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Iis1.eeza.csic.esThe Journal of Experimental Biology 214, 1463-1472 2011. Published by The Company of Biologists Ltddoi:10.1242/jeb.050153 RESEARCH ARTICLE
Benefits associated with escalated begging behaviour of black-billed magpie
nestlings overcompensate the associated energetic costs
David Martín-Gálvez1,2,*, Tomás Pérez-Contreras1,2,3, Manuel Soler2,3 and Juan José Soler1,2 1Departamento de Ecología Evolutiva y Funcional, Estación Experimental de Zonas Áridas (CSIC), 04120 Almería, Spain, 2Grupo de Coevolución, Unidad Asociada al CSIC, Universidad de Granada, 18071 Granada, Spain and 3Departamento de Biología Animal, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain *Author for correspondence (email@example.com) Several experimental results support the existence of costs associated with exaggerated begging behaviour, which are assumed
by some theoretical models of honest signalling in parent–offspring communication. However, to understand how honest begging
behaviour is evolutionarily maintained in nature, the long-term cost–benefit output associated with exaggerated signals should
also be estimated. As far as we know, the net cost–benefit balance of begging display has not previously been explored. Here, we
used an appetite stimulant, cyproheptadine hydrochloride, to increase the feeling of hunger in some magpie nestlings. Supporting
the use of cyproheptadine to manipulate hunger level and thereby begging behaviour, we found that experimental nestlings
increased the frequency of begging and received more food than their control nestmates. Contrary to the expectation that
physiological costs per se counteract the associated benefits of escalated begging signals, we found that near-fledging
experimental magpies showed a better physical condition than control nestlings. These findings stress the interesting question
of why magpie nestlings do not show to adults an escalated level of hunger if it implies an advantage. We discuss the
responsibility of inclusive fitness costs and indirect genetic effects for the maintenance of honesty in parent–offspring
Key words: begging behaviour, black-billed magpie, costs and benefits of begging, cyproheptadine, food allocation, honest signalling,parent–offspring conflict, Pica pica.
marginal fitness gain experienced by an offspring from receiving Offspring are genetically different to their parents, and gaining more the next unit of parental investment [but see Mock et al. (Mock et parental care than parents are selected to provide is of selective al., 2011) for a review about other two active-choice alternatives].
advantage (Trivers, 1974). Since this parent–offspring conflict was According to Godfray’s model, parents would preferentially feed proposed, several models have tried to explain its resolution in those offspring showing the most intense signals because they would general and the evolution of conspicuous and extravagant offspring signals during solicitation of care in particular (for reviews, see Mock Three predictions are generated from this hypothesis (see Kilner and Parker, 1997; Godfray and Johnstone, 2000; Budden and Wright, and Johnstone, 1997; Royle et al., 2002): (1) parental investment 2001; Johnstone and Godfray, 2002). A group of these models is regulated according to offspring signals; (2) the intensity of suggests that the conflict can be solved by the existence of costs the begging signal varies according to offspring need; and (3) associated with offspring solicitation signals that are constraining begging signals should be costly to produce. There exists a their expression. The alluded costs include physiological costs of substantial experimental and empirical body of results supporting production of signals and costs related to risk of predation and loss parent use of offspring begging behaviour to adjust feeding effort of indirect fitness (e.g. depriving relatives – either nestmates or future and to allocate food within the brood (e.g. Smith et al., 1988; unborn siblings – of food) (Godfray and Johnstone, 2000). In this Ottosson et al., 1997; Kilner and Johnstone, 1997; Burford et al., situation, dishonest begging behaviour would not be compensated 1998) (but see Clark and Lee, 1998). However, although by the extra benefits gained (i.e. more food). Parents, thus, would considerable evidence also exists about the relationship between be able to use these honest signals actively to properly adjust their begging intensity and levels of food deprivation and offspring effort as well as the food allocation among siblings (active-choice condition (Redondo and Castro, 1992b; Price et al., 1996; hypotheses) (sensu Mock et al., 2011), or passively by feeding the Iacovides and Evans, 1998; Sacchi et al., 2002), it is still not clear offspring that emerges victorious from scramble competition how food deprivation (i.e. hunger) and offspring condition are (Rodríguez-Gironés et al., 2001a; Parker et al., 2002) or negotiation related to offspring need as defined by Godfray (Godfray, 1991) (Roulin, 2002) among siblings. Between the active-choice models, [see Mock et al. (Mock et al., 2011) for a further disscussion about the most widely recognized is that developed by Godfray (Godfray, this issue]. With regard to costs associated with offspring begging 1991; Godfray, 1995a; Godfray, 1995b), which assumes that behaviour, its existence is not widely supported and sometimes begging signals could be reflecting offspring need, defined as the is controversial (reviewed in Kilner and Johnstone, 1997; Budden and Wright, 2001; Wright and Leonard, 2002; Moreno-Rueda, 1967). Cyproheptadine is broadly used in humans, including children, as a safe and effective appetite stimulant (Chinuck et al., Perhaps the clearest support for the costs of escalated begging 2007; Couluris et al., 2008; Mahachoklertwattana et al., 2009). It behaviour comes from several articles showing that predation risk is also often used in other animals, mainly in cats (see e.g. Plumb, can constrain begging expression (especially offspring vocalizations) 1999), and its property as appetite stimulant has also been manifested (e.g. Redondo and Castro, 1992a; Haskell, 1994; Haskell, 2002; in two bird species, domestic fowl (Gallus domesticus) (Injidi and Leech and Leonard, 1997; Briskie et al., 1999; Dearborn, 1999) and Forbes, 1987; Muralidharan et al., 1998) (but see Rao and from four articles giving support to the existence of physiological Varadarajulu, 1979) and domestic pigeons (Columba livia) costs associated with begging signals (Kilner, 2001; Rodríguez- (Gunturkun et al., 1989). The use of this methodology allows Gironés et al., 2001b; Moreno-Rueda, 2010; Noguera et al., 2010).
nestlings to be raised in their natural nests being fed by their parents.
Predation costs of begging signals are based on the possibility that Thus, and conversely to traditional methodologies based on food predators could more easily detect nests that contain nestlings that deprivation to increase begging signals, this experimental approach beg loudly. However, in broods with more than one nestling, allows us to estimate variables related to the net balance between predation costs would be shared by the entire brood because, once benefit and costs (e.g. fledging performance) associated with the a nest is located, all nestlings will probably be eaten. For this reason, predation costs would hardly explain individual differences in The aims of this experimental study were to test three different intensity of begging signals among nestlings within a brood and, methodological and theoretical predictions: (1) cyproheptadine, by thus, this cost would not prevent an escalation of dishonest nestling increasing the level of hunger of nestling, also increases the intensity behaviour [see Rodríguez-Gironés et al. (Rodríguez-Gironés et al., of begging signals (frequency and/or intensity of begging); (2) the 2001b) for further discussion]. Regarding the physiological cost of enhanced beggars receive more food from the parents than their control escalated begging, the first two articles from the four referred to nestmates; and (3) if physiological costs associated to signals are at above have shown the existence of negative effects on nestling least partially responsible for maintenance of honesty in signalling, growth in canaries (Serinus canaria) (Kilner, 2001) and black-billed the energetic net balance of an exaggerated begging behaviour should magpies (Pica pica) (Rodríguez-Gironés et al., 2001b); the third be negative for enhanced beggars. We addressed these predictions article gives support to the immunological cost of exaggerated by using four different methodological approximations. First, we begging signals in the house sparrow (Passer domesticus) (Moreno- video-recorded the behaviour of magpie nestlings and adults in their Rueda, 2010); and the fourth provides evidence about the oxidative nests in order to determine whether nestlings treated with cost of begging in yellow-legged gulls (Larus michahellis) (Noguera cyproheptadine beg more (frequency and/or intensity) and whether et al. 2010). In the first three studies, experimental nestlings were they are preferentially fed over their control nestmates. Second, in forced to beg for food at a higher rate than control nestlings (different laboratory conditions, we estimated the effect of experimental costs) and both groups received the same amount of food (same treatment and food deprivation time on the begging behaviour of benefits) (Kilner, 2001; Rodríguez-Gironés et al., 2001b; Moreno- nestlings. Third, in natural conditions, we quantified the amount of Rueda, 2010). The fourth study showed an increased begging display food received and change in body mass (used as an index of begging of nestlings experimentally supplemented with vitamin E, possibly effort) for control and experimental nestlings for a period of 2.5h.
because of the reduced oxidative cost of begging (Noguera et al., Finally, we estimated the effect of our experimental treatment on 2010). Nonetheless, these experimental approaches do not allow the variables related to nestling probability of recruitment, such as T-cell estimation of net cost–benefit output of the exaggeration of begging immune response (e.g. Møller and Saino, 2004; Cichon and Dubiec, behaviour, nor do they allow us to determine whether exaggerated 2005; Moreno et al., 2005), body size (lengths of tarsi, wings and begging behaviour is cost-effective in the sense of garnering more tails) and body mass (e.g. Magrath, 1991; Gebhart-Henrich and energy than the signal’s transmission expends. Parents, following Richner, 1998; Schwagmeyer and Mock, 2008).
the exaggerated signal of ‘selfish’ offspring could evenovercompensate energetic costs associated with the production of MATERIALS AND METHODS
the signal. Thus, it is possible that, even assuming extra costs, a Study area and species
certain level of exaggeration could still be advantageous for Fieldwork was performed in two close localities of southern Spain offspring. Theoretical studies modelling the evolution of honest during the springs of 2002–2003 and 2008: in the Hoya de Guadix begging behaviours refer to the net balance between benefits and (37°14ЈN, 3°11ЈW) and in Iznalloz (37°25ЈN, 3°33ЈW), respectively.
costs, rather than merely the costs of signals, as the keystone that The Hoya de Guadix is a high-altitude plateau (~1000m above sea predicts honesty in parent–offspring communication (e.g. Godfray level) with cereal crops (especially barley, Hordeum vulgare) that and Johnstone, 2000). Therefore, the experimental increase in alternate with more or less dispersed plots of almond groves offspring solicitation signals in natural conditions and the estimation (Prunus dulcis) or holm-oak trees (Quercus rotundifolia), which of the net physiological cost–benefit balance of this manipulation are preferentially used by magpies to build their nests (for details, are important for the understanding of the evolution of honest see Soler, 1990). Iznalloz has an undulated landscape mainly consisting of small patches of holm-oak forest coexisting with In the present study, we performed such an experiment in black- plantations of olive trees (Olea europea). In this population, magpies billed magpies, one of the two bird species in which the cost of prefer to build their nests mainly in holm-oak trees and secondarily exaggerated begging behaviour on nestling growth has been reported (Rodríguez-Gironés et al., 2001b). Briefly, we increased the hunger The black-billed magpie (Pica pica Linnaeus 1758) is a territorial, level in some nestlings within broods by the administration of an sedentary and relatively long-lived passerine bird with a well-studied appetite stimulant, cyproheptadine hydrochloride, which affected biology (reviewed in Birkhead, 1991). It occurs throughout large begging behaviour (see Results). This pharmaceutical is a type 2 parts of the Holarctic region where, in some places, it becomes serotonin receptor antagonist, which directly acts on the hunger abundant. Magpies lay a single clutch between March and May with centre at the hypothalamus (Stone et al., 1961; Chakrabarty et al., five to seven eggs (range3–10). The female starts to incubate before clutch completion (usually once the fourth egg is laid); this results (0.32mgml–1) (Injidi and Forbes, 1987). After the first dose (at first in broods that hatch asynchronously where some of the smallest weighing, 2–4days old), we revisited nests every 2days to recolor nestlings may starve soon after hatching (Birkhead, 1991).
the tarsi, weigh the nestlings and supply them with the experimentalor control doses. Survivor nestlings received the treatment with General field procedures
cyproheptadine or water on alternative days and on six occasions, At the beginning of the breeding season (end of March to early except those used for video recordings (see below), which received April), we intensively searched the study areas to find the location treatment on five occasions, i.e. until they were filmed.
of new nests. Once a new nest was detected, we visited it regularlyto determine the laying date. During the laying period, nests were Statistical analyses
checked at least twice per week to determine laying date, clutch When required for analysis, outcome variables were graphically size and the occurrence of brood parasitism by the great spotted checked for normal distribution of their frequencies (density and cuckoo (Clamator glandarius), which is common in these areas normal probability plots) and, if necessary, ln-transformed. Data (Soler and Soler, 2000). We used magpie nestlings from natural from video recordings, laboratory and neck-collar trials were non-parasitized broods (natural broods) and from experimental analysed mainly with linear mixed models (LMMs) and generalized parasitized broods, where two magpie and two great spotted cuckoo linear mixed models (GLMMs) in R version 2.9.2 (R Development nestlings shared the nest during development (parasitized broods).
Core Team, 2009) using lme4 (R package v.0.999375-31) (Bates Parasitized broods were also created to study the begging behaviour and Maechler, 2009), and were fitted by the restricted maximum of great spotted cuckoos in comparison with that of nestling hosts; likelihood and Laplace approximations, respectively. For LMMs, however, we only used data from magpie nestlings from these we used Markov-chain Monte Carlo (MCMC) simulations parasitized broods for the present study.
performed by using the pvals.fnc command of languageR (Baayen, Magpie nestlings in natural broods were weighed 2–4days after 2008) to compute the highest posterior density (HPD) 95% hatching (Pesola spring balance, accuracy 0.1g; Pesola Ag, Baar, confidence intervals (CI) of the model estimates and P-values from Switzerland) and marked by painting the tarsus with non-toxic MCMC simulations (PMCMC). The chain length for MCMC sampling waterproof pens of different colours (Mitsubishi Pencil, Rubí, was fixed at 10,000. For GLMMs, we used Wald-Z tests for Barcelona, Spain). Nestlings were ranked according to their mass hypothesis testing. All first-order interactions were initially included and alternately assigned to the experimental and control treatments.
in the model; the non-significant interactions were later removed Thus, we created pairs of experimental–control chicks of similar to improve model parsimony (Zuur et al., 2009). To analyse the mass within broods (nestling dyads). In this way, we could use paired effect of cyproheptadine on the physical condition of magpie statistical tests to reduce the within-nest variance due to other nestlings, we used general linear models (GLMs) with a repeated- variables such as body size and competitive abilities of nestlings.
measures design. GLMs were performed in Statistica version 8.0 Further, we alternated the order of assignment of the experimental (StatSoft Ibérica, Lisbon, Portugal). All tests were two-tailed and and control treatment between consecutive nests; thus, the heaviest values in text and tables are reported as means ± s.e.m.
nestling was assigned to the experimental treatment in some broodsand to the control treatment in others. Parasitized broods were also Video recordings in the field
created when nestlings were 2–4days old both in magpie nests that Between May and July 2008 and when nestlings were 10–12days were naturally parasitized by great spotted cuckoo (26 broods) and old, we filmed nestling begging behaviour and food delivery by in unparasitized magpie nests (18 broods). The magpie nestlings parents in 16 non-parasitized magpie nests (natural broods). We used used in each experiment came mainly from the same magpie brood a wireless microcamera (KPC-S500, black and white CCD camera, (in 39 of 44 experimental broods); they were randomly selected eSentia Systems Inc., Baton Rouge, LA, USA), a hard disk device from those that hatched within the first or second day of hatching media recorder (EMTEC, Gennevilliers, France) and a 3inch and had masses similar to those of cuckoo nestlings. However, and portable monitor. Before filming, nestlings were administered with because it is difficult to find two cuckoo nestlings of similar mass their fifth doses (cyproheptadine or water) and weighed. Nestlings in the same magpie nest, the two cuckoo nestlings came mainly were individually marked with a unique combination of white points from two different nests (36 of 44 experimental broods). Similar to drawn on the crown using correction fluid (Tipp-Ex). These marks natural broods, nestlings were ranked within species and we have a similar appearance to excremental remains, which sometimes alternated the order of assignment of experimental and control can be found on nestlings. The mean duration of the video recordings treatment between nests. In this case, we alternated the four possible was 3:27h (range2:23–4:39h), resulting in a mean of 5.5 adult combinations: the heaviest nestlings of each species received the visits per hour (range2.9–9.3visitsh–1).
same treatment or the heaviest nestlings of each species received We watched the video recordings using TMPGEnc DVD Author 3 software (Pegasys Europe, London, UK), which allows forward Treatments consisted of oral administration (by a plastic 1ml and backward frame stepping. During the visits by the adults, we syringe) of 0.1mg cyproheptadine hydrochloride (Acofarma, Inc., noted whether each nestling begged (begging) (i.e. if the nestling Barcelona, Spain) diluted in 0.25ml mineral water to the responded to the adult visit by at least opening its mouth towards experimental nestlings every 2days (i.e. 0.05mgday–1). Control the adult), received food or defecated (faeces). Magpies have a nestlings were administered with 0.25ml mineral water. We throat pouch used to carry food items to the nest, and thus we could calculated the dose of cyproheptadine based on that recommended not directly quantify the amount of food received by each nestling for children (0.4mgkg–1day–1) (Peisker, 2000) and extrapolated to from video recordings; however, this was estimated using neck a nestling of 100g. This dose was similar to that previously used collars (see below). We also ranked: (1) nestling body position in in chickens (average dose was ca. 0.5mgkg–1day–1) (Rao and the nest with respect to distance to adult location (nest position), Varadarajulu, 1979; Injidi and Forbes, 1987; Muralidharan et al., (2) height of nestling head whilst begging (height), and (3) the order 1998). Drug dilution was also the same as that used for children in which each nestling begged in relation to nestmates (order). Two (0.4mgml–1) (Peisker, 2000) and similar to that used in chicken or more nestlings had the same rank value when differences between them were not easily discernible. Because both brood sizes Table 1. Results from the analyses of video-recordings in magpie vary among nests (as does the number of begging nestlings within nests to assess the effect of treatment with cyproheptadine on nests), we standardized ranking values of nest position, height and begging behaviour and adult food allocation order by the formula: (rank–1)/(N–1) (modified from Smith and Montgomerie, 1991), where N equals brood size for the variable nest position, or the number of nestlings that begged during the adult visit for the variables height and order. Standardized values therefore vary between 0 and 1, so those values closest to 0 correspond to nestlings relatively close to the adult, the highest In order to reduce within-nest variance, we analysed the effect of cyproheptadine treatment within nestling dyads (i.e. paired tests); further, to homogenize nestling mass within dyads, we only used those dyads with nestling mass differences <15g (18 nestling dyads belong to 11 different nests). Differences in mass between nestlings within dyads were on average 6.37±0.98% of nestling mass.
Thereby, we compared nestlings with similar competitive abilities.
In such a situation, begging behaviour within a nestling dyad is assumed to reflect individual differences in interest in receiving food, independently of whether parent or offspring has total control over food distribution (see Royle et al., 2002). We have no data about whether parents or offspring control food distribution during video recordings. However, nestling behaviours associated with sibling negotiation or sibling competition should also be constrained by their associated costs [as is suggested by Royle et al. (Royle et al., 2002) and Johnstone and Roulin (Johnstone and Roulin, 2003)].
Therefore, the net balance of costs and benefits associated with escalated begging behaviour could be estimated using our Analyses are generalized mixed linear models (GLMMs) for begging (788 methodology independently of whether parent or offspring has observations in 229 adult visits) and linear mixed models (LMMs) for order control over food distribution. The effect of our treatment on begging (580 observations in 217 adult visits), height (573 observations in 217 and faeces was analysed using binomial GLMMs, and the effect of adult visits) and nest position (579 observations in 217 adult visits). Nest the experimental treatment on the nest position, height and order identity (11 different nests) and nestling dyad* (nested in nest identity, 18 was estimated using LMMs. Nest identity and nestling dyad (nested different nestling dyads) were used as two random factors, whereas in nest identity) were used as random factors, whereas experimental experimental treatment, time from the last feeding received (time withoutfood) and nestling mass were included as covariates. For GLMMs, we treatment, standardized time [(x–mean)/s.d.; in frames, also included the number of nestmates competing for food (nestmates 1frame0.04s) from the last feeding received (time without food) begging) as a further covariate. We used Wald z-tests [z-values and and nestling weight (g) were included as covariates. For GLMMs, Pr(>͉z͉)] for GLMMs, and for LMMs we estimated the highest posterior we also included the number of nestmates competing for food in a density 95% confidence intervals (HPD 95% CI) and P-values (PMCMC) given adult visit (nestmates begging) as a further covariate. The from Markov-chain Monte Carlo (MCMC) simulations.
success of nestlings begging for food was analysed using a GLM *Nestling dyad, one experimental and one control nestling from the same with the percentage of feedings each nestling received relative to the number of adult visits in which it begged as a dependent variable,experimental treatment as a within factor, the mass differences within piece of fabric to avoid any visual stimulation that could cause the nestling dyad as a covariable and nest identity as a fixed factor.
nestlings to beg. Early in the morning, each nestling was satiatedwith a mixture of commercial nestling food with water and raw cow Estimations of begging behaviour in the laboratory
heart. After 1h, each nestling was separately stimulated to beg for During May and June 2003 and late in the evening (between 20:00 food, but was not fed. We encouraged nestlings to beg by gently and 21:30h), 50 magpie nestlings (8–10days old) from 22 touching the border of the nest, whistling three times (the stimulus experimental nests (nine natural broods and 13 parasitized broods) that we previously used to feed the nestlings) and moving our fingers were moved to our laboratory (~30min by car). We used only one above their heads for 35s. Nestlings were encouraged to beg every magpie nestling dyad from the same nest, except for three natural 20min eight times (begging trials), except for three nests where broods where we used two nestling dyads per nest. When necessary, nestlings were only stimulated seven (one nest) and five times (two nestlings from non-experimental nests were placed in the nests). After each begging trial, we again covered the experimental experimental nests in place of those removed in order to prevent nestlings to prevent any further begging efforts by the experimental adults from abandoning the nests. Once in the laboratory, the nestlings. During these begging trials, we were blind to the treatment nestlings were administered with their fourth dose of cyproheptadine or water and weighed on a portable digital balance (Sartorius Nestling behaviour during begging trials was video-recorded.
Portable PT600, precision ±0.01g; Data Weighing Systems Inc., Afterwards, video recordings were watched to collect information Elk Grove, IL, USA). Nestlings were kept separately overnight in on the following variables: begging (nestling responded to the artificial nests in a soundless room and under a heating lamp (30 stimulus at least by opening its mouth towards our fingers); begging to 35°C). The artificial nests consisted of metal nest-shaped moulds calls (nestling emitted a sound when begging); body posture lined with raw cotton. Each artificial nest was covered with a small (nestling had its tarsi extended for begging); and begging duration [time (s) that the nestling spent begging in each trial]. After the Neck-collar trials were performed in 2002 (18 and 41 begging trials, nestlings were again fed until they were satiated.
experimental nests with data for biomass received and body mass Differences in begging behaviour between experimental and control loss, respectively) and 2003 (15 and 29 experimental nests with data nestlings were tested using binomial GLMMs (response variables: for biomass received and body mass loss, respectively). Nonetheless, begging, begging calls and body posture) and LMMs (response we decided to pool data from these two years because no significant variable: begging duration) with nestling treatment (cyproheptadine differences between years appeared with respect to biomass received or water) and brood type (i.e. natural or parasitized broods) as fixed per nestling (LMM, year effect: HPD 95% CI of ln-transformed factors, and time (min) from the beginning of the experiment and values–0.84 to –0.37mgh–1, PMCMC0.44) or body mass loss nestling mass as covariates. Nestling dyad was included in the model during the experiment (LMM, year effect: HPD 95% CI of ln- transformed values–0.38 to 0.03gh–1, PMCMC0.09). Furthermore, During all trials, faecal sacs were removed and weighed on a the effect of the experiment did not differ significantly between years portable digital balance (precision ±0.01g). Although nestlings for biomass received (LMM, interaction between year and treatment defecated mostly during the hand-feeding performed after begging factors: HPD 95% CI–1.10 to 0.54mgh–1, PMCMC0.46) or body trials, we used data from all faeces successfully weighed.
mass loss (LMM, interaction between year and treatment factors:HPD 95% CI–0.16 to 0.40 gh–1, PMCMC0.44). Besides treatment, Neck-collar trials
year and its interaction, nestling mass (g) before the neck-collar During May and June 2002 and 2003 and when nestlings were trial began was also included as a covariate in these analyses.
~10–12days old (i.e. after their fifth dose), we placed neck collars The food deprivation during begging trials performed in the on all the chicks in a group of parasitized and natural broods. The laboratory (see above) reproduces that suffered by nestlings with ligature wire around the neck was tight enough to hinder the neck collars in the field (i.e. begging but not getting food, see below).
swallowing of food and loose enough to avoid strangling the chicks In field conditions, adult magpies removed faecal sacs from the nest; [see Soler et al. (Soler et al., 1995) for a further description of the thus, it was not possible to know the mass of faecal sacs produced neck-collar method in magpies]. Nestlings wore the neck collar for during the neck-collar trials. By weighing faecal production of a period of 2 to 2.5h. This period is ~1h shorter than that previously experimental and control nestlings during the laboratory trials, we used in magpies (Soler et al., 1995). Although this shorter were able to test whether any change in body mass of experimental experimental period may reduce the hypothetical differences and control nestlings in natural nests with neck collars could be due between nestlings of different treatments, it may also reduce the to previous experience (i.e. food processing in the digestive tract), possibility of some nestlings regurgitating boluses of food. The food and whether faecal production was related to nestling body mass that was delivered by parents to each nestling during neck-collar (see below). We analysed the relationship between the mass of faecal trials was kept in absolute ethanol until it was analysed in the sacs produced during laboratory trials and nestling mass by laboratory. Biomass of food received by each nestling (mg) was estimating the correlation coefficient; moreover, we tested the estimated by dry mass of food samples. Food samples were placed possible effect of experimental treatment on faeces production (see in an oven at 60°C for a period of 24h (i.e. until a constant mass), above) by performing a GLM analysis with treatment as a fixed after which they were weighed (Adam equipment 120/0.0001g; factor, nestling mass as a covariable and mass of faecal sacs as the Adam Equipment Co. Ltd, Bletchley, UK). Nestlings were weighed outcome variable. Sample sizes were, in this case, reduced to 41 at the beginning and end of the neck-collar trial and change in body nestlings because in nine nestlings some of the faecal sacs leaked mass was calculated accordingly. Differences in body mass before they were weighed. Nestling mass was positively correlated (before–after the experiment) and biomass of food received were with the mass of the faecal sacs produced during the laboratory trials standardized by dividing by the duration of the experiment (h).
(r0.33, N41, t2.19, P0.035), whereas the experimental Because a gain in nestling body mass during neck-collar application treatment did not explain a significant proportion of the variance in would mean that the neck collar was not correctly placed, we only mass of faeces (F1,380.06, P0.81). Similarly, during the video used data from those nestlings that lost weight during neck-collar recordings of the magpie nests in natural conditions, we found that trials. In only 16 of 226 cases did nestlings maintain or gain mass probability of nestling defecation in a given adult visit was during neck-collar trials. Moreover, we only used in the analyses significantly and positively related to nestling mass [binomial those nests with data from at least one control and one experimental GLMM, nestling mass effect (estimate ± s.e.m.): 0.026±0.005, magpie nestling. Differences in body mass were also used as an z2.97, Pr(>|z|)0.003, 705 observations (i.e. whether a nestling estimate of begging effort of nestlings (e.g. Kilner, 2001) during defecated during a visit), 209 adult visits, 18 nestling dyads in 11 nests], whereas experimental treatment did not affect the Nests in which regurgitated boluses of food were detected in the probability of nestling defecation (binomial GLMM, treatment effect nest cup (i.e. they could not be assigned to nestlings) were not (estimate ± s.e.m.): –0.014±0.202, z–0.069, Pr(>|z|)0.95, 705 included in the analyses and, similar to body mass loss, we included observations, 209 adult visits, 18 nestling dyads in 11 nests).
in the analyses only those nests with data from at least one control Therefore, the inclusion in the models of the nestling masses before and one experimental magpie nestlings (i.e. excluding zeros).
the neck-collar experiment allowed us to statistically control the Consequently, sample sizes for biomass received (33 nests; 17 variation in faecal-sac production among nestlings during the neck- natural broods and 16 parasitized broods) and body mass loss (59 nests; 31 natural broods and 28 parasitized broods) differed. Weused LMMs to explain the effect of the experiment on ln(biomass Physical condition of magpie nestlings
of food received) (mg) and ln(change in body mass) (g) per hour During May and June 2002, we estimated the physical condition of and per nestling. Nest identity was included in the models as a control and experimental nestlings from natural broods close to random factor, brood type (i.e. natural or parasitized broods) as a fledging time (ca. 18days old). We measured nestling mass (spring second fixed factor and nestling mass (g) before the neck-collar trial balance, 300g ±1g), lengths of tarsi (with a digital calliper, ±0.01mm), wings and tail (using a ruler, ±0.1cm), and cell-mediated immune response estimated from values of skin swelling elicited by Table 2. Results of comparisons between begging behaviour of injection of the mitogen phytohemagglutinin (PHA) (reference no.
nestlings with an experimentally increased level of hunger and L8754, Sigma Chemical Co., St Louis, MO, USA) (see Tella et al., control nestlings with no treatment during the laboratory trials 2008). Briefly, we injected fledglings subcutaneously in the right wing web with 0.5mg of PHA dissolved in 0.1ml of physiological saline solution (Bausch and Lomb). As a control, the left wing web was injected with 0.1ml of physiological saline solution. Before and 24h after injection, we measured the thickness of each wing web at the injection site with a pressure-sensitive digital micrometer (model 547-301, ±0.01mm; Mitutoyo, Andover, Hants, UK). The immune response variable was then estimated as the change in swelling of the right wing web minus that of the left wing web (Lochmiller et al., 1993). We repeated measurements of each wing web three times and used the mean values in our analyses.
For these analyses, we used a repeated-measures analytical approach to check the possibility that the treatment effects were influenced by nestling hierarchy in body mass. We only used natural broods where at least the two heavier nestling dyads had survived to this age (N37 nests). In order to obtain the balanced statistical design needed for repeated-measures analyses, only data from these two nestling dyads were used. We included the experimental treatment as the first within factor, and the assigned rank in the body mass hierarchies of nestling (i.e. first or second heaviest nestlings within each treatment) as the second within factor. This design allowed us to determine whether the treatmenteffect was different for different nestling hierarchies (interaction factor). We used a Sigma-restricted model to code categorical factors and the type VI [the effective hypothesis method (Hocking, 1996)] to get the sums of squares. Moreover, we performed a repeated-measures multivariate ANOVA (RM-MANOVA) by using the same design but including all dependent variables Analyses are GLMMs for begging (374 observations in 25 groups), begging calls (332 observations in 25 groups) and body posture (327 observationsin 25 groups); and LMMs for begging duration (327 observations in 25 groups). Nestling treatment and brood type (i.e. natural or mixed broods)were included in the model as fixed factors, and time from the beginning of Effect of cyproheptadine on begging behaviour and food
the experiment and nestling mass were included as covariates. Nestling allocation during video recordings in magpie nests
dyad was considered as a random factor. For GLMMs, we used Wald z- We found a statistically significant association between tests for hypothesis testing [z and Pr(>͉z͉)]. For LMMs, we estimated the experimental treatment and probability of begging during adult visits (begging; Table1). Nonetheless, we did not find a significant effect of cyproheptadine on further variables related to nestlingbegging intensity (nest position, height and order; Table1). In CI0.02–0.04mgh–1, PMCMC<0.001] but lost more body mass relation to food allocation by magpie adults, we found that the [LMM, with ln(body mass lost) as the dependent variable, nestling proportion of parent visits where nestlings were fed relative to the mass effect: HPD 95% CI0.003–0.01 gh–1, PMCMC<0.001] during total of visits where they begged was greater for the experimental the standardized time of the experiment. After controlling for the nestlings (46.81±2.62%) than for their control nestmates allometric effects of nestling mass before trials and the possible (40.47±2.33%; GLM, treatment effect: F1,67.27; P0.036). This effect of brood type (see Materials and methods), experimental suggests that experimental nestlings were more efficient when nestlings received more food [LMM with ln(food received) as the begging for food than control nestmates.
dependent variable, treatment effect: HPD 95% CI1.82– 5.93mgh–1, PMCMC0.0004; Fig.1] but lost more body mass [LMM Effect of cyproheptadine on begging behaviour during
with ln(body mass lost) as the dependent variable, treatment effect: laboratory trials
HPD 95% CI0.03–0.31gh–1, PMCMC0.015; Fig.1] than control Results from the laboratory were similar to those obtained from video recordings in the field. Nestlings experimentally provided withcyproheptadine begged more frequently than their control nestmates Effect of cyproheptadine on the physical condition of magpie
(begging, treatment effect; Table2). The probability of emission of nestlings
sound (begging calls), body posture during begging and time of Magpie nestlings experimentally treated with cyproheptadine begging during a certain trial (begging duration) were not correlated showed a better physical condition than control nestlings when they with the experimental treatment (Table2).
were ~18days old (RM-MANOVA, Wilks’ 0.66, F5,272.79, P0.037). Experimental nestlings exhibited a higher immune Neck-collar trial
response than their control nestmates (experimental vs control, Heavier magpie nestlings received more food [LMM, with ln(food 1.25±0.06 vs 1.12±0.07mm, GLM: F1,344.14, P0.0497), had a received) as the dependent variable, nestling mass effect, HPD 95% longer tarsus (48.0±0.4 vs 47.2±0.4mm, GLM: F1,367.31, P0.010), Fig.1. Partial effect of treatments with cyproheptadine (experimental) or water (control) on biomass received [mgh–1, N33 nests, effect size (i.e. range)73.88mg] and body mass loss (gh–1, N59 nests, effect size0.25g) during neck-collar trials in magpie nestlings. Values were calculated by back-transformation of estimates obtained from linear mixed models with ln(biomass received) and ln(body massloss) as dependent variables. Values are means ± highest posterior density (HPD) 95% confidence intervals.
wing (81.9±1.5 vs 79.8±1.4mm, GLM: F1,365.24, P0.028) and and Forbes, 1987; Muralidharan et al., 1998) and domestic pigeons tail (24.6±0.9 vs 23.3±1.0mm, GLM: F1,344.35, P0.045) and a (Gunturkun et al., 1989), where it resulted in a significant increase higher body mass (138.3±2.7 vs 132.3±2.9g, GLM: F1,357.19, of food ingestion. Thus, the use of this drug as appetitive stimulant P0.011). Further, the effect of cyproheptadine was similar in the was well founded and we tested its effects on begging behaviour first and second nestling hierarchical dyads (interaction between treatment and rank of nestling dyad: in all cases P>0.35).
The use of cyproheptadine as appetite stimulant has several advantages. It is administered orally diluted in water, and thus the DISCUSSION
treatment can be easily done in the field and during short nest visits.
In the present study, by using an appetite stimulant (cyproheptadine Importantly, experimental nestlings can be administered during hydrochloride), we have for the first time explored the net development while they are being fed by their parents, who are the cost–benefit balance of escalated begging behaviour in natural receptors of the begging signals. This approach makes possible the conditions, where the extra costs of begging might be compensated estimation of variables related to the net cost–benefit balance by the acquisition of extra resources from parents. In accordance associated with the experimentally escalated begging behaviour. In with the widely manifested effects of the level of hunger on begging addition, experimental nestlings may also suffer from other possible behaviour, we found that experimental nestlings increased the sources of energetic costs not directly related to the production of frequency of begging (but not intensity) and received more food the begging signals and difficult to take into account in food than their control nestmates. Our main result was that the net benefit deprivation laboratory experiments. For example, the effect of of escalated begging behaviour was positive, i.e. experimental sibling competition (e.g. Johnstone, 1999; Rodríguez-Gironés, nestlings showed a better physical condition than control nestlings 1999; Royle et al., 2002; Neuenschwander et al., 2003), the possible close to the age of nest abandonment. This result suggests that the costs related to the nestling vigilance for parent arrival (Roulin, 2001) costs associated with exaggerated begging behaviour do not solely or those costs suggested for behaviours related to sibling negotiation counteract associated benefits as suggested and/or assumed by during the absence of parents (Johnstone and Roulin, 2003).
previous studies (e.g. Kilner, 2001; Rodríguez-Gironés et al., 2001b; Moreover, because cyproheptadine would exclusively affect hunger Moreno-Rueda, 2010; Noguera et al., 2010). Below we discuss the level, this experimental approach allow us to disentangle the effects methodological approach used for manipulating nestling begging of body condition and nestling hunger level on begging behaviors behaviour in natural conditions, as well as the resulting experimental (e.g. Clark, 2002) to address what offspring are really effects in a scenario of parent–offspring communication and honest communicating to their parents via their begging signals (need, quality or just hunger) (see Mock et al., 2011).
In accordance with previous results of the effects of hunger The use of cyproheptadine to increase begging behaviour
level on begging behaviour (e.g. Redondo and Castro, 1992b; Cyproheptadine is a type 2 serotonin receptor antagonist. It is Price et al., 1996; Iacovides and Evans, 1998; Sacchi et al., 2002), assumed that cyproheptadine acts directly on the hypothalamus – we found that nestlings provided with cyproheptadine begged cats administered with this drug demonstrated an increased neuronal more frequently than their control nestmates. In addition, we activity at this site (Chakrabarty et al., 1967) – by modifying the found that begging signals of experimental nestlings were more activity of serotonin on the feeding centre (Delitala et al., 1975).
effective in attracting parental feeding than those of their control Its action as appetite stimulant appears not to be due to a nestmates. This suggests the existence of a treatment effect on hypoglycaemic-induced hyperphagia or an increase in endogenous non-measured variables of begging intensity. The most important growth hormones (Bergen, 1964; Stiel et al., 1970). Cyproheptadine described side effect of cyproheptadine is the reduction of is broadly used in human patients, including children, that suffer physical activity because of transient drowsiness, which would from diseases such as cancer, HIV, cystic fibrosis and eating predict a reduced rather than an increased begging activity. Thus, disorders (see Chinuck et al., 2007; Couluris et al., 2008; the detected effects on begging behaviour are not explained by Mahachoklertwattana et al., 2009), where an increase in body mass the drug side effects but by the increased feeling of hunger of is desirable. Cyproheptadine is also used as an antihistaminic because it blocks H1 histamine receptors (Stone et al., 1961) and, similar to other antihistaminic substances, cyproheptadine has minimal side Evidence of short-term benefits and costs of escalated
effects, consisting mainly of transient drowsiness [see Homnick et begging behaviour
al. (Homnick et al., 2004) and references therein]. In birds, the effect Experimental nestlings received more food than their control of cyproheptadine has been tested in two species, chicken (Injidi siblings did during the neck-collar trials. Therefore, based on our results on the influence of hunger level on begging behaviour and resulting differences in mass loss during collar trials, this conclusion the extensive literature concerning this issue (e.g. Smith and Montgomerie, 1991; Cotton et al., 1996; Kacelnik et al., 1995;Kilner, 1995; Kölliker et al., 1998), relative to the use of begging Net cost–benefit balance of exaggerated begging behaviour
by parents to determine which chick to feed (e.g. Smith et al., during the nestling period
1988; Ottosson et al., 1997; Kilner and Johnstone, 1997; Burford If the costs associated with escalated begging behaviour surpass the et al., 1998) (but see Clark and Lee, 1998), the effect of our extra benefits, as is assumed by some theoretical models (for experimental treatment on biomass received during neck-collar reviews, see Mock and Parker, 1997; Godfray and Johnstone, 2000; trials by each nestling was probably mediated by a change in Budden and Wright, 2001; Johnstone and Godfray, 2002; Royle et begging behaviour associated with the experimentally increased al., 2002), and the direct costs are physiological and/or energetic, we should find a negative effect of our experiment on nestling The neck-collar approach may, however, have methodological phenotypic quality. Contrary to this expectation, experimental problems that could affect to the interpretation of the results. For nestlings showed a better physical condition than their control instance, feeding decisions of parents might depend on detecting siblings when abandoning the nest. Briefly, we found that after food in the nestling’s mouth, or adults might remove non-swallowed controlling for the effect of body-size hierarchy, experimental food from one nestling and place it in another chick’s mouth.
nestlings showed a higher immune response and larger body mass However, rather that predicting more food in the gullet of and tarsus, tail and wing lengths than their control siblings. Because cyproheptadine-treated nestlings, the above scenario predicts more these variables are related to the probability of nestling survival (see equally distributed food among nestlings. We compared Introduction and above), these results suggest that magpie nestlings experimental and control nestlings from the same nest, and thus our could escalate some aspect of their solicitation signals and gain more result of experimental nestlings receiving more food than their resources that are plausibly connected to enhanced personal fitness.
control siblings likely does arise from our experimental approach Therefore, these results gives rise to an interesting evolutionary (increased level of hunger). In accordance with this interpretation, question, namely, why has natural selection not shaped magpie results from video recordings indicate that parents selected experimental nestlings among nestlings that beg for food. Parents Apart from non-functional explanations including physiological should preferentially feed the hungriest nestlings in the nests (i.e.
or phylogenetic constraints, responses to this question could be those with more exaggerated begging behaviour); thus, because the related to the existence of costs other than the energetic costs hunger level of experimental nestlings was increased, a more associated with the escalation of begging behaviour and suffered efficient begging behaviour of cyproheptadine-treated nestlings can during development. One possibility is the existence of costs related be predicted. Moreover, during neck-collar trials, nestlings cannot to risk of predation, because predators would more easily detect be satiated by parents, and nestlings would therefore beg for food nests with nestlings that beg loudly (e.g. Redondo and Castro, 1992a; during more feeding visits than in natural conditions (i.e. video Haskell, 1994; Leech and Leonard, 1997; Briskie et al., 1999; recordings). This scenario is therefore the most plausible explanation Dearborn, 1999; Haskell, 2002). However, and as mentioned of the resulting larger biomass received by experimental nestlings previously, these costs are mainly suffered by the whole brood, and different costs for nestlings that beg and do not beg are necessary Rodríguez-Gironés et al. reported a delayed growth of magpie for explaining the evolution of begging behaviour as an honest signal nestling chicks in relation to experimentally increased begging [see Rodríguez-Gironés et al. (Rodríguez-Gironés et al., 2001b) for activity (Rodríguez-Gironés et al., 2001b), which is likely to affect further discussion], and thus this cost would not prevent an escalation probability of survival of magpie nestlings (Husby and Slagsvold, of dishonest nestling behaviour. Further, predation cost would only 1992; Ponz Miranda et al., 2007) (M. Molina-Morales and J. G.
constrain those signals whose exaggeration implies an increased nest Martínez, unpublished data). This suggests that escalated begging detectability for predators, as is the case for vocalizations, but not would be a costly behaviour for magpie nestlings. In our case, for other kinds of signals such as visual ones. Another cost that experimental nestlings lost more mass than their control nestmates might prevent escalation of begging behaviour is that related to the during the two and a half hours that nestlings wore neck collars loss of indirect fitness (e.g. Briskie et al., 1994; Johnstone, 1998; (Fig.1). This result cannot be explained by drowsiness, which is Lotem, 1998). Bergstrom and Lachmann (Bergstrom and Lachmann, the main side effect of cyproheptadine (see above) and from which 1998) argued that a cost-free signalling equilibrium is possible the expected influence on mass loss is just the opposite to that between parent and offspring if parents and/or siblings pay a detected (i.e. weight gain) (see Stiel et al., 1970). Another possibility disproportionally extra cost for dishonest signalling and these is that, because experimental nestlings may have ingested more food indirect fitness costs for escalated signallers outweigh the direct before the experiment, they may also have produced more and/or benefit that they gain. Other non-explored costs associated with larger faecal sacs during the time that nestlings wore neck collars.
escalated begging behaviour are those related to the costs of rearing However, this is unlikely for two reasons. First, our analyses were dishonest offspring. If begging behaviour has a genetic basis (see statistically controlled by body mass, a variable that is positively Kölliker et al., 2000; Kölliker and Richner, 2001; Dor and Lotem, related to the production of faecal sacs of nestlings in the laboratory 2009), nestlings with an escalated begging display would rear and in video recordings in the field. Second, experimental treatment offspring with an escalated begging display. This possibility was did not explain either faecal weight or probability of defecation. An proposed by Alexander, who stated that a certain allele causing the alternative explanation would be that because faecal sac production dishonest trait in an offspring that, because of its selfish behaviour, often occurs simultaneously with feeding, the greater mass loss of receives more than the optimum parental inversion could not spread experimental nestlings might be the direct consequence of being because the advantage of this allele to the young offspring would preferentially fed by parents. We have no data to discount this be counteracted by the disadvantage of producing dishonest possibility; therefore, although differences in begging effort between offspring when adult (Alexander, 1974). Although this idea was experimental and control nestlings is a tentative explanation of the controversial (e.g. Dawkins, 1976; Blick, 1977), costs of rearing offspring with inherited begging behaviour should be added as a Couluris, M., Mayer, J. L. R., Freyer, D. R., Sandler, E., Xu, P. and Krischer, J. P.
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