Hemolymph Cl Research Articles

Biochemical and physiological effects of nickel in the euryhaline crab Neohelice granulata (Dana, 1851) acclimated to different salinities

The estuarine crab Neohelice granulata was maintained under control condition or exposed to sublethal concentrations of dissolved Ni (measured: 128 and 1010μg/L) for 96h at different salinities (2 and 30ppt). After metal exposure, whole-body oxygen consumption was measured and tissue (hemolymph, gills, hepatopancreas and muscle) samples were collected. Control crabs acclimated to 2ppt salinity showed lower hemolymph concentrations of Na+ (33%), Mg2+ (19%) and K+ (30%), as well as increased LPO levels in anterior gills (379%), posterior gills (457%) and hepatopancreas (35%) with respect to those acclimated to 30ppt salinity. In crabs acclimated to 2ppt salinity, Ni exposure increased whole-body oxygen consumption (75%), hemolymph K+ concentration (52%), hemolymph (135%) and hepatopancreas (62%) LDH activity. Also, it reduced hemolymph Cl− concentration (16%) and muscle LDH activity (33%). In crabs acclimated to 30ppt salinity, Ni exposure increased LDH activity in hemolymph (195%), hepatopancreas (126%) and muscle (53%), as well as hemolymph osmolality (10%), Cl− (26%) and Ca2+ (20%) concentration. It also reduced hepatopancreas lipid peroxidation (20%) and hemolymph Mg2+ (29%) and K+ (31%) concentration. These findings indicate that N. granulata is hyper-osmoregulating in 2ppt salinity and hypo-regulating in 30ppt salinity, showing adjustments of hemolymph ionic composition and metabolic rates, with consequent higher oxidative damage to lipids in low salinity (2ppt). Ni effects are associated with metabolic (aerobic and anaerobic) disturbances in crabs acclimated to 2ppt salinity, while osmotic and ionoregulatory disturbances were more evident in crabs acclimated to 30ppt salinity.

The salinity tolerance of the invasive golden apple snail (Pomacea canaliculata)

ABSTRACTWe exposed snails of an invasive species of golden apple snail (Pomacea canaliculata) to five artificial sea water treatments at salinity levels of 0, 5, 10, 15 or 20 parts per thousand (ppt) to assess their salinity tolerance. We observed the behaviour, heart rate, total haemocyte counts, haemolymph ionic concentration and Na+/K+-ATPase activity in the mantle at 0, 12, 24, 48, 72 and 96 h post salinity exposures. The heart rate declined with increasing salinity, while Na+/K+-ATPase activity in the mantle presented a reverse trend, possibly to maintain normal osmolality. A trend of rising total haemocyte count was observed from 0 ppt and 5 ppt to 10 ppt salinities, while a sudden increase in the count was observed at 15 ppt and 20 ppt salinity groups. Furthermore, haemolymph Cl−, Na+ and K+ concentrations increased directly with elevated salinity. An additional trial was performed to assess the growth performance of the snails under exposure to low salinities. During a 1 month trial, snails grew better at 5 ppt salinity treatment. Taken together, our results demonstrate that P. canaliculata can tolerate salt stress to some extent. The finding also obviously implies a possible invasive risk to estuaries.

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.

Norepinephrine induces transient modulation of the physiological responses of whiteleg shrimp, Litopenaeus vannamei

Levels of glucose, lactate, Na +, K +, Cl −, protein, and oxyhemocyanin in the hemolymph and its osmolality were measured when the whiteleg shrimp, Litopenaeus vannamei (18.3∼21.6 g), were individually injected with saline or norepinephrine at 10 − 8 , 10 − 7 , or 10 − 6 mol shrimp − 1 . Results showed that elevations of hemolymph glucose and lactate occurred at between 2 and 4 h, increases in hemolymph osmolality, Na +, and total protein occurred at 2 h, and a reduction in hemolymph Cl − and K + occurred at 2 h after in shrimp which had received NE at > 10 − 8 , > 10 − 8 , and 10 − 6 mol shrimp − 1 , respectively. All physiological parameters had returned to the control values 2∼4 h after receiving the norepinephrine. The injection of norepinephrine at > 10 − 8 mol shrimp − 1 also significantly decreased the oxyhemocyanin / protein ratio of L. vannamei at 2 h as a result of elevation of hemolymph protein. These results suggest that stress-inducing norepinephrine causes a transient period of modulation of energy metabolism and osmoregulation, and a respiratory response in L. vannamei as it adapts to an environmental stress.

Putative involvement of crustacean hyperglycemic hormone isoforms in the neuroendocrine mediation of osmoregulation in the crayfish Astacus leptodactylus.

This study investigates the involvement of eyestalk neuroendocrine factors on osmoregulation in the crayfish Astacus leptodactylus maintained in freshwater. Eyestalk removal was followed by a significant decrease in hemolymph osmolality and Na(+) concentration and by a 50% increase in mass after one molting cycle. Several neurohormones have been isolated from the sinus gland through high-performance liquid chromatography (HPLC), and different crustacean hyperglycemic hormone (CHH)-related peptides, including stereoisomers (L-CHH and D-Phe(3) CHH), have been identified by direct enzyme-linked immunosorbent assay (ELISA). A glucose quantification bioassay demonstrated a strong hyperglycemic activity following injection of the immunoreactive chromatographic fractions and showed that the D-Phe(3) CHH was the most efficient. Destalked crayfish were then injected with purified CHH HPLC fractions. The D-Phe(3) CHH fraction significantly increased the hemolymph osmolality and Na(+) content 24 h after injection. Two other CHH-related peptides caused a smaller increase in Na(+) concentration. No significant variation was observed in hemolymph Cl(-) concentration following injection of any of the CHH isoforms. These results constitute the first observation of the effects of a CHH isoform, specifically the D-Phe(3) CHH, on osmoregulatory parameters in a freshwater crustacean. The effects of eyestalk ablation and CHH injection on osmoregulation and the identification of different CHH-related peptides and isoforms in crustaceans are discussed.

Physiological responses to acute silver exposure in the freshwater crayfish (Cambarus diogenes diogenes)—a model invertebrate?

Adult crayfish (Cambarus diogenes diogenes) exposed to 8.41 +/- 0.17 microg silver/L (19.4% as Ag+) in moderately hard freshwater under flow-through conditions for 96 h exhibited ionoregulatory disturbance, elevated metabolic ammonia (T(amm)) production and substantial silver accumulation in the gills, hemolymph, and hepatopancreas. The ionoregulatory disturbance included both a generally reduced unidirectional Na+ influx and an increased unidirectional Na+ efflux, leading to a substantial net loss of Na+ from the silver-exposed crayfish. The Na+ uptake in silver-exposed crayfish differed overall from controls, while the increased Na+ efflux recovered to control values 48 h into the 96 h of exposure. The general inhibition of Na+ uptake could be explained by a reduced sodium/potassium-adenosine triphosphatase (Na/K-ATPase) activity in terminally obtained gill samples from the silver-exposed crayfish. The silver-induced effect on Na+ uptake and loss translated to reduced hemolymph Na+ concentrations but not significantly reduced hemolymph Cl- concentrations. Hemolymph T(anim) and T(amm) efflux both increased in silver-exposed crayfish, indicating an increased metabolic T(amm) production. The present study demonstrates that the toxic mechanism of waterborne silver exposure in freshwater crayfish resembles that of freshwater teleost fish. The crayfish might therefore be a useful model system for extending current environmental regulatory strategies, currently based on teleost fish, to invertebrates.

Osmotic and ionic changes in Taiwan abalone Haliotis diversicolor supertexta at different salinity levels

Osmotic and ionic changes in Taiwan abalone Haliotis diversicolor supertexta (5.4±0.28 cm) were investigated after they were transferred individually from 33 practical salinity unit (psu) to 23, 28, 33 and 38 psu after 0.25, 0.5, 1, 2, 3, 6 and 9 days. Hemolymph osmolality of abalone stabilized within 2 days after they were transferred to different salinities from 33 psu. Hemolymph osmolality, Cl −, Na + and K + concentrations increased directly with medium salinity. Hemolymph osmolality was 652, 789, 945 and 1103 mOsm kg −1 for the abalone transferred to 23, 28, 33 and 38 psu, respectively. Hemolymph Cl − was 275, 323, 365, 409 mmol l −1, hemolymph Na + was 312, 376, 465, 569 mmol l −1 and hemolymph K + was 8.2, 10.1, 11.9, 12.8 mmol l −1 for the abalone transferred to 23, 28, 33, 38 psu, respectively. It is concluded that H. diversicolor supertexta are hypo-conformic and hypo-ionic with respect to Cl − and Na +, but hyper-ionic with respect to K + over ambient media in salinity ranges of 23–38 psu.

Effects of nitrite exposure on the hemolymph electrolyte, respiratory protein and free amino acid levels and water content of Penaeus japonicus

Penaeus japonicus which had been exposed individually for 3, 6, 12, 24 and 48 h to 0.001 (control), 0.07, 0.36, 0.72 and 1.43 mM nitrite at 30 ppt of salinity were examined for hemolymph electrolyte, oxyhemocyanin, protein, free amino acid (FAA) and urea levels and whole shrimp's nitrite uptake and ammonia excretion. Hemolymph oxyhemocyanin, protein, FAA, osmolality, Cl − and Na + levels were inversely related to ambient nitrite and exposure time. However, hemolymph urea level, osmotic differential (medium osmolality−hemolymph osmolality), nitrite uptake, ammonia excretion and water content increased directly with ambient nitrite and exposure time. It is suggested that when P. japonicus are exposed to nitrite, water uptake and nitrite uptake result in reductions of hemolymph Cl −, Na + and protein levels leading to a decrease of hemolymph osmolality. An increase of hemolymph urea and a decrease of arginine may indicate an occurrence of ureogenesis for the intracellular osmoregulation of P. japonicus under nitrite stress.

K+ current stimulation by Cl- in the midgut epithelium of tobacco hornworm (Manduca sexta)

Goblet cells in the midgut epithelium of the tobacco hornworm (Manduca sexta larva, 5th instar) actively secrete K+. This can be measured as short-circuit current (Isc) when the tissue is mounted in an Ussing chamber and bathed in K(+)-rich standard saline containing 32 mmol K+.l-1. Isc depends strictly on basolateral (i.e. haemolymph side) K+ and is therefore termed K+ current, IK. Basolateral, but not apical, chloride, bromide and iodide stimulate IK when compared to the baseline current recorded with gluconate-, nitrate- or thiocyanate-containing salines. So-called "Cl(-)-specific" transport inhibitors (frusemide, 9-anthracene carboxylic acid, diphenylamine carboxylic acid and 4,4'-diisothiocyana-to-stilbene-2,2'-disulphonic acid) reduce IK when added to the basolateral bath, whether Cl- or gluconate is the principal ambient anion. Cl- stimulates IK according to saturation kinetics. The Michaelis-Menten-type, K+ concentration-dependent, saturation of IK is altered in a highly specific manner when gluconate is replaced by Cl-: maximal K+ current, as well as the apparent Michaelis constant, are increased by a factor of 4. Since IK develops in these conditions exclusively via basolateral, Ba(2+)-blockable K+ channels, these results can be understood if it is assumed that haemolymph Cl- interferes with the K+ channel by simultaneously lowering the binding affinity for K+ ions and increasing their subsequent transfer rate across the basolateral goblet cell membrane.

The osmotic response of salmon louse,Lepeophtheirus salmonis (Copepoda: Caligidae), during the transition from sea water to fresh water

The osmotic changes in haemolymph and body tissues of the ectoparasitic salmon louse,Lepeophtheirus salmonis, have been studied upon transfer from sea water (SW) to dilute sea water (37% SW), and then to fresh water (FW). The parasite shows osmoconformity in SW but hyperosmotic regulation in 37% SW regardless of whether it is attached to the salmon host or free swimming in the water. The same conclusion is reached by haemolymph Cl− measurements. In FW, the osmotic tolerance and response of attached and free swimming parasites differ: Attached animals maintain steady haemolymph osmolality and Cl− concentration and survive for at least 1 week, while free swimming parasites quickly become diluted and start to die within 8 h.

Neuroendocrine control of Na+ balance in the fiddler crab Uca pugilator.

The hemolymph ionic composition and unidirectional Na+ fluxes were analyzed in fiddler crabs (Uca) acclimated to 500 mosmol seawater. Eyestalkless crabs had lower hemolymph osmotic, Na+, and Cl- concentrations. The Na+ efflux was increased from 13.6 +/- 0.6 in control crabs to 19.6 +/- 1.3 mueq.g-1.h-1 in eyestalkless crabs; this increase was shown to be due to a higher Na+ permeability in eyestalkless crabs. Hemolymph Na+ in eyestalkless crabs was increased by an injection of eyestalk extract. The Na+ influx was increased from 11.1 in intact to 15.8 mueq.g-1.h-1 in eyestalkless animals. A kinetic study showed that 1) the influx was higher at all external Na+ concentrations in eyestalkless and intact-Na+-depleted crabs than in intact controls, and 2) the influx mechanism exhibited allosterism. The elevated Na+ influx in eyestalkless crabs was decreased by an injected eyestalk extract. An injected brain extract caused in increase in the Na+ influx in intact animals. It is concluded that the Na+ uptake mechanism is activated by decreasing the hemolymph Na+ concentration and that a brain hormone mediates the effect.

Ventilatory behaviour and chloride regulation in relation to oxygen tension in the shrimpPalaemon adspersusRathke maintained in hypotonic medium

Abstract When exposed to gradually lowered PwO2 values, the brackish water shrimp Palaemon adspersus Rathke responds with an increased ventilation rate with maximum around PwO2 70–80 mm Hg below which the rate is rapidly lowered. Heart rate shows a steady decline with oxygen tensions. When PwO2 levels are subsequently increased again, ventilation rate shows a linear increase, but with a clear overshoot effect at PwO2 over 100 mm Hg. P. adspersus haemolymph has in normoxic condition a Cl− value of 408 mM · 1−1 (medium 250–275 mM · 1−1). The chloride of the haemolymph decreased when PwO2 was lowered below ca. 100 mm Hg. At PwO2 80 mm Hg (ca. Pcr) the haemolymph Cl− begins to show a steady decrease to a new, lower and stable value. At still lower tensions haemolymph Cl− decreased more rapidly and after 1–1.5 h Cl− reached isochlorinity in PwO2 25 mm Hg and less. This phenomenon is considered a re-allocation of energy from ion transport to other vital processes with the consequence of a gradual decrease in haemolymph chlorinity.

Effects of salinity fluctuation on the hemolymph composition of the blue crab Callinectes sapidus

The hemolymph of the blue crab Callinectes sapidus was hyperosmotic during 20-10-20‰ S and 30-10-30‰ S diurnal cycles. The hemolymph became isosmotic at 26‰ S and hyposmotic at 28‰ S in the 10-30-10‰ S diurnal cycle. Hemolymph Na+ was hyperionic to seawater throughout all cycles. Hemolymph Cl- was hyperionic below 24‰ S and either isionic or hypoionic from 24 to 30‰ S. Hemolymph K+ concentrations were hyperionic below 26‰ S and either isionic or hypoionic from 26 to 30‰ S. Hemolymph Mg++ values were hypoionic over the experimental salinity range (10 to 30‰). Hemolymph ninhydrin-positive substances (NPS) levels were directly related to ambient salinity.

Regulation of Ions in the Haemolymph of the Cockroach Periplaneta Americana During Dehydration and Rehydration

ABSTRACT Experiments using 22Na-uptake and serial sampling techniques showed that during dehydration Na was taken up by the fat body in adult Periplaneta americana, but not in nymphs. Generally, accumulation of Na in the fat body was highest in animals which had little reserve lipid and in those which excreted the smallest amounts of Na during the dehydration period. Dehydration caused little change in the Cl− concentration in the haemolymph. From results of 36Cl-uptake experiments, it appears that the fat body is not involved in the regulation of haemolymph Cl− during dehydration. In old adults, the hindgut was the tissue in which dehydration caused the greatest increase in 36C1 uptake. The mechanisms which enable cockroaches to withstand dehydrating conditions are discussed.

Effect of external chloride replacement on action potentials of giant axons in Periplaneta americana

1. 1. The hemolymph Cl − concentration of adult female Periplaneta americana was determined by automatic titration, and the effect of external Cl − replacement on action potentials of giant axons in intact nerve cords of adult P. americana was studied in vitro with external electrodes. 2. 2. The hemolymph Cl − concentration is 112 ± 6·07 mM. 3. 3. When bathed in high Cl − medium, the half-times of action potentials of adult male and female nerve cords are not significantly different. 4. 4. A chloride-free bathing medium with either acetate or SO 4 2− replacing Cl − significantly decreases the half-time of action potentials; no significant difference exists between half-times obtained with acetate and SO 4 2− solutions.