Afundamental consequence of sexual reproduction is that

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1 Behavioral Ecology Vol. 13 No. 2: Features of begging calls reveal general condition and need of food of barn swallow (Hirundo rustica) nestlings Roberto Sacchi, a Nicola Saino, b and Paolo Galeotti a a Dipartimento di Biologia Animale, Università degli Studi di Pavia, piazza Botta 9, I Pavia, Italy, and b Dipartimento di Biologia, Università degli Studi di Milano, via Celoria 26, I Milano, Italy Altricial offspring of birds solicit food provisioning by complex begging displays, implying acoustic and visual signals. Different components of begging behavior may function as reliable signals of offspring state and thus reproductive value, on which parents base optimal parental decisions about allocation of critical resources (e.g., food). We experimentally manipulated components of general condition of nestling barn swallows (Hirundo rustica) by (1) altering brood size by cross-fostering an unbalanced number of nestlings between pairs of synchronous broods and thus manipulating the level of within-brood competition for food, (2) injecting some nestlings with a harmless immunogen, simulating an infection, and (3) preventing part of the nestlings from receiving food for a short period while establishing control groups. We recorded rate of begging response by individual nestlings as parents visited the nest and recorded begging calls using a DAT recorder to analyze six sonagraphic features of vocalizations. Our factorial experiment revealed that nestlings deprived of food begged more frequently when parents visited the nest compared to their non food-deprived nest mates. Food deprivation increased duration of syllables forming begging calls, whereas brood size enlargement resulted in increased latency of response to parental calls. Heavy nestlings in good body condition vocalized at a relatively low peak frequency. To our knowledge, this is the first study in which begging rate and sonagraphic structure of begging calls are shown to reliably reveal a diverse set of components of offspring general state, on which parental decisions may be based. Key words: begging, competition, condition, food provisioning, Hirundo rustica, infection, parental care, spectrographic analysis, vocalizations. [Behav Ecol 13: (2002)] Afundamental consequence of sexual reproduction is that parents and offspring value themselves more than their relatives, paving the way to a conflict of interests between generations and among siblings over limiting resources (Dawkins and Krebs, 1979; Godfray, 1991, 1995a; MacNair and Parker, 1979; Trivers, 1974). This prominent effect of Mendelian segregation on relatedness leads to the prediction that parents should allocate fewer resources (e.g., food) to the offspring than their offspring are selected to obtain, whenever providing care imposes a cost in terms of future reproduction (Clutton-Brock, 1991; Lemon, 1991; Lindén and Møller, 1989; Partridge, 1989; Roff, 1992; Saino et al., 1999; Stearns, 1992). Moreover, offspring are selected to obtain more care than their siblings, to the extent to which this does not reduce their own inclusive fitness. Young mammals and birds have evolved morphological and behavioral characters that serve the function of soliciting parents to provide care (Kilner and Davies, 1998; Kilner and Johnstone, 1997; Lotem, 1998). Young passerines beg for food by giving typical displays, consisting of gaping, posturing, presenting their brightly colored gapes, and vocalizing loudly (see Kilner and Johnstone, 1997, for a review). Begging behavior has been envisaged as the evolutionary expression of the intergenerational conflict of interests over parental care, although at least two functional interpretations are plausible (Godfray, 1991, 1995a,b; Kilner and Johnstone, 1997; Mock and Parker, 1997; Parker et al., 1989). First, begging may con- Address correspondence to N. Saino. n.saino@mailserver. unimi.it. Received 29 January 2001; revised 23 April 2001; accepted 1 June International Society for Behavioral Ecology sist of manipulative signals that offspring address to their parents to obtain more care than is optimal for parents to provide. Alternatively, begging consists of reliable signals of state on which parents base optimal decisions on allocation of critical resources in relation to reproductive value of their individual offspring (Haig, 1990). Reliability of begging indicators of state may be enforced on nestlings by the cost of production of the signals or by physical or physiological constraints (Bretagnolle, 1996; Eberhardt, 1994; Godfray, 1991, 1995a,b; Gottlander, 1987; Grafen, 1990; Horn et al., 1995; Leonard and Horn, 1995; Reid, 1987). The theory of honest signaling predicts that parents may have selected for the evolution of indicators of state by adaptively tuning their investment in relation to offspring signals that cannot be faked, or, simply, have evolved the ability to base their decisions on reliable signals of quality. An example of the former case would be differential allocation to offspring displaying brightly colored gapes, which indicate the need of food or indicate good health (Götmark and Ahlström, 1997; Kilner, 1997; Saino et al., 2000a). The latter case would be exemplified by parents assessing offspring size by the pitch of their calls, which decreases as the size of the signaler increases because of the inverse relationship between size of the source and frequency of the sound produced (Bowman, 1979; Konishi, 1970; Morton, 1977; Ryan and Brenowitz, 1985). However, complexity of begging display involving different kinds of traits obviously raises the question of why such complexity arose. Offspring reproductive value may be influenced by diverse factors, such as general condition (see Saino et al., 2000b, for a definition), contingent need of food, or health (Saino et al., 2000a). Hence, it could be speculated that different elements of begging display reflect specific components of offspring state, as shown in experimental studies where intensity of begging reflected need or infection (e.g., Christe et al., 1996;

2 Sacchi et al. Begging vocalizations and condition in swallows 269 Kilner, 1995; Mondloch, 1995; Price and Ydenberg, 1995; Redondo and Castro, 1992), whereas mouth coloration has been demonstrated to reflect satiation or infection (Kilner, 1997; Saino et al., 2000a). In this field study of barn swallows (Hirundo rustica), we manipulated three components of nestling state and measured the effect of experimental treatments on frequency of begging events and several features of begging calls. Brood size was either increased or reduced to alter the level of competition for food (Saino et al., 1997b, 1999; Saino, unpublished data). Condition of some nestlings was depressed by injecting them with sheep red blood cells (SRBC) to mimic an infection by a pathogen eliciting a humoral immune response (Pinard-van der Laan et al., 1998; Roitt et al., 1996; Ros et al., 1997; Saino and Møller, 1996; Saino et al., 1997a), while the other nestlings were injected with physiological water to serve as controls. Finally, part of each brood was subjected to two short periods of food deprivation, simulating natural conditions of temporary unavailability of food as occurs, for example, during rainy days. Hence, our factorial design produced eight experimental groups of nestlings within each pair of partially cross-fostered broods, resulting from the brood size manipulation, injection with an antigen, and food deprivation treatments. After the second food deprivation period, we recorded begging vocalizations and measured the latency and frequency of response of nestlings to parental signals normally eliciting begging behavior and five sonagraphic components of begging calls (see Methods). We predicted that nestlings in enlarged broods and those that had been deprived of food had (1) relatively large rate and short latency of response to parental calls; (2) large duration and number of begging call elements (syllables) and short duration of intersyllable intervals because intense competition for food and food deprivation should result in more rapid and intense food solicitation. In addition, we predicted that (3) nestlings in enlarged broods produced calls at a relatively high peak frequency (Galeotti et al., 1997) because they are relatively small (Saino et al., 1997b, 1999), but also had lower maximal call frequency because they are in poor condition and thus unable to call at energetically expensive high frequencies. However, we had no clear expectation about the effect of SRBC injection. Finally, we analyzed covariation between body mass, body size, T-cell mediated immunity, and begging call features. The barn swallow is a socially monogamous, semicolonial, insectivorous passerine. Both parents provide food to 2 7 altricial nestlings (Møller, 1994). Nestlings give typical begging calls when parents visit their nest, while also raising their heads and widely opening their yellow to deep-orange gapes. METHODS We studied barn swallows in two breeding colonies located in stables in our study area east of Milano, northern Italy, during spring We visited nests at regular intervals to record breeding events and more frequently around the estimated day of hatching, on average 13 days after laying of the last egg. When two broods that completed hatching on the same day and colony were found, we performed an unbalanced cross-fostering of individually marked nestlings so that the size of one, randomly chosen brood in the pair (hereafter dyad ) of broods was enlarged by one nestling, while the other was reduced by one. The number of randomly chosen nestlings due to be swapped between broods was decided so as to minimize the within-brood difference in the number of resident versus introduced nestlings (see Saino et al., 2000b, for details). When nestlings were 6 days old, half the nestlings (or half the number of nestlings 0.5 in the case of an uneven brood size) in each brood were injected intraperitoneally with SRBC suspended in 30 l phosphate-buffered saline (PBS). SRBC are a multigenic antigen commonly used to elicit a humoral, T-cell dependent immune response in vertebrates (Deerenberg et al., 1997; Pinard-van der Laan et al., 1998; Roitt et al., 1996), and SRBC have been shown to act as an immunogen also in the barn swallow (Saino and Møller, 1996; Saino et al., 1977a). Previous studies on the barn swallow have shown that challenge of nestling immune system with SRBC diminishes the brightness of gape coloration (Saino et al., 2000a) and depresses other components of immunity (Sacchi et al., unpublished data). In addition, SRBC injection negatively affects other aspects of general condition in other animal models (Besedovsky et al., 1975). Nestlings that did not receive an injection of SRBC were injected with 30 l PBS to serve as controls. On the morning of the sixth day after injection, one or two randomly chosen individuals from the groups of SRBC- and sham-inoculated nestlings were subjected to 3 h of food deprivation by placing them in a part of the nest and covering them with a thin metal net. This food deprivation procedure was also repeated the next morning. In broods with a postmanipulation size of two or three nestlings, one or two experimental groups resulting in each brood from our factorial design were not represented. Three hours after the start of food deprivation, the metal net was removed. The food-deprivation procedure simulated short periods of starvation, which are relatively common in our study area when meteorological conditions reduce foraging success of parents. We recorded begging displays after the end of the second food deprivation period. On the first day of food deprivation we also initiated an in vivo test to assess T-cell mediated immunity by injecting the right wing web with a solution of 0.2 mg phytohemagglutinin (PHA; Sigma) dissolved in 0.04 ml PBS. The left wing web was injected with the same amount of PBS, after measuring the thickness of both wing webs with a pressure sensitive micrometer (Alpa S.p.A. Milano), which gives both accurate (0.01 mm) and highly repeatable measurements (Saino et al., 1997b, Sorci et al., 1997). PHA is a lectin that is mitogenic to T lymphocytes, and its injection results in thickening of the wing web. The difference between change in the thickness of the right wing web measured 24 h after injection of PHA minus the change in the thickness of the left wing web, which controlled for the effect of inoculation per se, is considered an index of T-cell mediated immunocompetence (Lochmiller et al., 1993). Body mass was measured using a spring balance (accuracy of 0.1 g), and tarsus length by a caliper (accuracy of 0.05 mm). An index of body condition was obtained as the residual body mass on tarsus length calculated from linear regression analysis. Begging call recording methods We recorded nestling vocalizations with a Sony TCD-D7 DAT recorder connected to a Sony ECM-155 super-tiny microphone. Nestling barn swallows generally beg vigorously in response to the high-pitched calls (contact or provisioning call) produced by adults visiting the nest. The microphone was placed on the border of the nest and directed toward its center. All nestlings were recorded without changing the position of the microphone and there was no apparent effect of the microphone on parental or nestling behavior. The microphone was connected to the DAT recorder by a long cable so that we could operate from a distant position, thus avoiding disturbance of the nest. However, it should be emphasized that barn swallows breeding inside the stables are well accus-

3 270 Behavioral Ecology Vol. 13 No. 2 Figure 1 Sonagraphic representation of a typical bout of begging calls by a nestling barn swallow aged 13 days after stimulation by the visiting parent. (a) indicates the high-pitched parent provisioning call, eliciting begging display and vocalizations. (b) is a begging call. 1, time (latency) elapsing between the provisioning call of the adult and the start of begging calls; 2, duration of a syllable; 3, duration of each interval between two consecutive syllables; 4, highest frequency of each syllable (Hz); 5, the frequency at which the maximum energy is concentrated (peak frequency) for each syllable. Number of syllables in this begging call is 8. tomed to humans and are thus not disturbed even when people closely approach their nest(s). At the end of the food deprivation period, we removed all nestlings from their nests and put them in a cloth bag. We then immediately extracted one nestling and put it back in the nest to record all vocalizations that it uttered when parents arrived to feed it. Three parental visits for each nestling were considered. After a nestling had been tested, it was removed from the nest. Nestlings were tested in a random sequence by an experimenter who was blind to the treatments. Sonagraphic analyses High-quality recordings (minimal background noise and high sound intensity) of begging obtained for each nestling were analyzed using COOL EDIT PRO computer software (Syntrillium Software Corporation). Nestlings usually produce more than one begging bout per parental visit to the nest. We analyzed an average of 7.36 (0.46 SE) bouts recorded during the three parental visits per nestling. The best resolution was achieved by analyzing vocalizations in the 0 22 khz frequency range (16 bit A/D converter), with a sample rate of 44,100 samples/s, band-width of 512 Hz, frequency resolution of 50 Hz, and time resolution of 1 ms. Begging vocalizations of barn swallows consist of a series of calls that have a broad range of frequency and are highly stereotyped within individuals (Sacchi et al., unpublished data). For each song we selected and measured directly on the sonagrams the following structural features: (1) time (latency) between the provisioning call of the adult and the start of begging calls (expressed in ms); (2) duration of each syllable (ms); (3) duration of each interval between two consecutive syllables (ms); (4) maximum frequency of each syllable (Hz); (5) peak amplitude frequency (hereafter peak frequency ) of each syllable (Hz); and (6) number of syllables per begging bout (see Figure 1). In our analyses we used mean values of variables 1 and 6 calculated across begging bouts of individual nestlings, and mean values of variables 2 5 computed across begging bouts and based on mean withinbegging bout values. A typical begging call in response to a provisioning call by parents and the structural variables measured are represented in Figure 1. Values of variables 1 3 and 6 were log 10 -transformed to obtain normality. We used multivariate analysis of variance to test for the effects of experimental treatments and dyad on the multivariate set of features of begging calls measured on sonagrams. In this analysis, dyad was considered as a random effect, whereas experimental treatments were fixed effects. Because no significant interaction between random and fixed effects existed, we tested the effect of fixed factors over the pooled error. RESULTS The 11 dyads of broods in our sample contained 93 nestlings, 91 of which were considered in the analyses of begging display. Sonagraphic analyses of recordings confirmed that when parents visit the nest, they produce a short, high-pitched call that apparently serves to elicit begging displays by nestlings (Figure 1). We used ANOVA to test for the effect of experimental manipulations on the number of parental feeding visits to individual nestlings that resulted in begging response. As factors we entered brood size manipulation (enlargement or reduction by one nestling), injection with SRBC or sham inoculation, and temporary food deprivation (yes or no), as well as their two-way interactions. In addition, we entered dyad of broods (random effect) to account for several potential sources of variation in begging behavior, such as seasonal change in food availability and thus satiation, meteorological conditions, as well as variation in microecological conditions among colonies. Only food deprivation had a significant effect on begging response (F 1, , p.003). Food-deprived nestlings responded by begging to visiting parents on average 2.45 (0.13 SE, n 53) times, whereas non food-deprived nestlings responded 1.63 (0.22 SE, n 38) times. Out of 38 nestlings that were allowed to continuously receive food, 24 (63%) produced vocalizations during any of the three parental visits to the nest, whereas 49 out of 53 (93%) food-deprived nestlings begged at least once during parental visits. We found no significant effect of brood size manipulation, injection with SRBC, interaction terms, and dyad of broods (p.05) on number of begging responses. Sonagraphic features of begging vocalizations were intercorrelated (Table 1). We used multivariate ANOVA to test for the effect of experimental manipulation on begging call sonagraphic features, while controlling for the effect of variation among dyads of broods. Brood size manipulation significantly affected the multivariate set of begging call features (Hotellings test; F 2.33, p.047), whereas the effects of injection with SRBC and food deprivation did not attain significance (SRBC injection: F 0.60; food deprivation: F 2.12, p.068). In addition, the effect of food deprivation depended on previous injection with SRBC (F 3.35, p.008). Univariate F tests on each dependent variable while con-

4 Sacchi et al. Begging vocalizations and condition in swallows 271 Table 1 Pearson correlation coefficients between sonagraphic begging call features in the whole set of nestlings (N 71) for which we have measurements of all spectrographic variables, in the 22 experimental broods No. of Intersyllable Syllable Peak Maximal syllables interval duration frequency frequency Latency p ns.001 No. of syllables p ns Intersyllable interval p.001 ns.006 Syllable duration p Peak frequency 0.28 p.017 See Methods for a detailed description of variables. trolling for the concomitant effect of the others were also run to identify the variables that significantly varied according to experimental treatment. Contrary to our expectations, we found that latency was significantly longer in enlarged compared to reduced broods (F 1, , p.04; Figure 2), whereas duration of syllables was significantly longer in vocalizations of food-deprived nestlings compared to those of nestlings that were allowed to receive food continuously, as predicted (F 1, , p.015; Figure 2). However, the effect of food deprivation on syllable duration was smaller for nestlings in reduced compared to enlarged broods, as indicated by the significant interaction of brood size manipulation and food deprivations (F 1, , p.034). No significant effect was observed in the other tests (Figure 2). An analysis of covariance with experimental treatments and dyad of broods as factors and the six spectrographic variables as covariates showed that body mass was significantly predicted by peak frequency of begging calls (t value for peak frequency 2.36, p.023), with heavier nestlings vocalizing at lower frequencies, as expected. In addition, a body condition index, calculated as residual body mass on tarsus length, reflecting skeletal body size, significantly and negatively covaried with peak frequency (t 2.20, p.033). However, no significant covariation was observed between length of the tarsometatarsus, an index of skeletal body size, or intensity of T-cell mediated immune response and sonagraphic features of begging calls. DISCUSSION In this experimental study we have shown that vocal begging display has the potential to reveal diverse components of general state of nestling barn swallows, which are likely to affect their fitness. Parental feeding visits to the nest were more likely to elicit begging displays when nestlings had been prevented from receiving food from their parents compared to a condition of continuous food provisioning, indicating that rate of begging display reflects the degree of hunger. The multivariate set of sonagraphic features of begging calls measured was significantly affected by brood size manipulation, which is known to influence the amount of food received per capita by nestlings and, thus, various components of body condition depending on nutrition. Indeed, brood size enlargement has been shown to result in relatively low body mass and diminished T-cell mediated immunity, possibly as a consequence of poor nutritional condition (Dietert and Golembowski, 1994; Gershwin et al., 1985; Saino et al., 1997b, 1999), which, in turn, may compromise survival after fledging (e.g., Bryant and Westerterp, 1983; Møller, 1994; see also Martin, 1987). Moreover, peak frequency of begging calls predicted not only body mass of nestlings, as expected, but also an index of body condition independent of size, thus providing parents with an acoustic cue to assess current size and a component of condition of their offspring. Nestling barn swallows subjected to the same experimental manipulations as in the present study were more likely to perform begging displays when artificially stimulated by the observer in the absence of parents if they had been deprived of food, but neither SRBC inoculation nor brood size manipulation significantly affected frequency of begging (Saino et al., 2000b). In the present study, however, begging response was measured when nestlings were individually confronted with their attending parents, instead of being artificially stimulated to beg, thus reproducing more natural conditions. In addition, the potentially confounding effect of perception of competition for food during a particular parental visit to the nest was controlled for by recording behavior of single nestlings instead of whole broods. The adoption of this procedure was also partly dictated by the impossibility of analyzing begging calls of individual nestlings in recordings of whole broods presented to their parents, owing to extensive spectrographic overlap among simultaneous calls of different nestlings. Hence, the present study confirms that the frequency of begging display is at least partly determined by current level of satiation of nestlings, but not brood size and thus long-term levels of competition for food, or current health conditions, as influenced by injection with an antigen. In addition, our results suggest that the method of eliciting begging display used elsewhere (Saino et al., 2000b) simulates natural conditions because the results obtained using the two methods, under the same experimental design, are qualitatively identical. Food deprivation had a marginally nonsignificant effect on the multivariate set of begging call spectrographic variables (p.07). However, food deprivation significantly enhanced duration of individual syllables in the begging call, as predicted under the general assumptions that duration of syllables is a direct component of intensity of the begging solicitation behavior and that hungry nestlings beg more than satiated ones (e.g., Price and Ydenberg, 1995). Hence, it is not only frequency of performance of begging display, but also its structure, in terms of duration of syllables, that potentially reveals nestlings need of food. Brood size manipulation had a significant effect on spectrographic variables. However, latency of response after parental

5 272 Behavioral Ecology Vol. 13 No. 2 Figure 2 Mean values ( SE) of the six sonagraphic variables measured on nestling barn swallow begging calls in relation to the three experimental treatments: (a) brood enlargement or reduction by one nestling, (b) injection with sheep red blood cells (SRBC) or sham inoculation, and (c) temporary food deprivation versus continuous food provisioning by parents (see Methods and Figure 1 for a description of measured variables and Results for statistical analyses). Measurement units have been adjusted to allow joint representation of variables. Latency, intersyllable interval, and syllable duration are in milliseconds/100, number of syllables is given as such, while peak and maximal frequencies are given in Hz acoustic stimulation was larger for nestlings in enlarged rather than in reduced broods, contrary to our expectation that nestlings that had experienced a regime of relatively intense competition should have responded more promptly to feeding visits of their parents. A possible a posteriori interpretation of this finding is that increased within-brood competition for food due to brood enlargement exhausts nestlings, negatively affecting their readiness to respond to parents. Alternatively, nestlings in enlarged broods might lower their basal metabolism to cope with reduced food supply which in turn influences their readiness to beg. Injection of a foreign antigen eliciting an immune response did not affect begging sonagraphic quality, providing additional information about results from a previous study of begging display frequency (Saino et al., 2000b). Parent barn swallows, however, do alter their feeding effort to individual nestlings in relation to inoculation of the same antigen we used here, indicating that they may rely on other components of begging display, such as coloration of nestling gape, which is depressed in SRBC-injected nestlings (Saino et al., 2000b). Indeed, parent barn swallows allocate more food to food-deprived nestlings (Saino et al., 2000b), but also to nestlings whose gapes have been artificially reddened (Saino et al., 2000a). In addition, sonagraphic features of begging calls do not covary with gape color of individual nestlings (Sacchi et al., unpublished data), corroborating the idea that different components of begging display (begging rate versus gape coloration) reflect different aspects of nestling state (e.g., hunger versus infection), but combine to determine parental feeding decisions, thus functioning as multiple signals of independent components of offspring general condition. Peak energy of begging calls was concentrated at a lower frequency for heavy compared to light nestlings. This was expected on the basis of physical constraints linking sound frequency to body size in birds and other vertebrates (e.g., Morton, 1977; Ryan and Brenowitz, 1985). This result implies that parents have an acoustic cue for assessing offspring mass. This may be particularly important for barn swallow parents, as well as for hole-nesting species, because most nests are in relatively dark sites, and some nestlings, particularly in crowded nests, may be partly concealed by their siblings, thus preventing parents from assessing nestling size via visual cues. In addition, we showed that body mass corrected for body size negatively covaried with peak frequency, implying that an index of condition, unrelated to skeletal body size, can be revealed by this prominent feature of begging calls. We conclude that different elements of begging behavior, including frequency of display, spectrographic features, and gape coloration, have the potential to reliably reveal a diverse set of partly independent components of barn swallow nestling condition. The results of this study are therefore compatible with the idea that behavioral and morphological components of begging display in the barn swallow function as reliable indicators of offspring general state. Parents may use these displays to assess offspring reproductive value and, thus, to make optimal decisions on allocation of resources critical to their own fitness and that of their offspring. We are grateful to several people who helped during field work. We are also grateful to T. Calinski, G. Marubini, A.P. Møller, M. Sari Gorla, and three anonymous referees for valuable suggestions. This study was supported by CNR and MURST grants to N.S. and P.G. REFERENCES Besedovsky HO, Sorkin E, Keller M, Muller J, Changes in blood hormone levels during immune response. Proc Soc Exp Biol Med 150:

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