DOI: 10.5061/dryad.sf7m0cght
title: Data from: Synchronising anti-predator behaviour in the red flour beetle Tribolium castaneum

description: In many animals, a phenomenon is often observed in which behaviour depends
 on population density and many individuals within the group synchronise
 their state of behaviour to some extent, and theoretical studies have
 suggested that this synchronisation phenomenon is adaptive for predation
 avoidance. Moreover, death-feigning behaviour (DF) has been observed as an
 anti-predator strategy in many animals. There are large individual
 differences in the duration of DF, and the optimal duration of DF often
 varies depending on the situation. Therefore, although it is expected that
 prey may synchronise with others around them for an optimal DF duration,
 there are few experimental studies testing this hypothesis. This study
 investigated whether DF duration varies with and without other
 individuals, and whether it synchronised with the DF duration of other
 individuals, in the red flour beetle Tribolium castaneum. This study used
 populations with genetically longer (L-population) and shorter
 (S-population) DF duration and measured DF duration when maintained alone
 and cohabitated with individuals from the L- and S-populations,
 respectively. The results showed that the DF duration of individuals
 living alone increased significantly compared to pre-treatment. Moreover,
 individuals that cohabitated with S populations were significantly shorter
 after cohabitation, but the presence of the L population did not cause any
 changes in how individuals synchronised their activities. When many
 individuals had shorter DF durations, DF was synchronised towards shorter
 durations. This is the first study to illustrate the synchronisation of
 anti-predator behaviour in terms of DF behaviour.
description: <strong>Insect and culture</strong>
 A stock culture of <em>T. castaneum</em>, in
 laboratories for more than 30 years, was used in this study. The beetles
 were reared on a mixture of wholemeal enriched with yeast in an incubator
 maintained at 25℃ under a 16:8 h (light: dark) photoperiod (lights on at
 7:00; lights off at 23:00). Distinct morphological characters of the
 abdomen were used in sex determination of beetles at the pupal stage.
 Then, single-sex groups were stored in a dish (diameter 90 mm, height 15
 mm) until experiments were performed. The focal individuals in each
 treatment were randomly collected from this stock culture.
 <strong>Selected populations for the duration of death
 feigning</strong> L-population with long DF
 duration and S-population with short DF duration in <em>T.
 castaneum</em> were established by a previous study (Miyatake et
 al., 2004), and these selection regimes have been maintained for over 30
 generations (Matsumura and Miyatake, 2018). More details of this selection
 were described in previous studies (Matsumura and Miyatake, 2018; Miyatake
 et al., 2004). Although approximately 20 years have passed since the
 selected populations were established, large differences in DF duration
 between the L- and S-populations have been maintained (Fig. 1).
 <strong>Experimental design</strong>
 Experimental design of this study is shown in Figure 2. Firstly,
 I measured DF duration (phase Ⅰ) according to a previously described
 method (Miyatake et al., 2004). Virgin beetles (21–35 days old) were used.
 The beetle body was stimulated by a woody stick. If the beetle showed
 immobility, the duration of this behaviour (until the beetle moved) was
 recorded by stopwatch. If the beetle did not show DF behaviour by stimuli,
 the stimulus was repeated up to three times for each beetle.
 On the next day of measurements of DF behaviour, each beetle from
 the stock culture was put into a plastic container (height 15 mm, width 10
 mm) with food, and three treatments were performed (alone; male: n = 24,
 female: n = 24, cohabitation with L; male: n = 38, female: n = 48,
 cohabitation with S; male: n = 38, female: n = 48). In the alone
 treatment, each beetle kept alone for an additional day. In the
 cohabitation treatment, a beetle (focal beetle) was cohabitated with five
 beetles from L- or S-populations in the plastic container. There was no
 significant difference in DF duration among the treatments at phase Ⅰ. To
 identify focal and cohabitant beetles, the elytra of <em>T.
 castaneum</em> was marked with a paint marker following methods
 described in previous studies (Matsumura and Miyatake, 2015; Matsumura et
 al., 2021). To eliminate the effect of marking on results, I marked
 cohabitants rather than focal beetles. All containers were kept for 30
 days in an incubator maintained at 25℃ and 16:8 h photoperiod. Each
 container was filled with 1.0 g of food, enough for six beetles to live
 for one month. To avoid influences of copulation, beetles were separated
 by sex in each treatment. After treatment, measurements of DF duration for
 focal beetles were repeated (phase Ⅱ).
 <strong>Statistical analysis</strong>
 To test the effects of treatment and cohabitated beetles on the
 DF duration, generalized linear mixed models (GLMMs) with gamma
 distribution (Ishikawa et al., 2023; Matsumura and Miyatake, 2023), phase,
 treatment, sex, and interactions of these factors as explanatory
 variables, beetle ID as a random effect were used for all DF duration
 data. The GLMM with the same model was also used to analyze the DF
 frequency (binary data of whether or not shown DF). I used chi-square test
 for each significance test. When a significant effect of interaction among
 factors was found, multiple comparisons using Bonferroni correction were
 performed. All analyses were conducted using R version 4.1.0 (RCoreTeam,
 2017) and statistical packages lme4 (Bates et al., 2015) and car (Fox and
 Weisberg, 2018).
description: # Synchronising anti-predator behaviour in the red flour beetle Tribolium
 castaneum **Description of the data and file structure** A *T. castaneum*
 stock culture, maintained for over 30 years, was used. Beetles were reared
 on yeast-enriched wholemeal at 25℃ under a 16:8 h light-dark photoperiod.
 Sex was determined at the pupal stage by abdominal morphology, and
 single-sex groups were stored until experiments. L- and S-populations with
 long and short death feigning (DF) durations were established and
 maintained for over 30 generations (Miyatake et al., 2004; Matsumura and
 Miyatake, 2018). Despite 20 years, the populations still show large
 differences in DF duration. In phase I, DF duration was measured in virgin
 beetles (21–35 days old) using a stick stimulus, recording immobility
 duration. On the next day, beetles were placed in plastic containers with
 food for three treatments: alone, cohabitation with L-population, and
 cohabitation with S-p **File:**
 Synchronising_anti-predator_behaviour_in_the_red_flour_beetle_Tribolium_castaneum.xlsx consists of column A, “treatment”; column B, “sex”; column C, “individual id”; column D, “duration of death feigning in phase I”; and column E, “duration of death feigning in phase II”, respectively.