Field work and blood sampling
Fieldwork was conducted at Fowlers Gap Arid Research Station, in
far-western New South Wales (31o05’S,
‘141o42’E) during the main part of the Austral
breeding season (August–December) in 2016. Data were collected from
zebra finch nestlings in nest boxes in their natural habitat at ‘Fowlers
Gap’ (details regarding field site characteristics can be found in
Griffith, Pryke, & Mariette, 2008). Given the mobility of the species
in the wild, we were working with a population in which most adults were
not banded, and we were unable to ascertain parents’ age or reproductive
history. Nest boxes were monitored periodically (every 2 days) during
the nest-building stage, and after the first egg was laid nest boxes
were monitored each morning until the entire clutch was laid, enabling
us to ascertain clutch size and projected hatch date. In the wild, we
have previously shown that parent zebra finches only initiate incubation
on the day that the last egg is laid (Gilby, Mainwaring, & Griffith,
2013). Consequently, we used the day of the last laid egg to represent
the first day of embryonic development and the usual incubation period
of ~12 days after the onset of incubation, to predict
clutch hatch date. Two days prior to a clutch’s estimated hatch date, we
monitored the nest three times per day between 06.00 and 17.00; our
first nest check was at ~06.00, our second nest check
was at ~11.30 and our third nest check was at
~17.00, which allowed us to identify the hatch date of
each nestling. The hatch date was used to calculate the post-hatch age
of each nestling (in days). Synchronous hatching meant that we could not
tie nestlings to specific eggs, we were unable to account for the order
in which eggs/nestlings were produced, something that has been shown to
impact telomere dynamics in the zebra finch (Noguera, Metcalfe, Reichert
& Monaghan, 2016).
To measure ambient developmental temperature, we obtained hourly
atmospheric temperature data in the shade (i.e. air temperature) from
the Australian Bureau of Meteorology’s automated weather station at
Fowlers Gap, located within 20km of the study site. We calculated the
mean temperature during each day of zebra finch development using hourly
data from 7am to 7pm. These data represent the period of the day when
the adults are periodically away from the nest foraging together
(Mariette & Griffith 2015), and the nestlings (or embryos) are
therefore subject to the greatest exposure to ambient (rather than
brooding/ incubation) temperatures; the duration of nestling exposure to
ambient temperature increases as the nestlings grow, and parents reduce
brooding. The daily temperature values were then averaged from day 3 to
day 11 of post-hatch development (prior to day 3, the ectothermic
nestlings are typically brooded or incubated for long periods during the
day, thus are not exposed to ambient temperatures, day 3 was also the
age at which the earliest blood sample was taken, see below). During our
sampling period (Aug-Nov), average temperatures across post-hatch
development ranged from 14.6- 27.6oC (average
20.3oC, SEM: 0.295). At day 3 a small patch of down
feathers was trimmed from a different area on each nestling allowing us
to identify individual nestlings throughout development. Trimming the
small patch of hair (approximately 0.5cm2) from each
nestling was unlikely to impose thermoregulatory costs.
At day 3 and day 11, we extracted blood (<20µl) from the
metatarsal vein of younger (day 3) nestlings, and the brachial vein of
older (day 11) nestlings with a hypodermic needle and capillary tube.
Blood was preserved in 0.5ml of 95% ethanol and stored at room
temperature in an Eppendorf tube that was labelled with a unique ID.
Growth rate (a measure of cell proliferation) can affect telomere
dynamics (Boonekamp et al., 2020; Ringsby et al., 2015). To account for
variation in growth rate between individual nestlings and broods we
measured tarsus length (a reliable metric of body size) at day 3 and day
11 using digital calipers. The associated effects of clutch and brood
size (i.e. parental care and resource acquisition) can also affect
telomere length (Costanzo et al., 2016; Boonekamp et al., 2014) and DNAm
changes (Sheldon et al., 2018), thus, we included clutch and brood size
in our model. We collected data on the clutch size of each nest daily
from the day of first lay to the onset of incubation, and on the two
days prior to the estimated day of hatch (clutch size ranged from 2 to
11 eggs). We collected data on the brood size of each nest on the day
the entire clutch hatched, day 3, and day 11 of post-hatch development
(brood size ranged from 2 to 8 nestlings). Due to egg/nestling
mortality, clutch/brood size often varied across development, thus we
averaged the clutch/brood size of each nest across the two time-points.