Drosophila stocks and maintenance A total of 127 lines of the Drosophila Genetic Reference Panel (DGRP) (doi:10.1101/GR.171546.113.) were obtained from Bloomington Drosophila Stock Center (NIH P40OD018537. The flies were maintained on a standard Drosophila oatmeal-sugar-yeast-agar medium (7 mL) at 20 °C, and a 12:12 h light/dark photoperiod (light on from 08:00a.m. to 08:00p.m.) for two generations before experimental start. The flies were pre-fed with additional dry yeast on the surface of the Drosophila medium before oviposition to generate flies used for the experiments. Experimental setup Experimental flies were produced from three replicate vials per line (100 DGRP lines were used). In each replicate vial, 15-20 randomly selected adult flies, comprising both males and females, were permitted to lay eggs for four 12-hour periods, with the flies being transferred to new vials every 12 hours. Within 48 hours of eclosion, flies were sexed under CO2 anesthesia and males were transferred to new vials with fresh food. Given the size of the experiment, it was not feasible to include both sexes. Moreover, since we cannot be certain that our experimental animals are virgins, we decided to use males because females allocate more energy into egg production compared to males’ less resource-expensive sperm production. Thus, including a mix of mated and unmated females in our study would introduce variation that we cannot account for. The problem is less with males and therefore we decided to use males. When the male flies were five days (± 24 h) old, 16 individuals from each line were transferred to 5 mm polycarbon tubes (TriKinetics, US) containing a droplet of Drosophila standard medium sealed with parafilm to prevent the media from drying out in one end and with a moist 5 mm long pipe cleaner in the other end. This was repeated on 3 experimental days. A total of 16 male flies per DGRP line were phenotypically assessed across 50 Drosophila Activity Monitors (DAM2, TriKinetics, US) on each of the three experimental days. The monitors quantify movement of animals over time by counting the number of times an animal crosses a laser centered at the middle of the polycarbon tube at a fixed time interval. The monitors were placed at an outdoor roofed field-site (57°00'52.6"N, 9°59'04.5"E) for 24 hours at three different experimental days in the Danish summer 2022 (May 4th, May 24th, and June 7th) where temperature and relative humidity differed markedly within and between days. Processing of locomotor activity The DAM monitors return the individual counts of infra-red laser beam breaks per 30 seconds. We refer to the count of breaks per time unit as the activity. Individuals showing no activity during field monitoring were considered dead and discarded from the analysis. The field activity was summarized for each individual by the total number of laser beam breaks per 20 minutes for the 24-hour monitoring period. In each comparison across experimental test days, we restricted subsequent statistical analyses to overlapping data points by restricting to activity data obtained between 10:00a.m. to 07:20a.m. the following morning (i.e., a total of 65 20-min bins). Besides the total activity per 20 minutes, we also computed the total activity across the entire experimental setup (the sum of activity across each 20-minute bin). For the subsequent statistical genetic analyses, the activity data was approximated to a Gaussian distribution by inverse-rank normalization.

In nature, organisms are exposed to variable and occasionally stressful environmental conditions. Responses to diurnal and seasonal fluctuations, such as temperature and food accessibility, involve adaptive behavioral and physiological changes. While much work has been done on understanding the genetic architecture and evolutionary potential of stress tolerance traits under constant thermal conditions, there has been less focus on the quantitative genetic background in variable environments. In this study, we use the Drosophila Genetic Reference Panel (DGRP) to investigate locomotor activity, a key behavioral trait, under variable natural thermal conditions during the summer in a temperate environment. Male flies from 100 DGRP lines were exposed to natural thermal and light conditions in Drosophila activity monitors across three experimental days. We found that activity was highly temperature- and time-dependent and varied between lines both within and between days. Further, we observed variation in genetic and environmental variance components, with low to moderate estimates of the heritability for locomotor activity, consistently peaking in the afternoons. Moreover, we showed that the estimated genetic correlations of locomotor activity between two time points decreased as the absolute differences in ambient temperature was increased. In conclusion, we find that the genetic background for locomotor activity is environment specific and we conclude that more variable and unpredictable future temperatures will likely have a strong impact on the evolutionary trajectories of behavioral traits in ectotherms.

# Data from: The Drosophila Genetic Reference Panel (DGRP) on locomotor activity across different environmental conditions ## Description of the data and file structure The data set contains individual measurements of a single fly's locomotor activity (summed in bins of 20 min) across 24 hours for three different experimental test days. The data set contains 9 columns (see description below) and 344,000 rows (corresponding to individual observations). The file is a comma seperated file (*.csv) `lineID` internal lab individual DGRP ID `RAL_full` DGRP id `stock` Bloomingstock line ID `datetime` indicator for date, and time of day the 20-min bins belong to. `batch` indicator variable for which of the three experimental test day the observation belong to. `diff` indicator variable of the 20-min bins ranging from zero to 1460. `tube` indicator variable of the individual DAM2 glass tubes. `Monitor` indicator variable of the DAM2 monitor ID. `act_sum` The sum of activity (number of crossings in the DAM2 tubes) within each 20-min bin.