DOI: 10.5061/dryad.ns1rn8pvz
title: Risk-taking coping style correlates with SERT SNP290 polymorphisms in free-living great tits

description: The coping style of an individual in relation to potentially dangerous
 situations has been suggested to be inherited in a polygenic fashion,
 being SERT one of the candidate genes. In this paper, we assessed in
 free-living great tits Parus major the association between SNP290 in the
 SERT promoter and three standard fear-related behaviors, namely the
 response of the birds to a black and white flag fixed to the top of the
 nest-box, distress calling rate of the birds in the hand once captured and
 the hissing call of incubating females when approached by a predator. We
 found a strong association between SNP290 polymorphism and the three
 risk-taking behaviors, with birds with genotype CT entering faster to the
 nest box with the flag and displaying more distress calls and less hissing
 calls. CT birds could therefore be described as more proactive than CC
 individuals. These results also suggest that hissing behavior should be
 regarded as a fear-induced shy behavior, and confirm that SERT has an
 important function in relation to risk aversion behaviors and coping
 style.
description: Methodology appears in detail in DOI:
 10.1242/jeb.243342. The data set contains data on
 genotypes in SERT SNP290 and on three experiments on coping style on free
 living Great tits Parus major: <strong>The first
 experiment</strong> was risk-taking behavior in reaction to a flag
 that was attached to the top of the nest box at the time of feeding of the
 nestlings. The method was previously used and tested by Cole and Quinn
 (2014), and reflects the proactivity of the birds in front of a new
 object. Time to enter into the flagged nest box correlated with additional
 assays with the same birds in captivity, in which exploration rate in a
 novel environment was measured. This novel environment assay additionally
 stresses the trade-off between risk-taking and chick provisioning. This
 experiment was conducted when the chicks had a median age of 9 days (range
 9 - 12) by video recording at the distance of 7-36 meters from the nests
 (median= 16 m), depending on the presence of vegetation around each nest.
 Distance from the observer to the nest was set as a covariate in the
 analysis.  To ascertain for the effect of the flag (novel object) in our
 experiment, we first recorded the normal latency of the birds to enter the
 nest-box without any flag. Then, we recorded the behavior of the birds
 with the flag attached at the top of the nest-box. We recorded the
 behavior of the birds with a video camera, and corrected for the distance
 of the camera from the nest, since this could affect to the time of
 appearance of the bird in the video. The flag was removed after 40
 minutes. This behaviour was tested in 34 individuals, using the birds that
 entered the nestbox during the control experiment (with no flag). However,
 when considering only the birds that entered the nestbox during the flag
 trial, sample size was reduced 24 birds. The trait has been shown
 previously to be repeatable (Cole and Quinn, 2014).
 <strong>The second test</strong> focused on distress
 calling rate during handling, which has also been previously recognized as
 a rate of proactivity (boldness): in black-capped chickadees (Poecile
 atricapillus), distress calling rate was positively related to exploration
 rate, which is one of the main characteristics of the proactive coping
 style (Guillette and Sturdy, 2011). In siskins (Carduelis spinus),
 individuals uttering more often distress calls also displayed bolder
 behaviors in front of a novel object (Mateos-González and Senar, 2012;
 Pascual and Senar, 2014). Distress calling has therefore been used as a
 proxy of proactivity in several studies (Andersen, 2012; Pascual and
 Senar, 2014; Richardson et al., 2016; Senar et al., 2017; Thorsteinsen,
 2015). This was tested during the capture of the parents, normally at age
 15 days of the chicks. Captured animals were kept in a ringing bag for a
 period of about 5 min to calm down. Just after extracting the bird from
 the ringing bag, distress calling rate was quantified by counting the
 number vocalized distress calls that were emitted by the birds during the
 next 15s of handling while holding their legs and moving a straight finger
 positioned at 1-2 cm from the beak of the focal bird (Markó et al., 2013;
 Senar et al., 2017). We recorded this behavior on a total of 64 birds. The
 trait has been shown previously to be repeatable (Senar et al.,
 2017). <strong>The third test</strong>
 focused on the propensity of incubating females to utter hissing calls
 towards a nest predator. This behaviour was first described in Great tits
 by Krams et al. (2014). We measured the hissing response of incubating
 females to a mouse model meanwhile we inserted the model head into the
 entrance of the nest box. The head of the mouse was kept in this position
 for 15 s. We counted the number of calls of incubating females in that
 period. As soon as 15 s was over, the mouse was removed and the observer
 silently moved away. The method has been used in several other papers
 although using a woodpecker as a nest predator (Krams et al., 2014; Thys
 et al., 2021; Timm et al., 2019). We recorded this behavior on a total of
 34 females. The trait has been found previously to be highly repeatable
 (Koosa and Tilgar, 2016; Krams et al., 2014). 
 <strong>DNA</strong> was extracted from blood samples
 using Ecogen MasterPure DNA purification kit (MCD85201). Primers were
 designed using the great tit genome (assembly 1.3, accession number:
 SRS1185780, (Laine et al., 2016) blasting with the reverse complement of
 the SERT promoter interval of the great tit (GenBank: accession number
 KP869099) (Riyahi et al., 2015). The primer sequences were as follows:
 Forward primer 5´-TTCAGCAATGCACAAAGTCCAG-3´, Reverse primer
 5´-ACTCCAGGTCTCCCTGTCCTC-3´. <strong>Genetic polymorphisms within
 the SERT loci</strong> were genotyped by standard PCR amplification
 and direct sequencing of the resulting amplicon. 50 ng genomic DNA was
 amplified in 25 µl reactions containing 10x reaction buffer. All the
 samples were sequenced at the Genomics Core Facility of the University of
 Pompeu Fabra (Barcelona, Spain) using an Applied Biosystems sequencing
 platform. All of the sequences were aligned using Sequencher v4.6 (Gene
 Codes Corporation, MI, Ann Arbor, USA) and visually checked for
 polymorphisms (SNPs). The names of the SNPs were given by the position of
 each SNP in the resulting amplicon.  
description: We intended to conduct the three behavioral experiments in our
 breeding pairs in all of the nest boxes. However, the number of
 individuals involved in each test varied because we were unable to perform
 some tests on some nests because the chicks died before conducting the
 experiment or because the parents abandoned the nests or were not
 captured. This is why sample sizes for the different experiments
 differ.