Hemolymph Chloride Research Articles

Osmotic and ionic regulation, and kinetic characteristics of a posterior gill (Na+, K+)-ATPase from the blue crab Callinectes danae on acclimation to salinity challenge

Salt tolerance reflects ecophysiological adaptation, and the wide-ranging distribution of the Brachyura mirrors their ability to adjust body fluid concentrations. The gill (Na+, K+)-ATPase underpins such hyper/hypo-regulatory mechanisms. We evaluate osmotic and chloride regulation in Callinectes danae after 10 days acclimation to a wide salinity range (5–50 ‰S), accompanying alterations in hemolymph osmolality and [Cl−] during hypo- (15 ‰S) or hyper- (45 ‰S) osmotic challenge. Further, we investigate posterior gill (Na+, K+)-ATPase kinetics, α-subunit immunolocalization and its mRNA and protein expression (15, 30 and 40 or 45 ‰S). The crab is a moderate, asymmetrical hyper/hypo-osmoregulator but is a strong, asymmetrical hyper/hypo-chloride regulator. Hyper-regulation at low salinity is sustained by a threefold increase in (Na+, K+)-ATPase activity, a 3.5-fold increase in α-subunit mRNA expression and 1.6-fold increase in protein expression. α-Subunit signal is highest in 15 ‰S-acclimated crabs, and is uniformly distributed throughout the ionocytes and pillar cells. Activity in 30- and 40% S-acclimated crabs is similar. Affinity for ATP and Na+ increases on high salinity acclimation but decreases for ouabain. K+ apparent affinity is independent of salinity, while that for Mg2+ decreases and for NH4+ increases with increasing salinity. A high-affinity ATP-binding site disappears on acclimation at any salinity. FOF1- and Na+- or K+-ATPase activities decrease with increased salinity. Hemolymph chloride hypo-regulation depends little on gill (Na+, K+)-ATPase activity. Hyper-, and hypo-osmotic and ionic regulatory capabilities in C. danae are intricate physiological processes underpinned by multifarious gill (Na+, K+)-ATPase kinetics and altered mRNA and protein expressions.

Effect of Carbon Dioxide-Induced Water Acidification on the Physiological Processes of the Baltic Isopod Saduria entomon

Survival, behavior, hemolymph pH, osmolality, and chloride ion concentration as well the total metabolic rate (heat dissipation rate) of the isopod Saduria entomon from the brackish Baltic Sea were investigated after exposure to carbon dioxide-induced water acidification (pH, 7.5, 7.0, and 6.5; control pH, 8.2) keeping other parameters constant (temperature, 10°C; salinity, 7). The short-term (12 h per each pH treatment) exposure to carbon dioxide-induced water acidification did not cause significant changes (P < 0.05) in the resting metabolic rate or the scope of activity in S. entomon; however, high interindividual variability was observed. The 2-wk exposure to lowered pH values did not affect either the activity of the isopods or their survival rate significantly (P > 0.05), which was greater than 90% in all pH treatments. The hemolymph pH increased significantly (P < 0.05) with a decrease of water pH from a control pH of 8.2 down to a pH of 7.0. Hemolymph osmolality increased significantly (P < 0.05) at pH 7.5, but exposure to pH 7.0 did not cause further increase in this parameter. Reduction of water pH did not affect the hemolymph chloride ion concentration. Obtained results indicated that S. entomon is adapted to large fluctuations of carbon dioxide levels in the water primarily to compensate for acid—base disturbances without additional energetic costs.

The physiological ecology of the supratidal amphipod Talorchestia longicornis

Physiology, behavior, habitat, and morphology are used to determine the degree of adaptation to life on land for amphipod species and systemization within the four functional groups of the family talitridae. Talorchestia longicornis is a semi-terrestrial amphipod found in the supratidal zone of estuaries. The present study investigates the physiological adaptations of this species to life on land through measurements of osmoregulation and respiration. Over the salinity range of 1–40‰, T. longicornis regulated its hemolymph hyperosmotically at low salinities and hypoosmotically at high salinities. The isosmotic point was about 27‰. Analogously, hemolymph chloride levels were well regulated being hyperionic at low salinities and hypoionic at high salinities. This species is capable of respiration in both air and water. Slopes ( b values) of the relationship between weight and oxygen uptake rates ranged from 0.316 to 0.590. Oxygen uptake rates were higher in air than water and at night versus day. Q 10 values were slightly below 2.0 for respiration in air for amphipods, irrespective of weight. These physiological adaptations, along with its behaviors, habitat, and morphology, place T. longicornis within the Group III sandhoppers of the Talitridae.

The influence of salinity on copper accumulation and its toxic effects in estuarine animals with differing osmoregulatory strategies

Copper is an important ionoregulatory toxicant in freshwater, but its effects in marine and brackish water systems are less well characterised. The effect of salinity on short-term copper accumulation and sublethal toxicity in two estuarine animals was investigated. The osmoregulating crab Hemigrapsus crenulatus accumulated copper in a concentration-dependent, but salinity-independent manner. Branchial copper accumulation correlated positively with branchial sodium accumulation. Sublethal effects of copper were most prevalent in 125% seawater, with a significant increase in haemolymph chloride noted after 96 h at exposure levels of 510 μg Cu(II) L −1. The osmoconforming gastropod, Scutus breviculus, was highly sensitive to copper exposure, a characteristic recognised previously in related species. Toxicity, as determined by a behavioural index, was present at all salinities and was positively correlated with branchial copper accumulation. At 100% seawater, increased branchial sodium accumulation, decreased haemolymph chloride and decreased haemolymph osmolarity were observed after 48 h exposure to 221 μg Cu(II) L −1, suggesting a mechanism of toxicity related to ionoregulation. However, these effects were likely secondary to a general effect on gill barrier function, and possibly mediated by mucus secretion. Significant impacts of copper on haemocyanin were also noted in both animals, highlighting a potentially novel mechanism of copper toxicity to animals utilising this respiratory pigment. Overall these findings indicate that physiology, as opposed to water chemistry, exerts the greatest influence over copper toxicity. An understanding of the physiological limits of marine and estuarine organisms may be critical for calibration of predictive models of metal toxicity in waters of high and fluctuating salinities.

Hyperventilation and loss of hemolymph Na+ and Cl- in the freshwater amphipod Gammarus fossarum exposed to acid stress: a preliminary study.

The effect of acidification on the acid-sensitive species Gammarus fossarum was investigated in the laboratory. The results showed that as mortality increased, mean hemolymph chloride and sodium concentrations decreased rapidly. Concomitantly, organisms hyperventilated during the first 24 h and then started to hypoventilate. These results demonstrated that exposure to acid stress in the acid-sensitive species G. fossarum led to ion-regulatory and respiratory failure as previously reported in fish and crayfish exposed to acid stress.

Environmental hypoxia affects osmotic and ionic regulation in freshwater midge-larvae

The effect of anaerobic metabolism on the osmotic and ionic regulation of the extracellular fluid was examined. Larvae of three species, characterized by different hypoxia tolerance, were studied: Chaoborus crystallinus, Culex pipiens and Chironomus gr. plumosus. The use of the capillary electrophoresis technique made it possible to determine approximately 15 different ions from individual hemolymph samples. The hemolymph concentration of both inorganic and organic anions and cations as well as the osmolality were measured. A correlation between the hypoxia tolerance and the capability to avoid net changes in the ion concentration or in the osmolality of the three species studied here is proposed: Culex larvae, which have the lowest hypoxia tolerance, show a very large and very rapid lactate accumulation in their hemolymph under experimental hypoxia. This lactate accumulation is not compensated for by a change in the concentration of any other ion. Chaoborus larvae, with a medium hypoxia tolerance, utilize their very large hemolymph malate pool as a source of anaerobic energy. It is converted into succinate, thus inducing little net changes in the sum of the anions. There is a marked increase of the hemolymph osmolality, though. Chironomus larvae have the highest hypoxia tolerance and there are remarkably little changes in their hemolymph under hypoxia. Although these larvae are described as relying mainly on ethanol fermentation under environmental anaerobiosis, we demonstrated a marked lactate fermentation in severe hypoxia. The lactate accumulation observed in our study was compensated by a concomittant decrease of the hemolymph chloride concentration.

Osmotic and ionic regulation in the giant Malaysian fresh water prawn, Macrobrachium rosenbergii (de Man), with special reference to strontium and bromine

Abstract Macrobrachium rosenbergii is able to exert a high degree of control over the osmotic pressure and the ionic concentration of its haemolymph when exposed to a wide range of salinity between fresh water and its isosmotic point. Sodium and chloride ions were regulated at almost identical concentrations over most of the salinity range tested. Calcium and potassium ions were hyperregulated at all salinities, whereas magnesium ions were always maintained at much lower concentrations than in the medium. The regulation of strontium ions has been demonstrated and appears to be linked to magnesium regulation. Haemolymph bromide concentration is also regulated, but not as strongly as haemolymph chloride, and the two regulatory mechanisms appear to be discriminatory. The regulation of both strontium and bromide ions suggests that both may be important in the physiology of this species.

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.

Responses of osmotic and chloride concentrations of Penaeus chinensis Osbeck subadults acclimated to different salinity and temperature levels

Hemolymph osmolality, chloride concentration and tissue water of Penaeus chinensis Osbeck subadults (16.40 ± 2.82 g) were determined after they were acclimated from 33 ppt to 10, 20, 30 and 40 ppt for 14 days at 24 °C, and then acclimated to 12, 18, 24 and 30 °C for 14 days. Hemolymph osmolality and chloride concentration increased with increased salinity, but decreased with increased temperature. Tissue water decreased with increased salinity. Hemolymph osmolality and chloride concentration were linearly related to medium osmolality and medium chloride concentration, respectively. The isosmotic point and isoionic point was 837, 811, 735, 707 mOsm/kg and 324, 299, 289, 278 mEq Cl −/l which was equivalent to 28.4, 27.5, 25.0, 24.1 ppt and 19.6, 18.1, 17.5, 16.9 ppt for shrimp acclimated to 12, 18, 24, 30 °C, respectively. Hemolymph chloride concentration increased with increased hemolymph osmolality, whereas tissue water decreased with increased hemolymph osmolality. Penaeus chinensis subadults at 24 and 30 °C had superior osmoregulation, and accepted less change of tissue water and hemolymph osmolality than those at 12 and 18 °C due to changes of salinity and temperature.

Sublethal physiological changes in freshwater crayfish, Astacus astacus, exposed to nitrite: haemolymph and muscle tissue electrolyte status, and haemolymph acid-base balance and gas transport

Crayfish, Astacus astacus, were exposed to 0.8 mM environmental nitrite for up to seven days to investigate nitrite-induced perturbations of crustacean homeostasis. Nitrite accumulated in the haemolymph to 8–10 mM within two days, whereafter the extracellular concentration remained constant. This was associated with a substantial decrease in haemolymph chloride and a smaller decrease in sodium, whereas other haemolymph ions (potassium, calcium, magnesium, bicarbonate and lactate) were unchanged. In muscle a pronounced decrease in potassium concentration occurred, whereas sodium, calcium and magnesium concentrations were unaffected. It is proposed that K + efflux from intracellular compartments constitute a general response to nitrite. The extracellular acid-base status changed towards a respiratory alkalosis. Haemolymph P O 2 increased and the haemocyanin concentration decreased. The respiratory pigment itself, however, appeared only marginally affected by nitrite. Oxygen transport accordingly was not severely perturbed, and lactate levels remained low. The mechanisms underlying the responses to nitrite are discussed, and physiological changes in crayfish are compared with corresponding nitrite-induced effects in teleost fishes.

THE CHLORIDE-STIMULATED Κ+-SECRETION BY INSECT MIDGUT AND ITS MODIFICATION IN THE PRESENCE OF OSMOTIC GRADIENTS: A SHORT-CIRCUIT CURRENT AND NOISE-ANALYSIS STUDY

K(+)-secretion in the midgut of the larval moth, Manduca sexta, was studied by measuring the kinetics of the lumen-directed short-circuit current (Isc) and the conduction noise from basolateral K+ channel block by Ba2+. Hemolymph chloride as well as hypotonicity both stimulate this K+ current (IK). The kinetic nature of the stimulation is, however, different in each case. Analysis of blocker noise supports, to a large degree, the interpretation obtained from kinetics, namely: chloride ions do not act via changes in cell volume but influence ion turnover and channel number.

A study of steady state and kinetic regulation of chloride ion and osmotic pressure in hemolymph of oysters,Crassostrea virginica, exposed to tri-n-butyltin

Two sets of experiments were made to determine if bis(tri-n-butyl)tin oxide (TBT) [IUPAC: hexabutyldistannoxane] influenced osmotic pressure or chloride ion concentration in hemolymph of adult American oysters, Crassostrea virginica. In the first set, oysters were acclimated to 5, 25 or 40%;, then exposed to bis(tri-n-butyl)tin oxide dissolved in seawater (0.5, 1.0 or 2.0 micrograms/L) for 11 days. Measurements of the total osmotic pressure and chloride ion concentration in hemolymph indicated that mean values of neither was significantly affected under steady state conditions. Oysters exposed to TBT exhibited a higher variation around the mean value for both total osmotic pressure and chloride ion concentration. There was significant mortality of oysters exposed to TBT in 25 and 40, but not in 5%. In a second experiment, adult oysters were acclimated to 25%, and simultaneously exposed to the TBT concentrations listed above for 10 days. Then, subgroups of oysters were abruptly moved to 5 or 40% and the time-course of adjustment of hemolymph osmotic pressure and chloride ion concentration was measured. During the first 25 hr, there was little adjustment to 5% in controls or TBT exposed oysters; apparently, they remained closed most or all of the time. In marked contrast, hemolymph of oysters moved to higher salinities rapidly readjusted. Exposure to TBT had a delaying effect on the schedule of the increase in hemolymph osmotic pressure and chloride ion concentration. Results of these experiments show that TBT has an indirect effect on osmotic pressure and chloride ion concentration adjustment in oysters; it does not appear to act strongly as an anionophore to influence anion transfer across epithelia.

Changes in gill ultrastructure and haemolymph chloride concentrations in the crayfish, Astacus astacus, exposed to de-acidified aluminium-rich water

Individuals of the crayfish, Astacus astacus, exposed to aluminium-rich, de-acidified water in a crayfish farm (acid-reactive aluminium fraction 180 μg All −1, labile aluminium fraction 20 μg All −1, pH 6.7) showed symptoms such as bulging of the integument between the carapace and the abdomen, and browning of the gills, generally followed by death in the pre-molt or molting stages. Exposed individuals had a significant decrease (≈35%) in haemolymph chloride concentrations compared to unexposed control individuals. Examination of the gills by transmission electron microscopy (TEM) revealed severe morphological changes in gill epithelia and underlying tissue. By X-ray microanalysis a pronounced accumulation of aluminium was detected on the gill surface of exposed specimens. This aluminium probably interferred with the normal ion-exchange mechanisms and led to crayfish death.

Osmotic and chloride regulation in the hemolymph of the tiger prawn Penaeus monodon during molting in various salinities

The effect of molting on osmotic and chloride concentrations in the blood of the prawn Penaeus monodon Fabricius (20±3 g) at various salinities was investigated. Prawns were obtained from ponds in Iloilo, Philippines, in 1984. They were stocked in salinities of 8, 20, 32 and 44‰, and their hemolymph was sampled during molt (Time 0), and then 0.125, 0.25, 0.5, 1, 2, 4, 6, 10 and 14 d after molting. Prawns during and immediately after molt tended to conform to the environmental osmolality. Subsequent postmolt (≧0.5 d) stages displayed more divergence from external salinity. The isosmotic point was higher (940±30 mOsm kg-1) during molt than during intermolt (663±8 mOsm/kg-1), suggesting different osmotic requirements in early molt. Hyperregulation of hemolymph chloride below 20‰ S, as well as isoionic point (301±6 mM), were independent of molting stage. At 20‰ S and above, newly molted (0 to 0.25 d post-molt) individuals tended to conform to the external chloride concentration while intermolt (≧0.5 d) post-molt individuals did not. Contribution of hemolymph chloride to hemolymph osmolality was greater during intermolt than during ecdysis, suggesting an important role for other negatively charged ions during molt. When molt occurred in 20‰ S (the test salinity most similar to the isoionic salinity), there was little or no change in hemolymph osmolality or chloride concentration from 0 to 14 d postmolt. At 8, 32 and 44‰ S, the change from molt to intermolt values in hemolymph osmotic and chloride concentrations was hyperbolic. Non-linear least-squares regression showed that prawns generally achieved intermolt values within 1 d after molting. Prawns at intermolt regulated hemolymph osmolality (620 to 820 mOsm kg-1) and chloride concentration (300 to 450 mM) at a much narrower range than during molt (520 to 1 170 mOsm kg-1 and 250 to 520 mM, respectively). Hemolymph osmolality was a more sensitive indicator of physiological response than hemolymph chloride concentration. Distribution and culture of P. monodon might be limited in low salinities by its ability to maintain a hemolymph osmolality ≧500 mOsm kg-1 during molt and ≧600 mOsm kg-1 in intermolt, and in high salinities by its capacity to reduce the hemolymph osmolality from values at molt to those in intermolt. Osmotic and chloride concentrations in the blood of P. monodon clearly varied with both molt stage and salinity of the medium. Dependence on external factors, however, gradually declined in older molt stages, suggesting a reduction in integument permeability and greater development of ion absorption/secretion mechanisms as the exoskeleton hardened.

Ion and CO₂ Regulation in the Freshwater Water Boatman, Cenocorixa blaisdelli (Hung.) (Hemiptera, Corixidae)

Isolated Malpighian tubules of the freshwater Cenocorixa blaisdelli secrete an alkaline urine following stimulation of fluid flow with cAMP. To determine the importance of this avenue of CO₂ loss for pH and ion regulation, corixids raised in fresh water were compared to those acclimated to alkaline (pH 9.8) conditions, with respect to whole organism ion balance. The high-pH insects exhibited an increase in hemolymph chloride concentration and a reduced rate of drinking. Urinary loss of ions and CO₂ could not balance the higher intake of sodium and CO₂ in the alkaline-acclimated insects, although hemolymph concentrations of these components were not significantly different from those concentrations in insects maintained in fresh water. Isolated Malpighian tubules from the two groups of insects did not exhibit differences in their ability to secrete ions or CO₂. Extrarenal avenues may account for the increased chloride concentration and the constant sodium and CO₂ concentrations. It is suggested that chlori...

Effect of salinity on the osmotic, chloride, total protein and calcium concentrations in the hemolymph of the prawn Peneaus monodon (fabricius)

1. 1. Osmolality and chloride concentrations in the hemolymph of Penaeus monodon became stable 1 day after molting in 32 ppt, while total protein and calcium concentrations remained stable throughout the molting cycle. When intermolt (≥ 36 hr postmolt) animals were transferred from control (32 ppt) to experimental (8–40 ppt) salinities, osmolality, chloride and total protein, but not calcium, concentrations in the hemolymph achieved steady state values 24–48 hr after transfer. 2. 2. The hemolymph osmolality was a linear function (slope = 0.28) of medium osmolality at salinities between 8 and 40 ppt. It was isosmotic to seawater at 698 mOsm (10 g prawns) and 752 mOsm (30 g), and was hyperosmotic to the medium below isosmotic concentrations, and hypoosmotic to those above. 3. 3. Hemolymph chloride concentration was isoionic to seawater at 334 mM, and was hyperregulated below isoionic concentrations, and hyporegulated to those above. 4. 4. P. monodon maintained its hemolymph calcium concentration between 6.4 and 10 mM when medium salinities increased from 8 to 40 ppt. 5. 5. Total protein concentration in the hemolymph was independent of medium salinity (8–40 ppt) and hemolymph osmolality (540–850 mOsm).

Water and ion balance in the tissues of the dehydrated cockroach, Periplaneta americana

Cockroaches dehydrated for 8 days lost nearly 50% of their haemolymph volume and approx 25% of their tissue water. Haemolymph osmolality and sodium, potassium, and chloride concentrations in the haemolymph and tissue water were all regulated within narrow limits. It is confirmed that sodium and potassium ions are sequestered within the fat body during periods of dehydration. The increase in sodium and potassium ions in the fat body is shown to arise from ionic regulation of haemolymph and other tissues. During periods of rehydration, sodium and potassium concentrations decrease in the fat body and haemolymph volume and ionic concentrations return to near original levels. A small proportion of the surplus haemolymph chloride ions is shown to be associated with the cuticle during times of water deprivation.

The role of branchial ventilation in hemolymph acid-base changes in the shore crabCarcinus maenas during hypoxia

1. When exposed to hypoxia in the ambient medium, the crabCarcinus maenas increased and sustained high levels of branchial ventilation for up to 70 h (Fig. 1). 2. The role of hyperventilation in establishing changes in hemolymph acid-base status during hypoxia was investigated. Hemolymph pH and\(P_{CO_2 } \) changes in hypoxic crabs (Figs. 2 and 4) were compared with crabs whose branchial chambers were artificially hyperventilated in normoxia by siphoning water through a mask attached to the carapace (Fig. 3). 3. Hyperventilation alone does not account for the observed alkalosis during hypoxia. It is suggested that changes in both CO2 production and ventilation may be responsible for altering hemolymph acid-base status. 4. Artificial hyperventilation in normoxic crabs resulted in a respiratory alkalosis which is fully compensated after 16 hours by a metabolic acidosis (Fig. 3). 5. Reduction of the hemolymph bicarbonate pool during hypoxia did not interfere with the ability of crabs acclimated to low salinity to regulate hemolymph chloride ion concentration (Fig. 5).

Crab Gill Intra-Epithelial Carbonic Anhydrase Plays a Major Role in Haemolymph CO2 and Chloride Ion Regulation

ABSTRACT The distribution and function of the enzyme carbonic anhydrase in the crab Pachygrapsus crassipes was investigated. Carbonic anhydrase was found in gill epithelial tissue but not in the haemolymph, muscle, heart, hepatopancreas or gonads of male crabs. Enzyme activity was completely inhibited in vitro by 2 × 10 −4M sodium acetazolamide (Diamox). Radiolabelled Diamox (2 ×10−4M) in sea water was found to equilibrate with the intact crab’s haemolymph within one hour. Haemolymph CO2 content, pH and chloride ion concentration were measured in crabs acclimated to different salinities and exposed to 2 × 10−4M Diamox. Haemolymph CO2 content increased at all salinities, especially low salinities, while pH remained unchanged, except at low salinities where it increased. Diamox impaired the regulation of haemolymph chloride at low salinities, but had no effect on chloride regulation at high salinities. Measurement of O2 uptake (estimating CO2 production) in crabs before and after Diamox exposure confirmed that elevated haemolymph CO2 was not due to increases in tissue CO2 production.

Steady state hemolymph osmotic and chloride ion regulation and percent body water in Clibanarius vittatus (decapoda: Anomura) from the texas gulf coast

1. 1. Clibanarius vittatus regulates its hemolymph hyperosmotic to the medium at salinities ranging from 5 to 50‰ The maintenance of a constant hemolymph osmolality at low salinities switches to the maintenance of an osmotic concentration a set difference above the medium at salinities greater than 20‰ 2. 2. Hemolymph chloride ion concentration is regulated hyperionic to the external medium in salinities below 15‰, and is hypoionic to the medium at higher salinities. 3. 3. When in its shell, the hermit crab is a good volume regulator as evidenced by no change in percent body water over a wide range of salinities. 4. 4. The shell offers protection to the crab and aids in the regulation of osmotic pressure and water balance in dilute media.