# Ultrasound tethering of insects

Gaillard Thomas, Contreras Victor, Martinez Dominique, Viollet Stéphane

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Gaillard Thomas, Contreras Victor, Martinez Dominique, Viollet Stéphane. Ultrasound tethering of insects. Multidisciplinary Biomechanics Journal, 2025, 50th congress of the., 10.46298/mbj.16144. hal-05191696v2

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Distributed under a Creative Commons Attribution 4.0 International License
# Ultrasound tethering of insects

Thomas Gaillard<sup>a*</sup>, Victor Contreras<sup>b</sup>,
Dominique Martinez<sup>a</sup>, Stéphane Viollet<sup>a</sup>

<sup>a</sup> Aix Marseille University, CNRS, Institute of Movement Sciences, Marseille, France

<sup>b</sup> Instituto de Ciencias Físicas, Universidad Nacional Autonoma de Mexico, Cuernavaca, Mexico

* Corresponding author: thomas.gaillard@univ-amu.fr

Received date: 06/04/2025

Accepted date: 27/06/2025

Publication date: 27/10/2025

**Keywords:** levitation, acoustic tethering, behaviour, fly, sensorimotor reflexes

© 2025 The Authors
Licence CC-BY 4.0
Published by Société de Biomécanique

## 1. Introduction

Studying sensorimotor response of flying or walking insects is usually done in tethered conditions (Verbe et al., 2020). Attaching an insect to a tether allows precise control of stimulus delivery and measurements of the steering response from the movements of the wings or legs (Minter et al., 2018). Yet, a rigid tether acts as mechanical constraints which may cause substantial behavioural artefacts such as unnatural wing kinematics and body posture. To overcome this issue, an ultrasound technology for insect tethering based on an acoustic levitator could be used (Marzo, Barnes, & Drinkwater, 2017). It relies on the emission of ultrasonic waves on both sides of the device, creating standing waves and forms pressure nodes that enable levitation (Contreras & Marzo, 2021). This affordable and accessible technology enables living insects to levitate in a stable manner during a long period of time and prevents them from flying away. We demonstrate its efficiency using hoverflies *Episyrphus balteatus* (Gaillard et al., 2025). We also show that it can be used with other insect species such as *Drosophila* and ants. By allowing insects to fly and rotate on the spot without any mechanical constraints, acoustic tethering could be considered as a new tool for behavioural analysis.

## 2. Methods

### 2.1 Acoustic levitator

The acoustic levitation is achieved by producing focused standing waves inside a cavity made of two arrays of 42

compact ultrasonic sources (piezoelectric transducers) (Fig.1.A). The 42 ultrasonic sources, operating with a frequency (f) = 40kHz, were embedded in 3D-printed spherical segments (SS). The ultrasonic sources were arranged in the SS by divided them into two identical halves, producing nodal planes along the axis of symmetry, achieving stable confinement of diverse samples of irregular shapes. The SS have vertical opening to allow optical access to the levitated samples and horizontal openings at top and bottom to allow the insects to fall freely.

### 2.1 Behavioural test

The hoverflies, *Episyrphus balteatus* were maintained by a tether in the centre of the levitator inside a white diffused box. The test was divided into 5 periods of 30 seconds with ultrasound and 5 periods of 30 seconds without ultrasound. The order was determined randomly. To study the response of the fly to the presence of ultrasound, we tracked the motion rate of three natural markers: motion rate of the legs, abdomen and tip of the wings. We also counted the number of wingbeat initiation, the latency before the first initiation of wingbeat and the duration of the first wingbeat sequence.

## 3. Results and discussion

To compare behaviour with and without ultrasound, we performed a PCA by considering the markers and the number of wingbeats to discriminate between the two classes, with and without ultrasound (Fig.1.B).
The PCA projection reveals no statistical difference (p-value > 0.05).

Concerning the effect of ultrasound on the three markers, our statistical models show an effect of ultrasound only for the wing motion rate (LMM wing motion rate; p-value=0.0393; adj.R2=0.5005).

Concerning the wingbeat analysis, we started by counting the initiation of wingbeat. Our model does not show any significant difference between the ultrasound and non-ultrasound groups (GLMM Poisson; p-value > 0.05; adj.R2=0.9424). We then studied the latency of the first initiation of wingbeat. The cox survival analysis does not show significant differences (Mixed effects cox model; p-value > 0.05). At last, we studied the duration of the first wingbeat sequence, our GLMM shows a significant difference between the two groups (GLMM Gamma; p-value < 0.001; adj.R2=0.6964).

Our behavioural tests showed that the levitator had little effect on the behaviour of the hoverflies: only the wing motion rate and the first wingbeat duration are higher. This difference might be caused by the fact that the hoverflies were not exactly between the pressure nodes when the ultrasound was activated/deactivated. This unstable position could lead to greater wing movements, which is not the case when the insect levitates.

Then, we wondered whether it would be possible to use the latter with different insect species. To answer these questions, we tested the levitator with several insects. We placed two fruit flies (*Drosophila*,<1mg) in two different pressure nodes (Fig.1.E). The largest insect levitated here was a dead bee weighing 58.6mg. The technique also was highly effective in levitating various living insects in a stable manner (right side up or upside down) without them flying away or struggling erratically: a German cockroach upside down (*Blattella germanica*, 10mg), an ant (7mg) (Fig.1.D) and the hoverflies used in the experiment (~30mg) (Fig.1.C).

## 4. Conclusions

We have shown here that acoustic levitation can be useful for levitating insects of various size (ranging from 1mm to 13.5mm) and various mass (ranging from less 1mg to 58.6mg) and levitation should not impact the behaviour of the hoverflies: the low impact on the flies' behaviour may be due to misalignment in the pressure nodes caused by the use of a mechanical tether in the experiments, which does not occur when the flies levitate freely.

<figure><img src="image_2.png" /><figcaption>**Figure 1.** Composite figure. (A) Photograph of the levitator. (B) PCA result (C) Photograph of a levitating hoverfly. (D) Photograph of a live levitating ant. (E) Photograph of two *Drosophila* in levitation.</figcaption></figure>

Credit: Gaillard Thomas (2024).

In further experiments, acoustic levitation will be used to prevent the fly from using their leg proprioception. The first tests will then be compared with the results of existing controls obtained from the literature (Verbe et al., 2020). Thus, further experiments will enable us to estimate fly's graviception during flight. Acoustic tethering certainly highlights new perspectives for insect behavioural studies.

## Acknowledgements

We are most grateful to Jean-Marc Ingargiola for developing the electronics. We are also very grateful to Leo Poughon and William Wilmot for their help in the implementation for the Schlieren imaging and the construction of the box.

## Conflict of Interest

None.

## Contributor Roles

Author contribution T.G. and S.V. drew up the research project; T.G. performed the experiments, T.G, D.M. and S.V. analysed the data; V.C. designed and made the levitator.

## Funding

This research was supported by CNRS, Aix Marseille University and the french defense agency for innovation (AID), Grant #2023940_IDEES.
The design and construction of the levitation sys-
tem was supported by UNAM through PAPIIT grant
IG101425

The editorial board thanks the authors for their
submission

Data, software, code availability

None.

References

Contreras, V., & Marzo, A. (2021). Adjusting single-axis acoustic levitators in real time using rainbow schlieren deflectometry. *Review of Scientific Instruments*, *92*(1). https://doi.org/10.1063/5.0013347

Gaillard, T., Contreras, V., Martinez, D., & Viollet, S. (2025) *JExp. Biol.*, submitted.

Marzo, A., Barnes, A., & Drinkwater, B. W. (2017). TinyLev: A multi-emitter single-axis acoustic levitator. *Review of Scientific Instruments*, *88*(8). https://doi.org/10.1063/1.4989995

Minter, M., Pearson, A., Lim, K. S., Wilson, K., Chapman, J. W., & Jones, C. M. (2018). The tethered flight technique as a tool for studying life-history strategies associated with migration in insects. *Ecological entomology*, *43*(4), 397–411. https://doi.org/10.1111/een.12521

Verbe, A., Varennes, L. P., Vercher, J. L., & Viollet, S. (2020). How do hoverflies use their righting reflex? *Journal of Experimental Biology*, *223*(13), jeb215327. https://doi.org/10.1242/jeb.215327