Animals can be permanently attached to a substrate in aerial environments at certain stages of their development. Pupa adhesion has evolved multiple times in insects and is thought to maintain the animal in a place where it is not detectable by predators. Here, we investigate whether pupa adhesion in Drosophila can also protect the animal by preventing potential predators from detaching the pupa. We measured the adhesion of Drosophila species sampled from the same area and found that pupa adhesion varies among species, which can be explained by different glue production strategies. Then, we compared attached and manually detached pupae in both field and laboratory assays to investigate the role of pupa adhesion to prevent predation. First, we found that attached pupae remain on site 30 % more than detached pupae in the field after three days, probably because they are less predated. Second, we observed that attached pupae are less efficiently predated by ants in the laboratory: they are not carried back to the ant nest and more ants are needed to consume them onsite. Our results show that pupa adhesion is crucial for Drosophila fly survival and can prevent the animal from being taken away by predators.

Figure S1. Distribution of the buckets in Bois de Vincennes. Each circle represents one bucket. Dashed lined circles indicate unused buckets and solid lined circles buckets used for the experiment. The number in the circle indicates the ID of the bucket. Crosses indicate the location of the trees. Orientation of the location is indicated in the top left corner.

Table S1. List of Drosophila flies collected in Bois de Vincennes. The number corresponds to the total number of individuals collected at each site. Traps were emptied twice, two days and five days after traps were set up in the field. GPS coordinates are shown for each trap and for the compost.

Table S2. Adhesion force and pupa-substrate interface measurements. Results of the adhesion assay performed on pupae from D. suzukii, D. simulans and D. hydei strain originating from Vincennes. Sample_ID column corresponds to a unique identifier for each pupa, Temperature_assay, Humidity, Pressure_mba indicate respectively the room temperature (°C), the room humidity (%) and the atmospheric pressure (mba) at the moment of the assay. date_measurement and time_measurement correspond to the day and hours of the assay. date_substrate and time_substrate correspond to the day and hours at which larvae were put on the substrate. force_detachment_mN corresponds to the maximum force reached during the experiment in mN. Comment_on_this_sample reports particular observations:  the pupa did not detach from the substrate (“not_detached”), the pupal case broke during the assay (“cuticle_broke”) or nothing special happened during the assay (“ok”). Area_px corresponds to the pupa-substrate contact surface measured in pixel and scale_px, scale_mm correspond respectively to the scale present on the picture in pixel and mm.

Table S3. Pupal size measurement. Measurements of the size of pupae of D. simulans, D. suzukii, D. hydei. ID column corresponds to a unique identifier for each pupa. Area corresponds to the area obtained by measuring the contour of the pupa in pixel. scale_px and scale_mm are defined as in Table S2.

Table S4. Pupal count in the field experiment. Results of the experiment performed in the field. bucket_ID column corresponds to a unique identifier for each bucket. Orientation_C and Orientation_NC give, respectively, the orientation of attached and detached pupa within the bucket. Count_C and Count_NC indicate, respectively, the number of attached and detached pupa in a dish. Time indicates the time at which pupae were counted: t0 after 0h (8 September 2020, day 1 morning, at 11 am) t1 after 6h30 (day 1 PM), t2 after 22h30 (day 2 AM), t3 after 31h (day 2 PM), t4 after 47h (day 3 AM), and t5 after 54h (day 3 PM).

Table S5. Animals observed in the dishes during the experiment with half-buried buckets in Bois de Vincennes. The number indicates the number of dishes where the respective animals were observed. In total, 56 dishes were examined at each time point.

Table S6. Ant count during predation assay in the laboratory. The number indicates the number of ants observed in direct contact with the pupa at each time point. The column “X_Y” corresponds to the count over one experiment with the ant colony X at the trial Y.

Table S7. Results table of the predation assay in the laboratory. Each row corresponds to the description of the experiment for one pupa. condition column indicates the initial state of the pupa (“attached” or “detached”), strategies indicates whether the pupa was brought to the nest over the duration of the experiment (“to_nest”) or was never brought to the nest and consumed outside the nest (“on_site”). Time corresponds to the time at which the pupa was brought to the nest or fully consumed outside the nest in min. max_ant corresponds to the maximum number of ants observed in contact with the pupa over the duration of the experiment. orientation indicates whether the pupa was initially on the left or on the right side of the glass slide at the beginning of the experiment. first_ant corresponds to the time in min at which the first interaction between the pupa and an ant is observed. max_ant_at_first_done corresponds to the maximum number of ants observed in direct interaction with the pupa from the beginning of the experiment to the time when the first pupa is brought to the nest or fully consumed outside the nest.

Table S8. Pupal count in the field experiment. Same as Table S4 except that one line corresponds to one individual. Status is either 0 (pupa still present) or 1 (pupa disappeared).

R script. Drosophila_glue_predation.R: R script used to prepare the figures and run the statistical tests.

pupa_prints: Folder containing pictures of the prints left by the pupae on glass slides after detachment during our adhesion assay force measurements.

pupal_size: Folder containing the pictures of pupae used to measure the size of the pupae of the different species.

Pictures_Videos_Vincennes: Folder containing pictures and videos taken during and after the experiments in the field.

Pictures_Videos_predation_lab: Folder containing pictures and video of the predation assays in the laboratory