Ontogeny of osmoregulation in the crayfish Astacus leptodactylus

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.     

Adaptive shifts in osmoregulatory strategy and the invasion of freshwater by brachyuran crabs: evidence from Dilocarcinus pagei (Trichodactylidae)

To evaluate putative adaptive changes underpinning the invasion of freshwater by the Brachyura, this investigation examines anisosmotic extra and isosmotic intracellular osmoregulatory capabilities in Dilocarcinus pagei, a neotropical, hololimnetic crab, including its embryonic and juvenile phases. All ontogenetic stages show a remarkable ability to survive a high salinity medium (25 per thousand, 750 mOsm/kg H2O, 350 mm Na+, 400 mM Cl-). Adults hyper-regulate hemolymph osmolality up to isosmoticity at 744 mOsm kg/H2O (24 per thousand), [Na+] and [Cl-] becoming isoionic at 449 (22 per thousand) and 256 mM (16 per thousand), respectively. Hemolymph (420+/-39 mOsm/kg H2O) and urine (384+/-44 mOsm/kg H2O) are isosmotic in adults held in freshwater, and after 5-days exposure to 25 per thousand (787+/-9 mOsm/kg H2O and 777+/-43 mOs/kg H2O, respectively); D. pagei does not produce dilute urine. Total free amino acid (FAA) concentrations in embryos (14.9+/-1.2), juveniles (32.8+/-0.1) and adult muscle (10.9+/-2.1 mmol/kg wet weight) in freshwater are 30-fold less than in brackish/marine Crustacea, suggesting that FAA constitute a useful parameter to evaluate adaptation to freshwater. On acclimation to 25 per thousand, total FAA increase by approximately 100% in embryos and in adult muscle and nerve tissue and hemolymph, owing to large increases in proline, arginine and/or alanine. However, effective FAA contribution to intracellular osmolality increases only in embryos, from 3 to 4.5%. These findings suggest that gill-based, anisosmotic extracellular regulation has supplanted isosmotic intracellular regulatory mechanisms during the conquest of freshwater by the Brachyura, and indicate that D. pagei may be an old, well-adapted inhabitant of this biotope.     

Occurrence of L- and D-crustacean hyperglycemic hormone isoforms in the eyestalk X-organ/sinus gland complex during the ontogeny of the crayfish Astacus leptodactylus.

We studied the ontogeny of the eyestalk structure and of the L-CHH and d-Phe3-CHH synthesis in the X-organ/sinus gland (XO/SG) complex by light microscopy and immunocytochemistry in the freshwater crustacean Astacus leptodactylus. The optic ganglia start to differentiate in embryos at EI 190 microm (EI: eye index; close to 410 microm at hatching). At EI 270 microm, the three medullae (externa, interna, and terminalis) and the lamina ganglionaris are present and are organized as in the adult eyestalk. The L-CHH was localized in perikarya of neuroendocrine cells, in their tracts, and in SG from the metanauplius stage to the adult. The d-Phe3-CHH was visualized in XO perikarya, in their tracts and in SG of embryos from EI 350 microm and in all later studied stages. Co-localization of both CHH stereoisomers always occurred in the d-Phe3-CHH-producing cells. These results show that the synthesis of CHH enantiomers starts during the embryonic life in A. leptodactylus, and that the d-isomer is synthesized later than its L-counterpart. We discuss the post-translational isomerization as a way to generate hormonal diversity and the putative relation between d-Phe3-CHH synthesis and the ability to osmoregulate, occurring late during the embryonic life of Astacus leptodactylus.     

Ontogeny of the antennal glands in the crayfish Astacus leptodactylus (Crustacea, Decapoda): immunolocalization of Na+,K+-ATPase

The involvement of the antennal urinary glands in the ontogeny of osmoregulatory functions was investigated during the development of Astacus leptodactylus by measurements of hemolymph and urine osmolality in juvenile and adult crayfish and by the immunodetection of the enzyme Na⁺,K⁺-ATPase. In stage II juveniles, 1-year-old juveniles, and adults, all of which were maintained in freshwater, urine was significantly hypotonic to hemolymph. In adults, chloride and sodium concentrations were much lower in urine than in hemolymph. During embryonic development, Na⁺,K⁺-ATPase was detected by immunocytochemistry in ionocytes lining the tubule and the bladder, at an eye index (EI) of 220–250 m, and in the labyrinth, at EI 350 m. In all regions, immunofluorescence was mainly located at the basolateral side of the cells. No immunofluorescence was detected at any stage in the coelomosac. In late embryonic stages (EI 410–440 m), in stage I juveniles, and in adults, strong positive immunofluorescence was found from the labyrinth up to and including the bladder. These results show that, as early as hatching, juvenile crayfish are able to produce dilute urine hypotonic to hemolymph. This ability originates from the presence of Na⁺,K⁺-ATPase in ion-transporting cells located in the labyrinth, the tubule, and the bladder of the antennal glands and constitutes one of the main adaptations of crayfish to freshwater.We thank the University of Tarbiat Modarres and Ministry of Science, Research and Technology, Islamic Republic of Iran for financial aid and support. Special thanks are also due to the Société Française dExportation des Ressources Educatives (SFERE) for the scholarship to S.K.     

Osmoregulation, immunolocalization of Na+/K+-ATPase, and ultrastructure of branchial epithelia in the developing brown shrimp, Crangon crangon (Decapoda, Caridea)

Aspects of osmoregulation including salinity tolerance, osmoregulatory capacity, location of transporting epithelia, and the expression of the enzyme Na+/K+-ATPase were investigated in the developing brown shrimp, Crangon crangon (L.), from the North Sea. Early developmental stages and large juveniles were exposed to a wide range of salinities for measurement of hemolymph osmolality and survival rates. In media ranging from 17.0 per thousand to 32.2 per thousand, salinity tolerance was generally high (survival rates: 70%-100%) in all developmental stages, but it decreased in media <10.2 per thousand. Zoeal stages and decapodids slightly hyperregulated at 17.0 per thousand and osmoconformed in media > or =25.5 per thousand. At 10.2 per thousand, these stages showed high mortality, and only juveniles survived at 5.3 per thousand. Juveniles hyperregulated at 10.2 per thousand and 17.0 per thousand, osmoconformed at 25.5 per thousand, and hyporegulated in media > or =32.2 per thousand. Large juveniles hyperregulated also at 5.3 per thousand. Expression of the Na+/K+-ATPase and ion-transporting cells was located through immunofluorescence microscopy and transmission electron microscopy. In zoeae I and VI, a strong immunoreactivity was observed in cells of the inner epithelia of the branchiostegites and in epithelial cells lining the pleurae. Their ultrastructure showed typical features of ion-transporting cells. In decapodids and juveniles, ionocytes and expression of Na+/K+-ATPase remained located in the branchiostegite epithelium, but they disappeared from the pleurae and appeared in the epipodites. In large juveniles, the cells of the gill shaft showed positive immunolabeling and ultrastructural features of ionocytes. In summary, the adult pattern of osmoregulation in C. crangon is accomplished after metamorphosis from a moderately hyperosmoconforming decapodid to an effectively hyper-/hyporegulating juvenile stage. Salinity tolerance and osmoregulatory capacity are closely correlated with the development of ion-transporting cells and the expression of Na+/K+-ATPase.     

Effect of osmolarity of the fixative on the ultrastructure of preimplantation rabbit embryos

In an attempt to clarify whether or not glutaraldehyde molecules contribute to the effective osmotic pressure of the fixative solution the ultrastructure of noncultured and in vitro-cultured day 1, 3 and 4 rabbit embryos was evaluated. Total osmolarity of the fixative solution (200-800 mosm) was only varied by changing the aldehyde concentration, whereas the vehicle osmolarity (145 mosm) remained unchanged. Optimum preservation in all embryonic stages was obtained when total osmolarity of the fixative solution was 285-340 mosm. Higher (480-800 mosm) or lower (250 mosm) osmolarities of the fixative solution led to alterations mainly in mitochondria and smooth-surfaced endoplasmic reticulum. Shrinkage of cells and condensation of the cytoplasm occurred only occasionally. Compared with early cleavage stage embryos blastocysts were generally more susceptible to hyperton and hypoton fixative solutions. In vitro culture for 24 h per se did not have any influence on the fixation.     

Expression and distribution of Na, K-ATPase in gill and kidney of the spotted green pufferfish, Tetraodon nigroviridis, in response to salinity challenge

Freshwater (FW) spotted green pufferfish (Tetraodon nigroviridis) were transferred directly from a local aquarium to fresh water (FW; 0 per thousand ), brackish water (BW; 15 per thousand ), and seawater (SW; 35 per thousand ) conditions in the laboratory and reared for at least two weeks. No mortality was found. To investigate the efficient mechanisms of osmoregulation in the euryhaline teleost, distribution and expression of Na,K-ATPase (NKA) in gill and kidney of the pufferfish were examined and the osmolality, [Na+] and [Cl-] of the blood were assayed. The lowest levels of both relative protein abundance and activity were found to be exhibited in the BW group, and higher levels in the SW group than FW group. In all salinities, branchial NKA immunoreactivity was found in epithelial cells of the interlamellar region of the filament and not on the lamellae. Relative abundance of kidney NKA alpha-subunit, as well as the NKA activity, was found to be higher in the FW pufferfish than fish in BW or SW. Renal NKA appeared in the epithelial cells of distal tubules, proximal tubules, and collecting tubules, but not in glomeruli, in fish groups of various salinities. Plasma osmolality and chloride levels were significantly lower in FW pufferfish than those in BW and SW, whereas plasma sodium did not differ among the groups. Although identical distributions of NKA were found in either gill or kidney of FW-, BW- or SW-acclimated spotted green pufferfish, differential NKA expression in fish of various salinity groups was associated with physiological homeostasis (stable blood osmolality), and illustrated the impressive osmoregulatory ability of this freshwater and estuarine species in response to salinity challenge.     

Comparison of calcium storage between a Baikalian gastropod and holarctic relatives

In Lake Baikal, extremely thin shells are reported as a typical feature of endemic gastropods. This statement derived only from observations; no experimental data were available up to now. Therefore, we quantitatively investigated the calcium distribution in the endemic prosobranch gastropod Benedictia baicalensis and compared the results with those of Lithoglyphus naticoides, a near relative, non-endemic, palaearctic species. The shell of the endemic mollusc B. baicalensis consists of 94.9+/-26.0 microg Ca(2+)/microl animal volume (n=43), and in L. naticoides 865.0+/-271.5 microg Ca(2+)/microl (n=10). Calcium contents in the tissue of B. baicalensis vary between different sampling stations and different sampling dates (from 9.4+/-5.1 (n=33) to 20.5+/-8.4 microg Ca(2+)/mg dry weight DW (n=16)) and are only 1/5-1/10 compared to L. naticoides (88.5+/-39.1 microg Ca(2+)/mg DW (n=9)). But the values for hemolymph calcium concentration and osmolality in both species are identical (B. baicalensis: osmolality: 84.4+/-5.3 mosm/kg (n=40); hemolymph calcium concentration: 4.6+/-1.7 mmol/l (n=40). L. naticoides: osmolality: 85.0+/-2.0 mosm/kg (n=8); hemolymph calcium concentration: 5.2+/-5.0 mmol/l (n=40).). This is the first experimental study demonstrating, that - besides a similar hemolymph ionic composition - the Baikalian species is characterized by significantly lower calcium storage in shell and tissue than the nearly related non-endemic species.     

Free amino acid pools as effectors of osmostic adjustment in different tissues of the freshwater shrimp macrobrachium olfersii (crustacea, decapoda) during long-term salinity acclimation

To examine osmotic regulation during long-term acclimation to a hyperosmotic medium, hemolymph osmolality, [Na⁺] and total protein, tissue hydration, and free amino acid (FAA) pools in abdominal muscle, gills, central nervous tissue and hemolymph were quantified in the diadromous freshwater (FW) shrimp, Macrobrachium olfersii, during direct exposure to 21‰S seawater over a 20-day period. Hemolymph osmolality and [Na⁺] reach stable maxima within 24 h while total protein is unchanged. Muscle and nerve tissues rapidly lose water while gills hydrate; all tissues attain maximum hydration (+5%) by 5 days, declining to FW values except for gills. Total FAA are highest in muscle, reach a maximum by 2 days (+64%), declining to FW values. Gill FAA increase by 110% after 24 h, diminishing to FW values. Nerve FAA increase 187% within 24 h, and remain elevated. Hemolymph FAA decrease (-75%) after 24 h, stabilizing well below the FW concentration. During acclimation, muscle glycine (+247%), gill taurine (+253%) and proline (+150%), and nerve proline (+426%), glycine (+415%) and alanine (+139%) increase, while hemolymph leucine (-70%) decreases. Total FAA pools contribute 10-20% to intracellular (22-70 mmol/kg) and 0.5-2.4% to hemolymph (3-7 mOsm/kg) osmolalities during direct acclimation from FW. These data emphasize the modest participation of FAA pools in intracellular osmotic regulation during physiological adaptation by M. olfersii to osmotic challenge, accentuating the role of anisosmotic extracellular regulation, suggesting that, during the invasion of freshwater by the Crustacea, dependence on intracellular adjustment employing FAA as osmotic effectors, has become progressively reduced.     

Ontogenic change in tissue osmolality and developmental sequence of mitochondria-rich cells in Mozambique tilapia developing in freshwater

We investigated a change in tissue fluid osmolality and developmental sequences of mitochondria-rich (MR) cells during embryonic and larval stages of Mozambique tilapia, Oreochromis mossambicus, developing in freshwater. Tissue osmolality, representing body fluid osmolality, ranged from 300 to 370 mOsm/kg during embryonic and larval stages. This suggests that tilapia embryos and larvae are also able to regulate body fluid osmolality to some extent, although the levels are somewhat higher and fluctuate more greatly in embryos and larvae than in adults. Na⁺/K⁺-ATPase-immunoreactive MR cells were first detected in the yolk-sac membrane 3 days before hatching (day − 3), followed by their appearance in the body skin on day − 2. Subsequently, MR cells in both the yolk-sac membrane and body skin increased in number, and most densely observed on days − 1 and 0. Whereas yolk-sac and skin MR cells decreased after hatching, MR cells in turn started developing in the gills after hatching. Thus, the principal site for MR cell distribution shifted from the yolk-sac membrane and body skin during embryonic stages to the gills during larval stages, and tilapia could maintain continuously their ion balance through those MR cells during early life stages.     

Plasma osmolyte concentrations and rectal gland mass of bull sharks Carcharhinus leucas, captured along a salinity gradient

Bull sharks (Carcharhinus leucas) were captured across a salinity gradient from freshwater (FW) to seawater (SW). Across all salinities, C. leucas were hyperosmotic to the environment. Plasma osmolarity in FW-captured animals (642 +/- 7 mosM) was significantly reduced compared to SW-captured animals (1067 +/- 21 mosM). In FW animals, sodium, chloride and urea were 208 +/- 3, 203 +/- 3 and 192 +/- 2 mmol l(-1), respectively. Plasma sodium, chloride and urea in SW-captured C. leucas were 289 +/- 3, 296 +/- 6 and 370 +/- 10 mmol l(-1), respectively. The increase in plasma osmolarity between FW and SW was not linear. Between FW (3 mosM) and 24 per thousand SW (676 mosM), plasma osmolarity increased by 22% or 0.92% per 1 per thousand rise in salinity. Between 24 per thousand and 33 per thousand, plasma osmolarity increased by 33% or 4.7% per 1 per thousand rise in salinity, largely due to a sharp increase in plasma urea between 28 per thousand and 33 per thousand. C. leucas moving between FW and SW appear to be faced with three major osmoregulatory challenges, these occur between 0-10 per thousand, 11-20 per thousand and 21-33 per thousand. A comparison between C. leucas captured in FW and estuarine environments (20-28 per thousand ) in the Brisbane River revealed no difference in the mass of rectal glands between these animals. However, a comparison of rectal gland mass between FW animals captured in the Brisbane River and Rio San Juan/Lake Nicaragua showed that animals in the latter system had a significantly smaller rectal gland mass at a given length than animals in the Brisbane River. The physiological challenges and mechanisms required for C. leucas moving between FW and SW, as well as the ecological implications of these data are discussed.     

Accumulation of cytoplasmic lipid droplets in bovine embryos and cryotolerance of embryos developed in different culture systems using serum-free or serum-containing media.

In this study, the quantitative fluctuation of cytoplasmic lipid droplets (LD) and cryotolerance were investigated in bovine embryos derived from in vitro-matured (IVM) and in vitro-fertilized (IVF) oocytes developed in different culture systems using serum-free or serum-containing media. The serum-free cultures were grown using IVMD101 medium in conjunction with bovine cumulus/granulosa cell (BCGC) cocultures or IVD101 medium without BCGC cocultures, and the serum-containing cultures were grown in the presence of BCGC cocultures using HPM199 medium supplemented with 5% calf serum (HPM199 + CS). Large numbers of sudanophilic LD were present in the cytoplasm of bovine embryos from 2-cell to hatched blastocyst stages, and the number and size differed between the embryos cultured in serum-free and serum-supplemented media. In the embryos cultured in HPM199 + CS, large (2-6 microm in diameter) sudanophilic LD increased significantly from the morula to the blastocyst stages. Throughout the embryonic development, the embryos developed in serum-free cultures with and without BCGC cocultures had numerous sudanophilic LD, but most of these droplets were small (<2 microm in diameter) and large LD were less numerous than those embryos cultured in HPM199 + CS. Giant LD (>6 microm in diameter) were frequently observed in morulae and blastocysts (including early blastocysts) developed in HPM199 + CS. Electron microscopic observations demonstrated that large LD were abundant in the cytoplasm of trophoblast and embryonic (inner cell mass) cells of blastocysts cultured in HPM199 + CS. These large LD were identified as osmophilic LD, an indication that these lipid inclusions contained a significant proportion of unsaturated lipids. Many elongated mitochondria were found in embryos developed in IVMD101 and IVD101 at the morula and early blastocyst stages, whereas many of the mitochondria in the morulae developed in HPM199 + CS were of an immature form such as spherical or ovoid shape. The survival and hatching rates of embryos (morulae, early blastocysts, and blastocysts) produced in serum-free media (both IVMD101 and IVD101) after post-thaw culture were superior to those of embryos produced in serum-containing medium. These results showed that bovine embryos cultured in serum-containing medium abnormally accumulated cytoplasmic lipids into their cytoplasm and the excess accumulation of cytoplasmic LD in embryos may affect the cryotolerance of embryos.     

Embryo tolerance and maternal control of the marsupial environment in Armadillidium vulgare (Isopoda: Oniscidea).

Marsupial development in terrestrial isopods subjects embryos to potential physiological stresses, including desiccation, osmotic variation, and high ammonia concentrations. In this study, we investigated tolerance of osmotic extremes, total ammonia, and pH in developmental stages of Armadillidium vulgare cultured in vitro. Marsupial stages were classified as stage 1 (chorionated eggs), stage 2 (having shed the chorion), and stage 3 (mancas). All stages showed wide but differing tolerance ranges. Stage 1 eggs possess the greatest ammonia tolerance, with high 7-d survival in 150 mM total ammonia, and a wide pH tolerance range. Mancas show the widest osmotic tolerance (100-1,400 mosm x kg(-1)) and display proficient hemolymph osmoregulation over this range. Stage 2 eggs reveal the narrowest tolerance ranges for all three parameters but still qualify as eurytopic. Silver staining revealed two distinct ion-transporting tissues in the developmental stages: a median band on the vitelline membrane of stage 1 and stage 2 eggs, corresponding in location to the embryonic dorsal organ, and the posterior three pairs of pleopodal endopodites in mancas. Gravid females do not downregulate ammonia but show efficient regulation of marsupial fluid pH and downregulation of osmolality during dehydration, both of which will provide additional protection to the marsupial young.     

Isovolumetric regulation of renal proximal tubules in hypotonic medium.

Isolated nonperfused proximal tubules maintained their cell volume at a constant level (isovolumetric regulation, IVR), when osmolality of the bathing medium was gradually decreased from 290 to 190 mosm at 1.5 and 5.0 mosm/min. Hypotonic IVR was blocked by inhibiting the Na(+)-K+ pump with ouabain (10(-4) M) when osmolality was decreased at 1.5 or 5 mosm/min. Concentration-dependent inhibition of cell volume maintenance was observed in the presence of the K+ channel blocker barium (10(-3)-10(-2) M) when osmolality decreased at 5 mosm/min. Quinine (10(-3) M), another K+ channel blocker, also inhibited IVR at osmolality decreases of 1.5 and 5 mosm/min. These results suggest that the maintenance of constant cell volume during gradual hypoosmotic exposure involves mechanisms that depend on intact Na-K-ATPase and the controlled loss of intracellular K+.     

The expression of gill Na,K-ATPase in milkfish,Chanos chanos,acclimated to seawater,brackish water and fresh water

Juvenile milkfish Chanos chanos (Forssk?l, 1775) were transferred from a local fish farm to fresh water (FW; 0 per thousand ), brackish water (BW; 10 per thousand, 20 per thousand ) and seawater (SW; 35 per thousand ) conditions in the laboratory and reared for at least two weeks. The blood and gill of the fish adapted to various salinities were analyzed to determine the osmoregulatory ability of this euryhaline species. No significant difference was found in plasma osmolality, sodium or chloride concentrations of milkfish adapted to various salinities. In FW, the fish exhibited the highest specific activity of Na, K-ATPase (NKA) in gills, while the SW group was found to have the lowest. Relative abundance of branchial NKA alpha-subunit revealed similar profiles. However, in contrary to other euryhaline teleosts, i.e. tilapia, salmon and eel, the naturally SW-dwelling milkfish expresses higher activity of NKA in BW and FW. Immunocytochemical staining has shown that most Na, K-ATPase immunoreactive (NKIR) cells in fish adapted to BW and SW were localized to the filaments with very few on the lamellae. Moreover, in FW-adapted milkfish, the number of NKIR cells found on the lamellae increased significantly. Such responses as elevated NKIR cell number and NKA activity are thought to improve the osmoregulatory capacity of the milkfish in hyposaline environments.     

Free amino acid pools as effectors of osmostic adjustment in different tissues of the freshwater shrimp macrobrachium olfersii (crustacea,decapoda) during long-term salinity acclimation

To examine osmotic regulation during long-term acclimation to a hyperosmotic medium, hemolymph osmolality, [Na⁺] and total protein, tissue hydration, and free amino acid (FAA) pools in abdominal muscle, gills, central nervous tissue and hemolymph were quantified in the diadromous freshwater (FW) shrimp, Macrobrachium olfersii, during direct exposure to 21‰S seawater over a 20-day period. Hemolymph osmolality and [Na⁺] reach stable maxima within 24?h while total protein is unchanged. Muscle and nerve tissues rapidly lose water while gills hydrate; all tissues attain maximum hydration (+5%) by 5 days, declining to FW values except for gills. Total FAA are highest in muscle, reach a maximum by 2 days (+64%), declining to FW values. Gill FAA increase by 110% after 24?h, diminishing to FW values. Nerve FAA increase 187% within 24?h, and remain elevated. Hemolymph FAA decrease (?75%) after 24?h, stabilizing well below the FW concentration. During acclimation, muscle glycine (+247%), gill taurine (+253%) and proline (+150%), and nerve proline (+426%), glycine (+415%) and alanine (+139%) increase, while hemolymph leucine (?70%) decreases. Total FAA pools contribute 10–20% to intracellular (22–70?mmol/kg) and 0.5–2.4% to hemolymph (3–7?mOsm/kg) osmolalities during direct acclimation from FW. These data emphasize the modest participation of FAA pools in intracellular osmotic regulation during physiological adaptation by M. olfersii to osmotic challenge, accentuating the role of anisosmotic extracellular regulation, suggesting that, during the invasion of freshwater by the Crustacea, dependence on intracellular adjustment employing FAA as osmotic effectors, has become progressively reduced.     

Ontogeny of osmoregulation and salinity tolerance in a mangrove crab, Sesarma curacaoense (Decapoda: Grapsidae)

The grapsid crab Sesarma curacaoense is believed to represent the closest saltwater relative to the ancestor which gave rise to an adaptive radiation of endemic freshwater and terrestrial species on the island of Jamaica. Living in mangrove swamps with variable salinity conditions and showing semiterrestrial behaviour, S. curacaoense exhibits ecological adaptations to non-marine conditions. In laboratory experiments, we studied the salinity tolerance during development from hatching to the end of the first juvenile stage. Successful development through metamorphosis occurred in the full salinity range tested (15-32 per thousand), although mortality was significantly enhanced and development delayed at 15 per thousand. In another series of experiments, we studied the ontogeny of the capability for osmoregulation, which is considered as the physiological basis of osmotic stress tolerance. Our results show that S. curacaoense is from hatching a fairly strong hyperosmoregulator in dilute media. This capability increased gradually from hatching throughout the larval and juvenile development. In seawater (32 per thousand) and at an enhanced salt concentration (44 per thousand), the zoeal stages remained hyperosmoconformers. The capability for hypoosmoregulation in concentrated media appeared first in the megalopa stage and increased thereafter. Adult crabs were observed to be strong hyper-hypo-osmoregulators in a salinity range from at least 1 per thousand to 44 per thousand. The unusually early appearance of strong regulatory capabilities, particularly in dilute media, is interpreted as a physiological preadaptation that should have facilitated the evolutionary process of adaptive radiation in non-marine environments on Jamaica.     

Development of respiratory rhythms in perinatal chick embryos

In chick embryos, gas exchange takes place via the chorioallantoic membrane (CAM) and the lungs at approximately 1 day prior to hatching. The present study was designed to elucidate the development of respiratory rhythms in the chick embryo during the whole pipping (perinatal) period with a condenser-microphone measuring system. The microphone was hermetically attached on the eggshell over the air cell on day 18 of incubation. It first detected a cardiogenic signal (i.e. acoustocardiogram), and then beak clapping and breathing signals (acoustorespirogram, ARG). The first signals of lung ventilation appeared intermittently and irregularly approximately once per 5 s among the clapping signals after the embryo penetrated its beak into the air cell (internal pipping, IP). The respiratory rhythm then developed irregularly, with a subsequent more regular rate. The envelope pattern of breathing from the onset of IP through external pipping (EP) to hatching was constructed by a specially devised procedure, which eliminated external and internal noises. The envelope patterns indicated that the IP, EP and whole perinatal periods of 10 embryos were 14.1+/-6.4 (S.D.), 13.6+/-4.0 and 27.6+/-5.4 h, respectively. In addition, they also indicated the period of embryonic hatching activity (i.e. climax) which was 48+/-19 min. The development of respiratory rhythm was also shown by the instantaneous respiratory rate (IRR) which was designated as an inverse value of two adjacent ARG waves.     

Dilute culture media as an environmental or physiological simulant in cultured gill epithelia from freshwater rainbow trout

The electrophysiological and ion-transporting properties of cultured gill epithelia from freshwater (FW) rainbow trout were examined in the presence of dilute cell culture media as an environmental or physiological simulant. Gill epithelia were cultured on cell culture inserts under symmetrical conditions (L15 apical-L15 basolateral) for 6-7 d. The following experiments were then conducted. (1) To mimic a gradual lowering of environmental salinity, apical L15 medium was progressively diluted with FW (first to 2/3 L15 for 8 h and then to 1/3 L15 for 6 h) before the introduction of apical FW (FW apical-L15 basolateral, analogous to a fish in a natural FW environment). Dilute apical media had no significant effect on the electrophysiological properties of preparations compared with symmetrical culture conditions, and no evidence for active Na(+) or Cl(-) transport was observed. Preparations subsequently exposed to apical FW exhibited a negative transepithelial potential and evidence of active Cl(-) uptake and slight Na(+) extrusion. (2) To mimic the extracellular fluid dilution that occurs in euryhaline fish after abrupt transfer from saline to FW, the osmolality or ionic strength (or both) of basolateral media was reduced by 20-40% (using either FW or FW + mannitol) while simultaneously replacing apical media with FW. Under these conditions, Na(+) and Cl(-) influx rates were low compared with efflux rates, while the Ussing flux ratio analysis generally indicated active Cl(-) uptake and Na(+) extrusion. The Na(+)-K(+) adenosine triphosphatase activity was not affected by alterations in basolateral osmolality. Our studies indicate that cultured trout gill epithelia are tolerant of media dilution from both the apical and the basolateral direction; however, neither treatment alone appeared to increase ion influx rates or stimulate active Na(+) uptake in cultured trout gill epithelia.