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Social complexity parallels vocal complexity: a comparison of three non-human primate species

Social factors play a key role in the structuring of vocal repertoires at the individual level, notably in non-human primates. Some authors suggested that, at the species level too, social life may have driven the evolution of communicative complexity, but this has rarely been empirically tested. Here, we use a comparative approach to address this issue. We investigated vocal variability, at both the call type and the repertoire levels, in three forest-dwelling species of Cercopithecinae presenting striking differences in their social systems, in terms of social organization as well as social structure. We collected female call recordings from twelve De Brazza's monkeys (Cercopithecus neglectus), six Campbell's monkeys (Cercopithecus campbelli) and seven red-capped mangabeys (Cercocebus torquatus) housed in similar conditions. First, we noted that the level of acoustic variability and individual distinctiveness found in several call types was related to their importance in social functioning. Contact calls, essential to intra-group cohesion, were the most individually distinctive regardless of the species, while threat calls were more structurally variable in mangabeys, the most “despotic” of our three species. Second, we found a parallel between the degree of complexity of the species' social structure and the size, diversity, and usage of its vocal repertoire. Mangabeys (most complex social structure) called twice as often as guenons and displayed the largest and most complex repertoire. De Brazza's monkeys (simplest social structure) displayed the smallest and simplest repertoire. Campbell's monkeys displayed an intermediate pattern. Providing evidence of higher levels of vocal variability in species presenting a more complex social system, our results are in line with the theory of a social-vocal coevolution of communicative abilities, opening new perspectives for comparative research on the evolution of communication systems in different animal taxa.

INTRODUCTION
Identifying the key determining factors guiding the evolution of communication systems in animals is still a matter of strong debate in the scientific community. The evolution of vocal communication has often been said to be inseparable from the evolution of social life (Marler, 1977, p. 46;Waser, 1982, p. 118;Snowdon and Hausberger, 1997), notably in non-human primates (Lemasson, 2011), but this evolutionary hypothesis is hard to test empirically. A means of testing it is to investigate to what extent social complexity has played a role in the structuring of a species' vocal repertoire, such as by comparing species that differ in their social system (Lemasson, 2011;Freeberg et al., 2012).
Animals have traditionally been categorised as either vocal learners, for those who need social tutor models to learn the structure of vocalizations from (e.g., oscines, cetaceans, humans), or non-vocal learners, for those whose vocal development is strongly genetically determined (e.g., non-human primates). Whereas the former present an extensible vocal repertoire (e.g., new complex song notes can be socially learned every year in certain songbirds: Hausberger, 1997), the latter present a comparatively limited number of fixed call types. However, within fixed call types, refined acoustic changes can still be found (Hammerschmidt and Fischer, 2008). Interestingly, a growing number of studies have highlighted that, within species, social factors play a key role in the structuring of individual vocal repertoires in terms of both production and usage, even in the so-called non-vocal learners. First, comparisons of different conspecific social groups showed that group membership can be encoded in the acoustic structure of vocalizations (e.g., primates: Crockford et al., 2004;Tanaka et al., 2006;bats: Boughman and Wilkinson, 1998;birds: Hopp et al., 2001;Wright and Wilkinson, 2001). Second, within a social group, inter-individual comparisons revealed that social bonding was reflected in the structure or usage of vocalizations. (e.g., primates: Lemasson and Hausberger, 2004;Lemasson et al., 2011a;cetaceans: Smolker and Pepper, 1999;birds: Hausberger et al., 1995;Brown and Farabaugh, 1997), as was the hierarchical dominance rank (e.g., primates: Mitani and Nishida, 1993;Fischer et al., 2004). Those vocalizations, functioning as "social badges," are typically contact calls or longdistance calls whose role is to maintain intra-group cohesion. Third, at the intra-individual level, social learning of vocal usage rules and adult-like acoustic structures has been demonstrated (e.g., primates: Snowdon et al., 1997;Lemasson et al., 2011b;cetaceans: McCowan and Reiss, 1997;birds: Nottebohm, 1970). Still at the intra-individual level, the assumption has been made that the social function of a call type may influence its level of acoustic variability ("call social function" hypothesis). In fact, Snowdon et al. (1997;p. 236) and Griebel and Oller (2008, p. 25) suggested that higher levels of acoustic variability may have been selected for in affiliative calls which are essential to the intra-group social functioning, while alarm calls may be more conservative. The structural variability expected in affiliative calls would be a means of encoding information about the caller's identity (inter-individual variability) or the context of emission (intra-individual variability), for example. Accordingly, the few attempts that have been made to compare the level of acoustic variability of functionally different call types throughout a species' vocal repertoire suggest a greater potential for identity coding in contact calls, compared to alarm or distress calls for example, in a few primate (Rendall et al., 1998(Rendall et al., , 2009Lemasson and Hausberger, 2011;Bouchet et al., 2012a) and bird (Charrier et al., 2001) species. At the inter-specific level, social complexity may have favoured the evolution of vocal complexity, in terms of both production and usage. The structural complexity of a system is defined as some function of the number of different parts it has and the irregularity of their arrangement; thus, heterogeneous, elaborate, or patternless systems are complex (McShea, 1991). Also, social complexity refers to the number of interacting individuals (i.e., group size, social network size), the different types of individuals (i.e., different types of social roles), and the frequency, diversity, and distribution of the interactions among them. Vocal complexity relates to the size and diversity of the vocal repertoire, its organisational structure (e.g., association patterns between sound units into calls, and calls into sequences), the level of intra-and inter-individual variability of the signals, and also the frequency and contextual diversity of usage of those signals. The species' social structure (i.e., group size and composition) has been hypothesized to account for the level of complexity of the vocal repertoire displayed by group members, in terms of both size (Blumstein and Armitage, 1997;McComb and Semple, 2005;Knotková et al., 2009;Gustison et al., 2012) and diversity (Freeberg, 2006;Freeberg and Harvey, 2008) ("sociallydriven repertoire complexity" hypothesis). In addition, it has been suggested that group size may also influence vocal activity (i.e., calling rates). When group size increases, the difficulty to perform "bodily grooming" would be compensated by "vocal grooming"; calling would thus serve the function of maintaining group cohesion (Dunbar, 1998;Griebel and Oller, 2008) ("vocal grooming" hypothesis). Nevertheless, to date, those evolutionary hypotheses have only been tested separately, through inter-species comparisons based on reviews of the existing literature (e.g., primates: Dunbar, 1998Dunbar, , 2012McComb and Semple, 2005;rodents: Blumstein and Armitage, 1997;Pollard and Blumstein, 2012;cetaceans: May-Collado et al., 2007;birds: Krams et al., 2012) or through inter-group comparisons (groups varying in size) at the intra-specific level (e.g., Carolina chickadees, Poecile carolinensis: Freeberg, 2006;Freeberg and Harvey, 2008). Also, those hypotheses remain to be formally tested jointly through a comparative study of multiple species.
To investigate the link between social and vocal complexity, we selected species presenting striking differences in their social systems, but being closely-related and originating from a similar habitat, as both phylogeny (Gautier, 1988;Cap et al., 2008;Thinh et al., 2011) and habitat (Brown et al., 1995;Daniel and Blumstein, 1998) are other factors known to constrain the shaping of animal vocal repertoires. Also, in the present study, we investigated vocal variability in three closelyrelated non-human primate species, namely De Brazza's monkey (Cercopithecus neglectus), Campbell's monkey (Cercopithecus campbelli) and red-capped mangabey (Cercocebus torquatus). They originate from comparable African tropical forests, and were studied here in similar, controlled captive conditions. They present interesting differences in their social systems, in both social organization (discrete hierarchy in guenons vs. strong hierarchy in mangabeys, rare physical interactions in guenons vs. relatively frequent physical interactions in mangabeys) and social structure (small family groups for De Brazza's monkeys, medium-size harems for Campbell's monkeys, and large multimale multi-female groups for red-capped mangabeys) (Gautier-Hion and Gautier, 1978;Mitani, 1989;Matthews and Matthews, 2002;Lemasson et al., 2006;Mwenja, 2006;Ouattara et al., 2009b;Dolado and Beltran, 2012). We decided to focus on females as we expected the influence of social factors on vocal variability to be more striking in this sex class whose role in mediating intra-group relationships is predominant (Rowell, 1988). Indeed, in all three species, sex differences in vocal as well as non-vocal behaviour are sizeable, especially in guenons. Females form the group "social core", they often interact socially and are very active vocally, whereas males are more peripheral both socially and spatially, they act mostly as group protectors and seldom vocalize (Gautier-Hion and Gautier, 1978;Oswald and Lockard, 1980;Rowell, 1988;Lemasson et al., 2006). Also, females frequently exchange contact calls with social partners, whereas males rather produce calls in response to external stimuli, such as a spotted danger (e.g., predator, tree fall, neighboring group) (Lemasson and Hausberger, 2004;Ouattara et al., 2009b;Bouchet et al., 2010Bouchet et al., , 2012b. Hence, we believe that if social factors guide vocal complexity in these species, females should be first impacted. We evaluated vocal variability at both the call type and the repertoire levels across our three species. First, we tested whether or not the species' social organization affects the relationship between the social function of a given call type and its level of acoustic variability, estimated here in terms of intra-individual structural variability and individual acoustic distinctiveness. We compared three functionally equivalent call types (contact, threat and alarm calls) in the three species through measurements of the same set of temporal and frequency parameters. Calls essential to the intra-group social functioning that is contact calls in all species, but also threat calls in more "despotic" species, were expected to display higher levels of both intra-and inter-individual acoustic variability than alarm calls ("call social function" hypothesis). Second, we tested whether or not the species' social structure influences its level of vocal activity, as well as the size and diversity of its vocal repertoire. Species living in larger groups were predicted to vocalize more frequently ("vocal grooming" hypothesis) and to display a structurally larger and richer vocal repertoire ("socially-driven repertoire complexity" hypothesis).

SUBJECTS AND HOUSING CONDITIONS
Call recordings were conducted with five groups of De Brazza's monkeys, one group of Campbell's monkeys and three groups of red-capped mangabeys housed in indoor-outdoor enclosures enriched with either perches, ropes, shrubs or trees, at the Station Biologique de Paimpont in France, and at Howletts and Port Lympne Wild Animal Parks in the UK (Table 1-for more details on the housing conditions, see Bouchet et al., 2010Bouchet et al., , 2012bLemasson and Hausberger, 2011). We considered females as sexually mature when more than 3 years old in De Brazza's and Campbell's monkeys (Gautier-Hion and Gautier, 1978;Galat and Galat-Luong, 1985), and when more than 4 years old in red-capped mangabeys (Hill, 1974). a In this group, observations (8 h15 in total) were conducted ad-libitum, thus those data have been used in structural analysis, but not for call rate analysis.
b Two adult females were carrying an infant, and in order to avoid any disturbance, they were not equipped with a telemetric harness (Lemasson and Hausberger, 2011 Cumulatively, we collected data from twelve De Brazza's monkey, six Campbell's monkey, and seven red-capped mangabey adult females. Subjects were provided with fruit, vegetables, and commercial monkey chow daily. Water was available ad-libitum. Animal care and research protocols used in this work complied with the current laws of France and the UK and were conducted under license from the Direction Départementale des Services Vétérinaires (DDSV n • 04672).

DATA COLLECTION
Observations were performed outdoors (to ensure high quality recordings suitable for acoustic analysis) between 09:00 and 18:00, outside of feeding times. Subjects were observed in a random order using the focal sampling method, sessions being distributed throughout the day for every subject. In De Brazza's monkeys and red-capped mangabeys, calls of focal (and occasionally of nonfocal) individuals were recorded using a directional microphone (Sony ECM-672 for the females housed in Paimpont, Sennheiser MKH-70 for the females housed in Howletts and Port Lympne) connected to a digital stereo recorder (Sony DAT TCD-D100 for the De Brazza's monkeys housed in Paimpont, Marantz PMD-660 for the other groups). In guenons, females frequently produce soft sounds with their mouths closed, which made it difficult to identify the caller within a multi-female group, even in captivity. Thus, sound recordings in Campbell's monkeys were performed through a telemetric system composed of a transmitter (fixed on a leather harness on the monkey), a receiver, and a digital stereo recorder (TASCAM DA-P1) (for technical details, see Lemasson and Hausberger, 2011).

Call types analysis
The vocal repertoires of the three species based on acoustic, contextual, and phylogenetic analyses have been described earlier (guenons: Gautier, 1988; De Brazza's monkeys: Bouchet et al., 2012b; Campbell's monkeys: Lemasson and Hausberger, 2011;red-capped mangabeys: Bouchet et al., 2010). We focused on three call types that are contextually equivalent across the three species but differ in their social function: a contact call (produced mostly during affiliative interactions), a threat call (uttered by aggressors during agonistic interactions) and an alarm call (elicited by external disturbances) (Figure 1).
Sonograms were drawn by a Fast-Fourier Transform (1024-pt FFT length containing 256 samples and zero padding, incremental step: 32 pt) using ANA acoustic software, a customized AMIGA micro-computer program (Richard, 1991) recently adapted to be run on PC implemented on LINUX. Calls recorded during both focal and non-focal observations were examined here, but signals with excessive background noise were excluded from the analyses. In order to best control for data set balance, we randomly selected, when possible, ten exemplars per call type and per individual (sample sizes are given in Table 2). We performed measurements at both the sound unit and the call levels (time resolution: 2.49 ms, frequency resolution: 50 Hz). A unit was defined as the basic element of a call, represented as a continuous tracing along the temporal axis of the sonogram (also referred to as "note" or "syllable" in the monkeys and birds literature, see Bouchet et al., 2010). For calls composed of more than one unit type ("CH", "WaHoo", "Un+(Uh)"), we performed measurements on the "principal unit" type only (i.e., the unit type introducing the call, as defined in Bouchet et al., 2010) (respectively, "On," "Wa," and "Un") ( Figure 1). Also, if a call consisted of several same-type units ("Tek+," "Wrr+," "RRA," "RRC," "Ro+," "Un+(Uh)"), we performed measurements on the second unit in the call because the first unit was often not clearly visible ( Figure 1). As we faced both noisy and tonal calls, we focused on five acoustic parameters that were shared by all call types: call duration (Dcall, ms), number of units (#units), unit duration (Dunit, ms), unit base frequency (Fbase, Hz; i.e., lowest-pitched reinforced frequency measured in the middle of the frequency band that is the fundamental frequency in tonal calls), and unit peak frequency (Fpeak, Hz; i.e., frequency at maximum energy, measured on the power spectrum) (following the same methodology as Bouchet et al., 2010Bouchet et al., , 2012a. In order to describe call types, as individuals contributed differently to the data set, we first calculated individual means and then averaged these scores. Standard deviations (SD) were calculated by averaging the data set SD of every individual and totaling it with the SD of the individual means ( Table 2).

Vocal activity analysis
For each individual, we calculated the global hourly call rate (all call types merged), by dividing the total number of calls given during focal samples by the sum of the total observation time (in hours) for that individual. Individual means were then averaged within each species.

Repertoire analysis
The structural acoustic complexity of each species' repertoire was assessed at the sound unit level, using vocalizations recorded during focal observations only (to control for relative proportions of each type within the entire repertoire). We noted whether each call within the entire repertoire was composed either of a single unit ("single"), of multiple units of the same type ("repeated"), or of units of different types ("combined"). We described all the "unit assembling patterns," and estimated their frequency of occurrence in each individual, then averaged within each species. We also computed the frequency of occurrence for the categories "single," "repeated," and "combined" patterns (whatever the unit types) at the individual level, then the species level.  Lemasson and Hausberger, 2011;red-capped mangabeys: Bouchet et al., 2010). Each sonogram is labeled according to the composition in units of the call (units are named using onomatopoeia), and the unit on which measurements have been performed is set in bold type. In the three species, alarm calls display a noisy pattern. In guenons, alarm calls ("Tek+" and "RRA") consist of repeated pulses, while red-capped mangabey alarm calls ("WaHoo") are systematically composed of two units of different types. Guenon threat calls ("Wrr+" and "RRC") display an acoustic pattern similar to that of their alarm calls, whereas red-capped mangabey threat calls ("Un+(Uh)") are composed of one or several short low-pitched tonal units, sometimes combined to an additional "Uh" unit. In the three species, contact calls are composed of one short, low-pitched, tonal unit which is uttered mostly singularly in De Brazza's monkeys ("On"), combined to a second arched-shape high-pitched unit in Campbell's monkeys ("CH"), and either singularly or repeated in red-capped mangabeys ("Ro+").

Call types analysis
To describe the pattern variability of each call type, we used coefficient of variation measurements. For each of the five acoustic parameters, and for every call type in each species, we computed the coefficient of intra-individual variation (CV intra = mean of individual CV values, with for each individual: CV = 100× SD/X), and then averaged it over the five acoustic parameters within each call type to compute an overall value: the call type intra-individual CV (CVmean intra = CV intra averaged over the five parameters). Then, we performed comparisons between call types and between species. Within each species, we tested whether  Lemasson and Hausberger, 2011;red-capped mangabeys: Bouchet et al., 2010).
the three call types differed in their level of intra-individual variability by performing Friedman tests on the five CV intra values. Likewise, for each call type, we tested whether the three species differed by performing Friedman tests on the five CV intra values. In both cases, Friedman tests were followed by post-hoc multiple comparisons (Siegel and Castellan, 1988).
To quantify the potential of each call type to encode identity, we used information capacity measurements, as described in the information analysis method developed by Beecher (1989) based on information theory (Shannon and Weaver, 1949). This approach has been used successfully in recent studies of birds (Searby et al., 2004) and mammals (Sèbe et al., 2010;Pollard and Blumstein, 2011). For each of the five acoustic parameters, and for every call type in each species, a One-Way analysis of variance (ANOVA, type III) was conducted with "caller identity' as the fixed factor. For variables giving a significant F (P ≤ 0.05), we then computed the stereotypy index Hs (Searby et al., 2004;Pollard and Blumstein, 2011). The information content of one parameter, Hs, is derived from the F-value found in the One-Way ANOVA, but, unlike F, Hs does not vary with sample size. Information capacity measurements are therefore more reliable when comparing samples. For a given parameter, Hs is expressed as: where F = result of ANOVA, n = number of calls, and k = number of individuals contributing to the dataset. Hs (in bits/signal) represents the number of binary decisions necessary to discriminate between N objects. The higher the value of Hs, the greater potential the parameter has for encoding individual identity. Parameters with an Hs < 1 can be considered as "lowinformative" (Searby et al., 2004). Then, we computed the total Hs information for each call type ( Hs) that is, the sum of the information in each of the independent variables. Since some of the acoustic parameters measured are likely to be inter-correlated, we combined the five variables into principal components (Principal Component Analysis with Varimax rotation), and retained those giving a significant F (P ≤ 0.05) to calculate the total Hs information (Beecher, 1989;Sèbe et al., 2010).

Repertoire analysis
To estimate the structural complexity of each species' vocal repertoire in terms of "unit assembling patterns", we computed a diversity index following a procedure introduced by Shannon and Weaver (1949). This involves the calculation of two indices: H i max represents the value if all signal types are uttered with the same frequency, while H i represents the actual observed values. The index of diversity DI is thus expressed as: where n = total number of different "unit assembling pattern" types, and p i = the probability of occurrence of each pattern. The smaller the value of DI, the less diverse the repertoire (i.e., the repertoire is dominated by one "unit assembling pattern" type).
We also compared the complexity of vocal production between the three species. We performed Kruskall-Wallis tests on the frequencies of utterance computed for the categories "single," "repeated," and "combined" patterns separately, followed by post-hoc multiple comparisons (Siegel and Castellan, 1988). Furthermore, within each species, we tested whether one category of pattern ("single," "repeated," or "combined") was more frequent than another by performing Friedman tests followed by post-hoc multiple comparisons (Siegel and Castellan, 1988).
Statistical analyses were performed using SPSS 13.0, Minitab 12.2 and Microsoft Excel. We set significance at α = 0.05.

CALL TYPES' LEVEL OF VARIABILITY AND INDIVIDUAL DISTINCTIVENESS
The overall level of structural intra-individual variability as well as the level of individual distinctiveness varied among the three functionally different call types (contact, threat and alarm; Figure 1) across the three species (Figure 2).

FIGURE 2 | Acoustic intra-individual variability and individual distinctiveness in contact, threat and alarm calls in each of three species of primate.
CVmean intra : call type intra-individual coefficient of variation (%) and Friedman test result ( * P < 0.05; results of the post-hoc multiple comparisons are illustrated by letters symbolizing homogeneous subsets). Hs: information content value (see the Statistical Analysis section for definitions). Dcall, call duration (ms); #units, number of units; Dunit, unit duration (ms); Fbase, unit base frequency (Hz); and Fpeak, unit peak frequency (Hz).

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July 2013 | Volume 4 | Article 390 | 7 Interestingly, the level of inter-individual variation did not correlate with the level of intra-individual variation found in the various call types. In the three species, the potential for identity coding was the highest in contact calls (highest Hs values) (Figure 2). However, while red-capped mangabey threat calls displayed a higher potential for identity coding (higher Hs value) than their alarm calls, the two guenon species displayed the opposite pattern (Figure 2). Regarding the ability to encode individual identity of the various acoustic parameters, it appeared that the number of units per call was low-informative (Hs < 1 in all cases but Campbell's monkeys' alarm calls) while the frequency parameters (base and/or peak frequencies) were informative regarding identity in most cases (one or both frequency parameters had a Hs > 1 in each call type, except threat calls in De Brazza's monkeys and contact calls in red-capped mangabeys) (Figure 2). Conversely to frequency parameters, no systematic pattern of variability was found across species for temporal features (call and unit duration), except that unit duration was informative (Hs > 1) for contact calls in the three species (Figure 2).

VOCAL ACTIVITY ACROSS SPECIES
Red-capped mangabeys and the two guenon species differed in their global vocal activity, the former calling more than twice as much as the two others (Kruskall-Wallis test: H 2 = 5.96, P = 0.051). The post-hoc one-tailed pairwise comparisons (chosen according to the "vocal grooming" hypothesis) revealed that call rates were significantly higher in red-capped mangabeys than Campbell's monkeys (P < 0.05), were higher in red-capped mangabeys than De Brazza's monkeys, although not significantly so (P < 0.075), and did not differ between the two guenon species (P > 0.15). However, we noted that one De Brazza's monkey female (102.5 calls/h) called ten times as much as the nine other conspecific females (X ± SE: 15.3 ± 3.6 calls/h; range: 4.8-38.5). Therefore, we excluded this outlier (Grubb's test: Z = 2.673, N = 10) and reran the analyses (Figure 3). The global test result did not change (Kruskall-Wallis test: H 2 = 7.75, P = 0.021), but the post-hoc one-tailed pairwise comparisons revealed that while call rates remained significantly higher in red-capped mangabeys than Campbell's monkeys (P < 0.05), the difference between red-capped mangabeys and De Brazza's monkeys was now significant as well (P < 0.025). We still did not observe a difference between the two guenon species (P > 0.15).

REPERTOIRE COMPLEXITY ACROSS SPECIES
When assessing the structural acoustic variability of each species' at the sound unit level, we identified 6 unit types in De Brazza's monkeys, 8 unit types in Campbell's monkeys, and 9 unit types in red-capped mangabeys (Figure 4). Those units were then concatenated into 9, 10 and 16 different "unit assembling patterns" respectively. Interestingly, the number of "combined" patterns was the lowest in De Brazza's monkeys (N = 1), intermediate in Campbell's monkeys (N = 3), and the highest in red-capped mangabeys (N = 6) (Figure 4).
The diversity index DI enabled us to estimate the structural complexity of each species' vocal repertoire in terms of "unit assembling pattern" types (Figure 4). As a result, it appeared that female De Brazza's monkeys displayed the less diverse repertoire (DI = 0.33), female red-capped mangabeys the most diverse FIGURE 4 | Relative proportion of the different "unit assembling patterns" produced by each of three species of primate. Below the species' name: number and name of unit types. Unit types are labeled as in previous publications for De Brazza's monkeys (Bouchet et al., 2012b) and red-capped mangabeys (Bouchet et al., 2010). Campbell's monkeys' repertoire, however, has been revised according to Bouchet et al. (2010) multi-level approach; the correspondence between this nomenclature and the call type names used in Lemasson and Hausberger (2011) is as follow: "On" = SH; "I" = ST; "Ra, RaRa, RaRaRa . . . " = RRA; "Wi" = SA; "RuRu, RuRuRu . . . " = RRC; "KekKek, KekKekKek . . . " = RSA; "OnHi" = CH; "IOn" = CT; "UnI" = RST. On the right-hand side: all the unit assembling patterns observed, grouped as "single" , "repeated" , "combined" (see the Data Analysis section for definitions). Below each diagram: DI, diversity index (Shannon and Weaver, 1949). The smaller the value of DI, the less diverse the repertoire.

DISCUSSION
Several findings emerge from this comparative study of the level of vocal variability in three closely related but socially distinct non-human primate species. Overall, we found evidence that the species' social structure and social organization covary with the level of complexity observed in its vocal repertoire, in terms of both structuring and usage; our results are in line with the predictions of the "social system-vocal variability" coevolution hypothesis. When comparing three functionally different call types (contact, threat and alarm), acoustic variability did not appear homogeneously within nor across species. Individual distinctiveness was the highest in contact calls regardless of the species examined. In contrast, threat calls were the least individually distinctive in the "tolerant' guenons, but displayed an intermediate pattern compared with contact and alarm calls in the "despotic" mangabeys. Meanwhile, female mangabey threat calls were more structurally variable than those of female guenons. These results support the "call social function" hypothesis. When investigating vocal activity, we found that calling rates of female mangabeys were more than twice as high as those of female guenons. Given that group size is larger in mangabeys compared with guenons in the wild, our result supports the "vocal grooming" hypothesis. Lastly, when investigating vocal repertoire structuring, we observed the largest and most diverse repertoire (i.e., more unit types, more assembling patterns, higher diversity index) in female red-capped mangabeys, the smallest and least diverse in female De Brazza's monkeys, and an intermediate pattern in Campbell's monkeys. All species enriched their repertoire diversity by producing their sound units singly, or in repeated or combined patterns. Interestingly, we found that FIGURE 5 | Hourly call rate of "single", "repeated" and "combined" unit assembling patterns in each of three species of primate. Number of calls/h: X ± SE. Results of the post-hoc multiple comparisons following the Kruskall-Wallis test ( * P < 0.05, NS, not significant).

Frontiers in Psychology | Comparative Psychology
July 2013 | Volume 4 | Article 390 | 10 the number of combinatorial patterns was the highest in female mangabeys, the lowest in De Brazza's monkeys, and intermediate in Campbell's monkeys. As repertoire complexity matches social structure complexity, these results support the "socially-driven repertoire complexity" hypothesis.

PERCEPTUAL SALIENCE OF THE OBSERVED ACOUSTIC VARIABILITY
It has to be acknowledged that the acoustic variability evidenced in this study, both at the call and the repertoire levels, remains to be tested for its relevance to listeners. Nonetheless, many studies have shown that non-human primates are able to discriminate between individuals from their calls (e.g., rhesus macaques, Macaca mulatta: Rendall et al., 1996; olive baboons, Papio hamadryas anubis: Lemasson et al., 2008; chacma baboons, Papio hamadryas ursinus: Rendall et al., 2009), can perceive subtle differences in acoustic structure (e.g., Campbell's monkeys: Lemasson et al., 2005), and can distinguish between sequences of sound units (e.g., cotton-top tamarins,  Lemasson et al., 2011a). Such evidence allows us to hypothesize that the acoustic variability observed here is perceptible to monkeys; future research must, however, determine its actual salience to listeners.

INFLUENCE OF SOCIAL ORGANIZATION ON CALL TYPE DESIGN
In accordance with the "call social function" hypothesis Griebel and Oller, 2008), we found the highest level of individual distinctiveness in contact calls, compared with alarm calls, in the three studied species. This is in agreement with the few studies in non-human primates and birds that have provided evidence of a higher level of individual distinctiveness in affiliative calls compared with distress, alarm, or courtship calls (Rendall et al., 1998(Rendall et al., , 2009Charrier et al., 2001;Lemasson and Hausberger, 2011;Bouchet et al., 2012a). Interestingly, we provided further evidence of an effect of the social organization (in terms of strong vs. discrete hierarchy) on the level of acoustic variability of the threat call. In fact, we found a greater level of intra-individual structural variability in the threat calls of the "despotic" red-capped mangabeys than in those of the two "tolerant" guenon species. Moreover, we found that identity coding was stronger in female mangabey threat vs. alarm calls, whereas in guenons threat calls were the least individually distinctive. Consequently, we suggest that the call's relevance to the species' social organization (i.e., whether or not the call mediates intra-group interactions, which are essential to social functioning), rather than the type of message it conveys (e.g., contact vs. threat vs. alarm), accounts for its level of acoustic variability. Furthermore, we found that the degree of individual distinctiveness of a call type did not correlate with its level of intra-individual acoustic variability. Also, it seems that in the case of mangabey threat calls, the realized acoustic variability could serve to encode information not only about the caller's identity but also about the context of emission (e.g., rhesus macaques: Gouzoules et al., 1984;chimpanzees, Pan troglodytes: Slocombe and Zuberbühler, 2007).
Whereas contact calls and alarm calls displayed similar frequency patterns (tonal and noisy respectively) in the three species we studied, threat call structural patterns differed across species (tonal as their contact calls in red-capped mangabeys vs. repeated noisy pulses as their alarm calls in guenons). Owren and Rendall (2001), suggested a "structure-affective processing" relationship: (1) call types used by callers to directly influence the affect of listeners would be characterized by peculiar acoustic features well-designed for capturing their attention (e.g., sharp onset, high-amplitude noisiness, repeated energy pulses) but not suitable for conveying individual distinctiveness; (2) call types used to indirectly influence the affect of listeners would give clear cues to caller identity (e.g., tonal harmonically-rich calls) that listeners would associate, through a conditioning process, with past positive or negative interactions with the caller. Accordingly, we found that identity coding was the strongest in the tonal contact calls regardless of the species, and that female mangabey tonal threat calls were more individually distinctive than their noisy alarm calls, whereas in guenons the noisy threat calls were the least individually distinctive. Owren and Rendall (2001) notably illustrated their "structure-affective processing" hypothesis with calls produced by baboons and macaques, contact and threat calls having indirect affective effects, distress and alarm calls having direct affective effects. Interestingly, we found a similar pattern in red-capped mangabeys whose social organization is based on relatively frequent peaceful and agonistic interactions and on a strong hierarchy like most baboons and macaques (Rowell, 1988;Dolado and Beltran, 2012). In contrast, guenons' social organization is based on rare physical interactions and on a discrete hierarchy (Gautier-Hion and Gautier, 1978;Rowell, 1988;Lemasson et al., 2006), and they displayed a different pattern here, with threat calls having an acoustic structure more likely to have direct affective effects on listeners. Consequently, we suggest that the call's relevance to the species' social organization (i.e., its role in mediating interactions that are crucial according to the species-specific social needs) accounts for its acoustic structure and its associated level of individual distinctiveness.
Taken together, these results suggest that the species-specific social needs have exerted a selective pressure on call structure, favouring acoustic variability (e.g., individual distinctiveness or context-related acoustic variability) in some particular calls involved in critical aspects of intra-group social functioning. Also, the theory of an influence of the call social function on its level of acoustic variability can be extended to an influence on the coevolution between its acoustic structure and the associated communicative capacities.

INFLUENCE OF SOCIAL STRUCTURE ON VOCAL REPERTOIRE STRUCTURING AND USAGE
In accordance with the "vocal grooming" hypothesis (Dunbar, 1998;Griebel and Oller, 2008), we found that female red-capped mangabeys called more than twice as much as female guenons. Also, in accordance with the "socially-driven repertoire complexity" hypothesis (Blumstein and Armitage, 1997;McComb and Semple, 2005;Freeberg, 2006;Freeberg and Harvey, 2008;Knotková et al., 2009;Gustison et al., 2012), we found that female mangabeys displayed the largest and most diverse vocal repertoire in terms of structural composition (greatest number of unit types and assembling patterns, highest diversity index: DI = 0.67), Campbell's monkeys showed an intermediate pattern (DI = 0.52), and De Brazza's monkeys produced the smallest and simplest repertoire (smallest number of unit types and assembling patterns, lowest diversity index: DI = 0.33). As redcapped mangabeys live in large multi-male multi-female groups, Campbell's monkeys in harem groups, and De Brazza's monkeys in small family units, our results suggest that the species' social structure account for the shaping of its vocal repertoire. While those evolutionary hypotheses have been, to date, tested separately in different taxa (e.g., primates: Dunbar, 1998Dunbar, , 2012McComb and Semple, 2005;Gustison et al., 2012;rodents: Blumstein and Armitage, 1997;Knotková et al., 2009;Pollard and Blumstein, 2012;cetaceans: May-Collado et al., 2007;birds: Freeberg, 2006;Freeberg and Harvey, 2008;Krams et al., 2012), our study highlights, by using a multi-level approach (sound unit-assembling patterns-repertoire) of vocal variability in terms of both production and usage, that those hypotheses are complementary .

FROM SINGLE SOUND UTTERANCE TO MORE COMPLEX VOCAL COMBINATION
It is interesting to look more thoroughly at the way differential levels of vocal complexity are achieved across the three species. Parallel to the increase in social complexity between our three species, we found an increase in the number of unit types, in the number of assembling patterns, as well as in the number of "combined" patterns (i.e., concatenation of units of different types). One possibility of "creating" acoustic variability is to have non-fixed acoustic parameters (e.g., red-capped mangabey threat calls), but this has some limit in non-human primates as the control they have over their vocal apparatus is restricted (Lemasson, 2011). Another possibility of "creating" acoustic variability is to use syntactic-like sound combinations. The perceptual salience of those sound combinations remains to be tested for our subjects, nevertheless a growing number of recent studies provided evidence that those higher-level acoustic structures can be meaningful in a proto-syntactic-like way. Guenon males use suffixation (Campbell's monkeys: Ouattara et al., 2009a) and call sequences (putty-nosed monkeys: Arnold and Zuberbühler, 2008;Campbell's monkeys: Ouattara et al., 2009b) to increase the number of context-specific alarm messages delivered. Even in apes, it has been reported that white-handed gibbons concatenate the same set of notes into different songs (Clarke et al., 2006), chimpanzees combine calls (Crockford and Boesch, 2005), and bonobos (Pan paniscus) combine calls into sequences (Clay and Zuberbühler, 2011) in context-specific ways.
The ability of monkeys and apes to combine sound units or calls into more complex structures parallels, in some respects, the combinatorial ability pushed to extremes in human languages. The three closely-related forest species we studied here all used this possibility of syntactic-like sound combinations, but redcapped mangabeys who live in the most complex social groups are the ones who made the most of it. Also, combinatorial ability could have been enhanced in species whose social needs require individuals to increase the number of messages that they can deliver.

SOCIAL COMPLEXITY AND THE EVOLUTION OF COMMUNICATIVE ABILITIES
Whereas broad inter-species comparisons based on a review of the existing literature suggest some patterns (e.g., repertoire size correlates with group size and time spent grooming in non-human primates: McComb and Semple, 2005), detailed comparative studies on a restricted number of closely-related species allow to take a closer look at how sociality has driven the evolution of communication . In a recent study, vocal repertoire size was compared between two species living in large social groups: geladas who aggregate into a complex multi-level society composed of small reproductive units within which one male forms long-term bonds with several females, and chacma baboons who live in a single-level multi-male multi-female society where cross-sex relationships consist mainly of temporary consortships (Gustison et al., 2012). The authors identified a number of homologous call types common to both species, but they found that geladas displayed a larger vocal repertoire than chacma baboons. The non-homologous (derived) call types specific to geladas function in cross-sex bonding, and they were produced primarily by males who are responsible for the maintenance of those long-term social bonds. Two interesting conclusions emerge from this study. First, social bonding turns out to be a key factor, more critical than the size of the reproductive unit, in the emergence of socially-driven vocal complexity in geladas. Second, the influence of social factors on vocal complexity appears to be more sizeable in males, as a result of their social role within the gelada society.
In our case, we focused on females whose role in mediating intra-group social relationships is predominant in the three species we studied. Therefore, we expected the influence of social factors on vocal variability to be most remarkable in this sex class. Overall, we found that female mangabeys, who are involved in a more complex social network than female guenons, display higher degrees of vocal complexity at several levels (acoustic variability, repertoire size, calling rate). Nevertheless, we do not rule out the possibility of socially-driven vocal complexity in males. It is notable that sex differences in vocal repertoire, though sizeable in the three studied species, are more striking in guenons (Campbell's monkeys : Gautier, 1988;Lemasson and Hausberger, 2011;De Brazza's monkeys: Bouchet et al., 2012b) than in red-capped mangabeys (Bouchet et al., 2010). Thus, relatively speaking, male mangabeys tend to equal the level of vocal complexity displayed by females (e.g., calling rate, vocal repertoire size). However, it is impossible here to discriminate between the effects of group size (larger in mangabeys than in guenons) vs. degree of involvement within the social network (male mangabeys are more socially active than male guenons). In the future, comparative studies on a small number of carefully chosen species might help uncover the relative influence of distinct aspects of sociality (e.g., group size, social network complexity, strength of social bonds) in the evolution of communicative abilities, as well as the extent to which it affects individuals unevenly according to their role within the social group.
So far, we have focused our discussion on comparative studies which, like ours, highlighted a parallel between social and vocal complexity in line with the theory of a social-vocal coevolution of communicative abilities. Interestingly, a study on gestural complexity in three species of macaques (Macaca sp.) showed a relationship between the complexity of social organization and the size of the gestural repertoire as well as the level of variability in communicative patterns (Maestripieri, 2007). More recently, a study on facial mobility in 12 non-human primate species revealed a correlation between group size and the variety of facial movements a species can produce (Dobson, 2009). Another study on chemical complexity of glandular secretions in eight species of brown lemurs (Eulemur sp.) that differ in their social system provided evidence of a correlation between social complexity and olfactory complexity (Del Barco-Trillo et al., 2012). Lastly, a study on the auditory system of 20 non-human primate species revealed a correlation between social complexity and enhanced hearing sensitivity (Ramsier et al., 2012). This set of comparative studies suggests that the theory of a social-vocal coevolution of communicative abilities might also apply to other communicative modalities at both the production and perception levels .
Our study points out the advantage of addressing multiple facets of communicative complexity in a small number of closelyrelated but socially distinct species. It opens new perspectives for comparative research on the evolution of communication in animal species. The existing literature suggests that sociallydriven vocal complexity is widespread in the animal kingdom (reviewed in Freeberg et al., 2012). Targeted detailed comparative studies would help uncover whether or not similar social pressures (e.g., group size, social bonding) have affected identical aspects of communication (e.g., calling rate, repertoire size), and led to comparable levels of vocal complexity in disparate taxonomic groups. This approach applied, more specifically, to representatives of the primate lineage would be of great interest to advance our understanding of the selective pressures that led to the emergence of a communicative system as complex as human language.


Autophagic Balance Between Mammals and Protozoa: A Molecular, Biochemical and Morphological Review of Apicomplexa and Trypanosomatidae Infections

Protozoa are unicellular eukaryotes that are able to live as parasites or as free-living organisms and interact with a great variety of environments and organisms, from bacte‐ ria to man; in addition, they represent one of most important sources of parasitic diseas‐ es. Every year, more than one million people die from complications from protozoal infections worldwide [1-5]. Of the medically relevant protozoa, Trypanosomatidae and Apicomplexa constitute a substantial group including the causative agents of several human diseases such as Chagas disease, sleeping sickness, leishmaniasis, malaria and toxoplasmosis [1,5,6]. The life cycles of these parasites are highly complex, involving different hosts and different specific interactions with a variety of cells and tissues [711]. Some of these parasites live in the extracellular matrix or blood of host mammals, but the majority of them infect host cells to complete their cycle. Despite the high infection and mortality rates of these protozoa, especially in low-income populations of developing regions such as Africa, Asia and the Americas, current therapies for these parasitic diseases are very limited and unsatisfactory. The development of efficient drugs is urgently necessary, as are serious public health initiatives to improve patients’ quality of life [12-16].

Introduction
Protozoa are unicellular eukaryotes that are able to live as parasites or as free-living organisms and interact with a great variety of environments and organisms, from bacteria to man; in addition, they represent one of most important sources of parasitic diseases. Every year, more than one million people die from complications from protozoal infections worldwide [1][2][3][4][5]. Of the medically relevant protozoa, Trypanosomatidae and Apicomplexa constitute a substantial group including the causative agents of several human diseases such as Chagas disease, sleeping sickness, leishmaniasis, malaria and toxoplasmosis [1,5,6]. The life cycles of these parasites are highly complex, involving different hosts and different specific interactions with a variety of cells and tissues [7][8][9][10][11]. Some of these parasites live in the extracellular matrix or blood of host mammals, but the majority of them infect host cells to complete their cycle. Despite the high infection and mortality rates of these protozoa, especially in low-income populations of developing regions such as Africa, Asia and the Americas, current therapies for these parasitic diseases are very limited and unsatisfactory. The development of efficient drugs is urgently necessary, as are serious public health initiatives to improve patients' quality of life [12][13][14][15][16].
The Trypanosomatidae family belongs to the order Kinetoplastida and is comprised of flagellated protists characterised by the presence of the kinetoplast, a DNA-enriched portion of the mitochondrion localised close to the flagellar pocket. The most studied pathogenic trypanosomatids are the following: (a) Trypanosoma brucei, which is responsible for sleeping sickness in Africa; (b) T. cruzi, which is the causative agent of Chagas disease in Latin America; and (c) a variety of Leishmania species that cause leishmaniasis in tropical and subtropical areas worldwide. These illnesses have been classified by the World Health Organization as neglected diseases, which affect people living in poverty in developing countries and for which no efficient therapy is available [17][18][19].
The Apicomplexa family encompasses a large group of protists, including approximately 5,000 known parasitic species, which are characterised by the presence of an apical complex containing a set of organelles involved in the infection process. Apicomplexan parasites infect invertebrate and vertebrate hosts, including humans and other mammals. The most serious parasitic disorder is caused by apicomplexan Plasmodium species, the etiological agent of malaria, which causes more than one million deaths annually [1]. Toxoplasmosis is another important disease caused by the apicomplexan parasite Toxoplasma gondii; it has been estimated that almost half of the human population worldwide is infected with this protozoa [20]. The life cycle of the apicomplexan parasites generally consists of complex asexual and sexual reproduction, but some differences are observable among distinct genera. Malaria transmission occurs during the blood feeding of the Anopheles mosquito, whereas toxoplasmosis is mainly transmitted by the ingestion of raw meat or contaminated cat feces.
Autophagy is a physiological self-degradative pathway essential for the maintenance of the metabolic balance in eukaryotes, leading to the turnover of cellular structures during both the normal cell cycle and during conditions of stress, such as starvation [21,22]. This process depends on double-membrane vesicles known as autophagosomes, which are responsible for the engulfment of macromolecules and organelles and the recycling of their components without an inflammatory response [23]. In eukaryotic cells, proteins known as Atgs contribute to the formation of autophagosomes and their targeting to lysosomes [24]. The autophagic machinery interfaces with many cellular pathways, such as that of the immune response and the inflammatory process, and acts as an inductor or suppressor of these processes [25]. Some molecules and organelles can undergo autophagy by specific proteins, such as in the selective pathway known as xenophagy, which is also observed in the degradation of intracellular pathogens [26,27]. The involvement of autophagy in this process has been demonstrated in the interactions of different pathogens with the host cells [28][29][30]. In protozoan infections, the role of autophagy has been debated in light of conflicting evidence presented in the literature, which tends to vary with the experimental model. Some studies suggest that parasites evade host cell defences using autophagy, while others suggest that the host uses autophagy to eliminate the pathogen [31][32][33][34][35]. However, there is no doubt that the autophagic machinery decisively influences the pathogenesis and virulence of protozoan infections; this machinery may therefore represent a promising target for drug discovery [36]. The autophagic process also occurs in the protozoa [37,38] and could occur in parallel to the host cell pathway, thus increasing the complexity of the phenomena. In the following sub-sections, the biology of Trypanosomatidae and Apicomplexa protozoa will be reviewed in relation to the role of autophagy during the infection of the host cells.

Trypanosomatids and autophagy
As previously mentioned, the transmission of neglected diseases caused by trypanosomatids (sleeping sickness, Chagas disease and leishmaniasis) depends on an insect vector, and the environmental change from one host to another is a drastic event for the protozoa. To complete its life cycle, many metabolic and morphological changes must occur for the parasite to survive in a new host [39][40][41][42]. In addition to the kinetoplast, other characteristic ultrastructural structures are present in these parasites, including a single mitochondrion, unique flagella, sub-pellicular microtubules, glycosomes, acidocalcisomes and reservosomes (the last one is present exclusively in T. cruzi) [8]. In the context of the remodelling of sub-cellular structures, autophagy is greatly involved in eukaryotic homeostasis (including in that of trypanosomatids). However, the deregulation of this pathway, which is induced by conditions of stress, also leads to the parasite's death ( Table 1). The sequencing of the complete genome of trypanosomatids has enabled the identification of parasitic genes [43][44][45]. Blast analysis comparing the trypanosome genome with yeast and mammalian genomes, with a particular emphasis on genes encoding autophagic machinery, has indicated the presence of some ATG genes in trypanosomatids [46,47]. However, the partial lack of a ubiquitin-like system, which is crucial for autophagosome formation, and the absence of cytoplasm-to-vacuole-targeting pathway orthologs suggest that these parasites have alternative autophagic features.

T. brucei
T. brucei is the etiological agent of sleeping sickness (or African trypanosomiasis) and is transmitted by the infected tsetse fly (Glossina sp.). After a blood feeding, procyclic trypomastigotes migrate from the insect midgut to the salivary gland where they undergo differentiation to infective metacyclic forms. Subsequently, these metacyclic trypomastigotes are inoculated into the mammalian host during the blood meal of the fly and differentiate into a proliferative bloodstream slender form. Interestingly, after a new differentiation, adapted short-stumpy forms evade the host immune system and disseminate the infection to the whole body; these forms are also able to cross the blood-brain barrier, which causes severe behavioural abnormalities, such as somnolence during daytime [48] (Figure 1). Unlike all other pathogenic trypanosomatids, which have an intracellular life-stage, T. brucei remains in the bloodstream of the mammalian host throughout the process of infection and, as such, is exposed to different environmental conditions that can trigger autophagy.

Role of autophagy in T. brucei
The first report on this parasite and autophagy was published in the 1970s by Vickerman and colleagues. These authors described the presence of myelin-like structures in different forms of the parasite observed by transmission electron microscopy [49,50]. Many years later, it was suggested that the autophagic pathway is involved in the turnover of glycosomes during protozoan differentiation [51]. Glycosomes are peroxysome-like organelles that perform early glycolytic steps and are also involved in lipid metabolism. It was demonstrated that glycosome contents are altered depending on the form of the parasite, with many of these organelles being close to glysosomes during the differentiation process. A similar phenomenon was observed after nutrient deprivation of the parasite, reinforcing the fact that differentiation may cause the degradation of glycosomes by pexophagy.
Further genomic and bioinformatic analyses were performed that identified in T. brucei many ATG orthologs to those of yeasts and mammals [47,52]. These genes are involved in different steps of the autophagic pathway, such as induction (ATG24, PEX14, TOR1 and TOR2, VAC8), vesicle nucleation (ATG6, VPS15 and VPS34) and vesicle expansion and completion (ATG3, ATG7, ATG9, two isoforms of ATG4 and ATG8). Two isoforms of Atg4 and two of Atg8 were recently characterised structurally [53], and it was postulated that Atg8.2 is essential for autophagosome formation and that Atg8 depletion is associated with delayed cell death [54].
It is thought that many drugs may trigger autophagy in African trypanosomes. Dihydroxyacetone (DHA), spermine (snake venom) and vasoactive intestinal peptide (VIP -a neuropeptide secreted by the immune system) induce the appearance of morphological features of autophagy in T. brucei [55][56][57][58]. DHA is an interesting compound to be used in therapy for sleeping sickness because its phosphorylation is DHA kinase-dependent, and DHA kinase is present in mammals and other eukaryotes but not in trypanosomes. After DHA uptake, this compound is not eliminated, leading to typical morphological characteristics of autophagy similar to those found in rapamycin treatment. In another report [59], the authors showed that hydrogen peroxide can produce the appearance of autophagic profiles, suggesting that the release of reactive oxygen species acts as a signal in the autophagic pathway in T. brucei, as it does in other eukaryotic cells [60][61][62].

T. cruzi
T. cruzi is the causative agent of Chagas disease. It is mainly transmitted by triatomine bugs, which are commonly known as "kissing bugs". In the insect midgut, proliferative forms of the parasite called epimastigotes differentiate to metacyclic trypomastigotes after migration to the posterior intestine. During the blood meal, triatomines eliminate urine and feces with infective trypomastigotes that then gain access to the vertebrate bloodstream. After internalisation in the host cell, trypomastigotes remain in parasitophorous vacuoles (PV) that fuse with lysosomes, allowing an acidification of this compartment, which is an essential step towards differentiation into proliferative amastigotes. In the cytosol, successive parasite cycles occur until a new intracellular differentiation to trypomastigotes occurs; it is these forms that are responsible for the infection and dissemination to other cells and tissues [8] (Figure 2).

Role of autophagy in T. cruzi
Ultrastructural evidence of autophagy in T. cruzi was observed after the treatment of epimastigotes and bloodstream trypomastigotes with drugs; the appearance of myelin-like figures was the most recurrent feature detected [63][64][65][66][67]. Recently, the synergistic combination of amiodarone and posoconazole was able to trigger autophagy in replicative amastigotes [68]. In this way, different classes of therapeutic agents are able to induce the formation of autophagosomes, an event associated with parasite-related autophagic cell death, being the interplay between other programmed cell death as apoptosis or necrosis not discarded [69]. Due to the limitations of cell models, previous studies of different parasite forms have employed alternative techniques, such as monodansylcadaverine (MDC) staining and ATG gene expression, to demonstrate autophagy in the parasite [66,67]. Unfortunately, T. cruzi molecular machinery does not allow the use of double-stranded RNA to knock down target RNAs [70]; in addition, the lack of recognition of protozoan proteins by anti-Atg commercial antibodies hampers the evaluation of autophagy in this parasite. In spite of the advances in molecular and cellular biology, transmission electron microscopy remains a gold standard for autophagy analysis [71,72].
Aside from the description of autophagosomes in all T. cruzi life stages, description of the Atg cascade involved in autophagosome formation is not complete. Almost all T. brucei ATG genes have ortholog genes in T. cruzi [37,47]. In this parasite, two isoforms of Atg8 were described, with only Atg8.1 localised in autophagosomes as expected. These data suggest that there is only partially shared autophagic machinery, as is observed in human Atg8 orthologs [37]. In another study [37], the authors described the participation of T. cruzi Atg4 and Atg8 isoforms under conditions of nutritional stress and in the differentiation process from epimastigotes to metacyclic trypomastigotes, a process known as metacyclogenesis. The authors observed a remarkable expression of Atg8.1 by immunofluorescence microscopy, which was suggestive of intense autophagy in differentiating epimastigotes. Moreover, Atg8 co-localised with reservosomes, which are pre-lysosomal compartments related to energy supply that are present only in epimastigotes [73,74]. The reservosomal content consumed during metacyclogenesis and the presence of Atg8 in this organelle strongly suggest that there is crosstalk between autophagy and reservosomes [75,76]. Transmission electron microscopy studies have produced images from endoplasmic reticulum profiles surrounding reservosomes that indicate the possible origin of preautophagosomal structures [66]. It is well known that PI3K inhibitors, such as 3-methyladenine and wortmannin, prevent autophagy in different experimental models [54,66]; however, these data are controversial due to a previous report demonstrating that treatment with kinase inhibitors staurosporine, genistein, 3-methyladenine and wortmannin led to the formation of autophagosomes [77]. The data indicate the necessity of careful use of PI3K inhibitors to block autophagy and the urgent need for the development of new specific autophagic inhibitors [78].

Host cell autophagy and T. cruzi infection
Though thought to be essential for parasite success, lysosomal fusion could be involved in autophagy during host cell interaction and might contribute to the process of degradation and elimination of T. cruzi. In 2009, the role of autophagy in parasite entry and co-localisation with the PV was described, resulting in increased infection of Chinese hamster ovary cells; this observation was subsequently confirmed in macrophage and heart cell lineages [34,79]. Starvation conditions and the addition of rapamycin led to an increase in the scale of the infection; this increase was partially reversed by 3-methyladenine, wortmannin and vinblas-tine, suggesting that autophagy favours the parasite during T. cruzi-host cell interactions. However, other groups demonstrated that classical autophagic stimuli (nutritional stress and rapamycin) did not produce an increase in parasite proliferation or even in the number of infected cells [33]. Recently, studies have emphasised role of autophagy in the control of T. cruzi infection using different cells and parasite strains ( Figure 3) [80,81]. Once more, the conflicting data presented in the literature need to be further debated in light of the complexity of the protozoal strains and host cell models employed.

Leishmania species
The other medically important trypanosomatids are Leishmania species. Leishmaniasis is transmitted to mammals by sandflies, mainly of the Phlebotomus and Lutzomia genuses. Amastigotes differentiate into replicative procyclic promastigotes in the digestive tract of these sandflies, proliferate in the Phlebotominae gut, and then migrate to the proboscis where a new differentiation occurs to metacyclic promastigotes, the infective forms of the parasite. During the sandflies' blood meals, metacyclic promastigotes are inoculated into mammalian tissue and are phagocytised by macrophages. Inside the host cells, promastigotes differentiate into amastigotes that replicate and are responsible for cell lysis and dissemination in the organism ( Figure 4). Currently, more than 20 species of Leishmania are known, each causing different clinical manifestations of the disease, including cutaneous leishmaniasis and visceral leishmaniasis (or Kala-azar). The pathogenicity depends on the Leishmania species and the host's immune response [8].

Role of autophagy in Leishmania sp.
Many groups have investigated autophagy cell death induced by drugs or antimicrobial peptides in various Leishmania species using electron microscopy and MDC staining [82][83][84][85][86][87][88][89]. Bioinformatics analysis has been a crucial checkpoint in the characterisation of ATG and TOR pathways in trypanosomatids [38,47,90]. In 2006, the role of autophagy in the differentiation process of L. major and L. mexicana was first evaluated [38,90]. The authors developed L. major VPS4, a mutant that could not complete the differentiation to the infective forms due to interference in autophagosome formation during conditions of starvation. The increase in Atg8 expression in differentiating forms supports the hypothesis that autophagy plays a pivotal role in metacyclogenesis [38,91]. In L. mexicana, the lack of cysteine peptidases CPA and CPB impairs autophagosomes formation and parasite differentiation; this finding is corroborated by the results of wortmaninn treatment and ATG deletion [90].
Recently, a subunit of protein kinase A in L. donovani that interferes with autophagy and protozoa differentiation was identified [92]. As observed in other trypanosomatids, the presence of Atg8-like proteins and their association with Atg4 in Leishmania species indicates that these proteins play a role in vesicle expansion [93]. Interestingly, the Atg5-Atg12 complex involved in autophagosome elongation was not previously detected [47], but recent studies have demonstrated its existence. It has also been shown that Atg5 deletion severally affects parasite homeostasis, producing a phenotype characterised by mitochondrial disruption, phospolipid accumulation and abnormal promastigote morphology [93,94]. Table 1 summarises the autophagic events in the three pathogenic trypanosomatids described in this chapter.

Host cell autophagy and L. amazonensis infection
The connection between the endosomal/lysosomal pathway and the PV results in macromolecules being taken up by the parasite, as demonstrated in T. cruzi infection [96]. In this context, a notable increase in the proliferation of L. amazonensis amastigotes was observed after autophagic induction by nutritional deprivation, rapamycin treatment or interferon-gamma. This mechanism was partially reversed by the autophagic inhibitors wortmaninn or 3methyladenine, which significantly reduced amastigote replication ( Figure 5) [33]. However, Figure 3. Autophagy in T.cruzi-host cell interaction. Romano et al [34] showed the co-localization of parasite vacuole with Atg proteins in the beginning of infection (1). Moreover, the replication of amastigotes is the same with or without autophagy induction (2) [33,34]. Rapamycin and starvation control infection reducing the number of amastigotes per cell (3) [80,81].
a recent report presented no correlation between the increase in LC3 expression and heightened L. amazonensis infection after treatment with autophagy inducers and inhibitors. In addition, macrophage autophagy was observed in inflammatory infiltrates of L. amazonensisinfected mice [97] and in natural human L. donovani infection [98].

Apicomplexa and autophagy
The phylum Apicomplexa comprises one of the most medically relevant groups of protists, which cause serious health and economic problems. Among these parasites, Toxoplasma gondii and Plasmodium species are well-known apicomplexans; it is estimated that malaria caused by P. falciparum kills over a million people annually. Another widespread disease is toxoplasmosis, which is caused by the apicomplexan parasite T. gondii; the severity of disease caused by this organism is directly related to patients´ immunosuppression and is characterised by congenital transmission. In this context, knowledge of the detailed mechanisms involved in parasite infection and survival, including the role of autophagy, could contribute important information to the development of novel strategies for controlling Apicomplexa infections. Autophagy is an evolutionarily conserved pathway found in all eukaryotes, from unicellular organisms to metazoans; orthologs for approximately 30% of autophagy-related genes have been detected in apicomplexan sequenced genomes [99].
Among the key molecules involved in early autophagy steps, Atg1/ULK complex, Atg8 and Atg9 play crucial roles in cargo selectivity and in autophagosome formation [100,101]. Unlike other cell models, in Apicomplexa protozoa, the Atg8 C-terminal appears to not undergo processing before its association with phosphatidylethanolamine (PE) in the membrane of autophagosomes, suggesting a different regulation of this Atg protein in these organisms than in mammals and fungi [102]. Using a technique to detect lipidated Atg8 in Plasmodium species, only a single band corresponding to ATG8 was observed, suggesting that this parasite's Atg8 exists predominantly in the PE-conjugated form [22].
Two important kinases have opposing roles in the autophagic process: TOR (target of rapamycin) and class III phosphatidylinositol3-kinase (PI3K) [78,103]. In well-established autophagic models, TOR and class III PI3K represent negative and positive regulators, respectively, that act through complexes with regulatory subunits orchestrated by signalling cascades. Analysis of the T. gondii genome revealed the presence of TOR and PI3K but not of other proteins crucial to the formation of these complexes [99]. Curiously, no genes for TOR complex machinery were found in the Plasmodium genome. Thus, it is possible that these unicellular eukaryotes have specific unknown proteins for several steps of the autophagic pathway instead of an absence of key proteins [22,104].

T. gondii
T. gondii is an obligate intracellular parasite with a complex life cycle involving one definitive feline host where the sexual phase occurs and intermediate hosts, such as birds, other mammals and man [105]. The main transmission routes to humans are the following: (i) the ingestion of raw meat containing tissue cysts (essentially bradyzoites forms); (ii) the ingestion of water and food contaminated with feline feces residue containing oocysts; and (iii) transplacentary pathway of tachyzoites [106]. After oral ingestion, tissue cysts or oocysts rupture, liberating the slow-replicating forms known as bradyzoites and sporozoites, respectively, which then invade intestinal epithelial cells. In the intracellular environment, the parasites differentiate into the fast-replicating tachyzoites that proliferate inside the host cell PV. The sustained infection depends on the modification of the PV membrane by the insertion of T. gondii secreted proteins, which prevent the fusion to lysosomes and, consequently, the elimination of the parasite ( Figure 6) [20,107].
In healthy adults, T. gondii cysts are established in the host cells mainly in the eyes, brain and muscles during the chronic phase of toxoplasmosis [108]; however, in immunocompromised patients, such as HIV-positive patients, or in congenital toxoplasmosis, the disease becomes much more severe, and its complications could lead to death [20,109,110]. Despite the high percentage of people infected, the available therapy for toxoplasmosis is effective only in the tachyzoite stage and presents limited efficacy against the tissue cyst, which is the latent form of the parasite [111]. In this context, many efforts are necessary to develop new drugs to treat T. gondii infection [17].

Role of autophagy in T. gondii infection
Only a few studies on the T. gondii autophagic pathway have been performed, and these studies suggest opposing roles of autophagy in the parasite infection [102,112]. The presence of TgAtg8 in autophagic vesicles was observed in tachyzoites during their intracellular replication; similarly, severe parasite growth arrest due to TgAtg3 knockdown and recent identifications of the presence of TgAtg1 and TgAtg4 in the parasite suggest a role for autophagy in T. gondii homeostasis, although long-term exposure to autophagic stimuli was found to be harmful to the parasite (Figure 7) [112; 113].
Tachyzoites divide by a process called endodyogeny, whereby two daughter cells are developed inside a mother cell and leave residual material at the end of division. During this process, autophagy might be involved in recycling the mother cell organelles, such as micronemes and rhoptries, which are synthesised de novo in the daughter cells; however the accumulation of organelles after endodyogeny has not been observed in TgATG3 knockout organisms, making other experiments necessary to confirm this hypothesis [113]. One important phenotype detected in autophagic mutants is the loss of mitochondrial integrity [102,112]. Mitophagy, which is the autophagy of mitochondria, regulates the mitochondrial number to match metabolic demand; this process represents a quality control that is necessary for the removal of damaged organelles [114]. Autophagic stimuli are able to direct the mitochondrial network of tachyzoites towards their autophagic pathway, but the molecular machinery involved in selective targeting of the organelle remains unclear [102,112]. Nutrient deprivation has been shown to be a classic stimulus for the autophagic pathway activation in a large variety of organisms [37,115]. In T. gondii tachyzoites, starvation induces autophagy in extracellular and intracellular parasites [102,112]. Furthermore, autophagosomes were observed in parasites after a long extracellular nutritional restriction, suggesting that autophagy can act as a mechanism of resistance to starvation for nutrient recycling until the infection of a new host cell [102]. The data presented here demonstrate possible functions of T. gondii autophagy in parasite homeostasis. However, it has been proposed that, when strongly induced, the autophagic pathway represents a self-destructive mechanism leading to protozoal death. The molecular pathway of autophagic cell death is still unknown, and it is debated whether the pathway is a type of programmed cell death or a survival response to death stimuli [119]. Intracellular starved tachyzoites showed systematic mitochondrial fragmentation and a defect in host cell internalisation. As T. gondii is an obligate intracellular protozoa, the loss of invasion capacity leads to parasite death. The impairment in infective ability was related to the loss of mitochondrial integrity because organelles from apical complexes, such as rhoptries and micronemes, which are usually associated with the invasion process, are intact in these parasites [112]. Interestingly, these authors also demonstrated that autophagic inhibitor 3-methylade-nine prevented mitochondrial fragmentation, suggesting autophagic involvement in T. gondii death.
While nutritional stress has been extensively used as a model for autophagy, this condition is not easily encountered in the host cells and tissues in vivo. However, parasites could be exposed to nutritional restriction in the extracellular environment. The viability of tachyzoites kept in an axenic medium for periods of up to 12 hours drastically decreases, but a significant number of parasites nevertheless differentiate into bradyzoites [120]. Indeed, these observations raise the hypothesis that autophagy could be an adaptive mechanism of T. gondii to survive for short periods in starvation conditions, allowing the parasite to recover when favourable conditions occur or even to differentiate into a cystic form. Another interesting point for discussion is the correlation between mitochondrial fragmentation in intracellular tachyzoites and the depletion of amino acids in the culture medium [112]. Activated macrophages infected with the parasite showed low availability of the essential amino acid tryptophan, a condition that directly contributes to the protozoa's death in these cells [121,122]. In this context, TOR kinase is a vital component of the amino acid sensing mechanism in eukaryotic cells, as suggested by the detection of TgTOR by bioinformatic approaches and the evaluation of the activity of the classical TOR inhibitor rapamycin. This inhibitor triggered mitochondrial fragmentation of intracellular tachyzoites in starved parasites, and this phenotype was reversed by adding 3methyladenine [112].

Host cell autophagy and T. gondii infection
As previously mentioned, T. gondii can infect any nucleated cell, but the parasite tropism principally involves nervous and muscular cells where the establishment of cystic forms is observed in chronic toxoplasmosis [111,123]. As was observed for T. cruzi-host cell interactions, controversial data on the importance of autophagy during T. gondii infection have been described in the literature; indeed, it has been suggested that autophagy can either control or facilitate parasite internalisation and proliferation [32,35,[124][125][126][127][128]. Despite the relevance of muscular and nervous cells for the establishment of infection and for the course of the disease, very little has been reported on the role of autophagy in the progression of infection. As we will discuss in the next paragraphs, previous studies on the connection between the autophagic pathway and T. gondii infection were performed in macrophages, which are cells that play an important role in the immune response against this parasite [129].
Previous reports have shown that cellular immunity mediated by CD40 stimulation redirects the T. gondii to a lysosomal compartment via the autophagic route, resulting in the antimicrobial activity of the macrophage in vitro and in vivo [124,125]. In vivo, parasite elimination was dependent on GTPase p-47, IFN-γ, IGTP, and PI3K and culminated in the rupture of the parasite's membrane [125] (Figure 8). Additionally, the relationship between autophagy and the fusion of lysosomes with the T. gondii PV seems to be dependent on the synergy between TRAF6 signalling downstream of CD40 and TNF-α [126]. However, the IFN-γ/p47 GTPasedependent elimination of the parasite by macrophages is independent of CD40/TNF signalling in vitro, demonstrating the primary role of IFN-γ in immunity against T. gondii in mice [127]. As observed in astrocytes, autophagy is activated to eliminate intracellular parasite debris and thus prevent the host cell death. Investigations in macrophages also indicated that the CD40-p21-Beclin 1 pathway is a CD40-dependent immunity route to mediating in vivo protection [128]. Similarly, Atg5 is required for damage to the PV membrane and removal of the parasite in primary macrophages stimulated by IFN-γ, despite the fact that no autophagosomes involving T. gondii have been detected. Atg5 also appeared crucial for in vivo p47 GTPase IIGP1 recruitment to the vacuole membrane induced by IFN-γ, suggesting an additional autophagyindependent role for Atg5 in the GTPase trafficking process [32]. In T. gondii infected astrocytes, the participation of autophagy has been shown to be indirect. The IFN-γ-stimulation of astrocytes infected with tachyzoites triggers the recruitment of p47 GTPases to the PV and usually leads to rupture of the vacuole and parasite membrane. In this case, autophagy acts by removing protozoal debris that accumulates in the cytoplasm and causes cell injury. Additionally, autophagy assists in antigen presentation through MHC class II in astrocytes, allowing an intracerebral immune response to parasite [130].
So far, little has been described regarding the involvement of autophagy in the interaction of T. gondii with nonprofessional phagocytes. In primary fibroblasts or Hela cells, infection with tachyzoites induced LC3 conjugation to PE, accumulation of LC3-containing vesicles close to the PV and an overexpression of beclin-1 and phosphatidylinositol-3-phosphate in the host cells in the mTOR-independent pathway. The infection of Atg5-deficient fibroblasts was reduced in physiological concentrations of amino acids, reinforcing the host cell autophagic role in the recovery of nutrients by the parasite. Because the classical function of autophagy involves recycling of various cellular components and because T. gondii depends on the uptake of many nutrients from the host cell, it has been proposed that the parasite may take advantage of the mammalian autophagic machinery to achieve successful infection [35]. Table 2 shows the host autophagic roles during T. gondii infection.  , where they migrate primarily to the liver. After internalisation in hepatocytes, the parasites convert from elongated sporozoites (invasion competent and motile) to round proliferative trophozoites (metabolically active), which start the asexual reproduction process known as schizogony. At the end of the reproductive process, the daughter cells (merozoites) initiate maturation for erythrocyte invasion. When the merozoites become mature, they are enclosed in a membrane (the merosome) and released from hepatocytes to invade red blood cells, causing clinical symptoms of malaria ( Figure 9). [135][136][137].

Role of autophagy in Plasmodium sp. infection
Recent publications have suggested that autophagy is involved in the differentiation of sporozoites to merosomes in hepatocytes [137,138]. The sporozoite-to-trophozoite differentiation is accompanied by the elimination of organelles unnecessary for schizogony and the production of merozoites in liver cells [137]. For example, micronemes and rhoptries are compartmentalised in the cytoplasm of sporozoites and sequestered in double-membrane structures resembling autophagosomes. In axenic conditions, the treatment of parasites with 3-methyladenine resulted in significant delay of the sporozoite differentiation process [139].
After sporozoite differentiation, Atg8 is present in autophagosomes during the replication phase, suggesting an additional independent role for this protein in autophagy [137,138,140].
The involvement of autophagy in Plasmodium-infected red blood cells has been poorly studied. One study demonstrated that erythrocytes infected with P. falciparum trophozoites and maintained in supplemented culture medium expressed Atg8 in the parasite cytosol. However, when these infected cells are submitted to restriction of glucose and amino acids, an increase in the number of autophagosomes labelled by Atg8 was observed, and these vesicles were found close to red blood cell membranes. Once erythrocytes no longer have organelles in the cytoplasm, the potential targets of autophagosomes in this cell model are debated. One hypothesis suggested that these autophagosomes target haemoglobin and blood nutrients to favour nutrient uptake by the parasite (Gaviria and colleagues, unpublished results). Surprisingly, no TOR ortholog was found in the P. falciparum genome (Sinai & Roepe, unpublished results), suggesting that under normal growth conditions, P. falciparum autophagy is configured as a survival process that is constitutively regulated by the acquisition of nutrients, although this route is unusual. Table 3 summarises the published reports on autophagic features in apicomplexans.
Little is known about the involvement of autophagy in the Plasmodium sp.-host cell interactions. So far, Plasmodium ATG8 knock-out resulted in a lethal phenotype, indicating that this gene is essential for the mammalian life-stage of the parasite [22]. However, there have been no studies on the importance of the host cell autophagic machinery during the infection.

Conclusion
The present chapter addresses the positive and negative regulations of the autophagic process of infected mammalian cells and the possible effects of these regulations on the in vitro and in vivo modulation of this process. This review also describes the autophagy pathway in pathogenic trypanosomatids and apicomplexans responsible for some of the most relevant neglected illnesses worldwide. The pivotal role of autophagy in pathogenicity and virulence was demonstrated in T. cruzi, T. brucei, Leishmania sp., T. gondii and Plasmodium sp., which suggests that autophagic machinery is a possible target for anti-parasitic intervention.