The cercal organ may provide singing tettigoniids a backup sensory system for the detection of eavesdropping bats

Abstract

Conspicuous signals, such as the calling songs of tettigoniids, are intended to attract mates but may also unintentionally attract predators. Among them bats that listen to prey-generated sounds constitute a predation pressure for many acoustically communicating insects as well as frogs. As an adaptation to protect against bat predation many insect species evolved auditory sensitivity to bat-emitted echolocation signals. Recently, the European mouse-eared bat species Myotis myotis and M. blythii oxygnathus were found to eavesdrop on calling songs of the tettigoniid Tettigonia cantans. These gleaning bats emit rather faint echolocation signals when approaching prey and singing insects may have difficulty detecting acoustic predator-related signals. The aim of this study was to determine (1) if loud self-generated sound produced by European tettigoniids impairs the detection of pulsed ultrasound and (2) if wind-sensors on the cercal organ function as a sensory backup system for bat detection in tettigoniids. We addressed these questions by combining a behavioral approach to study the response of two European tettigoniid species to pulsed ultrasound, together with an electrophysiological approach to record the activity of wind-sensitive interneurons during real attacks of the European mouse-eared bat species Myotis myotis. Results showed that singing T. cantans males did not respond to sequences of ultrasound pulses, whereas singing T. viridissima did respond with predominantly brief song pauses when ultrasound pulses fell into silent intervals or were coincident with the production of soft hemi-syllables. This result, however, strongly depended on ambient temperature with a lower probability for song interruption observable at 21°C compared to 28°C. Using extracellular recordings, dorsal giant interneurons of tettigoniids were shown to fire regular bursts in response to attacking bats. Between the first response of wind-sensitive interneurons and contact, a mean time lag of 860 ms was found. This time interval corresponds to a bat-to-prey distance of ca. 72 cm. This result demonstrates the efficiency of the cercal system of tettigoniids in detecting attacking bats and suggests this sensory system to be particularly valuable for singing insects that are targeted by eavesdropping bats.

Figure 1. Response to playback of repetitive ultrasound pulses to singing tettigoniids.

(A) Average verse duration of T. cantans males with and without repetitive ultrasound stimulation (25 ms and 50 ms pulse repetition period). Numbers in A represent the number of verses in each group. A significant difference (p<0.05) of stimulated verse durations from natural verse durations is indicated by *. B) Response of T. viridissima males to ultrasound stimulation in the form of song pausing (white bars) and the lack of such a response (black bars). 28°C: N = 131 (50 ms), N = 115 (25 ms); 21°C: N = 40 (50 ms), N = 38 (25 ms). All data was obtained from 8 T. cantans and 7 T. viridissima males. p<0.05 is indicated by *, p<0.001 is indicated by **; Upper and lower box limits in A represent 25 and 75 percentile and whiskers 10 and 90 percentile. Outliers are indicated by circles.

Figure 2. Examples of ultrasound stimulation of T. cantans and T. viridissima males.

(A) T. cantans males were stimulated with repetitive 30 kHz pulses during verse production. The same stimulus frequently elicited brief song pausing in T. viridissima (B). Stimulus level was 89 dBpe SPL.

Figure 3. Phase-dependent song pausing of T. viridissima males elicited by repetitive ultrasound pulses.

Histograms showing the phase of ultrasound pulses preceding song pausing in the stridulation cycle of T. viridissima. (A) 50 ms pulse repetition period: Two of three consecutive ultrasound pulses (labeled 2, 3) preceding song pausing were coincident with phases of relative silence. (B) The majority of ultrasound pulses presented with a pulse repetition period of 25 ms coincided with pauses between double-syllables (labeled 2) as well as with soft hemi-syllables separating syllable pairs (labeled 1,3) (B). Vertical lines in A and B indicate the average double syllable duration (40 ms). Data shown in histograms were obtained from 131 stimulus-associated song pauses (7 males tested at an ambient temperature of 28°C).

Figure 4. Response of a technical bat detector, an anemometer and wind-sensitive interneurons of T. cantans to bat attacks.

(A) The wind generated by an approaching bat measured by hot-wire anemometry. (B) The afferent activity of wind-sensitive interneurons in the ventral nerve cord of T. cantans was simultaneously recorded by a hook electrode. Three different neuronal units were clearly discernable by means of spike amplitude. (C) The neuronal activity of wind-sensitive interneurons (upper traces) of four different individual insect preparations recorded during bat attacks. Grey traces in C show the simultaneous activity of a technical ultrasound-based bat detector.

Figure 5. The minimum detection distance and minimum detection time derived from a first neuronal response of wind-sensitive interneurons to approaching bats.

(A) The minimum distance between bat and preparation eliciting a first response of wind-sensitive interneurons in 12 individuals of T. cantans and one individual of Ruspolia nitidula (R1). Numbers in A represent the number of bat approaches. (B) The average minimum time lag between a first nervous response of wind-sensitive interneurons to approaching bats and bat landing (contact with the insect preparation). Asterisks in B indicate p<0.05 which is the result of a two-way ANOVA on ranks followed by a Dunn's post hoc test. (C) A sector-wise comparison of such minimum detection times. Numbers in bars represent the number of bat approaches observed in each sector. * in C indicates p<0.05 as the result of a Kruskal Wallis ANOVA on ranks followed by a Dunn's post hoc test (N = 12 insects). (D) A schematic view of the flight arena and the definition of sectors used in C. The arrow in D indicates the direction of the remaining cercus. For an explanation of box plots see figure 3.

Figure 6. Neuronal response of wind-sensitive interneurons to bat attacks.

(A) The average number of spikes per burst fired by all three wind-sensitive interneurons plotted against the distance between bat and insect preparation. (B) The average maximum instantaneous spike rate of a population of three different wind sensitive units. Data were obtained from 52 platform landings (6 T. cantans individuals).

Figure 7. Analysis of the response of three different wind-sensitive interneurons to bats approaches.

Three different wind-sensitive interneurons were classified according to their extracellular spike amplitudes into small, medium, large units. (A) The first response of different interneuron classes shown as frequency histogram. Data summarized in A was obtained from 166 platform landings (15 T. cantans individuals). (B) The average number of spikes of different wind-sensitive interneurons counted in time windows of 100 ms (125 platform landings obtained from 15 T. cantans individuals). A significant negative correlation of the average spike count with bat-to-preparation distance was found for large and medium units (p<0.001, cc = −0.993 (medium), cc = −0.965 (large), N = 12, Spearman Rank Order correlation).

Figure 8. Singing posture of tettigoniids.

Singing posture of T. cantans (A), T. viridissima (B) and Mecopoda elongata (trilling species) (C). During singing males lower their abdomen and lift their front wings. Note that this singing posture increases the distance between cerci (arrow) and wings.

Figure 9. Setup for the measurement of the response of wind-sensitive interneurons to wind generated by approaching bats.

(A) Close-up of an insect preparation showing the remaining cercus (1), the recording electrode (3) and indifferent electrode (4). Inset in A: The cercal apparatus of a T. cantans male with wind-sensilla. (B) Bats were attracted to meal worms placed on top of a wire mesh covering a loud speaker (3) broadcasting beetle rustling noise. A preparation of T. cantans (1) was placed close to the loudspeaker near the tip of a hot-wire anemometer (2 in A and B). Echolocation calls emitted by bats were measured by means of a technical bat detector (4).