A nested analysis of similarity (ANOSIM) of the Euclidean distance matrix demonstrated a separation between signals produced by queens, soldiers, workers, Paussus favieri Pa, Pb, Pc and Myrmica queens and workers (overall: R = 0.994, P < 0.001). Patterns of similarity in the standardised Euclidean distances of stridulations revealed important interspecific similarities between Paussus and their host ants (Tables 1 – 2 ). In particular, Pa was equally similar to stridulations by P. pallidula soldiers and workers, and less similar to Pc and Pb ( Table 2 ). Pb was closer to the stridulations of workers’ than soldiers' or to Pa stridulations emitted by P. favieri itself ( Table 2 ). Pc stridulations were significantly more similar to P. pallidula queens than to soldier and to Pa stridulations ( Table 2 ). Paussus and Pheidole stridulations were always very different from those emitted by Myrmica queens and workers ( Table 2 ). Thus we can conclude that each of the three types of pulse emitted by P. favieri is always more similar to a stridulation emitted by a specific ant caste than to any other type of Paussus favieri pulses. Paussus trains of Pa and Pb are similar to those of either ant soldiers or workers (the shortest distance was found between Pb and Pheidole pallidula workers), while single pulses (Pc) are more similar to stridulations emitted by queens.

Combined effects of the three sound parameters (pulse length, frequency, and intensity) shown as the first and second component plot of a principal components analysis over all individual pulse measurements; ovals indicate 95% confidence intervals; filled squares indicate the centroid for each group. Sounds produced by Myrmica queen and worker ants are also plotted for comparison. The first component explained 57% of total variance and was mainly influenced by pulse length (waveform parameter). The second component accounted for 30% of total variance and significantly discriminated groups on the basis of peak frequency (spectral parameter). Component loadings of PC1: Pulse length: 0.893, Frequency: 0.602; Intensity: -0.749. Component loadings of PC2: Pulse length: -0.042, Frequency: 0.764; Intensity: 0.564. Univariate analysis of variance (ANOVA) performed on PCA factors showed significant difference among groups (see text).

The three sound parameters were also subject to a Principal Components Analysis (PCA) with stridulations of the ant Myrmica scabrinodis used as a control. A PCA of all individual measurements showed that sounds produced by Pheidole pallidula and Paussus favieri can be divided into 6 separate acoustic groups corresponding to the three ant castes, with only slight overlap between queens and soldiers, and to the three kinds of P. favieri pulses, with a deep separation of Pc from Pa and Pb ( Fig 3 ; results of ANOVA performed on the first two components extracted by the PCA: F 778,7 = 354.561, P < 0.001 for the first component; and F 778,7 = 179.204, P < 0.001 for the second component). As expected, stridulations emitted by Myrmica ants grouped apart from both Pheidole pallidula and Paussus favieri, suggesting the ability of the beetle to imitate its host ant stridulation more than other ants’ acoustical emissions.

A comparison of Paussus favieri and Pheidole pallidula sounds revealed that pulse Pa produced by the beetle had the same pulse length as those emitted by soldiers and workers, while pulse Pc emitted by the beetle had the same intensity and pulse length as that of the queens ( S2 Table ).

To account for individual differences in the sound emission, we analyzed variations in each sound component (Pulse length, Dominant Frequency and Intensity) by using Generalised Linear Models (GLM) in which beetle’s pulses (Pa, Pb, and Pc) were used as fixed factor and the “individual” was selected as random factor. We found no effect of individual identity (GLM results: F 482, 8 = 1.561, P = 0.0.183 for Pulse Length; F 482, 8 = 1.115, P = 0.400 for Frequency; F 482, 8 = 0.678, P = 0.705 for Intensity), whereas each sound component differed among Paussus pulses (F 482, 2 = 967.725, P < 0.001 for Pulse Length; F 482, 2 = 581.164, P < 0.001 for Frequency; F 482, 2 = 1937.577, P < 0.001 for Intensity). Univariate analysis of each sound component showed that the three types of pulses (Pa, Pb, and Pc) emitted by Paussus favieri were significantly different, except for the dominant frequency measured on Pa and Pc ( S2 Table ). Also Pheidole pallidula ants produced stridulations that did not differ among individuals (ANOVA: P > 0.05) but are cast specific (ANOVA: F 296,2 = 9.007, P <0.001 for Pulse length; F 296,2 = 13.447, P < 0.001 for Frequency; F 296,2 = 296.694, P <0.001 for Intensity).

All individuals of both sexes of Paussus favieri are able to emit stridulations ( Fig 2 and S4 File ). These stridulations are produced by a slightly different mechanism from that of ants. The ridges of the beetle’s file are located on the hind femora (Fig 1 , 1B2 and 1B5 ) and are about five times farther apart than those of the ant, which are located on the first gastral (fourth abdominal) segment (Fig 1 , 1A2 and 1A5 ). Moreover, the beetle uses a row of scrapers (placed on the proximal abdominal segment) (Fig 1 , 1B3 and 1B4 ) rather than the single scraper (placed on the postpetiole, i.e. the third abdominal tergite) used by the ant (Fig 1 , 1A3 and 1A4 ). These stridulations consist of three different kinds of pulses ( Fig 2 ), which we call Pa, Pb and Pc. Pa and Pb alternate in trains, while Pc pulses are emitted subsequently, forming a separate sequence. Sequences of pulses lasted approximately 3 seconds, on average. All studied beetles of both sexes emitted all three types of signals. The use of a Mann-Whitney test to compare pulse parameters (i.e., Pulse Length, Frequency and Intensity), as well as the various types of pulses (Pa, Pb and P), revealed no differences between female and male acoustics (P>0.05 in all cases).

Waveforms of stridulations emitted by three Pheidole pallidula castes (queen, soldier and worker) and by Paussus favieri (Pulses a-b-c) are shown in the upper part of the figure. Pulses a (Pa), which are more similar to the sounds emitted by soldiers and workers, and Pulses b (Pb), which are similar to the sounds emitted by workers, are found alternating in trains. Pulses c (Pc) are closer to queens’ stridulations and are emitted subsequently, to form different trains. The lower part of the figure shows the corresponding spectrograms: darker parts correspond to higher energy densities while lighter parts correspond to lower energy densities.

We found that members of all three castes (queen, workers, and soldiers) of Pheidole pallidula produce with their abdominal stridulatory organs (Fig 1 , 1A1 – 1A5 ) distinctive sounds ( Fig 2 and S1 – S3 Files). Stridulations consist of a series of pulses repeated from a few to about ten seconds, without any interruption. Trains of pulses of both queens and soldiers contain about twenty pulses, while the workers’ trains are generally longer with about thirty pulses.

Function and modularity of Paussus stridulations

To assess the effects of Paussus favieri stridulations on ant behaviour, we performed a series of playback experiments, where the only stimuli presented to workers were the acoustic emissions produced by one of the following: (1) the beetle (single pulses—Pc—and trains—Pa and Pb—as separate stimuli) and by (2) queens, (3) soldiers and (4) workers in separate experimental trials. As controls we used both a silenced headphone and a computer generated white noise.

In playback experiments, we did not observe any antagonistic or alarmed ant behaviour, but always non-aggressive responses involving attraction (walking, antennating and staying) and interaction with other ant castes (guarding, digging). A GLM analysis showed that workers’ reactions to the sound stimuli were significantly different from controls for all observed behaviours. Behaviours such as guarding, digging and staying were not produced by controls (Fig 4). Pheidole pallidula ants were attracted to and induced to “walk” on the speaker by all the sound stimuli, showing no differences in the frequency of responses to the beetle’s or ant castes’ stridulations. Interestingly, playback of P. favieri’s stridulations produced a number of antennations similar to that elicited by sounds emitted by Pheidole pallidula queens. Soldiers’ stridulations elicited a smaller amount of antennations, but not statistically different from those elicited by Paussus favieri single pulses or worker stridulations. Results for antennation are remarkable because this behaviour is known to be linked to nest-mate recognition, recruitment, or to facilitate trophallaxis or pheromone emission [25–27]. Guarding was only induced by Paussus favieri and queens’ sounds. Workers responded to these stimuli by assuming a posture similar to that adopted when they attended queens or objects of great value to their society [2,28]. The queen’s sounds produced the highest occurrences of guarding, which is consistent with the high status and protection afforded to queens in the colony’s hierarchy. One of the most stunning results of the present study is that the emission of single pulses (Pc) of Paussus favieri elicited in worker ants a guarding behaviour that is statistically no different than when presented with emissions of the queen (Fig 4). These findings, together with the highest similarity between Pc and queen stridulations (demonstrated by both uni- and multivariate analyses), support our hypothesis that single pulses are used by the beetle to be treated like a queen by its host ants.

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larger image TIFF original image Download: Fig 4. Playback experiments. Behavioural responses of Pheidole pallidula colonies to sound recordings of Paussus favieri (single pulses), P. favieri (trains), Pheidole pallidula queens, P. pallidula soldiers, P. pallidula workers, and to two controls (white noise and silence) are shown. Five benevolent behaviours were observed; no antagonistic behaviour was observed. GLM testing for the effect of sounds and colony showed a significant overall difference in responses occurred within five behaviours (N = 140; Walking: F stimuli = 22.623, df = 6, P < 0.001, F nest = 1.253, df = 9, P = 0.284; Antennate: F stimuli = 39.414, df = 6, P < 0.001, F nest = 1.221, df = 9, P = 0.302; Guarding: F stimuli = 12.942, df = 6, P < 0.001, F nest = 1.388, df = 9, P = 0.216; Digging: F stimuli = 2.667, df = 6, P = 0.024, F nest = 0.667, df = 9, P = 0.735; Staying: F stimuli = 5.083, df = 6, P = 0.004, F nest = 1.856, df = 9, P = 0.079). The letters above each column indicate significance (P < 0.05) in pairwise post hoc Tukey tests. The same letter indicates no significant difference within each type of behaviour. https://doi.org/10.1371/journal.pone.0130541.g004

A rarely elicited behaviour was digging, which is commonly induced in nest-mates by ants trapped under the soil, for instance after the collapse of a tunnel [29,30]. Atta ants, for example, are known to emit rescue calls when trapped [31]. Here we observed that both the sounds of P. favieri and those of queens and workers induced a digging behaviour in P. pallidula workers. Trains of P. favieri pulses seem to elicit digging, at least more often than the worker’s stridulations did (Fig 4). Except for the two controls, all sound stimuli caused workers to stand on the speakers. Sounds produced by queen ants induced a similar amount of staying on the speaker as the two sounds (trains and single pulses) emitted by Paussus favieri and a statistically higher amount of staying responses than those elicited by the conspecific soldiers’ and workers’ calls. We hypothesise that staying on the speaker might be related to the ant’s perception of vibrations, which is likely to occur along the legs, as shown in carpenter ants [32].

Findings provided by the analysis of ant-beetle acoustical patterns coupled with ethological observations reveal that a particularly complex form of communication occurs between the beetle (Paussus favieri) and the host ant (Pheidole pallidula). This represents the second known case of acoustic mimicry in myrmecophilous organisms (the first one being that between Maculinea butterflies and Myrmica ants), and the first one for beetles.

Although these models involve phylogenetically very distant taxa, and different life stages (larvae and pupae in lycaenids and adults in P. favieri), several similarities are surprisingly evident. In both cases queen’s stridulations are different from those of the other castes, in particular from those of workers. This clear distinction among castes generates the opportunity for the parasite to selectively imitate each specific caste [8]. Maculinea immatures exploit this opportunity to an extent by emitting stridulations that are more similar to those of the queen than to those of workers and thereby gain higher rank within the colony [8]. Also Myrmica’s sclerotised pupae used sounds to signal their social status within the colony’s hierarchy [9]. Queen-like stridulations produced by Maculinea immatures are an effective strategy to cope with their need to be foraged, attended and saved in case of colony disturbance during their development inside the ant nest. The butterfly larvae mostly remain still and hidden in the brood chambers, asking for food and care, thus encompassing a basically invariant interaction during all the time (11–23 months) the parasite lives within the ant colonies.

In contrast, the strategy of Paussus is much more complex, because it involves multiple types of interaction. Adult beetles need to enter the nest, move freely within it to find appropriate places for egg laying and larval development, feed on the ant broods without being attacked, rewarding the ants by supplying attractive substances from their glandular trichomes, and find their partners for mating inside the nest. Thus, they need to interact with different castes in different ways, due to the allocation of the behavioural repertoire of the different castes inside the society of the genus Pheidole [33, 34]. This beetle is able to use the same anatomical structure (possibly moving the hind legs singly or in combination or in any other way) to “speak” different “languages” by modulating the emissions of various types of pulses. Thus, the beetle can mimic the language of all the ant castes (Fig 3 and Table 2). Difficulties in rearing these insects prevented us from performing laboratory observations to assess if Paussus produces different stridulations in different circumstances (e.g. interactions with different ant castes) or if the beetle is able to choose which stridulation to emit as a function of its needs (e.g. for being accepted into the nest; for getting free access to the brood chamber, etc.). Our experiments coupled with the beetle's ability to emit at least two different types of stridulation, suggest an important adaptive role of acoustics in facilitating beetle parasitism.

In particular, the emission of single—pulse sounds (Pc) might elevate the beetle's social status to that of the queen, as suggested in bioassays where queens’ and beetle’s single pulse elicited the same “guarding” response in workers. This might allow the beetle to obtain more effective care and to gain free access to any chamber of the nest. Use of stridulations similar to those of soldiers might allow Paussus favieri to confound them and then to avoid their attacks [34,35]. Workers responded to each sound stimulus, both to conspecific castes and to Paussus favieri, by showing attraction and “nursing” behaviours [34,35]. All these results suggest that, thanks to the emission of pulse trains (Fig 4), Paussus favieri is mistaken for a worker, and hence rescued by worker nest-mates.

Our playback experiments reveal that workers are able to distinguish sounds produced by the three ant castes, as they react with different behaviours or different response frequency, as expected if acoustic signals are used to communicate inside the colony.