Hemolymph Ca2 Research Articles

Dietary calcium (Ca2+) impacts Ca2+ content and molecular expression of Ca2+-transporters in Malpighian tubules of the yellow fever mosquito, Aedes aegypti

The renal (Malpighian) tubules of insects play important roles in hemolymph Ca2+ regulation. Here we investigated how dietary Ca2+ loads from sucrose or blood meals affect the Ca2+ content and mRNA expression of Ca2+ transporters in Malpighian tubules of adult female mosquitoes. Using the yellow fever mosquito Aedes aegypti we found that feeding females 10% sucrose with elevated Ca2+ concentration ad libitum for 6 days led to increased Ca2+ content in Malpighian tubules. The increases of Ca2+ content correlated with up-regulations of mRNAs encoding intracellular Ca2+-ATPases (SERCA and SPCA), a plasma membrane Ca2+-ATPase (PMCA), and a K+-dependent Na+/Ca2+ exchanger (NCKX1). We also found that when adult females were fed blood, tubule Ca2+ content changed dynamically over the next 72 h in a manner consistent with redistribution of tubule Ca2+ stores to other tissues (e.g., ovaries). The changes in tubule Ca2+ were correlated with dynamic changes in mRNA abundances of SERCA, SPCA, PMCA, and NCKX1. Our results are the first to demonstrate that Malpighian tubules of adult female mosquitoes have a remarkable capacity to handle high dietary Ca2+ loads, most likely through the combination of storing excess Ca2+ within intracellular compartments and secreting it into the tubule lumen for excretion. Our results also suggest that the Malpighian tubules play key roles in supplying Ca2+ to other tissues during the processing of blood meals.

Exercise and emersion in air, and recovery in seawater in the green crab (Carcinus maenas): metabolic, acid-base, cardio-ventilatory and ionoregulatory responses.

In nature, the green crab exhibits emersion and terrestrial activity at low tide. Treadmill exercise in air (20-23°C) of crabs acclimated to 32ppt seawater (13°C) revealed an inverse relationship between velocity and duration: 2.0 body lengths (BL) s-1 was sustainable for several minutes, and 0.25BLs-1 was sustainable for long periods. Fatigue was not due to dehydration. Physiological responses over an 18h recovery in seawater after near-exhaustive exercise (0.25BLs-1, 1 h) in air were compared with responses after quiet emersion (1 h) in air. Exercising crabs exhibited transient scaphognathite slowing and progressive increases in heart rate, whereas emersed crabs exhibited persistent inhibition of ventilation and transient heart slowing. Upon return to seawater, all these rates increased above both control and treatment levels. Post-exercise disturbances were more marked and/or longer lasting (e.g. EPOC, hyperventilation, tachycardia, metabolic acidosis, lactate elevation, ionic disturbances) than those after simple air exposure. However, an increase in net acidic equivalent excretion to the environment occurred after emersion but not after exercise. Instead, post-exercise crabs relied on carapace buffering, signalled by elevated haemolymph Ca2+ and Mg2+. Prolonged lowering of haemolymph PCO2associated with hyperventilation also played a key role in acid-base recovery. EPOC after exercise was 3-fold greater than after emersion, sufficient to support resting ṀO2for >14 h. This reflected clearance of a large lactate load, likely by glycogen re-synthesis rather than oxidation. We conclude that the amphibious green crab uses a combination of aquatic and terrestrial strategies to support exercise in air, emersion in air and recovery in seawater.

Ca2+ levels in Daphnia hemolymph may explain occurrences of daphniid species along recent Ca gradients in Canadian soft-water lakes.

Calcium levels are declining in eastern North American and western European lakes. This widespread issue is affecting the composition of crustacean zooplankton communities, as the presence and abundance of several calcium-rich daphniid species are declining, while two other daphniids, D. catawba and D. ambigua, that apparently tolerate low calcium environments, are prospering. The physiological basis for low calcium tolerance of these daphniids is unknown. In this study the presence of one Ca-rich (D. pulicaria) and one Ca-poor (D. ambigua) daphniid species in Canadian Shield lakes is assessed in relation to lake water Ca levels. The occurrence of D. ambigua was independent of Ca levels in Ontario lakes, whereas D. pulicaria was more likely to occur in lakes with relatively more Ca. In the laboratory, D. ambigua maintained lower levels of hemolymph Ca2+ across a range of low Ca levels (0.7 to 7 mg l−1) compared with D. pulicaria. The hemolymph pH remained steady across this Ca gradient in D. ambigua while it was significantly more acidic in D. pulicaria in the two lowest Ca treatments. While Ca2+ uptake was observed adjacent to the surface of D. ambigua individuals, Ca2+ loss was observed for D. pulicaria assayed under moderately high Ca levels. Based on these observations we propose that D. ambigua is able to survive in low Ca lakes by maintaining low free ionic Ca2+ levels in the hemolymph which minimizes the Ca gradient across the body wall in low Ca water thus limiting overall Ca loss and facilitating Ca2+ uptake.

Cadmium bioaccumulates after acute exposure but has no effect on locomotion or shelter-seeking behaviour in the invasive green shore crab (Carcinus maenas).

Cadmium (Cd2+) is a non-essential metal ubiquitous in the environment due to industrial processes. However, little is known regarding the ability of Cd2+ to impact the behaviour of aquatic animals in receiving environments. Green shore crabs (Carcinus maenas) were exposed to waterborne Cd2+ [control (no Cd2+), low (0.30 μmol/L), medium (3.3 μmol/L) and high (63 μmol/L)], for 24 h, then, crabs were placed in an open field and shelter test to determine potential changes in locomotion and preference for shelter. Tissues (gill, haemolymph, stomatogastric ganglion) were taken for bioaccumulation analysis of Cd2+ and ion content. Behavioural testing was recorded with a motion-tracking software system and showed no impact of Cd2+ on any variable in either of the tests used. All three tissues accumulated Cd2+ in a concentration-dependent manner. Crabs exposed to low Cd2+ showed a small but significant decrease in haemolymph Ca2+, however, this effect was not present at higher Cd2+ exposures. Overall, the results indicate that short-term Cd2+ exposure, and the resulting Cd2+ accumulation, had no effect on locomotor and anxiety-related behaviour of green shore crabs.

Ocean acidification adversely influences metabolism, extracellular pH and calcification of an economically important marine bivalve, Tegillarca granosa

Oceanic uptake of CO2 from the atmosphere has significantly reduced surface seawater pH and altered the carbonate chemistry within, leading to global Ocean Acidification (OA). The blood clam, Tegillarca granosa, is an economically and ecologically significant marine bivalve that is widely distributed along the coastal and estuarine areas of Asia. To investigate the physiological responses to OA, blood clams were exposed to ambient and three reduced seawater pH levels (8.1, 7.8, 7.6 and 7.4) for 40 days, respectively. Results obtained suggest that OA suppresses the feeding activity and aerobic metabolism, but elevates proteins catabolism of blood clams. OA also causes extracellular acidosis and decreases haemolymph Ca2+ concentration. In addition, our data also suggest that OA impairs the calcification process and inner shell surface integrity. Overall, OA adversely influences metabolism, acid-base status and calcification of blood clams, subsequently leading to a decrease in the fitness of this marine bivalve species.

Physiological effects of short- and long-term exposure to elevated carbon dioxide on a freshwater mussel,Fusconaia flava

Zones of elevated carbon dioxide (CO2) have the potential to deter the movement of fishes. Should CO2be used as a barrier, non-target organisms, such as freshwater mussels, have the potential to be impacted. In this study, the physiological responses of adult Fusconaia flava exposed to elevated partial pressures of CO2(pCO2) over both short-term (6 h exposure with 6 h recovery) and long-term (4-, 8-, and 32-day exposure) periods were measured. A 6 h exposure to either ∼15 000 μatm (1 atm = 101.325 kPa) or ∼200 000 μatm caused an elevation in hemolymph Ca2+. Exposure to ∼200 000 μatm resulted in a decrease in hemolymph Cl−, and exposure to ∼15 000 μatm caused an increase in hemolymph Na+, whereas ∼200 000 μatm caused a decrease. Exposure to elevated pCO2for long-term periods caused a decrease in hemolymph Mg2+and an initial increase in hemolymph Ca2+. Body condition and hemolymph glucose were not significantly influenced by elevated pCO2for both experiments. This study shows that elevated pCO2had limited impacts on the physiological responses of adult freshwater mussels.

The response of two species of unionid mussels to extended exposure to elevated carbon dioxide

Changes in environmental conditions can act as stressors, with potential consequences for the health and fitness of organisms. Rising levels of carbon dioxide (CO2) is one potential environmental stressor that is occurring more frequently in the environment and can be a stressor for aquatic organisms. In this study, the physiological responses of two species of unionid mussel, Lampsilis siliquoidea and Amblema plicata, were assessed in response to exposure to two levels of elevated partial pressure of CO2 (pCO2) (~20,000 and ~55,000μatm) over a 28d period, followed by a subsequent 14d recovery period. Observations were consistent with responses associated with respiratory acidosis, as demonstrated by changes in hemolymph HCO3−, Ca2+, Cl−, and Na2+. Both species exposed to elevated pCO2 had elevated hemolymph HCO3– during the pCO2 treatment period compared to control mussels, but recovered once pCO2 was removed. Similarly, both species had elevated hemolymph Na+ during exposure to elevated pCO2, and this returned to control levels for A. plicata but remained elevated for L. siliquoidea once the pCO2 stimuli was removed. Changes in hemolymph Ca2+ and Cl− in response to elevated pCO2 were also observed, but these changes were species-specific. Additional physiological responses to elevated pCO2 (e.g., changes in hemolymph glucose and Mg2+) were consistent with a stress response in both species. This study demonstrates the importance of considering inter-specific differences in the response of organisms to stress, and also that responses to elevated pCO2 may be transient and can recover once the stress is removed.

Osmotic and ionic regulation and Na+/K+-ATPase, carbonic anhydrase activities in mature Chinese mitten crab, Eriocheir sinensis H. Milne Edwards, 1853 (Decapoda, Brachyura) exposed to different salinities

Mature Chinese mitten crabs, Eriocheir sinensis H. Milne Edwards, 1853, are exposed to brackish water or seawater as an obligatory part of their reproductive migration. We investigated the changes in haemolymph osmolality, haemolymph Na+, Cl−, Ca2+ and K+ concentrations, as well as gill Na+/K+-ATPase and carbonic anhydrase activity in both mature females and males, when in freshwater (0.3‰ salinity, 8 mOsm (kg H2O)−1) and after step-wise acclimation to salinities ranging from 7 to 35‰ (175-1044 mOsm (kg H2O)−1). Both females and males strongly hyper-regulated their haemolymph osmolality over a salinity range of 0.3-21‰, the isosmotic point approaching 28‰ salinity, and slightly hypo-regulated at 35‰ salinity. Haemolymph Na+ and Cl− levels correlated positively with haemolymph osmolality. Ca2+ and K+ levels, however, were maintained at a relatively narrow range and showed a contrary trend in their response to salinity. This indicated that Na+ and Cl− are the main contributors to increasing haemolymph osmolality. Reduced Na+/K+-ATPase and carbonic anhydrase activity in the posterior gills at elevated salinity suggests a direct physiological response to a decrease of Na+ intake. No sexual differences existed for all parameters except for significantly higher haemolymph Ca2+ and K+ levels and CA activity in females than in males at some salinity levels. It is suggested that mature Eriocheir sinensis is a strong hyper-osmoregulator but a weak hypo-osmoregulator. No sexual differences appeared to exist in osmotic and ionic regulation of mature Eriocheir sinensis exposed to different salinities.

Effects of lead on Na+, K+‐ATPase and hemolymph ion concentrations in the freshwater mussel Elliptio complanata

Freshwater mussels are an imperiled fauna exposed to a variety of environmental toxicants such as lead (Pb) and studies are urgently needed to assess their health and condition to guide conservation efforts. A 28-day laboratory toxicity test with Pb and adult Eastern elliptio mussels (Elliptio complanata) was conducted to determine uptake kinetics and to assess the toxicological effects of Pb exposure. Test mussels were collected from a relatively uncontaminated reference site and exposed to a water-only control and five concentrations of Pb (as lead nitrate) ranging from 1 to 245 μg/L in a static renewal test with a water hardness of 42 mg/L. Endpoints included tissue Pb concentrations, hemolymph Pb and ion (Na⁺, K⁺, Cl⁻, Ca²⁺) concentrations, and Na⁺, K⁺-ATPase enzyme activity in gill tissue. Mussels accumulated Pb rapidly, with tissue concentrations increasing at an exposure-dependent rate for the first 2 weeks, but with no significant increase from 2 to 4 weeks. Mussel tissue Pb concentrations ranged from 0.34 to 898 μg/g dry weight, were strongly related to Pb in test water at every time interval (7, 14, 21, and 28 days), and did not significantly increase after day 14. Hemolymph Pb concentration was variable, dependent on exposure concentration, and showed no appreciable change with time beyond day 7, except for mussels in the greatest exposure concentration (245 μg/L), which showed a significant reduction in Pb by 28 days, suggesting a threshold for Pb binding or elimination in hemolymph at concentrations near 1000 μg/g. The Na⁺, K⁺-ATPase activity in the gill tissue of mussels was significantly reduced by Pb on day 28 and was highly correlated with tissue Pb concentration (R² = 0.92; P = 0.013). The Na⁺, K⁺-ATPase activity was correlated with reduced hemolymph Na⁺ concentration at the greatest Pb exposure when enzyme activity was at 30% of controls. Hemolymph Ca²⁺ concentration increased significantly in mussels from the greatest Pb exposure and may be due to remobilization from the shell in an attempt to buffer the hemolymph against Pb uptake and toxicity. We conclude that Na⁺, K⁺-ATPase activity in mussels was adversely affected by Pb exposure, however, because the effects on activity were variable at the lower test concentrations, additional research is warranted over this range of exposures.

Short-term exposure to hypercapnia does not compromise feeding, acid–base balance or respiration ofPatella vulgatabut surprisingly is accompanied by radula damage

The effect of short-term (5 days) exposure to CO2-acidified seawater (year 2100 predicted values, ocean pH = 7.6) on key aspects of the function of the intertidal common limpetPatella vulgata(Gastropoda: Patellidae) was investigated. Changes in extracellular acid–base balance were almost completely compensated by an increase in bicarbonate ions. A concomitant increase in haemolymph Ca2+and visible shell dissolution implicated passive shell dissolution as the bicarbonate source. Analysis of the radula using SEM revealed that individuals from the hypercapnic treatment showed an increase in the number of damaged teeth and the extent to which such teeth were damaged compared with controls. As radula teeth are composed mainly of chitin, acid dissolution seems unlikely, and so the proximate cause of damage is unknown. There was no hypercapnia-related change in metabolism (O2uptake) or feeding rate, also discounting the possibility that teeth damage was a result of a CO2-related increase in grazing. We conclude that although the limpet appears to have the physiological capacity to maintain its extracellular acid–base balance, metabolism and feeding rate over a 5 days exposure to acidified seawater, radular damage somehow incurred during this time could still compromise feeding in the longer term, in turn decreasing the top-down ecosystem control thatP. vulgataexerts over rocky shore environments.

Osmotic and Ionic Concentrations of Scylla serrata (Forskål) Subjected to Different Salinity Levels

Scylla serrata (198.58 ± 35.98 g) were transferred from 34 ppt to 14, 24, 34 (control), and 44 ppt. Salinity, haemolymph osmolality, and ionic concentrations were examined after 1.5, 3, 6, 12, 24, 72, 120, and 240 hr. Haemolymph osmolality, and Cl−, Na+, K+, Ca2+, and Mg2+ concentrations of crabs transferred to 14, 24, and 44 ppt and reached constant levels within 5 days. The isosmotic value was 968.6 mOsm/kg (33.4 ppt), and the isoionic value was 365.4, 322.1, 8.4, 11.3, and 28.7 mmol/l (24.0, 22.3, 22.2, 33.4 and 21.3 ppt) for Cl−, Na+, K+, Ca2+, and Mg2+, respectively. S. serrata is hypo-osmotic, and regulates haemolymph Ca2+ hyperionic to the medium at salinities between 14 to 33.4 ppt, and osmoconforms above 33.4 ppt. Sodium and chloride ions accounted for 83.1, 87.3, 83.4, and 84.3% of haemolymph osmolality at 14, 24, 34, and 44 ppt, respectively. S. serrata is a hyper/hypo-osmotic osmoregulator, with osmotic regulation being largely accounted for by ion regulation.

Physiological responses to severe acid stress in four species of freshwater clams (unionidae)

Four species of freshwater clam,Anodonta anatina, A. cygnea, Unio pictorum, andU. tumidus were exposed for 2 weeks to acidified soft water (pH 4.0–4.5, Ca 4.6 mg/L) and for 4 weeks to acid in hard water conditions (Ca 18.5 mg/L). The exposures caused a decrease in Na+, K+, and Cl− ion and a rapid increase of Ca2+ in the hemolymph. The elevation of the hemolymph Ca2+ was positively correlated with the decrease in the hemolymph pH in all species studied. Low ambient [Ca2+] level accelerated the pH decrease and Ca2+ increase in the hemolymph. Na+ and Cl− ion concentrations changed less rapidly in the soft conditions. Although there were minor changes in the mineral composition of the calcium concretions in the gills, the amount of calcium in the concretions did not change during the exposure. There was no correlation between the thickness of the shell and the ionic response, but all four species responded to low ambient pH in the same way.

Effect of salinity fluctuation on the osmotic pressure and Na+, Ca2+ and Mg2+ ion concentrations in the hemolymph of bivalve molluscs

Specimens of Chlamys opercularis, Modiolus modiolus, Mytilus edulis, Crassostrea gigas, Scrobicularia plana and Mya arenaria were exposed to both gradual (sinusoidal) and abrupt (square-wave) salinity fluctuations and measurements made of osmotic, Na+, Mg2+ and Ca2+ concentrations in the hemolymph and where applicable in the mantle fluid. In both sinusoidal and square-wave regimes fluctuating between 100 and 50% seawater (100%=ca. 32‰ S), the hemolymph Na+, Mg2+, Ca2+ and osmotic concentrations followed the concentrations of the external medium in Chlamys opercularis. The hemolymph and mantle fluid osmotic Na+, Mg2+ and Ca2+ concentrations of Modiolus modiolus, Mytilus edulis, Crassostrea gigas and S. plana followed those of the external medium as long as the molluscs' shell valves remained open. There were no changes in the ionic or osmotic concentrations of the hemolymph or mantle fluid of any of these species during periods of shell-valve closure. The hemolymph osmotic, Na+ and Mg2+ concentrations of wedged-open Modiolus modiolus, Mytilus edulis, C. gigas and S. plana followed those of the external medium. Hemolymph Ca2+ concentrations showed a damped response in C. gigas and Mytilus edulis. The hemolymph osmotic, Na+, Ca2+ and Mg2+ concentrations of Mya arenaria fluctuated in a similar manner to the external medium, but were damped. Wedged-open Mytilus edulis exposed to fluctuating salinity and supplied with a constant supply of 10 mM Ca2+ showed greater changes in hemolymph ionic and osmotic concentrations than M. edulis exposed to the same salinity fluctuation without a constant Ca2+ supply. Chlamys opercularis and Modiolus modiolus survived in a 50% seawater minimum sinusoidal salinity fluctuation for 10 days; wedged-open M. modiolus survived only 3 days. Burrowing had no effect on the osmotic, Na+, Mg2+ or Ca2+ concentrations of the hemolymph of Mya arenaria or S. plana exposed to fluctuating salinities. All of the species studied were shown to be osmoconformers.