Physiology of An Atp Receptor in Labellar Sensilla of the Tsetse Fly Glossina Morsitans Morsitans Westw. (Diptera: Glossinidae)

Feeding behaviour in the tsetse fly is not a simple response to a single stimulus, but a series of responses to a sequence of stimuli. Initially the animal must fly to the host from variable distances, probably using visual and olfactory cues (Buxton, 1955). Once on the host the fly must be stimulated to probe the host’s tissues and actively suck up the blood meal, following probing. Two major stimuli are involved: temperature of the host skin (probing stimulus) (Dethier, 1954; Reinouts van Haga & Mitchell, 1976) and concentration of adenosine triphosphate (ATP) in the host tissue (feeding stimulus) (Galun & Margalit, 1969; Mitchell & Reinouts van Haga-Kelker, 1976). Further behavioural work leading to tentative conclusions regarding the physiology of the sense organs involved in ATP reception has been done by Margalit, Galun & Rice (1972) and Langley (1972). In addition, morphological and electrophysiological work on sensilla which possibly bear the ATP receptor was presented by Rice, Galun & Margalit (1973 a). In a brief report (Mitchell, 1976), I have presented initial electrophysiological results which indicate the presence of an ATP receptor in each of eight large, labellar sensilla (LR7 of Rice et al. 1973 a). Here, the physiology of these receptors is presented in more detail, and the results are compared with those of behavioural experiments.

Tsetse flies (Glossina morsitans morsitans Westw.) were from a laboratory colony which has been maintained on rabbits in this Department for 2 years. Flies for the colony were obtained from the Tsetse Research Laboratory, University of Bristol, U.K., in July 1973.

Prior to viewing in the SEM, the labellar lobes of an anaesthetized fly were everted by tightening a fine wire around the bulbous part of the haustellum. The haustellum was then removed, washed in mild detergent, and fixed in 70% ethanol. It was later dehydrated using a graded series of amyl acetate and ethanol, critical-point-dried and mounted on a stub. Single layers of carbon and gold were deposited on the mounted specimen before insertion into the SEM.

In preliminary experiments it was found that the most reliable preparations were obtained using teneral (unfed) flies between 4 and 15 h of age. In early experiments, the haustellum of the fly was removed entirely, after everting the labellum by tightening a fine wire around its bulbous, proximal part. It was then mounted by its proximal end on to the reference electrode. However, receptors in many of these preparations failed to respond.

For the present study, the fly was anaesthetized with CO2 and its wings and legs removed. It was then restrained, ventral side up, with a metal staple on a Plasticine mount. The distal two-thirds of the palpi were removed and a fine wire was tightened around the bulbous part of the haustellum with forceps. This procedure usually caused the labellar lobes to evert, exposing the LR7 sensilla. The wire and haustellum were then firmly fixed to the ventral surface of the head with a drop of molten, 1:1 beeswax-rosin mixture. This immobilized the haustellum with the labellar lobes everted. A minuten insect pin, serving as the indifferent electrode, was inserted into the abdomen of the fly and fixed in place by another drop of the beeswax-rosin mixture. The preparation retained more tissue than that used in the preliminary experiments, and provided a high success rate.

The prepared animal was inserted, abdomen first, up to the prothorax, into the large end of a trumpet-shaped glass tube that had been fashioned from the neck of a Pasteur pipette. It was held in place in the tube by another drop of beeswax-rosin mixture (Fig. 1).

The smaller end of the tube containing the preparation was filled with sufficient 0·15 M-NaCl solution to make contact with the minuten pin, and the whole was mounted on an electrode holder containing a ground wire that made contact with the NaCl solution. The electrode holder was mounted on a micromanipulator via two ball and socket mounts. With this arrangement, the preparation could be oriented to suit the sensillum under study.

A Leitz Ortholux compound microscope, fitted with a 50 · objective which had ST 6 mm working distance, was used to view the preparation. Total magnification was 780 x. Sufficient illumination was achieved with a fibre optics light guide, thus eliminating the electrical interference and temperature problems that would have resulted from using the microscope’s internal illumination system.

The tip-recording method described by Hodgson, Lettvin & Roeder (1955) was used for recording from the sensillum. With this technique, the recording pipetteelectrode also contains the chemical stimulus. The glass pipette-electrodes used had a tip diameter between 6 and 10μm and were fashioned with an Industrial Science Associates Mi micro-pipette puller. The solution in the pipette-electrode made contact with a silver wire that was connected to the input stage of a lab-constructed preamplifier (Fig. 2). This amplifier was small enough to be mounted on the micromanipulator, so that the connexion between the electrode and the amplifier input could be as short as possible. The output of the preamplifier was amplified, displayed on an oscilloscope, and recorded on magnetic tape. The experiments were also routinely recorded on a Honeywell 1858 oscillographic recorder, so that an immediate analysis of traces was possible. Experiments were carried out at 21·22 °C and approximately 40% R.H. Each stimulating-recording electrode was used within 1 min of filling. Tests with dilute dye solutions showed no visible evaporation from the small opening of the electrode over much longer times. Impulses/second in this paper means impulses from a receptor during the first second of response. With the amplifier used it was possible to begin counting impulses 10·20 ms after stimulus application.

All recordings were made from the largest labellar sensilla, designated the LR7 sensilla by Rice et al. (1973 a) (Fig. 3 A). The sensory dendrites apparently enter the sensillum via a single channel which curves a full 180· near the tip. A single pore opens into this channel (Fig. 3B). Jobling (1933) described this channel and Rice et al. (1973 a, b) have shown that it contains three dendrites near the base of the sensillum, two of which continue to the tip. They concluded that the one terminating in the base is a mechanoreceptor and that the two which continue to the tip are chemoreceptors.

Behavioural results have indicated that the ATP receptor in Glossina spp. is probably sensitive to concentrations of the chemical as low as 10⁻⁵ M (Galun & Margalit, 1969; Langley, 1972). Electrophysiological results have indicated that a cell in each LR7 sensillum responds to ATP. The response of the ATP receptor increases with ATP concentration (Figs. 4 and 5).

Experiments on the threshold of the ATP receptor are summarized in Fig. 4; some cells respond to concentrations as low as 10⁻⁶ M ATP. The variability of the responses from receptor to receptor, between flies and on the same fly, is quite great (Fig. 6), but this is not unusual for insect contact chemoreceptors (cf. Dethier, 1974). It seems not unlikely that information from one sensillum is sufficient to trigger sucking behaviour, because Getting (1971) has shown that proboscis extension in the blowfly, Phormia regina, can be induced by sucrose stimulation of a single labellar sensillum.

The pH of vertebrate blood is closely regulated at approximately 7·4 (Dittmer, 1961). One might expect a cell that normally functions in such an environment to be sensitive to changes in pH of the stimulating solution. In tsetse feeding experiments employing ATP, it has been shown that the pH optimum is around 7·2 and that the response drops rapidly at pH values below 5·6 (Galun & Margalit, 1970). The effect of pH upon the receptor was therefore studied to see if the receptor’s response was affected like the behavioural response. Chemoreceptors of other insects have been shown to be sensitive to pH, but only at extremes of the pH range (Gillary, 1966; Rees, 1970).

The effect of pH upon the ATP response of a number of receptors is summarized in Fig. 7, and the response of a single cell over the pH range is shown in Fig. 8. It is obvious that low pH did indeed affect the activity of the receptor, the response at pH 5·4 dropping, on average, to less than one-half of the response at pH 7·2. Some cells were quite resistant to a drop in pH to 5’4 while other cells were severely affected. At pH 4 and below, the response of all cells tested was considerably depressed.

Considering the variable response at pH 5·4, and remembering that each animal has 8 LR7 sensilla, it is quite possible that a sufficient number of these cells could fire in a given animal at this pH to account for the 70 % feeding response reported by Galun & Margalit (1970). There is also good agreement between behavioural results and the present electrophysiological results at pH 4, since Galun & Margalit (1970) obtained a feeding response equivalent to controls at this pH.

From pH 7·2·9·0 there was virtually no change in the response of the receptor to io⁻³ M ATP. At pH 9 0·10-6 some cells remained unaffected while others showed a marked increase in activity (on average 28 % greater than at pH 7·2). No cells showed a significant decrease in activity. This is at variance with behavioural results, which show a drop in feeding response from 88 to 70% over this range for G. austeni (Galun & Margalit, 1970), and a drop from approximately 90 to 50% for G. morsitans (Langley & Pimley, 1973). It is possible that other receptors than the one investigated here are responsible for the depression in feeding activity at high pH.

The increase in the response of some ATP receptors at pH 9·10-6 perhaps indicates injury since the frequency was the highest encountered in this study, but the effects could sometimes be reversed by the application of ATP solutions with pH below 9 (Fig. 9).

Since ATP can be ionized (Alberty, Smith & Bock, 1951), it was of interest to determine if the effect of pH on the response was due to a modification of the ATP molecule. Of the four ionizable groups on the ATP molecule, three are strongly ionized with pKa’ values of less than 2 (Alberty et al. 1951). The terminal phosphate, however, has a pKa’ of 6 48 in 0.5M-NaCl (Alberty et al. 1951) and 6·48 in 0.2 M-NaCl under rigidly controlled conditions (Smith & Alberty, 1956). It could have been the de-ionization of this part of the molecule that caused the reduction in response of the ATP receptor.

To test this, two pairs of solutions were prepared, the solutions in each pair having different pH but the same concentration of ATP molecules with their terminal phosphate ionized (Table 1). With both pairs, the solution with the near neutral pH was far more effective, even though [ATP^4-] was the same within the pairs and the [ATP] much less in the pH 7·4 solutions, so it seems unlikely that de-ionization of the ATP molecule has a significant effect on its stimulatory capacity. Although low pH decreases the response of the ATP receptor and de-ionizes the terminal phosphate group, the decrease in the receptor’s response is about 2·5 fold greater than would be expected solely on the basis of the addition of H+ to the ATP molecule. A more likely explanation is that the low pH affects the dendrite directly, perhaps at the receptor site.

pH, in the range discussed here, has been shown to reduce the sodium permeability of voltage-clamped frog nerves (Woodhull, 1973) and to reduce the potassium permeability of voltage-clamped crayfish giant axons (Shrager, 1974). Both these effects lead to a reduction in sensitivity. It is possible that the dendrite of the ATP receptor is also less sensitive at pH values below 7.2, and that what is being measured here is a general effect of pH on excitable tissues.

An interesting result of Morita (1959) may support this argument. The sucrose receptor in blowfly tarsal chemosensory hairs is clearly inhibited by 1.6 mm quinine hydrochloride (pH not given but probably around 6) and this inhibition increases with the concentration of the acid. By employing side-wall recording, Morita was able to show that the generator potential became more positive as the spike frequency decreased. This could be interpreted as a hyperpolarization of the dendrite (Dethier, 1971, p. 708). The effect was reversible.

Solutions of 0·15 M-NaCl at pH 11 were stimulatory to the ATP receptor to a similar degree as ATP solutions at pH 11, but apparently not to the other receptors in the sensillum. The response of the ATP receptor at pH values above 9 is probably due, at least in part, to the alkalinity rather than the ATP.

In other insect chemoreceptors that have been investigated, the effect of pH is mostly quite different. The water receptor in Phormia terranovae is relatively insensitive to pH changes between 5 and 11 (Rees, 1970). Below pH 5 the response of this receptor is severely depressed, reaching zero at pH 2. Rees suggested that the pH effect indicates that the transmembrane pores are lined with negatively charged groups with pKa’ of about 3·7.

The salt receptor of Phormia regina is unaffected over a pH range of about 3·10, is severely depressed between pH 2·3 and is stimulated by more acidic solutions (Gillary, 1966). Only at high pH is the response like that of the ATP receptor. At pH 10·11 there is a depression in the response (a feature not seen in the response of the ATP receptor) and above pH 11 there is a dramatic 50 % increase in the response. This response can also be elicited by high pH alone, and for short applications (1 s or less) is reversible upon application of salt solution at a pH in the plateau region of the response (Gillary, 1966).

A series of experiments was conducted with chemicals similar in molecular structure to ATP in an attempt to determine the specificity of the ATP receptor and, if specificity was shown, to gain some understanding of the parts of the molecule that are important in the reception process. Table 2 shows the results obtained with various adenosine phosphates and with CTP, GTP and ITP. Each chemical was tested on a receptor for which the response to ATP was also determined. A control stimulus of 0.5 M-NaCl was also required because, following stimulation with ATP or another adenosine phosphate, some receptors fired during 0.15 M-NaCl applications, though always at a low frequency. Perhaps some of the stimulating solution remained on the receptor following removal of the pipette. The details of this odd response could not be adequately described without recording continuously with a side-wall electrode. This technique is impracticable for the small receptors of G. morsitans.

It is apparent from Table 2 that the receptor was sensitive to adenine nucleotides in general. A comparison of the responses to AMP, ADP and ATP at 10 ⁻³ M points clearly to the importance of the length of the phosphate chain. The trend for increased effectiveness with extra phosphate did not continue to A-tetra-P, however, its effective ness lying somewhere between AMP and ADP. Apparently, three phosphate groups are optimal for the receptor. To be as effective a stimulus as 10 ⁻³ M ADP or ATP, AMP had to be at a ten times higher concentration. At this high concentration, AMS and c-AMP were somewhat less effective than AMP. Apparently, replacement of P with S reduced effectiveness, and the phosphate group was most effective in the 5’ position. In this regard the effectiveness of A 3’ 5’ DP at 10 ⁻³ is interesting. Apparently, the positioning of the phosphate groups in the di-phosphate need not greatly affect its effectiveness. Even adding a sugar molecule to ADP, as in ADP-ribose, did not completely destroy effectiveness, the response being approximately one-half the response to ADP.

Adenine appears to be an essential part of the molecule, as shown by the poor stimulatory capacity of CTP, GTP and ITP. GTP and ITP, both with purine bases, differ from ATP in having oxygen and a hydroxyl group, respectively, at carbon-6 of the purine ring instead of an amino group. The results recorded in Table 2 suggest that ITP, GTP and CTP are slightly stimulatory but, given the viability of the response of the receptor at low levels of stimulation, it would be difficult to demonstrate their true relative effectiveness.

The above data agree fairly well with the behavioural results of Galun & Margalit (1969). CTP, GTP, ITP and UTP were not effective as feeding stimulants, while ADP and AMP had similar effectiveness, relative to that of ATP, in both investigations (Table 3).

Arguing in favour of the conclusion that the ATP receptor investigated in this study mediates the behavioural responses of the animal to ATP solutions are the following four points of similarity between the present electrophysiological results and previous behavioural studies.

1. The sensitivity of the receptor.

2. The effect of low pH on the receptor.

3. The relative stimulating capacities of ATP, ADP and AMP.

4. The near ineffectiveness of other nucleotide phosphates as stimuli.

Molecular structure is extremely important if a chemical is to be stimulatory to this receptor. ATP was the optimal stimulus studied by virtue of its having an amino group at carbon-6 of the purine base, and three phosphate radicals in the 5’ position.

The pH effects on the receptor are those to be expected for nervous tissue. The effect of low pH values upon the ATP receptor of Glossina morsitans differs from other dipteran contact chemoreceptors that have been studied.

I am grateful to Dr R. H. Gooding for the tsetse flies, and to Dr Gooding and Dr B. S. Heming for their comments on the manuscript. I thank Mr J. Scott for drawing Fig.1 and Mr George Braybrook for his assistance with the SEM work. This work was supported by the US Army, Medical Research and Development Command.