Amorpha juglandis
Sound characteristics
Temporal characteristics were measured from the first three pulse trains from 10 animals. Trains ranged from 90 to 8764 ms (mean 2626±2339 ms; median 1907 ms) in duration and contained 1–8 pulses (Fig. 1A) (n=30). Within a given trial, no significant differences were observed in the durations of the first and third trains (Mann–Whitney U, P=0.88). Pulse durations ranged between 44 and 2060 ms (mean 440±272 ms; median 420 ms) (n=101), and the mean inter-pulse interval was 501±282 ms (n=72). Not all pulses were the same in their spectral characteristics, and therefore we divided them into one of three categories (Fig. 1; see supplementary Movie 1 and supplementary Audio 1). Type 1 pulses (Fig. 1B) contain one or two multiharmonic series occurring near the beginning or middle of the pulse, with 16–48 harmonics per series. Type 2 pulses (Fig. 1C) were more broadband than type 1 pulses throughout the entire pulse, often (67%) having a short whistle at the very beginning or end of the pulse. Type 3 pulses (Fig. 1D) were characterized as pure whistles, with one or two harmonic series with two to five harmonics per series. There was a trend with respect to where each type occurred during a train, with all type 1 pulses occurring in the first third of a train, whereas 42.6% and 68.9% of types 2 and 3 occurred in the middle and final third of the train, respectively (n=34 trains; N=11). Characteristics of the broadband, multi-harmonic and pure whistle sounds from the various pulse types are displayed in Fig. 1B–D and Table 1. Waveforms differed between multi-harmonic, pure whistles and broadband sounds, with pure whistles having a significantly higher element-repetition rate than the other two sounds (P=0.0001; Mann–Whitney U). Multi-harmonic and more broadband sounds had more complex waveforms, with repetition rates that also differed significantly from one another (P=0.0001; Mann–Whitney U) (see Table 1).
Sound pressure levels of 10 pulses selected at random from five animals ranged from 69 to 82 dB SPL at 5 cm. The relative amplitude of pulses in a typical train declined from the beginning to the end, with the first being 12.9 dB greater on average than the last (N=10). [1]
References
Sound production mechanism
Initial investigations into the mechanism of sound production involved videotaping body movements that accompanied sound production. Sound production was always accompanied by contraction of the anterior body segments (thorax and first two abdominal segments) (Fig. 2). The contraction lasted 404±193 ms and began 98±111 ms before the onset of the sound (N=5). Following the contraction, it took 156±36 ms to return to full extension.
Occlusion experiments were conducted to determine, first, whether spiracles were involved in sound production and then to identify which spiracles were involved. When all abdominal spiracles were obstructed, sound production was eliminated in 100% of caterpillars, but, when the spiracles were uncovered, sound production resumed in 100% of the individuals (N=5). In a second experiment, we applied latex to only the A8 spiracles, and sound production was eliminated in all trials (N=10). Once the latex was removed, sound production returned in all trials. The ability to signal was never lost in animals where another set of spiracles had been blocked (A7, N=5).
Laser recordings (N=5) confirmed that sound is produced by the movement of air through the A8 spiracles (Fig. 3). In all five individuals, the laser registered a large-amplitude signal when placed in front of A8 but not when placed in front of the control spiracle (A5). There was evidence of a small amount of vibration when recording over A5 (Fig. 3), but this occurred regardless of whether that spiracle was exposed or covered with latex. Therefore, we conclude that these vibrations were being picked up indirectly from sounds produced by A8.
Spiracle anatomy
External measurements of the spiracles in A. juglandis revealed that A8 was longer than the other seven abdominal spiracles (A8 and A7, P=0.0007; A8 and all other spiracles P<0.0001) (Fig. 3A), and wider than one spiracle (A8 and A1, P=0.001; N=5). This pattern was not repeated in P. myops, where there was only a significant difference between the length and width of spiracles A8 and A1 (P=0.0007 and 0.0056, respectively; N=5). See supplementary Table S1 for spiracle measurements in both species. [1]