Diel Changes in Potassium Activity in the Haemolymph of the Cockroach Leucophaea Maderae

The functioning of many tissues is crucially dependent on the activity of a small number of inorganic ions in the body fluids. In insects the concentration of the major inorganic ions in the blood is highly variable, both within one individual and between individuals and species (e.g. Pichon, 1970; Florkin & Jeuniaux, 1974). Such variation in potassium and sodium activity is likely to be of most significance to the muscles and peripheral nerves, since these, unlike the CNS, are not sheathed from the blood (see Treherne, 1974). The observations of Hoyle (1954) and Ellis & Hoyle (1954) indicated that the level of activity of locusts could be controlled by the potassium concentration of the blood, although this was not supported by the field observations of Chapman (1958) or subsequent laboratory experiments of Moorhouse (1968).

Although this variability in blood ion concentration is well known, it is not clear whether it exhibits a consistent temporal pattern within one species. Treherne, Buchan & Bennett (1975) demonstrated a seasonal variation in Na⁺ concentration in Periplaneta, although the Na⁺ activity remained relatively constant. Brady (1968) established that Periplaneta blood is a changing ionic environment throughout the day: he observed in some animals a fall of about 10% in potassium concentration during the first hour of darkness.

The motivation of Brady’s study was to establish whether potassium concentration, following Hoyle’s (1954) observations, could act as a link between the circadian ‘clock’ and the overt expression of rhythmic locomotor activity. The present investigation was begun to improve and extend Brady’s observations, using more modern techniques, in particular ion-selective electrodes. These have two important advantages over the whole-blood flame-photometry analysis used by Brady. First, they measure ionic activity of the plasma, not gross blood concentration, and therefore indicate the amount of ions freely available for physiological processes such as muscle contraction, whereas flame-photometry gives a measure of total ionic concentration, which includes, for example, ions that are bound. Second, ion-selective microelectrodes allow continuous measurements to be made on one particular cockroach; this eliminates the artefacts caused by repeatedly taking blood from one cockroach (Brady, 1968) and the great variability caused by sampling from different cockroaches.

The present study has three main aims. First, to develop methods for characterizing the ionic environment of cockroach blood using ion-selective microelectrodes. Second, to describe the diel changes in potassium and sodium activity in cockroach blood. Third, to relate such changes to known circadian rhythms, in particular locomotor activity.

We used Leucophaea maderae rather than Periplaneta americana because of the more precise and predictable locomotor activity of the former species (Harker, unpublished observations). Cultures of Leucophaea were maintained in large, light-tight boxes at a room temperature of 21–26 °C in a 16 h light:8 h dark cycle (LD 16:8). The light source was a 20 W fluorescent light producing 124 lux on the base of the box. Water was freely available. The cockroaches were fed ad libitum on a diet of the following composition: 350 g rolled oats, 350 g wheat feed, 50 g linseed cake, 120 g grass meal, 35 g casein, 25 g dried powdered yeast, 25 g sucrose, 50 ml linseed oil, 50 ml arachis oil and 11 g.W. salt mixture. In all experiments only adult males were used.

Ion-selective microelectrodes based on the design of Khuri, Agulian & Wise (1971) were used. A small amount of potassium liquid ion-exchanger (Corning no. 47132) was injected into the tip (ID 0·3 μ) of a siliconized micropipette (Pyrex redrawn tubing: OD 1·2 mm, ID 0·6 mm), which was then filled with 0·1 M-KCI. The mean resistance of the K⁺ selective electrodes was 5·8 + 0·45 x 10⁹ Ω (mean ± 1× s.e. ; n = 68).

In pure KC1 the potassium electrodes gave a slope of between 52 and 55 mV per potassium activity decade. The electrodes were calibrated (immediately before implantation) in the cockroach saline designed by Treherne, Buchan & Bennett (1975). Although devised for Periplaneta, this saline has similar concentrations of major cations to those of Leucophaea blood (Table 1). The activity coefficient (γ) of K+ and Na⁺ in this saline was calculated from a modified form of the Debye-Hückel equation,

where z = valency of the ion, I = ionic strength of the solution, A and b are constants (A = 0·507 at 20 °C, b = 0· z²; Robinson & Stokes (1959)). γ = 0·74 for K⁺ and Na⁺.

The selectivity of individual electrodes for potassium over sodium was between 45 and 70 to 1 and varied with the age of the electrode. The electrodes showed a reduced performance in cockroach saline as compared with pure KCl, and calculations show (see Eisenman, 1962) that this reduced performance can be accounted for by interference from sodium ions.

The stability of the electrodes is important, since they are intended for continuous use for at least 24 h whilst implanted in a living cockroach. The electrodes were always recalibrated in cockroach saline after removal from the experimental animal. The new calibration line had generally shifted a little and had a reduced slope (Fig. 1 a). Fig. 1 b shows the effect which this change in calibration line has on the calculated potassium activity in a typical experiment. The discrepancy between the two curves is most marked at the higher values, but the general pattern is similar. Potassium activities at intermediate stages of the experiment were in fact inferred by assuming that the calibration line changed linearly with time during the experiment (Fig. 1b). There is no direct justification for this, but the calibration line of electrodes in cockroach saline changed linearly with time.

The implanted electrodes eventually become encapsulated by haemocytes. The haemocytes do not appear to interfere with the behaviour of the electrode for about the first 24–60 h, the exact time depending on the individual electrodes. During this period the performance of an electrode in blood deteriorates in a broadly similar way to that of an electrode in cockroach saline. At some time after 24 h of implantation providing the electrodes do not fail for other reasons, the electrode response flattens out to give a stable potential record. Electrodes are invariably encapsulated at this point. When tested in cockroach salines of varying potassium concentrations, the encapsulated electrodes produce an unvarying potential reading. This encapsulation response of the cockroach sets a limit on the life of the electrode ; in favourable cases, the electrodes may remain workable for up to 96 h.

We used sodium-selective glass electrodes of ‘protruding-tip’ design (Hincke, 1959). Sealed-tip micropipettes of Na+-selective glass (NAS_11–18) were fixed into lengths of insulating glass with silicone rubber, so that about 0·5 mm of selective glass protruded at the tip. The micropipette was filled with 0·1 M-NaCl and an Ag-AgCl tipped silver wire was sealed into the end of the electrode with silicone rubber. The lectrodes were calibrated in solutions of pure sodium chloride and gave a slope of about 60 mV per activity decade. Given the composition of Leucophaea blood (Table 1), there is no danger of potassium interfering with the performance of the electrodes.

The sodium electrodes were not very stable, drifting by about 10 mV per 24 h. Intermediate values were inferred as for the potassium electrodes. The electrodes became encapsulated at about the same rate as did the potassium electrodes. The resistance of the sodium electrodes (3 x 10⁹·3 × 10¹⁰ Ω) increased by a factor of 10 during 24 h in cockroach haemolymph.

pH electrodes (Transidyne 805 ; tungsten carbide body with antimony film: supplied by Clark Electromedical Instruments) with a tip diameter of 5 μ and insulated in epoxy resin were calibrated in imidazole buffers.

Cockroaches were temporarily immobilized with a small dose of diethyl ether. Both pairs of wings were cut off near the base. The electrodes were inserted on either side of the midline of the metanotum through holes made with pins. The electrodes were held in position by a balsa wood harness (Fig. 2). The harness was glued to the thorax with sticky wax (Kerr Manufacturing Co., Detroit, Michigan).

During the experiment the cockroach was allowed to run free in a rectangular perspex box (12·5 ×5·5 × 5·5 cm) which was encased in sheets of pierced zinc to form Faraday cage. The end of the two insulated silver wires (about 15 cm long) from the electrodes were soldered to terminals in the centre of the box which was contained in a large light-tight box providing LD 16:8 at a temperature of 22–25 °C The light source was a 25 W tungsten light bulb, producing 66 lux on the base of the perspex box. The cockroach was not given food or water during the experiment.

Locomotor activity of the cockroach whilst in the box was measured with a light-activated switch (combined photodiode and monolithic integrated circuit).

The experimental treatment does not appear to have had a major effect on the timing of cockroach behaviour since the pattern of locomotor activity of individual cockroaches was broadly similar before, during and after the experimental treatment.

The potential of the electrodes was measured with a Keithley Electrometer 600B (input impedance 10¹⁴ Ω) and displayed on a Tekman dual-channel recorder. Locomotor activity was also displayed on this recorder. The electrometer was used to measure electrode resistance.

Blood was sampled from the bases of the mid and hind coxae with 2 μl disposable micropipettes. Night-time samples were taken in the dark using a red torch. The 2 μl sample was diluted in 2 ml of distilled water, and K⁺, Na⁺, Mg²⁺ and Ca²⁺ concentrations were measured with an atomic absorption spectrophotometer (Unicam SP 90A Series 2). Chloride was measured by electrometric titration (Ramsay, Brown & Croghan, 1955). Osmotic pressure was measured with a Nanolitre Osmometer (Clifton Technical Physics, N.Y.).

For the measurement of haematocrit, haemolymph was sampled from heat-fixed cockroaches in 2 μl micropipettes and spun at about 3000 g for 15 min in a haematocrit centrifuge. The volume of the cell fraction was then measured under a microscope.

The concentration of the major inorganic ions and the osmotic concentration of Leucophaea blood are shown in Table 1. The values for Na⁺ and Ca²⁺ are similar to those obtained by Todd (1958), but the concentrations of K⁺ and Mg²⁺ in the present study are considerably higher. The osmotic and ionic concentrations in Leucophaea are broadly similar to those in the Periplaneta saline devised by Treherne et al. (1975). This saline was used for calibrating the electrodes in the present study.

The pH of the blood of one individual Leucophaea, measured with an implanted electrode, varied between 6·90 and 7·24 over a 22 h period (Fig. 3). There were two peaks, at dusk and dawn, corresponding to the periods of maximum locomotor activity, but it would be unwise to infer too much from the record of one individual.

Preliminary measurements of haematocrit indicate that between 2 and 6% of the haemolymph volume is occupied by haemocytes (14 measurements from 7 male Leucophaea) this compares with Brady’s (1967b) figure of 2·6% from 16 animals.

The diel changes in potassium activity of three adult male Leucophaea in LD 16:7 are shown in Fig. 4. There is a dramatic variation in K⁺ activity throughout the day: the maximum range for an individual cockroach in 24 h was from 4–5 to 25·0 mm. There is a consistent pattern in this diel change in potassium activity; a minimum about an hour before lights-off, rising to a peak during the hours of darkness, falling to a second low point about 2 h after dawn, and rising to a second peak during the day. A composite curve from 14 cockroaches, with activity expressed as a % change from the maximum for each individual, is shown in Fig. 5.

Fig. 5 also shows a composite curve of locomotor activity of 15 cockroaches. In six of these, locomotor activity and potassium activity were measured simultaneously.

The remaining cockroaches were sham-operated as though for measurement of potassium activity, but only locomotor activity was measured. There is a sharp peak in locomotor activity in the hour after lights-off. Activity remains relatively high during the remainder of the dark period with a small broad peak in the hours around lights-on. The major peak in locomotor activity occurs about 1 h after the first low point in K⁺ and the small dawn peak in locomotor activity some hours before the second low point in potassium activity.

The individual potassium activity records indicate that the observed pattern is probably not the result of some artefact of implantation (e.g. wounding). Individual cockroaches showed the same pattern of K⁺ activity no matter what time of day the electrodes were implanted (Fig. 4). Wounding causes an increase in haemocyte numbers in Periplaneta(Brady, 1967 b), and this leads to an increase in whole-blood K+ concentration of the order of 2–0 mm (Brady, 1967b). The effect of this on K⁺ activity would probably be of little importance.

Diel changes in sodium activity were measured chiefly to establish whether these changes could interfere with the operation of the potassium microelectrode. Records from 4 individuals show that there may be marked changes in sodium activity during the day (maximum range, 52–92 mm) but reveal no clear overall pattern (Fig. 6). Assuming a potassium electrode with a 60:1 selectivity for potassium over sodium, this maximum range in sodium activity (40 mm) would be equivalent to a change in potassium activity of only 0·67 mm.

There are marked fluctuations in sodium concentration during the day (Fig. 7). The lowest values were found during the middle of the light period with a second minimum at the end of the light period, and the highest values occur shortly after lights-off, but there is a considerable scatter about the mean for each value and a less consistent pattern than for K⁺.

Whole blood potassium concentration rises to a peak about 5 h before lights-off, falls a little an hour before lights-off, rises again at the beginning of the dark period and falls away to low values during the remainder of the dark period (Fig. 8b). The fluctuations in potassium concentration and activity follow a roughly similar pattern (Fig. 8 a).

The present observations clearly emphasize the accepted view (e.g. Pichon, 1970) that insect blood is a highly variable ionic environment. The observations further demonstrate, for the first time, a marked and consistent diel rhythm in the activity of a major blood ion, potassium. This rhythm is probably of particular significance to the functioning of excitable tissues – nerve and muscle. Although the insect CNS and larger peripheral nerves are protected behind the so-called ‘blood-brain’ barrier (Treherne & Pichon, 1972; Treherne, 1974), the smaller peripheral nerves (Lane & Treherne, 1973) and the muscles and neuromuscular junctions (Osborne, 1970, 1975) appear not to be sheathed from the blood that bathes them. There is unfortunately no published information on the electrophysiology of Leucophaea nerve or muscle, but the present observations may be related to electrophysiological analysis of excitable tissues in other insects.

Preliminary, day-time (12·00–18·00 h) measurements of potassium activity in 13 individual Periplaneta americana with ion-selective electrodes gave a mean value of 9 4 mm (range 7·7–11·5 mm) (W. A. Foster, unpublished observations). A diel fluctuation in blood potassium activity in Periplaneta comparable to that shown by Leucophaea (say 60%, Fig. 5 a) would cause severe disturbance of central nervous function, were it not for the existence of an effective blood-brain barrier (Thomas & Treherne, 1975). If the smaller peripheral nerves in Leucophaea are, by analogy with those of Periplaneta, freely accessible to small ions from the blood, then the observed diel variation in K+ activity could have a profound effect on axonal function.

Changes in blood potassium activity of the magnitude described here (e.g. 4·5–25·0 mm) might be expected to produce axonal depolarization that would induce significant inactivation of the inward current of the action potential (see, Hodgkin & Huxley, 1952). In desheathed cockroach giant axons, for example, elevation of the potassium level from the supposed extracellular concentration of 3·0 mm in intact connectives caused substantial reduction of the amplitude of the recorded action potential at 10 mm and conduction block at around 12·5 mm-K⁺ (Thomas & Treherne, 1975) It is possible, therefore, that any exposed small peripheral axons might be specialized to enable them to function at high potassium concentrations, either because their resting potential is unusually insensitive to external potassium concentrations in this physiological range, or because sodium-inactivation following large depolarizations is insufficient to block action potentials, as has been demonstrated in the giant axon of a serpulid worm (Carlson & Treherne, 1977).

The neurohaemal regions in the peripheral nerve of the insect Rhodmus prolixus are unprotected by perineurial cells and the neurosecretory terminals are accessible to exogenously applied lanthanum (Lane, Leslie & Swales, 1975). In Rhodnius and Glossina, step changes in external potassium concentration similar to the diel changes observed in the present investigation have been shown to depolarize the neurosecretory terminals, causing the release of diuretic hormone from the neurohaemal organs (Maddrell & Gee, 1974). If the neurohaemal areas in Leucophaea are accessible, as in Rhodnius, then it follows that the diel changes in potassium activity could profoundly affect the release of neurosecretory products into the blood and could, as a result, influence a variety of bodily functions. It is, of course, impossible to predict the extent of such an effect since it is likely to depend upon the rate of depolarization and the kinetics of calcium inactivation (see Baker & Rink, 1975 ; Nordmann, 1976).

The resting potentials of cockroach muscle (Usherwood, 1969; Wareham, Duncan & Bowler, 1974) and locust muscle (Hoyle, 1954) are sensitive to changes in external concentrations of potassium. Wareham et al. (1974) demonstrated that there is a linear relation between external potassium concentration and muscle resting potential in Periplaneta, with a slope of 43 mV per decade change in potassium concentration. In locusts, Hoyle (1954) showed that changes in potassium concentration of up to 60% could follow a short period of starvation. This could in turn account for an increase in the muscle resting potential of about 12 mV and an improvement in the mechanical responses of the muscles to nervous stimulation. In Leucophaea, changes of this magnitude apparently occur twice daily and will almost certainly influence muscle resting potential and also muscle performance.

It has been suggested that potassium may have an important general role in the control of circadian rhythm (Eskin, 1972; Njus, Sulzman & Hastings, 1974; Satter, Galston & Racusen, 1975). Several authors have noted a connexion between blood potassium concentration and insect locomotor activity (Hoyle, 1954; Ellis & Hoyle, 1954; Asperen & Esch, 1956; Pichon & Boistel, 1963). Brady (1968) examined whether changes in potassium concentration could form a link between the ‘clock’ of an insect and the overt expression of locomotor activity. His results were inconclusive; there was a drop of about 2 mm (10%) in blood K+ concentration at the time of the onset of locomotor activity, but this was not apparent in all the series of animals. The present techniques, which allow continuous measurement in one animal of the freely available potassium in cockroach haemolymph, show that diel changes in potassium activity are of an order which could affect the general level of excitability or trigger circadian activity via an effect on muscle or nerve resting potential. It is conceivable that rhythms in K⁺ activity could play a role in the observed transmission of circadian rhythms between insects in parabiosis (Harker, 1956; Cymborowski & Brady, 1972). However, the present results show that any effects of blood potassium on circadian locomotory behaviour are likely to be complex. Thus, although the ‘dusk’ increase and subsequent decline in locomotory movement parallel the changes in potassium activity, there was no equivalent correlation with the ‘dawn’ decline in potassium activity (Fig. 5). The present results do not, therefore, establish that potassium activity is a direct link between the circadian clock and the expression of rhythmic behaviour. They do, however, suggest that further experiments to test the possibility of a more complex relation between potassium activity and rhythmic behaviour would be worth while. It is important, for example, to attempt to establish whether this K+ activity is truly circadian and what phase relation exists between the free-running K⁺ and locomotor’ activity rhythms.

It is not clear what mechanisms directly control the variations in potassium activity. Broadly speaking, K⁺ activity could change either by movement of K⁺ between compartments within the haemolymph or by movement in and out of the haemolymph. K⁺ may become associated with soluble blood proteins or with the haemocytes, which in cockroaches may contain a significant proportion (sometimes more than 50%) of the total blood potassium (Brady, 1967 a, b). It is clear from observations on the dehydration and rehydration of Periplaneta that there is considerable capacity for storage of Na⁺ and K⁺ in sites outside the haemolymph (Wall, 1970). An increased rate of Malpighian tubule secretion could reduce total K⁺ by the temporary accumulation of a potassium-rich fluid in the tubules during solute recycling.

A comparison of diel changes in total blood K⁺ (Fig. 8) and K+ activity (Fig. 8 a) gives some indication of the movement of K⁺ within the blood. A plot of the apparent activity coefficient (see Treherne et al. 1975), derived from values in Fig. 8, gives an idea of the diel changes in the proportion of freely available potassium in the blood (Fig. 9). Assuming that the blood is broadly similar, in terms of ionic strength and available water content, to the calibration saline (Table 1), should not exceed 0·74. The fact that it does suggests, for example, that at certain times of day the effective amount of solvent water in the blood is reduced by sequestration in some compartment. The haemocytes are unlikely to form a large part of this compartment since they occupied only some 2–6 % of the blood volume.

The pre-dusk (19·00–23·00 h) fall in K⁺ activity is accompanied by a fall in total blood K⁺ concentration and by a significant fall in ⁠; this suggests that there is a net movement of K+ from the plasma to a store outside the blood. The post-dawn (07·00–11·00 h) fall in K⁺ activity is accompanied by a fall in but there is no significant fall in total blood K⁺; this suggests that there is a net movement of K⁺ from the free to the bound potassium compartments within the blood. The sharp rise in total K+ and the fall in at 19·00 h with no associated change in free K⁺ suggests that some bound store of K⁺ is replenished from a source outside the blood. It is perhaps suggestive that at about this time, the blood pH begins to rise (Fig. 3); this would tend to make blood proteins more negative and hence attractive to K⁺. Confirmation of the pH change would be of great interest. Further measurements, particularly paired readings of K⁺ activity and whole-blood K⁺ in the same individual, are required before the trends outlined in Fig. 9 can be confirmed.

The fact that the baseline potassium activity of insect blood may fluctuate in a diel fashion is of considerable practical importance to those who study the physiology of insect tissues, in particular excitable tissues. For example, it may well be important to standardize the time of day of an experiment and to take diel variations into account in the elaboration of insect salines.

It is hoped that the present observations will provoke further work on the diel variations in insect blood ions, particularly in relation to the role of potassium in the expression of circadian locomotor activity.

This work was supported by a Science Research Council Grant to Dr J. E. Harker. We are indebted to Dr R. B. Moreton for helpful discussions. Mr P. C. Steam gave valuable practical assistance. Mr J. W. Rodford prepared Fig. 2.