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Accurate Epigenetic Aging in Bottlenose Dolphins (Tursiops truncatus), an Essential Step in the Conservation of at-Risk Dolphins

Authors:

Abstract

Epigenetics, specifically DNA methylation, allows for the estimation of animal age from blood or remotely sampled skin. This multi-tissue epigenetic age estimation clock uses 110 longitudinal samples from 34 Navy bottlenose dolphins (Tursiops truncatus), identifying 195 cytosine-phosphate-guanine sites associated with chronological aging via cross-validation with one individual left out in each fold (R2 = 0.95). With a median absolute error of 2.5 years, this clock improves age estimation capacity in wild dolphins, helping conservation efforts and enabling a better understanding of population demographics.
Brief Report
Accurate Epigenetic Aging in Bottlenose Dolphins
(Tursiops truncatus), an Essential Step in the Conservation of
at-Risk Dolphins
Ashley Barratclough 1, *, Cynthia R. Smith 1, Forrest M. Gomez 1, Theoni Photopoulou 2, Ryan Takeshita 1,
Enrico Pirotta 3,4 , Len Thomas 2, Abby M. McClain 1, Celeste Parry 1, Joseph A. Zoller 5,6 , Steve Horvath 5,6
and Lori H. Schwacke 1


Citation: Barratclough, A.; Smith,
C.R.; Gomez, F.M.; Photopoulou, T.;
Takeshita, R.; Pirotta, E.; Thomas, L.;
McClain, A.M.; Parry, C.; Zoller, J.A.;
et al. Accurate Epigenetic Aging in
Bottlenose Dolphins
(Tursiops truncatus), an Essential Step
in the Conservation of at-Risk
Dolphins. J. Zool. Bot. Gard. 2021,2,
416–420. https://doi.org/10.3390/
jzbg2030030
Academic Editor: Lance Miller
Received: 2 July 2021
Accepted: 27 July 2021
Published: 6 August 2021
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4.0/).
1Conservation Medicine Division, National Marine Mammal Foundation, San Diego, CA 92106, USA;
cynthia.smith@nmmf.org (C.R.S.); forrest.gomez@nmmf.org (F.M.G.); ryan.takeshita@nmmf.org (R.T.);
abby.mcclain@nmmf.org (A.M.M.); celeste.parry@nmmf.org (C.P.); lori.schwacke@nmmf.org (L.H.S.)
2Centre for Research into Ecological and Environmental Modelling, University of St Andrews,
St Andrews KY16 9LZ2, UK; tp14@st-andrews.ac.uk (T.P.); len.thomas@st-andrews.ac.uk (L.T.)
3Department of Mathematics and Statistics, Washington State University, Vancouver, WA 98686, USA;
pirotta.enrico@gmail.com
4School of Biological, Earth and Environmental Sciences, University College Cork, T23 N73K Cork, Ireland
5Department of Biostatistics, Fielding School of Public Health, University of
California, Los Angeles, CA 90095, USA; jaz18@g.ucla.edu (J.A.Z.); SHorvath@mednet.ucla.edu (S.H.)
6Department of Human Genetics, David Geffen School of Medicine, University of California,
Los Angeles, CA 90095, USA
*Correspondence: ashley.barratclough@nmmf.org
Abstract:
Epigenetics, specifically DNA methylation, allows for the estimation of animal age from
blood or remotely sampled skin. This multi-tissue epigenetic age estimation clock uses
110 longitudinal
samples from 34 Navy bottlenose dolphins (Tursiops truncatus), identifying
195 cytosine
-phosphate-
guanine sites associated with chronological aging via cross-validation with one individual left out in
each fold (R
2
= 0.95). With a median absolute error of 2.5 years, this clock improves age estimation
capacity in wild dolphins, helping conservation efforts and enabling a better understanding of
population demographics.
Keywords: DNA methylation; epigenetics; aging; bottlenose dolphin; chronological age
1. Introduction
Determining chronological age in cetaceans is an ongoing challenge, with knowledge
of age being critical to interpreting biological data, understanding population demograph-
ics, and predicting survival. Previous age estimation methodologies have involved invasive
tooth extraction for growth layer analysis, morphometrics, and pectoral flipper radiogra-
phy [
1
,
2
]. These methods require physical examinations and vary in accuracy according
to age demographic. Interdisciplinary approaches to conservation medicine, through the
application of tools developed in human medicine and applied to marine mammals, are
needed to accurately expand the capacity of aging. Epigenetic aging is a novel technol-
ogy utilizing DNA methylation patterns as biomarkers of age [
3
]. DNA methylation is
the addition of methyl groups CH
3
to cytosine–phosphate–guanine sites (CpG sites) in
the DNA sequence. In humans, analysis of the CpG site modification demonstrated a
close correlation with chronological age; therefore, analysis of epigenetic modifications
can provide an estimation of age [
3
,
4
]. Epigenetic age acceleration occurs when the es-
timated age exceeds the chronological age [
5
]. Exploring DNA methylation in humans
has identified drivers of epigenetic age acceleration including environmental stressors,
lifestyle and disease. The initial application for chronological age estimation of bottlenose
dolphins (Tursiops truncatus) focused on two cytosine–phosphate–guanine sites (CpG) (of
J. Zool. Bot. Gard. 2021,2, 416–420. https://doi.org/10.3390/jzbg2030030 https://www.mdpi.com/journal/jzbg
J. Zool. Bot. Gard. 2021,2417
17 screened) producing an R
2
of 0.74 and a root mean squared error of 5.14 years when
estimating chronological age [
6
]. Similarly, for nine odontocete species combined, a median
absolute age prediction error of 2.57 years was produced using 142 CpG sites [
7
]. Creating
species-specific epigenetic clocks with a wide range of known age animals improves the
accuracy of age estimation [
8
]. The present study stands out in its use of a longitudinal
dataset from the U.S. Navy Marine Mammal Program (Navy). Since 1959, the Navy has
expanded knowledge in bottlenose dolphin health and physiology [
9
,
10
]. The extensive
tissue archive, paired with daily observational and medical records for individual dolphins,
provides a unique opportunity for scientific research. The application of DNA methylation
technology to bottlenose dolphins promises to improve our understanding of species-
specific aging drivers, as well as potential preventative measures for the reversal of DNA
methylation and increased survival [11].
2. Materials and Methods
A total of 110 samples (101 blood buffy coat and 9 skin) were analyzed from
34 different
dolphins (19 female, 15 male). Of these, 24 had exact birth dates, and the remaining
10 were
estimated (within 2–4 years) via morphometric measurement and, where available, com-
bined with tooth growth layer group analysis (n= 2). Sample ages ranged between 1 month
and 58 years. Each dolphin was selected according to life history and health status, with
2–5 samples per dolphin spaced at least 5 years apart. Dolphins were selected according to
their age and lifespan to ensure a representative coverage of bottlenose dolphin lifespan
(up to 60 years of age). The longitudinal measures helped to validate expected changes
over the lifespan. Health status was assessed, and dolphins were classified as healthy
or unhealthy. Due to epigenetic changes occurring over longer periods of time, chronic
health concerns were defined as having a duration of > 6 months; therefore, acute episodes
of illness were not included. Samples were collected during routine animal care, under
the authorization of U.S. Code, Title 10, USC 7524. The Navy is accredited by AAALAC
International, and adheres to the national standards of the U.S. Public Health Service Policy
on the Humane Care and Use of Laboratory Animals and the Animal Welfare Act. Ethical
approval was granted by the University of St Andrews’ Animal Welfare and Ethics Commit-
tee (SEC20015). Archived blood buffy coat samples were from 1992 to 2020. Skin samples
were collected from fresh carcasses during necropsy using standard protocols. Buffy coat
and skin samples were archived at
80
C. Samples were submitted to the Technology
Center for Genomics and Bioinformatics, University of California at Los Angeles, for DNA
extraction using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). Each DNA
sample was 20 µL with a concentration of 250 ng.
DNA methylation data were generated using a custom mammalian methylation array
(HorvathMammalMethylChip40) with 37,492 CpGs [
12
]. An elastic net regression model
was used to select the CpG sites associated with chronological age [
13
]. The elastic net was
run on twenty random training sets (each including 2/3 of the data) to select the elastic
net mixing parameter (
α
). The value of
α
that returned the lowest mean squared error
for the corresponding test sets was selected. The regularization parameter (
λ
) was then
chosen via cross-validation (CV), using individual IDs as the folds to account for repeated
measurements (cross-validation with one individual left out in each fold, LOIOCV). Details
are given in the Supplementary Material.
3. Results
The final model retained 112 CpG sites for the blood clock and 195 CpG for the multi-
tissue clock (Figure 1, Figures S1 and S2). The lists of CpG sites selected in each clock are
provided in the supplemental information. R
2
(coefficient of determination) values for the
LOIOCV predictions were 0.97 and 0.95 for the blood and multi-tissue clocks, respectively,
compared to R
2
= 0.74 previously reported for both dolphin and beluga clocks [
6
,
10
]. The
final epigenetic clocks were highly accurate, with a median absolute LOIOCV prediction
error of 2.0 years for the blood clock and 2.5 years for the multi-tissue clock. The accuracy
J. Zool. Bot. Gard. 2021,2418
was likely achieved by longitudinal samples from a wide range of ages, facilitated by the
long lifespans of Navy dolphins.
J. Zool. Bot. Gard. 2021, 2, FOR PEER REVIEW 3
compared to R2 = 0.74 previously reported for both dolphin and beluga clocks [6,10]. The
final epigenetic clocks were highly accurate, with a median absolute LOIOCV prediction
error of 2.0 years for the blood clock and 2.5 years for the multi-tissue clock. The accuracy
was likely achieved by longitudinal samples from a wide range of ages, facilitated by the
long lifespans of Navy dolphins.
Figure 1. Regression of known chronological age against estimated age for blood (n = 101, R2= 0.97,
median absolute error of 2.0 years) and multi-tissue (n = 110, R2= 0.95, median absolute error of 2.5
years) clocks generated by leave-one-individual-out-cross-validation (LOIOCV). Each point repre-
sents a single bottlenose dolphin sample. Solid lines represent the 1:1 line; dashed lines represent
the fitted line. The grey ribbon is the 95% prediction interval.
4. Discussion
This robust epigenetic clock validates the use of blood and skin tissue to support the
precise estimation of age in bottlenose dolphins. In wild, free-ranging dolphins, blood can
be obtained during a hands-on veterinary examination, but skin can be sampled remotely
without requiring restraints. Estimation of chronological age from remotely sampled skin
is pivotal in advancing cetacean conservation, particularly in large, free-swimming
whales where temporary capture and restraint are currently not feasible. Knowledge of
animals’ age aids conservation and management efforts by improving our understanding
of population demographics, as well as age-specific rates of morbidity, mortality and re-
production.
To establish the chronological aging clock in bottlenose dolphins, this study focused
on blood buffy coat samples to ensure the most accurate results, due to the expected
higher quality of DNA obtained from this tissue. An additional objective was to validate
the use of DNA extracted from skin for epigenetic aging analysis. Whilst our sample size
for skin was small, it provided us with validation, from known-age dolphins, to move
forward with the next phase of the project, which is analyzing DNA extracted from wild
dolphin skin samples. Currently, the blood clock is more accurate than the multi-tissue
clock; however, a skin-specific clock can be produced with additional samples and com-
pared with the multi-tissue clock to see which is most accurate for future use. The recently
published odontocete clock produced an R2 of 0.81 for a skin-only clock with a mean ab-
solute error of 7.76 years [7]. We hypothesize that a more accurate species-specific skin
clock with an error equivalent to the multi-tissue clock of approximately 2 years can be
Figure 1.
Regression of known chronological age against estimated age for blood (n= 101,
R2= 0.97
,
median absolute error of 2.0 years) and multi-tissue (n= 110, R
2
= 0.95, median absolute error
of 2.5 years) clocks generated by leave-one-individual-out-cross-validation (LOIOCV). Each point
represents a single bottlenose dolphin sample. Solid lines represent the 1:1 line; dashed lines represent
the fitted line. The grey ribbon is the 95% prediction interval.
4. Discussion
This robust epigenetic clock validates the use of blood and skin tissue to support the
precise estimation of age in bottlenose dolphins. In wild, free-ranging dolphins, blood can
be obtained during a hands-on veterinary examination, but skin can be sampled remotely
without requiring restraints. Estimation of chronological age from remotely sampled skin
is pivotal in advancing cetacean conservation, particularly in large, free-swimming whales
where temporary capture and restraint are currently not feasible. Knowledge of animals’
age aids conservation and management efforts by improving our understanding of popula-
tion demographics, as well as age-specific rates of morbidity, mortality and reproduction.
To establish the chronological aging clock in bottlenose dolphins, this study focused
on blood buffy coat samples to ensure the most accurate results, due to the expected higher
quality of DNA obtained from this tissue. An additional objective was to validate the use
of DNA extracted from skin for epigenetic aging analysis. Whilst our sample size for skin
was small, it provided us with validation, from known-age dolphins, to move forward with
the next phase of the project, which is analyzing DNA extracted from wild dolphin skin
samples. Currently, the blood clock is more accurate than the multi-tissue clock; however,
a skin-specific clock can be produced with additional samples and compared with the
multi-tissue clock to see which is most accurate for future use. The recently published
odontocete clock produced an R
2
of 0.81 for a skin-only clock with a mean absolute error
of 7.76 years [
7
]. We hypothesize that a more accurate species-specific skin clock with an
error equivalent to the multi-tissue clock of approximately 2 years can be created with
an increased skin sample size. This study confirmed, as demonstrated in the cluster
dendrogram (Figure S1), methylation patterns are tissue-specific in cetaceans; therefore,
creating both species- and tissue-specific clocks will provide the most accurate results.
J. Zool. Bot. Gard. 2021,2419
Establishing this accurate bottlenose dolphin species-specific clock was facilitated by
the high proportion of known-age Navy dolphins and long lifespans, enabling longitudinal
sampling. With some dolphins contributing up to five samples and a maximum age of
58 years
old, this longitudinal approach has allowed repeated measures to be accounted for
within our statistical analysis, producing the highly correlated R
2
of 0.95. This longitudinal
sample approach has not been feasible in previous cetacean studies due to the lack of
known-age individuals or, in wild cases, lack of access to repeated samples. Using only
known-age dolphins would have reduced both our sample size and our range of samples
across the dolphin lifespan. While it may have produced a more accurate clock for a smaller
age demographic, our aim was to apply the clock across the full lifespan; therefore, older
animals without exact birth dates were included. Sample selection for this study was
focused on establishing the chronological clock; however, the selection of both healthy and
unhealthy individuals will enable the next phase of the project.
The next phase of this project will investigate the biological aging component of DNA
methylation, using additional wild dolphin samples and health information to identify
CpG sites associated with specific health parameters and cumulative stress. Due to the
standard of care provided to the Navy dolphins, chronic, advanced health conditions are an
unusual occurrence. Utilizing wild dolphin samples in the next phase of this project, will
likely aid in the identification of CpG sites that are methylated in association with biological
aging. Wild dolphins may have more advanced disease states than Navy dolphins due to
lack of veterinary intervention and increased environmental stress. A comparison between
known healthy individuals and unhealthy individuals will aid the identification of CpG
sites involved in biological aging. If future conservation efforts are able to determine
biological age from a skin biopsy, this could provide new insight into population health
without hands-on veterinary examinations.
Finally, future epigenetic research should aim to predict individual dolphin lifespan
by estimating the average remaining lifespan from DNA methylation patterns similar to
what has been accomplished for humans [
14
]. These epigenetic estimators of mortality and
morbidity risk could become useful for identifying environmental stress factors. This would
enable epigenetics to provide insight into survivability by improving the understanding of
demographics, and potential for population growth [
14
]. From a conservation perspective,
knowledge of age for threatened and endangered species is one of the biological keys to
determining population survival.
Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10
.3390/jzbg2030030/s1, Additional Statistical methods description with associated figures:
Figure S1
:
Cluster Dendrogram for data quality control, Figure S2: Mean squared error plotted against the
elastic net mixing parameter,
α
, for blood and mixed tissue, Figure S4: Modelled median relationship
between absolute prediction error and known age resulting from a quantile non-parametric additive
model. References [10,13,1517] were used in Supplementary Materials.
Author Contributions:
Conceptualization, A.B., C.R.S., F.M.G., S.H., and L.H.S.; methodology, A.B.,
S.H., L.H.S.; formal analysis T.P., E.P., J.A.Z., S.H., L.T., R.T.; investigation, A.B., L.H.S.; resources,
F.M.G., A.B.; data curation, F.M.G., C.P., A.M.M., C.R.S.; writing—original draft preparation, A.B.
and C.R.S.; writing—review and editing, all co-authors.; visualization, A.B.; supervision, C.R.S. and
L.H.S.; project administration, A.B.; funding acquisition, A.B., C.R.S., F.M.G. and L.H.S. All authors
have read and agreed to the published version of the manuscript.
Funding:
This research was funded by SERDP grant RC20-C2-1097 awarded to Peter Tyack and
Prescott Award NA20NMF4390132 awarded to Ashley Barratclough.
Institutional Review Board Statement:
Samples were collected during routine animal care, under
the authorization of U.S. Code, Title 10, USC 7524. The Navy is accredited by AAALAC International,
and adheres to the national standards of the U.S. Public Health Service Policy on the Humane Care
and Use of Laboratory Animals and the Animal Welfare Act. Ethical approval was granted by the
University of St Andrews’ Animal Welfare and Ethics Committee (SEC20015).
Informed Consent Statement: Not applicable.
J. Zool. Bot. Gard. 2021,2420
Data Availability Statement: Not applicable.
Acknowledgments:
The authors thank Eric Jensen, Mark Xitco, and the U.S. Navy Marine Mammal
Program staff for their support; Sam Ridgway and the National Marine Mammal Foundation animal
care and veterinary staff who facilitated this study. This is scientific contribution number 311 from
the National Marine Mammal Foundation.
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.
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