Experimental set-up.

<p>A schematic drawing of the experimental setup showing the initial position of the male on the top of the nettle plant and the positions of vibration exciters (not drawn to scale).</p

A linalool background alters orientation of male <i>Spodoptera littoralis</i> to pheromone source.

<p>Walking activity and orientation behaviour of male <i>Spodoptera littoralis</i> were measured on a locomotion compensator. Males were submitted to the main pheromone component, Z9E11–14:Ac during 2 min, <b><i>1a:</i></b> a neutral background was maintained for the duration of the test (n = 30). <b><i>1b</i></b>: in addition to pheromone, a linalool background was applied after 1 min (n = 42). Mean upwind speed: average of the distances per second walked by males in direction of the source (solid line) and mean ± SEM (dashed lines). Orientation index: oi = cos (θ) * ρ, where θ is the mean angle of track sample and ρ the length of the mean vector at t_i. Horizontal grey bar = linalool background.</p

Effects of a linalool background on the decay of the firing response to a single pheromone pulse.

<p>The mean firing rate of Ph-ORNs in response to the 1st pulse of a pheromone pulse train at 1 pps in neutral background (dashed line, mean of n = 54 recordings) or linalool background (solid line, n = 45) was calculated. Then, we estimated the exponential decay function in neutral (inset: dashed line) or linalool background (solid line) in normalized scale; black points in the inset: 90% fall decay.</p

The Effect of Timing of Female Vibrational Reply on Male Signalling and Searching Behaviour in the Leafhopper <i>Aphrodes makarovi</i>

<div><p>Sexual communication in animals often involves duetting characterized by a coordinated reciprocal exchange of acoustic signals. We used playback experiments to study the role of timing of a female reply in the species-specific duet structure in the leafhopper <i>Aphrodes makarovi</i> (Hemiptera: Cicadellidae). In leafhoppers, mate recognition and location is mediated exclusively by species- and sex-specific substrate-borne vibrational signals and a female signal emitted in reply to male advertisement calls is essential for recognition and successful location of the female. In <i>A</i>. <i>makarovi</i>, males have to initiate each exchange of vibrational signals between partners, and in a duet the beginning of a female reply overlaps the end of the male advertisement call. Results of playback treatments in which female replies were delayed and did not overlap with the male call revealed that in order to trigger an appropriate behavioural response of the male, female reply has to appear in a period less than 400 ms after the end of the initiating male call. Results also suggest that males are not able to detect a female reply while calling, since female reply that did not continue after the end of male call triggered male behaviour similar to behaviour observed in the absence of female reply. Together, our results show that vibrational duets are tightly coordinated and that the species-specific duet structure plays an important role in mate recognition in location processes.</p></div

The effect of delay of female reply on signalling and searching behaviour of <i>A</i>. <i>makarovi</i> males.

<p>(a) male advertisement call duration; (b) calling rate; (c) proportion of males searching for the source of female reply; (d) proportion of calling males locating the source; (e) proportion of searching males locating the source. (a, b) box and whisker plots show the median (black line), the 25–75% interquartile range (boxes), the lowest and the highest data points still within 1.5 of interquartile range (whiskers) and outliers (circles). Values obtained in the F₁₀ treatment are shown as median (thick white line) together with 95% confidence interval for median (gray area). *, ** and *** indicate significant difference from the F₁₀ treatment (Lme model followed by Dunnett’s multiple comparisons test, p < 0.05, p < 0.01 and p < 0.001, respectively). (c-e) determined proportion (black circle) together with 95% confidence interval for proportions is shown. Proportion obtained in the F₁₀ treatment (thick white line) is shown together with 95% confidence interval for proportions (gray area). *, ** and *** indicate values that are significantly lower than in the F₁₀ treatment (Regwq multiple comparisons test, p < 0.05, p < 0.01 and p < 0.001, respectively). N = number of males included in the analyses. F₁₀: N = 20 (a-d), N = 18 (e).</p

Monitoring of the odorant in the walking track recording device with a miniature Photo Ionisation Detector.

<p>To check the shape of the odour panache in the locomotion compensator, the probe of the PID was positioned either at the output of the stimulus delivering tube or over the sphere while a flow of air odorized with linalool (10% in mineral oil) was delivered.</p

Responses of the PID to pulse trains of linalool.

<p>To trace the dynamics of the odorant concentration in air from the stimulation device in the electrophysiological set-up, the PID signal was recorded while linalool was delivered as ten 50 ms pulses at 1, 2, 4, 7 and 10 pps.</p

Background and pulse rate affect the firing response of Ph-ORNs to pulses.

<p>The curves presents the mean firing rates of the Ph-ORNs whose individual responses appear in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026443#pone-0026443-g003" target="_blank">figure 3</a> responding to pulse trains at 1, 4 and 10 pps in neutral (4a) and in linalool backgrounds (4b).</p

The effect of hidden female reply on signalling and searching behaviour of <i>A</i>. <i>makarovi</i> males.

<p>(a) male advertisement call duration; (b) calling rate; (c) proportion of males searching for the source of female reply; (d) proportion of calling males locating the source; (e) proportion of searching males locating the source. (a, b) box and whisker plots show the median (black line), the 25–75% interquartile range (boxes), the lowest and the highest data points still within 1.5 of interquartile range (whiskers) and outliers (circles). *, ** and *** indicate significant difference between treatments (Lme model followed by Tukey’s all pair comparisons test, p < 0.001). (c-e) determined proportion (black circle) together with 95% confidence interval for proportions is shown. * indicate values that are significantly lower than in the F5 treatment (one-tailed Fisher’s exact test, p < 0.05). Values obtained in the F₀ treatment (white circles) shown for comparison indicate the number of males changing their position during the trial (c) and the number of males arriving to the leaves (d, e) and were not included in statistical analyses.</p

Representative male-female duet in <i>A</i>. <i>makarovi</i>.

<p>Spectrogram (above) and waveform (below) are shown. Me0-Me3: species-specific elements as described in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139020#pone.0139020.ref025" target="_blank">25</a>].</p