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Suppression of phototaxis in silkworm larvae
Anim. Behav., 1981, 29, 873-877
SUPPRESSION OF PHOTOTAXIS IN SILKWORM LARVAE B'z H. INOKO, M. KATSUKI & I. WATANABE
Department of Molecular Biology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo, Japan Abstract. A study of the olfactory and visual organs of the larvae of the silkworm (Bombyx mori), using electrophysiological and surgical techniques, indicates that olfactory stinmli from mulberry leaves, conducted through the antennae or the maxillary palps, cause continuous suppression of the phototactic response, and that the central nervous system plays an important role in this 'control' of phototaxis. Such phototactic suppression lasts for 30 h in fifth instar larvae, even after mulberry leaves have been removed. Phototactic Assays A 40-W clear tungsten lamp was employed as the light source for estimating the phototactic response. Monochromatic yellow light 0~max585 rim, half peak w i d t h - - 12.8 nm) obtained with an interference filter (KL58, Toshiba Chemical Industries), or yellow light obtained through yellow cellophane ()~m,• = 610 rim, half peak width = 50 nm), was projected into a black box. Larvae placed on a starting line 40 cm from the lamp, where the intensity of illumination was 1.0 lx/m 2, were permitted to run for 10 min. Each larva was run three or four times. The test box was always kept free from the odour of mulberry leaves. The phototactic index was defined as the average value of the distance moved (cm) towards the light in 10 min by 40 tested larvae.
While insect behaviour involving phototaxis is largely under the control of genes (Hotta & Benzer 1970, 1972; Bentley 1975; Hall & Greenspan 1979; Quinn et al. 1979), its expression must be regulated in some manner by environmental factors (e.g. light, temperature, odour) and/or physiological states (e.g. hunger, pupation) (Mast 1911 ; Dethier 1970). Studies on these regulatory systems in insects may be expected to provide an insight into the neurophysiological mechanisms underlying integration in the central nervous System. Larvae of the silkworm Bombyx mori kept starved for a period of hours display positive phototaxis, moving vigorously towards ultraviolet (357 rim), green (557 rim) and yellow (585 nm) light (Shimizu et al. 1976). This phototactic response to all the three wavelengths is rapidly suppressed after the larvae are fed on mulberry leaves or exposed to their odour, and the suppression of phototaxis continues subsequently for some hours even in the absence of mulberry leaves (Shimizu & Kato 1978). Interestingly, electric shock treatment causes immediate recovery of the phototactic response (Shimizu & Kato 1978). We are currently studying phototaxis and its neural control in B. mori, attempting to elucidate the neurophysiological mechanisms underlying the suppression of phototactic responses mediated by olfactory cues. Here, we report that the suppression caused by the odour of mulberry leaves persists for as long as 30 h in fifth instar larvae, implying an effect upon the central nervous system continuing after the sensory cue has gone and controlled by an integrated mechanism to inhibit phototaxis.
Eleetroretinograms (ERG) of the Stemmata ERGs of the stemmata in response to light were recorded from the optic nerve which was cut proximal to the brain and hung over a silver wire electrode for recording, as described previously (Hirao et al. 1972). The intensity of the yellow light used was adjusted to 1.0 lx/m 2 on the preparation for electrophysiological recording. Results Effects of Inactivation of the Olfactory Organs Fifth instar larvae which had been starved after moulting showed a strong phototactic response to yellow light (phototactic index: > 8), but the response disappeared abruptly after feeding on mulberry leaves (see control larvae in Fig. 1). Phototaxis was also suppressed with almost the same kinetics by exposure to the odour of mulberry leaves alone (unpublished data). By contrast, geotactic and chemotactic responses continued to be elicited almost normally in larvae which had bitten or smelt mulberry leaves. These results indicate that loss of photo-
Materials and Methods The silkworms used throughout the experiments were larvae of the C108 strain. The larvae were grown on mulberry leaves at 25 C.
873
ANIMAL
874
BEHAVIOUR,
taxis is not due to a general inactivation of the motor neurons or muscles, and confirm the earlier studies by Shimizu & Kato (1978). Both the antennae and the maxillary palps of silkworm larvae are known to be olfactory receptors (Hirao et al. 1972). The antennae are mainly involved in chemotaxis, and the maxillary palps in the facilitation of feeding by activating the biting movements of the mandibles. Shimizu & Kato (1978) reported that removal of the antennae prevents suppression of phototaxis by odour. We also examined the effects on phototaxis of inactivating the olfactory organs by amputation or the application of hydrochloric acid (HC1). Unexpectedly, inactivation of either olfactory organ alone exerted no appreciable effect on the kinetics of the suppression of the phototactic response evoked by feeding on mulberry leaves (Fig. 1). By contrast, larvae with both the antennae and maxillary palps inactivated maintained the pre-feeding phototactic response for as long as 24 h after feeding. During these experiments, larvae with inactivated olfactory organs showed approximately normal feeding behaviour (i.e. like control larvae), probably because ample mulberry leaves were made available in such close contact to the individuals that it was easy for them to find and bite leaves in spite of their inability to perceive the odour. These results clearly indicated that the loss of phototaxis is not due to the production of
29,
3
a new metabolite after feeding, nor to the neural stimuli of feeding itself. It is apparently mediated by some neural system affected by olfactory stimuli via either the antennae or the maxillary palps, for both the antennae and the maxillary palps are found to participate in suppression of phototaxis by odour. Involvement of some neural systems is supported by the fact that the phototactic response of larvae subjected to electric shock is not suppressed after exposure to the odour of mulberry leaves (Shimizu & Kato 1978). Continued Suppression of the Phototactie Response in the Absence of Mulberry Leaves The suppression of the phototactic response in fifth instar larvae lasted for 20 h even when they were transferred to a glass dish free from the odour of mulberry leaves (Fig. 2a). The response then gradually returned and was fully relO
a o
8
(
,o
o o o~ 9
0
5
10
15
20 25 Time ( h )
i
I
50
35
40
Fig, 2(a)
10 ..........S ..................... ~- .......... ~ ........ o
8
10
~6
8
~4 "5
\ .\ \
~6
.
EL
?4
I
0 84 -1
o
'0-
0
1
2 Time ( h )
?~
24
Fig. 1. Effect of inactivation of the olfactory organs on the phototactic response after feeding on mulberry leaves. Newly moulted, fifth instar larvae which had been starved and shown a strong phototactic iesponse to yellow light ('lead-in' section) were fed on mulberry leaves at time zero. Forty larvae were removed and assayed for phototaxis in a test box free from the odour of mulberry leaves at the times indicated. Squares: larvae with the antennae inactivated by amputation or application of HC1; triangles; larvae with the maxillary palps inactivated; open circles: larvae with both the antennae and maxillary palps inactivated; filled circles: control larvae.
2 0
1
'2 Time(h)
3
4
Fig. 2(b) Fig. 2. (a) Recovery of the phototactic response of fifth instar larvae following starvation. Newly-moulted fifth instar larvae were fed on mulberry leaves, which suppressed their phototactic response for one day. They were then shifted to a glass dish free from the odour of mulberry leaves at the time zero and removed for phototactic assay at the times indicated. (b) Recovery of the phototactic response of first instar larvae treated as above.
I N O K O ~ E T AL. : S U P P R E S S I O N O F P H O T O T A X I S
stored at about 30 h after the transfer. In the case of the first instar, the duration of no response to light was as short as one hour, and complete restoration was attained after only three hours (Fig. 2b). These findings show that different instar larvae behave differently after smelling mulberry leaves, and indicate that the mechanism which suppresses phototaxis operates for longer in older instars. Recordings from the Optic Nerve It is possible that the odour of mulberry leaves might cause the loss of the phototactic response by making the larvae blind. To test this possibility, optic nerve responses initiated by light stimulation of the stemmata were recorded extracellularly with silver wire electrodes by the method of Ishikawa & Hirao (1969). Sustained potentials were recorded from the optic nerve of control, starved larvae in response to light stimulation, as reported previously (Ishikawa & Hirao 1969; Morohoshi ct al. 1977) (Fig. 3a), suggesting that visual information from the stemmata is transmitted electrotonically through the optic nerve to the brain (Morohoshi et al. 1977). Essentially the same responses were observed in non-phototactic larvae (Fig. 3b): the mean optic nerve potential for 10 individuals tested was 7.3 mV, compared to 6.9 mV in 10 control larvae. No marked differences were thus noted between these groups, and the loss of phototaxis cannot be accounted for by any abnormalities in the input pathway of optic stimuli from the stemmata to the brain. Effect of Inactivation of the Olfactory Organs Another possibility is that certain chemical substances from mulberry leaves which stimulate l O mV
b
a
___~-
"L
-- L i g h t r a d i a t i o n
-
J - - - - -
Fig. 3.(a) Typical record of the optic nerve responses from phototactic, unfed, fifth instar larvae. (b) Typical record of the optic nerve responses from non-phototactic, fifth instar larvae after being fed on mulberry leaves.
875
the olfactory organs might remain tightly bound to the olfactory receptor cells of the antennae or the maxillary palps even after the odour has been removed, and repeatedly elicit reflex mechanisms through the antennae or maxillary nerve to suppress phototaxis, until they diffuse away from the receptor cells. To exclude this possibility, fifth instar larvae which became unresponsive to light after exposure to the odour were subjected to removal or inactivation with HC1 of both the antennae and maxillary palps. Such larvae continued to show no phototactic response after the operation (the average value for the distance moved towards light in 10 min by 40 operated larvae was 0.3 cm, compared to 0.2 cm in control larvae). These results indicate that incessant stimulation of the olfactory receptors by certain chemical substances derived from mulberry leaves is not responsible for the suppression of phototaxis. Discussion After exposure to the odour of mulberry leaves, phototaxis in silkworm larvae is specifically suppressed. This suppression is not due to incapacity of the general motor system, since geotaxis and chemotaxis are normal. The fact that the phototactic response induced by optical stimuli is selectively affected by a stimulus through a different modality strongly implies the involvement of the central nervous system. From the present experiments it can be concluded that peripheral responses by the optic or olfactory organs do not play an important role. In contrast with the finding of Shimizu & Kato (1978) that removal of the antennae alone prevented suppression of phototaxis by odour, our present studies show that both the antennae and maxillary palps must be removed. The reason for this discrepancy is unknown; it may be due to the different procedures for phototactic assay, or the different silkworm strains employed. Odour stimuli are known to be transmitted from the antennae to the brain via the antennal nerve or from the maxillary palps to the suboesophageal ganglion via the maxillary nerve (Hirao et al. 1972). The optical stimuli are transmitted from the stemmata to the brain via the optic nerve (Ishikawa & Hirao 1969) (Fig. 4). Thus the central nervous system response to the odour of mulberry leaves through either of the two olfactory pathways probably involves integration at the suboesophageal ganglion to 'inhibit' the visual pathway. Phototaxis is thus suppressed for a certain period, the length of
ANIMAL
876
BE H A V I O U R ,
Maxillary polps
erve
29,
3
system are elucidated, the control of phototaxis in the silkworm may offer a useful model system for genetic and physiological studies of the molecular mechanisms of neuronal plasticity, since abundant data on the genetics of the silkworm are available (Chikushi 1972).
Acknowledgments
Antennal nerve
i Maxillary nerve
Motor ngrves
The present work was supported in part by grants from the Ministry of Education, Science and Culture of Japan. We also wish to thank Dr T. Hirao and Dr A. Kaneko for their valuable technical comments regarding the electrophysiological recordings, and Dr I. Shimizu for his helpful discussions.
REFERENCES Fig. 4. Schematic diagram of the connections to the central nervous system involved in the control of phototaxis in response to sensory inputs from the sternmata, antennae and maxillary palps in larvae of the silkworm Bombyx mori. SG: Suboesophageal ganglion. which depends on the larval instar (about 3 h for the first instar, and 30 h for the fifth). The experiments to examine the recovery patterns of the phototactic responses of second, third and fourth instar larvae after transfer to a glass dish free from the odour of mulberry leaves did not give clear results, mainly because these instar larvae do not show so strong a phototacfic response as do first or fifth instar larvae. There was, however, a tendency for older instars to take longer to attain complete restoration of the phototactic response. This dependency on age may result from growth or capacity of the central nervous system, but since the suppression outlasts the stimulus, it might involve other physiological influences, such as hormonal changes. Further research is clearly needed to elucidate the underlying mechanisms. The biological meaning of the loss of phototaxis has been discussed previously by Shimizu & Kato (1978), who suggested that suppression of phototaxis might be advantageous in keeping larvae in close proximity to mulberry leaves. The time courses of changes in neuronal activity and behaviour found in silkworm larvae have some similarities to other phenomena such as the habituation and facilitation of the gill-withdrawal reflex in Aplysia (Castellucci & Kandel 1974, 1976), and olfactory and visual discriminative learning in Drosophila melanogaster (Quinn et al. 1974) and in Apis mellifera (Menzel et al. 1974). When the neural elements concerned in the
Bentley, D. 1975. Single gene cricket mutations: effects on behavior, sensillar, sensory neurones and identified interneurons. Science, N.Y., 180, 760764. Castellucci, V. F. & Kandel, E. R. 1974. A quantal analysis of the synaptic depression underlying habituation of the gill-withdrawal reflex in Aplysia. Proc. nat. Acad. Sci. U.S.A., 71, 50045008. Castellucci, V. & Kandel, E. R. 1976. Presynaptic facilitation as a mechanism for behavioral sensitization in Aplysia. Science, N.Y., 194, 1176-1178. Chikushi, H. 1972. Genes and Genetical Stocks of the Silkworm. Tokyo: Keigaku Publishing Company. Dethier, V. G. 1970. Control of Insect Behavior by Natural Products (Ed. by D. L. Wood, R. M. Silverstein & M. Nakajima), pp. 21-28. London: Academic Press. Hall, J. C. & Greenspan, R. J. 1979. Genetic analysis of Drosophila neurobiology, Ann. Rev. Genet., 13, 127-195. Hirao, T., Ishikawa, S. & Arai, N. 1972. Studies on mechanism of feeding in the silkworm, Bombyx mori L. I. The role of the olfactory organs in chemotaxis and feeding. J. sericult. Sci. Japan, 41, 413-417. Hotta, Y. & Benzer, S. 1970. Genetic dissection of the Drosophila nervous system by means of mosaics. Proc. nat. Acad. ScL U.S.A., 67, 1156-1163. Hotta, Y. & Benzer, S. 1972. Mapping of behaviour in Drosophila mosaics. Nature, Lond., 240, 527-535. Ishikawa, S. & Hirao, T. 1969. Electrophysiological studies on vision of the silkworm, Bombyx mori. (I) Electroretinogram of stemmata. J. serictdt. Sci. Japan, 29, 8-14. Mast, S. O. 1911. Light and the Behavior of Organisms. New York: John Wiley. Menzel, R., Erber, J. & Masuhr, T. 1974. Learning and memory in the honeybee. In: Experimental Analysis of lnsect Behaviour (Ed. by L. Barton Browne), pp. 195-217. Berlin-Heidelberg-New York: Springer. Morohoshi, S., Morita, Y. & Tabata, M. 1977. The control of growth and development in Bombyx mori. XXXVIII. Input pathway of light information to the brain of silkworm larvae. Proc. Japan Acad., 53, 14-17.
INOKO ET AL.: SUPPRESSION OF PHOTOTAXIS Quinn, W. G., Harris, W. A. & Benzer, S. 1974. Conditioned behavior in Drosophila melanogaster. Proc. nat. Acad. Sci. U.S.A., 71, 708-712. Quinn, W. G., Sziber, P. P. & Booker, R. 1979. The Drosophila memory mutant amnesiac. Nature, Lond., 277, 212-214. Shimizu, I. & Kato. M. 1978. Loss of phototaxis in silkworm larvae after smelling mulberry leaves,
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and recovery after electroconvulsive shock. Nature, Lond., 272, 248-249. Shimizu, I., Kitabatake, S. & Kato, M. 1976. Loss of phototactic behavior in silkworm larvae reared on synthetic diet. Proc. Japan Acad., 52, 137-140.
(Received 15 July 1980; revised 3 November 1980; MS. number: 2033)
SUPPRESSION OF PHOTOTAXIS IN SILKWORM LARVAE B'z H. INOKO, M. KATSUKI & I. WATANABE
Department of Molecular Biology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo, Japan Abstract. A study of the olfactory and visual organs of the larvae of the silkworm (Bombyx mori), using electrophysiological and surgical techniques, indicates that olfactory stinmli from mulberry leaves, conducted through the antennae or the maxillary palps, cause continuous suppression of the phototactic response, and that the central nervous system plays an important role in this 'control' of phototaxis. Such phototactic suppression lasts for 30 h in fifth instar larvae, even after mulberry leaves have been removed. Phototactic Assays A 40-W clear tungsten lamp was employed as the light source for estimating the phototactic response. Monochromatic yellow light 0~max585 rim, half peak w i d t h - - 12.8 nm) obtained with an interference filter (KL58, Toshiba Chemical Industries), or yellow light obtained through yellow cellophane ()~m,• = 610 rim, half peak width = 50 nm), was projected into a black box. Larvae placed on a starting line 40 cm from the lamp, where the intensity of illumination was 1.0 lx/m 2, were permitted to run for 10 min. Each larva was run three or four times. The test box was always kept free from the odour of mulberry leaves. The phototactic index was defined as the average value of the distance moved (cm) towards the light in 10 min by 40 tested larvae.
While insect behaviour involving phototaxis is largely under the control of genes (Hotta & Benzer 1970, 1972; Bentley 1975; Hall & Greenspan 1979; Quinn et al. 1979), its expression must be regulated in some manner by environmental factors (e.g. light, temperature, odour) and/or physiological states (e.g. hunger, pupation) (Mast 1911 ; Dethier 1970). Studies on these regulatory systems in insects may be expected to provide an insight into the neurophysiological mechanisms underlying integration in the central nervous System. Larvae of the silkworm Bombyx mori kept starved for a period of hours display positive phototaxis, moving vigorously towards ultraviolet (357 rim), green (557 rim) and yellow (585 nm) light (Shimizu et al. 1976). This phototactic response to all the three wavelengths is rapidly suppressed after the larvae are fed on mulberry leaves or exposed to their odour, and the suppression of phototaxis continues subsequently for some hours even in the absence of mulberry leaves (Shimizu & Kato 1978). Interestingly, electric shock treatment causes immediate recovery of the phototactic response (Shimizu & Kato 1978). We are currently studying phototaxis and its neural control in B. mori, attempting to elucidate the neurophysiological mechanisms underlying the suppression of phototactic responses mediated by olfactory cues. Here, we report that the suppression caused by the odour of mulberry leaves persists for as long as 30 h in fifth instar larvae, implying an effect upon the central nervous system continuing after the sensory cue has gone and controlled by an integrated mechanism to inhibit phototaxis.
Eleetroretinograms (ERG) of the Stemmata ERGs of the stemmata in response to light were recorded from the optic nerve which was cut proximal to the brain and hung over a silver wire electrode for recording, as described previously (Hirao et al. 1972). The intensity of the yellow light used was adjusted to 1.0 lx/m 2 on the preparation for electrophysiological recording. Results Effects of Inactivation of the Olfactory Organs Fifth instar larvae which had been starved after moulting showed a strong phototactic response to yellow light (phototactic index: > 8), but the response disappeared abruptly after feeding on mulberry leaves (see control larvae in Fig. 1). Phototaxis was also suppressed with almost the same kinetics by exposure to the odour of mulberry leaves alone (unpublished data). By contrast, geotactic and chemotactic responses continued to be elicited almost normally in larvae which had bitten or smelt mulberry leaves. These results indicate that loss of photo-
Materials and Methods The silkworms used throughout the experiments were larvae of the C108 strain. The larvae were grown on mulberry leaves at 25 C.
873
ANIMAL
874
BEHAVIOUR,
taxis is not due to a general inactivation of the motor neurons or muscles, and confirm the earlier studies by Shimizu & Kato (1978). Both the antennae and the maxillary palps of silkworm larvae are known to be olfactory receptors (Hirao et al. 1972). The antennae are mainly involved in chemotaxis, and the maxillary palps in the facilitation of feeding by activating the biting movements of the mandibles. Shimizu & Kato (1978) reported that removal of the antennae prevents suppression of phototaxis by odour. We also examined the effects on phototaxis of inactivating the olfactory organs by amputation or the application of hydrochloric acid (HC1). Unexpectedly, inactivation of either olfactory organ alone exerted no appreciable effect on the kinetics of the suppression of the phototactic response evoked by feeding on mulberry leaves (Fig. 1). By contrast, larvae with both the antennae and maxillary palps inactivated maintained the pre-feeding phototactic response for as long as 24 h after feeding. During these experiments, larvae with inactivated olfactory organs showed approximately normal feeding behaviour (i.e. like control larvae), probably because ample mulberry leaves were made available in such close contact to the individuals that it was easy for them to find and bite leaves in spite of their inability to perceive the odour. These results clearly indicated that the loss of phototaxis is not due to the production of
29,
3
a new metabolite after feeding, nor to the neural stimuli of feeding itself. It is apparently mediated by some neural system affected by olfactory stimuli via either the antennae or the maxillary palps, for both the antennae and the maxillary palps are found to participate in suppression of phototaxis by odour. Involvement of some neural systems is supported by the fact that the phototactic response of larvae subjected to electric shock is not suppressed after exposure to the odour of mulberry leaves (Shimizu & Kato 1978). Continued Suppression of the Phototactie Response in the Absence of Mulberry Leaves The suppression of the phototactic response in fifth instar larvae lasted for 20 h even when they were transferred to a glass dish free from the odour of mulberry leaves (Fig. 2a). The response then gradually returned and was fully relO
a o
8
(
,o
o o o~ 9
0
5
10
15
20 25 Time ( h )
i
I
50
35
40
Fig, 2(a)
10 ..........S ..................... ~- .......... ~ ........ o
8
10
~6
8
~4 "5
\ .\ \
~6
.
EL
?4
I
0 84 -1
o
'0-
0
1
2 Time ( h )
?~
24
Fig. 1. Effect of inactivation of the olfactory organs on the phototactic response after feeding on mulberry leaves. Newly moulted, fifth instar larvae which had been starved and shown a strong phototactic iesponse to yellow light ('lead-in' section) were fed on mulberry leaves at time zero. Forty larvae were removed and assayed for phototaxis in a test box free from the odour of mulberry leaves at the times indicated. Squares: larvae with the antennae inactivated by amputation or application of HC1; triangles; larvae with the maxillary palps inactivated; open circles: larvae with both the antennae and maxillary palps inactivated; filled circles: control larvae.
2 0
1
'2 Time(h)
3
4
Fig. 2(b) Fig. 2. (a) Recovery of the phototactic response of fifth instar larvae following starvation. Newly-moulted fifth instar larvae were fed on mulberry leaves, which suppressed their phototactic response for one day. They were then shifted to a glass dish free from the odour of mulberry leaves at the time zero and removed for phototactic assay at the times indicated. (b) Recovery of the phototactic response of first instar larvae treated as above.
I N O K O ~ E T AL. : S U P P R E S S I O N O F P H O T O T A X I S
stored at about 30 h after the transfer. In the case of the first instar, the duration of no response to light was as short as one hour, and complete restoration was attained after only three hours (Fig. 2b). These findings show that different instar larvae behave differently after smelling mulberry leaves, and indicate that the mechanism which suppresses phototaxis operates for longer in older instars. Recordings from the Optic Nerve It is possible that the odour of mulberry leaves might cause the loss of the phototactic response by making the larvae blind. To test this possibility, optic nerve responses initiated by light stimulation of the stemmata were recorded extracellularly with silver wire electrodes by the method of Ishikawa & Hirao (1969). Sustained potentials were recorded from the optic nerve of control, starved larvae in response to light stimulation, as reported previously (Ishikawa & Hirao 1969; Morohoshi ct al. 1977) (Fig. 3a), suggesting that visual information from the stemmata is transmitted electrotonically through the optic nerve to the brain (Morohoshi et al. 1977). Essentially the same responses were observed in non-phototactic larvae (Fig. 3b): the mean optic nerve potential for 10 individuals tested was 7.3 mV, compared to 6.9 mV in 10 control larvae. No marked differences were thus noted between these groups, and the loss of phototaxis cannot be accounted for by any abnormalities in the input pathway of optic stimuli from the stemmata to the brain. Effect of Inactivation of the Olfactory Organs Another possibility is that certain chemical substances from mulberry leaves which stimulate l O mV
b
a
___~-
"L
-- L i g h t r a d i a t i o n
-
J - - - - -
Fig. 3.(a) Typical record of the optic nerve responses from phototactic, unfed, fifth instar larvae. (b) Typical record of the optic nerve responses from non-phototactic, fifth instar larvae after being fed on mulberry leaves.
875
the olfactory organs might remain tightly bound to the olfactory receptor cells of the antennae or the maxillary palps even after the odour has been removed, and repeatedly elicit reflex mechanisms through the antennae or maxillary nerve to suppress phototaxis, until they diffuse away from the receptor cells. To exclude this possibility, fifth instar larvae which became unresponsive to light after exposure to the odour were subjected to removal or inactivation with HC1 of both the antennae and maxillary palps. Such larvae continued to show no phototactic response after the operation (the average value for the distance moved towards light in 10 min by 40 operated larvae was 0.3 cm, compared to 0.2 cm in control larvae). These results indicate that incessant stimulation of the olfactory receptors by certain chemical substances derived from mulberry leaves is not responsible for the suppression of phototaxis. Discussion After exposure to the odour of mulberry leaves, phototaxis in silkworm larvae is specifically suppressed. This suppression is not due to incapacity of the general motor system, since geotaxis and chemotaxis are normal. The fact that the phototactic response induced by optical stimuli is selectively affected by a stimulus through a different modality strongly implies the involvement of the central nervous system. From the present experiments it can be concluded that peripheral responses by the optic or olfactory organs do not play an important role. In contrast with the finding of Shimizu & Kato (1978) that removal of the antennae alone prevented suppression of phototaxis by odour, our present studies show that both the antennae and maxillary palps must be removed. The reason for this discrepancy is unknown; it may be due to the different procedures for phototactic assay, or the different silkworm strains employed. Odour stimuli are known to be transmitted from the antennae to the brain via the antennal nerve or from the maxillary palps to the suboesophageal ganglion via the maxillary nerve (Hirao et al. 1972). The optical stimuli are transmitted from the stemmata to the brain via the optic nerve (Ishikawa & Hirao 1969) (Fig. 4). Thus the central nervous system response to the odour of mulberry leaves through either of the two olfactory pathways probably involves integration at the suboesophageal ganglion to 'inhibit' the visual pathway. Phototaxis is thus suppressed for a certain period, the length of
ANIMAL
876
BE H A V I O U R ,
Maxillary polps
erve
29,
3
system are elucidated, the control of phototaxis in the silkworm may offer a useful model system for genetic and physiological studies of the molecular mechanisms of neuronal plasticity, since abundant data on the genetics of the silkworm are available (Chikushi 1972).
Acknowledgments
Antennal nerve
i Maxillary nerve
Motor ngrves
The present work was supported in part by grants from the Ministry of Education, Science and Culture of Japan. We also wish to thank Dr T. Hirao and Dr A. Kaneko for their valuable technical comments regarding the electrophysiological recordings, and Dr I. Shimizu for his helpful discussions.
REFERENCES Fig. 4. Schematic diagram of the connections to the central nervous system involved in the control of phototaxis in response to sensory inputs from the sternmata, antennae and maxillary palps in larvae of the silkworm Bombyx mori. SG: Suboesophageal ganglion. which depends on the larval instar (about 3 h for the first instar, and 30 h for the fifth). The experiments to examine the recovery patterns of the phototactic responses of second, third and fourth instar larvae after transfer to a glass dish free from the odour of mulberry leaves did not give clear results, mainly because these instar larvae do not show so strong a phototacfic response as do first or fifth instar larvae. There was, however, a tendency for older instars to take longer to attain complete restoration of the phototactic response. This dependency on age may result from growth or capacity of the central nervous system, but since the suppression outlasts the stimulus, it might involve other physiological influences, such as hormonal changes. Further research is clearly needed to elucidate the underlying mechanisms. The biological meaning of the loss of phototaxis has been discussed previously by Shimizu & Kato (1978), who suggested that suppression of phototaxis might be advantageous in keeping larvae in close proximity to mulberry leaves. The time courses of changes in neuronal activity and behaviour found in silkworm larvae have some similarities to other phenomena such as the habituation and facilitation of the gill-withdrawal reflex in Aplysia (Castellucci & Kandel 1974, 1976), and olfactory and visual discriminative learning in Drosophila melanogaster (Quinn et al. 1974) and in Apis mellifera (Menzel et al. 1974). When the neural elements concerned in the
Bentley, D. 1975. Single gene cricket mutations: effects on behavior, sensillar, sensory neurones and identified interneurons. Science, N.Y., 180, 760764. Castellucci, V. F. & Kandel, E. R. 1974. A quantal analysis of the synaptic depression underlying habituation of the gill-withdrawal reflex in Aplysia. Proc. nat. Acad. Sci. U.S.A., 71, 50045008. Castellucci, V. & Kandel, E. R. 1976. Presynaptic facilitation as a mechanism for behavioral sensitization in Aplysia. Science, N.Y., 194, 1176-1178. Chikushi, H. 1972. Genes and Genetical Stocks of the Silkworm. Tokyo: Keigaku Publishing Company. Dethier, V. G. 1970. Control of Insect Behavior by Natural Products (Ed. by D. L. Wood, R. M. Silverstein & M. Nakajima), pp. 21-28. London: Academic Press. Hall, J. C. & Greenspan, R. J. 1979. Genetic analysis of Drosophila neurobiology, Ann. Rev. Genet., 13, 127-195. Hirao, T., Ishikawa, S. & Arai, N. 1972. Studies on mechanism of feeding in the silkworm, Bombyx mori L. I. The role of the olfactory organs in chemotaxis and feeding. J. sericult. Sci. Japan, 41, 413-417. Hotta, Y. & Benzer, S. 1970. Genetic dissection of the Drosophila nervous system by means of mosaics. Proc. nat. Acad. ScL U.S.A., 67, 1156-1163. Hotta, Y. & Benzer, S. 1972. Mapping of behaviour in Drosophila mosaics. Nature, Lond., 240, 527-535. Ishikawa, S. & Hirao, T. 1969. Electrophysiological studies on vision of the silkworm, Bombyx mori. (I) Electroretinogram of stemmata. J. serictdt. Sci. Japan, 29, 8-14. Mast, S. O. 1911. Light and the Behavior of Organisms. New York: John Wiley. Menzel, R., Erber, J. & Masuhr, T. 1974. Learning and memory in the honeybee. In: Experimental Analysis of lnsect Behaviour (Ed. by L. Barton Browne), pp. 195-217. Berlin-Heidelberg-New York: Springer. Morohoshi, S., Morita, Y. & Tabata, M. 1977. The control of growth and development in Bombyx mori. XXXVIII. Input pathway of light information to the brain of silkworm larvae. Proc. Japan Acad., 53, 14-17.
INOKO ET AL.: SUPPRESSION OF PHOTOTAXIS Quinn, W. G., Harris, W. A. & Benzer, S. 1974. Conditioned behavior in Drosophila melanogaster. Proc. nat. Acad. Sci. U.S.A., 71, 708-712. Quinn, W. G., Sziber, P. P. & Booker, R. 1979. The Drosophila memory mutant amnesiac. Nature, Lond., 277, 212-214. Shimizu, I. & Kato. M. 1978. Loss of phototaxis in silkworm larvae after smelling mulberry leaves,
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and recovery after electroconvulsive shock. Nature, Lond., 272, 248-249. Shimizu, I., Kitabatake, S. & Kato, M. 1976. Loss of phototactic behavior in silkworm larvae reared on synthetic diet. Proc. Japan Acad., 52, 137-140.
(Received 15 July 1980; revised 3 November 1980; MS. number: 2033)