Hemolymph pH Research Articles

Changes in oxygen consumption and nitrogen metabolism in the dragonfly Somatochlora cingulata exposed to aluminum in acid waters

Oxygen consumption, ammonia excretion and changes in O:N ratios by the dragonfly Somatochlora cingulata were measured in four nymphal growth stages, relative to aluminum concentrations and low pH. A differential reduction in respiration and ammonia excretion rates resulted in an increase in ON ratios for all nymphal stages. The earlier stages, however, were the most sensitive. The ratios obtained were indicative of a decreased dependence on protein reserves and increased utilization of carbohydrates or lipid reserves. Also observed was an increase in the haemolymph pH and glutamate levels with a concomitant accumulation of tissue ammonia.

Respiratory properties of the haemolymph of the intertidal prawn Palaemon elegans (Rathke)

AbstractThe properties of the haemolymph of Palaemon elegans for oxygen and carbon dioxide transport were studied with respect to the considerable fluctuation in the environmental conditions normally experienced by this prawn in intertidal rock pools. The haemolymph was found to have a high oxygen affinity (P50 = 3.6 Torr, 15°C, pH 8.0) and a large oxygen carrying capacity (1.17 mmol.L−1), as well as a marked Bohr shift (φ = ‐ 1.7). Under some conditions, the affinity appeared to be independent of temperature as did the n50 value (n50 = 2.8). Experiments in which the pH of the haemolymph was varied using fixed acid together with two different CO2 tensions demonstrated a specific CO2 effect on O2 affinity, which did not affect the Bohr factor. The nonbicarbonate buffer value of the haemolymph was found to be high (16.02 mmol.L−1.pH unit−1). The in vivo postbranchial haemolymph pH at 15°C was 7.89 with a calculated Pco2 of 1.26 Torr. A large functional Haldane coefficient was observed (1.25), which was only moderately temperature dependent. The haemolymph exhibited an extremely high capacitance for CO2 at low Pco2 values (βCO2 = 8.4 mmol.L−1.Torr−1). Some functional implications of linkage between the large Bohr and Haldane effects are discussed in light of present knowledge.

Acid-base regulation during exercise and recovery in the blue crab, Callinectes sapidus

During swimming activity Callinectes sapidus incurred a severe hemolymph acidosis due to elevated levels of P CO 2 and lactate. Although hemolymph lactate concentration rose steadily during 1 h of exercise, hemolymph pH was maximally depressed within the first 15 min. A discrepancy between the quantities of lactate and H + released from the tissues into the hemolymph is explained by a large apparent efflux of H + into the ambient seawater, presumably via a branchial ion exchange process. Ammonia excretion increased 6 fold during exercise, but it is not clear if this contributed to the excretion of H +. The total quantity of H + excreted into the environment during exercise and recovery far exceeded that which could be attributed to hemolymph lactic acid, indicating that some of the excreted H + must have originated from other sources, such lactic acid which dissociated intracellularly, with the lactate anions remaining in the cells. Because lactate anions increase hemocyanin O 2 affinity when unopposed by the Bohr shift, the excretion of a large portion of the metabolic H + load, leaving lactate behind in the hemolymph, has important consequences for the regulation of O 2 transport during swimming.

31P-NMR studies of the freeze-tolerant larvae of the gall fly, Eurosta solidaginis.

31P NMR was applied to an examination of the freeze-tolerant larvae of the gall fly, Eurosta solidaginis. Resonances from sugar phosphates, inorganic phosphate, adenylates and arginine phosphate were identified. Two peaks of Pi were identified corresponding to intracellular and extracellular Pi. Anoxia produced an expected decrease in peak intensities of ATP and arginine phosphate while the peak of intracellular Pi was enhanced and shifted to indicate intracellular acidification during anoxia. Spectra of whole larvae were monitored over a temperature range from -30 degrees to +25 degrees C. No abrupt alterations in the spectra were seen at the point of extracellular freezing which occurs at about -8 degrees C but temperature had dramatic effects upon the peak intensities of ATP and arginine phosphate. A reversible increase/decrease in peak intensities, relative to Pi, was observed as temperature was raised/lowered. At 15 degrees and -20 degrees C, the beta peak of ATP was 64% and 2% of the peak intensity of Pi while that of arginine phosphate was 78% and 11%, respectively. This temperature effect was not an artifact of instrumentation (as model solutions containing Pi, ATP and arginine phosphate did not show this effect) or a result of changes in the total amounts of these compounds in the cell with temperature. Rather it is apparent that these molecules become restricted in their rotational movement as temperature is lowered perhaps via binding to subcellular components. Changes in the amounts of freely soluble ATP and arginine phosphate with temperature could have important implications for metabolism and its control. Analysis of the effect of temperature on the chemical shift of Pi was also used to determine pH in the intracellular and extracellular compartments. Temperature change had no effect on extracellular (hemolymph) pH which remained constant at 6.1-6.3. Intracellular pH varied with temperature, however, from pH 6.8 at 15 degrees C to pH 7.3 at -12 degrees C with a change, delta pH/delta 0, of -0.0185 degrees C consistent with alphastat regulation.

Hemolymph pH of the larvae of three species of mosquitoes, and the effect of Romanomermis culicivorax parasitism on the blood pH of Culex pipiens

The hemolymph pHs of late fourth-instar Culex pipiens, Aedes taeniorhynchus, and Toxorhynchites amboinensis were measured with pH-sensitive glass microelectrodes and were slightly alkaline; pH 7.51, 7.62, and 7.37, respectively. The hemolymph pH remained relatively constant during the development of C. pipiens larvae through the third and early and late fourth instars. The hemolymph pH in C. pipiens larvae parasitized with the mermithid nematode Romanomermis culicivorax was unaltered. These measurements provide an approximate pH (ca 7.4) which is normal for the hemolymph of larval mosquitoes, and should be useful for further development of a culture medium for R. culicivorax.

Carbon dioxide excretion and carbonic anhydrase function in the Red Rock CrabCancer productus

The function of carbonic anhydrase (CA) in the Red Rock Crab,Cancer productus Randall, was investigated. CA activity was found to varying degrees in the gills and many other tissues but not in hemolymph. Crabs injected with acetazolamide, a specific CA inhibitor, demonstrated a significant hemolymph acidosis. Hemolymph CO2 tension (\(Pa_{CO_2 }\)) and CO2 content (\(Ca_{CO_2 }\)) also increased and remained significantly elevated for 96 h following treatment. No significant changes could be detected in either hemolymph oxygenation or ionic status (except for HCO 3 − ) as a result of acetazolamide treatment. Crabs treated with acetazolamide, and also exposed to air, exhibited a more pronounced hemolymph acidosis with significantly increased respiratory (\(P_{CO_2 }\)) and metabolic (lactate) components compared with the control group. Upon reimmersion acetazolamide treated crabs showed a slower recovery of hemolymph pH compared with the control group and no significant removal of the total CO2 load induced by air exposure. No significant differences between experimental and control groups during air exposure and recovery could be detected in hemolymph oxygenation, ionic status, NH3+NH 4 + levels or respiratory and cardiac pumping frequency and so the effects of acetazolamide treatment were apparently limited to CO2 removal across the gills. These results indicate that branchial CA facilitates the removal of CO2 from the hemolymph of SW adaptedC. productus largely by catalyzing the dehydration of hemolymph HCO 3 − to molecular CO2 at the gill. It is also recognized that gill CA may also serve to hydrate molecular CO2 to H+ and HCO3/− for use as counterions for ionic uptake mechanisms. Crab gill CA thus appears to play an important role in CO2 excretion as well as hemolymph ionic regulation.

The physical characteristics of the haemolymph in Porcellio spinicornis say (Porcellionidae, Isopoda).

Absolute haemolymph volume in Porcellio spinicornis, as determined by the 14C-dilution and the haemolymph sodium estimation techniques, ranged, on the average, from 8.1 microliter in the 6th growth--stage to 15.06 microliter in the 9th growth--stage. The mean haemolymph volume per cent for all growth--stages (6th-9th) amounted to 33.78. The haemolymph pH varied from 6.42 to 6.54, osmotic pressure from 221 to 256 mM NaCl equivalents and the specific gravity from 1.031 to 1.052.

Water carbonate alkalinity as a determinant of hemolymph acid-base balance in the shore crab,Carcinus maenas: a study at two different ambientP CO 2 andP O 2 levels

1. Hemolymph acid-base balance (pH,PCO2 and bicarbonate concentration) was measured in shore crabs,Carcinus maenas, exposed first for 17–18 h to normoxic seawater at three levels of titration alkalinity, TAw 0.95, 2.5 and 4.5 meq·l−1, either at low (ca. 0.3 Torr) or at high (ca. 4.3 Torr) ambientPCO2 (PwCO2), and subsequently exposed acutely for 2 h to hyperoxia in the same ambient acid-base conditions. WaterPCO2 was controlled with a pH-CO2-stat system in all experiments. 2. At low constantPwCO2 (0.3 Torr), decreased TAw led to an uncompensated hypercapnic acidosis with hemolymphPCO2 increased and hemolymph pH decreased. At highPwCO2 (4.3 Torr) however, hemolymph acid-base state was independent of TAw. 3. Acute hyperoxia induced hypercapnic acidosis in all conditions. At lowPwCO2, the hyperoxia-induced hypercapnia was more marked at low than at high TAw whereas at highPwCO2 it was of the same magnitude whatever the TAw. 4. The internal acid-base state thus depends on TAw at constant low inspiredPwCO2, probably via related changes of the water CO2 capacitance coefficient, ΔCwCO2/ΔPwCO2, due to buffering of excreted CO2 by carbonate ions at high water pH. Therefore, all the acid-base properties of the ambient water, pH,PCO2 and carbonate alkalinity, must be taken into account in studying acid-base balance of water-breathers.

Some In Vivo and In Vitro Characteristics of Aplysia Californica Haemolymph*

ABSTRACT The nervous system of the opisthobranch mollusc, Aplysia californica Cooper has been extensively studied and a great deal is known about the electrophysiological properties of a number of identifiable neurones (see Kandel, 1976, 1979). A variety of synaptic and electrical connections have been mapped, permitting identification of the mode in which nervous control is exerted over certain motor functions. Substantially less is known regarding other physiological systems of Aplysia (Kandel, 1979), particularly the respiratory system, and this limits the description of how nervous control is affected by internal physiological state. Chalazonitis & Nahas (1965) reported data for haemolymph pH, CO2 tension and O2 tension in the related species, Aplysia fasciata, and Bevelaqua et al. (1975) identified the respiratory pigment, haemocyanin, in the haemolymph of A. californica. However, there has been no detailed study of in vivo oxygen levels or acid-base status of A. californica. Neither are there any data for the in vitro properties of A. californica haemolymph, such as oxygen carrying capacity, oxygen affinity or buffering properties. Such data are of particular importance in A. californica, for two reasons. Firstly, motor function, such as the often-studied gill withdrawal reflex, may be directly influenced by factors such as pH, or as are the respiratory performances of other marine invertebrates (Mangum & Burnett, 1975; McMahon & Wilkens, 1975; Bayne, 1976; Batterton & Cameron, 1978). Secondly, Chalazonitis (1974) has shown clearly that membrane potential, spike frequency and membrane resistance of many single cells in A. fasciata are directly affected by pH, and of the bathing fluid. Therefore, the present study was undertaken to describe some of the in vivo and in vitro properties of the haemolymph of A. californica with regards to acid-base status and oxygenation characteristics.

Active HCO3- Secretion in the Rectal Salt Gland of A Mosquito Larva Inhabiting NaHCO3–CO3 Lakes

ABSTRACT Aedes dorsalis larvae are one of the few organisms capable of inhabiting hypersaline NaHCO3-CO3 lakes. Under laboratory conditions larvae can survive and develop normally in saline media with pH values of 10·5, HCO3− concentrations of 250 mm, or CO32− concentrations of 100 mm. Despite ingestion of alkaline media at a rate equal to 130% of larval body wt/day, these insects regulated haemolymph pH (7·55–7·70) and HCO3− concentration (8·0–18·5 mm) within narrow limits. Analysis of fluid obtained by micropuncture or microcannulation indicated that rectal secretion is an important mechanism of pH and HCO3− regulation. The lumen-to-haemocoel HCO3− and CO32− gradients generated by isolated, microcannulated recta were 21 :1 and 241 :1, respectively. Transepithelial potential in cannulated recta was – 14·2 to –23·3 mV (lumen negative), demonstrating that net HCO3− secretion occurs against a large electrochemical gradient.

Changes in the hemolymph pH and specific gravity of the silkworm larvae, Bombyx mori, infected with Beauveria bassiana

Changes in the hemolymph pH and specific gravity of the silkworm larvae, Bombyx mori, infected with Beauveria bassiana

Effect of Maintained Hypoxic Exposure on the Crayfish Orconectes Rusticus

ABSTRACT Oxygen uptake in the crayfish Orconectes rusticus was maintained at nor-moxic levels throughout 6 days exposure to an ambient oxygen tension of 45–55 torr. This was attributed to compensatory responses of the ventilatory and cardiovascular pumps. The first 72 h of hypoxic exposure were characterized by a transitory 3-fold increase in both scaphognathite rate and cardiac output above initial normoxic values. During the latter 72 h of hypoxic exposure both scaphognathite pumping and cardiac output were significantly below maximum values but were maintained above initial normoxic levels. Thus, as reflected by the increased convection requirements for both water and haemolymph, the increased scaphognathite and heart activity served to increase oxygen delivery to both the branchial chambers and the tissues. Additionally, the increased branchial ventilation and gill perfusion facilitated removal of dissolved carbon dioxide from the haemolymph, effecting a respiratory alkalosis. The increased haemolymph pH elevated the oxygen affinity of the haemocyanin via the Bohr affect, enabling a greater volume of oxygen to be picked up at the gills by the haemocyanin despite the reduced pressure gradient.

The hemoglobin of the snail Biomphalaria glabrata. The absence of sulfhydryl groups (SH), presence of disulfide bonds (SS), and their relation to ligand properties

1. 1. The hemoglobin of Biomphalaria glabrata (Mollusca, Planorbidae), was devoid of SH groups, its two cysteinil residues forming an SS bridge for each 3.4 × 10 4 daltons of assumed molecular mass. 2. 2. The molecule dissociated incompletely, and no evidence of chain separation was obtained. 3. 3. The natural osmolality of the laboratory-reared snail's hemolymph was 0.085 ± 0.003 osmolal. 4. 4. The anaerobic pH of hemolymph was 7.22 ± 0.02. 5. 5. The O 2 affinity was related to both concentration and pH, and decreases sharply with SS breaking. 6. 6. The Bohr effect was concentration-dependent, and increases slightly with SS rupture. 7. 7. The cooperativity n was 1.6–1.7 at the natural conditions of pH and osmolality. It was related to pH and concentration, decreasing to near 1 with rupture of SS bonds.

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).

Responses ofHelix pomatia to anoxia: Changes of solute activity and other properties of the haemolymph

1) The following variables were measured in the haemolymph ofHelix pomatia, during periods of anoxia lasting up to 45 h and during recovery in air after 24 h of anoxia:d-lactate, succinate, propionate, acetate, Na+, K+, Ca2+, Mg2+, Cl−, protein, total osmolarity. 2) d-lactate and succinate appear in the haemolymph immediately after the onset of anoxia and may reach concentrations of 60 and 40 mM, respectively. In the period 10 to 30 h following the onset of anoxia the two endproducts accumulate in the haemolymph at approximately the same rate. Propionate is not formed. Acetate may occur in the liver but its concentration is unrelated to the duration of anaerobiosis. 3) Complete recovery after 24 h of anoxia takes a further 24 h. Changes in the concentration ofd-lactate and succinate suggest that these two end-products are metabolized in the liver. 4) No significant changes of Na+, Cl− and protein occur during anoxia, suggesting that haemolymph volume remains more or less constant. However, the concentration of K+ in the haemolymph increases up to 3-fold. 5) Ca2+ increases at a rate which corresponds to the rise ofd-lactate and succinate concentrations. Mg2+ shows only a modest increase and remains constant after 10 h of anoxia. 6) Solute activity during anoxia increases initially, from an average of 160 to 270–280 mOsm, and then drops to approximately 220 mOsm. After 40 h of anoxia the difference between solute activity and concentration of all measured solutes amounts to approximately 160 mM. 7) In the haemolymph of anaerobic snails the activity of Ca2+ is reduced by up to 60% but this is not sufficient to account for the reduction of total solute activity. 8) In a buffer system resembling the haemolymph of anaerobic snails (containing 50 mM NaCl, 60 mM Ca2+, 60 mMd-lactate, 40 mM succinate, pH 6.9) the haemocyanin ofH. pomatia displays a negative Bohr shift (Δlogp50/ΔpH-0.14) with respect to aerobic haemolymph (pH 7.8) over the whole\(P_{O_2 } \) range considered. Cooperativity is identical under both conditions. 9) It is concluded that none of the speculations so far advanced concerning mechanisms which might control the flow of metabolites along the major pathways of anaerobiosis, can completely explain the responses ofH. pomatia to anoxia.

Post‐exercise acid‐base balance and ventilatory control in Birgus latro, the coconut crab

AbstractThere is a postexercise acidosis in the coconut crab, Birgus latro, which is initially due to an elevation in the hemolymph P, and a metabolic acidosis which is only partially due to lactic acid. Hyperventilation following exercise causes a respiratory alkalosis that helps to restore acid‐base balance. The ventilatory receptors appear to be internally located, and ventilation is stimulated by a drop in the hemolymph pH or [HCO], but not by P or P. Amelioration of the metabolic acidosis in normal crabs occurs in 1 hour despite elevated lactate levels. Crabs with their gills removed showed only a transient lag in both respiratory and metabolic recovery from acidosis, indicating the dominance of the branchial lung of Birgus in gas exchange. The data suggest that intra‐ and extracellular transfers may be significant in acid‐base regulation.

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.

Effect of Bacillus thuringiensis on the pH of gut and haemolymph as well as on the sodium‐potassium concentration in the haemolymph of Achoea janata L. and Spodoptera litura F. larvae (Lepid., Noctuidae)

AbstractA comparative study of the pathogenicity of Bacillus thuringiensis to Achoea janata and Spodoptera litura showed that S. litura is less susceptible to B. thuringiensis action. Further, the data revealed that the two insect species differed in their response to B. thuringiensis with respect to change in sodium‐potassium concentration and ratio in the haemolymph. The results strongly suggest that changes in pH of gut and haemolymph as well as disturbance of Na+ and K+ concentration and their ratio in the haemolymph are not related to the toxicity of B. thuringiensis.ZusammenfassungZur Wirkung von Bacillus thuringiensis auf den pH‐Wert des Darmes und der Haemolymphe sowie auf die Natrium‐Kalium‐Konzentration der Haemolymphe von Achoea janata L. und Spodoptera litura F.‐Raupen (Lepid. Noctuidae)Eine vergleichende Untersuchung der Pathogenität von Bacillus thuringiensis auf Achoea janata und Spodoptera litura ergab, daß S. litura wenig empfindlich gegenüber B. thuringiensis war. Weiterhin zeigten die Experimente, daß die 2 Insektenarten bezüglich ihrer Reaktion gegenüber B. thuringiensis auf Veränderungen der Natrium‐Kalium‐Konzentration und ‐menge in der Haemolymphe unterschiedlich reagierten. Die Ergebnisse zeigen deutlich, daß weder Veränderungen im pH‐Wert des Darmes und der Haemolymphe noch Störungen der Na‐K‐Konzentration sowie deren Menge in der Haemolympe zur Toxizität von B. thuringiensis in Beziehung standen.

Effects of temperature acclimation on crayfish hemocyanin oxygen binding

Crayfish, Pacifastacus leniusculus, were acclimated to 10, 20, and 25 degrees C for 1 mo. Hemocyanin from animals at these three acclimation temperatures showed distinctly different oxygen binding patterns. At any particular set of test temperature and pH, hemocyanin from 10 degrees C-acclimated animals had the lowest oxygen affinity and the greatest cooperativity, whereas hemocyanin from 25 degrees C-acclimated animals had the highest oxygen affinity and the lowest cooperativity. When tested at their own acclimation temperature, and at normal hemolymph pH for that temperature, all three hemocyanins showed oxygen pressure for half-saturation of hemoglobin of 6-7 Torr. Thus acclimation keeps oxygen affinity centered around a narrow range of values. The acclimation response probably eliminates hemocyanin oxygen affinity as a major factor in the decline of oxygen uptake ability in the crayfish above 20 degrees C. The structural basis for the observed functional changes in the hemocyanin is not yet clear.

Hemolymph acid-base balance of the crayfish Astacus leptodactylus as a function of the oxygenation and the acid-base balance of the ambient water

The acid-base balance of the prebranchial hemolymph of the crayfish Astacus leptodactylus was studied at various acid-base balances and levels of oxygenation of the ambient water at 13°C. The water acid-base balance was controlled automatically by a pH-CO 2-stat. Into water of constant titration alkalinity, TA, this device intermittently injects carbon dioxide to maintain the pH at a present value. Water pH was reduced to the same value either by hypercapnia (at constant TA) of by adding HCl or H 2SO 4 to decrease the TA (at constant CO 2 tension). Decrease of hemolymph pH and increase of hemolymph P CO 2 were similar for the acidic waters. Water oxygenation changes strongly affected hemolymph ABB. In crayfish living in hyperoxic water ( P O2 ⋍ 600 Torr ) compared to those in hypoxic water ( P O 2 ⋍ 40 Torr ), hemolymph pH was 0.3 to 0.4 unit lower and hemolymph P CO 2 several times higher, the exact values of pH and P CO 2 depending on the controlled ambient acid-base balance. In any study of the hemolymph acid-base balance of the crayfish, it is as important to control ambient water's acid-base balance and oxygenation as it is to control its temperature, a conclusion which probably holds true for studies on all water breathers.