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Electrophysiological investigations on tarsal chemoreceptors of the spruce budworm, Choristoneura fumiferana (Lepidoptera)
J. Insect Physiol., 1974, Vol. 20, pp. 1209 to 1218. Pergamon Press. Printed in Great Britain
ELECTROPHYSIOLOGICAL INVESTIGATIONS ON TARSAL CHEMORECEPTORS OF THE SPRUCE BUDWORM, CHORISTONEURA FUMIFERANA (LEPIDOPTERA) B. K. MITCHELL*
and W. D. SEABROOK
Department of Biology, University of New Brunswick, Fredericton, N.B., Canada
(Received 7 December 1973) Abstract-Electrophysiological investigations show that chemosensitive cells in tarsal setae of the spruce budworm are sensitive to at least two salts and possibly water. Two cells are involved in the salt response and one in the possible water response. Each seta also has a cell which responds to mechanical deformation. Stimulation with four sugars did not show that a specific sugar sensitive cell is present in these setae. Results are compared with those from other arthropods. INTRODUCTION
As PART of a widespread search for effective means of biological control of the spruce budworm, studies on the sensory capabilities of the adult animal have recently begun. ALBERT and SEABROOK(1971) have described the site of pheromone reception in this animal and the distribution of sensory receptors on the antenna (ALBERTand SEABROOK,1973). MITCHELL and SEAEJROOK (1972) have studied the morphology of tarsal chemoreceptors in the adult female. The present study is a continuation of the work on the tarsal receptors. It was undertaken to supply information on the basic gustatory capabilities of the adult female. The technique necessary to record electrophysiological events occurring in insect contact chemoreceptors was first introduced by HODGSONet al. (1955), on the labellar setae of the blowfly, Phormiu regina (Meig.). A similar technique was reported for the tarsal contact chemoreceptors of the butterfly P’a,essu indica (MORITA et al, 1957). Since then, many investigations on the electrophysiology of insect contact chemoreceptors have been undertaken, but most of these studies have been on the labellar and tarsal setae of various Diptera (reviews by DETHIER, 1963 ; HODGSON,1965). Detailed electrophysiological investigations on contact chemoreceptors of adult Lepidoptera are confined to five papers on V. indica (MORITAet al., 1957; MORITA and TAKEDA,1957; MORITAand TAKEDA,1959; TAKEDA,1961; KUWABARA, 1963), * Present address: Alberta, Canada.
Department
of Entomology, 1209
University
of Alberta,
Edmonton,
1210
B. K. MITCHELLANDW. D. SEABR~~K
and a recent paper on the large white butterfly Pieris brassicae (MA and SCHOONHAVEN,1973). HODGSON (1958), in a survey of receptors in various arthropods, recorded from the tarsal receptors of several butterflies. However, he was unable to obtain records from three species of moths. MATERIALS AND METHODS The technique used to record from the tarsal sensilla was similar to that described by HODGSONand ROEDER(1956). The signal was passed via the solution in a stimulating-recording pipette to a silver-silver chloride electrode inserted part way down the large end of the pipette. This electrode was connected by a coaxial cable to a negative capacitance preamplifier. A 10 pF capacitor was inserted between the recording electrode and the input of the preamplifier to reduce the amplifier block caused by the potential difference existing between the recording and the indifferent electrodes (HODGSONand ROEDER,1956; WOLBARSHT,1958). From the preamplifier, the signal was amplified, viewed, and recorded in the usual fashion. To prepare an animal for recording, its abdomen, wings, and head were removed. The thoracic ganglia were then destroyed with forceps; in most cases this immobilized the legs and partially extended them. The reference electrode was inserted into the thorax. This method was found superior to placing a single leg on the reference electrode (MORITA et al., 1957), as the life of the preparation was considerably lengthened. A total of 136 experimental animals were used, all adult females. Most of the electrophysiological studies were carried out using a pair of setae on the ventral part of the fifth tarsal segment (Fig. 1). These setae were most prominent when the animal was set up for recording, and were among the longest chemosensory setae on the tarsus. Their length, about 0.7 mm, is not apparent in the micrograph in Fig. 1 due to the angle of observation. RESULTS AND DISCUSSION Stimulation with low salt concentrations The recording method used in this investigation precluded the use of pure water as a test stimulus. It was possible, however, to obtain recordings using a salt concentration of 5 mM in the stimulating-recording pipette. This concentration was used to investigate the possibility of there being a cell in the tarsal chemoreceptors that responds to water. Adult animals that had no access to free water frequently gave electrophysiological responses to 5 mM NaCl. The results were often complex, and the responses from specific cells were difficult to distinguish. However, in several records two to three spikes were distinguishable. One of these predominated: its frequency was highest immediately after stimulation and rapidly declined. This cell was possibly responding to water, and was was designated w (Fig. 2a and b). A second cell, which may be one of the salt sensitive cells to be described later, appears in these traces.
1211
FIG. 1. Scanning electron micrograph of the distal part of the fifth tarsal segment and pretarsus of C.fumiferana. Ventral view. The pair of setae labelled c were used extensively in electrophysiological studies.
INVESTIGATIONS
ON TARSAL CHBMORBCBPTORS OF THE SPRUCE BUDWORM
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A water-sensitive cell was reported in the labellar contact chemoreceptors of the blowfly by EVANSand MELLON (1962a, b). In this animal the well-known cation response occasionally becomes evident during stimulation with as low a concentration as 50 mM NaCl. However, at this salt concentration the water cell was predominant in the response (DETHIER and HANSON, 1968). In the tarsal chemoreceptors of V. in&a, two cells responded to stimulation with 15 mM NaCl (MORITAand TAKEDA,1959). One of these cells could have been a water receptor. The use of the sidewall recording technique first introduced by MORITA and YAMASHITA(1959) could perhaps decide the question of a water sensitive cell in these setae. The tedious nature of this technique did not justify its use in this preliminary investigation. Stimulation with higher salt concentrations The response to higher salt concentrations is complex. Experiments were conducted in which three concentrations of NaCl were applied to the same seta. As in all experiments where a single seta was used for successive applications of stimulus, a 3 to 4 min period was given between stimuli. This length of time was sufficient to yield reproducible results, since it allowed time for the cells to disadapt completely. With 100 mM NaCl as the stimulus, a cell (m) begins firing soon after stimulation and adapts to a low level within 1 set (Fig. 2~). The response from a second cell (1) can also be seen, but its frequency is not as great as that of the m cell, nor is adaptation evident. The spikes of the I cell are always larger in amplitude than those of the m cell. With 500 mM NaCl as a stimulus, the spikes from the m cell appear again but adapt sooner (Fig. Zd). The I cell is also active. One to two set after the onset of stimulation, a response which is not common in insect salt receptors occurs. The m cell rapidly increases its frequency in a volley that can last for 10 to 12 sec. The unadapted cell fires at a peak frequency of about 150 spikes per set and falls to one-half this frequency in about 4 sec. Its amplitude also decreases as it adapts. A similar effect occurs when 1.0 M NaCl is applied to the tip of the seta, with an even greater reduction in the number of spikes preceding the large increase in frequency of the m cell (Fig. 2e). Successive stimulations of the m cell in the same hair with 500 mM KCl, show that its peak frequency decreases if the stimuli follow one another closely. When the stimuli are applied in rapid succession (4-13 set apart) the initial peak frequency of the m cell is never reached on succeeding stimulations. However, when a 4 min disadaptation period is given between stimuli, 95 per cent of the initial peak frequency can be recovered, indicating the unusual response is not a result of injury from the high salt concentration. The m and I cells are both sensitive to KCl. The I cell occasionally fires at a moderate frequency (lO/sec) when the stimulus is 500 mM KCl, and always fires at a low frequency (1-2/set) when the stimulus is 500 mM NaCl. This may indicate that the Ecell is slightly more sensitive to KC1 than to NaCl.
1214
dj++++++.;
e
#;
‘;..
m
; 1
1 m
t :
;
..
‘. .I.
FIG. 2. (a-b) Responses of tarsal chemoreceptors on different insects to 5 mM NaCl. In this and all recordings, the artifact near the beginning of each trace indicates application of the stimulus. w, Possible spikes of the water sensitive cell. (c-e) Responses of a single tarsal chemoreceptor to successive stimulations with increasing concentration of NaCl. Stimulus concentrations: c, 100 mM ; d, 500 mM ; e, 1 a0 M. 712,Spikes of the m cell; 1, spikes of the I cell ; me, spikes of the mechanoreceptive cell. The arrows in c and d indicate the point at which the seta was bent.
When the stimulus is 100 to 250 mM KCl, the spikes of the m cell are prominent. This response adapts to a low level. The frequency of both the m and I cells is greater at 500 mM KC1 and greater still at 750 mM KCl. At 1-O M KC1 the m cell fires for 2 to 3 set, then its frequency increases rapidly as with NaCl stimulation (Fig. 3a-e). The salt receptors in different setae can have different sensitivities. The delayed volley of activity of the m cell can be detected in some setae at as low a stimulus concentration as 500 mM KCl, though it is seldom seen below this concentration. It usually appears at a concentration of at least 1-O M, whether the stimulating salt is NaCl or KCl. The delayed response of the m cell is unusual. However, there is evidence in the literature that arthropod chemoreceptors can react in this manner. The response of the sense organs of the dactylopodide of the first walking leg in some crustacea to trimethylamine oxide (TMO) shows a gradual increase in frequency (LAVERACK, 1963). In an attempt to define the sensitivity of the ‘fifth cell’ in the blowfly, DETHIER and HANSON (1968) applied solutions of fatty acids of various chain lengths to the receptor. In some cases, after prolonged stimulation (2-3 min), the salt cell fired in a delayed volley much like that reported here. With long chain fatty acid salts (C&i,) an exposure of 2 set was sometimes sufficient to induce a volley from the salt cell. Some salts stimulated two cells to volley. The response of more than one cell per receptor seta to simulation by salt is in agreement with the results of MORITA and TAKEDA (1959). They described a cell in V. in&u that responded to concentrations of NaCl below 250 mM, and which
INVl?.STIGATIONS ON TARSAL CHEMORRCEPTORS OF THE SPRUCE BUDWORM
C
1215
.
e
FIG. 3. (a-e) Responses of a single tarsal chemoreceptor to successive stimulations with increasing concentrations of KCI. Stimulus concentrations: a, 100 mM; b, 250 mM; c, 500 mM; d, 750 mM; e, I.0 M. m, Spikes of the m cell; I, spikes of the 1 cell; me, spikes of the mechanoreceptive cell. The arrow in c indicates the point at which the seta was bent.
predominated at a concentration of 15 mM. This, as explained above, could have been a water-sensitive cell. A second cell responded over a range of NaCl concentrations from 15 mM to 1-OM, and predominated at 60 mM. At 250 mM NaCl, a third cell was observed. In P. brassicae two cells were active in many setae when stimulated by 0.1 M NaCl (MA and SCHOON~O~, 1973). The situation in the blowfly, which for a time seemed relatively simple, is now complicated by the presence of a ‘fifth’ cell which responds to high salt concentrations (ST~~~KOW, 1963; DETHIER and HANSON, 1968; MCCUTCHAN, 1969). Although this cell responds to salt, there is some doubt that this is its adequate stimulus (DETHIERand HANSON,1968). In C. f~~z~~~~~, the evidence seems to indicate that the m cell has salt as its adequate stimulus and the I cel1, while perhaps mildly stimulated by salt, has some other chemical, perhaps a plant constituent, as its most adequate stimulus. In P. brassr’cae, for instance, a cell in some B-type tarsal setae responds to some mustard oil glucosides which are oviposition stimulants for the female butterfly (MA and SCHOONHOVEN, 1973). It we assume that no new mechanism of salt reception is operating at the membrane level, the increased frequency of the m cell 2 to 3 set after the stimulus application could be explained if this were the time required for a sufficient number of ions to come in contact with the receptor surface. It could represent the
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B. K. MITCHELLANDW. D. SEABROOK
time required for ions to diffuse through the pore in the seta tip, and the solution surrounding the dendrite. The answers to these questions, and the identification of the undefined spikes in the records, can only come from further research on these receptors. Comparison of NaCl, KC& and CaCZ, as stimulants The responses to relatively high concentrations of three salts, NaCl, KCl, and CaCI, were compared. Fig. 4(a-f) shows the results of an experiment with two concentrations of each of these salts. The responses to NaCl and KC1 are similar to those described above. CaCl, stimulates much less activity at the concentrations tested (Fig. 4e-f). In one experiment (not shown) 500 mM CaCl, did stimulate the m cell to a much delayed volley at a much lower frequency than similar concentrations of KC1 or NaCl. CaCl, has been described as less stimulating than NaCl (GOTHILF et al., 1971), non-stimulator-y (EVANSand MELLON, 1962b), or inhibitory (WOLBARSHT,1965) in other insects. I
b &u&n&~+
I’,!
! m! s; !! ! I!‘! 4;
!!
‘! ! !!I,! I ‘:I
I! ! :’
!!
I II
I
0.2,*c e
:
FIG. 4. (a-f) Responses of a single tarsal chemoreceptor to successive stimulations with two concentrations of three salts. Stimulus concentrations: a, 100 mM NaCl; b, 500 mM CaCl,; c, 100 mM KCI; d, 500 mM KCl; e, 100 mM CaCl,; f, 500 mM CaCl *. m, Spikes of the m cell; I, spikes of the 1 cell.
Stimulation with salt and sugar mixtures Experiments were carried out to determine if a cell which responds specifically Fructose (10-500 mM), sucrose (500 mM), to sugar could be demonstrated. ratEnose (100 mM), and dextrose (500 mM) all in 100 mM KC1 were tested because they are the more abundant sugars found in balsam fir needles (LITTLE, 1970), a principal host plant of C. fum+rana. The only truly reproducible result of the sugar salt stimulation was the reduction in spike amplitude, and in some cases, spike frequency, when compared with
INVESTIGATIONS ON TARSAL
CHEMORECEPTORS OF
THE SPRUCEBUDWOBM
1217
pure salt stimulation. The amplitude reduction must be a direct effect of the sugar on the conductance of the stimulating solution. The salt response was normal following sugar-salt stimulation. Depression of the salt response by sugar has been reported in the blowfly (HODGSON,1958). MA and SCHOONHOVEN(1973), could find no electrophysiological evidence for a cell responding to sugars in the B-type tarsal sensilli of P. brassicae. Stimulation by mechanical deformation The chemosensory setae on the tarsi of the spruce budworm almost invariably respond to mechanical deformation, and a distinct mechanosensory cell can be seen in many of the records (Figs. 2c, 3~). This cell is designated ‘me’. Most of these mechanoreceptors are of the tonic type, although a few which responded only to motion were observed. The me cell can also fire when the sita is not being disturbed by the electrode, but does so at a reduced frequency. It is therefore important to establish the identity of this cell (usually smaller in amplitude than either the m or the I cells) to avoid confusion. CONCLUSIONS From the results, it is concluded that in the tarsal contact chemoreceptors of the spruce budworm there is at least one type of cell which responds either to water, or at least to extremely low salt concentration. There is also a response to salt that increases with concentration of the stimulus. This response may be mediated by two cells, one of which may be more sensitive to KC1 than to NaCl. A comparison of three salts at similar molarities indicates that NaCl and KC1 are highly stimulatory, while CaCl, is much less stimulatory. The presence of a cell specifically sensitive to sugars in the tarsal chemoreceptors of the spruce budworm is not established. It is apparent that the presence of sugar reduces the response to salt. In the adult budworm, the only observed feeding that does occur seems to involve the occasional uptake of moisture. Thus we would not expect the strong responses to sugars found in more active feeding Lepidoptera. The possibility that the tarsal chemoreceptors of C. fumiferana may respond to compounds important in behavioural activities such as oviposition cannot be ruled out. The sensilla which house the chemosensory cells also contain a cell which responds to mechanical deformation. Acknowledgements-This work was supported by a grant from the Canadian Forestry Service, Environment Canada, to W. D. S. REFERENCES ALBERTP. J. and SBABROOK W. D. (1971) The antennae as the site of pheromone receptors in the eastern spruce budworm, Choristonaru fumiferunu (Lepidoptera: Tortricidae). Can. Enr. 102, 1610-1612. ALBERTP. J. and SBABROOK W. D. (1973) Morphology and histology of the antenna of the male eastern spruce budworm, Choristoneuru fumiferunu (Clem.) (Lepidoptera: Tortricidae). Cun.J. Zool. 51, 443-448. DETHIERV. G. (1963) The Physiology of Insect Senses. Wiley, New York. 40
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DETHIERV. G. and HANSON F. E. (1968) Electrophysiological responses of the chemoreceptors of the blowfly to sodium salts of fatty acids. Proc. nat. Acad. Sci. U.S.A. 60, 1296-1303. EVANSD. R. and MELLON DE F. (1962a) Electrophysiological studies of a water receptor associated with the taste sensilla of the blowfly. J. gen. Physiol. 45, 487-500. EVANSD. R. and MELLON DE F. (1962b) Stimulation of a primary taste receptor by salts. J. gen. Physiol. 45, 651-661. GOTHILF S., GALUNR., and BAR-&N M. (1971) Taste reception in the Mediterranean fruit fly: electrophysiological and behavioural studies. r. Insect Physiol. 17, 1371-1384. HODGSONE. S. (1958) Electrophysiological studies of arthropod chemoreception-III. Chemoreceptors of terrestrial and fresh water arthropods. Biol. Bull., Woods Hole 115, 114-125. HODG~ONE. S. (1965) The chemical senses and changing viewpoints in sensory physiology. In Viewpoints in Biology (Ed. by CARTHYJ. D. and DUDDINGTON C. L.), Butterworths, London. HODGSONE. S., LETTVINJ. Y., and ROEDERK. D. (1955) Physiology of a primary chemoreceptor unit. Science, Wash. 122, 417-418. HODGSONE. S. and ROEDERK. D. (1956) Electrophysiological studies of arthropod chemoreception-I. General properties of the labellar chemoreceptors of Diptera. J. cell. con@. Physiol. 48,51-76. KUWABARA M. (1963) Tarsal chemoreception in the butterfly, Vanessa indica. Proc. 16th int. Congr. Zool. 3, 96-97. LAVERACK M. S. (1963) Aspects of chemoreception in Crustacea. Comp. Biochem. Physiol. 8, 141-151. LITTLE C. H. A. (1970) Seasonal changes in carbohydrate and moisture content in needles of balsam fir (Abies bahamea). Can.J. Bot. 48,2021-2028. MCCUTCHANM. C. (1969) Behavioural and electrophysiological responses of the blowfly, Phormia regina Meigen, to acids. Z. vergl. Physiol. 65,131-152. MA, WEI-CHUN and SCHOONHOVEN L. M. (1973) Tarsal contact chemosensory hairs of the large white butterfly Pieris brassicae and their possible role in oviposition behaviour. Entomologia exp. appl. 16, 343-357. MITCHELL B. K. and SBABROOK W. D. (1972) Morphological investigations on the tarsal receptors of the spruce budworm, Choristoneura fumrferana (Lepidoptera: Tortricidae). Can. Ent. 104, 1931-1935. MORITAH., DOIRAS., TAKEDAK., and KVWABARA M. (1957) Electrical response of contact chemoreceptor on tarsus of the butterfly, Vanessa indica. Mem. Fat. Sci. Kyusku Univ., Ser. E (Biol.). 2, 119-139. MORITAH. and TAKEDAK. (1957) The electrical resistance of the tarsal chemosensory hair of the butterfly Vanessa indica. _Y.Fat. Sci. Hokkaido Univ. Ser. VI, Zool. 13, 465-469. MORITAH. and TAK~DAK. (1959) Initiation of spike potentials in contact chemosensory hairs of insects-II. The effect of electric current on tarsal chemosensory hairs of Vanessa. J. cell. camp. Physiol. 54, 177-187. MORITAH. and YAMA~HITA S. (1959) G eneratior potental of insect chemoreceptor. Science, Wash. 30, 922. STURCKOW B. (1963) Electrophysiological studies of a single taste hair of the fly during stimulation by a flowing system. Proc. 16th int. Congr. Zool. 3, 102-104. TAKEDAK. (1961) The nature of impulses of single tarsal chemoreceptors in the butterfly, Vanessa indica. J. cell. camp. Physiol. 58, 233-244. WOLBARSHT M. L. (1958) Electrical activity in the chemoreceptors of the blowfly-II. Responses to electrical stimulation. r. gen. Physiol. 42, 413-428. WOLBARSHT M. L. (1965) Receptor sites in chemoreceptors. CoZd Spr. Harb. Symp. quant. Biol. 30, 281-288.
ELECTROPHYSIOLOGICAL INVESTIGATIONS ON TARSAL CHEMORECEPTORS OF THE SPRUCE BUDWORM, CHORISTONEURA FUMIFERANA (LEPIDOPTERA) B. K. MITCHELL*
and W. D. SEABROOK
Department of Biology, University of New Brunswick, Fredericton, N.B., Canada
(Received 7 December 1973) Abstract-Electrophysiological investigations show that chemosensitive cells in tarsal setae of the spruce budworm are sensitive to at least two salts and possibly water. Two cells are involved in the salt response and one in the possible water response. Each seta also has a cell which responds to mechanical deformation. Stimulation with four sugars did not show that a specific sugar sensitive cell is present in these setae. Results are compared with those from other arthropods. INTRODUCTION
As PART of a widespread search for effective means of biological control of the spruce budworm, studies on the sensory capabilities of the adult animal have recently begun. ALBERT and SEABROOK(1971) have described the site of pheromone reception in this animal and the distribution of sensory receptors on the antenna (ALBERTand SEABROOK,1973). MITCHELL and SEAEJROOK (1972) have studied the morphology of tarsal chemoreceptors in the adult female. The present study is a continuation of the work on the tarsal receptors. It was undertaken to supply information on the basic gustatory capabilities of the adult female. The technique necessary to record electrophysiological events occurring in insect contact chemoreceptors was first introduced by HODGSONet al. (1955), on the labellar setae of the blowfly, Phormiu regina (Meig.). A similar technique was reported for the tarsal contact chemoreceptors of the butterfly P’a,essu indica (MORITA et al, 1957). Since then, many investigations on the electrophysiology of insect contact chemoreceptors have been undertaken, but most of these studies have been on the labellar and tarsal setae of various Diptera (reviews by DETHIER, 1963 ; HODGSON,1965). Detailed electrophysiological investigations on contact chemoreceptors of adult Lepidoptera are confined to five papers on V. indica (MORITAet al., 1957; MORITA and TAKEDA,1957; MORITAand TAKEDA,1959; TAKEDA,1961; KUWABARA, 1963), * Present address: Alberta, Canada.
Department
of Entomology, 1209
University
of Alberta,
Edmonton,
1210
B. K. MITCHELLANDW. D. SEABR~~K
and a recent paper on the large white butterfly Pieris brassicae (MA and SCHOONHAVEN,1973). HODGSON (1958), in a survey of receptors in various arthropods, recorded from the tarsal receptors of several butterflies. However, he was unable to obtain records from three species of moths. MATERIALS AND METHODS The technique used to record from the tarsal sensilla was similar to that described by HODGSONand ROEDER(1956). The signal was passed via the solution in a stimulating-recording pipette to a silver-silver chloride electrode inserted part way down the large end of the pipette. This electrode was connected by a coaxial cable to a negative capacitance preamplifier. A 10 pF capacitor was inserted between the recording electrode and the input of the preamplifier to reduce the amplifier block caused by the potential difference existing between the recording and the indifferent electrodes (HODGSONand ROEDER,1956; WOLBARSHT,1958). From the preamplifier, the signal was amplified, viewed, and recorded in the usual fashion. To prepare an animal for recording, its abdomen, wings, and head were removed. The thoracic ganglia were then destroyed with forceps; in most cases this immobilized the legs and partially extended them. The reference electrode was inserted into the thorax. This method was found superior to placing a single leg on the reference electrode (MORITA et al., 1957), as the life of the preparation was considerably lengthened. A total of 136 experimental animals were used, all adult females. Most of the electrophysiological studies were carried out using a pair of setae on the ventral part of the fifth tarsal segment (Fig. 1). These setae were most prominent when the animal was set up for recording, and were among the longest chemosensory setae on the tarsus. Their length, about 0.7 mm, is not apparent in the micrograph in Fig. 1 due to the angle of observation. RESULTS AND DISCUSSION Stimulation with low salt concentrations The recording method used in this investigation precluded the use of pure water as a test stimulus. It was possible, however, to obtain recordings using a salt concentration of 5 mM in the stimulating-recording pipette. This concentration was used to investigate the possibility of there being a cell in the tarsal chemoreceptors that responds to water. Adult animals that had no access to free water frequently gave electrophysiological responses to 5 mM NaCl. The results were often complex, and the responses from specific cells were difficult to distinguish. However, in several records two to three spikes were distinguishable. One of these predominated: its frequency was highest immediately after stimulation and rapidly declined. This cell was possibly responding to water, and was was designated w (Fig. 2a and b). A second cell, which may be one of the salt sensitive cells to be described later, appears in these traces.
1211
FIG. 1. Scanning electron micrograph of the distal part of the fifth tarsal segment and pretarsus of C.fumiferana. Ventral view. The pair of setae labelled c were used extensively in electrophysiological studies.
INVESTIGATIONS
ON TARSAL CHBMORBCBPTORS OF THE SPRUCE BUDWORM
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A water-sensitive cell was reported in the labellar contact chemoreceptors of the blowfly by EVANSand MELLON (1962a, b). In this animal the well-known cation response occasionally becomes evident during stimulation with as low a concentration as 50 mM NaCl. However, at this salt concentration the water cell was predominant in the response (DETHIER and HANSON, 1968). In the tarsal chemoreceptors of V. in&a, two cells responded to stimulation with 15 mM NaCl (MORITAand TAKEDA,1959). One of these cells could have been a water receptor. The use of the sidewall recording technique first introduced by MORITA and YAMASHITA(1959) could perhaps decide the question of a water sensitive cell in these setae. The tedious nature of this technique did not justify its use in this preliminary investigation. Stimulation with higher salt concentrations The response to higher salt concentrations is complex. Experiments were conducted in which three concentrations of NaCl were applied to the same seta. As in all experiments where a single seta was used for successive applications of stimulus, a 3 to 4 min period was given between stimuli. This length of time was sufficient to yield reproducible results, since it allowed time for the cells to disadapt completely. With 100 mM NaCl as the stimulus, a cell (m) begins firing soon after stimulation and adapts to a low level within 1 set (Fig. 2~). The response from a second cell (1) can also be seen, but its frequency is not as great as that of the m cell, nor is adaptation evident. The spikes of the I cell are always larger in amplitude than those of the m cell. With 500 mM NaCl as a stimulus, the spikes from the m cell appear again but adapt sooner (Fig. Zd). The I cell is also active. One to two set after the onset of stimulation, a response which is not common in insect salt receptors occurs. The m cell rapidly increases its frequency in a volley that can last for 10 to 12 sec. The unadapted cell fires at a peak frequency of about 150 spikes per set and falls to one-half this frequency in about 4 sec. Its amplitude also decreases as it adapts. A similar effect occurs when 1.0 M NaCl is applied to the tip of the seta, with an even greater reduction in the number of spikes preceding the large increase in frequency of the m cell (Fig. 2e). Successive stimulations of the m cell in the same hair with 500 mM KCl, show that its peak frequency decreases if the stimuli follow one another closely. When the stimuli are applied in rapid succession (4-13 set apart) the initial peak frequency of the m cell is never reached on succeeding stimulations. However, when a 4 min disadaptation period is given between stimuli, 95 per cent of the initial peak frequency can be recovered, indicating the unusual response is not a result of injury from the high salt concentration. The m and I cells are both sensitive to KCl. The I cell occasionally fires at a moderate frequency (lO/sec) when the stimulus is 500 mM KCl, and always fires at a low frequency (1-2/set) when the stimulus is 500 mM NaCl. This may indicate that the Ecell is slightly more sensitive to KC1 than to NaCl.
1214
dj++++++.;
e
#;
‘;..
m
; 1
1 m
t :
;
..
‘. .I.
FIG. 2. (a-b) Responses of tarsal chemoreceptors on different insects to 5 mM NaCl. In this and all recordings, the artifact near the beginning of each trace indicates application of the stimulus. w, Possible spikes of the water sensitive cell. (c-e) Responses of a single tarsal chemoreceptor to successive stimulations with increasing concentration of NaCl. Stimulus concentrations: c, 100 mM ; d, 500 mM ; e, 1 a0 M. 712,Spikes of the m cell; 1, spikes of the I cell ; me, spikes of the mechanoreceptive cell. The arrows in c and d indicate the point at which the seta was bent.
When the stimulus is 100 to 250 mM KCl, the spikes of the m cell are prominent. This response adapts to a low level. The frequency of both the m and I cells is greater at 500 mM KC1 and greater still at 750 mM KCl. At 1-O M KC1 the m cell fires for 2 to 3 set, then its frequency increases rapidly as with NaCl stimulation (Fig. 3a-e). The salt receptors in different setae can have different sensitivities. The delayed volley of activity of the m cell can be detected in some setae at as low a stimulus concentration as 500 mM KCl, though it is seldom seen below this concentration. It usually appears at a concentration of at least 1-O M, whether the stimulating salt is NaCl or KCl. The delayed response of the m cell is unusual. However, there is evidence in the literature that arthropod chemoreceptors can react in this manner. The response of the sense organs of the dactylopodide of the first walking leg in some crustacea to trimethylamine oxide (TMO) shows a gradual increase in frequency (LAVERACK, 1963). In an attempt to define the sensitivity of the ‘fifth cell’ in the blowfly, DETHIER and HANSON (1968) applied solutions of fatty acids of various chain lengths to the receptor. In some cases, after prolonged stimulation (2-3 min), the salt cell fired in a delayed volley much like that reported here. With long chain fatty acid salts (C&i,) an exposure of 2 set was sometimes sufficient to induce a volley from the salt cell. Some salts stimulated two cells to volley. The response of more than one cell per receptor seta to simulation by salt is in agreement with the results of MORITA and TAKEDA (1959). They described a cell in V. in&u that responded to concentrations of NaCl below 250 mM, and which
INVl?.STIGATIONS ON TARSAL CHEMORRCEPTORS OF THE SPRUCE BUDWORM
C
1215
.
e
FIG. 3. (a-e) Responses of a single tarsal chemoreceptor to successive stimulations with increasing concentrations of KCI. Stimulus concentrations: a, 100 mM; b, 250 mM; c, 500 mM; d, 750 mM; e, I.0 M. m, Spikes of the m cell; I, spikes of the 1 cell; me, spikes of the mechanoreceptive cell. The arrow in c indicates the point at which the seta was bent.
predominated at a concentration of 15 mM. This, as explained above, could have been a water-sensitive cell. A second cell responded over a range of NaCl concentrations from 15 mM to 1-OM, and predominated at 60 mM. At 250 mM NaCl, a third cell was observed. In P. brassicae two cells were active in many setae when stimulated by 0.1 M NaCl (MA and SCHOON~O~, 1973). The situation in the blowfly, which for a time seemed relatively simple, is now complicated by the presence of a ‘fifth’ cell which responds to high salt concentrations (ST~~~KOW, 1963; DETHIER and HANSON, 1968; MCCUTCHAN, 1969). Although this cell responds to salt, there is some doubt that this is its adequate stimulus (DETHIERand HANSON,1968). In C. f~~z~~~~~, the evidence seems to indicate that the m cell has salt as its adequate stimulus and the I cel1, while perhaps mildly stimulated by salt, has some other chemical, perhaps a plant constituent, as its most adequate stimulus. In P. brassr’cae, for instance, a cell in some B-type tarsal setae responds to some mustard oil glucosides which are oviposition stimulants for the female butterfly (MA and SCHOONHOVEN, 1973). It we assume that no new mechanism of salt reception is operating at the membrane level, the increased frequency of the m cell 2 to 3 set after the stimulus application could be explained if this were the time required for a sufficient number of ions to come in contact with the receptor surface. It could represent the
1216
B. K. MITCHELLANDW. D. SEABROOK
time required for ions to diffuse through the pore in the seta tip, and the solution surrounding the dendrite. The answers to these questions, and the identification of the undefined spikes in the records, can only come from further research on these receptors. Comparison of NaCl, KC& and CaCZ, as stimulants The responses to relatively high concentrations of three salts, NaCl, KCl, and CaCI, were compared. Fig. 4(a-f) shows the results of an experiment with two concentrations of each of these salts. The responses to NaCl and KC1 are similar to those described above. CaCl, stimulates much less activity at the concentrations tested (Fig. 4e-f). In one experiment (not shown) 500 mM CaCl, did stimulate the m cell to a much delayed volley at a much lower frequency than similar concentrations of KC1 or NaCl. CaCl, has been described as less stimulating than NaCl (GOTHILF et al., 1971), non-stimulator-y (EVANSand MELLON, 1962b), or inhibitory (WOLBARSHT,1965) in other insects. I
b &u&n&~+
I’,!
! m! s; !! ! I!‘! 4;
!!
‘! ! !!I,! I ‘:I
I! ! :’
!!
I II
I
0.2,*c e
:
FIG. 4. (a-f) Responses of a single tarsal chemoreceptor to successive stimulations with two concentrations of three salts. Stimulus concentrations: a, 100 mM NaCl; b, 500 mM CaCl,; c, 100 mM KCI; d, 500 mM KCl; e, 100 mM CaCl,; f, 500 mM CaCl *. m, Spikes of the m cell; I, spikes of the 1 cell.
Stimulation with salt and sugar mixtures Experiments were carried out to determine if a cell which responds specifically Fructose (10-500 mM), sucrose (500 mM), to sugar could be demonstrated. ratEnose (100 mM), and dextrose (500 mM) all in 100 mM KC1 were tested because they are the more abundant sugars found in balsam fir needles (LITTLE, 1970), a principal host plant of C. fum+rana. The only truly reproducible result of the sugar salt stimulation was the reduction in spike amplitude, and in some cases, spike frequency, when compared with
INVESTIGATIONS ON TARSAL
CHEMORECEPTORS OF
THE SPRUCEBUDWOBM
1217
pure salt stimulation. The amplitude reduction must be a direct effect of the sugar on the conductance of the stimulating solution. The salt response was normal following sugar-salt stimulation. Depression of the salt response by sugar has been reported in the blowfly (HODGSON,1958). MA and SCHOONHOVEN(1973), could find no electrophysiological evidence for a cell responding to sugars in the B-type tarsal sensilli of P. brassicae. Stimulation by mechanical deformation The chemosensory setae on the tarsi of the spruce budworm almost invariably respond to mechanical deformation, and a distinct mechanosensory cell can be seen in many of the records (Figs. 2c, 3~). This cell is designated ‘me’. Most of these mechanoreceptors are of the tonic type, although a few which responded only to motion were observed. The me cell can also fire when the sita is not being disturbed by the electrode, but does so at a reduced frequency. It is therefore important to establish the identity of this cell (usually smaller in amplitude than either the m or the I cells) to avoid confusion. CONCLUSIONS From the results, it is concluded that in the tarsal contact chemoreceptors of the spruce budworm there is at least one type of cell which responds either to water, or at least to extremely low salt concentration. There is also a response to salt that increases with concentration of the stimulus. This response may be mediated by two cells, one of which may be more sensitive to KC1 than to NaCl. A comparison of three salts at similar molarities indicates that NaCl and KC1 are highly stimulatory, while CaCl, is much less stimulatory. The presence of a cell specifically sensitive to sugars in the tarsal chemoreceptors of the spruce budworm is not established. It is apparent that the presence of sugar reduces the response to salt. In the adult budworm, the only observed feeding that does occur seems to involve the occasional uptake of moisture. Thus we would not expect the strong responses to sugars found in more active feeding Lepidoptera. The possibility that the tarsal chemoreceptors of C. fumiferana may respond to compounds important in behavioural activities such as oviposition cannot be ruled out. The sensilla which house the chemosensory cells also contain a cell which responds to mechanical deformation. Acknowledgements-This work was supported by a grant from the Canadian Forestry Service, Environment Canada, to W. D. S. REFERENCES ALBERTP. J. and SBABROOK W. D. (1971) The antennae as the site of pheromone receptors in the eastern spruce budworm, Choristonaru fumiferunu (Lepidoptera: Tortricidae). Can. Enr. 102, 1610-1612. ALBERTP. J. and SBABROOK W. D. (1973) Morphology and histology of the antenna of the male eastern spruce budworm, Choristoneuru fumiferunu (Clem.) (Lepidoptera: Tortricidae). Cun.J. Zool. 51, 443-448. DETHIERV. G. (1963) The Physiology of Insect Senses. Wiley, New York. 40
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DETHIERV. G. and HANSON F. E. (1968) Electrophysiological responses of the chemoreceptors of the blowfly to sodium salts of fatty acids. Proc. nat. Acad. Sci. U.S.A. 60, 1296-1303. EVANSD. R. and MELLON DE F. (1962a) Electrophysiological studies of a water receptor associated with the taste sensilla of the blowfly. J. gen. Physiol. 45, 487-500. EVANSD. R. and MELLON DE F. (1962b) Stimulation of a primary taste receptor by salts. J. gen. Physiol. 45, 651-661. GOTHILF S., GALUNR., and BAR-&N M. (1971) Taste reception in the Mediterranean fruit fly: electrophysiological and behavioural studies. r. Insect Physiol. 17, 1371-1384. HODGSONE. S. (1958) Electrophysiological studies of arthropod chemoreception-III. Chemoreceptors of terrestrial and fresh water arthropods. Biol. Bull., Woods Hole 115, 114-125. HODG~ONE. S. (1965) The chemical senses and changing viewpoints in sensory physiology. In Viewpoints in Biology (Ed. by CARTHYJ. D. and DUDDINGTON C. L.), Butterworths, London. HODGSONE. S., LETTVINJ. Y., and ROEDERK. D. (1955) Physiology of a primary chemoreceptor unit. Science, Wash. 122, 417-418. HODGSONE. S. and ROEDERK. D. (1956) Electrophysiological studies of arthropod chemoreception-I. General properties of the labellar chemoreceptors of Diptera. J. cell. con@. Physiol. 48,51-76. KUWABARA M. (1963) Tarsal chemoreception in the butterfly, Vanessa indica. Proc. 16th int. Congr. Zool. 3, 96-97. LAVERACK M. S. (1963) Aspects of chemoreception in Crustacea. Comp. Biochem. Physiol. 8, 141-151. LITTLE C. H. A. (1970) Seasonal changes in carbohydrate and moisture content in needles of balsam fir (Abies bahamea). Can.J. Bot. 48,2021-2028. MCCUTCHANM. C. (1969) Behavioural and electrophysiological responses of the blowfly, Phormia regina Meigen, to acids. Z. vergl. Physiol. 65,131-152. MA, WEI-CHUN and SCHOONHOVEN L. M. (1973) Tarsal contact chemosensory hairs of the large white butterfly Pieris brassicae and their possible role in oviposition behaviour. Entomologia exp. appl. 16, 343-357. MITCHELL B. K. and SBABROOK W. D. (1972) Morphological investigations on the tarsal receptors of the spruce budworm, Choristoneura fumrferana (Lepidoptera: Tortricidae). Can. Ent. 104, 1931-1935. MORITAH., DOIRAS., TAKEDAK., and KVWABARA M. (1957) Electrical response of contact chemoreceptor on tarsus of the butterfly, Vanessa indica. Mem. Fat. Sci. Kyusku Univ., Ser. E (Biol.). 2, 119-139. MORITAH. and TAKEDAK. (1957) The electrical resistance of the tarsal chemosensory hair of the butterfly Vanessa indica. _Y.Fat. Sci. Hokkaido Univ. Ser. VI, Zool. 13, 465-469. MORITAH. and TAK~DAK. (1959) Initiation of spike potentials in contact chemosensory hairs of insects-II. The effect of electric current on tarsal chemosensory hairs of Vanessa. J. cell. camp. Physiol. 54, 177-187. MORITAH. and YAMA~HITA S. (1959) G eneratior potental of insect chemoreceptor. Science, Wash. 30, 922. STURCKOW B. (1963) Electrophysiological studies of a single taste hair of the fly during stimulation by a flowing system. Proc. 16th int. Congr. Zool. 3, 102-104. TAKEDAK. (1961) The nature of impulses of single tarsal chemoreceptors in the butterfly, Vanessa indica. J. cell. camp. Physiol. 58, 233-244. WOLBARSHT M. L. (1958) Electrical activity in the chemoreceptors of the blowfly-II. Responses to electrical stimulation. r. gen. Physiol. 42, 413-428. WOLBARSHT M. L. (1965) Receptor sites in chemoreceptors. CoZd Spr. Harb. Symp. quant. Biol. 30, 281-288.