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.

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: The first experiment 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).

The second test 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).

The third test 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). 

DNA 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´. Genetic polymorphisms within the SERT loci 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.  

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.