The age structure of populations is an important and often unresolved factor in ecology
and wildlife management. Parameters like onset of reproduction and senescence, reproductive
success and survival rate are tightly correlated with age. Unfortunately, age information of wild
animals is not easy to obtain, especially for birds, where few anatomical markers of age exist.
Longitudinal age data from birds banded as chicks are rare, particularly in long lived species.
Age estimation in such species would be extremely useful as their long life span typically
indicates slow population growth and potentially the need for protection and conservation.
Telomere length change has been suggested as a universal marker for ageing vertebrates
and potentially other animals. This method, termed molecular ageing, is based on a shortening of
telomeres with each cell division. In birds, the telomere length of erythrocytes has been reported
to decline with age, as the founder cells (haematopoietic stem cells) divide to renew circulating
red blood cells. I measured telomere length in kakapo, the world largest parrot and four other
bird species (Buller’s albatross, kea, New Zealand robin and saddleback) using telomere
restriction fragment analysis (TRF) to assess the potential for molecular ageing in these species.
After providing an overview of methods to measure telomere length, I describe how one
of them (TRF) measures telomere length by quantifying the size distribution of terminal
restriction fragments using southern blot of in-gel hybridization (Chapter 2). Although TRF is
currently the ‘gold standard’ to measure telomere length, it suffers from various technical
problems that can compromise precision and accuracy of telomere length estimation. In addition,
there are many variations of the protocol, complicating comparisons between publications. I
focused on TRF analysis using a non-radioactive probe, because it does not require special
precautions associated with handling and disposing of radioactive material and therefore is more
suitable for ecology laboratories that typically do not have a strong molecular biology
infrastructure. However, most of my findings can be applied to both, radioactive and nonradioactive
TRF variants. I tested how sample storage, choice of restriction enzyme, gel
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electrophoresis and choice of hybridization buffer can influence the results. Finally, I show how
image analysis (e.g. background correction, gel calibration, formula to calculate telomere length
and the analysis window) can not only change the magnitude of estimated telomere length, but
also their correlation to each other. Based on these findings, I present and discuss an extensive
list of methodological difficulties associated with TRF and present a protocol to obtain reliable
and reproducible results.
Using this optimized protocol, I then measured telomere length of 68 kakapo (Chapter 3).
Almost half of the current kakapo population consists of birds that were captured as adults,
hence only their minimum age is known (i.e. time from when they were found +5 years to reach
adulthood). Although molecular ageing might not be able to predict chronological age
accurately, as calibrated with minimum age of some birds, it should be able to compare relative
age between birds. Recently, the oldest kakapo (Richard Henry) was found to show signs of
reproductive senescence. The age (or telomere length) difference to Richard Henry could have
been used to approximate the remaining reproductive time span for other birds. Unfortunately,
there was no change of telomere length with age in cross sectional and longitudinal samples.
Analysis of fitness data available for kakapo yielded correlations between telomere length
and fledging success, but they were weak and disappeared when the most influential bird was
excluded from analysis. The heavy management and small numbers of kakapo make conclusions
about fitness and telomere length difficult and highly speculative. However, telomere length of
mothers and their chicks were significantly correlated, a phenomena not previously observed in
any bird.
To test if the lack of telomere loss with age is specific to kakapo, I measured telomere
length of one of its closest relatives: the kea (Chapter 4). Like kakapo, telomere length did not
show any correlation with age. I then further assessed the usefulness of molecular ageing in birds
using only chicks and very old birds to estimate the maximum TL range in an additional long
lived (Buller’s albatross) and two shorter lived species (NZ robin and saddleback). In these
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species, telomere length was on average higher in chicks than in adults. However, age matched
individuals showed high variations in telomere length, such that age dependent and independent
telomere length could not be distinguished. These data and published results from other bird
species, coupled with the limitations of methodology I have identified (Chapter 2), indicate that
molecular ageing does not work in most (if not all) birds in its current suggested form.
Another way to measure telomere length is telomere Q-PCR, a real-time PCR based
method. Measurement of the same kakapo samples with TRF and Q-PCR did not result in
comparable results (Chapter 4). Through experimentation I found that differences in
amplification efficiency between samples lead to unreliable estimation of telomere length using
telomere Q-PCR. These differences were caused by inhibitors present in the samples.
The problem of differential amplification efficiency in Q-PCR, while known, is largely
ignored by the scientific community. Although some methods have been suggested to correct for
differing efficiency, most of these introduce more error than they eliminate. I developed and
applied an assay based on internal standard oligonucleotides that was able to corrected EDTA
induced quantification errors of up to 70% with high precision and accuracy (Chapter 5). The
method, however, failed when tested with other inhibitors commonly found in DNA samples
extracted from blood (i.e. SDS, heparin, urea and FeCl3). PCR inhibition was highly selective in
the probe-polymerase system I used, inhibiting amplification of genomic DNA, but not
amplification of internal oligonucleotide or plasmid standards in the same reaction. Internal
standards are a key feature of most diagnostic PCR assays to identify false negatives arising from
amplification inhibition. The differential response to inhibition I identified greatly compromises
the accuracy of these assays. Consequently, I strongly recommend that researchers using PCR
assays with internal standards should verify that the target DNA and internal standard actually
respond similarly to common inhibitors.
| Identifer | oai:union.ndltd.org:canterbury.ac.nz/oai:ir.canterbury.ac.nz:10092/3329 |
| Date | January 2008 |
| Creators | Horn, Thorsten |
| Publisher | University of Canterbury. School of Biological Sciences |
| Source Sets | University of Canterbury |
| Language | English |
| Detected Language | English |
| Type | Electronic thesis or dissertation, Text |
| Rights | Copyright Thorsten Horn, http://library.canterbury.ac.nz/thesis/etheses_copyright.shtml |
| Relation | NZCU |
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