967; Magniette, 1979; cited in Morritt and Spicer, 1995; the dorsal organ has also been described in Orchestia cavimana by Meschenmoser, 1989) to the coxal gills (Morritt and Spicer, 1996c, 1998). Young hatchlings remain within the female s marsupium, where they are osmotically protected as were the early embryos, until their ability to osmoregulate increases again. They thus would attain a level of physiological competency in osmoregulation before leaving the marsupium (Morritt and Spicer, 1999). In summary, the strategy of Orchestia gammarellus toward confronting variable salinity associates osmoprotection in the marsupium of the female (for early embryos and young hatchlings) and the development of autonomous osmoregulation in embryos and in later post-embryonic stages, based on two successive osmoregulatory sites. Similar adaptations involving the ventral chamber in the control of the embryonic osmotic environment have been reported in terrestrial amphipods, and they suggest that the ventral chamber can be viewed as a preadaptation to the colonization of land by the Talitridae (Morritt and Richardson, 1998). The apparent ability to regulate the marsupial fluid osmolality has also been reported in other systematic groups, particularly in the euryhaline mysids Neomysis integer (Ralph, 1965, cited in Morritt and Spicer, 1996b) and Praunus flexuosus (McLusky and Heard, 1971), and in some terrestrial isopods (Hoese, 1984; Hoese and Janssen, 1989). Most freshwater calanid copepods spawn in ovisacs and it has been suggested that the ovisacs would provide osmotic protection to the eggs and that they might be considered as a major adaptation to saline conditions in continental waters (Bernard, 1971). ONTOGENY OF OSMOREGULATION IN EMBRYOS: EXTERNAL DEVELOPMENT We will now consider the case of embryos which develop in eggs directly exposed to the external medium (Table 1). The eggs can be kept in open brood pouches or attached to the pleopods of the female, as in pleocyematan decapods, or freely released in the environment, as in penaeid shrimps. In Cladocera, beside the cases of internal development considered above, the brood chamber remains open to the environment in many, including most brackish and freshwater genera and some species living in hypersaline continental waters. The osmolality of the embryonic fluid is most generally

5 1082 G. CHARMANTIER AND M. CHARMANTIER-DAURES regulated, hyper-osmotically in freshwater and hypo-osmotically under hypersaline conditions (Aladin and Potts, 1995). The egg envelopes would protect the embryo from the hostile environment, at least until embryonic or larval organs of osmoregulation have developed. In freshwater forms, these include the nuchal gland, or neck organ, with ion transporting cells for salt uptake, and excretory organs for water excretion. The neck organ degenerates at hatch in those forms which develop epipodites but is otherwise retained (Halcrow, 1982; Aladin, 1983; Aladin and Valdivia Villar, 1987; Aladin and Potts, 1995). The anostracan branchiopod Artemia sp. is known for its adaptability to hypersaline media. Depending on environmental conditions, it can shift from viviparous to oviparous reproduction, with production of encysted and dehydrated embryos. In rehydrated cysts, embryos are protected against the external high salinity by the cyst envelopes which are permeable to water but impermeable to ions (Dutrieu, 1960); the embryos rely on a trehalose-glycerol mechanism for hyper-osmoregulation (review in Thuet, 1982). At hatch, the emerging nauplius is usually confronted with very high salinities. It reacts through strong hypo-osmoregulation permitted by active ion excretion mediated by Na -K ATPase and effected by a dorsal organ or salt gland, which develops in late pre-naupliar embryonic stages and becomes apparently functional shortly before hatching. Osmoregulation later shifts to coxal gills (Conte et al., 1972, 1977; Peterson et al., 1978; Russler and Mangos, 1978; reviews in Thuet, 1982; Conte, 1984). The amphipod Gammarus duebeni lives in habitats subjected to high salinity changes, from coastal freshwater streams to intertidal and evaporating pools. Adults are hyper-iso-osmoregulators, i.e., they hyperregulate at low salinity and are osmoconformers in sea water and at high salinity (review in Morritt and Spicer, 1995). The eggs are carried by the female in an open marsupium. Experiments carried out on in vitro-cultured eggs showed that early embryos (stage 2/3) are able to regulate their peri-embryonic fluid in a hyper-isosmotic pattern. In medium and late embryos (stages 5, 6, 7) regulation is hyper-hypo-osmotic, before reverting to hyper-isosmotic in hatchlings (Morritt and Spicer, 1995). The significance of the temporary ability to hyper-hypo-regulate is unknown, although it may be associated with the appearance of the coxal gills, the presumed primary osmoregulatory organs in juveniles and adults, and with the subsequent degeneration of the dorsal organ. This organ, and/or the vitelline membrane, might be the site(s) responsible for embryonic osmoregulation (Vlasbom and Bolier, 1971; Bregazzi, 1973; review in Morritt and Spicer, 1995). Another studied case of species using an open marsupium for egg development is the isopod Cyathura polita. This euryhaline species, usually found in brackish waters, can tolerate salinities ranging from freshwater to 40, and embryonic development can proceed in waters from 0 to 30 salinity (Kelley and Burbanck, 1976). Each embryo is surrounded, from exterior to interior, by the chorion, the vitelline membrane and the embryonic membrane (Strömberg, 1972). The vitelline fluid (inside the vitelline membrane) is hyper-osmotically regulated in stage 1 of embryonic development, then regulation progressively changes to hyper-hypo-osmotic. Following the first hatching, which corresponds to the shedding of the two outer membranes, the embryonic fluid regulation (inside the embryonic membrane) is hyper-osmotic in stage 5 and becomes hyper-hypo-osmotic in the following stages, through the second (last) hatching and in post-hatch marsupial and free juvenile stages (Kelley and Burbanck, 1976). From these observations and following experiments involving membrane removals, the authors concluded that the vitelline and embryonic membranes are essential to osmoregulation during development. In addition embryonic organs, which are present in all isopods except the epicarids, are represented by one dorsal and two dorsolateral organs in Cyathura polita that develop at different times of the embryonic life (Strömberg, 1972). The structure of their cells, including apical infoldings, and their apical communication with the vitelline fluid (dorsolateral organs) or with the

6 OSMOREGULATION IN CRUSTACEAN EMBRYOS 1083 water in the female marsupium (dorsal organ) suggest a role in osmoregulation. In decapod crustaceans, the effect of salinity on embryonic development has been investigated in several species (review in Bas and Spivak, 2000) but information on the embryonic ontogeny of osmoregulation is scarce. In the thalassinid ghost shrimp Lepidophthalmus louisianensis (formerly Callianassa jamaicense louisianensis) which lives in burrows in estuarine habitats of low and variable salinity (Felder et al., 1986), larvae are released in the parental burrows from eggs carried by the female. Hyper-osmotic ability is present at the time of hatching. The activity of the Na -K ATPase was measured in four stages, S1 S4, covering the embryonic development and in larvae. The enzyme activity was low in stages S1 S2, increased in stage S3, reached a peak in stage S4 immediately preceding hatching, and decreased to a still high level in larval stages. Hyper-osmotic ability, which is present at the time of hatching (Felder et al., 1986), probably occurs late in the embryonic development, as shown by the increase in Na -K ATPase activity. The ontogeny of osmoregulation in embryos has also been studied in two species of Astacidea. The homarid lobster Homarus americanus differs from all other species cited above since its ontogeny of osmoregulation belongs to pattern 3: lobster larvae are osmoconformers before the acquisition of the ability to osmoregulate at metamorphosis (review in Charmantier et al., 2001). It is thus not suprising that experiments involving transfer to dilute media of intact or artificially opened eggs have shown that the embryos are unable to osmoregulate by themselves; they are osmotically protected by the outer egg membrane inside which the embryo remains hyper-osmotic at low salinity (Charmantier and Aiken, 1987). Crayfish are known for their strong ability to hyper-osmoregulate in freshwaters which constitute their most common habitat (reviews in Mantel and Farmer, 1983; Wheatly and Gannon, 1995; Susanto and Charmantier, 2000). In Astacus leptodactylus, this ability is already existent in juveniles at hatch (hemolymph osmolality: 290 mosm/kg), although at a lower level compared to adults (hemolymph osmolality: 420 mosm/kg) (Susanto and Charmantier, 2000). This species, as probably crayfish in general, thus presents a pattern 2 ontogeny of osmoregulation and offers a convenient decapod model to study the occurrence of osmoregulation during the embryonic development. The eggs, carried by the female on its pleopods, are directly exposed to the external medium. The embryonic development can be followed through the measurement of the eye dimensions, giving an eye index which is close to 450 m at hatch. The large size of the eggs, approximately mm in diameter, permits experimental approaches including the measure of the osmolality of the peri-embryonic fluid and, when available, of the embryo s hemolymph. The peri-embryonic fluid and/or hemolymph osmolality remain stable at mosm/kg from early to late embryos and it decreases only prior to hatching, probably following an osmotic uptake of water. This demonstrates the osmotic protection offered by the egg envelope during the majority of the duration of the embryonic development. Artificial opening and removal of the egg envelope, followed by direct exposure to freshwater, demonstrated that the ability to hyper-osmoregulate, and consequently to survive, in freshwater appears only a few hours or days (according to temperature) before hatching in embryos with eye index 410 m (Susanto and Charmantier, 2001). The ability to osmoregulate probably originates at least partly from the occurrence at eye index m of ionocytes in the gills where Na -K ATPase mediates active ion uptake as observed through electron microscopy and immunofluorescent microscopy (unpublished data, J. H. Lignot, G. N. Susanto and G. Charmantier). SYNTHESIS AND DIRECTIONS FOR FUTURE WORK Considering the three patterns of ontogeny of postembryonic osmoregulation, it is clear that the studied species classed in pattern 1 (all stages are weak osmoregulators or osmoconformers) are marine stenohaline osmoconformers (Charmantier, 1998). Their

7 1084 G. CHARMANTIER AND M. CHARMANTIER-DAURES FIG. 2. Synthesis of ontogeny of osmoregulation during the embryonic phase in pattern 2 species of crustaceans, established from the published data. See text and Figure 1 for definition of pattern. See text and Table 1 for list of species and references. The shaded bars represent the outset and end of events, with lighter shades for timing changes according to species. Dotted lines indicate that differentiated organs and ability to osmoregulate are retained in the postembryonic phase. internal osmolality is close to that of sea water, approximately 1,000 mosm/kg. Although, to our knowledge, no experimental data are available regarding their eggs or embryos osmolality, it can be safely assumed that it is also close to such values. No particular osmotic protection and no autonomous ability to osmoregulate are thus required during embryonic development. In species belonging to pattern 3 (metamorphosis marks the appearance of the adult type of osmoregulation), the adults may live under conditions of variable salinities. The embryos thus require osmotic protection, provided by the egg envelopes, at least in the documented case of Homarus gammarus (Charmantier and Aiken, 1987) and probably in most other species. Since the larvae are usually osmoconformers or weak regulators until metamorphosis, an export strategy to stable-salinity sea water habitats may be temporarily required for their development and completion of their life cycle. In pattern 2 species, the adult type of osmoregulation is established at hatch. All or most of their developmental stages live under variable or extreme salinity conditions. From the available examples reviewed above, the strategy selected for the success of the life cycle of these species associates an osmotic protection of the embryos and the development of autonomous osmoregulation in embryos (Fig. 2). Hatchlings are thus able to confront harsh salinity conditions immediately following the emergence from the eggs. This feature is often associated with direct development.

8 OSMOREGULATION IN CRUSTACEAN EMBRYOS 1085 Osmoprotection of embryos can be accomplished in two main ways. Embryos can be enclosed in more or less tightly closed chambers (incubating pouch, brood pouch, marsupium) in which they are bathed by a fluid whose osmolality is controlled by the female. This is the case in several Cladocera (Aladin and Potts, 1995), in the isopod Sphaeroma serratum (Charmantier and Charmantier-Daures, 1994), in the amphipod Orchestia gammarellus (Morritt and Spicer, 1996b, c), probably in the latter case through direction of urine isosmotic to hemolymph to the marsupium (Morritt and Spicer, 1996b). Comparable mechanisms have yet to be studied in Cladocera or in isopods, although a possible function in nourishment of embryos (see above) and in ion transport has been postulated in relation to emergence to land (review in Warburg and Rosenberg, 1996). In other species, embryos are often enclosed in eggs which are directly exposed to the external environment. The osmotic protection of embryos is provided by the egg envelopes. The mechanisms underlying this osmotic protection are still the object of different hypotheses, for crustaceans as well as other marine groups such as molluscs (reviews in Pechenik, 1986; Rawlings, 1999) and elasmobranchs (review in Kormanik, 1993). One of them is based on the variable degree of permeability of the egg envelopes, which would be highly impermeable during most or part of development, preventing for instance water invasion and ion loss at low salinities or in freshwater (Krogh, 1939; Strömberg, 1972; Kelley and Burbanck, 1976; Valdes et al., 1991; Noblitt and Payne, 1995; Glas et al., 1997; Susanto and Charmantier, 2001; reviews in Charmantier and Aiken, 1987; Saigusa, 1996; Saigusa and Terajima, 2000). The permeability of the envelopes would tend to increase toward the end of embryogenesis, perhaps following the action of proteolytic enzymes in some cases (De Vries and Forward, 1991). The increased permeability would favor an osmotic uptake of water resulting in an osmotic facilitation of hatching (Davis, 1968, 1981; Strömberg, 1972; Morritt and Spicer, 1995; Susanto and Charmantier, 2001; reviews in Kelley and Burbanck, 1976; Saigusa, 1996; Saigusa and Terajima, 2000). However, if this hypothesis of limited and variable permeability to water and ions can be verified, it must also accommodate high permeability of the egg envelopes for gas exchanges, since crustaceans rely on diffusive transport for gas exchange during the embryonic development (Reiber, 1997). Other hypotheses involve charged inorganic molecules which would lead to the accumulation of ions in the peri-embryonic fluid, and hence to its high osmolality; this would invoke a Donnan effect as has been shown to occur in fish eggs (Peterson and Martin-Robichaud, 1986) and in Cnidaria mematocysts (Weber, 1989), and/or an increase of colloidal osmotic pressure resulting in osmotic uptake of water (Prosser, 1991). However, both these mechanisms could account for only slight increases in internal osmolality of the eggs, in the range of a few tens of mosm/kg (J.-P. Truchot, personal communication), but not for the high osmolalities measured in the egg periembryonic fluid of some species, e.g., mosm/kg in the crayfish Astacus leptodactylus maintained in freshwater (Susanto and Charmantier, 2001). Considering the high but probably not absolute impermeability of egg envelopes, the high osmolality of the egg internal fluid probably generates an osmotic uptake of water which in turn leads to the build-up of a high internal hydrostatic pressure. In eggs of Astacus leptodactylus, its value inferred from Van t Hoff s equation would be approximately 8.6 Atmos (J.-P. Truchot, personal communication). Comparable high pressures have been reported in nematocysts of Cnidaria (Weber, 1989). One of the main mechanisms permitting the osmoprotection of embryos would thus be based on the restricted volume of the egg by the tensile strength of the bounding egg envelopes, a mechanical feature of the egg membranes, the significance of which has been emphasized for the crayfish Procambarus zonangulus (Noblitt and Payne, 1995) and in insects (Hinton, 1981). During the course of embryogenesis in pattern 2 species, embryos osmoregulating autonomously result in hatchlings that os-

9 1086 G. CHARMANTIER AND M. CHARMANTIER-DAURES moregulate at hatch. The ability to osmoregulate may appear early in embryonic development, as for instance in Orchestia gammarellus (Morritt and Spicer, 1996c), and its level may be temporarily higher than in later postembryonic stages, as in Gammarus duebeni (Morritt and Spicer, 1995). Alternatively, embryos may osmoregulate later in their development, shortly before hatching, as in Sphaeroma serratum (Charmantier and Charmantier-Daures, 1994) and Astacus leptodactylus (Susanto and Charmantier, 2001). The causes leading to such differences are unclear and might originate from different levels, from low to high, of osmotic protection between, i) semi-closed (Orchestia gammarellus) or open (Gammarus duebeni) marsupium and, ii) closed incubating pouch (Sphaeroma serratum) or efficient egg envelopes (Astacus leptodactylus). The development of the ability to osmoregulate in embryos is based, as in adult (reviews in Mantel and Farmer, 1983; Péqueux, 1995) and larval (review in Charmantier, 1998) crustaceans, on the occurrence of ion-transporting cells in specialized sites. Osmoregulatory structures are temporary in some species, where they form dorsal or nuchal or neck organs. They are replaced, later in the embryonic development or following hatch, by definitive organs, usually gills, located on other parts of the body. Documented examples include several Cladocera (Aladin and Potts, 1995), Artemia sp. (Conte, 1984), Orchestia gammarellus (Meschenmoser, 1989; Morritt and Spicer, 1996b, c, 1998) and Gammarus duebeni (Morritt and Spicer, 1995). In other species, osmoregulatory structures occur only once at their definitive location, as in embryos of Astacus leptodactylus (unpublished data, J. H. Lignot, G. N. Susanto and G. Charmantier). The presence or activity of Na -K ATPase also indicates the osmoregulatory ability of embryos in Artemia sp. (Conte, 1984), Lepidophthalmus louisianensis (Felder et al., 1986), Astacus leptodactylus (unpublished data, J. H. Lignot, G. N. Susanto and G. Charmantier). Future work must take two factors into account: the as yet limited number of species that have been investigated and the technical difficulties inherent in the small size of embryos and the difficult access to these stages enclosed in brood chambers or in egg envelopes. Given the interest in comparative approaches in biology (Bartholomew, 1987; Schmidt-Nielsen, 1997), new examples of embryonic ontogeny of osmoregulation should be investigated in species with internal or external development. This applies particularly to decapods living under variable or extreme conditions of salinity, which have been very seldom studied. A wider knowledge of osmoprotection and osmoregulation of embryos may also lead to renewed insight on adaptation to terrestrial conditions, particularly in amphipods, isopods and decapods (review on land adaptation in crabs in Anger, 1995). The functions of the brood, incubating and marsupial pouches should be further investigated in relation to their osmoprotective and perhaps also trophic roles for the embryos. The mechanisms of osmoprotection effected by the egg envelopes are still hypothetical and should be further studied. One promising area for future research is the development of osmoregulatory abilities in embryos, in several directions: evaluation of extracellular regulation through micro-osmometry, determination of intracellular isosmotic regulation, localization and structural study of osmoregulatory sites (extrabranchial and branchial structures, excretory organs especially in freshwater species), involvement of enzymes other than Na -K ATPase such as carbonic anhydrase and of other transporters. The cellular differentiation of the ionocytes and molecular aspects of the enzymes structure and expression control should be studied at selected stages of embryonic development. Since osmoregulation is known to be under neuroendocrine control, the ontogeny of this control will also have to be investigated. ACKNOWLEDGMENTS We are grateful to Professor Jean-Paul Truchot for stimulating discussions. We also thank the sponsors of the symposium, NSF, TCS, and SICB s DIZ and DEE, Dr.

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