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

Review for “Drosophila learn to prefer immobile spherical objects through repeated physical interaction”

He Yang and Yoshinori Aso

Preprint doi: https://doi.org/10.1101/2025.06.09.658381

In this preprint, the authors used Drosophila as a model organism to study how animals physically interact with objects, taking advantage of its extensive genetic toolkit and accessibility. With a novel technique, they visually guided freely walking fruit flies to approach small balls and analyzed the subsequent behaviors for over 1000 times per fly. Flies displayed complex and structured patterns of interaction, which could be categorized into five distinct behavioral motifs. The ratio of these behavioral motifs shifted over time. Flies also developed a preference for immobile over mobile objects. They then examined the behaviors of flies expressing Kir2.1 with driver lines for different hD neuron types and observed driver-specific loss or enhancement of selective behavioral motifs and the preference to immobile balls. The evidence presented convincingly supports the conclusion that flies are capable of learning about the physical properties of objects and adjusting future decisions based on prior experience, thereby addressing the gap in the field to understand how flies interact with their environment.

Strengths:

The authors developed an innovative way to analyze fly-object interactions by employing the “pinwheel” cue to guide flies toward inanimate objects. This new approach enabled the authors to observe hundreds of object interactions for individual flies within a reasonable timeframe and how they developed over time.  The authors collected movies of nearly 30,000 interaction events and defined five behavioral motifs. This highly efficient way of data collection provided a novel framework for quantifying and comparing complex behavioral patterns across time and examining the effect of cell type-specific perturbation.

Weakness:

The main conclusion of this study is that flies develop object interaction motifs and learn to prefer immobile balls over time. The data presented in Figure 3 and Figure 5 convincingly show that flies change these behaviors over time. However, these changes could partly arise from changes in the physiological status of flies rather than learning physical properties of objects. As detailed below, providing more details on methods, experimental design and data interpretation would enhance the manuscript's clarity, reproducibility, and overall impact.

Major comments:

1.     In this behavioral assay, flies are forced to keep walking for ~18 hours until death in a chamber without food or water. The observed changes in patterns of interaction events (Figure 3) over time may therefore correlate not only with learning but also with the fly's physiological state - transitioning from an eager search for resources to eventual weakness. For instance, even guided flies take ~6 hours to start showing enhanced ball displacement (Figure 1C and D). This could be a period necessary for learning to interact with balls. Alternatively, flies may need to keep walking ~6 hours in response to pinwheel stimulus to change their status. One possible control experiment to rule out the latter possibility is to guide them to walk to a fixed point with pinwheel for 6 hours without a ball and then introduce a ball. If the latter scenario is true, flies without prior experience with a ball may show enhancement of ball displacement or preference to immobile balls immediately after the introduction of the ball. Another possible control experiment is to capture experienced flies and retesting them after feeding.

2.     In the methods, the authors described that a “contact” was defined as a period during which the fly maintained any physical contact with the ball, with a fly–ball distance of zero. Clarify how this fly-ball distance was defined in methods. Is it a distance between the surface of fly’s body and the surface of the ball? If flies could extend legs and touch a ball and examine the mobility of the ball from non-zero distance, Figure 5 data could be explained without hypothesizing that “flies may associate spatial locations with object properties, and that these associations could guide future interactions.” Instead, flies may have developed a quick way to access the mobility of balls with light touch from non-zero distance. To rule out this possibility, the authors may show analysis of leg-ball contact in a greater detail. One idea for an additional control experiment is to change the location of balls during the experiments. The authors could set up two mobile balls and two immobile balls in the arena. Initially flies are guided to one set of mobile and immobile balls until they develop stable preference to the immobile ball, and then they can be guided to another set of balls. If they need to form a new association between the location and the properties of the ball, it should take many trials to re-establish preference to the immobile ball.

3.     For Figure 6 and Figure 7, it is generally recommended to attribute behavioral phenotypes to a particular cell type by replicating the phenotype with multiple driver lines. Although these driver lines are highly specific to the hΔ neurons in adult brains, the observed phenotypes could be due to off-targeted expression during development or driver specific genetic backgrounds. It would also be a good practice to validate the expression of Kir-eGFP in the targeted cell types by immunolabeling and confocal microscope, because the expression patterns of 10XUAS-IVS-eGFPKir2.1 could be different from that of 20xUAS-CsChrimson in attP18 used by Wolff et al., 2025 (doi: 10.7554/eLife.104764).

Minor comments:

1.     The videos referenced in the text are currently missing from the preprint. Including them would greatly assist readers in appreciating the nuanced behaviors described. Overlaying segmentations of fly body parts and balls on movies would be also helpful to understand the definition of “fly-ball contact”.

2.     Various interaction scores (cumulative / mean / global) are used as important parameters to quantify fly-ball interactions. For improved readability, consider including a brief explanation of how these scores are calculated in the main text or figure legends (e.g., in Figure 1F-G and Figure 4G) when they are first introduced, even if the full details are in Methods.

3.     When introducing the “pinwheel”, the authors may include a very brief background introduction to explain that this is a projected visual cue, with which clockwise rotation guides the fly to turn right and counterclockwise rotation guiding left. This way readers can understand the basics of this key experimental component without checking the previous paper.

4.     The authors summarized a good review of how animals generally explore other objects in the environment in the Introduction. In either the Introduction or Discussion, the authors may consider discussing whether the preference for immobile objects is observed in any other animal to determine if the behavioral principles observed in flies are conserved across animals, thereby broadening the impact of this work within the field of animal behavior.

5.        Consider an alternative hypothesis about the contents of learning in Figure 5. The authors favored a hypothesis that “flies may associate spatial locations with object properties, and that these associations could guide future interactions”. Instead, flies may associate spatial locations with jumping, a potentially aversive action. Amin et al. (doi: https://doi.org/10.1101/2025.07.07.663268) reported that backward walking via moonwalker neurons can activate punishment-representing dopaminergic neurons and thereby induce aversive olfactory memory. Similarly, interaction with mobile balls may result in jump-induced activation of punishment pathway to promote avoidance of the location around the mobile balls. It would improve reader understanding if the authors could comment on the alternative hypotheses in the discussion.

Competing interests

The authors declare that they have no competing interests.