Supplementary material
Study area and populations
300 nest boxes have been installed in deciduous forests in the surroundings of the
University of Lausanne (Switzerland) where we have breeding populations of Starlings and
Great tits. The nest boxes are made of an internal wooden box with a thick and opaque plastic
cover (Heeb et al. 2003). From early March 2003 onwards, the breeding activity of the birds
was followed every day from nest building onwards. For each breeding attempt, the date of
laying of the first egg, clutch size and date of hatching was recorded. The growth of the
nestlings was followed from hatching to fledging from the nest. A second starling population
breeding in southern England was used to provide independent measurements of reflectance
spectra and nest irradiance.
Measures of flange and skin reflectance
For both species in Switzerland, reflectance spectra (280 – 700 nm) were recorded on
six day old nestlings using an S2000 spectrophotometer, with a DH-2000 deuterium-halogen
light source and 2.5 mm diameter coaxial reflectance probe, and the OOIBase32 TM
spectrophotometer operating software (Ocean Optics, Inc., Dunedin, FL, USA). Gapes were
gently held open while the probe was held perpendicularly at a fixed distance to a tangent to
the surface of the flange. The strobe illuminated an area of about 1.5 mm in diameter. The
reflectance was calculated relative to a 99% white Spectralon reflection standard sampled
every eight scans. For each Starling nestling we collected a total of eight spectra on the upper
and lower flanges without any treatment and eight spectra with either +UV or UV treatment
(see below). We also measured one spectrum of the skin of the back of the body, one
spectrum where the legs connect to the body and one spectrum on the forehead between the
eyes (we measured each of these spectra again for the +UV and UV treatments). The number
of spectra measured for the curves shown in Fig. 1ab are: natural flanges: 21 individuals, 168
spectra; flanges –UV: 12 individuals, 92 spectra; flanges + UV: 9 individuals, 69 spectra; skin
natural: 21 individuals, 54 spectra; skin –UV: 10 individuals, 29 spectra; skin +UV: 7
individuals, 20 spectra.
Spectra obtained from nestlings in the English Starling population were measured by
using an Ocean optics PX-2 light source and by holding the probe onto the sample. The
irradiance spectra in the nest environment was measured in 5 nests (4 spectra per nest, Fig
1e). Spectra for nestling flanges were measured in 5 nests (n=10 chicks, 4 spectra per chicks
aged 3-5 days old). Body skin of chicks was measured on the back of head, body back and
throat (n=3 nests, n=6 chicks, 4 spectra per chick per region). Methods used for measurement
of these spectra and irradiance are reported in Hunt et al (2003).
For Great tits, we measured for each nestling a total of four spectra of the upper and
lower flanges without any treatment and four spectra with either +UV or UV treatment, (Fig
1cd). We measured one spectrum of the throat skin and one spectrum of the skin above a hind
leg with no treatment (flanges: n = 25 individuals, 109 spectra; body skin: n = 22, 39
spectra); +UV (n = 10, 42 spectra); -UV (n = 10, 42 spectra).
For the Swiss data we calculated the median of the spectra values and the standard
deviation every 20 m. During the measurements we removed nestling from the nest and
kept them in warmed boxes. We always made sure that at least one or two nestlings remained
in the nest. Before replacing the young back in the nest they were fed ad libitum with pieces
of mealworms.
Calculation of the reflectance peaks on the skin
We measured immune responses in a number of Starling nestlings and calculated the
median of their spectra of flanges and body skin. From this median spectra we obtained for
flanges and body skin the median reflectance values between 320 and 360 nm (M1), between
440 and 480 nm (M2) and between 540 and 700 nm (M3). M1 represents the median maximal
value of UV reflectance, M2 gives the median baseline reflectance value, and M3 stands for
the median reflectance in the visible spectrum. For each nestling peak flange reflectance,
illustrating spectral brightness was obtained as (M1 – M2) + (M3 – M2). To this value we
added (M1 – M3) for the body skin and used the final sum to examine the relationship
between brightness of skin reflectance and morphological traits (see below).
Modifications of flange and skin reflectance
We carried out one experiment with six days old Starling nestlings from the second
broods. We removed the young from the nest and had them defecate before measuring their
body mass to the nearest 0.1 g with a portable "Sartorius" electronic balance. After two hours
of experiment we measured again body mass of nestlings. Within each brood, we randomly
assigned nestlings in a group that reflected in the UV (+UV) and in a second group where
flange and body skin UV reflectance was specifically reduced (-UV). Individuals were
individually marked by plucking down feathers on the head and wings and ranked according
to their relative mass within the brood. After ranking the nestlings in relation to their body
mass, they were assigned alternatively to one of the two treatments (+UV or -UV) in each
brood order to obtain similar overall mean body masses between treatments. The order of the
treatments was randomized between broods, thus ensuring that heaviest nestlings appeared as
often in the two treatments. In this experiment the body and mouth flanges were covered with
a small quantity of petroleum jelly (+UV) or petroleum jelly with UV protection (-UV). A
total of 40 nestlings in 12 broods (21 in -UV and 19 in +UV), were tested in this experiment.
Nestlings in the two groups did not differ in their body mass before the experiment (t-test: t =
0.1, DF = 38, P = 0.92) and they significantly gained mass during the experiment (mass gain:
1.05 ± 0.19 (g ± se), n = 40; t-test = 5.61, P = 0.0005).
For the +UV treatment, we spread a small quantity of petroleum jelly (Petroleum:
93.95%, Cetiol B: 6%, BHT: 0.05%) with a cotton swab on both inner and outer sides of the
flanges and on the whole of their body. For the -UV treatment we blocked UV reflectance by
spreading on the body and mouth flanges petroleum jelly mixed with UV protection
(Petroleum jelly: 79.95%, Cetiol B: 6%, BHT: 0.05%, Parsol 1789: 3%, Parsol MCX: 6%,
Eusolex OS: 5%; Roche). In both treatments, the excess product was gently removed from
the skin with a clean paper tissue. Spectra in Figures 1a-c show that our -UV treatment
reduced skin reflectance between 280 and 400 nm whilst the +UV treatment allowed for
reflectance in these wavelengths. UV blocking substances used by Roche have been chosen
for their neutral effect on vertebrate skin. However, any potential effect of petroleum jelly
applied on the skin on nestling begging performance is unknown.
An examination of spectra for a number of nestlings revealed that after 180 minutes, the
differences between nestlings in the -UV and +UV treatments were still present on the
flanges. However, the body skin had reduced UV reflectance presumably due to close body
contact between nestlings in the brood, and between nestlings and the nest.
We performed a second experiment when Starling nestlings of the first brood and
Great tit broods were six days old. In this experiment petroleum jelly was only applied to
mouth flanges. We followed the same procedure as described above with a difference in that
the larger brood sizes in Great tits allowed us to assign young in a third treatment where the
young were handled in the same way but where petroleum jelly was not applied and thus
acted as a second control. A total of 103 Starling nestlings from 27 broods and 333 Great tits
in 53 broods were included in this experiment. There were no significant differences in initial
body mass between Starlings in the two treatments (F1,101 = 0.04, P = 0.84). Nestlings
significantly gained mass during the two hour interval of the experiment (mean mass gain:
1.33 ± 0.17 (g ± se), n = 103 nestlings; t-test = 7.93, P < 0.0001). In Great tits, there were no
significant differences in initial body mass between nestlings in the three treatments (F2,330 =
0.084, P = 0.92). Nestlings significantly gained mass during the two hour interval of the
experiment (mass gain: 0.29 ± 0.016, n = 333 nestlings; t-test = 17.7, P < 0.0001).
In all experiments, immediately after applying the petroleum jelly, all the young were
replaced in the nests for 120  2 minutes. After this time interval, the young were taken out of
the nest and weighed again to the nearest 0.1 g in order to determine individual changes in
body mass (see below).
Assessment of nestling immunocompetence
The immune response of 14 day old Starling nestlings towards phytohaemaglutinin
(PHA) was tested. Nestlings were subcutaneously injected in the right wing web with 0.1 mg
of PHA dissolved in 0.02ml of phosphate-buffered saline (PBS). The thickness of the wing
web was measured at the injection site with a pressure-sensitive micrometer (Mitutoyo model
2046F, accuracy 0.01 mm) before and 24 h (± 1h) after the injection. A control injection of
PBS in the left wing was not done as this has previously been shown to be unnecessary (Smits
et al. 1999). We estimated the T-cell-mediated immune response or wing web index as the
change in thickness of the right wing web (Brinkhof et al. 1999). Nestling immune response
was not significantly correlated with their body mass (F1,13= 1.22, P = 0.29) or with their
tarsus length (F1,13= 1.47, P = 0.25).
Statistical analyses
For each brood, we calculated the mean mass gained by nestlings in the brood (sum of
all individual masses gained divided by brood size). We then calculated for each nestling the
difference in mass gain from the brood mean mass gain. These calculations allowed us to
correct for inter-brood variation in total brood mass gained and use the value of each young as
a data point in a population of mass deviations around a mean of zero mass gains (for the
three experiments P > 0.60). In the analyses we included the effect of nest as a random effect
and present the effect of treatment as a factor on individual relative mass gain. All analyses
were made with JMP (Sall & Lehmann 1996).
Ethical note
The experiments were carried out according to local regulations and had no detectable
long term effect on the nestlings. We found no significant difference in mass difference
between days 6 and 14 in nestlings in the different treatments.
References
Brinkhof, M.G.W., Heeb, P., Kölliker, M., & Richner, H. 1999. Immunocompetence of
nestling great tits in relation to rearing environment and parentage. Proc. Roy. Soc.
Lond. 266, 2315 – 2322.
Sall, J. & Lehman, A. 1996. JMP Start Statistics. A guide to statistics and data analysis using
JMP and JMP IN software. Belmont, California: Duxbury Press.
Smith, J. E. et al. 1999. Simplifying the phytohemaglutinin skin testing technique in studies of
avian immunocompetence. Funct. Ecol. 13: 567-577.