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Virtual reality experiments on a digital servosphere: guiding male silkworm moths to a virtual odour source
Computers and Electronics in Agriculture 35 (2002) 243–254 www.elsevier.com/locate/compag
Virtual reality experiments on a digital servosphere: guiding male silkworm moths to a virtual odour source Masayuki Sakuma Laboratory of Insect Physiology, Graduate School of Agriculture, Kyoto Uni6ersity, 606 -8502 Kyoto, Japan
Abstract A digital ‘servosphere’ locomotion-compensation apparatus has been developed for use in experiments on the mechanisms of spatial orientation in ambulatory animals. The sphere is driven by a pair of digital AC-servo motors, which provide a negative feedback response to any displacements of the test animal. A high-speed video tracker continually reports the position of the animal to a computer, which controls the servo-motors via a motor-control board and also logs the position of the motor axes. Movements of the servos define the path of the test animal on a virtual plane. The computer also generates odour cues in real time via a relay-control board and an airflow system. Odour presentation is programmed to occur in response to the animal’s movements on the virtual plane, and can result in the animal being led to a virtual source. The servosphere was first used to investigate orientation of a walking male silkworm moth (Bombyx mori ) towards a female in still air. When a calling female moth was placed beside the sphere, the male moth beat its wings and walked straight towards the female. Unilaterally dewinged males also walked in a straight line, though slightly to one side of the direction of the female, but bilaterally dewinged moths remained stationary. This indicates that in still air the male moths orientate to pheromone sources by fanning air from in front of them over their antennae with their wings. If a moth continually reverses its turning movements when it encounters a train of pheromone concentration peaks, it will maintain a course towards the source and eventually arrive there. This idea was demonstrated on the servosphere by incorporating a computer-controlled solenoid valve which releases pheromone when a moth points towards a virtual odour source. In this sensory field, both intact and bilaterally dewinged males reached the source irrespective of the wind direction. This result supports the proposed orientation mechanism and also demonstrates
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the applicability of the servosphere virtual-reality environment to the experimental investigation of insect orientation mechanisms. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Bombyx mori; Servosphere; Locomotion compensator; Orientation; Pheromone
1. Introduction When orientating, animals use external cues to modulate their behaviour in a manner that guides them towards a target. To investigate the cause of this type of behaviour, cues should be presented to the animals being tested in a controlled way (Kennedy, 1977). Conventional experiments in a wind tunnel or arena, however, allow test animals some degree of freedom, so that the cues they receive are not fully predetermined. An experimental arrangement that overcomes this problem is the ‘servosphere’, a locomotion-compensating device originated by E. Kramer and P. Heinecke (Thiery and Visser, 1986). Servospheres have been widely used in studies of orientation in insects and other small animals (Kramer, 1975; Bell and Kramer, 1979; Weber et al., 1981; Schal et al., 1983; Thiery and Visser, 1986; Wendler and Vlatten, 1993; McMahon and Guerin, 2000). The servosphere is a form of treadmill that compensates every locomotive movement of an ambulatory test animal. The animal walks freely on the top of a large sphere, which is rotated by a pair of motors orthogonally arranged at the sphere’s equator. A feedback system continually rotates the sphere about both axes so that the freely walking test animal is kept at the same spatial position (the top of the sphere), thus cues can be presented in a precisely controlled manner. If this apparatus is combined with an actuator that generates the cue, a virtual sensory field can be created and the orientation manoeuvring of the animal in response to it can be investigated in a fully automated experiment. In this paper, the construction of (1) a servosphere system that employs modern, digital, technology, and (2) a system for real-time control of an odour cue, are described. The paper also provides examples of the use of these systems in a virtual-reality investigation of the orientation behaviour of male silkworm moths homing in on an odour source. A more complete analysis of the orientation behaviours identified during this experiment will be reported elsewhere.
2. A digital-technology servosphere system The servosphere consists of a high-speed video tracker (G-240, OKK Inc., Tokyo, Japan), a personal computer (PC-9821, NEC, Tokyo, Japan) with motorcontrol board (QPG-45, Cosmotechs Co. Ltd., Yamato, Japan), a pair of digital AC-servo motors (MINAS MSM021-A1A, Matsushita Electric Industrial Co. Ltd., Kadoma, Japan) and drive wheels, a hollow perspex sphere supported by a universal ball bearing, and a steel frame (Fig. 1). The sphere has an outside
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diameter of 505 mm and a black suede-texture coating (Nextel 3101, Mankiewicz Gebr. & Co., Hamburg, Germany). It is made from a pair of hemispherical ceiling-lamp shields (YKA4387401, Matsushita Electric Industrial Co. Ltd., Kadoma, Japan) designed for use in streets and weighs 1.77 kg. The universal ball bearing is a 60 mm diameter plastic billiards ball. The drive wheels (SP442, Tamiya Inc., Shizuoka, Japan), which are fitted with 60 mm diameter sponge-rubber tires, are directly connected to the drive axles of the servo-motors. The entire system is controlled digitally. The video tracker captures the video image of the test animal at the top of the sphere, calculates its location relative to the centre of the image, and transmits the (x, y) coordinates to the computer through a DIO parallel interface (AZI-2746, Interface Corp., Hiroshima, Japan). This process is repeated continually at a rate of either 120 Hz (9 ms delay time) or 240 Hz (5 ms delay). The image captured by the tracker is displayed on an NTSC video monitor. The computer converts the coordinates to pulse counts for the motor-control board, which generates pulses to rotate the two servomotors so that the sphere is turned, about both axes, just sufficiently to take the animal back to the highest point. The amount of movement is controlled by reference to the outputs of high-resolution encoders (10 000 pulses per rotation) on the servomotor axes. The positional shifts of the axes, as pulse counts, are logged temporarily to the computer’s memory every 100 ms. They are later recorded in a file on the computer’s hard disk and converted to displacements of the sphere’s surface, from which the track of the test animal can be reconstructed. The use of a video tracker has several advantages. Since, the form of the captured image depends on the threshold exposure value for binary conversion, it can be
Fig. 1. The digital servosphere system.
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controlled to some extent by simply changing the aperture of the tracker’s lens. This may allow selection of either the animal’s whole body or just a small highlight. Attachment of a small reflector (Scotchlite, Sumitomo 3M Ltd., Tokyo, Japan) on the notum of an insect will create a highlight close to the insect’s turning axis, so that changes in course angle are easily and quickly identifiable. The speed and precision of the feedback control is adjustable by altering the zoom factor of the tracker’s lens and the multiplication factor for generating the motor-control pulses. By these means, tracking performance can be tuned specifically for each experiment. In the motor-control board, new data repeatedly override old ones, so the servomotors are always sent the pulse sequences required to return the animal directly to the highest point. The tracker’s resolution (6400× 4160 pixels) and sampling rate (120 or 240 Hz) are sufficient to allow persistent, smooth, and accurate tracking of a running insect. Delegation of these tracking and compensation tasks to intelligent peripheral devices frees up the PC’s CPU for processing of displacement data and the control of cue-presentation devices. The control software for this system is written in the C (ANSI) language (Quick-C, Microsoft, Seattle, USA) and runs under the MS-DOS 6.2 (Microsoft, Seattle, USA) operating system. The acquired data are analysed by a second program, written in the C + + language (Visual C+ + version 6 and Microsoft Foundation Class Library, Microsoft, Seattle), that is run under the WINDOWS 98 (Microsoft, Seattle, USA) operating system. Both programs were developed by the author.
3. Odour-cue control system Application of a servosphere (or any form of locomotion compensator) in behavioural research will usually involve the presentation of cues to a test animal, and observation of its responses. In many studies, cues have been presented independently of the animal’s movement, but more precise experiments have employed a feedback or closed-loop system. For example, Kramer (1976) exposed a honeybee, Apis melifera, to an odour cue that varied in concentration according to the bee’s position, and Bell (1986) exposed a walking male warehouse beetle, Trogoderma 6ariabile, to sex pheromone only while the male remained within a programmed corridor. In these experiments, the cue was controlled by the animal’s absolute position, but this may not reflect the cue perception of the animal which is more likely to be influenced by relative and directional factors. A programmed system for cue presentation in real time may make it possible to produce a more realistic representation of the animal’s perceived environment. The aim is to conduct a virtual-reality experiment, by presenting odour to the test animal in exactly the way it would occur during actual manoeuvring to a natural odour source. The digital servosphere system described here incorporates real-time control of cues. The main program computes the position, walking speed and direction of the test animal at a rate of 10 Hz, and controls presentation of odour by switching a
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Fig. 2. Cylindrical wind tunnel with a transparent cover used in the virtual-reality experiments (plan view). A three-way solenoid valve controlled by the PC switches the airflow between the odoured and bypass channels. Air is exhausted from the tunnel outlet at the same rate that it is supplied at the inlet.
solenoid-controlled valve via a Photo-MOS relay-module board (PRY-32, CONTEC Co. Ltd., Osaka, Japan). To insulate the experimental arena from outside influences, a cylindrical perspex cover (10 cm internal diameter× 3 cm deep) produces a horizontal-flow wind tunnel over the top of the sphere (Fig. 2). The ceiling of the cover is transparent, to allow imaging by the tracker. Illumination is by a ring fluorescent light (40 W, excitation at 40 kHz) when simulating daytime conditions and by IR light-emitting diodes (IREDs) when simulating night. These lights are suspended above the perimeter of the tunnel, to avoid obscuring the tracker’s view or causing reflections from the perspex cover. The airflow originates at a compressor and passes through glass fibre and then charcoal filters for the removal of condensed oil mists and organic contaminants (Fig. 2). A flowmeter (model RK200, Kofloc, Tokyo, Japan) in the line monitors the air-flow rate, which can be precisely adjusted by an attached miniature needle valve. The air is then remoistened in a bubbler, and its relative humidity (RH) is adjusted manually by changing a bypass ratio over the bubbler with a pair of needle valves while a humidity sensor (CHS-UGS, TDK, Tokyo, Japan) monitors the RH. A solenoid-controlled valve just before the inlet to the wind tunnel (Fig. 3) switches the flow between odoured and bypass channels. The odour source used in the experiment is a pheromone gland freshly removed from a calling female silkworm moth. The air is then removed from the tunnel through an exhaust port opposite the inlet by using an aspirator. The rate of suction is adjusted manually to the same
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rate as it enters by a flowmeter-jointed needle valve, producing a practically closed airflow within the tunnel. In the present experiment, the airflow comes from a single fixed inlet and the odour cue is controlled simply by switching between the odoured and bypass channels. Visualisation of the airflow with titanium-dioxide smoke shows it to be confined to a narrow stream between the inlet and outlet and to pass directly over the test animal. The speed of the flow, measured by a hot-wire anemometer ( c6312 airflow transducer with c0965-03 probe, Kanomax Co. Ltd., Suita, Japan), indicates considerable variation across the stream. Nevertheless, the arrangement, which certainly achieves the essential requirement of allowing odour to be presented only when the test animal orientates towards the (virtual) odour source, appears to produce the required virtual sensory field. Experimental evidence for this is presented in the next section.
4. Example: homing of male silkworm moths to a virtual odour source As an illustration of how the digital servosphere can be used for virtual-reality experiments, some results from a trial in which walking male silkworm moths (Bombyx mori ) responded to a computer-generated odour field are presented here. The response of a male silkworm moth to sex pheromone is characterised by a turning motion and wing beating, a behaviour referred to as the ‘mating dance’. Although it has previously been suggested that the function of wing beating is to draw in pheromone-laden air (Obara, 1979), cues responsible for the homing of the moth to an odour source and the orientation responses by which homing is achieved have not yet been fully identified.
Fig. 3. The servosphere, air flow and illumination systems.
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Fig. 4. Tracks in the virtual plane of a male moth walking for 10 min on the servosphere in still air at distances, d, of (a) 10 cm, (b) 20 cm and (c) 40 cm from a calling female placed on the right. The radius of the dotted circle is 2.0 m. Open circles on the tracks indicate 30 s time intervals. (d) The linearity of the tracks, is represented by the mean vector length, r.
In a preliminary experiment, the servosphere was used to observe walking of male moths towards a calling female in still air, i.e. without making use of the wind tunnel described in the previous section. When a calling female moth on a perch was placed 70–100 mm from a male moth on the servosphere, and a smoke source placed near the female’s pheromone gland was used to visualise the resulting pheromone plume, it was observed that the plume flowed over the antennae of the male as it walked towards the female. The male moth was beating its wings, and this appeared to have the effect of drawing in air from in front of it and fanning this air over its antennae. Modulation of the odour concentration in the fanned air as the moth turns provides a cue that allows the moth to orientate itself towards the source. Tracks shown in Fig. 4 are those of a male moth in still air at various distances from a female. The linearity of the tracks, represented by the mean vector length r (Batschelet, 1981), decreased as the distance increased. Fig. 5 shows the tracks made by three male moths: (a) intact, (b) right wing amputated, (c) both wings removed. A calling female was placed on a perch 10 cm to the right of the top of sphere. It can be seen that moths (a) and (b) walked along a nearly straight track, although (b) was deflected to the right of the direct line to the moth, presumably because of unbalanced fanning. Moth (c) remained stationary, again suggesting the importance of fanning for orientation.
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Fig. 5. Tracks in the virtual plane of male moths: a (intact), b, (right wing amputated) and c (de-winged). The dotted circle indicates 1.0 m and the open circles on the tracks show 30 s time intervals.
From these observations, the following orientation mechanism may be inferred (Fig. 6). While turning, a male moth beats its wings to draw-in air from in front of it and passes the air over its antennae (Fig. 6a). The moth will thus be most exposed to pheromone when it is heading towards the source, in other words, the timing of
Fig. 6. Orientation mechanism of male B. mori moths inferred from results of Fig. 5. (a) In the presence of sex-pheromone odour, the moth keeps turning while beating its wings to draw air from in front of it over its antennae. (b) When the moth faces an odour source, turning is repeatedly reversed in response to pulses of odour concentration, and the turning is transformed into a straight walk towards the source.
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pheromone perception coincides with the moth’s orientation being towards the source. There is increasing evidence that moths perceive pheromone odour as an intermittent stimulus reflecting a pulsed cloud of pheromone rather than continuous presence of pheromone in a concentration gradient (Baker et al., 1985). A recently proposed neural mechanism working in the moth’s central nervous system suggests that each pulse of pheromonal stimulation triggers the switching between the high and low excitation states of certain descending interneurons (Olberg, 1983; Kanzaki et al., 1994; Kanzaki, 1998). It has been noted that some of these interneurons in the left and right connectives show an antiphasic pattern of excitation similar to that occurring in a ‘flip-flop’ electronic circuit. Thus flipflopping in these interneurons could cause reversal of the turning direction as each pulse of pheromone is detected (Mishima and Kanzaki, 1998), and repeated reversals while the moth is pointing directly at the source should result in the moth walking straight towards it (Fig. 6b). This possible mechanism can be examined by modulating the odour concentration automatically within the wind-tunnel arena at the top of the servosphere. In this experiment, a ‘virtual’ odour source is created by switching the solenoid valve to release pheromone when the moth’s course is towards the (previously designated) virtual position of the source. The main program computes the virtual position of the travelling moth every 100 ms, and estimates its instantaneous course direction by comparing sequential positions. The direction of the virtual odour source from the moth’s current position is also computed, and if this lies within 922.5° of the moth’s course, pheromone is released. Although the program does not intrinsically provide pulsed odour, the moth’s frequent turning effectively generates pulses of 0.2 s duration at a frequency of 1.0 Hz. The resulting tracks, together with time series of the male’s speed and orientation, are shown in Fig. 7. When the source was placed at virtual coordinates of (2.0, 2.0) m relative to the male moth’s starting position, the male proceeded almost straight towards the source and stayed very close to it after arriving 3 min later (Fig. 7a). The track and the graphs of velocity and orientation all indicate that the moth moved almost directly to the source at (2, 2) m and stayed there after arriving 3.0 min after setting out. The darker sections of the tracks and graphs indicate when the moth was heading within 9 22.5° of the source, and so causing pheromone to be dispensed. Orientation was thus successfully controlled by the automatic release of pheromone, whereas the walking speed changed little through the experiment. Anemotaxis (Kramer, 1975) appears to have contributed little to the results, because the direction of the airflow was fixed and different from that of the source, and because the moth remained very close to the source after arriving there, even though it continued to experience a directed airflow. After amputation of both pairs of wings, the moth was still able to walk directly to the virtual source under the guidance of the automatic odour-release system (Fig. 7b), i.e. a dewinged male, although unable to generate pulses of odour concentration by itself, could reach the virtual source with artificially
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Fig. 7. Tracks of male moths in response to a virtual odour source; (a) intact, (b) de-winged, (c) de-winged and downwind source. The radius of the dotted circle is 0.5 m, and the dotted arrow indicates the direction of the 2.0 ms − 1 airflow.
pulsed odour stimulation. Moreover, the wingless male could even be led to a source placed downwind at the same distance (Fig. 7c). This clearly indicates that upwind anemotaxis is not a major orientation mechanism for a walking male silkworm moth. However, the greater time taken for the moth to reach the downwind source suggests that a secondary role for upwind anemotaxis cannot be ruled out. The orientation mechanism mentioned above works at close range to the source, but the same mechanism probably also functions to locate a pheromone plume. In preliminary experiments conducted in laminar airflow, moths drew in, by fanning, an odour plume passing close by them and moved predominantly towards the plume. They would have soon reached the main stream of the plume if they had not been dynamically tethered by the servosphere. After entering the plume, they could be expected to follow it in the same manner as they locate a point odour source. It seems also possible for flying moths to draw air by wing-beating. Although the forward motion of flying moths through the air seem to preclude the need for it, the coincidence of timing of pheromone perception with the moth’s orientation being towards a cloud of pheromone might play a role either as a component of, or in addition to, the various orientation mechanisms already established for them (Baker and Vickers, 1998; Carde´ and Mafra-Neto, 1998). This is a topic for future work.
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5. Conclusion The digital servosphere, wind-tunnel arena, and computer-controlled real-time odour-release system described here have made it possible to create a programmed odour environment on an endless virtual plane. This virtual-reality environment appears to reproduce the cues and to stimulate the manoeuvring responses that cause male B. mori to home on a calling female in natural conditions. These trial results suggest that this apparatus will find application in the experimental investigation of the mechanisms of orientation behaviour in this and other ambulatory, odour-responsive, species.
Acknowledgements Dr J.H. Visser (Plant Research International, The Netherlands), OKK Inc. (Tokyo, Japan), Cosmotechs Co. Ltd. (Yamato, Japan), and Kanomax Co. Ltd. (Suita, Japan) provided invaluable advice on, and support for, the construction of the digital servosphere. I thank the editors of this special issue for their critical reading and editing of the manuscript. This work was supported by a Grant-in-Aid for Scientific Research (C)(2) No.10660046 from the Japan Society for the Promotion of Science.
References Baker, T.C., Vickers, N.J., 1998. Pheromone-mediated flight in moths. In: Carde´ , R.T., Minks, A.K. (Eds.), Insect Pheromone Research New Directions. Chapman & Hall, New York. Baker, T.C., Willis, M.A., Haynes, K.F., Phelan, P.L., 1985. A pulsed cloud of sex pheromone elicits upwind flight in male moths. Physiol. Entomol. 10, 257 – 265. Batschelet, E., 1981. Circular Statistics in Biology. Academic Press, London. Bell, W.J., 1986. Responses of arthropods to temporal chemical stimulus changes: simulation of a humidity differential and pheromone plume. In: Payne, T.L., Birch, M.C., Kennedy, C.E.J. (Eds.), Mechanisms in Insect Olfaction. Clarendon Press, Oxford. Bell, W.J., Kramer, E., 1979. Search and anemotactic orientation of cockroaches. J. Insect Physiol. 25, 632 – 640. Carde´ , R.T., Mafra-Neto, A., 1998. Mechanisms of flight of male moths to pheromone. In: Carde´ , R.T., Minks, A.K. (Eds.), Insect Pheromone Research New Directions. Chapman & Hall, New York. Kanzaki, R., 1998. Pheromone processing in the lateral accessory lobes of the moth brain: flip-flopping signals related to zigzagging upwind walking. In: Carde´ , R.T., Minks, A.K. (Eds.), Insect Pheromone Research New Directions. Chapman & Hall, New York. Kanzaki, R., Ikeda, A., Shibuya, T., 1994. Morphological and physiological properties of pheromonetriggered flipflopping descending interneurons of the male silkworm moth, Bombyx mori. J. Comp. Physiol. A 175, 1 –14. Kennedy, J.S., 1977. Behaviorally discriminating assays of attractants and repellents. In: Shorey, H.H., McKelvey, J.J. Jr (Eds.), Chemical Control of Insect Behavior: Theory and Application. Wiley-Interscience, New York. Kramer, E., 1975. Orientation of the male silkmoth to the sex attractant bombykol. In: Denton, D.A., Coghlan, J.P. (Eds.), Olfaction and Taste V. Academic Press, New York.
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Kramer, E., 1976. The orientation of walking honeybees in odour fields with small concentration gradients. Physiol. Entomol. 1, 27 – 37. McMahon, C., Guerin, P.M., 2000. Response of the tropical bont tick, Amblyomma 6ariegatus (Fabricius), to its aggregation-attachment pheromone presented in an air stream on a servosphere. J. Comp. Physiol. A 186, 95 –103. Mishima, T., Kanzaki, R., 1998. Coordination of flipflopping neural signals and head turning during pheromone-mediated walking in a male silkworm moth Bombyx mori. J. Comp. Physiol. A 183, 273 – 282. Obara, Y., 1979. Bombyx mori mating dance: an essential in locating the female. Appl. Entomol. Zool. 14, 130 – 132. Olberg, R.M., 1983. Pheromone-triggered flip-flopping interneurons in the ventral nerve cord of the silkworm moth, Bombyx mori. J. Comp. Physiol. A 152, 297 – 307. Schal, C., Tobin, T.R., Surber, J.L., Vogel, G., Tourtellot, M.K., Leban, R.A., Sizemore, R., Bell, W.J., 1983. Search strategy of sex pheromone-stimulated male German cockroaches. J. Insect Physiol. 29, 575 – 579. Thiery, D., Visser, J.H., 1986. Masking of host plant odour in the olfactory orientation of the Colorado potato beetle. Entomol. Exp. Appl. 41, 165 –172. Weber, T., Thorson, J., Huber, F., 1981. Auditory behavior of the cricket. I. Dynamics of compensated walking and discrimination paradigms on the Kramer treadmill. J. Comp. Physiol. 141, 215 – 232. Wendler, G., Vlatten, R., 1993. The influence of aggregation pheromone on walking behaviour of cockroach males (Blattella germanica L.). J. Insect Physiol. 39, 1041 – 1050.
Virtual reality experiments on a digital servosphere: guiding male silkworm moths to a virtual odour source Masayuki Sakuma Laboratory of Insect Physiology, Graduate School of Agriculture, Kyoto Uni6ersity, 606 -8502 Kyoto, Japan
Abstract A digital ‘servosphere’ locomotion-compensation apparatus has been developed for use in experiments on the mechanisms of spatial orientation in ambulatory animals. The sphere is driven by a pair of digital AC-servo motors, which provide a negative feedback response to any displacements of the test animal. A high-speed video tracker continually reports the position of the animal to a computer, which controls the servo-motors via a motor-control board and also logs the position of the motor axes. Movements of the servos define the path of the test animal on a virtual plane. The computer also generates odour cues in real time via a relay-control board and an airflow system. Odour presentation is programmed to occur in response to the animal’s movements on the virtual plane, and can result in the animal being led to a virtual source. The servosphere was first used to investigate orientation of a walking male silkworm moth (Bombyx mori ) towards a female in still air. When a calling female moth was placed beside the sphere, the male moth beat its wings and walked straight towards the female. Unilaterally dewinged males also walked in a straight line, though slightly to one side of the direction of the female, but bilaterally dewinged moths remained stationary. This indicates that in still air the male moths orientate to pheromone sources by fanning air from in front of them over their antennae with their wings. If a moth continually reverses its turning movements when it encounters a train of pheromone concentration peaks, it will maintain a course towards the source and eventually arrive there. This idea was demonstrated on the servosphere by incorporating a computer-controlled solenoid valve which releases pheromone when a moth points towards a virtual odour source. In this sensory field, both intact and bilaterally dewinged males reached the source irrespective of the wind direction. This result supports the proposed orientation mechanism and also demonstrates
E-mail address: [email protected] (M. Sakuma). 0168-1699/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 9 9 ( 0 2 ) 0 0 0 2 1 - 2
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M. Sakuma / Computers and Electronics in Agriculture 35 (2002) 243–254
the applicability of the servosphere virtual-reality environment to the experimental investigation of insect orientation mechanisms. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Bombyx mori; Servosphere; Locomotion compensator; Orientation; Pheromone
1. Introduction When orientating, animals use external cues to modulate their behaviour in a manner that guides them towards a target. To investigate the cause of this type of behaviour, cues should be presented to the animals being tested in a controlled way (Kennedy, 1977). Conventional experiments in a wind tunnel or arena, however, allow test animals some degree of freedom, so that the cues they receive are not fully predetermined. An experimental arrangement that overcomes this problem is the ‘servosphere’, a locomotion-compensating device originated by E. Kramer and P. Heinecke (Thiery and Visser, 1986). Servospheres have been widely used in studies of orientation in insects and other small animals (Kramer, 1975; Bell and Kramer, 1979; Weber et al., 1981; Schal et al., 1983; Thiery and Visser, 1986; Wendler and Vlatten, 1993; McMahon and Guerin, 2000). The servosphere is a form of treadmill that compensates every locomotive movement of an ambulatory test animal. The animal walks freely on the top of a large sphere, which is rotated by a pair of motors orthogonally arranged at the sphere’s equator. A feedback system continually rotates the sphere about both axes so that the freely walking test animal is kept at the same spatial position (the top of the sphere), thus cues can be presented in a precisely controlled manner. If this apparatus is combined with an actuator that generates the cue, a virtual sensory field can be created and the orientation manoeuvring of the animal in response to it can be investigated in a fully automated experiment. In this paper, the construction of (1) a servosphere system that employs modern, digital, technology, and (2) a system for real-time control of an odour cue, are described. The paper also provides examples of the use of these systems in a virtual-reality investigation of the orientation behaviour of male silkworm moths homing in on an odour source. A more complete analysis of the orientation behaviours identified during this experiment will be reported elsewhere.
2. A digital-technology servosphere system The servosphere consists of a high-speed video tracker (G-240, OKK Inc., Tokyo, Japan), a personal computer (PC-9821, NEC, Tokyo, Japan) with motorcontrol board (QPG-45, Cosmotechs Co. Ltd., Yamato, Japan), a pair of digital AC-servo motors (MINAS MSM021-A1A, Matsushita Electric Industrial Co. Ltd., Kadoma, Japan) and drive wheels, a hollow perspex sphere supported by a universal ball bearing, and a steel frame (Fig. 1). The sphere has an outside
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diameter of 505 mm and a black suede-texture coating (Nextel 3101, Mankiewicz Gebr. & Co., Hamburg, Germany). It is made from a pair of hemispherical ceiling-lamp shields (YKA4387401, Matsushita Electric Industrial Co. Ltd., Kadoma, Japan) designed for use in streets and weighs 1.77 kg. The universal ball bearing is a 60 mm diameter plastic billiards ball. The drive wheels (SP442, Tamiya Inc., Shizuoka, Japan), which are fitted with 60 mm diameter sponge-rubber tires, are directly connected to the drive axles of the servo-motors. The entire system is controlled digitally. The video tracker captures the video image of the test animal at the top of the sphere, calculates its location relative to the centre of the image, and transmits the (x, y) coordinates to the computer through a DIO parallel interface (AZI-2746, Interface Corp., Hiroshima, Japan). This process is repeated continually at a rate of either 120 Hz (9 ms delay time) or 240 Hz (5 ms delay). The image captured by the tracker is displayed on an NTSC video monitor. The computer converts the coordinates to pulse counts for the motor-control board, which generates pulses to rotate the two servomotors so that the sphere is turned, about both axes, just sufficiently to take the animal back to the highest point. The amount of movement is controlled by reference to the outputs of high-resolution encoders (10 000 pulses per rotation) on the servomotor axes. The positional shifts of the axes, as pulse counts, are logged temporarily to the computer’s memory every 100 ms. They are later recorded in a file on the computer’s hard disk and converted to displacements of the sphere’s surface, from which the track of the test animal can be reconstructed. The use of a video tracker has several advantages. Since, the form of the captured image depends on the threshold exposure value for binary conversion, it can be
Fig. 1. The digital servosphere system.
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controlled to some extent by simply changing the aperture of the tracker’s lens. This may allow selection of either the animal’s whole body or just a small highlight. Attachment of a small reflector (Scotchlite, Sumitomo 3M Ltd., Tokyo, Japan) on the notum of an insect will create a highlight close to the insect’s turning axis, so that changes in course angle are easily and quickly identifiable. The speed and precision of the feedback control is adjustable by altering the zoom factor of the tracker’s lens and the multiplication factor for generating the motor-control pulses. By these means, tracking performance can be tuned specifically for each experiment. In the motor-control board, new data repeatedly override old ones, so the servomotors are always sent the pulse sequences required to return the animal directly to the highest point. The tracker’s resolution (6400× 4160 pixels) and sampling rate (120 or 240 Hz) are sufficient to allow persistent, smooth, and accurate tracking of a running insect. Delegation of these tracking and compensation tasks to intelligent peripheral devices frees up the PC’s CPU for processing of displacement data and the control of cue-presentation devices. The control software for this system is written in the C (ANSI) language (Quick-C, Microsoft, Seattle, USA) and runs under the MS-DOS 6.2 (Microsoft, Seattle, USA) operating system. The acquired data are analysed by a second program, written in the C + + language (Visual C+ + version 6 and Microsoft Foundation Class Library, Microsoft, Seattle), that is run under the WINDOWS 98 (Microsoft, Seattle, USA) operating system. Both programs were developed by the author.
3. Odour-cue control system Application of a servosphere (or any form of locomotion compensator) in behavioural research will usually involve the presentation of cues to a test animal, and observation of its responses. In many studies, cues have been presented independently of the animal’s movement, but more precise experiments have employed a feedback or closed-loop system. For example, Kramer (1976) exposed a honeybee, Apis melifera, to an odour cue that varied in concentration according to the bee’s position, and Bell (1986) exposed a walking male warehouse beetle, Trogoderma 6ariabile, to sex pheromone only while the male remained within a programmed corridor. In these experiments, the cue was controlled by the animal’s absolute position, but this may not reflect the cue perception of the animal which is more likely to be influenced by relative and directional factors. A programmed system for cue presentation in real time may make it possible to produce a more realistic representation of the animal’s perceived environment. The aim is to conduct a virtual-reality experiment, by presenting odour to the test animal in exactly the way it would occur during actual manoeuvring to a natural odour source. The digital servosphere system described here incorporates real-time control of cues. The main program computes the position, walking speed and direction of the test animal at a rate of 10 Hz, and controls presentation of odour by switching a
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Fig. 2. Cylindrical wind tunnel with a transparent cover used in the virtual-reality experiments (plan view). A three-way solenoid valve controlled by the PC switches the airflow between the odoured and bypass channels. Air is exhausted from the tunnel outlet at the same rate that it is supplied at the inlet.
solenoid-controlled valve via a Photo-MOS relay-module board (PRY-32, CONTEC Co. Ltd., Osaka, Japan). To insulate the experimental arena from outside influences, a cylindrical perspex cover (10 cm internal diameter× 3 cm deep) produces a horizontal-flow wind tunnel over the top of the sphere (Fig. 2). The ceiling of the cover is transparent, to allow imaging by the tracker. Illumination is by a ring fluorescent light (40 W, excitation at 40 kHz) when simulating daytime conditions and by IR light-emitting diodes (IREDs) when simulating night. These lights are suspended above the perimeter of the tunnel, to avoid obscuring the tracker’s view or causing reflections from the perspex cover. The airflow originates at a compressor and passes through glass fibre and then charcoal filters for the removal of condensed oil mists and organic contaminants (Fig. 2). A flowmeter (model RK200, Kofloc, Tokyo, Japan) in the line monitors the air-flow rate, which can be precisely adjusted by an attached miniature needle valve. The air is then remoistened in a bubbler, and its relative humidity (RH) is adjusted manually by changing a bypass ratio over the bubbler with a pair of needle valves while a humidity sensor (CHS-UGS, TDK, Tokyo, Japan) monitors the RH. A solenoid-controlled valve just before the inlet to the wind tunnel (Fig. 3) switches the flow between odoured and bypass channels. The odour source used in the experiment is a pheromone gland freshly removed from a calling female silkworm moth. The air is then removed from the tunnel through an exhaust port opposite the inlet by using an aspirator. The rate of suction is adjusted manually to the same
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rate as it enters by a flowmeter-jointed needle valve, producing a practically closed airflow within the tunnel. In the present experiment, the airflow comes from a single fixed inlet and the odour cue is controlled simply by switching between the odoured and bypass channels. Visualisation of the airflow with titanium-dioxide smoke shows it to be confined to a narrow stream between the inlet and outlet and to pass directly over the test animal. The speed of the flow, measured by a hot-wire anemometer ( c6312 airflow transducer with c0965-03 probe, Kanomax Co. Ltd., Suita, Japan), indicates considerable variation across the stream. Nevertheless, the arrangement, which certainly achieves the essential requirement of allowing odour to be presented only when the test animal orientates towards the (virtual) odour source, appears to produce the required virtual sensory field. Experimental evidence for this is presented in the next section.
4. Example: homing of male silkworm moths to a virtual odour source As an illustration of how the digital servosphere can be used for virtual-reality experiments, some results from a trial in which walking male silkworm moths (Bombyx mori ) responded to a computer-generated odour field are presented here. The response of a male silkworm moth to sex pheromone is characterised by a turning motion and wing beating, a behaviour referred to as the ‘mating dance’. Although it has previously been suggested that the function of wing beating is to draw in pheromone-laden air (Obara, 1979), cues responsible for the homing of the moth to an odour source and the orientation responses by which homing is achieved have not yet been fully identified.
Fig. 3. The servosphere, air flow and illumination systems.
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Fig. 4. Tracks in the virtual plane of a male moth walking for 10 min on the servosphere in still air at distances, d, of (a) 10 cm, (b) 20 cm and (c) 40 cm from a calling female placed on the right. The radius of the dotted circle is 2.0 m. Open circles on the tracks indicate 30 s time intervals. (d) The linearity of the tracks, is represented by the mean vector length, r.
In a preliminary experiment, the servosphere was used to observe walking of male moths towards a calling female in still air, i.e. without making use of the wind tunnel described in the previous section. When a calling female moth on a perch was placed 70–100 mm from a male moth on the servosphere, and a smoke source placed near the female’s pheromone gland was used to visualise the resulting pheromone plume, it was observed that the plume flowed over the antennae of the male as it walked towards the female. The male moth was beating its wings, and this appeared to have the effect of drawing in air from in front of it and fanning this air over its antennae. Modulation of the odour concentration in the fanned air as the moth turns provides a cue that allows the moth to orientate itself towards the source. Tracks shown in Fig. 4 are those of a male moth in still air at various distances from a female. The linearity of the tracks, represented by the mean vector length r (Batschelet, 1981), decreased as the distance increased. Fig. 5 shows the tracks made by three male moths: (a) intact, (b) right wing amputated, (c) both wings removed. A calling female was placed on a perch 10 cm to the right of the top of sphere. It can be seen that moths (a) and (b) walked along a nearly straight track, although (b) was deflected to the right of the direct line to the moth, presumably because of unbalanced fanning. Moth (c) remained stationary, again suggesting the importance of fanning for orientation.
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Fig. 5. Tracks in the virtual plane of male moths: a (intact), b, (right wing amputated) and c (de-winged). The dotted circle indicates 1.0 m and the open circles on the tracks show 30 s time intervals.
From these observations, the following orientation mechanism may be inferred (Fig. 6). While turning, a male moth beats its wings to draw-in air from in front of it and passes the air over its antennae (Fig. 6a). The moth will thus be most exposed to pheromone when it is heading towards the source, in other words, the timing of
Fig. 6. Orientation mechanism of male B. mori moths inferred from results of Fig. 5. (a) In the presence of sex-pheromone odour, the moth keeps turning while beating its wings to draw air from in front of it over its antennae. (b) When the moth faces an odour source, turning is repeatedly reversed in response to pulses of odour concentration, and the turning is transformed into a straight walk towards the source.
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pheromone perception coincides with the moth’s orientation being towards the source. There is increasing evidence that moths perceive pheromone odour as an intermittent stimulus reflecting a pulsed cloud of pheromone rather than continuous presence of pheromone in a concentration gradient (Baker et al., 1985). A recently proposed neural mechanism working in the moth’s central nervous system suggests that each pulse of pheromonal stimulation triggers the switching between the high and low excitation states of certain descending interneurons (Olberg, 1983; Kanzaki et al., 1994; Kanzaki, 1998). It has been noted that some of these interneurons in the left and right connectives show an antiphasic pattern of excitation similar to that occurring in a ‘flip-flop’ electronic circuit. Thus flipflopping in these interneurons could cause reversal of the turning direction as each pulse of pheromone is detected (Mishima and Kanzaki, 1998), and repeated reversals while the moth is pointing directly at the source should result in the moth walking straight towards it (Fig. 6b). This possible mechanism can be examined by modulating the odour concentration automatically within the wind-tunnel arena at the top of the servosphere. In this experiment, a ‘virtual’ odour source is created by switching the solenoid valve to release pheromone when the moth’s course is towards the (previously designated) virtual position of the source. The main program computes the virtual position of the travelling moth every 100 ms, and estimates its instantaneous course direction by comparing sequential positions. The direction of the virtual odour source from the moth’s current position is also computed, and if this lies within 922.5° of the moth’s course, pheromone is released. Although the program does not intrinsically provide pulsed odour, the moth’s frequent turning effectively generates pulses of 0.2 s duration at a frequency of 1.0 Hz. The resulting tracks, together with time series of the male’s speed and orientation, are shown in Fig. 7. When the source was placed at virtual coordinates of (2.0, 2.0) m relative to the male moth’s starting position, the male proceeded almost straight towards the source and stayed very close to it after arriving 3 min later (Fig. 7a). The track and the graphs of velocity and orientation all indicate that the moth moved almost directly to the source at (2, 2) m and stayed there after arriving 3.0 min after setting out. The darker sections of the tracks and graphs indicate when the moth was heading within 9 22.5° of the source, and so causing pheromone to be dispensed. Orientation was thus successfully controlled by the automatic release of pheromone, whereas the walking speed changed little through the experiment. Anemotaxis (Kramer, 1975) appears to have contributed little to the results, because the direction of the airflow was fixed and different from that of the source, and because the moth remained very close to the source after arriving there, even though it continued to experience a directed airflow. After amputation of both pairs of wings, the moth was still able to walk directly to the virtual source under the guidance of the automatic odour-release system (Fig. 7b), i.e. a dewinged male, although unable to generate pulses of odour concentration by itself, could reach the virtual source with artificially
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Fig. 7. Tracks of male moths in response to a virtual odour source; (a) intact, (b) de-winged, (c) de-winged and downwind source. The radius of the dotted circle is 0.5 m, and the dotted arrow indicates the direction of the 2.0 ms − 1 airflow.
pulsed odour stimulation. Moreover, the wingless male could even be led to a source placed downwind at the same distance (Fig. 7c). This clearly indicates that upwind anemotaxis is not a major orientation mechanism for a walking male silkworm moth. However, the greater time taken for the moth to reach the downwind source suggests that a secondary role for upwind anemotaxis cannot be ruled out. The orientation mechanism mentioned above works at close range to the source, but the same mechanism probably also functions to locate a pheromone plume. In preliminary experiments conducted in laminar airflow, moths drew in, by fanning, an odour plume passing close by them and moved predominantly towards the plume. They would have soon reached the main stream of the plume if they had not been dynamically tethered by the servosphere. After entering the plume, they could be expected to follow it in the same manner as they locate a point odour source. It seems also possible for flying moths to draw air by wing-beating. Although the forward motion of flying moths through the air seem to preclude the need for it, the coincidence of timing of pheromone perception with the moth’s orientation being towards a cloud of pheromone might play a role either as a component of, or in addition to, the various orientation mechanisms already established for them (Baker and Vickers, 1998; Carde´ and Mafra-Neto, 1998). This is a topic for future work.
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5. Conclusion The digital servosphere, wind-tunnel arena, and computer-controlled real-time odour-release system described here have made it possible to create a programmed odour environment on an endless virtual plane. This virtual-reality environment appears to reproduce the cues and to stimulate the manoeuvring responses that cause male B. mori to home on a calling female in natural conditions. These trial results suggest that this apparatus will find application in the experimental investigation of the mechanisms of orientation behaviour in this and other ambulatory, odour-responsive, species.
Acknowledgements Dr J.H. Visser (Plant Research International, The Netherlands), OKK Inc. (Tokyo, Japan), Cosmotechs Co. Ltd. (Yamato, Japan), and Kanomax Co. Ltd. (Suita, Japan) provided invaluable advice on, and support for, the construction of the digital servosphere. I thank the editors of this special issue for their critical reading and editing of the manuscript. This work was supported by a Grant-in-Aid for Scientific Research (C)(2) No.10660046 from the Japan Society for the Promotion of Science.
References Baker, T.C., Vickers, N.J., 1998. Pheromone-mediated flight in moths. In: Carde´ , R.T., Minks, A.K. (Eds.), Insect Pheromone Research New Directions. Chapman & Hall, New York. Baker, T.C., Willis, M.A., Haynes, K.F., Phelan, P.L., 1985. A pulsed cloud of sex pheromone elicits upwind flight in male moths. Physiol. Entomol. 10, 257 – 265. Batschelet, E., 1981. Circular Statistics in Biology. Academic Press, London. Bell, W.J., 1986. Responses of arthropods to temporal chemical stimulus changes: simulation of a humidity differential and pheromone plume. In: Payne, T.L., Birch, M.C., Kennedy, C.E.J. (Eds.), Mechanisms in Insect Olfaction. Clarendon Press, Oxford. Bell, W.J., Kramer, E., 1979. Search and anemotactic orientation of cockroaches. J. Insect Physiol. 25, 632 – 640. Carde´ , R.T., Mafra-Neto, A., 1998. Mechanisms of flight of male moths to pheromone. In: Carde´ , R.T., Minks, A.K. (Eds.), Insect Pheromone Research New Directions. Chapman & Hall, New York. Kanzaki, R., 1998. Pheromone processing in the lateral accessory lobes of the moth brain: flip-flopping signals related to zigzagging upwind walking. In: Carde´ , R.T., Minks, A.K. (Eds.), Insect Pheromone Research New Directions. Chapman & Hall, New York. Kanzaki, R., Ikeda, A., Shibuya, T., 1994. Morphological and physiological properties of pheromonetriggered flipflopping descending interneurons of the male silkworm moth, Bombyx mori. J. Comp. Physiol. A 175, 1 –14. Kennedy, J.S., 1977. Behaviorally discriminating assays of attractants and repellents. In: Shorey, H.H., McKelvey, J.J. Jr (Eds.), Chemical Control of Insect Behavior: Theory and Application. Wiley-Interscience, New York. Kramer, E., 1975. Orientation of the male silkmoth to the sex attractant bombykol. In: Denton, D.A., Coghlan, J.P. (Eds.), Olfaction and Taste V. Academic Press, New York.
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Kramer, E., 1976. The orientation of walking honeybees in odour fields with small concentration gradients. Physiol. Entomol. 1, 27 – 37. McMahon, C., Guerin, P.M., 2000. Response of the tropical bont tick, Amblyomma 6ariegatus (Fabricius), to its aggregation-attachment pheromone presented in an air stream on a servosphere. J. Comp. Physiol. A 186, 95 –103. Mishima, T., Kanzaki, R., 1998. Coordination of flipflopping neural signals and head turning during pheromone-mediated walking in a male silkworm moth Bombyx mori. J. Comp. Physiol. A 183, 273 – 282. Obara, Y., 1979. Bombyx mori mating dance: an essential in locating the female. Appl. Entomol. Zool. 14, 130 – 132. Olberg, R.M., 1983. Pheromone-triggered flip-flopping interneurons in the ventral nerve cord of the silkworm moth, Bombyx mori. J. Comp. Physiol. A 152, 297 – 307. Schal, C., Tobin, T.R., Surber, J.L., Vogel, G., Tourtellot, M.K., Leban, R.A., Sizemore, R., Bell, W.J., 1983. Search strategy of sex pheromone-stimulated male German cockroaches. J. Insect Physiol. 29, 575 – 579. Thiery, D., Visser, J.H., 1986. Masking of host plant odour in the olfactory orientation of the Colorado potato beetle. Entomol. Exp. Appl. 41, 165 –172. Weber, T., Thorson, J., Huber, F., 1981. Auditory behavior of the cricket. I. Dynamics of compensated walking and discrimination paradigms on the Kramer treadmill. J. Comp. Physiol. 141, 215 – 232. Wendler, G., Vlatten, R., 1993. The influence of aggregation pheromone on walking behaviour of cockroach males (Blattella germanica L.). J. Insect Physiol. 39, 1041 – 1050.