Cloning and functional studies of the epithelial sodium channel of guinea-pig colon

Schnizler, M.; Mastroberardino, L.; Weber, W.-M.; Verrey, F. and Clauss, W.

Institute of Animal Physiology, Justus-Liebig-University Giessen, Wartweg 95, D-35392 Giessen, FRG and Institute of Physiology, University Zürich, CH-8057 Zürich, Switzerland

Our previous electrophysiological work has shown that guinea-pig (gp, Cavia porcellus) distal colon, as a tight epithelium, possesses an amiloride-sensitive electrogenic Na⁺ transport. As an exceptional feature guinea-pigs, in opposite to other species, require no previous physiologic stimulus, as i.e. low-salt diet or aldosterone-injection, to develop colonic amiloride-blockable Na⁺ conductances. Amiloride-sensitive epithelial Na⁺ channels (EnaC) are heteromultimeric proteins, made of homologous a, b and g subunits. Guinea-pig EnaC (gpENaC) can be expressed in Xenopus oocytes from mRNA of distal colon of low-Na⁺ diet fed animals. Interestingly, the resulting amiloride-sensitive Na⁺ current is considerably increased by intracellular cAMP (Liebold et al. 1996, Pflügers Arch. Eur. J. Physiol.; 431, 913-922). Such a stimulation has not been reported for other EnaCs expressed in Xenopus oocytes. To address the question whether this difference is due to structural differences within the channel molecule, we cloned a cDNA (2.8 kB) encoding the a subunit of gpENaC. The deduced amino acid sequence exhibits 80% identity with that of previously cloned mammalian aENaCs. It indicates that gpaENaC has the same overall organization as its homologues, namely a cytoplasmic NH₂- and COOH-terminal domains, two transmembrane domains and a large extracellular loop. Several structural motifs are highly conserved. Since expression of aENaCs in oocytes alone exhibits only small amiloride-blockable Na⁺ currents, we coexpressed gpaENaC with rat b and g subunits (rbg).

Supported by DFG Cl 63/12-1

Control of blood flow in the swimbladder rete mirabile

Schwerte, Thorsten; Pelster, Bernd

Institute for Zoology and Limnology, University of Innsbruck, Technikerstr. 25A, A-6020 Innsbruck, Austria

Gas secretion in the fish swimbladder seems to be modified by both, control of gas gland cell metabolism and control of perfusion, and the presence of three capillary beds in series requires a sophisticated regulation of swimbladder perfusion. While the presence of hormonal control mechanisms for the regulation of swimbladder perfusion was established a long time ago, the importance of nervous control was demonstrated only in a recent study. In eel swimbladder adrenaline induced a decrease of swimbladder blood flow, and using videoanalytical techniques the a-adrenergic resistance vessels were located close to the arterial entrance into the rete mirabile. In blood-perfused preparations electrical stimulation of the nervus vagus produced a decrease, and vagotomy an increase in perfusion pressure, suggesting that swimbladder blood flow is under vagal tonic control. Vasoactive intestinal peptide (VIP) and sodium nitro prusside (SNP) induced a significant vasodilatation, and Nwnitro-L-arginine, a competitive inhibitor of NO-synthase, significantly increased perfusion pressure induced by an increase in flow rate. The existence of VIP- and NOS-like fibers was demonstrated by immunohistochemical techniques. A vasodilatation was also induced by a decrease in blood pH. This led to the conclusion that the acid release of gas gland cells also contributes to perfusion control, while lactate seems to have no direct effect on blood flow.

The role of extracardiac haemocoelic pulsations in insect respiration.

Sláma, Karel

Institute of Entomology, Czech Academy of Sciences, Drnovsk 507, 16100 Prague, Czech Republic

The widely accepted diffusion theory of insect respiration claims that insect respiration is based on a simple passive diffusion of oxygen and carbon dioxide through spiracles. The theory has been often criticized but due to a lack of reliable experimental data it has never been unequivocally disproved.

Several years ago, I have invented micro-respirographic, micro-anemometric and thermographic methods for recording respiratory functions in very small insects. The results obtained with various species and different developmental stages revealed that insect respiration does not depend completely on passive gaseous diffusion. The sphingid or cetoniid giants as well as the smallest fleas or ants are able to perform active, precisely regulated, real breathing of air. They can actually inspire or expire through just one or a group of selected spiracles. The convective stream of gas, which periodically passes up and down throughout the whole tracheal system, is achieved by rhythmic pulsations in haemocoelic (internal, intratracheal) pressure. These extracardiac pulsations in haemocoelic pressure are produced by contractions of intersegmental or dorsoventral abdominal musculature (abdominal pressure pump). The frequency of extracardiac pulsations is infrasonic, ranging mostly from 0.1 Hz to 4 Hz, with the amplitude from 10 Pa to 800 Pa. The amplitude of extracardiac pulsations in haemocoelic pressure is usually 100- to 500- fold greater than amplitude of the heartbeat. Spiracular valves usually open in twinkles with intervals in millisecond range. During extracardiac pulsation, selected spiracular valves are opened or twinkle in synchrony with the ascending or the descending phases of each pressure pulse ( fluttering spiracles). This results in unidirectional stream of air inside or outside of the body, respectively. The active insect breathing is fully integrated by an autonomic, cholinergic, parasympathetic-like nervous system (coelopulse), which is composed from: a) Nervous input centre located in thoracic ganglia of the ventral nerve cord, b) Muscular output executed by contractions of intersegmental or dorsoventral abdominal muscles, and c) Physiological feedback represented by a system of metameric perisympathetic neurohaemal organs. Some examples illustrating active regulation of breathing by the coelopulse system will be presented in several insect species.

Life of the shallow water fish species Zoarces viviparus under different climatic conditions: Ecological and physiological background

Ulleweit, J.; Hardewig, I.; Tesch, C.; Knust, R.; Pörtner, H.-O.

Alfred-Wegener Institute für Polar und Marine Research, Biology I / Ecophysiology, Columbusstr., Bremerhaven, Germany

The size of the distribution area of a boreal marine fish species depends on the adaptation ability on different environmental conditions. One important factor is temperature but other abiotical factors such as light, salinity and hydrodynamics may be limiting as well. Populations at distribution borders can be characterized by ecological parameters e.g. growth, behaviour and reproduction. Differences in these parameters found in populations of different geographic origin could physiological determined by the limits of metabolic regulation.

For this study investigations on the ecology and physiology of eelpout (Zoarces viviparus, L.) were carried out. Field data and test animals were collected from the North Sea (German Bight) at the southern border of distribution and from the White Sea (Kandalaksha Bay) at the northern border of distribution. Clear differences in growth and reproduction were found. The production of eelpout calculated by growth rate and reproduction rate at the White Sea is low in comparison to the North Sea. The mean water temperature at the White Sea as a subpolar region is distinct lower compared with the German Bight. Presumable low temperature may cause a higher maintenance metabolic rate in specimen living at the White Sea. Oxygen measurements at different temperatures of seasonal adapted North Sea animals show a higher metabolic rate of cold adapted animals. Increasing temperature results in an increase in muscle lactate at a lower critical temperature in White Sea eelpouts than in North Sea animals. These results will be discussed in context to adaptation mechanisms.

About heartbeat in diapausing Pieris brassicae pupae

Vanatoa, A.; Tartes, U.

Institute of Zoology and Botany, Riia 181, Tartu EE2400, Estonia

The present study analyses heart activity in diapausing Pieris brassicae pupae investigated by the means of infrared transmission described by Hetz (1994). The electronic scheme was modified to fit our needs. Two infrared emitters and sensors were used, one on the thorax and another on first abdominal segment. Experiments were carried out at room temperature (20°C).
We distinguished five types of heartbeat patterns: slow forward beat (about once per minute), fast intermittent forward beat (circa 20 times per minute), slow backwards beat (about once per two minutes), fast intermittent backwards beat (circa 30 times per minute), and heart inactivity. All heartbeat patterns described above, were present during the diapause.
In the beginning of the intermittent fast beat period (approximately 5 to 10% of all), the cardiogram differs from that of the rest: the diastole of the beginning is not as clearly registered as in the rest of the cycle. The likely cause of this is, that in the beginning of the intermittent fast beat period, the hemolymph is standing relatively still in the body cavity, but the diastole represents the influx of the hemolymph into the heart, thus dilating the aorta. When the hemolymph starts to move, the diastole will be more potent.
We also estimated the speed of peristaltic wave for forward and backward pulse. The speed of this pulse was approximately 43 millimetres per second for forward pulse and about 8 millimetres per second for backward pulse.
The mechanics and functioning of heartbeat in P. brassicae is discussed according to our data. Also, the role of backward heartbeat is analysed.

Hetz, S. K. 1994 Untersuchungen zu Atmung, Kreislauf und Säure-Basen-Regulation an Puppen der tropischen Schmetterlingsgattungen Ornithoptera, Troides und Attacus. Den Naturwissenschaftlichen Fakultäten der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades.

Mechanisms of seasonal temperature adaptation in enzymes from Nordic krill Meganyctiphanes norvegica

Vetter, R.-A.H.¹; Thatcher, B.J.²; Storey, K.B.³; Buchholz F.¹

¹Biologische Anstalt Helgoland, Meeresstation, D-27498 Helgoland, Germany, ²Clinical Neuroscience Branch, NIH, Bethesda MD 20892 4405, USA, ³Carleton University, 1125 Colonel By Drive, Ottawa K1S 5B6, Ontario, Canada

Two isoforms of pyruvate kinase (PK I and PK II) from Nordic krill Meganyctiphanes norvegica were partly purified and characterized. Both PK variants were present in summer and winter specimens with a tissue specificity in abdomen (PK I) and cephalothorax (PK II). Obvious differences were found in chromatographic and kinetic characteristics: Enzymatic adaptations to low temperatures were found in PK I only, whereas PK II did not contribute to seasonal temperature adaptation. In winter specimens, the activation energy of PK I decreased significantly from 53.2 ± 1.5 to 50.2 ± 1.2 kJ·mol^-1. The affinity of PK I to phosphoenolpyruvate was higher in winter (K_M = 0.024 ± 0.002 mmol·l^-1) compared to summer (K_M = 0.033 ± 0.003 mmol·l^-1). Both effects lead to an increased efficiency of this enzyme isoform in the cold. In contrast, K_M values of PK II showed no significant differences between summer (K_M = 0.181 ± 0.014 mmol·l^-1) and winter specimens (K_M = 0.193 ± 0.015 mmol·l^-1).

In addition, the biochemical mechanisms of seasonal differences were investigated in PK I only. One of the most common mechanisms of regulating PK by reversible phosphorylation can be excluded for the PK of M. norvegica, because the krill enzyme lacks phosphorylation sites. However, the seasonal change of kinetic parameters of PK I was followed by a reduction of the P_i-value from pH 7.02 in summer to 6.89 in winter. The same decrease in K_M from summer to winter values was observed after reversible in vitro denaturation of summer PK I with urea. Conclusively, the seasonal changes in catalytic properties of PK I might be driven by attaching removable, low molecular weight substances to the winter enzyme resulting in the summer isoform. In contradiction, after isoelectro focusing elution profiles of urea-treated summer enzyme and untreated winter enzyme differed distinctly. Accordingly, the possible mechanism of regulation by attached biomolecules must be excluded. Further results of biophysical properties (e.g. molecular weight, amino acid composition, hydrophobic interactions) showed small differences between the summer and winter enzyme suggesting the existence of two different isoforms. These isoforms might be regulated via so-called qualitative adaptation.

The red blood cells of rainbow trout and common carp exhibit differential tolerances to sulfide

Völkel, S.

Animal Physiology, Humboldt University Berlin, D-10115 Berlin, Germany

Hydrogen sulfide may accumulate in freshwater habitats when the organic content is high and when oxygen is depleted. Sulfide is toxic for fish and other organisms mainly because of its inhibition of the cytochrome c oxidase. In addition, sulfide can cause the formation of sulfhemoglobin, a hemoglobin derivative which is practically non-functional in oxygen transport (Carrico RJ, Peisach J, Alben JO (1978) J Biol Chem 253, 2386). The rainbow trout (Oncorhynchus mykiss) is quite sulfide-sensitive dying at sulfide concentrations of a few µM. In contrast, the common carp (Cyprinus carpio) can survive several hundred µM sulfide for at least 30 days. In this study some effects of sulfide on the blood of both species were investigated. Washed red blood cells were oxygenated and were then exposed to various sulfide concentrations (0.2 - 5 mM) for one hour. In the trout red blood cells sulfide equilibrated across the erythrocyte membrane during the first 5 min of exposure. Although extracellular sulfide concentrations decreased during 1 hour, intracellular sulfide remained at high levels during the whole period of incubation. In contrast, intracellular sulfide concentrations in the carp were 3 - 10 times lower than in the plasma. The different intracellular sulfide concentrations in trout and carp erythrocytes was not due to a higher sulfide oxidising capacity in the carp. Thiosulfate as the main product of sulfide oxidation was not enhanced in carp blood as compared to trout.

In the trout red blood cells 10 % of total hemoglobin was converted to sulfhemoglobin after 1 hour of sulfide incubation (2.5 mM). At the same time the oxygen binding capacity was reduced by 20 %. Under the same conditions no sulfhemoglobin was detected in the carp red blood cells and the oxygen binding capacity was not affected. The carp's ability to keep low intracellular sulfide concentrations seem to provide some protection against sulfhemoglobin formation during exposure to sulfide.

Ventilation and gas exchange in varanid lizards during treadmill exercise

Wang, T.¹; Farmer, C.²; Carrier, D.R.³ and Hicks, J.W.²

¹Univ. of Odense (DK), ²Univ. of California, Irvine, CA (USA) and ³Univ. of Utah, UT (USA)

The effects of exercise on the cardiorespiratory physiology of reptiles has not been studied in great detail and it is, therefore, uncertain how reptiles respond to elevated metabolic rate and which factors limit gas exchange. Furthermore, the extent to which lizards ventilate their lungs during locomotion has been a matter of controversy. Recent measurements on Varanus exanthematicus and Iguana iguana show that although locomotion dramatically alters the ventilatory pattern in both species, Varanus effectively ventilate its lungs during locomotion, whereas Iguana may not be able to match the increased oxygen demand with adequate ventilation at moderate and higher speeds (J. Exp. Biol. 200: 2629-39).

The reptilian lung is, in general, characterised by a low oxygen diffusing capacity. MIGET studies on Varanus show that pulmonary gas exchange is diffusion limited during exercise (J. Exp. Biol 198:1783-89). Thus, to investigate whether the structurally simple reptilian lung constrain maximum oxygen uptake (VO₂max), we determined the effects of hypoxia on gas exchange during treadmill exercise at 1 km h^-1 in Varanus. Lizards were equipped with air-tight face masks for simultaneous measurements of ventilation, oxygen uptake and _CO2 excretion. Physiological parameters were determined 3-5 min following the onset of exercise in normoxia, whereafter F_IO₂ was reduced to 0.18 or 0.15 for an additional 5 min. At these levels of hypoxia, oxygen uptake was reduced (P<0.001) by 12 and 26% respectively, whereas ventilation and _CO2 excretion were not affected. The sensitivity of VO₂max to mild hypoxia is pronounced compared to most mammals and it, therefore, appear that the septate lung of reptiles is severely diffusion limited. Thus, it is possible that periods of relative hypoxia during the geologic past may have been an important selective force in the evolution and distribution of reptiles.

Supported by the Danish Research Council and NSF.

The hypertonicity-induced Na⁺ conductance of rat hepatocytes: A novel mechanism of cell volume regulation

Wehner, Frank; Kirschner, Udo; Böhmer, Christoph and Tinel, Hanna

Max-Planck-Institut für molekulare Physiologie, Abteilung Epithelphysiologie, Rheinlanddamm 201, D-44139 Dortmund

In confluent primary cultures, rat hepatocytes under hypertonic stress (+100 mosmol/l) exhibit a significant regulatory volume increase (RVI) that, after 10 min, amounts to some 45% of initial cell shrinkage. This RVI is mainly mediated by an increase in cell Na⁺ leading to an activation of Na⁺/K⁺-ATPase and, consequently, to an increase in cell K⁺. Intracellular microelectrode recordings and cable analysis, microfluorometry of cell pH, measurements of time-dependent ⁸⁶Rb⁺ uptake, as well as confocal laser-scanning microscopy reveal that an increase in Na⁺ conductance rather than Na⁺/H⁺ exchange or Na⁺-K⁺-2Cl^- symport is the predominant mechanism of volume-activated Na⁺ influx. The relative contribution of the above pathways to initial Na⁺ entry is found to be approximately 4:1:1 with an overall Na⁺ influx that would tend to increase cell Na⁺ by 34.3 mmol/l^-1 · (10 min)^-1. Monitoring the volume-sensitive Na⁺ conductance in intracellular recordings by means of low Na⁺ pulses reveals inhibition of activation by ethylisopropylamiloride (EIPA), amiloride, benzamil, and phenamil with apparent K_i values of 230 nmol/l, 6.0 mmol/l, 240 mmol/l, and 1.1 mmol/l, respectively. This pharmacological profile is confirmed in Xenopus oocytes injected with total rat liver mRNA where amiloride and its analogues inhibit volume-activated inward currents with similar differences in efficacy. Moreover, in solitary rat hepatocytes in primary culture, whole-cell patch-clamp recordings reveal hypertonicity-induced increases in Na⁺ conductance that are reduced by 70% after addition of 10 mmol/l amiloride. It is concluded that a hypertonicity-induced Na⁺ conductance is the main mechanism initiating RVI in rat hepatocytes. This pathway reflects a low (amiloride) affinity type of Na⁺ channel that exhibits an inverse pharmacological profile compared to the epithelial Na⁺ channels (ENaCs). Induction of a Na⁺ conductance represents a novel mechanism in cell-volume regulation observed in an increasing number of cells.

Effect of UV-B on respiratory and circulatory parameters of developing plaice (Pleuronectes platessa L.)

Wiemer, M.; Steeger, H.-U.; Paul, R.J.

Institut für Zoophysiologie, Westfälische Wilhelms-Universität Münster, Hindenburgplatz 55, 48143 Münster, Germany

The egg stages of the plaice (Pleuronectes platessa L.) are floating at the sea-surface during the whole period of their development. During this time (10 to 13 days at 10°C) they are exposed to solar radiation and may be harmed by the increased intensity of UV-B (280-320 nm) caused by the reduction of stratospheric ozone.

The use of optophysiological methods which characterize vital functions makes it possible to detect effects of UV-B irradiation on early life stages. This approach, the optical measurement of circulatory and respiratory parameters, is a non invasive and very sensitive method. It does not impair the ontogeny of the embryo and allows repeated measurements at later stages of development.

The heart rate of plaice eggs irradiated with a combination of sunlight-like and UV-B emitting fluorescent lamps and a daily dose of 4.86 kJm^-2 (present outdoor level in April) after gastrulation (stage II) was lower than that of the control animals, but the difference was not significant. A bradycardia which was generated in the embryos by a sine-shaped hypoxia (oxygen range: 100 % to 5 % air saturation) showed no significant differences between UV-B irradiated and control animals either. Irradiation of early embryos (stage I - before gastrulation) led to 100 % mortality.

A significantly lower heart rate and a trend to a prolonged bradycardia was observed in embryos irradiated from stage II on for five days with a daily dose of 8.64 kJm^-2, resembling the highest present outdoor level in May. Sublethal effects, like lower heart rate and poor ability of respiratory control could still be detected in these animals 2 month after the irradiation.

As a result of the ongoing loss of the ozone layer critical UV-B doses will be reached earlier in spring. Depending on the latitude, spawning of plaice starts in January at the Southern Bight and lasts until the end of March at the northern spawning grounds. Under unfavourable weather conditions (cloudless sky, calm weather), UV-B induced impairments may then occur in plaice eggs at the northern spawning grounds German Bight and Fisher Bank.

Patterns of respiration in hibernation in edible dormice (Myoxus glis, L.)

Wilz, Michael and Heldmaier, Gerhard

Philipps Universität Marburg, FB Biologie/Zoologie, Karl v. Frisch Str., 35032 Marburg

The hibernation period of edible dormice lasts for seven months, from September through May. The entire hibernation period is composed out of hibernation bouts. The bout length is highly variable and lasts from 3 to 28 days. During the entry into a hibernation bout the metabolic rate is reduced to a small fraction of basal metabolic rate and body temperature decreases close to ambient temperature.

We examined hibernation patterns at different ambient temperatures (T_a). We measured six edible dormice in hibernation under controlled conditions in the laboratory. The animals were housed in 1m x 0.4m x 0.4m cages in a climate chamber (T_a= 2°C-18°C) without food and water ad lib. Body temperature was measured by telemetry, using implanted temperature sensitive transmitters. Ambient temperature was monitored with thermocouples and metabolic rate was measured by indirect calorimetry in an open system. All data were collected by a computer system. Breathing patterns were additionally observed using an infra-red video camera.

The minimum metabolic rate in hibernation varied between 0.044 ml O₂ *g^-1*h^-1 and 0.005 ml O₂ *g^-1*h^-1. With the beginning of a hibernation bout the dormice rapidly lowered their metabolic rate and the body temperature decreases. After about six hours we see a shift from continuous breathing to intermittent breathing, prior to reaching minimum metabolic rate. At this transition from continuous to intermittent breathing T_b is still about 20°C. During intermittent breathing the non-ventilatory period was temperature-dependent and lasted about 8 minutes at T_a = 16°C and increased up to 60 minutes at T_a = 3°C. At T_a's below 3°C non-ventilatory period was decreased again. The infra-red camera showed that the non-ventilatory periods are followed by ventilation-bursts, which lasted 7(± 2) seconds with 7(± 2) ventilations at T_a = 13°C.

Physiological and metabolic adaptations to sulphide and hypoxia in the lugworm Arenicola marina

Wohlgemuth, S.; Grieshaber, M.K.

Heinrich-Heine-Universität Düsseldorf, Germany

The lugworm A. marina is a common inhabitant of tidal flats of the North and Baltic Sea. The worm lives in a L-shaped burrow which extends about 20 cm deep into the black reduced zone of the sediment. Peristaltic muscular movements of the animals body wall serve to irrigate the burrow (Wells GP 1945: J Mar Biol Ass UK 26, 170-207). In the sediment or the overlying water column sulphidic and hypoxic conditions can develop which may affect the animal in its burrow.

In this study we investigated the effect of sulphide and hypoxia on physiological and metabolic functions of A. marina. Animals incubated in artificial burrows increased their ventilation threefold with decreasing PO₂ down to 6.3 kPa in the seawater whereafter it decreased when the oxygen content diminished further. The increased ventilation rate compensated for the moderate hypoxia down to a PO₂ of 6.3 kPa. Thereafter anaerobiosis was observed (Pc). The addition of 0.025 mM sulphide during hypoxia induced a non-significant depression of ventilation compared to hypoxic controls. A significant decrease was seen in the presence of 0.12 mM sulphide which furthermore shifted the Pc towards a higher PO₂.

In simultaneous measurements of heat production and oxygen consumption in a flowthrough calorespirometer a correlated decrease of heat dissipation and oxygen consumption was observed when PO₂ fell below 16 kPa. Heat production was only slightly affected in the presence of sulphide £ 0.18 mM.

Localisation of carbonic anhydrase in swimbladders of European eel (Anguilla anguilla) and perch (Perca fluviatilis)

Würtz, Jürgen; Salvenmoser, Willi; Pelster, Bernd

Institute of Zoology and Limnology, University of Innsbruck, Technikerstrasse 25A-6020 Innsbruck

Carbonic anhydrase activity is essential for the gas secretion in the gas gland of fishes. To localise the distribution of carbonic anhydrase histochemical staining was carried out on swimbladder tissue of the European eel (Anguilla anguilla) and the perch (Perca fluviatilis) according to Hansson (1967). In the European eel the gas gland cells are distributed over the whole secretory part of the swimbladder as a single layered epithelium. Strong staining of carbonic anhydrase was found mainly in the membrane of the basal labyrinth and the lateral membrane. Additionally the endothelium and the erythrocytes showed a positive reaction. No staining was visible at the apical membrane towards the lumen of the swimbladder. The gas gland of the perch consists of a compact, richly vascularised “multilayered” epithelium, in which the gas gland cells have contact to the swimbladder lumen via small tunnels. Similar to the gas gland cells of the eel, perch gas gland cells in direct contact to blood vessels always show richly folded membranes towards the vessels. A strong positive reaction was mainly found in these membrane foldings, but also in the endothelia of the blood vessels. All other membranes were only slightly stained. Swimbladder epithelial cells outside the gas gland showed no positive staining of carbonic anhydrase. The fact, that membranes facing blood vessels showed the highest staining of carbonic anhydrase with the apical membrane towards the lumen was not stained clearly demonstrates, that a rapid equilibrium of the CO₂ – HCO₃^- reaction in the intracellular as well as in the extracellular space is essential for the swimbladders function.

Hansson, P.J. 1967 Histochemical Demonstration of carbonic anhydrase activity. Histochem. 11, 112-128.

Supported by Österreichischer Fonds zur Förderung der wissenschaftlichen Forschung (FWF P11837-BIO)

Osmotic concentrations in littoral and abyssal gammarids captured from different water depths in Lake Baikal

Zerbst-Boroffka, I.; Takhteev, V.; Mekhanikova, I.

Institut für Tierphysiologie, Freie Universität Berlin, Grunewaldstr. 34, 12165 Berlin Limnological Institute Irkutsk, Ulan Batorskaja 3, Irkutsk 664033, Russia

Recently we could show that in abyssal amphipods of the ancient fresh water Lake Baikal, trawled from 1000 m water depth, the osmotic concentration decreased during the first day at the water surface (DZG 1998). The reduction of osmotic concentration of 40-60 mosm/kg was due to the reduction of NaCl concentration more than that of lactate which was not higher than 6 mmol/l in specimens immediately after capture. In order to study the question whether abyssal amphipods have different osmotic concentrations when living in different water depths, we investigated specimens of different species immediately after capture from different water depths respectively.

Results:
1. In Parapallasea lagowski the osmotic concentration was significantly higher in specimens captured by trap than by trawl from the same water depth. Thus we assume that trawling can cause a certain reduction in the osmotic concentration.
2. In 6 species we succeeded in getting healthy and active specimens trawled from different water depths respectively. No correlation between osmotic concentration and water depths of capture could be demonstrated. In 3 species, however, specimens captured from the highest water depths had a significantly higher osmotic concentration. Although experimental conditions probably influence the measured osmotic concentrations, this demonstrates that amphipods tend to develop higher osmotic concentrations in deep water.
3. Comparing 4 species also studied by Bazikalova et al (1946), we registered higher osmotic concentrations in 2 species and lower ones in the others. The difficulty to get reproducible results was probably due to different experimental conditions. But the influence of depths of capture, trawling or trapping, and the time until hemolymph sampling cannot be compared since corresponding information is lacking in the paper of Bazikalova et al 1946.
4. The nearly related abyssal Ceratogammarus dybowski and the supra-abyssal C. cornutus sympatrically abundant in Bukhta Chivyrkui had identical osmotic concentrations when trawled from the same water depth. Thus, a changed osmotic concentration as specialisation during phylogenetic adaptation to the abyssal cannot be concluded from this example.

Intertidal respiration of the springtail Anurida maritima

Zinkler, D. & Biefang, M.

Lehrstuhl für Tierphysiologie, Ruhr Universität Bochum, FRG

The marine littoral Collembola Anurida maritima is a colonizer of the intertidal zone. One characteristic feature of this physiological stressful environment is the occurrence of short, predictable periods of low and high tide. The air-breathing springtail rely on cuticular respiration. Due to the dramatic differences between the oxygen concentration in air and seawater the oxygen supply of submerged animals is limited.
Oxygen consumption of single specimens was monitored by a Strathkelvin oxygen electrode in a microrespiration cell filled with seawater. The efficiency of the crude gas gill was studied by local and temporal PO₂ measurements with microneedle electrodes in seawater as well as in small bubbles of air at saturation water vapour pressure. To prevent respiratory failure A. maritima has developed specific adaptation mechanisms:

(1) down-regulation of oxygen consumption during air-breathing in response to declining ambient oxygen tensions. The normoxic O₂ -uptake rate (269 ± 49 µl O₂ · g FW^-1 · h^-1, at 18°C) decreases gradually. A relatively constant level of the original metabolic activity (approx. 46%,) is maintained at low PO₂ between and 75 to 7.5 Torr (Rüssbeck & Zinkler 1989).
(2) air-breathing within a compressible gas gill enclosed by seawater. The gas gill principle extends the lifetime of the air bubble in unstirred seawater by more than eight-fold in the case of a bubble/liquid volume ratio of 1:10⁵. In contrary, at a 1:10² ratio condition the efficiency of the gas gill is drastically reduced (only 1.5-fold). A bubble of N₂ remains. Obviously, only the first result is in accordance with the mathematical model of the physical principle of a compressible gas gill (Rahn & Paganelli 1968). Nevertheless, still water conditions and low bubble/seawater volume ratios prevail in A. maritima refuge cavities during high tide.
(3) exploitation of low oxygen concentrations from seawater. Specific structures of the epicuticle (hydrophobic microtubercles) trap an air-film which serves as an incompressible gas gill.
(4) Glycogenolysis with anaerobic ATP-production via formation of ethanol.

Rahn H & C V Paganelli 1968: Resp Physiol 5, 145 - 164.
Rüssbeck R & D Zinkler 1989: Verh Dtsch Zool Ges 82, 233.