DOI: 10.5061/dryad.z34tmpgf6
title: The telomere regulatory gene POT1 responds to stress and predicts performance in nature: implications for telomeres and life history evolution

description: Telomeres are emerging as correlates of fitness-related traits and may be
 important mediators of ecologically relevant variation in life history
 strategies. Growing evidence suggests that telomere dynamics can be more
 predictive of performance than length itself, but very little work
 considers how telomere regulatory mechanisms respond to environmental
 challenges or influence performance in nature. Here, we combine
 observational and experimental datasets from free-living tree swallows
 (Tachycineta bicolor) to assess how performance is predicted by the
 telomere regulatory gene POT1, which encodes a shelterin protein that
 sterically blocks telomerase from repairing the telomere. First, we show
 that lower POT1 gene expression was associated with higher female quality,
 i.e. earlier breeding and heavier body mass. We next challenged mothers
 with an immune stressor (lipopolysaccharide injection) that led to
 ‘sickness’ in mothers and 24h of food restriction in their offspring.
 While POT1 did not respond to maternal injection, females with lower
 constitutive POT1 gene expression were better able to maintain feeding
 rates following treatment. Maternal injection also generated a one-day
 stressor for chicks, which responded with lower POT1 gene expression and
 elongated telomeres. Other putatively stress-responsive mechanisms (i.e.
 glucocorticoids, antioxidants) showed marginal responses in stress-exposed
 chicks. Model comparisons indicated that POT1 mRNA abundance was a largely
 better predictor of performance than telomere dynamics, indicating that
 telomere regulators may be powerful modulators of variation in life
 history strategies.
description: We test the hypothesis that telomere regulatory mechanisms
 respond to stress and predict performance in wild animals, evaluated with
 a focus on POT1 ('protection of telomeres 1'). First, we ask
 whether POT1 gene expression is associated with three markers of
 individual quality in breeding female tree swallows (<i>Tachycineta
 bicolor</i>), including first egg date, body mass, and wing length
 (Winkler et al., 2020). We captured females and measured their body mass
 and wing length when they were rearing approx. 5-day old chicks. At the
 same time, we also collected whole blood samples from each female for
 telomere length and POT1 gene expression analyses. Second, we
 experimentally disrupted relationships between POT1 and performance by
 exposing breeding females to an ecologically relevant stressor
 (lipopolysaccharide injection), which leads to ‘sickness’ in mothers, and
 24h of food restriction in their offspring. We assessed whether a
 female's telomere length and/or POT1 gene expression predicted
 fluctuations in body mass and visitation rates following injections. We
 also assessed effects of this stressor on their offspring. We measured
 chick growth and key aspects of telomere biology (i.e. change in relative
 telomere length, POT1 gene expression), as well as other traits that have
 been linked to telomere dynamics in previous work (i.e. glucocorticoids,
 antioxidants), mostly sampled one week after maternal injections. Last, we
 recorded fledging success and recruitment of chicks into the breeding
 population to assess whether telomere length and/or POT1 gene expression
 predict survival-related metrics. Female
 visitation rates: We quantified maternal visitation to the nest
 using PIT tags and RFID readers positioned at the entrance of the nest
 box. Relative telomere length: DNA
 was extracted from whole blood using a Promega Maxwell RSC instrument, and
 telomere length was quantified using qPCR. For questions relating to
 changes in chick telomere length over time, we use a corrected metric
 accounting for initial telomere length at 5-days old (corrected for
 regression to the mean). Gene
 expression: RNA was extracted using a Trizol-based method. We
 then quantified antioxidant and POT1 gene expression using qPCR.
 Corticosterone: We extracted hormone from
 plasma using ether extractions and quantified CORT levels using an enzyme
 immunoasssay kit (Cayman #501320). 
 Datasets: The "FemaleRNA" excel
 sheet is used to ask 1) whether female POT1 gene expression, telomere
 length, and other variables predicts several metrics of quality, and 2)
 whether POT1 and telomere length predict her responses to injection with
 saline or LPS. The "ChickTelomereData" excel sheet is used to
 ask 1) how chick phenotypes respond to maternal injections and 2) whether
 chick phenotypes predict fledging success and recruitment into the
 breeding population.
description: Please see the "Notes" tab on each excel datasheet for
 more details about the columns/variables. Because of
 the nature of field work, each variable of interest does have missing data
 for some individuals. That information (# missing per variable and other
 subsetting) can be found in the methods section of the published
 manuscript.