DOI: 10.5061/dryad.mgqnk995s
title: Data from: Forecasting the future? Differential allocation of maternal hormones in function of experimentally manipulated social contexts in blue tits Cyanistes caeruleus

description: Sociability, i.e. the tendency to interact with other individuals, varies
 significantly within populations, with some individuals being consistently
 more sociable than others. This variation may be maintained because more
 sociable individuals can thrive better in certain conditions, in which
 social interactions facilitate information exchange or cooperation, but
 not so if social encounters increase aggressive disputes or infection
 risk. At the proximate level, apart from genes, mothers transfer
 non-genetic compounds to their offspring that can influence the
 development of social skills. In this context, they may adjust their
 offspring’s sociability to match the social environment they will
 experience after birth, for example, via prenatal hormones. To test this,
 we experimentally manipulated the social density as perceived by blue tit
 females before egg laying. We subsequently measured yolk testosterone
 concentrations and social interactions among family members post-hatching.
 Females that were exposed to a simulated high social density transferred
 less testosterone to their eggs than control females. Network degree
 (i.e., the number of social interactions of the brood) was not affected by
 the social density treatment, but broods with lower yolk testosterone
 concentration showed a higher network degree. This suggests that mothers
 experiencing an environment with high social density (but not increased
 resource competition) deposit less yolk testosterone to produce offspring
 that are likely less aggressive but more sociable.
description: <strong>General methods</strong>
 The study was carried out in Miraflores de la Sierra, Madrid,
 central Spain (40° 48′ N, 03° 47′ W) during the spring of 2021. We studied
 a wild blue tit population breeding in nest-boxes located in a deciduous
 forest, mainly dominated by Pyrenean oak (<em>Quercus
 pyrenaica</em>), at an elevation of 1250 m. The experimental
 manipulation took place from mid-March to the beginning of May, i.e.,
 until most clutches were completed. The study area was divided into four
 plots, two high-density and two control, which included, respectively, 83
 and 87 nest-boxes distributed across a similar-sized area. From mid-March
 onwards, almost three weeks prior to the laying of the first egg in the
 population, we exposed blue tit adults daily to either a recording of
 social stimuli simulating high-social density or to control playback. For
 the high-social density treatment, we broadcasted vocalizations of male
 and female blue tits. These included male songs and female calls
 (excluding alarm calls). For the control plots, we broadcasted
 vocalizations of male and female common chaffinches (<em>Fringilla
 coelebs</em>), which are common in the study area, but do not breed
 in nest-boxes and do not compete for food with blue tits. All recordings
 were obtained from Xeno-Canto (www.xeno-canto.org). Once laying started, i.e., when the first egg (or the two first eggs) was (were) detected, nests were visited daily to collect the third egg of each clutch on the day it was laid. Collected eggs were kept cool during transport and stored at -80 °C within the same day of collection until analysis.  In the second week after hatching (days 9-12), we trapped both adults when entering the nest-box for feeding the nestlings, and weighed them with a digital scale to the nearest 0.01 g. Their sex was determined based on plumage characteristics and the presence of an incubation patch. Thus, we could distinguish male and female parents during video observations. On day 12, nestlings were ringed, weighed with a digital scale to the nearest 0.01 g, and we marked individually on the head or wings with a white permanent marker (Edding 751). We also collected a blood sample from the brachial vein for molecular sexing. <strong>Behavioural observations</strong> On day 12, we replaced the original nest-box of each nest with a recording nest-box to familiarize blue tit breeding adults with it prior to video recordings. On day 13, we placed a night-vision video camera (wide-angle 8-LED IR DVR camcorder, DX, China) on the recording nest-box, approximately 10 cm above the nest. We recorded the behaviour of all family members during 30 min, excluding the first 30 and the last 10 min of the video to avoid possible interferences due to the presence of researchers when placing and removing the camera. A single observer analysed all video recordings and was unaware of the social density manipulation. For each feeding event, we registered whether the adult was marked or not and the nestlings’ begging intensity.  Begging intensity was rated on a 5-point scale following Kölliker et al. (1998): 0 = calm, 1 = weak gaping, 2 = gaping and neck stretched, 3 = gaping, neck stretched, and standing, and 4 = gaping, neck stretched, standing, and wing flapping. For each nestling, we calculated the average overall begging intensity across all feeding events directed to both parents together. Additionally, we obtained sex specific scores by averaging begging intensities for each parent separately. Also, for each parent, we extracted the number of feeding events and the average time spent in the nest.  We scored the social behaviour of the nestlings in 10 events between the parental visits, that is, when no parent was present (hereafter: parent-absent events). We considered that a parent-absent event started at least 5 seconds after the parents had left the nest, and as soon as all chicks stayed quiet and lasted until the parents returned. Nestlings change positions mainly during or soon after feeding events (personal observation), and thus we recorded nestling positions at a single time point immediately after each parent-absent event had started. To this end, the video was paused and each nestling was assigned an individual number. We considered a social interaction as the direct physical contact between two nestlings. With this information, we created an N x N matrix where N is the number of individuals, and each cell indicates the existence of an interaction between two individuals (1) or the absence of interaction (0). As a proxy of sociability, we calculated <em>network degree</em>, that is, the average number of physical interactions among all nestlings within a nest. <strong>Yolk hormone analysis</strong> On the day of hormone analyses, each egg was slowly defrosted to isolate the yolk, which was weighed with a high-precision scale to the nearest 0.001 g. Subsequently, each yolk was diluted (1:2 w/v) in double distilled water. We took half of the sample and extracted steroids by adding 3 ml of a mixture of petroleum and diethyl ether (30:70) to the diluted yolk sample, vortexed it for 10 min, and then centrifuged the suspension for 10 min (4 °C, 1500 rpm). The ether phase was decanted after snap-freezing the tube in an ethanol bath with dry ice. This procedure was repeated a second time. Both ether phases were combined in a single tube and evaporated to dryness in a heating block at 50 °C under a stream of nitrogen. To remove protein residues, the extract was resuspended in 0.5 ml of 90% ethanol, vigorously mixed, and kept at -20 °C overnight. On the following day, the extract was centrifuged for 10 min (4 °C, 1500 rpm), the supernatant was then decanted to a new clean tube, dried again in a heating block at 50 °C under a stream of nitrogen and subsequently resuspended in 0.5 ml of steroid-free serum (DRG Labs, Germany). After a 10-min vortexing, the samples were transferred to Eppendorf tubes and frozen at -20 °C until analysis. To measure testosterone concentrations, we used an enzyme immunoassay (EIA) according to protocol (Testosterone ELISA; ref. EIA-1559; DRG, Germany). A Synergy HT Multi-Mode Microplate Reader (Biotek, USA) at 450 nm was used to measure changes in absorbance according to EIA procedure. We multiplied the obtained concentrations by the yolk weight of each sample to calculate the absolute values contained in each egg. <strong>Molecular sexing of nestlings</strong> DNA was extracted from blood samples using the Qiagen DNeasy Blood and Tissue kit (Qiagen Inc, Valencia, CA, USA). Sex identification was performed by polymerase chain reaction (PCR) amplification of the CHD-W and CHD-Z genes with primers P2 and P8. An initial denaturizing step at 94 °C for 4.5 min was followed by 40 cycles of 94 °C during 30 s, 49 °C during 45 s and 72 °C during 45 s. A final run of 72 °C during 10 min completed the program. Amplification was carried out in a total volume of 10 µl. Each PCR sample contained 2 µl DNA, 0.08 µl Taq polymerase (TaKaRa BIO Inc, Japan), 0.8 µl dNTP 2.5 mM, 0.5 µl of each primer 10 µM, 1 µl of 10X PCR buffer, and 5 µl of sterilized distilled water. The sex of 2 chicks from 2 nests could not be determined due to unsuccessful DNA extraction, and were hence excluded from analyses that included nestling sex.
description: # Data from: Forecasting the future? Differential allocation of maternal
 hormones in function of experimentally manipulated social contexts in blue
 tits Cyanistes caeruleus ## Description of the data and file structure The
 data is divided into four different files: *
 eggData_ForecastingFuture.csv: here is the data related to the analyses of
 yolk hormones and other variables (including clutch size). *
 networkData_ForecastingFuture.csv: here is the data related to the
 analyses of the network degree. * nestlingData_ForecastingFuture.csv: here
 is the data related to the analyses of the begging intensity and body
 mass. * parentData_ForecastingFuture.csv: here is the data related with
 the analyses of the number of feeding events and the time spent in the
 nest. You can find a description of the variables below: * nest: ID of the
 nestbox. * treatment: social density manipulation. * 0: control. * 1: high
 density. * minDistSpk: minimun distance to the closest speaker in meters.
 * lDate: laying date of the clutch. * CS: clutch size (number of eggs). *
 weightE3: weight of the third egg in grams. * yolkWeight: weight of the
 yolk from the third egg in milligrams. * testo.conc: concentration of yolk
 testosterone in the third egg in picograms/milligrams. * testo.absVal:
 absolute quantity of yolk testosterone in the third egg (yolkWeight x
 testo.conc) in picograms. * BS: brood size. * dateVideo_julian: date of
 video recording in julian format. * networkDegree: average number of
 physical interactions in the nest. * brood_meanbeggingIntensity: mean
 begging intensity of the brood. * nestling: number assigned to each
 nestling in the nest. * ID: nestling ID in the database (nest_nestling). *
 sex.n: sex of the nestling. * dateRinging_julian: date the individual was
 ringed in julian format. * beggingIntensity: begging intensity of the
 individual. * sex.p: sex of the adult. * meanTimeInNest: mean time spent
 in the nest by the adult in seconds. * feedings: number of feeding events.
 In some columns there are cells with 'NA', indicating that the
 data is not available. This occurs in: * eggData_ForecastingFuture.csv: In
 the columns "weightE3", "yolkWeight" and
 "testo.absVal". This occurs in the row corresponding to nest
 206, which is an outlier for egg and yolk weight (and, consequently, for
 the absolute value of testosterone). We therefore decided not to include
 these data in the analysis. * nestlingData_ForecastingFuture.csv: * In the
 column  "sex.n": the sexing was unsuccessful for some nestlings.
 * In the columns "sex.p" and "beggingIntensity": some
 nests were not recorded. Thus, the data regarding begging behaviour is not
 available for these nestlings. **Contact** If you have any question,
 please, contact me. Alejandro García Antón
 [alejandro.garciaanton@uantwerp.be](mailto:alejandro.garciaanton@uantwerp.be) Departement Biologie Behavioural Ecology & Ecophysiology (BECO) Campus Drie Eiken - D.1.28 Universiteitsplein 1 2610 Antwerp (Wilrijk), Belgium [www.uantwerp.be](http://www.uantwerp.be)