Osmoregulation was studied throughout the embryonic development of Astacus leptodactylus. Egg-carrying females were held in freshwater (FW) and in three dilute seawater media (200, 400, 600 mosm kg(-1), 6.8, 13.6, 20.4 per thousand salinity). In FW, changes in peri-embryonic fluid (PEF) and (when available) embryonic hemolymph osmolality were followed from newly-laid eggs to hatching (for an embryonic eye index, EI, of 430-450 microm) and in first-stage juveniles. The PEF and/or hemolymph osmolality remained stable at about 360-380 mosm kg(-1) from early to late (EI 410 microm) embryos; it decreased prior to hatching (EI 420 microm) and in newly-hatched juveniles, down to 290 mosm kg(-1). Artificial opening and removal of the egg membranes, followed by direct exposure to FW, demonstrated that the ability to hyper-osmoregulate, and consequently to survive, in FW appears in embryos with EI > or = 410 microm, i.e., only a few hours or days before hatching. Following a transfer to the dilute seawater media, the PEF/hemolymph osmolality increased slowly over 18-20 days and became isosmotic with the external media at 13.6 and 20.4 per thousand. The embryos died at EI 380-395 microm in these media, and only at 6.8 per thousand was the development completed until successful hatch. These results demonstrate that (1) the embryos become able to osmoregulate in FW shortly before hatching, (2) the embryos are osmo-protected in the eggs during their development, (3) embryonic development and hatching are possible up to a salinity of 7 per thousand. These results are discussed in relation to freshwater adaptation of crayfish.

Osmoregulation was studied during the postembryonic development of Astacus leptodactylus Eschscholtz 1823 in juvenile stages 1-8 and in adults. Juveniles hatch and later stages develop in freshwater or in moderately saline waters. The time of acclimation from freshwater to a saline medium increased from early juveniles to adults. At all stages, it was longer than in comparable stages of marine crustaceans, reflecting the high impermeability of the teguments to water and ions. All stages were able to hyperisoosmoregulate. In freshwater, the ability to hyperosmoregulate was established at hatching and increased during development. The hemolymph osmolality increased from 286 mosm kg-1 in stage 1 juveniles to 419 mosm kg-1 in adults. All stages also hyperregulated at low salinities (7 per thousand and 13 per thousand salinity) and were osmoconformers at higher salinities up to 21 per thousand salinity. The lowest isosmotic salinity tended to increase with the developmental stages. The ability to osmoregulate at hatch and throughout postembryonic development is probably a key physiological adaptation in this and other freshwater crayfish.

The effect of salinity on survival, osmoregulation and oxygen consumption was determined on coastal youngof-the-year (YOY) largemouth bass Micropterus salmoides by exposing them to saline media of 0, 4, 8, 12, and 16%o. The data indicated a decrease in survival with longer exposure time and increased salinity . There were no significant differences in plasma osmolality with increased salinity from 0 to 8%o but osmolality was significantly greater at 12%o than 0%o and osmolality from 16%o was greater than all other salinity treatments . No significant differences in hematocrits were detected between 0 and 12%o, but hematocrits in the 16%o treatment were significantly reduced compared to all other treatments . YOY M. salmoides are good osmoregulators up to 8%o but increased salinity caused measurable osmoregulatory dysfunction . Oxygen consumption rate increased significantly as salinity increased, suggesting that adaptation of this species to hypersaline media is in part accompanied by increased energy expenditure . 119

The ontogeny of osmoregulation was examined in the branchial cavity of embryonic and early postembryonic stages of the crayfish Astacus leptodactylus maintained in freshwater, at the sub-cellular level through the detection of the Na + ,K + -ATPase. The embryonic rate of development was calculated according to the eye index (EI) which was 430-450 µm at hatching. The distribution of the enzyme was identified by immunofluorescence microscopy using a monoclonal antibody IgGα 5 raised against the avian α-subunit of the Na + ,K + -ATPase.Immunoreactivity staining indicating the presence of Na + ,K + -ATPase appeared in the gills of late embryos (EI ≥ 400 µm), i.e. a few days before hatching time, and steadily increased throughout the late embryonic and early postembryonic development. The appearance of the enzyme correlates with the ability to osmoregulate which also occurs late in the embryonic development at EI 410-420 µm and with tissue differentiation within the gill filaments. These observations indicate that the physiological shift from osmoconforming embryos to hyperregulating late embryos and post-hatching stages in freshwater must originate partly from the differentiation in the gill epithelia of ionocytes which are the site of ion pumping, as suggested by the location of Na + ,K + -ATPase. Only the gills were immunostained and a lack of specific staining was noted in the lamina and the branchiostegites. Therefore, osmoregulation through Na + active uptake is likely achieved in embryos at the gill level; all the newly-formed gills in embryos function in ion regulation; other parts of the branchial chamber such as the branchiostegites and lamina do not appear to be involved in osmoregulation.2