Branchial carbonic anhydrase activity and ninhydrin positive substances in the Pacific white shrimp, Litopenaeus vannamei, acclimated to low and high salinities

The Pacific white shrimp, Litopenaeus vannamei, acclimated to 30 ppt salinity, was transferred to either low (15 and 5 ppt), or high (45 ppt) salinity for 7 days. Hemolymph osmolality, branchial carbonic anhydrase activity, and total ninhydrin-positive substances (TNPS) in abdominal muscle were then measured for each condition. Hemolymph osmotic concentration was regulated slightly below ambient water osmolality in shrimp acclimated to 30 ppt. At 15 and 5 ppt, shrimp were strong hyper-osmotic regulators, maintaining hemolymph osmolality between 200 and 400 mOsm above ambient. Shrimp acclimated to 30 ppt and transferred to 45 ppt salinity were strong hypo-osmotic and hypo-ionic regulators, maintaining hemolymph osmolality over 400 mOsm below ambient. Branchial carbonic anhydrase (CA) activity was low (approximately 100 micromol CO(2) mg protein(-1) min(-1)) and uniform across all 8 gills in shrimp acclimated to 30 ppt, but CA activity increased in all gills after exposure to both low and high salinities. Anterior gills had the largest increases in CA activity, and levels of increase were approximately the same for low and high salinity exposure. Branchial CA induction appears to be functionally important in both hyper- and hypo-osmotic regulations of hemolymph osmotic concentrations. Abdominal muscle TNPS made up between 19 and 38% of the total intracellular osmotic concentration in shrimp acclimated to 5, 15, and 30 ppt. TNPS levels did not change across this salinity range, over which hemolymph osmotic concentrations were tightly regulated. At 45 ppt, hemolymph osmolality increased, and muscle TNPS also increased, presumably to counteract intracellular water loss and restore cell volume. L. vannamei appears to employ mechanisms of both extracellular osmoregulation and intracellular volume regulation as the basis of its euryhalinity.     

Hypoosmotic volume regulation in the sea anemone metridium senile

1. The anemone Metridium senile survives salinities from seawater (950 mOsm) to 55% SW (520 mOsm) for at least two weeks. Animals exposed to 40% SW (380 mOsm) die within three days.2. The tissue amino acid content of M. senile acclimated to 950 mOsm, 807 mOsm, 665 mOsm and 520 mOsm for two weeks is respectively, 444, 382, 331 and 251 μmol/g dry wt. A decrease in the concentration of taurine accounts for nearly all of the decrease in the free amino acid pool.3. Tissue hydration increases in M. senile acclimated to dilute seawater, but the increase was not proportional to the change in ambient salinity, indicating that the anemones partially regulate volume in dilute media.4. Mathematical analyses of changes in tissue hydration as a function of ambient salinity in M. senile, Haliplanella lineata, and Diadumene leucolena suggest that the effectiveness of volume regulation increases in individuals of these species acclimated to progressively more dilute media. The volume regulatory capability of Bunodosoma cavernata does not change in dilute media.     

The response of contractile and non-contractile vacuoles of Paramecium calkinsi to widely varying salinities.

Paramecium calkinsi from tidal marshes survive a wide salinity range. Fluid output of contractile vacuoles of these cells decreased as salinity of the medium to which they were acclimated increased, and both pulse rate and vacuole volume were used to regulate output. When cells were first exposed to more dilute medium, contractile vacuoles greatly increased volume so that fluid output increased even though pulse rate decreased. In cells shifted to a more concentrated medium, contractile vacuole output decreased by decreasing pulse rate. The contractile vacuole is surrounded by a set of collecting structures which change form as the salinity changes. Distensible ampullae are found in media of low salinity and collecting canals are found in media of high salinity. When cells are shifted from high salinity to low, the number of ampullae increases and the number of canals decreases. When cells are shifted from low salinity to high, the number of ampullae decreases and the number of canals decreases. Other non-contracting vacuoles also appear in response to a hypoosmotic shock. These include vacuoles within the cell as well as "blisters" on the surface. The number and frequency of blisters increases with the size of the hypoosmotic shock. They detach from cells without resulting in any visible loss of cytoplasm. Non-contractile vacuoles may play a role in sequestering and removing excess water that the contractile vacuoles cannot handle.     

The Response of Contractile and Non-Contractile Vacuoles of Paramecium calkinsi to Widely Varying Salinities

ABSTRACT. Paramecium calkinsi from tidal marshes survive a wide salinity range. Fluid output of contractile vacuoles of these cells decreased as salinity of the medium to which they were acclimated increased, and both pulse rate and vacuole volume were used to regulate output. When cells were first exposed to more dilute medium, contractile vacuoles greatly increased volume so that fluid output increased even though pulse rate decreased. In cells shifted to a more concentrated medium, contractile vacuole output decreased by decreasing pulse rate. The contractile vacuole is surrounded by a set of collecting structures which change form as the salinity changes. Distensible ampullae are found in media of low salinity and collecting canals are found in media of high salinity. When cells are shifted from high salinity to low, the number of ampullae increases and the number of canals decreases. When cells are shifted from low salinity to high, the number of ampullae decreases and the number of canals decreases. Other non-contracting vacuoles also appear in response to a hypoosmotic shock. These include vacuoles within the cell as well as "blisters" on the surface. The number and frequency of blisters increases with the size of the hypoosmotic shock. They detach from cells without resulting in any visible loss of cytoplasm. Non-contractile vacuoles may play a role in sequestering and removing excess water that the contractile vacuoles cannot handle.     

Sting Nematode,Belonolaimus longicaudatus,Immotility Induced by Extracts of Composted Municipal Refuse

Water extracts from saturated composted municipal refuse rendered sting nematodes, Belonolaimus longicaudatus, immotile after immersion for 12 hr. Extract concentrated to 33% o f its original volume rendered all o f the 50 sting nematodes tested immotile in 3 hr. The effect of compost extract was slightly reduced by cation exchange and greatly reduced by peroxide digestion of the organic fraction. Immotile nematodes were transferred from compost extract to distilled water after 24 hz and 60% regained motility, but after 144 hr none regained motility.     

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.     

Early carbonic anhydrase induction in the gills of the blue crab, Callinectes sapidus, during low salinity acclimation is independent of ornithine decarboxylase activity

Carbonic anhydrase (CA) induction in the gills of the euryhaline blue crab, Callinectes sapidus, was measured in response to lowered environmental salinity. Simultaneous measurements of ornithine decarboxylase (ODC) activity were made in gills and nonbranchial tissues to determine whether ODC activity and the resultant synthesis of polyamines played a role in the initiation and regulation of CA induction. CA induction in the seventh gill pair (G7) was proportional to the decrease in ambient salinity, but activity in the third gill pair (G3) remained unchanged. Induction began by 24 hr after low salinity transfer, much earlier than previously reported, and peaked after 4 days. The magnitude of salinity change affected the magnitude of CA induction only, not the time course. A general cell volume regulatory response, as measured by the appearance of total ninhydrin-positive substances (TNPS) in the hemolymph, was initiated within 4 hr of low salinity transfer and was complete by 24 hr post-transfer. General cell swelling may be the initial signal in the pathway of CA induction. ODC activity in the gills of acclimated animals was not influenced by salinity. For crabs transferred from 35 to 25 ppt, ODC activity did not change significantly over the time course of acclimation. There was an early but transient increase in ODC activity in all tissues for crabs acclimated to 28 ppt and transferred to 15 ppt. Induction of ODC activity does not appear to be a precursor for CA induction; therefore, it does not appear that polyamines are substantially involved in the up-regulation of transport enzyme activity in low salinity. ODC, and resultant polyamine synthesis, may, however, have a role in cell volume regulation.     

Schistosoma mansoni: increasing saline concentration signals cercariae to transform to schistosomula

Cercariae of S. mansoni shed the surface glycocalyx, form a double lipid bilayer on their surface, and transform to schistosomula when tails are removed and parasites are transferred from pond water to 300 mOsm phosphate-buffered saline. To determine whether the absolute concentration of saline or the relative change in saline concentration was the signal for surface transformation, cercariae were isolated from the snail hepatopancreas, sheared to remove the tails, and incubated in defined media for 3 hr at 37 degrees C. Surface transformation was assayed using the binding of the fluorescein-conjugated lectin concanavalin A to the schistosomular double unit membrane but not to the cercarial glycocalyx. An increase in salinity either from 18 mOsm (artificial pond water) to 120 mOsm (the snail osmolarity) or from 120 to 300 mOsm (the mammalian osmolarity) triggered transformation to schistosomula. Organisms constantly exposed to 120 mOsm or shifted from 120 mOsm to pond water did not transform their surfaces. The signal for transformation appeared to be increasing salinity rather than increasing osmolarity because cercarial bodies did not become schistosomula in 300 mOsm mannitol. Surface transformation was inhibited when cercariae were incubated with the acetylcholinesterase inhibitor eserine sulfate during a 10 min time when the osmolarity was raised. We conclude that increasing salinity rather than the absolute saline concentration is the signal for surface transformation and that eserine sulfate may inhibit the receipt of this signal.     

Contribution of organic and inorganic osmolytes to volume regulation in rat brain cells in culture

In this work we examined the time course and the amount released, by hyposmolarity, for the most abundant free amino acids (FAA) in rat brain cortex astrocytes and neurons in culture. The aim was to evaluate their contribution to the process of cell volume regulation. Taurine, glutamate, andd-aspartate in the two types of cells, -alanine in astrocytes and GABA in neurons were promptly released by hyposmolarity, reaching a maximum within 1–2 min. after an osmolarity change. A substantial amount of the intracellular pool of these amino acids was mobilized in response to hyposmolarity. The amount released in media with osmolarity reduced from 300 mOsm to 150 mOsm or 210 mOsm, represented 50%–65% and 13%–31%, respectively, of the total amino acid content in cells. In both astrocytes and neurons, the efflux of glutamine and alanine was higher under isosmotic conditions and increased only marginally during hyposmotic conditions.⁸⁶Rb⁺, used as tracer for K⁺, was released from astrocytes, 30% and 11%, respectively, in hyposmotic media of 150 mOsm or 210 mOsm but was not transported in neurons. From these results it was calculated that FAA contribute 54% and inorganic ions 46% to the process of volume regulation in astrocytes exposed to a 150 mOsm hyposmotic medium. This contribution was 55% for FAA and 45% for K⁺ and Cl^– in cells exposed to 210 mOsm hyposmotic solutions. These results indicate that the contribution of FAA to the process of cell volume regulation is higher in astrocytes than in other cell types including renal and blood cells.Special issue dedicated to Dr. Claude Baxter.     

The response of Mytilus edulis to short duration hypoosmotic stress

• 1.1. Valve movements monitored from Mytilus edulis acclimated to 34‰ SW during exposure to low salinity indicate that the shell is not used to isolate the mussel from hypoosmotic SW when the salinity is 17‰ or greater.
• 2.2. The water content of the whole animal, gill and posterior adductor muscle tissue; hemolymph osmotic pressure, Na +, and Cl⁻ concentrations were determined hourly for 9 hr in mussels with valves propped open and exposed to five hypoosmotic salinities from 25.5 to 3.4‰.
• 3.3. Water loading was essentially complete within the first 2hr of exposure: whole animal water content increased 26.51 g H₂O/100g wet tissue in 11.2‰ while the gill tissue water content increased only 2.48 g H₂O/100g wet tissue. During the first 2hr of exposure hemolymph osmotic pressure decreased 200–250 mOsm, regardless of salinity, and at all salinities below 25.5‰ the hemolymph remained hyperosmotic to the medium for the 9 hr of the experiment.
• 4.4. When taken together, these data suggest physiological mechanisms other than valve closure are responsible for tolerance of short duration hypoosmotic stress in Mytilus edulis.

     

Osmotic and ionic haemolymph concentrations in the Baltic Sea amphipod Gammarus oceanicus in relation to water salinity

The aim of this work was to determine the osmotic and ionic (Na(+), K(+), Ca(2+), Mg(2+) and Cl(-)) haemolymph concentrations in Gammarus oceanicus at different salinity levels. Being a species of marine origin it inhabits brackish waters of the Baltic Sea. G. oceanicus specimens were collected in January 2003 from the Gulf of Gdansk (salinity 7 psu). The animals were gradually acclimated to eight different salinity levels (5, 7, 14, 20, 25, 30, 35 and 41 psu) at a temperature of 5 degrees C and 100% oxygen saturation. The haemolymph osmolalities correlated positively with external salinity, from 545.4+/-17.3 mOsm in 5 psu to 1185.9+/-34.6 mOsm in 41 psu. G. oceanicus hyperregulated within the 5-31.5 psu range; above 31.5 psu it hyporegulated its body fluids in comparison to the external medium. At 31.5 psu (1017 mOsm) the haemolymph concentration of G. oceanicus was isoosmotic with the habitat. The haemolymph concentrations of all the studied ions, except K(+), correlated positively with their concentrations at the various salinity.     

The Influence of water salinity on the free amino acid concentration in muscle and hepatopancreas of adult shrimps, Penaeus japonicus

Variations of the total free amino acid (FAA) pool and the content of specific amino acids have been measured in the muscle and hepatopancreas of adult shrimps, Penaeus japonicus, acclimatized at five water salinities: 38, 32, 26, 20 and 14%‰ The FAA content is always higher in muscle than in hepatopancreas at all tested salinites. On the other hand, the hepatopancreas exhibits the highest concentrations of essential amino acids. Two steps in the evolution of FAA content can be observed, the first one regarding decrease in salinity from 38 to 20%‰ and the second one, when salinity goes below 20%_°. The first step can be characterized by a 16% decrease of total FAA content in the muscle and a 36% increase in the hepatopancreas. In muscle, the variations are mainly due to changes in non-essential FAA content, whereas in the hepatopancreas, they are linked to variations in essential FAA content. The other step is characterized by a drastic increase in moisture and decrease in FAA content in both studied organs when water salinity is 14%‰ The total FAA content is about 40% lower in shrimps at 14%_° compared to 38%‰ seawater salinity. During adaptation, the FAA pool (mainly NEFAAs) of muscle seems to be directly related to osmoregulation, whereas in the hepatopancreas, its evolution seems to be linked with energy expenditure and protein synthesis. The results are evaluated in order to elucidate the role of FAA in intracellular osmoregulation and in relation to animal ecology.     

Salinity-mediated carbonic anhydrase induction in the gills of the euryhaline green crab, Carcinus maenas

The euryhaline green crab, Carcinus maenas, is a relatively strong osmotic and ionic regulator, being able to maintain its hemolymph osmolality as much as 300 mOsm higher than that in the medium when the crab is acclimated to low salinity. It makes the transition from osmoconformity to osmoregulation at a critical salinity of 26 ppt, and new acclimated concentrations of hemolymph osmotic and ionic constituents are reached within 12 h after transfer to low salinity. One of the central features of this transition is an 8-fold induction of the enzyme carbonic anhydrase (CA) in the gills. This induction occurs primarily in the cytoplasmic pool of CA in the posterior, ion-transporting gills, although the membrane-associated fraction of CA also shows some induction in response to low salinity. Inhibition of branchial CA activity with acetazolamide (Az) has no effect in crabs acclimated to 32 ppt but causes a depression in hemolymph osmotic and ionic concentrations in crabs acclimated to 10 ppt. The salinity-sensitive nature of the cytoplasmic CA pool and the sensitivity of hemolymph osmotic/ionic regulation to Az confirm the enzyme's role in ion transport and regulation in this species. CA induction is a result of gene activation, as evidenced by an increase in CA mRNA at 24 h after transfer to low salinity and an increase in protein-specific CA activity immediately following at 48 h post-transfer. CA gene expression appears to be under inhibitory control by an as-yet unidentified repressor substance found in the major endocrine complex of the crab, the eyestalk.     

Seasonal variability of free amino acids in two marine bivalves, Macoma balthica and Mytilus spp., in relation to environmental and physiological factors

The seasonal variability of the intracellular free amino acid (FAA) concentration was studied in 5 Macoma balthica populations and 7 Mytilus spp. populations along their European distribution. Because of the well known physiological role of FAA as organic osmolytes for salinity induced cell volume regulation in marine osmoconformers, FAA variations were compared in bivalve populations that were exposed to high vs. low intraannual salinity fluctuations. In general, seasonal FAA variations were more pronounced in M. balthica than in Mytilus spp. In both bivalve taxa from different locations in the Baltic Sea, highest FAA concentrations were found in autumn and winter and low FAA concentrations were measured in summer. Seasonal patterns were less pronounced in both taxa at locations with constant salinity conditions. In contrast to Baltic Sea populations, Atlantic and Mediterranean bivalves showed high FAA concentrations in summer and low values in winter, regardless of seasonal salinity fluctuations. Significant seasonal FAA variations at locations with constant salinity conditions showed that salinity appeared not to be the main factor in determining FAA concentrations. The seasonal patterns of the main FAA pool components, i.e. alanine, glycine and taurine, are discussed in the context of seasonal variations in environmental factors (salinity, temperature) and physiological state (glycogen content, reproductive stage).     

Volume regulation in salt-acclimated toad (Bufo viridis): the role of urea and the urinary bladder

Body water (weight) was studied in the euryhaline toad Bufo viridis during high salt (500 mOsm NaCl) acclimation. Plasma osmolality was greatly increased upon salt acclimation mainly by urea, and was always hyperosmotic to the ambient solution. Water content was regulated quite efficiently in slowly acclimated undisturbed toads. Repeatedly catheterized toads behaved like osmometers when transferred to hyperosmotic solutions. Total urea loss was greatly reduced in salt acclimated toads, suggesting urine was not voided under these conditions. It is concluded that urea accumulation, inhibition of the urine voiding response and the urine in the bladder are the principal factors involved in volume regulation under conditions of salt acclimation.     

Heat shock protein of Hsp70 in Paramecium nephridiatum and its role in adaptation to environmental salinity changes

The level of Hsp70 was studied in the cells of eurihaline ciliate Paramecium nephridiatum after the environmental salinity changes. Two types of treatment were applied. "Shock": ciliates were placed for 1 h to the medium with stress salinity, then transferred back to the medium, they were acclimated to, for 2 h; "adaptation": ciliates were placed for 3 h into stress salinity. It has been shown, that ciliates, acclimated to fresh water (0%) have the higher constitutive level of Hsp70, than those, acclimated to 10%. Transfer from fresh water to 10% does not cause the increase of Hsp70 synthesis in protists, whereas the reciprocal transfer results in induction of Hsp70 in the cells. "Adaptation" results in induction of Hsp70 in both "directions" of salinity changes. The results obtained allow to presume that the possibility to survive in the media of various salinity in eurihaline ciliates is somehow determined by the higher initial level of Hsp70 in their cells, than in stenohaline representatives of the same genus.     

Depression of prolylendopeptidase activity in the delayed hypersensitive guinea pig skin lesion induced by bovine gamma-globulin

Prolylendopeptidase activity was increasingly depressed with time from 6 to 24 hr after the start of sensitization in the delayed hypersensitive guinea pig skin lesion induced by bovine gamma-globulin as an antigen. The remarkably depressed activity of the enzyme in the violently inflamed skin began to be restored slowly 48 hr after sensitization, and its activity was ultimately recovered to the original level by 504 hr after a single sensitization in vivo. Depression of the enzymatic activity is caused by a novel prolyendopeptidase inhibitor, whose amino acid composition is 7 Glu, 1 Ser, 2 Gly, 1 Ala, 2 Pro, and 1 Val, generated by inflammation.     

Regulatory volume decrease (RVD) by isolated and in situ bovine articular chondrocytes

Articular chondrocytes in vivo are exposed to a changing osmotic environment under both physiological (static load) and pathological (osteoarthritis) conditions. Such changes to matrix hydration could alter cell volume in situ and influence matrix metabolism. However the ability of chondrocytes to regulate their volume in the face of osmotic perturbations have not been studied in detail. We have investigated the regulatory volume decrease (RVD) capacity of bovine articular chondrocytes within, and isolated from the matrix, before and following acute hypotonic challenge. Cell volumes were determined by visualising fluorescently-labelled chondrocytes using confocal laser scanning microscopy (CLSM) at 21 degrees C. Chondrocytes in situ were grouped into superficial (SZ), mid (MZ), and deep zones (DZ). When exposed to 180mOsm or 250mOsm hypotonic challenge, cells in situ swelled rapidly (within approximately 90 sec). Chondrocytes then exhibited rapid RVD (t(1/2) approximately 8 min), with cells from all zones returning to approximately 3% of their initial volume after 20 min. There was no significant difference in the rates of RVD between chondrocytes in the three zones. Similarly, no difference in the rate of RVD was observed for an osmotic shock from 280 to 250 or 180mOsm. Chondrocytes isolated from the matrix into medium of 380mOsm and then exposed to 280mOsm showed an identical RVD response to that of in situ cells. The RVD response of in situ cells was inhibited by REV 5901. The results suggested that the signalling pathways involved in RVD remained intact after chondrocyte isolation from cartilage and thus it was likely that there was no role for cell-matrix interactions in mediating RVD.     

Free amino acids in the clam Macoma balthica L. (Bivalvia,Mollusca) from brackish waters of the southern Baltic Sea

Fourteen acidic and neutral free amino acids (FAA) were investigated in soft tissue of Macoma balthica from different depth zones of the Gulf of Gdansk (Baltic Sea) over a full seasonal cycle. The dry weight of the bivalves and physico-chemical parameters of overlying bottom water and surface sediments were measured simultaneously at each site. In the brackish waters of the Baltic, the main pool of FAA is composed of Ala, Gln, Arg, Gly and Orn which represent approximately 80% of the total. Compared to the full saline environments, the composition of FAA in the clams from the Baltic differs substantially. The differences can be attributed to the lower salinity of the Baltic. In the Baltic, Gly appears to play a most important role in regulating intracellular osmolarity in the clams, a function performed primarily by Tau in Atlantic and North Sea populations. Spatio-temporal variations of the FAA are affected by biotic and environmental parameters; their respective influence differs with the amino acids. The concentration of Arg depends on its uptake from the external medium. However, its level might be temporarily modified by stress-induced metabolic transformation (e.g. hydrolysis to Orn) caused by changes in the ambient environment. The concentration of Ala increases with depth, probably because of physiological adaptations of the animal to diminishing oxygen concentration through anaerobic glucose catabolism. Biosynthesis of Ala, similarly to Gln, in the shallower zone is generally related to the physiological state of an organism. The concentration of Gly is most likely regulated by internal mechanisms driven by gonadal development and reproduction.