Peer review of: Behavioral and neuronal underpinnings of safety in numbers in fruit flies (2019)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
Ferreira and Moita present a behavioral and neurogenetic analysis of the impact of group size in 
Drosophila on the probability that an individual animal will exhibit a defensive freezing behavior in 
response to a threatening (looming) visual cue. The authors demonstrate that when in larger groups 
(i.e., higher density), individual flies spend less timing in a freezing state in response to such a cue, 
because of higher probability of exiting this state. They provide evidence that this is due to detection 
of motion of other flies in the group, mediated, at least in part, by the LC11 visual projection neurons 
(previously shown to respond physiologically to small moving objects). 
 
This is an original, fascinating, rigorously analyzed and clearly presented study. The results will be of 
general interest because of its wider implications for the social influences on individual animal 
behavior, and the potential of future exploitation of this tractable system to understand the 
neurobiological underpinnings of this phenomenon. 
 
I have only a few minor comments: 
 
Figure 1B: the grouping of behavioral responses into three categories (i.e., 1 fly, 2-5 flies and 6-10 
flies), as opposed to a linear impact of group size (animal density) is intriguing. Can the authors 
comment on what might define the apparently threshold between groups of 5 flies and groups of 6 
flies? 
 
Line 90: typo in “were” 
 
Figure 2A: I don’t understand the color key; in the plot there are orange bars and dark orange bars, 
but the key indicates orange, black and grey. 
 
Figure 5B: the Kir2.1 control parental line show higher freezing behavior than in panel A (and the 
control Gal4 line). Is this just a fluke of this set of experiments? 
 
The use of auditory cues as defense signals in other species made me wonder whether this sensory 
modality contributes to social regulation of freezing behavior in flies. (As a precedent, analysis of 
courtship has shown a male responds to auditory cues from the footsteps of females (PMID: 
18802468)). While visual cues are most likely to be the most important signal, can the authors rule in 
or out other modalities? (No experiments are needed, but they could comment on this in the 
Discussion). 
 
Discussion: this feels a bit long - the conclusions and broader implications of the work would come 
across stronger with a bit of pruning. For example, lines 216-231 are more background material 
(which might fit better in the (currently brief) introduction), and in lines 257-267, the highlighting of 
potential parallels between fly and visual processing feels a bit labored (and not really key for this 
paper). 
 
Line 320: the web-link given is uninformative (I was expecting to have access the software). 
 
 
 
Reviewer #2: 
Remarks to the Author: 

In the manuscript by Ferreira and Moita, authors investigate how freezing behavior in response to 
looming stimuli is regulated in groups using Drosophila melanogaster as a model organism. They first 
show that freezing response of flies decrease with increase in group size. This decrease is not a 
general unresponsiveness to a looming stimulus, but due to an increase in the probability of stopping 
freezing. Using fly-sized magnets, authors show when objects surrounding a fly transition from 
moving to freezing, that individual fly is more likely to freeze as well. Based on their results, authors 
hypothesized that motion cues from other flies or surrounding objects might mediate exit from 
looming-triggered freezing. To test this hypothesis, they formalized the motion signals perceived by a 
focal fly using three parameters; speed, size, and distance. Then, they showed that focal flies which 
had higher motion cue values were more likely to resume movement. To further examine the effect of 
motion cues on freezing, they manipulated the motion cues in the focal fly by using blind flies, that are 
insensitive to the looming stimulus and therefore do not change walking behavior during stimulus 
presentation. They found that the amount of time a fly froze decreased when surrounded by blind 
flies, compared to that of groups of wild-types, suggesting flies at least in part use motion cues of 
others to decide whether to stay frozen or exit freezing. Finally, they found that a group of visual 
projection neurons, LC11, mediate group effect on freezing responses; silencing of LC11 neurons 
make flies less sensitive to the motion of others. 
 
Overall, I enjoyed reading the manuscript and I find the results interesting. This paper shows that flies 
in groups display a reduction in freezing responses that scales with the group size. However, I am not 
certain the reduction in freezing in groups is due to only motion sensing. There might be 
proprioceptive or chemosensory cues that are involved in the inhibition of looming responses that are 
not addressed in the manuscript. 
I have major and minor points indicated below. 
 
Major points: 
1. In figure 1, authors showed that flies in groups of 6 to 10 were less likely to enter to a freezing 
state compared to individuals and groups of 2 to 5 flies. The difference between groups of 2-5 and 
groups of 6-10 is a step wise increase rather than a gradual difference. Is this an artifact of the 
chamber size or is this a biologically relevant result? If authors increase the size of the chamber would 
they expect different results in their assays? 
 
2. In supplementary figure 2, authors investigate whether mechanosensory interactions contribute to 
freezing behavior and found minor impact of collisions on freezing behavior. Based on these 
preliminary tests, authors conclude that freezing behavior is not related to mechanosensation. I think 
these experiments are a good start however they need to be expanded to exclude the possibility that 
any proprioceptive feedback is not involved in this behavior. Authors also ignore, any chemosensory 
signal that can impact this behavior. In many species, alarm pheromones play a key role in directing 
group behavior to predators or threats in the environment. These experiments are simple, fly mutants 
are available to test impact of olfactory or taste signal on freezing behavior in fly groups. I think 
authors either need to conduct these experiments or explain in the text why they exclude the 
possibility that chemosensation or proprioception can regulate freezing behavior. 
 
3. In figure 5, authors showed that inhibiting LC11 neurons by Kir2.1 expression reduces group effect 
on looming-triggered freezing behavior and they argue that LC11 neurons are involved in processing 
motion cues of other flies to regulate freezing response. In these experiments Kir2.1 is expressed 
throughout the development and LC11 neurons have been constantly hyperpolarized. These 
experiments need to be repeated by silencing LC11 neurons only in adults using temporal inactivation 
of these neurons with available reagents such as temperature controlled shibire. In addition, they can 
also activate LC11 neurons and examine whether freezing behavior will be suppressed in individual 
flies when LC11 neurons are active during looming stimulation. If LC11 neurons indeed mediate 

freezing responses in grouped flies, activation of these neurons should induce the opposite phenotype 
caused by neural silencing. 
 
4. In figure 5B, there is a significant difference between two control groups in freezing behavior; LC11 
split-GAL4 and UAS-Kir2.1. What is the source of this variation in freezing behavior? Did they observe 
any behavioral or genetic problems in the LC11 split-GAL4 strain? Please clarify. 
 
Minor points: 
1. Please revise the gray color scheme in Figure 2. It is very hard to distinguish different experimental 
groups especially in Figure 2C. Also, please indicate the control groups in the figures more clearly. 
Stopped magnets and freezing magnets sounds too similar and confusing for the reader. 
 
2. In the line 279, authors mentioned that they used mated females for their experiments. Mating 
could influence various aspects of animal behaviors including foraging, egg laying, courtship, and 
receptivity. Would they expect differences in freezing behavior if virgin females or males are used? 
 
3. Authors use blind flies in the Figure 4, but they do not describe the genotype or the strain used in 
the methods. Please indicate which blind fly strain is used in the paper. 
 
 
 
Reviewer #3: 
Remarks to the Author: 
Drs. Ferreira and Moita Show, for the first time, changes in defensive behaviour of fruit flies that are 
depending on group size. Authors used a looming stimulus that would elicit escape jumps in open 
space and freezing in the enclosed environment used here. Flies were tested individually or in groups 
of 2 to 10 flies. Individual flies froze most often, groups of 6 to 10 flies least often, groups of 2-5 flies 
were intermediate. Freezing occurs within a 1s time window after stimulus onset and peaks around 
0.5s. It is argued that this is enough time for taking freezing of other individuals into account. The 
interdependence is statistically shown and elegantly demonstrated in a setup with one real and four 
dummy flies that could be moved and stopped (frozen) at will. The higher the summed retinal speed 
caused by the other flies, the less likely a test fly will enter freezing, and the more likely a frozen test 
fly will give up freezing. This is nicely demonstrated on a seeing fly with four blind companions, who 
do not freeze upon looming. Finally, lobula columnar neurons LC11 were inactivated using 
neurogenetic tools. This measure does not affect the reaction of individuals’ freezing upon looming 
stimuli. However, it reduces the probability of exiting freezing. Authors conclude that LC11 neurons 
are involved in the perception of the motion of companion flies. Overall, the findings are novel, 
substantiated by data and will find the interest of a large community. 
 
Major 
Fig.3 B right side x-y-plots. Y-axis labels are missing and plots are not explained in the legend. What 
is the difference (despite x-axis range)? Shouldn’t the 3rd contain the 2nd from -4s to 0s and the 2nd 
the first from -0.6s to 0s?. If this is “Summed motion signal”, then what is the unit? How many flies 
are summed up? 
 
Discussion is missing on the evidentiary value of the neurogenetic experiments. Kir/+ controls are 
vastly different in 5A and 5B. Why that discrepancy? Suppl.S5 Kir/+ control looks like 5B, not like 5A 
(same data set as 5B?). The split-GAL4 line shown in Fig.5 does contain LC-11 as one component. 
Then there might still be an expression outside LC11 causing the changes in behaviour. 
 
Minor 

Method: The description of the setup does not say whether the flies can walk under the ceiling of the 
chamber (could be prevented by coating). The behaviour is different when the flies’ actions are 
restricted to the floor. 
 
Error bars in all figures and suppl. figures are not defined in the legends. 
 
Fig.3 D left/right. I suggest using an additional letter E. 
L.30 “…wide prevalence, the…” (comma) 
L.358 to stay frozen 
L.368 variables βi and βs are not definded.