Ionic composition of the rectal contents and excreta of the reduviid bug Triatoma infestans.

We have investigated the chemical composition of the rectal contents, faeces and urine of the blood-sucking bug Triatoma infestans. This is the environment in which the important disease-causing organism, Trypanosoma cruzi, lives. Directly after feeding of Triatoma infestans, the pH of the excreta switched from an acidic to an alkaline pH and, 1 day later, back to a slightly acidic pH. The osmolality varied in the initial excreta and in the rectal contents on the day following the meal between 300 and 460 mosmol/kg H(2)O, but after an additional day it increased to 350-970 mosmol/kg H(2)O. Determinations by ion capillary electrophoresis showed that sulphate and phosphate dominated the rectal contents in unfed bugs. After feeding, the first four drops of fluid excreta were mainly a sodium chloride solution (>150 mM for each). One to 10 days after feeding strong individual variations in the concentrations of individual ions were evident, especially for potassium and sodium. Mean concentrations of chloride remained at about 70 mM; sulphate and phosphate showed an increase within the first 1 or 2 days and then reached a level of about 160 and 210 mM, respectively. The rectal contents of long-term starved bugs contained high concentrations of phosphate and potassium; sulphate and sodium were slightly lower.

Hemolymph ion composition and volume changes in the supralittoral isopod Ligia pallasii Brandt, during molt.

We analyzed ion composition and volume of the hemolymph of Ligia pallasii in four different stages of the molt cycle using capillary electrophoresis and 3H-inulin. The main ions in the hemolymph were Na+, K+, Mg2+ , Ca2+, and Cl-. The Ca2+ concentration increased significantly during the molt by 47% from intermolt to intramolt and by 37% from intermolt to postmolt, probably due to resorption of Ca2+ from the cuticle and sternal CaCO3 deposits. The K+ concentration increased significantly by 20% during molt. The hemolymph volume normalized to the dry mass of the animals decreased by 36% from intermolt to late premolt. This was due to a reduction in the hemolymph volume and to an increase in dry mass of the animals during premolt. A sudden increase in the hemolymph volume occurring between late premolt and intramolt served to expand the cuticle. Since the Na+, K+, Mg2+, and Cl- concentrations did not change significantly from late premolt to intramolt, the increase in hemolymph volume suggests an uptake of seawater rather than freshwater.

Comparison of calcium storage between a Baikalian gastropod and holarctic relatives.

In Lake Baikal, extremely thin shells are reported as a typical feature of endemic gastropods. This statement derived only from observations; no experimental data were available up to now. Therefore, we quantitatively investigated the calcium distribution in the endemic prosobranch gastropod Benedictia baicalensis and compared the results with those of Lithoglyphus naticoides, a near relative, non-endemic, palaearctic species. The shell of the endemic mollusc B. baicalensis consists of 94.9+/-26.0 microg Ca(2+)/microl animal volume (n=43), and in L. naticoides 865.0+/-271.5 microg Ca(2+)/microl (n=10). Calcium contents in the tissue of B. baicalensis vary between different sampling stations and different sampling dates (from 9.4+/-5.1 (n=33) to 20.5+/-8.4 microg Ca(2+)/mg dry weight DW (n=16)) and are only 1/5-1/10 compared to L. naticoides (88.5+/-39.1 microg Ca(2+)/mg DW (n=9)). But the values for hemolymph calcium concentration and osmolality in both species are identical (B. baicalensis: osmolality: 84.4+/-5.3 mosm/kg (n=40); hemolymph calcium concentration: 4.6+/-1.7 mmol/l (n=40). L. naticoides: osmolality: 85.0+/-2.0 mosm/kg (n=8); hemolymph calcium concentration: 5.2+/-5.0 mmol/l (n=40).). This is the first experimental study demonstrating, that - besides a similar hemolymph ionic composition - the Baikalian species is characterized by significantly lower calcium storage in shell and tissue than the nearly related non-endemic species.

Osmotic and ionic hemolymph concentrations of bathyal and abyssal amphipods of Lake Baikal (Siberia) in relation to water depth.

We studied the adaptive variations of the hemolymph concentrations in relation to water depth and pressure using deep-dwelling amphipods from Lake Baikal. Hemolymph osmolality was determined in six bathyal and abyssal species immediately after capture when values come closest to the habitat concentrations. In three species, hemolymph osmolalities correlated positively with depth of capture. Prevalent ions in the hemolymph are sodium and chloride. Lactate, our indicator for capture stress, was highest after trawling (2-6 mM) and lowest after retrieval from cages (0-0.6 mM). Acclimation to different pressure was studied by exposing the specimens to different water depths over several days. Hemolymph concentrations did not change after acclimation to surface pressure in the sublittoral Acanthogammarus albus, a native also to shallow water, but decreased by 30-80 mosmol/kg H2O in the bathyal and abyssal species Acanthogammarus grewingki, Acanthogammarus reicherti, and Parapallasea lagowskii. Similarly, hemolymph osmolality decreased in A. reicherti and P. lagowskii originating from deep water, when acclimated to reduced water depth, and, in A. reicherti hemolymph osmolality reached its original high value when returned to the depth of capture. Higher hemolymph osmolalities and NaCl concentrations, demonstrated here for the first time, may provide selective advantages to abyssal species.

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.

Chloride secretion drives urine formation in leech nephridia.

The transport mechanisms underlying urine formation in leech nephridia were investigated in situ and in isolated preparations using pharmacological, electrophysiological and micropuncture techniques. Canalicular cells, which secrete the primary urine, function as a Cl(-)-secreting epithelium. An apical Cl- conductance contributes to the lumen-negative potential which drives transcellular K+ transport and paracellular Na+ transport. On the basolateral side, a ouabain-sensitive Na+/K(+)-ATPase contributes substantially to the cellular and transcellular potential and provides the Na+ gradient necessary for a bumetanide-sensitive Na+/K+/2Cl- cotransport. Final urine is formed by subsequent reabsorption of ions along the central canal, where KCl and NaCl are reabsorbed in different portions. The postprandial diuresis is not a consequence of the changes in blood osmolality or ion concentrations. Similar changes in the ionic environment do not promote diuresis in isolated nephridia. Apparently, the composition and volume of the primary urine cannot be separately controlled. Any increase in fluid secretion by leech canalicular cells involves upregulation of the paracellular pathway and stimulation of Cl- entry, which thereby changes the normally K(+)-enriched primary urine to the Na(+)-enriched primary urine characteristic of leeches in diuresis.

Anaerobic metabolism in the leech (Hirudo medicinalis L.): direct and indirect calorimetry during severe hypoxia.

Anaerobic metabolism in the limnic annelid Hirudo medicinalis L. was investigated by direct and indirect calorimetry. During long-term severe hypoxia, the rate of heat dissipation was reduced up to 13% of the aerobic rate. At the same time, the rate of ATP turnover was reduced to about 30% of the aerobic rate, indicating that metabolic depression is an important mechanism to ensure survival of the leech during environmental anaerobiosis. Heat dissipation during hypoxia was monitored under two experimental conditions, favouring either concomitant hypocapnia (continuous N2 bubbling) or hypercapnia (self-induced hypoxia). The reduction in heat dissipation during hypocapnic hypoxia was less pronounced than during hypercapnic hypoxia, indicating that the different experimental conditions may influence anaerobic metabolism and the extent of metabolic depression. Biochemical analysis of known anaerobic substrates and endproducts provided the basis for indirect calorimetry during self-induced hypoxia. From changes in metabolites, the expected heat dissipation was calculated for initial (0-8 ,h) and long-term severe hypoxia (8-72 h). During the initial period, the calculated heat dissipation fully accounted for direct calorimetric determination. During long-term hypoxia, only 71% of the measured heat production could be explained from biochemical analysis of metabolites. Therefore, an additional unknown endproduct cannot be excluded, especially when anaerobic ammonia production and analysis of the carbohydrate balance are considered.

Hyperosmotic acclimation in the leech, Hirudo medicinalis L.: energy metabolism, osmotic, ionic and volume regulation.

1. Acclimation of Hirudo medicinalis L. to hyperosmotic medium (12/1000 salinity) is characterized by changes in energy metabolism. A transient succinate fermentation is followed by malate accumulation in the tissue from the control level of 41.4 +/- 7.6 to 82.8 +/- 15.4 mumol/g dry weight after 9 days. 2. Initial osmotic water loss of 40% is restored in the intravasal space by isosmotic volume regulation after 9 days, but restoration is incomplete in the tissue even after 6 weeks of acclimation. 3. Elevation of SCCA anions in the blood is a mechanism of chloride hyporegulation.

Recovery after anaerobic metabolism in the leech (Hirudo medicinalis L.).

Medicinal leeches (Hirudo medicinalis L.) responded to self-induced hypoxia (72 h) with typical anaerobic metabolism characterized by a decrease in adenylate energy charge, utilization of the substrates glycogen and malate, and accumulation of the main anaerobic end-products succinate and propionate. Propionate was also excreted into the medium. Ammonia excretion was suppressed. Aerobic recovery resulted in a profound O2 debt. Resynthesis of ATP was completed within 30 min. Disposal of succinate and restoring of malate required 2-3 h, and clearance of propionate and recharging of glycogen 6-12 h. Ammonia excretion did not exceed normoxic rates and excretion of propionate during recovery accounted for only 10% of total propionate accumulated during hypoxia. It is postulated that the clearance of succinate and propionate involves oxidation but also resynthesis of malate and glycogen. During hypoxia and recovery blood osmolality remained constant. The Na+ and Cl- ion concentrations in blood, the decrease of which was nearly equimolar during hypoxia, were re-established following different time-courses. Na+ concentration returned to normoxic levels after 2-3 h. The delayed increase in Cl- concentration, however, correlating with 6-12 h necessary to clear blood propionate, is interpreted as an anion regulating effect.

Osmotic and ionic regulation during hypoxia in the medicinal leech, Hirudo medicinalis L.

The concentrations of inorganic and organic ions and osmolality in the blood of the medicinal leech, Hirudo medicinalis, were determined during normoxia and hypercapnic and hypocapnic hypoxia. In normoxic animals, the blood sodium concentration was 124.5 +/- 4.2 mmol/l and the total cation concentration was 132.2 +/- 4.3 mEq/l (mean +/- S.D.). Major anionic compounds were chloride (40.8 +/- 1.6 mmol/l), bicarbonate (8.4 +/- 1.3 mmol/l), and organic anions (42.5 +/- 2.3 mEq/l). Among the latter, malate accounts for 30.4 +/- 2.2 mEq/l. The nature of the remaining anion fraction, which balances cation and anion concentrations in leech blood, remains unknown. Within 96 h of hypercapnic hypoxia, the amount of organic osmolytes in leech tissue increased from the control level of 56.6 +/- 9.1 to 158.3 +/- 19.5 mumol/g dry weight. An even higher amount of organic acids was accumulated within 96 h of hypocapnic hypoxia (218.0 +/- 53.7 mumol/g dry weight). A possible reason for this is that lactate, which is a major end-product of hypocapnic hypoxia, cannot be excreted to the external medium as easily as propionate. The accumulation of blood organic acids generating osmotic stress in the animals was compensated by an equimolar decrease in sodium and chloride ion concentrations. In hypercapnic animals these changes resulted in a constant osmotic concentration of the blood (200 mosmol/kg H2O) during the experimental period. Between 24 and 96 h of hypocapnic hypoxia, however, the increase in the osmotic gradient between animal and medium was correlated with further net water uptake and the obvious deterioration of the volume- and ion-regulatory mechanisms in these animals.

[Studies on the rectal pads of the honey bee Apis mellifca]. / Untersuchungen an Rektalpolstern der Honigbiene Apis mellifica

According to earlier investigations young bees show a rectal fluid hypo-osmotic to the haemolymph. It had been assumed, however, that in certain situations honey bees have to be economical with water and simultaneously form a rectal fluid hyperosmotic to the haemolymph. The latter has been confirmed by the present investigation.