DISCUSSION
Our study is the first investigation of the relationship between
telomere length and DNAm dynamics in wild animals exposed to naturally
variable early life conditions. In our longitudinal analysis we found a
negative relationship between individual changes in telomere length and
changes in the level of methylated CpG loci (92 CpG loci in our study)
across early life, such that individuals with accelerated rates of
telomere shortening were associated with increases in DNAm levels. The
observational nature of our field data means that we were unable to
establish whether the negative relationship between telomere length and
DNAm changes reflected coincidental or causal effects. That is, whether
DNAm and telomere length respond comparably to the same
intrinsic/extrinsic conditions experienced across early life and/or
whether potential regulatory effects between DNAm and telomere length
may exist. How changes in telomere length and DNAm relate to each other
and respond to early life conditions warrants further investigation
since it may help identify potentially distinct aspects of biological
age that DNAm and telomere length reflect. It is important to note that
while variation in DNAm levels may reflect variation in the early life
environment (Angers, Castonguay, & Massicotte, 2010; Sheldon et al.,
2018; 2020; Makinen, van Oers, Eeva, Laine & Ruuskanen, 2020), the
relevance of early-life, ‘genome-wide’ hypo or hyper methylation in the
context of biological age remains unclear. Thus, while our MS-AFLP
analyses are useful to compare DNAm and telomere length associations in
different environments, next-generation sequencing data (used to
generate an ‘epigenetic clock’) are necessary to compare DNAm and
telomere length predictors of biological age in different environments
(Horvath & Raj 2018; Banszerus et al., 2019). We did not detect a
relationship between cross-sectional measures of DNAm and telomere
length at either day 3 or day 11 post-hatch. However, telomere length at
day 3 was weakly, but significantly positively associated with clutch
size. Given this relationship was positive rather than negative, it
suggests that the within clutch decline in telomere length across the
laying order that has been seen in other studies did not have much
effect here. The positive relationship could perhaps be due to parental
age if older parents had a reduced clutch size and their offspring
inherited shorter telomere lengths or lost more up to this point
(Monaghan and Metcalfe, 2019).
Evidence is accumulating to suggest that longitudinal measures of
within-individual change in telomere length may represent a more useful
biomarker of ageing/individual condition than static, cross-sectional
telomere length measures (Wood and Young, 2019; Boonekamp et al., 2014;
Tricola et al., 2018; Wilbourn et al., 2018. However, to our knowledge,
no other study has conducted longitudinal analyses on the relationship
between within-individual change in telomere length and DNAm across
early development, in any species. Although it may be expected that
factors associated with telomere length would also be associated with
telomere length changes , telomere length at a given timepoint is
the outcome of both initial telomere length and subsequent
attrition/elongation, which may not be associated. Indeed, in our
supplementary analysis (Supplementary Analysis 2) the initial length of
telomeres (at day 3) did not affect the rate of telomere length change
across early life after controlling for effects of regression to the
mean (Verhulst et al., 2013) (however, other studies have detected this
trend (Salomons et al., 2009; Aviv et al., 2009)). Hence, variation in
initial telomere length, which may be more influenced by heritable
genetic factors (Broer et al., 2013, but see Voillemot et al., 2012),
may confound the relationship between environmental factors associated
with telomere length versus telomere length change , and indeed,
may confound the accuracy of telomere length as a biomarker of
individual condition later in life (Boonekampp et al., 2014; Wood &
Young 2019). In line with this, in our study we found that genetic
and/or familial effects (represented by nest ID) explained the greatest
amount of variance in our cross-sectional telomere length measures at
both day 3 and day 11, while telomere length change was better
described by DNAm changes and growth rate, factors that are extremely
sensitive to the environment.
Contrary to other studies (Stier et al., 2020; Fitzpatrick et al.,
2019), we did not find an effect of ambient temperature on telomere
length or telomere length changes across development. Temperature has
been shown to effect within individual changes in DNAm (Sheldon et al.,
2020) and telomere length (Stier et al 2020; Fitzpatrick et al 2019)
however, it is unclear if temperature effects these DNA modifications
comparably. The temperatures (average daily maximum of
27.6oC) in our study may not have been ‘stressful’
enough to impact telomere dynamics, and indeed temperature has not
affected telomere length in studies on other taxa (McLennan et
al., 2018; Boonekamp et al, 2020).
In our longitudinal analyses, we also detected a significant, negative
relationship between tarsus growth and change in telomere length, such
that individuals that grew faster lost more telomere length across the
measured period of their early life. This relationship has been detected
in previous studies, and could be due to increases in cell division,
energy expenditure and oxidative stress associated with increased growth
rate (Monaghan & Ozanne, 2018). The relationship between telomere
length and tarsus growth occurred independently of temperature in our
study. However, previous studies have shown that embryonic growth rate,
manipulated by small variations (+-1oC) in incubation
temperature, negatively affect telomere length in the laboratory (Stier
et al., 2020). We did not detect a relationship between cross-sectional
measures of telomere length and tarsus length in our attempt to explore
proximate effects within individuals. However, in a study examining the
same relationship (between telomere length and tarsus length) at an
ultimate level, across generations, in a selection experiment on body
size in the house sparrow, a relationship was detected (Ringsby et al.,
2015).
In our cross-sectional analysis we detected a positive correlation
between telomere length at day 3 and clutch size. We have previously
shown that DNAm was affected by brood size during early life of wild
zebra finches (Sheldon et al., 2018), and experimental work has also
shown that telomeres are generally shorter when brood size is increased
during early life (Voillemot et al., 2012; Reichert et al.,
2014), potentially as a response to the stress of lower per capita food
delivery rates to offspring (Costanzo et al., 2016). The positive
relationship detected in our analysis is thus interesting and may
reflect higher quality parental care (Bichet et al., 2020), lower rates
of nestling/egg mortality, or optimal environmental factors among chicks
developing in larger (unmanipulated) clutches (Van Noordwijk & de Jong,
1986).
In our study, telomere length was found to increase as well as decrease
from day 3 to day 11 of post-hatch development. Increasing evidence from
a range of avian taxa suggests this could be a somewhat common
occurrence (Wood & Young, 2019; Kotrschal et al., 2007; Young et al.,
2013;
Uivari
& Madsen, 2009; Haussmann & Mauck, 2008) possibly due to the actions
of telomerase (Haussmann, Winkler, Huntington, Nisbet, & Vleck, 2004;
2007; Jaskelioff et al., 2011; Bateson & Nettle, 2017; Hoelzl et al.,
2016). The extent to which mechanisms underlying telomere length
elongation may weaken the utility of within‐individual telomere length
variation and, indeed, the utility of telomere length as a biomarker of
biological ageing, is unclear, as telomere repair by telomerase may
itself be inhibited by the same factors effecting telomere length (e.g.
oxidative damage; Ahmed et al. 2008).
Our measurements of absolute telomere length in wild zebra finches gave
telomere lengths similar to those in captive birds measured by the same
in-gel TRF method; measurements in the same lab and using the same
equipment and protocol for laboratory birds (varying in age from 7 to
120 days) ranged from 10.1 to 16.35kb (Millet & Salmon, pers comm).
These data suggest that, in contrast to the laboratory mouse which
typically has telomeres several times longer than its wild counterparts
possibly due to inbreeding (Manning et al 2002), but this is not the
case for the zebra finch.
In conclusion, our study of wild zebra finches has confirmed previously
documented effects in captive zebra finches of both growth rate and
clutch size on telomere length dynamics during early life. This
consistency is important given the differences in resources and
variability in the environment of wild compared to captive animals. Most
significantly, our study detected a negative relationship between early
life telomere and DNAm dynamics, such that individuals with accelerated
rates of telomere shortening have associated increases in DNAm levels.
The nature of this relationship warrants further investigation since it
may shed light on potentially clinically/ecologically distinct aspects
of biological age reflected by DNAm and telomere length. It would be
valuable for future work to focus on next-generation sequencing
techniques (e.g., reduced representative bisulfite sequencing (RRBS)) to
compare DNAm and telomere length measures of biological age across
different environments and/or to compare potentially functional relevant
or regulatory associations between DNAm and telomere length across
different environments. Our results provide a base for further
investigations to link and/or disentangle the relationship between the
early life environment and DNA biomarkers of biological age.