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Developmental ecophysiology of the beachflea Orchestia gammarellus (Pallas) (Crustacea: Amphipoda) II. Embryonic osmoregulation
JOURNAL OF EXPERIMENTAL MARINE BIOLOGY AND ECOLOGY
Marine Biology and Ecology, 207 (1996) 205-216
Journal
of Experimental
ELSEVIER
Developmental ecophysiology of the beachflea Orchestia gammarellus (Pallas) (Crustacea: Amphipoda) II. Embryonic osmoregulation David Merritt*,
John I. Spicer
Department of Animal and Plant Sciences, University of Sheffield. Sheffield SIO 2TN, UK Received
12 January
1996; revised 24 April 1996; accepted
3 May 1996
Abstract The osmotic concentrations of the periembryonic fluid (PF) of a number of key embryonic stages of the semi-terrestrial beachflea Orchestiu gammarellus, cultured in vitro, were measured over a wide range of external concentrations ( 150- 1250 mOsm . kg I ). Similarly the haemolymph concentration of immediate post-hatch ( < 24 h) individuals was measured over the same concentration range. All embryonic stages displayed a well-developed hyper-hypo-osmotic regulation pattern, maintaining the PF between concentrations of 500-900 mOsm . kg-’ over an external concentration range of 150- 1250 mOsm . kg- ’ . In post-hatch individuals, however, hyper-hypo-regulation was much weaker (approaching isosmotic in higher concentrations), the haemolymph being maintained at concentrations between 320 and 1100 mOsm . kg-’ over a concentration range of 150- 1250 mOsm . kg- ’ . Experiments in which early embryos were cultured in a number of seawater dilutions (25%-50% sea water) showed >50% survival and normal development of 0. gammarellus embryos in culture only occurred at concentrations of 240% sea water. The results are discussed in relation to the female control of the embryonic environment and the environmental osmotic stresses experienced by the different developmental stages. It is suggested that fluctuating osmotic stress has acted as a strong selection pressure on early developmental stages thus affecting the ontogeny of osmoregulation in 0. gammarellus embryos. Keywords:
Amphipoda;
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1. Introduction The semi-terrestrial beachflea Orchestia gammarellus (Pallas) is a highly successful occupant of supralittoral strandline habitats in NW Europe where it is often the dominant detritivore (Lincoln, 1979). The ecophysiology of adult 0. gammarellus has been widely studied (Spicer et al., 1987) and we have much information concerning respiratory biology and gaseous exchange (Spicer and Taylor, 1987a,b, 1994; Taylor and Spicer, 1986), desiccation tolerance, osmoregulation and ionic regulation (Moore and Francis, 1985; Spicer and Taylor, 1987~; Morritt, 1987, 1988, 1989) and environmental tolerances (Moore and Francis, 1986). The supralittoral habitats occupied by 0. gammarellus are frequently subject to major changes in salinity due to inundation by sea (or estuarine) water during high tides, land run-off, rainfall and also concentration due to evaporation, especially during periods of neap tides during the summer. Adult 0. gammarellus display a well-developed hyper-hypo-osmotic osmoregulatory ability (Moore and Francis, 1985; Morritt, 1988). This pattern is characteristic of crustaceans living in conditions of fluctuating salinities and is seen in other supralittoral talitrids (Morritt, 1988; Koch, 1991; Moore et al., 1995) supralittoral fiddler crabs (Rabalais and Cameron, 1985) and estuarine prawns (Kirkpatrick and Jones, 1985) amongst others. Whilst osmoregulation in adult crustaceans has been well documented (e.g., Mantel and Farmer, 1983) the development of the osmoregulatory ability is poorly understood and the ontogeny of osmoregulation in direct-developing (non-metamorphic) crustaceans in general has only recently attracted the attentions of ecophysiologists (Charmantier and Charmantier-Daures, 1994; Morritt and Spicer, 1995, 1996a). In amphipods the eggs are laid directly into the brood pouch (see accompanying paper) where they develop and subsequently hatch into what appear to be miniature versions of the adult. They do not, therefore, undergo the drastic morphological changes associated with the metamorphosis from larva to post-larva seen in many commonly studied decapod crustaceans such as shrimps, e.g., Penaeus japonicus, lobsters, e.g., Homarus americanus (Charmantier et al., 1988) and crabs e.g., Cancer magister (Brown and Terwilliger, 1992). Studying the ontogeny of physiological regulation in peracarids such as amphipods (Morritt and Spicer, 1995, 1996a; Spicer, 1995; Spicer and Morritt, 1996) or isopods (Kelley and Burbanck, 1976; Charmantier and Charmantier-Daures, 1994) thus removes the added complication of gross morphological change when considering ontogenic physiological changes. For a brief discussion of the rationale behind the ontogenic approach to ecophysiology see Burggren (1992). Observations on the survival of 0. gammarellus eggs cultured in vitro on media of a range of salinities showed that high mortalities occurred in early embryos on dilute salinity media (25% sea water or less) (Vlasblom and Bolier, 1971; Morritt and Spicer, 1996a). Hatching success was improved in medium concentrations greater than 50% sea water or by introducing embryos into culture at a later developmental stage (Morritt and Spicer, 1996a). This contrasted with the observation that 0. gammarellus maintained under dilute sea water (10%) conditions were able to reproduce and the females were capable of producing normal broods (Morritt and Stevenson, 1993). Following on from these observations the ability of females to control the fluid concentration to which the developing embryos are exposed has been tested (Morritt and Spicer, 1996b). Females
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do have the ability to influence the marsupial fluid concentration and this was especially important when animals were exposed to dilute media. The possibility of maternal control of the marsupial fluid may then have implications for the development of osmoregulation of the embryos in the marsupium. To complete the story, the present study describes the development of the osmoregulatory ability in the embryos of 0. gammarellus.
The only previous work on the developmental changes in osmoregulation in amphipods, apart from the limited observations of Vlasblom and Bolier (1971) for 0. gammarellus and Gammarus ( = Marinogammarus) marinus, is our own work on the brackish water species Gammarus duebeni (Morritt and Spicer, 1995). In G. duebeni at least two changes in the pattern of osmoregulation were reported; intermediate stage embryos demonstrating a well-developed hyper-hypo-osmotic pattern whereas early stages and later stages/hatchlings exhibited hyper-iso-osmotic regulation also characteristic of the adult animal (e.g., Lockwood, 1961; Sutcliffe, 1971). A similar developmental series has been described for the estuarine isopod, Cyathura polita (Kelley and Burbanck, 1976) whereas Charmantier and Charmantier-Daures (1994) have shown that pre-hatch embryos of another estuarine isopod species, Sphaeroma serratum had little or no osmoregulatory ability. A number of other studies have described changes in the salinity tolerance of embryos at different developmental stages, including in vitro studies, e.g., estuarine mysid Mesopodopsis slabberri (Greenwood et al., 1989), although in these cases embryonic osmoregulation was not investigated. The semiterrestrial species, 0. gammarellus represents a direct contrast with the fully aquatic species such as G. duebeni as it spends the majority of its life, by choice, away from standing bodies of water.
2. Material and methods Individuals were collected by hand from strandline debris lying at the top of the boulder beach at Farland Bight, Great Cumbrae Island, Firth of Clyde (OS Grid Ref. NS 173542). Ovigerous females were removed within 1 h of collection and those carrying eggs at early developmental stages (egg with white spotted purple colouration, c.f. Magniette and Ginsburger-Vogel (1982) stage BG-PG) were debrooded and the eggs from the broods equally divided into six batches. Each batch was rinsed thoroughly in 5 ml of one of six experimental seawater dilutions (nominally 10% ( 14256 mOsm * kg-‘), 25% (26324 mOsm. kg-‘), 50% (502+3 mOsm * kg-‘), 75% (75726 mOsm. kg-‘), 100% (98227 mOsm *kg-‘) and 125% (119628 mOsm . kg-‘)). These experimental concentrations were made by dilution of Millport sea water with distilled water or by the addition of Sigma Sea Salts to sea water to produce a concentrated brine (125%). After rinsing eggs were transferred to experimental cultures each comprising of a clean Petri dish containing a filter paper disc (Whatman No. 1) soaked with 4 ml of the appropriate concentration of sea water. The culture dishes were then sealed with Nescofilm and maintained at 152 1 “C. Cultures were examined daily and dead eggs removed when detected; media were replaced every 2-3 d. Embryos were removed from Petri dish
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cultures at a number of recognised developmental stages (sensu Lalitha et al., 1989, see also Magniette and Ginsburger-Vogel, 1982) and these were sampled as described below. 2.1. Sampling Embryos were carefully removed from culture media using watchmakers forceps and immediately transferred to a small volume of mineral oil (Cargille Type A) held in a siliconised watchglass. Any traces of adherent water were removed using a fine glass capillary. After drying the vitelline membrane was gently ruptured using fine forceps in the ventral region of the embryo. This allowed the periembryonic fluid (PF) to leak out and form a distinct droplet under the mineral oil. Samples of PF (0.02-0.05 l.~l) were taken using siliconised microcapillaries, duplicate measurements made for each sample using a direct reading nanolite osmometer and a mean value calculated (see Morritt and Spicer, 1996b accompanying this paper for details). The concentrations of the experimental media (8 pl samples) were measured both periodically during the acclimation period and at each sampling occasion using a vapour pressure osmometer. Both osmometers were regularly calibrated using verified NaCl standards and also checked against each other. Preliminary studies indicated that it was not possible to obtain viable samples from Stage 1 embryos, despite the presence of a small fluid-filled space in the embryo due to contamination of the PF with vitellogenic material. Consequently the earliest developmental stage from which PF samples could be obtained was Stage 2 (c.f. Gammarus duebeni in Morritt and Spicer, 1995). For each of the six experimental dilutions samples were obtained for the following stages; 2-3, 4, 5 and 7-8 and also hatchlings ( < 24 h post hatch). Haemolymph from hatchlings was sampled by excision of the second antenna of a dried hatchling under mineral oil and collection of the haemolymph as it emerged as a distinct droplet. The number of replicates for each treatment was dependant on the availability of embryos of appropriate developmental stages and also sampling success. 2.2. Tolerance
to low dilutions
As early eggs maintained on dilute media (10 and 25% sea water) died and disintegrated, it was thus not possible to obtain data for osmoregulation of embryos at these dilute concentrations using the technique described above. In order to circumvent this problem a series of cultures were established, similar to those described above, except that eggs were introduced into the cultures at a later developmental stage i.e., after formation of the dorsal organ/stage 1 (sensu Lalitha et al., 1989). Survival of these later embryos was much improved and they appeared to develop normally. This facilitated the sampling of PF from later developmental stages (stages 4 through to hatchling) in dilute salinities, although not from stage 2/3 embryos in which acclimation time (between introduction into culture at stage 1 and reaching stage 2) would have been short ( < 5 d.). All embryos used had been cultured under experimental conditions for 2 5 days prior to sampling. In order to pinpoint the critical salinity at which mortality increased significantly
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when eggs/embryos were cultured on dilute media the following experiment was carried out. Early eggs (white spotted purple - see above) were removed from a total of 10 ovigerous females and each brood (mean brood size = 13.1) was equally divided between one of six experimental treatments. The six treatments employed were sea water dilutions between 25% and 50% sea water at 5% increments. The culture method was identical to that described above. Twenty eggs were used per treatment. Cultures were examined daily, survival (i.e., eggs not completely disintegrated) was noted and any dead eggs removed when detected.
3. Results Fig. 1 and 2 depict the relationships between periembryonic fluid (PF) or haemolymph concentration and concentration of the culture medium, for the different developmental stages investigated. The approximate ages of the developmental stages are given in Table 1. The concentration of the culture medium used in the data presentation is given as the mean value of the culture media over the experimental acclimation period. There was no significant difference between the medium con-
1. The relationship between the osmotic concentration of the periembryonic fluid (PF) and the concentration of the medium for different embryonic stages of 0. gammarellus. (a) Stage 2/3, (b) Stage 4, (c) Stage 5 and (d) Stage 7/8. Each data point = mean2S.D. Figures indicate number of replicates at each data point. Broken line indicates line of no osmoregulation (isosmotic line).
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1200
600 600 400
3
,’
0
,’
,’
0
,’
,’
I’
200
,’
400
600
Medium osmolality
600
1000
1200
(mOsm/kg)
Fig. 2. Relationship between the osmotic concentration of the haemolymph and the concentration medium for immediate post-hatch ( < 24 h) 0. ~wrwuu-ellus. Conventions as for Fig. I.
of the
centrations for each developmental stage at the same dilutions (Kruskal Wallis, P > 0.05 in each case). The earliest stage from which clean PF samples could be taken (stage 2/3 embryos) displayed a well-developed hyper-hypo-osmotic regulation of the PF (Fig. la). Thus the osmolality of the PF was maintained within reasonably narrow limits (approx. 500-850 mOsm . kg-‘) over a range of external concentrations from approx. 250 -1200 mOsm * kg-‘. This resulted in the embryo, within the vitelline membrane(s) inhabiting a relatively constant environment. With a few minor differences this pattern was also exhibited by stages 4 and 5 and also stage 7/8 (Fig. 1 b,c and d). The isosmotic point for all the embryonic stages was between 700-800 mOsm . kg- ‘. The osmoregulation pattern exhibited by individuals immediately post-hatch ( < 24 h) was considerably different from that seen in the embryonic stages (Fig. 2). Whilst there was a degree of hyper-hypo-osmoregulation this was considerably less well-developed than seen in the earlier stages and tended towards being isosmotic at higher external concentrations. Table 2 shows the results of analyses of variance (Kruskall-Wallis) comparing the osmolality of PF/haemolymph of the five different developmental stages Table I Approximate age of 0. gammarellus embryos at different developmental stages (using the stages of (a) Lalitha et al., 1989 and (b) Magniette and Ginsburger-Vogel, 1982) when cultured at 15+1 “C and L:D 14:lO h Stage”
Stageh
Approximate
I
C
213 4 5 718 Hatchling
RI R2 R3lY 0 E
8-10 I O-14 14-16 16-18 19-21 22-24
age/days
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Table 2 Results of non-parametric analysis of variance (Kruskal-Wallis) comparing different developmental stages at the same external concentration
PF/haemolymph
Medium
H
df
P
Difference
10% 25% 50% 75% 100% 125%
9.28 14.42 19.67 14.31 22.88 24.64
3 4 4 4 4 4
CO.05 eo.05 CO.05 CO.05
4, = 5,, = 6/7,, = hatch,, 611, = 5,, = 4,, = 2/3,, = hatch, 6/7* = 4, = 2/3,, = 5d,, = hatch, hatch, = 6/7,, = 213a, = 5,, = 4, hatch4 = 617, = 5, = 4, = 213, hatch, = 4,, = 213, = 5, = 617,
211
concentrations
of
between treatments
Developmental stage with highest median value on the left in difference between treatments column: stages which share a subscript letter in common are not significantly different (non-parametric multiple comparisons based on method of Dunn [see Zar, 19841).
at the same medium concentration. There was no consistent pattern between the different stages although hatchling haemolymph had the lowest concentration in 10, 25 and 50% sea water and the haemolymph concentration was significantly lower than the concentration of PF of at least one other developmental stage at these external concentrations. In external concentrations of 75%, 100% and 125% sea water the haemolymph of the hatchlings consistently had a greater concentration than the concentration of the PF of the earlier developmental stages and again the haemolymph concentration was significantly greater than at least one of the earlier developmental stages. The difference between haemolymph osmotic concentration and that of the PF of embryonic stages was particularly marked in the higher external concentrations (100 and 125% sea water). The results of the experiments designed to establish the critical salinity at which hypo-osmotic stress resulted in a significant increase in early embryo mortality are shown in Fig. 3. Culture in salinities below 30% sea water ( = approx. 300 mOsm . kg-‘) resulted in significant ( > 50%) mortality within 72 h. Mortality in 35 and 40% sea water increased steadily with time: 50% mortality at 9 days in 35% sea water. Good survival and development of the embryos ( > 75%) was only seen in 45 and 50% sea water (approximating to 450-500 mOsm . kg-‘). The experiment was terminated at 11 days at which point all the surviving embryos were developing normally (approximately stage l/2).
4. Discussion The ontogeny of osmoregulation reported here for the embryos and hatchlings of the semi-terrestrial beachflea, Orchestia gammarellus contrasts sharply with that described for the gammarid Gammarus duebeni (Morritt and Spicer, 1995) in that the hyper-hypoosmotic pattern in the former is established by stage 2/3 and is retained throughout the rest of embryonic development. Upon eclosion, however, this well-developed hyperhypo-regulatory ability appears to be lost temporarily post-hatch, although we know it must be subsequently regained as adult 0. gammarellus have a well-developed hyper-
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Days in culture Fig. 3. Survival of early embryos of 0. gammarellus with time for six experimental salinities. Symbols: crosses = 25% sea water, open triangles = 30%, open circles = 35%, squares = 40%, solid triangles = 45% and solid circles = 50%.
hypo-osmotic regulatory ability (Moore and Francis, 1985; Morritt, 1988). In fact the observed pattern of hyper-hypo-osmotic regulation in these embryonic 0. gammarellus was better developed than that described for adults. The maintenance of the PF, which surrounds the developing embryos inside the vitelline membrane, within fairly narrow concentration limits during embryonic development may be related to, or be a prerequisite for successful organogenesis, e.g., the ontogeny of the cardiovascular structure and function (Spicer and Morritt, 1996). Of particular interest is that external concentrations (500-1000 mOsm . kg-‘) over which the PF is maintained within the narrowest limits correspond very closely with the concentration range reported for the marsupial fluid (630-965 mOsm . kg-‘) of ovigerous females when the females were maintained in a wide range of external concentrations, from 25-100% sea water (see Morritt and Spicer, 1996b). Thus the developing embryos of 0. gammarellus have the ability to maintain the PF osmolality within extremely narrow limits over the external concentration range which they are most likely to experience in the marsupium. Thus we have a situation where the combination of female control of the embryonic environment and a near perfect osmoregulatory ability of the embryos within the range of osmotic concentrations experienced in the marsupium results in the effective maintenance of an extremely stable internal milieu for embryonic organogenesis and development over a wide range of external concentrations. This might be especially important in an organism such as the semi-terrestrial 0. gammarellus which regularly experiences a wide range of salinity conditions (see Introduction). These findings also help explain the capacity of female 0. gammarellus to produce normal broods when maintained over a wide range of external salinities, from 10 to 100% sea water (Morritt and Stevenson, 1993). The hyper-hypo-osmotic pattern of regulation exhibited by the embryos of 0. gammarellus is frequently associated with crustaceans that inhabit conditions of fluctuating osmotic
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stress (see Introduction). It could be suggested that fluctuating osmotic stress has acted as an important selection pressure on the ontogeny of osmoregulatory physiology in 0. gammarellus. The fact that the intermediate embryonic stages of the brackish water gammarid Gammarus duebeni, which regularly experience salinity changes from O-35%0 over a tidal cycle, also have a pronounced hyper-hypo-osmotic pattern (Morritt and Spicer, 1995) tends to add weight to this argument. It is possible, therefore, that even embryos within the marsupium have been subject to selection pressures leading to the development of the physiological mechanism for the maintenance of a relatively constant PF concentration over a wide range of salinities. The most striking change in osmoregulatory patterns reported here is the change between the well-developed hyper-hypo-osmotic regulation of the embryonic stages and the weak hyper-hypo-osmotic regulation, tending towards isosmotic in higher concentrations, of the immediate post-hatch hatchlings. This pattern is also considerably less efficient than the osmoregulation displayed by the adult stage (Morritt, 1988). These differences were most pronounced in the more dilute and most concentrated external concentrations investigated. Thus we have a situation where the most complex and efficient (in terms of maintaining a constant PF concentration) pattern of osmoregulation is associated with early, embryonic stages of the life cycle (c.f. Morritt and Spicer (1995) for G. duebeni) and not with the hatchling or indeed the adult. Furthermore, whilst the juvenile appears to hatch as a miniature adult it does not have the fully developed adult physiological mechanism for osmoregulatory control - adults have a well developed hyper-hypo-osmotic regulation pattern (Moore and Francis, 1985; Morritt, 1988). Perhaps the mechanism develops post-hatch whilst the hatchling is still afforded some protection by the marsupium? The change between pre-hatch osmoregulation of PF and post-hatch control of haemolymph may represent a switch in primary osmoregulatory structures, from a membrane-based system, dependent on the vitelline membrane and intimately associated dorsal organ (Meschenmoser, 1989), to a tissuebased system involving the coxal gills and medial surfaces of the coxal plates. The direction of the change, i.e., from a strong hyper-hypo-osmoregulator to a weak one upon hatching, contrasts somewhat with the situation in some of the euryhaline decapods studied where metamorphosis from larva to post-larva is associated with a concomitant increased salinity tolerance and capability for hyper-hypo-osmotic regulation (e.g., Rabalais and Cameron, 1985; Charmantier et al., 1988). These changes can be related to the increased activities of osmoregulatory enzymes, i.e., Naf-K’-ATPase and carbonic anhydrase (Bouaricha et al., 1991) and also to the development of the adult osmoregulatory structures (Bouaricha et al., 1994). In some cases changes in the physiology can also be related to changes in habitat and the associated osmotic stresses (Rabalais and Cameron, 1985). Similarly in the isopod Sphaeroma serratum, although embryonic stages (in the incubating pouch) showed little osmoregulatory ability, progressive juvenile stages up to the adult showed a gradual improvement in hyperosmotic regulation in dilute media (Charmantier and Charmantier-Daures, 1994). The experiments designed to establish the critical salinity demonstrated that good survival and development of the embryos was not possible below 45 and 50% sea water (approximating to 450-500 mOsm . kg-‘). This data again supports the hypothesis that female control of the marsupial fluid is especially important for newly laid eggs if the
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female finds herself in hypo-osmotic conditions. It is of interest that the lowest mean PF value recorded was 482 mOsm . kg ’ for stage 7/8 embryos in 10% sea water which is exactly within the range of external concentrations where good survival was recorded for early embryos in the critical point experiments. Adults of the freshwater amphipod species Gammarus pulex and Orchestia cavimana maintain haemolymph osmolalities of 274 and 350-400 mOsm . kg-’ respectively in fresh water (Lockwood, 1961; Morritt, 1988) suggesting that concentrations in this range are necessary for cell function. The lowest haemolymph concentration recorded here for newly hatched 0. gammarellus in 10% sea water was 311 mOsm* kg-‘. It is perhaps not surprising, therefore, to discover that for newly laid eggs (in which there is probably little or no osmoregulatory ability certainly the dorsal organ, which probably plays an important osmoregulatory role (Meschenmoser, 1989), is yet to appear) external concentrations below 400 mOsm . kg ’ result in osmotic swelling of the egg and disintegration. In conclusion, therefore, it seems that through a combination of maternal control of the brood pouch environment (Morritt and Spicer, 1996b) and the early development of an efficient osmoregulatory system in the embryos which persists to adulthood, 0. gammarellus is well adapted throughout the life cycle to live in conditions of fluctuating osmotic stress. One might predict that the ultimate suite of selection pressures on the developmental ecophysiology of talitrid amphipod embryos would be presented by the fully terrestrial leaf litter habitat. This might be particularly relevant in terms of hypo-osmotic stress in a group which arguably does not appear to have included a freshwater stage in its colonisation of land (Morritt, 1988; Little, 1990). Spicer and Taylor (1994) have suggested that the retention of exosomotic water within the ventral chamber or brood pouch by talitrid amphipods is the key to retaining an essentially aquatic physiology whilst adopting a terrestrial existence. The talitrids have successfully colonised the terrestrial domain, being independent of water for reproduction, food and dispersal, (Friend and Richardson, 1986; Spicer et al., 1987) and yet what we know of the adult ecophysiology is restricted to a handful of, largely synanthropic, species and we know nothing of the brood pouch environment or embryonic physiology. The investigation of these areas may well reveal previously overlooked physiological adaptations to the colonisation of land by the Amphipoda.
Acknowledgments We would like University Marine (U.M.B.S.M.) are carried out whilst
to thank Prof. J. Davenport for the provision of facilities at the Biological Station, Millport. Mr T.D.I. Stevenson and Mr S. Parker thanked for their unstinting help with equipment. This work was DM was in receipt of a NERC Advanced Fellowship (GT5/94/ALS/
2).
References Bouaricha, N., P. Thuet, G. Charmantier, M. Charmantier-Daures and J.-P. Trilles, 1991. Na’-K’-ATPase and carbonic anhydrase activities in larvae, postlarvae and adults of the shrimp Penaeus japonicus (Decapoda, Penaeidea). Camp. Biochem. Physiol., Vol. IOOA, pp. 433-437.
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Bouaricha, N., M. Charmantier-Dames, I? Thuet, J.-P. Trilles and G. Charmantier, 1994. Ontogeny of osmoregulatory structures in the shrimp Penaeus japonicus (Crustacea, Decapoda). Biol. Bull., Vol. 186, pp. 29-40. Brown, A.C. and N.B. Terwilliger, 1992. Developmental changes in ionic and osmotic regulation in the Dungeness crab, Cancer magister. Biol. Bull., Vol. 182, pp. 270-277. Burggren, W.W., 1992. The importance of an ontogenic perspective in physiological studies; Amphibian cardiology as a case study. In, Strategies of Physiological Adaptation, Respiration, Circulation and Metabolism, edited by SC. Wood, R. Weber, A. Hargens and R. Millard, Dekker, New York, pp. 235-253. Charmantier, G. and M. Charmantier-Daures, 1994. Ontogeny of osmoregulation and salinity tolerance in the isopod crustacean Sphaeroma, serratum. Mar. Ecol. Prog. Ser., Vol. 114, pp. 93-102. Charmantier, G., M. Charmantier-Daures, N. Bouaricha, P Thuet, D.E. Aiken and J.-P. Trilles, 1988. Ontogeny of osmoregulation and salinity tolerance in two decapod crustaceans: Homarus americanus and Penaeus japonicus. Biol. Bull., Vol. 175, pp. 102-I 10. Friend, J.A. and A.M.M. Richardson, 1986. Biology of terrestrial amphipods. Ann. Rev. Entomol.,Vol. 31, pp. 25-48. Greenwood, J.G., M.B. Jones and J. Greenwood, 1989. Salinity effects on brood maturation of the mysid crustacean Mesopodopsis slabheri. J. Mar. Biol. Assoc. U.K., Vol. 69, pp. 683-694. Kelley, B.J. and W.D. Burbanck, 1976. Responses of embryonic Cyathura politu (Stimpson) (Isopoda: Anthuridae) to varying salinities. Chesapeake Sci., Vol. 17, pp. 159-167. Kirkpatrick, K. and M.B. Jones, 1985. Salinity tolerance and osmoregulation of a prawn, Pulaemon af3nis Mime Edwards (Caridea: Palaemonidea). J. Exp. Mar. Biol. Ecol., Vol. 93, pp. 61-70. Koch, H., 1991. Salinity tolerance and osmoregulation of Traskorchestia transkiana (Stimpson, 1857) (Amphipoda, Talitridae). Crustaceana, Vol. 61, pp. 21-37. Lalitha, M., K. Shyamasundari and K.H. Rao, 1989. Studies on the embryonic development of Talorchestia martensii (Weber) (Crustacea: Amphipoda). Arch. Ital. Anat. Embriol., Vol. 94, pp. 185-195. Lincoln, R.J., 1979. British Marine Amphipoda: Gammaridea, British Museum (Natural History), 658 pp. Little, C., 1990. The terrestrial invasion: an ecophysiological approach to the origins of land animals. Cambridge Univ. Press, 304 pp. Lockwood, APM., 1961. The urine of Gammarus duebeni and G. pulex. .I. Exp. Biol., Vol. 38, pp. 647-658. Magniette, F. and T. Ginsburger-Vogel, 1982. Etablissement d’une table chronologique de developpement embryonnaire, a differentes temperatures, chez Orchestia gammarellus (Pallas) (Crustace, Amphipode). Bull. Sot. Zool. France, Vol. 107, pp. 101-l 10. Mantel, L.H. and L.L. Farmer, 1983. Osmotic and ionic regulation. In, Biology of Crustacea, edited by D.E. Bliss, Vol. -5: Internal anatomy and physiological regulation, edited by L.H. Mantel, Academic Press, New York, pp. 373-429. Meschenmoser, M., 1989. Ultrastructure of the embryonic dorsal organ of Orchestia cavimaria (Crustacea, Amphipoda); with a note on the localisation of chloride and on the change in calcium-deposition before the embryonic moult. Tiss. Cell, Vol. 21, pp. 431-442. Moore, P.G. and C.H. Francis, 1985. On the water relations and osmoregulation of the beachhopper Orchestia gammarellus (Pallas) (Crustacea: Amphipoda). J. Exp. Mar. Biol. Ecol., Vol. 94, pp. 131-150. Moore, P.G. and C.H. Francis, 1986. Environmental tolerances of the beach-hopper Orchestia gammarellus (Pallas) (Crustacea: Amphipoda). Mar. Environ. Res., Vol. 19, pp. 115-129. Moore., P.G., H.E. MacAlister and A.C. Taylor, 1995. The environmental tolerances and behavioural ecology of the sub-Antarctic beach-hopper Orchestia scutigerula Dana (Crustacea: Amphipoda) from Husvik, South Georgia. J. Exp. Mar. Biol. Ecol., Vol. 189, pp. 159-l 82. Merritt, D., 1987. Evaporative water loss under desiccation stress in semi-terrestrial and terrestrial amphipods (Crustacea: Amphipoda: Talitridae). J. Exp. Mar. Biol. Ecol., Vol. I1 1, pp. 145-157. Morritt, D., 1988. Osmoregulation in littoral and terrestrial talitroidean amphipods (Crustacea) from Britain. J. Exp. Mar. Biol. Ecol., Vol. 123, pp. 77-94. Monitt, D., 1989. Ionic regulation in littoral and terrestrial amphipods (Crustacea: Amphipoda: Talitridae). J. Exp. Mar. Biol. Ecol., Vol. 132, pp. 53-67. Morritt, D. and J.I. Spicer, 1995. Changes in the pattern of osmoregulation in the brackish water amphipod Gammarus duebeni Lilljeborg (Crustacea) during embryonic development. J. Exp. Zool., Vol. 273, pp. 271-281. Morritt, D. and J.I. Spicer, 1996a. The culture of eggs and embryos of amphipod crustaceans: implications for brood pouch physiology. J. Mar. Biol. Assoc. U.K., 76: 361-376.
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Merritt, D. and J.I. Spicer, 1996b. Developmental ecophysiology of the beachftea Orchesria gammarellus (Pallas) (Crustacea: Amphipoda). I. Female control of the embryonic environment. J. Exp. Mar. Biol. Ecol. Vol. 207, pp. 191-203. Merritt, D. and T.D.I. Stevenson, 1993. Factors influencing breeding initiation in the beachflea Orchesticr gammarellus (Pallas) (Crustacea: Amphipoda). /. Exp. Mar. Biol. Ecol., Vol. 165, pp. 191-208. Rabalais, N.N. and J.N. Cameron, 1985. The effects of factors important in semi-arid environments on the early development of Uca suhcylindrica. Biol. Bull., Vol. 168, pp. 147- 160. Spicer, J.I., 1995. Ontogeny of respiratory function in crustaceans exhibiting either direct or indirect development, J. Exp. Zool., Vol. 272, pp. 413-418. Spicer, J.I. and D. Merritt, 1996. Ontogenic changes in cardiac function in crustaceans. Camp. Biochem. Physiol., (In Press). Spicer, J.I., P.G. Moore and A.C. Taylor, 1987. The physiological ecology of land invasion by the Talitridae (Crustacea: Amphipoda). Proc. R. Sot. Land. Ser. B, Vol. 232, pp. 95-124. Spicer, J.I. and A.C. Taylor, 1987a. Carbon dioxide transport and acid-base regulation in the blood of the beach-hopper Orchesfia gummarellus (Pallas) (Crustacea: Amphipoda). Ophelia, Vol. 28, pp. 49-61, Spicer, J.I. and A.C. Taylor, 1987b. Respiration in air and water of some semi- and fully terrestrial talitrids (Crustacea: Amphipoda: Talitridae). J. Exp. Mar. Biol. Ecol., Vol. 106, pp. 265-277. Spicer, J.I. and A.C. Taylor, 1987~. Ionic regulation and salinity related changes in haemolymph protein in the semi-terrestrial beachflea Orchesriu gammarellus (Pallas) (Crustacea: Amphipoda). C~~mnp. Bioc~hrm. Physiol., Vol. EEA, pp. 243-246. Spicer, J.I. and A.C. Taylor, 1994. The origin and metabolic significance of exosomatic water in the semi-terrestrial beachflea Urcheslia gammarcllus (Crustacea: Amphipoda). J. 2001. (London), Vol. 232, pp. 617-632. Sutcliffe, D.W., 1971. Sodium influx and loss in freshwater and brackish-water populations of the amphipod Cammurrus duebeni Lilljeborg. J. Exp. Biol., Vol. 54, pp. 255-268. Taylor, A.C. and J.I. Spicer, 1986. Oxygen transporting properties of the blood of two semi-terrestrial amphipods, Orchesfia gammarel1u.s (Pallas) and 0. mediterranuu (Costa), J. Exp. Mar. Biol. Ecol., Vol. 97, pp. 135-150. Vlasblom, A.G. and G. Bolier, 1971. Tolerance of the embryos of Murinogammarus marinus and Orchrstiu gammarella to lowered salinity. Neth. J. Sea Res., Vol. 5, pp. 3346341. Zar, J.H., 1984. Biostatisricul analysis. Prentice-Hall, NJ, second edition, 718 pp.
Marine Biology and Ecology, 207 (1996) 205-216
Journal
of Experimental
ELSEVIER
Developmental ecophysiology of the beachflea Orchestia gammarellus (Pallas) (Crustacea: Amphipoda) II. Embryonic osmoregulation David Merritt*,
John I. Spicer
Department of Animal and Plant Sciences, University of Sheffield. Sheffield SIO 2TN, UK Received
12 January
1996; revised 24 April 1996; accepted
3 May 1996
Abstract The osmotic concentrations of the periembryonic fluid (PF) of a number of key embryonic stages of the semi-terrestrial beachflea Orchestiu gammarellus, cultured in vitro, were measured over a wide range of external concentrations ( 150- 1250 mOsm . kg I ). Similarly the haemolymph concentration of immediate post-hatch ( < 24 h) individuals was measured over the same concentration range. All embryonic stages displayed a well-developed hyper-hypo-osmotic regulation pattern, maintaining the PF between concentrations of 500-900 mOsm . kg-’ over an external concentration range of 150- 1250 mOsm . kg- ’ . In post-hatch individuals, however, hyper-hypo-regulation was much weaker (approaching isosmotic in higher concentrations), the haemolymph being maintained at concentrations between 320 and 1100 mOsm . kg-’ over a concentration range of 150- 1250 mOsm . kg- ’ . Experiments in which early embryos were cultured in a number of seawater dilutions (25%-50% sea water) showed >50% survival and normal development of 0. gammarellus embryos in culture only occurred at concentrations of 240% sea water. The results are discussed in relation to the female control of the embryonic environment and the environmental osmotic stresses experienced by the different developmental stages. It is suggested that fluctuating osmotic stress has acted as a strong selection pressure on early developmental stages thus affecting the ontogeny of osmoregulation in 0. gammarellus embryos. Keywords:
Amphipoda;
*Corresponding author. [email protected] 0022.0981/96/$15.00 PII SOO22-098
Crustacea;
Tel.:
Copyright
1(96)02635-4
Embryo;
( + 44-114)
0
Osmoregulation
282-4632;
fax:
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2760159;
e-mail:
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1. Introduction The semi-terrestrial beachflea Orchestia gammarellus (Pallas) is a highly successful occupant of supralittoral strandline habitats in NW Europe where it is often the dominant detritivore (Lincoln, 1979). The ecophysiology of adult 0. gammarellus has been widely studied (Spicer et al., 1987) and we have much information concerning respiratory biology and gaseous exchange (Spicer and Taylor, 1987a,b, 1994; Taylor and Spicer, 1986), desiccation tolerance, osmoregulation and ionic regulation (Moore and Francis, 1985; Spicer and Taylor, 1987~; Morritt, 1987, 1988, 1989) and environmental tolerances (Moore and Francis, 1986). The supralittoral habitats occupied by 0. gammarellus are frequently subject to major changes in salinity due to inundation by sea (or estuarine) water during high tides, land run-off, rainfall and also concentration due to evaporation, especially during periods of neap tides during the summer. Adult 0. gammarellus display a well-developed hyper-hypo-osmotic osmoregulatory ability (Moore and Francis, 1985; Morritt, 1988). This pattern is characteristic of crustaceans living in conditions of fluctuating salinities and is seen in other supralittoral talitrids (Morritt, 1988; Koch, 1991; Moore et al., 1995) supralittoral fiddler crabs (Rabalais and Cameron, 1985) and estuarine prawns (Kirkpatrick and Jones, 1985) amongst others. Whilst osmoregulation in adult crustaceans has been well documented (e.g., Mantel and Farmer, 1983) the development of the osmoregulatory ability is poorly understood and the ontogeny of osmoregulation in direct-developing (non-metamorphic) crustaceans in general has only recently attracted the attentions of ecophysiologists (Charmantier and Charmantier-Daures, 1994; Morritt and Spicer, 1995, 1996a). In amphipods the eggs are laid directly into the brood pouch (see accompanying paper) where they develop and subsequently hatch into what appear to be miniature versions of the adult. They do not, therefore, undergo the drastic morphological changes associated with the metamorphosis from larva to post-larva seen in many commonly studied decapod crustaceans such as shrimps, e.g., Penaeus japonicus, lobsters, e.g., Homarus americanus (Charmantier et al., 1988) and crabs e.g., Cancer magister (Brown and Terwilliger, 1992). Studying the ontogeny of physiological regulation in peracarids such as amphipods (Morritt and Spicer, 1995, 1996a; Spicer, 1995; Spicer and Morritt, 1996) or isopods (Kelley and Burbanck, 1976; Charmantier and Charmantier-Daures, 1994) thus removes the added complication of gross morphological change when considering ontogenic physiological changes. For a brief discussion of the rationale behind the ontogenic approach to ecophysiology see Burggren (1992). Observations on the survival of 0. gammarellus eggs cultured in vitro on media of a range of salinities showed that high mortalities occurred in early embryos on dilute salinity media (25% sea water or less) (Vlasblom and Bolier, 1971; Morritt and Spicer, 1996a). Hatching success was improved in medium concentrations greater than 50% sea water or by introducing embryos into culture at a later developmental stage (Morritt and Spicer, 1996a). This contrasted with the observation that 0. gammarellus maintained under dilute sea water (10%) conditions were able to reproduce and the females were capable of producing normal broods (Morritt and Stevenson, 1993). Following on from these observations the ability of females to control the fluid concentration to which the developing embryos are exposed has been tested (Morritt and Spicer, 1996b). Females
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do have the ability to influence the marsupial fluid concentration and this was especially important when animals were exposed to dilute media. The possibility of maternal control of the marsupial fluid may then have implications for the development of osmoregulation of the embryos in the marsupium. To complete the story, the present study describes the development of the osmoregulatory ability in the embryos of 0. gammarellus.
The only previous work on the developmental changes in osmoregulation in amphipods, apart from the limited observations of Vlasblom and Bolier (1971) for 0. gammarellus and Gammarus ( = Marinogammarus) marinus, is our own work on the brackish water species Gammarus duebeni (Morritt and Spicer, 1995). In G. duebeni at least two changes in the pattern of osmoregulation were reported; intermediate stage embryos demonstrating a well-developed hyper-hypo-osmotic pattern whereas early stages and later stages/hatchlings exhibited hyper-iso-osmotic regulation also characteristic of the adult animal (e.g., Lockwood, 1961; Sutcliffe, 1971). A similar developmental series has been described for the estuarine isopod, Cyathura polita (Kelley and Burbanck, 1976) whereas Charmantier and Charmantier-Daures (1994) have shown that pre-hatch embryos of another estuarine isopod species, Sphaeroma serratum had little or no osmoregulatory ability. A number of other studies have described changes in the salinity tolerance of embryos at different developmental stages, including in vitro studies, e.g., estuarine mysid Mesopodopsis slabberri (Greenwood et al., 1989), although in these cases embryonic osmoregulation was not investigated. The semiterrestrial species, 0. gammarellus represents a direct contrast with the fully aquatic species such as G. duebeni as it spends the majority of its life, by choice, away from standing bodies of water.
2. Material and methods Individuals were collected by hand from strandline debris lying at the top of the boulder beach at Farland Bight, Great Cumbrae Island, Firth of Clyde (OS Grid Ref. NS 173542). Ovigerous females were removed within 1 h of collection and those carrying eggs at early developmental stages (egg with white spotted purple colouration, c.f. Magniette and Ginsburger-Vogel (1982) stage BG-PG) were debrooded and the eggs from the broods equally divided into six batches. Each batch was rinsed thoroughly in 5 ml of one of six experimental seawater dilutions (nominally 10% ( 14256 mOsm * kg-‘), 25% (26324 mOsm. kg-‘), 50% (502+3 mOsm * kg-‘), 75% (75726 mOsm. kg-‘), 100% (98227 mOsm *kg-‘) and 125% (119628 mOsm . kg-‘)). These experimental concentrations were made by dilution of Millport sea water with distilled water or by the addition of Sigma Sea Salts to sea water to produce a concentrated brine (125%). After rinsing eggs were transferred to experimental cultures each comprising of a clean Petri dish containing a filter paper disc (Whatman No. 1) soaked with 4 ml of the appropriate concentration of sea water. The culture dishes were then sealed with Nescofilm and maintained at 152 1 “C. Cultures were examined daily and dead eggs removed when detected; media were replaced every 2-3 d. Embryos were removed from Petri dish
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cultures at a number of recognised developmental stages (sensu Lalitha et al., 1989, see also Magniette and Ginsburger-Vogel, 1982) and these were sampled as described below. 2.1. Sampling Embryos were carefully removed from culture media using watchmakers forceps and immediately transferred to a small volume of mineral oil (Cargille Type A) held in a siliconised watchglass. Any traces of adherent water were removed using a fine glass capillary. After drying the vitelline membrane was gently ruptured using fine forceps in the ventral region of the embryo. This allowed the periembryonic fluid (PF) to leak out and form a distinct droplet under the mineral oil. Samples of PF (0.02-0.05 l.~l) were taken using siliconised microcapillaries, duplicate measurements made for each sample using a direct reading nanolite osmometer and a mean value calculated (see Morritt and Spicer, 1996b accompanying this paper for details). The concentrations of the experimental media (8 pl samples) were measured both periodically during the acclimation period and at each sampling occasion using a vapour pressure osmometer. Both osmometers were regularly calibrated using verified NaCl standards and also checked against each other. Preliminary studies indicated that it was not possible to obtain viable samples from Stage 1 embryos, despite the presence of a small fluid-filled space in the embryo due to contamination of the PF with vitellogenic material. Consequently the earliest developmental stage from which PF samples could be obtained was Stage 2 (c.f. Gammarus duebeni in Morritt and Spicer, 1995). For each of the six experimental dilutions samples were obtained for the following stages; 2-3, 4, 5 and 7-8 and also hatchlings ( < 24 h post hatch). Haemolymph from hatchlings was sampled by excision of the second antenna of a dried hatchling under mineral oil and collection of the haemolymph as it emerged as a distinct droplet. The number of replicates for each treatment was dependant on the availability of embryos of appropriate developmental stages and also sampling success. 2.2. Tolerance
to low dilutions
As early eggs maintained on dilute media (10 and 25% sea water) died and disintegrated, it was thus not possible to obtain data for osmoregulation of embryos at these dilute concentrations using the technique described above. In order to circumvent this problem a series of cultures were established, similar to those described above, except that eggs were introduced into the cultures at a later developmental stage i.e., after formation of the dorsal organ/stage 1 (sensu Lalitha et al., 1989). Survival of these later embryos was much improved and they appeared to develop normally. This facilitated the sampling of PF from later developmental stages (stages 4 through to hatchling) in dilute salinities, although not from stage 2/3 embryos in which acclimation time (between introduction into culture at stage 1 and reaching stage 2) would have been short ( < 5 d.). All embryos used had been cultured under experimental conditions for 2 5 days prior to sampling. In order to pinpoint the critical salinity at which mortality increased significantly
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when eggs/embryos were cultured on dilute media the following experiment was carried out. Early eggs (white spotted purple - see above) were removed from a total of 10 ovigerous females and each brood (mean brood size = 13.1) was equally divided between one of six experimental treatments. The six treatments employed were sea water dilutions between 25% and 50% sea water at 5% increments. The culture method was identical to that described above. Twenty eggs were used per treatment. Cultures were examined daily, survival (i.e., eggs not completely disintegrated) was noted and any dead eggs removed when detected.
3. Results Fig. 1 and 2 depict the relationships between periembryonic fluid (PF) or haemolymph concentration and concentration of the culture medium, for the different developmental stages investigated. The approximate ages of the developmental stages are given in Table 1. The concentration of the culture medium used in the data presentation is given as the mean value of the culture media over the experimental acclimation period. There was no significant difference between the medium con-
1. The relationship between the osmotic concentration of the periembryonic fluid (PF) and the concentration of the medium for different embryonic stages of 0. gammarellus. (a) Stage 2/3, (b) Stage 4, (c) Stage 5 and (d) Stage 7/8. Each data point = mean2S.D. Figures indicate number of replicates at each data point. Broken line indicates line of no osmoregulation (isosmotic line).
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1200
600 600 400
3
,’
0
,’
,’
0
,’
,’
I’
200
,’
400
600
Medium osmolality
600
1000
1200
(mOsm/kg)
Fig. 2. Relationship between the osmotic concentration of the haemolymph and the concentration medium for immediate post-hatch ( < 24 h) 0. ~wrwuu-ellus. Conventions as for Fig. I.
of the
centrations for each developmental stage at the same dilutions (Kruskal Wallis, P > 0.05 in each case). The earliest stage from which clean PF samples could be taken (stage 2/3 embryos) displayed a well-developed hyper-hypo-osmotic regulation of the PF (Fig. la). Thus the osmolality of the PF was maintained within reasonably narrow limits (approx. 500-850 mOsm . kg-‘) over a range of external concentrations from approx. 250 -1200 mOsm * kg-‘. This resulted in the embryo, within the vitelline membrane(s) inhabiting a relatively constant environment. With a few minor differences this pattern was also exhibited by stages 4 and 5 and also stage 7/8 (Fig. 1 b,c and d). The isosmotic point for all the embryonic stages was between 700-800 mOsm . kg- ‘. The osmoregulation pattern exhibited by individuals immediately post-hatch ( < 24 h) was considerably different from that seen in the embryonic stages (Fig. 2). Whilst there was a degree of hyper-hypo-osmoregulation this was considerably less well-developed than seen in the earlier stages and tended towards being isosmotic at higher external concentrations. Table 2 shows the results of analyses of variance (Kruskall-Wallis) comparing the osmolality of PF/haemolymph of the five different developmental stages Table I Approximate age of 0. gammarellus embryos at different developmental stages (using the stages of (a) Lalitha et al., 1989 and (b) Magniette and Ginsburger-Vogel, 1982) when cultured at 15+1 “C and L:D 14:lO h Stage”
Stageh
Approximate
I
C
213 4 5 718 Hatchling
RI R2 R3lY 0 E
8-10 I O-14 14-16 16-18 19-21 22-24
age/days
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Table 2 Results of non-parametric analysis of variance (Kruskal-Wallis) comparing different developmental stages at the same external concentration
PF/haemolymph
Medium
H
df
P
Difference
10% 25% 50% 75% 100% 125%
9.28 14.42 19.67 14.31 22.88 24.64
3 4 4 4 4 4
CO.05 eo.05 CO.05 CO.05
4, = 5,, = 6/7,, = hatch,, 611, = 5,, = 4,, = 2/3,, = hatch, 6/7* = 4, = 2/3,, = 5d,, = hatch, hatch, = 6/7,, = 213a, = 5,, = 4, hatch4 = 617, = 5, = 4, = 213, hatch, = 4,, = 213, = 5, = 617,
211
concentrations
of
between treatments
Developmental stage with highest median value on the left in difference between treatments column: stages which share a subscript letter in common are not significantly different (non-parametric multiple comparisons based on method of Dunn [see Zar, 19841).
at the same medium concentration. There was no consistent pattern between the different stages although hatchling haemolymph had the lowest concentration in 10, 25 and 50% sea water and the haemolymph concentration was significantly lower than the concentration of PF of at least one other developmental stage at these external concentrations. In external concentrations of 75%, 100% and 125% sea water the haemolymph of the hatchlings consistently had a greater concentration than the concentration of the PF of the earlier developmental stages and again the haemolymph concentration was significantly greater than at least one of the earlier developmental stages. The difference between haemolymph osmotic concentration and that of the PF of embryonic stages was particularly marked in the higher external concentrations (100 and 125% sea water). The results of the experiments designed to establish the critical salinity at which hypo-osmotic stress resulted in a significant increase in early embryo mortality are shown in Fig. 3. Culture in salinities below 30% sea water ( = approx. 300 mOsm . kg-‘) resulted in significant ( > 50%) mortality within 72 h. Mortality in 35 and 40% sea water increased steadily with time: 50% mortality at 9 days in 35% sea water. Good survival and development of the embryos ( > 75%) was only seen in 45 and 50% sea water (approximating to 450-500 mOsm . kg-‘). The experiment was terminated at 11 days at which point all the surviving embryos were developing normally (approximately stage l/2).
4. Discussion The ontogeny of osmoregulation reported here for the embryos and hatchlings of the semi-terrestrial beachflea, Orchestia gammarellus contrasts sharply with that described for the gammarid Gammarus duebeni (Morritt and Spicer, 1995) in that the hyper-hypoosmotic pattern in the former is established by stage 2/3 and is retained throughout the rest of embryonic development. Upon eclosion, however, this well-developed hyperhypo-regulatory ability appears to be lost temporarily post-hatch, although we know it must be subsequently regained as adult 0. gammarellus have a well-developed hyper-
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Days in culture Fig. 3. Survival of early embryos of 0. gammarellus with time for six experimental salinities. Symbols: crosses = 25% sea water, open triangles = 30%, open circles = 35%, squares = 40%, solid triangles = 45% and solid circles = 50%.
hypo-osmotic regulatory ability (Moore and Francis, 1985; Morritt, 1988). In fact the observed pattern of hyper-hypo-osmotic regulation in these embryonic 0. gammarellus was better developed than that described for adults. The maintenance of the PF, which surrounds the developing embryos inside the vitelline membrane, within fairly narrow concentration limits during embryonic development may be related to, or be a prerequisite for successful organogenesis, e.g., the ontogeny of the cardiovascular structure and function (Spicer and Morritt, 1996). Of particular interest is that external concentrations (500-1000 mOsm . kg-‘) over which the PF is maintained within the narrowest limits correspond very closely with the concentration range reported for the marsupial fluid (630-965 mOsm . kg-‘) of ovigerous females when the females were maintained in a wide range of external concentrations, from 25-100% sea water (see Morritt and Spicer, 1996b). Thus the developing embryos of 0. gammarellus have the ability to maintain the PF osmolality within extremely narrow limits over the external concentration range which they are most likely to experience in the marsupium. Thus we have a situation where the combination of female control of the embryonic environment and a near perfect osmoregulatory ability of the embryos within the range of osmotic concentrations experienced in the marsupium results in the effective maintenance of an extremely stable internal milieu for embryonic organogenesis and development over a wide range of external concentrations. This might be especially important in an organism such as the semi-terrestrial 0. gammarellus which regularly experiences a wide range of salinity conditions (see Introduction). These findings also help explain the capacity of female 0. gammarellus to produce normal broods when maintained over a wide range of external salinities, from 10 to 100% sea water (Morritt and Stevenson, 1993). The hyper-hypo-osmotic pattern of regulation exhibited by the embryos of 0. gammarellus is frequently associated with crustaceans that inhabit conditions of fluctuating osmotic
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stress (see Introduction). It could be suggested that fluctuating osmotic stress has acted as an important selection pressure on the ontogeny of osmoregulatory physiology in 0. gammarellus. The fact that the intermediate embryonic stages of the brackish water gammarid Gammarus duebeni, which regularly experience salinity changes from O-35%0 over a tidal cycle, also have a pronounced hyper-hypo-osmotic pattern (Morritt and Spicer, 1995) tends to add weight to this argument. It is possible, therefore, that even embryos within the marsupium have been subject to selection pressures leading to the development of the physiological mechanism for the maintenance of a relatively constant PF concentration over a wide range of salinities. The most striking change in osmoregulatory patterns reported here is the change between the well-developed hyper-hypo-osmotic regulation of the embryonic stages and the weak hyper-hypo-osmotic regulation, tending towards isosmotic in higher concentrations, of the immediate post-hatch hatchlings. This pattern is also considerably less efficient than the osmoregulation displayed by the adult stage (Morritt, 1988). These differences were most pronounced in the more dilute and most concentrated external concentrations investigated. Thus we have a situation where the most complex and efficient (in terms of maintaining a constant PF concentration) pattern of osmoregulation is associated with early, embryonic stages of the life cycle (c.f. Morritt and Spicer (1995) for G. duebeni) and not with the hatchling or indeed the adult. Furthermore, whilst the juvenile appears to hatch as a miniature adult it does not have the fully developed adult physiological mechanism for osmoregulatory control - adults have a well developed hyper-hypo-osmotic regulation pattern (Moore and Francis, 1985; Morritt, 1988). Perhaps the mechanism develops post-hatch whilst the hatchling is still afforded some protection by the marsupium? The change between pre-hatch osmoregulation of PF and post-hatch control of haemolymph may represent a switch in primary osmoregulatory structures, from a membrane-based system, dependent on the vitelline membrane and intimately associated dorsal organ (Meschenmoser, 1989), to a tissuebased system involving the coxal gills and medial surfaces of the coxal plates. The direction of the change, i.e., from a strong hyper-hypo-osmoregulator to a weak one upon hatching, contrasts somewhat with the situation in some of the euryhaline decapods studied where metamorphosis from larva to post-larva is associated with a concomitant increased salinity tolerance and capability for hyper-hypo-osmotic regulation (e.g., Rabalais and Cameron, 1985; Charmantier et al., 1988). These changes can be related to the increased activities of osmoregulatory enzymes, i.e., Naf-K’-ATPase and carbonic anhydrase (Bouaricha et al., 1991) and also to the development of the adult osmoregulatory structures (Bouaricha et al., 1994). In some cases changes in the physiology can also be related to changes in habitat and the associated osmotic stresses (Rabalais and Cameron, 1985). Similarly in the isopod Sphaeroma serratum, although embryonic stages (in the incubating pouch) showed little osmoregulatory ability, progressive juvenile stages up to the adult showed a gradual improvement in hyperosmotic regulation in dilute media (Charmantier and Charmantier-Daures, 1994). The experiments designed to establish the critical salinity demonstrated that good survival and development of the embryos was not possible below 45 and 50% sea water (approximating to 450-500 mOsm . kg-‘). This data again supports the hypothesis that female control of the marsupial fluid is especially important for newly laid eggs if the
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female finds herself in hypo-osmotic conditions. It is of interest that the lowest mean PF value recorded was 482 mOsm . kg ’ for stage 7/8 embryos in 10% sea water which is exactly within the range of external concentrations where good survival was recorded for early embryos in the critical point experiments. Adults of the freshwater amphipod species Gammarus pulex and Orchestia cavimana maintain haemolymph osmolalities of 274 and 350-400 mOsm . kg-’ respectively in fresh water (Lockwood, 1961; Morritt, 1988) suggesting that concentrations in this range are necessary for cell function. The lowest haemolymph concentration recorded here for newly hatched 0. gammarellus in 10% sea water was 311 mOsm* kg-‘. It is perhaps not surprising, therefore, to discover that for newly laid eggs (in which there is probably little or no osmoregulatory ability certainly the dorsal organ, which probably plays an important osmoregulatory role (Meschenmoser, 1989), is yet to appear) external concentrations below 400 mOsm . kg ’ result in osmotic swelling of the egg and disintegration. In conclusion, therefore, it seems that through a combination of maternal control of the brood pouch environment (Morritt and Spicer, 1996b) and the early development of an efficient osmoregulatory system in the embryos which persists to adulthood, 0. gammarellus is well adapted throughout the life cycle to live in conditions of fluctuating osmotic stress. One might predict that the ultimate suite of selection pressures on the developmental ecophysiology of talitrid amphipod embryos would be presented by the fully terrestrial leaf litter habitat. This might be particularly relevant in terms of hypo-osmotic stress in a group which arguably does not appear to have included a freshwater stage in its colonisation of land (Morritt, 1988; Little, 1990). Spicer and Taylor (1994) have suggested that the retention of exosomotic water within the ventral chamber or brood pouch by talitrid amphipods is the key to retaining an essentially aquatic physiology whilst adopting a terrestrial existence. The talitrids have successfully colonised the terrestrial domain, being independent of water for reproduction, food and dispersal, (Friend and Richardson, 1986; Spicer et al., 1987) and yet what we know of the adult ecophysiology is restricted to a handful of, largely synanthropic, species and we know nothing of the brood pouch environment or embryonic physiology. The investigation of these areas may well reveal previously overlooked physiological adaptations to the colonisation of land by the Amphipoda.
Acknowledgments We would like University Marine (U.M.B.S.M.) are carried out whilst
to thank Prof. J. Davenport for the provision of facilities at the Biological Station, Millport. Mr T.D.I. Stevenson and Mr S. Parker thanked for their unstinting help with equipment. This work was DM was in receipt of a NERC Advanced Fellowship (GT5/94/ALS/
2).
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