Ripple effects in a communication network: Anti-eavesdropper defence elicits elaborated sexual signals in rival males
Creators: name: Leavell, Brian
name: Pantoja-Sanchez, Hoover
name: Vélez, Viviana
name: Hemingway, Claire
name: Wilhite, Kyle
name: Halfwerk, Wouter
name: Bernal, Ximena
Publisher: name: Zenodo
Year: 2023
Subjects: communication
Foraging
male-male competition
predator-prey
vibratory cue
Sexual selection

description: Emitting conspicuous signals into the environment to attract mates comes with the increased risk of interception by eavesdropping enemies. As a defence, a commonly described strategy is for signallers to group together in leks, diluting each individual's risk. Lekking systems are often highly social settings in which competing males dynamically alter their signalling behaviour to attract mates. Thus, signalling at the lek requires navigating fluctuations in risk, competition, and reproductive opportunities. Here, we investigate how behavioural defence strategies directed at an eavesdropping enemy have cascading effects across the communication network. We investigated these behaviours in the túngara frog (Engystomops pustulosus), examining how a calling male's swatting defence directed at frog-biting midges indirectly affects the calling behaviour of his rival. We found that the rival responds to swat-induced water ripples by increasing his call rate and complexity. Then, performing phonotaxis experiments, we found that eavesdropping fringe-lipped bats (Trachops cirrhosus) do not exhibit a preference for a swatting male compared to his rival, but females strongly prefer the rival male. Defences to minimize attacks from eavesdroppers thus shift the mate competition landscape in favour of rival males. By modulating the attractiveness of signalling prey to female receivers, we posit that eavesdropping micropredators likely have an unappreciated impact on the ecology and evolution of sexual communication systems.
description: Funding provided by: National Science FoundationCrossref Funder Registry ID: https://ror.org/021nxhr62Award Number: IOS-1433990
Funding provided by: National Science FoundationCrossref Funder Registry ID: https://ror.org/021nxhr62Award Number: DGE-1842166
Funding provided by: Smithsonian Tropical Research InstituteCrossref Funder Registry ID: https://ror.org/035jbxr46Award Number:
description: Rival male experiment

In August 2017, we collected calling male túngara frogs 1 to 4 hours after sunset from breeding sites within 1.5 km of Smithsonian Tropical Research Institute facilities (Gamboa, Panamá; 9°07.0'N, 79°41.9'W). Frogs took part in behavioural experiments in a large outdoor flight cage (5×5×2.5 m) within 3 hours following capture and before midnight. Following protocols established by the American Society of Ichthyologists and Herpetologists (https://asih.org/animal-care-guidelines), after each experiment we weighed, measured and toe-clipped the frog (to prevent testing the same individual multiple times), and released him the following night at his collection location. The research reported here complied with University of Texas at Austin #s AUP-2015-00048, AUP-2017-00051 and AUP-2020-00034; STRI IACUC protocols 2017-0102-2020-A8, 2018-0411-2021; and República de Panamá permits SE/AO-1-17, SE/A-44-18, SE/A-40-19, SE/A 69-15 and SE/AH-2-6.

Field recordings

To sample the natural variation of swat and call ripples from calling male túngara frogs, we recorded ripples from six males (n = 2 in situ; n = 4 in a planter tray filled with water from the breeding site, placed by the original calling site). We measured the frequency, velocity and displacement of the water ripples with a digital laser vibrometer (LDV; Polytech PDV-100; Velocity = 20 mm/s, Low Pass = 22 kHz, High Pass = none). The laser was focused on a reflective marker floating on the water surface; its reflection is detected by the LDV, allowing measurement of the surface vibrational velocities. We recorded these measurements from the digital output of the LDV using a Marantz audio recorder (48 kHz sample rate, 24 bit). See Supplementary Materials for a full description of the recording setup. We analysed call ripples (52 samples from 6 males; range: min = 5, max = 13 per male) and swat ripples (66 samples from 6 males; range: min = 5, max = 15 per male) using the velocity measurements recorded from the LDV digital output. We also converted velocity data to displacement values for comparison with related studies that have used this variable to characterize call ripples in this species (e.g. [32,36]). All ripple characterization analyses were performed in MATLAB R2016a (https://www.mathworks.com) using custom code (Supplementary Materials). 

The vertical velocity of a swat ripple differed depending on the location of the sensor relative to the side of the body that swats (Figure S1, Supplementary Materials and R scripts for details of the analysis). We therefore tested calling behaviour in response to both high- and low-velocity swat ripples to determine male responses to the range of swat ripple intensities encountered in the wild. We used the point estimate of the median maximum velocity of all recorded swat ripples (5.84 mm/sec. peak-to-peak), which was between the 95% credible intervals for the median low- and high-intensity swat values, as a guide to calibrate low- and high-velocity swat playbacks. While there was variation across playbacks, the vast majority of calibration measurements were within the range measured from the field recordings (see Figures S2-3 and associated R scripts for further details). 

Experimental setup

For our ripple playback files, we used a total of three field recordings. These files represented the median dominant frequency and median maximum velocity of either a i) high-velocity swat, ii) low-velocity swat, or iii) call ripple. As playback of call ripples always co-occurred with an airborne call playback, we ensured that the call ripple accurately coincided with the airborne call component by synchronizing their playback to match the temporal profile of the field recording. For the airborne component of the multimodal call playback, we generated a call comprised of a whine with one chuck, as this is the most common call complexity observed in natural settings (see Supplementary Materials for details) [37,38]. 

Prior to an experiment, a male (n = 29 frogs; no males were from field recordings) was placed in a pool (2.6 × 2.1 m) of dechlorinated tap water. The male was contained within a ripple cage but could otherwise call freely. This ripple cage, used in a previous study of call-induced ripples, is acoustically transparent to low frequency ripples [33]. The cylindrical ripple cage (10.5 cm total diameter) consisted of a plastic, circular base and top held in place by three evenly spaced, vertical screws (~0.4 cm diam.). Monofilament fishing line (~0.36 mm thick) was strung vertically, encircling all sides of the plastic base and top (spaced ~0.25 cm apart creating an acoustically transparent cylinder 8 cm. in diameter) to prevent the frog from escaping. Once the male was consistently calling, he was presented with the first of four trials, which were presented in random order and separated by at least 1 minute of silence. The order of presentation did not affect male responses (Tables S1-2). Each trial consisted of a 1-minute silent control playback and 1-minute playback of one of the following treatments: i) high-intensity swats, ii) low-intensity swats, iii) multimodal calls (airborne calls and call ripples), and iv) multimodal calls alternating with high-intensity swats (Figure 1). Each stimulus was played back every 2 seconds, which is consistent with the average natural call rate [37] and within the natural range of swat rates [19]. For a subset of the males (n = 15) we performed an additional trial at the end of the experiment, playing back the multimodal call stimulus once every four seconds (i.e. "slow multimodal call", Figure 1) to control for the reduced call rate of the treatment in which the multimodal call alternated with a high-intensity swat ripple.

For each frog (n = 29 frogs; # trials/frog: range = 1 to 5, median = 4; 106 trials total), we compared call rate (average # calls/sec) and complexity (average # chucks/whine) between control conditions (i.e., silent playback) and treatment playback. Males called during all silent control conditions (106/106 trials), but sometimes ceased calling (9/106 trials) or only produced a single call (2/106 trials) in response to a treatment playback. All 11 of these latter trials were excluded from call rate analysis, whereas only the 9 trials in which the male ceased calling were excluded from complexity analysis. We also excluded 2 treatment playbacks due to playback error or a sudden, loud stimulus from the environment. All acoustic analyses were performed in Adobe Audition CS6. 

Bat foraging & female choice experiments

The effectiveness and consequences of calling strategies are ultimately defined by the responses of the receivers. We therefore investigated how frog-biting midges impact decision-making in foraging bats and female túngara frogs by modulating the calling behaviours of male frogs and their rivals. Bats (n = 5; 10-20 trials/bat per experiment) and female túngara frogs (n = 24; 1 trial/female per experiment) were presented with two-choice tests of airborne (no ripples) frog call playbacks to assess preferences (see Supplementary Materials for detailed descriptions of methods). 

To evaluate relative preferences between a calling, swatting male and a rival responding to his swat ripples (i.e. the scenario examined in the male behavioural experiment), we broadcast calls of a male who intermittently swats – i.e. representative of the call rate and complexity of a frog that swats every 2 seconds; 0.34 calls/second, 1.00 chucks/call [19]- versus a calling rival male responding to high-intensity swat ripples (i.e. representative of the call rate and complexity of frogs that were exposed to a high-intensity swat ripple once every 2 seconds; 0.5 calls/second, 1.75 chucks/call). In a second two-choice test, to investigate if and to what extent responding to swat ripples alters receiver preferences relative to a typical, non-swatting male, we broadcast calls of a characteristic non-swatting, calling male (i.e. representative of the call rate and complexity of a rival frog before being exposed to high-intensity swat ripples; 0.45 calls/second, 1.00 chucks/call) and calling male responding to high-intensity swat ripples (same as in previously mentioned two-choice test). A third two-choice test was given to a single bat (n = 1; 20 trials), in which we broadcast calls of a male who intermittently swats, but does not append ornamental chucks (i.e. approximate call rate of a frog that swats every 2 seconds, but without ornamental chucks; 0.34 calls/second, 0 chucks/call) versus a calling rival male responding to high-intensity swat ripples (same as above).

Statistical analyses

For our analyses, we formulated generalized linear mixed models fit under a Bayesian framework using the brms package version 2.14.4 [39] and R version 4.0.3 [40]. We used weakly informative priors to ensure that draws from the prior could be from any hypothetical dataset [41]. In this way, these priors include probability mass around potential, but not implausible extreme values. We ran 4 chains with 3000 iterations each and discarded the first 500, resulting in 10,000 posterior samples. To ensure model convergence, we visually examined traceplots for good mixing of chains and confirmed that   < 1.1 for all parameters. We performed posterior predictive checks to assess model fit with the bayesplot package version 1.8.1 [42]. Models were compared by assessing expected log pointwise predictive densities with the loo package [43]. See Table S3 for details specific to each model. Effects were determined using the hypothesis function in brms, first with two-side tests to determine an effect and directionality and subsequently a post-hoc one-sided test. Comparisons of effects between treatments were determined by subtracting the posterior probability densities from one another. 

(See manuscript and supplementary materials for references, figures and tables.)