Aspects of chemoreception in crustacea

Comp. Biochem. Physiol., 1963, VoL 8, pp. 141 to 151. PergamonPress Ltd., London. Printed in Great Britain

ASPECTS OF CHEMORECEPTION IN CRUSTACEA M. S. LAVERACK The Garry Marine Laboratory, St. Andrews (Received 2 October 1962)

Abstract--1. Action potentials have been monitored from the dactylopodite of the walking legs of Carcinus maenas, Portunus puber and Homarus vulgaris in response to the addition of chemical stimuli. 2. Aspartic acid, tryptamine, glutathione, urea, tyrosine, y-aminobutyric acid, glycogen, glycine, cysteine, methionine, serine, leucine and histamine were all without action. Glutamic acid and glutamine had occasional effects. Trimethylamine oxide and betaine were consistently stimulatory. 3. Three types of receptor activity are described in response to stimulation. 4. The significance of these findings with regard to the natural occurrence of these chemicals in marine animals is discussed. INTRODUCTION HODGSON (1958) was the first to apply electrophysiological methods to an analysis of the chemosensory mechanisms of crustacea, using freshwater and terrestrial forms. Action potentials arising from sensory cells were monitored by means of pipette electrodes placed to record directly from the cut end of the antennule of Carabarus and various hair projections from other surfaces of the body. Case & Gwilliam (1961) have also recorded impulses from chemoreceptor organs by means of wire electrodes which they used to pick up fine bundles of nerve fibres originating in the dactylopodite of the walking legs of various species of crabs. Other arthropods which have been investigated by a variety of methods include many insects (Hodgson & Roeder, 1956; Wolbarsht & Dethier, 1958; Hodgson, 1958; Evans & Mellon, 1961; Roys, 1958; Browne & Hodgson, 1962)and Liraulus (Barber, 1961). Stimulation of the sensory mechanism has been shown to be fairly specific in terms of chemical substances. Thus blowflies and butterflies respond to a few sugar solutions and salts (Wolbarsht & Dethier, 1958; Hodgson & Roeder, 1956) and to proteins (WalKs, 1961) and the aquatic arthropods which have been investigated seem to be sensitive towards glycine (Liraulus) (Barber, 1961) and other amino substances such as glutamic acid (Cambarus, Carcinus and Libinia) (Case et al., 1960; Case & Gwilliam, 1961). Specificity with regard to optical isomerism is also indicated within any one group of chemical substances since L-amino adds are apparently more stimulatory than D-isomers. In the present paper it will be shown that other chemical substances, not previously considered, can be stimulatory on crustacean sense organs, and may, by reason of their natural occurrence, be important in stimulating marine crustacea in their normal environment. 141

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MATERIALS AND METHODS Experiments were carried out on the first walking legs of Carcinus maem~s, Portunus puber and Homarus vulgaris. In the latter species the dactylopodite forms a sub-chelate joint, whilst in Carcinus and Portunus the last segment of the lcg is single and entire. Less success attended experiments on the second and third pairs of walking legs. Similar results have been obtained from each species and the following account can be taken as typical of each. The dactylopodite-propodite section of the first walking leg was isolated from the animal. T h e propodite was pinned firmly to a wax-dish surface so that the dactylopodite was held in a trough cut in the wax (Fig. 1). The indifferent electrode was inserted through the cut end of the propodite which was not further dissected.

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FIG. 1. Diagram to show arrangement of limb in the experimental dish and the area from which the exoskeleton is removed before chemical stimulation was applied. Next, the surface of the dactylopodite was carefully removed starting at the articulated membrane of the joint and proceeding distally for a distance of about in. This procedure exposed the flesh within, and careful teasing with fine tungsten needles exposed numerous bundles of nerve fibres. These represent the innervation of the sensory armament of the dactylopodite since no muscular innervation is present at the periphery of the dactylopodite. The nerve bundles were readily separated one from another and could be easily picked up on platinum electrodes. Thinning of bundles was carried out infrequently since the slightest degree of stretching destroyed all signs of activity in the bundle. In a few cases a nerve was successfully thinned to contain only a small number of active fibres, but as the naturally occurring nerves contain few fibres anyway this is not normally necessary. Display of potentials was via a conventional amplifier and oscilloscope set-up, with concurrent tape-recording for later reference. Chemical stimulation was carried out by gently running solutions from a pipette against the end of the dactylopodite as it lay in the trough. Activity in

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mechanoreceptors, which are very numerous on this segment of the leg, was often pronounced in response to tactile stimulation as the solution impinged on the limb. No amount of previous tactile stimulation was found to completely adapt or fatigue the mechanoreceptors. However, the latency shown by the chemoreceptors is considerably longer than that of the mechanoreceptors as will be seen later, and this facilitated the distinction between the types of activity. All solutions were made up in sea water at concentrations between 0.001-0.1 M. Chemicals used were: glutamic acid (unbuffered and buffered to pH 7.2), aspartic acid, glutamine, tryptamine, glutathione, urea, tyrosine, ~,-amino-butyric acid, glycogen, glycine, cysteine, methionine, serine, leucine, histamine acid phosphate, betaine hydrochloride (unbuffered and buffered) and trimethylamine oxide hydrochloride (unbuffered and buffered). All amines were used as the L-isomers. RESULTS Considerable activity could be obtained from the nerves of the dactylopodite in the absence of any known stimulus. As the distal end of the limb was frequently exposed to the drying influence of the atmosphere it is possible that some of the basal activity was due to a humidity or salinity detector, but there is no evidence for this. As mentioned by Case & Gwilliam (1961) under the experimental conditions described above it is very difficult to obtain bundles of nerve fibres that show responses completely divorced from mechanoreceptor activity. Tactile stimuli are detected by many hairs that project from the surface of the dactylopodite. A wide range of spike sizes may be obtained in response to stimulation by such means as small water drops or manual manipulation. From experiments in which such stimuli were presented frequently and rapidly there is little indication that fatigue occurs after many repeated applications. Rapid adaptation to any_ one stimulus takes place but recovery is also swift and series of water drops are readily monitored via the tactile sense organs. A jet of water also fails to fatigue mechanoreceptors. The time course of the responses indicates that while many units fire only whilst actively stimulated, ceasing as soon as the stimulus is removed, other units are more slowly adapting. These show short repetitive volleys of potentials that sometimes persist for up to 2 sec. Photographic records of such unit activity reveal a gradually adapting frequency (Fig. 2). The significance of this type of tactile response will be discussed later. With regard to chemical stimulation several discrepancies with the results of Case & Gwilliam (1961) have been seen. These authors reported that L-glutamic acid was active on sense organs at concentrations of higher than 5-0 x 10-5 M. Other amino acids and amines were also stimulatory but with rather higher threshold. In the early part of the present work the commonly occurring amino acids listed earlier were used as stimulants, together with extracts of ground-up mussels (Mytilus edulis) and cockles (Cardium edule). Over thirty experiments were carried out without any unequivocal response to chemical stimulation being observed. In the majority of cases tactile responses obscured any short-lasting

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chemoresponses that may have occurred and nothing lasting longer than 1 or 2 sec was noted. Glutamic acid, which is not readily soluble in cold sea water, was used often as a saturated solution with no effect. Glutamine (0.1 M) gave two positive results in thirty trials, but glutathione, histamine, cysteine, leucine, aspartic acid, y-amino-butyric acid and glycine had no noticeable effect. Mussel extracts, prepared by grinding the soft parts in sea water and by boiling in sea water, also were without obvious stimulatory effects. Two chemicals, however, have been observed to have profound effects on chemosensory units in the dactylopodite of the three species of crustaceans investigated. These are betaine and trimethylamine oxide. Both these substances were obtained as hydrochlorides, and when dissolved in sea water gave acid solutions. In both cases, therefore, these solutions were also made up in sea water buffered to pH 7.2. This was without effect upon the pattern of response. The effects of these synthetic chemicals were mimicked by extracts prepared from fresh fish tissues. In most cases, twenty-five out of thirty-two, responses were obtained to these substances. In many cases the activity was monitored from the same units in the bundle, in a minority of cases different units were involved. Response patterns of three types were seen. The first type, seen most frequently, is that shown in Fig. 3. The preparation illustrated was the only one in which a single active unit was isolated. More than one fibre was present but the remainder were inactive. The spike potential of the unit was about 400/~V, considerably greater than the diameter of most chemoreceptor nerve fibres would lead one to expect. The unit was totally inactive before stimulation, and the reaction of this unit to chemical stimulation was very prolonged. A latency of some 10-15 sec (Fig. 3) was seen, after which a gradually mounting frequency of impulses was recorded. There was a maximum frequency of approximately 5/see after 35-40 sec treatment with trimethylamine oxide (TMO), and 10/see after 40-50 sec of betaine. These responses were long lasting, up to 90 sec, and diminished rapidly only when washed with sea water. This unit did not respond to any other chemical. Although other single units were not obtained, many preparations contained receptors that responded in this fashion, i.e. a long latent period, a gradual rise in frequency, slow adaptation and quick cessation of activity when the stimulation was removed. The second pattern of activity is illustrated in Fig. 4. These nerves carry impulses from rapidly adapting mechanoreceptors that obscure the initial stages. As far as can be determined for the chemoreceptor there is a latency lasting 5001000 msec before the response occurs. The activity is typically that of an adapting sense organ. The initial frequency is very high, and this is followed by a period of adaptation, complete after 10-30 sec. Unit 2 (Fig. 4) demonstrates this type of activity but Unit 1 has a much slower response, rising slowly to a maximum frequency of 8-12/see in a manner similar to the unit in Fig. 3, differing mainly in the fact that the latency is considerably shorter and the initial response

FIG. 6. Comparison of the effect of (a) TMO, initially and 20 set later, with (b) fresh fish extracts, initially and 20 set later. Time base 20/set. Portums puber.

FIG. 2. Response of an organ slowly adapting in response to mechanical stimulation. A drop of sea water After an initial volley of impulses at high frequency the organ was run against the side of the dactylopodite. showed repetitive discharge which adapted over a period of approximately 14 sec. This was repeatable many times. Curcinus maenas. Time marks = 0.25 sec.

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is indistinguishable from a burst of mechanoreceptor spikes. Both units plotted in Fig. 4 respond to T M O and to betaine, the response to T M O being rather more pronounced. The third type of activity pattern is shown in Fig. 5 (in which a single unit is plotted). This unit was tonically active throughout the period prior to experimentation. The frequency of action potentials was approximately constant at Washed

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stimulatory effect on the dactylopodite receptors, and this is shown in Fig. 6 in which it is compared with the response of the same preparation to TMO.

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FIG. 4. Graphical representation of record to illustrate differences in the first two types of response (see text). DISCUSSION Hodgson (1958) recorded electrical activity in the antennule of Cambarus bartonii when a glass micropipette containing 0.25 M glutamic acid as stimulant was placed over the cut end of the appendage. Repetitive discharges lasting several seconds were observed. Stimulation may have been direct upon nerves within the antennules. Similar activity was found to occur in single hairs of setal-tufts when stimulated by 0.25 M glycine (see Barber, 1961). Responses of another arthropod chemoreceptor system to amino acids has been reported by Barber (1961) for Limulus which shows sensitivity towards solutions of glycine, as well as towards extracts of bivalve tissues. Case et al. (1960) and Case & Gwilliam (1961) examined four species of marine crustaceans and found that glutamic acid was the most effective stimulant for chemoreceptor populations on first walking legs of Carcinus ( Carcinides) maenas, Libinia emarginata, Callinectes sapidus and Pagurus pollicaris. Other amines were less active in stimulating these animals, though many were able to do so to some degree.

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Low concentrations of active substances evidently are sufficient for stimulation. Hodgson (1958) used 0.25 M solutions to demonstrate activity, probably far above threshold and certainly considerably higher than is liable to be obtained directly 60

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FIG. 5. Activity of a tonic unit. At "on" TMO (0"1 M) was added. There was a tactile response followed'by a depression of the firing rate. This was later followed by an increased frequency showing the effect of TMO. Portunus puber. from animal tissues such as constitute the food of Cambarus. Much lower concentrations were used by Barber (1961) who obtained a threshold sensitivity of the gnathobase spine hairs towards glycine of between 0.01 and 0.001 M concentration although records that closely paralleled responses to minced clam extract were only seen in response to 0.5 M glycine. In the case of marine crustaceans threshold sensitivities of as low as 5.0 x 10-5 M for L-glutamic acid were claimed by Case et al. (1961). In the present work no response to commonly occurring amines was recorded save in two isolated cases when glutamine was stimulatory, and one occasion when saturated glutamic acid had an action upon a preparation. A possible explanation for this lack of response compared with the work of other authors may be that within the dactylopodite run many fine strands of nervous tissue, each consisting of a bundle of fibres. As the number of these bundles is large, about fifty, it is conceivable that the bundles carrying glutamic acid-sensitive fibres have never been monitored. This is unlikely since over thirty legs were examined and in some of these at least 75 per cent of the complement of nerves was looked at. In no case did fibres responding unequivocally to solutions of amino acids occur.

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It has already been mentioned that it is extremely difficult to separate chemosensory fibres from the very numerous mechanoreceptor units that are present. Case & Gwilliam (1961) also mention this difficulty, stating that they endeavoured to adapt these units before adding chemical solutions. No amount of prior stimulation has sufficed to adapt such units in the present work although the records of the authors mentioned above show only one spike originating from a mechanoreceptor cell which they use as a stimulus marker to indicate the time at which stimulation was applied. The entire response to chemical application was completed in less than 2 sec, after which adaptation towards amino acids was complete. As will be readily seen from reference to Fig. 2 which has been photographed on a time scale comparable to that of Case & Gwilliam certain tactile units fire repetitively when water drops are run over them. This is repeatable many times, and the course of adaptation is very like that shown by Case & Gwflliam (1961), lasting for approximately the same length of time. It does not, however, explain the fact that these authors obtained different responses when using different dilutions of stimulant, though from the published records for glutamic acid stimulation different units respond to different concentrations, and not the same unit in different ways. Failure to obtain results to amino acid stimulation led to a consideration of other materials that may be of importance chemosensorily. Decapod crustaceans can be noted by observation in an aquarium to be scavengers, feeding on dead animal fragments, and often on live tissues such as bivalves that have been injured and exposed to the sea water. Detection occurs from a distance so it is evident that a highly soluble chemical is carried by water currents and detected by the crustacean. Among substances that are commonly present in certain animal tissues, and which might readily leach out of damaged tissues, are trimethylamine oxide, which is present in considerable amounts in fish, and betaine in invertebrates (though it is absent from mussels, Norris & Benoit, 1945). Trimethylamine oxide (TMO) has been reported as occurring in marine fish, CH:~ TMO

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The threshold concentrations of material required to stimulate chemoreceptors in both cases lie between 0.1-0.001 M, suggestive that the sense organs may be contact receptors, reacting only when stimulatory substances actually touch the limb, and not distant chemoreceptors that respond to very low dilutions of chemicals emanating from a distant source. Certain anomalies are evident when the behaviour of crabs and lobsters is considered since mussels are readily eaten when the legs touch the tissues, a phenomenon noted by Case & Gwilliam (1961) and explained by them on a basis of amino acid sensitivity. There appears to be little orientation from a distance. Fish tissues placed in a tank containing crabs and lobsters almost immediately elicit movement toward the food material. It therefore seems likely that more sensitive distance chemoreceptors are present elsewhere on the body surface. Responses to fish extracts have been monitored from nerves in the dactylopodite, but in contrast to the experiments of Case & Gwilliam (1961) no reaction to mussel extracts was ever seen. Close chemical similarity exists between the active substances mentioned above, T M O and betaine, and quaternary ammonium salts. Betaine and T M O possess the grouping (CH3)3 N in the molecule whilst quaternary ammonium compounds have (CH3)4N in the molecule. These latter materials, such as tetramethyl and tetraethyl-ammonium (TMA and TEA), are known to induce repetitive firing in crustacean motor axons (Burke, Katz & Machne, 1953) after stimulation electrically. If the chemosensory fibres discussed here end freely at the cuticular surface it is possible that repetitive activity could be induced in these axons in an analogous manner. A small number of experiments in which TEA and T M A have been applied to experimental limbs indicate that no response takes place. On the other hand, choline, also used to displace sodium in experiments of motor axons (Burke et al., 1953) but which produces no after-discharge, contains the (CHz)3N moiety and when tested in the present experiments gave rise to trains of potentials. To test the probability that (CH3)3N groupings are of importance several experiments were also carried out utilizing N.N dimethyl glycine (CH3)zN, sarcosine (CH3N) and glycine (NH2.CH2). These all proved negative. It is therefore suggested that crustacean chemosensitivity relies greatly upon the presence of (CH3)3N groups. Sensitivity towards commonly occurring amino acids is not indicated by these observations and further investigation is required in this respect. Three distinct types of receptor pattern were seen in active units. These are summarized in Fig. 7. First, a long latent period followed by a gradually mounting frequency of impulses. Second, an initial volley of impulses that adapts over a period of a few seconds. Last, a post-stimulation inhibition in a tonic unit that is followed by a raised frequency before a return to the original tonic level. None of these responses are paralleled by stimulation with other chemicals or sea water. The identification of the end organs involved in these responses remains a mystery. Hodgson (1958) obtained responses to high concentrations of glutamic acid from organs he termed setal tufts, which are possibly the "Biischelorganen" of Luther (1930). No response to mechanical stimulation was seen. Case &

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Gwilliam (1961) also suggest a chemosensory role for these hair-tufts on account of their distribution and because of the high permeability of these structures. Laverack (1962) has found that sense organs bearing a close resemblance to those previously mentioned are active in monitoring water currents and pressure waves but not chemicalsl This may indicate that apparently morphologically similar

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FIG. 7 .Graph summarizing the three types of activity noted in response to chemical stimulation. organs are distinguishable physiologically, or that sensitivity changes from time to time towards different parameters. T h e long mechanoreceptor hairs situated on the dactylopodite may also carry chemosensory endings and in this case one might expect to obtain a burst of action potentials in response to tactile and chemical stimulation. Recordings of this type have been described in this paper. A third type of ending, not mentioned by Case & Gwilliam (1961) but figured by L u t h e r (1930), is represented by the funnel canals or pores that extend into the cuticle from the surface and which are innervated along the side walls. T h e nerve fibres end at the surface of the cuticle (Barber, 1961). It is conceivable that these canal nerves are in some way active as chemoreceptors. In view of the fact that some responses arise only after considerable latency the time taken for material to diffuse through the pore into the funnel canal may be of importance. REFERENCES BARBER S. B. (1961) Chemoreception and Thermoreception. Physiology of Crustacea, Vol. II (Edited by WATERMANT. H.), pp. 109-131. Academic Press, New York.

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BROWNE L. B. & HODGSON E. S. (1962) Electrophysiological studies of arthropod chemoreception. IV. Latency independence and specificity of labeUar chemoreceptors of the blowfly, Ludlia. 37. Cell. Comp. Physiol. 59, 187-202. BURKE W., KATZ B. & MACHNE X. (1953) T h e effect of quaternary ammonium ions on crustacean nerve fibres. 37. Physiol. 122, 588-598. CASE J. & GWILLIAM G. F. (1961) Amino acid sensitivity of the dactyl chemoreceptors of Carcinides maenas. Biol. Bull., Woods Hole 121, 449-455. CASE J., GWILLIAM G. F. & HANSON F. (1960) Dactyl chemoreceptors of brachyurans. Biol. Bull., Woods Hole 119, 308. EVANS D. R. & MELLON DE F. (1961) Electrophysiological studies of a water receptor associated with the taste sensilla of the blowfly..7. Gen. Physiol. 45, 487-500. HOI~GSON E. S. (1958) Electrophysiological studies of arthropod chemoreception. I I I . Chemoreceptors of terrestrial and fresh water arthropods. Biol. Bull., Woods Hole 115, 114-125. HO~)GSON E. S. & ROEDER K. D. (1956) Electrophysiological studies of arthropod chemoreception. I. General properties of the labellar chemoreceptors of diptera. 37. Cell. Comp. Physiol. 45, 51-75. KRAVITZ B. A., POTTER D. D. & VAN GELDEa N. M. (1962) Gamma-aminobutyric acid and other blocking substances extracted from crab muscle. Nature, Lond. 194, 382-383. KUTSCHER F. & ACKE~ANN D. (1933) Comparative biochemistry of invertebrates. Ann. Rev. Biochem. 2, 355-376. LAVERACKM. S. (1962) Responses of cuticular sense organs of the lobster Homarus vulgarus. II. Hair-fan organs as pressure receptors. Comp. Biochem. Pkvsiol. 6, 137-145. LUTHER W. (1930) Zur Frage der Chemoreception der Brachyuren und Anomuren. Zool. Anz. 94, 147-153. MATHL~S A. P., Ross D. M. & SCHACHTER M. (1960) T h e distribution of 5-hydroxytryptamine, tetramethylammonium, homarine and other substances in sea anemones. J . Physiol. 151, 296-311. NORRIS E. R. & BENOIT G. J., Jr. (1945) Studies on trimethylamine oxide. I. Occurrence of trimethylamine oxide in marine organisms. J. Biol. Chem. 158, 433-438. Rovs C'. C. (1958) A comparison between taste receptors and other nerve tissues of the cockroach on their responses to gustatory stimuli. Biol. Bull., Woods Hole 115, 490-507. WALLIS D. I. (1961) Responses of the labellar hairs of the blowfly, Phormia regina Meigen, to protein. Nature, Lond. 191, 917. WOLBARSHT M. L. & DETHmR V. G. (1958) Electrical activity in the chemoreceptors of the blowfly. I. Responses to chemical and mechanical stimulation. J. Gen. Physiol. 42, 393-412.