Alarm calls evoke a visual search image of a predator in birds

Study Site and Subjects.

Experiments were conducted on Japanese tits (P. minor) in mixed deciduous–coniferous forests in Nagano and Gunma (36°19′–25′ N, 138°27′–39′ E), Japan. In this study site, there were three species of snakes, including Japanese rat snakes, Japanese forest rat snakes (E. conspicillata), and tiger keelbacks (Rhabdophis tigrinus). Only Japanese rat snakes climb up trees and prey on birds including eggs, young, and adults. Experiments were conducted between May 18 and May 30, 2017 (Experiment 1) and April 22 and May 12, 2014 (Experiment 2), during the early breeding season of tits. Within each experiment, trials were conducted at different sites (n = 36 in Experiment 1, n = 48 in Experiment 2), which were separated by at least 350 m. This distance was enough to ensure that independent data were collected from different individuals, which was confirmed by observations of color-ringed Japanese tits in a previous study (30). Trials took place under calm and dry weather between 0830 and 1600 hours (Japan Standard Time).

Experimental Stimuli.

Sticks (n = 12; 18-cm length, 1.5-cm diameter) were cut from dead branches and were then connected to a thin, black string. Playback stimuli for the three call types were constructed from previous recordings of Japanese tits’ vocalizations (13, 14) and were edited by using Adobe Audition 3.0 software (Adobe Corporation). For each playback exemplar, one call was chosen from an individual on the basis of sound quality (i.e., when the signaler was close to the microphone and there was low background noise) and was repeated in a sound file at a rate of five calls per 12 s (one call every 2.4 s), imitating a natural calling rate in Japanese tits (13, 14). Snake-specific alarm calls are composed of a single type of note, whereas general alarm calls can vary subtly with their note composition (13). I used general alarm calls composed of four types of notes (a string of A, B, and C notes followed by 7–10 D notes) since this combination is a typical composition of general alarm calls and recruits individuals to scan for danger (17). Recruitment calls are composed of 7–10 D notes, which recruit individuals to nondangerous situations (17). Previous playback studies have shown that all three of the calls attract Japanese tits to within 2 m of the speaker (15, 17, 31). I filtered out low-frequency (<1 kHz) noise and amplified all of the calls to be played back at a standardized volume (75 dB re: 20 μPa at 1 m from the speaker measured using an SM-325 sound-level meter; AS ONE Corporation). I saved all sound files in .WAV format (16-bit depth, 48.0-kHz sampling rate) to an SD memory card. For each treatment, 12 exemplars were constructed using the calls of 12 individuals that had been recorded with an LS370 parabolic microphone (Fuji Planning Corporation) connected to an R-09HR digital audio recorder (16-bit depth, 48.0-kHz sampling rate; Roland Corporation) (13, 14).

Experiment 1.

This experiment investigated the responses of tits to a stick moving up a tree trunk during the playback of snake-specific alarm calls, general alarm calls, or recruitment calls. At each experimental site, an AT-SPG50 speaker (Audio-Technica Corporation) was hung from a tree branch at 1.5 ± 0.1 m above the ground. The speaker was connected to an R-09HR digital audio recorder using EXC-12A extension cords (JVC KENWOOD Corporation) to control the playbacks from an observation position ∼10 m away from the speaker tree. A stick was fixed at a height of 1.1 ± 0.2 m (mean ± SD, n = 24) on the trunk of a different tree. This tree was carefully chosen with the following criteria: (i) it allowed the stick to be pulled up the tree trunk in a straight line to imitate a snake moving up the trunk, (ii) it allowed the stick to be visible from the position of the speaker, ensuring that an approaching, focal bird was exposed to the stick, and (iii) it allowed the stick to be located at a distance of 3.2 ± 0.2 m (mean ± SD, n = 36) from the speaker.

Playbacks were started when no birds were visible around the speaker tree and no birds were calling. When a focal tit flew to the speaker tree and approached within 2 m above the speaker where it could see the stick, the movement of the stick was initiated by pulling up the thin string 20 cm and continually repeating this movement five times for 12 s. I then recorded whether the focal tit flew directly toward the moving stick during the first 24 s. Observations were made ∼10 m from the tree with the stick and the trials were video-recorded using a GZ-EX350 digital video camera (JVC KENWOOD Corporation). After each trial, I used a tape measure to measure the minimum approach distance of the focal bird to the stick. I confirmed the exact location at which the focal bird made the closest approach by checking the video recording at each experimental site. When the focal tit flew toward the moving stick, it also approached within 1 m of the stick, so I used this distance to determine approach behavior. In consideration for the possibility of observer bias in behavioral data (32), I conducted an interobserver reliability test with a second rater who was naive to the hypothesis but had been accustomed to observing Japanese tit behavior. The second rater observed randomly selected video clips (n = 18, 50% of the trials) and estimated whether tits approached within 1 m of the stick or not. The results obtained from the author and the second rater showed a high degree of agreement, indicating that there was no observer bias (kappa statistic of 1.0) (32).

The order of trials (snake alarm, general alarm, and recruitment calls) was counterbalanced across treatments so that responses to all treatments were observed under largely similar conditions. In seven trials, the first bird to approach the call playback was from a heterospecific species, such as a coal tit (Periparus ater) (n = 6) or a willow tit (Poecile montanus) (n = 1). To account for the possibility that Japanese tits copied the behavior of these other birds, I only used the data from instances where the first individual to approach the speaker was a Japanese tit. Otherwise, I repeated the same treatment at the next site. Unique exemplars of calls (n = 12) were used for each trial to completely avoid pseudoreplication (33). Unique exemplars of sticks (n = 12) were also used for each trial within each treatment.

Experiment 2.

This experiment investigated the responses of tits to two types of stick movements (along the ground and swinging on a shrub) in combination with playbacks of either snake-specific or general alarm calls. As in Experiment 1, an AT-SPG50 speaker was hung from a tree branch at 1.4 m above the ground, and was connected to an R-09HR digital audio recorder using EXC-12A extension cords. For the snake-like movement along the ground, the stick was positioned on the ground at a distance of 2.5 m from the speaker, and the string was extended from the stick to the observation position to control the movement of the stick from a distance. To generate the non–snake-like swinging movement, the stick was attached to a small shrub (1.0 ± 0.2 m high, mean ± SD, n = 24) at a distance of 2.5 m from the speaker and the string was extended to the observation position, so that I could swing the stick from a distance.

Playbacks were started when no birds were visible around the speaker tree and no birds were calling. When a tit flew to the speaker tree and approached within 2 m above the speaker where it could see the stick, the movement of the stick (dragging on the ground or swinging on a shrub) was initiated by pulling the thin string 20 cm and continually repeating this movement five times for 12 s. I then recorded whether the focal tit approached within 1 m of the moving stick during the first 24 s. I also measured the minimum distance at which the focal tit approached the stick, as in Experiment 1. Interobserver reliability tests between the author and a second naive rater were also conducted for these analyses using randomly selected video clips (n = 12 for each movement type, 50% of the trials). The results showed a high degree of agreement between the two raters, indicating that there was no observer bias (kappa statistic: 1.0 for both) (32).

Trials were conducted in 12 blocks, with all 4 treatments (2 call types × 2 movement types) presented in a randomized order. As in Experiment 1, unique call exemplars and unique sticks were used for each block to avoid pseudoreplication (33). When the first bird to approach an alarm call playback was from a heterospecific species (n = 40 trials), I repeated the same treatment at the next site.

Statistical Analysis.

Fisher’s exact probability tests were used to compare the probability of approach to the movement of the stick. All tests were two-tailed, and statistical significance was set at α = 0.05. When making multiple comparisons, a false discovery rate control was applied to adjust P values (34). All of the statistical analyses were performed in R (35) using the stats package.

Ethics Statement.

All experiments were performed in accordance with relevant guidelines and regulations. All experimental protocols were approved by the Animal Care and Use Committees at The Graduate University for Advanced Studies, and adhered to the Guidelines for the Use of Animals of the Association for the Study of Animal Behaviour/Animal Behavior Society (36). This research was performed with permission from the Ministry of the Environment and the Forestry Agency of Japan.