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Vocal production learning (VPL) is the experience-driven ability to produce novel vocal signals through imitation or modification of existing vocalizations. A parallel strand of research investigates acoustic allometry, namely how information about body size is conveyed by acoustic signals. Recently, we proposed that deviation from acoustic allometry principles as a result of sexual selection may have been an intermediate step towards the evolution of vocal learning abilities in mammals. Adopting a more hypothesis-neutral stance, here we perform phylogenetic regressions and other analyses further testing a potential link between VPL and being an allometric outlier. We find that multiple species belonging to VPL clades deviate from allometric scaling but in the opposite direction to that expected from size exaggeration mechanisms. In other words, our correlational approach finds an association between VPL and being an allometric outlier. However, the direction of this association, contra our original hypothesis, may indicate that VPL did not necessarily emerge via sexual selection for size exaggeration: VPL clades show higher vocalization frequencies than expected. In addition, our approach allows us to identify species with potential for VPL abilities: we hypothesize that those outliers from acoustic allometry lying above the regression line may be VPL species. Our results may help better understand the cross-species diversity, variability and aetiology of VPL, which among other things is a key underpinning of speech in our species. This article is part of the theme issue ‘Voice modulation: from origin and mechanism to social impact (Part II)’.

Acoustic allometry occurs when features of animal vocalisations can be predicted from body size measurements. Despite this being considered the norm, allometry sometimes breaks, resulting in species sounding smaller or larger than expected for their size. A recent hypothesis suggests that allometry-breaking mammals cluster into two groups: those with anatomical adaptations to their vocal tracts and those capable of learning new sounds (vocal learners). Here we test which mechanism is used to escape from acoustic allometry by probing vocal tract allometry in a proven mammalian vocal learner, the harbour seal (Phoca vitulina). We test whether vocal tract structures and body size scale allometrically in 68 young individuals. We find that both body length and body mass accurately predict vocal tract length and one tracheal dimension. Independently, body length predicts vocal fold length while body mass predicts a second tracheal dimension. All vocal tract measures are larger in weaners than in pups and some structures are sexually dimorphic within age classes. We conclude that harbour seals do comply with anatomical allometric constraints. However, allometry between body size and vocal fold length seems to emerge after puppyhood, suggesting that ontogeny may modulate the anatomy-learning distinction previously hypothesised as clear-cut. We suggest that seals, like other species producing signals that deviate from those expected from their vocal tract dimensions, may break allometry without morphological adaptations. In seals, and potentially other vocal learning mammals, advanced neural control over vocal organs may be the main mechanism for breaking acoustic allometry.

Partial response to chemotherapy leads to disease resurgence. Upon treatment, a subpopulation of cancer cells, called drug-tolerant persistent cells, display a transitory drug tolerance that lead to treatment resistance 1,2. Though drug-tolerance mechanisms remain poorly known, they have been linked to non-genomic processes, including epigenetics, stemness and dormancy 2–4. 5-fluorouracil (5-FU), the most widely used chemotherapy in cancer treatment, is associated with resistance. While prescribed as an inhibitor of DNA replication, 5-FU alters all RNA pathways 5–9. Here, we show that 5-FU treatment leads to the unexpected production of fluorinated ribosomes, exhibiting altered mRNA translation. 5-FU is incorporated into ribosomal RNAs of mature ribosomes in cancer cell lines, colorectal xenografts and human tumours. Fluorinated ribosomes appear to be functional, yet, they display a selective translational activity towards mRNAs according to the nature of their 5’-untranslated region. As a result, we found that sustained translation of IGF-1R mRNA, which codes for one of the most potent cell survival effectors, promoted the survival of 5-FU-treated colorectal cancer cells. Altogether, our results demonstrate that “man-made” fluorinated ribosomes favour the drug-tolerant cellular phenotype by promoting translation of survival genes. This could be exploited for developing novel combined therapies. By unraveling translation regulation as a novel gene expression mechanism helping cells to survive a drug-challenge, our study extends the spectrum of molecular mechanisms driving drug-tolerance.

Communicating species identity is a key component of many animal signals. However, whether selection for species recognition systematically increases signal diversity during clade radiation remains debated. Here we show that in woodpecker drumming, a rhythmic signal used during mating and territorial defense, the amount of species identity information encoded remained stable during woodpeckers’ radiation. Acoustic analyses and evolutionary reconstructions show interchange among six main drumming types despite strong phylogenetic contingencies, suggesting evolutionary tinkering of drumming structure within a constrained acoustic space. Playback experiments and quantification of species discriminability demonstrate sufficient signal differentiation to support species recognition in local communities. Finally, we only find character displacement in the rare cases where sympatric species are also closely related. Overall, our results illustrate how historical contingencies and ecological interactions can promote conservatism in signals during a clade radiation without impairing the effectiveness of information transfer relevant to inter-specific discrimination., Animal signals often encode information on the emitter’s species identity. Using woodpecker drumming as a model, here the authors show that limited signal divergence during a clade radiation does not impair species discrimination, as long as the signals are adapted to local ecological requirements.

Tissue vibrations in the larynx produce most sounds that comprise vocal communication in mammals. Larynx morphology is thus predicted to be a key target for selection, particularly in species with highly developed vocal communication systems. Here, we present a novel database of digitally modeled scanned larynges from 55 different mammalian species, representing a wide range of body sizes in the primate and carnivoran orders. Using phylogenetic comparative methods, we demonstrate that the primate larynx has evolved more rapidly than the carnivoran larynx, resulting in a pattern of larger size and increased deviation from expected allometry with body size. These results imply fundamental differences between primates and carnivorans in the balance of selective forces that constrain larynx size and highlight an evolutionary flexibility in primates that may help explain why we have developed complex and diverse uses of the vocal organ for communication., Tissue vibrations in the larynx produce most sounds that comprise vocal communication in mammals. A multidisciplinary study reveals that the primate larynx is larger, more decoupled from body size, and under faster rates of evolution than the carnivoran larynx, providing evidence of evolutionary flexibility that may help explain why primates have developed such diverse and complex uses of the vocal organ.

Summary This study investigates the effect of the ventricular folds on fundamental frequency (fo) in the voice production of domestic pigs (Sus scrofa domesticus). The excised larynges of six subadult pigs were phonated in two preparation stages, with the ventricular folds present (PS1) and removed (PS2). Vocal fold resonances were tested with a laser vibrometer, and a four-mass computational model was created. Highly significant fo differences were found between PS1 and PS2 (means at 93.7 and 409.3 Hz, respectively). Two tissue resonances were found at 115 Hz and 250–290 Hz. The computational model had unique solutions for abducted and adducted ventricular folds at about 150 and 400 Hz, roughly matching the fo measured ex vivo for PS1 and PS2. The differing fo encountered across preparation stages PS1 and PS2 is explained by distinct activation of either a high or a low eigenfrequency mode, depending on the engagement of the ventricular folds. The inability of the investigated larynges to vibrate at frequencies below 250 Hz in PS2 suggests that in vivo low-frequency calls of domestic pigs (pre-eminently grunts) are likely produced with engaged ventricular folds. Allometric comparison suggests that the special, mechanically coupled “double oscillator” has evolved to prevent signaling disadvantages. Given these traits, the porcine larynx might – apart from special applications relating to the involvement of ventricular folds – not be an ideal candidate for emulating human voice production in excised larynx experimentation.

A fundamental issue in the evolution of communication is the degree to which signals convey accurate (“honest”) information about the signaler. In bioacoustics, the assumption that fundamental frequency (fo) should correlate with the body size of the caller is widespread, but this belief has been challenged by various studies, possibly because larynx size and body size can vary independently. In the present comparative study, we conducted excised larynx experiments to investigate this hypothesis rigorously and explore the determinants of fo. Using specimens from eleven primate species, we carried out an inter-specific investigation, examining correlations between the minimum fo produced by the sound source, body size and vocal fold length (VFL). We found that, across species, VFL predicted minimum fo much better than body size, clearly demonstrating the potential for decoupling between larynx size and body size in primates. These findings shed new light on the diversity of primate vocalizations and vocal morphology, highlighting the importance of vocal physiology in understanding the evolution of mammal vocal communication.

Summary Acoustic allometry consists of looking at how an organism’s body size scales with the characteristics of its vocalizations. A typical finding based on this framework is that across mammals body size is reflected in the fundamental frequency (fo) of vocalizations, whereby lower fo indicates larger body size [1] . This relationship holds owing to the fact that vocal fold length generally scales with body size [2] . Cross-species comparisons allow for the identification of interesting outliers from the body size–f0 regression [3] . Such cases are of particular relevance as they can provide insight into the selective forces potentially driving deviations from standard allometric principles [2] . In a recent study in Current Biology, Grawunder et al. [4] argue that selective pressure for higher f0 has led to the evolution of shorter vocal folds in bonobos than in chimpanzees. Thus, they claim, vocal fold length has evolved independently of body size in bonobos for the purposes of signal diminution (i.e., reducing the impression of body size that they advertise through their calls). However, considering both the existing literature and their own data, this conclusion does not appear to be supported for several reasons.

Ribosomal RNAs (rRNAs) are main effectors of messenger RNA (mRNA) decoding, peptide-bond formation, and ribosome dynamics during translation. Ribose 2′-O-methylation (2′-O-Me) is the most abundant rRNA chemical modification, and displays a complex pattern in rRNA. 2′-O-Me was shown to be essential for accurate and efficient protein synthesis in eukaryotic cells. However, whether rRNA 2′-O-Me is an adjustable feature of the human ribosome and a means of regulating ribosome function remains to be determined. Here we challenged rRNA 2′-O-Me globally by inhibiting the rRNA methyl-transferase fibrillarin in human cells. Using RiboMethSeq, a nonbiased quantitative mapping of 2′-O-Me, we identified a repertoire of 2′-O-Me sites subjected to variation and demonstrate that functional domains of ribosomes are targets of 2′-O-Me plasticity. Using the cricket paralysis virus internal ribosome entry site element, coupled to in vitro translation, we show that the intrinsic capability of ribosomes to translate mRNAs is modulated through a 2′-O-Me pattern and not by nonribosomal actors of the translational machinery. Our data establish rRNA 2′-O-Me plasticity as a mechanism providing functional specificity to human ribosomes.

While the call repertoire and its communicative function is relatively well explored in Japanese macaques (Macaca fuscata), little empirical data is available on the physics and the physiology of this species' vocal production mechanism. Here, a 6 year old female Japanese macaque was trained to phonate under an operant conditioning paradigm. The resulting “coo” calls, and spontaneously uttered “growl” and “chirp” calls, were recorded with sound pressure level (SPL) calibrated microphones and electroglottography (EGG), a non-invasive method for assessing the dynamics of phonation. A total of 448 calls were recorded, complemented by ex vivo recordings on an excised Japanese macaque larynx. In this novel multidimensional investigative paradigm, in vivo and ex vivo data were matched via comparable EGG waveforms. Subsequent analysis suggests that the vocal range (range of fundamental frequency and SPL) was comparable to that of a 7-10 year old human, with the exception of low-intensity chirps, whose production may be facilitated by the species' vocal membranes. In coo calls, redundant control of fundamental frequency in relation to SPL was also comparable to humans. EGG data revealed that growls, coos, and chirps were produced by distinct laryngeal vibratory mechanisms. EGG further suggested changes in the degree of vocal fold adduction in vivo, resulting in spectral variation within the emitted coo calls, ranging from “breathy” (including aerodynamic noise components) to “non-breathy”. This is again analogous to humans, corroborating the notion that phonation in humans and non-human primates is based on universal physical and physiological principles.

Vocal communication is a crucial aspect of animal behavior. The mechanism which most mammals use to vocalize relies on three anatomical components. First, air overpressure is generated inside the lower vocal tract. Second, as the airstream goes through the glottis, sound is produced via vocal fold vibration. Third, this sound is further filtered by the geometry and length of the upper vocal tract. Evidence from mammalian anatomy and bioacoustics suggests that some of these three components may covary with an animal’s body size. The framework provided by acoustic allometry suggests that, because vocal tract length (VTL) is more strongly constrained by the growth of the body than vocal fold length (VFL), VTL generates more reliable acoustic cues to an animal’s size. This hypothesis is often tested acoustically but rarely anatomically, especially in pinnipeds. Here, we test the anatomical bases of the acoustic allometry hypothesis in harbor seal pups Phoca vitulina. We dissected and measured vocal tract, vocal folds, and other anatomical features of 15 harbor seals post-mortem. We found that, while VTL correlates with body size, VFL does not. This suggests that, while body growth puts anatomical constraints on how vocalizations are filtered by harbor seals’ vocal tract, no such constraints appear to exist on vocal folds, at least during puppyhood. It is particularly interesting to find anatomical constraints on harbor seals’ vocal tracts, the same anatomical region partially enabling pups to produce individually distinctive vocalizations.

A fundamental assumption in bioacoustics is that large animals tend to produce vocalizations with lower frequencies than small animals. This inverse relationship between body size and vocalization frequencies is widely considered to be foundational in animal communication, with prominent theories arguing that it played a critical role in the evolution of vocal communication, in both production and perception. A major shortcoming of these theories is that they lack a solid empirical foundation: rigorous comparisons between body size and vocalization frequencies remain scarce, particularly among mammals. We address this issue here in a study of body size and vocalization frequencies conducted across 91 mammalian species, covering most of the size range in the orders Primates (n = 50; ~0.11–120 Kg) and Carnivora (n = 41; ~0.14–250 Kg). We employed a novel procedure designed to capture spectral variability and standardize frequency measurement of vocalization data across species. The results unequivocally demonstrate strong inverse relationships between body size and vocalization frequencies in primates and carnivores, filling a long-standing gap in mammalian bioacoustics and providing an empirical foundation for theories on the adaptive function of call frequency in animal communication.

Pancreatic ductal adenocarcinoma (PDAC) is a very aggressive malignancy characterized by an excessive resistance to all known anticancer therapies, a still largely elusive phenomenon. To identify original mechanisms, we have explored the role of post-translational modifications (PTMs) mediated by members of the ubiquitin family. Although alterations of these pathways have been reported in different cancers, no methodical search for these kinds of anomalies has been performed so far. Therefore, we studied the ubiquitin-, Nedd8-, and SUMO1-specific proteomes of a pancreatic cancer cell line (MiaPaCa-2) and identified changes induced by gemcitabine, the standard PDAC's chemotherapeutic drug. These PTMs profiles contained both known major substrates of all three modifiers as well as original ones. Gemcitabine treatment altered the PTM profile of proteins involved in various biological functions, some known cancer associated genes, many potentially cancer-associated genes, and several cancer-signaling networks, including canonical and noncanonical WNT and PI3K/Akt/MTOR pathways. Some of these altered PTMs formed groups of functionally and physically associated proteins. Importantly, we could validate the gemcitabine-induced PTMs variations of relevant candidates and we could demonstrate the biological significance of such altered PTMs by studying in detail the sumoylation of SNIP1, one of these new targets.

The information conveyed in acoustic signals is a central topic in mammal vocal communication research. Body size is one form of information that can be encoded in calls. Acoustic allometry aims to identify the specific acoustic correlates of body size within the vocalizations of a given species, and formants are often a useful acoustic cue in this context. We conducted a longitudinal investigation of acoustic allometry in domestic piglets (Sus scrofa domesticus), asking whether formants of grunt vocalizations provide information concerning the caller's body size over time. On four occasions, we recorded grunts from 20 kunekune piglets, measured their vocal tract length by means of radiographs (X-rays) and weighed them. Controlling for effects of age and sex, we found that body weight strongly predicts vocal tract length, which in turn determines formant frequencies. We conclude that grunt formant frequencies could allow domestic pigs to assess a signaler's body size as it grows. Further research using playback experiments is needed to determine the perceptual role of formants in domestic pig communication., Summary: Longitudinal investigation of acoustic allometry in domestic piglets indicates that grunt formant frequencies allow conspecifics to assess a signaler's body size.

Determining whether a species' vocal communication system is graded or discrete requires definition of its vocal repertoire. In this context, research on domestic pig (Sus scrofa domesticus) vocalizations, for example, has led to significant advances in our understanding of communicative functions. Despite their close relation to domestic pigs, little is known about wild boar (Sus scrofa) vocalizations. The few existing studies, conducted in the 1970s, relied on visual inspections of spectrograms to quantify acoustic parameters and lacked statistical analysis. Here, we use objective signal processing techniques and advanced statistical approaches to classify 616 calls recorded from semi‐free ranging animals. Based on four spectral and temporal acoustic parameters—quartile Q25, duration, spectral flux, and spectral flatness—extracted from a multivariate analysis, we refine and extend the conclusions drawn from previous work and present a statistically validated classification of the wild boar vocal repertoire into four call types: grunts, grunt‐squeals, squeals, and trumpets. While the majority of calls could be sorted into these categories using objective criteria, we also found evidence supporting a graded interpretation of some wild boar vocalizations as acoustically continuous, with the extremes representing discrete call types. The use of objective criteria based on modern techniques and statistics in respect to acoustic continuity advances our understanding of vocal variation. Integrating our findings with recent studies on domestic pig vocal behavior and emotions, we emphasize the importance of grunt‐squeals for acoustic approaches to animal welfare and underline the need of further research investigating the role of domestication on animal vocal communication.

Directionality is a key feature of vocalizations in birds and can provide insight into vocalizations° function and propagation. Despite recent interest in birds' production of nonvocal acoustic signals, little is known about the directionality of these sounds. Here, we examine the directionality of the drumming display of male Ruffed Grouse (Bonasa umbellus), a nonvocal acoustic signal used in mate attraction and territorial defense. We recorded drumming males with a low-frequency microphone and measured sound-pressure levels with a sound-level meter from three orientations: front (0°), behind (180°), and side (90°). The sound-pressure level was significantly higher in front of and behind a drumming male than on the sides, indicating that the drumming sound radiates primarily along the displaying bird's longitudinal axis. Spectral analyses of the recordings did not, however, yield consistent differences in frequency among the three orientations. The acoustic radiation pattern of the drumming dis...

Individual variation in vocalizations is a common feature of many forms of long-distance communication in vertebrates. The extent to which individual variation occurs in non-vocal, long-distance acoustic communication has not, however, been tested. Here, we examine the spectral and temporal characteristics of a non-vocal acoustic signal, the wingbeating drumming display of the male Ruffed Grouse (Bonasa umbellus, L.), and test whether its structure varies more among individuals than within them. Drumming displays were recorded over two field seasons, and we measured several temporal and spectral features of these recordings. Each drumming display consists of 39‐50 pulses produced over a period of 9‐10 s with most of the energy concentrated at frequencies below 100 Hz. We calculated the potential for individual coding of several temporal and spectral features, and both the number of pulses and pulse rate were highly individually specific. This was corroborated by analyses of variance, bivariate plots of pulse number and rate, and discriminant function analyses. Overall, we conclude that male Ruffed Grouse produce individually specific drumming displays in a similar fashion to vocal individuality in other birds. The extent to which these individual differences persist from one season to the next is unclear, but individual differences in the number of pulses and pulse rate could provide information on individual identity to conspecifics.

Red deer stags (Cervus elaphus) give two distinct types of roars during the breeding season, the “common roar” and the “harsh roar.” Harsh roars are more frequent during contexts of intense competition, and characterized by a set of features that increase their perceptual salience, suggesting that they signal heightened arousal. While common roars have been shown to encode size information and mediate both male competition and female choice, to our knowledge, the specific function of harsh roars during male competition has not yet been studied. Here, we investigate the hypothesis that the specific structure of male harsh roars signals high arousal to competitors. We contrast the behavioral responses of free ranging, harem-holding stags to the playback of harsh roars from an unfamiliar competitor with their response to the playback of common roars from the same animal. We show that males react less strongly to sequences of harsh roars than to sequences of common roars, possibly because they are reluctant to escalate conflicts with highly motivated and threatening unfamiliar males in the absence of visual information. While future work should investigate the response of stags to harsh roars from familiar opponents, our observations remain consistent with the hypothesis that harsh roars may signal motivation during male competition, and illustrate how intrasexual selection can contribute to the diversification of male vocal signals.

It is well established that in humans, male voices are disproportionately lower pitched than female voices, and recent studies suggest that this dimorphism in fundamental frequency (F0) results from both intrasexual (male competition) and intersexual (female mate choice) selection for lower pitched voices in men. However, comparative investigations indicate that sexual dimorphism in F0 is not universal in terrestrial mammals. In the highly polygynous and sexually dimorphic Scottish red deer Cervus elaphus scoticus, more successful males give sexually-selected calls (roars) with higher minimum F0s, suggesting that high, rather than low F0s advertise quality in this subspecies. While playback experiments demonstrated that oestrous females prefer higher pitched roars, the potential role of roar F0 in male competition remains untested. Here we examined the response of rutting red deer stags to playbacks of re-synthesized male roars with different median F0s. Our results show that stags’ responses (latencies and durations of attention, vocal and approach responses) were not affected by the F0 of the roar. This suggests that intrasexual selection is unlikely to strongly influence the evolution of roar F0 in Scottish red deer stags, and illustrates how the F0 of terrestrial mammal vocal sexual signals may be subject to different selection pressures across species. Further investigations on species characterized by different F0 profiles are needed to provide a comparative background for evolutionary interpretations of sex differences in mammalian vocalizations.

With the development of high-throughput gene expression profiling technologies came the opportunity to define genomic signatures predicting clinical condition or cancer patient outcome. However, such signatures show dependency on training set, lack of generalization, and instability, partly due to microarray data topology. Additional issues for analyzing tumor gene expression are that subtle molecular perturbations in driver genes leading to cancer and metastasis (masked in typical differential expression analysis) may provoke expression changes of greater amplitude in downstream genes (easily detected). In this chapter, we are describing an interactome-based algorithm, Interactome-Transcriptome Integration (ITI) that is used to find a generalizable signature for prediction of breast cancer relapse by superimposition of a large-scale protein-protein interaction data (human interactome) over several gene expression datasets. ITI extracts regions in the interactome whose expression is discriminating for predicting relapse-free survival in cancer and allow detection of subnetworks that constitutes a generalizable and stable genomic signature. In this chapter, we describe the practical aspects of running the full ITI pipeline (subnetwork detection and classification) on six microarray datasets.