Cooperative Behavior Emerges among Drosophila Larvae Mark Dombrovski, Leanne Poussard, Kamilia Moalem, Lucia Kmecova, Nic Hogan, Elisabeth Schott, Andrea Vaccari, Scott Acton, Barry Condron  Current Biology  Volume 27, Issue 18, Pages 2821-2826.e2 (September 2017) DOI: 10.1016/j.cub.2017.07.054 Copyright © 2017 The Authors Terms and Conditions

Figure 1 Vision Is Required for Clustering (A) Typical larval cluster. All larvae feed with heads down to the edge of the liquid phase (darker layer) and breathing spiracles at rear and inserted into the air cavity. A typical cluster will have 10–100 larvae and can last for many hours. (B) A larval cluster rapidly breaking up when larvae lose access to air. (C) Summary of cluster frequency (measured for “crude” vials in the original directed genetic screen), averaged for days 5 to 25 after hatching, for a number of genotypes. The bars represent the average, and errors bars represent the SEM. Number of observations are shown in bold numbers for each genotype. (D) Summary of cluster frequency after 200 L2 larvae are placed in a pre-processed vial. Indicated are the averages, and error bars represent the SEM. Number of observations are shown in bold numbers for each genotype. (E) Summary of cluster lifespans, measured for crude vials and pre-processed vials (both wild-type and blind GMR-hid1 larvae, including side and top view). Cluster lifespan time and error were derived from average clustering frequency. Indicated are the averages, and error bars represent the SEM. Number of observations are shown in bold numbers for each genotype and condition. Statistical significance was calculated by ANOVA using Tukey’s method for (C)–(E): ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figure S1 and Movie S1. Current Biology 2017 27, 2821-2826.e2DOI: (10.1016/j.cub.2017.07.054) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Properties of 2D Clusters (A) An example of a larval cluster in a 2D configuration. Two wild-type clusters (indicated with arrows) form within an hour after transplantation in pre-processed food. (B) Properties of clusters in 2D configuration. Blue bars represent the average digging depths of 30 or 15 wild-type larvae, blind (GMR-hid1) larvae, and wild-type larvae that were flat reared, dark reared, or reared in isolation. Depths are expressed as percent distance into 38 mm of pre-processed food averaged over all larvae. Both blind and isolated larvae, as well as larvae reared in darkness and in a thin layer of food, display reduced digging efficiency similar to 15 wild-type larvae. Red bars represent cluster formation efficiency expressed as percentage of larvae in clusters. Both blind and isolated wild-type larvae, along with larvae reared in the darkness and in a thin layer of food, display significantly reduced percentage of larvae in clusters. Indicated are the averages, and error bars represent the SEM. Bold numbers represent the number of measures. Statistical significance was calculated by ANOVA using Tukey’s method: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. (C) Example of a single larva transplantation experiment. Individual larvae of different genotypes were placed in blue food-colored pre-processed food, then washed with water, and placed over an established cluster of a given genotype in the 2D apparatus. (D) Residing time of transplants. Individual larvae of a given genotype were transplanted into a cluster, and their residing time was measured. Wild-type into wild-type is the most stable combination. Indicated are the averages, and error bars represent the SEM. Bold numbers represent the number of measures. Statistical significance was calculated by ANOVA using Tukey’s method: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figure S2 and Movie S2. Current Biology 2017 27, 2821-2826.e2DOI: (10.1016/j.cub.2017.07.054) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Inter-larval Coordination within Clusters (A) Different phases of larval coordinated movements within a cluster. During the down phase (left), larval spiracles pull the meniscus. During the rising phase (middle) occurring every 2–4 min, larvae shuffle up alongside each other by exhibiting coordinated backward contractions. Visually impaired (GMR-hid1) larvae form smaller clusters with poorly coordinated movements (right). (B) Measures of the timing of spiracle contractions between individual larvae in 3D clusters in pre-processed vials (samples were measured in the front end of a cluster in a vial). In each case, three adjacent larvae were chosen, and for each contraction of the middle larva, the next contractions of the left and right neighbors are measured. Indicated are the averages, and standard errors with numbers of measures are shown in bold. As a negative control, “CS separated” represents three separated and independently backward-crawling larvae in a vial, and the timing shown is the closest to the middle animal. Visually impaired larvae (NorpAP41, conditional mutants GMR-GAL4 > UAS-NaChBac and wild-type in the darkness) all display significantly increased time disparities between neighbors’ movements. Indicated are the averages, and error bars represent the SEM. Statistical significance was calculated by ANOVA using Tukey’s method: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. (C) Measures of the timing of spiracle contractions between individual larvae in 2D clusters. All measurements were performed using the same approach described for Figure 2B. Consistent with data from 3D clusters, visually impaired larvae (GMR-hid1) display significantly increased time disparities and so do wild-type larvae grown in isolation or reared in the darkness. Larvae reared in a thin layer of food display an intermediate phenotype. In addition, same measurements were done for individually transplanted larvae (same combinations described in Figure 2D). CS larvae transplanted into CS clusters behave the same way as non-transplanted CS, while all other transplant combinations display significantly decreased time disparities. Indicated are the averages, and error bars represent the SEM. Statistical significance was calculated by ANOVA using Tukey’s method: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figure S3 and Movie S3. Current Biology 2017 27, 2821-2826.e2DOI: (10.1016/j.cub.2017.07.054) Copyright © 2017 The Authors Terms and Conditions