DOI: 10.5061/dryad.m0cfxpp16
title: No, you go first: phenotype and social context affect house sparrow neophobia

description: Novel object trials are commonly used to assess aversion to novelty
 (neophobia), and previous work has shown neophobia can be influenced by
 the social environment, but whether the altered behaviour persists
 afterwards (social learning) is largely unknown in wild animals. We
 assessed house sparrow (Passer domesticus) novel object responses before,
 during, and after being paired with a conspecific of either similar or
 different behavioral phenotype. During paired trials, animals housed with
 a similar or more neophobic partner demonstrated an increased aversion to
 novel objects. This change did not persist a week after unpairing, but
 neophobia decreased after unpairing in birds previously housed with a less
 neophobic partner. We also compared novel object responses to non-object
 control trials to validate our experimental procedure. Our results provide
 evidence of social learning in a highly successful invasive species, and
 an interesting asymmetry in the effects of social environment on neophobia
 behavior depending on the animal’s initial behavioral phenotype.
description: <b><i>2.1
 Subject capture & housing</i></b>             We captured 24 house
 sparrows (12 males, 12 females) in Iberville and Lafourche Parishes,
 Louisiana, USA, using mist nets near bird feeders in November 2019. We
 individually housed sparrows in cages in a vivarium at Louisiana State
 University with unlimited access to mixed seeds, grit, a vitamin-rich food
 supplement (Purina Lab Diet for small birds), and water. Light cycle was
 synchronized with capture date (10.5L:13.5D) and maintained at this level
 for the duration of the experiment. Sparrows were visually but not
 acoustically isolated from one another to prevent observations of their
 neighbours’ novel object trials. Sparrows received an aluminum band with a
 unique number and were acclimated to captivity for 8-10 weeks until novel
 object trials began in mid-January. <b><i>2.2 Study
 design</i></b>             This study took place
 over five weeks. <i>Week 1, test individual phenotypes:</i> We
 exposed individual house sparrows to three randomly selected novel objects
 and two control days (no object) in a randomized order. <i>Week 2,
 assess individual phenotypes and create pairs:</i> We observed
 recordings for each sparrow’s time to feed from its food dish. Based on
 average feeding time, we created house sparrow pairs that were similar in
 approach time (matched phenotype controls; 5 pairs; Table S1 in
 Supplementary Material). The remaining birds were paired with a sparrow
 with a different phenotype (mixed phenotype pairing; 7 pairs; Table S1).
 We introduced pairs to one another in a new cage to reduce the possibility
 of territoriality. One individual from each pair received a second
 aluminum band to distinguish individuals in recordings. We acclimated
 pairs together for three days before continuing novel object trials.
 <i>Week 3, paired trials:</i> We exposed pairs to three
 randomly selected objects, novel to both individuals, and two control
 conditions in a random order. <i>Week 4, individual housing
 acclimation: </i>We separated pairs and returned individuals to
 their original cage during the fourth week to allow acclimation before
 beginning the final novel object exposures. <i>Week 5, re-test
 individual phenotypes: </i>We exposed sparrows to three new novel
 objects and two control conditions in a random order to test whether
 having a cage mate had lasting effects on neophobia behaviour (evidence of
 social learning). <b><i>2.3 Novel
 object exposure procedure</i></b> Over the five
 weeks, we exposed sparrows to a total of 15 trials, six controls and nine
 unique novel objects: a blinking light, a white cover over part of the
 dish, yellow pipe cleaners, a purple plastic egg, a red-painted dish, a
 tinfoil hood, three gold bells, pink puffs, and an opened blue cocktail
 umbrella (images in Supplementary Material). Objects were chosen to
 maximize the diversity of textures, colours, and shapes of novel objects,
 and all objects were placed on, in, or immediately above the food dish.
 Sparrows saw each object once. These objects were based on objects shown
 to elicit delayed approach and feeding in another songbird species [32],
 but altered slightly to share fewer common features (e.g., red color) that
 might incidentally target ecologically-relevant cognitive biases
 [33]. The night
 before a trial, we removed food dishes from cages 30 min before lights off
 and fasted sparrows overnight. We replenished seed in the dishes and
 outfitted them with an object (or no object, for controls). The next
 morning, 30 min after lights on, researchers entered the room, began a
 video recording of all cages (12 cameras, 2 birds per camera), replaced
 food dishes according to individual treatments, and left for 1 h.
 Researchers were present in the room &lt; 4 min. Sparrows were
 therefore fasted for a total of 15 h (1 h light, 14 h dark) before
 neophobia or control treatments. This fasting period ensured that sparrows
 would be motivated to approach the food dish. Upon re-entering the room,
 the video recording was stopped, and objects removed from food dishes.
 Videos were scored for the time each sparrow took to eat from the food
 dish. Two different observers scored all videos. To ensure intercoder
 reliability, these observers re-watched 14 videos from paired trials (n =
 28 feeding events) one month later, and ANOVA tests showed no significant
 differences within (F<sub>2,81</sub>&lt;0.001; p = 0.99)
 or between (F<sub>1,54</sub>&lt;0.001, p=0.99)
 observers. <b><i>2.4 Statistical
 approach</i></b> We split
 sparrows into three categories: (1) paired with an individual of similar
 neophobia response (control; n = 10), (2) paired with a less neophobic
 individual (partner faster to feed in the presence of novel objects; n =
 7), (3) paired with a more neophobic individual (partner slower to feed in
 the presence of novel objects; n = 7; Table S1). See Supplementary
 Material for datapoint losses. We used Cox proportional hazard models to
 investigate the mere effect of having a cage mate (1) as well as having a
 cage mate with a different neophobia phenotype (2 and 3) separately using
 the ‘coxme’ package [34] in R Studio version 3.6.3 [35]. Using a survival
 analysis approach [36] avoids creating arbitrary threshold values when a
 subject does not perform the expected behaviour during the allotted time
 period, which may bias alternative statistical approaches – i.e., giving
 subjects a time of 3600 s if they do not feed during a 60 min trial. Each
 model included subject as a random effect, week as a fixed effect and were
 interpreted as full models. Analyses were performed for control trials (to
 detect effects of the experimental procedure) and novel object trials (to
 detect neophobia effects) separately, resulting in six models. Novel
 object trials also included object as a fixed effect. Because effects of
 neophobia were detected in models run using novel object trials only, the
 contrasts among objects were arbitrarily against the object coded as ‘1’,
 so we do not report these contrasts. Instead, to validate that each object
 significantly increased latency to feed, we ran a seventh model that
 included all trials (all individuals, control, and object trials). This
 seventh model included subject as a random effect and object (or not) as a
 fixed effect.<br> <br>
 <em><strong>References:</strong></em><br>
 [32] de Bruijn R, Romero LM. 2019 Prior restraint stress inhibits
 habituation to novel objects in the European starlings
 (<i>Sturnus</i> <i>vulgaris</i>). <i>J. Exp.
 Zool. Part A Ecol. Integr. Physiol.</i> , doi:10.1002/jez.2327.
 (doi:10.1002/jez.2327)<br> [33] Greggor AL, Thornton A, Clayton NS.
 2015 Neophobia is not only avoidance: improving neophobia tests by
 combining cognition and ecology. <i>Curr. Opin. Behav.
 Sci.</i> <b>6</b>, 82–89.
 (doi:10.1016/j.cobeha.2015.10.007)<br> [34] Therneau T. 2020 coxme:
 mixed effect cox models. , URL: https://CRAN.R-project.org/package=coxme.
 (doi:10.1017/CBO9781107415324.004)<br> [35] R Core Team. 2020 R: A
 language and environment for statistical computing. <i>R Found.
 Stat. Comput. , Vienna, Austria.</i> , URL
 https://www.R-project.org/.[36] Stankowich T, Coss RG. 2007 The
 re-emergence of felid camouflage with the decay of predator recognition in
 deer under relaxed selection. <i>Proc. R. Soc. B Biol.
 Sci.</i> <b>274</b>, 175–182.
 (doi:10.1098/rspb.2006.3716)  
 <b>Additional
 information about animal husbandry</b> Sparrows were provided with a
 variety of perches and dust baths as enrichment. On the rare occasions
 that animals escaped from cages in the bird room when cage doors were
 opened during animal husbandry or neophobia tests, they were immediately
 and safely recaptured using a handheld net and returned to their
 housing.<b> </b>Animals were solo housed for part of this
 study, but all birds were housed in the same room and birds could hear
 each other. Because we were interested in individual animal personalities
 and how personality can change based on social learning, it was necessary
 to first test the animals individually before testing them in a group
 (paired) context. We also wished to understand if changes in personality
 persisted outside of the group context. Solo housing was therefore
 absolutely essential for the design of our study. Because this was only a
 behavioral study and did not involve any physiological manipulations, we
 requested and received permission from our university’s Institutional
 Animal Care And Use Committee (IACUC) to transfer the animals to another
 protocol after the published project was finished, so as to minimize
 animal numbers required for our research. Animals were therefore not
 euthanized at the end of this study. However, one control bird died of
 natural causes during week 4 (see Datapoint losses below). The age of wild
 sparrows brought into captivity is not known beyond “juvenile” and
 “adult,” nor is it known whether sparrows have any chronic illnesses or
 parasites, which are common in wildlife. Therefore, the occasional death
 due to natural causes in wild birds is expected. 
description: <b>Datapoint
 Losses:</b> Control Pairs:
 One individual escaped during object exposure during week 3 paired trials.
 We removed these datapoints for both birds (control pairing; week 3 object
 trials n = 28). Week 5 (unpaired) novel object videos were lost (three
 trials) for one control bird (control pairing; week 5 object trials n =
 27, week 5 control trials n = 18). Mixed Pairs:
 Due to a camera failure, one trial was lost during week 3 (paired trials;
 n = 20).   <b>Guide to
 datafiles:</b> Files are split by pairing type
 and by control or object trials. Pairing
 Effects: <b><i>Control Trials
 (no object presented)</i></b><br> R Code:
                       “R Code week comparisons by pairing_control trials
 only.R”<br> Data:   [control pair]   “Approach times_controls all
 weeks_control trials only.csv”<br>             [more neo]     
 “Approach times_more neophobic all weeks_control trials
 only.csv”<br>             [less neo]        “Approach times_less
 neophobic all weeks_control trials only.csv” <b><i>Object
 Trials</i></b><br> R Code:                       “R Code
 week comparisons by pairing_no control trials.R”<br> Data:  
 [control pair]   “Approach times_controls all weeks_no control
 trials.csv”<br>             [more neo]      “Approach times_more
 neophobic all weeks_no control trials.csv”<br>             [less
 neo]        “Approach times_less neophobic all weeks_no control
 trials.csv” Object Effects (seventh
 model): R Code:                       “R
 Code all weeks all phenotypes for object effects.R”<br>
 Data:                           “Approach times_all phenotypes all
 weeks.csv” Hazard Ratio 95% CI
 calculations: “Calculating Hazard Ratio
 95CIs.xls”