Fig. 4.
Behavioral analysis of rumpel. (A–E) 150 third instar larvae of the respective genotypes were recorded in groups of 15 animals for 3 min at 25°C or 32°C, as indicated. Larvae were always placed at the middle of the tracking plate. (A–D) For heatmap analyses, the 2048×2048 px image of the agar plate is divided in 50×50 px squares. The number of larval appearances per square is determined and indicated in blue shading using R. Darker blue colors indicate less frequent appearance, while lighter blue ones more. (A) Heatmap analysis of control w1118 larvae at 25°C. Wild-type larvae crawl in every direction and spread evenly on the agar plate at 25°C (indicated by fewer lighter blue squares). (B) Heatmap analysis of rumpelC40 larvae. rumpelC40 larvae shows wild type-like distribution on the agar plate at 25°C (indicated by similar number of lighter blue squares). (C) Wild-type larvae spread evenly on the agar plate at 32°C. (D) At 32°C rumpelC40 larvae spread less on the agar plate (indicated by more light blue squares in the middle). (E) Quantification of the mean distance to origin of wild-type versus rumpelC40 larvae at 25°C and 32°C. At 25°C no significant difference is indicated (Mann–Whitney U-test P>0.05, n=150). Mean distance to origins of wild type and rumpelC40 are 439.4 and 381.4 px, respectively at 32°C. Wild-type larvae spread significantly more on the agar plate at 32°C compared to rumpelC40 larvae (Mann–Whitney U-test P=0.023, n=150). (F) To monitor the effects of rumpel on sleep behavior rumpelΔ+cherry flies were backcrossed 10 times to white1118. The activity of 40 flies was tracked over 7 days in the ethoscope (Geissmann et al., 2017). (G) rumpelΔ+cherry flies sleep significantly more during the day (P=2e-16, Wilcoxon rank sum), whereas night sleep is not affected. (H) Heat shock assay of 5-day-old male and mated female flies of wild type [Canton S], [GMR83E12-Gal4AD; repo-Gal4DBD, UAS-GFPdsRNA], [UAS-rpr; GMR83E12-Gal4AD; repo-Gal4DBD, UAS-hid] (each genotype n=100). Flies are heat shocked in a water bath for 2 min at 40°C and were immediately recorded at room temperature. Not moving flies lying on their back are considered as paralyzed. Recording was stopped after 240 s. Error bars indicate standard deviation. (I) Average sleep time over seven days summarized for 24 h (shown in %). Flies lacking ensheathing glia show an increased day time sleep compared to the control. (J) Summary of the fraction of time sleeping over 7 days (shown in %). Loss of ensheathing glia leads to an increased sleeping time during the day (P=9.991e-07, Wilcoxon rank sum). (K) The rapid iterative negative geotaxis (Ring) assay (Gargano et al., 2005) shows the climbing ability of females with the genotypes: [GMR83E12-Gal4AD; repo-Gal4DBD], or [GMR83E12-Gal4AD; repo-Gal4DBD, UAS-GFPdsRNA], or [UAS-rpr; GMR83E12-Gal4AD; repo-Gal4DBD, UAS-hid]. The age of tested flies is indicated. Flies are heat shocked in a water bath for 2 min at 40°C and immediately recorded at room temperature for 240 s. Non-moving flies lying on their back are considered to be paralyzed. Both control and ensheathing glia ablated flies show a similar age-related decline of locomotor abilities. P-values are: 5-day-old flies: P pEG>+ / EG>GFPdsRNA=0.0014, P pEG>+ / EG>rpr,hid=0.0009, P EG>GFPdsRNA / EG>rpr,hid >0.9999, 12-day-old flies: P EG>GFPdsRNA / EG>hid=0.0043. All other P-values are >0.05=non-significant (ns). Error bars indicate standard deviation. Quantification was done using a two-way ANOVA multiple comparison. (L) Longevity assay. 200 males and 200 virgin females of the genotypes indicated were kept on sugar only food. rumpel mutant males live 28% shorter than w1118 control flies, rumpel mutant females live 8% shorter than w1118 control flies (Pmales=2.43× e-34; Pfemales=2×e-9).