Appendix 1
Telomere analysis. We used the telomere restriction fragment assay, a modified Southern blot that
visualizes terminal telomere repeat densities at all molecular weights (see figure below). We calculated
the average telomere length in the entire smear, according to a protocol detailed in Salomons et al. [1]
and Haussmann and Mauck [2]. Briefly, 5 µL of packed red blood cells, stored frozen in a glycerol
buffer, were extracted into agarose plugs using a kit (CHEF Genomic DNA Plug Kit, Bio-Rad). Plugs
were digested with a mixture of 3 U Hinf I, 15 U Hae III, and 40 U Rsa I (Roche Applied Science) in a
restriction enzyme buffer (NEBuffer 2, New England Biolabs). Half plugs were separated using
pulsed-field gel electrophoresis (CHEF Mapper, Bio-Rad) on a 0.8% agarose gel. Run parameters were
21 hours at 3 V/cm and 0.5 (initial) to 7 (final) s switch times. The circulating 0.5x TBE buffer was
kept at 14 °C. Gels were dried and then hybridized overnight at 37 °C with 3,000,000 cpm of the
telomere-specific 32P-labeled oligo (CCCTAA)4. After hybridization, rinsing and visualization
followed Haussmann and Mauck [2]. ImageJ software (version 1.46r) was used to extract telomere
smear densities. Lane-specific background was subtracted from each density value. Lower and upper
cut-off followed Salomons et al. [1]. Samples were analyzed in pairs (i.e. the 2011 sample of an
individual next to its 2012 sample), in random order on four gels, and control samples (from a large
sample of a standard individual used in all our lab's analyses) were run twice in each gel to determine
inter- (8.7%) and intra-assay variability (<3.6%).
Typical gel with telomere smears of
black-legged kittiwakes. Telomeres
were assayed with the telomere
restriction fragment method. Repeated
samples of an individual (2011, 2012)
were arranged on the gel side-by-side.
The molecular weight marker is the
central line of the gel. Two control
samples (i.e. lines 7 & 23, counting
from the left) were run on each gel to
control for inter- and intra-gel
variations.
Appendix 2
rank
1
2
3
4
5
6
7
8
9
10
11
Results of model selection, sorted by model rank, for effects of wintering time (days spent south of
70ºN), stress treatment during reproduction (stress hormone versus control implant), and sex (male
versus female) on change in telomere length (%) in black-legged kittiwakes. AICc = Akaike
Information Criterion value for finite sample size, ∆AICc = difference in AICc value between this and
the model with lowest AICc value, wi = AICc weights, i.e. ratio of AICc values for this relative to the
whole set of models.
wintering
wintering
time x
time x
wintering
stress
stress
wintering
stress
stress
time x
treatment x treatment x
intercept
time
treatment sex treatment
sex
sex
sex
AICc
∆AICc
x
x
x
x
81.21
0.00
x
x
x
81.91
0.70
x
x
x
x
x
84.99
3.78
x
x
x
x
x
87.37
6.16
x
x
88.29
7.08
x
x
x
89.03
7.82
x
x
94.73
13.52
x
x
x
94.87
13.66
x
x
95.05
13.84
x
95.44
14.23
x
x
x
x
x
x
x
x
117.78
36.57
Results of model averaging for models with ∆AICc < 2. Time spent at the wintering grounds was
scaled before averaging.
model term
model-averaged estimate
standard error
95% confidence intervals
intercept
-0.050
1.10
-2.21, 2.11
wintering time
3.56
0.88
1.85, 5.28
stress treatment
-4.38
1.19
-6.71, -2.05
sex
-3.55
1.57
-6.64, -0.46
wi
0.512
0.360
0.077
0.024
0.015
0.010
0.001
0.001
0.001
<0.001
<0.001
Appendix 3
Telomere loss in kittiwakes did not appear to reflect an increase in their oxidative stress levels (as
reflected in titers of reactive oxygen metabolites in plasma) in response to corticosterone treatment.
Thus, our empirical evidence currently does not support the hypothesized direct link between the
adrenocortical and oxidative stress [3].
We obtained plasma samples for the analysis of oxidative damage immediately before and
after the implantation experiment. Samples were kept on ice in the field and stored at -80 ºC within
hours, until further analysis. We measured oxidative damage through levels of reactive oxygen
metabolites using a kit (d-ROM, Diacron International), and following a protocol for bird plasma [4].
Briefly, 10 µL of plasma were diluted into 200 µL of chromogen-buffer-solution, and incubated at
37 ºC for 75 min. Absorbance was measured at 490 nm using a plate reader (SpectraMax
Plus384, Molecular Devices) and converted to concentrations (Carratelli Units, CARR U), using
the provided calibrator and the formula CARR U = absorbance sample/absorbance calibrator ×
concentration of calibrator. All samples were run in duplicate. Inter-plate and intra-plate CV were
calculated using the kit standard, and were 1.9% and 2.5%, respectively.
We observed no effect of glucocorticoid treatment on either total levels of oxidative damage
after implantation (F1,16 = 0.008, p = 0.931) or the change in oxidative damage from before to after
implantation (F1,15 = 0.096, p = 0.761).
Appendix 4
Results for the effects of migration timing on changes in telomere length (%). Timing of autumn
departure rather than spring arrival—defined as the date on which individuals crossed latitude 70ºN
south- and northbound, respectively—correlated with changes in telomere length. A later departure was
associated with higher telomere loss. The effect did not differ between stress treatments, as indicated
by the non-significant interaction between autumn departure and treatment. These results suggest that
autumn rather than spring conditions may be most influential on telomere dynamics in kittiwakes.
examined event
autumn departure
spring arrival
explanatory terms
autumn departure (date crossing 70ºN)
stress treatment
autumn departure × stress treatment
spring arrival (date crossing 70ºN)
stress treatment
spring arrival × stress treatment
estimate ± s.e.
-0.381 ± 0.143
-2.262 ± 6.022
-0.082 ± 0.203
0.325 ± 0.230
-3.750 ± 5.748
0.028 ± 0.325
F(d.f.)
9.058(1,11)
13.117(1,11)
0.164(1,11)
1.816(1,10)
3.101(1,10)
0.008(1,10)
p
0.012
0.004
0.693
0.208
0.109
0.933
Appendix 5
Raw data
animal id
6146199
6146538
6146542
6202402
6217946
6217949
6223753
6223908
6223910
6223911
6223914
6223916
6223917
6223918
stress treatment
corticosterone
corticosterone
control
corticosterone
control
corticosterone
control
corticosterone
control
corticosterone
corticosterone
control
control
corticosterone
wintering time (days)
157
174
189
198
169
166
174
179
153
173
178
165
168
180
sex
m
m
m
f
m
m
f
f
f
f
f
f
f
f
telomere length 2011
(basepairs)
6317.56
8630.85
7834.69
8203.76
7128.48
9805.32
9508.99
8314.11
7084.67
8211.09
9442.68
7631.93
7702.72
6951.70
telomere length 2012
(basepairs)
5456.09
8277.21
8138.01
8321.38
7284.49
8892.12
9681.73
8382.95
6912.34
7886.43
9370.51
7636.39
7936.40
7304.86
References for the electronic supplementary material
1.
Salomons H.M., Mulder G.A., van de Zande L., Haussmann M.F., Linskens M.H.K., Verhulst
S. 2009 Telomere shortening and survival in free-living corvids. Proc R Soc B 276(1670), 3157-3165.
(doi:10.1098/Rspb.2009.0517).
2.
Haussmann M.F., Mauck R.A. 2008 New strategies for telomere-based age estimation. Mol
Ecol Resour 8(2), 264-274. (doi:10.1111/j.1471-8286.2007.01973.x).
3.
Costantini D., Marasco V., Moller A.P. 2011 A meta-analysis of glucocorticoids as
modulators of oxidative stress in vertebrates. J Comp Physiol B 181(4), 447-456. (doi:10.1007/S00360011-0566-2).
4.
Haussmann M.F., Longenecker A.S., Marchetto N.M., Juliano S.A., Bowden R.M. 2012
Embryonic exposure to corticosterone modifies the juvenile stress response, oxidative stress and
telomere length. Proceedings of the Royal Society B-Biological Sciences 279(1732), 1447-1456.
(doi:10.1098/Rspb.2011.1913).