SUPPLEMENTARY MATERIAL - Prenatal environmental effects match offspring begging to parental provisioning
DETAILED METHODS
Hormonal correlates of plasticity
Collection of female faecal samples during laying
After pairing, 3-5 faecal samples were collected from females each morning until the third egg had been laid. The cage’s metal barrier was reinserted, temporarily isolating the female over a plastic sheet for up to 25 min until she produced roughly 1cm3 faeces, which was immediately frozen for subsequent analysis (see below for details). For statistical analysis, we took the mean of the three daily faecal metabolite measures collected before the day each egg was laid, just as in Schwabl (1996a), and then derived the mean for the first three eggs in the clutch.
Testosterone metabolite and plasma analyses
Faecal samples were extracted in ethanol and assayed using direct radioimmunoassay with anti-testosterone serum (see Buchanan et al. 2007). The nestling samples were run in two assays and the breeding female samples were run in four assays. The interassay coefficient of variation (CV) was 22.4% calculated from four pooled faecal samples run twice in three assays and once in three assays and 10.1% calculated from six pooled plasma samples run in six assays. The mean 50% binding was 6.23 pg/tube, and the mean detection limit for the three assays was 0.012 ng/ml for duplicate 20µl aliquots of reconstituted extract.
Nestling begging intensity
Five days after hatching, nestlings were temporarily transferred from their nest in the breeding room to an identical, heated nest in the testing room. After food deprivation for 40 min, they were fed 0.50 ml Nectarblend rearing mix, mixed to a standard concentration (6.00 g + 15.0 ml warm tap water) and dispensed through plastic syringes. Each nestling was then each transferred to its own heated nest, housed within a testing box, which supported a videocamera overhead, was illuminated with a 12V halogen light bulb, and offered access to the nestling through a sleeve on the side of the box (further details in Kilner & Davies 1998). At 40 and 80 min after feeding, nestlings were induced to beg with a standard tapping stimulus and their behaviour was filmed. After filming at 80 min, nestlings were fed to satiation with Nectarblend rearing mix and returned to their nest, where their (foster) parents, meanwhile, had been tending the rest of the brood. From the videotapes, blind to information about the chick’s true parentage, we quantified postural begging intensity, an estimate of begging vigour that increases with nestling hunger (Kilner 1995), predicts parental provisioning behaviour (Kilner 1995; Kilner 2002) and has associated costs (Kilner 2001). Each second of the begging trial, for the first 12 s, we paused the videotape and described begging intensity at that moment with an ordinal posture rank (from 0 (weak) to 4 (vigorous), details of ranking in Kilner 2001). The ranks were then summed to derive an overall measure of begging vigour, ranging from 0 to 48.