Avalilação PREreview de Drosophila learn to prefer immobile spherical objects through repeated physical interaction

Summary:

In this study, the authors investigate how Drosophila fruit flies interact with spherical objects using a new behavioral paradigm. They provide compelling evidence supporting their main claims and include appropriate controls. The study demonstrates that using a visual stimulus to guide flies to the vicinity of a spherical object (ball) increases both the probability and duration of interaction with the object. The authors further provide solid evidence that flies employ a set of different behavioral motifs to interact with the object, and that the mixture of these motifs changes over time. They also present compelling evidence that flies exhibit different interaction levels with a movable versus immovable ball. Finally, they report on silencing experiments screening for the role of different hΔ central complex neurons in the behavioral motifs exhibited during interaction with objects. This very important study lays the groundwork for a mechanistic understanding of the neural substrate controlling object interaction behaviors in Drosophila.

Strengths: The manuscript is very well written, with clear structure and language. The claims are substantiated and supported by appropriate controls, minimizing potential confounds.

 Weaknesses: Some minor adjustments in figure labeling and captions, along with clarifications in methodology and the main text, would help the readers better appreciate the reported results. Specific suggestions are provided below.

 Major points:

• The authors quantified the flies' preference for the mobile and the immobile ball by counting the number of contacts with the ball per visit (lines 186–191). A contact event was registered when the fly-to-ball distance reached zero. However, it is unclear whether the tracking software was tracking the fly's centroid or also the position of the legs. If only the centroid was tracked (as is typical for most tracking software), the flies could have been touching the ball with their legs and getting feedback about its mechanical properties before a contact event was registered. The interpretation of the data shown in Figures 4D-G depends on this detail, so clarifying exactly how a zero distance was defined in the methods section would help the readers better appreciate the figures.

• The finding that flies interacted little with the ball during the first few hours of the experiments is intriguing. It would be valuable to include a discussion of the possible reasons. Does this initial period reflect a familiarization and learning phase, or a motivational state that changes after some time in the arena? An earlier version of the preprint included an additional plot (Figure S3) showing that the animals' locomotion speed followed a similar temporal trajectory, increasing after a few hours. This pattern would suggest a change in motivation state, with the flies ignoring the visual guidance stimulus for the initial period. A full analysis of this would be beyond the scope of this study, but a discussion of authors’ observations about this salient feature would be valuable.

• In lines 107–108, the text states: “Flies were not required to engage with the object and could leave without penalty.” However, one possible interpretation of the data is that the flies avoided the rotating pinwheel stimulus. The only location where the pinwheel was turned off was at the ball and a 5-mm zone around it. Upon leaving this area, the rotating pinwheel was projected again. If the flies perceive the rotating pinwheel as aversive, then it would constitute a penalty for leaving the ball vicinity. In this case, it would be preferable to remove the statement that the flies could leave “without penalty”. Alternatively, the authors could design a control experiment to test whether the flies avoided or followed the rotating pinwheel stimulus. For example, one or more rotating pinwheels could be projected at random locations in an empty arena (remaining in place instead of tracking the fly), and the occupancy of the arena surface by the fly over time could be analyzed. If the regions with projected rotating pinwheels are less occupied than other areas, it would suggest aversion to the stimulus. If the rotating pinwheels are not less occupied the statement that the flies were not penalized for leaving the ball vicinity could be justified. Another possible control would be to delay turning on the rotating pinwheel stimulus until the fly moves further away from the ball than the 5-mm zone used in the current experiments. If the visual stimulus is not aversive, the number of contacts per visit should remain similar to the currently reported values.

Minor points:

• In a few figures, the labeling could be made more descriptive to help readers interpret the presented data. Specifically, consider replacing “Interaction score” with “Ball displacements per visit” in Figure 1F, and with “Contacts per visit” in Figures 4F–G, 5E–G.

• In Figures 3B and 3C, there appears to be a discrepancy between the y-axis label, “Event #,” and the figure caption. Please clarify whether these panels report counts or frequencies.

• Although not necessary, it would be interesting for the authors to comment on potential reasons for the sharp dip in the score early in the experiment (around the 8th–10th visit) shown in Figure 5G.

• In Figure S1A, please define in the figure caption what the “active time” percentage displayed on the x-axis represents.

• In Figure S5H, the plot does not show a pronounced dip like the one in Figure 5H. The caption mentions a dip, which could be revised for consistency with the plot.

• In line 140: Looking at Figure 2B, the wording could perhaps be clarified as “with varying degrees of fly displacement”.

Finally, a more detailed discussion of the study’s limitations, especially regarding the silencing of the hΔ neurons, would further strengthen the already well-presented manuscript.

Competing interests

The authors declare that they have no competing interests.