Haemolymph Sodium Concentrations Research Articles

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.

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.

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.

Effect of ammonia on survival and osmoregulation in different life stages of the lobsterHomarus americanus

Acute toxicity of ammonia was determined for cultured larval, postlarval, and wild adult lobsters (Homarus americanus) in 1988. Ammonia tolerance was found to increase with ontogenetic development. Based on 96-h LC50 values of 58 mg l−1 NH4 + + NH3 l−1 seawater (0.72 mg NH3 l−1) for Stage I larvae, 87 mg NH4 + + NH3 l−1 (1.7 mg NH3 l−1) for Stage II larvae, 125 mg NH4 + + NH3 l−1 (2.13 mg NH3) for Stage III larvae, 144 mg NH4 + + NH3 l−1 (2.36 mg NH3 l−1) for Stage IV postlarvae, 377 mg NH4 + + NH3 l−1 (5.12 mg NH3 l−1) for adult lobsters at 5°C and 219 mg NH4 + + NH3 l−1 (3.25 mg NH3 l−1) for adult lobsters at 20°C, recommendations for “safe” levels of total ammonia and un-ionized ammonia were calculated using an application factor of 0.1. Effects of ammonia on osmoregulatory capacity were studied on postlarvae and adults. Ability of postlarvae and adults to hyper-regulate in low-salinity media decreased after exposure to ammonia. In postlarval lobsters, osmoregulatory capacity was significantly affected in ammonia concentrations exceeding 32 mg l−1. Osmoregulatory capacity in adult lobsters (5 and 20°C) was affected at 150 mg l−1. In postlarval lobsters, a minimum exposure time of 12 h was required to impair osmoregulatory capacity. The decrease in hemolymph osmotic pressure was caused by lower hemolymph sodium concentrations. The presence of ammonia in the external medium could markedly affect the Na+/NH4 + transport mechanism by permanently, temporarily, or partially impairing the transport sites for sodium.

Osmotic and Ionic Regulation in the Ghost Crab Ocypode'saratan in Hypoionic and HyperionicMedia and Following Desiccation in Air

1.Sodium. potassium and magnesium ion concentrations in the blood of Ocypode were hypoionic (0 normal and concentrated sea water. 2. Over the entire salinity range tested calcium ion concentration was found to remain fairly constant and hyperionic to all media. 3. The changes in haemolymph sodium concentration reflect the changes in tQtal osmotic concentration. 4. Death from desiccation related directly to weight loss,indicatiogthaf wa~er loss probably as circulatin& ha~molymph volume was the critical factor, rather than ionic concentration.

Influence of environmental salinity on oxygen consumption and ammonia excretion of the arctic under-ice amphipodOnisimus glacialis

Changes in salinity affect the metabolic rate of the sympagic amphipodOnisimus glacialis collected from the Barents Sea in 1986 and 1988. When transferred from 35 to 5 ppt S, oxygen consumption and ammonia excretion both increase three-fold during the first 5 h of exposure, and they remain high throughout the rest of the experimental period (26 h). During 24-h acclimation to various salinities (5 to 45 ppt), the amphipods exhibit a respiratory and excretory response to hyper- and hypoosmotic stress; however, a rather constant O:N atomic ratio (around 15) was obtained at the experimental salinities, indicating protein/lipids as metabolic substrate. Both rates of oxygen consumption and ammonia excretion increased with an increasing osmotic difference (0 to 650 mOsm) between the haemolymph and the environmental medium, indicating higher energy requirements for osmotic and ionic regulation at low salinities. In amphipods abruptly transferred from 35 to 5 ppt, a minor decrease of the haemolymph sodium concentrations together with an increased ammonia excretion output indicate a counter-ion regulation of NH4+ and Na+ during hyposmotic stress.

Seasonal aspects of sodium, potassium, and water balance in the predaceous diving beetle Dytiscus verticalis

A previous study showed that food availability i the laboratory can dramatically affect sodium balance of the predaceous diving beetle Dytiscus verticalis. Because this species inhabits small ponds that dry up in the summer, it seemed likely that wild beetles would undergo annual changes in ody ion content. Seasonally collected predaceous diving beetles were analyzed for dry body mass, body water, body sodium and potassium, hemolymph sodium concentration, and hemolymph osmolality. Beetles varied in all characteristics over a 16-month period, and variation was seasonally cyclic for body water, body sodium, hemolymph sodium, and hemolymph osmolality, but not for dry mass and body potassium. A laboratory experiment and two field-enclosure experiments determined possible mechanisms underlying these cycles. Beetles in enclosures with food had greater dry mass than nonfed beetles in enclosures, but not greater than that of wild beetles. The number of tadpoles killed by beetles in enclosures correlated well with beetle dry mass. Because food availability directly affects dry mass, wild beetles must not suffer seasonal periods of food limitation. Body sodium and potassium levels also appear related to food availability, but not closely enough to explain the seasonal variation in sodium, the nonseasonal variation in potassium, and the nonparallel variation in the two cations. Food intake can be important for increasing both hemolymph sodium concentration and hemolymph osmolality, but these characteristics can be regulated independently of food intake and of each other. Seasonal variation in hemolymph sodium was out of phase with body sodium variation, suggesting that sodium supply is not the cause of hemolymph sodium cycles. Hemolymph osmolality was greatly increased in winter months, perhaps reflecting elevated levels of free amino acids in the hemolymph.

The effect of food consumption on sodium and water balance in the predaceous diving beetle,Dytiscus verticalis

Sodium and water balance ofDytiscus verticalis in fresh water were investigated under three feeding regimes: unfed, and fed a diet either low or high in sodium chloride. Unfed sodium influx was 0.13 and sodium efflux was 0.74 μmoles/100 gwm·h. These values are low in comparison with most freshwater animals. The electrical potential difference across the integument in artificial soft water (ASW) was about 150 mV smaller than the potential necessary to maintain sodium balance in the absence of active transport. However, sodium influx did not show saturation kinetics over an external concentration range of 91 to 1725 μM. Unfed beetles failed to arrest net sodium loss to baths that were initially distilled water or ASW, even when bath sodium concentrations reached 75–298 μM. The long-term rate of net sodium loss ranged from 0.61 to 4.4 μmoles/100 gwm·h for four sets of animals. Beetles decreased sodium efflux during a period of fasting. During subsequent feeding, beetles fed a high sodium diet (HSD) increased sodium efflux while beetles fed a low sodium diet (LSD) maintained low rates of sodium efflux. HSD fed beetles increased body sodium and hemolymph sodium concentration, and expanded extracellular fluid, relative to LSD fed beetles. Thus beetles cannot achieve sodium balance in fresh water without dietary sodium input, although they are able to regulate sodium loss.

Ionic regulation and salinity related changes in haemolymph protein in the semi-terrestrial beachflea Orchestia gammarellus (Pallas) (Crustacea: Amphipoda)

1. 1. Orchestia gammarellus maintained in air and provided with food in the form of agar was found to be very tolerant of changes in the ionic content of the food and was shown to have well-developed powers of ionic regulation over the salinity range 5–40‰ at 10°C. 2. 2. There was an inverse relationship between haemolymph protein and acclimation salinity. 3. 3. The concentration of sodium and protein ions in the haemolymph of O. gammarellus from above high water mark (H.W.M.) was markedly different from animals collected below H.W.M. Individuals taken from above H.W.M. characteristically had low haemolymph sodium but elevated haemolymph protein concentrations.

Osmotic and cationic urine concentrations/blood concentrations ratios in hyporegulating Ucides cordatus

1. 1. In crabs exposed to sea-water for 96 hr, the haemolymph sodium, potassium, calcium, magnesium and osmotic concentrations have risen, but to levels below those found in the medium. 2. 2. Blood and urine were isosmotic for all exposure times. 3. 3. Urine sodium concentration was higher than that of blood during the first 24 hr. 4. 4. Urine, potassium and magnesium concentrations were higher than those of blood during every exposure period. 5. 5. Na U/B ratios and K U/B ratios significantly decreased, whereas Mg U/B ratios significantly increased along the sea-water exposure time. 6. 6. The role of the antennal gland is discussed.

Variation of cation concentrations in the haemolymph of the silkworm, Bombyx mori, with diet and larval-pupal development

The concentrations of haemolymph cations were studied from the early 5th instar to a new pupal stage in the silkworm, Bombyx mori, reared on either mulberry leaves or an artificial diet. 1. 1. The potassium level in the haemolymph changed little during feeding and mature stage and] averaged 50 μequiv/ml in the silkworm larvae reared on both diets. It increased during larval-pupal development to 90 μequiv/ml at pupation. 2. 2. The magnesium level in the haemolymph of the silkworm larvae reared on mulberry leaves increased as the larval growth proceeded. On the other hand in the silkworm on artificial diet the level was maintained at a steady-state level of 100 μequiv/ml. In both groups it declined remarkably during larval-pupal development. 3. 3. The haemolymph sodium concentration was higher in the larvae on the artificial diet than those fed on mulberry leaves. 4. 4. The calcium level was higher in the larvae on the mulberry leaves diet than those on the artificial one during the feeding stage, reflecting the content of diets.