Morphology
Dendroctonus valens males have ridges on the ventral posterior margins of each elytron, with most ridges occurring on the left elytron (Fig. 2A,B,D). It is not understood whether one or both elytra contribute to sound production. Males also have a sclerotized protrusion on their seventh abdominal tergite, hypothesized to function as a stridulatory plectrum (Fig. 2A,C) although this has not been directly tested. These putative sound-producing structures were examined in seven males using light microscopy (Olympus SZX12, Tokyo, Japan) and scanning electron microscopy (SEM; JOEL JSM-6400). In preparation for SEM, elytra were cleaned and placed on aluminium stubs, while abdomens were prepared following the procedure outlined by Rumph and Turner (1998) and placed on separate stubs. Specimens were sputter coated with gold–palladium and examined.
Contributions of the file and plectrum to sound production
To assess whether one or both elytra contribute to chirp production, sound recordings were conducted prior to and following ablation of the left (n=5) or the right elytron (n=6). Ablation was performed by clipping the posterior tip of either elytron, the region of the elytron containing the proposed file ridges, using dissection scissors. Sound recordings (30 s duration) were performed during disturbance trials (set-up as above) both pre- and post-ablation and recordings were analysed for the number of chirps and pulses per chirp.
To view the sound production mechanism, the right elytron was completely removed in seven males and disturbance sounds evoked as described above. Note that the elytron ablation experiment described above confirmed that the right elytron did not contribute to sound production (see Results). Stridulation was filmed at high speed using a GoPro Hero3 camera (frame rate: 240 frames s−1, resolution: 848×480, GoPro Inc.) connected to an external microphone (ECM-MS908C, Sony) and mounted on a two- headed observation light microscope (Leica, Wild M3Z). Sounds were simultaneously recorded by an Earthworks microphone on a data recorder as described above. Audio was matched to the high- speed video using Raven Pro 1.5 by aligning several pure tone test sounds made throughout the recording.
Mechanism of pulse production
In Dendroctonus spp., each sound pulse in a chirp is proposed to result from the plectrum striking one tooth on the file. However, this has not been confirmed for any elytro-tergal stridulator. We tested this using two methods. First, we counted the number of teeth on the file measured along the file midline (see Fig. 2E) using SEMs of 15 males, and compared this with the mean number of sound pulses per chirp. Because there were more teeth than sound pulses (see Results), we then more specifically estimated the number of teeth impacted by the plectrum during chirp production. This was done by examining the location of the plectrum on the left elytron at the first and last pulse of each chirp for 10 simple chirps from the high-speed video footage of each of seven individual males (see above). The distance the plectrum travelled during the chirp was measured from higher resolution images of the stridulatory mechanism taken with a camera (Zeiss AxioCam MRc5, 1.4 megapixels, 1388×1040) mounted on a light microscope (Olympus SZX12) using Zeiss AxioVision digital image processing software. The number of teeth over that distance was then counted using SEMs (see above) of the left files from each male.
Mechanics of chirp production and variability
Simple chirps – that consist of continuous pulse production with regular intervals – may be produced by one of two hypothesized mechanisms: (1) an ‘escapement mechanism’ type of action, whereby the plectrum and abdominal movements are tightly coupled as the plectrum passes the file; in this case, we predicted that abdominal movement would be coupled with plectrum movement and sound production; (2) spring stridulation, where the plectrum temporarily catches on a tooth of the file, is stretched and then released; in this case, we predicted that the plectrum would move independently from the abdomen. To quantify this, we measured the angle between the plectrum (on the seventh abdominal tergite) and the eighth abdominal tergite to establish whether the position of the plectrum against the abdomen was rigid or pliable. From the high-speed video recordings, we analysed each video frame during the production of five simple chirps for each of seven males and then calculated plectrum–abdomen angle measurements for each frame.
Interrupted chirps could result by two hypothesized mechanisms: by rapid repetition of the file–plectrum cycle or by one file–plectrum cycle that is intermittently stopped. Of the seven males with high-speed video trials, one male was observed to produce multiple interrupted chirps. We analysed each video frame during the production of the interrupted chirp and identified the point of contact of the plectrum on the elytral file for each video frame to determine plectrum movement. This was repeated for five interrupted chirps. [1]