Quantification of Drosophila melanogaster Grooming Behavior for Evaluation of Excessive Grooming Phenotypes

Summary

The method presented here involves the manual annotation of footage acquired of Drosophila melanogaster for specific grooming behaviors. It allows for quantification of both the number of grooming bouts and overall time spent grooming to evaluate for atypical self-grooming phenotypes.

Abstract

Observable changes in stereotyped grooming are applied translationally in model organisms. These changes are representative of pathologies that elicit similar deviations in human behavior; for example, excessive grooming acts as a proxy for obsessive and compulsive behaviors present in conditions like Tourette Syndrome or obsessive-compulsive disorder. The grooming assay presented allows for the evaluation of abnormal self-grooming phenotypes in Drosophila melanogaster. Flies are recorded for a period of 10 min, and these recordings are observed and annotated blind for previously defined grooming behaviors. Quantitative measures of both grooming bout frequency and the time spent engaging in self-grooming can be obtained by manually annotating the footage. The assay is relatively inexpensive, requires few materials not already available in laboratory environments, and is easily adaptable to fit the specific needs of any given study aiming to observe grooming. Additionally, the low level of skill needed to perform the assay, as compared to computer science-heavy automated methods, makes the protocol well-suited for small labs and students. We discuss in detail the steps required to perform this assay and its present limitations.

Introduction

Drosophila melanogaster is a well-established model organism in behavioral and neurobiological studies, providing insight into mechanisms driving analogous human behaviors. Self-grooming in this organism is a highly regulated and well-defined behavior, following stereotypical patterns that are easily distinguished from one another¹. The separate grooming behaviors exhibited by the fly can generally be classified by anatomical region², most easily being defined as posterior or anterior. Drosophila grooming will initially focus on the anterior region and subsequently transition to the posterior end

Protocol

NOTE: An overview of the protocol is presented in Figure 1.

1. Preparation for filming

1. Place four three-well dishes side by side on a white surface, covering each with a glass slide.
2. Position a camera (here, a Raspberry Pi system, but any camera capable of high-definition video is sufficient) above the dishes.
   1. Check to ensure that all dishes are in frame and focus.
   2. Check to ensure that there is no glare on the slides that will limit the visibility of flies in the dish. Eliminate the glare by adjusting the plate position or dimming the lights in the r....

Results

This assay produces quantitative data measuring the time and frequency of grooming behaviors from annotated footage. A representative image of the setup and how behaviors are defined is outlined in Figure 2. Given the subjectivity introduced by video analysis, all annotations of videos should be blinded to the researcher performing the analysis.

This method was used to evaluate the role the Drosophila gene Atg8a has in the development of grooming.......

Discussion

Overall, this easily executable and cost-effective assay allows for robust characterization of Drosophila melanogaster grooming behavior. This technique gives insight into the frequency, time spent engaging in, and anatomical distribution of a large number of previously identified grooming behaviors. A good indicator that grooming will occur or is underway is changes in leg placement, specifically raising any of the 6 legs. Taking note specifically of behaviors after leg movement can assist in identifying groomi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
16 GB Micro SD card	Adafruit	1294	For use in introducing the OS
Display	Adafruit	3578	Any display able to be connected by HDMI or flex cable is sufficient
Drosophila Atg8a-RNAi UAS line	Bloomington Stock Center	34340	Line used in representative data
Drosophila Ok6 Driver	N/A	N/A	Line used in representative data
Glass slides	Fisher Scientific	12-550-A3	Keeps flies in containment
Heatsinks	Adafruit		Prevents overheating of the computer
Lens	Adafruit	4563	Used with HD camera only
PiCamera	Adafruit	4561	The HD version was used here but a standard camera can be used under budget constraints
Raspberry pi 4	Adafruit	4292	Computer PiSpy is run on
Ribbion Cable	Adafruit	1648	For use in connecting camera and display
SB components Case	Adafruit	4301	Protects the computer
Spot Plate	Fisher Scientific	S99406	Pyrex versions were already avaliable for our use but cell culture dishes or plastic spot plates are sufficient as well
Tripod	Best Buy	6355959	For suspension and positioning of camera, any apparatus capable of this is sufficient
USB C Power Supply	Adafruit	1995	Supplies power to the computer