Abstract
Here we describe a simple method to measure larval muscle contraction and locomotion behavior. The method enables the user to acquire data, without the necessity of using expensive equipment (Rotstein et al., 2018). To measure contraction and locomotion behaviour, single larvae are positioned at the center of a humidified Petri dish. Larval movement is recorded over time using the movie function of a consumer digital camera. Subsequently, videos are analyzed using ImageJ (Rueden et al., 2017) for distance measurements and counting of contractions. Data are represented as box or scatter plots using GraphPad Prism (©GraphPad Software).
Keywords: Locomotion, Video analysis, Drosophila melanogaster, Crawling, Muscle contraction
Background
It is well known that despite other factors, the composition of the surrounding extracellular matrix (ECM) is of great relevance for proper organ functionality. Changes in its composition can ultimately lead to organ malfunction or failure. Using the muscles of third instar wandering larvae as a model, we can assess the influence of the concentration of single ECM proteins on meshwork flexibility or strength with larval locomotion behavior as a readout. In general, this is also possible for first or second instar larvae, but we decided to use wandering third instar larvae due to their large body size. For precise aging of Drosophila larvae, we refer to the descriptions given by Demerec (1950).A wide range of methods for measuring Drosophila melanogaster larvae crawling has arisen in recent years. Techniques such as FIM2c (Risse et al., 2017) enable the user to study locomotion in a detailed manner; however, they require a specialized set of equipment. The simple test described herein allows the recording of differences in crawling speed and muscle contractibility with tools that can most likely be found in every laboratory or students classroom. The method is based on a protocol published by Nichols et al. (2012). However, instead of using agar-filled plates, we conducted our experiments in humidified, empty Petri dishes. Thereby, we eliminate the problem of larvae digging into the agar, which can negatively influence the measurement of Z-direction crawling, leading to a loss of information.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Notes
Acknowledgments
We would like to thank Mareike Panz, Benjamin Hallier, Ronja Schiemann and Heiko Harten for initial experiments with this method. We also thank Caldwell et al. (2003) and Nichols et al. (2012) who published an earlier version of this method and the SFB 944 for funding. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study. All authors declare no conflicting or competing interests.
References
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