Haemolymph Sodium Research Articles

Osmoregulatory power influences tissue ionic composition after salinity acclimation in aquatic decapods

Decapod crustaceans show variable degrees of euryhalinity and osmoregulatory capacity, by responding to salinity changes through anisosmotic extracellular regulation and/or cell volume regulation. Cell volume regulatory mechanisms involve exchange of inorganic ions between extra- and intra-cellular (tissue) compartments. Here, this interplay of inorganic ions between both compartments has been evaluated in four decapod species with distinct habitats and osmoregulatory strategies. The marine/estuarine species Litopenaeus vannamei (Lv) and Callinectes danae (Cd) were submitted to reduced salinity (15‰), after acclimation to 25 and 30‰, respectively. The freshwater Macrobrachium acanthurus (Ma) and Aegla schmitti (As) were submitted to increased salinity (25‰). The four species were salinity-challenged for both 5 and 10 days. Hemolymph osmolality, sodium, chloride, potassium, and magnesium were assayed. The same inorganic ions were quantified in muscle samples. Muscle hydration (MH) and ninhydrin-positive substances (NPS) were also determined. Lv showed slight hemolymph dilution, increased MH and no osmotically-relevant decreases in muscle osmolytes; Cd displayed hemolymph dilution, decreased muscular NaCl and stable MH; Ma showed hypo-regulation and steady MH, with no change in muscle ions; As conformed hemolymph sodium but hypo-regulated chloride, had stable MH and increased muscle NPS and ion levels. Hemolymph and muscle ions (especially chloride) of As were highly correlated (Pearson, +0.83). Significant exchanges between hemolymph and muscle ionic pools were more evident in the two species with comparatively less AER regulatory power, C. danae and A. schmitti. Our findings endorse that the interplay between extracellular and tissue ionic pools is especially detectable in euryhaline species with relatively lower osmoregulatory strength.

Dopamine binding directly up-regulates (Na+, K+)-ATPase activity in the gills of the freshwater shrimp Macrobrachium amazonicum

We examined the effects of exogenous dopamine on gill (Na+, K+)-ATPase activity in vitro in microsomal preparations from juvenile or adult freshwater shrimp, Macrobrachium amazonicum. Dopamine had no effect on enzyme activity in juveniles but stimulated activity in adult shrimp gills by ≈35%. Stimulation of the gill (Na+, K+)-ATPase in adult shrimps by 100 mmol L−1 dopamine was characterized kinetically by varying ATP, MgATP, and Na+ and K+ concentrations, together with inhibition by ouabain. Dopamine stimulated ATP hydrolysis by ≈40% obeying Michaelis-Menten kinetics, reaching VM = 190.5 ± 15.7 nmol Pi min−1 mg−1 protein, KM remaining unaltered. Stimulation by Na+ (≈50%) and K+ (≈25%) revealed distinct kinetic profiles: although KM values were similar, Na+ stimulation followed cooperative kinetics, contrasting with the Michaelian kinetics seen for K+. Stimulation by MgATP increased activity by ≈30% with little change in KM. Similar saturation profiles were seen for ouabain inhibition with very similar calculated KI values. Our findings suggest that dopamine may be involved in hemolymph sodium homeostasis by directly binding to the gill (Na+, K+)-ATPase at a site different from ouabain, thus stimulating enzyme activity in an ontogenetic stage-specific manner. However, dopamine binding does not affect enzyme affinity for cations and ouabain. This is the first report of the direct action of dopamine in stimulating the crustacean gill (Na+, K+)-ATPase.

Effect of Microplastic on the Gills of the Shore Crab Carcinus maenas

Microscopic plastic debris (microplastics, <5 mm in diameter) is ubiquitous in the marine environment. Previous work has shown that microplastics may be ingested and inhaled by the shore crab Carcinus maenas, although the biological consequences are unknown. Here, we show that acute aqueous exposure to polystyrene microspheres (8 μm) with different surface coatings had significant but transient effects on branchial function. Microspheres inhaled into the gill chamber had a small but significant dose-dependent effect on oxygen consumption after 1 h of exposure, returning to normal levels after 16 h. Ion exchange was also affected, with a small but significant decrease in hemolymph sodium ions and an increase in calcium ions after 24 h post-exposure. To further asses the effects on osmoregulation, we challenged crabs with reduced salinity after microplastic exposure. Neither microspheres nor natural sediments altered the crab's response to osmotic stress regardless of plastic concentration added. Carboxylated (COOH) and aminated (NH2) polystyrene microspheres were distributed differently across the gill surface, although neither had a significant adverse impact on gill function. These results illustrate the extent of the physiological effects of microplastics compared to the physiological resilience of shore crabs in maintaining osmoregulatory and respiratory function after acute exposure to both anthropogenic plastics and natural particles.

Physiological Indicators of Stress and Morbidity in Commercially Handled AbaloneHaliotis iris

ABSTRACT Abalone are enigmatic, displaying subtle responses to stress, including a delayed onset of morbidity, and death after severe stress. The present study therefore sought to further elucidate the nature of stress responses in the blackfoot abalone Haliotis iris and develop practical indices to quantify stress and forecast survival. Abalone were challenged with air exposure (16°C, 4–120 h). Physiological responses were assessed at three organizational levels: whole organism (heart rate), extracellular (hemolymph metal concentrations), and intracellular (hemocyte lysosome stability). Hemolymph sodium:potassium ratio showed a strong negative correlation with emersion time (R2 = 0.82) and a reasonable capacity to predict subsequent survival (R2 = 0.50); a [Na]:[K] of 23 was representative of an undisturbed abalone, whereas values less than 15 typically indicated morbidity. Hemocytes tended to lyse before lysosome destabilization could be quantified by neutral red loss; the stability assay therefore fail...

Hemolymph chemistry and histopathological changes in Pacific oysters (Crassostrea gigas) in response to low salinity stress

This study described seasonal differences in the histopathological and hemolymph chemistry changes in different family lines of Pacific oysters, Crassostrea gigas, in response to the stress of an abrupt change to low salinity, and mechanical grading. The most significant changes in pallial cavity salinity, hemolymph chemistry and histopathological findings occurred in summer at low salinity. In summer (water temperature 18°C) at low salinity, 9 (25.7% of full salinity), the mean pallial cavity salinity in oysters at day 3 was 19.8±1.6 (SE) and day 10 was 22.8±1.6 (SE) lower than oysters at salinity 35. Associated with this fall in pallial cavity salinity, mean hemolymph sodium for oysters at salinity 9 on day 3 and 10 were 297.2mmol/L±20(SE) and 350.4mmol/L±21.3(SE) lower than oysters at salinity 35. Similarly mean hemolymph potassium in oysters held at salinity 9 at day 3 and 10 were 5.6mmol/L±0.6(SE) and 7.9mmol/L±0.6 (SE) lower than oysters at salinity 35. These oysters at low salinity had expanded intercellular spaces and significant intracytoplasmic vacuolation distending the cytoplasm of epithelial cells in the alimentary tract and kidney and hemocyte infiltrate (diapedesis) within the alimentary tract wall. In contrast, in winter (water temperature 8°C) oyster mean pallial cavity salinity only fell at day 10 and this was by 6.0±0.6 (SE) compared to that of oysters at salinity 35. There were limited histopathological changes (expanded intercellular spaces and moderate intracytoplasmic vacuolation of renal epithelial cells) in these oysters at day 10 in low salinity. Mechanical grading and family line did not influence the oyster response to sudden low salinity. These findings provide additional information for interpretation of non-lethal, histopathological changes associated with temperature and salinity variation.

Osmoregulation in Hypogean Populations of the Freshwater Amphipod, Gammarus pulex (L.)

Abstract The freshwater amphipod Gammarus pulex is widely distributed in freshwater streams and rivers of Europe. This amphipod also has isolated hypogean populations, which are transparent in appearance, suggestive of adaptation to their cave environment. Since cave habitats are often food limited, physiological adaptations have been observed that reduce the energy expenditure of cave organisms. Osmoregulation is an energetically expensive mechanism that allows gammarids to survive in fresh water. This study tested the hypothesis that differences in osmoregulation existed between hypogean and epigean populations of G. pulex. The osmoregulatory parameters measured were haemolymph cation concentrations, water and sodium fluxes and gill Na+/K+-ATPase activity. The hypogean G. pulex had significantly lower haemolymph sodium and potassium concentrations, but had a significantly higher haemolymph ammonium concentration than the epigean G. pulex. The low food availability in the hypogean environment was conside...

Parasitic infection manipulates sodium regulation in the freshwater amphipod Gammarus pulex (L.)

The acanthocephalan parasite Polymorphus minutus induces both physiological and behavioural effects in its intermediate host, Gammarus pulex. The net effect of parasite infection is to increase the likelihood of transmission to the definitive host. Osmoregulation is an energetically expensive mechanism that allows G. pulex to survive in dilute media. Any factor influencing osmoregulation is thus likely to affect the allocation of resources to other areas. This study investigated whether P. minutus infection alters sodium regulation in G. pulex. Haemolymph sodium concentration, water permeability and sodium fluxes were measured over the salinity acclimation range of G. pulex. Water permeability was unaltered by either acclimation salinity or parasite infection. Acclimation to 12‰ significantly raised the haemolymph sodium concentration, reduced the sodium influx, and increased the sodium efflux, to the same extent in both uninfected and infected G. pulex. However, parasite infection induced a significant increase in haemolymph sodium concentration in G. pulex acclimated to 6‰, which was not observed in uninfected G. pulex acclimated to the same salinity. Also, both sodium influx and sodium efflux were significantly lower in parasitized G. pulex acclimated to 6‰, when compared to uninfected G. pulex acclimated to the same salinity. It was concluded that the parasite induced disturbances to sodium regulation in G. pulex acclimated to 6‰ were a functional consequence of the manipulative strategy employed to alter behaviour, rather than a primary target.

Na/K-ATPase activity and osmo-ionic regulation in adult whiteleg shrimp Litopenaeus vannamei exposed to low salinities

Na/K-ATPase activity and osmo-ionic regulation were examined in Litopenaeus vannamei exposed to low salinities. The hemolymph osmolality of shrimp exposed to very low salinities (0.5 ppt and 1 ppt) decreased rapidly after 6 h from 800 mOsm to 540–560 mOsm; these individuals could not survive beyond 1 day. Under exposure to 7 ppt, osmolality levels decreased rapidly to 630 and 570 mOsm after 6 h and 1 day respectively, whereas in shrimp exposed to 18 ppt, levels declined slowly from 800 mOsm to 700 mOsm after 6 h and thereafter remained stable for 7 days until the end of the experiment. Hemolymph sodium ion levels showed a pattern of change similar to that of hemolymph osmolality, with initial levels being 380 mmol/L and decreasing to 180 mmol/L under very low salinity. Changes in calcium, potassium, and magnesium ion concentrations were also investigated. Na/K-ATPase activity was 3.98 μmol ADP/mg protein/hour in the gills of control individuals maintained at 28 ppt for the experimental duration. Levels increased in individuals exposed to 18 ppt, 7 ppt and 3 ppt for 7 days to 5.06, 6.51, and 5.81 μmol ADP/mg protein/hour, respectively. Based on the above, it is suggested that L. vannamei could not survive in low salinity (< 1 ppt) due to the loss of osmoregulatory capacity when the shrimps were transferred directly from high salinities to low salinities.

Tolerance of Three European Native Species of Crayfish to Hypoxia

Species that can act as indicators of ecosystem health offer a valuable tool in the management of natural resources. Crayfish have been suggested as bioindicators of water quality in Europe and at least one species (Austropotamobius pallipes) has been studied to determine its tolerance to pollution and its potential as a bioindicator. The genus Austropotamobius includes three crayfish species native to western Europe: A. pallipes, A. italicus and A. torrentium. It was hypothesised that because of their geographical and habitat distribution, the three Austropotamobius species might vary in their value as a bioindicator of water quality. Crayfish of species A. pallipes and A. italicus were subjected to three different treatments: hypoxia (treatment 3, approx 3 mg l−1 O2), light hypoxia (treatment 2, approx 5.5 mg l−1 O2) and normoxia (treatment 1, control, approx 8.5 mg l−1 O2). A. torrentium crayfish were only subjected to treatment 1 (control) and 3. Variations in haemolymph sodium, calcium and chloride were used as a biomarker and concentrations were measured before and after treatment to evaluate hypoxia-induced stress. Significant differences in the concentrations of sodium between the control groups (treatment 1, normoxia) and the experimental groups (treatment 3, 3 mg l−1 O2) were found in the species A. pallipes and A. torrentium. Groups of A. italicus did not show any significant difference between treatments in sodium concentrations but in chloride concentrations. Crayfish of all three species demonstrated a disruption in the ion exchange process in hypoxia, but all tolerated very low oxygen concentration for an extended period of time.

Gill Na +, K +-ATPase in a series of hyper-regulating gammarid amphipods. Enzyme characterisation and the effects of salinity acclimation

The enzyme Na +, K +-ATPase was investigated in the gills of selected hyper-regulating gammarid amphipods. Gill Na +, K +-ATPase was characterised with respect to the main cation and co-factor concentrations for the freshwater amphipod Gammarus pulex. The optimum cation and co-factor concentrations for maximal gill Na +, K +-ATPase activity in G. pulex were 100 mM Na +, 15 mM K +, 15 mM Mg 2+ and 5 mM ATP, at pH 7.2. The effects of salinity acclimation on gill Na +, K +-ATPase activity and haemolymph sodium concentrations was investigated in selected gammarid amphipods from different salinity environments. Maximal enzyme activity occurred in all gammarids when acclimated to the most dilute media. This maximal activity coincided with the largest sodium gradient between the haemolymph and the external media. As the haemolymph/medium sodium gradient decreased, a concomitant reduction in gill Na +, K +-ATPase activity occurred. This implicates the involvement of gill Na +, K +-ATPase in the active uptake of sodium from dilute media in hyper-regulating gammarids.

The effect of copper on osmoregulation in the freshwater amphipod Gammarus pulex

The influence of copper on osmoregulation in the freshwater amphipod Gammarus pulex was determined from the analysis of water permeability, haemolymph sodium concentration, sodium influx and gill Na +/K + ATPase and Mg 2+ ATPase activity. Exposure to nominal copper concentrations of 100 μg l −1 or greater caused a significant reduction in both haemolymph sodium concentration and sodium influx within 4 h. Measurements of water permeability, expressed as the half-time of exchange of body water ( t 1/2), excluded structural gill damage as the cause of this fall in haemolymph sodium. Copper at 10 μg l −1 or above in the assay solution significantly reduced gill Na +/K + ATPase activity. In contrast gill Mg 2+ ATPase activity was markedly less affected by copper. These differences in enzyme sensitivity were considered with respect to the potential mechanisms of copper toxicity.

Effects of extracts from the cyanobacterium Microcystis aeruginosa on ion regulation and gill Na+,K+-ATPase and K+-dependent phosphatase activities of the estuarine crab Chasmagnathus granulata (Decapoda, Grapsidae).

Recent discoveries indicate that microcystins affect enzymes, such as Na(+),K(+)-ATPase, involved in ion regulation of aquatic animals, through K(+)-dependent phosphatase inhibition. In vitro studies showed the inhibitory effect of Microcystis aeruginosa extracts on Na(+),K(+)-ATPase and K(+)-dependent phosphatase activities in gills of Chasmagnathus granulata (Decapoda, Grapsidae). Extracts of M. aeruginosa were prepared from lyophilized or cultures cells of the cyanobacterium. For lyophilized cells, IC(50) values were estimated as 0.46 microg/L (95% confidence interval [CI]=0.40-0.52 microg/L) and 1.31 microg/L (95% CI=1.14-1.51 microg/L) for Na(+),K(+)-ATPase and K(+)-dependent phosphatase, respectively. However, extracts prepared from cultured cells presented a much lower inhibitory potency against both enzymes. Gas chromatography revealed long-chain fatty acids in the lyophilized cell extracts, indicating that they are in part responsible for the enzyme inhibition. In vivo studies showed that the toxin inhibited Na(+),K(+)-ATPase activity in anterior gills, whereas an increased augmented activity of glutathione-S-transferase was observed in both kind of gills, indicating that the crab has increased its ability to conjugate the toxin. No significant differences in hemolymph sodium or chloride concentration were detected. This result is in agreement with the lack of effects of microcystin on Na(+),K(+)-ATPase activity of posterior (osmoregulating) gills.

Osmoregulatory capacity according to nutritional status, molt stage and body weight in Penaeus stylirostris

Hypo-osmoregulatory capacity (or hypo-OC, i.e., the difference between the osmolality of the haemolymph and that of sea water), haemolymph glycemia and haemolymph sodium and chloride concentrations of Penaeus stylirostris were studied in experimental tanks according to the size, the molt stage and the nutritional status of the shrimp. Osmolality and haemolymph glycemia of fed and starved P. stylirostris were also studied in earthen ponds in fluctuating sea water according to time. In experimental tanks, the absolute hypo-OC of shrimp decreased linearly with increasing wet weight for each molt stage considered. For specimens weighing 16.6±1.7 g, the absolute hypo-OC was maximum in stage C (217±17 mosm kg −1) and significantly lower in stages B (165±16 mosm kg −1), D 0 (181±18 mosm kg −1), D 1 (146±20 mosm kg −1) and D 2 (135±13 mosm kg −1). The hypo-OC and haemolymph glucose concentration varied significantly after the food supply. No variations in haemolymph sodium and chloride concentrations were observed. In earthen ponds, haemolymph osmolality and haemolymph glucose concentration of molt stage C shrimp increased shortly after the food supply. When shrimp were kept starved for 24 and 48 h, haemolymph osmolality remained constant and haemolymph glucose concentration varied only slightly according to time. In light of these results, the use of shrimp hypo-OC (and/or haemolymph osmolality) and haemolymph glycemia in aquaculture as potential physiological indicators of disturbance in the aquatic environment is discussed.

Haemolymph osmoregulation and the fate of sodium and chloride during dehydration in terrestrial isopods

Osmoregulation of the haemolymph during dehydration was investigated in a selection of temperate oniscidean isopods. Inulin tracer studies show that the haemolymph contributes approximately 69% of water losses in Porcellio scaber, significantly more than predicted from the volume of this compartment (42% of total water). Haemolymph osmolality increases linearly as a function of haemolymph dehydration but at a significantly lower rate than predicted from the change in haemolymph fluid volume. Similar results for Oniscus asellus show that both species display efficient osmoregulation until lethal dehydration. Osmoregulation is associated with significant hyporegulation of haemolymph sodium and chloride. These findings indicate that: (1) cell water is conserved at the expense of the haemolymph; and (2) haemolymph dehydration is associated with the removal of Na + and Cl − contributing to net osmoregulation. During dehydration, accumulations of both Na + and Cl − are seen in the hindgut, with significant accumulations of electrolytes also seen in the luminal fluid of the hepatopancreas. Low fluid volumes in the foregut and hindgut suggest macromolecular association as the most plausible mechanism of ion sequestration. Evidence refutes ion excretion and haemocyte sequestration as osmoregulatory mechanisms. Sequestration of Na + as urate salts, as shown for Periplaneta and generally assumed for other insects, is insignificant in isopods.

Regulation of hemolymph osmolytes and gill Na +/K +-ATPase activities during acclimation to saline media in the freshwater shrimp Macrobrachium olfersii (Wiegmann, 1836) (Decapoda, Palaemonidae)

To evaluate the mechanisms of long-term osmotic adaptation to saline media, total hemolymph osmolytes, hemolymph sodium and chloride concentrations, and gill Na +/K +-ATPase activities were measured in the freshwater shrimp Macrobrachium olfersii (Wiegman) after acclimation for 10 or 20 days to media of <0.5, 21 and 28‰ salinity. Total hemolymph osmolytes are maintained strongly hyperosmotic to the external medium in low and moderate salinities, becoming slightly hyperosmotic at high salinity. In contrast, however, hemolymph [Na +] and [Cl −] are distinctly hyporegulated at the higher salinities. This difference in response pattern to acclimation apparently results from the presence of free amino acids in the hemolymph as a consequence of the synthesis of intracellular organic osmolytes. The resulting hyperosmotic regulation both avoids water loss from the animal in saline media and provides an osmotic gradient, allowing the uptake of water from the medium for excretion of the salt load. Gill Na +/K +-ATPase activities decrease by ≈35% after acclimation to saline media, suggesting that the rates of the cellular mechanisms responsible for salt uptake in freshwater become reduced, avoiding excessive salt loading. A Na +-ATPase activity is salinity independent. These data are examined with regard to the intrinsic coupling between the physiological alterations taking place in hemolymph osmolytes during acclimation to saline media and the concomitant ultrastructural rearrangements in the salt transporting tissues of the gills, and their possible neurosecretory control mechanisms.

Changes of osmotic and electrolyte concentrations in the haemolymph of Penaeus japonicus exposed to ambient ammonia

The osmolality and sodium, potassium, calcium, magnesium and chloride concentrations in the haemolymph of Penaeus japonicus individually exposed for 24 h in a salinity of 30 at 25 °C to 0.004 (control), 0.076, 0.356, 0.784, and 1.476 mmol/L ammonia-N (molecular ammonia plus ammonium as nitrogen) were examined. The haemolymph osmolality and sodium concentration of shrimps exposed to 0.356 mmol/L ammonia-N, and haemolymph concentrations of sodium, calcium, magnesium and chloride of shrimps exposed to 1.476 mmol/L were significantly lower (P < 0.05) than those in the control animals. It is concluded that P. japonicus is iso-osmotic in control water, and becomes hypo-osmotic to the environment due to significant decreases of haemolymph electrolytes under ammonia stress at concentrations as high as 1.476 mmol/L.

Extracellular and intracellular ionic changes in crayfish Astacus astacus exposed to nitrite at two acclimation temperatures

Freshwater crayfish, Astacus astacus, were exposed to two concentrations of ambient nitrite (0.5 and 1.0 mM) at two acclimation temperatures (6°C and 16°C) for 1 week. The concentration of nitrite in the haemolymph increased with increasing temperature and ambient level of nitrite. The haemolymph/water concentration ratio was the same at the two ambient nitrite concentrations but increased with temperature. Haemolymph nitrite reached concentrations that were 12 and 16 times higher than the water values at 6°C and 16°C, respectively. Nitrite accumulation induced a decrease in the haemolymph chloride concentration that far exceeded the rise in haemolymph [NO 2]. Haemolymph sodium concentration decreased significantly but less than haemolymph chloride. A large decrease in muscle tissue potassium concentration was seen in nitrite-exposed animals at both temperatures. In midgut gland tissue, the potassium concentration tended to increase, suggesting that the K + efflux mechanism that was stimulated in muscle tissue was absent (or less affected) in midgut gland tissue, and that part of the potassium lost from muscle may have been taken up by the midgut gland.

The effect of eyestalk ablation on growth, haemolymph composition and gill Na +, K +-ATPase activity of Penaeus monodon juveniles

1. 1. Eyestalk unablated and unilaterally ablated Penaeus monodon juveniles had survival rates after 5 months of 75–72.5 and 67.5–60%, respectively. 2. 2. Unilaterally ablated shrimps had significantly higher ( P < 0.05) growth rate than unablated shrimps. 3. 3. Eyestalk-ablatement resulted in a decrease in the haemolymph sodium concentration and an increase in the potassium and calcium concentration of shrimps. 4. 4. The osmolarity of haemolymph and total protein concentration of unablated shrimps were demonstrated to be higher than those of unilaterally ablated shrimps. 5. 5. The eyestalk-ablated shrimps possess higher total ATPase and Na +,K +-ATPase activities in the gill than those of unablated shrimps.

Effects of mercury exposure on ionic regulation in the crayfish Orconectes propinquus.

Hemolymph sodium, potassium and calcium concentrations were determined in crayfish (Orconectes propinquus) exposed to (203)HgCl(2) mixed with food to a concentration of 1 microg Hg g(-1). Dummy-fed animals were exposed to Hg-dosed food wrapped in dialysis tubing to control for mercury reaching the animals via leaching from food to water. Hemolymph analyses were made following 14-day Hg exposures and again after a further 21-day 'depuration' period during which all animals were fed Hg-free food. After 14 days, the mercury reached a concentration of 0.175 microg g(-1) in the hepatopancreas and approximately half this level in the gills of Hg-fed animals. No depuration occurred from the hepatopancreas although the gills lost approximately two-thirds of their labelled mercury during the depuration period. Hemolymph sodium concentrations in Hg-exposed crayfish, both fed and dummy-fed, after 14 days were significantly lower than in Hg-free controls and remained low following the 21-day depuration period. Hemolymph calcium concentrations were lower in Hg-fed animals than in dummy-fed and control animals after 14 days although calcium levels rose in all treatments after 35 days. This may have been due to the incidence of pre-molt animals in all experimental groups, although the relationship between this and mercury exposure was not established unequivocally. Hemolymph potassium levels showed no differences between treatments.

The ecophysiology of under-ice fauna

During exposure to low salinity, the under-ice amphipods Gammarus wilkitzkii and Onisimus glacialis appeared as euryhaline osmoregulators, displaying regulation of haemolymph concentrations of sodium and chloride. Free amino acids took part in the regulation. During freezing and brine formation, the amphipods were freeze-sensitive and did not tolerate being frozen into solid ice. However, they could stay in the vicinity of the ice, conforming osmotically to the ambient brine and thus lowering the melting point of the amphipods' body fluids. This prevented internal ice formation in the absence of antifreeze agents (THF) in the haemolymph. When G. wilkitzkii, O. glacialis and Apherusa glacialis were exposed to dilute seawater, elevated rates of oxygen consumption and ammonia excretion were observed. The O:N atomic ratio was kept nearly constant during hyposmotic stress, indicating protein/lipids as metabolic substrate. Rates of oxygen consumption and ammonia excretion increased with increasing osmotic differences between the haemolymph and the medium, indicating higher energy requirements for osmotic and ionic regulation at low salinities. A minor decrease in haemolymph sodium concentrations coincided with the increased ammonia output during hyposmotic stress, indicating a possible counter ion regulation of NH+4 and Na+. An increased rate of oxygen consumption, ammonia excretion and 0:N ratio versus temperature was observed for all species.