DOI: 10.5061/dryad.b8gtht7kk
title: Immune challenge reduces begging behavior and modifies begging call structure in spotless starling nestlings

description: Symptoms of illness offer insights into an organism’s condition, altering
 social signals that affect others' behavior. Nestling birds employ
 begging signals to solicit parental care, but the extent to which begging
 reflects nestling health beyond hunger remains controversial. We
 investigated how experimentally induced changes in health affect begging
 signals in spotless starling (Sturnus unicolor) nestlings. To alter health
 status, we administered an immune challenge by injecting
 lipopolysaccharide (LPS) or a control substance (PBS) and observed begging
 behavior under controlled food deprivation conditions. LPS-treated
 nestlings exhibited delayed gaping, reduced stretching, and less begging
 time compared to control nestlings. Moreover, LPS-treated nestlings
 produced calls with higher spectral entropy, particularly when deprived of
 food for longer. Our findings indicate that begging signals are sensitive
 to induced sickness. However, addressing mechanistic issues is crucial for
 effectively testing whether begging reflects nestling conditions as a
 signal of quality in future experimental setups.
description: <strong>Experimental design</strong>
 We conducted the study in a nest-box breeding population of
 spotless starlings located in central Spain (Soto del Real, Madrid). In
 our study population, spotless starlings, medium-sized colonial
 passerines, typically have two asynchronous clutches per season, with a
 modal clutch size of 5 eggs (López-Rull et al. 2007; Salaberria et al.
 2014). We determined the hatching date of the nests through regular
 inspections. We chose 25 nests from the first wave of
 clutches in the breeding season. On the day before the experiment, at the
 age of 5 days, we selected two chicks of intermediate-mass rank from each
 brood, ensuring that at least two nestlings remained to prevent parental
 desertion. The nestlings were transported in a warm container to the
 laboratory, where they were placed together in artificial nest cups with
 controlled temperature (approximately 35°C) and humidity (45%). The
 nestlings were familiarized with a begging stimulus, which was a playback
 of a parental feeding call recorded from the same population while being
 provided ad libitum with super worms (<em>Zophobas
 morio</em>). On the following morning (day 6 of
 age), we weighed the nestlings with a digital balance (accuracy = 0.1 g),
 placed them in individual nest cups side by side with the nest cup of
 their sibling, and provided them with three different meals to ensure a
 uniform nutritional condition. In the first two meals, nestlings had ad
 libitum access to superworms and crickets (<em>Acheta
 domestica</em>), respectively, with a 45-minute gap between
 feedings, during which we recorded the number of insect items consumed.
 These intervals were determined based on the mean intervals of parental
 feeding rates obtained from a random video sample of 48 nests in our study
 population (mean interval = 59.05 min ± 4.27 SE; our data). We calculated
 the average weight of each superworm and cricket by weighing 10 pools of
 10 units of each insect type. On average, one superworm larva weighed 0.5
 g, and one cricket weighed 0.14 g. In order to estimate the total amount
 of food consumed by nestlings, we multiplied these weights by the number
 of insects of each type consumed per nestling. After 45 min, we offered
 nestlings high-quality food (i.e. boiled egg, Moreno-Rueda and Redondo
 2012) in an amount equivalent to 3.5% of their body mass. This food
 quantity was estimated using the allometric relationship from Weathers
 (1996): daily food intake = 0.98 M<sup>0.814</sup>, where M
 represents nestling body mass in grams, divided into 14 daily portions.
 Egg quantity was also weighed to estimate the total food consumed by
 nestlings. Following the last meal, nestlings were individually
 transported to a recording chamber to monitor their begging behavior. This
 first test was used to determine baseline begging behavior. Begging
 behavior was recorded at 30, 60, and 90 min after the last meal. During
 each begging trial, nestlings were stimulated twice using the same
 stimulus as before (feeding call playback), allowing begging to cease
 completely between both stimuli. Postural begging was recorded using a
 digital video camera (Panasonic HC-V180), and acoustic begging was
 recorded using a digital audio recorder (Marantz Professional PMD620)
 placed 5 cm from the nest cup. After the final begging trial, nestlings
 were provided ad libitum with super worms, and siblings of each dyad were
 randomly assigned to either an LPS or control treatment. Nestlings in the
 LPS treatment received an intraperitoneal injection of 50 μl of LPS (ref:
 L2880, Sigma-Aldrich) suspended in a phosphate-buffered saline solution
 (PBS) at a concentration of 1 mg/kg body weight. Control nestlings
 received the same volume of PBS alone. This dosage was selected based on
 the dose-dependent results reported by Armour et al. (2020) in the
 northern bobwhite quail (<em>Colinus virginianus</em>) and was
 the same dose used by Serra et al. (2012) in European Starling
 (<em>Sturnus vulgaris</em>) nestlings. 1h
 and 15 min later, we initiated feeding to equalize the short-term food
 deficit, following the same protocol described above, with feeding every
 45 min. This ensured that the second begging test occurred 3-4.5 h after
 the injection, allowing the chicks to remain influenced by LPS while
 avoiding the peak effect of the challenge (for justification, see below).
 We deliberately chose not to conduct the post-treatment tests at the peak
 effect of the challenge (1-2 hours after the injection) as we anticipated
 that, at that point, nestlings might be in a weakened state, potentially
 yielding unreliable responses (see below). Post-injection test was carried
 out in the same manner as the initial one, recording the behavior of the
 nestlings at 30, 60, and 90 min following the last meal (i.e., at 3 h 15
 min, 3 h 45 min, and 4 h 15 min after the LPS/Control injection). This
 approach allowed us to observe changes in the behavior of a focal nestling
 in response to both LPS and Control treatments, thereby testing both
 within- and between-subject effects. At the conclusion of the experiment,
 we feed nestlings again with super worms recording the number of insects
 to estimate the amount of food consumed as before, we weighed nestlings,
 collected a blood sample (approximately 50 µl) from the brachial vein for
 genetic sexing, and returned them to their original nests by the end of
 the day. Nestlings were weighed again on day 8 of age (i.e., 48 hours
 after the laboratory experiment ended) to detect any potential medium-term
 effects of the experimental treatment on their mass.
 The timing of the post-injection begging test was synchronized
 with the effect of LPS on nestling temperature, based on a prior pilot
 test involving 17 nestlings of the same age as those used in the
 experiment (see Supplementary Material for additional details). In this
 pilot assay, we subjected nestlings to the same LPS injection protocol and
 doses mentioned previously and measured variations in cloacal temperature.
 The most pronounced effects of LPS on temperature were observed within the
 initial 3 hours following the injection, with the peak temperature
 difference between LPS and Control nestlings occurring at 2 hours
 post-injection (see Supplementary Figure S1). These results were
 consistent with previous studies employing similar injection protocols
 (Owen-Ashley et al. 2006; Burness et al. 2010), suggesting that the peak
 effects of LPS on body temperature, interleukin-mediated inflammation, and
 corticosterone levels (Nakamura et al. 1998; De Boever et al. 2008;
 Burness et al. 2010) manifest within 1-2 hours after injection.
 <strong>Analysis of postural and acoustic
 traits</strong> An external trained observer,
 blind to the experiment's purpose and chick treatments, analyzed
 videos at a reduced speed (0.125x) using the Solomon coder software
 (Version: beta 19.08.02; Péter 2013). Postural begging intensity was
 assessed on a five-level ordinal scale, indicating the degree of body
 stretching: 0 (no begging), 1 (gaping, lying body), 2 (gaping, neck
 extended, lying body), 3 (neck extended, body separated from nest floor,
 back reclining), and 4 (gaping fully stretched, vertical back, sometimes
 including wing flapping) (adapted from Redondo and Castro 1992). At each
 sampling time, we recorded the time spent by the nestling at each postural
 intensity level and calculated the total duration of the begging display
 by summing the durations of each posture. Additionally, we computed the
 mean and maximum postural ranks for the whole begging display. The mean
 postural intensity was computed by multiplying the duration of each
 begging level by its rank and then dividing it by the total duration
 (Leech and Leonard 1996; Kilner 2001). We also recorded the latency time
 from the start of each stimulus to the onset of the begging (precision =
 0.1 s). We used Avisoft SAS-Lab Pro v. 5.1.23 software
 (Specht 2002) to analyze begging vocalizations. Spectrograms were created
 with the following settings: 256 FFT, FlatTop window, frame 100%, overlap
 50%, a bandwidth of 162 Hz, and a high-pass filter at 2 kHz, resulting in
 a time resolution of 11.6 ms and a frequency resolution of 43 Hz. From
 these spectrograms, we extracted six temporal and spectral measurements
 commonly examined in nestling begging behavior studies that are related to
 offspring cryptic need (hunger) and that are known to vary with
 modifications of their internal state (e.g. Sacchi et al. 2002; Reers and
 Jacot 2011; Klenova 2015). We manually measured the total vocal bout
 duration (in ms) from the start of the first call to the end of the last
 one and the total number of calls. The calling rate was calculated by
 dividing the total number of calls by the bout duration. We selected a
 subsample of the first 10 calls in each bout following each playback
 stimulus to obtain spectral measurements. For each begging bout, we
 measured call duration, peak frequency, maximum and minimum frequency,
 call entropy (energy dispersion across the frequency spectrum), and the
 lower (25%) and upper quartiles (75%) of energy distribution across the
 frequency spectrum. Spectral variables were measured at the point of
 maximum amplitude of the element, both instantaneously and after
 specifying a minimum hold duration of 40 ms (peak hold mode). We also
 obtained measures for spectral variables considering either the Max or
 Mean spectrum of the entire element. Due to a recording system failure, no
 acoustic measurements were obtained in the two nestling dyads.
 <strong>Statistical analysis</strong>
 We performed statistical analyses using R 4.1.3 (R Core Team
 2022). To enhance model stability, the likelihood of model convergence,
 and the accuracy of parameter estimates, all independent variables were
 Z‐transformed (mean-centered with an SD of 1) (Harrison et al.
 2018). To assess the impact of the experimental
 treatment on nestling conditions, we examined short- and medium-term
 effects on body mass. We used a linear mixed model to analyze differences
 in body mass at the end of the experiment (day 6), controlling for the
 initial mass (day 5). Additionally, we conducted two more analyses to
 explore medium-term effects on body mass when chicks returned to their
 nests after the laboratory procedures. For this purpose, we used a model
 with body mass on day 8 (two days after returning to the nest) as the
 response variable, controlling for the initial mass (day 5), and a
 separate model controlling for the mass at the end of the laboratory
 procedures (day 6). In the last two models, we included brood size as a
 covariate to account for differences in individual feeding rates due to
 sibling competition. Experimental treatment (LPS or Control) served as the
 main predictor in all these models. To evaluate the effect of treatment on
 nestling food intake, we used the total amount (g) of food consumed before
 each test period as the response variable. In this model, we included
 experimental treatment (LPS or Control) interacting with the test period
 (before or after the injection) as the main predictor and the initial body
 mass as a covariate. The nutritional values, chemical and chitin
 compositions, digestibility, and handling times differed among the three
 food types (superworms, crickets, and boiled egg) fed to nestlings
 (Kaspari and Joern 1993; Banbura et al. 1999; Finke 2002; Moreno-Rueda and
 Redondo 2012; Oonincx and Finke 2021). As this may affect nestling
 predisposition to feed and their mass gain, we conducted three additional
 models with each food type separately as the response variables. We also
 conducted an additional analysis to evaluate the effect of LPS on the
 amount of superworms consumed at the end of the experiment (after the
 second begging test). To analyze the effects of LPS
 treatment on postural begging, we employed linear mixed models with
 latency time to gape, mean begging intensity, and duration of the begging
 display as response variables. The maximum postural intensity was examined
 using an ordinal mixed model (Christensen 2022). In all these models we
 included the experimental treatment interacting with the test period as
 the main effect. To account for the influence of hunger, we included time
 of food deprivation (30, 60, or 90 min since the last feeding) as a
 three-level factor covariate in a three-way interaction with treatment and
 test period. If this interaction proved non-significant, it was excluded
 from the final model. We incorporated initial body mass and order of
 stimulus (1 or 2) as covariates. For analyses of acoustic begging, we
 applied the same approach as described above to assess calling rate and
 call duration. We also conducted individual Principal Components Analysis
 (PCA) for peak frequencies, maximum frequencies (including quartile 75%),
 minimum frequencies (including quartile 25%), and entropies. This allowed
 us to combine various measurements obtained from different sections of a
 call, including the point of peak amplitude with and without peak hold
 mode, as well as the Max and Mean spectra of the entire element
 (Supplementary Table S2). We used the scores on the first component (PC1)
 as response variables and analyzed them using the same approach.
 Additionally, we included the sequential order of calls as a covariate
 since we analyzed up to 10 consecutive calls per playback stimulus. The
 effect of nestling sex was examined in all models as a covariate; however,
 it did not significantly influence any of the response variables and was
 therefore omitted from the final models. We determined
 the optimal random-effect structure for each of the aforementioned models
 using a top-down strategy. Nested models were compared through likelihood
 ratio tests (Zuur et al. 2009). We included chick nested within a nest of
 origin and both nested within the date of the experiment as random
 intercept effects.  Experimental treatment, test period, and time of food
 deprivation (considering their effects alone, additive, or interacting)
 were included as random slope effects. To address the
 issue of multiple-testing with the experimental treatment and time of food
 deprivation predicting various begging variables, we incorporated a
 P-value correction (Benjamini-Hochberg method) for postural and acoustic
 analyses separately, to control for false discovery rate in the
 statistical tests (Benjamini and Hochberg 1995; Storey and Tibshirani
 2003).
description: # Immune challenge reduces begging behavior and modifies begging call
 structure in spotless starling nestlings Parejo-Pulido, D, Redondo, T,
 Pérez-Rodríguez, L. Contact:
 [dapapu96@gmail.com](mailto:dapapu96@gmail.com) Datasets are saved as .txt
 files. Missing data are indicated as “NA". ### File list *
 Parejo-Pulido_et_al_Pilot: data of nestling temperature according to the
 treatment. * Parejo-Pulido_et_al_mass: data of nestling mass and food
 consumed according to the treatment. * Parejo-Pulido_et_al_posture: data
 of nestling postural begging performance. * Parejo-Pulido_et_al_acoustic:
 data of nestling acoustic begging performance. ### Description of the
 files #### Parejo-Pulido\_et\_al\_Pilot.txt This file contains the data
 used to analyze the effect of the LPS or Control treatment on
 nestlings' cloacal temperature in time. Columns: * "nest":
 identification code of nest. * "ID": identification code of
 nestling, which combines the nest of origin (e.g. 309) and the identifier
 (e.g. A). * "treatment": experimental treatment (coded as:
 "LPS"; "Control"). * "hour": time in hours
 since the LPS/Control administration. *
 "temperature_difference": difference in cloacal temperature of
 each chick with respect to the average temperature of the brood at a given
 time. * "mass": chick body mass (g) at day 6 post-hatching. ####
 Parejo-Pulido\_et\_al\_mass.txt This file contains the data used to
 analyze the effect of the LPS or Control treatment on nestling mass and
 food consumed. Columns: * "nest": identification code of nest. *
 "ID": identification code of nestling, which combines the nest
 of origin (e.g. 125) and the treatment identifier (e.g. Control). *
 "treatment": experimental treatment (coded as: "LPS";
 "Control"). * "test_period": before (coded as:
 "PRE") or after (coded as: "POST") the LPS/Control
 administration. * "initial_mass": initial chick body mass (g) at
 day 5 post-hatching. * "final_mass": chick body mass (g) at the
 end of the laboratory procedures, day 6 post-hatching. *
 "mass_8d": chick body mass (g) at day 8 post-hatching. *
 "total_food_consumed": weight (g) of total food consumed. *
 "Zophobas": weight (g) of the amount of Zophobas items consumed.
 * "crickets": weight (g) of amount of crickets items consumed. *
 "egg": weight (g) of the amount of boiled egg consumed. *
 "Zophobas_fin": weight (g) of the amount of Zophobas items
 consumed at the end of the experiment (after the last begging test). *
 "sex": sex of nestlings (“1"=male; “2"=female). *
 “broodsize": number of chicks in the brood. * “date": experiment
 date, whereby “1" corresponds to the day of the first experimental
 brood. #### Parejo-Pulido\_et\_al\_posture.txt This file contains the data
 used to analyze the effect of the LPS or Control treatment on
 nestlings' postural begging performance. Columns: * “nest":
 identification code of nest. * “ID": identification code of nestling,
 which combines the nest of origin (e.g. 125) and the treatment identifier
 (e.g. Control). * “treatment": experimental treatment (coded as:
 "LPS"; "Control"). * "test_period": before
 (coded as: "PRE") or after (coded as: "POST") the
 LPS/Control administration. * "deprivation": time (minutes) of
 food deprivation ("30", "60", "90"). *
 "stimulus": begging stimulus ("1" or "2"). *
 "latency": latency time (seconds) from the start of each
 stimulus to the onset of begging. * “max_intensity": maximum level of
 postural begging intensity reached by a nestling, measured as an ordinal
 scale of the degree of body stretching ("4"=highest degree of
 body stretching). * “mean_intensity": mean level of postural begging
 intensity measured as the duration of each begging level multiplied by its
 rank and then divided by the total duration. * "duration":
 duration (seconds) of total begging bout. * “initial_mass": initial
 chick body mass (g) at day 5 post-hatching. * "sex": sex of
 nestlings (“1"=male; “2"=female). * “broodsize": number of
 chicks in the brood. * “date": experiment date, whereby “1"
 corresponds to the day of the first experimental brood. ####
 Parejo-Pulido\_et\_al\_acoustic.txt This file contains the data used to
 analyze the effect of the LPS or Control treatment on nestlings acoustic
 begging performance. Columns: * “nest": identification code of nest.
 * “ID": identification code of nestling, which combines the nest of
 origin (e.g. 125) and the treatment identifier (e.g. Control). *
 “treatment": experimental treatment (coded as: "LPS";
 "Control"). * "test_period": before (coded as:
 "PRE") or after (coded as: "POST") the LPS/Control
 administration. * "deprivation": time (minutes) of food
 deprivation ("30", "60", "90"). *
 "stimulus": begging stimulus ("1" or "2"). *
 "filter_callingrate": filter to obtain the dataset used for
 calling rate analyses (1 data per stimulus) * "calling_rate":
 calls/total duration of acoustic display. * "spectral_filter":
 filter to select data to be used in the analyses of acoustic variables
 except for that of the calling rate. * "sequence": order of the
 calls analyzed per stimulus (maximum 10 calls). *
 "call_duration": duration of each call. * "peak_peak":
 peak frequency at the point of peak amplitude without peak hold mode. *
 "min_peak": minimum frequency at the point of peak amplitude
 without peak hold mode. * "max_peak": maximum frequency at the
 point of peak amplitude without peak hold mode. *
 "entropy_peak": call entropy at the point of peak amplitude
 without peak hold mode. * "peak_peakhold": peak frequency at the
 point of peak amplitude with peak hold mode. * "min_peakhold":
 minimum frequency at the point of peak amplitude with peak hold mode. *
 "max_peakhold": maximum frequency at the point of peak amplitude
 with peak hold mode. * "entropy_peakhold": call entropy at the
 point of peak amplitude with peak hold mode. * "peak_max": peak
 frequency at the point of maximum spectra of the entire element. *
 "min_max": minimum frequency at the point of maximum spectra of
 the entire element. * "max_max": maximum frequency at the point
 of maximum spectra of the entire element. * "entropy_max": call
 entropy at the point of maximum spectra of the entire element. *
 "peak_mean": peak frequency at the point of mean spectra of the
 entire element. * "min_mean": minimum frequency at the point of
 mean spectra of the entire element. * "max_mean": maximum
 frequency at the point of mean spectra of the entire element. *
 "entropy_mean": call entropy at the point of mean spectra of the
 entire element. * "peak_PC1": first principal component loadings
 of peak frequency values. * "min_PC1": first principal component
 loadings of minimum frequency values. * "max_PC1": first
 principal component loadings of maximum frequency values. *
 "entropy_PC1": first principal component loadings of call
 entropy values. * “initial_mass": initial chick body mass (g) at day
 5 post-hatching. * "sex": sex of nestlings (“1"=male;
 “2"=female). * “broodsize": number of chicks in the brood. *
 “date": experiment date, whereby “1" corresponds to the day of
 the first experimental brood