DOI: 10.5061/dryad.3ffbg79xn
title: Data from: Repeated experimental challenges do not shorten adult telomeres in a songbird

description: Responding to environmental challenges can have both positive and negative
 effects on fitness, but the mechanisms and/or biomarkers underlying these
 links are not well understood. Telomeres, repetitive sections of DNA that
 form protective caps at chromosome ends, which enhance genome integrity
 and often predict organismal lifespan, may be important in this context.
 Some evidence suggests that stress exposure shortens telomeres, but most
 studies are correlational and/or conducted during early life; thus, how
 stress exposure influences telomeres in adulthood is not well known. Here
 we experimentally exposed wild-captured adult house sparrows (Passer
 domesticus) to a series of standardized protocols of repeated, rotating
 stressors (including threats, unpredictable food availability, and immune
 challenges) designed to increase stress levels. Although there were some
 effects of the treatment on glucocorticoid levels and mass dynamics, there
 were no effects on telomeres during any of the 6-11 month studies. Taken
 together, these results suggest that telomeres are robust or resilient to
 a range of stressors in adulthood. The effects of stress exposures on
 telomeres are likely to be highly context-dependent and vary depending on
 the timing, intensity, and duration of the stress exposures as well as an
 individual’s life stage.
description: We completed 4 experiments in sequence, which reflected a
 combination of three agendas. First, space limited sample size for any one
 experiment, so replicates were needed. The first experiment was conducted
 inside, whereas the 2nd-4th were conducted in outdoor aviaries in small
 flocks, which for house sparrows is a more natural context than indoors,
 and so we thought our treatments would better reflect natural, albeit
 elevated challenges. Finally, we were also interested in assessing the
 potential for different types of stressors to have different effects;
 hence, we varied the potential threats within the first 3 experiments and
 shifted to different types of stressors in the last experiment
 (below). <em>Captive housing
 conditions</em> The first experiment was
 conducted in an indoor aviary using local male house sparrows captured
 from Fargo, North Dakota, and three others were done in an outdoor aviary
 facility using local house sparrows of both sexes captured from Lexington,
 KY. In North Dakota, mist nets were used to capture 30 adult male house
 sparrows in early 2017. Males were individually housed in wire cages (23”
 wide x 16” deep x 16” tall) on racks in two rooms at North Dakota State
 University. All cages were visually and acoustically open to the other
 birds in the room, and the male cage racks were positioned about 3 feet
 apart with a centrally located cage stand housing 4 females, which were
 visible to all males. Food (Kaytee supreme mixed seed for finches), water,
 and cuttlebones were provided <em>ad libitium</em>, and
 supplemental food (broccoli, baby spinach, peas and carrots, or Quicko
 Classic Egg Food) was provided once per week. The bird rooms were kept on
 a short-day cycle of 8h light: 16 h dark for 27 weeks. After the period of
 stress exposure, the rooms were shifted to 16 h light: 8 h dark.
 In Kentucky, we captured free-living house sparrows using mist
 nets at several locations between October and December in 2017, 2018, and
 2019. Subject birds were then released into aviaries in groups of 4
 consisting of 2 males and 2 females. We provided each aviary with a
 retired Christmas tree for cover with the quality of cover (more needles
 on the tree) higher in control aviaries than experimentals, and
 <em>ad libitum</em> food (white millet and chicken starter
 mix), water, and sand grit. Assignment of birds to aviaries was based on
 filling aviaries in order by their identity number (matched by eventual
 treatment) as subjects of each sex were caught producing a random
 assignment by body size and capture date between treatments.
 <em>Details of stress protocols</em>
 The experiments were modeled on previous research. For the first
 three, we were informed by prior work using a standardized series of
 rotating stressors designed to induce chronic stress in house sparrows and
 other songbird species. They differed in the exact rotating stressors
 employed, their schedules, and their duration (Figure 1 of published
 paper). In the fourth experiment, we shifted to repeatedly stimulating the
 immune system and to manipulate the predictability of food. In total,
 these 4 experiments served as replicates of generalized stress exposure,
 but because they were conducted across some variation in context, we could
 potentially assess a broader array of stressor intensity and type than
 could be accomplished in any one experiment. Our specific protocols for
 each are described in detail below and summarized in Figure 1 of the
 published paper. <em>Experiment 1</em> - We
 assigned North Dakota birds randomly to either experimental or control
 treatment upon capture (n = 15 males per treatment). One week after the
 last bird was captured, we began the stress protocol to all birds in one
 room. We exposed subjects to one of five stressors daily, applied in a
 randomly generated order and at a random time each day between the hours
 of 10am and 10pm. Each stressor was applied for 30 minutes. At the same
 time as the initiation of the stressor each day, the control room received
 a nontreatment protocol of opening and immediately closing the door
 without entry. The five experimental stressors were selected to mimic
 stressful events that house sparrows might be exposed to in the wild: a
 live avian predator (common kestrel, <em>Falco
 tinnunculus</em>), a live feline predator (domestic cat,
 <em>Felis catus</em>), a mounted avian predator (merlin,
 <em>Falco</em> <em>columbarius</em>), loud radio
 (to represent anthropogenic acoustic disturbance), and a human rattling
 and shaking of the cage racks. We inserted the live predators into the
 stress order twice, such that the birds received 7 stress events per cycle
 per week with the two live predators represented twice. This protocol was
 repeated for 27 weeks. <em>Experiment
 2</em> – Subjects were captured between 26 September 2017 – 5
 December 2017. Birds were assigned treatment by aviary, and aviaries were
 assigned treatments based on aviary location. Aviaries were arrayed around
 four separate central chambers accessed through a door adjacent to 1 or 2
 of the aviaries. Birds were given food and water from inside the chamber,
 so this meant the aviaries adjacent to the door were disturbed more than
 other aviaries each time food and water were provided. These aviaries were
 assigned to the elevated stress treatment. Aviaries on the back side of
 each chamber were assigned the control treatment (no stressors), and a few
 other less disturbed aviaries were randomly assigned with the goal of
 generating equal sample sizes. We began stress treatments on 5 December
 2017. Each aviary was stressed twice per day at a random time in the
 morning and in the afternoon for 30 min. We chose among six stressor
 types: a human moving around just outside the aviary, an owl model with
 moveable head sitting on a platform inside the aviary, a model cat with
 moveable head and tail on the floor of the aviary, a rubber snake (1 of 2
 types) draped realistically in the refuge tree in the aviary, one of 10
 different novel objects (determined randomly) placed in the food dish, and
 a radio-operated streamer placed near the roost tree that periodically
 jerked back and forth. We assigned stressors in a mixed procedure that
 included random assignment but with biases toward balancing the number of
 times we presented each stressor and some constraints (such as the
 proximity of two aviaries to each other). We employed the stress protocol
 for 6 days each week until March 30, 2018 for a total of 16.5
 weeks. Experiment 3 – New subjects were captured
 starting on 18 September 2017 and continued until 11 October 2018. They
 were assigned to aviaries as in 2017. Treatments were assigned to aviaries
 similarly as well. We began the experimental treatment on 21 October 2018.
 The protocol was similar to the 2017 experiment with a few key
 differences. First, we stressed birds 3 times daily at random times within
 three 3-hour blocks throughout the day. We also added an additional
 stressor; a taxidermy mount of a Cooper’s hawk (<em>Accipiter
 cooperii</em>) placed on a platform inside the aviary. We also added
 a capture-restraint-sampling protocol, experienced only by the
 experimental group, twice (on 16-17 November and 20-21 January) in the
 middle of the experiment. The stressor schedule continued until 26 March
 2019 (22 weeks) when all birds were captured again and sampled.
 <em>Experiment 4</em>– A third set of Kentucky
 subjects were captured from 20 October to 29 November 2019. Birds were
 measured, sampled, and assigned to aviaries as in the previous two years.
 In this replicate, we altered the nature of the stressors. We divided
 aviaries into three groups, with one group assigned as control and treated
 as in the other two years (and in similar aviary positions given the
 disturbance of providing food and water at the other aviaries). Both other
 groups were injected intradermally with 0.1 mL of PBS containing 25 ug
 bacterial lipopolysaccharide (LPS) on 4 occasions, approximately every 3-4
 weeks between January 13, 2020 to the end of March 2020. At the end of
 February, we also held experimental subjects in both groups for 30 min and
 collected blood. One experimental group was additionally treated to a 6 h
 food deprivation regime starting 2 days after each injection, starting at
 a random time in the morning and repeated 3 times in 5 days. Each time
 birds were caught we also recorded their mass. In
 experiments 2-4, subjects were kept after the end of the stress protocol
 in aviaries under minimal disturbance so that they would breed. This
 included supplementing their diet with additional protein and sources of
 calcium and providing nesting material. Some birds did breed and those
 were provided with insect food to raise their offspring. Non-breeders,
 regardless of treatment, were removed to other aviaries either in hopes
 they would breed or to keep until release in August of each year. At the
 end of the summer, all remaining subjects were blood sampled and weighed
 again and released back into the wild.
 <em>Sampling</em> Timing of
 sampling for all four experiments is summarized in Figure 1 of the
 published paper. In experiment 1, we measured mass and took blood samples
 from all birds before the experimental protocol began on 19 April 2017,
 and then again at the end of the experiment on 9 October 2017. Birds in
 control and stress treatments were also weighed on 3 May and 7 June 2017.
 To measure if stressors elevated corticosterone as intended, birds on the
 stress protocol were also captured, weighed, and blood sampled on 3 May
 and 7 June after 30 min exposures to the live cat, again on 25 September
 when they were sampled at baseline and after a 30 min restraint. On 9
 October the treated birds were exposed to the live kestrel mount for 30
 min, blood sampled and sperm collected through cloacal massage. All birds
 were then euthanized and tissue from heart, liver, and pectoralis muscle
 collected. At each blood sampling event approximately 75 μL of whole blood
 was collected via brachial venipuncture into heparinized micro-hematocrit
 tubes. The blood was stored on ice for 5-120 min, centrifuged, separated
 into plasma and red blood cell fractions, and frozen at -80 °C until
 measurement. In experiments 2-4, we collected blood
 samples from the bird’s brachial vein in heparinized microcapillary tubes
 from all subjects upon initial capture, at the end of treatment, and upon
 release. In experiment 2, 5 birds were sampled 4 times in two days in the
 period between the treatment ending and being released as part of another
 experiment; we used the first sample from that series in the present
 analysis. In experiment 3, we also took samples during the
 capture-restraint protocol. Not all birds in experiments 2-4 were sampled
 at all time points due to either death, escape or failed DNA extraction.
 In experiment 3, a set of excess birds were sampled once but not used as
 subjects. Details on sample sizes in the analysis are reported in Table S1
 linked to the published paper. <em>Telomere
 measures</em> DNA was extracted from red blood
 cells and organs using commercially available extraction kits
 (Macherey-Nagel NucleoSpin Blood #740951, Macherey-Nagel NucleoSpin Tissue
 #740952) and following the manufacturer’s protocol. DNA was extracted from
 whole diluted semen samples using commercially available extraction kits
 with modifications. After extraction, DNA concentration and purity were
 assessed with a Nanodrop 8000 spectrophotometer (Thermo Scientific,
 Waltham, MA, USA) and DNA quality was verified by electrophoresis on a 2%
 agarose gel. Relative telomere length was measured
 using quantitative PCR on a Strategene Mx3000P (Stratagene, San Diego, CA,
 USA). The relative telomere length of samples was calculated as the ratio
 (T/S) between telomere repeat copy number (T) and single copy control gene
 number (S) relative to the reference sample. Glyceraldehyde-3-phosphate
 dehydrogenase (GAPDH) was used as the single-copy control gene. The
 telomere forward and reverse primers used were: Tel 1B
 (5′-CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT-3′), Tel2b
 (5′-GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT-3′). Zebra finch GAPDH primers
 were used to amplify the control gene: GAPDH-F
 (5’-AACCAGCCAAGTACGATGACAT-3’), GAPDH-R (5’CCATCAGCAGCAGCCTTCA-3’). The
 telomere and GAPDH reactions were run in duplicate, and each reaction
 contained 20 ng of DNA, telomere or GAPDH primers at a concentration of
 200 nM forward/200 nM reverse, and 12.5 ml of perfeCTa SYBR green supermix
 Low ROX (Quantabio) for a total volume of 25 ml. The thermal profiles for
 the telomere reactions were 10 min at 95°C, followed by 27 cycles of 15 s
 at 95°C, 30 s at 58°C, and 30 s at 72°C, finishing with 1 min at 95°C, 30
 s at 58°C, and 30 s at 95°C. The thermal profiles for the GAPDH reactions
 were 10 min at 95°C, followed by 40 cycles of 30 s at 60°C, finishing with
 1 min at 95°C, 30 s at 55°C, and 30 s at 95°C. The
 number of PCR cycles (Ct) required for the products to accumulate enough
 fluorescence to cross a threshold was determined. Samples from a given
 experiment were randomized across plates but balanced with respect to
 treatment. Each plate also included a serially diluted 5 point standard
 curve (40, 20, 10, 5, 2.5 ng) that was run in triplicate created from a
 pooled house sparrow reference sample to measure reaction efficiencies.
 The average reaction efficiencies were 93.30 ± 0.97 (mean ± s.e.m.) for
 telomere plates and 98.59 ± 0.86 (mean ± s.e.) for GAPDH plates. The 20 ng
 point from the standard curve was used to as the reference sample to
 calculate relative telomere lengths for the unknown samples. Relative
 telomere lengths 2^-ΔΔC^, where ΔΔCt =
 (Ct <sub>telomere</sub> – Ct <sub>GAPDH</sub>)
 focal sample - (Ct <sub>telomere</sub> –
 Ct <sub>GAPDH</sub>) reference sample. We previously
 established that the repeatability of the T/S ratio is high (0.86, p
 &lt; 0.001, 95% CI[0.72, 0.93) by running samples from 28 adult house
 sparrows in random well locations across two plates.
 <em>Corticosterone measures</em>
 To measure corticosterone levels in our samples, we used a
 commercially available CORT enzyme immunoassay kit (Enzo Life Sciences
 ADI-901-097) following a protocol that was previously validated in house
 sparrows. Briefly, we added 5 mL of 10% steroid displacement buffer (SDB)
 to our 5 mL of plasma samples. After 5 minutes, we then added 240 mL of
 Assay Buffer 15 to each sample, thoroughly vortexed and then aliquoted 100
 mL duplicates into a 96 well plate. A standard curve ranging from 32 –
 200,000 pg/ml was aliquoted in triplicate on each plate. All wells were
 incubated with conjugated CORT and antibody on a shaker; wells were then
 washed, and substrate was added before a second incubation. Stop solution
 was added, and the plate was read at 405 nm (corrected at 580 nm per
 manufacturer’s recommendation). Individuals were randomized and treatments
 were balanced across plates. We ran a total of 4 plates and the inter- and
 intra-assay variation were 8.07% and 2.43% respectively.
 <em>Statistical Analysis</em> We
 constructed two datasets. The first contained telomere measures from the
 five tissues at the end of the North Dakota experiment. The second dataset
 contained the repeated measures of blood sample telomeres, corticosterone,
 and mass from all four experiments. This included 60 telomere measures
 from 30 subjects in experiment 1, 153 measures of telomeres from 54
 subjects in experiment 2, 206 measures from 64 subjects in experiment 3,
 and 173 measures from 58 subjects in experiment 4 (see Table S1 linked to
 published paper for the distribution of subjects with different numbers of
 measures). Also included in this dataset was the elapsed time since the
 start of the experiment in fraction of a year, the bird’s mass at each
 capture, and the treatment they experienced. We subset this dataset to
 analyze individual experiments but used the whole dataset for some
 measures of power and to facilitate generating some figures.
 All univariate analyses involved using a mixed model in the R
 package <em>lme4</em> with subject identity as a random
 effect. Corticosterone measures following threat presentations were
 natural log transformed and compared with baseline measures within a mixed
 model, and we also compared the corticosterone following the two cat
 presentations separated by 35 days to assess habituation. We calculated a
 separate raw repeatability for telomeres using the R program
 <em>rptR</em> for each experiment as the proportion of total
 variance in telomere length that was among-subjects. For experiment 1, we
 calculated repeatability for the two blood telomeres measures alone and
 also across all 6 tissue types. We adjusted this repeatability by
 including assay unit (the pair of plates containing a subset of samples)
 in all experiments as a fixed effect, which removed all potential variance
 due to methodological differences between assays. We retained this base
 model structure in all subsequent analyses. We then investigated the
 impact of elapsed time from initial capture on telomere length regardless
 of treatment; we treated this variable as a fixed effect to obtain the
 mean change in telomeres over time, and for experiments 2-4, included a
 subject by time random effect to estimate the variance in the effect of
 time across subjects. We then added treatment and a treatment by time
 interaction to assess the impact of treatment on telomere dynamics.
 Similar models were constructed for mass, except we also included a
 quadratic of time for all experiments except experiment 2 which did not
 have enough repeated observations of mass. We assessed
 power to detect different effect sizes reported in the literature which we
 could standardize and, ideally assess as a rate per year. For instance,
 one result (Hau et al. 2015), obtained after 1 year, is reported only in a
 figure, so we measured the observed effect size from the published figure,
 standardized it by the estimated standard deviation, calculated from
 standard errors given in the figure and sample sizes given in original
 publication. We then assessed our ability to detect an effect that size on
 a standardized scale. For our data, we obtained standardized effect sizes
 (by standardizing telomere measures and rerunning relevant models) and
 then retrieved standard deviations for the slope of telomere length by
 time for controls in each experiment separately and overall. We used an
 online calculator for power (statskingdom.com) of a two-sample t-test with
 the observed standard deviation and sample sizes of our control and
 experimental treatments. We also used values from the meta-analysis of
 Chatelain et al. (2020) which were already in standardized
 units. References Chatelain M.,
 Drobniak S.M., Szulkin M. 2020 The association between stressors and
 telomeres in non-human vertebrates: a meta-analysis. <em>Ecology
 Letters</em> <strong>23</strong>(2), 381-398.
 (doi:10.1111/ele.13426). Hau M., Haussmann M.F.,
 Greives T.J., Matlack C., Costantini D., Quetting M., Adelman J.S.,
 Miranda A.C., Partecke J. 2015 Repeated stressors in adulthood increase
 the rate of biological ageing. <em>Frontiers in Zoology</em>
 <strong>12</strong>. (doi:10.1186/s12983-015-0095-z)
description: # Data from: Repeated experimental challenges do not shorten adult
 telomeres in a songbird Dataset DOI:
 [10.5061/dryad.3ffbg79xn](10.5061/dryad.3ffbg79xn) ## Description of the
 data and file structure This document provides metadata for the data files
 used in the MS entitled “Repeated experimental challenges do not shorten
 adult telomeres in a songbird”. Two files (.csv format) are described. ###
 Files and variables #### File: Alldata.csv **Description:** This file
 contains data from 4 experiments, details of which are in the methods
 section. Empty (blank) cells indicate missing data, the reasons for this
 depend on the variable and are described in brackets for each. #####
 Variables * BirdID: Unique code for each subject * Experiment: A label for
 each of the four experiments * Treatment: A label for which treatment each
 subject received. Options are: Control – Left alone as much as possible
 Threat stress – Received the rotating stressors Immune – Received repeated
 injections of LPS Immune & Deprive - Received repeated injections of
 LPS and a food deprivation * Threat: Code for type of threat. [Relevant
 for only 2017 North Dakota Experiment and all other experiments are blank
 and so are missing] * ExpType: Either Stress or Control * Type: Code for
 2017 North Dakota experiment indicating type of sample for Corticosterone,
 either BL = Baseline, SI = Stress induced. [All other experiments are
 blank=missing] * Date: Date of sampling * Duration: Time in days since
 treatments began * DurYear: Time in years since treatments began * Sex: M
 = male, F = Female * Assay: Code indicating the telomere assay unit of
 plates. [This is blank = missing for any rows in which telomeres were not
 measured] * Telomere: Measure of telomere length as TS Ratio [Blanks
 indicate missing data, which comes from rows having other data but no
 telomere measure or if DNA quality was poor and a reliable telomere could
 not be obtained] * Mass: Weight in grams [Blanks indicate missing data
 from captures when mass was not measured or recorded] * InitMass: Mass at
 start [Blanks indicate missing data from an initial capture when mass was
 not measured or recorded] * Cort: Observed level of corticosterone (in
 pg/mL). [Blanks indicate missing data from captures when a sample was not
 taken (all experiments except for North Dakota 2017 and some captures in
 2017, or if the sample was insufficient to reliably measure
 Corticosterone)] * lnCort: Natural log of Cort. [Blank cells indicate
 missing data for the same reasons as for the Cort variable] #### File:
 Code_for_review.Rmd **Description:** Contains annotated R code for all
 analyses and figures in the main text and ESM for the paper.  #### File:
 Tissue.csv **Description:** Contains data on telomere lengths from 5
 tissues collected at the end of a stress experiment (except for Blood1,
 which was collected at the start). Empty (blank) cells indicate missing
 data. In this file this is due to either an inadequate sample (Tissue =
 Sperm) or poor DNA quality preventing a proper telomere assay. #####
 Variables * Bird: Number indicating bird identity * Tissue: Source of DNA
 for telomere measurements Blood1: Blood sample collected at start of
 experiment Blood2: Blood sample collected at end of experiment Heart:
 Collected at end of experiment Liver: Collected at end of experiment Pec:
 Pectoralis muscle collected at end of experiment Sperm: Collected at end
 of experiment * Treatment: Either Stress or Control, with the details of
 these treatments described in the linked paper * Telo: Telomere measure as
 the TS Ratio. ## Code/software R Computing Environment - Programs used:
 lme4, ggplot2, rptR, cowplot, gridExtra, grid, MASS, dplyr ## Access
 information Other publicly accessible locations of the data: None