Buenos Aires – 5 to 9 September, 2016 Acoustics for the 21st Century… PROCEEDINGS of the 22nd International Congress on Acoustics Animal Bioacoustics: Paper ICA2016-908 Phonotactic response depends on trackball surface texture in Gryllus bimaculatus (Gryllidae, Orthoptera) Edith Julieta Sarmiento-Ponce(a), Berthold Hedwig(a), Michael Sutcliffe(b) (a) University of Cambridge, Department of Zoology, Downing Street, Cambridge, CB2 3EJ, United Kingdom, [email protected], [email protected] (b) University of Cambridge, Department of Engineering, Trumpington Street, Cambridge, CB2 1PZ, United Kingdom, [email protected] Abstract Mating behaviour in crickets is driven by acoustic communication. Phonotaxis is the behavioural process in which females are attracted and orient to male calling songs. We tested the female phonotactic response when the animals were walking on trackballs with different surface textures. Textures were measured with profilometry and were characterised as smooth, medium, or rough, with pore sizes of ~150, ~460, and ~800 micrometer, respectively. Female crickets walk better and have a higher phonotactic response on a rough or medium trackball surface, with numerous and large pores. A smooth surface, with small or few pores, prevents female crickets from walking properly, resulting in a significant decrease of their phonotactic response. Cricket claws are crucial for walking. Crickets hold on to the trackball by inserting their claws into the surface pores. If the surface is smooth or slippery, the crickets slide their feet and claws over the surface but cannot make proper mechanical contact. These findings may inform other studies that use trackball or treadmill systems, or arena experiments. The surface on which crickets are walking is crucial to obtain the optimal phonotactic response in behavioural studies. Keywords: field cricket, animal behaviour; profilometry; depth profile; treadmill study 22nd International Congress on Acoustics, ICA 2016 Buenos Aires – 5 to 9 September, 2016 Acoustics for the 21st Century… Phonotactic response depends on trackball surface texture in Gryllus bimaculatus (Gryllidae, Orthoptera) 1 Introduction For more than 30 years behavioural studies on crickets have been using treadmills or trackballs to measure phonotactic behaviour1–4. However, the texture or material of the ball is rarely mentioned. We found that the texture of the trackball surface is very important for the phonotactic performance of the crickets. In this study we discovered that crickets walk better on trackballs with a rough surface, resulting in faster and longer phonotactic responses, as compared to the weak auditory responses in experiments with smooth trackballs. Ants, bees, beetles, cockroaches, and bush crickets have special pretarsal adhesive organs in their legs to be able to walk on slippery or smooth surfaces 5,6. Gryllus bimaculatus is a ground living species that uses its claws to enhance the mechanical contact when the legs generate thrust and the pretarsal pads to stabilize its walking. In this study we also measured the leg force that crickets can produce on the three trackball surfaces. Our findings may have important implications on arena and trackball experiments analysing cricket phonotaxis. 2 Methods 2.1 Animals and trackball system Female crickets G. bimaculatus were taken as last mature stages from a colony at the Cambridge Department of Zoology and were raised individually to maintain phonotactic responsiveness 7. Individual crickets were kept on woodchips in 1.5l plastic containers at 25- 28°C with a 12-h light:dark cycle. They were fed protein-rich diet and water. After the final moult, a metal pin (32 mg; cricket, 1.2g) was attached vertically with wax to the first abdominal tergite, close to the animal’s centre of gravity. A trackball system was used to perform phonotaxis experiments 4,8; 25 female crickets aged 7-20 days after the final moult were tested three to five times. All experiments were performed in the dark at a temperature of 25-28°C. 2.2 Sound stimuli Sound stimuli were generated with Cool Edit 2000 (Syntrillium, now Adobe Audition) and were presented by two speakers (Neo 13s, Sinus live, Conrad Electronic, Germany). Speakers were positioned in front of the cricket at a distance of 57 cm and at an angle of 45º to the left and right of the animal’s longitudinal axis. Sound intensities were adjusted to 75 dB SPL relative to 10-5 Nm-2 at the position of the cricket4,8. Our standard calling song sound pattern had a frequency of 4.8 kHz, 75 dB SPL and 34 ms pulse period, and was presented for 30 s from the left and right side (Figure 1a). The phonotactic walking component towards the active speaker (i.e. the lateral 2 22nd International Congress on Acoustics, ICA 2016 Buenos Aires – 5 to 9 September, 2016 Acoustics for the 21st Century… deviation) was measured from the trackball rotations. The experiments were performed inside a sound-proofed chamber. N = 25 crickets tested 3 times each, for each of the three trackballs, resulting in 225 tests. 2.3 Trackball surfaces Three types of Rohacell trackballs (Evonik, Darmstadt, Germany) with different pore diameters were used: Rohacell 31 HF with ~ 150 µm, Rohacell 31 IG with ~ 460 µm, and Rohacell 31 IG-F with ~ 800 µm (Figure 2). The smooth trackball was additionally covered with conductive paint to make the surface smoother, decreasing the pore size to 40-80 µm as well as the overall number of pores. 2.4 Profilometer measurements An Olympus BX51 optical microscope with 5 objective was used to measure z-stack images of three Rohacell trackballs. Montage images of the three surfaces are shown in Figure 2; these have been constructed within Olympus LAS software by merging the focussed pixels from each image. Height data was calculated from the image stack using bespoke Matlab software following the general shape-from-focus (SFF) technique described by Pertuz9. The method selects, for a given pixel in the image, the image from a z-stack which has the best degree of focus. Firstly the Laplacian of Gaussian measure of focus was calculated for all the pixels in the z-stack. This data was then smoothed within each image in the lateral dimensions using a square averaging filter and downsampled in the in-plane dimensions of the images by a factor of 4 to make the calculations more manageable. The peak in the focus measure as a function of z position for each pixel was then found, after removing slowly-varying changes in the focus measure. Smoothing of the variation of focus measure with height, and use of a quadratic fit to the peak, were used to improve the accuracy and robustness of the estimate. Finally a median filter was applied to the resulting variation of height data with in-plane dimensions to remove ‘salt and pepper’ noise in the estimated height variation. The resulting height contours with line plots running horizontally across the centre of each of the figures are combined in Figure 2. To quantify the roughness of each surface the height data for each sample, as per Figure 3, was split into 10 strips. The Rq roughness for each of these ten strips was then evaluated. 2.5 Statistical analysis All data was analysed in R software (Ver. 3.2.2). We used one-way ANOVA to quantify whether trackball texture influenced female response to acoustic stimuli. We used Tukey HSD tests to ascertain significant differences among the three trackball textures when ANOVA results were significant. We used Matlab to perform a 2-sided t-test comparing the roughness amplitude data for the three surfaces. The significance level was set at 0.01. 3 22nd International Congress on Acoustics, ICA 2016 Buenos Aires – 5 to 9 September, 2016 Acoustics for the 21st Century… 3 Results 3.1 Phonotactic behavioural analysis Female crickets walking on three trackball surfaces exhibited different phonotactic responses (Figure 1). The highest phonotactic response, measured as the lateral deviation towards the active speaker for the duration of the test period, was generated on the rough surface (43.9±9.4 cm, Figure 1), followed by the response on the medium surface (36.6±10.1 cm, Figure 1). The phonotactic response significantly decreased on the smooth surface (6.7±5.9 cm, Figure 1). The phonotactic response on the smooth trackball was significantly lower (n=25; ***P<0.001, ANOVA with Tukey's post hoc test, Figure 1b) in comparison to the medium and rough trackballs. However, the phonotactic responses for the rough and medium surfaces were significantly different (n=25; **P<0.01, ANOVA with Tukey's post hoc test, Figure 1b). Figure 1: A. Female cricket phonotactic lateral deviation towards the active speaker on the three trackball surfaces at 75 dB SPL, 34 ms pulse period, and 4.8 kHz; sample responses of one animal. B. Boxplot revealing how the three trackball surfaces affect phonotaxis. Lateral deviation (cm), including Tukey HSD post-hoc analysis. 4 22nd International Congress on Acoustics, ICA 2016 Buenos Aires – 5 to 9 September, 2016 Acoustics for the 21st Century… 3.2 Trackball surfaces The rough surface presented had the largest and deepest pores (Figure 2). The mean Rq roughness for the smooth, medium and rough surfaces were 0.0053, 0.013 and 0.11 µm, respectively, with the t-tests showing significant differences between these roughness amplitudes (***p < 10-8). Figure 2: A. Trackball surface texture of a rough, medium, and smooth trackball. B. Depth profile of the three trackball textures. C. Probability density function (1/mm) of the three trackball textures. 5 22nd International Congress on Acoustics, ICA 2016 Buenos Aires – 5 to 9 September, 2016 Acoustics for the 21st Century… 4 Discussion Trackball experiments are crucial for analysing and understanding in detail phonotactic behaviour not only in crickets, but also in other insect species.