J. exp. Biol. 130, 425-432 (1987) 4 2 5Printed in Great Britain © The Company of Biologists Limited 1987

SHORT COMMUNICATION

A NEW MICROCOMPUTER-BASED METHOD FORMEASURING WALKING PHONOTAXIS IN FIELD

CRICKETS (GRYLLIDAE)

BY JOHN A. DOHERTY* AND ANTHONY PIRES

Section of Neurobiology and Behavior, Seeley G. Mudd Hall, Cornell University,Ithaca, NY 14853, USA

Accepted 13 March 1987

Calling songs of male crickets attract sexually mature, conspecific females formating (for recent reviews see Eisner & Popov, 1978; Huber & Thorson, 1985;Doherty & Hoy, 1985). This communication system has been the subject of manybehavioural studies on the relevant properties of the male's calling song forrecognition by females (e.g. Walker, 1957; Popov & Shuvalov, 1977; Pollack & Hoy,1979, 1981; Thorson, Weber & Huber, 1982; Stout, DeHaan & McGhee, 1983;Doherty, 19856; Doolan & Pollack, 1985). The behavioural investigations haverequired some means of measuring the female's phonotaxis (her locomotion orturning towards a sound source). These include 'closed-loop' methods in which theanimal moves freely in acoustic space and 'open-loop' methods in which the animal istethered and is not allowed to experience changes in sound intensities as it runs in thedirection of the sound source. Closed-loop methods include free walking in arenas(e.g. Murphey & Zaretsky, 1972; Paul, 1976; Hoy, Hahn & Paul, 1977; Pollack &Hoy, 1979; Stout et al. 1983); open-loop methods include tethered flight (Moiseff,Pollack & Hoy, 1978; Pollack & Hoy, 1979, 1981; Pollack, Huber & Weber, 1984),tethered walking on Y-maze globes (Hoy & Paul, 1973) and free walking on aspherical locomotion compensator (Wendler, Dambach, Schmitz & Scharstein,1980; Thorson & Huber, 1981; Thorson et al. 1982; Pollack et al. 1984; Doherty,I985a,b,c; Schmitz, 1985).

Here we describe a new open-loop method for quantifying cricket phonotaxis.This method uses inexpensive microcomputer technology, is completely automatedand therefore rapid and objective, and could be adapted for studying locomotorymovements of other animals. The data obtained are in such a form that they are easilycompared to data generated by the spherical locomotion compensator (Kramertreadmill, see Kramer, 1976; Wendler^ al. 1980; Weber et al. 1981), which has beenused for quantifying phonotaxis in the field cricket, Gryllus bimaculatus (Doherty,1985a, b,c).

•Present address: Department of Biology, Villanova University, Villanova, PA 19085, USA.

Key words: crickets, phonotaxis, communication, microcomputer.

426 J. A. DOHERTY AND A. PlRES

The spherical locomotion compensator or Kramer treadmill was first developed byscientists in West Germany (Kramer, 1976; Wendlere* al. 1980; Weber et al. 1981;Thorson et al. 1982). It uses an infrared detection system to monitor movements ofan untethered cricket on top of a sphere. This positional information feeds back toservomotors that move the sphere in the opposite direction. These compensatorymovements 'fix' the cricket at the top of the sphere as it performs walking phonotaxis.The colloquial name for this locomotion compensator is 'Kugel' (German translationof the word 'sphere'). Because our device for measuring walking phonotaxis utilizesthe Apple Macintosh computer and is similar in appearance to the German Kugel,we call our new device the 'MacKugeP. The main mechanical difference betweenthese two devices is that in the MacKugel system the cricket is tethered and its ownwalking movements provide the power to rotate the sphere.

The MacKugel is a complete stimulus control and data acquisition system for on-line studies of cricket phonotaxis. Data on cricket movements during phonotaxisexperiments are passed directly to an Apple Macintosh microcomputer, which canalso synthesize acoustic stimuli or send control signals to an external acousticsynthesizer. In designing this system, we took advantage of ROM routines in theMacintosh for quantifying movements of the Apple 'mouse'.

The mouse is a mechanical, optoelectronic device that converts rotationalmovements of a rubber ball to changes in the coordinates of a cursor displayed on thecomputer's CRT monitor. The ball bears against the operator's desk surface, andagainst three rollers inside the ball's housing. One roller is for mechanical supportonly. The other two, representing orthogonal x- and y-axes, are connected to rotatingvanes which interrupt light beams between LED—phototransistor pairs. Thephototransistors produce electronic signals which are sent to the computer.

We adapted the mouse to study cricket phonotaxis by expanding the distancebetween the rollers and replacing the small rubber ball (2-5 cm diameter) with alarger and much lighter sponge-rubber ball (10 cm diameter, 8-7g). This larger ball(a hollowed-out, toy 'Nerf ball' from Parker Bros, Beverly, MA, USA) was mountedin a frame along with the roller/phototransistor modules from the mouse (Fig. 1). Inthis way the rollers could be actuated by a tethered cricket walking on top of the ball.As the cricket ran in one direction (i.e. towards an attractive acoustic stimulus), theball was moved in the opposite direction and the movements were transduced intochanging pixel coordinates on the Macintosh screen. The minimum force required tomove the Nerf ball ranged from 1-117 to 2-793xlO~2N. A plastic sphere (10 cmdiameter, 12-8 g) is currently under development and this improved version onlyrequires forces ranging from 0-69 to 1-05x10" N. High-level computer languages(Macintosh Pascal®) were used for data acquisition and analysis (program availablefrom author). By sampling pixel coordinates once every second, we were able tocalculate instantaneous velocity and direction profiles for the cricket's movements.These profiles, along with their corresponding 'vector plots' (see below), weredisplayed simultaneously on the Macintosh screen, and the data were saved onmicro-floppy disks for subsequent data analysis.

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Fig. 1. Side (A) and top (B) view of MacKugel device. TM, tether mount; F, frame;RT, rotating tether; PR, passive roller; T, , x-axis tachometer (composed of photo-transistor module, extension axle and roller); Ty, y-axis tachometer; FB, frictionlessbearing (composed of a 1 cm Teflon ball riding on a film of air); AS, air supply (air routedthrough a threaded aluminium cylinder used to adjust the height of the Nerf ball);S, sphere.

To establish the usefulness and validity of our method, we repeated somelocomotion compensator experiments run previously on the field cricket, G. bimacu-latus (Doherty, 1985a,b). In both methods, walking phonotaxis in crickets can bequantified by measuring time profiles of the cricket's walking velocity and direction.Fig. 2 shows these time profiles for one MacKugel experiment in which a syntheticcalling song was presented to a female G. bimaculatus from one of two loudspeakersseparated by 180°. This calling song was played from the left speaker for 2min andthen from the right speaker for another 2min. A 30-s silent period preceded andfollowed this playback. The female tracked the stimulus, as shown by her narrowmeandering about the direction of the active speaker. Her walking velocity alsoincreased when the stimulus was presented, compared to the preceding silent period.

The time profiles shown in Fig. 2 were generated by sampling and storing theposition of the animal once each second during an experiment. This method of datacollection easily lent itself to another way of representing phonotactic movementsvisually. For each 1-s interval, a vector was calculated that showed the animal'swalking direction relative to the direction of the active speaker (vector angle) and itsvelocity (vector length). These vectors were cumulatively plotted as shown in Fig. 2.These 'vector plots' show that when the stimulus was presented from either the left orright speaker, the vectors were clustered about the direction of the active speaker.

428 J. A. DOHERTY AND A. PlRES

The direction component of cricket walking phonotaxis is a sensitive indicator ofthe attractiveness of an acoustic stimulus. To investigate this sensitivity further, weran phonotaxis experiments in which acoustic stimuli with different pulse periodswere presented in a sequential, to-and-fro paradigm. As in earlier experiments ofcricket phonotaxis on a locomotion compensator (Doherty, 1985c), pulse periodsranging from 30 to 50 ms yielded the best tracking on the MacKugel (i.e. bestorientation to active speaker direction, see Fig. 3).

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Fig. 2. Phonotaxis of a female Gryllus bimaculatus on the MacKugel device. A syntheticcalling song composed of 4-pulse chirps repeated at a constant rate (40 ms pulse period,PP; 4 pulses per chirp, PN; 22 ms pulse duration, PD; 400 ms chirp period, CP; shown inbox) was presented from one of two speakers for 4 min. The cricket's movements weretranslated into cursor movements on the CRT of a microcomputer. The cursor's position(in x—y pixel coordinates) was sampled once every second and vector plots and timeprofiles of the cricket's velocity and angular direction were calculated and plotted duringthe experiment. The two vector plots show the accumulation of vectors during stimulusplaybacks from the two loudspeakers. Each vector has an angle and a length. Vector angleis the direction the animal was moving during the sample interval, relative to the positionof the active speaker. Vector length is directly proportional to the animal's velocity. Everyvector was plotted with its origin at the centre of the vector plot circle. The radius of thiscircle corresponds to a velocity of 3-4cms~'. The solid dot beside each vector plotindicates the position of the active speaker (left or right speaker; LS, RS), whichcorresponds to the active speaker position (serrated horizontal line at either 90° or 270°)in the angular direction profile below. The numbers above each vector plot are the vectorscore (left-hand number, defined in text) and the percentage of the stimulus presentationtime that the animal was moving (right-hand number). The vector score (2426) and thepercentage of time spent moving (97 %) were both higher when the stimulus was playedfrom the right speaker.

Measurement of phonotaxis in crickets 429

In these same phonotaxis experiments on the MacKugel we found slight effects ofpulse period on the walking velocity profiles. Females walked for a greater percentageof the stimulus presentation time when pulse periods were optimal (i.e. 30-50ms)

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Fig. 3. Effects of synthetic calling songs with different pulse periods on the accuracy oforientation to the speaker. Orientation was quantified as follows: for each 1-s interval of atrial in which the animal was moving, the cosine of the vector angle was calculated. Amean cos (angle) was then obtained for all such intervals within a trial. An averagecos (angle) was computed for all trials of each pulse period and plotted as orientation tospeaker. If the cricket ran directly towards the loudspeaker (defined as 0°), the cos (angle)was 1; this value was — 1 when it ran directly away from the speaker and 0 when it ranperpendicular to the sound source. The points are means for each pulse period and thevertical lines are standard errors. Data were based on to-and-fro scans of pulse period for21 female Gryllus bimaculatus. Sample sizes are shown.

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Fig. 4. Comparison of two different methods of quantifying stimulus efficacy insequential, to-and-fro scans of pulse period (see text and Doherty, 1985a,c). The twomethods were (A) manual measurements of the percentage of stimulus presentation timethat females tracked the stimulus and (B) microcomputer measurements of vector score.The criteria for manual scoring of stimulus tracking are found in Doherty (1985a,c). Thepoints are mean values and the vertical lines are standard errors. Nine Gryllusbimaculatus females were tested. Sample sizes are the same in A and B.

80

430 J. A. DOHERTY AND A. PlRES

and walked less in response to stimuli with pulse periods outside this range.Furthermore, mean walking velocity was highest in response to pulse periods of 35and 40 ms, whether or not pauses in walking were included in the calculation.

Because both walking velocity and direction were affected by acoustic stimuli, wedevised a numerical 'vector score' which incorporates both of these components andserves as a quick measure of the phonotactic efficacy of different sound stimuli. Thisscore is defined as:

Vector score = 2[cos(vector angle) X vector length].

In calculating the vector score, the angular direction of the active speaker is alwaysdefined as 0° and the summation is over all the sample intervals within a trial. Whenthe animal runs towards the loudspeaker the vector score increases, when it runsaway from the loudspeaker the score decreases. By definition, movements perpen-dicular to the speaker axis (cos 90° or cos 270°) and no movements at all (vectorlength = 0) result in vector scores approaching zero. For example, in Fig. 2, thevector score in response to calling song from the left speaker was lower than that forthe right speaker. This difference was due to less accurate tracking, widermeanderings about the speaker direction and more pauses in walking.

The vector score is comparable to other measures of stimulus efficacy in cricketphonotaxis. In earlier studies of G. bimaculatus phonotaxis on a locomotioncompensator, the attractiveness or efficacy of an auditory stimulus was quantified bymeasuring the percentage of the stimulus presentation time that the female clearly'tracked' the stimulus. Tracking has been defined as meandering within a certainangle 'window' (±60°) about the angular direction of the active speaker (see Thorsonet al. 1982; Doherty, 1985a,b,c). These same criteria were used in our study formeasuring the percentage of time spent tracking in a group of nine G. bimaculatusfemales. In to-and-fro sequential experiments, tracking scores and vector scores inresponse to calling songs with different pulse periods were comparable (Fig. 4). Bothscores showed that songs with pulse periods between 35 and 50 ms were mosteffective in eliciting positive phonotaxis.

The MacKugel system is also useful for quantifying cricket phonotaxis in two-stimulus (choice) playback experiments. The total vector score was a sensitive,objective measure of the relative attractiveness of alternative acoustic stimuli. Whengiven a choice between alternating chirps with different pulse periods (PP), femaleG. bimaculatus clearly preferred the standard chirp stimulus (40 ms PP) to thealternative chirp stimuli with pulse periods of 20, 30, 50 and 60 ms. The strength ofthe female's preference for the standard over the alternative pulse period was alsoreflected in the mean vector score. These results were consistent with those ofprevious choice experiments on a locomotion compensator (Doherty, 1985c).

The MacKugel system is an efficient, rapid and objective method for measuringthe efficacy of different acoustic stimuli in eliciting walking phonotaxis of crickets.Using this system the data can be displayed to give a qualitative, visual represen-tation of an individual cricket's movements. Complicated time profiles of movementvelocity and direction can be reduced objectively to a numerical score that can be

Measurement of phonotaxis in crickets 431

used as a rough basis of comparison between individuals and between experimentaltreatments. Data analysis can be done 'on-line' as the animal is behaving. Otherstrengths of the MacKugel are its low price, simplicity of design, and efficientimplementation of existing microcomputer technology.

This research was supported by grants from NSF and NIH to JAD, AP andRonald R. Hoy. We thank Alan Cohen and Rick Nicoletti for technical assistance andWendy Sussdorf for preparing Fig. 1. Ronald Hoy, Peter Brodfuehrer and JudCrawford read earlier drafts of this manuscript.

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