After a few days of feeding in agar-molasses food vials, Drosophila larvae liquefy the upper food layer. To reach the deeper and potentially higher-quality food, single larvae make brief dives and breathe through an air tunnel created by their descent. Such tunnels, though, can easily collapse or be destroyed by the passage of other larvae. Cooperative behavior emerges within groups of larvae as a more efficient way of digging. Under crowded conditions, wild-type Canton S (CS) larvae congregate to form organized groups, termed here “clusters,” all oriented with their breathing spiracles directed up and their heads down, and make coordinated dives ( Figures 1 A and S1 A; Movie S1 ). By pulling a large common meniscus, they can often continue feeding at the lower half of the vial for many hours before group breakups occur ( Figure 1 B). All observed cluster breakups occur when access to air is lost. This coherent motion is observed to rise and fall a few body lengths every few minutes, and animals appear to be moving closely together (CS clusters move 1.12 ± 0.22 mm every 176 ± 44 s; n = 37; see Figure S1 B). As this appears to be a model example of cooperative behavior, further experiments were carried out to examine its properties.

(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.

(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.

(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.

(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.

Clusters were defined as groups of more than 4 larvae pulling a meniscus down more than half a body length from the surface ( Figure 1 A). In a vial, clusters are quantified when pressed against the clear plastic where only the front row of larvae is counted. Clusters can be identified from above as a cavity. A cluster in a vial is estimated to contain 10–100 larvae. In vials with about 50 egg-laying adults, cluster frequency was measured using the average number of clusters per vial, against the vial side, counted 3 times a day, and assayed over 4 weeks. Cluster frequency was measured over 2 weeks and compared to the depth of liquefaction ( Figure S1 C), which was measured by evaluating the darkening of the agar ( Figure 1 A). Clustering was observed to closely correlate with the liquefaction depth and to decrease after 2 weeks as vials began to dry out ( Figures S1 C and S1D). To further understand the dynamics of clusters that form and break up continuously in vials, we conducted a directed genetic screen and quantified the clustering properties of several mutants using the parameters described above ( Figure 1 C shows the average clustering frequency for each mutant). The results show that a transgenic control background Rh5-GAL4 and the smell-blind mutant orcoboth clustered with frequencies similar to CS, whereas the morphological mutant tubby, which has a round body shape, failed to cluster. The mechanosensory mutant nompC clustered less frequently as did three independent blind mutants, norpA, GMR-hid1, and GMR-hid2. This means that vision and mechanosensation play important roles in clustering. Gustation [] and IR-class olfaction [] were not tested. In addition, vials of CS, cultured in the dark, also clustered less frequently ( Figure 1 C). Because of experimental tractability, the contribution of vision to clustering was chosen for further study.

To control for the process of food liquefaction and for larval number, we “pre-processed” agar vials with about 100 CS larvae produced over 24 hr from 50 adults and incubated them up to the time when pupation started. Vials were frozen for 24 hr so that all resident larvae died. This produced a vial with about 50% liquefaction. Adding 200 s instar (L2) larvae back to such a pre-processed vial produces robust clustering ( Figure S1 D). Clustering frequency was found to peak at the end of the larval stages, independently of the age at which animals are added, and also depends on the number of larvae added ( Figure S1 E). The contribution of vision was reexamined using these pre-processed vials, and cluster formation frequency was again greatly reduced in visually impaired larvae ( Figure 1 D). Compared to wild-type CS, two transgenic white-minus backgrounds (GMR-GAL4 and UAS-NaChBac) clustered normally. Crossing two of these backgrounds produces larvae that are visually compromised due to excess activity in visual neurons []. Blocking vision using UAS-NaChBac resulted in few clusters (3 clusters out of 3 vials observed over 4 weeks, with resulting frequency per vial = 0.05 ± 0.03, n = 58). Decreased clustering was also observed in two independent lines where photoreceptors were ablated (GMR-hid1, 2) as well as in blind NorpAmutants and in wild-type CS reared in the dark ( Figure 1 D). These results support a role of vision in clustering behavior. We measured digging movement frequency of CS larvae in the light and in the darkness ( Figure S1 F). Our data indicate that no significant differences in the number of backward digging movements per minute are observed between two groups of larvae. Therefore, the absence of light does not affect general digging activity in larvae but at the same time causes alterations in clustering. Interestingly, we observed that once clusters have formed in visual mutants, they tend to survive just as long as wild-type ( Figure 1 E). However, the distribution of cluster lifespans, for wild-type and blind larvae, is a skewed distribution, indicating a more complex dynamical process ( Figure S1 F). Given that visually compromised larvae have reduced cluster frequencies but similar cluster lifespans, when compared to wild-type, vision appears to be important for cluster initiation.

While clusters in vials probably represent a closer experimental model to how this behavior occurs naturally on rotting fruit, only the front row of larvae of a cluster can be imaged in this experimental scenario ( Movie S1 ). In addition, because not all larvae can be monitored, the percentage in clusters or average digging depth cannot be measured. To visualize all larvae and obtain these measurements, we developed 2D configuration ( Figure 2 A). 30 larvae from a pre-processed vial are added to pre-processed liquefied food sandwiched between two glass slides (see Figure S2 A for a general outline of experimental setup). Clusters form within minutes ( Movie S2 ), and both wild-type and blind (GMR-hid1) larvae in 2D configuration show similar movements as those in vials ( Figure S2 B). However, similar to vials, few clusters form for blind larvae (GMR-hid1). Cluster formation was quantified for 2D by measuring percentage of larvae in clusters. Digging depth was also measured by calculating the average depth of all larvae in the food. Digging depth and average cluster membership is reduced for blind larvae when compared to wild-type ( Figures 2 B, S2 C, and S2D). However, similar to vial-formed clusters, blind clusters, once formed, survive about as long as wild-type (CS: 363 ± 139 min, n = 20; GMR-hid1: 241 ± 184 min, n = 36, p = not significant [ns]; see Figure S2 E). As clusters tend to break up when the sides of the tunnels collapse, lifespan is probably also dependent on the mechanics of the food in addition to larval coordination.

(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.

(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.

(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.

In previous experiments, larvae were found to go through an early third instar critical period in which they acquire visual recognition of movements of other larvae []. To test the role of early visual experience in clustering, we isolated larvae at L2, grew them up to the middle of L3, and grouped them in the 2D apparatus. These isolated larvae rarely cluster when compared to their socially reared sibs ( Figures 2 B and S2 C). As isolation could affect either visual or mechanosensory development, we performed two specific critical-period rearing experiments to distinguish between the two possibilities. First, we reared CS larvae in pre-processed vials in complete darkness until late L3 stage and then tested them for clustering. This approach would allow larvae to obtain mechanosensory-based, but not visual, experience. When these larvae were tested, both percentage of larvae in clusters and digging efficiency were reduced ( Figures 2 B and S2 C), but not to the level of total isolation. We next tested group rearing in a thin layer of processed food in a Petri dish and in the light. This preserves all conditions of a processed vial except that the flat arrangement means that clustering cannot happen and larvae should lack group mechanosensory experience. These animals also showed reduced digging and percentage of larvae in clusters but at a more moderate level ( Figures 2 B and S2 C) than visually deprived larvae. This indicates that the group experience likely involves both mechanosensory and visual component, though the impact of the latter seems to be more significant. To further examine the role of vision and experience on clustering, we labeled individual larvae from wild-type clusters, blind larvae (GMR-hid1), or those reared in isolation with food-coloring ( Figure 2 C) and placed them into preformed 2D wild-type or blind clusters. All larvae almost immediately joined a cluster over which they crawled. Once in a cluster, the labeled larvae were followed to determine how long they spent as members. Wild-type larvae spent more time in wild-type clusters than did any of the other combinations, indicating that vision and experience contribute to more stable cluster membership ( Figure 2 D). The role of vision raises the hypothesis that general cluster stability is related to how individual larvae use sensory cues to coordinate their movements in relation to each other. We therefore further examined the dynamics of individual larvae in clusters.

Role of Inter-larval Coordination in Clustering

6 Slepian Z.

Sundby K.

Glier S.

McDaniels J.

Nystrom T.

Mukherjee S.

Acton S.T.

Condron B. Visual attraction in Drosophila larvae develops during a critical period and is modulated by crowding conditions. Figure 3 Inter-larval Coordination within Clusters Show full caption (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. ∗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 2 B. 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 2 D). 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 Higher-resolution videos of neighboring larvae were examined to extract information about the movements between individuals. To analyze larval movements, we determined head and tail position in time using ImageJ kymograms, which allowed us to identify positions of larval front and rear ends (see Figures S1 B, S2B, and S3A). A Python-based machine vision for larval tracking was used to measure positions over time (see Figure S3 E). The infrequent and smaller contractions during the downward motion are contrasted by longer and vigorous contractions during the backward crawling (see rising and diving phases in Figure 3 A). In backward crawling, larvae move about 1/3 of their body lengths in each contractile cycle (see kymogram in Figure S3 A and Movie S3 ). Because of this, only upward movements were analyzed. The first objective was to determine whether the backward larval locomotion cycle was different between single larvae and those in clusters and how vision affected this movement. Locomotion cycles were measured, in 2D and 3D, for single larvae, pairs, and clusters. Visually impaired larvae (GMR-hid1) were also examined in 2D clusters. Each upward locomotion cycle, just over 2 s long ( Figure S3 B), was observed to be consistent across genotypes, numbers of larvae, and spatial configuration. The contraction cycle can be subdivided into a compression phase, in which a contractile wave begins at the anterior thorax and moves posterior and ends with spiracle withdrawal ( Figure S3 B), followed by upward extension, and finally a variable delay before the next cycle. When compared to 2D grouped larvae, times are similar except that 3D grouped compression and extension phases are faster ( Figure S3 C). This might be related to the fact that in 3D, larvae are moving more against each other and less against the glass surface as in 2D configuration. Visually compromised individual larvae have the same contractile parameters as CS. Finally, in a free-range crawling test in a 10 cm Petri dish with no distinct visual cues [], visually compromised larvae move the same distance as CS ( Figure S3 D). The conclusion from these experiments is that visual function does not measurably affect locomotion and that the general backward locomotion cycle is not affected by vision, group size, or the spatial environment.

P41 mutants are indistinguishable from the spaced control. The background strains are not different from CS (GMR-GAL4 and UAS-NaChBac). When imaged with a dim red light, which Drosophila larvae do not sense, CS larvae are as uncoordinated as their blind counterparts ( To measure how closely larvae coordinated their movements, the posterior positions of individual larvae were tracked. In 3D vial clusters, only larvae of the front row were tracked, while the 2D configuration allowed tracking of all larvae simultaneously ( Figure S3 E). The timing differences of spiracle retractions between three adjacent larvae were measured in runs of 2–3 backward contractions. The differences in timing of spiracle contraction for wild-type larvae were compared between the middle larva and its left and right neighbor ( Figure S3 F). As a negative control where no temporal linking is expected, the timing differences between three single larvae, spaced out and undergoing backward locomotion, were measured. The average time difference, expressed as a fraction of the 2 s normal contraction cycle time, was plotted for different pairs as a relative temporal difference. For the separated control, the smallest time difference between two of the three neighbors was chosen and corresponds to about 1/3 of a cycle with high variation ( Figure S3 F). In contrast, the closest neighbor in time for 2D and 3D clusters is significantly less than the control. While one neighbor is paired closely in time, the other neighbor is no more paired than the negative control. This suggests that for any larval contraction, only one other larva, either to the left or to the right, is closely following in time with its own contraction. When the same measures were performed for the blind (GMR-hid1) larvae, no significant differences were observed between negative control (spaced larvae) and any combination of the neighbors ( Figure S3 G), suggesting that vision plays a key role in regulating close timing between neighbor contractions. Larval coordination was further measured for 3D clusters of different genotypes using the same method ( Figure 3 B). Compared to CS, visually compromised larvae (GMR-GAL4 > UAS-NaChBac) show over twice the time delay, and blind NorpAmutants are indistinguishable from the spaced control. The background strains are not different from CS (GMR-GAL4 and UAS-NaChBac). When imaged with a dim red light, which Drosophila larvae do not sense, CS larvae are as uncoordinated as their blind counterparts ( Figure 3 B). These data again suggest that larvae are dependent on vision to match the timing of contractions between larval pairs. Similar measures were performed for 2D clusters, and results were consistent with previous data ( Figure 3 C). Importantly, wild-type larvae reared either in isolation or in the darkness are also as uncoordinated as the blind counterparts, while larvae reared in a thin layer of food show no significant differences with the wild-type ( Figure 3 C). This indeed suggests that prior visual rather than mechanosensory experience is specifically important for neighbor movement coordination. We next analyzed the timing differences for transplanted larvae. CS larvae transplanted into CS are indistinguishable from non-transplanted CS larvae within clusters ( Figure 3 C). Transplanting does not change this temporal coordination. However, all other combinations break down this coordination. This indicates that coordination of timing is likely a mutually timed behavior and requires two-way signaling between any larval pair. To further examine the relationship between movement coordination and cluster formation, we have measured the relationship between time disparities and percentage of larvae in clusters as well as cluster residing time ( Figures S3 H and S3I). Our data show that these parameters are closely related, indicating that high coordination of neighbors’ digging movements determined by visual cues is a key to cluster size and stability.

11 Stamps J.A.

Yang L.H.

Morales V.M.

Boundy-Mills K.L. Drosophila regulate yeast density and increase yeast community similarity in a natural substrate. 12 Fischer C.N.

Trautman E.P.

Crawford J.M.

Stabb E.V.

Handelsman J.

Broderick N.A. Metabolite exchange between microbiome members produces compounds that influence Drosophila behavior. 13 Giuggioli L.

Potts J.R.

Rubenstein D.I.

Levin S.A. Stigmergy, collective actions, and animal social spacing. 14 Lihoreau M.

Clarke I.M.

Buhl J.

Sumpter D.J.

Simpson S.J. Collective selection of food patches in Drosophila. 15 Ramdya P.

Schneider J.

Levine J.D. The neurogenetics of group behavior in Drosophila melanogaster. 1 Alexander R.D. The evolution of social behavior. 2 Noë R. Cooperation experiments: coordination through communication versus acting apart together. 13 Giuggioli L.

Potts J.R.

Rubenstein D.I.

Levin S.A. Stigmergy, collective actions, and animal social spacing. 16 Fleury F.

Ris N.

Allemand R.

Fouillet P.

Carton Y.

Boulétreau M. Ecological and genetic interactions in Drosophila-parasitoids communities: a case study with D. melanogaster, D. simulans and their common Leptopilina parasitoids in southeastern France. 17 Carton Y.

Sokolowski M.B. Interactions between searching strategies of Drosophila parasitoids and the polymorphic behavior of their hosts. 18 Carton Y.

David J.R. Relation between the genetic variability of digging behavior of Drosophila larvae and their susceptibility to a parasitic wasp. Our experiments show that clustering emerges at late stages of larval development and requires, among other senses, vision and social experience. Clustering may serve a number of functions in larvae. The simplest role is to dive to better food when the surface has become liquefied. This is a likely scenario on rotting fruit where the complex and dynamic microbial milieu can both influence and be managed by Drosophila larvae []. Larval clustering may serve to tightly manage certain microbial communities into distinct beneficial patches, which might also provide an organizational role, or stigmergy []. The sharing of these constructed patches should require some cooperation such that the generated common resource is appropriately distributed []. Finally, up to 90% of wild Drosophila larvae can be infected with parasitoid wasp eggs [], and digging depth provides protection from these lethal attacks []. Cooperation provides a clear way to dig deeper and is therefore likely to provide a selective advantage in avoiding parasitoid wasps.