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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

The present protocol assesses the locomotor activity of Drosophila by tracking and analyzing the movement of flies in a hand-made arena using open-source software Fiji, compatible with plugins to segment pixels of each frame based on high-definition video recording to calculate parameters of speed, distance, etc.

Abstract

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and diverse behavioral features. Identifying behavioral deficiency in animal models is a crucial measure of disease severity, for example, in neurodegenerative diseases where patients often experience impairments in motor function. However, with the availability of various systems to track and assess motor deficits in fly models, such as drug-treated or transgenic individuals, an economical and user-friendly system for precise evaluation from multiple angles is still lacking. A method based on the AnimalTracker application programming interface (API) is developed here, which is compatible with the Fiji image processing program, to systematically evaluate the movement activities of both adult and larval individuals from recorded video, thus allowing for the analysis of their tracking behavior. This method requires only a high-definition camera and a computer peripheral hardware integration to record and analyze behavior, making it an affordable and effective approach for screening fly models with transgenic or environmental behavioral deficiencies. Examples of behavioral tests using pharmacologically treated flies are given to show how the techniques can detect behavioral changes in both adult flies and larvae in a highly repeatable manner.

Introduction

Drosophila melanogaster provides an excellent model organism for investigating cellular and molecular functions in neuronal disease models created by gene modification1, drug treatment2, and senescence3. The high conservation of biological pathways, physical properties, and disease-associated homolog genes between humans and Drosophila makes the fruit fly an ideal mimic from the molecular to the behavioral level4. In many disease models, behavioral deficiency is an important index, providing a helpful model for various human neuropathies5....

Protocol

W1118 adult flies and third instar larvae were used for the present study.

1. Experimental preparation

NOTE: An open-field arena for Drosophila locomotion tracking is made withacolorless and odorless silica gel.

  1. Mix reagent A and reagent B at a ratio of 1:10, according to the manufacturer's instructions for the silica kit (see Table of Materials). Ensure that sodium bicarbonate is added t.......

Representative Results

In the present study, locomotor deficits in adult flies and third instar larvae treated with rotenone were examined and compared in their motor activity to that of a control fly fed with the drug solvent dimethyl sulfoxide (DMSO). Treatment with rotenone in Drosophila has been shown to cause dopaminergic neuron loss in the brain22 and lead to significant locomotor deficits23. As shown in Figure 11 and Figure 12

Discussion

We have designed a method, based on the open-source material AnimalTracker API compatible with the Fiji image processing program, that can enable researchers to systematically evaluate locomotor activity by tracking both adult and individual larval flies. AnimalTracke is a tool written in Java that can be easily integrated into existing databases or other tools to facilitate the analysis of application-designed animal-tracking behavior24. Upon a frame-by-frame analysis by a softw.......

Acknowledgements

This work was supported by a special launch fund from Soochow University and the National Science Foundation of China (NSFC) (82171414). We thank Prof. Chunfeng Liu's lab members for their discussion and comments.

....

Materials

NameCompanyCatalog NumberComments
Animal trackerHungarian Brain Research Programversion: 1.7pfficial website: http://animaltracker.elte.hu/main/downloads
Camera softwareMicrosoftversion: 2021.105.10.0built-in windows 10 system
ComputerDELLVostro-14-5480a comupter running win 10 system is available
Drosophila carbon dioxide anesthesia workstationWu han Yihong technology#YHDFPCO2-018official website: http://www.yhkjwh.com/
Fiji softwareFiji teamversion: 1.53vofficial website: https://fiji.sc/
Format factory softwarePcfreetimeversion: X64 5.4.5official website: http://www.pcfreetime.com/formatfactory/CN/index.html
Graph pad prismGraphPad Softwareversion: 8.0.2official website: https://www.graphpad-prism.cn
Hight definition cameraTTQJingwang2 (HD1080P F1.6 6-60mm)official website: http://www.ttq100.com/product_show.php?id=35
Office softwareMicrosoftversion: office 2019official website: https://www.microsoftstore.com.cn/software/office
Petri dishBkman110301003size: 60 mm
Silica gelDOWSYLGARD 184 Silicone Elastomer KitMix well according to the instructions
Sodium bicarbonateMacklin#144-55-8Mix well with silica gel

References

  1. Ham, S. J., et al. Loss of UCHL1 rescues the defects related to Parkinson's disease by suppressing glycolysis. Science Advances. 7 (28), (2021).
  2. Algarve, T. D., Assmann, C. E., Aigaki, T., da Cruz, I. B. M.

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DrosophilaLocomotor ActivityOpen source SoftwareVideo TrackingBehavioral AnalysisSilica ArenaCamera SetupFiji SoftwareVideo ConversionTracking Parameters

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